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Emine Ercikan Abali

The Cancer Institute of New Jersey, Robert Wood Johnson Medical School,
University of Medicine and Dentistry of New Jersey, New Brunswick,
New Jersey 08903
Yehuda G. Assaraf
To whom correspondence should be addressed; Email:
Carol E. Cass
Department of Experimental Oncology, Cross Cancer Institute, Edmonton,
Alberta T6G 1Z2, Canada and Department of Oncology, University of Alberta,
Edmonton, Alberta T6G IZ2, Canada
Hilal Celikkaya
The Cancer Institute of New Jersey, Robert Wood Johnson Medical School,
University of Medicine and Dentistry of New Jersey, New Brunswick,
New Jersey 08903
Karen E. Christensen
Montreal Childrens Hospital Research Institute, Montreal, QC, Canada H3Z 2Z3
James K. Coward
Departments of Medicinal Chemistry and Chemistry, University of Michigan,
3813 Chemistry, 930 N. University, Ann Arbor, Michigan 48109
Vijaya L. Damaraju
Departments of Experimental Oncology, Cross Cancer Institute, Edmonton,
Alberta T6G 1Z2, Canada and Department of Oncology, University of Alberta,
Edmonton, Alberta T6G 1Z2, Canada
Jeremy P. Derrick
Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, University of
Manchester, Manchester, United Kingdom
Chheng-Orn Evans
Department of Neurosurgery and Laboratory of Molecular Neurosurgery and
Biotechnology, Emory University School of Medicine, Atlanta, Georgia 30322
Michael Fenech
CSIRO Human Nutrition, Adelaide BC, Adelaide, South Australia 5000

xiv Contributors

Jennifer T. Fox
Graduate Field of Biochemistry, Molecular and Cellular Biology, Cornell
University, Ithaca, New York 14853
Zhanjun Hou
Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute,
Wayne State University School of Medicine, Detroit, Michigan 48201
Yi-Ching Hsieh
The Cancer Institute of New Jersey, Robert Wood Johnson Medical School,
University of Medicine and Dentistry of New Jersey, New Brunswick,
New Jersey 08903
Ilan Ifergan
The Fred Wyszkowski Cancer Research Laboratory, Department of Biology,
Technion-Israel Institute of Technology, Haifa 32000, Israel
Ann L. Jackman
Institute of Cancer Research, Section of Medicine, Sutton, Surrey, SM2 5NG
United Kingdom
Christopher P. Leamon
Endocyte, Inc., West Lafayette, Indiana 47906
David LaBorde
Department of Neurosurgery and Laboratory of Molecular Neurosurgery and
Biotechnology, Emory University School of Medicine, Atlanta, Georgia 30322
Zigmund Luka
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville,
Tennessee 37232
Robert E. MacKenzie
Department of Biochemistry, McGill University, Montreal, QC, Canada H3G 1Y6
Larry H. Matherly
Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute,
Wayne State University School of Medicine, Detroit, Michigan 48201
John J. McGuire
Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263
H. F. Nijhout
Department of Biology, Duke University, Durham, North Carolina 27705
Nelson M. Oyesiku
Department of Neurosurgery and Laboratory of Molecular Neurosurgery and
Biotechnology, Emory University School of Medicine, Atlanta, Georgia 30322
Contributors xv

Stephen W. Ragsdale
Department of Biological Chemistry, University of Michigan Medical School,
Ann Arbor, Michigan 48109-0606
M. C. Reed
Department of Mathematics, Duke University, Durham, North Carolina 27705
Michael B. Sawyer
Departments of Experimental Oncology, Cross Cancer Institute, Edmonton,
Alberta T6G 1Z2, Canada and Department of Oncology, University of Alberta,
Edmonton, Alberta T6G 1Z2, Canada
Thomas B. Shea
Center for Cellular Neurobiology and Neurodegeneration Research,
UMassLowell, Lowell, Massachusetts 01854
Nancy E. Skacel
The Cancer Institute of New Jersey, Robert Wood Johnson Medical School,
University of Medicine and Dentistry of New Jersey, New Brunswick,
New Jersey 08903
Patrick J. Stover
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853
Flaubert Tchantchou
University of Maryland, Baltimore, Maryland
Philip Thomas
CSIRO Human Nutrition, Adelaide BC, Adelaide, South Australia 5000
C. M. Ulrich
Cancer Prevention Program, Fred Hutchinson Cancer Research Center, Seattle,
Washington 98109
Congjun Yao
Department of Neurosurgery and Laboratory of Molecular Neurosurgery and
Biotechnology, Emory University School of Medicine, Atlanta, Georgia 30322

For many years, folic acid, its relatives, and antifolates have been the center
of much attention in one-carbon metabolism and in relation to cancer
research. So much progress has occurred in the last decade in relation to
cancer research and the mechanism of enzyme action as well as in the
mechanism by which folate is transported that the overall subject deserves
a current review.
We begin with contributions on one-carbon metabolism, the methio-
nine cycle, and folate deficiency. The first of these papers is from J. T. Fox
and P. J. Stover entitled Folate-mediated one carbon metabolism.
F. Nijhout, M. C. Reed, and C. M. Ulrich report on Mathematical
models of folate-mediated one-carbon metabolism. This is followed by a
treatise on Folate deprivation, the methionine cycle, and Alzheimers
disease by F. Tchantchou and T. B. Shea. Authors I. Ifergan and Y. G.
Assaraf complete the introductory section with Molecular mechanisms of
adaptation to folate deficiency.
The next group of papers deals with the folate transporter and receptor.
Structure and function of the reduced folate carrier: A paradigm of a major
facilitator superfamily mammalian nutrient transporter is contributed by
L. H. Matherly and Z. Hou. Authors V. L. Damaraju, C. E. Cass, and M. B.
Sawyer review Renal conservation of folates: Role of folate transport
proteins. C. P. Leamon and A. L. Jackman present Exploitation of the
folate receptor in the management of cancer and inflammatory disease.
Another report on cancer is given by C.-O. Evans, C. Yao, D. Leborde, and
N. M. Oyesiku: Folate receptor expression in pituitary adenomas: Cellular
and molecular analysis.
The third and final section concentrates on enzymes. In the first of these,
E. E. Abali, N. E. Skacel, H. Celikkaya, and Y.-C. Hsieh have written on
Regulation of human dihydrofolate reductase activity and expression.
S. W. Ragsdale covers Catalysis of methyl group transfers involving tetra-
hydrofolate and B12. Methyltetrahydrofolate in folate-binding protein
glycine N-methyltransferase is authored by Z. Luka. J. K. Coward and
J. J. McGuire review Mechanism-based inhibitors of folylpoly-g-gluta-
mate synthetase and g-glutamyl hydrolase: Control of folylpoly-g-glutamate
homeostasis as a drug target. P. Thomas and M. Fenech report on
Methyltetrahydrofolate reductase, common polymorphisms, and relation
to disease. This is followed by a contribution from K. E. Christensen
and R. E. McKenzie on Mitochondrial methylenetetrahydrofolate

xviii Preface

dehydrogenase, methylenetetrahydrofolate cyclohydrolase, and formylte-

trahydrofolate synthetases. The final treatise is on The structure and
mechanism of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase
by J. P. Derrick.
The structure on the cover comes from the Protein Data Bank and is,
1HFP, the assumed biological molecular structure of human dihydrofolate
reductase from V. Cody et al. (1998). Anti-Cancer Drug Des. 13, 307315.
After these manuscripts have been reviewed by the editor and assembled,
the Preface and front and back matter are prepared and forwarded to the
publisher. Then, with the guidance of Renske van Dijk and Tari Broderick,
the production process begins. In my view, Academic Press/Elsevier does a
commendable job of getting these volumes into print.

Gerald Litwack
Toluca Lake, California
December 8, 2007

Folate-Mediated One-Carbon
Jennifer T. Fox* and Patrick J. Stover*,

I. Overview 2
II. Introduction to Cytoplasmic One-Carbon Metabolism 4
A. Enzymes that generate one-carbon units 5
B. Folate-interconverting enzymes 13
C. Biosynthetic enzymes 18
D. Folate-binding proteins 24
III. Introduction to Mitochondrial One-Carbon Metabolism 24
A. Enzymes that generate one-carbon units 25
B. Folate-interconverting enzymes 27
C. Biosynthetic enzymes 28
IV. Nuclear Folate-Mediated One-Carbon Metabolism 28
Acknowledgments 29
References 29

Tetrahydrofolate (THF) polyglutamates are a family of cofactors that carry and
chemically activate one-carbon units for biosynthesis. THF-mediated one-
carbon metabolism is a metabolic network of interdependent biosynthetic path-
ways that is compartmentalized in the cytoplasm, mitochondria, and nucleus.
One-carbon metabolism in the cytoplasm is required for the synthesis of
purines and thymidylate and the remethylation of homocysteine to methionine.
One-carbon metabolism in the mitochondria is required for the synthesis of
formylated methionyl-tRNA; the catabolism of choline, purines, and histidine;
and the interconversion of serine and glycine. Mitochondria are also the primary
source of one-carbon units for cytoplasmic metabolism. Increasing evidence
indicates that folate-dependent de novo thymidylate biosynthesis occurs in
the nucleus of certain cell types. Disruption of folate-mediated one-carbon
metabolism is associated with many pathologies and developmental anomalies,

* Graduate Field of Biochemistry, Molecular and Cellular Biology, Cornell University, Ithaca,
New York 14853
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853

Vitamins and Hormones, Volume 79 # 2008 Elsevier Inc.

ISSN 0083-6729, DOI: 10.1016/S0083-6729(08)00401-9 All rights reserved.

2 Jennifer T. Fox and Patrick J. Stover

yet the biochemical mechanisms and causal metabolic pathways responsible

for the initiation and/or progression of folate-associated pathologies have yet
to be established. This chapter focuses on our current understanding of mam-
malian folate-mediated one-carbon metabolism, its cellular compartmentation,
and knowledge gaps that limit our understanding of one-carbon metabolism
and its regulation. 2008 Elsevier Inc.

I. Overview
The reduced tetrahydrofolates (THFs) serve as a family of enzyme
cofactors that chemically activate and carry one-carbon units on the N5 and/
or N10 of THF at the oxidation level of formate (e.g., 10-formylTHF),
formaldehyde (e.g., 5,10-methyleneTHF), or methanol (e.g., 5-methylTHF)
(Appling, 1991; Girgis et al., 1997; Schirch and Strong, 1989; Wagner, 1995).
Folate derivatives also contain a covalently bound polyglutamate peptide of
varying length. Serum folates contain a single glutamate residue, whereas
intracellular folates contain a polyglutamate peptide usually consisting of five
to eight glutamate residues that are polymerized through unusual g-linked\
peptide bonds (Moran, 1999; Shane, 1995). The polyglutamate pep-
tide increases the affinity of THF cofactors for folate-dependent enzymes
and-binding proteins, and prevents their efflux from the cell and intracellular
organelles (Schirch and Strong, 1989). THF polyglutamates are coenzymes
that donate or accept one-carbon units in a network of reactions known as
one-carbon metabolism that occurs in three specific and isolated cellular
compartments: the mitochondria, nucleus, and cytoplasm (Fig. 1.1; Porter
et al., 1985; Shane, 1989; Woeller et al., 2007a). The one-carbon forms of
THF can be interconverted enzymatically (Fig. 1.1), although each cofactor
form is specific to a particular biosynthetic pathway. The formyl group of
10-formylTHF is incorporated into the C2 and C8 of the purine ring
in the cytoplasm and is used to synthesize formylated methionyl-tRNA
in mitochondria (Fig. 1.1). The one-carbon moiety of 5,10-methyleneTHF
is required to convert uridylate to thymidylate, and the one carbon
carried by 5-methylTHF is required to remethylate homocysteine to
methionine. The cellular concentration of folate-binding proteins exceeds
that of folate derivatives, and therefore the concentration of free folate
in the cell is negligible (Schirch and Strong, 1989; Strong et al., 1990;
Suh et al., 2001). This implies that each folate-dependent biosynthetic
pathway competes for a limiting pool of folate cofactors (Scott et al., 1981;
Suh et al., 2001).
Epidemiological studies implicate impaired folate metabolism in several
pathologies and developmental anomalies including neural tube defects
(NTDs) (Scott, 2001; van der Put and Blom, 2000), cardiovascular disease
(Gerhard and Duell, 1999; Lindenbaum and Allen, 1995; Ueland et al., 2000),
Mitochondria Cytoplasm

THF 10
5,10-MethenylTHF 10-FormylTHF
Formate 10-FormylTHF Purines
fMet-tRNA 7 8 Formate 9
THF 13 16
Histidine 5-formylTHF
Purines 14,15 5,10-MethenylTHF
5,10-MethyleneTHF THF
THF 17
Dimethylglycine 4 1
Serine Thymidylate
3 2 Serine 5,10-MethyleneTHF
Glycine 16 19
Sarcosine glycine
Sarcosine Glycine 20
Glycine CO2,NH3 5-MethylTHF Methionine
Nucleus Homocysteine AdoMet
Sequestered AdoHcy
Glycine dUMP
16 19 Methylation reactions

Figure 1.1 Compartmentation of folate-mediated one-carbon metabolism in the cytoplasm, mitochondria, and nucleus. One-carbon
metabolism in the cytoplasm is required for the de novo synthesis of purines and thymidylate and for the remethylation of homocysteine to
methionine. One-carbon metabolism in mitochondria generates one-carbon units for cytoplasmic one-carbon metabolism by generating
formate from serine, glycine, sarcosine, and dimethylglycine. One-carbon metabolism in the nucleus synthesizes dTMP from dUMP and
serine. 1, Mitochondrial serine hydroxymethyltransferase; 2, Aminomethyltransferase; 3, Sarcosine dehydrogenase; 4, Dimethylglycine
dehydrogense; 5, 5,10-Methylenetetrahydrofolate dehydrogenase (NAD-dependent); 6, 5,10-Methenyltetrahydrofolate cyclohydrolase;
4 Jennifer T. Fox and Patrick J. Stover

and cancer (Ames, 2001; Blount et al., 1997; Choi and Mason, 2000; Kim,
1999; Pogribny et al., 1995). One-carbon metabolism can be impaired by
folate and other vitamin B deficiencies and/or common, penetrant genetic
mutations and polymorphisms (Bailey, 1995; McNulty, 1995; Scott, 1998;
van der Put and Blom, 2000). However, the biochemical mechanisms and
causal metabolic pathways responsible for the initiation and/or progression of
folate-associated pathologies have yet to be established. In fact, there are still
major gaps in our fundamental understanding of one-carbon metabolism and
its regulation, including the potential for identifying putative missing
enzymes and their associated genes whose discovery may be necessary to
complete the assembly of the folate-dependent metabolic network. This
chapter focuses on our current understanding of mammalian folate-mediated
one-carbon metabolism, its cellular compartmentation, and knowledge gaps
that limit our understanding of folate metabolism and its regulation.

II. Introduction to Cytoplasmic One-Carbon

Folate-mediated one-carbon metabolism in the cytoplasm is a meta-
bolic network of interdependent biosynthetic pathways that are required for
the biosynthesis of purines and thymidylate, and the remethylation of
homocysteine to methionine (Fig. 1.1). Methionine can be adenosylated
to S-adenosyl methionine (AdoMet), a cofactor, and methyl group donor for
numerous methylation reactions including the methylation of neurotransmit-
ters and other small molecules, phospholipids, proteins including histones,
RNA, and cytosine bases within CpG islands in DNA. Many AdoMet-
dependent methylation reactions, including those involved in chromatin
methylation, serve regulatory functions by affecting gene transcription
(Miranda and Jones, 2007), protein localization (Winter-Vann et al., 2003),
and the catabolism of small molecules (Stead et al., 2004). The sources of
one-carbon moieties for cytoplasmic one-carbon metabolism include
formate, serine, histidine, and purines.

7, Methionyl-tRNA formyltransferase; 8, 10-Formyltetrahydrofolate synthetase;

9, 10-Formyltetrahydrofolate synthetase; 10, 10-Formyltetrahydofolate dehydroge-
nase; 11 and 12, Phosphoribosylglycinamide formyltransferase and Phosphoribo-
sylaminoimidazolecarboxamide formyltransferase; 13, 5,10-Methenyltetrahydrofolate
cyclohydrolase; 14 and 15, Glycine formiminotransferase/formimidoyltetrahydrofolate
cyclodeaminase and Glutamate formiminotransferase/formimidoyltetrahydrofolate cyclo-
deaminase; 16, Cytoplasmic serine hydroxymethyltransferase; 17, Methenyltetrahydrofo-
late synthetase; 18, 5,10-Methylenetetrahydrofolate dehydrogenase (NADP-dependent);
19, Thymidylate synthase; 20, Methylenetetrahydrofolate reductase; 21, Methionine
synthase; 22, Glycine N-methyltransferase; 23, Dihydrofolate reductase.
Folate-Mediated One-Carbon Metabolism 5

Proteins involved in folate metabolism can be classified into four func-

tional categories, although many folate-dependent enzymes exhibit two or
more of these activities: (1) One-carbon generating enzymes, (2) THF one-
carbon interconverting enzymes, (3) THF-dependent biosynthetic enzymes,
and (4) noncatalytic THF-binding proteins (Table 1.1). This section details
the mechanisms, regulation, and physiological functions of the enzymes that
are involved in cytoplasmic one-carbon metabolism, as well as common
genetic variants that affect enzyme function and the one-carbon network.

A. Enzymes that generate one-carbon units

1. Cytoplasmic serine hydroxymethyltransferase
a. Reaction Serine hydroxymethyltransferase (SHMT) catalyzes the
reversible and PLP-dependent interconversion of serine and glycine. Mam-
mals express cytoplasmic (SHMT1) and mitochondrial (SHMT2) SHMT
isozymes; the human isozymes share 63% amino acid sequence identity
and are encoded on separate genes (Garrow et al., 1993). When catalyzing
serine cleavage, SHMT1 transfers the C3 of serine to THF generating
glycine and 5,10-methyleneTHF, the cofactor required for thymidylate
biosynthesis. The one-carbon moiety of 5,10-methyleneTHF can also
support homocysteine remethylation when converted to 5-methylTHF
by methylenetetrahydrofolate reductase (MTHFR) (Fig. 1.1). SHMT1-
derived one-carbons are not believed to make significant contributions to
purine biosynthesis because the reductive environment (NADPH/
NADP ratio) in the cytoplasm does not support the conversion of
5,10-methyleneTHF to 10-formylTHF (Christensen and MacKenzie,
2006). When catalyzing serine synthesis, SHMT1 depletes methyleneTHF
pools for AdoMet synthesis and regenerates unsubstituted THF for purine
biosynthesis (Herbig et al., 2002; Strong and Schirch, 1989). The SHMT1
may also play a role in gluconeogenesis; glycine is a glucogenic amino acid
through its conversion to serine, although it is not known which SHMT
isozyme functions in this capacity (Nijhout et al., 2006).

b. Mechanism The SHMT1 protein is a homotetramer consisting of two

obligate dimers. Residues from each subunit of the obligate dimer contrib-
ute to the formation of a single active site on each subunit, where Lys257 is
covalently bound to the PLP cofactor (Renwick et al., 1998; Schirch and
Szebenyi, 2004; Szebenyi et al., 2000). The definitive mechanism for the
reaction is still subject to debate (Schirch and Szebenyi, 2004). The pro-
posed retroaldol cleavage mechanism involves a base-catalyzed proton
abstraction from the C3-hydroxyl group of serine to form glycine and a
formaldehyde intermediate. The SHMT-bound formaldehyde then
Reactions catalyzed in folate-mediated one-carbon metabolism

Table 1.1

Folate-dependent enzymes EC No. Localization Reaction catalyzed

One-carbon activating
10-Formyltetrahydrofolate Cytoplasm ATP formate tetrahydrofolate ADP phosphate
synthetase 10-formyltetrahydrofolate
10-Formyltetrahydofolate Cytoplasm 10-Formyltetrahydrofolate NADP H2O
dehydrogenase tetrahydrofolate CO2 NADPH H
Cytoplasmic serine Cytoplasm and 5,10-Methylenetetrahydrofolate glycine H2O
hydroxymethyltransferase nucleus tetrahydrofolate L-serine
Glycine formiminotransferase Cytoplasm N-formiminoglycine tetrahydrofolate ) glycine
Glutamate Cytoplasm N-formiminoglutamate tetrahydrofolate ) glutamate
formiminotransferase 5-formiminotetrahydrofolate
Mitochondrial serine Mirochondrian 5,10-Methylenetetrahydrofolate glycine H2O
hydroxymethyltransferase tetrahydrofolate L-serine
Sarcosine dehydrogenase Mitochondrian Sarcosine acceptor H2O glycine formaldehyde
reduced acceptor
Dimethylglycine Mitochondrian N,N-dimethylglycine acceptor H2O sarcosine
dehydrogense formaldehyde reduced acceptor
Aminomethyltransferase Mitochondrian [Protein]-S8-aminomethyldihydrolipoyllysine
tetrahydrofolate [protein]-dihydrolipoyllysine
5,10-methylenetetrahydrofolate NH3
One-carbon interconverting enzymes
5,10-Methylenetetrahydrofolate Cytoplasm 5,10-Methylenetetrahydrofolate NADP 5,10-
dehydrogenase (NADP- methenyltetrahydrofolate NADPH H
5,10-Methylenetetrahydrofolate Mitochondrian 5,10-Methylenetetrahydrofolate NAD 5,10-
dehydrogenase (NAD- methenyltetrahydrofolate NADH H
5,10-Methenyltetrahydrofolate Cytoplasm 5,10-Methenyltetrahydrofolate H2O
cyclohydrolase 10-formyltetrahydrofolate
Methenyltetrahydrofolate Cytoplasm ATP 5-formyltetrahydrofolate ADP phosphate 5,10-
synthetase methenyltetrahydrofolate
Methylenetetrahydrofolate Cytoplasm 5-Methyltetrahydrofolate NAD(P) 5,10-
reductase methylenetetrahydrofolate NAD(P)H H
Dihydrofolate reductase Cytoplasm and 5,6,7,8-Tetrahydrofolate NADP 7,8-dihydrofolate
nucleus NADPH H
Formimidoyltetrahydrofolate Cytoplasm 5-Formimidoyltetrahydrofolate 5,10-
cyclodeaminase methenyltetrahydrofolate NH3
Biosynthetic enzymes
Phosphoribosylglycinamide Cytoplasm 10-Formyltetrahydrofolate N1-(5-phospho-D-ribosyl)
formyltransferase glycinamide tetrahydrofolate N2-formyl-N1-
Phosphoribosylaminoi Cytoplasm 10-Formyltetrahydrofolate 5-amino-1-(5-phospho-
midazolecarboxamide D-ribosyl)imidazole-4-carboxamide tetrahydrofolate
formyltransferase 5-formamido-1-(5-phospho-D-ribosyl)imidazole-
Methionine synthase Cytoplasm 5-Methyltetrahydrofolate L-homocysteine
tetrahydrofolate L-methionine
Thymidylate synthase Cytoplasm and 5,10-Methylenetetrahydrofolate dUMP dihydrofolate
nucleus dTMP
Methionyl-tRNA Mitochondrian 10-Formyltetrahydrofolate L-methionyl-tRNAfMet H2O
formyltransferase tetrahydrofolate N-formylmethionyl-tRNAfMet
Other folate-binding proteins
Glycine N-methyltransferase Cytoplasm S-adenosyl-L-methionine glycine S-adenosyl-L-
homocysteine sarcosine
8 Jennifer T. Fox and Patrick J. Stover

condenses with THF to form 5,10-methyleneTHF through an N5 imi-

nium cation intermediate. However, this mechanism is not consistent
with the structure of the Bacillus stearothermophilus SHMT (bsSHMT)
serine complex (Trivedi et al., 2002) and metabolic labeling experi-
ments (Tatum et al., 1977), both of which indicate that the C3-hydroxyl
group of serine is oriented in the synperiplanar configuration rather than
the antiperiplanar configuration required for a retroaldol mechanism.
In addition, the catalytic base has never been identified. A second puta-
tive direct displacement mechanism was revealed from the structures of
bsSHMT complexed with serine and with 5-formylTHF and glycine
(Trivedi et al., 2002). This proposed mechanism proceeds with the N5
of THF displacing the C3 hydroxyl of serine to form a covalent interme-
diate. However, Szebenyi et al. provide evidence that the reverse reaction
could not proceed by direct displacement and that the position of N5
of THF is unfavorable for nucleophilic attack. Rather, they propose a
third mechanism whereby the N5 of THF attacks the C3-hydroxy
group of serine to form N5-hydroxymethyleneTHF, glycine, and possibly
a transient formaldehyde intermediate (Schirch and Szebenyi, 2005;
Szebenyi et al., 2004).

c. Regulation In addition to their primary catalytic function, both SHMT

isozymes catalyze the irreversible conversion of 5,10-methenylTHF to
5-formylTHF (Fig. 1.1). 5-FormylTHF is not a cofactor for folate-
dependent one-carbon transfer reactions, but rather is an inhibitor of several
folate-dependent reactions including SHMT (Stover and Schirch, 1990,
1993). The SHMT isozymes may also play roles as folate-binding proteins
(Stover and Schirch, 1991). Both 5-formylTHF and 5-methylTHF poly-
glutamates are tight-binding SHMT inhibitors.
Unlike many enzymes involved in cytoplasmic folate-mediated one-
carbon metabolism, SHMT1 is not ubiquitously expressed in tissues, but it is
abundant in the liver, kidney, and colon and is also found in the brain
(Girgis et al., 1998). Its expression and/or activity are regulated by several
nutrients and metabolic factors including pyridoxal phosphate (vitamin B6),
retinoic acid, zinc, and ferritin. Vitamin B6 deficiency was shown to
decrease SHMT1 activity in rat liver (Scheer et al., 2005) and protein levels
in cultured cells (Perry et al., 2007). Retinoic acid, which inhibits prolifera-
tion and induces differentiation during vertebrate development, greatly
reduces SHMT1 mRNA levels (Nakshatri et al., 1996). In contrast, zinc
induces SHMT1 transcription by acting through a metal regulatory element
present within the promoter (Perry et al., 2005). The heavy chain subunit
of the iron-storage protein ferritin was also shown to increase SHMT1
protein levels (Oppenheim et al., 2001) by stimulating the cap-independent
translation of the transcript (Woeller et al., 2007b).
Folate-Mediated One-Carbon Metabolism 9

d. Physiological function/gene variants Although the SHMT1 and

SHMT2 isozymes exhibit similar catalytic and physical properties, they
have distinct physiological functions. Loss of the mitochondrial SHMT2
isozyme creates a glycine auxotrophy in Chinese hamster ovary cells
(Chasin et al., 1974), indicating that SHMT1 is not a primary source of
glycine and that SHMT1 cannot substitute for SHMT2 function. Stable
isotope tracer studies using cultured cells indicate that SHMT1-derived
5,10-methyleneTHF is preferentially directed to thymidylate biosynthesis
relative to homocysteine remethylation (Herbig et al., 2002; Oppenheim
et al., 2001). This preferential partitioning of SHMT1-derived one-carbons
to thymidylate synthesis may be achieved through the cell cycle-dependent
partitioning of the thymidylate synthesis pathway in the nucleus (Anderson
et al., 2007) (Fig. 1.1; see Section IV). The SHMT1 protein has also been
demonstrated to be a 5-methylTHF tight-binding protein in cultured cells;
increased expression of SHMT1 increased cellular levels of 5-methylTHF at
the expense of other one-carbon forms of folate while depleting AdoMet
levels (Herbig et al., 2002). This latter observation is consistent with
SHMT1 serving as a 5-methylTHF-binding protein in the cytoplasm and
thereby limiting the availability of 5-methylTHF for homocysteine
remethylation (Fig. 1.1; Herbig et al., 2002).
A common SHMT1 single nucleotide polymorphism (SNP), C1420T,
has been shown to be protective against adult acute lymphocytic leukemia
(ALL) (Skibola et al., 2002) and malignant lymphoma (Hishida et al., 2003).
This SNP results in an amino acid substitution of leucine to phenylalanine at
position 474 of the protein (L474F) and prevents SHMT1 SUMOylation
(Woeller et al., 2007a). The SHMT1 C1420T gene variant has also been
shown to be associated with elevated plasma and red cell folate levels (Heil
et al., 2001), and some studies report that it protects against NTDs (Relton
et al., 2004a,b). When present in combination with the MTHFR C677T
polymorphism (see Section II.B.4), SHMT1 C1420T is a risk factor for
cardiovascular disease (Lim et al., 2005).

2. 10-FormylTHF synthetase
a. Reaction 10-FormylTHF synthetase (FTHFS) is a formate-activating
enzyme found in a wide variety of organisms including bacteria, plants, insects,
nematodoes, yeast, and mammals. In eukaryotes, FTHFS activity is found on
the C-terminal domain of the trifunctional enzyme, C1-THF synthase, which
also contains 5,10-methenylTHF cyclohydrolase (MTHFC) and 5,10-methy-
leneTHF dehydrogenase (MTHFD) activities (see Section II:B.1; Howard
et al., 2003). C1-THF synthase is encoded by the Mthfd1 gene. FTHFS
catalyzes the ATP-dependent conversion of THF and formate to
10-formylTHF, ADP, and inorganic phosphate. The reaction is reversible
(Appling, 1991). The enzyme requires monovalent cations (NH4, K, or
Rb) to achieve maximal activity; in Clostridium cylindrosporum and Clostridium
10 Jennifer T. Fox and Patrick J. Stover

acidiurici, these cations serve to maintain the quaternary structure of the enzyme
(Welch et al., 1968) and decrease the Km of formate by stabilizing its
negative charge (Scott and Rabinowitz, 1967). The enzyme also requires a
divalent metal ion, usually Mg2, which is coordinated between the b- and
g-phosphates of the ATP substrate (Himes and Cohn, 1967).

b. Mechanism The FTHFS reaction proceeds by a random sequential

mechanism, based on results from steady-state kinetic measurements
( Joyce and Himes, 1966) and partial exchange reactions (McGuire and
Rabinowitz, 1978). The reaction initiates with the formation of a formyl-
phosphate intermediate generated through a nucleophilic attack by formate
on the g-phosphate of MgATP. The activated formyl group is then trans-
ferred directly to N10 of THF with the loss of phosphate to form
10-formylTHF (Mejillano et al., 1989; Song et al., 1993). Involvement of
the formylphosphate intermediate is supported by the transfer of 18O from
formate to inorganic phosphate (Himes and Rabinowitz, 1962) and the
synthesis of ATP from ADP and carbamoyl phosphate, a structural analogue
of formylphosphate (Buttlaire et al., 1976). However, the most conclusive
proof comes from experiments that successfully used synthetic formyl
phosphate as a substrate for the enzyme (Mejillano et al., 1989).

c. Regulation To date, there have been few reports regarding the regula-
tion of FTHFS expression and activity. The enzyme is inhibited by THF and
purine nucleotides (Leaphart et al., 2002; Mackenzie and Baugh, 1980).
Perry et al. (1980) demonstrated that nitrous oxide-induced vitamin B12
deficiency stimulates FTHFS activity in rats. However, these results were
not confirmed by an independent group who showed that nitrous oxide
exposure decreased hepatic C1-THF synthase expression (Barlowe and
Appling, 1988). Mammalian Mthfd1 that encodes all three activities of
cytoplasmic C1-THF synthase is expressed ubiquitously and is transcription-
ally upregulated in response to conditions that require increased DNA
synthesis (Christensen and MacKenzie, 2006). The promoter region of the
rat C1-THF synthase gene contains several transcription factor-binding sites
through which this regulation could occur, including NF-kB, HNF-4a1,
RARa1, C/EBP, and PPAR. However, the rat promoter region does not
share significant homology with the human Mthfd1 promoter region
(Howard et al., 2003). In mice, the upregulation of C1-THF synthase
expression is thought to occur through insulin-like growth factor-1, which
increases the stability of the mRNA transcript (Peri and MacKenzie, 1991).
In yeast, C1-THF synthase mRNA levels are decreased in the presence of
adenine, histidine, methionine, and pantothenic acid (Appling and
Rabinowitz, 1985).
Folate-Mediated One-Carbon Metabolism 11

d. Physiological function/gene variants Cytoplasmic FTHFS activity is

believed to be the primary entry point of one-carbons for cytoplasmic
folate-dependent biosynthetic reactions. Once formed, 10-formylTHF
can be used as cofactor for purine biosynthesis (Smith et al., 1981), or the
one-carbon can be sequentially dehydrated and reduced for use in the
biosynthesis of thymidylate and methionine (Fig. 1.1). However, studies
using Saccharomyces cervisiae have raised the possibility that FTHFS may play
other roles in purine biosynthesis in addition to its catalytic activity. Bar-
lowe et al. observed that yeast lacking the ADE3 gene that encodes C1-
THF synthase are auxotrophic for purines; yeast carrying point mutations in
ADE3 that inactivated all three of its enzymatic activities did not require
purines for growth, suggesting that adequate cytoplasmic 10-formylTHF
was produced in the absence of FTHFS activity. In addition, the heterolo-
gous expression of C. acidiurici FTHFS in an ade3 deletion strain did not
restore the wild-type phenotype (Barlowe and Appling, 1990). Collec-
tively, these studies indicate that C1-THF synthase possesses other poten-
tially noncatalytic activities required for pure biosynthesis. However, Song
et al. reported that a monofunctional FTHFS activity can restore the wild-
type phenotype in the ade3 deletion strain (Song and Rabinowitz, 1993), a
result consistent with the hypothesis that the catalytic activity of cytoplasmic
FTHFS is involved in purine biosynthesis.
A common SNP in human Mthfd1, G1958A, results in the substitution
of glutamine for arginine at position 653 that encodes the FTHFS domain
of C1-THF synthase. The effect of this substitution on the physical or
catalytic properties is not known. Although R653Q does not affect
homocysteine levels, plasma folate levels, or red blood cell folate levels
(Brody et al., 2002), it was found to increase a mothers risk of having a
child with an NTD in several different populations (Brody et al., 2002;
De Marco et al., 2006; Parle-McDermott et al., 2006a). This polymor-
phism has also been identified as a maternal risk factor for severe placenta
abruption and unexplained second trimester loss (Parle-McDermott et al.,

3. Glutamate formiminotransferase and glycine

a. Reaction The catabolism of histine and purines generates one-carbon
units that enter the cytoplasmic folate-activated one-carbon pool as 5,10-
methenylTHF (Fig. 1.1). During their catabolism, the imidazole ring of
histidine, adenine, and guanine is converted to a formimino group that can
be transferred to THF forming 5-formiminoTHF.

b. Mechanism In mammalian liver cells, formiminoglutamic acid, an interme-

diate in histidine catabolism, reacts with THF to form 5-formiminoTHF in a rea-
ction catalyzed by glutamate formiminotransferase (Revel and Magasanik, 1958).
12 Jennifer T. Fox and Patrick J. Stover

formimidoyltetrahydrofolate cyclodeaminase activity on the C-terminal domain of
the protein, which allows for the rapid conversion of 5-formiminoTHF to 5,10-
methenylTHF (Mackenzie, 1984; Tabor et al., 1952). The bifunctional enzyme is
assembled as a circular tetramer of dimers and channels folate polyglutamates
between catalytic sites (Murley and MacKenzie, 1995). Similarly, formiminogly-
cine, which is a product of purine ring degradation, is also a source of
5-formiminoTHF through the activity of glycine formiminotransferase. As in
histidine catabolism, 5-formiminoTHF is converted to 5,10-methenylTHF and
is available for one-carbon transfer reactions in the cytoplasm (Pricer and
Rabinowitz, 1956).

c. Regulation Histidine and purine catabolism occurs in the liver in mam-

malian cells and can be influenced by vitamin B12 levels, metals, and THF.
Vitamin B12 deficiency has been shown to increase the urinary excretion of
formiminoglutamate, presumably because unsubstituted THF is not available
during severe vitamin B12 deficiency for the formiminotransferase reaction
(see Section II.C.3). Both glutamate formiminotransferase (Miller and
Waelsch, 1957) and glycine formiminotransferase (Uyeda and Rabinowitz,
1965) are inhibited by various cations, including Mn2 and Zn2, and the
cyclodeaminase activity of the bifunctional enzyme is inhibited by THF.

d. Physiological function/gene variants The quantitative contribution of

purine and histidine catabolism to the cytoplasmic folate-activated one-
carbon pool is not known. However, severe inborn errors of metabolism are
associated with impairments in histidine and purine catabolism. Histidine-
mia and glutamate formiminotransferase deficiency are autosomal recessive
disorders resulting from mutations in the histidase (HAL) and formimino-
transferase/cyclodeaminase (FTCD) genes, respectively. Both have been
characterized by mental retardation, speech impairment, and developmental
delay; severe glutamate formiminotransferase deficiency is also associated
with elevated serum folate (Hilton et al., 2003). Three disease-causing
mutations that significantly reduce glutamate formiminotransferase activity
have also been found (Hilton et al., 2003). The R135C mutation is located
within an extended loop of the formiminotransferase domain that is involved
in folate-binding; R229P is thought to disrupt the formiminotransferase
dimerization interface. The 1033insG mutation results in the production
of a monofunctional enzyme that allows the transfer of the forminimo
group to THF, but cannot catalyze the second reaction, the production of
Folate-Mediated One-Carbon Metabolism 13

B. Folate-interconverting enzymes
1. 5,10-MethenylTHF cyclohydrolase and 5,10-methyleneTHF
a. Reaction As mentioned above, mammalian C1-THF synthase is a
homodimer and trifunctional enzyme consisting of two functionally inde-
pendent domains encoded by Mthfd1. The C-terminal domain cont-
ains FTHFS activity whereas the N-terminal domain contains MTHFC
and MTHFD activities (Paukert et al., 1976; Tan et al., 1977). MTHFC
catalyzes the reversible interconversion of 10-formylTHF and 5,10-methe-
nylTHF, whereas MTHFD catalyzes the NADP-dependent and reversible
interconversion of 5,10-methenylTHF and 5,10-methyleneTHF.

b. Mechanism MTHFC and NADP-dependent MTHFD share an

overlapping active site on C1-THF synthase that allows for the intercon-
version of folate-activated one-carbon units between the formate and
formaldehyde levels of oxidation (Cohen and Mackenzie, 1978; Schirch,
1978). There is evidence that the folate substrates are channeled between
the MTHFD and MTHFC active sites without dissociating from the
complex and equilibrating with the cytoplasmic milieu. Exogenous methe-
nylTHF does not compete as a substrate for the cyclohydrolase reaction
with the methenylTHF produced in the dehydrogenase reaction
(Mackenzie and Baugh, 1980; Wasserman et al., 1983). Substrate channeling
permits increased metabolic rates in the presence of low substrate concen-
trations and protects reaction intermediates from competing reactions or
degradation [reviewed by Spivey and Merz (1989)]. Within the bifunctional
complex, the reaction catalyzed by 10-methenylTHF cyclohydrolase is rate-
limiting in the overall conversion of 10-formylTHF to 5,10-methyleneTHF
(Pawelek and MacKenzie, 1998).

c. Regulation The regulation of C1-THF synthase is described above for

FTHFS; no specific regulation of the cyclohydrolase or dehydrogenase
activities apart from regulation of Mthfd1 is known.

d. Physiological function/gene variants The reversible reactions cata-

lyzed by MTHFC and MTHFD are essential for the provision of folate-
activated one-carbons for thymidylate biosynthesis and homocysteine
remethylation when the dehydrogenase reaction proceeds in the reductive
direction (Fig. 1.1). An SNP in Mthfd1 has been identified that affects the
MTHFD/MTHFC domain and has an association with disease. Although
its functional significance is currently unknown, R134K was shown to be
associated with a significant increase in risk for postmenopausal breast cancer
(Stevens et al., 2007).
14 Jennifer T. Fox and Patrick J. Stover

2. 10-FormyTHF dehydrogenase
a. Reaction 10-FormylTHF dehydrogenase (FDH) catalyzes the irrevers-
ible and NADP-dependent oxidation of 10-formylTHF to THF and
CO2. FDH consists of two functionally distinct domains connected by an
intermediate linker. The C-terminal domain catalyzes an NADP-depen-
dent aldehyde-dehydrogenase reaction, and the N-terminal domain cata-
lyzes the hydrolysis of 10-formylTHF to THF and formate (hydrolase
reaction) (Cook et al., 1991; Donato et al., 2007). Although the two
domains can function independently, the two active sites work in concert
through a 40 -phosphopantetheine swinging arm that is bound through a
phosphoester bond to Ser354; the swinging arm transfers formate between
the two active sites (Donato et al., 2007).

b. Mechanism The FDH reaction initiates with a hydrolase reaction

where a water molecule, activated by aspartate 142, acts as a nucleophile
by attacking the formyl carbon atom of 10-formylTHF to produce a
hydrated aldehyde intermediate (Donato et al., 2007; Tsybovsky et al.,
2007). In the absence of NADP, this intermediate can be further cleaved
to release formate. In the presence of NADP, the reaction continues
through an aldehyde-dehydrogenase-like mechanism where the formyl
group of the intermediate is cleaved with oxidation to CO2.

c. Regulation FDH is one of the most abundant folate enzymes but is

expressed primarily in the liver, kidney, and the central nervous system
(Mackenzie, 1984). FDH displays product inhibition by THF and also
contains a second THF tight-binding site that is separate from its active
site (Fu et al., 1999). The product inhibition by THF can be suppressed by
both SHMT1 and C1-THF synthase (Kim et al., 1996), presumably by
channeling the THF polyglutamate cofactor to these acceptor proteins.

d. Physiological function/gene variants It is not known if the individual

reactions catalyzed by the N-terminal and C-terminal domains of FDH
have any physiological significance. The dehydrogenase reaction, on the
other hand, has been proposed to have several important metabolic roles.
These include: (1) recycling THF cofactors by removing excess
10-formylTHF, (2) protecting the cell from formate toxicity through its
conversion to CO2, (3) regulation of de novo purine biosynthesis, (4) removal
of excess one-carbon units from folate metabolism in the form of CO2, and
(5) sequestering and storing cellular folate in the form of THF. These
proposed physiological functions were investigated in human neuroblas-
toma (Anguera et al., 2006) and no evidence was found that FDH seques-
tered THF nor that it regulated de novo purine biosynthesis. FDH was
shown, however, to regulate cellular concentrations of 10-formylTHF
Folate-Mediated One-Carbon Metabolism 15

and the homocysteine remethylation cycle, presumably by regulating the

supply folate-activated one-carbon units.
Two SNPS within the FDH gene have been shown to alter the risk of
developing postmenopausal breast cancer. One gene variant [rs2276731 (T/C)]
is associated with an increased risk, while the other [rs2002287 (T/C)] is
associated with a decreased risk. Both SNPs are located within introns, and
therefore they may exist in linkage disequilibrium with a coding SNP such as
V812I, G481S, or F330V (Stevens et al., 2007).

3. 5,10-MethenylTHF synthetase
a. Reaction 5,10-MethenylTHF synthetase (MTHFS, also referred to as
5-formylTHF cycloligase) catalyzes the ATP-dependent and irreversible
conversion of 5-formylTHF to 5,10-methenylTHF. It is the only enzyme
identified to date that utilizes 5-formylTHF as a substrate. Like FTHFS,
MTHFS activity requires Mg2 that is involved in the binding of the ATP
substrate to the enzyme (Chen et al., 2005). The MTHFS reaction and the
SHMT1-catalyzed synthesis of 5-formylTHF from 5,10-methenylTHF
constitute a futile cycle that serves to buffer intracellular 5-formylTHF
concentrations (Stover et al., 1993).

b. Mechanism The MTHFS-catalyzed reaction occurs via a sequential

mechanism with a nucleophilic attack by the 5-formyl oxygen on the
g-phosphate of ATP to form an N5-imminium phosphate intermediate.
This intermediate undergoes cyclization via nucleophilic attack by N10 to
form a phosphoimidazolidine tetrahedral intermediate, which is the rate-
limiting step in catalysis (Huang and Schirch, 1995; Kounga et al., 1995).
The N10 attack on the N5-imminium phosphate is made possible through
the hydrophobic and aromatic properties of a conserved active site Tyr.
This residue defines the architecture of the MTHFS active site, forming a
pocket that restricts the motion of N10 (Field et al., 2007). The above steps
in the MTHFS mechanism are all reversible. The final step is the irreversible
step, in which the phosphoimidazolidine tetrahedral intermediate is broken
down through phosphate elimination to generate the 5,10-methenylTHF
product (Huang and Schirch, 1995).

c. Regulation In humans, MTHFS is expressed in all tissues, with the

highest mRNA levels found in the liver, heart, and kidney, and the lowest
levels found in the brain (Anguera et al., 2003). MTHFS enzymatic activity
is regulated primarily by folate coenzymes. 5-MethylTHF and
10-formylTHF, the latter of which is in chemical equilibrium with the
product of the MTHFS reaction, act as tight-binding inhibitors of MTHFS
(Field et al., 2006).
16 Jennifer T. Fox and Patrick J. Stover

d. Physiological function/gene variants To date, two metabolic roles

have been ascribed to MTHFS. Anguera et al. (2003) found that the expres-
sion of human MTHFS cDNA in cell culture models led to an increase in the
catabolism of monoglutamate forms of folate, indicating that MTHFS may
regulate cellular folate concentrations by affecting rates of folate turnover.
MTHFS is also thought to regulate de novo purine biosynthesis through two
distinct mechanisms. First, MTHFS activity reduces levels of 5-formylTHF,
an inhibitor of the purine-synthesizing enzyme phosphoribosylaminoi-
midazolecarboxamide formyltransferase (AICARFT) (Bertrand and Jolivet,
1989). Second, MTHFS expression enhances purine biosynthesis either by
enriching cellular 10-formylTHF pools or by channeling 10-formylTHF to
AICARFT and/or phosphoribosylglycinamide formyltransferase (GARFT)
(Field et al., 2006).
One MTHFS variant allele has been associated with a clinical outcome
(Matakidou et al., 2007). The MTHFS T202A variant allele was associated
with poorer prognosis in individuals with same-stage lung cancer. The
functional significance of this polymorphism, as well as its effect on folate
levels and purine biosynthesis, has yet to be determined.

4. 5,10-MethyleneTHF reductase
a. Reaction MTHFR is a flavoprotein consisting of two identical sub-
units. The C-terminal domain of each subunit contains the binding site for
AdoMet, an allosteric inhibitor; the N-terminal domain catalyzes the
NADPH-dependent reduction of 5,10-methyleneTHF to 5-methylTHF
for use in the remethylation of homocysteine to methionine. The MTHFR
reaction is virtually irreversible in vivo and therefore commits one-carbon
units to methionine biosynthesis (Appling, 1991; Wagner, 1995).

b. Mechanism The reaction catalyzed by MTHFR proceeds by two half

reactions. In the reductive half-reaction, the 4S-hydrogen of NADPH is
transferred as a hydride to N5 of the flavin adenine dinucleotide (FAD)
cofactor. After NADP dissociates from the enzyme, 5,10-methyleneTHF
binds. In the oxidative half-reaction, 5,10-methyleneTHF is protonated at
the N10 position by a general acid catalyst (Trimmer et al., 2001), leading to
the opening of the imidazolidine ring and generating the N5-iminium
cation intermediate. Transfer of a hydride from N5 of the reduced FAD
to C11 of the methylene group results in the production of 5-methylTHF
(Sumner and Matthews, 1992).

c. Regulation Regulation of MTHFR is critical for AdoMet-dependent

methylation reactions and to prevent elevated homocysteine levels in the
cell. The complexity of the MTHFR transcript allows for the regulation of
its expression at several levels. Exon 1 of MTHFR undergoes extensive
alternative splicing, generating transcripts that vary in the length of their
Folate-Mediated One-Carbon Metabolism 17

50 UTR (Tran et al., 2002). The length of the 50 UTR has been shown to
influence translational efficiency, as longer, more GC-rich UTRs slow the
scanning of the translation initiation machinery (van der Velden and
Thomas, 1999). Multiple polyadenylation signals result in MTHFR tran-
scripts that vary in the length of their 30 UTR. Additionally, two distinct
promoters and translation start sites generate two isoforms of the MTHFR
protein. Transcription initiation from the upstream promoter followed by
translation from the downstream AUG results in the production of a 70 kDa
protein; transcription initiation from the downstream promoter followed by
translation from the upstream AUG generates a 77 kDa protein (Tran et al.,
2002). MTHFR expression from the downstream promoter has been
shown to be regulated by NF-kB in a tissue-specific manner (Pickell
et al., 2005). At the protein level, MTHFR activity is regulated by the
AdoMet/AdoHcy ratio in the cell (Kutzbach and Stokstad, 1971). AdoMet
preferentially binds to MTHFR in the inactive T state and thus increases the
T/R ratio in the cell ( Jencks and Mathews, 1987). Although AdoHcy does
not itself alter MTHFR enzymatic activity, it can reverse the inhibitory
effect of AdoMet by competing for its binding site (Kutzbach and Stokstad,
1971). Recently, phosphorylation of the MTHFR N-terminal domain at
Thr34 was shown to reduce the inhibition of enzymatic activity by AdoMet
by altering the equilibrium between the T and R states of the protein so that
it favors the active R state (Yamada et al., 2005). NADPH, the reducing
equivalent in the MTHFR reaction, binds to R subunits, and thus acts as an
AdoMet antagonist ( Jencks and Mathews, 1987).

d. Physiological function/gene variants MTHFR serves as the link

between nucleotide biosynthesis and AdoMet-dependent methylation reac-
tions. Its activity depletes one-carbon units that can be used for DNA
synthesis and increases the concentration of one-carbon units available for
the remethylation of homocysteine to methionine and for the subsequent
production of AdoMet. Thus, although MTFHR is expressed ubiquitously,
its mRNA levels are the highest in the testis, where DNA methylation is critical
for germ cell maturation and genomic imprinting (Gaughan et al., 2000). Mild
MTHFR deficiency, as characterized by an enzyme with 3545% residual
activity, is the most common inborn error of folate metabolism, affecting
520% of North Americans and Europeans (Pejchal et al., 2006). The primary
cause is a common C to T substitution at nucleotide 677, which results in the
amino acid change A222V in the catalytic domain of the protein (Shivapurkar
et al., 1995). The C677T SNP does not affect the kinetic properties of
MTHFR but rather enhances the loss of the FAD cofactor by displacing
helix a5 (Guenther et al., 1999; Pejchal et al., 2006; Yamada et al., 2001).
This creates a thermolabile protein (Kang et al., 1988). Mild MTHFR defi-
ciency is associated with mild hyperhomocysteinemia, especially in those with
low folate concentrations ( Jacques et al., 1996), and decreased plasma and red
18 Jennifer T. Fox and Patrick J. Stover

cell folate levels (Molloy et al., 1997; Parle-McDermott et al., 2006b). Clini-
cally, C677T has been shown, in some cases, to be associated with an increased
risk for cardiovascular disease (Klerk et al., 2002; Kluijtmans et al., 1996; Morita
et al., 1997), NTDs (Christensen et al., 1999; Ou et al., 1996; van der Put et al.,
1995), cleft lip and palate (Mills et al., 1995; Zhu et al., 2006), thrombosis
(Keijzer et al., 2002; Quere et al., 2002; Zalavras Ch et al., 2002), and schizo-
phrenia (Lewis et al., 2005; Muntjewerff et al., 2005, 2006; Scher et al., 2006). It
has also been shown to be protective against several types of cancers, including
ALL (Skibola et al., 1999), childhood acute leukemia (Wiemels et al., 2001),
and colorectal cancer (Chen et al., 1996; Ma et al., 1997).
Another common MTHFR SNP, A1298C (E429A), exists in strong
linkage disequilibrium with C677T (Stegmann et al., 1999). Unlike C677T
which is located in the N-terminal domain of the protein, A1298C affects
the regulatory (C-terminal) domain of the protein and is therefore catalyti-
cally indistinguishable from the wild-type enzyme (Yamada et al., 2001).
Individuals with the A1298C polymorphism exhibit increased red cell folate
levels but have no significant change in vitamin B12, plasma folate, or
homocysteine levels (Parle-McDermott et al., 2006b). Clinically, the poly-
morphism was shown to be associated with a decreased risk for ALL
(Skibola et al., 1999) and childhood acute leukemia (Wiemels et al., 2001).

C. Biosynthetic enzymes
a. Reaction De novo purine biosynthesis is a 10-step reaction whereby
5-phosphoribosylpyrophosphate is converted to inosine monophosphate
(IMP), the precursor of adenine and guanine nucleotides. Of the 10 steps
involved in de novo purine biosynthesis, 2 are catalyzed by folate-dependent
enzymes. In the third reaction, GARFT transfers the formyl group of
10-formylTHF to glycinamide ribotide (GAR) to form formylglycinamide
ribonucleotide (FGAR) and THF. In the ninth reaction, AICARFT trans-
fers the formyl group of 10-formylTHF to aminoimidazole carboxomide
ribotide (AICAR) to form formylaminoimidazole carboxomide ribonucle-
otide (FAICAR) and THF. In eukaryotic cells, GARFT and AICARFT
activities are part of multi-functional enzymes. GARFT activity comprises
the C-terminal domain of a protein that also contains the active sites of
GAR synthetase (GARS) and aminoimidazole ribotide synthetase (AIRS)
(Aimi et al., 1990; Schild et al., 1990), and AICARFT resides on the same
polypeptide as IMP cyclohydrolase. Substrate channeling among GARFT,
GARS, and AIRS drives the AICARFT reaction forward by coupling the
energetically unfavorable production of FAICAR to the highly favorable
cyclohydrolase reaction (Wall et al., 2000).
Folate-Mediated One-Carbon Metabolism 19

b. Mechanism Despite performing similar reactions, GARFT and

AICARFT act through distinct mechanisms. The reaction catalyzed by
GARFT occurs by an ordered sequential mechanism (Caperelli, 1989),
with 10-formylTHF binding to the active site first through interactions
with His108 and Asn106. His108, which has a high the pKa due to the
formation of a salt bridge with Asp144, aids in the nucleophilic attack of
GAR on 10-formylTHF by withdrawing electrons from the formyl group
of the cofactor. A water molecule hydrogen bonded to Asp144 then
catalyzes the transfer of a proton from the amino group of GAR to the
N10 of THF (Klein et al., 1995; Qiao et al., 2004).
Unlike GAR, AICAR contains a relatively non-nucleophilic C5 amine
that must first be activated in order for the formylation reaction to occur
(Bulock et al., 2002; Wall et al., 2000; Yamazaki, 1978). In one proposed
mechanism, activation of the C5 amine results from Phe542 orienting the
AICAR carboxamide upward and out of the imidazole ring plane, allowing
the carboxamide to hydrogen bond to the C5 amino group and thus
increases the nucleophilicity of the amine. Proton abstraction from the
amino group via His268 then occurs concomitant with the nucleophilic
attack of this group on 10-formylTHF. The transition state of this reaction is
thought to be stabilized by Lys267, a residue that may also play a role in the
subsequent protonation of THF (Wolan et al., 2002). The findings of
Wolan et al. are in partial disagreement with the earlier proposed
mechanisms (Shim et al., 2001).

c. Regulation To date, little is known about the about the tissue specific
or genetic regulation of GARFT and AICARFT expression. The putative
promoter region of the gene encoding GARFT was found to have four SP1
sites, but the importance of these sites in transcriptional control has yet to be
determined. GARFT is developmentally regulated in the human cerebel-
lum, with high expression found during prenatal development, and no
expression detected in that tissue shortly after birth (Brodsky et al., 1997).

d. Physiological function GARFT and AICARFT play a key role in

de novo nucleotide biosynthesis by catalyzing the incorporation of formate
into the C8 and C2 positions of the purine ring, respectively. Once formed,
purine nucleotides function as precursors for DNA, RNA, coenzymes,
energy transfer molecules, and regulatory factors. Although the salvage
pathway is thought to be the major source of purines in differentiated
mammalian cells (Meredith et al., 1995), the de novo pathway was found to
supply most of the adenine and guanine nucleotides during human embry-
onic development (Brodsky et al., 1997).
Compared to normal cells, cancer cells have an increased dependence on
de novo purine biosynthesis for adenine and guanine nucleotides. Thus, both
20 Jennifer T. Fox and Patrick J. Stover

GARFT and AICARFT are chemotherapeutic targets. 6-R-dideazatetra-

hydrofolate (DDATHF, Lometrexol) is an antifolate that specifically targets
GARFT. In contrast to the natural substrate of the enzyme, DDATHF
contains carbon atoms at positions 5 and 10 that render it unable to serve as a
substrate (Beardsley, 1989; Erba, 1994). The antimetabolate methotrexate
(4-amino-10-methylpteroyglutamic acid) inactivates several folate-
dependent enzymes, including both GARFT and AICARFT, by depleting
10-formylTHF (Allegra et al., 1986). Inhibition of AICARFT by metho-
trexate and dihydrofolate polyglutamates results in an anti-inflammatory
response (Cronstein et al., 1993; Gadangi et al., 1996; Szabados et al., 1994).
A common SNP in the AICARFT gene, C347G (Thr116Ser), is associated
with a better therapeutic response to methotrexate in patients with rheu-
matoid arthritis, although the mechanism by which the polymorphism
influences drug efficacy remains unknown (Dervieux et al., 2004).
Another AICARFT gene variant, A1277G (K426R), completely
abolishes AICARFT enzymatic activity, presumably by disrupting the
binding of a potassium ion that plays a key role in tertiary structure stabili-
zation. To date, this mutation has only been identified in one allele of a
4-year-old girl who presented with profound mental retardation, epilepsy,
dysmorphic features, and congenital blindness. Her other allele showed a
frameshift in exon 2 due to a duplication/deletion event (125129dup
GGGAT; 130132 delGCT) that resulted in mRNA instability (Marie
et al., 2004).

2. Thymidylate synthase
a. Reaction Thymidylate synthase (TS) catalyzes the 5,10-methyleneTHF-
dependent conversion of deoxyuridine monophosphate (dUMP) to the DNA
precursor deoxythymidine monophosphate (dTMP). This is the only folate-
dependent reaction whereby the folate cofactor serves both as a one-carbon
donor and source of reducing equivalents. When 5,10-methyleneTHF is
limiting in the cell, TS must compete with MTHFR for this cofactor. Thus,
in addition to its role in DNA synthesis, TS expression may also indirectly
influence homocysteine levels (Trinh et al., 2002).

b. Mechanism The TS reaction initiates with the opening of the imidazole

ring and activation of the methyleneTHF cofactor to the reactive 5-iminium
cation. Ring opening is facilitated through either the TS-assisted protonation
of N10, a protonated water molecule that acts as a general acid catalyst, or the
formation of a hydrogen bond between N10 and an active site residue such as
Glu60. dUMP must also be activated by the thiol group of Cys198, which
either directly attacks C6 of the substrate to produce a nucleophilic enolate
or transfers a hydrogen to a water molecule that acts as a base. The enolate
then attacks C6 of the iminium cation to form a ternary covalent interme-
diate complex. Tyr146-assisted deprotonation of C5 (Liu et al., 1999)
Folate-Mediated One-Carbon Metabolism 21

followed by the elimination of THF from this complex results in an

exocyclic methylene intermediate. Hydride transfer from THF to this
intermediate yields the products dTMP and dihydrofolate (Carreras and
Santi, 1995).

c. Regulation TS is a housekeeping gene but its expression is increased

markedly in dividing cells (Ayusawa et al., 1986; Conrad, 1971). Although
protein and mRNA levels increase as cells progress from the G1 to the
S-phase of the cell cycle, TS gene transcription remains constant, suggesting
that regulation occurs primarily at the posttranscriptional level (Ash et al.,
1995; Jenh et al., 1985; Navalgund et al., 1980). The cell cycle-directed
regulation of TS is thought to be controlled by a spliceable intron located
downstream of the transcription start site (Ash et al., 1993; Ke et al., 1996)
and several transcriptional control elements in the promoter region (Dong
et al., 2000). The transcription factor GABP, acting synergistically with Sp1,
stimulates TS promoter activity by binding to the Ets site (Rudge and
Johnson, 2002). The mouse LSF element has been shown to be necessary
for the S-phase-specific expression of the gene in growth-stimulated cells
(Powell et al., 2000). In the G0 and G1 stages, E2F interacts with the
retinoblastoma tumor suppressor, histone deacetylase, and SWI/SF chro-
matin remodeling proteins, forming a repressor complex that inhibits
the enzymes transcription (Angus et al., 2002). This inhibition can be
overcome by the ectopic expression of E2F (DeGregori et al., 1995).
In addition to regulation by various promoter elements, TS expression can
also be controlled through polyadenylation, site-specific cleavage, and trans-
lational repression. TS contains two polyadenylation signals that affect the
length of the 30 UTR and thus have an impact on mRNA stability (Takeishi
et al., 1985). A naturally occurring antisense RNA (rTSa) produced from a
gene (rTS) that overlaps with the 30 end of the TS gene downregulates TS
expression by inducing the site-specific cleavage of TS RNA (Chu and
Dolnick, 2002). In the absence of bound folate cofactors, TS can also bind
to its own mRNA and repress its translation (Chu et al., 1991).

d. Physiological function/gene variants The reaction catalyzed by TS is

the only source of de novo dTMP synthesis, making TS indispensable for
DNA replication and repair. Impairments in TS enzymatic activity,
whether due to polymorphisms or pharmacological agents, are associated
with decreased DNA synthesis, increased uracil misincorporation into
DNA, chromosome damage, fragile site induction, and apoptotic cell
death (Hori et al., 1984).
Because of its importance in DNA synthesis, TS is the target of several
antineoplastic agents including the fluoropyrimidines 5-fluorouracil and
5-fluoro-2-deoxyuridine, and the antifolates ralitrexed, premetrexed, and
methotrexate. These chemotherapeutic drugs generate metabolites that
22 Jennifer T. Fox and Patrick J. Stover

inhibit TS enzymatic activity and have been effective in the treatment of

head, neck, breast, stomach, and colon cancers (Takemura and Jackman,
1997). Although these agents decrease TS catalytic function, they also
increase its intracellular concentration (Gorlick et al., 1998; Van der Wilt
et al., 1992) either by inhibiting the binding of TS to its mRNA or by
decreasing the rate of ubiquitin-independent enzyme degradation
(Forsthoefel et al., 2004; Kitchens et al., 1999). This phenomenon is thought
to lead to cellular resistance and decreased drug efficacy.
Within the 50 UTR of TS, there is a common 28-nucleotide G/C-rich
tandem repeat polymorphism that is thought to influence a patients
response to TS-based chemotherapy. The number of these repeats can
vary, with the most common number being two (2R) or three (3R).
These repeat regions contain USF-1 transcription factor-binding sites
(Mandola et al., 2003) and act as TS promoter enhancers; they also serve
to increase its translation. Thus, individuals with the 2R/2R geno-
type produce significantly less TS protein than those with the 3R/3R
genotype (Horie et al., 1995; Kawakami and Watanabe, 2003) and show a
better response to fluoropyrimidine and mexotrexate therapy. However,
they also suffer increased toxicity because of cytotoxic damage to normal
tissues (Krajinovic et al., 2002; Pullarkat et al., 2001). Within the second
repeat of the 3R allele and the first repeat of the 2R allele, there is a G ! C
polymorphism that results in a reduction in TS expression, presumably due
to the disruption of a USF-1-binding site (Kawakami et al., 1999; Lincz
et al., 2007; Mandola et al., 2003).
Another TS gene variant results from a six-base pair insertion/deletion
in the 30 UTR of the transcript (Ulrich, 2000). This deletion affects mRNA
stability and translation (Chu, 2002) and results in the reduction of TS
expression (Mandola, 2004). It also leads to an increase in red blood cell
folate concentrations and a decrease in homocysteine levels (Kealey, 2005).
The homozygous insertion genotype has been found to be associated with
an increased risk for spina bifida, especially when present in combination
with the 2R/2R genotype (Volcik, 2003).

3. Methionine synthase
a. Reaction Methionine synthase (MS) is a cobalamin (vitamin B12)-
dependent enzyme that, in mammalian tissue, functions within the trans-
methylation cycle by catalyzing the 5-methylTHF-dependent remethylation
of homocysteine to methionine. The MS-catalyzed reaction occurs via three
separate methyl transfer reactions that take place in different binding domains
of the four functional modules that comprise MS. The N-terminal module
utilizes a (Cys)3Zn2 cluster to bind homocysteine. A second module binds
and activates 5-methylTHF for methyl transfer. A third module binds cobal-
amin. The C-terminal module binds S-adenosyl methionine (AdoMet)
and is required for reductive reactivation of the cobalamin cofactor
Folate-Mediated One-Carbon Metabolism 23

(Drennan et al., 1994; Goulding et al., 1997; Katrina Peariso et al., 1998;
Ludwig and Matthews, 1997). Each methyl transfer requires a different
arrangement of modules that is made possible by the interdomain connectors
of the enzyme (Gokhale and Khosla, 2000).

b. Mechanism The catalytic mechanism initiates with the methylation of

cob(I)alamin by 5-methylTHF to form an enzyme-bound methylcob(III)
alamin intermediate and THF. Methyl transfer from methylcob(III)alamin
to homocysteine produces methionine and regenerates cob(I)alamin for use
in subsequent methylation cycles (Banerjee et al., 1990). Cob(I)alamin and
methylcob(III)alamin are susceptible to oxidation and photolysis, respec-
tively, resulting in the occasional formation of a cob(II)alamin species that
inactivates the enzyme. Mammalian MS is reactivated through reducing
equivalents that are generated by MS reductase, a P450-reductase like
protein that binds NADPH, FAD, and FMN (Leclerc et al., 1998).

c. Regulation MS expression is regulated by several factors including

vitamin B12, cis-acting elements located within its mRNA, and nitrous
oxide. Vitamin B12 was found to stimulate MS translation by interacting
(via an auxiliary protein) with an internal ribosome entry site located within
the 50 UTR of the transcript (Oltean and Banerjee, 2005). The 50 leader
sequence of human MS mRNA also contains two upstream open reading
frames that recruit the 40 S ribosomal subunit and cause it to stall on the
UTR, thus inhibiting the translation of MS (Col et al., 2007).
Loss of vitamin B12, due to nutritional deficiency or nitrous oxide
exposure, inhibits nucleotide biosynthesis because of the accumulation of
cytoplasmic folate cofactors as 5-methylTHF. The effect of vitamin B12
deficiency on 5-methylTHF accumulation is referred to as methyl trap
(Lassen et al., 1956; Shane and Stokstad, 1985). 5-MethylTHF accumulates
because the MTHFR reaction is irreversible in vivo and MS is the only
5-methylTHF utilizing enzyme. When cellular vitamin B12 levels are
adequate, the regulation of MTHFR by AdoMet protects against a methyl
trap by inhibiting the 5-methylTHF synthesis and preventing the depletion
of 5,10-methyleneTHF pools required for thymidylate biosynthesis. The
feedback inhibition of AdoMet also ensures that, during times when methi-
onine is abundant, one-carbon units are spared for the synthesis of DNA
precursors (Kutzbach and Stokstad, 1971).

d. Physiological function/gene variants MS serves three important phys-

iological functions: (1) the regeneration of the THF cofactor, (2) the
synthesis of the essential amino acid methionine, and (3) the removal of
cellular homocysteine, which is a risk factor for cardiovascular disease
(Refsum et al., 1998), NTDs (Mills et al., 1995), and Alzheimers disease
(Clarke et al., 1998). MS is an essential enzyme as evidenced by the
24 Jennifer T. Fox and Patrick J. Stover

embryonic lethality of the MS knockout mouse (Swanson et al., 2001).

Although betaine homocysteine methyltransferase can also remethylate
homocysteine to form methionine, its expression is limited primarily to
the liver and kidney, whereas MS displays ubiquitous expression (Chen
et al., 1997).
Rare mutations in the MS gene, such as P1173L, result in an autosomal
recessive disease that is associated with homocysteinemia, homocysteinuria,
hypomethioninemia, megaloblastic anemia, neural dysfunction, and mental
retardation (Gulati et al., 1999). More subtle clinical outcomes are associated
with the common polymorphic variant, A2756G, that affects the domain
involved in methylation and reactivation of the B12 cofactor (Leclerc et al.,
1996) and results in decreased plasma homocysteine levels (Harmon et al.,
1999). A2756G was found to be positively associated with aberrant methyla-
tion in patients with colorectal, breast, or lung tumors (Paz et al., 2002) and
has been implicated as a risk factor for systemic lupus erythematosus
(Burzynski et al., 2007), bipolar disorder, schizophrenia (Kempisty et al.,
2007), and for having a child with spina bifida (Doolin et al., 2002), oralfacial
clefts (Mostowska et al., 2006), and Down syndrome (Bosco et al., 2003).

D. Folate-binding proteins
1. Glycine N-methyltransferase
Glycine N-methyltransferase (GNMT) is a relatively abundant methytrans-
ferase that catalyzes the AdoMet-dependent methylation of glycine to
sarcosine. Its metabolic role is to govern transmethylation reactions by
regulating and buffering the AdoMet/AdoHyc ratio. GNMT activity is
allosterically regulated by 5-methylTHF, which is a tight-binding inhibitor
of GNMT. Under conditions of adequate AdoMet concentrations,
AdoMet inhibits MTHFR and limits 5-methylTHF synthesis to decrease
rates of methionine synthesis. GNMT remains active under these conditions
and metabolizes excess AdoMet. In contrast, when AdoMet levels are low,
the production of 5-methylTHF by MTHFR inhibits GNMT activity
and conserves the limited amount of methionine for essential methylation
reactions (Porter et al., 1985).

III. Introduction to Mitochondrial

One-Carbon Metabolism
Relatively little is known about one-carbon metabolism in the mito-
chondria compared with one-carbon metabolism in the cytoplasm, and
virtually nothing is known about its regulation. Interestingly, many of
the enzyme activities associated with the interconversion of THF-activated
Folate-Mediated One-Carbon Metabolism 25

one-carbons in the cytoplasm are also found in the mitochondrial compart-

ment. However, unlike the cytoplasm, the interconversion of one-carbon
substituted folates in mitochondria is driven in the oxidative direction toward
formate production and/or differs with respect to the source of reducing
equivalents (Appling, 1991; Christensen and MacKenzie, 2006). Approxi-
mately 40% of total cellular folate polyglutamates are present in mitochondria
as a stable pool that does not exchange with the cytoplasmic compartment
(Horne et al., 1989; Lin et al., 1993). The primary functions of mitochondrial
one-carbon metabolism are (1) to generate one-carbon units in the form of
formate for cytoplasmic one-carbon metabolism, (2) to generate the amino
acid glycine, and (3) to synthesize formylmethionyl-tRNA for protein
synthesis. Communication between mitochondrial and cytoplasmic folate
metabolism is facilitated through the exchange of one-carbon donor
substrates including serine, glycine, and formate (Appling, 1991).
The essentiality of mitochondrial folate metabolism for glycine synthesis
was revealed when complementation groups of glycine auxotrophs were
isolated from mutagenic screens of Chinese hamster ovary (CHO) cells.
Cell lines were identified with mutations in the genes that encode the
mitochondrial folate-dependent proteins SHMT2 ( glyA) (Pfendner and
Pizer, 1980) and the mitochondrial folate transporter ( glyB) (Titus and
Moran, 2000). Other studies have demonstrated that mitochondria effec-
tively convert serine to glycine and formate; isolated mitochondria from rats
are capable of synthesizing formate from serine (Barlowe and Appling,
1988; Garcia-Martinez and Appling, 1993). However, the definitive path-
way for mitochondrial synthesis of formate from serine has yet to be
established (Christensen and MacKenzie, 2006), and not all the enzymes
required for this pathway have been identified.

A. Enzymes that generate one-carbon units

1. Mitochondrial serine hydroxymethyltransferase
Serine is a primary source of one-carbon units carried by THF for folate-
dependent biosynthetic reactions in humans (Davis et al., 2004). The
metabolism of serine to formate and glycine in mitochondria is initiated
by the pyridoxal-phosphate-dependent mitochondrial isozyme of serine
hydroxymethyltransferase (SHMT2). Although the cytoplasmic and mito-
chondrial isozymes share similar physical and catalytic properties, their
physiological functions appear to be distinct. As mentioned previously,
CHO cells lacking SHMT2 are autotrophic for glycine; the C3 of serine
is also a primary source of one-carbon units for cytoplasmic one-carbon
metabolism in human MCF-7 cells (Herbig et al., 2002; Pfendner and Pizer,
1980). SHMT2 may also function in the conversion of glycine to serine
during gluconeogenesis (Christensen and MacKenzie, 2006; Nijhout et al.,
26 Jennifer T. Fox and Patrick J. Stover

Little is known about the regulation of SHMT2 expression and activity.

Unlike the cytoplasmic SHMT1 isozyme, SHMT2 expression is ubiqui-
tously expressed in human tissues (Girgis et al., 1997). Its activity is sensitive
to pyridoxal-phosphate levels (Anguera et al., 2006; Scheer et al., 2005).
SHMT2 transcription is myc responsive consistent with its role in generating
one-carbons for cytoplasmic metabolism; expression of the SHMT2 cDNA
in c-myc-null cells partially complements growth inhibition associated with
the loss of myc expression (Nikiforov et al., 2002).

2. Glycine cleavage system and aminomethyltransferase

The glycine cleavage system (GCS) is a multienzyme complex that catalyzes
the reversible oxidation of glycine to CO2, ammonia, and 5,10-methylene-
THF (Okamura-Ikeda et al., 2005). The complex consists of four proteins:
(1) the P-protein, which catalyzes the pyridoxal-phosphate-dependent
decarboxylation of glycine; (2) the H-protein, a lipoic acid-requiring
hydrogen carrier; (3) the T-protein, which is a THF-dependent amino-
methyltransferase (AMT); and (4) the L-protein, a lipoamide dehydroge-
nase. This complex is located in the inner mitochondrial membrane and
expressed in the liver, kidney, the glia-astrocyte lineage of the brain, and
the neuroepithelium during development (Ichinohe et al., 2004). Recent
stable isotope tracer studies in human subjects demonstrate that GCS
accounts for nearly 40% of overall glycine flux and that the 5,10-methylene-
THF produced from glycine catabolism makes major contributions to cyto-
plasmic THF-dependent purine and thymidylate biosynthesis (Lamers et al.,
Little is known regarding the regulation of GCS but has been shown to
be essential for normal embryonic development. Nonketotic hyperglycine-
mia (NKH) is an autosomal recessive inborn error of metabolism whose
clinical manifestations include severe mental retardation, seizures, apnea,
and hypotonia and result from the accumulation of glycine in all tissues
including the central nervous system. NKH is usually associated with
mutations in the P-protein or the T-protein (Dinopoulos et al., 2005).

3. Dimethylglycine dehydrogenase and sarcosine dehydrogenase

The oxidative catabolism of choline occurs through the sequential conver-
sion of choline ! betaine ! dimethylglycine ! sarcosine ! glycine;
dimethylglycine and sarcosine catabolism occurs in liver mitochondria
matrix through the activity of dimethylglycine dehydrogenase (DMGDH)
and sarcosine dehydrogenase (SDH), respectively. Both enzymes contain a
covalently bound FAD and are major folate-binding proteins in liver
(Wittwer and Wagner, 1980). The reaction mechanisms are not established
(Porter et al., 1985) but the electrons generated are transferred ultimately to
the electron-transport chain.
Folate-Mediated One-Carbon Metabolism 27

The quantitative contribution of choline degradation to the cytoplasmic

folate-activated one-carbon pool is not known.
Inborn errors of metabolism are associated with both DMGDH and
SDH deficiency. DMGDH deficiency results in muscle fatigue and body
odor; sarcosinemia is a rare autosomal disorder with a broad and variable
spectrum of symptoms including mental retardation and growth failure
(Binzak et al., 2001).

4. 10-FormylTHF synthetase
The final step in the putative conversion of the hydroxymethyl group of
serine to formate in mitochondria requires the generation of formate from
10-formylTHF (Appling, 1991). This reaction can occur in mitochondria
through the reverse reaction of FTHFS, driven by a favorable ADP/ATP
ratio in mitochondria (Appling, 1991). Mitochondria contain a monofunc-
tional FTHFS enzyme that is encoded by MthfdL1, which is expressed
ubiquitously in mammalian cells (Christensen et al., 2005; Prasannan et al.,
2003; Walkup and Appling, 2005). Further studies of this recently identified
FTHFS enzyme will determine if its primary function is to generate formate
from 10-formylTHF.

B. Folate-interconverting enzymes
1. 5,10-MethenylTHF cyclohydrolase and 5,10-methyleneTHF
Human mitochondria contain isozymes of MTHFD and MTHFC activities
encoded by a single gene, Mthfd2, which is believed to have evolved
through gene duplication and mutation of Mthfd1 (Di Pietro et al., 2002,
2004). Mthfd2 does not encode FTHFS activity, and the mitochondrial
MTHFD activity is distinguished from its cytoplasmic counterpart by its
NAD-dependence that serves to drive the reaction in the oxidative direc-
tion to generate 10-formylTHF (Christensen and MacKenzie, 2006).
Mthfd2 is an essential gene during mouse development but is not found in
adult tissues. Its expression appears to be limited to embryonic and trans-
formed cells (Peri and MacKenzie, 1993). Deletion in murine embryonic
fibroblasts creates a glycine auxotrophy, indicating a role for this enzyme in
generating unsubstituted THF for SHMT2 and potentially generating
fomate from serine. Therefore, while a complete folate-dependent pathway
for generating formate from serine exists in embryonic cells, the lack of
identified MTHFD and MTHFC activities in adult tissues represents a gap
in our understanding of mitochondrial folate metabolism and/or the inter-
action between cytoplasmic and mitochondrial one-carbon metabolism in
adult tissues.
28 Jennifer T. Fox and Patrick J. Stover

C. Biosynthetic enzymes
1. Methionyl-tRNAfMet formyltransferase
Protein synthesis in mitochondria and prokaryotes is initiated with formyl-
methionyl-tRNA (fMet-tRNA), which is formed by the 10-formylTHF-
dependent formylation of Met-tRNA-catalyzed methionyl-tRNAfMet
formyltransferase (MFT) (Bianchetti et al., 1977; Takeuchi et al., 1998). This
is the only known biosynthetic reaction that occurs in mitochondria, other
than amino acid interconversion reactions (Fig. 1.1). Although MFT-deficient
Saccharomyces cerevisiae displays normal mitochondrial function and mitochon-
drial protein synthesis (Tibbetts et al., 2003), MFT does offer selective advan-
tage under severe growth conditions (Vial et al., 2003). Formylation of
Met-tRNA confers specificity to its interaction with initiation factor
2 (IF-2); bovine IF-2 binds fMet-tRNA with 25-fold greater affinity than
Met-tRNA and mitochondrial ribosomes bind fMet-tRNA 50-fold tighter
than Met-tRNA in the presence of IF-2 (Spencer and Spremulli, 2004).

IV. Nuclear Fol ate-Mediated One-Carbon

There is increasing evidence that folate-mediated thymidylate syn-
thesis occurs in both the nucleus and the cytoplasm (Fig. 1.1). Approxi-
mately 10% of cellular folate is present in the nucleus, and TS and
SHMT1 have been localized to the nucleus in several mammalian cell
types in S-phase (Anderson et al., 2007; Bissoon-Haqqani et al., 2006;
Brown et al., 1965; Prem veer Reddy and Pardee, 1980; Samsonoff et al.,
1997; Wong et al., 2001). The three enzymes that constitute the TS cycle
[SHMT1, TS, and dihydrofolate reductase (DHFR)] are all substrates for
UBC9-mediated modification with the small ubiquitin-like modifier
(SUMO), which targets proteins for nuclear localization during S-phase
(Anderson et al., 2007; Woeller et al., 2007a). Nuclear TS was shown to form
part of a putative replitase complex along with DNA polymerase a,
ribonucleotide reductase, thymidylate kinase, NDP kinase, the folate-
dependent enzyme DHFR (Boorstein and Pardee, 1983; Noguchi et al.,
1983; Prem veer Reddy and Pardee, 1980), and possibly SHMT1 (Woeller,
2007a). Because SHMT1 exhibits a narrow range of tissue-specific expres-
sion compared with TS and DHFR, it is unlikely that all cells synthesize
thymidylate in the nucleus. Although the biological significance of nuclear
dTMP synthesis at the cellular level remains unclear, it has been hypothesized
that its association with the replitase complex allows for de novo thymidylate
synthesis directly at the replication fork during S-phase and may lower uracil
misincorporation into DNA.
Folate-Mediated One-Carbon Metabolism 29

This work was supported by PHS DK58144 to PJS.

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Mathematical Models of
Folate-Mediated One-Carbon
H. F. Nijhout,* M. C. Reed, and C. M. Ulrich

I. Introduction 46
II. Structure and Function of the Cycles 49
III. Why Mathematical Modeling? 51
A. Previous modeling efforts 52
B. Why modeling? 54
C. Difficult issues in modeling 55
D. Advantages of mathematical models 57
E. Kinetics, parameter values, and model structure 59
IV. Model Development 61
V. Blood Versus Intracellular Metabolite Concentrations 66
VI. Modeling GeneGene and GeneEnvironment Interactions 67
VII. Modeling and Simulation have Revealed Novel Homeostatic
Mechanisms 70
VIII. Steady States and Fluctuations 75
IX. Conclusions 77
Acknowledgments 78
References 78

Folate-mediated one-carbon metabolism is an unusually complex metabolic
network, consisting of several interlocking cycles, and compartmentation
between cytosol and mitochondria. The cycles have diverse functions, the
primary being thymidylate synthesis (the rate limiting step in DNA synthesis),
the initial steps in purine synthesis, glutathione synthesis, and a host of methyl
transfer reactions that include DNA and histone methylation. Regulation within
the network is accomplished by numerous allosteric interactions in which

* Department of Biology, Duke University, Durham, North Carolina 27705

Department of Mathematics, Duke University, Durham, North Carolina 27705
Cancer Prevention Program, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109

Vitamins and Hormones, Volume 79 # 2008 Elsevier Inc.

ISSN 0083-6729, DOI: 10.1016/S0083-6729(08)00402-0 All rights reserved.

46 H. F. Nijhout et al.

metabolites in one part of the network affect the activity of enzymes elsewhere
in the network. Although a large body of experimental work has elucidated the
details of the mechanisms in every part of the network, the multitude of
complex and non-linear interactions within the network makes it difficult to
deduce how the network as a whole operates. Understanding the operation of
this network is further complicated by the fact that human populations maintain
functional polymorphisms for several enzymes in the network, and that the
network is subject to continual short and long-term fluctuations in its inputs as
well as in demands on its various outputs. Understanding how such a complex
system operates is possible only by means of mathematical models that take
account of all the reactions and interactions. Simulations with such models can
be used as an adjunct to laboratory experimentation to test ideas and alterna-
tive hypotheses and interpretations quickly and inexpensively. A number of
mathematical models have been developed over the years, largely motivated
by the need to understand the complex mechanisms by which anticancer drugs
like methotrexate inhibit nucleotide synthesis and thus limit the ability of cells
to divide. More recently, mathematical models have been used to investigate
the regulatory and homeostatic mechanisms that allow the system to accom-
modate large fluctuations in one part of the network without affecting critical
functions elsewhere in the network. 2008 Elsevier Inc.

I. Introduction
The origin of our mathematical modeling work stems from an interest in
understanding how genes and the environment interact in the biochemistry of
cells. This led us to study folate and methionine metabolism because this part of
cell metabolism is linked to a diversity of human diseases that have both genetic
and environmental contributing factors. Folate and other B vitamins play
critical roles in the biochemical reactions of one-carbon metabolism that are
related to amino acid metabolism, nucleotide synthesis, and numerous
methyl-transferase reactions, including DNA and protein methylation.
Defects in folate-mediated one-carbon metabolism (FOCM; Table 2.1
lists the acronyms and abbreviation used in this chapter), either due to
mutations in the genes that code for enzymes in the pathway or to deficien-
cies in vitamin cofactors, are associated with megaloblastic anemia, spina
bifida and other neural tube defects, cardiovascular disease, increased sensi-
tivity to oxidative stress, and a variety of neuropsychiatric disorders. FOCM
is also involved in the etiology of colorectal and other types of cancer, and
chemotherapeutic agents, such as methotrexate and 5-fluorouracil, target
FOCM and play a central role in cancer treatment. FOCM is highly com-
plex. It consists of a set of interlocked biochemical cycles (Fig. 2.1) whose
enzymes are subject to complex allosteric regulations. The function of this
complex network is further complicated by the fact that there are genetic
polymorphisms for many of the enzymes in the network, and the functions
Mathematical Models of Folate-Mediated One-Carbon Metabolism 47

of the network are sensitive to the input of various amino acids (glycine,
serine, methionine, and cysteine), B vitamins (folic acid, B6 and B12), and is
affected by environmental factors such as alcohol intake in intricate ways that
alter the normal operation of the network and the risk of disease.
Considerable research over the past 40 years has identified most if not all of
the important details of FOCM. However, a limiting factor of these critical
studies is that they have primarily focused on single reactions and on small
portions of the pathway, and thus provide no means for understanding the
overall functioning of the system. The multiple cycles and pathways of FOCM
together are part of a complex nonlinear system, which is difficult to capture
using purely experimental methods. Mathematical modeling is an approach
that has been particularly useful in the study of complex biological systems
(Edelstein-Keshet, 1988; Murray, 1989). Below we will review how mathe-
matical modeling has been able to confirm key hypotheses about the operation
of various portions of FOCM, and how modeling has provided novel insights
into the properties and consequences of various regulatory mechanisms that
stabilize portions of the network against environmental perturbations.

Table 2.1 Abbreviations and acronyms used in the text and figures

Acronym Name
10f-THF 10-Formyltetrahydrofolate
10f0DHF 10-Formyldihydrofolate
5fTHF 5-Formyltetrahydrofolate (leucovorin)
5mTHF 5-Methyltetrahydrofolate
AICAR P-ribosyl-5-amino-4-imidazole carboxamide
AICART Aminoimidazolecarboxamide ribonucleotide transferase
BET, Bet Betaine
BHMT Betaine-homocysteine methyltransferase
CBS Cystathionine b-synthase
CH:NHTHF 5-Formiminotetrahydrofolate
CHTHF 510-Methenyltetrahydrofolate
CH2-THF 510-Methylenetetrahydrofolate
CTGL g-Cystathionase
Cys Cysteine
Cyst Cystathionine
DHF Dihydrofolate
DHFR Dihydrofolate reductase
DHFS Dihydrofolate synthase
DHPR Dihydropteridine reductase
DMG Dimethylglycine
DMGD Dimethylglycine dehydrogenase
DNMT DNA-methyltransferase
48 H. F. Nijhout et al.

Table 2.1 (continued)

Acronym Name
dTMP Deoxythymidine monophophate
dUMP Deoxyuridine monophophate
FOCM Folate-mediated one-carbon metabolism
FR-RFC Folate receptor reduced folate carrier
FTD 10-Formyltetrahydrofolate dehydrogenase
FTD 10-Formyltetrahydrofolate dehydrogenase
FTS 10-Formyltetrahydrofolate synthase
GAR Glycinamide ribonucleotide
GCS g-Glutamylcysteine synthetase
GDC Glycine decarboxylase (glycine cleavage system)
Glut Glutamate
Glut-Cys Glutamyl-cysteine
Gly Glycine
GNMT Glycine N-methyltransferase
GPX Glutathione peroxidase
GR Glutathione reductase
GS Glutathione synthetase
GSH Reduced glutathione
GSSG Oxidized glutathione disulfide
H2CO Formaldehyde
H2O2 Hydrogen peroxide
HCOOH Formate
Hcy Homocysteine
MAT-I Methionine adenosyl transferase I
MAT-II Methionine adenosyl transferase II
MAT-III Methionine adenosyl transferase III
Met Methionine
MS Methionine synthase
MTCH 5,10-Methenyltetrahydrofolate cyclohydrolase
MTD 5,10-Methylenetetrahydrofolate dehydrogenase
MTHFR 5,10-Methylenetetrahydrofolate reductase
MTS 5,10-Methenyltetrahydrofolate synthetase
NADPH Nicotinamide adenine dinucleotide phosphate
NE non-enzymatic conversion
PGT Phosphoribosyl glycinamidetransformylase
SAH S-adenosylhomocysteine
SAHH S-adenosylhomocysteine hydrolase
SAM S-adenosylmethionine
Sarc Sarcosine
SDH Sarcosine dehydrogenase
Ser Serine
SHMT Serinehydroxymethyltransferase
TS Thymidylate synthase
THF Tetrahydrofolate
Mathematical Models of Folate-Mediated One-Carbon Metabolism 49

One-Carbon Metabolism
Folate Methionine Blood
FR-RFC Metin


Purine Met SAM
Sarc vSer GNMT
10f-THF Ser Ser 10f-THF MS BHMT
SDH Sarc
vGly NE Methylation
Gly Gly Gly DHF reactions

CH=THF Pyrimidine CH=THF 5mTHF

GDC synthesis
Ser Adenosine H O
Mitochondria CTGL
Oxidative stress
H2O2 Cys
Serin Glyin Cysin

Serine Glycine Cysteine

GSSG Glutathione(GSH)


Figure 2.1 Diagram of mammalian hepatic folate-mediated one-carbon metabolism.

This pathway includes the mitochondrial compartmentation, reduced glutathione
synthesis, and transport of some metabolites from the blood. Metabolites that are
variables in the model are enclosed in boxes, and enzymes are in ellipses. Full names
of the acronyms of enzymes and metabolites are given in Table 2.1.

II. Structure and Function of the Cycles

FOCM consists of three functional modules: the folate cycle, the
methionine cycle, and the glutathione synthesis pathway. In this chapter,
we do not consider the role of the polyamine synthesis pathway which has
recently been modeled by Rodriguez-Caso et al. (2006).
The function of FOCM is to pick up carbons from amino acids, primarily
serine, but also glycine and methionine, and deliver them (as methyl groups)
for the synthesis of purines and pyrimidines and for a variety of methylation
reactions (e.g., DNA, tRNA, and histones) (Clarke and Banfield, 2001; Cook,
2001; Shane, 1995; Wagner, 1995). Serine enters the reactions as a substrate
for SHMT which transfers one carbon to THF, yielding glycine and CH2-
THF. The glycine can enter the mitochondria where it is processed by the
glycine cleavage system, transferring one of its carbons to CH2-THF in the
50 H. F. Nijhout et al.

mitochondrial folate cycle. The mitochondrial cycle then releases its carbons
to the cytosol as formate (HCOOH in Fig. 2.1). Glycine is also used in the
synthesis of sarcosine by GNMT. Sarcosine, in turn, also enters the mitochon-
dria and eventually yields all but one of its carbons to the mitochondrial folate
cycle. Serine is also used by the CBS reaction which complexes it with
homocysteine to yield cystathionine, which, in turn, is used for the synthesis
of cysteine and glutathione. The one-carbon units held by CH2-THF have
three fates: they can be passed to 5mTHF by MTHFR and subsequently to the
methionine cycle where they are used in a great diversity of methylation
reactions; they can also be passed to 10f-THF and subsequently used for
purine synthesis; finally they can be passed to TS and used to synthesize
dTMP from dUMP. Thus, FOCM plays a critical role in nucleotide synthesis,
and the TS reaction is the rate-limiting step in DNA synthesis (Fukushima
et al., 2003). The cytosolic and mitochondrial SHMT reactions are reversible.
In the forward direction they use serine, and in the backward direction they
use glycine and one-carbon units from the mitochondrial folate cycle to
synthesize serine, which can serve as the basis for gluconeogenesis. Methionine
enters the methionine cycle and is adenosylated by MAT-I and MAT-III
(in the liver; MAT-II is the adenosyl transferase used in other tissues).
S-adenosyl methionine (SAM) serves as the general methyl donor for the
majority of methylation reactions in the cell. About half of the mass of
methionine that enters the methionine cycle leaves via the transulfuration
pathway to cystathionine and cysteine (Finkelstein, 1990; Finkelstein and
Martin, 1986), and the other half is remethylated to methionine by MS and
BHMT, using methyl groups from 5mTHF and betaine, respectively.
If the reactions illustrated in Fig. 2.1 were the only pertinent ones, this
would be a case of complicated but standard biochemistry. However, many
of the metabolites in this system are allosteric activators or inhibitors of
enzymes at some distance in the network. For example, SAM inhibits
BHMT and MTHFR and activates CBS; DHF inhibits MTD, MTCH,
and MTHFR; 5mTHF inhibits GNMT and SHMT (Finkelstein, 2003;
Finkelstein and Martin, 1984; Finkelstein et al., 1972; Jencks and Matthews,
1987; Kluijtmans et al., 1996; Ou et al., 2007; Yamada et al., 2001; Yeo and
Wagner, 1992). Many reactions also depend strongly on the cellular status
for folate and vitamins B6 and B12. In addition, the velocities of many
reactions depend on the concentrations of the substrates that are controlled
by dietary inputs of glycine, serine, glutamate, cysteine and methionine.
These inputs naturally undergo enormous fluctuations so the system is often
far away from steady state (Nijhout et al., 2007b).
It is a significant challenge to understand the biological reasons for the
complicated interlocking cycles, the compartmentalization to the mitochon-
dria, and the multiple reactions by which one substrate can be transformed
into another. Since many parts of the network of FOCM share enzymes and
metabolites, there must be mechanisms that ensure that large variation in a
Mathematical Models of Folate-Mediated One-Carbon Metabolism 51

particular region of the network does not compromise the function in other
regions. Furthermore, the many critical reactions in the network must be
buffered against large and irregular hourly and daily fluctuations in inputs of
amino acids. The overall network is too complex to understand these
regulatory functions by inspection of the reaction diagram alone, or to
deduce the integration of its various functions with any degree of certainty.
The system is well-enough understood that it is possible to develop a
mathematical description of the kinetics of the various individual reactions,
and couple these together into a single mathematical simulation model that
can be used to explore questions about structure and function.
Over the past 5 years, we have developed mathematical models for the
pathways shown in Fig. 2.1, which represents mammalian hepatic one-
carbon metabolism. Hepatic FOCM is what we might call the complete
pathway, in that all known enzymes and reactions operate in the liver. This
is not true for most other tissues. While most FOCM enzymes are also
expressed in the kidney, most other tissues in the body express only a subset
of the enzymes and thus operate on what we might called reduced
FOCM. FOCM is an ancient pathway and we have recently developed
a model for the structure and kinetics of folate metabolism in bacteria
(Leduc et al., 2007 and Fig. 2.6).

III. Why Mathematical Modeling?

We view a mathematical model as an experimental tool, much like
electrophoresis or PCR or gene knockout. Like all experimental tools,
models have their own particular strengths and limitations and these should
be understood if the tool is used to address a particular problem. A model is a
mathematical description of a specific system. One of the particular
strengths of a model is that it is completely explicit about what is in the
system and what is not. In addition, a model is explicit about all
the assumptions that are made about the properties of the components of
the system and about their interactions.
The mathematical models we are dealing with here are not theoretical
models in the sense that they attempt to discover necessary and sufficient
conditions for the behavior of a particular system, or attempt to estimate
parameter values for the system. Rather, they are strict quantitative descrip-
tions of properties that have been determined experimentally by investiga-
tors. Ideally, a model represents the state of our understanding of the
properties and interactions among the component parts of a system, and
allows one to examine the behavior of the ensemble, and the consequences
for the system as a whole of various assumptions one makes about how the
components behave and interact. The model is tested against as much
52 H. F. Nijhout et al.

experimental data as possible. Ideally, the model reproduces the results of a

broad diversity of experiments both qualitatively and quantitatively.
If it does, then it can be used as an experimental tool to ask questions and
do experiments that would be difficult, or expensive, or unethical to do
in a real living system. In particular, a model provides a means to rapidly and
inexpensively test the effects of specific perturbations and of alternative
experimental strategies before committing time and resources to potentially
expensive, and possibly inconclusive laboratory experiments. In its most
useful guise, simulations with a model should interact with laboratory
experimentation in a mutually illuminating exploration of FOCM.
A modeling approach is useful when the system one wishes to study is
large and complex, with nonlinear interactions. Nonlinear systems produce
context-dependent and nonintuitive responses to perturbations, and a sim-
ple examination of the connectivity diagram is seldom able to reveal
anything useful about the dynamics of the system, nor its response to
perturbation. FOCM is such a large, complex, and nonlinear system, con-
sisting of several interlocking cycles with multiple inputs and outputs
(Fig. 2.1). In addition, many of the enzymes in this system are subject to
complex allosteric regulation by metabolites that are many steps removed in
the network. These long-range regulatory interactions provide important
homeostatic functions (see Section VII), which can only be evaluated by
simulation studies.

A. Previous modeling efforts

A number of investigators have developed mathematical modes for various
parts of FOCM (Harvey and Dev, 1975; Jackson and Harrap, 1973;
Morrison and Allegra, 1989; Seither et al., 1989; Vorontzov et al., 1980;
Werkheiser et al., 1973) and the methionine cycle (Martinov et al., 2000;
Prudova et al., 2005). Perhaps the best known among these are the extensive
studies of R. C. Jackson and his associates ( Jackson, 1980, 1984, 1986,
1993, 1995; Jackson and Harrap, 1973, 1979).
Almost without exception the models of folate metabolism have been
aimed at understanding the mechanism of action and the kinetics of anti-
cancer drugs, particularly methotrexate and 5-fluorouracil. In most cases the
models focused on the portions of the system that were most relevant for
their investigations. Jackson (1980, 1986; Jackson and Harrap, 1979) devel-
oped what is probably the most extensive model for folate metabolism
(Fig. 2.2A), consisting of more than 60 reactions that also included the
kinetics of membrane transport of folate and methotrexate, and more
detailed reactions for the synthesis of purines, pyrimidines, RNA, and
DNA. This model made specific predictions about the rates of DNA
synthesis and the amount of time required to replicate all the DNA in a
cell, and was thus able to estimate the maximal rate of cell division under
Mathematical Models of Folate-Mediated One-Carbon Metabolism 53

Pyrimidine HCOOH
Glycine SH Purine
5,10-CH2-THF 5 mTHF

FDS Serine
Purine Methionine
AICART synthesis
Pyrimidine HCOOH AICAR
synthesis M
Glycine SH

dUMP 10f-THF


5,10-CH2-THF 5 mTHF

Figure 2.2 Diagrams of two early models of folate metabolism. A, the model of
Jackson (1980). This model also includes synthesis of nucleotides, RNA and DNA, as
well as the transport of folates and methotrexate into the cell, not shown in this
diagram. B, the model of Morrison and Allegra (1989). Full names of the acronyms
of enzymes and metabolites for this and other figures, and the text, are given in
Table 2.1. Redrawn from Jackson (1980) and Morrison and Allegra (1989).

various methotrexate treatment regimes. The simulated results closely

matched experimentally observed data on the inhibition of cell division
by various methotrexate treatments. The Morrison and Allegra model
(1989), shown in Fig. 2.2B, dealt specifically with the kinetics of folate
metabolism in the MCF-7 breast cancer cell line, and included the effects of
methotrexate polyglutamation and the consequent improved cellular reten-
tion of methotrexate. In spite of the fact that these models typically dealt
only with subsections of one-carbon metabolism, and did not include any of
the allosteric interactions that regulate and stabilize metabolite and fluxes,
54 H. F. Nijhout et al.

they were generally able to simulate the correct pool sizes of several of the
metabolites, and the time course of inhibition of nucleotide synthesis rates
by treatment with antifolate cancer drugs.

B. Why modeling?
The traditional negative view of mathematical modeling is the following.
If the biology and biochemistry are well understood, then there is no reason
for models. On the other hand, if the biology or biochemistry is not well
understood then there is not enough information to make an accurate
model. Therefore, in either case, mathematical modeling is useless. And,
of course, this negative view is reinforced by poor modeling or modelers
who do not want deal with the full complexity of biological systems. In fact,
many biological systems are partly understood in the sense that there is
good information about many of the components of the systems but
incomplete information about how the components work together to
give rise to functional system properties. This is exactly the case with
FOCM where a great deal of information is available on individual reactions
but there is not much understanding of how the whole system works
together. It is in this intermediate partly understood situation that a
mathematical model can be a valuable, indeed necessary, investigative
tool. To illustrate this point, it is helpful to face how difficult it really is to
understand FOCM.
It is possible to understand a moderately sized traditional biochemical
reaction diagram by walking the diagram. If substrate A goes up then, since
A makes B, we expect B to go up and so forth. However, the existence of
allosteric interactions by which substrates in one part of the network activate
or inhibit enzymes in other parts makes this type of simple reasoning
impossible or at best inconclusive. For example, SAM activates the enzyme
CBS and inhibits the enzyme MTHFR. So, if we moderately increase the
methionine input to the system, will the homocysteine concentration go up
or down? Well, we would expect more mass in all the methionine cycle
metabolites, so [Hcy] should go up. On the other hand, when methionine
input goes up SAM rises appreciably and this will activate CBS, which will
draw down [Hcy]. But since SAM is up, it will inhibit MTHFR, which
will lower [5mTHF]. Since [5mTHF] is lower, the MS reaction will run
more slowly and thus [Hcy] is not used as rapidly and thus should go up. So
what will happen to [Hcy]? It is clear that no amount of verbal reasoning is
going to answer this question, especially since the reactions and the allosteric
interactions are nonlinear. One has to make calculations (and experiments)
about the relative strengths of the competing influences on [Hcy]. Two
other issues make the question even more daunting. First, the answer
may depend on the overall context of the rest of FOCM (see below).
Second, the allosteric activation of CBS and inhibition of MTHFR may
Mathematical Models of Folate-Mediated One-Carbon Metabolism 55

have evolved to stabilize [Hcy] concentration in the face of moderate

changes in methionine input, in which case the answer to the question of
whether [Hcy] goes up or down is neither: it doesnt change much.
It is now well understood that gene expression is a stochastic process that
leads to phenotypic protein differences even among identical cells
(Elowitz et al., 2002; Sigal et al., 2006). Not only do the protein levels
vary by as much as 1530% from the mean from cell to cell, but also the
levels vary over time even in individual cells. This variation has conse-
quences for the understanding of FOCM. First, it does not make sense to
ask for the exact value of a given parameter (a Vmax, a Km, or a Ki). Those
values will vary substantially from cell to cell and from time to time in any
given cell. Second, specific questions like Does [Hcy] go up or down?
may have answers that depend on the context of all the other enzymes in the
system. Third, some of the most important properties of FOCM (or indeed
of all of cell metabolism) are regulations, not obvious from the standard
biochemical reaction diagram, that allow the system to function despite
these large variations. The allosteric interactions mentioned above are
examples of such regulations. Thus, FOCM should not be thought of as a
single fixed system but a whole family of systems with large variations in
important parameters. It is difficult to see how one could understand
function in the face of such variation without mathematical modeling.
In Nijhout et al. (2007b), we show how many of the concentrations and
reaction velocities of hepatic FOCM react to the daily inputs of amino acids
due to meals. Some concentrations and velocities fluctuate wildly while
others are protected by regulatory mechanisms. More recent calculations
with the full model depicted in Fig. 2.1 (see Fig. 2.10) show the same
behavior. This is the reason, of course, that many experimental and clinical
measurements are done in the fasting state. Because of the difficulty of
making many simultaneous measurements of concentrations and velocities
as functions of time in living cells it is difficult to see how such dynamic
fluctuating behavior could be investigated experimentally. Thus, mathe-
matical modeling has a central role in elucidating the regulatory mechanisms
that allow cells to adapt to such dramatic changes in inputs.

C. Difficult issues in modeling

Suppose that one wants to investigate a particular phenomenon in FOCM
seen experimentally or clinically, for example, the behavior of homocyste-
ine under methionine loading or the stability of the glutathione pool in the
face of daily meals. Which variables and interactions should be included?
If the model is too small, one may have excluded (or rather held constant)
just those variables and interactions that are crucial to understanding the
phenomenon. If the model is too large it may be too unwieldy to experi-
ment with and the noise from all the approximations that one makes may
56 H. F. Nijhout et al.

obscure the phenomenon that one wants to study. So, how large should a
model be? This is always a difficult question (though usually not admitted by
modelers), and every modeling attempt answers it explicitly by what is
included and what is excluded. Since our goal is not only to reproduce
experimental or clinical results but also to use the model to understand how
and why they arise, our philosophy is to start with smaller models and
expand to larger models when the smaller ones are well understood and the
expansion to more variables is necessary. Thus, as we outline below in
Section IV, we began with a model of methionine metabolism that had only
four variables (Reed et al., 2004). Then we made a model of the folate cycle
(Nijhout et al., 2004) so we could study the inhibition of DHFR by
methotrexate and the allosteric binding of folates to folate enzymes. Then
we made a larger model combining to two smaller ones so that we could
study the effects of the inhibition of MTHFR by SAM and the inhibition of
GNMT by 5mTHF (Nijhout et al., 2006). There has been a long discussion
in the literature or the role of the folate cycle in mitochondria (Appling,
1991; Christensen and MacKenzie, 2005) so to investigate these questions
we added compartmentation and the mitochondrial reactions (Nijhout
et al., 2007a). At each stage we had to make difficult (imperfect) decisions
about which variables and interactions to include in the models.
We note that this difficulty of knowing where to draw the boundaries is
also a difficulty for the interpretation of laboratory experiments or clinical
observations. In an experiment one changes the system by, say, knocking
out a gene or introducing a chemical that binds to a particular enzyme. One
then measures the changes in a few variables (the results of the experi-
ment) and then makes conclusions about how the system functions. Implic-
itly, one is assuming that everything else besides what one measures is the
same (or can be considered the same), that the knocked out gene did not
affect other genes or that the inhibitor has no other effects but the intended
one. The interpretation of the experiment typically involves implicitly
drawing the boundaries of a model (in the experimenters head) of
which variables are allowed to be included in the interpretation. Thus,
the interpretation of experimental results must always face and answer
(albeit implicitly) the same difficult question of boundaries faced explicitly
in modeling.
The next difficulty is deciding what level of detail to include for individ-
ual reactions in FOCM. An enormous amount of information is available
about enzymes, their genes and conformations and the way that they bind to
substrates. How much of this detail should be included? Our approach is to
use simple Michaelis-Menten kinetics and simple kinetic forms for activa-
tions and inhibitions unless we have good reason to believe that a more
detailed treatment is necessary for important biological functions of FOCM.
For example, we could have modeled the synthesis of SAM from methionine
in liver cells by a simple Michaelis-Menten formula. But we knew from the
Mathematical Models of Folate-Mediated One-Carbon Metabolism 57

experiments of Finkelstein et al. (1982) and Finkelstein and Martin (1984,

1986) that the methionine levels are fairly stable under methionine loading
whereas SAM increases enormously, and that Corrales et al. (2002) had
suggested that this is a result of the different kinetics of the two isoforms
MAT-I and MAT-III. Because we believe that the stabilization of methio-
nine and the many regulations by SAM are biologically important, we
decided to include the rather complicated special kinetics of MAT-I and
MAT-III in the model. Subsequently, we were able to show (unpublished)
that the suggestion of Corrales et al. (2002) was completely correct.
Finally, of course, one must choose Vmax, Km, and Ki values. This is not
such an easy matter since there are few measurements of Vmax values and the
reported measurements of Vmax, Km, and Ki values show large variation.
Given the stochastic variation in gene expression discussed above and the
dependence of protein conformation and function on the in vivo context,
this variation is not surprising. We try to choose Km, and Ki values within
reasonable experimental ranges and adjust the Vmax values so that the values
of the metabolite concentrations are in the experimental ranges. It is always
a question whether the results of in silico experiments would have been
different if we had chosen different parameters. We have found that most of
our qualitative results are quite insensitive to variations in parameter values.
In some sense, it has to be that way because FOCM must have evolved to be
able to continue to function in the face of the stochastic variation in
gene expression discussed above. Nevertheless, all three difficulties that
we have discussed necessarily temper the confidence that one has (that we
have!) in model results.

D. Advantages of mathematical models

Although models have difficulties and limitations, they also have advantages
and it is worthwhile to state them explicitly. First, to formulate a model one
has to be explicit about ones assumptions. If A inhibits B one must say how
much B is inhibited at different concentrations of A and how this may
or may not depend on other variables in the system. Secondly, once one has
a model, in silico experimentation is cheap, fast, and easy. One does not need
animals, IRB protocols, or technicians. Third, and most important, when
the model behaves in the same way as interesting experimental results, one
can take the model apart and put it back together (by removing reactions or
inhibitions, for example) until one understands the causal chain of events
that gives rise to the observed behavior. Thus, experiments with the model
can give real biological understanding of the phenomena under study.
Finally, in the model, one can follow the time course of all concentrations
and velocities and determine how the system reacts to outside influences
or changes in internal parameters. This is impossible to do by in vivo
58 H. F. Nijhout et al.

Every experimental scientist is a modeler because every hypothesis is

based on a conceptual model of how a system ought to behave. A mathe-
matical model is simply a way of making a conceptual model explicit by
describing and connecting all the underlying knowledge and assumptions.
If a mathematical model does not reproduce the known behavior of a
system then, obviously, the model is wrong. But if the model is based on
all known data, then the ancillary conclusion is that the knowledge of the
system must be inadequate. Thus, a model can reveal the inadequacy of
current data or concepts. The model can then be used to test hypotheses
about what kind of additional (or different) information can yield the
correct behavior, and this can stimulate research to verify those predictions.
Another important use of a model is to test hypotheses about mechan-
isms that are difficult to study experimentally. We will give two examples
from FOCM. The first comes from a series of studies by Finkelstein and
Martin (1984, 1986) and Finkelstein (1990, 2001) who studied the allosteric
effect of SAM on the CBS and BHMT. They suggested that the concen-
tration of SAM rose with methionine input and that the allosteric stimula-
tion of CBS and inhibition of BHMT by SAM would result in an increased
transsulfuration of homocysteine, which removes the excess methionine
from the system. Thus, the allosteric regulations by SAM constitute a
homeostatic mechanism that stabilizes the mass in the methionine cycle.
Our simulations with a model of the methionine cycle (Reed et al., 2004)
show that variation in methionine input is completely absorbed by variation
in the concentration of SAM. The model also shows that the allosteric
regulation of BHMT and CBS by SAM increases the transsulfuration rate in
such a way that total mass in the methionine cycle, and the flux around the
methionine cycle, remain stable in the face fluctuating methionine input, as
first hypothesized by Finkelstein and Martin (1984).
The second example involves the role of the mitochondrial bifunctional
enzyme. In the mitochondria, the MTD MTCH reactions are catalyzed
by a single bifunctional enzyme (Mejia and MacKenzie, 1986; Peri et al.,
1989). This enzyme is not normally expressed in adult cells; it is expressed
only during embryonic development and in cancer cells (Di Pietro et al.,
2004; Smith et al., 1990), so its expression appears to be restricted to cells
undergoing high rates of cell division. On the basis of interpretation of a
series of radiotracer and gene knockout experiments, Christensen and
MacKenzie (2005) hypothesized that the bifunctional enzyme provides a
metabolic switch that controls the flow of one-carbon units to determine,
for example, the degree to which mitochondria produce formate and/or
convert glycine to serine. This hypothesis was confirmed by our mathe-
matical model (Nijhout et al., 2007a). Elimination of the mitochondrial
bifunctional enzyme in the model did not show a runaway accumulation of
CH2-THF, as might be expected. Instead, the GDC reaction slowed down,
the production and export of formate stopped entirely, and most
Mathematical Models of Folate-Mediated One-Carbon Metabolism 59

importantly, the mitochondrial SHMT reaction reversed direction and now

ran toward serine synthesis. Thus, in the presence of the bifunctional
enzyme, a situation typical of embryonic and cancer cells, the mitochondria
export large quantities of formate that are directed to purine and TS in the
cytosol. When the bifunctional enzyme is not expressed, as in adult cells that
do not divide, the mitochondrial reactions become strong producers of
serine, which is exported to the cytosol and where it is directed toward
gluconeogenesis and other reactions. The bifunctional enzyme switch in
effect transforms the mitochondria from formate factories into serine fac-
tories, and may thus be an adaptation to the very different metabolic and
biosynthetic needs of rapidly growing embryonic cells and more quiescent
adult cells, as suggested by Christensen and MacKenzie (2005).

E. Kinetics, parameter values, and model structure

The reported diversity of parameter values for the same enzyme can be due
to various reasons: (1) the orthologous enzymes from different species can
have different kinetic properties; (2) enzyme expression differs in different
tissues, in particular some enzymes are up-or downregulated in cancers as
well as in tissues of animals undergoing chronic nutrient or vitamin depri-
vation or excess; (3) different semipurified enzyme preparations may con-
tain different, and unknown, concentrations of allosteric activators or
inhibitors; (4) enzyme preparations made at different times of day can
contain different concentrations of metabolites and allosteric effectors;
(5) in bimolecular reactions the values of the Kms depend on the concen-
tration of both substrates (Segel, 1975), but it is common to maintain one of
the substrates constant, resulting in the measurement of an apparent Km that
can differ depending on the preparation used.
Our approach to modeling the kinetics of one-carbon metabolism is to
restrict our use of reported kinetics to those measured in mammals, prefera-
bly humans, and we differentiate between parameters measured in different
tissues by building different models that specifically deal with hepatic
one-carbon metabolism and epithelial one-carbon metabolism (Figs. 2.3
and 2.5). Although measures of kinetic parameter values can vary signifi-
cantly, fortunately metabolite concentrations can be measured with great
accuracy and consistency, and the actual flux through a particular reaction,
or the relative dimensions of the fluxes through different portions of the
pathway are sometimes known. We start our modeling by choosing a value
of each Km and Ki roughly in the middle of the reported range, and vary the
Vmax to obtain the reported metabolite concentrations and fluxes. We have
found experimental Vmax values to be largely not useful for modeling
purpose since they are typically reported in units of rate/mg protein,
without stating how protein was determined. We use values of the kcat in
those few cases where in vivo enzyme concentrations are known. We have
60 H. F. Nijhout et al.

Folate cycle Methionine cycle

Serine Purine Methionine SAM
Pyrimidine synthesis
synthesis DNA
dTMP M H2C=O Glycine
Glycine SH


dUMP Methylation
Sarcosine reactions
Betaine DNA-CH3
5,10-CH2-THF 5 mTHF Serine

Figure 2.3 Diagram of our model of the coupled hepatic folate and methionine cycles.
Not shown in this figure are all the allosteric interactions between metabolites and
various enzymes in the two cycles. Full names of the acronyms of enzymes and
metabolites for this and other figures, and the text, are given in Table 2.1. Redrawn
from Reed et al. (2006).

found that the choices of Vmax values are often constrained by the require-
ment that the model produce the right combination of known metabolite
concentrations, relative flux rates, half-lives, and time-dependent responses
to perturbations in the experimental literature.
When the kinetic mechanism of an enzyme is known we use the
conventional equations for the relevant uni- or bimolecular reaction as
described by Segel (1975). Allosteric activation or inhibition of enzymes
often does not admit to one of the conventional equations. In such cases, we
do a nonlinear regression on the published experimental data and use that as
the empirical equation. In these, as in all, cases, we ensure that the model
operates within the limits of the experimental data. Our models assume that
certain substrates (dUMP, GAR) and energetic metabolites (ATP, NADP,
and NADPH) are constant, so that their effect is absorbed by the Vmax for
the reaction.
At present the model does not contain terms for polyglutamation and
deglutamation. The model also does not contain a nuclear compartment,
although it is known that nuclear compartmentation is important (Appling,
1991; Woeller et al., 2007). We set the total folate level in the cell by
defining the overall size of the folate pool. If we start the simulation with all
folates in one form (e.g., THF or 5mTHF), the reaction kinetics rapidly
redistribute the folates, and the system comes to equilibrium for the differ-
ent folate species in 56 h. The half-life for folate in the body is about 90
days, and the mean residence time for folate is 124212 days (Gregory and
Quinlivan, 2002; Gregory et al., 1998), so for short-term studies like the
ones we do, the assumption of a constant intracellular folate pool seems
Mathematical Models of Folate-Mediated One-Carbon Metabolism 61

The model then, consists of a set of kinetic formulas, one for each
enzyme, that describe the velocity of the reaction as a function of the
concentrations of substrates, products and allosteric regulators, plus a set of
differential equations, one for each variable metabolite, that contain the
kinetic formulas for its synthesis and degradation. In addition, we have
transport functions for amino acids into and out of the cell, and or amino
acids and formate into and out of the mitochondria. The overall system is
solved by numerical integration using a stiff ode solver (because different
quantities tend to vary at very different rates), implemented in MatLab
(The MathWorks). The program allows us to vary inputs of amino acids
and vitamins over time and follow the time-dependent responses of all
metabolites and reaction rates. In addition, the model allows us to simulate
the effects of mutations and of vitamin deficiency (or excess). We have
modeled mutations primarily by altering the Vmax values of the relevant
enzymes. This would correspond to mutations that affect the amount of
active enzyme present (e.g., mutations that affect enzyme expression or
activation). Likewise, we model the effects of variation in non-folate vita-
min cofactors, such as B12 and B6, by altering the Vmax of the corresponding
enzyme(s), assuming in effect that the activity of the enzyme is a function of
the amount of cofactor available.

IV. Model Development

Previous models of folate metabolism, outlined above, were devel-
oped in the 1970s and 1980s. Much new information and understanding
have become available in the intervening 25 years, which have guided our
approach. We began by first developing a model for the methionine cycle
(Reed et al., 2004). This model built on the prior work of Martinov et al.
(2000) who had studied the properties of a model for a portion of the
methionine cycle that did not include the MS, BHMT, and CBS reactions
and used simplifying assumptions about inputs into the cycle. Our model
closed the cycle and added the CBS reaction and several allosteric effects of
SAM. This model was able to reproduce the observed dependence of the
transsulfuration reaction on the concentration of SAM described by
Finkelstein and Martin (1984), and the effects of variation in CBS and MS
activity on homocysteine, methionine, and SAM (Finkelstein, 1990;
Finkelstein et al., 1974; Janosik et al., 2001; Pogribna et al., 2001;
Rosenblatt, 2001). Perhaps the most interesting finding with this model
was that SAM acts as a buffer for methionine input: that is, variation in
methionine input has little effect on the methionine and homocysteine
concentrations but is mostly absorbed by variation in the concentration of
SAM. Furthermore the allosteric effect of SAM on CBS provides a
62 H. F. Nijhout et al.

mechanism for stabilizing mass in the methionine cycle so that the flux out
of the methionine cycle via CBS matches the rate of methionine input into
the cycle without much change in the homocysteine concentration. If it
were not for the allosteric effect of SAM, the homocysteine concentration
would have to rise to drive the CBS reaction.
Our next step was to develop a model for the folate cycle that
contained what at the time we understood to be the important reactions
of that metabolic network (Nijhout et al., 2004). This model incorporated
the finding that folates bind to and inhibit many of the enzymes in the
folate cycle. This binding was believed to provide a reservoir of folates. The
model allowed us to resolve the puzzle as to why enzymes of the folate
cycle should be inhibited by allosteric binding of folates. The model shows
that this nonenzymatic binding greatly reduces the sensitivity of the system
to folate deficiency, because as the total pool of folate diminishes, more
enzyme is released from inhibition, and the reaction velocities are main-
tained because of the increased enzyme activity (Nijhout et al., 2004).
We next modeled the allosteric interactions between the folate and methi-
onine cycles (Fig. 2.11) in order to test the hypothesis of Wagner et al.
(1985) that these interactions serve to stabilize the DNA methylation
reaction rates (Nijhout et al., 2006). Some results of these experiments are
outlined in Section VII below. We subsequently merged our models for the
folate and methionine cycles (Fig. 2.3) to produce an integrated model of
one-carbon metabolism (Reed et al., 2006). This model also incorporated
allosteric interactions between the folate and methionine cycles (inhibition
of MTHFR by SAM and SAH, and inhibition of GNMT by 5mTHF) and
added the ability to vary the rate of input of betaine. We used this model to
simulate the interaction between folate deficiency and the MTHFR C677T
polymorphism and the interaction between folate and vitamin B12 deficien-
cies. Experimentation with this model showed that the inverse relationship
between folate status and homocysteine level is strongest at low folate levels
and disappears at high folate levels. Furthermore, the model shows that as
folate levels in the cell rise, the reactions of the folate cycle slow down. This
is due to the allosteric inhibition of enzymes in the folate cycle by folate
metabolites. This is a consequence of the homeostatic mechanism described
by Nijhout et al. (2004). This mechanism stabilizes the folate cycle at low
and intermediate folate levels, but also predicts that as folate levels rise, the
reaction rates in the folate cycle will slow down. Thus, a prediction of the
model is that a high intracellular folate level can have the same effect as a
folate deficiency. This prediction of the model is now supported by a variety
of clinical and experimental data that show that high doses of folate can have
detrimental effects (Akoglu et al., 2001; Czeizel, 2004; Morris et al., 2005;
Sunder-Plassman et al., 2000; Troen et al., 2006).
We then expanded the model of Reed et al. (2006) to include com-
partmentation of the folate cycle between cytosol and mitochondria
Glycine Serine Methionine



synthesis ATP
Sarc NE Methionine SAM
Sarc 10f-THF Ser Ser 10f-THF NADPH
Gly Gly BHMT
Gly 5,10-CH=THF 5,10-CH=THF Sarc
Pyrimidine DNA-CH
synthesis Methylation
MTD MTD dTMP reactions
5,10-CH2-THF 5,10-CH2-THF
dUMP Homocysteine SAH
CBS Adenosine
Mitochondria MTHFR 5 mTHF


5 mTHF

Figure 2.4 Diagram of our model of hepatic FOCM including the mitochondrial compartmentation. Boxed substrates are variables in the
model. Substrates without boxes are constants. Full names of the acronyms of enzymes and metabolites for this and other figures, and the
text, are given in Table 2.1. Redrawn from Nijhout et al. (2007a).
64 H. F. Nijhout et al.

(Nijhout et al., 2007a). This model included terms for the glycine cleavage
system and the metabolism of sarcosine and dimethylglycine in the mito-
chondria, mechanisms for transport of serine and glycine into the cell and
between the cytosol and mitochondria, and terms for the transport of
formate between cytosol and mitochondria (Fig. 2.4). As discussed above,
we discovered that in rapidly dividing cells mitochondria act primarily to
supply formate to the cytosol for purine and pyrimidine synthesis, whereas
in adult cells the mitochondria export no formate but are excess producers
of serine, targeted for gluconeogenesis. We also found that the rate of export
of formate from the mitochondria to the cytosol is remarkably insensitive to
fluctuations in serine and glycine input. This is because both mitochondrial
and cytosolic SHMT reactions are reversible and the rates at which they run
are highly responsive to the relative concentrations of glycine and serine.
The model was used to investigate the effect of varying the relative inputs of
glycine and serine on the rate and direction of the mitochondrial and
cytosolic SHMT reactions, and showed that both SHMT reactions can
reverse and run in the serine synthesis direction when external glycine is
increased replicating the results of Kastanos et al. (1997). This model was
also used to successfully simulate the experiments of MacFarlane et al. (2005)
and Herbig et al. (2002) on the effect of SHMT expression and glycine
availability on SAM.
To investigate the characteristics of FOCM in nonhepatic tissues we
developed a model for epithelial FOCM, which is representative of most
tissues except liver and kidney. Extrahepatic tissues do not express all
enzymes of FOCM, and some enzymes are active at much lower levels
than in the liver (dashed arrows in Fig. 2.5). Epithelia thus run on a reduced
version of the network. This model also includes a term for export of
homocysteine, which is typically exported from extrahepatic tissues for
remethylation in the liver. With this model we have explored the interac-
tion of multiple genetic polymorphisms and the interaction of genetic
and environmental variation on the level of homocysteine, the rates of
methylation, and purine and pyrimidine synthesis (Ulrich et al., 2008).
We have also created a model (Fig. 2.1) that includes the synthesis of
reduced glutathione and exchange of substrates with the blood (Reed
et al., 2008).
FOCM is an ancient pathway and occurs, with variations, in animals,
plants, fungi, and bacteria. Recently, we have developed a model of bacte-
rial FOCM for Rhodobacter capsulatus (Leduc et al., 2007), motivated by the
discovery of a novel flavin-dependent thymidylate synthase (ThyX) that
produces THF rather than DHF upon methylation of dUMP (Fig. 2.6).
This model was used to examine the relative roles of ThyA (TS), ThyX, and
FolA (DHFR) in the mechanism of resistance to antifolates such as
Folate cycle Methionine cycle
Serine MAT-II
Purine Methionine SAM
Pyrimidine synthesis
synthesis AICART
TS Methylation METH
GAR MS reactions
dUMP 10f-THF


5,10-CH2-THF 5 mTHF Serine
CBS Adenosine


Figure 2.5 Diagram of our mode of epithelial FOCM. This model does not include mitochondrial compartmentation, but does include all
allosteric regulations (not shown in this diagram). Dashed arrows indicate enzymes of low activity in epithelial tissues. Full names of the
acronyms of enzymes and metabolites for this and other figures, and the text, are given in Table 2.1.
66 H. F. Nijhout et al.

DHF Pyrimidine THF
dTMP Purine
AICART synthesis
Pyrimidine HCOOH PurH Methionine
synthesis ThyX
SHMT Glycine
Glycine MetH
Ammonia + CO2
5f-THF 10f-THF

GlyA Hcy


FolD FolD

5,10-CH2-THF 5 mTHF

Figure 2.6 Diagram of our model of bacterial folate metabolism. This mechanism can
synthesize thymidylate but bypass DHF. Redrawn from Leduc et al. (2007).

V. Blood Versus Intracellular Metabolite

The models we have developed are for intracellular metabolism, and
thus deal with intracellular concentration and pool sizes. However, almost all
of our understanding of the relationships between folate status and disease is
based on measurements of the concentrations of folate, homocysteine, SAM,
SAH, and methionine in the blood, plasma, or red blood cells. Red blood
cell measurements are believed to reflect the metabolite status at the time the
red blood cells differentiated: in a mixed-age population of cells this pre-
sumably represents an average or long-term metabolic status. Blood and
plasma concentrations may be in equilibrium with overall cellular cytosolic
concentrations, though it is more likely that they result from the interaction
between uptake from the digestive system, export by some tissues (like
epithelial cells, kidneys, muscle, and nervous system), import by others
(like the liver), and excretion by the kidneys. Whether these processes are
ever a steady state is an open question. The half-life of folate in the body is
about 90 days, and about 500 days are required for folate levels to come to a
new steady state (Gregory et al., 1998). Methionine loading experiments
shows that the methionine and homocysteine levels in the blood require
1224 h to return to steady state after a perturbation (Bianchi et al., 2000;
Mathematical Models of Folate-Mediated One-Carbon Metabolism 67

[SAM] 7500
Concentrations and velocities 200 [cCys]

150 6500



0 4000
0 10 20 30 40 50
Time (h)

Figure 2.7 Response of selected metabolite concentration and reaction velocities to

fasting. The system was allowed to come to steady state, and at 3 h the input of amino
acids (glycine, serine, methionine, and cysteine) was reduced to 0.25 of their normal values.
Different components of FOCM decline at different rates to a new steady state. Some
approachsteady state after 56 h, other take more than 48 h to approach thenew steady state.

Silberberg and Dudman, 2001). In our models, the time required for differ-
ent components of the system to relax to equilibrium is in the order of hours
to days (Fig. 2.7). Given that variation in input into the system is in the order
of hours, it is unlikely that the system is ever at steady state and may actually
exist far from equilibrium most of the time (Nijhout et al., 2007b).
Thus, blood measurements represent some average of what is going on in
different cell types, and one would therefore expect a variable and context-
dependent correlation between blood components (particularly for meta-
bolites that are used in many processes) and the state of a given organ or
cellular metabolic system. Our current intracellular models accurately simu-
late intracellular responses to experimental or clinical intervention, and it is
obviously desirable for a model to also simulate how the levels of commonly
measured blood metabolites will respond. We are beginning to approach this
difficult question of whole body modeling of folate metabolism by allowing
our cytosolic models for hepatic and epithelial FOCM to communicate with
a blood compartment that is subject to dietary input and excretory output.

VI. Modeling GeneGene and GeneEnvironment

One advantage of models is that they can be used to investigate the
effects of simultaneous variation of many variables. In the case of FOCM,
the models can be used to study the effect of simultaneous genetic
68 H. F. Nijhout et al.


Relative concentration or rate

DNMT synthesis Purine

20 [5 mTHF] rate synthesis

[SAH] [Hcy]
T/T (low folate)

Figure 2.8 Simulated response of various biomarkers to the MTHFR 677CT polymor-
phism. Folate deficiency exacerbates the effect of the T/T genotype for most biomarkers,
but reverses the effect of the T/T genotype on purine and pyrimidine synthesis.

polymorphisms, or the interaction between a polymorphism and an envi-

ronmental variable such as an amino acid, vitamin B12, or folate. For
instance, the interaction of the MTHFR C677T polymorphism with low
folate status is shown in Fig. 2.8. The T/T genotype is known to diminish
the risk of colon cancer under high folate, but it enhances risk for cardio-
vascular disease (Curtin et al., 2004; Frosst et al., 1995). In the model, the T/
T genotype lowers the concentration of SAM and the DNMT reaction rate
but raises homocysteine levels and both thymidylate and purine synthesis
rates. Folate deficiency enhances the effects of the T allele on most biomar-
kers, with the exception that it reverses the effect on thymidylate and purine
synthesis. Although these simulated changes in biomarkers correspond to
those observed in practice, it is not yet clear how these metabolic effects
translate into differential risks for colon cancer and cardiovascular disease.
The interaction between variation in the Vmax of MS and of MTHFR is
illustrated in Fig. 2.9. The variation along the MS axis in Fig. 2.9 can be
interpreted in several ways. It can represent variation in the expression level
of MS which could be due to a regulatory mutation (e.g., in the promoter
region of the MS gene), or it could be due to mutations in a structural gene
that affects the kcat. Variation could also be due to variation in the level of
vitamin B12, which is a cofactor for MS. In the first two cases variation is
genetic, and in the latter case the variation is environmental (e.g., due to a
vitamin B12 deficiency). Allowing parameters to vary continuously makes it
possible to explore a broad range of possibilities (corresponding to a range of
alleles with minor effects, or a range of environmental exposures) around
the normal or wild type, indicated by the open circles in Fig. 2.9. The shape
Mathematical Models of Folate-Mediated One-Carbon Metabolism 69

Figure 2.9 Bivariate graphs showing the interaction of various enzymes in FOCM on
selected traits (Z axes). X and Y axes show variation in the Vmax of the respective
enzymes. This variation could be due to allelic variation or, in the case of MS, to variation
in vitamin B12. The circle shows the location of the normal or wild-type phenotype.

of the interaction relationship is clearly nonlinear, so the effect of variation

in MS depends on the exact value of MTHFR activity, and vice versa.
The effects of the interaction of MS and SHMT activity on purine
synthesis and homocysteine concentration are shown in Fig. 2.9. These
relationships are also nonlinear, as indeed are all relationships between
variables within FOCM. In many cases, such as the ones illustrated here,
the wild-type values lie on a relatively flat and horizontal region of the
phenotypic surface. This indicates that the wild type is relatively insensitive
to variation in parameter values, because modest variation of the variables or
parameter values (x and y axes) has little effect on the phenotype (z axis).
As the parameter values move far away from the normal, or wild type, the
effect of their variation increases dramatically.
The finding that many wild-type phenotypes lie in regions of the
phenotypic surface that are relatively flat and horizontal, implies that the
70 H. F. Nijhout et al.

system is relatively insensitive to the exact values of those parameters. From

an evolutionary perspective one would therefore not expect strong selection
to maintain those parameter values within close tolerances, because moder-
ate variation has little effect on the phenotype. This observation may help
explain why reports on parameter values from different preparations are
often inconsistent. Although differences in protocols and experimental
errors surely play a role, it is not unreasonable to assume that some of this
variation may be real. It is possible that many genes accumulate small-effect
mutations in their regulatory region, or their coding region, that would be
neutral to selection. Indeed, human genes exhibit abundant single nucleo-
tide polymorphism (SNP) variation. The HapMap Project has uncovered a
polymorphic SNP on average every 825 base pairs, and on the average
2 nonsynonymous SNPs per gene (International HapMap Consortium,
2007; McVean et al., 2005). In addition, there is a large amount of regu-
latory variation in the promoter region of genes that leads to variation in the
level of expression (Rockman and Wray, 2002; Yan et al., 2002). A survey
of naturally occurring polymorphisms in the promoter regions of 107
human genes showed that 60% caused more than a twofold difference in
expression, and 11% caused more than a tenfold difference in expression
(Rockman and Wray, 2002). Finally, within a genetically identical popula-
tion of cells the concentration of a given protein can vary by as much as 30%
from cell to cell and from time to time (Elowitz et al., 2002; Sigal et al., 2006).
Thus, there is far more individual genetic variation and individual
variation in gene expression than is typically assumed. This, together with
the fact that the metabolites and allosteric effectors involved in FOCM vary
among individuals and from time to time (e.g., Fig. 2.10), suggests that
much of this variation is without significant effect on fitness, and is therefore
not under selection, and may therefore explain some of the observed
interindividual variability.

VII. Modeling and Simulation have Revealed

Novel Homeostatic Mechanisms
FOCM has many functions that must continue to operate normally in
face of variation in the demand on specific reactions and variation in the
input of metabolites. For instance, the expression levels of TS and DHFR
are upregulated more than 100-fold during the S-phase of the cell cycle,
when there is an increased demand for nucleotide synthesis (Bjarnason et al.,
2001; Obama et al., 2002; Slansky et al., 1993; Wade et al., 1995). At the
same time there will be an increased demand for DNA methylation to
maintain the correct methylation pattern of the newly synthesized DNA
strands. FOCM is also subject to great hourly and daily variation in amino
acid input, which varies with meals and nutrition. The amino acids serine
Mathematical Models of Folate-Mediated One-Carbon Metabolism 71

A [Methionine]

140 100*[Hcy]

Concentrations (mM) 120







Reaction rates (mM/h)

50 mSHMT




0 5 10 15 20
Time (h)

Figure 2.10 Simulated response of selected hepatic FOCM metabolite concentrations

and reaction velocities to periodic pulses of amino acid input. (A) Concentration
profiles of methionine cycle metabolites. (B) Velocity profiles of the CBS reaction
and the mitochondrial and cytosolic SHMT reactions (mSHMT and cSHMT, respec-
tively). (C) Velocity profiles of the DNMT, TS, and AICART reactions and the rate of
export of formate (vHCOOH) from the mitochondria. Modified from Nijhout et al.
72 H. F. Nijhout et al.

and glycine are the primary methyl donors for FOCM, and methionine is
both a methyl donor and an essential amino acid linking the folate and
methionine cycles. An interesting question is whether and how the stability
of critical reactions in the cycle are maintained when there are large
localized changes in demand, or large localized changes in input.
Perhaps the best way to illustrate the relative stability of some reactions
in the face of variation in inputs is by simulating a day in the life of FOCM.
After each meal with protein, the human body experiences a pulse of amino
acids that lasts about 3 h. We simulated this by pulsing the four amino acids
that serve as inputs for the model (Fig. 2.10). It is evident from the
simulations shown in Fig. 2.10 that some variables change dramatically
with each meal, while others are almost unaffected. The TS and DHFR
reactions are quite stable as are the DNMT rate and the rate of export of
formate from the mitochondria. By contrast, the SHMT reactions fluctuate
greatly as do the concentrations of SAM and homocysteine.
As discussed above (Section III.D), the stability of formate export from
the mitochondria arises from the dynamical interplay between the mito-
chondrial and cytosolic SHMT reactions, whose magnitude and direction
vary with serine and glycine input. The fluctuations in SHMT velocity are a
dynamic homeostatic mechanism that dampens the effects of fluctuations in
glycine and serine input (Nijhout et al., 2007a).
The methylation of DNA is an important function of FOCM, and it seems
reasonable to stabilize these reactions against a variable and often unpredictable
input of methyl groups. Simulations with our models show that the DNMT
reaction is extraordinarily stable against variation in input, and that this stability
arises from two allosteric interactions between the folate cycle and the methi-
onine cycle: SAM inhibits MTHFR and 5mTHF inhibits GNMT (see
Fig. 2.11). Wagner et al. (1985), and Wagner (1995) suggested that the purpose
of these interactions might be to stabilize the rate of DNA methylation. The
general idea of how this mechanism works is easy to understand. If the
concentration of SAM goes up, then MTHFR is inhibited, which causes the
concentration of 5mTHF to fall. When 5mTHF is lower, the inhibition of
GNMT is released causing the rate of the GNMT reaction to go up, utilizing
the extra SAM and allowing the DNMT rate to remain stable. The reverse
scenario explains what happens if SAM goes down.
We experimented with the model shown in Fig. 2.11 by adding and
removing the long-range allosteric regulations in various combinations.
Figure 2.12 shows how the [SAM]/[SAH] ratio and the DNMT reaction
rate vary as a function of methionine input under two scenarios: with all
allosteric interaction present, and with all allosteric interactions absent. It is
clear that the allosteric interactions stabilize the SAM/SAH ratio and the
DNMT reaction rate against variation in methionine input, and that the
effect is most pronounced at low methionine input. This is because under an
optimal supply of methionine the DNMT reaction runs close to saturation,
Mathematical Models of Folate-Mediated One-Carbon Metabolism 73


THF Methionine SAM

(folate cycle) DNA






5 mTHF Homocysteine SAH

Adenosine H2O


Figure 2.11 Model of the allosteric regulatory interactions within the methionine
cycle and between the folate and methionine cycles used to study the stability of the
DNMT reaction against fluctuations in methionine input. Thick lines show the alloste-
ric interactions of SAM and 5mTHF. Arrow indicates activation and bars indicate
inhibition. Redrawn from Nijhout et al. (2006).

so the main benefit of these regulations appears to be to protect the DNMT

reaction against periods of protein starvation.
Finally, as noted above, the expression of TS and DHFR vary a 100-fold
or more with various stages of the cell cycle, and we have shown, by
simulation, that this variation has little or no effect on the reaction velocities
and metabolite concentrations elsewhere in the folate and methionine
cycles (Nijhout et al., 2004). An implication of this funding is that FOCM
should be relatively insensitive to inhibition of TS and DHFR by chemo-
therapeutic drugs such as methotrexate (which inhibits DHFR) and
5-fluoro-uracil (which inhibits TS). This is indeed the case. When the
Vmax of DHFR is lowered (corresponding, e.g., to treatment with metho-
trexate) the velocity of the DHFR reaction remains virtually constant until
there is almost no free enzyme left. The reason for this remarkable stability
of the DHFR reaction is that the normal concentration of its substrate,
DHF, is exceptionally low, typically 0.02 mM out of a total folate pool of
20 mM. Thus, the concentration of DHF can rise more than a 100-fold
74 H. F. Nijhout et al.


Methylation rate (mM/h)



60 Unregulated


B 20
0 20 40 60 80 100 120 140
Methionine input (mM/h)



15 Regulated
SAM/SAH ratio


0 20 40 60 80 100 120 140
Methionine input (mM/h)

Figure 2.12 Effect of allosteric regulation by SAM and 5mTHF on the response of
(A) the [SAM]/[SAH] ratio and (B) the DNMT reaction to variation in methionine
input. Solid line shows the response when all allosteric regulations are in place, and the
dotted line shows the response without allosteric regulation. Allosteric regulation
stabilizes both the [SAM]/[SAH] ration and DNMT reaction at low methionine
input, and thus may be an adaptation to protein starvation.

(and drive the DHFR reaction by substrate accumulation) without substan-

tially depleting the folate pool and disrupting the reaction rates elsewhere in
FOCM. The inhibition of DHFR by methotrexate in effect creates a DHF
Mathematical Models of Folate-Mediated One-Carbon Metabolism 75

trap. The rate at which this DHF trap develops is determined by the rate of
the TS reaction. In a rapidly dividing cancer cell, where TS is highly up-
regulated, the DHF trap will develop rapidly, whereas in a non-cancerous,
non-dividing cell it will develop very slowly if at all.

VIII. Steady States and Fluctuations

The mathematical models allow us to calculate how long it takes for
FOCM to return to steady state after a perturbation. The interlocking cycles
of FOCM are complex and different reactions return to steady state at
different rates. An example is shown in Fig. 2.7 where we show the
simulated response to fasting. It takes 610 h for some reactions to go to
steady state while others take more than 2 days. Given a normal pattern of
eating, these findings imply that FOCM is never at steady state (Nijhout
et al., 2007b). Indeed many reaction rates and metabolite concentrations are
likely to always be far from steady state (Fig. 2.10).
This calls into question the utility of standard metabolic control analysis
to understand the operation of this system. In metabolic control analysis one
typically lets the system come to steady state, then perturbs it by changing
one parameter by a small amount, and lets the system come to the new
steady state (Fell, 1992). The fractional change in the reaction velocities and
metabolite concentrations at this new steady state is then taken to be a
measure of the sensitivity of each component of the system to the parameter
that was changed. This method is used to deduce how control is distributed
among the reactions of a system, and the relative control any given enzyme
has over the operation of the system. Metabolic control analysis is, in effect,
a sensitivity analysis preformed by perturbing the steady state. When a
system normally operates far from steady state, and its reaction velocities
and metabolite concentrations are continually changing, a steady-state
sensitivity analysis is not a useful way of obtaining insight into the operation
of the system.
Instead, it is more natural to see how the system responds to large scale
fluctuations. We have been using such fluctuations in different ways. First,
we have been using fluctuations to make quantitative statements about the
effects of particular homeostatic regulatory mechanisms. For example, in
Nijhout et al. (2006) we added to the normal methionine input (100 mM/h)
a continuous stochastic fluctuation with standard deviation 30 mM/h. The
standard deviation in the velocity of the DNA methylation reaction was
exceptionally small, because of the long-range allosteric interactions dis-
cussed above. We then removed allosteric interactions one by one to see
which ones and which combinations had the greatest effects. When all four
are removed, the standard deviation of the velocity of the DNMT reaction
76 H. F. Nijhout et al.

goes up by a large factor. We have also used such fluctuation analysis to

show that it is the unusual kinetics of MAT-I and MAT-III that stabilizes
the methionine concentration at the expense of large fluctuations in SAM
(unpublished). In Nijhout et al. (2007), we applied stochastic fluctuations to
the serine and glycine inputs and showed that the production of formate by
the mitochondria remains remarkably stable. This stability is caused by the
parallel SHMT reactions in the cytosol and the mitochondria that make
glycine from serine and vice versa.
Secondly, we often use external stochastic fluctuations as a probe of
system behavior. It is very interesting to fluctuate an input or a Vmax
(corresponding to gene up- and downregulation) and then observe which
concentrations and velocities fluctuate a lot, a moderate amount, or hardly
at all. Our experience is that when a concentration or velocity hardly
fluctuates at all, there is usually a good biological reason why this is so.
We can then take the system apart to discover the mechanisms that cause the
homeostatic behavior. Usually, some other concentrations and velocities
change a lot so that the homeostatic ones can remain stable.
Finally, we have been conducting a mathematical analysis of the way
in which general fluctuations propagate through biochemical networks.
In Anderson et al. (2007), we showed that the variances of reactions
velocities are always strictly decreasing down linear chains. The biological
significance of this result is that if it is important to stabilize the output of a
chain of biochemical reactions against fluctuations in the input, then the
chain should be long. It was also shown that side reaction systems and
feedback loops decrease the variations of the velocities in downstream
reactions. In Anderson and Mattingly (2008), many of these results are
proven in the case of Michaelis-Menten chains. Efforts are underway to
prove how more complicated network geometries and different kinds of
kinetics affect the ways in which fluctuations propagate.
Finally, we note that metabolic networks do not arise fully formed. They
evolve over time by the addition and elimination of reactions and by
changes in the kinetics of existing reactions. In evolutionary biology, it is
typically assumed that natural selection acts to maximize flux through a
pathway (e.g., Hartl et al., 1985; Wagner, 2005), in effect making reactions
more efficient in some way. But if a system normally experiences con-
tinual and large fluctuations of input, and continuous and large changes in
the demand for many different synthetic reactions, then a more likely target
for natural selection would be those reactions or connections that stabilize
certain parts of the system against the effects of those changes. That is,
evolution would not necessarily favor faster and more efficient pathways,
but rather would favor pathways that operate stably and reliably under
variation. Eating imposes enormous hourly and daily fluctuations, as well
as unpredictable long-term deficiencies in specific nutrients, and normal
daily and seasonal activities impose large variation in demand. This is true of
Mathematical Models of Folate-Mediated One-Carbon Metabolism 77

FOCM, and it must be true of most if not all of metabolism. The key
regulatory features of metabolic systems are thus those that stabilize func-
tion, and those that prevent local perturbations from propagating through
the system. As is the case in FOCM, these regulatory mechanisms are not
the emergent properties of large networks, but are evolved adaptations for
specific functions.

IX. Conclusions
FOCM is one of the best studied metabolic systems: all or almost all
enzymes and metabolites in the system are known, as is the structure of
the reaction network. This network is complex and consists of several
intersecting cycles and a large number of complex allosteric regulatory
interactions between metabolites and enzymes. The reactions in this system
are nonlinear, which makes it exceptionally difficult to deduce the proper-
ties of the overall system, the way it is regulated, and the effects of mutations
and nutrient and vitamin deficiencies from the connectivity diagram alone.
The most direct way to understand the function of different parts of a
complex system like FOCM is through computer simulation with a mathe-
matical model. Because FOCM has been so well studied, it has been
possible to construct models that accurately simulate metabolite pools and
reaction velocities, as well as the effects of mutations and vitamin deficien-
cies on markers like homocysteine, TS and methylation capacities.
A mathematical model is an experimental tool that can be used as a
complement to laboratory experimentation or clinical investigation to do
pilot experiments and test hypotheses quickly and inexpensively. When a
new interaction is discovered, or suspected, it can be incorporated into a
preexisting model to determine its effect. We expect that our mathematical
models will evolve in three ways: first by progressive improvement of the
accuracy of the existing models by incorporating details like polyglutamation,
substrate channeling, and compartmentalization; second, by extending the
models to include other related aspects of metabolism, like insulin signaling;
third, by developing additional tissue-specific models, for instance, for the brain
and transport across the bloodbrain barrier, and by linking models for multiple
organ systems together through the circulatory system.
One important purpose of studying FOCM is to understand the rela-
tionship between genetic and environmental variables and disease out-
comes. There are two large steps necessary for this understanding. First,
one needs to understand how genetic and the environmental perturbations
affect the system behavior of FOCM. Second, one needs to understand how
the system changes in FOCM lead to the various disease states. Both are
very difficult questions. Most of our work outlined above has been
78 H. F. Nijhout et al.

dedicated to understanding the regulatory system properties of FOCM and

how the behavior of FOCM changes in the presence of genetic polymorph-
isms and changes in environmental input. It remains a formidable challenge
to understand the pathway by which inadequacies or malfunctions of the
processes regulated by FOCM contribute to the development of such
diverse diseases as colon cancer, psychiatric disorders, cardiovascular disease,
and neural tube defects.

We thank Marian Neuhouser, Jess Gregory, Barry Shane, Jill James, and Jon Mattingly for
their advice during the development of the mathematical models of FOCM. This work was
supported by grant DMS-0616710 from the National Science Foundation, and grant RO1
CA 105437 from the National Institutes of Health.

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Folate Deprivation, the Methionine

Cycle, and Alzheimers Disease
Flaubert Tchantchou* and Thomas B. Shea

I. Introduction 84
II. Folate Metabolism, the Transmethylation Pathway, and AD 85
III. The Transsulfuration PathwayHomocysteine Elimination and
Glutathione Metabolism 90
References 94

Folate deficiency is associated with increase in homocysteine levels. Abnormal
plasma levels of that neurotoxic nonproteinogenic amino acid is implicated in
many pathological conditions including cardiovascular diseases, neural tube
defects, and is now recognized as a risk factor in Alzheimers disease (AD)
dementia. Homocysteine elimination is regulated by two metabolic pathways,
namely, the transmethylation and the transsulfuration pathways. Its elimi-
nation via these two metabolic pathways is modulated by folate, a member
of the B-vitamin family. Folate provides, via its metabolic end product
5-methyltetrahydrofolate, a methyl group that is used to reconvert homocyste-
ine back to methionine through the transmethylation pathway. The efficiency of
folate metabolism has an impact on the availability of S-adenosylmethionine,
a compound that is known to activate homocysteine flux through the transsul-
furation pathway and is necessary for utilization of a downstream antioxidant
called glutathione under the catalysis of glutathione S-transferase enzyme.
In this review, we will explore the impact of folate deprivation on the regulation
of the methionine cycle and exhaustively describe different biochemical reac-
tions that are implicated in the regulation of homocysteine elimination and that
folate deficiency influences in AD neuropathology. 2008 Elsevier Inc.

* University of Maryland, Baltimore, Maryland


Center for Cellular Neurobiology and Neurodegeneration Research, UMass Lowell,
Lowell, Massachusetts 01854

Vitamins and Hormones, Volume 79 # 2008 Elsevier Inc.

ISSN 0083-6729, DOI: 10.1016/S0083-6729(08)00403-2 All rights reserved.

84 Flaubert Tchantchou and Thomas B. Shea

I. Introduction
Folate is a member of the B-vitamin family and a carrier of one-carbon
fragments, which it transfers to various biochemical targets. Folate is impor-
tant for the functioning of the central nervous system (CNS) at all ages
(Bottiglieri et al., 1995; Reynolds, 2002). Its metabolism provides a methyl
group, via its metabolite 5-methyltetrahydrofolate, which is necessary for
the remethylation of the neurotoxic amino acid homocysteine back to
methionine, an essential amino acid that plays a key role in the generation
of methyl groups required for numerous biochemical reactions. Substantial
scientific evidence associates folate deficiency to Alzheimers disease (AD).
The deficiency of this B vitamin induces homocysteine accumulation. This
sulfur-containing nonproteinogenic amino acid transitionally exists at the
intersection of the transmethylation and the transsulfuration pathways,
which regulate its elimination (Selhub, 1999).
Hyperhomocysteinemia is associated with an increased risk of several
pathological conditions including vascular diseases and vascular dementia
and has been confirmed in patients with AD and mild cognitive impairment
(MCI) where they represent an independent risk factor (Seshadri et al.,
2002; Shea and Rogers, 2002a). HCY levels in several biological fluids
and tissues represent a predictive index for the incidence of AD and other
dementias. Substantial evidence has established a connection between HCY
metabolism and cognitive function. Abnormal levels of HCY have been
related to multiple cognitive dysfunctions including age-related memory
loss, vascular dementia, and AD (Malaguarnera et al., 2004; OSuilleabhain
et al., 2004; Sachdev et al., 2003). Deficiencies in folic acid are often
observed in the elderly population with a resultant increase in HCY.
They are proposed to be owing to an increasing prevalence of atrophic
gastritis type B, which occurs with a frequency of up to 50% in elderly
subjects (Wolters et al., 2004). The link between increase in homocysteine
levels and AD resulted from the growing recognition that cerebrovascular
disease may promote AD. This idea was taken from studies of HCY and
heart disease research and is being extended to cerebral disorders. This
correlation lays on the fact that plasma HCY maybe directly toxic to
vascular endothelial cells or induces their dysfunction, leading to the loss
of the bloodbrain-barrier function and altered production of nitric oxide.
In addition, HCY crossing the bloodbrain barrier or being released by cells
within the brain could act as a potent neurotoxin (Miller, 1999).
Such neurotoxic effects may be due to the direct interaction of HCY
with plasma membrane components or to the intracellular accumulation
of S-adenosylhomocysteine (SAH). This latter metabolite inhibits the
methylation of catechol substrates resulting in the generation of oxyradicals
and other chemically reactive products that are cytotoxic. Moreover,
Folate Deprivation, the Methionine Cycle, and Alzheimers Disease 85

homocysteine as sulfhydryl compound is an electron donor, which acts with

the transition metal ions, iron and copper, to generate hydrogen peroxide
(Kruman et al., 2002). HCY also has the ability to induce the storage of iron
from ferritin, and this could explain the increase in redox-active iron in AD
neurons and concomitant oxidative stress, which subsequently triggers
deposition of amyloid plaques in the AD brain (Ulrich et al., 2002).
Considering the deleterious effect of HCY accumulation in the brain, its
continuous elimination is necessary, and hence, the importance of the
methyl group provided by the folate (also known as vitamin B9) as it
provides 5-methyltetrahydrofolate, required for the reconversion of HCY
to methionine via the transmethylation pathway.
Another major consequence of folate deficiency is a decline in
S-adenosylmethionine (SAM; the major methyl donor). This decline in
SAM, which is endogenously generated from methionine, is responsible for
increased DNA breakage in mouse models (Kruman et al., 2000, 2002) and
the gradual hypomethylation of DNA accompanies aging and AD
(Morrison, 1996; Seshadri et al., 2002). The depletion of SAM can also
lead to overexpression of presenilin-1 (PS-1; Fuso et al., 2005; Scarpa et al.,
2003), which is associated to abnormal processing of the amyloid precursor
protein that results into the formation of the b-amyloid protein (Parihar and
Hemnani, 2004). Furthermore, this principal methyl donor mediates the
enzymatic reaction utilizing an endogenous antioxidant and downstream
metabolite of HCY metabolism via the transsulfuration pathway called
glutathione, under the catalysis of glutathione S-transferase enzyme
(Tchantchou et al., 2006b). Therefore, the utilization of glutathione by
glutathione S-transferase would promote HCY elimination via the trans-
sulfuration pathway. This clearly highlights the important role that folate
plays in the elimination of HCY via both the transmethylation and the
transsulfuration pathways.

II. Folate Metabolism, the Transmethylation

Pathway, and AD
The transmethylation pathway is derived from the conjunction of two
biochemical pathways, namely, the folate and the methionine metabolic
pathways. The transmethylation pathway consists of transferring a methyl
group (CH3) to HCY by the end product of folate metabolism,
5-methyltetrahydrofolate, or betaine to form methionine. Folate is a mem-
ber of the B-vitamin family and a carrier of one-carbon fragments, which it
transfers to various biochemical targets (Chen et al., 2004). Its metabolism
starts with its deconjugation in the cells of the intestinal wall to the mono-
glutamate form. This form is further reduced to dihydrofolate and then to
86 Flaubert Tchantchou and Thomas B. Shea

tetrahydrofolate (THF) via the catalytic actions of folate and dihydrofolate

reductase enzymes, respectively (Fig. 3.1; reactions 1 and 2, respectively).
Both of these enzymes require NAPDH as a cofactor. The resulting THF
receives a hydroxymethyl group from the combination of a serine molecule
with a pyridoxal-50 -phosphate to form 5,10-methylene tetrahydrofolate (5,10-
methyleneTHF) and glycine in a reaction catalyzed by the enzyme
serine hydroxymethyltransferase (Fig. 3.1, reaction 3). 5,10-MethyleneTHF

Diet intake Folate

FR; Rx-1
Acetylcholine NADP+

Acetyl-CoA DR; Rx-2
PPi + Pi THF
MAT; Rx-6 HMT; Rx-3
CH3-Ac SAM ChaT, Rx15
5,10-methylene THF
CH3- SAMT; Rx-7 Betaine MS/Vit B12; Rx-5


SAHH; Rx-8
Homocysteine 5-MeTHF
CBS/Vit B6; Rx-9 (Circulating folate)

Cg L;
Cystathionine Cysteine

GS; Rx-11
H2O2 2H2O

GPx; Rx-12

GsT(E); Rx-14

GR; Rx-13


Figure 3.1 Pathway regulating homocysteine elimination and glutathione metabolism

in the brain.
Folate Deprivation, the Methionine Cycle, and Alzheimers Disease 87

is of central importance in several biological events. It is the precursor of

the metabolically active 5-methyltetrahydrofolate, which is involved in
HCY metabolism, and methylene tetrahydrofolate, which is involved in
purine synthesis (Brotto and Yang, 2000; Raber et al., 1998; Selhub, 1999).
With relevance to the transmethylation pathway, 5,10-methyleneTHF
produces 5-MTHF in a reaction catalyzed by methylene tetrahydrofolate
reductase (MTHFR) (Fig. 3.1, reaction 4). Variations in the gene encoding
for the MTHFR enzyme can decrease folate metabolism and subsequently
result in an increase in HCY levels. Patients with congenital MTHFR
deficiency have reduced levels of several important biological metabolites
such as methionine and SAM in the cerebrospinal fluid (CSF) and show
demyelination in the brain which might be due to decreased methylation.
MTHFR polymorphisms that exhibit decreased activity are present in as
much as 20% of some populations (Brotto and Yang, 2000). Diminished
activity of this enzyme also reduces the production of SAM (required for
DNA methylation). MTHFR deficiency and the presence of ApoE4 may
represent synergistic risk factors for AD. This is a plausible extension of the
known association of diminished folate metabolism with AD, in that indi-
viduals who are homozygote carriers of MTHFR polymorphic gene are at
particular risk for other folate-related neural defects when plasma folate is at the
low end of the normal range (Shields, 1999). Moreover, individuals
affected with AD who are also homozygote carriers of MTHFR poly-
morphisms show elevated HCY levels compared to nonhomozygous
AD patients despite the presence of equal levels in other considerable biological
compounds such as folate in both groups. It has also been demonstrated that
mice heterozygous for the MTHFR defect are vulnera-
ble to hyperhomocysteinemia when fed with low folate diets and have altered
tissue methylation capacity and impaired endothelial function in
cerebral microvessels (Devlin et al., 2004). APOE/, APOE/, and
APOE/ mice exposed to oxidative stress inducing diet deficient in folate
show increased transcription and activity levels of MTHFR when com-
pared to APOE/ mice maintained on folate supplemented diet. This is
indicative of the need for folate, whose metabolic product 5-MTHF is the
principal methyl donor for remethylation of HCY back to methionine in
the CNS in a reaction catalyzed by methionine synthase (MS) (also known
as 5-methyltetrahydrofolatehomocysteine S-methyltransferase; Fig. 3.1, reac-
tion 5), as betaine, the alternative methyl donor, is absent in the brain. Previous
studies showed that folate supplementation with or without vitamins B6 or B12
to individuals with hyperhomocysteinemia lower their homocysteine levels
(Anonymous, 1998), and recently, a 2-year term double-blind placebo-
controlled randomized clinical trial showed no association between homocys-
teine lowering and cognitive performance (McMahon et al., 2006). The
methodological quality of that study is questionable in that the investigators
recruited for their trial, healthy elderly people who were showing no signs of
88 Flaubert Tchantchou and Thomas B. Shea

cognitive dysfunction and had no means of determining whether they were

prone to developing cognitive impairment during the trial. The erroneous
sampling of the targeted population rendered the results of their findings
opened to serious critical comments. During homocysteine reconversion to
methionine, THF can be regenerated as the second product of the reaction.
This reaction is a precursor to the regeneration of 5-MTHF via 5,10-methy-
leneTHF (Bronstrup et al., 1998). Folate deficiency mediates neurotoxicity in
part by increasing levels of HCY. This nonproteinogenic amino acid over-
stimulates N-methyl-D-aspartate (NMDA) receptors, potentiates glutamate
accumulation and amyloid-b aggregation and neurotoxicity, and induces
DNA breakage and lipid peroxidation (Al-Gazali, 2001; Ho, 2001). Mouse
models of AD and Parkinsons disease as well as wild-type mice subjected to
folate deficiency show elevated HCY and place neurons at the risk of
degeneration and endothelial damage. The mechanism whereby homo-
cysteine leads to endothelial cell damage has been found to be via its
auto-oxidation to homocysteine and H2O2 (Loscalzo, 1996). Electron
microscopic studies of the brains of folate-deprived rats revealed that
hyperhomocysteinemia induced by folate deprivation was accompanied by
ultrastructural degenerative changes in the cerebral microvasculature,
including endothelial and pericytic degeneration, mitochondrial destruc-
tion, and cytoplasmic dissolution (Kim et al., 2002). These alterations were
similar to the previously reported microvascular degenerative features typi-
cally found in cerebral diseases such as AD, Parkinsons disease, and aging
processes (Farkas et al., 2000; Ureno et al., 2001). Oxidative damage due to
folate deficiency is potentiated by lack of apolipoprotein E gene and iron
supplementation as pro-oxidant. The extent of the damage to brain cells can
be determined by the measure of thiobarbituric acid reactive species
(TBARs) levels, which is an index of oxidative damage induced by lipid
peroxidation (Ho et al., 2003; Mattson and Haberman, 2003; Shea et al.,
2001). The expression status of MS is easily altered under oxidative stress.
The transcription levels and/or the activity of the enzyme it encodes for is
significantly decreased under different oxidizing conditions. Vitamin B12
is an indispensable cofactor in the transmethylation reaction in the brain. This
reaction is of great importance in the regulation of serum HCY levels and is the
only reaction in the body in which folate and vitamin B12 are co-participants
(Mattson et al., 2002; Miller and Kelly, 1997). A decrease in MS transcription
and activity observed in normal mice under oxidizing conditions can be viewed
as a natural downregulating compensatory process, which attempts to avoid
further regeneration of methionine from HCY, which would infinitely be
demethylated to reform HCY. Consistent with this line of thought, other
investigations have shown that the increased HCY flux through the transsul-
furation pathway could result from an increase in the levels of methionine
adenosyltransferase and/or cystathionine-b-synthase or a decrease in methio-
nine synthase activity (Mosharov et al., 2000; Tchantchou et al., 2006a). The
Folate Deprivation, the Methionine Cycle, and Alzheimers Disease 89

methyl group transferred from 5-MTHF to HCY to form methionine con-

tributes to the formation of SAM in a one-step reaction in which an ATP
molecule is involved (Fig. 3.1, reaction 6) and that represents the end point of
the transmethylation pathway. In the reaction mechanism of the remethylation
of HCY to methionine, a methyl group is transferred to the MS cofactor, cob(I)
alamin, which is then activated by forming methylcobalamin. The activation of
cob(I)alamin to form methylcobalamin requires the methyl donor SAM. But
once the first molecule of methylcobalamin is formed and used for the recon-
version of HCY to methionine, subsequent molecules could be regenerated by
using 5-MTHF as methyl donor to serve the same purpose. The absence of the
alternative betaine remethylation pathway in the CNS greatly reduces the
methylation capacity. Therefore, folate deprivation would inhibit transmethy-
lation reactions by reducing SAM and further potentiates HCY accumulation
in the CNS. Considering that the action of vitamin B12 plays a role in HCY
metabolism that is similar to that of folate (Mattson and Shea, 2003), and
because folate and vitamin B12 deficiencies retard methionine regeneration,
SAM levels are also reduced as a consequence of lack of folate or deficiency in
vitamin B12 action (Selhub and Miller, 1992). Impaired methylation has been
implicated in many neurological and psychological disorders, including
dementia, depression, and psychosis. Decreased intracellular methylation reac-
tions can also result in an increase of SAH. This line of reasoning is supported by
the demonstration that HCY induces DNA breakage and resultant apoptosis,
and that co-treatment with SAM prevented homocysteine-induced apoptosis.
The participation of SAM in methylation reactions results in the production of
SAH under the catalysis of S-adenosylmethionine methyltransferase (Cantoni,
1986) (Fig. 3.1, reaction 7). The hydrolysis of SAH catalyzed by SAH hydrolase
produces HCY and adenosine (Fig. 3.1, reaction 8). The compensatory down-
regulation of the reconversion of homocysteine to methionine and the absence
of the betaine metabolic pathway in the brain further excludes the possibility of
efficiently regenerating methionine from homocysteine. Choline, a precursor
of betaine in other organs, could therefore condense with acetyl-coA to
optimally produce the neurotransmitter acetylcholine in the brain, in a reaction
catalyzed by choline acetyltransferase (ChaT) (Fig. 3.1, reaction 15) (Fisher
et al., 2002). Findings of a recent study demonstrates that dietary folate defi-
ciency in middle age adult mice decreased levels of acetylcholine, which were
restored by dietary supplementation with SAM even in the absence of folate.
Adult mice heterozygously lacking 50 ,100 -methylene tetrahydrofolate reduc-
tase (which are impaired in folate usage and have reduced SAM levels), or
homozygously lacking apolipoprotein E (which have reduced SAM due to
oxidative stress) and aged normal mice each display reduced acetylcholine levels
as compared to normal adult mice. Dietary folate deficiency further reduced
acetylcholine and induced cognitive impairment in each of these mice, while
supplementation with SAM in the absence of dietary folate restored acetylcho-
line levels and cognitive performance to levels observed in the presence of folate
90 Flaubert Tchantchou and Thomas B. Shea

(Chan et al., 2006). That observation underscores the importance of folate and
the principal methyl donor SAM in the regulation of acetylcholine levels,
which represents an important the rapeutic approach for age-related neurode-
generation such as AD.

III. The Transsulfuration PathwayHomocysteine

Elimination and Glutathione Metabolism
The transsulfuration pathway is another path for HCY elimination via
which about 60% of HCY is believed to be metabolized (Storch et al.,
1988). It comprises several reaction sequences which start with the forma-
tion of cystathionine followed by those of cysteine and glutathione.
Cystathionine formation is derived from the condensation of L-serine
with homocysteine, in a reaction catalyzed by the heme and vitamin B6-
dependent cystathionine-b-synthase (CBS). Cystathionine is subsequently
cleaved to yield cysteine and 2-ketobutyrate in a reaction catalyzed by
cystathionine g-lyase (Mosharov et al., 2000). But, cystathionine g-lyase is
absent or has a limited presence in the brain therefore, levels of cystathio-
nine found in brain tissues is putatively due to the action of CBS since it is
considered the rate-limiting enzyme in HCY transsulfuration. CBS genetic
knockout mice model first developed by Watanabe et al. (1995) exhibit
increased HCY levels. Among them, CBS homozygote knockout mice
developed significantly elevated levels of plasma total HCY compared
to heterozygote knockout which showed mildly elevated concentration
of total plasma HCY, which are closely similar to those in humans
with heterozygous CBS deficiency. Those findings suggest that partial
impairment in homocysteine transsulfuration produces similar effects on
HCY metabolism in humans and mice. A recent study aiming at evalu-
ating the in vivo effect of high serum homocysteine concentration on
amyloid-b-peptide (Ab) levels in the brain and in relation to AD neuropa-
thology using a mice model carrying the well-established amyloidosis
mutant genes APP/PS1 (Holcomb et al., 1998) and heterozygous for a
cystathionine-b-synthase mutation (APP/PS1/CBS); therefore, resulting
in deficient CBS activity and high homocysteine levels. The mouse
model showed significant elevations of serum homocysteine levels com-
pared to the double transgenic APP/PS1 model of amyloidosis. Results
showed that female (but not male) APP/PS1/CBS mice exhibited signifi-
cant elevations of Ab40 and Ab42 levels in the brain. Correlations between
homocysteine levels in serum and brain Ab levels were statistically signifi-
cant (Pacheco-Quinto et al., 2006). Deficiency in CBS leading to
Folate Deprivation, the Methionine Cycle, and Alzheimers Disease 91

homocysteinuria results in multiple organs/systems damage, severe vascular

disease, and mental retardation (Mudd et al., 1995). Human hepatoma cell
line demonstrates increase synthesis of cystathionine under oxidative stress
conditions. The increase synthesis of cystathionine is followed by an
increase in HCY flux through the transsulfuration pathway (Mosharov
et al., 2000). Increased transcription levels of CBS gene are present in
apolipoprotein E homozygote and heterozygote knockout mice in a
gene dosage manner. This increase is potentiated by folate deprivation
(Tchantchou et al., 2006a). The immediate consequence of this increase
in cystathionine levels is that it could lead to an increase in the concentra-
tions of the downstream metabolites, cysteine and glutathione (GSH). The
transsulfuration reaction thus provides a direct link between homocysteine
and glutathione, the major endogenous redox buffer in mammalian cells.
It is therefore not surprising that a number of enzymes at this metabolic
nexus display sensitivity to redox changes (Mosharov et al., 2000). Thus,
changes in the levels of expression or functional activity of CBS can
affect levels of HCY (Mattson and Shea, 2003). Such a regulatory switch
could be rationalized as representing a self-correcting response to depleted
glutathione levels in cells faced with an oxidative challenge (Loehrer et al.,
1996). This highlights the overwhelming importance of the transsulfuration
pathway, which under oxidative stress has a dual beneficial impact
since its upregulation would, in addition to accelerating homocysteine
elimination, also contribute to the increase synthesis of the antioxidant
glutathione in many cell and tissue types. In so doing, the transsulfuration
pathway contributes at least indirectly in preventing or quenching oxidative
damage to the brain and other organs. GSH systematically called g-gluta-
mylcysteinylglycine is a ubiquitous tripeptide, formed from the amino acids
glutamate, glycine, and cysteine by two ATP-dependent enzymatic reac-
tions (Schulz et al., 2000). GSH is a major intracellular antioxidant and
its antioxidant activity depends upon the thiol group within the molecules.
GSH is crucial in the free radical scavenging of singlet oxygen and the
OH radical (Larsson et al., 1983). GSH plays this crucial role in detoxifying
peroxides and/or electrophilic toxins such as 1-chloro-2,4-dinitrobenzene
(CDNB) used as substrates in reactions catalyzed by GSH peroxidase
(Fig. 3.1, reaction 12) and glutathione S-transferase (Fig. 3.1, reaction 14),
respectively. Intracellular GSH is maintained in its thiol form by glutathione
disulfide (GSSG) reductase, which requires NADPH molecule (Fig. 3.1,
reaction 13). The availability of cysteine is critical for the synthesis of
GSH in most cells (Ceballos-Picot et al., 1996). Glutathione levels and acti-
vity of glutathione synthase (GS) are increased under oxidative stress
conditions induced by dietary deficiency (folate and vitamin E). This increase
in glutathione levels is substantiated by apoliprotein E deficiency (Gilgun-
92 Flaubert Tchantchou and Thomas B. Shea

Sherki et al., 2001; Shea et al., 2002). Deficiency in this gene was shown to
promote the increase of oxidative stress (Ramassamy et al., 1999). Moreover,
experimental elevations in glutathione in AD brain were capable of reducing
oxidative damage and therefore represented an attempt to compensate for
increased ROS induced by dietary and genetic deficiency (Huang et al., 2000).
Furthermore, this increase in GSH levels in the nervous system is triggered by
the upregulation of the transcription and activity profile of glutathione
synthase. Glutathione synthase gene and activity displayed differential
compensatory responses to dietary folate and apolipoprotein E deficiency.
A significant increase in transcription of GS is observed only in APOE/
mice and only when they were maintained on folate-deficient diet, suggesting
that the combined impact of diet-induced and genetically induced oxidative
stress is required to induce an increase in transcription. The magnitude of this
combined impact is reflected by a synergistic increase in thiobarbituric acid-
reactive substance levels in brain tissue of APOE/ mice maintained under
folate deficiency. Maintenance of normal mice on dietary folate deficiency also
induces a significant increase in the combined impact of the absence of APOE
and the dietary folate deficiency results in a dramatic increase in TBARs in
brain tissue (Shea and Rogers, 2002b), and further indicates a synergistic
deleterious impact of these dietary folate and genetic deficiencies. Therefore,
the increase in GS transcription and activity in APOE/ mice subjected to
oxidative stress inducing diet correlate with the synergistic increase in TBARs.
But, these increases in both activity and transcription of GS in the brain of
APOE/ mice maintained on folate-deficient diet are unable to compensate
fully for the synergistic increase in oxidative damage. This observation under-
scores the extent of oxidative damage that diet-induced and genetically
induced oxidative stress could cause to brain tissue (Tchantchou et al.,
2004a). These findings highlight the fact that distinct compensatory responses
in an antioxidant-generating enzyme can be invoked depending on the nature
and extent of oxidative stress. The combined efficacy of these responses was
reflected by steady-state levels of glutathione, in that both diet-induced and
genetically induced oxidative stress individually elevated glutathione levels,
whereas the combined impact of both induced an apparent additive increase
(Shea and Rogers, 2002b; Tchantchou et al., 2004a). The cumulative increase
of GSH levels in brain tissues under oxidative damage is suggestive of a possible
alteration of the activity of enzymes that help use glutathione to quench
reactive oxygen species and toxins that induce oxidative damage to the
brain. A recent clinical trial with a small number of cognitively impaired
patients demonstrated that the therapeutic combination of N-acetylcysteine
and B-vitamin supplements (folate and vitamin B12) improved cognitive
status of these hyperhomocysteinemic patients (McCaddon, 2006). In that
combinatorial therapy, which has homocysteine levels lowering properties,
NAC will increase the flow of homocysteine through the transsulfuration
Folate Deprivation, the Methionine Cycle, and Alzheimers Disease 93

pathway leading to an increase in GSH formation while folate and vitamin B12
will regulate homocysteine remethylation via the transmethylation pathway.
The use of N-acetylcysteine in this combinatorial therapy correlates with
previous findings demonstrating that the administration of N-acetylcysteine
to ApoE-deficient mice deprived of folate alleviated oxidative damage and
cognitive decline, and their restored glutathione synthase and GSH levels to
those of normal mice maintained in the presence of folate (Tchantchou et al.,
2005). The activity and the transcription profile of GSH peroxidase which
catalyzes the reaction in which GSH is used to eliminate hydrogen peroxide
and results in the formation of the oxidized form of glutathione (GSSG) and
that of GSH reductase, which catalyzes the reconversion of GSSG to GSH
are elevated in hippocampus and inferior parietal lobule of AD patients
(Aksenov et al., 1998; Lovell et al., 1998). This might reflect the protective
gene response to the increased peroxidation in the brain regions showing
severe AD pathology (Aksenov et al., 1998; Tchantchou et al., 2004a).
The levels of glutathione S-transferase, a protective enzyme against aldehydes
and especially 4-hydroxynonenal (HNE, a marker of lipid peroxidation) are
decreased in the brain and ventricular CSF of autopsied AD (Lovell et al.,
1998). APOE / mice maintained on folate-deficient diet demonstrate
similar increase in the activity of glutathione peroxidase and glutathione
reductase than that of APOE / mice on the complete diet. By contrast,
but consistent with observations made in AD patient brains, APOE / mice
display a significant decrease in glutathione S-transferase activity. The decrease
might be due to the methylation status of this enzyme. The similar increase in
GPx and GR activity, which contributes in recycling the oxidized glutathione
(GSSG) back to the reduced form (GSH), combined with the significant
decrease in GST activity constitutes a justification for the increase GSH levels
in mice brain under oxidative damage (Shea and Rogers, 2002b; Tchantchou
et al., 2004a). The supplementation of APOE / mice with a potent methyl
donor, S-adenosylmethionine, when maintained on folate-deficient diet,
restores GST, GPx, and GR activity (Tchantchou et al., 2006b). This high-
lights the importance of potent methyl donors in the regulation of enzymes
that catalyze reactions involving the utilization of glutathione.
Considering the direct correlation between folate deprivation and
increase homocysteine levels, which exerts its neurotocixity via several
frameworks that include its ability to trigger increase b-amyloid deposition,
free radical formation, or its direct interaction with the plasma membrane,
combinatorial therapeutic approaches (McCaddon, 2006; Tchantchou et al.,
2004b) aiming at preventing homocysteine accumulation while maintaining
a normal methylation status provide a real hope to the management of
AD onset and at least part of the diseases symptoms.
94 Flaubert Tchantchou and Thomas B. Shea

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Molecular Mechanisms of Adaptation

to Folate Deficiency
Ilan Ifergan* and Yehuda G. Assaraf

I. Folate Metabolism 101
II. Pathological States Associated with Folate Deficiency or
Nutritional Folate Insufficiency 105
A. Folate deficiency, neural tube defects and congenital
heart defects 105
B. Folate deficiency, homocysteinemia, and atherosclerotic
cardiovascular disease 106
III. Molecular Mechanisms of Adaptation to Folate Deprivation 108
A. The role of folate-dependent enzymes in adaptation
to folate deficiency 108
B. Cellular retention of folates: The key role of polyglutamylation 110
C. Overexpression of folate influx systems 112
D. Downregulation of folate efflux systems 123
References 131

Folic acid is an essential vitamin for a wide spectrum of biochemical reactions;
however, unlike bacteria and plants, mammals are devoid of folate biosynthesis
and thus must obtain this cofactor from exogenous sources. Therefore, folate
deficiency may impair the de novo biosynthesis of purines and thymidylate and
thereby disrupt DNA and RNA metabolism, homocysteine remethylation, methi-
onine biosynthesis, and subsequent formation of S-adenosylmethionine (the
universal methyl donor) which in turn may lead to altered methylation reac-
tions. This impaired folate-dependent intracellular metabolism can lead to
several key pathologies including, for example, megaloblastic anemia, homo-
cysteinemia, cardiovascular disease, embryonic defects, in particular neural
tube defects (NTDs), congenital heart defects, and possibly cancer. The current
review presents and evaluates the up-to-date knowledge regarding the

* The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of
Technology, Haifa 32000, Israel
To whom correspondence should be addressed; Email:

Vitamins and Hormones, Volume 79 # 2008 Elsevier Inc.

ISSN 0083-6729, DOI: 10.1016/S0083-6729(08)00404-4 All rights reserved.

100 Ilan Ifergan and Yehuda G. Assaraf

molecular mechanisms underlying cellular survival and/or adaptation to folate

deficiency or insufficiency. These mechanisms of adaptation to folate deficiency
generally associated with folate uptake, intracellular folate retention, folate-
dependent metabolism, and active folate efflux specifically include: (a) Up- or
downregulation of various folate-dependent enzymes like dihydrofolate reduc-
tase (DHFR) and thymidylate synthase (TS), (b) Cellular retention of folates via
polyglutamylation by the enzyme folylpoly-g-glutamate synthetase (FPGS),
(c) Overexpression of folate influx systems including the reduced folate carrier
(RFC), folate receptor (FR) as well as the proton-coupled folate transporter
(PCFT), a recently identified intestinal folate influx transporter optimally func-
tioning at the acidic microclimate of the upper intestinal epithelium, (d) Down-
regulation of ATP-driven folate efflux transporters of the multidrug resistance
protein (MRP; ABCC) family and breast cancer resistance protein (BCRP; ABCG2)
that belong to the multidrug resistance (MDR) efflux transporters of the ATP-
binding cassette (ABC) superfamily. Moreover, the intricate interplay between
various components of the adaptive response to folate deprivation is also
discussed. 2008 Elsevier Inc.


ABC ATP-binding cassette

AICARTF aminoimidazole carboxamide ribonucleotide
ALL acute lymphoblastic leukemia
BCRP breast cancer resistance protein
cDNA complementary DNA
CFD cerebral folate deficiency
CHO Chinese hamster ovary
CNS central nervous system
CSF cerebrospinal fluid
DHF dihydrofolate
DHFR dihydrofolate reductase
FPGS folylpoly-g-glutamate synthetase
FR folate receptor
GARTF glycinamide ribonucleotide transformylase
GGH g-glutamyl hydrolase
GSH glutathione
HFM hereditary folate malabsorption
RFC reduced folate carrier
IMP inosine monophosphate
LF low folate
MDR multidrug resistance
Adaptation to Folate Deprivation 101

MFS major facilitator superfamily

MRPs multidrug resistance proteins
MSDs membrane-spanning domains
MTHFR methylene-tetrahydrofolate reductase
MTX methotrexate
NBF nucleotide-binding fold
NTD neural tube defect
PABA p-amino benzoic acid
pB promoter B
PCFT proton-coupled folate transporter
Pgp P-glycoprotein
PI3K phosphoinositol-3-kinase
ROS reactive oxygen species
SAM S-adenosylmethionine
SLC solute carrier family
SHMT serine hydroxymethyltransferase
THF tetrahydrofolate
TMD transmembrane domain
TS thymidylate synthase

I. Folate Metabolism
Folic acid, the oxidized form of vitamin B9, and its reduced derivative,
tetrahydrofolate (THF; Fig. 4.1), are water-soluble vitamins commonly
termed folates. The term folate stems from the Latin word folium which
means leaf; indeed, folates are present in substantial amounts in green leafy
vegetables. THF cofactors play an essential role as one-carbon donors and
acceptors in several crucial intracellular metabolic reactions. Folic acid is
composed of three covalently linked components: a pteridine ring, p-amino
benzoic acid (PABA) and a glutamate residue (Fig. 4.1). Mammals are able
to synthesize the pteridine ring. However, unlike bacteria and plants,
mammals are devoid of the enzymatic capacity of coupling pteridine to
PABA (Birn, 2006) and thus are absolutely dependent on folate uptake from
exogenous sources. These sources include intestinal uptake of folates bio-
synthesized by intestinal bacterial flora as well as dietary folate intake
particularly from green leafy vegetables, fruits, grain, yeast, liver, and dairy
products (Birn, 2006).
Biologically active folates exist predominantly in the reduced form (i.e.,
the two double bonds on the pteridine ring are enzymatically reduced),
thereby resulting in THF (Adams et al., 1970); Figs. 4.1 and 4.2). Inside the
cell, THF cofactors function as both acceptors and donors of one-carbon
102 Ilan Ifergan and Yehuda G. Assaraf

Chemical structures Chemical category

Pteridine ring p-amino benzoic acid Glutamyl residue

3 N 6 CH2 NH C NH CH
CH2 Oxidized folate
H 2N 2 N N 7
1 8 Folic acid CH2



CH2 Reduced folate


CH2 Reduced folate
(leucovorin) C OH



N CH2 CH2 Reduced folate

5,10-CH2-THF C OH


N Antifolate
(DHFR inhibitor)

Methotrexate C OH

Figure 4.1 Chemical structures of oxidized and reduced folates as well as antifolates.
The one-carbon units donated during purine and thymidylate biosynthesis are
highlighted in gray.
Adaptation to Folate Deprivation 103

units. These one-carbon units are covalently linked to the THF cofactor
and exist as a wide range of oxidized and reduced forms of the one-carbon
moiety, including methyl, formyl and methylene derivatives (Figs. 4.1 and
4.2). The primary source of one-carbon units for mammalian folate metab-
olism is first donated from serine to the pteridine ring of THF thereby
resulting in the formation of glycine and 5,10-methylene-THF, respectively
(Fig. 4.2). The various THF cofactors include 5,10-methylene-THF,
10-formyl-THF, and 5-methyl-THF (the major folate form in the plasma)
serve as one-carbon donors in various biochemical reactions (Carmel et al.,
2003; Qureshi et al., 1994; Scott, 1999) as depicted in Fig. 4.2; specifically,
cytosolic methionine synthase transfers a methyl group from 5-methyl-THF
to homocysteine in a methyl-cobalamin-dependent reaction, thereby result-
ing in the formation of methionine (Fig. 4.2). The latter is further converted
to S-adenosylmethionine (SAM). In the methylation cycle which occurs in
all nucleated cells, SAM serves as the key methyl group donor of most
biological methylation reactions including that of CpG island DNA methyl-
ation that regulates gene expression (Kim, 2004, 2005), protein methylation



Serine DHF dTMP


Met acid

Figure 4.2 Simplified scheme of intracellular folate metabolism in mammalian cells
with emphasis on the de novo biosynthesis of purines and thymidylate. Abbreviations
of the enzymes and intermediate compounds that are involved in these pathways:
AICARTF, aminoimidazole carboxamide ribonucleotide transformylase; DHF, dihy-
drofolate; DHFR, dihydrofolate reductase; FPGS, folylpoly-g-glutamate synthetase;
GARTF, glycinamide ribonucleotide transformylase; GGH, g-glutamyl hydrolase;
Hcy, homocysteine; IMP, inosine monophosphate; Met, methionine; MS, methionine
synthase; MTHFR, methylene-tetrahydrofolate reductase; PRPP, Phosphoribosyl
pyrophosphate; SAM, S-adenosylmethionine; SHMT, serine hydroxymethyltransfer-
ase; THF, tetrahydrofolate; TS, thymidylate synthase.
104 Ilan Ifergan and Yehuda G. Assaraf

which serves as an important posttranslational modification, and lipid meth-

ylation important for their biosynthesis including that of phosphatidylcholine
(Stokstad) (Fig. 4.2). Moreover, methylation is a crucial step in the metabo-
lism of neurotransmitters and detoxification of xenobiotics. Hence, folate
deficiency may lead to elevated homocysteine levels due to decreased utili-
zation of this amino acid as well as to altered intracellular methylation
processes. In addition to the above-mentioned role of THF cofactors in
the biosynthesis of glycine and methionine along with the metabolism of
serine and homocysteine, respectively, THF coenzymes play a key role in the
catabolism of histidine and formic acid. Importantly, THF coenzymes are
essential for cellular proliferation due to their primary role in the de novo
biosynthesis of purines and thymidylate that are essential for DNA replication
(Fig. 4.2). Several key enzymes use reduced folates as cofactors in these
biosynthetic pathways (Fig. 4.2). Specifically, thymidylate synthase (TS)
catalyzes the conversion of dUMP to dTMP through the transfer of a methyl
group from 5,10-methylene-THF to dUMP. Similarly, the cofactor
10-formyl-THF contributes one-carbon units in each of two key de novo
biosynthetic transformylase reactions of purine metabolism (Fig. 4.2). The
first reaction is catalyzed by the enzyme glycinamide ribonucleotide trans-
formylase (GARTF) which transfers a formyl group from 10-formyl-THF
resulting in the formation of the imidazole ring of purines. Whereas, a more
downstream reaction involves an additional formyl transferase reaction that is
carried out by aminoimidazole carboxamide ribonucleotide transformylase
(AICARTF), resulting in the formation of the purine intermediate inosinic
acid which is commonly termed inosine monophosphate (IMP) (Fig. 4.2).
The latter serves as a crucial intermediate in a biochemical Y-junction which
leads to the regulated and balanced formation of the purine nucleotides AMP
and GMP. Importantly, the folate cofactor is utilized in a cyclic manner
as follows: dihydrofolate (DHF), the by-product of the TS-dependent
reaction, is recycled to the biologically active THF by the enzyme dihydro-
folate reductase (DHFR) in an nicotinamide adenine dinucleotide
phosphate (NADPH)-dependent reduction. The latter THF as well as the
THF by-product of the purine metabolism can be further converted to 5,10-
methylene-THF and 10-formyl-THF. The latter two folate cofactors are
reused in the de novo biosynthesis of purines and thymidylate precursors of
nucleic acids as has been previously described (Fig. 4.2).
On the basis of plethora of folate-dependent metabolic processes, a
deficiency in intracellular THF pools may therefore impair nucleotide
biosynthesis, homocysteine remethylation, methionine biosynthesis, as
well as cellular methylation processes. This altered intracellular biochemical
status can lead to several disorders including megaloblastic anemia, leuco-
penia and thrombocytopenia, homocysteinemia, cardiovascular disease,
embryonic defects, in particular neural tube defects (NTDs) and congenital
heart defects and possibly cancer (Stanger, 2002). These pathological states
Adaptation to Folate Deprivation 105

associated with folate deficiency are discussed in greater detail in the

following chapters. Moreover, a similar effect of folate deprivation can be
achieved by using folic acid antagonists known as antifolates that inhibit the
biosynthesis of purines and/or thymidylate. This rationale has been
exploited for the introduction of a wide spectrum of antifolate-based
therapies of various diseases that are characterized by an abnormal cellular
proliferation including cancer, rheumatoid arthritis, and psoriasis ( Jukes,
1987). Methotrexate (MTX) (Fig. 4.1), the first and most studied antifolate,
binds stoichiometrically and tightly (KD 1 pM) to the target enzyme
DHFR with an affinity that is a million fold higher than the natural
substrate, DHF (Km 1 mM) (Bertino, 1993; Ozaki et al., 1981). However,
at least 95% of intracellular DHFR must be inhibited by MTX in order to
block DNA replication (Jackson and Harrap, 1973). In addition to inhibi-
tion of DNA synthesis, antifolate treatment may result in misincorporation
of 20 -deoxyuridine 50 -triphosphate (dUTP) into DNA, thereby leading to
DNA strand breaks and cell death (Richards et al., 1986). MTX is currently
used for the combination chemotherapeutic treatment of various human
malignancies including childhood acute lymphoblastic leukemia (ALL),
non-Hodgkins lymphoma, osteosarcoma, head and neck cancer, choriocar-
cinoma, small cell lung cancer, and breast cancer (Bertino, 1993; Schornagel
and McVie, 1983). A detailed discussion of the currently available antifolates
is beyond the scope of this review; however, this has been the subject of
a recent review from this laboratory (Assaraf, 2007). Indeed, several key
folate-dependent enzymes have been successfully targeted by one or various
antifolates that are widely used in the combination chemotherapeutic treat-
ment regimen of various human cancers of hematolymphoid lineage or
epithelial origin (Walling, 2006).

II. Pathological States Associated with

Folate Deficiency or Nutritional
Folate Insufficiency
A. Folate deficiency, neural tube defects and congenital
heart defects
The closure of the neural tube occurs during early stages of embryogenesis.
This developmental process heavily relies on the interactions between
genetic, environmental, and nutritional factors. Failure of neural tube
closure is a common congenital malformation resulting in significant mor-
bidity and mortality. NTDs are complex congenital malformations of the
central nervous system (CNS) and the most common (with a prevalence of
1:1000 births) and severe forms of NTDs include anencephaly and spina
bifida (Blom et al., 2006). Anencephaly is an NTD that occurs when there is
106 Ilan Ifergan and Yehuda G. Assaraf

failure of neurulation, in which the neural folds do not fuse at the cranial
end of the developing embryo, and hence, these infants lack most or all of
the brain tissue. Infants with anencephaly are stillborn or die shortly after
birth. Spina bifida is a posterior NTD that is caused by the failure of the
neural tube to fuse at the caudal end. Consequently, infants with Spina
bifida can have an open lesion on their spine, where significant damage to
the nerves and spinal cord has occurred or the lesion can be closed. Infants
with spina bifida can survive but are at high risk of developing psychosocial
maladjustments. Maternal nutrition factors contribute significantly to the
various etiologies of NTDs. In a seminal study reported on 1976, Smithells
and colleagues (Smithells et al., 1976) discovered that the diets and postpar-
tum blood of women who had given birth to a fetus with an NTD were
deficient in several micronutrients, particularly folic acid. Over the past two
decades, it has been well established that women can reduce their risk of
having an NTD-affected pregnancy by as much as 5075% by taking folic
acid supplementation. Small nonrandomized studies in women who previ-
ously had NTD-affected pregnancies revealed that intake of folic acid supple-
ments during the periconceptional period resulted in a fourfold reduction in
the NTD recurrence risk (Laurence et al., 1981; Seller and Nevin, 1984;
Smithells et al., 1976, 1981, 1983; Vergel et al., 1990). Moreover, in a
thorough, double-blind, placebo-controlled, randomized trial performed by
the Medical Research Council in the UK it was confirmed that supplemen-
tation with a daily dose of 4 mg folic acid resulted in a beneficial effect of a
threefold reduction in the recurrence risk of NTDs (Group, 1991). However,
it is still unclear as to what are the molecular mechanisms by which folic acid
supplementation markedly decreases the risk of an infant being born with an
NTD or other congenital malformations. Moreover, it is completely unclear
why a significant fraction of women who do take folic acid supplementation
during the periconceptional period give birth to NTD-harboring infants.

B. Folate deficiency, homocysteinemia, and atherosclerotic

cardiovascular disease
As detailed above, reduced folates are cofactors that are essential for the
biosynthesis of purine and thymidine nucleotides. Furthermore, folates are
also necessary for the biosynthesis of methionine from homocysteine and
5-CH3-THF in a methyl-cobalamin (i.e., methyl-B12)-dependent reaction
catalyzed by methionine synthase (Fig. 4.2). Impairment of folate-mediated,
one-carbon metabolic pathways can result from folate deficiency and/or nutri-
tional folate insufficiency (i.e., a diet that is relatively poor in folates), single
nucleotide polymorphisms and inactivating mutations in folate-dependent
enzymes and folate transporters. These result in homocysteinemia and
increased risk of various pathologies including atherosclerotic cardiovascular
Adaptation to Folate Deprivation 107

disease and vascular thrombosis (Arnesen et al., 1995; Durand et al., 2001;
Graham et al., 1997; Rasouli et al., 2005; Ubbink et al., 1993). Data suggest
that folate deficiency was the most common vitamin deficiency in the United
States, thereby affecting 10% of the population; furthermore, more than 50%
of the children and elderly that live in poverty were folate deficient (Bailey et al.,
1982). These statistics of folate deficiency were found to be true also for other
non-Western countries including, for example, Thailand (Assantachai and
Lekhakula, 2007) and rural areas of India (Pathak et al., 2004). However,
since the mandated fortification of processed grains with folic acid in the United
States and Canada in 1998, the incidence of folate deficiency in most popula-
tions in these countries has dramatically declined ( Joelson et al., 2007).
Epidemiological evidence suggests that hyperhomocysteinemia is an
independent single risk factor for arterial thrombotic diseases such as acute
myocardial infarction, stroke, peripheral ischemic occlusive disorders as
well as venous thromboembolism (Arnesen et al., 1995; Durand et al.,
2001; Graham et al., 1997; Rasouli et al., 2005). In this respect, panoply
of epidemiological studies has accumulated over the past decade; these
studies establish a tight association between homocysteinemia and the
significantly increased risk of atherosclerotic cardiovascular disease. It has
been well established that folate deficiency results in elevated levels of
serum homocysteine (i.e., homocysteinemia). Hence, in folate-deficient
individuals, decreased levels of THF cofactors (e.g., 5-CH3-THF) limit
metabolic flux through the methionine synthase reaction, with conse-
quent accumulation of homocysteine, the substrate of this enzyme.
Importantly however, dietary folate supplementation can readily normalize
plasma homocysteine levels and may thereby reduce the risk of coronary
artery disease (Graham and OCallaghan, 2000). Studies with animal models
of atherosclerosis and thrombosis have provided evidence that even mod-
erate elevation of homocysteine levels can produce damage to vascular
endothelium and can enhance platelet aggregation (Sauls et al., 2004,
2007). These vasotoxic and other deleterious effects of homocysteine
are believed to result from the following characteristics (Ramakrishnan
et al., 2006):
(a) During the oxidation of excess homocysteine to homocystine and
disulphides, reactive oxygen species (ROS) which are potent oxidants
are generated in the blood which can cause severe endothelial injury.
For example, endothelial cell injury due to copper-catalyzed hydrogen
peroxide formation from cytseine has been demonstrated in vitro
(Starkebaum and Harlan, 1986).
(b) Homocysteine can directly interact in a thiolation reaction by binding
to thiol groups of proteins and thereby form disulphides which can
markedly alter the function of various proteins.
108 Ilan Ifergan and Yehuda G. Assaraf

(c) Homocysteine can be converted to a highly reactive thiolactone which

could react with proteins resulting in the formation of NH-CO-
adducts, thereby altering the function of various proteins and enzymes.
For instance, a potential mechanism for the thrombotic tendency in
hyperhomocysteinemic patients has been recently identified; thus,
modification of fibrinogen by homocysteine thiolactone increased its
resistance to fibrinolysis (Sauls et al., 2006).

III. Molecular Mechanisms of Adaptation

to Folate Deprivation
This chapter reviews and discusses the up-to-date knowledge regard-
ing the molecular mechanisms that regulate folate homeostasis under folate
replete and deplete conditions. Our specific aim is to describe the role of
folate-dependent enzymes, folate polyglutamylation as well as folate influx
and efflux transport systems in cellular survival and/or adaptation to folate
deficiency and/or insufficiency.

A. The role of folate-dependent enzymes in adaptation

to folate deficiency
De novo biosynthesis of purines and thymidylate precursors of nucleic acids is
absolutely dependent on THF cofactors. Hence, cellular proliferation will be
eventually blocked under conditions of severe folate deficiency. One possi-
ble mechanism of adaptation to folate deprivation lies within the folate
metabolic pathway. Specifically, the DHF by-product is recycled to the
biologically active THF cofactor by the enzyme DHFR; the resultant
THF cofactor can be further converted to 5,10-methylene-THF and
10-formyl-THF for utilization in the de novo biosynthesis of purines and
thymidylate as has been previously described (Fig. 4.2). Hence, augmented
activity of DHFR should theoretically increase the recycling rate of DHF,
at least in some cells, and thus compensate, at least partially, for conditions in
which folate is deprived from the growth medium. Indeed, analysis of three
independent clonal variants of Chinese hamster lung fibroblasts including
FA3, FA7, and FA14 selected under low folate (LF) conditions [15 pM
5-formyl-THF; (Lamers et al., 2006)], revealed that each clone had a fivefold
to sixfold increase in both DHFR activity and protein levels (Zhu et al.,
2002). Similarly, DHFR gene amplification was documented after treatment
with MTX both in cultured cells (Schimke, 1984) and in clinical samples
from patients that were treated with this antifolate (Horns et al., 1984; Trent
et al., 1984). For certain concentrations of MTX, the significant increase in
the intracellular levels of DHFR produces sufficient free enzyme molecules
Adaptation to Folate Deprivation 109

to catalyze its biosynthetic reaction resulting in the production of THF.

Therefore, increased activity of DHFR plays a key role under conditions of
folate deficiency or DHFR inhibition by antifolates, thereby producing
more enzyme molecules to minimize the disruption of folate metabolism.
TS activity was also examined under conditions of folate deficiency; as
has been previously mentioned, TS catalyzes the conversion of dUMP
to dTMP by transferring a single methyl group from the cofactor 5,10-
methylene-THF to dUMP (Fig. 4.2). In this study, four colon cancer cell
lines (C26-A, C2610, C26-G, and WiDr) and three squamous cell carci-
noma lines of the head and neck (HNSCC) (11B, 14C, and 22B) were
adapted to grow in a culture medium containing LF levels (i.e., 0.52.5 nM
leucovorin) (Backus et al., 2000). This study revealed that whereas folate
deprivation was associated with up to threefold increased catalytic activity of
TS in squamous cell carcinoma of the head and neck, the folate-depleted
colon cancer cells showed up to sevenfold decreased catalytic activity of
TS when compared to their folate-replete parental controls. The increased
activity of TS in the folate-depleted squamous carcinoma cells of the head
and neck may compensate for the folate-deprived condition, thus facilitat-
ing an enhanced utilization of folates for the biosynthesis of dTMP. How-
ever, the decreased activity of TS in the folate-deprived colon cancer cells
may serve as an important mechanism for rebalancing the intracellular
concentration of nucleotides for cellular utilization. Specifically, the
decreased activity of TS in these colon cancer cells may release more THF
cofactors for de novo purine biosynthesis; moreover, decreased TS activity
may result in diminished levels of dTMP, the latter of which is presumably
less required as the proliferation rate is decreased due to the severe folate
deficiency. Indeed, folate deprivation of cultured cells has been reported to
increase the intracellular ratio of dUMP to dTMP by as high as tenfold
( James et al., 1994; Melnyk et al., 1999). Consistent with the hypothesis that
certain cells downregulate DNA synthesis under folate-deprived conditions,
folate-deficient human colon adenocarcinoma HCT116 cells appeared
to favor a preferential shuttling of the flux of one-carbon units to the
methionine cycle (upregulation of methylene-tetrahydrofolate reductase,
MTHFR) and thereby suppress the nucleotide biosynthetic pathway
(including downregulation of the enzymes SHMT, TS, and DHFR;
Fig. 4.2) (Hayashi et al., 2007). Therefore, suppression of the pathway of
nucleotide biosynthesis, at least partially, may be strategically exercised by
cells as an adaptation and/or survival mechanism under folate deprivation.
Additional studies are necessary to reveal the role of folate-dependent
enzymes in the complex intracellular biochemical network of folate-
dependent pathways under folate-deprived conditions. Moreover, compu-
terized simulations of de novo biosynthesis of purines and thymidylate
precursors of nucleic acids currently became available (Assaraf et al., 2006;
Seither et al., 1989); these computer-aided models may be used to analyze
110 Ilan Ifergan and Yehuda G. Assaraf

the nature of this important pathway under conditions of folate deficiency

and thereby predict the possible role of specific folate-dependent enzymes
in adaptation to conditions of folate deprivation.

B. Cellular retention of folates: The key role

of polyglutamylation
Whereas the monoglutamate form of 5-CH3-THF is the primary circulatory
folate, the polyglutamylated forms of 5-CH3-THF as well as other reduced
folates serve as the abundant intracellular THF cofactors. The enzyme folyl-
poly-g-glutamate synthetase (FPGS) which exists in both the cytosol and
mitochondria (McGuire et al., 2000), catalyzes the polyglutamylation of
folates and antifolates through the addition of multiple equivalents of gluta-
mate units to the g-carboxyl residue of THF cofactors and certain hydrophilic
antifolates (Fig. 4.3). In contrast to FPGS, the enzyme g-glutamyl hydrolase
(GGH) catalyzes the hydrolysis of these terminal glutamate residues from
polyglutamylated folates and antifolates (Fig. 4.3) (Shane, 1995). Intracellular
folates exist mainly as poly-g-glutamate derivatives, with the parent THF
being elongated by 710 glutamyl residues (McGuire et al., 1980; Shane,
1989). Importantly, the long chain (n > 3) polyglutamylated (anti)folate
derivatives are no longer substrates of efflux systems such as multidrug
resistance proteins (MRPs) (Wielinga et al., 2005; Zeng et al., 2001), breast
cancer resistance protein (BCRP) (Volk and Schneider, 2003) as well as
bidirectional folate transporters including the reduced folate carrier (RFC)
(Matherly and Goldman, 2003). Hence, polyglutamylation is the primary
determinant of intracellular retention and sequestration of THF cofactors and
polyglutamatable antifolates. Moreover, folate polyglutamate congeners dis-
play higher affinities for various folate-dependent enzymes relative to their
non-polyglutamylated forms (Allegra et al., 1985, 1987). Therefore, folylpo-
lyglutamylation increases the efficiency of THF cofactor utilization. Addi-
tionally, a mitochondrial FPGS species exists which allows mitochondria to
accumulate folylpolyglutamates thereby resulting in the biosynthesis of gly-
cine (Lin et al., 1993). The FPGS-dependent entrapment of intracellular
THF cofactors suggests that increased cellular activity of this enzyme should
be highly beneficial to cells subjected to conditions of folate deprivation.
Several studies lend support to this hypothesis:
(a) An increased FPGS activity was documented in several tumors as well as
in the liver and kidney of mice that were subjected to a LF diet for two
weeks (Mendelsohn et al., 1996).
(b) Consistently, mice injected i.v. with a single dose of the antifolate
lometrexol (5,10-dideaza-tetrahydrofolate), an inhibitor of GARTF,
accumulated more drug in their livers when compared to untreated
controls. Moreover, analysis of antifolate polyglutamates revealed that
Adaptation to Folate Deprivation 111


Folate(glu1) Glutamate
+ O


ADP + Pi


Figure 4.3 The reaction of folate polyglutamylation by FPGS and hydrolysis of folate
polyglutamates by GGH. Note that various reduced folate derivatives undergo these
reactions including 5-methyl-THF as depicted in this scheme.

longer polyglutamate forms of lometrexol (hepta- and octa- species of

lometrexol) appeared earlier and persisted longer in liver of the folate-
deprived mice when compared to the control group of mice
(Mendelsohn et al., 1996). The increased activity of FPGS may there-
fore enhance (anti)folate polyglutamylation and thereby improve intra-
cellular folate retention. Moreover, the markedly increased affinities of
various folate-dependent enzymes for polyglutamylated folate deriva-
tives relative to the non-glutamylated forms (Allegra et al., 1985, 1987)
should be particularly important under conditions of folate deprivation.
(c) An additional study aimed at understanding the molecular mechanisms
underlying adaptation of cultured breast cancer cells to states of
folate deficiency also supports the important role that increased polygluta-
mylation plays in adaptation to folate deficiency (Ifergan et al., 2004). In this
study, MCF-7 breast carcinoma cells and their mitoxantrone (an anticancer
112 Ilan Ifergan and Yehuda G. Assaraf

drug which is a specific BCRP substrate)-resistant MCF-7/MR subline,

with BCRP (an ATP-driven efflux transporter of mitoxantrone and
folates) overexpression were gradually deprived (3.5 months) of folic
acid from 2.3 mM to 3 nM resulting in the sublines MCF-7/LF and
MCF-7/MR-LF, respectively. Consistent with the above results, folate
deprivation resulted in statistically significant increases of 63% and 20% in
FPGS activity of MCF-7/MR-LF and MCF-7/LF cells, respectively,
relative to their folate-replete counterparts (Ifergan et al., 2004). A decrease
in one or more folate efflux systems along with increased FPGS activity
were associated with an augmented ability of the LF-adapted sublines to
accumulate folates.
(d) Furthermore, a study with colonic epithelial cells aimed at determining
the alterations occurring at the transcript level of various genes involved
in intracellular folate metabolism and one-carbon transfer reactions
under folate-deprived conditions was recently published (Hayashi
et al., 2007). Consistent with previously mentioned studies, a real-
time quantitative RTPCR analysis revealed that the steady-state
mRNA levels of FPGS were 400% and 16% higher in folate-deficient
HCT116 and Caco2 human colon adenocarcinoma cells, respectively,
relative to their folate-replete counterparts (Hayashi et al., 2007). These
results indicate that transcriptional upregulation of FPGS and/or
decreased degradation of FPGS transcripts (i.e., increased mRNA sta-
bility) are plausible mechanisms of cellular adaptation to folate defi-
ciency. Taken in toto, these cumulative results strongly suggest that
increased FPGS activity may shift the intracellular equilibrium of folates
toward the long chain, hyperpolyglutamylated congeners, thereby
decreasing active folate export via various ATP-driven folate efflux
trasnporters; for a recent review see Assaraf (2006). Hence, increased
FPGS activity appears to play a major protective role and is apparently an
important survival factor in cellular adaptation to folate deficiency.

C. Overexpression of folate influx systems

1. Cellular uptake of folates and MTX
Whereas a balanced diet may contain both folate monoglutamates and
polyglutamates, the latter are enzymatically hydrolyzed to the monogluta-
mate form by glutamate carboxypeptidase II (GCPII), an exopeptidase
anchored to the intestinal apical brush border membrane (Chandler et al.,
1986). The monoglutamate form of 5-methyl-THF, additional reduced
folate derivatives as well as the oxidized folate form, folic acid, are divalent
anions that cannot traverse lipid bilayers by simple diffusion (Dembo and
Sirotnak, 1984; Goldman and Matherly, 1986; Sirotnak and Tolner, 1999).
Therefore, folate monoglutamate uptake into mammalian cells must be
facilitated by specialized carrier-mediated systems (Dembo and Sirotnak,
Adaptation to Folate Deprivation 113

Intestinal lumen Upper intestinal Blood Choroid plexus CSF Brain

epithelial cell
(acidic pH)
H+ H+
Folate Folate
(oxidized and (mM)
reduced )

Intestinal Folate Folate Folate

epithelium (nM) (mM) (mM)

(neutral pH)

Reduced Folate
folates (mM)
(i.e., 5-CH3-THF )

- PCFT : proton-coupled symport of folates and protons.

- RFC : anion-exchange-based transport of reduced folates.

- FR: high-affinity (K D = 0.1 nM)-based endocytosis of oxidized and reduced folates.

Figure 4.4 Putative transport pathways of folates from the intestinal lumen through
the blood into the cerebrospinal fluid and the brain.

1984; Goldman and Matherly, 1986; Ratnam and Freisheim, 1992)

(Figs. 4.4 and 4.5). Indeed, carrier-mediated systems for the uptake of folates
across biological membranes are necessary for intestinal folate absorption
and renal reabsorption as can be found in the upper small intestine and
proximal renal tubules, respectively, as well as for folate uptake into cells
located within the intact embryo and the various tissues of adult organisms
(Lee et al., 1992; Matherly and Goldman, 2003; Ratnam and Freisheim,
1992; Rubin et al., 1967; Selhub and Rosenberg, 1978, 1986; Sirotnak and
Tolner, 1999). Three specialized transport systems exist that can accommo-
date the transport of folates and antifolates across biological membranes
(Matherly and Goldman, 2003):
(i) The RFC is a major uptake route for the transport of folates into
mammalian cells (Matherly and Goldman, 2003) (Figs. 4.4 and 4.5).
RFC (SLC19A1) functions as a bidirectional anion exchanger
(Goldman, 1971; Henderson and Zevely, 1981) with a high affinity
(Km 1 mM) for reduced folates and hydrophilic antifolates including
MTX (Km 510 mM) but low affinity (Km 200400 mM) for folic
acid (Goldman, 1971; Sirotnak, 1985, 1987). RFC is a member of the
solute carrier family (SLC) of facilitative carriers which currently com-
prises 300 genes that have been classified into 43 subfamilies (http:// The SLC family belongs to an
even larger superfamily known as the major facilitator superfamily
114 Ilan Ifergan and Yehuda G. Assaraf

folate Folic acid 5-CH3-THF



Reduced Reduced FPGS

folate folate Folate(glun)



Figure 4.5 Proposed model for major adaptive responses to folate deficiency. Note
that upward arrows denote upregulation whereas downward arrows denote down-
regulation of the relevant components activity. AFD-Absolute folate deficiency.

(MFS) of transporters (Saier et al., 1999). Human RFC contains 591

amino acids with a core molecular weight of 64 kDa; however,
although it contains a single consensus site for N-linked glycosylation,
RFC undergoes an extensive glycosylation thereby resulting in a
molecular mass of 85 kDa (Drori et al., 2000). Putative hydropathy
plots and membrane topology studies suggested that RFC contains 12
transmembrane domains (TMDs) with a short cytosolic N-terminus and a
long cytosolic C-terminus (Ferguson and Flintoff, 1999; Liu and
Matherly, 2002). In contrast to most known folate transport systems,
RFC can neither bind nor hydrolyze ATP in order to drive folate
substrate translocation across the plasma membrane. Rather, the RFC-
dependent uphill influx of folates and antifolates is coupled to the
downhill efflux of organic phosphates including thiamine
monophosphate and pyrophosphate (Zhao et al., 2002) that are
synthesized and retained in the cytoplasm. However, when compared
Adaptation to Folate Deprivation 115

to transporters including MRPs, RFC is considered a low-capacity

transporter. Therefore, the net efflux of organic phosphates via the
RFC is negligible when compared to the rate of the biosynthesis of
these organic phosphates (Goldman, 1971). The central developmental
role that mammalian RFC plays has been established in RFC knockout
mice studies; whereas RFC-null embryos died in utero before embryonic
day 9.5 (E9.5), the rescue of the nullizygous embryonic lethal phenotype
was achieved by supplementation of pregnant monoallelic RFC dams
with 1 mg daily subcutaneous doses of folic acid (Zhao et al., 2001).
Furthermore, the rescued RFC nullizygous embryos died within 12
days after birth due to a failure of the hematopoietic organs (Zhao et al.,
(ii) The second route of folate uptake involves the small family of folate
receptors (FRs) (Figs. 4.4 and 4.5). This group of receptors is encoded
by three distinct genes known as FRa, FRb, and FRg, respectively
(Elnakat and Ratnam, 2004, 2006; Elwood, 1989; Lacey et al., 1989;
Ratnam et al., 1989; Sadasivan and Rothenberg, 1989; Shen et al.,
1994, 1995). An additional FR isoform (i.e., FRd) has been putatively
identified from genome database mining; however, neither its tissue
expression nor its folate-binding activity has been experimentally
established (Spiegelstein et al., 2000). Whereas the high-affinity
folate-binding membrane glycoproteins FRa and FRb are glycosyl-
phosphatidylinositol (GPI)-anchored receptors, FRg is a secreted pro-
tein which lacks a GPI anchor. FRa displays a high affinity for folic acid
and 5-CH3-THF (KD 0.110 nM) but lower affinity (KD 10
300 nM) for other reduced folates and MTX (Antony, 1992; Brigle
et al., 1994; Wang et al., 1992; Westerhof et al., 1995). The FR-
dependent uptake of folates proceeds via a classical mechanism of
receptor-mediated endocytosis. Moreover, whereas RFC exhibits a
relatively wide pattern of tissue expression, the expression of FRa,
the most abundant FR isoform in adults, is restricted to few tissues
including the apical (luminal) surface of epithelial cells. Moreover,
membrane-associated FRb is expressed and binds folate in the placenta
(Ratnam et al., 1989). A specific pathological state of loss of function of
FRa has been described recently; auto-antibodies against FRa have
been associated with cerebral folate deficiency (CFD) in the presence of
normal folate blood levels (see paragraph below discussing Cerebral folate
deficiency syndrome and auto-antibodies against folate receptors). It therefore
appears that FRa is a key mediator of folate uptake into the brain
(Ramaekers et al., 2005; Spector and Johanson, 2006). Additionally,
gene knockout studies have established that FRa is vital for normal
nerve tube development of mouse embryos; importantly, maternal folic
acid supplementation prevents the development of NTDs in FRa
knockout mouse embryos (Piedrahita et al., 1999) (see the paragraph
116 Ilan Ifergan and Yehuda G. Assaraf

below entitled Knockout mice lacking the folic acid-binding protein Folbp1,
currently termed Folr1, the murine homologue of the human FRa). In contrast
to the severe morphogenetic abnormalities of FRa knockout embryos
that die in utero, FRb knockout mouse embryos have been shown to
develop normally (Piedrahita et al., 1999).
Cerebral folate deficiency syndrome and auto-antibodies against folate receptors:
Cerebral folate deficiency (CFD) is defined as any neuropsychiatric syn-
drome associated with low levels of 5-CH3-THF in the cerebrospinal fluid
(CSF) (the active THF cofactor in the blood and CSF) in the presence of
normal folate metabolism outside the CNS, as evidenced by normal folate
levels in serum and erythrocytes, normal hematological values and normal
homocysteine levels. Infantile-onset CFD is a neurological syndrome that
presents 4 to 6 months after birth; the primary manifestations of this type
of CFD are significant irritability, slow head growth, cerebellar ataxia,
psychomotor retardation, pyramidal tract signs in the legs, dyskinesias, and
sometimes seizures (Ramaekers and Blau, 2004; Ramaekers et al., 2002).
Later in infancy, central visual disturbances may become apparent thereby
leading to blindness. Despite these numerous and severe clinical manifesta-
tions, the sole biochemical anomaly consistently identifiable in these CFD
patients is low levels of 5-CH3-THF in the CSF. Maintenance of normal
folate levels in the CSF is primarily attributable to the transport activity of
high affinity FRs that are anchored to the plasma membrane of various
epithelial and mesenchymal cells via a GPI moiety (Fig. 4.4). Membrane-
associated FRa is expressed at substantial levels in the basolateral surface of
the choroid plexus epithelium (Fig. 4.4). 5-CH3-THF binds to FRa with a
very high affinity (KD 0.1 nM) and is then internalized into choroid
epithelial cells via receptor-mediated endocytosis (Fig. 4.4). As discussed
above, reduced folate transport from the plasma into the CSF is apparently a
concentrative process as the ratio of 5-CH3-THF levels in the CSF versus
the plasma is 3:1 (Spector, 1989; Weitman et al., 1992). Following endocy-
tosis and dissociation of 5-CH3-THF from FRa in the cytosol, a fraction of
the endocytotic membrane-bound FRa recycles back to the basolateral cell
surface of the choroid plexus epithelium (Holm et al., 1991); whereas,
another fraction of FRa is sorted out to the apical surface of the choroid,
where it is cleaved from its GPI anchor. Hence, receptor molecules are
released into the CSF while retaining their functional capacity to bind
5-CH3-THF. Then, at the apical surface of the choroid, folate efflux into
the CSF is believed to occur via the bidirectional anion exchanger RFC that
displays a folate transport Km at the micromolar range, consistently reflect-
ing the intracellular concentration of reduced folate cofactors (Assaraf et al.,
2006) (Fig. 4.4). Once in the spinal fluid, 5-CH3-THF is transported into
neuronal tissues via the RFC. Taken together, the above physiological
characteristics clearly establish the central role that FRa plays in the
Adaptation to Folate Deprivation 117

transport of 5-CH3-THF across the choroid plexus epithelial cells. There-

fore, this raised the hypothesis that CFD may potentially be associated with
alterations (i.e., decrease) in the capacity of FRa to transport reduced folates
across the choroid epithelium. Direct clinical evidence to support this
hypothesis was first obtained by Rothenberg and colleagues (Rothenberg
et al., 2004) who initially discovered auto-antibodies against FRs in women
with a pregnancy complicated by NTDs. In this study, it was found that 9 of
12 women with a current or previous affected pregnancy with an NTD
contained neutralizing auto-antibodies against FRs. Moreover, these auto-
antibodies were capable of blocking the binding of radiolabeled folic acid to
FRs on isolated membranes from FRa-rich cells as well as in intact KB cells
that highly express FRa. Consistently, folic acid uptake by KB cells was
potently blocked in the presence of these auto-antibodies to FRs.
Subsequent studies by Ramaekers and associates (Ramaekers et al., 2005)
revealed that sera from 25 out of 28 children with CFD contained high-
affinity blocking auto-antibodies against membrane-bound FRs present in
choroid plexus epithelial cells. Oral replacement therapy with folinic acid
improved the levels of 5-CH3-THF in the CSF and led to clinical amelio-
ration. Hence, these important studies demonstrated for the first time
that CFD is a CNS disorder which may be caused by auto-antibodies
abolishing the FRa-dependent transport of 5-CH3-THF from the plasma
into the CSF.
Knockout mice lacking the folic acid-binding protein Folbp1, currently termed
Folr1, the murine homologue of the human FRa: Early embryonic lethality was
observed in knockout mice lacking the folic acid-binding protein Folbp1
(currently termed Folr1), the murine homologue of the human FRa
(Piedrahita et al., 1999). Histological examination of transverse sections of
Folbp/ nullizygous E8.5 embryos revealed defects in the neuroepithelium;
in the cephalic neural tube, neither the forebrain nor the optic vesicles were
formed. The neuroepithelium was only one to two cells thick. At the level
of the midbrain, the neuroepithelium of both the basal and planar plates was
also limited to no more than two cells in thickness. Remarkably, supple-
menting pregnant Folbp1/ dams with folinic acid reversed the embryonic
lethality and the neuroepithelium defects in 80% of the nullizygous pups.
Moreover, administration to pregnant rats of an antiserum against FRs
(da Costa and Rothenberg, 1996) resulted in resorption of embryos or
multiple embryonic developmental abnormalities (da Costa et al., 2003).
Specifically, the antiserum displayed a dose-dependent effect on embryo
viability and organogenesis. Administration of folinic acid prevented tera-
togenicity resulting from low doses of antiserum, but failed to protect
against high antibody concentrations that inflicted embryo damage by
immune-mediated cell lysis. Moreover, resorption of embryos with larger
doses of antiserum was prevented by the immunosuppressant dexametha-
sone. Furthermore, cardiovascular abnormalities were observed in 100%
118 Ilan Ifergan and Yehuda G. Assaraf

of Folr1 knockout mice lacking a functional Folr1 (Zhu et al., 2007).

A dose-response study with folinic acid was also performed in order to
determine the impact of maternal folate supplementation on cardiac devel-
opment in Folr1 nullizygous fetuses. Hence, partially rescued preterm Folr1
fetuses were found to harbor outflow tract defects, aortic arch artery
abnormalities, and isolated dextrocardia (i.e., a peculiar anatomic anomaly
in which the heart is positioned on the right side of the chest). Consistent
with the above results, maternal supplementation with folinic acid rescued
embryonic lethality and the observed cardiovascular abnormalities in a
dose-dependent manner. This study further establishes the beneficial effects
of folate supplementation in the prevention of congenital heart defects. The
latter may be possibly mediated via the folate supplementation impact on
neural crest cells and regulation of genes associated with signaling pathways
resulting in normal development of pharyngeal arches and the secondary
heart field. Moreover, these studies clearly demonstrate the important
developmental role of the Folr1 in folate homeostasis.
(iii) The third route of folate uptake is the low pH folate transporter that
has been recently cloned and termed proton-coupled folate transporter
(PCFT; heme-carrier protein, HCP-1; SLC46A1) [Qiu et al., 2006);
Figs. 4.4 and 4.5]. This transport system functions optimally at acidic
pH (5.5) but poorly at physiological pH (7.4). PCFT recognizes folic
acid, reduced folates (e.g., 6S-5-CH3-THF) and MTX as transport
substrates with comparable high affinities (Km 0.52 mM) (Assaraf
et al., 1998; Henderson and Strauss, 1990; Kumar et al., 1997; Sierra
et al., 1997). Hence, PCFT plays a key role in the intestinal absorption
of folates within the acidic microclimate of the upper small intestine,
notably the duodenum and the upper jejunum (Qiu et al., 2006)
(Fig. 4.4). Human PCFT is a 459 amino acids membrane glycoprotein
(50 kDa) with 12 predicted transmembrane helices. PCFT belongs
to the solute carrier superfamily (i.e., SLC) of transporters (Saier et al.,
1999). PCFT is a classic representative of proton-coupled folate trans-
porters and low pH transport systems mediating intestinal absorption of
various essential nutrients including amino acids, peptides, metal ions
and various organic anions (Boll et al., 2002; Fei et al., 1994; Gunshin
et al., 1997; Nozawa et al., 2004). Although initially deposited into the
GenBank as a heme carrier protein (HCP1) (Shayeghi et al., 2005),
further kinetic studies at pH 5.5 revealed that the predominant (if not
the sole) transport function of PCFT is high-affinity transport of folic
acid [Km 0.83 mM; (Qiu et al., 2006)] as well as reduced folates such
as (6S)55-CH3-THF [Km 0.53 mM; (Qiu et al., 2006)]. At this
acidic pH, PCFT was also shown to mediate an extremely high affinity
transport of the novel antifolate pemetrexed [Alimta; Km 0.09 mM;
(Wang et al., 2004)] as well as high affinity for MTX [Km 2.0 mM;
Adaptation to Folate Deprivation 119

(Qiu et al., 2006)]. PCFT displayed an optimal folic acid transport

activity at pH 5.5 and the transport Vmax markedly declined as the pH
increased toward the physiological pH of 7.4. PCFT was shown to
function as an inward symporter cotransporting protons and folates and
thus proved to be an electrogenic transporter. Hence, the downhill
gradient of protons in the acidic microclimate of the proximal small
intestine drives the uphill uptake of oxidized and reduced folates.
Consistent with the important role that PCFT plays in intestinal folate
absorption, it was recently discovered that inactivating mutations in
this folate transporter result in hereditary (congenital) folate malab-
sorption (HFM) (Qiu et al., 2006; Zhao et al., 2007) (See the detailed
paragraph below entitled Loss-of-function mutations in the PCFT gene and
HFM ). Moreover, in the presence or absence of restoration of normal
folate levels in the circulation of HFM patients via oral folate replace-
ment therapy, these patients also exhibit a folate transport defect from
the blood into the CNS (Geller et al., 2002).
Loss-of-function mutations in the PCFT gene and HFM: Consistent with the
acidic pH of the upper small intestine (Mason, 1994; McEwan et al., 1990;
Selhub and Rosenberg, 1981), intestinal folate transport occurs primarily via
PCFT (Qiu et al., 2006). HFM (OMIM 229050HFM) first described by
Lubhy et al. in 1961 (Lubhy, 1961) is a rare autosomal recessive disorder
caused by impaired intestinal folate absorption and defective folate uptake
into the CNS with an early onset of a few months after birth. The disease
manifests itself with very low levels of folate in the blood as well as in the
CSF. HFM patients typically suffer from some or all of the following
symptoms: megaloblastic anemia, recurrent or chronic diarrhea, failure to
thrive, immune deficiency, recurrent infections (e.g., upper respiratory
infections), seizures, neurogical deficits as well as moderate to severe mental
retardation. The underlying deficit in this syndrome has been shown as a
severe defect in intestinal folate absorption (Geller et al., 2002) reflected in
an abnormal ratio (i.e., markedly decreased) of CSF:serum folate level.
As mentioned above, in healthy individuals, the normal folate level ratio
of CSF:serum is typically 3:1 (Alperin and Haggard, 1970), whereas in
HFM patients this ratio was found to range from 1:10 to 1:1 (Geller
et al., 2002). Importantly, if diagnosed early, most of the symptoms of this
disorder could be resolved by parenteral administration of large doses of
lecucovorin (Geller et al., 2002; Matherly and Goldman, 2003; Poncz and
Cohen, 1996). However, it should be noted that although the signs and
symptoms of this disorder could be obviated by folate replacement therapy
and the CSF:blood folate level ratio is improved, the normal CSF:blood
folate ratio of 3:1 was never restored. Recently, multiple loss-of-function
mutations were identified in the PCFT gene in five families with HFM
(Qiu et al., 2006; Zhao et al., 2007). These PCFT inactivating mutations
120 Ilan Ifergan and Yehuda G. Assaraf

were largely homozygous and were spread out throughout the entire coding
region. Introduction of these mutations into a PCFT expression vector and
their stable transfection into a HeLa subline that is doubly deficient in both
PCFT as well as RFC transport activities allowed for the assessment of the
impact of these congenital PCFT mutations on folate transport at acidic pH.
A complete loss of folic acid transport was observed in four of the five HFM
families; this loss of folate transport was due to decreased transporter stability
and/or plasma membrane trafficking defects inflicted by the amino acid
substitutions in highly conserved transporter regions. Whereas, a few
mutant PCFT transporters retained some residual folate transport activity
at low pH. Moreover, folate transport at acidic pH was markedly impaired
in immortalized lymphocytes from HFM patients. Taken together, these
pioneering studies establish the key role that inactivating PCFT mutations
play in the pathogenesis of HFM. Moreover, it is imperative that these
molecular studies will facilitate the rapid diagnosis and treatment of this
disorder in infants as well as the prenatal identification of families harboring
the mutant PCFT gene and thereby offer proper genetic counseling.

2. The role of influx transporters in adaptation to folate deficiency

Several lines of evidence support the notion that an increased activity of
folate influx systems is a crucial cellular adaptive response to folate
(a) The facility of RFC to mediate an efficient influx of reduced folates
should be beneficial to cells under conditions of folate deprivation
(Fig. 4.5). An increased activity of this reduced folate transporter may
serve as an important cellular adaptive response under conditions in
which folates are present in the growth medium at limiting concentra-
tions. Indeed, the T-cell leukemia subline CCRF-CEM-7A which was
gradually adapted to grow in 150-fold decreased leucovorin concen-
tration relative to its parental CCRF-CEM counterpart, displayed a
dramatic overexpression of the RFC ( Jansen et al., 1990). This over-
expression was associated with a consistently increased Vmax of MTX
influx [95-fold; (Assaraf et al., 1998)] when compared to parental
CCRF-CEM cells. Moreover, the expression of MRP1, an ATP-
dependent folate efflux transporter, was nearly completely lost in
these LF-adapted CCRF-CEM-7A cells (Assaraf et al., 2003). These
alterations were consistently associated with a marked decrease in both
folic acid and leucovorin growth requirements in CCRF-CEM-7A
(b) In independent studies, four colon cancer cell lines (C26-A, C2610,
C26-G, and WiDr) as well as three squamous cell carcinoma lines of the
head and neck (HNSCC) (11B, 14C, and 22B) were adapted to grow
in a culture medium containing LF levels (i.e., 0.52.5 nM leucovorin)
Adaptation to Folate Deprivation 121

(Backus et al., 2000). Consistent with the efficient folate influx ability of
the RFC, a two- to sevenfold increased transport activity of RFC in
LF-adapted cell lines was observed when compared to their folate-
replete counterparts. Hence, the increased activity of RFC appears to
serve as an important adaptive response to folate deficiency. The
importance of RFC transport activity in folate deplete growth condi-
tions should be studied in regards to the mechanism underlying upre-
gulation or downregulation of this folate transporter gene. Whereas
several transcript variants of the human RFC (regulated by several
promoters) have been identified, promoter B (pB) of the human
RFC was found to drive the expression of one variant which serves
as the major form of the intestinal human RFC (Gong et al., 1999;
Nguyen et al., 1997; Sirotnak and Tolner, 1999; Williams and Flintoff,
1998; Zhang et al., 1998). A recent study aimed at identifying the
minimal promoter region required for basal transcriptional activity of
this promoter undertook a deletion construct analysis of the human
RFC pB and determined their transcriptional activity in colon cancer-
derived Caco-2 cell transfectants (Subramanian et al., 2003). The mini-
mal region required for basal activity of human RFC pB in Caco-2 cells
was identified as a sequence between nucleotides 1088 and 1043.
Moreover, a sequence between nucleotides 141 and 2016 (i.e.,
outside the minimal region of the pB) was found to be responsive to
folate deficiency in Caco-2 cells; this promoter region led to a signifi-
cant and specific upregulation in folate uptake under conditions of
folate deficiency. This upregulation was associated with a parallel
increase in human RFC mRNA and protein levels as well as in the
transcriptional activity of human RFC pB. The identification of puta-
tive folate responsive elements in the human RFC pB may pave the
way for detailed studies aimed at pinpointing the exact role that
transcriptional upregulation of the human RFC plays in adaptation to
cellular conditions of folate deficiency.
(c) As with enhanced RFC transport activity, increased FR levels should
also play a contributing role in the adaptation to folate deficiency
(Fig. 4.5). This is particularly true as FRs mediate the unidirectional
influx of folates and are devoid of the folate efflux component displayed
by the RFC. Consistent with this presumption, it has been recently
shown that introduction of an FRa cDNA into cells that normally lack
receptor expression induced higher proliferation rates and increased
cellular survival under conditions of folate deprivation relative to their
parental counterparts (Antony, 1996). Indeed, a 5.5-fold increased
density of FRs was documented in human tumor xenografts implanted
in folate-deprived mice (Mendelsohn et al., 1996). Interestingly, the
increased density of FRs was associated with a modest reduction in
the affinity of this receptor to folic acid in four out of five tumors,
122 Ilan Ifergan and Yehuda G. Assaraf

suggesting an increased expression of the second FR with lower affinity

(i.e., FRb) in these tumors (Mendelsohn et al., 1996). Additionally, an
increased FPGS activity was also documented in these tumors. The
increased levels of FRs should augment the rate of unidirectional folate
uptake. Once taken up into cells, folates will be efficiently retained in
the cancer cells due to the increased activity of FPGS, the enzyme
responsible for folylpolyglutamylation (Fig. 4.5).
(d) In another study, a severe folate restriction (the growth medium
contained only 15 pM leucovorin; Lamers et al., 2006) was imposed
on Chinese hamster lung fibroblasts DC-3F/FA3 cells (Zhu et al.,
2002). Consistent with the above-mentioned experiments, these LF-
adapted cells also exhibited FRa overexpression. Moreover, three
independent LF-selected clones (i.e., FA3, FA7, and FA14) displayed
a five- to sixfold increased DHFR protein levels as well as catalytic
activity (Zhu et al., 2002). Thus, increased FRa levels should enhance
the rate of folate uptake following which the internalized folates could
be efficiently reduced and further utilized due to the increased activity
of DHFR (Fig. 4.5).
(e) In yet another study, LF [15 pM leucovorin (Lamers et al., 2006)]-
selected Chinese hamster lung fibroblasts were found to overexpress
FRa mRNA by more than 500-fold; furthermore, the mechanisms
underlying this dramatic increase in FRa transcripts were found to be
mediated via a significant gene amplification as well as an increase in
transcript half-life (Zhu et al., 2001). As has been discussed above, FR
levels have been shown to be upregulated under LF levels (Kane et al.,
1988; McHugh and Cheng, 1979). This inverse relationship was found
to occur at the translational level in cervical carcinoma cells (Sun and
Antony, 1996); specifically, it was suggested that the folate-dependent
translational regulation of FRa is attributable to an interaction of a
46 kDa cytosolic protein, recently identified as hnRNP E1(Xiao et al.,
2001), with an 18-base cis-element in the 50 -untranslated region of the
human FRa transcript (Sun and Antony, 1996). In contradistinction to
these results, a recent study demonstrated that 48 and 72 h of exposure
of HepG2 cells to a folate-deficient medium resulted in 12% and 43%
decreased FR protein expression, respectively (Abdel Nour et al.,
2007). Similarly, FR expression in kidney tubules was shown to be
downregulated in response to a LF diet (da Costa et al., 2000; Gates
et al., 1996). The molecular mechanisms underlying this decreased FR
protein expression under conditions of folate deficiency are yet to be
(f ) Chinese hamster ovary (CHO) cells were exposed to a stepwise selec-
tion to the lipophilic antifolate pyrimethamine resulting in the estab-
lishment of the PyrR100 subline (Assaraf and Slotky, 1993). These
pyrimethamine-resistant cells displayed a fourfold increase in the folic
Adaptation to Folate Deprivation 123

acid influx mediated by the low pH folate transporter currently identi-

fied as PCFT (HCP-1/SLC46A1) (Assaraf et al., 1998; Qiu et al., 2006).
These lipophilic antifolate-resistant cells also displayed the loss of several
folate efflux transporters including MRP1 (Stark et al., 2003). The
increased activity of the folate influx transporter PCFT along with the
loss of several ATP-driven folate efflux transporters should increase
folate uptake and decrease folate efflux, respectively, thereby increasing
the intracellular folate pool (Fig. 4.5). Indeed, PyrR100 cells displayed a
17-fold increase in the intracellular folic acid pool relative to parental
CHO AA8 cells (Assaraf and Goldman, 1997; Stark et al., 2003).
Of special note was the finding that the expansion in the intracellular
folate pool in these cells resulted in high levels of resistance to lipophilic
antifolates via competitive negation of the antifolate inhibitory effect at
the level of folate-dependent enzymes. Hence, it appears that over-
expression of PCFT may serve as a cellular adaptive response under
folate-deprived conditions; however, further studies are warranted in
order to identify the mechanisms underlying PCFT overexpression
under conditions of folate deficiency and/or antifolate inhibition.

D. Downregulation of folate efflux systems

1. The ABC superfamily of transporters
The term ABC transporters was first introduced in 1992 by Chris Higgins in
a memorable review (Higgins, 1992). The acronym ABC represents the
highly conserved ATP-binding cassette signature of the 49 known members
of this large superfamily of human transporters (Dean and Allikmets, 2001);
the ABC is utilized by these transporters to bind and hydrolyze ATP,
thereby energizing the vectorial transport of various substrates across
biological membranes (Borst et al., 2000; Dean, 2002; Gottesman et al.,
2002). Additional terminologies have been used for this superfamily includ-
ing Traffic ATPases and P-glycoproteins (Pgps). The basic structure of the
ABC transporters is often based on minimal data; however, the putative
membrane topology of Pgp (ABCB1), one of the most famous and well-
studied members of this group of transporters, is thought to consist of 12
TMDs and two ATP-binding sites and is 1,280 amino acids long. Some
ABC transporters may dimerize to form two nearly equal (e.g., BCRP) or
unequal halves (ABCG5 and ABCG8), resulting in a homodimeric or
heterodimeric ATP-dependent transport systems, respectively. Several
ABC transporters contain more than 12 hydrophobic transmembrane helices
and may be much larger than Pgp; for example, MRP1 is a 1522 amino acids
membrane glycoprotein with as many as 17 transmembrane helices.
ABC transporters mediate the ATP-dependent transport of various
drugs and their conjugates and thereby confer a multidrug resistance
124 Ilan Ifergan and Yehuda G. Assaraf

(MDR) phenotype upon cancer cells (Szakacs et al., 2006). This transport
activity is clearly exemplified by several MDR transporters including Pgp,
MRP1 (ABCC1), or BCRP (ABCG2/MXR/ABCP), each of which has
been already shown to mediate MDR in cancer cells. The identified genetic
variations of several ABC transporters (Fromm, 2002) may alter the
response to drug treatment. Several members of the ABC superfamily play
a key role in preventing the intestinal absorption of toxic compounds,
including many drugs and food components. Moreover, these transporters
play a key role in a second defense line by protecting vital organs in the body
including the brain, CSF, testis, as well as protecting the fetus against various
highly toxic xenobiotics. Consistent with this protective role of ABC
transporters, knockout mouse models of ABC transporter genes (e.g.,
ABCB1) resulted in an altered (i.e., decreased) bloodbrain barrier function
(Schinkel et al., 1997), intestinal drug absorption ( Jonker et al., 2002;
Sparreboom et al., 1997), fetal drug exposure (Smit et al., 1999), and
drug-induced damage to testicular tubules (Wijnholds et al., 2000). Addi-
tional physiological roles of several ABC transporters include the excretion
of endogenous metabolites from mammalian secretory epithelia, even
against a steep concentration gradient (Borst and Elferink, 2002). Accord-
ingly, ABC transporters play a key physiological role in liver excretion of
bile salts (transported by BSEP, the bile salt export pump, ABCB11),
phosphatidylcholine (MDR3 P-glycoprotein, ABCB4), bilirubin glucuro-
nides (MRP2, ABCC2), and various hydrophobic cytotoxic drugs (MDR1
P-glycoprotein, ABCB1) (Borst and Elferink, 2002). Moreover, ABC
transporters are capable of mediating hydrophobic peptide export such as
gramicidin D or cyclosporin A (transported by ABCB1) (Borgnia et al.,
1996; Sarkadi et al., 1994), peptides for antigen presentation (transported by
heterodimeric ABC transporters TAP1 and TAP2) (Schmitt and Tampe,
2000), and mitochondrial peptides (transported by ABC transporter related
to TAP) (Young et al., 2001). Nuclear receptors play a key role in the
transcriptional regulation of many mammalian ABC transporters; this is
clearly the case for MDR1 and MRP2 (Borst and Elferink, 2002).

2. The MRP (ABCC) family of MDR efflux transporters

Human MRPs constitute an important ABCC subfamily comprising 13
members including the cystic fibrosis conductance regulator (CFTR/
ABCC7) and the sulfonylurea receptors (SUR1/ABCC8 and SUR2/
ABCC9) (Deeley et al., 2006). Several MRPs mediate the ATP-driven
efflux of both endogenous compounds and various drugs as well as their
conjugated metabolites including Vinca alkaloids, anthracyclines, epipodo-
phyllotoxins, camptothecins, taxenes, antifolates, nucleoside and nucleotide
analogues, peptide-based cytotoxins, platinum compounds, and tyrosine
kinase inhibitors (Cools et al., 2005; Szakacs et al., 2006). The detoxification
process of many cytotoxic drugs requires their conjugation to glutathione
Adaptation to Folate Deprivation 125

(GSH), glucuronate, or sulfate before their binding to- and extrusion by

MRPs. However, the hydrophilic nature of the resultant conjugates pre-
vents them from diffusing back into the cell through biomembranes. Hence,
the ATP-dependent efflux of these drug conjugates must be mediated by
dedicated transporters, as first pointed out by Ishikawa (1992). As discussed
above, various members of the MRP family mediate the export of these
drug conjugates. Furthermore, the broad spectrum of MRP substrates also
includes phosphate as well as glutamate conjugates of organic compounds
(Borst et al., 2004; Keppler et al., 1997; Kruh and Belinsky, 2003; Wielinga
et al., 2005). MRP1 (ABCC1) was the first GS-X pump to be identified in
cells selected for MDR (Cole et al., 1992). This 190 kDa N-glycosylated
transmembrane protein is the founding member of the MRP family, first
cloned in the laboratory of Cole et al. (1992). Transport experiments carried
out with purified membrane vesicles from MRP1-overexpressing cells
established that MRP1 mediates the transport of various drugs conjugated
to GSH, sulfate, or glucuronate. MRP2 (ABCC2) was cloned in 1996
(Konig et al., 1996; Paulusma et al., 1996) and MRP3, MRP4, and
MRP5 soon followed (Kool et al., 1997) due to the identification of 21
potential human ABC transporters by Allikmets et al. (1996). Additional
studies identified new MRPs including MRP6, MRP7 (Hopper et al.,
2001), MRP8, and MRP9, respectively (Tammur et al., 2001). MRP4
(ABCC4) and MRP5 (ABCC5) were found to mediate the transport of
cyclic nucleotides and nucleotide analogs and thus confer resistance to base-
and nucleoside analogues used in the chemotherapy of cancer and viral
diseases such as HIV and AIDS. Whereas these 9 MRPs have been identi-
fied experimentally, an additional MRP pseudogene has been identified
during a computerized human genome search, suggesting that all human
MRPs have been already identified.
The MRP family is composed of two major structural types, one with 17
hydrophobic transmembrane segments (MRP1, -2, -3, -6, and -7), and one
with 12 transmembrane segments (MRP4, 5, -8, -9, and -10). Moreover,
the membrane-spanning domains (MSDs) of various MDR transporters
play a key role in substrate binding as was revealed in studies using affinity
probes as well as site-directed mutagenesis (Hafkemeyer et al., 1998; Loo
and Clarke, 1994).

3. BCRP (ABCG2), a MDR efflux transporter

Breast cancer resistance protein (BCRP) is a member of the small ABCG
subfamily of transporters which is composed of five members including
ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8, respectively. Whereas
Pgp and MRP1 confer drug resistance upon most cell lines, the mechanism
underlying high level mitoxantrone-resistance and lower level resistance to
anthracyclines and camptothecins could not be attributed to these transpor-
ters (Borst and Elferink, 2002). This initially mysterious and atypical
126 Ilan Ifergan and Yehuda G. Assaraf

drug resistance phenotype was found to be a result of decreased drug

accumulation which could be explained by transport activity of an uniden-
tified efflux transporter. Finally, a 72 kDa membrane glycoprotein was
cloned by Doyle et al. (1998) and termed BCRP. The latter is a half-size
ABC transporter which is expressed at substantial levels in breast cancer
cells. Allikmets et al. (1998) termed this transporter ABCP, a transporter
present at high levels in the placenta. BCRP cDNA was subsequently
cloned from human placenta (Allikmets et al., 1998) and mitoxantrone-
resistant human colon carcinoma cells (Miyake et al., 1999). The BCRP
gene is composed of 16 exons and 15 introns spanning 66 kb and has been
mapped to chromosome 4q22 (Bailey-Dell et al., 2001). Initial studies of the
BCRP promoter region revealed that it is TATA-less with five putative Sp1
sites downstream from a CpG island and contains several AP1 sites as well
as a functional estrogen response element (ERE) (Bailey-Dell et al., 2001;
Ee et al., 2004).
Members of the ABCG subfamily are considered half-transporters
with a single hydrophobic MSD and two cytosolic nucleotide-binding
folds (NBF; i.e., ATP-binding sites). It has been found that the transport
activity of these transporters is activated upon dimerization (Polgar et al.,
2004, 2006). BCRP transports a wide array of both positively and nega-
tively charged compounds including various chemotherapeutic cytotoxic
agents. Similarly to Pgp, BCRP does not require GSH for translocation of
electroneutral amphipathic drugs (Maliepaard et al., 2001). The broad
spectrum of BCRP substrates includes anticancer drugs such as anthracy-
clines (e.g., mitoxantrone and doxorubicin), epipodophyllotoxins, irinote-
can (SN-38) and topotecan, bisantrene, imatinib, flavopiridol, hydrophilic
antifolates (e.g., MTX and pemetrexed) and lipophilic antifolates (e.g.,
trimetrexate and pyrimethamine) and their di- and triglutamate conjugates
as well as the nucleoside analogues lamivudine and zidovudine as has been
previously reviewed (Krishnamurthy and Schuetz, 2006). Additionally,
BCRP exports various dietary toxic compounds such as the carcinogen
PhIP, the chlorophyll derivative pheophorbide as well as porphyrins.
BCRP also extrudes fluorescent compounds including Hoechst 33342,
lysotracker, rhodamine 123, and novel receptor-targeted agents such
as imatinib and gefitinib (Morgillo and Lee, 2005; Ozvegy et al., 2001).
A recent study demonstrated that BCRP is expressed in mammary gland
alveolar epithelial cells during pregnancy and lactation ( Jonker et al., 2005).
In contrast to the detoxifying role of BCRP in adult humans ( Jonker et al.,
2002; van Herwaarden et al., 2003), BCRP expressed in the mammary
gland was found to secrete a variety of drugs, toxins, and carcinogens into
milk and thus expose suckling newborn and young to xenotoxins ( Jonker
et al., 2005). The contradiction between the detoxifying and the newborn
contaminating role of BCRP in humans has been partially resolved by an
additional finding from this group demonstrating that BCRP secretes
Adaptation to Folate Deprivation 127

riboflavin (vitamin B2) into milk, thereby supplying the suckling infants and
young with this important micronutrient (van Herwaarden et al., 2007).

4. The role of efflux transporters in folate homeostasis

and adaptation to folate deficiency
The intracellular folate pool can be modulated by the above-mentioned
folate influx systems, by FPGS activity as well as by several ATP-driven
folate efflux transporters of the ABC superfamily (Borst and Elferink, 2002).
Various studies demonstrated that inverted membrane vesicles isolated from
cell lines overexpressing MRP1 (ABCC1) through MRP5 (ABCC5) have
the capacity to accumulate folate and MTX in an ATP-dependent manner
(Chen et al., 2002; Hooijberg et al., 1999; Kool et al., 1999; Wielinga et al.,
2005; Zeng et al., 1999, 2001). Moreover, kinetic analyses with inside-out
membrane vesicles isolated from MRP3- and MRP5-overexpressing cells
revealed that these MRPs mediate the transport of both folic acid and
leucovorin with Km values in the millimolar range (Km 0.62 mM)
(Zeng et al., 2001). cMOAT (MRP2) has been found to mediate the
transport of various reduced folate cofactors including THF, 5-CH3-THF,
5,10-CH2-THF, and 5-CHO-THF (Kusuhara et al., 1998). Kinetic studies
with inside-out membrane vesicles isolated from MRP4-overexpressing
cells documented a higher affinity for folic acid (Km 0.17 mM) and
leucovorin (Km 0.64 mM) than MRP3 and MRP5 (Chen et al., 2002).
The higher affinity of MRP4 for these folate derivatives is consistent with
the relatively high affinity of this efflux transporter for MTX (Km
0.22 mM) (Chen et al., 2002). Moreover, folic acid transport by MRP3,
MRP4, and MRP5 occurs with a relatively high Vmax of 0.71.7 nmol/mg
protein/min. Thus, the ATP-dependent transport of folic acid and leucov-
orin via MRP3, MRP4, and, MRP5 occurs with a low affinity, yet high
Kinetic studies with inside-out vesicles isolated from BCRP-overex-
pressing HEK293/ABCG2 cells documented an ATP-dependent transport
of radiolabeled folic acid with a transport rate of 87 pmol/mg protein/min
(Chen et al., 2003). In contrast to the wild-type BCRP, inside-out vesicles
isolated from HEK293/G482 ABCG2 cells overexpressing the mutant
Gly482 BCRP were devoid of folic acid transport capability. The activity
of folate efflux systems may decrease the intracellular folate pool; indeed,
overexpression of MRP1, MRP2, and MRP3 in human ovarian carcinoma
2008 cells resulted in a 3238% decrease in the intracellular folate pools
under folate-replete growth conditions (Hooijberg et al., 2003). Consis-
tently, a marked increase in the folic acid (4 h pulse) growth requirement
was documented for MRP1- and MRP3-overexpressing cells relative
to parental 2008 ovarian carcinoma cells. These results suggest that
MRP1, MRP3 as well as additional folate efflux transporters may decrease
intracellular folate pools and thereby exert a deleterious effect on cells,
128 Ilan Ifergan and Yehuda G. Assaraf

particularly under conditions of folate deficiency. Several studies lend

support to the role of various MRPs as well as BCRP in adaptation to
folate deficiency:
(a) The capacity of MRP1 to transport folates should be deleterious to cells
under conditions of folate deprivation. This possible contribution of
MRP1 to cellular folate homeostasis could be readily examined in
CCRF-CEM leukemia T-cells lacking MRP2, MRP3, and BCRP
expression but barely expressing MRP4 and MRP5 (Assaraf et al.,
2003). Accordingly, gradual folate deprivation of this cell line resulted
in the CCRF-CEM-7A cell line which can grow in about 150-fold
decreased leucovorin concentration (i.e., 0.25 nM) ( Jansen et al., 1990);
these cells showed dramatic decreases in both folic acid and leucovorin
growth requirements. As expected, MRP1 expression was nearly
completely lost (Assaraf et al., 2003) along with a dramatic overexpres-
sion of the RFC ( Jansen et al., 1990). However, the poorly expressed
transporters MRP4 and MRP5 retained their low expression level in
this LF-adapted cell line. Consistently, the loss of MRP1 expression was
associated with diminished folate efflux capability in CCRF-CEM-7A
cells; these cells showed a fivefold fall in the folic acid efflux rate
constant relative to parental CCRF-CEM cells. A similar fall in the
folic acid efflux rate was achieved by incubating parental CCRF-CEM
cells with probenecid, an MRP1 transport inhibitor (Hooijberg et al.,
1999). These results suggest that under conditions of folate deprivation,
loss of MRP1 expression serves as an adaptive response aimed at pre-
venting the deleterious folate depletion effect of this ATP-driven folate
efflux transporter (Fig. 4.5).
(b) The loss of MRP1 protein levels under folate-deprived conditions
raised the possibility that a folate-dependent bidirectional regulatory
mechanism controlling MRP1 expression is operative in these cells.
Therefore, folate-replete conditions (5 nM leucovorin) were imposed
on CCRF-CEM-7A cells which, as shown above, have completely lost
MRP1 expression under folate deplete conditions. Indeed, under these
folate-replete conditions, CCRF-CEM-7A cells displayed a complete
restoration of parental MRP1 levels (Assaraf et al., 2003). Similarly,
RFC-defective CCRF-CEM sublines (CEM/MTX-LF and CEM/
GW-70LF) lost MRP1 expression under low folic acid conditions
(25 nM folic acid). Consistently, full restoration of MRP1 expression
was achieved upon growth of these cell lines under folate-replete
conditions (2.3 mM folic acid; Assaraf et al., 2003). These compelling
evidences suggest that changes in extracellular folate status can modu-
late MRP1 levels. The molecular mechanism underlying this folate-
dependent bidirectional regulatory mechanism of MRP1 expression is
yet to be revealed.
Adaptation to Folate Deprivation 129

(c) An increased intracellular folate pool serves as a resistance mechanism

against lipophilic antifolates such as pyrimethamine and trimetrexate by
competitively negating the inhibitory effect of these antifolates. This
study supports the putative intracellular folate depletion effect of MRP1
and MRP5. Specifically, CHO cells were exposed to a stepwise selec-
tion with the lipophilic antifolate pyrimethamine, resulting in the
establishment of the PyrR100 subline (Assaraf and Slotky, 1993). These
pyrimethamine-resistant cells displayed a complete loss of MRP1
expression and a marked decrease in MRP5 levels (Stark et al., 2003)
along with a fourfold increase in PCFT folate influx activity (Assaraf
et al., 1998; Qiu et al., 2006). The lack of MRP1 and MRP5 expression
along with the poor expression of other MRPs, was associated with a
fivefold decreased efflux of both folic acid and cholic acid as well as
17-fold increase in the intracellular pool of folic acid relative to parental
CHO cells (Assaraf and Goldman, 1997; Stark et al., 2003). Consis-
tently, this resulted in a 14-fold decreased folic acid and leucovorin
growth requirements; moreover, a very low concentration of 100 pM
leucovorin was sufficient to support the growth of PyrR100 cells. These
results strongly suggest that downregulation of MRP1 and MRP5 may
be crucial components of cellular adaptation to folate deficiency.
(d) Despite the above body of evidence, folate deprivation does not neces-
sarily result in the loss of MRP1 expression as has been found with
MRP1-overexpressing 2008/MRP1 cells subjected to a pulse exposure
in folate-free medium (Hooijberg et al., 2004). Consistent with previous
studies, these folate-deprived cells showed a decreased efflux of dauno-
rubicin, a typical reflection of decreased MRP1 activity (Hooijberg
et al., 2004). Moreover, full restoration of MRP1-dependent daunoru-
bicin efflux activity was achieved upon a 48 h pulse replenishment of
folate-deprived cells with 2.5 mM leucovorin. However, this folate-
dependent MRP1 activity occurred in the absence of any alterations
in MRP1 protein levels. This folate-dependent modulation of MRP1
activity may be possibly explained by putative posttranslational modifi-
cations of MRP1 (Hooijberg et al., 2004). Indeed, a cyclic phosphory-
lation (and dephosphorylation) of MRP1 has been previously suggested as
a posttranslational modification that could modulate MRP1-dependent
anthracycline efflux activity (Ma et al., 1995). Accordingly, these
researchers showed that treatment of MRP1-overexpressing HL60/
ADR cells with the protein serine kinase inhibitors H-7 and stauros-
porine completely abolished MRP1 serine phosphorylation; in turn, the
lack of MRP1 serine phosphorylation was associated with a major
decrease in MRP1 drug efflux activity and a marked increase in drug
accumulation. The suggestion that posttranslational modifications of
MRP1 could modulate its transport activity is consistent with studies
demonstrating that blocking phosphoinositol-3-kinase (PI3K/Akt)
130 Ilan Ifergan and Yehuda G. Assaraf

activity with LY294002 in MRP1-overexpressing colon carcinoma

cells resulted in a marked increase in doxorubicin accumulation and
cytotoxicity (Abdul-Ghani et al., 2006). Yet, further studies are neces-
sary to determine whether phosphorylation (and depohosphorylation)
of MRP1 and/or other posttranslational modifications can modulate
MRP1 transport activity in a bidirectional manner thereby serving as a
delicate and well-controlled folate homeostasis mechanism.
(e) The role of BCRP in folate homeostasis was studied as well; in these
studies, MCF-7 breast cancer cells, with low BCRP protein levels, and
their MR (i.e., mitoxantrone, an anticancer drug which is a specific
BCRP substrate)-resistant MCF-7/MR subline, with BCRP overex-
pression, were gradually deprived (over 3.5 months) of folic acid from
2.3 mM to 3 nM resulting in the sublines MCF-7/LF and MCF-7/
MR-LF, respectively. These LF-adapted sublines displayed only resid-
ual BCRP mRNA consistent with poor BCRP protein levels and
transport activity along with a significant increase in FPGS activity
(Ifergan et al., 2004). The loss of BCRP and MRP1 expression along
with the increased FPGS activity were associated with enhanced cellu-
lar accumulation of folates. These results suggest that significant expres-
sion of BCRP and MRP1 may induce intracellular folate depletion.
Thus, BCRP and MRP1 are likely to undergo downregulation upon
folate deprivation (Fig. 4.5). Additional support to this hypothesis was
gained by conducting experiments of short-term (2 weeks) exposure of
MCF-7/MR cells to folate-free medium followed by one week of
adaptation to low folic acid (1 nM)-containing medium (Ifergan et al.,
2005). The short-term folate-deprived cells were characterized by a
selective confinement of BCRP to the endoplasmic reticulum instead
of the plasma membrane as was apparent to some extent before folate
deprivation. Moreover, consistent with the effects of long-term folate
deprivation, these cells also displayed a threefold decrease in BCRP and
MRP1 protein levels, thereby resulting in a 5-fold increased cellular
accumulation of folic acid. These data suggest that lack of plasma mem-
brane targeting of BCRP may be selected or regulated upon short-term
folic acid deprivation due to the folate-depleting effect of this efflux
transporter. One possible mechanism underlying translocation of BCRP
from the plasma membrane to the cytoplasmic compartment may be
associated with downregulation of the PI3K-Akt signaling pathway as
has been documented in hematopoietic stem cells (Mogi et al., 2003).
However, further studies are required to determine whether the con-
finement of BCRP to the cytoplasmic compartment in the short-term
folate-deprived breast cancer cells may be a result of decreased activity of
the PI3K-Akt signaling pathway. Hence, consistent with the folate
transport capability of both MRP1 and BCRP, downregulation of
these ATP-driven folate efflux transporters, increased FPGS activity
Adaptation to Folate Deprivation 131

and selective confinement of BCRP to the endoplasmic reticulum

appear to be crucial components of the preservation of the precious
cellular folate pools that are particularly shrunken under conditions of
folate deficiency (Assaraf et al., 2003; Liani et al., 2003; Rothem et al.,
2002). Moreover, it has been reported recently that the folic acid trans-
port rate via BCRP is 2- to 5-fold higher at pH 5.5 than at pH 7.3
(Breedveld et al., 2007). Hence, it is possible that the role of BCRP in
folate homeostasis is mostly important in many tumor tissues, where
the extracellular pH is more acidic than in most normal mammalian
cells and may be as low as pH 5.8 (Tannock and Rotin, 1989) due to the
high rate of lactic acid production, even under aerobic conditions
(Boyer and Tannock, 1992; Gatenby and Gillies, 2004; Tannock and
Rotin, 1989).
(f ) As has been previously discussed, RFC is a nonconcentrative, facilitative
bidirectional anion-exchanger that equally displays a high-affinity (i.e.,
at the micromolar range) influx and efflux activities for reduced folate
cofactors. Hence, under conditions of extreme folate deprivation, RFC
activity may be downregulated in order to protect cells from further
loss of intracellular folates (Fig. 4.5). Indeed, we have recently found
(I. Ifergan and Y. G. Assaraf, unpublished data) a 15-fold decrease in
the viable cell number of RFC-overexpressing CHO C5/RFC cells
when compared to the RFC-deficient C5 cells after 6 days of in-
cubation in folic acid-free medium. Moreover, both the moderately
RFC-expressing as well as the RFC-overexpressing MCF7/MR and
CCRF-CEM/7A cell lines, respectively, showed 2.5-fold decreased
RFC mRNA levels after 7 and 3 days of incubation in folic acid-free
medium, respectively. These decreased mRNA levels were associated
with a marked decrease in RFC transport activity. Hence, it appears that
whereas increased activity of RFC may serve as an important mecha-
nism of adaptation to conditions of folate deprivation ( Jansen et al.,
1990), the folate efflux component of the RFC may be detrimental to
cells under the complete lack of folates in the growth medium. Hence,
downregulation of RFC transport activity may serve as a novel
component of adaptation to conditions of severe folate deficiency.

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Structure and Function of the

Reduced Folate Carrier: A Paradigm
of a Major Facilitator Superfamily
Mammalian Nutrient Transporter
Larry H. Matherly*, and Zhanjun Hou

I. Introduction 146
II. MFS of Transporters 147
III. Folate Transport in Tissue Folate Homeostasis and Physiology:
Role of Multiple Transport Systems for Folate Uptake and Efflux 150
IV. Role of RFC in Antifolate Chemotherapy 152
V. Functional Properties of RFC 154
VI. Biochemistry of RFC 156
VII. Cloning of RFC cDNAs That Restore Transport to
Transport-Impaired Cultured Cells 158
VIII. Topological Structure of RFC 159
A. Topological structure by HA epitope accessibility
and N-glycosylation scanning mutagenesis 159
B. Topological structure by scanning cysteine
accessibility methods 163
IX. Insights into Structural and Functional Determinants of RFC from
Studies of Mutant RFC Proteins 164
A. Identification of structurally or functionally important amino
acids by selecting for mutant RFCs with antifolate inhibitors 165
B. Identification of structurally or functionally important
amino acids in RFC by homology comparisons and
site-directed mutagenesis 166
C. Deletional and insertional mutagenesis of RFC 167
D. Localization of a substrate-binding domain
by radioaffinity labeling 168

* Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University
School of Medicine, Detroit, Michigan 48201
Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University
School of Medicine, Detroit, Michigan 48201

Vitamins and Hormones, Volume 79 # 2008 Elsevier Inc.

ISSN 0083-6729, DOI: 10.1016/S0083-6729(08)00405-6 All rights reserved.

146 Larry H. Matherly and Zhanjun Hou

E. SCAM for mapping the substrate translocation pathway 169

F. Mapping helix packing associations in hRFC 172
X. Conclusions 173
Acknowledgment 175
References 175

Folates are essential for life and folate deficiency contributes to a host of health
problems including cardiovascular disease, fetal abnormalities, neurological
disorders, and cancer. Antifolates, represented by methotrexate, continue to
occupy a unique niche among the modern day pharmacopoeia for cancer along
with other pathological conditions. This article focuses on the biology of the
membrane transport system termed the reduced folate carrier or RFC with a
particular emphasis on RFC structure and function. The ubiquitously expressed
RFC is the major transporter for folates in mammalian cells and tissues. Loss of RFC
expression or function portends potentially profound physiological or develop-
mental consequences. For chemotherapeutic antifolates used for cancer, loss of
RFC expression or synthesis of mutant RFC protein with impaired function results in
antifolate resistance due to incomplete inhibition of cellular enzyme targets and
low levels of substrate for polyglutamate synthesis. The functional properties for
RFC were first documented nearly 40 years ago in murine leukemia cells. Since
1994, when RFC was first cloned, tremendous advances in the molecular biology of
RFC and biochemical approaches for studying the structure of polytopic membrane
proteins have led to an increasingly detailed picture of the molecular structure of
the carrier, including its membrane topology, its N-glycosylation, identification
of functionally and structurally important domains and amino acids, and helix
packing associations. Although no crystal structure for RFC is yet available,
biochemical and molecular studies, combined with homology modeling, based
on homologous bacterial major facilitator superfamily transporters such as LacY,
now permit the development of experimentally testable hypotheses designed to
establish RFC structure and mechanism. 2008 Elsevier Inc.

I. Introduction
Folate is the generic term for water-soluble members of the B class of
vitamins that are required for normal tissue growth and development. Folic
acid is the synthetic form of the metabolically important folates found in cells
that differ in the level of oxidation of the pteridine ring, the nature of the one-
carbon substituent at the N5 and N10 positions, and the extent of g-glutamate
conjugation (Stokstad, 1990). The biological importance of reduced folates
derives from their essential roles in one-carbon transfer leading to thymidylate,
purine nucleotides, serine, and methionine, and in biological methylation
reactions from S-adenosyl methionine (Stokstad, 1990).
Structure and Function of the Reduced Folate Carrier 147

Folates are hydrophilic molecules that are anions at physiological pH and

thus cross biological membranes only poorly by diffusion. Reflecting this,
mammalian cells have evolved sophisticated uptake systems for facilitating
cellular uptake of folate cofactors (Matherly and Goldman, 2003). The
reduced folate carrier (RFC) or SLC19A1 is expressed ubiquitously and is
recognized to be the major transport system for folates in mammalian cells
and tissues (Matherly and Goldman, 2003). In addition to its generalized
role in folate transport, RFC performs certain specialized tissue functions
including absorption across intestinal/colonic epithelia (Balamurugan and
Said, 2006; Chiao et al., 1997; Said, 2004), transport across the basolateral
membrane of renal proximal tubules (Kneuer et al., 2005), transplacental
transport of folates (Sweiry and Yudlievich, 1985), and folate transport
across the bloodbrain barrier (Spector and Johanson, 2006). Reflecting
these important physiological roles, low levels of RFC can be envisaged to
contribute to a number of pathophysiological states associated with folate
deficiency, including cardiovascular disease, fetal abnormalities, neurological
disorders, and possibly cancer (Matherly, 2004). Susceptibilities to these
conditions could be exacerbated with folate deficiency. Importantly, mem-
brane transport by RFC is also important for the antitumor activities of
antifolate therapeutics used for cancer chemotherapy such as methotrexate
(MTX) and pemetrexed (Matherly et al., 2007; Fig. 5.1).
This chapter focuses on the biology of the RFC with a particular
emphasis on RFC structure and function. The functional properties for
RFC were first documented nearly 40 years ago in murine leukemia cells
(Goldman et al., 1968). However, it is only since the cloning of the rodent
RFCs in 1994 (Dixon et al., 1994; Williams et al., 1994) and the human
RFC (hRFC) in 1995 (Moscow et al., 1995; Prasad et al., 1995; Williams
and Flintoff, 1995; Wong et al., 1995), and the application of molecular
biology approaches to engineer RFC for biochemical studies, that a detailed
picture of the molecular structure of this physiologically important carrier
has emerged, including its membrane topology, its N-glycosylation, and
identification of functionally and structurally important domains and amino
acids. In this chapter, we review the considerable progress in this area,
drawing significantly from the literature for RFC since 1994, along with
structural inferences from structurally homologous bacterial major facilitator
superfamily (MFS) members for which crystal structures were recently
reported (Abramson et al., 2003; Huang et al., 2003; Yin et al., 2006).

II. MFS of Transporters

The MFS of transporters is represented in animals, plants, fungi, lower
eukaryotes, bacteria, and eukaryotic organelles and transports a diverse
assortment of substrates in a uniport, symport, or antiport fashion, including




Folic Acid Methotrexate





5-Methyl Tetrahydrofolate Pemetrexed




5-Formyl Tetrahydrofolate Tomudex (Ratitrexed)

Figure 5.1 Transport substrates for the reduced folate carrier (RFC). Structures are shown for folate and antifolate substrates for RFC.
Structure and Function of the Reduced Folate Carrier 149

amino acids, neurotransmitters, sugars, vitamins, nucleosides, and organic

phosphates (Saier et al., 1999). The MFS includes over 2000 sequenced
members (Chang et al., 2004), making it the largest secondary active
transporter family. MFS proteins typically contain 400600 amino acids
and a structural motif composed of two halves, each half composed of six
transmembrane-spanning a-helices connected by a large hydrophilic loop,
with cytosolic N- and C-termini.
Protein structural information is a prerequisite for understanding the
mechanism of membrane transport. It is remarkable that, given their abun-
dance and biological importance, there is a paucity of structural information
on the MFS proteins. This in part reflects difficulties in isolating sufficient
quantities of purified proteins and in crystallizing membrane proteins for
X-ray diffraction. Accordingly, structural insights have relied on an exten-
sive array of sophisticated biochemical and biophysical approaches, along
with homology structural modeling from the crystal structures that have
been reported.
To date, X-ray crystal structures have been reported for four MFS
proteins. The first structural evidence approaching atomic resolution was
for the oxalate transporter (OxlT) at 6.5 A, generated by cryoelectron
microscopy (Hirai et al., 2002, 2003). In 2003, X-ray crystallographic
structures of the MFS proteins, the lactose/proton symporter (LacY)
(Abramson et al., 2003), and the inorganic phosphate/glycerol-3-phosphate
antiporter (GlpT) (Huang et al., 2003) were reported at resolutions of 3.5
and 3.3 A, respectively. In 2006, a crystal structure of the Escherichia coli
multidrug transporter EmrD was reported (Yin et al., 2006). For GlpT,
LacY, and OxlT, the proteins were crystallized in cytoplasmic orientations,
whereas EmrD appears as an intermediate conformation.
In spite of their very different substrates and mechanisms, and limited
sequence homologies, the overall structures of these four MFS transporters
are quite similar. In some cases, most notably LacY (Kaback, 2005), the
structure data corroborates an abundance of biochemical and biophysical
data. All four MFS structures exhibit symmetrical structures in which two
bundles of six helices surround a large central cavity. In both the GlpT and
LacY structures, hydrophilic cavities accommodate the substrate-binding
sites formed by helices-I, -II, -IV, and -V of the N-terminal domain, and
helices-VII, -VIII, -X, and -XI of the C-terminal domain (Abramson et al.,
2003; Huang et al., 2003). Helices III, VI, IX, and XII do not directly
participate in substrate binding and are embedded in the lipid bilayer. The
helices that form the hydrophilic cavity are irregular in that they contain
numerous proline and glycine residues, likely permitting structural
While no crystal structures are available, mammalian MFS proteins have
also been studied by biochemical methods and modeling based on the GlpT
and LacY structures. Examples include the human glucose transporter
150 Larry H. Matherly and Zhanjun Hou

(GLUT1) (Hruz and Mueckler, 2001; Salas-Burgos et al., 2004), the human
glucose-6-phosphate transporter (Almqvist et al., 2004; Fiser and Sali,
2003), the rat organic cation transporter-1 (OCT1) (Popp et al., 2005),
the rabbit organic cation transporter-2 (OCT2) (Zhang et al., 2005), and, as
described below, hRFC (Hou et al., 2006; Matherly et al., 2007).
Biochemical and structural data with LacY were used to argue for a
model for lactose/H symport in which the hydrophilic substrate-binding
cavity is alternately accessible to either side of the membrane (alternating
access model) (Abramson et al., 2003). Direct support for this model was
recently described in studies in which almost every residue of Lac Y was
replaced individually with cysteine and tested for reactivity with N-ethyl
maleimide (Kaback et al., 2007). In this report, alkylation of substituted
cysteine residues with NEM was alternately increased on the periplasmic
side of the LacY sugar-binding site in the presence of ligand, accompanying
decreased reactivity on the cytoplasmic side, consistent with a model in
which the sugar-binding site is alternately exposed to either side of the
membrane during transport (Kaback et al., 2007). Similar models were
proposed for GlpT (Huang et al., 2003) and EmrD (Yin et al., 2006).

III. Folate Transport in Tissue Folate

Homeostasis and Physiology: Role of
Multiple Transport Systems for Folate
Uptake and Efflux
Mammalian cells cannot synthesize folates de novo, so these derivatives
must be acquired from foods. Although folates are absorbed throughout the
intestine, absorption occurs primarily in the duodenum and upper jejunum.
Excellent sources of folate include orange juice, liver, dried beans and peas,
dark green leafy vegetables, and strawberries. A proton-coupled folate
transporter (PCFT; SLC46A1) has been implicated as the major transport
system at the acidic pH in the upper small intestine (Qiu et al., 2006).
However, the expression of RFC throughout the intestine (Balamurugan
and Said, 2006; Chiao et al., 1997; Said, 2004; Wang et al., 2001) implies its
contribution to intestinal folate uptake as well, particularly in the lower
intestine. Folates are also absorbed by RFC in the colon and monoglutamyl
folate synthesized by the intestinal microflora can be nutritionally significant
(Rong et al., 1991). Following intestinal absorption, folates are transported
across the basolateral membrane [via the multidrug resistance-associated
proteins (MRPs) 1 and 3] of enterocytes (Mutch et al., 2004) and delivered
via the hepatic portal system to the liver, where they are stored as polyglu-
tamates. Folates are eventually released from the liver (primarily 5-methyl
tetrahydrofolate monoglutamate) into the bloodstream, whereupon they
are transported via specific transport systems into peripheral tissues.
Structure and Function of the Reduced Folate Carrier 151

Specific transport systems for folates include RFC and PCFT, both of
which are reported to be widely expressed (Qiu et al., 2006; Whetstine
et al., 2002; Zhao and Goldman, 2007), but are functionally distinct in that
transport by RFC occurs optimally at neutral pH (7.4) whereas transport
by PCFT is optimal at acidic pH (5.56.5) (Matherly and Goldman, 2003;
Zhao and Goldman, 2007). Other uptake systems include the high-affinity
folate receptors (FRs) (a and b), glycosyl-phosphoinositol-linked proteins
that accumulate folates via an endocytotic process (Matherly and Goldman,
2003; Salazar and Ratnam, 2007), and the organic anion transporters
(OATs) that are expressed in epithelial tissues such as kidney and transport
organic anions in addition to folates (Matherly and Goldman, 2003;
Miyazaki et al., 2004; Zhou and You, 2007), such as bromosulfopthalein,
taurocholate, and probenecid.
Renal tubular secretion and reabsorption of folates in proximal tubules
involve specific roles for folate transporters on the basolateral (e.g., OAT1,
OAT3, and RFC) and apical (e.g., OATP1, FRa, MRP2, and MRP4)
membranes (Nozaki et al., 2004; Russel et al., 2002). Folates are filtered via
the glomerulus and reabsorbed by an FRa-mediated endocytotic process,
then transported into the bloodstream by folate transporters localized to the
basolateral membrane. Both FRa and RFC are also involved in transpla-
cental transport of folates (Barber et al., 1999; Sweiry and Yudlievich, 1985).
The choroid plexus separates the blood compartment from the cerebral
spinal fluid (CSF). 5-Methyl tetrahydrofolate is typically present in CSF at
approximately four times the concentration found in plasma (Spector and
Lorenzo, 1975). FRa is localized to the basal (blood) side of choroid plexus
and likely mediates uptake of folates from blood (Spector and Lorenzo,
1975; Suleiman and Spector, 1981). RFC is localized on the apical mem-
brane of the bulbous microvili of the choroid plexus epithelium adjacent to
the ventricular membrane (Wang et al., 2001), suggesting its role in trans-
porting folates into the CSF at the apical surface. The recent finding that at
least some cases of hereditary folate maladsorption syndrome are accompa-
nied by low levels of central nervous system and plasma folates and a loss of
functional PCFT (Qui et al., 2006) suggests a role of PCFT in CNS folate
transport along with those of FRa and RFC. The possibility that RFC may
also contribute to hereditary folate maladsorption syndrome has been sug-
gested (Said, 2004). Interestingly, RFC was detected in axons and dendrites,
and on the apical membrane of the spinal canal (Wang et al., 2001),
suggesting that this carrier is an important mode of folate transport in
neuronal cells.
For mice in which RFC was inactivated by targeted homologous
recombination (knockout mice), RFC was obligatory for development
because targeting both alleles was embryonic lethal (Zhao et al., 2001b).
However, 10% of RFC-null mice could be brought to live birth by
supplementing the dams with folic acid. These mice subsequently died
152 Larry H. Matherly and Zhanjun Hou

within 1 or 2 weeks due to the failure of hematopoietic organs such as the

bone marrow, thymus, or spleen (Zhao et al., 2001b), consistent with in vitro
immunohistochemistry detection of RFC in the red pulp of the spleen
(Wang et al., 2001).
RFC knockout mice have begun to provide insights into the possible
role of RFC in cancer etiology. For instance, APCmin/ mice in which one
copy of RFC was inactivated developed significantly fewer intestinal ade-
nomas than mice with two functional RFC alleles (Lawrance et al., 2007).
Conversely, ablation of one RFC allele in mice decreased plasma S-adenosyl
methionine/S-adenosyl homocysteine, increased colonocyte proliferation,
increased transcripts for colon cancer-related genes (e.g., Cdh1, Cdx1, Igf2,
and Ptgs2) regulated by methylation, and increased susceptibility to carcin-
ogen (azoxymethane), as reflected in the numbers of aberrant crypt foci in
colon (Ma et al., 2005). While it is not easy to reconcile the seemingly
disparate findings of these reports, they presumably reflect the model system
(i.e., carcinogen-treated versus genetically modified Apc min/ mouse) or the
localization of the tumors (colon versus intestine). Regardless, these results
raise the intriguing possibility that levels of RFC can profoundly impact the
neoplastic process.
Thus, while the RFC is ubiquitously expressed and plays an integral role
in in vivo folate homeostasis and tissue-specific folate transport, this is often in
concert with other folate transport systems such as PCFT and/or high-
affinity FRa. Altered RFC levels and function could easily exacerbate effects
of dietary folate deficiency, thereby contributing to cardiovascular disease,
fetal abnormalities, neurodegenerative disease, and cancer (Matherly, 2004).
Alterations in folate membrane transport by RFC may be further com-
pounded by gene polymorphisms that result in changes in the catalytic
activities of folate-dependent interconverting and biosynthetic enzymes
such as 5,10-methylene tetrahydrofolate reductase (MTHFR) that impact
the intracellular distribution of individual reduced folate forms (Matherly,
2004). The recent generation of a humanized mouse in which the hRFC
gene locus has replaced the mouse RFC gene (Patterson et al., 2008) should
provide an opportunity to study the regulation and function of hRFC in
relation to folate homeostasis and polymorphisms in hRFC or other critical
genes, and in response to dietary interventions with folic acid.

IV. Role of RFC in Antifolate Chemotherapy

MTX continues to be an important component of the chemothera-
peutic arsenal for a number of cancers including pediatric acute lymphoblastic
leukemia, osteogenic sarcoma, lymphoma, and breast cancer (Monahan and
Allegra, 2001). Raltitrexed is used throughout much of the world outside of
Structure and Function of the Reduced Folate Carrier 153

the United States for advanced colorectal cancer (Chu et al., 2003). In 2004,
pemetrexed was approved for pleural mesothelioma in the United States
(Hazarika et al., 2004), and subsequently as a second-line treatment for
nonsmall cell lung cancer (Cohen et al., 2005). MTX has found other
clinical applications including treatment of autoimmune diseases and psori-
asis (Chladek et al., 1998; Giannini et al., 1992).
These classical antifolates are all excellent substrates for cellular uptake
by RFC (Goldman and Matherly, 1985; Goldman and Zhao, 2002; Jansen,
1999; Matherly et al., 2007). Membrane transport of antifolates such as
MTX is critical to drug activity because sufficient intracellular drug is
required to sustain suppression of enzyme targets and to support synthesis
of polyglutamates required for high-affinity inhibition of intracellular
enzymes and sustained drug effects as plasma drug levels decline (Goldman
and Matherly, 1985). Not surprisingly, impaired active transport of MTX
has been identified as an important mechanism of MTX resistance (Goldman
and Matherly, 1985; Matherly et al., 2007; Zhao and Goldman, 2003).
Impaired MTX transport was reported as early as 1962 in MTX-resistant
L5178Y mouse leukemia cells (Fischer, 1962). Decreased MTX transport
by RFC has been reported in cultured murine and human tumor cells
selected in vitro with antifolate (in some cases, with prior exposure to
carcinogen) (Drori et al., 2000; Gong et al., 1997; Jansen et al., 1998;
Rothem et al., 2002, 2003; Roy et al., 1998; Sadlish et al., 2000; Schuetz
et al., 1988; Wong et al., 1999; Zhao et al., 1998a,b, 1999) and in vivo in
MTX-resistant murine leukemia cells from mice treated with MTX che-
motherapy (Sirotnak et al., 1981). In 42 primary osteosarcoma samples from
patients who experienced poor responses to chemotherapy including MTX,
65% showed low-level RFC expression (Guo et al., 1999). Similarly, in
primary acute lymphoblastic leukemia, low levels of RFC were associated
with a poor prognosis (Ge et al., 2007; Gorlick et al., 1997; Levy et al.,
The MTX-resistant phenotype is frequently complex and involves
elevated or kinetically altered dihydrofolate reductase, and/or decreased
synthesis of MTX polyglutamates in addition to impaired MTX transport
(Goldman and Matherly, 1985; Zhao and Goldman, 2003). Impaired trans-
port that results in a loss of sensitivity to standard doses of antifolate should,
at least in part, be circumvented by increasing extracellular concentrations
of drug. This forces the drug into tumor cells expressing mutated or low
levels of RFC, and involves alternate uptake routes and/or passive diffusion
to a sufficient extent to inhibit intracellular enzymes and/or to support
antifolate polyglutamate synthesis. However, a point is often achieved for
which these elevated extracellular antifolate concentrations are no longer
capable of significantly increasing intracellular drug levels. This is due to
the saturability of RFC, electrical restrictions on net drug accumulation,
and the presence of high-capacity efflux pumps such as the MRPs.
154 Larry H. Matherly and Zhanjun Hou

Thus, relatively small increases in intracellular target enzymes or decreased

levels of antifolate uptake can result in a requirement for intracellular drug
that cannot be achieved clinically.

V. Functional Properties of RFC

Detailed functional studies of RFC transport date back to the late
1960s (Goldman et al., 1968; Sirotnak et al., 1968) and have included a wide
range of both rodent and human (mostly tumor) cell lines in culture
(Sirotnak, 1985). Whereas folates such as 5-methyl or 5-formyl tetrahydro-
folate (leucovorin) and classical antifolates such as MTX, pemetrexed, and
raltitrexed are all RFC substrates (Fig. 5.1), most functional studies have
used radioactive MTX as a surrogate substrate. This reflects its commercial
availability and efficient unidirectional transport over short intervals (due to
its rapid and tight binding to intracellular dihydrofolate reductase)
(Goldman et al., 1968). Further, in contrast to reduced folates such as
5-formyl tetrahydrofolate, MTX is not appreciably metabolized over the
short intervals used for assaying transport. At steady state, bound intracellular
MTX can be easily distinguished from free intracellular drug by simple
efflux into MTX-free media (Goldman et al., 1968). This permits calcula-
tion of transmembrane gradients and uphill transport from the membrane
For most reduced folate and many antifolate substrates for RFC, uptake
is saturable at low micromolar concentrations (Kt  15 mM) (Goldman and
Matherly, 1985; Jansen, 1999; Matherly and Goldman, 2003; Sirotnak,
1985). Folic acid is generally a poor substrate for RFC (Kt > 100 mM) in
physiological buffers. RFC transport is not stereospecific for 5-methyl
tetrahydrofolate (Sirotnak and Donsbach, 1974; White et al., 1978). Because
leucovorin is racemic mixture of (6R) and (6S ) stereoisomers of 5-formyl
tetrahydrofolate, it is of interest that the (6R) isomer of 5-formyl tetrahy-
drofolate has a far lower affinity than the natural (6S ) stereoisomer (Sirotnak
et al., 1979). A benzoquinazoline antifolate, GW1843U89, is a surprisingly
poor substrate for the murine RFC (Vmax/Kt 0.25) and one of the best
known substrates for the human carrier (Vmax/Kt 20.3) (Duch et al.,
1993). This is the only example of a substantial substrate disparity between
the rodent and human RFCs.
A consistent feature of RFC substrates is their anionic character (Fig. 5.1).
For folates, the glutamate is of particular significance in that its a- and
g-carboxyl groups are ionized at physiological pH, thus limiting diffusion
across biological membranes. For transport by RFC, modifications of the
glutamic acid (e.g., 2-amino-4-phosphonobutanoic acid, L-homocysteic
acid, ornithine) are generally not well tolerated (Westerhof et al., 1995).
Structure and Function of the Reduced Folate Carrier 155

However, modifications of the glutamate g-carboxyl group in the antifo-

lates ZD9331 and PT523 are extremely well tolerated ( Jansen, 1999).
Interestingly, for a series of diamino furo[2,3-d ]pyrimidine antifolates
with substituted (e.g., methyl) a- or g-carboxyl groups, analogues with
only single a-carboxyl and no g-carboxyl were potent inhibitors of MTX
transport by hRFC, whereas analogues with only g-carboxyl group but no
a-carboxyl group were poor inhibitors (Deng et al., 2008). This indicates
that the a-carboxyl group is essential for binding to hRFC.
Transport by RFC is temperature-dependent and sodium-independent
(Goldman and Matherly, 1985; Jansen, 1999; Matherly and Goldman, 2003;
Sirotnak, 1985). Although a neutral pH appears to be optimal for RFC
transport in leukemia cells (Goldman et al., 1968), in prostate carcinoma
(Horne and Reed, 2001) and intestinal epithelial cells (Balamurugan et al.,
2006; Chiao et al., 1997) an acidic pH optimum (pH 5.56.5) for RFC
transport was reported. This low-pH transport activity was accompanied by
altered specificity for certain substrates (e.g., folic acid). Very recent studies
suggest that the low-pH transport of (anti)folate substrates in intestine is
likely due to expression of PCFT rather than RFC (Qiu et al., 2006; Zhao
and Goldman, 2007).
RFC is an anion transporter and thus is highly sensitive to its anionic
environment. For instance, replacing anionic buffers with nonanionic
HEPES-sucrose buffers results in concentrative MTX uptake (Henderson
and Zevely, 1983a). This is due to a decreased competition for binding to
RFC in the absence of anions because MTX influx via RFC is competi-
tively inhibited by inorganic anions such as chloride, bicarbonate, or phos-
phate in physiological buffers. Likewise, transport is inhibited by structurally
diverse organic anions such as adenine nucleotides and thiamine phosphates
(Goldman, 1971a).
In studies with membrane vesicles loaded with sulfate or phosphate anions,
uptake of MTX in the trans compartment was dramatically stimulated via a
counter-transport mechanism (Yang et al., 1984). Similarly, influx of radiola-
beled MTX by RFC is significantly enhanced (trans-stimulated) in cells
preloaded with high concentrations of 5-formyl or 5-methyl tetrahydrofolate
(Goldman, 1971a,b). In anion-free buffers without glucose, the rate of MTX
efflux from cells is inhibited but can be stimulated with both inorganic and
organic anions (e.g., folic acid, 5-formyl tetrahydrofolate, AMP, ADP, thia-
mine pyrophosphate, phosphate, sulfate, and chloride) (Henderson and
Zevely, 1980, 1981, 1983b). The anion concentrations required for half-
maximal stimulation of efflux were similar to their Ki values for inhibition of
influx by RFC.
Thus, large electrochemical anion gradients accelerate the movement
of RFC within the plasma membrane and are likely to provide the
driving force for the concentrative uptake of folate substrates by the carrier.
Most probably, this involves an extrusion of intracellular organic anions into
156 Larry H. Matherly and Zhanjun Hou

the extracellular medium and down a concentration gradient that somehow

drives uptake of folate substrates into cells. Consistent with this model are
recent studies with phosphorylated derivatives of thiamine that show that
these anionic species are good substrates for RFC in murine L1210 cells, and
that efflux of these forms is dramatically enhanced in cells with increased
expression of RFC (Zhao et al., 2001a). However, neither the identification
of the actual physiological counteranion(s), the binding sites for dianionic
folate substrates and the putative transport counteranion(s), nor the mecha-
nism by which the bidirectional fluxes are coupled is firmly established
(see below).
As described above, RFC shows a striking structural homology to
bacterial transporters of the MFS for which crystal structures have recently
been reported [lactose/proton symporter (LacY) (Abramson et al., 2003),
inorganic phosphate/GlpT (Huang et al., 2003), and multidrug transporter
EmrD (Yin et al., 2006)]. By analogy with these bacterial proteins, transport
of folates by RFC into mammalian cells would be expected to involve a
physical movement of the carrier within the plasma membrane accompa-
nied by alternate accessibility of the aqueous substrate-binding cavity to the
intra- and extracellular sides of the plasma membrane. For RFC, as noted
above, this is driven by extrusion of anions down a large (intra- to extracel-
lular) concentration gradient. As described below, recent studies have begun
to explore the three-dimensional (3-D) structure of the hRFC molecule
including identification of substrate-binding residues and the transmem-
brane translocation pathway for anionic folates and antifolates (Hou et al.,
2005, 2006).

VI. Biochemistry of RFC

The low levels of RFC in most tissues and mammalian cell lines for
many years limited its characterization mostly to functional kinetic assays of
folate and antifolate uptake. However, commencing in the early 1980s,
a number of novel biochemical strategies were developed to identify and
study the RFC protein that would subsequently facilitate its cloning and
structural characterization.
Sirotnak et al. (1984) developed a strategy to select L1210 murine
leukemia cells with upregulated RFC. Selection was based on the notion
that carrier-mediated uptake of reduced folate cofactors is rate-limiting to
their utilization in biosynthetic reactions and involved growing cultures
in folate-free culture medium with growth-limiting concentrations of
leucovorin. Under these conditions, only cells that exhibited enhanced
capacities for reduced folate transport were capable of sustained growth.
Structure and Function of the Reduced Folate Carrier 157

Analogous RFC transport-upregulated K562 (Matherly et al., 1991), HL60

(Yang et al., 1992), and CCRF-CEM ( Jansen et al., 1990) leukemia sublines
were generated by selection under folate-limiting growth conditions.
Another key advance involved development of approaches for identify-
ing and quantitating low levels of RFC protein. Specific binding of radi-
olabeled RFC substrates (5-methyl tetrahydrofolate, MTX, or aminopterin)
to surface RFC at 0  C (corresponding to the difference between bound
ligand in the absence and presence of high concentrations of a competing
unlabeled ligand) in intact cells provides an overall estimate of RFC levels
(Henderson et al., 1980a). However, it was not until development of affinity
ligands for covalently modifying the carrier that the molecular characteristics
of the RFC protein could be studied. A number of RFC affinity reagents have
been reported including 8-azidoadenosine-50 -monophosphate (Henderson
et al., 1979), 4,40 -diisothiocyanostilbene-2,20 -disulfonate (Henderson and
Zevely, 1982), carbodiimide-activated antifolates (Henderson et al., 1980b),
3,30 -dithiobissulfosuccinimidyl propionate ( Jansen et al., 1989), N-hydroxy-
succinimide (NHS)-MTX ester (Henderson and Zevely, 1984), and N a-(4-
amino-4-deoxy-10-methylpteroyl)-N e-4-azido-5-salicylyl)-L-lysine (APA-
ASA-Lys) (Freisheim et al., 1992). Whereas treatment of cells with all of
these reagents irreversibly inhibited 3H-MTX uptake by RFC, only APA-
ASA-Lys and NHS-MTX showed the sensitivity and specificity needed for
radioaffinity labeling the carrier.
NHS esters of 3H-MTX and 3H-aminopterin have been used extensively
for covalently labeling RFC (Henderson and Zevely, 1984; Hou et al.,
2005; Matherly et al., 1991; Schuetz et al., 1988; Witt et al., 2004; Yang
et al., 1992) because these inhibitors are simple to prepare from NHS and
commercially available radioactive antifolates. Both of these reagents show
a relatively high specificity for RFC. In studies of transport-upregulated
K562 human erythroleukemia cells (designated K562.4CF) treated with
NHS-3H-MTX, tritium was incorporated into a broadly migrating
7685 kDa band that was increased (7-fold) over parental K562 cells
and could be completely blocked by unlabeled MTX or (6S) 5-formyl
tetrahydrofolate, establishing specificity (Matherly et al., 1991). Additional
experiments confirmed that radioaffinity-labeled hRFC protein was glyco-
sylated because treatment of K562.4CF plasma membranes with endo-
b-galactosidase resulted in a shift to a substantially lower molecular mass
(58 kDa). The mouse RFC from L1210 leukemia cells treated with
NHS-3H-MTX or NHS-3H-aminopterin typically migrated on SDS gels
or gel filtration (with 0.1% SDS) as a 4248 kDa species (Schuetz et al.,
1988; Yang et al., 1992). However, more recent studies with antibody to
the mouse RFC identified a 58 kDa RFC protein (Zhao et al., 2000b),
suggesting that the smaller molecular mass species must have arisen
from proteolytic degradation of a larger RFC form. A very recent study
with a series of classical diamino furo [2,3-d ] pyrimidine analogues with
158 Larry H. Matherly and Zhanjun Hou

methyl-substituted a- and g-carboxyl groups established that the g-carboxyl

NHS ester of antifolate substrates is far more reactive with nucleophilic
amino acid(s) than is the a-carboxyl NHS ester (Deng et al., 2008). This is
interesting because the a-carboxyl group rather than the g-carboxyl group is
essential for high-affinity substrate binding to hRFC (Deng et al., 2008).
APA-125I-ASA-Lys is a radioiodinated photoaffinity ligand originally
used for labeling dihydrofolate reductase (Price et al., 1986) that was
subsequently adapted for labeling RFC (Freisheim et al., 1992; Wong
et al., 1995). Ultraviolet activation of a reactive nitrene in APA-125I-ASA-
Lys results in a covalent modification of proteins to which it is bound. The
advantages of APA-125I-ASA-Lys over NHS-3H-MTX include its
increased specificity for MTX-binding proteins, resulting from its decreased
reactivity in the absence of ultraviolet irradiation, and its greater sensitivity,
reflecting the incorporation of the 125I radionuclide rather tritium.
Freisheim et al. (1992) also used APA-125I-ASA-Lys to label 8085 kDa
glycosylated hRFC protein from transport-upregulated CCRF-CEM cells
(Freisheim et al., 1992).

VII. Cloning of RFC cDNAs That Restore

Transport to Transport-Impaired
Cultured Cells
In 1994, a mouse RFC cDNA was isolated by expression cloning and
found to restore MTX transport activity and sensitivity to transport-
impaired ZR75-1 human breast cancer cells (Dixon et al., 1994). This was
followed by a report from Flintoff and coworkers that a homologous
hamster cDNA could restore MTX transport to transport-impaired Chinese
hamster ovary (CHO) cells (Williams et al., 1994). By early 1995, there
were four published reports on the characteristics of the homologous
human cDNAs (Moscow et al., 1995; Prasad et al., 1995; Williams and
Flintoff, 1995; Wong et al., 1995). An identical cDNA from human intestine
was reported in 1997 (Nguyen et al., 1997).
These homologous cDNAs consistently restored in vitro antifolate sensi-
tivities and transport properties typical of the endogenously expressed RFCs
to transport-impaired cell lines. These include characteristic uptake patterns
of radioactive folate and antifolate substrates, inhibition by known substrate
competitors (e.g., 5-formyl tetrahydrofolate, raltitrexed), irreversible inhibi-
tion by NHS-MTX, and a capacity for trans-stimulation by preloading with
reduced folates (Wong et al., 1995, 1997). For transport-impaired CHO or
K562 cells transfected with hRFC cDNAs, a cDNA-encoded 8592 kDa
protein was detected by photoaffinity labeling with APA-125I-ASA-Lys
Structure and Function of the Reduced Folate Carrier 159

( Wong et al., 1995, 1997) or antibodies raised to a peptide sequence

predicted from the hRFC cDNA sequence (Wong et al., 1998). This
species was deglycosylated with N-glycosidase F to 65 kDa, in close
agreement with the predicted size of the full-length hRFC protein
(64,873 Da) and the size of the deglycosylated NHS-3H-MTX-labeled
hRFC protein originally identified in transport-upregulated K562.4CF
cells (Matherly et al., 1991).
By hydropathy analysis of the predicted amino acid sequence for hRFC,
the hRFC cDNA encoded a protein that conformed to a model expected
for an integral membrane protein, with up to 12 stretches of mostly
hydrophobic, a-helix-promoting amino acids, internally oriented N- and
C-termini, an external N-glycosylation site at Asn58, and a large central
loop domain connecting transmembrane domains (TMDs) 16 and 712
(Fig. 5.2). Much of this topology structure has been confirmed experimen-
tally (Cao and Matherly, 2004; Ferguson and Flintoff, 1999; Flintoff et al.,
2003; Liu and Matherly, 2002) (see below). The predicted amino acid
sequence is reasonably conserved between species ranging from Xenopus
laevis to mice and humans, with the highest homologies in the TMDs
(Fig. 5.3). Sequence homology is substantially decreased in the most N- or
C-terminal regions and in the TMD6/TMD7 connecting loop domain. The
RFC sequence for primates (humans, chimpanzee) includes 5086 more
amino acids than that for the other species (Fig. 5.3).
Glycosylation at Asn58 is responsible for the aberrant migration of
hRFC on SDS protein gels (Wong et al., 1998). However, N-glycosylation
is not essential for RFC function because the murine RFC is not glycosylated.
A transport competent 65 kDa hRFC protein was identified by Western
blots prepared from hRFC-null K562 cells transfected with an hRFC cDNA
in which Asn58 was replaced by glutamine (Wong et al., 1998). When tagged
with a C-terminal hemagglutinin (HA) epitope (YPYDVPDYASL), Gln58
hRFC transport function was largely preserved, accompanying efficient
plasma membrane targeting (Wong et al., 1998). These results suggest that
the N-glycosylation of hRFC does not significantly alter either membrane
targeting or transport function.

VIII. Topological Structure of RFC

A. Topological structure by HA epitope accessibility
and N-glycosylation scanning mutagenesis
The original report by Cowan and coworkers (Dixon et al., 1994) suggested
that the mouse RFC amino acid sequence conformed to a 12 TMD
structure, closely resembling that for other MFS proteins such as GLUT1
(Salas-Burgos et al., 2004). The demonstration of N-glycosylation at Asn58

K N N58
S N Extracellular F
R V H T R H P 360
40 Q 73 S L 117 A 123 E 178 G 186 N 288 V 310 D 355 H S 418 V S 436
W 1 L 2 L 3 V 4 V 5 L 6 P 7 V 8 L 9 F 10 L 11 L 12
24 S 91 R V 95 R 145 A 159 K 204 R 264 K 328 L 337 L 379 E 394 R 456
Cytosol Y Y R D G L E R L A S S R
P S S P V M1 F P L L H
591 Q N V N

Figure 5.2 Topological model for hRFC showing conserved residues between seven species. Topology model for hRFC, depicting 12
TMDs, internally oriented N- and C-termini, an externally oriented N-glycosylation site at Asn-58, and a cytosolic loop connecting TMDs 6
and 7. Amino acids conserved between RFCs from different species as summarized in Fig. 5.3 are depicted as black circles.
Human -------------------- -------------------- ------MVPSSPAVEKQVPV EPGPDPEL-RSWRRLVCYLC FYGFMAQIRPGESFITPYLL 53
Chimpanzee -------------------- -------------------- ------MVPSSPAVEKQVPV EPGPDPEL-RSWRRLVCYLC FYGFMAQIRPGESFITPYLL 53
Cattle -------------------- -------------------- ------MALSVPEVEKQMPA EPQPGHEQ-QSWWCLVFFLC FYGFMAQMRPGESFITPYLL 53
Mouse -------------------- -------------------- ------MVPTGQVAEKQAYE EPRQDHEL-KSWRCLVFYLC FFGFMAQLRPGESFITPFLL 53
Rat -------------------- -------------------- ------MVPTGQVAEKQACE EPRQDREL-KSWRWLVFYLC FFGFMAQLRPGESFITPYLL 53
Hamster -------------------- -------------------- ------MVPTGQVAEKQACE EPRQDREL-KSWRCLVFYLC FFGFMAQLRPGESFITPYLL 53







Figure 5.3 Species homologies for RFC proteins. GenBank accession numbers are Homo sapiens (human) NP 001069921; Pan troglodytes
(chimpanzee) XP 001157360; Gallus gallus (chicken,) NP 001006513; Danio rerio (zebrafish) XP 687261; Bos taurus (cow)NP 001069921;

Rattus norvegicus (Norway rat) NP 001030309; Cricetulus griseus (Chinese hamster) U17566; Mus musculus (mouse) NP 112473; Xenopus laevis
(African clawed frog) NM 001092530.


Xenopus LAVLYLGAAAFVIIKHYHAE RLKEK--PQIPPKVESEHKT TS------------------ ---SSAVTCESRA------- -------------------- 516
Zebrafish LTVIYLGCSAFIITCHYRNQ RAGAESTEEAVSTELSPIAT AA------------------ ---SESTTVHNGTSIKT--- -------------------- 528
chicken LALVYLLMAMVVIVRHSRRA QP-----PELIPTEG-QVQE KS------------------ ---PEAATVQA--------- --------------------


Cattle -------------------- -------------------- ----------
Mouse -------------------- -------------------- ----------
Rat -------------------- -------------------- ----------
Hamster -------------------- -------------------- ----------
Xenopus -------------------- -------------------- ----------
Zebrafish -------------------- -------------------- ----------
chicken -------------------- -------------------- ----------

Figure 5.3 (continued)

Structure and Function of the Reduced Folate Carrier 163

for hRFC provided direct confirmation of an extracellular orientation for

the loop domain connecting TMDs 1 and 2 (Fig. 5.2). HA epitope accessi-
bility methods, in which HA epitopes were inserted into the hRFC mole-
cule followed by immunofluorescence detection with HA-specific antibody
in the presence and absence of low levels of Triton X-100 (0.1%) were used
to map the hRFC topological structure (Ferguson and Flintoff, 1999; Liu
and Matherly, 2002). In these studies, intracellular orientations were
confirmed by inserting HA epitopes into the hRFC N-terminus (Pro20,
Gly17), the connecting loop between TMDs 6 and 7 (Ser225, Glu226), and
TMDs 8 and 9 (Ala332). Because HA epitopes in the connecting loops
between TMDs 3 and 4 (Gln120) and between TMDs 7 and 8 (Glu294,
Pro297) were accessible to antibody without permeabilization, these likely
had extracellular orientations. By N-glycosylation scanning mutagenesis, in
which an N-glycosylation consensus sequence [NX(S/T)] was inserted into
putative loop domains, followed by Western blotting of functional constructs
to confirm glycosylation status, the TMD 5/6 loop of hRFC was confirmed
as having an extracellular orientation (Liu and Matherly, 2002).

B. Topological structure by scanning cysteine

accessibility methods
The development of sulfhydryl reagents with reactivities amenable to use
with intact cells, typified by the alkylthiosulfonates and maleimides, has
revolutionized the structural analysis of membrane-spanning ion channels
and membrane transporters (Frillingos et al., 1998; Karlin and Akabas,
1998). Thus, by inserting cysteine residues into functional cysteine-less
membrane proteins and treating with cysteine-active reagents, it is possible
to establish aqueous accessibilities and, by inference, determine membrane
topologies (Hu and Kaplan, 2000; Loo and Clarke, 1995; Nicoll et al.,
1999), identify amino acids that line the transmembrane translocation
pathway (Dodd and Christie, 2001; Loo and Clarke, 2000; Slotboom
et al., 2001), and confirm the spatial relationships between domains
(Kwaw et al., 2001; Loo and Clarke, 2001; Zeng et al., 1999).
In hRFC, there are 11 cysteine residues. A functional cysteine-less
hRFC was generated by deleting 56 C-terminal amino acids including
4 cysteines (Cys546, 549, 563, and 580) and replacing the remaining 7 cysteines
(Cys30, 33, 220, 246, 365, 396, and 458) with serines (Cao and Matherly,
2003). A Myc-His6 epitope was added to the truncated C-terminus and single-
cysteine hRFC mutants, including Ser301Cys (TMD7/8 loop), Ala332Cys
(TMD8/9), Ser360Cys (TMD9/10), Ala388Cys (TMD10/11), Ser390Cys
(TMD10/11), and Arg429Cys (TMD11/12) hRFCs, were expressed in
RFC-null CHO cells (Cao and Matherly, 2004). Cells were treated
with thiol-reactive biotin maleimide [3-(N-maleimidylpropionyl)biocytin]
with or without membrane-impermeant stilbenedisulfonate maleimide
164 Larry H. Matherly and Zhanjun Hou

(4-acetamido-40 -maleimidylstilbene-2,20 -disulfonic acid). Plasma membrane

proteins were solubilized, the hRFC-Myc-His6 immunoprecipitated, and the
immunoprecipitates analyzed on Western blots. Biotinylated thiols were
detected with peroxidase-linked streptavidin. Consistent with their predicted
extracellular orientations (Fig. 5.2), Ser301Cys, Ser360Cys, and Arg429Cys
hRFC mutants were all highly reactive with biotin maleimide and labeling was
significantly blocked with membrane-impermeant stilbenedisulfonate malei-
mide. Whereas Ala388Cys and Ser390Cys mutants, located in the middle of
the putative conserved TMD1011 loop domain, were completely unreactive
toward biotin maleimide, these positions (as well as Ala332Cys) were all labeled
following permeabilization with Streptolysin O. An identical strategy was used
by Flintoff et al. (2003) for hamster RFC to localize the connecting loops
between TMD1/2 (Ser46), TMD4/5 (Ser152), TMD5/6 (Ser179), TMD6/7
(Cys224), TMD7/8 (Leu300), TMD 9/10 (Tyr355), and TMD11/12
(Lys430), and the C-terminal domain (Leu475).
Thus, the patterns of biotin maleimide reactivity and protection
by stilbenedisulfonate maleimide, combined with previous findings of N-gly-
cosylation at Asn58 and the results of HA epitope insertion and scanning
glycosylation mutagenesis (see above), strongly support a 12 TMD topology
structure for hRFC with cytosolic orientations for the N- and C-termini and
TMD6/7 loop domain (Fig. 5.2).

IX. Insights into Structural and Functional

Determinants of RFC from Studies of Mutant
RFC Proteins
An important goal of RFC structurefunction studies has been to
identify amino acids and/or domains that contribute to binding and/or
translocation of folate and antifolate substrates, along with TMD helix
packing associations that facilitate folate substrate binding and translocation.
For RFC, experimental approaches have included: (1) identifying mutant
amino acids in RFC from drug-resistant cells selected with antifolate drugs;
(2) targeting specific amino acids for site-directed mutagenesis based on
amino acid charge or hydrophilic character, along with homology and
membrane topology considerations; (3) deletion and insertion mutagenesis
of potentially functional domains; (4) radioaffinity labeling with specific
radiolabeled affinity inhibitors and identification of the labeled domain/
amino acid by selective proteolysis and site-directed mutagenesis; (5) scan-
ning cysteine accessibility methods (SCAM) with thiol reactive reagents;
and (6) protein cross-linking of cysteine-insertion hRFC mutants with
homobifunctional thiol-reactive cross-linkers. These studies are described
in the following sections.
Structure and Function of the Reduced Folate Carrier 165

A. Identification of structurally or functionally important

amino acids by selecting for mutant RFCs with
antifolate inhibitors
The largest concentration of RFC mutant studies has involved residues
localized in or around TMD1, following initial reports of MTX-resistant
mutants with defective RFC including Glu45Lys (Zhao et al., 1998a)
and Ser46Asn (Zhao et al., 1998b) (unless specifically noted, all numbers
designate the positions in hRFC).
In the hRFC structure, Glu45 flanks TMD1 and is highly conserved
among different species (Fig. 5.3). The first description of the Glu45Lys
mutant was in MTX-resistant L1210 cells treated with N-methyl-N-nitro-
sourea and selected in the presence of MTX (Zhao et al., 1998a). This
Glu45Lys-RFC phenotype included a global decline in carrier mobility,
decreased Kts for folic acid and 5-formyl tetrahydrofolate, respectively, an
unchanged Kt for 5-methyl tetrahydrofolate, and a markedly increased Kt for
MTX. Virtually identical transport phenotypes were attributed to Glu45Lys
hRFC in separate CCRF-CEM sublines selected for MTX resistance (Gifford
et al., 2002; Jansen et al., 1998) and in a number of CCRF-CEM sublines
selected for resistance to the benzoquinazoline antifolate, GW1843U89
(Drori et al., 2000). When Glu45 was systematically mutated by site-directed
mutagenesis, all position 45 mutants were functional for MTX uptake; how-
ever, there were substantial differences in maximal transport rates (Vmax) for
different substitutions. Increased affinities were measured for 5-formyl tetra-
hydrofolate and folic acid, accompanying some amino acid replacements (Gln,
Arg) but not others (Asp, Leu, Trp) (Zhao et al., 2000c). This implies that
amino acid size rather than the charge at position 45 of RFC is the most
important determinant of RFC substrate specificity. For certain substrates such
as 5-formyl tetrahydrofolate, MTX, or folic acid, adverse effects of particular
amino acid replacements on binding affinities were disproportionate to those
seen when 5-methyl tetrahydrofolate was used as transport substrate. Although
these mutagenesis data do not convincingly argue for a direct role of Glu45 in
substrate binding, they, nonetheless, imply that position 45 somehow plays a
role in transport. The role of the exofacial stretch of amino acids flanking
TMD1 and including Glu45 is further considered below.
Ser46 was also suggested to be critical to RFC function because replace-
ment of this amino acid with asparagine in MTX-resistant L1210 cells
resulted in a decreased rate of carrier mobility (Vmax) with MTX but no
change in MTX Kt (Zhao et al., 1998b). The Vmax effect was substantially
greater for MTX than for reduced folates such as 5-formyl tetrahydrofolate
or 5-methyl tetrahydrofolate. Further evidence of a functionally important
role of Ser46 involves detection of Ser46Ile in CCRF-CEM cells selected
for resistance to the benzoquinazoline antifolate GW1843U89 (Drori et al.,
2000) and of Ser46Asn in a primary osteosarcoma specimen treated with
MTX (Flintoff et al., 2004; Yang et al., 2002).
166 Larry H. Matherly and Zhanjun Hou

Gly44Arg was identified in MTX-resistant CCRF-CEM T-cell acute

lymphoblastic leukemia cells by Wong et al. (1999) and was associated with
an 11-fold increased Kt for MTX. The same mutation was reported in a
separate CCRF-CEM subline selected for resistance to the hemiphthaloyl
ornithine antifolate, PT523 (Drori et al., 2000). An Ile48Phe mutation was
detected (in combination with Trp105) in mouse RFC from L1210 cells
selected for resistance to 5,10-dideazatetrahydrofolate (Tse et al., 1998).
Ile48Phe was associated with a marked increase in the accumulation of
folic acid, due to a selective decrease in the Kt for folic acid compared with
5,10-dideazatetrahydrofolate, resulting in an expansion of cellular folate pools.
Mutant studies in murine cells have implicated amino acids localized to
other TMDs as functionally or structurally important to RFC function.
These include Ser309 (Ser313 in hRFC) and Ser297 (not conserved in
hRFC) in TMD8 (Roy et al., 1998; Zhao et al., 1999), Val104 (Val106 in
hRFC) (Zhao et al., 2000a) and Trp105 (Trp107 in hRFC) (Tse et al.,
1998) in TMD3, and Ala130 (Ala132 in hRFC) (Brigle et al., 1995) in
TMD4. In hRFC, Ser127 in TMD4 was implicated as functionally
important ( Wong et al., 1999). The impact of these mutations ranges
from effects on carrier mobility without an effect on the Kt for MTX
binding (Ala130Pro in mouse RFC) (Brigle et al., 1995) to decreases in
both Kt and Vmax for MTX (Ser127Asn in hRFC) ( Wong et al., 1999).
For certain mutants (Trp105Gly and Ser309Phe in mouse RFC), different
transport phenotypes were seen for different transport substrates (Tse et al.,
1998; Zhao et al., 2000a), similar to findings for Ser46Asn and Ile48Phe
(see above). For instance, Trp105Gly increased transport of folic acid
compared with 5,10-dideazatetrahydrofolate by mouse RFC. Ser309Phe
in mouse RFC resulted in an increased (5-fold) Kt for MTX and
5-formyl tetrahydrofolate without a significant change in the affinities for
folic acid and 5-methyl tetrahydrofolate.

B. Identification of structurally or functionally important

amino acids in RFC by homology comparisons and
site-directed mutagenesis
From membrane topology and comparisons of homologies between hRFC
and RFCs from other species, a number of highly conserved charged amino
acids are found to map within the TMDs (Figs. 5.2 and 5.3). These include
Asp88 (TMD2), Arg133 (TMD4), Arg373 (TMD10), Lys411 (TMD11),
and Asp453 (TMD12). While replacement of Asp88 in hRFC with gluta-
mate partially preserved transport activity for both MTX and 5-formyl
tetrahydrofolate, substitution with valine abolished activity. Conversely,
valine replacement of Asp453 had only a small effect on carrier activity
(Liu and Matherly, 2001). For murine RFC, transport activity was abolished
by replacement of conserved Arg131 (Arg133 in hRFC) and Arg363
Structure and Function of the Reduced Folate Carrier 167

(Arg373 in hRFC) residues with leucine (Sharina et al., 2001). Similar

results were obtained with aliphatic amino acid substitutions at Arg 133
and Arg373 in hRFC (Deng et al., 2007; Liu and Matherly, 2001). With
hamster RFC, Arg373 was suggested to be functionally important because
systematic replacement at this position with lysine, histidine, glutamine, or
alanine progressively decreased the capacity of the position 373 mutant
RFCs to complement a transport defective hamster phenotype in support-
ing colony formation in the presence of low levels of 5-formyl tetrahydro-
folate (Sadlish et al., 2002b). In direct transport assays, these substitutions
had a much more pronounced adverse effect on carrier translocation (Vmax)
than on substrate binding and also decreased RFC stabilities and intracellular
trafficking. Replacement of Lys404 in mouse RFC with leucine resulted in
a selective loss of binding and transport of reduced folates over MTX
(Sharina et al., 2001). For hRFC, both conservative and non-conservative
substitutions at Lys411 were tolerated; however, Lys411Glu hRFC showed
a substantial decrease in drug uptake (Deng et al., 2008).
Thus, a negative charge at position 88 and positive charges at positions
133 and 373 are essential for high levels of MTX transport by RFC. While
the situation for Lys411 is more complex and differs with different transport
substrates, it nonetheless appears that Lys411 is at least somewhat important
for binding hRFC substrates. As described below, very recent results suggest
that Lys411 in TMD11 of hRFC is the major target for covalent modifica-
tion by NHS-MTX (Deng et al., 2008), consistent with a role of this
conserved cationic amino acid in binding to the carboxyl group(s) of folate
substrates. Finally, recent site-directed mutagenesis results for Ser313
(TMD8), Tyr136 (TMD4), and Tyr281 (TMD7) in hRFC (Hou et al.,
2006) imply critical structural or functional roles of these residues. As
described above, from the results of RFC mutant studies, important roles
for Ser313 and a large portion of TMD4 are likewise suggested.

C. Deletional and insertional mutagenesis of RFC

Another useful strategy to identify and characterize functionally important
domains in RFC has involved deletional mutagenesis. Thus, deletion of
27 N-terminal amino acids (residues 127) from hRFC (Marchant et al.,
2002) or removal of 16 residues (amino acids 722) from hamster RFC
(Sadlish et al., 2002a) had at most minor effects on membrane targeting or
transport function. Deletion of 58 C-terminal residues from hamster RFC
(residues 461518) (Sadlish et al., 2002a) or 139 C-terminal residues from
hRFC (residues 453591) (Marchant et al., 2002) had a slight effect on
transport function and/or surface targeting; for murine RFC, loss of the
C-terminus (residues 445512) resulted in a complete loss of membrane target-
ing (Sharina et al., 2002). As expected, larger deletions (e.g., 302591, 1301),
including entire TMDs, completely abolished plasma membrane targeting of
168 Larry H. Matherly and Zhanjun Hou

hRFC (Marchant et al., 2002). Collectively, these studies argue that neither
the cytosolic facing N- nor C-terminus is directly involved in substrate
binding and that, in general, they only slightly influence membrane targeting
and insertion of the carrier.
A prominent feature of the MFS family of transporters involves a con-
necting loop between TMDs 6 and 7 (Fig. 5.2). Both deletion and insertion
mutagenesis strategies have been used to explore the functional and struc-
tural role of this region in RFC. Thus, deletion of 31 of the 66 amino acids
from the TMD6/TMD7 connecting loop in murine RFC (Sharina et al.,
2002) or deletion of up to 45 of the 67 amino acids in the TMD6/TMD7
loop domain from hamster RFC (Sadlish et al., 2002a) preserved membrane
targeting and transport activity. However, larger deletions in the TMD6/
TMD7 linker domain (57 and 5355 amino acids, respectively) abolished
transport activity.
While deletions of 49 or 60 amino acids from the TMD6/TMD7 linker
of hRFC (amino acids 215263 and 204263, respectively) completely
ablated transport of MTX and 5-formyl tetrahydrofolate, replacement of
the deleted segments with nonhomologous 73 or 84 amino acid segments
from another MFS protein, SLC19A2 (transports thiamine; 18% homolo-
gous to hRFC for the TMD6/TMD7 linker region), restored transport (Liu
et al., 2003). Interestingly, maximal transport activities for these insertional
mutants were absolutely dependent on the presence of the highly conserved
204214 peptide and deletion of the 204214 segment alone completely
abolished transport (Liu et al., 2003).
Thus, the primary purpose of the TMD6/TMD7 linker domain is to
ensure the optimal spacing between the two halves of the RFC protein for
carrier function. This appears to be essentially independent of amino acid
sequence, although an important role for amino acids 204214 is implied.
Most recently, TMD16 and TMD712 hRFC half molecules were
cotransfected into hRFC-null K562 cells (Witt et al., 2004). Coexpressed
hRFC half molecules were targeted to the membrane surface where they
restored transport activity with normal kinetics, showed sensitivity to inhi-
bition by NHS-MTX, and exhibited a capacity for trans-stimulation by
preloading with 5-formyl tetrahydrofolate (Witt et al., 2004).

D. Localization of a substrate-binding domain

by radioaffinity labeling
The ability to restore functional RFC transport by coexpression of hRFC
half molecules provided a unique tool to localize substrate-binding
domains. Thus, coexpression and NHS-3H-MTX radioaffinity labeling of
hRFC TMD16 and TMD712 half molecules localized covalent labeling
to TMD712 (Witt et al., 2004). Treatment of radioaffinity-labeled
TMD712 with 2-nitro-5-thiocyanato benzoic acid cleaved adjacent to
Structure and Function of the Reduced Folate Carrier 169

cysteine residues and localized binding of the radioligand to between amino

acids 394 and 457, corresponding to TMDs 11 and 12 (Hou et al., 2005).
More recent results localized affinity labeling of NHS-MTX to Lys411 in
TMD11 because Lys411Ala could effectively transport MTX yet abolished
MTX transport inhibition by unlabeled NHS-MTX and covalent incor-
poration from NHS-3H-MTX (Deng et al., 2008). While this directly
implicates Lys411 in carboxyl binding for RFC substrates, its role is para-
doxical because Lys411 in hRFC could be replaced by nonconservative
amino acid substitutions with comparatively modest effects on transport
(Deng et al., 2008; Witt and Matherly, 2002).

E. SCAM for mapping the substrate translocation pathway

The availability of a functional cysteine-less hRFC (Cao and Matherly,
2003) permitted corroboration of mutagenesis and affinity labeling results
by SCAM, which identified amino acids that were aqueous accessible and
were likely involved in forming the putative membrane translocation path-
way for anionic folate substrates. Thus, for hRFC, 282 cysteines were
individually inserted into TMDs 112 of a cysteine-less hRFC template
and hRFC cysteine mutants were expressed in hRFC-null HeLa cells (Hou
et al., 2005, 2006). Altogether, 272 of the 282 single cysteine mutants were
functional for MTX transport, the only exceptions being Arg133, Ile134,
Ala135, Tyr136, and Ser138 in TMD4; Gly163 in TMD5; Tyr281 in
TMD7; Ser313 in TMD8; Arg373 in TMD10; and Gly401 in TMD11.
For the 272 functional mutants, aqueous accessibilities of the cysteine
insertions were confirmed by monitoring losses of transport activity and
the protective effects of substrate (i.e., leucovorin) in the presence of the
membrane-impermeable hydrophilic sodium (2-sulphonatoethyl) metha-
nethiosulfonate (MTSES). The results of these studies strongly supported a
role for amino acids localized to TMDs 4, 5, 7, 8, 10, and 11 in forming the
putative substrate-binding pocket of hRFC, in excellent agreement with
the results of RFC mutant studies.
Exofacial residues flanking TMD1 including positions 40, 44, and 48
(but not 45 or 46), corresponding to a region suggested to be functionally
important from mutant studies (see above), were likewise implicated as
involved in substrate binding by earlier SCAM experiments with MTSES
and cysteine insertion hRFC mutants expressed in transport defective CHO
cells (Cao and Matherly, 2003). Similarly, biotinylation by biotin maleimide
of cysteines inserted at positions 41, 46, 70, and 71, including the exofacial
spanning region connecting TMDs 1 and 2 in hamster RFC, was prevented
by prior treatment with MTX or leucovorin, suggesting that these sites form
part of a substrate-binding domain (Flintoff et al., 2003).
A three dimensional model based in part on SCAM biochemical data for
hRFC is shown in Fig. 5.4 that depicts TMDs 1, 2, 4, 5, 7, 8, 10, and 11 as
170 Larry H. Matherly and Zhanjun Hou

B 2

7 1 3

8 5

Extracellular TMD7 TMD8

5.7 Ser313

K411 Tyr281

TMD11 Arg373

Tyr136 Ile134


Cytosol TMD4

Figure 5.4 Proposed 3-D models of hRFC based on solved crystal structures of LacY
and GlpT and SCAM analysis, and the hypothesized substrate-binding site of hRFC.
A 3-D hypothetical model for hRFC is presented based on structure alignments
between hRFC and LacY and GlpT and fine-tuned based on experimental SCAM
data. Modeling was performed with the Modeller 8v1 auto mode (Marti-Renom
et al., 2000). All models were drawn by PyMol (DeLano, 2002). Panel A depicts a
side view of hRFC for which the extended C-terminal segment is truncated at Lys479.
TMDs 1, 2, 4, and 5 of the N-terminal region and TMDs 7, 8, 10, and 11 of the
C-terminal region are hypothesized to be involved in the formation of the hydrophilic
Structure and Function of the Reduced Folate Carrier 171

components of the putative aqueous membrane-spanning translocation

pathway flanked by TMDs 3, 6, 9, and 12. These results are consistent
with the published crystal structures of the LacY and GlpT MFS homo-
logues (Abramson et al., 2003; Huang et al., 2003). Based on data showing
the nearly complete ablation of transport activity upon cysteine substitutions
into cysteine-less hRFC, it appears that a number of amino acids are
structurally or functionally important including Arg133, Ile134, Ala135,
Tyr136, and Ser138 in TMD4; Tyr281 in TMD7; Ser313 in TMD8; and
Arg373 in TMD10. A functionally important role was also suggested for
Lys411 in TMD11 (Deng et al., 2008; Sharina et al., 2001; Witt and
Matherly, 2002). Studies with the activated NHS-MTX ester established
covalent modification at this site, thus implicating Lys411 in binding the
glutamate portion of folate substrates (Deng et al., 2008).
Notably, all these amino acids are highly conserved between species as
diverse as Xenopus, zebrafish, mice, humans, and cattle (Fig. 5.3). Compel-
ling evidence from RFC mutant studies (see above) established that Ser313
and Arg373 are functionally important and may contribute to substrate-
binding specificity (Deng et al., 2007; Hou et al., 2006; Sadlish et al., 2002b;
Sharina et al., 2001; Zhao et al., 1999). Lys411, Ser313, and Arg373 may
easily comprise a hydrophilic binding pocket for anionic folate substrates
(Hou et al., 2006) (Fig. 5.4). Reflecting its juxtaposition to both Ser313 and
Arg373 in this model, Tyr281 may also participate in substrate binding. The
finding that individual cysteine replacements of Arg133, Ile134, Ala135,
Tyr136, and Ser138 abolished transport activity is entirely consistent with
previous reports that functionally important amino acids (Ser127, Ala132,
and Arg133) were localized to TMD4 (Brigle et al., 1995; Liu and Matherly,
2001; Sharina et al., 2001; Wong et al., 1999).
The role of the highly conserved residues at positions 40, 44, 45, 46, and
48 in RFC transport remains a paradox. Whereas mutant studies suggested a
possible functional importance for amino acids located at positions 44, 45, 46,
and 48 in hRFC (Tse et al., 1998; Wong et al., 1999; Zhao et al., 1998a,b),
only positions 40, 44, and 48 were implicated as contributing to a substrate-
binding domain by SCAM (Cao and Matherly, 2003). Similarly, in hamster
RFC, positions 41, 46, and 49 flanking TMD1 along with positions 70 and

cavity for anionic substrate binding (colored as black). TMDs 3, 6, 9, and 12 are likely
buried in the lipid bilayer and do not directly participate in substrate binding (colored as
gray). Panel B depicts a cytosolic view of only the TMD segments (numbered 112 as in
Fig. 5.2) of the hRFC molecule so that the order of helix packing can be easily seen.
TMD shading is the same as in Panel A. Panel C shows an enhanced view of the
hypothetical substrate-binding site, including Lys411, Ser313, Tyr281, and Arg373, as
described in the text. Other residues that may contribute to the substrate-binding
pocket are also shown and include Arg133, Ile134, Ala135, Tyr136, and Ser138. The
physical distances between a-carboxyl groups of Lys411, Ser313, Tyr281, and Arg373
are shown in Angstrom. Adapted from Hou et al. (2006).
172 Larry H. Matherly and Zhanjun Hou

71 in or flanking TMD2 were implicated in substrate binding by the ability

of RFC substrate to protect from the inhibitory effects of biotin maleimide
(Flintoff et al., 2003).

F. Mapping helix packing associations in hRFC

Characterizing conserved charged residues in or flanking TMDs by mutant
analysis can shed light on tertiary structural elements in membrane proteins
including interactions between distal domains. Interpretation is in part based
on the notion of an energetic unfavorability associated with uncompensated
charged amino acids localized within the lipid bilayer. However, should
there be a salt bridge between residues of opposite charge, charged amino
acids localized to hydrophobic environments can be substantially stabilized
(Barril et al., 1998). Salt bridges between oppositely charged residues in
separate domains can serve to orient TMDs for membrane insertion and/or
for optimal transport function (Dunten et al., 1993; Merickel et al., 1997).
For hRFC, neutralization of the positive charge on Arg133 (TMD4) by
substitution with leucine or the negative charge on Asp88 (TMD2) by
replacement with valine abolished transport activity (Liu and Matherly,
2001). However, when both mutations were present in the same construct
(i.e., Asp88Leu/Arg133Val), transport activity was restored. This suggests
that disruption of the charge-pair by replacing either Arg133 or Asp88,
individually, with a neutral amino acid results in an unstable, unpaired
charge. However, simultaneous neutralization of both charged amino
acids results in a restoration of high levels of transport activity. These results
strongly imply that Arg133 and Asp88 form a salt bridge complex that
stabilizes the association between TMDs 2 and 4 in the hRFC tertiary
structure. Other reports have also explored RFC tertiary structure at the
level of putative charge-pairs. For murine RFC, a structural or functional
interaction between Glu45 (flanks TMD1) and Lys404 (TMD11) was
suggested because the properties of the double Glu45Lys/Lys404Glu
murine RFC mutant more closely resembled the properties of the Glu45Lys
mutant than those for the Lys404Glu mutant (Zhao et al., 2003). Further,
a cross-linking analysis of hamster RFC suggested that Arg373 (in TMD10)
is in proximity to Glu394 (flanks TMD11), implying juxtaposition of these
domains and the possible formation of a charge-pair (Sadlish et al., 2002b).
Yu et al. (1995) have described a method for assessing TMD helix
packing in polytopic membrane proteins based on disulfide formation
between paired cysteine residues in purified segments of the visual pigment
rhodopsin. This method was subsequently adapted to assess helix proximities
and tilts of the bacterial MFS transporter LacY (Kaback and Wu, 1999).
For hRFC, in situ site-directed thiol cross-linking was applied to study
the proximities and tilts of neighboring transmembrane helices 2, 5, 8, and 11
(Z. Hou and L. H. Matherly, manuscript in preparation), based on their
Structure and Function of the Reduced Folate Carrier 173

proposed orientations toward the putative hRFC hydrophilic cavity and their
relative proximities in 3-D models (Hou et al., 2006; Matherly et al., 2007;
Fig. 5.4). As described above, Ser313 in TMD8 was previously implicated in
the binding of antifolate substrates for RFC (Deng et al., 2007; Zhao et al.,
1999) and was identified as irreplaceable by scanning cysteine mutagenesis
(Hou et al., 2006). TMD8 abuts TMD5 and, by SCAM, both these regions
are aqueous-accessible and contribute to the substrate-binding pocket in
hRFC (Hou et al., 2006). TMD11 includes Lys411, the primary target for
covalent radioaffinity labeling with NHS-3H-MTX (Deng et al., 2008), and is
aqueous-accessible by SCAM (Hou et al., 2005). Finally, TMD2 flanks
TMD11 and, by homology with LacY (Abramson et al., 2003), lines the
substrate translocation pathway and includes at least one residue (Asp88) that
is essential for transport (Liu and Matherly, 2001).
In initial studies, cysteine-less hRFC was expressed as two (TMD16 and
TMD712) half molecules, each with a cysteine residue inserted at a defined
position in the TMD16 (TMDs 2 or 5) or TMD712 (TMDs 8 or 11)
portions. Altogether, 19 cysteine-substituted TMD16/TMD712 pairs
(175/311, 174/314, 172/315, 171/317, 168/318, 167/321, 164/322,
163/325, 161/326, and 160/326 in TMDs 5/8; 74/415, 74/412, 74/411,
75/408, 78/405, 81/404, 82/404, 84/404, and 85/404 in TMDs 2/11)
were selected for coexpression and cross-linking with homobifunctional
cross-linkers of different lengths [1,1-methanediyl bismethanethiosulfonate,
3 A; o-phenylenedimaleimide, 6 A; p-phenylenedimaleimide, 10 A; and
1,6-bis(maleimido)hexane, 16 A], so to assess helix proximities and tilts in
relation to the putative hRFC hydrophilic cavity. The results unequivocally
establish that the helices of TMDs 5 and 8 are relatively close together at
their extracellular ends (within 10 A), then tilt away from each other toward
the cytoplasmic ends; TMDs 2 and 11 are in proximity at both their
extracellular and cytoplasmic ends (within 10 A). Pro82 in TMD2 may
cause a bend in TMD2, resulting in a lack of cross-linking between the
middle segments of TMDs 2 and 11.

X. Conclusions
Folates are essential for life and folate deficiency contributes to a host of
health problems including cardiovascular disease, fetal abnormalities, neuro-
logical disorders, and cancer (Lucock, 2000; Matherly, 2004). Antifolates,
represented by MTX, continue to occupy a unique niche among the modern
day pharmacopoeia for cancer along with other pathological conditions
(Matherly et al., 2007).
This chapter focuses on the biology of the membrane transport system
termed the reduced folate carrier or RFC with a particular emphasis on
174 Larry H. Matherly and Zhanjun Hou

structure and function. The ubiquitously expressed RFC is the major

transporter for folates in mammalian cells and tissues (Matherly and
Goldman, 2003). Loss of RFC expression or function portends potentially
profound physiological and developmental consequences (Matherly, 2004).
For chemotherapeutic antifolates used for cancer, loss of RFC expression or
synthesis of mutant RFC protein results in antifolate resistance due to
incomplete inhibition of cellular enzyme targets and low levels of antifolate
substrate for polyglutamate synthesis (Goldman and Matherly, 1985;
Goldman and Zhao, 2003).
Protein structural information is a prerequisite for understanding
mechanisms of membrane transport. However, for mammalian MFS trans-
porters, this information has not been widely available due to difficulties in
isolating sufficient quantities of purified proteins and in crystallizing proteins
for X-ray diffraction studies. Since 1994, when RFC was first cloned,
tremendous advances in molecular biology and biochemical approaches
for studying the structures of polytopic membrane proteins have led to an
increasingly detailed picture of the molecular structure of the carrier,
including its membrane topology, its N-glycosylation, identification of
functionally and structurally important domains and amino acids, and
helix packing associations. Although no crystal structure for RFC is yet
available, biochemical and molecular studies, combined with homology
modeling based on homologous bacterial MFS transporters such as LacY,
now permit the development of experimentally testable hypotheses
designed to establish RFC structure and mechanism.
Of course, significant challenges remain. For instance, it is essential to
further identify critical amino acids and domains that comprise the
substrate-binding sites and translocation pathways by biochemical studies
and eventually by X-ray crystallography. Mechanistic studies are needed to
further characterize the energetics of RFC transport, namely how
counter transport by an unidentified physiological counteranion drives
folate substrate accumulation against a concentration gradient, including
the relationship between counteranion and substrate binding. Greater focus
in RFC protein structure studies needs to be on key substrate-specific deter-
minants of binding and translocation, as a prelude to the design of new
antifolate inhibitors that rely on RFC for cellular entry, or with substantially
enhanced transport by other folate transporters such as FRs or PCFT over
RFC. It will be necessary to extend static structural studies of helix packing
by protein cross-linking to dynamic structural changes involving function-
ally important TMD helices that accompany substrate binding and translo-
cation. Likewise, it will be important to expand simple considerations of
secondary and tertiary structures for RFC to potential oligomeric quater-
nary associations, including possible homomeric and heteromeric protein
protein associations that may be significant to transport mechanism or
regulation. Indeed, insights from RFC structurefunction studies may
Structure and Function of the Reduced Folate Carrier 175

eventually foster the development of strategies for biochemically

modulating the carrier that could be therapeutically exploited in the context
of nutritional interventions or antifolate chemotherapy.

This work was supported by Grant CA53535 from the National Cancer Institute, National
Institutes of Health.

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Renal Conservation of Folates: Role

of Folate Transport Proteins
Vijaya L. Damaraju,*, Carol E. Cass,*, and Michael B. Sawyer*,

I. Introduction 186
II. Physicochemical Properties, Protein Binding,
and Water Solubility 186
III. Role of Folates in Genomic Stability 188
IV. Folates and the Kidney 188
V. Folate Transport Proteins 189
A. a-Folate receptor 189
B. Reduced folate carrier 190
C. Proton-coupled folate transporter 191
D. Organic anion transporters and multidrug resistance protein 192
VI. Localization of Putative Folate Transporters in Kidney 192
VII. Clinical Studies of Renal Handling of Folates and Antifolates 193
A. Folates 193
B. Antifolates 194
VIII. Role of Renal Folate Conservation in Ethanol-Related
Folate Deficiency 196
IX. Role of Folate Transport Processes in Renal Conservation
of Folates and Antifolates 197
References 198

Folates play vital roles in one-carbon metabolism that produces the early
substrates necessary for nucleotide synthesis and salvage. Folates are essen-
tial vitamins in that humans cannot synthesize them and are totally dependent
on the diet to obtain them. As water-soluble vitamins, they would be easily
filtered by the kidney and lost to the tubular fluid but for a highly efficient renal
conservation mechanism. This renal folate trap is made up of a-folate recep-
tors and reduced folate carriers. The locations of these transporters are such

* Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada

Department of Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada
Department of Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada

Vitamins and Hormones, Volume 79 # 2008 Elsevier Inc.

ISSN 0083-6729, DOI: 10.1016/S0083-6729(08)00406-8 All rights reserved.

186 Vijaya L. Damaraju et al.

that they direct folate transport from the apical/luminal sides of kidney cells to
the basolateral/plasma sides. In addition, other transporters such as organic
anion transporters and multidrug resistance proteins are also found in kidney
cells and play a role in renal elimination of folate analogues such as antifolate
cancer chemotherapy drugs. This chapter discusses how these transporter
activities manifest themselves in folate and antifolate pharmacokinetics. It also
discusses effects of alcohol on renal reabsorption of folates. 2008 Elsevier Inc.

I. Introduction
This chapter reviews the current understanding of renal handling of
folates in mammals and the molecular processes underlying folate homeo-
stasis. Various folate analogues and their chemical structures and properties
are described briefly followed by a description of the roles that folates play in
one-carbon metabolism, especially their pivotal role in thymidine synthesis.
Then the early studies that demonstrated the kidneys role in maintaining
the bodys folate stores are reviewed.
Roles of several folate transport proteins including a-folate receptors
(aFRs), reduced folate carriers (RFCs), proton-coupled folate transporters
(PCFTs), organic anion transporters (OATs), and multidrug resistance pro-
teins (MRPs) in renal reabsorption are then reviewed. Clinical implications
of renal reabsorption of folates and antifolates are also discussed.

II. Physicochemical Properties, Protein

Binding, and Water Solubility
The term folate can have two definitions: the anion of folic acid or the
group of structurally related naturally occurring vitamins. Naturally occur-
ring folates are composed of seven members, the chemical structures of
which are presented in Fig. 6.1. Folates consist of a 2-amino-4-hydroxy-
pteridine (pterin) group conjugated by a methylene group to r-amino
benzoic acid, which in turn is linked to one or more glutamic acid residues.
The form most commonly used clinically is folic acid, also known as
pteroylglutamate (PteGlu), which is the fully oxidized form of folate and
is the most stable. The anionic and hydrophilic folate (vitamin B9, isolated
from spinach leaves in 1941) occurs physiologically in its reduced form (Mr
440) and belongs to the group of water-soluble B vitamins. Although
the pteridine ring can be synthesized by mammals, inability to couple it
to r-aminobenzoic acid makes folate an essential vitamin in mammals (Birn,
2006). The first step in absorption of dietary folates is hydrolysis of the
glutamate residues conjugated to the PteGlu backbone by g-glutamylhydro-
lase (glutamate carboxypeptidase II) producing the monoglutamate form.
Renal Handling of Folates and Antifolates 187


OH 5 O
N 10


(Folic acid)

Position Substituent Derivative Metabolic step

5,6,7,8 -H Tetrahydrofolate Generation of formate

N5 -CH3 5-Methyltetrahydrofolate Homocysteine to methionine
N5 -CNH2 Formiminotetrahydrofolate Histidine metabolism
N5 -CHO Folinic acid Synthesis of purines
N10 -CHO 10-Formyltetrahydrofolate Synthesis of purines
N510 -CH- 5,10-Methenyltetrahydrofolate Synthesis of purines
N510 -CH2- 5,10-Methylenetetrahydrofolate Synthesis of thymidylate


N H2 5 O
N 10


4-amino-10-methyl folic acid (methotrexate)

Figure 6.1 Structures of folic acid and derivatives and their role in metabolism.

Some folates are metabolized by intestinal cells by adding a methyl group to

the fifth nitrogen of the pterin ring to produce 5-methyltetrahydrofolate (5-
methylTHF). 5-MethylTHF is the most common form that is found in
plasma. Serum concentrations of the major circulating form,
5-methylTHF, range from 5 to 30 nM. Intracellular pools of reduced tetra-
hydrofolates serve as cofactors in several metabolic processes (Stover, 2004).
The vitamin is present in serum either free or bound to carrier proteins like
the folate-binding protein (Holm et al., 1980) or other serum proteins like
albumin (Soliman and Olesen, 1976). In humans, there is a wide variation in
the fraction of protein-bound folate from 20% to 65% (Zettner and Duly,
1978). Serum folates exist mostly in the monoglutamate form, which is the
form that can be readily transported across cell membranes. Once inside cells,
folates are converted to polyglutamate forms by addition of several glutamic
acid residues by folylpolyglutamate synthetase (Balinska et al., 1982). This leads
to retention of folate pools inside the cells for subsequent steps in metabolism.
188 Vijaya L. Damaraju et al.

III. Role of Folates in Genomic Stability

Folate is an essential vitamin that gives rise to cofactors in one-carbon
transfer reactions required for the de novo synthesis of purines and pyrimidines.
Various folates serve as essential donors and receptors for one-carbon metabo-
lism reactions. Possibly, the most important and crucial of these is methylation
of deoxyuridine monophosphate (dUMP) to deoxythymidine monopho-
sphate (dTMP). In this reaction, 5,10-methylene-H4PteGlun donates a methyl
group to dUMP to form dTMP, a reaction that is catalyzed by thymidylate
synthase. The dihydrofolate (H2PteGlun) produced is reduced by dihydrofo-
late reductase to tetrahydrofolate (H4PteGlun). 5,10-Methylene tetrahydrofo-
late is regenerated by serine hydroxymethyl transferase. Folates are not only
involved in pyrimidine metabolism but 5,10-methenyl-H4PteGlun and
10-formyl-H4PteGlun are also involved in synthesizing the inosine monopho-
sphate that is used to synthesize adenosine monophosphate and guanosine
monophosphate. Folates therefore play a crucial role in synthesis of nucleotides.
Low folate pools result in reduced availability of nucleotides for DNA
synthesis and repair. Chronic low folate levels lead to uracil misincorporation
in place of thymine that results in a futile repair cycle that causes frequent
damage to DNA and chromosomes leading to malignant transformation
(Blount and Ames, 1994; Reidy, 1987). Folate also has important roles in
regulating gene expression through its role in the methylation of cytosine
residues and in DNA repair. S-adenosylmethionine is the methyl donor for
methylation of cytosines in DNA and in other cellular biochemical reactions.

IV. Folates and the Kidney

Folates are hydrophilic anionic molecules that require tightly regu-
lated processes for facilitating the transport of natural folates and antifolates.
One of the challenges that the body faces is maintaining adequate folate
stores and this is complicated by folates physiochemical characteristics that
in the absence of reabsorption mechanisms would result in folate being
freely filtered at the renal glomerulus and lost in the urine. Plasma
5-methylTHF is the major form of folate and unbound 5-methylTHF is
freely filtered at glomeruli followed by reabsorption in proximal tubules
(Goresky et al., 1963). Plasma concentrations of 5-methylTHF are 20 ng/ml
and because the kidneys glomerular filtration rate is 125 ml/min, 2000 mg
of 5-methylTHF is filtered at glomeruli each day, with only 1050 mg being
lost in urine (Birn, 2006; Birn et al., 1997). The central role of the kidney in
maintaining the 5-methylTHF pool was first suggested by Johns et al. (1961).
In their study, they administered increasing doses of radiolabeled folic acid to
Renal Handling of Folates and Antifolates 189

healthy volunteers and found that at high doses, renal excretion of folic acid
approximated renal excretion of inulin, which provides a measure of renal
glomerular filtration. The same researchers confirmed the earlier study more
accurately using [3H] folic acid with a higher specific activity in studies
using dogs. They also demonstrated that methotrexate (MTX), a folate
analogue used in cancer treatment, displaced [3H] folic acid from the binding
sites for folic acid.
Mechanisms of renal folate reabsorption remained unsolved until the
studies of Selhub et al. (1987) who investigated the roles of aFRs and RFCs
in the process of renal reabsorption. Selhub et al. (1987) showed that renal
clearance was less than that of inulin, suggesting reabsorption. Renal con-
servation of folate occurs by specialized processes that depend on circulating
folate concentrations and appear to involve FRs, RFCs, and possibly the
PCFT/HCP1 systems (Bhandari et al., 1991; Kamen and Caston, 1975;
McMartin et al., 1992; Morshed et al., 1997; Qiu et al., 2006; Sikka and
McMartin, 1998). In addition to the above-mentioned transporters, OATs
and MRPs were shown to have a role in renal folate transport processes.
A concerted action of these proteins is required to provide mammalian cells
with adequate folate cofactors for nucleotide synthesis. Folate uptake pro-
cesses in apical versus basolateral membranes are important determinants of
the vectorial flow of substrates across cells. In addition to maintaining
cellular folate homeostasis, folate membrane transporters play an important
role in antitumor activity of many antifolates used in cancer therapy.

V. Folate Transport Proteins

A. a-Folate receptor
It is accepted as fact that folate homeostasis is maintained by renal reabsorp-
tion and that aFRs play a major role in this process. The aFR gene of
humans is found on chromosome 11q13 (Ragoussis et al., 1992) and
encodes a 1.28-kb mRNA (Sadasivan and Rothenberg, 1989). aFR is a
single polypeptide of 225 amino acids with a molecular mass of 26,252
(Sadasivan and Rothenberg, 1989). aFRs are anchored to apical brush
border membranes by glycosyl-phosphatidylinositol and are found in
cholesterol-rich membrane domains called caveolae (Lacey et al., 1989;
Rothberg et al., 1990). The major folate-binding protein of kidney, the
aFR, is responsible for 5-methylTHF reabsorption.
The role of the aFR, which was initially termed folate-binding pro-
tein (Selhub and Rosenberg, 1978), in folate renal handling was indicated
when it was identified in kidney proximal tubules (Kamen and Caston,
1975). Selhub and Franklin (1984) determined the proteins mass and
composition and demonstrated that it bound MTX. They also showed
190 Vijaya L. Damaraju et al.

that the affinity of the aFR for a folate derivative was inversely related to its
clearance and, although aFR had the lowest affinity for MTX of the folate
derivatives tested, MTX was also reabsorbed. McMartin et al. (1992) studied
folate transport by human renal proximal tubule epithelial cells (RPTEC)
and demonstrated that aFRs were responsible for folate reabsorption and
showed that this reabsorption was pH dependent being maximal at pH 5.0.
Morshed et al. studied the transepithelial transport of folates using renal
proximal tubule epithelial cells grown on collagen-coated inserts
(McMartin et al., 1992; Morshed et al., 1997). They showed (using probene-
cid as an RFC inhibitor) that folate uptake at apical membranes was due to
aFRs and RFCs, whereas folate transport at basolateral membranes was due
to RFCs. aFRs were localized to brush border membranes of proximal
tubular epithelial cells in human kidney (Weitman et al., 1992). More
recently, the importance of aFRs in renal folate transport and conservation
was demonstrated in mice in which the genes folbp1 and folbp2 were deleted
(Birn et al., 2005). Elevated (100 times higher than in wild type) folate renal
clearances at low and high folate intakes were demonstrated in mice in whom
folbp1 (similar to aFRs in humans) was knocked out thus implicating folbp1 in
folate renal reabsorption. aFRs have high affinities for folic acid and MTX
with Km values of 1 and 100 nM, respectively ( Jansen et al., 1999). aFRs
have higher affinities for a number of folate and antifolate compounds than
bFRs (Brigle et al., 1994). Differences in binding affinities were shown to be
due to amino acid sequence differences in the binding sites between bFRs
(Leu-49, Phe-104, and Gly-164) and aFRs (Ala, Val, and Glu) (Maziarz et al.,
1999). Folate uptake by membrane bound aFRs was postulated to occur by
endocytotic processes. Folates bound to aFRs form vesicles that are then
endocytosed into cytosol, after which acidification releases folates from the
receptor complexes followed by transport into cytosol (Kamen et al., 1988;
Rothberg et al., 1990). The role of potocytosis, which is another uptake
process that has been described in cultured cells (Anderson et al., 1992), in
cellular uptake of folates is considered more controversial than the more
generally accepted endocytotic processes.

B. Reduced folate carrier

The RFC (or SLC19A1) is another membrane protein that plays a major
role in folate transport in mammalian cells with ubiquitous expression in
normal and malignant tissues, consistent with its role in tissue folate homeo-
stasis. The human RFC gene is found at position 21q22.222.3 (Moscow
et al., 1995), and has seven exons that encode a 21.4-kb mRNA
(Tolner et al., 1998). The gene encodes a 65-kDa protein that is hea-
vily glycosylated. Although there are five RFC transcript variants that
arise from alternate splicing of the 50 untranslated region (Moscow et al.,
1995; Nguyen et al., 1997; Prasad et al., 1995; Williams and Flintoff, 1995;
Renal Handling of Folates and Antifolates 191

Wong et al., 1995), they encode the same protein. RFC exhibits broad
tissue distribution and human RFC transcripts were detected in 68 human
tissues (Whetstine et al., 2002). Immunohistochemistry studies in mice have
demonstrated RFC proteins in brush-border membranes of the jejunum,
ileum, duodenum, and colon and in basolateral membranes of renal tubular
epithelia (Wang et al., 2001). Cell-specific localization and expression of
RFC may reflect its differential roles in relation to tissue needs. RFC is a
facilitative carrier (i.e., nonconcentrative) for folates and other anionic
compounds. RFCs have different affinities for folates compared with
aFRs. Human RFCs have higher affinities for MTX and reduced folates
(Km  15 mM) than for folic acid (Km  100200 mM). In contrast,
aFRs have higher affinities for folic acid (Kd  1 nM) and reduced folates
(5-formyltetrahydrofolate and 5-methylTHF, Kd  1040 nM) than MTX
(Kd  150 nM) (Spinella et al., 1995). For an extensive review of RFCs, see
Matherly (2001) and Matherly and Goldman (2003).

C. Proton-coupled folate transporter

For many years, studies of uptake of folates in mammals were mainly
focused on FRs and RFCs [for recent reviews, see Matherly and
Goldman (2003)], although a low pH transport activity that differs from
both aFRs and RFC-mediated activities had been demonstrated in many
cell types. Folate transport activity with a low pH optimum was first
identified in intestinal segments, isolated cells, and membrane vesicles
(Said, 2004). The distinguishing feature of this low pH transport activity is
its similar specificity at low pH toward both oxidized and reduced folates, in
marked contrast to RFCs, which have very low affinities toward oxidized
folates (Zhao and Goldman, 2007). This low pH activity was shown to be
present in cells devoid of RFC activity, thus suggesting that it is distinct
from the RFC activity (Zhao et al., 2004, 2005). Recent cloning and
functional expression of the human gene that encodes low pH activity
demonstrated the existence of the PCFT and confirmed its role in folate
transport and absorption in intestine (Qiu et al., 2006). The PCFT gene is
found on chromosome 17 in humans and the coding region has five exons.
Prior to this discovery, this carrier was reported to be a heme carrier protein,
hence the gene has been referred as PCFT/HCP1 (Qiu et al., 2006). PCFT is
found apically in brush border membranes in duodenum and upper jejunum
(Zhao and Goldman, 2007). A low-pH transport activity was demonstrated in
rat kidney brush border membrane vesicles (BBMVs) (Bhandari et al., 1988).
In addition to its role in intestinal folate absorption, PCFT may have a role in
FR-mediated endocytosis (Anderson et al., 1992) in kidney, central nervous
system, and other tumor tissues. FRs present on cell membranes bind extra-
cellular folates, which are internalized in endocytotic vesicles. Folates
are released from the FRs into the cytosol upon acidification of vesicles.
192 Vijaya L. Damaraju et al.

This release of folate from vesicles to cytosol may be mediated by PCFT

present on vesicle membranes driven by high transvesicular pH gradient
(Murphy et al., 1984; Prasad et al., 1994).

D. Organic anion transporters and multidrug

resistance protein
In addition to aFRs and RFCs, additional proteins transport antifolates in
the kidney: (1) OAT2 and OAT3 are found in basolateral membranes of
RPTECs (Matherly, 2001; Qiu et al., 2006; Sun et al., 2001; van Aubel
et al., 2002; Wang et al., 2001); (2) OAT4 is found in apical membranes of
RPTECs (Takeda et al., 2002); and (3) MRP4 (van Aubel et al., 2002) and
MRP2 (Hooijberg et al., 1999) (also known as cMOAT) are found in apical
membranes of RPTECs. In contrast to rat OAT1 (Takeuchi et al., 2001),
the human homologue hOAT1 does not transport MTX (Hirohashi et al.,
1999; Lu et al., 1999). Lu et al. expressed hOAT1 in HeLa cells and showed
that MTX was not a substrate for hOAT1 (Hirohashi et al., 1999; Lu et al.,
1999). In contrast, when Takeda et al. (2002) transfected mouse proximal
tubule cells with hOAT1, MTX transport was observed, although mice
lacking the gene encoding mouse OAT1 were not used. Evidence for
MRP-mediated transport of folates was derived from cells transfected
with cDNAs expressing the genes for MRPs (Chen et al., 2002; Hirohashi
et al., 1999; Hooijberg et al., 1999; Kusuhara et al., 1998; Zeng et al., 2000,
2001). Studies with diglutamate and higher polyglutamate derivatives of
folates demonstrated that these compounds were not substrates for these
exporters (Chen et al., 2002; Zeng et al., 2001). The major role of MRPs is
to enhance substrate export from cells and thus it may play a role in the rate
and extent of folate retention in cells by regulating intracellular concentra-
tions of monoglutamyl folates. Although MRP3 does transport MTX
(Hooijberg et al., 1999), it is not found in RPTECs, but instead is localized
to distal convoluted tubules (Scheffer et al., 2002).

VI. Localization of Putative Folate

Transporters in Kidney
Renal cells transport folates vectorially from the apical to basolateral
sides of proximal tubule cells. Resulting net fluxes and intracellular con-
centrations are a function of the activities of folate transporters in apical or
basolateral membranes of proximal tubule cells in kidney cortex. From the
integration of information obtained in different experimental systems, a
model for localization of different folate transport proteins in proximal
tubule cells is now emerging (Matherly and Goldman, 2003). aFRs are
present on brush border membranes of renal tubular epithelial cells
Renal Handling of Folates and Antifolates 193

MRP 2/4


Proximal tubule
cell MRP1



Figure 6.2 Vectorial localization of putative folate transporters in kidney.

(Weitman et al., 1992). OAT-K1, a renal organic anion transporter, med-

iates MTX uptake and is localized to brush-border membranes from rat
kidneys (Masuda et al., 1997). In recent studies, Chen et al. (2002) showed
that in addition to MRP1 and MRP3, MRP2 and MRP4 also mediate
MTX efflux. MRP2 was localized to apical brush border membranes of rat
proximal tubule cells (Schaub et al., 1997). In mice, RFC1 was localized to
basolateral membranes of renal tubular epithelia (Wang et al., 2001). Human
MRP1 and MRP3 were localized to basolateral membranes of kidney
proximal tubules (Evers et al., 1996; Scheffer et al., 2002). Some anion
transporters, rOAT1 and hOAT3, were also present on basolateral mem-
branes of kidney tubules (Cha et al., 2001; Tojo et al., 1999). Interplay of
these various transport systems would favor reabsorption of folates from the
tubular lumen. Development of a cell culture model system (see Fig. 6.2)
with transporters identified thus far that play a role in folate renal transport
might give invaluable insight into roles of each transport process in renal
reabsorption versus secretion, thus enabling selective modulation of these
processes based on cellular needs.

VII. Clinical Studies of Renal Handling

of Folates and Antifolates
A. Folates
It has been well demonstrated that folic acid and other folate derivates are
renally reabsorbed in healthy volunteers who are not aggressively hydrated.
Johns et al. (1961) studied folic acid absorption kinetics using [3H] folic acid
194 Vijaya L. Damaraju et al.

(with relatively low activity) in 16 adult males whose age ranged from 22 to
29 years. When they administered 1 mg/kg to subjects, less than 2% of
radioactivity was recovered in the urine after 2 h. After administering
15 mg/kg, 25% of the radioactivity was recovered in the urine. At higher
doses of 150 and 1430 mg/kg, 60% to 70% of the administered radioac-
tivity was recovered in the urine. Based on these results, they concluded that
folic acid was reabsorbed by the kidney. They subsequently replicated these
experiments in vitro using dogs and [3H] folic acid as reported by Goresky
et al. (1963) In this later study, dogs were anaesthetized, their ureters were
catheterized, and [3H] folic acid was injected directly into the renal artery
along with inulin, the latter to serve as a measure of the glomerular filtration
rate. At least 25 urine samples were then collected over the ensuing 10 min.
At high doses, folic acid elimination paralleled and closely approximated
inulin filtration; whereas at low doses, folic acid secretion was substantially
less than that of inulin filtration, suggesting renal reabsorption. Interestingly
when MTX was coadministered with inulin and low dose [3H] folic acid,
the folic acid elimination approximated inulin filtration, suggesting that
MTX competed with folic acid to be reabsorbed. Selhub et al. (1987)
have studied other folate analogues in rats. They administered [3H]
folic acid, 5-methylTHF, or MTX to rats by either intravenous bolus
or continuous infusion found that urinary clearance was least for
5-methylTHF at 0.026 ml/min, followed by 0.343 ml/min for folic acid
and 1.51 ml/min for MTX. Comparing these folate urinary clearances to
the apparent urinary clearance for inulin, it was concluded that all three
folates (folic acid, 5-methylTHF, and MTX) were reabsorbed.

B. Antifolates
In contrast to studies with folates in healthy volunteers which clearly
showed reabsorption, studies in cancer patients receiving therapeutic doses
of antifolates have been less clear with many contradicting results. Calvert
et al. (1977) studied 18 cancer patients receiving MTX treatment either as
continuous intravenous infusion or as bolus. In all but 1 of the 18 patients,
renal MTX clearance was less than the measured glomerular filtration rate,
suggesting renal reabsorption. Hendel and Nyfors (1984) studied renal
clearance of low-dose MTX in 12 patients with psoriasis who were treated
with MTX with doses ranging from 7.5 to 30 mg. They found MTX had its
highest renal clearance at high initial concentrations but as plasma concen-
trations fell so did renal MTX elimination. Similarly in the phase I study of
ZD9331, a folate analogue that cannot be polyglutamated, Goh et al. (2001)
found as the dose of ZD9331 increased, the clearance increased, suggesting
renal reabsorption. As well, Sawyer et al. (2003) in a study of the oral
formulation of ZD9331 found that elevated blood urea nitrogen levels,
Renal Handling of Folates and Antifolates 195

which are a marker of increased renal reabsorption, were associated with

increased risk of toxicity from ZD9331.
In contrast, when MTX was administered to patients or animals with
hydration or urine alkalinization, the kidney secreted MTX (Huang et al.,
1979; Monjanel et al., 1979). Bourke et al. (1975) studied MTX elimination
in monkeys and found that MTX elimination exceeded glomerular filtra-
tion rates. Eleven monkeys were administered MTX to achieve plasma
MTX levels ranging from 0.04 to 32 mg/m. At concentrations exceeding
1.7 mg/m, MTX elimination was approximately equal to the glomerular
filtration rate. In contrast when MTX concentrations were less than 1.7 mg/ml,
elimination was threefold higher than the glomerular filtration rate. The
investigators administered probenecid, which blocks OATs, to six addi-
tional monkeys in an attempt to determine if a saturable renal excretion
process was involved. Addition of probenecid resulted in lower MTX
elimination that approximated glomerular filtration rates as assessed by
inulin clearance, suggesting saturable renal secretion. In all of these experi-
ments, the monkeys were aggressively hydrated with Ringers lactate,
a buffered lactate hydration solution. Huang et al. (1979) studied MTX
elimination in rhesus monkeys and dogs and again found that MTX elimi-
nation exceeded inulin clearance. In the study by Huang et al., the animals
were aggressively hydrated even more than the animals in the study by
Bourke et al. (1975). Liegler et al. (1969) studied effects of salicylates, para-
aminohippuric acid, and sulfisoxazole on MTX in 15 cancer patients treated
with MTX. Patients were treated with MTX to which a known quantity of
[3H]MTX was added. Fourteen patients were found to have normal renal
function as measured by inulin and of these, 12 had MTX clearances greater
than the inulin clearance. In patients administered high-dose para-
aminohippuric acid, the MTX clearance dropped as was the case in the
patients administered salicylates. In contrast, sulfisoxazole had little effect on
MTX clearance.
Clinical studies of renal handling of antifolates showed great contra-
dictions compared with studies of folate renal conservation. The major
difference between studies that showed renal reabsorption and studies that
showed renal secretion was that in the latter studies patients or study
animals were aggressively hydrated and their urine was alkalinized. Hydra-
tion and alkalinization were undertaken due to the belief that antifolates
can cause renal failure because of precipitation of MTX crystals in renal
tubules, leading to mechanical obstruction. No study had adequately
addressed whether antifolates could be absorbed by proximal tubule cells
via the same processes intended for folate renal conservation. Damaraju
et al. (2005) undertook studies with brush BBMVs prepared from renal
cortex to try to resolve these contradictions. BBMVs bound all anti-
folates tested, including CB3717, ZD9331, raltitrexed, aminopterin, and
MTX. Binding of antifolates was likely due to aFRs because treatment of
196 Vijaya L. Damaraju et al.

BBMVs with phosphatidylinositol phospholipase, which cleaves glycosyl-

phosphatidylinositol linkers of aFRs, abolished binding of antifolates to
BBMVs. In contrast to naturally occurring folates that demonstrated
maximal binding to BBMVs at a pH of 6, antifolate binding to BBMVs
increased with increasing pH in many cases or stayed constant. These
results suggested that potentially all antifolates could be reabsorbed under
certain conditions, especially in the setting of dehydration or vomiting,
which would cause the urine to become alkaline. Although the alkaline
pH in many of the studies in cancer patients or laboratory animals should
have substantially increased renal reabsorption, there was net antifolate
secretion, suggesting that hydration and rapid passage of antifolates
through proximal tubules likely caused net renal secretion of antifolates
despite the presence of aFRs in proximal tubules.

VIII. Role of Renal Folate Conservation in

Ethanol-Related Folate Deficiency
Acute and chronic alcohol ingestion has been associated with folate
deficiency. Several mechanisms have been put forward as contributing
to this folate deficiency: (1) decreased dietary intake, (2) decreased folate
absorption, and (3) increased folate renal excretion because of decrea-
sed renal reabsorption (Eichner and Hillman, 1971, 1973; Halsted, 1980).
Tamura and Halsted (1983) studied folate metabolism in monkeys using
intramuscular [3H] folic acid and found increased excretion of folic acid in
monkeys chronically fed alcohol compared with control monkeys fed
alcohol-free diets. Russell et al. (1983) confirmed the increased renal elimi-
nation of folates during acute alcohol ingestion in five patients with chronic
alcoholism. They did their studies in a crossover fashion in which each
patient served as his/her own control. They found that acute ethanol
ingestion was associated with a 2040% increase in renal folate losses.
McMartin et al. (1986) have extensively studied the effects of ethanol on
renal folate conservation in rat models and primary cultures of human
proximal tubule cells. They demonstrated that rats fed alcohol acutely for
14 days had increased urinary excretion of folates that was maximal 8 h
after alcohol ingestion. Alcohols effect on urinary folate excretion was
similar whether the rats had been fed for 14 days, suggesting that rats
could not adapt to alcohols effects. Muldoon and McMartin (1994) showed
in a set of experiments using isolated perfused rat kidneys that infusing
alcohol into the kidney significantly increased 5-methylTHF excretion
compared to controls. Based on studies of the isolated perfused rat kidneys,
Ross and McMartin (1996) studied alcohols effects on binding of folates to
rat brush border membranes and did not find an effect of alcohol on folate
Renal Handling of Folates and Antifolates 197

binding. Hamid and Kaur (2005) studied the effect of chronic alcohol
administration on binding of folates to rat renal brush border membranes
and, in contrast to the work of McMartin et al., found a decreased Bmax
without any change in the affinity (Kd). Romanoff et al. (2007) revisited the
issue of alcohols effect on folate transport using cultured human proximal
tubule cells. In their experiments, cells were exposed to alcohol in culture
media either for 1 h or for 5 days. Transport experiments were done on
membrane inserts so that alcohol effects on apical and basolateral transport
could be dissected. They found that alcohol decreased apical transport by
2025% without affecting basolateral transport. They did not find an effect
of alcohol on the Bmax or Kd values of brush border membranes for folate.
After 5 days of exposure, they found an upregulation of both FRs and RFC
levels. In terms of the effects that alcohol has on renal conservation of folate,
there is evidence that acute ethanol exposure decreases renal reabsorption of
folate based on both in vivo and in vitro studies. The in vitro studies have not
yet produced a convincing explanation of the cause of the altered renal

IX. Role of Folate Transport Processes in Renal

Conservation of Folates and Antifolates
Folates are essential vitamins that cannot be synthesized in the human
body. They are very water soluble and have modest protein binding, which
means that circulating plasma folates are subject to extensive filtration by
glomeruli in the kidneys. The amount of folate that would be lost in the
urine would be up to tenfold the average intake of folate in the North
American diet were it not for the extensive reabsorption of folates by
proximal tubule cells. In proximal tubule cells, aFRs work together with
RFCs serving as filters to prevent loss of folates into urine. The circulating
folate pool is maintained by the folate trap in the proximal tubule cells of
the kidney; this is evidenced by the fact that aFRs Kd values for
5-methylTHF at pH 6.07.0 are 62.47.6 nM, which encompasses typical
plasma values for 5-methylTHF.
Renal conservation of folates may be a significant factor in antifolate
pharmacokinetics. Some of the contradictions of antifolate clinical studies
may be explained by the roles aFRs play in folate reabsorption in renal
proximal tubule cells. Antifolate pharmacokinetics would be more predict-
able by considering that an administered antifolate such as MTX displaces
naturally occurring folates from the circulating plasma pool and the anti-
folate is then subject to the folate trap in the kidney. Additionally, aggressive
hydration along with administration of a naturally occurring folate would
substantially decrease renal antifolate reabsorption and possibly reduce
198 Vijaya L. Damaraju et al.

interpatient variability of antifolate pharmacokinetics. We hypothesize that

addition of folic acid inhibits the renal reabsorption of antifolates preventing
prolonged and unwanted retention of antifolates that can lead to excess
toxicity. Considering that antifolates are integrated into the folate pool and
incorporating this concept into models of antifolate pharmacokinetics may
allow better prediction of antifolate pharmacokinetics. Further studies are
clearly warranted to examine the interplay between folate conservation and
metabolism and antifolate pharmacokinetics and renal excretion.

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Exploitation of the Folate Receptor

in the Management of Cancer
and Inflammatory Disease
Christopher P. Leamon* and Ann L. Jackman

I. Introduction 204
II. Aspects of the FR 205
A. FR isoforms 205
B. FR tissue expression 206
III. Exploitation of the FR for Disease Management 208
A. FR role as a biomarker 208
B. Cancer therapy 212
C. Inflammation therapy 223
D. Modulation of FR expression levels 224
IV. Future Prospects 225
References 226

Over the last 25 years, the folate receptor (FR) has emerged as an attractive
tumor biomarker with the potential to be exploited for therapeutic purposes.
Increasing evidence suggests that this endocytosing protein can functionally
mediate the cellular uptake and retention of natural folates, certain antifolates,
and folate-drug conjugates; the consequences of the latter two events could
result in biological modulation, including (but not limited to) tumor-targeted
cytotoxicity. Because its tissue expression profile appears to be somewhat
limited to either tissues responsible for whole body retention of folates (e.g.,
kidney and placenta), or certain pathologic tissues (e.g., tumors or activated
macrophages), the FR is believed to be a useful biological target for disease
management. Indeed, recent years have been peppered with reports of novel
FR-targeted therapies, and many have demonstrated impressive in vivo potency,
particularly against tumor xenografts, without the undesirable toxicity that often

* Endocyte, Inc., West Lafayette, Indiana 47906

Institute of Cancer Research, Section of Medicine, Sutton, Surrey, SM2 5NG United Kingdom

Vitamins and Hormones, Volume 79 # 2008 Elsevier Inc.

ISSN 0083-6729, DOI: 10.1016/S0083-6729(08)00407-X All rights reserved.

204 Christopher P. Leamon and Ann L. Jackman

accompanies nontargeted drug regimens. This chapter will provide essential

details on the properties of the FR, including where it is expressed and how it
has been successfully manipulated for therapeutic benefit. 2008 Elsevier Inc.

I. Introduction
Folic acid (FA), or vitamin B9, is an essential nutrient required by all
living cells for proper metabolic maintenance of one-carbon pathways as
well as for nucleotide biosynthesis (Clifford et al., 1998). This ligand displays
extremely high affinity (KD  100 pM) for a cell surface-oriented glyco-
protein called the folate receptor (FR), which is a glycosylphosphatidylino-
sitol (GPI)-linked protein that captures its ligands from the extracellular
milieu (Luhrs and Slomiany, 1989). The FR also binds the biologically
active and reduced form of FA, namely 5-methyltetrahydrofolate, with high
affinity (Kamen and Capdevila, 1986). Immediately after binding, the plasma
membrane surrounding the FRligand complex will invaginate to form an
internal vesicle, called an endosome (see Fig. 7.1). The pH of the vesicle
lumen is somewhat lowered through the action of proton pumps that are
colocalized in the endosome membrane, and this acidification presumably
mediates a conformational change in the FR protein to release its bound
ligand to allow for cytosolic entry (Leamon and Reddy, 2004). Whereas all
eukaryotic cells also express an anionic transporter called the reduced folate
carrier (RFC) that functions in a low-affinity, high-capacity mode to deliver
folates to the cytoplasm [see Matherly et al. (2007) for an excellent review],
expression of the FR is believed to afford cells a selective growth advantage
by enabling the capture and utilization of folates, particularly under low
folate physiological conditions (Leamon and Low, 2001).
The FR is also a recognized tumor antigen (Coney et al., 1991; Ross
et al., 1994; Weitman et al., 1992a, 1994), and because of this, methods to
exploit its presence and function have been explored for possible therapeu-
tic value. For example, some laboratories are developing anti-FR antibodies
for diagnostic and therapeutic applications (Buijs et al., 1998; Konner et al.,
2006; van Zanten-Przybysz et al., 1999), whereas others are developing
high-affinity antifolates that can be delivered inside the cell via the FR to
inhibit critical proliferative functions (Gibbs et al., 2005; Henderson et al.,
2006; Jackman et al., 2004; Theti and Jackman, 2004; Theti et al., 2003).
Finally, the FA ligand itself can be covalently modified to deliver a toxic
drug payload to FR expressing cells (Leamon and Low, 2001; Leamon
and Reddy, 2004; Leamon et al., 2007a; Reddy et al., 2005).
The purpose of this chapter is to provide the reader with a basic
understanding of FR biology, what tissues express it, and how this protein
can be exploited for the uptake and/or delivery of active pharmaceutical
Folate Receptor-Based Therapies 205



Late endosome

H+ H+ Recycling


Figure 7.1 Schematic representation of the trafficking of folates, antifolates, and

FA-conjugates by the FR-mediated endocytosis pathway. Exogenously added ligands
bind specifically to the FR protein with high affinity. The plasma membrane invaginates
around the ligandFR complex to form an intracellular vesicle (early endosome). As
the lumen of the maturing endosome acidifies, the receptor changes conformation and
releases the ligand. Eventually, the fates of released ligands, drug cargo, and the FR are
determined during a sorting process within late endosomal elements near the peri-
nuclear region. The reduced folate carrier (RFC), which unlike the FR is an anion
transporter, can shuttle folate molecules and certain antifolates inside the cell. FAdrug
conjugates, however, are not substrates for the RFC.

agents. Our review will not exhaustively cover this ever expanding field. For
example, topics such as folate-targeted liposomes, nanoparticles, antisense/
gene formulations, or antibody-mediated therapies will not be covered in
this volume. Instead, we intend to focus only on small molecule-based
applications that are currently being evaluated in the clinic, or those that
are at the preclinical stage of development.

II. Aspects of the FR

A. FR isoforms
Several isoforms of the FR have been reported, and they appear to have
distinct expression patterns in normal and pathological tissues as well as
different properties. The two FR isoforms most relevant to therapeutic
206 Christopher P. Leamon and Ann L. Jackman

exploitation are FR-a and FR-b, both of which possess a GPI anchor and
both are functional for high-affinity FA binding, although they can display
some differences with respect to their affinity for reduced folate cofactors or
antifolates (Salazar and Ratnam, 2007). FR-g is an isoform that can be
detected in some hematopoeitic cells; however, because it is not anchored
to the plasma membrane, this protein is constitutively excreted. For this
reason, FR-g will not be discussed further. Following capture of folate
molecules, membrane-associated FR-a and FR-b appear within endocytic
compartments before they are recycled back to the cell surface. Studies
reviewed by Salazar and Ratnam (2007) suggest that there may be cell type
differences for receptor recycling kinetics that may impact the delivery
efficiency of folate or folate conjugates from the cell surface to the cytoplasm.

B. FR tissue expression
1. Methods
There are a number of relevant reports, some with small sample numbers,
that describe the tissue distribution of FRs. Five broad methods have been
used to compare FR expression levels. These are (1) FR mRNA levels
measured by PCR or in situ hybridization, (2) FR-a protein levels measured
by immunohistochemistry (IHC) using mono- and polyclonal antibodies,
(3) functional FR levels as measured by 3H-FA binding to solubilized
tissue membrane preparations, (4) assessing FR tissue expression in real time
using radiodiagnostic imaging, and (5) quantifying FR expression ex vivo by
flow cytometric methods. Importantly, each method has its own particular
merits and pitfalls. For example, FR expression has been reported to be
controlled at the translational level, at least partly in some cells, thus it might
be expected that mRNA and protein levels may not always correlate. IHC is
less amenable to precise quantitation, particularly when comparing the
results from different studies, but it has the advantage in that FR distribution
within a tissue can be visualized; the latter is probably essential when
considering therapeutic exploitation of FR expression because in normal
polarized tissues, expression of this protein is restricted to the apical (luminal)
membrane surface rather than the blood-accessible, basolateral surface. Total
tissue receptor measurement using 3H-FA has the advantage of measuring
FR that is functional for ligand binding, but this method may be less useful
for (1) polarized normal tissues, (2) assessing the degree of homogeneity
of receptor expression within a tumor, or (3) distinguishing between differ-
ent receptor isoforms. Imaging tissues for FR expression after injection
of an animal or human with a radioactive high-affinity folate-conjugate
has the advantage of measuring FR that is accessible within the blood
stream. Therefore, this latter approach is of high therapeutic relevance
(see Section III.A.2 below); but, it unfortunately cannot quantitate absol-
ute levels of FR protein. Finally, the FR can also be measured by flow
Folate Receptor-Based Therapies 207

cytometry on either disaggregated tumor material (Toffoli et al., 1998) or

surface of epithelial tumor cells in effusions, such as ascitic fluid (Forster
et al., 2007). One disadvantage of this method, though, is that the tissue to
be analyzed requires isolation and ex vivo manipulation.

2. Normal tissue expression

FR-a distribution in normal adult tissue is restricted to the apical membrane
surface of some polarized epithelial cells, including lung, choroid plexus,
and some glandular tissue (Weitman et al., 1992a,b). Expression is also high
in placental trophoblasts and on the luminal surface of proximal tubule
kidney epithelial cells, the latter probably being important for the reabsorp-
tion of folates from the urine (Birn et al., 1993, 1997; Garin-Chesa et al.,
1993; Holm et al., 1992; Prasad et al., 1994; Rettig et al., 1985; Weitman
et al., 1992b). FR-b is expressed on some normal hematopoeitic cells of
myelomonocytic lineage. Interestingly, this isoform is functional when
expressed on activated monocytes and macrophages, but it is nonfunctional
(unable to bind folate) when present on mature neutrophils or CD34 stem
cells (Nakashima-Matsushita et al., 1999; Paulos et al., 2004; Reddy et al.,
1999; Ross et al., 1999; Turk et al., 2004).

3. Cancer expression
Elevated expression of the FR-a occurs in several cancer types, although the
actual frequency and degree of expression reported is variable, in part due to
the different methods used and the small sample numbers in some studies.
Nonmucinous ovarian cancer (the majority of ovarian cancers) was the tumor
type first to be associated with overexpression (Campbell et al., 1991).
Initially, this was in the form of reports that a monoclonal antibody (mAb;
MOv18) raised against a membrane preparation from an ovarian tumor,
detected a glycoprotein present in most ovarian tumors (Miotti et al., 1987;
Veggian et al., 1989); furthermore, it was later shown that this antigen was a
high-affinity folate-binding protein (FBP) with an amino acid sequence
identical to FBPs found in placenta and KB (human nasopharyngeal carci-
noma) tumor cells (Campbell et al., 1991; Coney et al., 1991). Several studies
confirmed that 8090% of these tumors overexpress FR-a (Parker et al.,
2005; Toffoli et al., 1997; Wu et al., 1999). Other gynecological cancers (e.g.,
50% of serous uterine tumors) also overexpress the receptor (Allard et al.,
2007; Dainty et al., 2007; Marziarz et al., 1999; Wu et al., 1999). Although the
endometrioid histological subtype may express the receptor less frequently, it
is by far the most common form of uterine cancer and therefore a consider-
able number of uterine cancer patients may benefit from some form of
FR-targeted therapy. Other tumors reported to overexpress FR-a to varying
frequencies include pediatric ependymal brain tumors, breast, colon, renal
and lung tumors, and mesothelioma (Parker et al., 2005).
208 Christopher P. Leamon and Ann L. Jackman

Current knowledge also suggests that functional FR-b overexpression in

cancer is restricted to myeloid leukemia and perhaps head and neck carci-
noma (Ross et al., 1994, 1999). Taken together, the total number of tumors
with FR overexpression is very large; thus, FR-targeted strategies could
have significant impact on cancer treatment for patients diagnosed with FR

III. Exploitation of the FR for

Disease Management
A. FR role as a biomarker
As discussed above, the FR is expressed on the apical membrane of polar-
ized epithelia in a limited number of normal tissues; however, many
different types of malignant tissues have also been found to express this
protein, and in large quantities (Coney et al., 1991; Parker et al., 2005; Ross
et al., 1994; Weitman et al., 1992a, 1994). Importantly, not all human
cancers within a particular indication will express the FR; ovarian cancer
is close at 90%, but the total positives of most other cancer types is
lower. For example, 64% (102 of 160) of renal cell carcinoma specimens
are positive, and that number decreases to less than 20% for primary
colorectal carcinoma (unpublished data, Endocyte, Inc.). Because novel
FR-targeted therapies are now being tested clinically (Amato et al., 2005;
Konner et al., 2006; Leamon et al., 2007a; Messmann et al., 2007; Reddy
et al., 2007a; Sausville et al., 2007), the FR may be considered a useful tissue-
based biomarker. Therefore, having the ability to screen patients for
FR-positive disease could certainly increase the efficiency of and decrease
the time for clinical investigations of FR-targeted therapies.
At present, two principal methods have been utilized for assessing
a patients FR status. These include an invasive tissue-based immunohisto-
chemical assay, and a noninvasive radiodiagnostic approach. The merits of
both techniques will be discussed below.

1. Immunohistochemistry
IHC has proven to be beneficial for the selection of patients that are more
likely to respond to a given therapy. Indeed, the HercepTest IHC screen,
which is used for selecting patients with Her-2 expressing breast cancer
( Jacobs et al., 1999), enables the clinician to predict with reasonable cer-
tainly which patients may not benefit from Herceptin therapy. A related test
has also been developed for the immunohistochemical detection of the FR.
Thus, polyclonal rabbit antiserum was produced using bovine milk soluble
folate-binding protein as the antigen. Due to the high degree of homology
between the human FR and folate-binding proteins from various sources,
Folate Receptor-Based Therapies 209

the affinity-purified IgG fraction from this antiserum (herein called PU17)
adequately detects the presence of FR in formalin-fixed, paraffin-embedded
tissues (Endocyte, Inc.). PU17 was selected as the probe to validate the first
FR IHC method to be clinically used. Importantly, the scoring methodol-
ogy is based on a 0 to 3 scale (similar to HercepTest ), and the technique
is both robust and sensitive. Normal kidney cortex serves as the positive
tissue control (3), whereas normal skeletal muscle is the negative tissue
control (zero). To date, this assay has been used to screen well over 100
patients for eligibility related to the clinical testing of potent folate-targeted
agents (vide infra).

2. Radiodiagnostic imaging
Nuclear medicine has both therapeutic and diagnostic value. The latter can
be important for the noninvasive measuring of FR expression within a
given tissue or organ. Applications with anti-FR monoclonal antibodies
or derivatives have been tried clinically with limited success due to their
(1) prolonged circulation times owing to their rather large molecular size,
(2) potential immunogenicity (possibly requiring humanization), (3) cost of
goods, and (4) suboptimal tumor to nontarget tissue ratios (T/NT) (Buijs
et al., 1998; Colcher et al., 1990; Modorati et al., 1994; Seccamani et al.,
1989; van Zanten-Przybysz et al., 1999). Thus, more focus has recently been
directed toward the use of smaller FA-based imaging agents that do not
suffer from such limitations. Numerous folate-targeted small molecule
agents have been produced to date (Antich et al., 1994; Guo et al., 1999;
Ilgan et al., 1998; Leamon et al., 2002; Linder et al., 2000; Mathias and
Green, 1998; Mathias et al., 1996, 1998, 2000; Muller et al., 2004, 2006c;
Reddy et al., 2004; Wang et al., 1996), but one of the earlier compounds to
be investigated was a diethylenetriaminepentaacetic acid derivative of FA
(DTPA-folate). This molecule was found to efficiently chelate Indium-111
to produce a highly water-soluble, FR-targeted radiodiagnostic agent that
rapidly bound to the FR-expressing tissues but also rapidly cleared from the
circulation following intravenous administration (Mathias et al., 1998).
111In-DTPA-folate was clinically tested through Phase 2 and found to be

safe and useful for the identification of FR-positive lesions (Siegel et al.,
2003). Most importantly, the performance of this agent provided the
needed proof of concept data to support ongoing efforts toward the devel-
opment of FA-based therapeutics. Interestingly, DTPA-folate was not
developed any further; instead, it was replaced with a peptidic analogue of
FA that could efficiently chelate Technetium-99m. This new agent, herein
called EC20, is cheaper to produce, and a greater radiation dose can be
given to patients (to increase image resolution) because 99mTc has a much
shorter half-life compared to 111In (6 h vs 2.8 days, respectively) (Leamon
et al., 2002; Reddy et al., 2004). To date, more than 250 patients in Phase
1 and 2 trials have safely received 99mTc-EC20 for the purpose of identifying
210 Christopher P. Leamon and Ann L. Jackman

FR-positive lesions. In fact, 99mTc-EC20 (FolateScan) is currently being

used for selecting FR-positive cancer patients before they enter clinical trials
for the testing of folate-targeted therapeutics (Endocyte, Inc.).
Because multiple methods exist for FR detection in human tissues, one
may question the utility for using one versus another. As summarized in
Table 7.1, the FolateScan (e.g., 99mTc-EC20) and IHC methods are com-
pared relative to five performance parameters. For each situation, it appears
that the radiodiagnostic imaging method has a distinct advantage over IHC.
Specifically, IHC permits the staining identification of selected archived
tissue. In fact, it is not uncommon for tissue samples to be stained many
months to even years after the specimen was obtained. Conversely, imaging
allows for the noninvasive, real-time assessment of functionally active and,
very importantly, accessible FRs. The latter point is further illustrated in
Fig. 7.2 where FRs are correctly identified on the membranes of normal
human alveolar cells, yet no uptake of an intravenously administered folate-
based radiodiagnostic agent is observed in the lungs of a cancer-free patient.
Actually, unless there is malignant disease, no uptake of FolateScan has ever
appreciably been seen in human lung (Endocyte, Inc.). Taken together,
these arguments suggest that radiodiagnostic scanning (likely together with
computerized tomography) is the preferred technique for assessing the FR
status of malignant lesions.
Although it has been known for many years, the image in Fig. 7.2
distinctly shows that normal human kidney expresses a fair amount of
functional FR protein (Birn et al., 1993, 1997; Weitman et al., 1992a,b).
The FR is predominantly expressed on the apical membrane of the proxi-
mal tubules, and its function within this organ is believed to be for

Table 7.1 Reasons FolateScan may be better than IHC for detecting the FR in tissues

criteria FolateScan IHC
Assessment of Real-time tissue analysis Archived tissue
FR status
Specimen Whole body imaging Selected tissue specimen
Functionality Binds to functional FR Detects functional and
nonfunctional FR
Specificity Can bind to FR-a and Validated FR-b IHC is
FR-b not available
Accessibility Reveals accessible tissue Indiscriminant (e.g.,
sites normal lung)a
see Fig. 7.1.
Folate Receptor-Based Therapies 211

Figure 7.2 FolateScan versus IHC. Top panel, FolateScan image of a cancer-free
patient showing no uptake in normal lung and marked uptake in normal kidney.
Bottom panel, IHC image of normal human lung tissue stained with an anti-FR
probe. White arrow, alveolar cell wall; scale bar, 20 mm.

resorption of glomerularly filtered folates to allow for homeostatic concen-

trations of this vitamin to persist in the serum. Because of this highly
localized uptake and potential for unwanted tissue damage, the amount of
injectable radiation (from FolateScan) is typically kept to less than 30 mCi
per patient. Attempts to competitively block FolateScan uptake in the
kidney with preinjected, unlabeled folates have been found to be effective;
however, the uptake in FR-positive tumors is also typically reduced under
these conditions (Leamon et al., 2002; Reddy et al., 2004). Recently, Muller
et al. (2006a, 2007) reported an interesting finding that preinjected, high-
dose pemetrexed (Alimta; pemetrexed; LY231514) selectively reduced
kidney uptake of folate-based radiodiagnostic agents in mice (by >80%)
without adversely affecting uptake in FR-positive tumors (Muller et al.,
2006a, 2007). While the mechanism responsible for this apparent selectivity
212 Christopher P. Leamon and Ann L. Jackman

is not yet known, this approach could easily be tested clinically since the
safety of pemetrexed in humans has been established. Admittedly, if this
maneuver (or a related one) should work in humans, higher doses of
radiation could potentially be administered for (1) increasing the resolution
of imaging FR-positive tumors and possibly, (2) enabling the development
of folate-targeted radiotherapy.

3. Prognostic applications
The FR is well-known to be upregulated in select cancers of epithelial
origin (Parker et al., 2005). Furthermore, higher expression levels have also
been associated with aggressively growing cancers (Bottero et al., 1993;
Campbell et al., 1991; Hartmann et al., 2007; Ross et al., 1994; Toffoli
et al., 1997, 1998). Recently, it was proposed that this relationship may
possibly be used for prognostic purposes (Hartmann et al., 2007). For
example, using a sensitive IHC staining method, the intensity of FR
expression was found to strongly correlate with therapeutic outcome of
patients with invasive breast cancers. Thus, cancer in 81% of women
bearing high expressing malignancy was found to recur as opposed to 38%
of women bearing low expressing disease (Hartmann et al., 2007). Similarly,
high FR expression was observed more often in poorly differentiated
endometrial cancers and tumors with serous histology (Allard et al., 2007).
The authors concluded that FR expression is both a biomarker associated
with endometrial cancer as well as a prognostic factor associated with
adverse outcome. Likewise, FR expression seemingly predicted the recur-
rence of colorectal carcinomas (Nitzkorski et al., 2007). Notably, while the
prognostic importance of FR expression may not be universal among all
human cancer types (Zhai et al., 2007), it may find utility in helping the
clinician choose the best treatment option for his/her patients.

B. Cancer therapy
1. High-affinity antifolates
a. History There has been a history of research in folate metabolism and
the discovery of antifolate drugs that goes back to the 1940s, when cytotoxic
drug therapy was in its infancy. The first reported antifolate, aminopterin,
was quickly succeeded by its N10-methylated counterpart, methotrexate
(MTX), a molecule that remains widely used in clinical practice to this day.
These drugs mimic the structure of key folate intermediates to inhibit the
activity of dihydrofolate reductase (DHFR). 5-Fluorouracil (5-FU) has a
pyrimidine rather than a folate structure, and one of its active metabolites
(FdUMP) potently inhibits thymidylate synthase (TS). This enzyme
requires the cofactor, 5,10-methylene tetrahydrofolate, for activity and
this folate-dependency has been exploited for the development of the
folate-based TS inhibitors, CB3717, raltitrexed (Tomudex; ZD1694),
Folate Receptor-Based Therapies 213

pemetrexed, and plevitrexed (see Figs. 7.3 and 7.4). While there is no doubt
that drugs targeted at folate metabolism continue to make important con-
tributions to cancer treatment, the fact remains that they are cytotoxic
molecules that can not adequately distinguish tumor from normal tissue.
In fact, little opportunity exists for producing tumor-selective cytotoxicity
with this class of agents because (1) both DHFR and TS are expressed in
normal and malignant proliferating tissue and (2) the major plasma

TS inhibitors

5, 10-CH2FH4
FH2 salvage

de novo pathway

dUrd (plasma) (DNA synthesis)

Figure 7.3 Pathways for thymidylate synthesis. Thymidylate can be synthesized via
the de novo and the salvage pathways. When TS is inhibited, the substrate dUMP
accumulates in cells and effluxed into the plasma. TS, thymidylate synthase; TK,
thymidine kinase; 5,10-CH2FH4, 5,10-methylene tetrahydrofolic acid; FH2, dihydro-
folate; Thd, thymidine; TMP, thymidylate; TTP, thymidine triphosphate, dUMP,
deoxyuridylate; dUrd, deoxyuridine.

Raltitrexed (TomudexTM; ZD1694) Plevitr exed (BGC 9331; ZD9331)
BGC 945
Pemetrexed (AlimtaTM; LY231514)

Figure 7.4 Structures of folate-based thymidylate synthase inhibitors.

214 Christopher P. Leamon and Ann L. Jackman

membrane transporter for these drugs is the RFC that happens to be

ubiquitously expressed. However, it was recognized that the cellular folate
content could regulate the degree of FR expression, and that the extremely
high FA content found in most commercial media was competitively
inhibiting antifolate binding (Kamen and Capdevila, 1986); thus, studies
soon followed showing that the FR was indeed capable of transporting
antifolates inside cells.
The relative contribution that the FR provides (compared with other
transporters) toward antifolate activity has largely been studied in cell
culture systems. Early pivotal studies demonstrated that the FR was indeed
an antifolate transporter, and they often employed experimental conditions
with only partial clinical relevance. For example, McHugh and Cheng
published a paper in 1979 providing good evidence that a high-affinity
membrane associated folate binder could be detected on human tumor
cells, most notably KB cells, but they were cautious about ascribing a
transport function to this protein. Interestingly, both, MTX and aminop-
terin were found to inhibit 3H-FA binding to that protein (to varying
degrees). Studies from several other laboratories began to suggest that this
protein could indeed mediate the accumulation of folates (5-methyltetra-
hydrofolate) into cells under physiological conditions and that MTX, in
spite of binding relatively weakly to this protein, could also be transported
inside the cells via this system (Antony et al., 1985; Elwood et al., 1986;
Kane et al., 1986). Henderson et al. (1980) provided evidence for a similar
protein on the surface of L1210 mouse leukemia cells. Later, L1210 cells
were adapted to low folate conditions (1 nM folate), and isolated sublines
were found to express an unregulated FR that could bind folates and MTX
(again more weakly) and to also mediate their uptake (Henderson et al.,
1988). Jansen et al. (1989) also isolated an L1210 variant (L1210B73) with an
upregulated FR that was downregulated after transfer to high folate medium
( Jansen et al., 1989). Further studies with this cell line, which coexpressed
the RFC, suggested that the FR was minimally responsible for MTX uptake
under near physiological conditions, but that it may be an important
transport route for the quinazoline TS inhibitor, CB3717, as well as an
additional route for other novel antifolates such as raltitrexed and peme-
trexed that have much higher affinities for the FR than MTX ( Jansen et al.,
1999; Westerhof et al., 1991, 1995b). Another FR-expressing cell line,
L1210-FBP, devoid of a functional RFC, was found to be highly sensitive
(IC50s  1 nM) to a number of antifolate drugs including CB3717, ralti-
trexed, BW1843U89, plevitrexed, and pemetrexed, when tested under low
folate conditions (see structures in Fig. 7.4). That the non-RFC mechanism
of uptake was the FR had been demonstrated by the reduced activity of the
drugs when 20 nM FA was added to the test medium. Raltitrexed, but
particularly CB3717, displayed some FR-a-mediated activity against KB
cells that had been propagated in low folate conditions (e.g., 1 nM folinic acid)
Folate Receptor-Based Therapies 215

as demonstrated by decreased sensitivity when 20 nM FA was added

(Westerhof et al., 1995a). No FR-a-mediated effects could be detected in
epithelial IGROV-1 (ovarian tumor) or MA104 (mouse kidney) cells, both
of which express lower levels of the FR compared to KB. Thus, the
conclusion from these studies was that the FR-a had the potential to
substitute for the RFC as a folate transporter or to cotransport some
antifolate drugs in tumor cell lines expressing high levels of the FR.
Notably, similar conclusions were drawn for pemetrexed by Shih and
Thornton (1999) using ZR-75-1 human breast carcinoma sublines with
different transport characteristics.
The relevance of the FR to antifolate action under physiological folate
conditions was studied by Theti and Jackman (2004) using A431 cells trans-
fected with the FR (A431-FBP) and KB cells. These highly FR-expressing
tumor cell lines, which also express the RFC, were constantly exposed to
FA-free medium supplemented with 20 nM folinic acid (physiological
folate concentration) as the only folate source. To distinguish between
FR-versus RFC-mediated growth inhibition, antifolate activity was
also measured in the presence of 1 mM FA to competitively inhibit FR-
mediated uptake. In addition, activity was measured in the isogenic A431
cell line in which the FR was not expressed. Up to an eightfold increased
growth, inhibitory activity for raltitrexed, plevitrexed, pemetrexed, and
lometrexol (but not MTX) in at least one of the two cell lines was seen
under conditions where the FR was expressed and not blocked by
FA (Fig. 7.5). With the exception of the nonpolyglutamatable TS inhibitor,
plevitrexed, the exposure time did not affect the relative contribution that
the FR made toward the activity of these drugs. Interestingly, plevitrexed
did display considerable FR-a-mediated activity when the exposure period
was short. This was ascribed to the FR/endosomal apparatus serving as a
selective trap for the drugs. These data demonstrated the potential for some
antifolate drugs to be transported via the FR in tumors highly overexpres-
sing the receptor. Unfortunately, there is little evidence to suggest that cells
expressing low amounts of the FR will be sensitive to the high-affinity
antifolates already in clinical use (Gibbs et al., 2005). Furthermore, RNA
interference in HeLa cells (to knock down FR-a) was not found to affect
their sensitivity to pemetrexed (Chattopadhyay et al., 2004).

b. Development of selective FR-targeted antifolates Elements of FR-

targeting observed with the high-affinity antifolates suggested that it might
be possible to design an antifolate with a high degree of FR targeting giving
a much reduced exposure to normal tissues. Of all the high-affinity anti-
folates drugs studied in cell lines, CB3717 was the most specific for the FR,
as ascribed to its lower affinity for the RFC. Clinical activity in platinum-
refractory ovarian cancer had been reported with this drug, suggesting that
the FR might be implicated in the activity (Clarke et al., 1993). Thus,
216 Christopher P. Leamon and Ann L. Jackman

100 A431-FBP

Cell growth (% control)




0.0001 0.001 0.01 0.1 1 10 100
BGC945 concentration ( M)

Growth inhibitory IC50 ( M)

A431-FBP + FA
1 KB + FA

0.1 KB
















Figure 7.5 Relative selectivity of folate-based thymidylate synthase inhibitors for

folate receptor positive cell lines. Top panel, representative doseeffect curves for
BGC 945 against A431 (FR-negative) and A431-FBP (FR-positive) tumor cell lines.
Cells were exposed to BGC 945 for 3 days, and the endpoint for growth inhibition was
assessed using the MTT assay. The dotted line indicates the IC50 values. Bottom panel,
growth inhibitory IC50 values for TS inhibitors. A431 cells are FR-a-negative while
A431-FBP and KB cells are FR-a-positive. IC50 are also given for KB and A431-FBP
cells that had been coexposed to the TS inhibitors in the presence of 1 mM folic acid (to
competitively inhibit FR binding).

a group at the Institute of Cancer Research set out to design a TS inhibitor

with a range of properties that they believed would lead to the first truly
FR-targeted antifolate. Desirable properties included potent TS inhibition,
high affinity for the FR, low affinity for the RFC, and therefore growth
inhibition that was selective for FR-a overexpressing cell lines ( Jackman
et al., 2004). Importantly, substrate activity for folylpolyglutamyl synthetase
(FPGS) was considered undesirable. This was because non-FR-mediated
uptake into normal tissues was predicted to occur when exposure was high
immediately after iv dosing in vivo. For a polyglutamatable drug, this would
lead to the formation and retention of highly active polyglutamate forms in
proliferating tissues such as gut and bone marrow.
Folate Receptor-Based Therapies 217

Subsets of quinazoline-based TS inhibitors emerged that had many of

the desirable features, the most promising of which was a subset of cyclo-
penta[g]quinazolines with dipeptide ligands that included CB300638 (BGC
638) and CB300945 (BGC 945) (Bavetsias et al., 2000, 2002; Gibbs et al.,
2005; Henderson et al., 2006; Jackman et al., 2004; Theti et al., 2003). BGC
945, in particular, possessed exceptional FR and TS targeting against tumor
cell lines and against KB and IGROV-1 tumor xenografts (Gibbs et al.,
2005; Jackman et al., 2007a). BGC 945 (Fig. 7.4) is virtually a nonsubstrate
for the RFC with a Km 2-orders of magnitude higher than that of
conventional antifolates. The structural feature that contributes most to
this property is the dipeptide (L-Glu-g-D-Glu) moiety. The same modifi-
cation to the more usual single L-glutamate found in folates and most
antifolates also contributes to the potent TS inhibition (Ki 1.4 nM).
Further, the second glutamate is in the D rather than the L configuration
to prevent (1) metabolism by FPGS and (ii) hydrolysis of the peptide bond
by systemic hydrolases. Affinity for the FR-a is of the same order (50%) as
the high-affinity FA ligand and the conventional antifolate TS inhibitors
(50150%). These properties combine to give high and low cytotoxic
potency against tumor cell lines that are FR-a-positive and -negative,
respectively (Fig. 7.5) (Gibbs et al., 2005). For example, the growth inhibi-
tory IC50 for BGC 945 is 1 nM in FR-a-transfected A431-FBP cells, and it
is 3 orders of magnitude lower than the IC50 in the parental A431 cells (IC50,
6.9 mM). Similarly, FR-a-positive KB cells are highly sensitive to this agent
(IC50, 3.3 nM). That the FR is the uptake mechanism for BGC 945 was
demonstrated by an 1000-fold increase in IC50 when cells were supplied
with an excess of FA (1 mM) to competitively and selectively inhibit binding
of BGC 945 to the FR. This concentration of FA has no effect on the IC50 of
BGC 945 against the FR negative A431 cells (Gibbs et al, 2005).
Evaluation of TS inhibitor efficacy in rodent systems is complicated by
the 100-fold higher plasma thymidine (dThd) levels compared with humans
( Jackman et al., 1984; Li et al., 2007). Salvage of dThd by thymidine kinase
leads to circumvention of TS inhibition and consequently low efficacy in
tumor models and low levels of mechanism-based toxicity ( Jackman et al.,
1984). This can be overcome partially by chronic administration schedules
of conventional TS inhibitors, for example, daily 514 days ( Jackman
et al., 2007b). However, these schedules in rodents overpredict exposure
required for efficacy and mechanism-related toxicity toward proliferating
tissues in species such as dogs and humans that have low dThd levels. For
this reason, the preclinical development of BGC 945 has largely been
dependent on the use of pharmacodynamic endpoints of TS inhibition in
mouse tumor and normal proliferating tissues to determine the optimal
exposure required to give a high therapeutic index. Tumor dUrd (KB and
IGROV-1) and plasma dUrd measurements have been used as markers of
localized and normal proliferating tissue TS inhibition, respectively, and
218 Christopher P. Leamon and Ann L. Jackman

they have indicated that the therapeutic window is encouragingly large

(Fig. 7.6) (Forster et al., 2005; Jackman et al., 2007a). In addition, the
increased flux through the dThd salvage pathway that occurs when TS is
inhibited has been exploited by the use of isotopically labeled dThd or dThd
analogues. For example, it was demonstrated that 100 mg/kg 6-R,S-BGC
945 induced tumor-selective TS inhibition, whereas the less FR-selective
agent, BGC 9331, induced additional effects on proliferating tissues such as
gut (Gibbs et al., 2005). Furthermore, very compelling data for the unique
FR-targeted nature of BGC 945 came from positron emission tomography
studies in which 18F-dThd (FLT) was used to image TS inhibition in KB
tumor-bearing mice (Prof. E. Aboagye, Imperial College, London, personal
communication). Pharmacokinetic analysis demonstrated remarkably slow
clearance from KB tumor (t1/2 28 h) so that drug levels fell to 1 mM at 8 h
and then remained at approximately this level for at least 72 h. Plasma and liver

10 * 10
Plasma Plasma

8 8
Plasma dUrd (mM)
Plasma dUrd (mM)

6 6

4 4

2 2

0 0
0 50 0 50
BGC 9331 (mg/kg) BGC 945 (mg/kg)

40 Tumor 40 Tumor
* *
Tumor dUrd (mM)
Tumor dUrd (mM)

30 30

20 20

10 10

0 0
0 50 0 50
BGC 9331 (mg/kg) BGC 945 (mg/kg)

Figure 7.6 Plasma and tumor deoxyuridine (dUrd) levels 24 h after dosing KB tumor-
bearing mice with 50 mg/kg BGC 9331 (plevitrexed) or BGC 945. Each bar represents
the mean  SEM for n 1018 mice from three to four independent experiments. *p <
0.05 compared with controls. Elevated plasma dUrd is a pharmacodynamic endpoint of
TS inhibition in proliferating tissues such as gut and bone marrow, while elevated
tumor dUrd is a marker of TS inhibition in the tumor.
Folate Receptor-Based Therapies 219

levels were less than 25 nM at 16 h (Gibbs et al., 2005). Phase 1 clinical studies
with BGC 945 are planned for 2008, and some of the issues relating to
evaluation of this first in man FR-targeted TS inhibitor are discussed in a
recent review ( Jackman et al., 2007b).

2. Folate-targeted chemotherapy
a. Modular design The success of FolateScan (99mTc-EC20; see Section
III.A.2) helped to prove that an FA conjugate could rapidly and easily
penetrate solid tumor tissue in patients. This finding prompted some to
investigate folates ability to deliver potent therapeutic molecules to can-
cer. Yet, initial attempts toward producing active and specific folate-drug
conjugates met with limited success (Leamon and Reddy, 2004). Endo-
cyte, Inc. has remained very active in this area of research, and following
years of troubleshooting and numerous structure-activity studies, some
general rules for producing successful lead conjugates have emerged.
For example, many small molecule cytotoxics (both naturally occurring
and synthetic) are highly lipophilic. This property is typically beneficial to
an agent that must penetrate through the bilayer of a cell before exerting
biological activity, but it becomes an unfavorable trait as one considers
(1) the difficulties of formulating the agent for parenteral administration
and (2) the potential off-target toxicities that usually emerge when the
drug indiscriminately enters normal cells. Whereas FA is appreciably water
soluble at physiological pH owing to its two glutamyl-based carboxylic
acid groups, direct covalent attachment of a drug to either one of those
carboxyl sites does not ensure aqueous solubility of the resulting conju-
gate. Such a result is in fact quite rare. Instead, a hydrophilic spacer can
be placed in-between the folate and drug moieties to effectuate high
water solubility (Leamon et al., 2006). A second benefit that a spacer offers
is to physically separate the targeting moiety (folate) from the drug payload
to maximize its potential to bind to the FR. The spacers composition
can widely vary because peptides, polymers, and even polycarboxylic
acid structures have been found to be very effective (Endocyte, Inc.,
unpublished data).
In addition to a spacer, all highly potent small molecular weight folate
drug conjugates have thus far been constructed with a biologically cleavable
linker (Ladino et al., 1997; Leamon et al., 2005, 2006, 2007a,b; Reddy et al.,
2006, 2007a,b). Referring back to Fig. 7.1, folate conjugates are endocy-
tosed into the cell after binding to the FR. Within the vesicular compart-
ments, the conjugates are exposed to a mildly acidic as well as reducing
environment (Leamon and Reddy, 2004). It was once assumed that the low
pH inside the endosomes triggered a conformational change in the FR to
afford release of the bound folate (or folatedrug conjugate). This event
likely occurs for some of the reduced folates that can bind to the FR with
reasonable affinity, like 5-methyltetrahydrofolate; but for FA, which is the
220 Christopher P. Leamon and Ann L. Jackman

fully oxidized, highest affinity ligand to the FR (and the form of folate that
is most commonly used in the construction of folatedrug conjugates),
endosomal release from the FR may not be very efficient. Evidence
supporting this theory has come from acid-stripping recycling studies with
3H-FA (Kamen et al., 1988), and more recently, with studies using FA-

based fluorescence resonance energy transfer probes (Yang et al., 2006,

2007). Overall, the predominant route for FA conjugates appears to be
high-affinity binding followed by endocytosis and recycling of the FR
folate complex back to the cell surface under normal conditions. Indirect
but supportive data has also come from the observations that folatedrug
conjugates constructed with stable linkers (e.g., amide bonds) do not pro-
duce pharmacological activity whereas their releasable linker-containing
counterparts do (Leamon et al., 1993). Considering all of the aforemen-
tioned research, a modular design for constructing folatedrug conjugates
evolved, as exemplified in Fig. 7.7. Here, pteroic acid (Pte) typically func-
tions as Module 1, while the drug moiety is placed in the Module 4 position.
A Glu moiety is usually placed within the spacer (Module 2) at a position
juxtaposed to Pte. Importantly, the combination of the Pte and Glu moi-
eties produces FA. Therefore, these molecules are typically referred to as
folate conjugates. However, we and others have found that the Glu
residue of FA is not critical for FR recognition (Leamon et al., 1999,
2003; Muller et al., 2004, 2006b). Finally, Module 3 is reserved for
a cleavable bond.

b. Preclinical pharmacology Both acid-labile and reducible (i.e., disul-

fide) linkers have yielded active folate conjugates. Over the last few years,
the Endocyte team has published on the synthesis and preclinical pharma-
cology for novel folate conjugates of mitomycin C [MMC; (Leamon et al.,
2005; Reddy et al., 2006)], desacetylvinblastine monohydrazide [DAVLBH;
(Leamon et al., 2006, 2007b; Reddy et al., 2007a)], and maytansinoid DM1

Spacer Linker Drug

Module 1 Module 2 Module 3 Module 4

(targeting ligand) (spacer) (cleavable bond) (base drug)

Figure 7.7 Modular design of folatedrug conjugates. A folatedrug conjugate con-

sists of four basic modules: (1) a pteroate ligand, (2) a spacer, (3) a biologically cleavable
bond, and (4) a potent drug moiety.
Folate Receptor-Based Therapies 221

(Reddy et al., 2007b). Throughout this unrelenting venture, this team has
repeatedly observed marked antitumor effect against FR-expressing tu-
mors across multiple animal models using well-tolerated treatment regimens
(e.g., cures are typically observed under conditions that cause little to no
weight loss). A typical example of this phenomenon is shown in Fig. 7.8.
Here, a folate conjugate of a powerful antimicrotubule agent was adminis-
tered intravenously to mice bearing well-established subcutaneous human
tumor xenografts. Following a brief three times per week, 2-week schedule,
animals were declared tumor-free (Panel A). In fact, the tumors never
recurred up to the 110 day study endpoint (not shown); furthermore, the
activity was not accompanied by any weight loss (Panel B), and it was

A 1600

Tumor volume (mm3)

1000 0/5 cures
400 5/5 cures
10 20 30 40 50 60
PTI (days)

B 25

(%) Weight change


10 20 30 40 50 60
PTI (days)

Figure 7.8 Antitumor activity of a folatedrug conjugate. FR-positive human KB cells

were inoculated sc in nu/nu mice. Eleven days later, animals were treated intravenously
with a folatedrug conjugate on days 11, 13, 15, 18, 20, and 22 either alone () of with a
40-fold excess of free folate (). A control cohort () was not treated. Tumor volume
was assessed every 23 days. The dotted vertical line represents the day of final dosing.
Data represent the average  1 SD (n 5 animals per cohort). Panel A, activity; Panel
B, change in body weight. PTI, post-tumor implantation.
222 Christopher P. Leamon and Ann L. Jackman

competable with an excess of coinjected folates (i.e., an indicator of FR-tar-

geted specificity). The consequence of such findings typically allows for an
increase in therapeutic index (over the unconjugated drug) as well as for the
potential to dose folate-drug conjugates more frequently (e.g., dose-dense
regimens) (Reddy et al., 2007a). Experience has further shown that anti-
tumor activity is more often observed when the conjugated drug is intrinsi-
cally very potent, such as those with single digit nanomolar IC50 values
in vitro (Leamon and Reddy, 2004; Leamon et al., 2005, 2006). This latter
concept also adds value in enabling some highly toxic compounds to be
dosable (as a folate conjugate).
The Endocyte research team has also recognized the probable benefit for
tethering not only multiple copies of the same drug to folate but also the
simultaneous tethering of different drug molecules to this vitamin. An
example of the latter was recently published. Here, the molecule called
EC0225 represents the first in class multidrug, folate-targeted agent to be
disclosed. It is constructed with a single folate moiety, extended by a
hydrophilic peptide-based spacer, which in turn is attached to Vinca alkaloid
and mitomycin units via two distinct disulfide-containing linkers. Despite
its large, bulky size (molecular weight 2327 g/mol), EC0225 retained its
high affinity for FR-positive cells, and it was found to produce potent dose-
responsive activity in vitro via an endocytic mechanism. EC0225 further
proved to be very active against syngeneic and xenograft in vivo models,
with curative activity occurring with the administration of well-tolerated
regimens (Leamon et al., 2007a).

c. Clinical experience To date, two folate-drug conjugates have entered

clinical trials. The first, EC145, is a folate-Vinca alkaloid conjugate that was
evaluated in 2006 for safety and pharmacokinetics in a Phase 1 trial
(Sausville et al., 2007). The study treated refractory cancer patients with
either an intravenous (IV) bolus dose or a 1 h IV infusion on days 1, 3, 5
(week 1) and 15, 17, 19 (week 3) of a 4-week cycle. EC145 was generally
found to be well tolerated at doses up to 2.5 mg. Interestingly, one ovarian
cancer patient reportedly achieved a partial response, with declining
CA125, and she remained on study for more than 9 months; another
ovarian cancer patient exhibited stable disease for less than 5 months with
declining CA125. Importantly, Phase 2 trials were initiated in mid-2007
and interim results should be available in early 2008.
A Phase 1 trial for a second agent, EC0225 (the targeted dual-drug
conjugate; see above), began in March of 2007. This study is currently
evaluating bolus IV administration of EC0225 on days 1, 3, and 5 (i.e., week 1)
and on days 8, 10, and 12 (i.e., week 2) of a 4-week cycle. It is a standard dose
escalation study designed to identify the dose and schedule that will be used
in future Phase 2 (efficacy) trials.
Folate Receptor-Based Therapies 223

3. Folate-targeted immunotherapy
A successful cancer immunotherapy may be one that teaches the immune
system to recognize and destroy malignant tumor cells while simultaneously
building a long-lasting antitumor memory (Blattman and Greenberg, 2004).
To date, one particular approach has been reported whereby folate is used to
deliver a hapten (fluorescein, or FITC) molecule to FR-positive tumors
(Lu and Low, 2002; Lu et al., 2004, 2005, 2006). This immunotherapy is
composed of an FITC-based vaccine, the targeted small molecule conjugate,
EC17 (folateFITC), and immunostimulatory cytokine(s). The vaccine
component is formulated with a Th1-biased adjuvant to stimulate the
production of antihapten antibodies. Following the formation of an anti-
FITC titer, which generally takes a few weeks, the folateFITC conjugate is
administered subcutaneously to stimulate the formation of a bispecific
molecular bridge between the tumor surface FR and endogenous anti-
FITC antibodies. This process effectively marks the tumor cells for
immune recognition and also initiates a specific antitumor response that
can be potentially enhanced upon costimulation with the cytokines inter-
leukin-2 and interferon-a (Lu and Low, 2002; Lu et al., 2004, 2005, 2006).
A Phase 1 safety trial for this program, called FolateImmune, was
successfully completed in 2007 (Amato et al., 2005; Messmann et al.,
2007). At present, evaluation of this targeted hapten therapy continues in
Phase 2 clinical trials of renal cell carcinoma, an immune-responsive cancer
with 64% FR positivity (Endocyte, internal communications).

C. Inflammation therapy
As discussed above (Section IIA), the two principal tissue-associated FR
isoforms are FR-a and FR-b. Despite their different tissue expression
profiles, both isoforms can efficiently bind the FA ligand as well as drug
conjugates thereof. FR-a is indeed more prevalent among epithelial-based
pathologies, but its b counterpart has been found in some solid cancers as
well as AML samples (Ross et al., 1994, 1999). Interestingly, FR-b was also
identified on the surface of activated (not resting) macrophages, particularly
in the inflamed tissues from rheumatoid arthritis patients (Nakashima-
Matsushita et al., 1999).
While its role, or physiological relevance, has not yet been firmly
established, it is known that FR-b can be targeted with folatedrug con-
jugates. For example, 99mTc-EC20 (FolateScan; see Section III.A.2) was
reported to concentrate in the livers, spleens, and arthritic extremities of
adjuvant-induced arthritic rats via a folate-dependent mechanism (Turk
et al., 2002). The uptake within these tissues was also shown to be due to
resident ED2-positive macrophages. Similarly, near-infrared fluorescent-
labeled folate probes were reported to accumulate within the inflamed
224 Christopher P. Leamon and Ann L. Jackman

arthritic joints of mice via a folate-targeted mechanism (Chen et al., 2005),

and activated peritoneal macrophages were found to capture 10 times more
folate-labeled liposomes than their coresident FR-positive tumor cells
(Turk et al., 2004).
Knowing that activated, TNF-a producing macrophages express FR-b,
and that they are targetable in vivo, quickly allowed for the testing of possible
FR-based therapies. The first reported approach was actually a modified
form of the targeted immunotherapy described in the preceding section.
Thus, paw swelling in rats immunized against a hapten (prior to the induc-
tion of adjuvant arthritis) was profoundly decreased in a manner that was
attributed to folatehapten targeting (Paulos et al., 2004). While no supple-
mental cytokine therapy was needed to observe this effect, this therapeutic
approach was found to be reproducible in multiple rodent models, and was
at least comparable in activity to that produced by methotrexate, etanercept,
anakinra, and celecoxib (Paulos et al., 2006). A second therapeutic approach
was also reported recently that utilized an FR-b-specific monoclonal anti-
body conjugated with the potent protein synthesis inhibitor, Pseudomonas
exotoxin A (Nagayoshi et al., 2005). Interestingly, this immunotoxin was
observed to induce apoptosis and reduce the numbers of macrophages in
RA synovial tissue engrafted into SCID mice (Nagai et al., 2006). To date,
there have been no publications regarding inflammation therapy with small
molecule folatedrug conjugates. However, such programs are currently
being resourced and preclinical leads may soon follow.

D. Modulation of FR expression levels

Resistance to some of the aforementioned FR-targeted therapies will
almost certainly be, in part, due to inadequate expression of the FR. For
example, there is a relationship between the growth inhibitory IC50 for
BGC 945 and FR expression levels in a small panel of cell lines (Gibbs et al.,
2005). A similar relationship also exists for some folate conjugates, in
particular those that are constructed with less-potent drug payloads
(C. Leamon, personal observations). It is now known that the amount of
FR expressed within a tissue is regulated transcriptionally and posttranscrip-
tionally by discrete regulatory elements in the genes or the mRNA (Elnakat
and Ratnam, 2006; Matherly and Goldman, 2003). Increased transcription
can be observed in FR-a expressing cultured cells in response to their
transfer to subphysiological concentrations (low or subnanomolar) of folate.
Notably, folate-dependent translational regulation, involving a 46 kDa
cytosolic protein (hnRNP E1), has been reported in cervical carcinoma
cells (Pillai et al., 2003).
Some studies suggest that it may be possible to increase endogenous FR
expression levels in tumors with the use of clinically approved agents. For
example, the FR-a promoter is negatively regulated by the estrogen
Folate Receptor-Based Therapies 225

receptor, particularly in the presence of the ligand, estrogen. Tamoxifen, via

its antagonizing action of estrogen, has been shown to derepress transcrip-
tion and increase FR-a expression up to 36-fold in HeLa-I-1 cells that
express both the ER and FR-a (Kelley et al., 2003). Another steroid that
increases transcription of the FR-a is dexamethasone, a positive regulator of
the FR-a gene. While known to be mediated via its agonistic activity on the
glucocorticoid receptor, dexamethasones effects on the FR-a appear to be
indirect (Tran et al., 2005). Interestingly, these nuclear receptors appear to
only modulate expression of the FR-a in cells in which this protein is
already transcriptionally active. Histone deacetylase (HDAC) inhibitors,
such as valproic acid or SAHA, particularly when combined with dexa-
methasone, have also been shown to increase FR-a expression. This is
presumably related to the fact that histone acetylation is important for the
transcriptional activity of nuclear receptors because nuclear receptor cor-
epressors recruit class II HDACs, thereby inducing a transcriptionally
repressed state (Tran et al., 2005).
The expression level of FR-b can also be modulated. Here, all-trans
retinoic acid appears to be useful for increasing FR-b in AML cells. This
effect is mediated via the retinoid nuclear receptors, RARa, RARb, and
RARg that act on a common downstream target (Elnakat and Ratnam,
2004). Importantly, transcriptional upregulation of this nonclassical target
can be further potentiated by HDAC inhibitors.
Finally, there is accumulating evidence from mouse xenograft studies to
suggest that modulation of FR expression by these nuclear receptor ligands,
possibly in combination with HDAC inhibitors, may be feasible in a clinical
situation for tumors that express the appropriate nuclear receptors.
Although this concept has not yet been evaluated clinically, it may prove
to be a useful addition to some antifolate and even folatedrug conjugate
therapies, especially for those patients bearing disease with limited levels of
FR expression.

IV. Future Prospects

The FR is viewed as a potentially useful biological target, certainly
for the management of many human cancers. To date, there are several
different therapeutic approaches being evaluated clinically that exploit the
function and/or presence of the FR in human malignancies, and a few more
are yet to come. Preliminary Phase 1 evidence suggests that both the small
molecule and biological approaches may some day yield promising thera-
peutic alternatives for patients having FR-positive disease. The FR may be
utilized either to deliver potent drugs inside cells or as an immunological
marker; in both cases, it is clearly thought to be a rational target for the
226 Christopher P. Leamon and Ann L. Jackman

oncology and possibly inflammation sectors of drug development. As more

knowledge is gained from clinical evaluation and new insights on modulat-
ing the expression levels of this protein emerge, FR-based medicines could
provide the clinician with critical therapeutic alternatives for disease

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Folate Receptor Expression in

Pituitary Adenomas: Cellular
and Molecular Analysis
Chheng-Orn Evans,* Congjun Yao,* David LaBorde,*
and Nelson M. Oyesiku*

I. Introduction 236
II. Methods 238
A. Cell culture 238
B. Stable transfection 238
C. Western blotting procedures 238
D. Cell proliferation assay 239
E. Soft agar colony assay 239
F. BrdU incorporation assay 239
G. Flow cytometric assessment of PCNA expression 239
H. Folic acid binding 240
I. Quantitative RT-qPCR 240
J. Statistical analyses 240
III. Result 242
A. Tumor classification 242
B. FRa mRNA expression in NF adenomas 242
C. Expression of FRa protein 242
D. Assessment of FRa-binding capacity 246
E. Immunohistochemical analysis of FRa expression 247
F. Selection of clones of aT3-1 cells stably expressing FRa
and mFRa 249
G. FRa induces cell proliferation in aT3-1 cells 250
H. FRa induces cell cycle progression and PCNA expression 251
I. FRa promotes growth in soft agar assay 251
J. [3H]Folic acid binding 252
K. FRa induces aT3-1 cell growth through NOTCH3 pathway 252
L. In vivo imaging of FR expression 258

* Department of Neurosurgery and Laboratory of Molecular Neurosurgery and Biotechnology,

Emory University School of Medicine, Atlanta, Georgia, 30322

Vitamins and Hormones, Volume 79 # 2008 Elsevier Inc.

ISSN 0083-6729, DOI: 10.1016/S0083-6729(08)00408-1 All rights reserved.

236 Chheng-Orn Evans et al.

IV. Discussion 259

Acknowledgments 263
References 263

Clinically nonfunctional pituitary adenomas cause hypopituitarism or compres-
sion of regional structures. Unlike functional tumors, there is no available
medical treatment or specific imaging technique for these tumors. We have
recently discovered that both folate receptor (FR)a mRNA and protein are
uniquely overexpressed in nonfunctional pituitary tumors, but not in functional
adenomas. We hypothesized that FRa may hold significant promise for medical
treatment by enabling novel molecular imaging and targeted therapy. Here, we
used murine pituitary tumor cell line aT3-1 as a model to investigate the
biological significance of FRa and its mutant FR67. We demonstrate that over-
expression of FR facilitated tumor cell growth and anchorage-independent
growth in soft agar. More colonies were observed in FR overexpressing cells
than in mutant FR67 clones in soft agar. Cell proliferation rate was increased,
the percentage of cells in S-phase was increased, and high PCNA staining was
detected in cells overexpressing the receptor. In aT3-1 cells transfected with
mutant FR67, cell proliferation rate was reduced, the percentage of cells resid-
ing in S-phase was slightly decreased, and low PCNA staining was observed. By
real-time quantitative PCR, the genes involved in NOTCH3 pathway including
NOTCH3, HES-1, and TLE2 were altered; the mRNA expression of FGFR1 was
upregulated, and ERb mRNA was downregulated in FR overexpressing cells. Our
findings suggest that FRa plays a role in pituitary tumor formation, and this
effect may in part be due to its regulation of the NOTCH3 pathway. 2008
Elsevier Inc.

I. Introduction
Pituitary tumors are mostly benign adenomas arising from adenohypo-
physeal cells in the anterior pituitary. They comprise 10% of all brain tumors
and occur in 20% of the population. They cause significant morbidity by
compression of regional structures and the inappropriate expression of
pituitary hormones (Asa, 1998; Greenman and Melmed, 1996). Functional
tumors, such as GH and ACTH adenomas, give rise to severe life-threatening
clinical syndromes, such as Acromegaly or Cushings disease, and PRL
adenomas result in impaired reproduction. However, 30% of all anterior
pituitary adenomas are termed nonfunctional (NF) pituitary adenomas due
to their lack of clinical hormone hypersecretion (Asa and Kovacs, 1992).
Clinically, NF tumors manifest as hypopituitarism or visual field defects due
to regional compression of the optic chiasm (Asa and Ezzat, 1998; Asa and
Kovacs, 1992; Black et al., 1987; Katznelson et al., 1993). The NF tumors are
uniquely heterogeneous (Table 8.1). They typically are quite large and cause
Folate Receptor Expression in Pituitary Adenomas 237

Table 8.1 Classification of NF adenomas by cell of origin

Cell type Hormone expression NF tumors (%)

Null cell None 17
Oncocytoma None 6
Silent corticotroph ACTH 8
Silent somatotroph GH 3
Gonadotrophs Intact LH/FSH or subunits 4079

hypopituitarism or blindness from regional compression (Greenman and

Melmed, 1996). Despite the lack of clinical hormone hypersecretion,
immunocytochemical staining for hormones reveals evidence for hormone
expression in 79% of these tumors, and we refer to these as immunohisto-
chemically positive (NF). The remainder is negative for hormone expression
(Asa et al., 1992; Katznelson et al., 1993) and these are referred to as
immunohistochemically negative (NF).
In a previous study (Evans et al., 2001), we used cDNA microarray
analysis and real-time quantitative PCR (RT-qPCR) to compare expression
profiles of 7075 genes in the normal pituitary with that in different adeno-
mas, including NF, PRL-producing adenomas, GH-producing adenomas,
and ACTH-secreting adenomas. In those experiments, we found that the
folate receptor (FR)a gene was significantly overexpressed in NF adeno-
mas. Next, we have discovered that both FRa mRNA and protein are
uniquely overexpressed in NF tumors, but not in functional adenomas
(Evans et al., 2003; Moreno et al., 2005). But whether or how FRa plays a
role in pituitary tumorigenesis is unclear. The FR genes are located on
chromosome 11q13.313.5, a region commonly amplified in carcinomas
of the head and neck and breast (Rijnboutt et al., 1996). There are three
isoforms of FR (FRa, FRb, and FRg) that vary in sequence, ligand
preference, and tissue distribution (Miotti et al., 1987; Shen et al., 1994).
FRa (GenBank U20391) is the major isoform mediating folate transport and
is the subject of this study. The FRa receptor is absent or weakly expressed in
most normal tissues. FRa is vastly overexpressed in tumors such as ovarian,
renal, breast, and colorectal carcinomas, as well as anaplastic ependymomas,
choroid plexus, and pituitary tumors (Evans et al., 2001; Kane et al., 1986;
Mathias and Green, 1998; Mathias et al., 1998; Miotti et al., 1987).
It is suggested that elevated levels of FRa induce cell proliferation not
only by mediating folate uptake but also by generating other regulatory
signals. In vitro studies demonstrate that cellular overexpression of the FRa
results in enhanced proliferation and survival by providing enhanced folic
acid uptake (Antony, 1992; Chung et al., 1993; Kane et al., 1986; Luhrs
et al., 1992; Ross et al., 1994).
238 Chheng-Orn Evans et al.

Cell culture studies also demonstrate that some NF tumors secrete

hormone in vitro (Yamada et al., 1989). Unlike functional tumors, currently,
there is no available medical treatment or specific imaging technique for
these NF tumors.
In order to study the possible influence of FRa overexpression in NF
pituitary tumors cell growth, we analyzed the biochemical and the
biological characteristics of the murine aT3-1 cell transfected with FRa
cDNA and a mutant FR67 cDNA. We took advantage of this mutant FR67
in our current study because it significantly inhibited folate binding and
uptake in one tumor cell line in other studies (Orr and Kamen, 1994, 1995).
We also examined molecules that might physically and functionally associ-
ate with FRa in order to elucidate the involvement of the receptor in signal
transduction of pituitary adenoma pathogenesis.

II. Methods
A. Cell culture
Cells (aT3-1) were maintained in monolayer culture in high-glucose
DMEM (Invitrogen, Carlsbad, California) with 10% FBS and were grown
in folate-free RPMI with 5% FBS for all the experiments in humidified
5% CO2 at 37  C. Cells were routinely passaged with 0.5 mmol/liter
EDTA in phosphate-buffered saline. Cell numbers were determined by

B. Stable transfection
The cells were plated in 35-mm culture wells at 5  105 cell/ml and cultured
for 1 day. The following day, the transfections were performed by a lipofection
method (LipofectamineTM 2000, Invitrogen). DNA (4 mg) was mixed
with 10 ml of LipofectamineTM 2000 in 500 ml of Opti-MEM , and added
to the cell. The cells were passaged at 1:10 dilution into fresh growth medium
24 h after transfection. Zeocin, 300 mg/ml was added for stable selection.

C. Western blotting procedures

For total cell extracts, cells were washed twice with PBS, scraped, and cen-
trifuged at 1000 rpm for 5 min. The pellet was suspended in denaturation
buffer (10 mmol/liter Tris, 150 mmol/liter NaCl, 2 mmol/liter EDTA,
pH 7.4, and 0.2% SDS and were denatured at 100  C for 5 min. Protein
concentration was determined using the bicinchoninic acid protein assay
and proteins were resolved on a 15% SDS acrylamide gel and transferred to a
nitrocellulose membrane. FRa was detected by incubating the membrane
Folate Receptor Expression in Pituitary Adenomas 239

with polyclonal antihuman FRa antibody, followed by HRP-conjugated

anti-rabbit IgG secondary antibody. Visualization was carried out on X-ray
film by enhanced chemiluminescence. b-Actin expression was detected to
ensure equal protein loading.

D. Cell proliferation assay

Cells growing in log phase were lifted using EDTA and seeded in 24-well
plates (1  104 cells/well in a final volume of 500 ml) in triplicates and
incubated at 37  C in 5% CO2. The cells were incubated for 10 days and the
media were changed every 3 days. Every other day, the cells were counted
with Trypan blue in hematocytometer. Each experiment was repeated three

E. Soft agar colony assay

Cells (5  103) from each cell line collected using EDTA were suspended in
0.3% agar in DMEM containing 10% FBS and plated on 0.7% solidified agar
in 35 mm dishes. After 15 days of culture, colonies with more than 20 cells
were counted and photographed.

F. BrdU incorporation assay

BrdU is only incorporated into the DNA of proliferating cells. BrdU
incorporation assay was carried by pulse labeling the cells with BrdU for
30 min. The levels of BrdU incorporated into cellular DNA were quanti-
fied by anti-BrdU antibody; total DNA was stained by 7-AAD. The samples
were analyzed by BD FACScan and data were evaluated using Flowjo
software. This two-color flow cytometric analysis permits the enumeration
and characterization of cells that are actively synthesizing DNA (BrdU
incorporation) in terms of their cell cycle position (i.e., G0/1-, S-, or
G2/M-phases, defined by 7-AAD staining intensities).

G. Flow cytometric assessment of PCNA expression

PCNA is a nuclear antigen that is only expressed in proliferating cells and
absent in resting cells. For PCNA analysis, cells were washed with 1% FBS/
PBS and fixed with cold 70% ethanol overnight in 50 ml tube. After
removing ethanol, the cells were incubated with an anti-PCNA,
fluorescein-conjugated monoclonal antibody, or isotype-matched control
IgG (Pharmingen, Becton Dickinson, Jan Hose, California) for 30 min at
room temperature. The stained cells were then washed and fixed with
formaldehyde. Fluorescence data from 104 cells were collected and histogram
analysis was performed with the Flowjo software.
240 Chheng-Orn Evans et al.

H. Folic acid binding

[3H]folic acid purification and solubilized folic acid binding were performed
as described (Doucette and Stevens, 2001). Cells were grown in T-25 in
folate-free RPMI for 3 days and were selected with 300 mg/ml of Zeocin
when applicable. After washing twice with PBS and centrifugation for
5 min at 1000 rpm, the pellet was solubilized in 1-ml ice-cold solubilization
buffer (25 mmol/liter TrisHCl, pH 7.5, 150 mmol/liter NaCl, 5 mmol/
liter EDTA, and 1% (v/v) Triton X-100) for 20 min. [3H]folic acid
(20175 nmol/liter, depending on the cell line) was added and the samples
were incubated at 37  C for 1 h in shaking water bath to allow ligand
binding to the receptor. The samples were then placed on ice for 5 min,
and 1 ml of dextran-coated charcoal solution (25 mmol/liter TrisHCl, pH
7.5, 150 mmol/liter NaCl, 5 mmol/liter EDTA, 8 mol/liter dextran, and
80 mol/liter activated charcoal) was added to each tube to absorb unbound
radiolabel. The samples were vortexed and incubated on ice for 15 min,
followed by centrifugation for 30 min at 3400 rpm at 4  C. Aliquot of 1 ml
of each supernatant was counted by scintillation counting. Nonspecific
binding was determined in each experiment by measuring [3H]folic
acid binding in the presence of 750-fold excess unlabeled folic acid. Specific
binding values were determined by subtracting the nonspecific value from
the respective total radioactivity.

I. Quantitative RT-qPCR
Expression of the selected genes was quantified using RT-qPCR analysis.
Briefly, total RNA was isolated using the Trizol reagent (Invitrogen) and
was reverse transcribed using Supersript II RT (Invitrogen). Specific pri-
mers were designed using Primer3 software (MIT Whitehead Institute, and listed in
Table 8.2. All PCR reactions were performed in GeneAmp 5700 sequence
detection system (Applied Biosystems) and repeated in three separate
experiments, each sample was run in duplicate. b-Actin and b-tubulin
were used as internal control. Cycle threshold (Ct) values were obtained
and the relative differences in expression between groups were determined
using the 2(ddCT) formula, where ddCT (Ct gene of interest  Ct
actin or tubulin in experimental sample)  (Ct gene of interest  Ct actin or
tubulin in untransfected, untreated aT 3-1 cells sample).

J. Statistical analyses
Results are expressed as mean  SEM. Differences were assessed by one-
way Anova to all results except the binding assay where student t-test was
used. p < 0.05 was considered to be statistically significant.
Table 8.2 Primer sequences used for quantitive RT-qPCR

Gene Forward primer Reverse primer

Wang et al. (2007).
Gao et al. (2004).
242 Chheng-Orn Evans et al.

III. Result
In a previous study (Evans et al., 2001), we used cDNA microarray
analysis and RT-qPCR to compare expression profiles of 7075 genes in the
normal pituitary with that in different adenomas, including NF, PRL-
producing adenomas, GH-producing adenomas, and ACTH-secreting ade-
nomas. In those experiments, we found that the FRa gene was significantly
overexpressed in NF adenomas. Next, we characterized the expression of
FRa in NF and NF, PRL, GH, and ACTH pituitary adenomas (Evans
et al., 2003). The identification of FRa may further elucidate the pathways
of pituitary oncogenesis and provide effective therapeutic treatment to
pituitary tumors.
We show some of the importance results of our previous study (Evans
et al., 2003) in following sections.

A. Tumor classification
The clinical and pathological characteristics of the 39 adenomas used in the
study are listed in Table 8.3. Ten of the NF adenomas were not positive
with anterior pituitary hormone histochemistry and were designated immu-
nohistochemically negative (NF) tumors. Thirteen NF tumors stained
with one or more anterior pituitary hormones and were designated immu-
nohistochemically positive (NF). Six were classified as PRL, five as GH,
and five as ACTH-positive adenomas. With the exception of two with
cavernous sinus invasion, all tumors were noninvasive as defined by histo-
logical and radiological criteria. Other clinical features related to the tumor
are noted in Table 8.3. Five normal pituitary controls were obtained from
the National Hormone and Pituitary Program, National Institute of Diabe-
tes and Digestive and Kidney Diseases.

B. FRa mRNA expression in NF adenomas

We investigated the relative expression levels of FRa mRNA in NF
adenomas by RT-qPCR. We found that there was significant overexpres-
sion of FRa mRNA in 13 NF adenomas from 17- to 174-fold compared
with the controls ( p 0.001) (Fig. 8.1).

C. Expression of FRa protein

Whether the high levels of FRa mRNA resulted in overexpression of FRa
protein was investigated with Western blotting of 39 pituitary tumors and 5
normal pituitaries using a highly specific polyclonal antibody against this
Folate Receptor Expression in Pituitary Adenomas 243

Table 8.3 Clinical and pathological characteristics of adenomas from patients used
in the study (Evans et al., 2003)

Patient ID Sex, Age Clinical features/tumor size IHC

93 M, 57 NF, visual loss, cavernous Nega
sinus invasion, 1.5 cm
74 M, 67 NF, hypogonadism, Neg
2.5 cm
164 M, 35 NF, visual loss, 4 cm Neg
153 M, 72 NF, hypogonadism, 2 cm Neg
155 M, 66 NF, headache, visual loss, Neg
1.5 cm
104 F, 61 NF, visual loss, 1.5 cm Neg
105 F, 48 NF Neg
75 M, 60 NF, visual loss, 2 cm Neg
68 M, 74 NF Neg
191 F, 44 NF Neg
65 F, 54 NF FSH 1, LH 2
77 M, 67 NF FSH 2
174 F, 53 NF FSH 1, LH 1
143 M, 60 NF FSH 1, LH 1,
89 M, 62 NF FSH 2
91 M, 56 NF FSH 2, TSH 1
100 F, 65 NF FSH 1, LH 1,
112 F, 58 NF FSH 3, PRL 2,
LH 2
69 M, 67 NF FSH 2, LH 3
60 F, 90 NF FSH 1,
LH 23
198 M, 58 NF FSH 1, LH 1
208 F, 47 NF LH1
138 M, 60 NF FSH 12, LH
183 M, 36 Hyperprolactinemia, PRL 3
visual loss, 2 cm
192 M, 41 Hyperprolactinemia, PRL 3
visual loss, 3 cm
151 M, 52 Hyperprolactinemia PRL 3, GH 1
240 M, 39 Hyperprolactinemia PRL 3
244 F, 40 Hyperprolactinemia PRL 3
244 Chheng-Orn Evans et al.

Table 8.3 (continued)

Patient ID Sex, Age Clinical features/tumor size IHC

213 M, 24 Hyperprolactinemia PRL 3
123 M, 30 Acromegaly, 2 cm GH 3, TSH 3,
FSH 3, LH 3
196 F, 69 Acromegaly, 2 cm NDb
168 F, 39 Acromegaly, 1 cm GH 3, PRL 23
218 F, 33 Acromegaly GH 3, PRL 23
145 M, 34 Acromegaly GH 3, PRL
23, TSH 1
232 F, 39 Cushings disease ACTH 2
137 F, 41 Cushings disease ND
239 F, 55 Cushings disease, cav ACTH 4
sinus invasion
126 F, 29 Cushings disease, 1.6 cm ACTH 3
233 M, 11 Cushings disease ACTH 3,
Neg, immunostaining for GH, ACTH, PRL, FSH, LH, and TSH were negative.
ND, not determined.
Sex, age of patient, and a brief description of tumor type are given.

FR expression in NF immunohistochemically
positive by RT-qPCR
FR message (relative to control)





Con 65 77 174 143 89 91 100 112 69 60 198 208 138
NF immunohistochemically positive samples

Figure 8.1 Relative expression levels of FRa mRNA in NF adenomas by RT-

qPCR. Expression levels of each tumor were normalized to the 18S RNA of the
same sample. Fold difference was the ratio of the normalized value of each sample to
controls as described in Methods and Materials (Evans et al., 2003). Con, five normal
controls; 65138, NF adenomas. There was a significant overexpression of FRa
mRNA in 13 NF adenomas from 17- to 174-fold compared with the controls ( p
0.001). Notably, sample 208 exhibited the highest FRa mRNA expression among the
13 samples (174-fold).
Folate Receptor Expression in Pituitary Adenomas 245

receptor. This antibody was produced by rabbits immunized with a recom-

binant FRa-glutathione S-transferase fusion protein and has been shown to
recognize the same protein as the Mab LK26 (data not shown). The results
for two normal pituitaries (N1 and N3), one NF adenoma (#65), and
9 NF adenomas are shown in Fig. 8.2A and for the 13 NF adenomas are
shown in Fig. 8.2B. Two PRL-secreting adenomas (PRL #183 and 192)
and three GH-secreting adenomas (GH #123, 196, and 168) are shown
in Fig. 8.2C, whereas four ACTH-secreting (ACTH #232, 137, 239,
and 126) adenomas are shown in Fig. 8.2D. Adenoma #65, which was
NF, was included in all of the blots to provide a basis of comparison for
the different gels. These results show that the majority (7 of 10 tumors
showing FRa expression) of the NF tumors and all of the NF tumors
overexpress FRa relative to the normal pituitaries and functional adenomas.
The results of the Western blot analysis are summarized in a box plot
shown in Fig. 8.3. The horizontal line in each box represents the median
value of FRa expression of each adenoma group and controls. The box