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Therapeutic Reactivation of the p53 Tumor Suppressor Protein

in HPV-Positive Cervical Cancer Cells by the Creosote Bush Lignan

3-O-Methyl-Nordihydroguaiaretic Acid

A dissertation submitted to Kent State University


in cooperation with Northeastern Ohio Universities College of Medicine
in partial fulfillment of the requirements for the degree of Doctor of Philosophy

By

Kristi Lynne Allen

November 2006
Dissertation written by

Kristi Lynne Allen

B.S., Youngstown State University, 1997

Ph.D. Kent State University, 2007

Approved by

______________________ Dr. Angelo L. DeLucia


Chair, Doctoral Dissertation Committee

______________________ Dr. John J. Docherty, Doctoral Dissertation Committee

______________________ Dr. J. Gary Meszaros, Doctoral Dissertation Committee

______________________ Dr. James Hardwick, Doctoral Dissertation Committee

______________________ Dr. Diane Stroup, Graduate Faculty Representative

Accepted by

______________________ Dr. Robert Dorman,


Director, School of Biomedical Sciences

______________________ Dr. John Stalvey


Dean, College of Arts and Sciences

ii
TABLE OF CONTENTS

Dedication and Acknowledgements...........vii

List of figures........ix

List of tables........xiii

List of abbreviations......xiv

Chapters

I. Introduction

1. Introduction to human papillomaviruses....1

2. Pathophysiology of Human Papillomaviruses....5

3. HPV Virology
3.1 HPV classification...8
3.2 HPV genome structure.....10
3.2.1 HPV E2...13
3.2.2 AP-1.15
3.2.3 Sp1...16
3.3 High Risk HPV Life Cycle...17
3.4 Structures and functions of HPV gene products
3.4.1 HPV E1 protein......22
3.4.2 HPV E2 protein......24
3.4.3 HPV E1-E4 proteins..25
3.4.4 HPV E5 protein......26
3.4.5 HPV E6 protein......27
3.4.6 HPV E7 protein..32
3.4.7 HPV L1 and L2 proteins...40

iii
3.5 Escape of human papillomaviruses from the immune
system......41

4. Molecular Biology of Carcinogenesis


4.1 p53...43
4.1.1 p53 as a transcription factor47
4.1.2 Transcriptionally independent roles of p53..51
4.1.3 Decision on cell cycle arrest versus apoptosis by p53 and
its targets....53
4.1.4 p53 and human cancers....55
4.2 Apoptosis...56
4.2.1 Regulation of the Mitochondrial (Intrinsic) Pathway to
Apoptosis...58
4.3 Other important players in the apoptotic pathway
4.3.1 Survivin..58
4.3.2 p38/ MAP kinase...60

5. HPV and cancers


5.1 Coexpression of HPV E6 and E7 facilitates carcinogenesis64
5.2 p53 status of cervical cancers.......65
5.3 Current treatments for HPV Induced Cancers of the uterine
cervix...65

6. Natural Products as Anticancer Agents.67

7. Specific aims for this study...73

II. Materials and methods

1. Reagents..76

2. Cell Culture.77

3. alamar Blue Assay..77

4. MTT Assay..79

iv
5. Trypan Blue Assay.....79

6. Western Blots......80

7. Immunofluorescence Assay for p53... 81

8. Transfection Study.....82

9. Caspase Assays.......82

10. Flow cytometry...83

11. TUNEL Assay.....84

12. Assay for inhibition of lipoxygenase activity....85

III. Results

1. 3-O-methyl-NDGA shows selective toxicity to cells containing HPV


DNA and wild type p53 protein..87

2. The plant lignan, 3-O-methyl nordihydroguaiaretic acid (NDGA),


stabilizes p53 tumor suppressor protein in treated HPV positive
cervical carcinoma cell lines.....91

3. The p53 protein stabilized by 3-O-methyl NDGA treatment is


transcriptionally active..98

4. 3-O-methyl-NDGA fails to activate p21 protein in HPV-positive


cervical cancer cells..101

5. 3-O-methyl NDGA activates caspases within treated HPV-positive


cervical carcinoma cell lines...101

v
6. The Caspase-3 Inhibitor ZAD-FMK Rescues lignan treated cells from
the apoptotic pathway.....110

7. 3-O-methyl NDGA induces apoptosis in HPV-positive cervical


carcinoma cell lines..110

8. The Bax and Puma genes are induced within lignan treated CaSki
cells.....118

9. The lignan reduces E7 protein levels within treated CaSki cells..118

IV. Discussion......124

V. References.......132

vi
DEDICATION

This work is dedicated to my mother, Jan Allen, and to the memory of my


late maternal grandmother, Cynthia Hoek. The bravery, dignity, and grace with
which you both faced the horrors of cancer, combined with the deeply personal
experiences with the disease you kindly shared with me during your illness, are
the sole inspiration for my career as a cancer researcher. I thank God every day
that you survived, and it is my hope that one day, others will not have to suffer
as you did and that the word cancer will inspire no more fear than the
common cold. I love you and thank you both.

ACKNOWLEDGEMENTS

I owe my utmost gratitude to the many people whose support and


guidance were indispensable in the completion of this project. First and
foremost I would like to acknowledge my advisor and mentor, Dr. Angelo
DeLucia. He exemplified and mentored me not only in being a skilled and
meticulous technician in the lab, but also in how to think like a scientist. He
challenged me to be a skeptic and realize that the data is not always as
impressive as it initially appears. Even in difficult times, his ongoing feedback
and guidance kept me on task and made this work a true achievement. More
than anything, he provided candid insights and discussions on many topics from
science to life, for which I respect him and am truly grateful. Thank you for
believing in me even though it was not always easy.
I would also like to thank my dissertation committee, Dr. John Docherty,
Dr. James Hardwick, Dr. Gary Meszaros, and Dr. Diane Stroup for support,
encouragement, and scientific advice. I also am also grateful to my fellow
students in the DeLucia lab, Keytam Awad and Deidra Tschantz. Thank you for
your encouragement and friendship. I hope that I will find more people down
the road that are as pleasurable to work with as you; I will miss you both.

I could not have made it to the finish line with out the unwavering love
support of my husband and better half, Dr. Mathew Lesniewski. I am truly
blessed to find in you not only my soul mate, but a fellow nerd who loves science
as much as I do. I love that you are always available to discuss my work, the
meaning of the universe, and how to create wormholes. I love you with every
ounce of my being.

vii
I would also like to acknowledge my parents, Doug and Jan Allen. Thank
you for your unending help and support, both emotional and financial, and for
always believing in me. You are both my rock and truly encouraged me to see
this endeavor to completion. I am grateful for your encouragement to pursue my
dream and for loving me unconditionally as the bookworm I am. Dad, you
taught me to see the world through different eyes and imparted me with the
steadfastness to never give up. Mom, from you I got my ability to pay close
attention to detail and you taught me to be patient and meticulous in reaching
my goals. These are all qualities that will make me the best scientist I can be. I
love you both.
I would like to thank my sister Amy Allen and my brother Aarin Allen for
their support and encouragement. I am blessed to have you both as part of my
life.
Finally, I would like to extend my gratitude to the pack and the pride. There
is nothing sweeter than a friend who is pleased to see you at the end of a long
day. Your purrs and cuddles kept me sane.

Nothing in life is to be feared, it is only to be understood. Now is the time to


understand more, so that we may fear less. -Marie Curie

viii
LIST OF FIGURES

Figure

1. The course of events that, over time, can lead to the development of
precancerous and cancerous cervical tumors.....9

2. Circular diagram of HPV genome and representative open reading frames..11

3. Linear representation of HPV genome..12

4. Structure of HPV-16 LCR and expanded view of associated transcription


factors..14

5. Differentiation of normal squamous epithelium compared to HPV infected


epithelial tissue..18

6. Illustration of HPV life cycle.......19

7. Transcriptional targets of p53 protein in the pathways of growth arrest and


apoptosis ...29

8. Phosphorylation of retinoblastoma protein by cyclin/CDK complexes...34

9. Role of pRb phosphorylation on E2F target gene transcription.36

10. Role of HPV E7....38

11. Diagram of the domain structure of p53 protein...45

12. Mdm2 control of p53 levels in normal cells....46

13. A partial list of the many roles of p53..48

14. PUMA protein and the activation of cytoplasmic p53..52

15. Downstream targets of p53 in the induction of apoptosis....54

ix
16. Intrinsic and extrinsic pathways to caspases activation....50

17. Processes involved in HPV-induced carcinogenesis.63

18. Naturally occurring polyphenols that have anticancer properties .....69

19. Chemical structures of the creosote bush lignans..70

20. Hypothetical mechanism of action of 3-O-methyl-NDGA......74

21. Toxicity of the lignan in CaSki cells at 24 hours post treatment as


determined by the MTT assay...88

22. Toxicity of the lignan in CaSki cells at 24 hours post treatment as


determined by Trypan Blue assay....89

23. Toxicity results obtained from the alamar Blue assay......90

24. p53 protein is stabilized in CaSki cells treated with 30 M 3-O-Methyl-


NDGA..94

25. p53 protein is stabilized in HeLa cells treated with 30 M 3-O-Methyl-


NDGA.95

26. p53 protein levels do not change in C33-A cells treated with 30 M 3-O-
methyl-NDGA...96

27. Immunocytochemical staining of p53 in CaSki cells after lignan treatment


confirms that p53 levels are increased in lignan-treated cells..97

28. Structure of the p53-luciferase vector used to transfect HeLa cells with
p53.........99

29. 15 M 3-O-methyl-NDGA stabilizes p53 within HeLa cells transfected


with p53-luciferase....100

x
30. 15 M 3-O-methyl-NDGA stabilizes p53 within CaSki cells transfected
with p53-luciferase....102

31. 15 M 3-O-methyl-NDGA stabilizes p53 within SiHa cells transfected


with p53-luciferase103
32. Western blot analysis of p21 induction in CaSki cells by 30 M 3-OMe-
NDGA.104

33. Western blot analysis of p21 induction in CaSki cells by 30 M 3-OMe-


NDGA.105

34. 30 M 3-O-methyl-NDGA activates caspase-3 in treated HPV-containing


cervical cancer cells, but not in HPV-negative C33-A cells........106

35. C33-A cells have an intact caspase-3 pathway.....108

36. 30 M 3-O-methyl-NDGA activates caspase-9 in treated HPV-containing


cervical cancer cells, but not in HPV-negative C33-A cells....109

37. Addition of the caspase-3 inhibitor Z-VAD-FMK inhibits caspase-3 activation


in lignan treated cells......111

38. Lignan treated cells are undergoing apoptosis as assayed using flow
cytometry and annexin V-FITC and propidium iodide staining...113

39. Analysis of apoptosis in HPV-16 positive CaSki cells by TUNEL assay


showing induction of cell death by 30 M 3-O-methyl-NDGA....114

40. Analysis of apoptosis in HPV-16 positive SiHa cells by TUNEL assay


showing induction of cell death by 30 M 3-O-methyl-NDGA....115

41. Analysis of apoptosis in HPV-negative C33-A cells by TUNEL assay116

42. Analysis of apoptosis in HT-3 cells by TUNEL assay ...117

43. Western blot analysis shows that bax protein is induced by 30 M 3-O-
Methyl-NDGA in CaSki cells.118

xi
41. Western blot analysis shows that bax protein is induced by 30 M 3-
O-Methyl-NDGA in HeLa cells.....120

42. Western blot analysis shows that both PUMA protein is induced by 30 M
3-O-Methyl-NDGA in HPV DNA containing cells.....121

43. E7 protein levels in lignan treated CaSki cells......122

xii
LIST OF TABLES

Table

1. HPV types in benign and malignant disease..3

2. Functions of HPV Early and Late Proteins....23

3. Cellular Targets of HPV E6 Oncoprotein......31

4. Cellular Targets of HPV E7 Oncoprotein..39

5. Cellular Targets of p53 in the Activation of Apoptosis...49

6. Cell lines used in this study and their HPV and p53 statuses....78

7. Growth arrest induction capability of 3-O-methyl-NDGA in various cell


lines.....92

xiii
LIST OF ABBREVIATIONS

3-O-methyl-NDGA 3-O-methyl-nordihydroguaiaretic acid


AD Actinomycin D
CMV cytomegalovirus
DEVD aspartic acid-glutamic acid-valine-aspartic acid
DME Dulbeccos modified Eagles medium
DMSO dimethylsulfoxide
DNA deoxyribonucleic acid
DNase deoxyribonucleotide-ase
dUTP deoxyuridine triphosphate
E6-AP E6 associated protein
EDTA ethylenediaminetetraacetic acid
FBS fetal bovine serum
FITC fluorescein isothiocyanate
HPV human papillomavirus
LEHD leucine-glutamic acid-histidine-aspartic acid
MDM2 murine double minute 2
mRNA messenger ribonucleic acid
nM nanomolar
PBS phosphate buffered saline
PI propidium iodide
PMSF phenylmethylsulphonylfluoride
pNA paranitroaniline
pRB retinoblastoma protein
PUMA p53 upregulated modulator of apoptosis
PVDF polyvilylidene fluoride
RIPA radio immune precipitation assay
RNA ribonucleic acid
SDS sodium dodecylsulfate
TUNEL terminal dUTP nick end labeling
uM, M micromolar

xiv
xv
CHAPTER ONE

INTRODUCTION

BACKGROUND AND SIGNIFICANCE

1. Introduction to human papillomaviruses

Human papillomaviruses (HPVs) are the most common of the human

pathogens and are the leading causative agent of sexually transmitted disease

worldwide. Papillomaviruses are small, non-enveloped, circular, double-

stranded DNA viruses. HPVs are members of the Papillomaviridae family and

are in the genus Papillomavirus which contains more than 100 types of the virus

classified by genotype. A larger number likely exist due to the presence of

subgenomic HPV amplicons (de Villiers et al., 2004). HPVs are ubiquitous and

infect a variety of hosts, however, humans and bovines are the most extensively

studied.

Papillomaviruses infect the basal keratinocytes of squamous epithelium


and may cause benign (noncancerous) tumors called warts, or papillomas,

although some infections never produce clinical symptoms. The abundance of

viral types and wide variety of lesions HPV infection can generate are illustrated

in table 1. The papillomaviruses that cause the common warts that grow on

hands or feet (plantar warts) differ from the HPVs that cause growths in the

throat or genital regions. All HPVs trigger proliferation of the cells they infect,

but only a few types are associated with the risk of developing cancer.

Sexually transmitted HPVs are divided into low-risk and high-risk types

based on their likelihood of causing cancer. Cervical cancer is strongly

associated with high-risk HPV types 16 and 18; nevertheless, the majority of

high-risk HPV infections are cleared without treatment. A major advancement

in the prevention of cervical cancer and other anogenital tumors occurred with

the advent of a vaccine against the HPV types responsible for a majority of these

tumors. The tetravalent vaccine Gardasil (Merck) against high risk HPV-16 and -

18 and HPV-6 and -11, the types responsible for a majority of genital warts, has

recently been approved for use by the FDA. Glaxosmithkline also has a vaccine

against HPV-16 and -18 in late stage clinical trials. Results from vaccine trials

have shown that the three-dose vaccine protects against persistent HPV infection

over a period of 2 to 4 years (Schmiedeskamp and Kockler 2006). However, the

2
Table 1. HPV types in benign and malignant disease.

Manifestation HPV Types

Common Less Common

Skin warts
Plantar warts 1 2,4,63
Common warts 2,27 1,4,7,26,28,29,57,60,65
Flat warts 3,10 2,26,27,28,29,41,49

Anogenital lesions
Condyloma acuminata 6,11 2,16,30,40,41,42,44,45,54,55,61
CIN, VIN, VAIN, PIN, 16,18,31 6,11,30,34,35,39,40,42-45,51,52,
PAIN 56-59,61,62,64,66,67,69

Malignancies
Cervical cancer 16,18,31,45 6,10,11,26,33,35,39,51,52,55,56,
58,59,66,68
Other anogenital cancers 6,16,18 11,31,33
Laryngeal cancer 6,11 16,18,35
Oral cancer 16,18 3,6,57,16
Subungual, digital

Adapted from DeVilliers et al., 2004 and http://www.arhp.org.

3
vaccines will not eliminate the need for cervical cancer screening since they will

not protect the millions of women who are already infected with HPV who have

the potential to develop uterine cervical cancer (UCC) in the future.

Cancer of the uterine cervix represents the second most common cancer

in females worldwide and is a major cause of morbidity and mortality (Moore et

al., 2006). In 2006, 9,710 women were diagnosed with cervical cancer and 3,700

women died from the disease (American Cancer Society, www.cancer.org).

Worldwide, morbidity and mortality rates are far higher with an incidence of

490,000 new cases and 273,000 deaths occurring in 2005, according to the World

Health Organization. About 80 percent of these cases occur in developing

countries (Cox 2006; WHO data 2005).

High-risk HPV infection is the primary risk factor for the development of

cervical carcinoma (zur Hausen, 1999). Over 90 percent of cancers of the

uterine cervix and at least 50 percent of other anogenital tumors contain high-

risk HPV DNA (zur Hausen 2001). However, infection with high-risk HPV does

not guarantee a patient will develop cervical cancer because most infections clear

within 12-18 months, and there have been very rare instances where women

have developed cervical cancers that are HPV-negative (Avrich et al., 2006).

Cervical tumors can be resistant to traditional courses of chemotherapy and

4
those patients who do respond positively to chemotherapy have tumors that can

recur (Alexander et al., 2005; Dreyer et al., 2005; Lindeque 2005; Tewari and

Monk 2005). Therefore, the primary goal of this study is to identify novel

chemotherapeutic agents that are effective and specifically target HPV

oncogenes.

2. Pathophysiology of Human Papillomaviruses

Papillomaviruses primary host tissue is squamous epithelial tissues such

as skin or the mucosal surfaces of the genital tract, anus, mouth, or upper

respiratory tract (Doorbar 2005). HPVs can be divided into cutaneous skin tropic

viruses and mucosal skin tropic viruses. HPV infections trigger the benign or

malignant hyperproliferation of cells in these tissues. HPVs can produce a

variety of lesions including common warts, plantar warts, flat warts, laryngeal

papillomas, genital condylomas, and cancerous tumors of the uterine cervix and

anus, and also the head and neck (zur Hausen 1999).

Low-risk cutaneous HPV infections are ubiquitous and are usually spread

environmentally through casual skin-to-skin contact (Antonsson et al., 2000).

Some cutaneous papillomaviruses produce benign tumors called warts, or

papillomas. These include HPV-1 and HPV-2 which cause plantar warts on the

5
soles of the feet and common warts on the hands, respectively (Akgul et al.,

2006). Common warts and plantar warts appear as raised lesions that have a

distinct cauliflower-like appearance. Cutaneous warts commonly affect children

and typically emerge and regress randomly over a period of weeks to months

(Fox and Tung 2005). Recurring cutaneous warts appear in about 10 percent of

adults as well. Low-risk HPVs can also cause subungual and periungual warts

that form under and around the fingernails and cuticles, or flat warts that can

emerge on the arms, face, forehead, or trunk. A few cutaneous HPVs, such as

HPV-5, can cause persistent subclinical infections that persist for a lifetime

without ever displaying any visible lesions or symptoms. Papillomavirus types

associated with the lesions described above are not usually linked with an

increased risk of developing cancer (Hazard et al., 2006).

A distinct category of approximately 30 HPV types infect mucosal skin

and are transmitted through sexual contact. However, HPV types that have a

tendency to elicit genital warts are not the same ones that can cause cancer. For

this reason, sexually transmitted HPVs are divided into two groups, those

associated with the development of benign anogenital warts (condylomas) such

as types 6 and 11 (low-risk), and those that carry a risk of developing

precancerous and cancerous lesions of the anogenital tract such as types 16, 18,

6
31, 33, 35, and 45 (high-risk) (Stanley et al., 2001).

Genital HPV infection is widespread; estimates show that as many as 75% of

women will become infected with one or more of the sexually-transmitted HPV

types during her lifetime (Baseman and Koutsky 2005). Genital or anal warts

(condylomata acuminata or venereal warts) are the hallmark of symptomatic

genital HPV infections. HPV-6 and HPV-11 are responsible for a majority of

cases but other HPV types are known to cause this type of infection (Greer et al.,

1995). Nevertheless, other HPV types that infect the genital tract do not cause

overt symptoms. In addition, roughly 80 percent of individuals who acquire

sexually transmitted HPV infections clear the virus rapidly without ever

developing warts or other clinical symptoms (Dunne and Markowitz 2006). It is

important to note that these people are able to pass the virus to others even if

they don't show obvious signs of infection.

Some sexually transmitted human papillomaviruses are associated with the

development of cancer and are termed high-risk HPVs. High-risk HPV infection

that is not cleared by the immune system can lead to malignant transformation of

infected cells and cancer progression. Though progression to malignancy is not

the typical consequence of HPV infection, carcinomas triggered by a handful of

high-risk HPV types, especially uterine cervical tumors, kill several hundred

7
thousand people each year and represent a major disease burden in women

worldwide (Schiffman and Castle 2003). Persistent HPV infection is critical for

the development of over 90 percent of cervical cancers, though a very small

percentage of these tumors do not contain HPV DNA (Walboomers et al., 1999;

Herrington 1999). A number of individuals with persistent high risk HPV

infections also develop other types of anogenital cancers or malignant head and

neck tumors. Known high-risk HPVs include types 16, 18, 31, 33, 35, 39, 45, 51,

52, 56, 58, 59, 68, 69, and possibly a few others (zur Hausen 2000, 2002).

Cervical cancer develops from precursor lesions called cervical

intraepithelial neoplasias (CIN) (Wiley and Masongsong 2006; Cox, 2006).

Progression from normal epithelium to a CIN lesion to cancer takes decades and

may be due to changes induced by the virus as well as other environmental

factors (smoking, HIV infection, oral contraceptive use, and family history). The

typical time course of events that leads to the development of CIN and cervical

cancer is illustrated in figure 1.

3. HPV Virology

3.1 HPV Classification

HPVs are classified according to genome sequence and are numbered in

8
High Risk HPV Persistance
Additive epigenetic events (oncogenes)

Progression

Regression
HPV Clearance

MONTHS YEARS DECADES

Normal Persistent CIN I/CINII CIN II/CIN III Invasive


Early Late
Cervical HPV Precancerous
Cervical
Precancerous
Epithelium Infection Cells Cells Carcinoma

Figure 1. The course of events that, over time, can lead to the development of

precancerous and cancerous cervical tumors. Induction of cancerous tumors

takes decades and is preceded by the development of early and late precancerous

lesions called cervical intraepithelial neoplasias (CIN), which may regress.

Persistent HPV infection is required for progression to carcinoma.

(Adapted from zur Hausen 2002).

9
the order of discovery. HPV types differ from one another by at least a

10%difference in the gene sequences of E6, E7, and L1. Subtypes, or variants,

denoted by the HPV type followed by a, b, c, etc., are defined as a difference in

sequence from the primary type by less than 2-5% (DeVilliers et al., 2004).

3.2 HPV genome structure

The papillomavirus genome is encased in an icosahedral capsid composed

of 360 protein monomers assembled into 72 pentameric capsomers. HPVs

contain a closed circular, double stranded genome that is approximately 8000

base pairs in length. A map of the HPV genome is shown in figure 2. The

genome is divided into three segments, an early region, a late region, and a long

control region (LCR) (Zheng and Baker 2006). The three regions are separated

by two polyadenylation (polyA, pA) sites (see figure 2). The early region of the

HPV genome contains six open reading frames (ORFs): E1, E2, E4, E5, E6, and E7.

There is no E3 gene. Each ORF codes for an individual protein with the

corresponding name. The late region contains 2 ORFs, L1 and L2, that encode

the major and minor capsid proteins, respectively. A linearized map of the

genome containing size of each of the genes is shown figure 3.

High risk HPV-16 will be used to illustrate the typical genome

10
Long control region (LCR)
TATA Signal
1,2
Poly A Signal 2

E6
65-556

E7
544-859

L1
5528-7153
HPV-16 E1
7904 base pairs 859-2811

L2 E4
4135-5655 3333-3617 E2
2726-3850
E5
3809-4098

Poly A Signal 1
Figure 2. Circular diagram of HPV-16 genome and open reading frames.

The three separate regions of the HPV genome along with early genes E1-E7 and

late genes L1 and L2 are shown. (Adapted from zur Hausen 1999).

11
HPV-16 Genome Organization

URR

E7 E1 E5 L2

544 859 2811 3809 4098/4135 5655

E6 E6 L1

65 556 2726 3850 5528 7153

E4

3333 3617

0 7905 Base Pairs 7905

Figure 3. Linear representation of HPV genome. Relative sizes of HPV early

(E1-E7) and late (L1-L2) genes are shown.

(Adapted from: www-ermm.cbcu.cam.ac.uk/smc/fig002smc.htm)

12
organization and regulation of the HPV life cycle. The LCR region is about

850base pairs long and it contains the origin of replication as well as binding sites

for multiple transcription factors that regulate viral gene expression (Bernard

2002). The structure of the LCR showing associated transcription factors is

illustrated in figure 4. Both viral and cellular transcription factors, such as Sp1

and AP-1, are required for viral gene transcription.

The HPV-16 genome contains two promoters, the P 97 promoter that

controls early gene expression (prior to viral DNA replication) and the P670

promoter that controls late gene expression (following viral DNA replication).

The P97 promoter is active in both basal and differentiated keratinocytes and is

tightly regulated by upstream cis elements located in the LCR (Zheng and Baker

2006). These cis elements include four HPV E2 binding sites and binding sites for

host cellular transcription factors such as Sp1 and AP-1. Some of the

transcription factors that bind to the early promoter are depicted in figure 4 and

are described as follows.

3.2.1 HPV E2

HPV E2 proteins can function as transcription factors. Binding of E2 to

DNA induces a bend in the DNA that facilitates transcription of E2 mediated

13
Human papillomavirus Long Control
Region (LCR)

Enhancer region Replication


C
origin E6/E7 promoter
E O
E B
C E E E
YY1 2 GR P
T 2 E1 2 2 E6
YY1
AP1 AP1 AP1
NF1 NF1
Sp1 TFDII

Figure 4. Structure of HPV-16 LCR and expanded view of promoter-associated

transcription factors. Binding sites of viral and cellular transcription factors are

shown relative to the P97 promoter.

14
genes (Zimmerman et al, 2003). A dimeric -barrel motif in the DNA binding

domain of E2 represents the DNA recognition region. This area is connected

with activation domains by linker sequences of 40200 amino acids. The E2

DNA binding consensus sequence is 5'-ACCGNNNNCGGT.

The DNA of all HPVs contains multiple E2 binding sites. The E2 DNA

binding site sequences all contain a conserved but variable 4 nucleotide sequence

(N4 spacer). The makeup of this sequence determines E2 binding affinity. For

example, the N4 sequence 5ACGT is associated with lower E2 binding affinity

than 5TTAA (8-fold lower) or 5AATT (33-fold lower) (Zimmerman et al, 2003).

3.2.2 AP-1

The cellular transcription factor AP-1 is a heterodimer that contains the

subunits c-jun and c-fos. AP-1 binds to the DNA consensus sequence

5'-TGAGTCA-3'. The DNA binding region of the protein contains a leucine

zipper in which hydrophobic interactions between leucine residues located every

seventh amino acid in an alpha helix stabilize the c-jun/c-fos interaction as well as

binding to DNA. DNA binding of AP-1 also involves ionic interactions between

DNA and groups of arginine residues located within each subunit. AP-1 controls

the transcription of a multitude of cellular genes including those involved in cell

proliferation (Glover and Harrison 1995).

15
3.2.3 Sp1

Sp1 is a transcription factor that controls many genes involved in early

development. Sp1 binds to the DNA consensensus sequence

5'-(G/T)GGGCGG(G/A)(G/A)(C/T)-3' (called a GC box element) via a Cys2/His2

zinc finger domain. Sp1 is known to be important in transcription of genes from

a variety of viruses, including SV40 and HPV (Dreier et al, 2001).

It has been postulated that the tissue specificity of HPVs may, in part, be

due to the fact that the required set of transcription factors are only present in

basal keratinocytes. HPV is not known to infect any other tissue.

Early mRNA transcripts that arise from the early promoter are

polyadenylated and each contains three exons and two introns that are capable

of undergoing alternative RNA splicing. Alternative splice sites can produce 14

unique mRNA species that each have different coding potential. Viral proteins

are translated from mRNA via ribosomal scanning (Sen et al., 2004; Deng et al.,

2003).

The P670 late promoter that controls transcription of the L1 and L2 genes is

only active late in the life cycle of the virus and solely in differentiated

keratinocytes. Lytic DNA replication is required for promoter activation. The

16
P670 promoter controls expression of the L1 and L2 genes whose gene products

are components of the viral capsid (Finnen et al., 2003).

3.3 High Risk HPV Life Cycle

Papillomaviruses have a tropism for squamous epithelial tissues such as the

skin, or the mucosal surfaces of the genital tract, anus, mouth, or airways, and

are not known to infect any other tissue type (Doorbar 2005). HPVs stimulate the

expansion of the infected cell population but HPV infections do not result in the

lysis and death of the host cell. Thus, papillomaviruses utilize an alternative

mechanism for transmission. The virus resides in, replicates in, and stimulates

the host cell to divide and differentiate. The mature HPV virions are then

released during the sloughing of dead, surface squamous epithelial cells

(koilocytes) and this serves as the vector of transmission (Ackgul et al., 2006).

Figure 5 illustrates differences in the cellular processes between normal and

HPV- infected epithelium and provides an overview of the viral life cycle.

Mature HPV virions infect surrounding undifferentiated basal keratinocytes

of squamous epithelium through a microtrauma in the skin. The life cycle of

human papillomaviruses (illustrated in figure 6) begins when a virus particle

attaches to a receptor on the surface of a host cell, which has been identified as

17
Normal Squamous Epithelium HPV Infected Epithelium
Stratum
Corneum Keratin Release of
Envelopes mature virions
Stratum
Granulosum Keratins
Accumulate Capsid synthesis
and virion
Keratin assembly
Stratum Synthesis and
Spinosum Nuclear Late promoter
Breakdown active; genome
amplification

Stratum Cell Division Infection;


Basale and DNA activation of
Synthesis early promoter

Figure 5. Differentiation of normal squamous epithelium compared to HPV

infected epithelial tissue. HPV infects keratinocytes in the basal layer.

Activation of viral genes keeps cells dividing for genome amplification and viral

assembly and mature virions exit as dead cells are sloughed off. (Adapted from

Hebner and Laimins 2006).

18
Sloughing of virus
HPV Infection laden cells from
upper epithelial
layers

Normal Keratinocyte

Papilloma
Formation

Episomal HPV
DNA in nucleus
of infected cell

Figure 6. Illustration of HPV life cycle. HPV DNA copy number is low in

basal keratinocytes. A lesion develops and viral copy number increases in cells

that are closest to the surface. Mature virions are assembled in these cells and are

sloughed from upper epithelial layers creating the vector for transmission.

(Adapted from zur Hausen 1999).

19
an integrin (Yoon et al, 2001). The virus then penetrates the cell membrane.

Inside the cell, the virus uncoats and viral DNA is released from the capsid.

The viral DNA is delivered to the nucleus of the infected cell. The closed,

circular, double stranded genome encodes eight open reading frames (ORFs)

that are transcribed as polycistronic mRNAs from a single strand of DNA.

Once the viral DNA reaches the nucleus, cellular transcription factors interact

with the non-coding viral regulatory region (LCR) and transcription of two

transforming early genes, E6 and E7, begins (Zheng and Baker 2006).

The viral E1 and E2 genes are expressed concomitantly with the E6 and

E7 genes and function to regulate viral DNA replication and transcription. Both

gene products are DNA binding proteins; E1 is a helicase and E2 functions as a

transcription factor. E2 binds to the consensus sequence 5'-ACCGNNNNCGGT

in duplex DNA and controls transcription of E6 and E7, each of which have

multiple functions that stimulate growth of infected cells (Zheng and Baker

2006). The virus replicates in a rolling circle replication mode. The progression

of the viral replication and maturation are closely linked with the differentiation

of the infected basal keratinocytes. As the basal cells containing HPV divide and

begin to migrate toward the surface, the daughter cells carry multiple copies of

the HPV genome. During the early phase of infection, the viral genome persists

20
extrachromasomally as a slowly replicating plasmid, and the viral copy number

per cell is kept relatively low at fewer than 100 copies per cell (DiMaio and Liao

2006).

As infected cells mature, the remainder of the HPV early genes is

expressed, including E4 and E5. The E4 protein is believed to have a role in the

activation of the productive phase of the viral life cycle and may have a role in

viral genome amplification (Hebner 2006). Though little is known about its

function, E5 is a viral protein believed to be involved in transformation of the

infected cell (Straight et al., 1993; Gu and Matlashewski 1995; Hebner 2006).

As the HPV-infected cells reach terminal differentiation, the late genes L1

and L2 that encode viral capsid proteins, are transcribed from the P670 late

promoter. At this point of the viral life cycle, the viral copy number per cell is

greatly increased from several hundred to several thousand per cell. L1 and L2

proteins self assemble into virus particles by encasing a viral genome (Sapp et al.,

1995; Finnen et al., 2003). Little is currently understood regarding how HPV

DNA is packaged into the virion or the process by which the mature virus is

released from the cell. Infected cells approach the surface of the skin and are

sloughed off, and in the process, newly synthesized HPV virus particles are

21
released into the surrounding area where the virions have the potential to infect

other cells or spread to other hosts (Buck et al., 2005).

3.4 Structures and functions of HPV gene products

The structures and key functions of HPV proteins are reviewed in table 2.

The proteins are described in detail in following sections.

3.4.1 HPV E1 protein

Among all HPV types, the HPV E1 and E2 gene products are highly

conserved (Ustav and Stenlund 1991; Ching et al., 1993; Sverdrup and Khan 1994;

Hebner et al., 2006). Both proteins are absolutely essential for replication of the

viral genome (Ustav and Stenlund 1991). The E1 gene codes for an

approximately 80 kDa protein that has sequence similarities to SV40 T antigen

and functions as a DNA-binding DNA helicase/ATPase that is involved in the

initiation of DNA replication. Structure prediction studies indicate that E1 forms

two distinct domains: an N-terminal domain (residues 1-125), and a highly

structured C-terminal domain (residues 170-649), with an intermediate region

(residues 125-170) that serves as a link between the N and C termini (Hughes and

Romanos 1993; Hebner et al., 2006). The N terminal region functions in DNA

binding and the C-terminal has enzymatic functions (Sverdrup and Khan 1994).

22
Table 2. Functions of HPV Early and Late Proteins

(Adapted from Tommasino and Crawford, 1995)

Gene Product Size (aa) Function(s)

Early Proteins

E1 694 aa Replication of viral DNA,


helicase activity

E2 365 aa Replication, transcriptional


factor, maintenance of episomal
copy number, encapsidates viral
genome

E4 92 aa Assembly and release of mature


virions

E5 83 aa Upregulation of EGFR, induction


of cell growth pathways, weak
transforming activity

E6 151 aa Binding and degradation of p53,


cell immortalization

E7 98 aa Binds pRb, deregulation of G1/S


checkpoint, cell immortalization

Late Proteins

L1 505 aa Major capsid protein

L2 473 aa Minor capsid protein

23
The E1 protein binds to AT rich viral DNA regions close to the start site of

early gene transcription. E1 also forms protein complexes with HPV E2 protein

that bind to HPV DNA at sequence specific sites located in the vicinity of the AT

rich sites. Once it is bound to DNA, the E1 protein forms a hexameric ring

structure around the viral DNA and begins to unwind the viral DNA for

replication (Hebner et al., 2006). Recently, E1 has been shown to be

phosphorylated via interaction with cyclin/CDK complexes in the host cell. This

modification serves as a means of regulating the proteins activity and it is

essential for efficient viral replication (Ma et al., 1999; Deng et al., 2004).

3.4.2 HPV E2 Protein

The ~50 kDa HPV E2 protein has a role in viral replication and in the

maintenance of episomal copy number, as well as in the regulation of HPV early

gene expression. The N terminal region of E2 consists of a transactivation

domain and the C terminal region contains both E1 protein and DNA binding

sequences (Hebner et al., 2006). E2 protein forms a homodimer that binds to

DNA in a beta barrel structure (Hegde et al., 1992; Antson et al., 2000; Kim et al.,

2000; Hebner et al., 2006). The protein binds to the HPV DNA consensus

sequence ACCN6GGT (Steger and Corbach 1997). Cooperative binding of E2

homodimers to these sequences can both activate and repress transcription of the

24
HPV early genes from the P97 promoter, depending on the amount of E2 protein

that is present. E2 regulates viral transcription by activating transcription at low

E2 protein concentrations, and inhibiting it when E2 levels are high. E2 has an

especially critical role in the control of E6 and E7 gene expression, because the

loss of E2 control over these genes appears to be a major contributor to the

induction of malignant transformation of an infected cell (Hebner et al., 2006).

3.4.3 HPV E1^E4 Proteins

The term E1^E4 describes several proteins that are gene products formed

by the alternative splicing of mRNA sequences from the HPV E1 gene and the E4

ORF. These proteins are the most highly expressed of the HPV proteins and

their expression occurs throughout the lifecycle of the virus (Hebner et al., 2006).

The E1^E4 proteins localize in the infected cells cytoplasm and may form

multimers (Sterling et al., 1993; Pray and Laimins 1995; Roberts et al., 1997; Wang

et al., 2004). The exact function of these gene products is not currently known.

Some studies suggest the proteins may have a role in the exit of the mature HPV

virion from the infected cell, since E1^E4 has been shown to bind to cellular

cytokeratins (Doorbar et al., 1991; Roberts et al., 1993; Wang et al,, 2004). It has

also been proposed that the proteins may influence genome amplification by

blocking the infected cells entry into mitosis (Hebner et al., 2005). Recent studies

25
show that overexpression of E1^E4 protein causes arrest at the G2-M cell cycle

checkpoint by altering the localization of cellular cdk1/cyclin B1 complexes,

which are critical for progression to mitosis (Davy et al., 2005).

3.4.4 HPV E5 Protein

The HPV E5 protein is a small, hydrophobic, membrane-associated

protein approximately 12 kDa in size that is localized in the Golgi apparatus,

endoplasmic reticulum, and, in small concentrations, in the membrane of the

host cell. Like the E1^E4 proteins, the functions of this gene product are not

entirely clear at this time and most of the speculated functions currently

attributed to the protein come from the results of overexpression studies. For

example, overexpression studies suggest HPV E5 may augment the activity of

epidermal growth factor (EGF) receptor or enhance downstream receptor

signaling, including the mitogen-activated protein (MAP) kinase pathway

(Straight et al., 1993; Gu and Matlashewski 1995).

E5 protein may be involved in cellular transformation since it appears

to enhance the transforming properties of the E6 and E7 proteins (Hebner et al.,

2006). The E5 protein is known to target the epidermal growth factor receptor

(EGF), and affect other cell signaling pathways. (Straight et al., 1993; Crusius et

al., 1997; Crusius et al., 1998; Hebner et al., 2006). Studies show cells

26
overexpressing E5 have increased amounts of cell surface phosphorylated EGFR.

The interaction of E5 with EGFR also alters endocytosis and endosomal

trafficking pathways, so it may affect the recycling and degradation of EGFR

(Hebner et al., 2006).

E5 also appears to be critical for immune system evasion by the virus.

E5s alterations in the endosomal pathways have been predicted to alter host cell

antigen presentation by inducing retention of MHC (HLA) class I complexes in

the Golgi apparatus and impeding transport of the MHC-peptide complex to the

cell membrane (Ashrafi et al., 2005). There is evidence for a direct interaction of

E5 and HLA I heavy chain (HC) through the first hydrophobic domain of E5. E5

also desensitizes infected cells to pro-apoptotic stimuli by downregulation of the

CD95 death receptor on the cell surface. This is yet another mechanism the virus

uses to overcome host defenses and favors propagation of the viral infection

(Ashrafi et al., 2005; Hebner and Laimins 2006).

3.4.5 HPV E6 Protein

The functions of the E6 protein will be described in the context of high risk

HPV infection, since the protein is critical for the malignant transformation of the

infected cell by high risk HPVs. Low risk HPV E6 protein does not have the

ability to transform infected cells. High risk HPV-16 E6 protein is 106 amino

27
acids in length and contains two C-X-X-C zinc finger domains (Hebner et al.,

2006). The protein is found in both the nuclei and the cytoplasm of HPV-infected

cells. E6 has a variety of functions, though its ability to bind to the p53 and

target it for proteasomal degradation is certainly the most characterized one, and

may be the most critical of its functions for induction of carcinogenesis (Werness

et al., 1990; Keen et al., 1994; Hebner et al., 2006).

p53, also known as the guardian of the genome, is a tumor suppressor

protein that controls both the G1/S and G2/M checkpoints of the cell cycle. It

functions to induce the transcription of genes, such as p21 (cip1/waf1) that

induce cell cycle arrest upon cell stress or genotoxic assault, and allows for either

damage repair or induction of apoptosis (figure 7). High risk E6 protein binds to

p53 in a heterotrimer with the cellular E6-Ap protein and the complex acts as a

p53 specific ubiquitin ligase that facilitates the degradation of p53 via the

ubiquitin protesome pathway (Sheffner et al., 1998). This dramatically decreases

the half-life of the p53 tumor suppressor protein in infected cells and p53-

mediated regulation of the cell cycle is lost. E6 decreases the half-life of p53 in

vitro from several hours to less than 20 minutes. The E6-mediated loss of p53

removes restrictions on DNA synthesis in the infected cell and allows HPV DNA

replication to proceed unchecked. In addition to the loss of p53 protein, the

28
G1 arrest
DNA damage Cyclin/
MDM2
Cell stress CDK

p53 p53 p21


Inactive Active p-Rb + E2F
Rb
Increased levels, G1 phase S phase
Stabilization GADD45
PCNA
DNA polymerase
Transcriptional activation

Transcriptional repression

Bax

FAS Apoptosis

IGP-BP3

Figure 7. Transcriptional targets of p53 protein in the pathways of growth

arrest and apoptosis. p53 activates some genes and represses others when

the protein is stabilized. Stabilized p53 can initiate the processes of growth

arrest and apoptosis. (Adapted from Michalak 2005).

29
expression of E6 protein facilitates uncontrolled proliferation of the infected cell

through the activation of cellular cyclins E and A (Malanchi et al., 2002).

E6 protein also interacts with other cellular proteins (summarized in

table 3). The vast majority of the proteins E6 binds to contain domains called

PDZ domains. PDZ domains, or post synaptic density protein domains, are

diverse, but specific, protein-protein interaction domains between 80 and 90

amino acids in length that are found in proteins involved in signal transduction

pathways (Ranganathan and Ross 1997). The PDZ domain-containing proteins

that E6 binds to have roles in the cell cycle and tumor suppression, cell adhesion,

and cell signaling pathways (James et al., 2006; Hebner et al., 2006). The

variability of the sequence of amino acids that lies directly in the peptide-binding

groove of a PDZ domain is what confers diversity of binding specificity.

The ability of E6 to bind to PDZ domain-containing proteins may perpetuate its

function in oncogenesis, since some of the proteins to which it binds, including

p53, are tumor suppressors or putative tumor suppressors. The interruption of

tumor suppressor pathways can interfere with a cells control over the cell cycle

and cell division. In a study involving transgenic mice, the PDZ domain binding

function of E6 was required for the initiation of precancerous cervical

intraepithelial neoplasia (Doorbar 2006).

30
Table 3. Cellular Targets of HPV E6 Oncoprotein

Adapted from Scheurer et al, 2005

Target Effect

p53 Escape from growth arrest, apoptosis

Paxillin Disruption of actin cytoskeleton

Bak Antiapoptotic

E2F/Cyclin A complex Disruption of cell cycle regulation

IRF-3 Decreased IFNexpression

PDZ domain proteins Increased cell proliferation

p300/CBP Disruption of TGFgrowth arrest

p48 and IRF1 Inhibition of transcription of cell


proteins

Unknown Increased telomerase activity

31
Another key role for E6 in carcinogenesis is the ability of the protein to

activate telomerase (Veldman et al., 2003). In uninfected cells undergoing

continuous rounds of cell division, the DNA telomeres shorten until the resulting

chromosomal instability inhibits the cell from undergoing further divisions and

the cell becomes senescent. By activating telomerase and inducing the

lengthening of the host cells telomeres, the E6 protein inhibits senescence and

prolongs the life cycle of the cell for the perpetuation of progeny viruses.

3.4.6 HPV E7 protein

HPV E7 protein is 107 amino acids in length. Like E6, it contains 2 Cys-

X-X-Cys zinc finger domains. E7 has three conserved regions: the CR1

domain in the N terminal region, the middle CR2 domain which contains the

consensus sequence LXCXE that is essential for E7 binding to Retinoblastoma

protein (pRB), and the C terminal CR3 domain that contains the zinc fingers

(Hebner et al., 2006). The protein also has phosphorylation sites, where the

protein can be modified during the G1 and S phases of the cell cycle, that are

essential for the role of E7 in maintenance of a continually dividing infected cell.

The function of E7 that is central to HPV induced carcinogenesis is its

ability to bind to and inactivate Retinoblastoma protein (pRB) and related family

member proteins p107 and p130 (Felisani et al., 2006; Ying and Xiao 2006). E7

32
forms a covalent complex with the active hypophosphorylated form of pRB and

promotes its proteolytic destruction (Munger et al., 1989; Felisani et al., 2006).

pRB is a tumor suppressor protein that is approximately 110 kDa in size.

When functional pRb is transfected into tumor cells with a mutant pRB gene, the

result is growth arrest, indicating that the function of pRB as a tumor suppressor

is to restrict cell proliferation (Felisani et al., 2006). The most relevant target of

pRB is the cellular transcription factor E2F. E2F binds to specific DNA sequences

as a heterodimer with the protein DF and controls the transcription of E2F

responsive genes (Giacinti and Giordano 2006). Active E2F is a potent stimulator

of cell proliferation and supports entry of the cell into the S phase of the cell

cycle. Three general classes of genes are under E2F control. These include the

following: genes such as cyclins A and E, c-myc, pRb, and p107, all of which are

upregulated at the G1/S transition; genes that are required for DNA replication

during S phase, such as DNA polymerase and PCNA; and genes involved in

the induction of apoptosis such as p19ARF (Felisani et al., 2006). In normal cells,

pRB is regulated by phosphorylation in a cell cycle dependent manner. In the G1

phase, pRB exists in a hypophosphorylated (active) form where it is tightly

bound to the E2F transcription factor, acting as a repressor of the transcription of

E2F sensitive genes (figure 8). As the cell cycle proceeds, phosphorylation of pRB

33
P P

Cyclin D pRb Cyclin E


Cdk2/4 Cdk2
P

pRb
P
P P

pRb
G1 P
P

Cyclin A
Cdk2
pRb P
M S

P
P

pRb
P

G2 P
P

PP1
P
P P

P pRb P

P P
P

Figure 8. Phosphorylation of retinoblastoma protein by cyclin/CDK

complexes. Beginning in G1, cyclin/CDK complexes phosphorylate pRb

resulting in its release from the E2F transcription factor. PP1 protein regenerates

the hypophosphorylated form in G2/M. (Giacinti and Giordano 2006).

34
is upregulated (Felisani et al., 2006). pRB is phosphorylated by serine/threonine

kinases called cyclin dependent kinases (CDKs), which associate with regulatory

subunits called cyclins. In total, there are approximately 15 CDK

phosphorylation sites on pRB. Phosphorylation of pRB inhibits its interaction

with E2F, and, once pRB is dissociated from E2F, transcription of E2F active

genes is permitted (figure 9). Phase specific phosphorylation of pRb occurs as

follows (figure 8):

Early in G1, cyclin D protein heterodimerizes with CDKs 4 and 6 to

form active cyclin/CDK enzyme complexes that perform the first

phosphorylation of pRB in the G1 phase.

Later in G1, cyclin E associates with CDK 2 and this enzyme complex

phosphorylates pRb at additional residues near the restriction point.

Cyclin A associates with CDK2 in the S phase of the cell cycle and

hyperphosphorylates pRB.

As the cell reaches mitosis (M phase), the phosphates are removed by

protein phosphatase1-like protein (PP1) and pRB is recycled to its

hypophosphorylated state, it again binds to E2F, and the cycle of

phosphorylation begins again when the cell reenters G1 (Giacinti and

Giordano 2006).

35
P
P

pRb
P

Cyclin D P
Cdk2/4 P
P

pRb P

X
Cyclin E
E2F DP Cdk2 E2F DP

TTTCGCGC TTTCGCGC

Active repression. pRb bound Transactivation. pRb released.


to E2F. Target genes off. target genes transcribed.

Figure 9. Hypophosphorylated Retinoblastoma protein blocks transcription of

E2F target genes. Phosphorylation of pRb by CDK/cyclin complexes removes its

repressive function and allows genes to be transcribed at the appropriate point in

the cell cycle (Munger et al, 1992).

36
HPV E7 overrides checkpoint control of the cell by pRB by inducing degradation

of the hypophosphorylated form of pRB (E2F repressor), and E2F is left

constitutively active and unregulated (Munger et al, 1992). Unbound to pRB,

E2F promotes unchecked DNA synthesis and cell division. The process by which

E7 abolishes the function of pRB is shown in figure 10.

Table 4 shows the known cellular targets of HPV E7 oncoprotein. E7

protein can also bind to and inactivate the cyclin-dependent kinase (CDK)

inhibitors p21/Cip1 and p27/Kip1 and inhibit their ability to induce cell growth

arrest (Westbrook et al., 2002). E7 protein can modify the cell cycle in other

ways via its ability to bind to cyclin-kinase complexes, such as the cyclin A/cdk2

and cyclin E/cdk2 complexes, and cyclin dependent kinase inhibitors (CDKIs),

and also through its ability to increase cyclin A and cyclin E levels within HPV-

infected cells. Since both cyclins A and E act to phosphorylate pRB, the ability of

the viral protein to inhibit cyclin destruction and increase cyclin levels is another

mechanism the virus uses to disrupt the interaction of hypophosphorylated pRB

with E2F (Hebner et al., 2006).

Another procarcinogenic consequence of E7 expression is the induction of

genomic instability in HPV infected cells (Felisani et al., 2006). E7 expressing

cells exhibit a variety of abnormal karyotypes, such as tetrasomy, and also are

37
Transcriptional E7
Repressor E7
Complex E2F
pRb
E2F
pRb

G1 Transcriptional
E2F activator

PO4 PO4
pRb M S pRb
PO4 PO4

G2
PO4
pRb
PO4

Figure 10. Role of HPV E7. E7 oncoprotein functions through forming a

complex with and promoting proteolysis of hypophosphorylated pRB, the active

form of retinoblastoma tumor suppressor gene protein. Formation of E7/pRB

complex interferes the binding of pRB with E2F. As a result, the E2F

transcription factor is freed and continues its work as a to promote DNA

synthesis and cell proliferation.

38
Table 4. Cellular Targets of HPV E7 Oncoprotein

Adapted from Scheurer et al, 2005

Target Effect

pRb Disruption of cell cycle regulation

p107 Disruption of cell cycle regulation

p130 Disruption of cell cycle regulation

E2F/Cyclin A complex Disruption of cell cycle regulation

Cyclin E Disruption of cell cycle regulation

p21 Impairs growth arrest pathways;


diminished effectiveness of TNF

p27 Disruption of TGFgrowth arrest

p48 and IRF1 Alteration of interferon signaling

Unknown Abnormal duplication of centrosomes

39
known to have abnormal numbers of centrosomes. Observations of

chromosomal abnormalities appear to be independent of the interaction of E7

protein with pRB, and undoubtedly contribute to the formation of precancerous

lesions and malignant tumors of the uterine cervix (Hebner et al., 2006).

3.4.7 HPV L1 and L2 proteins

Little is known about the products of the HPV late promoter. HPV late

proteins L1 and L2 make up the components of the viral capsids. Both proteins

are expressed late in the life cycle of the virus; L1 and L2 self assemble into viral

capsids that capture nascent HPV genomes to form complete progeny virions.

The mature virus particles exit the cell when it dies (Finnen et al., 2003; Zheng

and Baker 2006). The 600 Angstrom HPV capsids are composed of 72 pentameric

capsomers arranged in a T = 7 icosahedral lattice structure (Finnen et al., 2003).

Each capsomer contains 5 L1 major capsid protein monomers (~55 kDa) and 12

L2 minor capsid proteins (~74 kDa). The exact structural interactions between

the proteins are unknown, although extensive studies of the structures of virus-

like particles (VLPs) have shed light on the approximate structure of native

virions (Finnen et al., 2003; Buck et al., 2005).

L1 and L2 proteins have been studied for use as targets for HPV

vaccine studies, although their expression late in the viral life cycle may make

40
them less useful than early gene products for this purpose (Tomson et al., 2004).

3.5 Escape of human papillomaviruses from the immune system

The development of immunity to papillomaviruses is type-specific,

meaning that an individual can be immunologically resistant to one HPV type

while remaining susceptible to others. All papillomaviruses, whether high risk

or low risk, establish long term latent infections within the stem cells of the basal

layers of the skin. Most often, the host immune system is capable of clearing the

virus after persistence of the infection for 12-18 months, although in some cases,

the infection may never be fully eliminated. The few patients who are unable to

resolve the infection are the ones who are believed to be at high risk for

developing cancer (Stanley 2006). It is believed that the ability of the virus to

escape detection by the immune system is central to establishing persistent

infections that lead to the development of precancerous and cancerous lesions

(zur Hausen 2000).

Immunological suppression of the virus is believed to block the

appearance of clinical symptoms such as warts. Early infections are successfully

eliminated by MHC class I presentation of viral antigens and activation of CD8+

(cytotoxic) T lymphocytes (CTLs). However, once the E6 and E7 proteins are

expressed, CTLs appear to no longer recognize infected cells. Indeed, the virus

41
has evolved a number of mechanisms by which it can successfully evade and

subvert the host immune system (Stanley 2006).

The ability of the virus to successfully escape immune detection by both

the innate and acquired immune systems is related in part to all of the following

processes:

HPV E5 protein mediated down regulation of MHC class I expression,

confinement of MHC class I molecules to the Golgi apparatus, and loss

of the MHC class I associated transporter protein TAP 1; all of the

above impair the ability of the infected cell to present viral antigens to

CTLs. HPV-16 E5 protein selectively downregulates HLA-A and

HLA-B, the molecules that are used by the cell to present viral peptides

to cytotoxic T cells, but not HLA other subtypes. Selective

downregulation of cell surface HLA class I molecules may allow the

virus to establish infection by avoiding immune destruction of virus-

infected cells by CTLs, the cells that would normally slaughter cells

displaying aberrant peptides in HLA-A and HLA-B molecules.

HPV induced alterations in cytokine expression result in the inability

of infected cells to communicate properly with cells of the host

immune system.

42
Infected cells have the ability to ignore apoptotic stimuli stemming

from the tumor necrosis factor alpha (TNF-pathway.

The virus suppresses the induction of interferon alpha (IFN-

inducible genes. Interferons are chemicals released by virus infected

cells and induce antiviral and protective processes in surrounding cells

through the activation of Jak/Stat signaling pathways (Stanley 2006).

4. Molecular Biology of Carcinogenesis

The term carcinogenesis defines the process by which normal cells are

transformed into malignant tumor cells. Cancer is the loss of cellular control

over cell division. At the cellular level, two types of genes, positive regulators, or

inducers, called oncogenes, and negative regulators called tumor suppressors,

control the process of carcinogenesis. Tumor suppressors have critical functions

in protecting cells from becoming cancer cells and killing the host. p53 is

perhaps the most important tumor suppressor protein. p53 and the molecules it

regulates are described below.

4.1 p53

p53 is perhaps the most intensively studied protein in history. Over

10,000 journal papers are published each year related to p53. The p53 tumor

43
suppressor gene (TP53) is the most commonly mutated gene in human cancers;

more than 50 percent of all cancers contain a mutation in this gene (Yee and

Vousden 2005; Bouchet et al., 2006). The TP53 gene codes for a 53 kilo Dalton

protein that has several evolutionarily conserved domains. These include a

sequence-specific DNA binding domain, a tetramerization domain, an SH3

binding domain, and conserved C-terminal and N-terminal region sequences.

The binding domains of p53 are illustrated in figure 11 (Yee and Vousden 2005).

A myriad p53 functions have been described including transcriptional

activation, regulation of apoptosis, permeabilization of the mitochondrial

membrane, exonuclease activity, DNA repair, and regulation of angiogenesis

(Vousden and Lu 2002; Manfredi 2003; Meek 2004; Sengupta and Harris 2005).

The p53 tumor suppressor protein normally remains in the cell at low levels in an

inactive form. An autoregulatory feedback loop keeps p53 protein at low levels

because Mdm2 protein binds to p53 protein and targets it for degradation via

ubiquitin proteolysis. The regulation of cellular p53 levels by Mdm2 is illustrated

in figure 12 (Momand et al., 1992; Picksley and Lane 1993).

p53 protein can be activated and stabilized during periods of cellular

stress. Unlike other proteins, the increase in p53 protein levels and its activation

during times of cellular stress are most often a result of post translational

44
Nuclear
Oligimerization
Export
Signal

1 97 300 393
N C

Proline Sequence specific


Rich Nuclear
DNA binding and
Nuclear Export
binding of BCL2
Localization Signal
Transactivation family proteins
Signal
Rich

Figure 11. Diagram of the domain structure of p53 protein. The N terminus

contains the transactivation domain and a proline rich region required for

apoptosis. The N terminal region also contains the nuclear export signal,

phosphorylation sites, and the binding site for Mdm2. The central region

contains the DNA binding domain and binding sites for BCL2 family member

proteins. The C terminus contains a nuclear export signal, the oligimerization

domain, and sites for various posttranslational modifications. (Adapted from

Yee and Vousden 2005).

45
p53 degraded by
p53 UBQ proteolysis
Mdm2
Proteosome

Mdm2

p53
Mdm2

Mdm2
Nucleus
p53

Figure 12. Mdm2 control of p53 levels in normal cells. p53 levels are kept low

in normal cells because Mdm2 protein binds to p53 and shuttles it to the

cytoplasm where p53 protein is degraded by ubiquitin proteolysis. Mdm2

protein is then recycled (Picksley and Lane 1993).

46
modification of the p53 protein by phosphorylation or acetylation rather than

genetic transcriptional or translational upregulation (Vousden 2000). For

example, p53 protein can be stabilized by phosphorylation at the N-terminus,

which inhibits its interaction with Mdm2 and subsequent degradation. Once p53

protein is stabilized and activated, it has a multitude of functions. These

functions can be divided into two general categories: transcription dependent

and transcription-independent functions (Yee and Vousden 2005).

4.1.1 p53 as a transcription factor

Of all its functions, the most well defined role of p53 is its operation as a

transcription factor. By governing the transcription of countless genes, p53

makes life or death decisions about the fate of a cell. Hundreds of p53 target

genes have been identified, and a partial list of these genes and their functions is

shown in figure 13 and table 5 (Michalak et al 2005; Yu and Zhang 2006).

p53 binds to DNA as a homotetramer. p53 binds the to a repeat DNA

palindrome with the sequence RR(CA/TA/TG)YYY(0-14 nucleotides) (Kitayner et

al, 2006).

Transcriptional activation by p53 is becoming well understood as the

number of studies describing the roles of p53 as a transcription factor continues

to expand. Phosphorylation or acetylation of p53 at the C and N termini

47
p53
MDM2

ATM p300/pCAF

P
P

MDM2 p53
P Acetyl
p53 stabilized via
posttranslational modification

NOXA
P21 PUMA
BAX IGFR FAS/APO1 -Binds to mitochondria
14-3-3-
ROS/PIGs BCL-2 DR5 -Modulates BH3 proapoptotic
Cyclin G
p53AIP1 Survivin PIDD proteins ie., BAX
PTGF-

Mitochondria Death Receptor

GROWTH
ARREST
APOPTOSIS

Transcriptionally dependent Transcriptionally


independent

Figure 13. A partial list of the many roles of p53. Both transcriptionally

dependent roles and transcriptionally independent roles in the processes of

growth arrest and apoptosis are shown (Yu and Zhang 2006).

48
Table 5. Cellular Targets of p53 in the Activation of Apoptosis

Adapted from Michalak et al, 2005

Target Effect

ALDH4 Enzyme-proline degratation

APAF1 Activation of caspase-9

BAX Proapoptotic BCL-2 family member

BID Proapoptotic BCL-2 family member (BH3 only)

FAS/APO-1 Death receptor

FDXR Steroid biosynthesis

IGF-BP3 IGF inhibitor, antimitogenic

NOXA Proapoptotic BCL-2 family member (BH3 only)

P53AIP1 Dissipates mitochondrial membrane potential

P53DINP1 Kinase cofactor for p53 Ser-46 phosphorylation

PIDD Death domain protein/ caspase-2 activation

PIG3 Redox regulation

PIG8/ei24 Redox regulation

PTEN Tumor suppressor/ phosphatase

PUMA Proapoptotic BCL-2 family member (BH3 only)

SIVA Death domain protein

WIP1 Serine/ threonine kinase

49
augments binding to specific DNA targets and upregulates transcription of p53

responsive downstream genes. Results of p53 transcriptional activation can

result in two cellular processes, growth arrest and apoptosis. The first target

gene of p53 to be discovered was p21 (Cip1/Waf1). Upregulation of the p21 gene

by p53 results in the inhibition of CDKs and causes growth arrest (Foijer and

te Riele 2006). This allots the cell time for DNA damage repair or activation of

programmed cell death pathways (apoptosis).

A second class of key transcriptional targets of p53 is genes involved in

the pathway to apoptosis. The upregulation of certain genes by p53 is critical for

the induction of apoptosis. Studies show that transcriptionally impaired mutants

of p53 are unable to induce apoptosis and respond appropriately to oncogene

overexpression (Erster and Moll 2005). p53 can modulate the extrinsic pathway

to apoptosis by upregulating the expression of the death receptors Fas, DR4, and

DR5 (Yu and Zhang 2006). In the intrinsic apoptotic pathway, p53 controls the

expression of pro- apoptotic genes such as Bax, and genes that function upstream

of Bax, such as PUMA and NOXA (Vousden 2005).

In contrast to transcriptional activation, the roles of p53 as a

transcriptional repressor are less understood. It is currently accepted that genes

repressed by p53 include BCL2, BCL-XL, and survivin. The role of suppression

50
of these molecules by p53 likely prevents the anti-apoptotic function of these

proteins from interfering with the pathway to apoptosis, but the mechanisms by

which these repressions occur is not currently understood will be determined in

ongoing studies (Yu and Zhang 2006).

4.1.2 Transcriptionally independent roles of p53

It is known that p53 fragments that lack the DNA binding domain retain

the ability to induce apoptosis independent of upregulation of p53 translation

(Yee and Vousden 2005). It is now known that p53 has a number of

transcription-independent roles in the induction of apoptosis (shown in figure

13) (Vousden 2005).

p53 is able to function similarly to proapoptotic members of the BCL-2

family that contain sequence regions called Bcl-2 homology 3 (BH3) domains.

Though it does not contain a BH3 domain itself, p53 is capable of binding to

several anti-apoptotic members of the BCL-2 family in the cytoplasm including

BCL-2 and BCL-XL. By binding to these proteins and abrogating their pro-

survival roles, p53 shunts the cell toward apoptosis (Vousden 2005).

The induction of a protein called p53 upregulated modulator of apoptosis

(PUMA) by nuclear p53 is essential for the function of cytoplasmic p53 (figure

14). Activation of the TP53 gene results in the translation of p53 protein that

51
Pro-Apoptotic Events
BCL-2
bax
cytochrome c release
p53 caspase activation

Nucleus
Mitochondria

p53
PUMA p53
BCL-XL
BCL-XL

Figure 14. PUMA protein and the activation of cytoplasmic p53. Stabilization

of nuclear p53 results in its activation of the PUMA gene. In the cytosol, PUMA

protein facilitates the release of cytoplasmic p53 from the grip of the anti-

apoptotic BCL-XL protein. Cytosolic p53 then activates apoptotic pathways at

the mitochondria (Vousden 2005).

52
accumulates in both the nucleus and cytosol of the cell. Cytosolic p53 is bound

to and inactivated by the antiapoptitic BCL-XL protein. As nuclear p53 is

stabilized and activated, the transcription of the PUMA gene is induced by

nuclear p53 (Vousden 2005). The function of PUMA protein is to release

cytosolic p53 from the grip of BCL-XL in the so that it can activate the chain of

events in the cytoplasm that trigger apoptosis. These events affect the

mitochondria through downregulation of BCL-2, activation of the pro-apoptotic

Bax protein, cytochrome c release, and caspase activation (Vousden 2005). The

overall picture of p53 and molecular players in apoptosis is shown in figure 15.

4.1.3 Decision between cell cycle arrest versus apoptosis by p53 and its targets

How p53 determines the fate of a cell when it is stabilized and activated

remains an intensely studied topic. The choice between growth arrest versus

apoptosis appears to be governed by the subset of targets activated by p53 at a

particular time. There are 2 models of how activation of p53 leads to the choice

of cell cycle arrest over apoptosis. The first model, the p53 dumb model, states

that activation of p53 always results in the same cellular response, which is a

signal for apoptosis. This pathway can then be blocked by independently-

induced survival signals resulting in cell growth arrest, or in other cases, the

apoptotic signal can be amplified to reach a required threshold level for

53
dATP
cyt. C
Procaspase-9
apaf -1

Procaspase -3

Apoptosome Caspase-9
APOPTOSIS

Caspase-3
Cytochrome C
BCL-XL

GROWTH
BCL-2 pRb ARREST

Bax

cdk
p53
p21

DNA Damage, cell stress,


chemotherapeutic agents

Nucleus

Figure 15. Downstream targets of p53 in the induction of apoptosis. The

stabilization of p53 can result in growth arrest or the activation of apoptosis.

54
induction of apoptosis by different independent signals. The second model, the

p53 smart model, suggests that p53 is able to govern the cellular response to its

activation. In this model, p53 is able to function differently in cells undergoing

growth arrest versus cells destined to die via apoptosis (Vousden 2005). Studies

have revealed that p53 levels themselves can affect the response of cells to p53

activation; low levels of p53 tend to favor growth arrest whereas high p53 levels

activate apoptosis. Promoter binding of p53 may also be affected by p53 post-

translational modifications such as phosphorylation or acetylation (Vousden

2005). Moreover, the presence of PUMA protein may be required for p53 to

induce apoptosis as well (Vousden 2005).

4.1.4 p53 and human cancers

A disruption of the normal protective and adaptive mechanisms of p53

can be initiated in several ways. For example, mutations in the TP53 gene can

result in the expression of an abnormal protein that is not functional in DNA

binding, or accelerated loss of p53 protein can occur via proteasomal degradation

induced by the presence of high risk HPV E6 protein or overexpression of Mdm2

protein. Mechanisms such as these effectively allow DNA-damaged cells to

continue dividing without checkpoint controls, and hence, obstruction of p53

function can initiate carcinogenesis and the formation of malignant tumors.

55
4.2 Apoptosis

Apoptosis, or programmed cell death, is a process in which a cell commits

suicide via the destruction of vital cellular components or DNA. Cells

undergoing apoptosis condense and fragment into membrane-bonded apoptotic

bodies, which are then ingested and removed by phagocytes (Cereghetti and

Scorano 2006). The process involves a number of signaling pathways and can be

activated by several different mechanisms. The rate at which cells undergo

apoptosis is tightly controlled since severe pathological conditions, such as

autoimmune disorders, neurodegenerative diseases and cancer, can result from

abnormal rates of apoptosis, be they to rapid or too slow (Alirol and Martinou

2006).

Apoptosis can be broken down into three phases, each of which are

regulated by specific molecules. The phases of apoptosis are described as

follows (Pollack and Leeuwenburgh 2001):

(1) Induction phase Pro apoptotic signals trigger pro-apoptotic signal

transduction pathways during the induction phase. Examples of pro-

apoptotic signals include the following: reactive oxygen and nitrogen

56
intermediates, TNF-, ceramide, intracellular calcium pathway

overactivation, and Bcl-2 family members.

(2) Effector phase - Key signals commit the cell to die in the effector phase.

These regulatory signals include the following pathways: cell membrane

bound death receptors (FAS), nuclear activators (i.e. p53), endoplasmic

reticulum pathways, mitochondrial-induced pathways (i.e. cytochrome c)

(3) Degradation phase Activation of caspases and cellular endonucleases

results in the degradation of cellular proteins and DNA.

In addition to being divided into phases, apoptosis can occur via 2 unique

pathways, the intrinsic pathway and the extrinsic pathway. The extrinsic

pathway is triggered by signals from cell surface TNF-family receptors which are

carried over a network of adapter proteins. The activation of the signaling

pathway results in the activation of initiator cysteine protease caspase-8. The

intrinsic (mitochondrial) pathway is entirely intracellular and its activation is

dependent upon signaling from activated p53 protein. p53 activates a number of

down stream signals such as the release of cytochrome C from the mitochondria.

This, in turn, triggers the activity of initiator caspase-9. Once the initiator

caspases are activated, the pathways converge upon the same effector caspase-3,

and its activation results in proteolytic degradation of key cellular components in

the process of apoptosis (Pollack and Leeuwenburgh 2001). The intrinsic and

57
extrinsic apoptotic pathways are depicted in figure 16.

4.2.1 Regulation of the Mitochondrial (Intrinsic) Pathway to Apoptosis

p53 and BCL-2 family proteins regulate the mitochondrial pathway to

apoptosis through both inhibitory and provocative mechanisms. Anti-apoptotic

proteins from the BCL-2 family, such as Bcl-2 and BCL-XL, block the pathway to

apoptosis. Conversely, pro-apoptotic members such as Bax and Bad induce

apoptosis (Fadeel 2003). The mitochondrial pathway to apoptosis is mediated by

the signaling from the pro-apoptotic BCL-2 family protein Bax which translocates

to the mitochondrial membrane and induces the release of cytochrome C from

the mitochondria. Cytochrome C is a key regulator of the effector phase of

apoptosis. Once cytochrome C is released from the mitochondria, the cell is

irreversibly committed to apoptosis. Cytochrome c complexes with Apaf-1,

initiator caspase-9, and ATP to form a structure called the apoptosome. Effector

caspase-3 is activated by the apoptosome complex, and caspase-3, in turn

initiates the destructive caspase cascade and the irreversible final degradation

phase of apoptosis (Cereghetti and Scorano 2006).

4.3 Other important molecular players in the apoptotic pathway

4.3.1 Survivin

Survivin is a bifunctional protein that both controls cell division and

58
Ligand
Survivin, a member of the inhibitor of apoptosis family of proteins (IAP), is a
INTRINSIC EXTRINSIC
PATHWAY p53 PATHWAY
Fas

Mitochondrion

Cytochrome C release

Procaspase-9 Active Caspase-9 Active Caspase-8 Procaspase-8

Procaspase-3 Active Caspase-3

Figure 16. Intrinsic and extrinsic pathways to caspase activation. The p53

dependent intrinsic apoptotic pathway is triggered by the activation of initiator

caspase-9. The extrinsic pathway to apoptosis is activated by initiator caspase-8.

Both pathways result in the activation of effector caspase-3.

59
inhibits apoptosis. The IAP family is a group of proteins that have at least one

BIR domain (a protein fold that binds zinc ion) and suppress apoptosis when

overexpressed in cell lines (Reed et al., 2001). Some IAP family members have

the ability to bind to and inhibit caspases. Studies have shown that survivin

may have the ability to bind to and block caspase-3 activity, but comparisons of

the structure of survivin with other known direct caspase inhibitors such as XIAP

do not show a direct structural explanation for how this may be possible (Reed et

al., 2001).

Survivin is of great interest as a potential target for anticancer therapies

because its expression is highly tumor specific. Most normal adult cells do not

contain abundant amounts of survivin mRNA or protein, whereas cancerous

tumors express high levels of the survivin protein. This indicates that the

survivin gene may be reactivated when normal cells become malignant.

Scientists are currently investigating the relationship between survivin

expression and carcinogenesis.

4.3.2 p38/ MAP kinase

Mitogen-activated protein (MAP) kinases are serine/threonine-specific

protein kinases. MAP kinases respond to extracellular signals called mitogens

and activate a signal transduction pathway that regulates cellular processes

60
including as gene expression, mitosis, differentiation, and cell survival versus

apoptosis decisions (Pearson et al., 2001). The p38 MAP kinase pathway can be

activated by cellular stress. The result of p38 MAP kinase activation varies

depending on the tissue type; in many cells, the pathway triggers apoptosis,

whereas in other tissues, its activation promotes survival. In some cases, MAP

kinase signaling activates p53 (Pearson et al., 2001)

5. HPV and cancers

The current hypothesis for induction of uterine cervical tumors by HPV is

that during the course of viral infection, the HPV genome randomly becomes

integrated into the hosts genome (Jeon et al., 1995; Corden et al., 1999). If the

integration of the viral genome results in disruption of the viral E2 gene, then

there is a loss of its control over expression of viral oncoproteins E6 and E7.

Uncontrolled expression of these two proteins is a consequence, and E6 and E7

interfere with functions of tumor suppressor proteins which control both the cell

cycle and apoptosis, resulting in initiation of pre-cancerous lesions and cancer

(Steger and Corbach 1997). E6 protein binds to p53 via the E6-Ap protein and

facilitates the degradation of p53 through the ubiquitin proteasome pathway

(Keen et al., 1994; Scheffner 1998). This dramatically decreases the half-life of the

61
p53 tumor suppressor protein in infected cells, and p53-mediated regulation of

the cell cycle is lost (Scheffner 1998). Additional evidence for deregulation of the

p53 pathway by HPV E6 is that HPV-positive cell lines are resistant to p53-

dependent chemotherapy drugs such as cisplatin (Padilla et al., 2002).

In addition to the loss of p53 protein, the overexpression of E6 and E7

proteins facilitates uncontrolled proliferation of the infected cell through the

activation of cellular cyclins E and A (Malanchi 2002; Caldeira 2000). E7 protein

also interferes with the functions of the cellular tumor suppressor protein

retinoblastoma (pRB). Viral E7 protein binds to pRb and inhibits the interaction

of pRB with E2F and other factors associated with the cell cycle (Ying and

Xiao 2006). An illustration of the processes that contribute to HPV induced

carcinogenesis is depicted in figure 17.

As previously stated, the committed step in the transformation of an HPV

infected cell to one capable of becoming cancerous is believed to be the

integration of the viral genome into host DNA. The process by which this,

integration occurs is poorly understood but it appears to be an infrequent event

since there is a large discrepancy between the occurrence of HPV infection and

the incidence of cervical cancer. Upon its integration into host chromosomes,

HPV DNA frequently breaks in the E1/E2 gene region. In the episomal state, the

62
E7 proliferation E6
Ubiquitin
E6 Proteolysis
p53

E6
E7 p53

X
E5 immortalization
E6Ap
E6

Growth Arrest
Apoptosis
Loss of cell polarity,
E6 DNA damage

E6 Loss of cell adhesion,


DNA damage

Metastasis

Distant Organs

Figure 17. Processes involved in HPV-induced carcinogenesis.

63
E2 gene controls both the upper regulatory region (URR), which governs viral

transcription and replication, and also the E6 and E7 oncogenes, the two viral

genes responsible for cell transformation. Integration and loss of E2 is critical to

carcinogenesis becauseE2 protein is such an important regulator of the E6 and E7

oncogenes. Integration that interrupts the E2 gene locus also causes a loss of E2

mediated transcriptional control over both transcription and replication of the

virus (Ying and Xiao 2006).

5.1 Coexpression of HPV E6 and E7 facilitates carcinogenesis

High risk E6 and E7 proteins are usually coexpressed within infected cells.

By simultaneous disruption of the functions of the functions of key cellular

tumor suppressors p53 and pRb, high-risk HPV E6 and E7 proteins immortalize

and transform human cells, setting the stage for cancer progression. As

previously stated, E7 interferes with the cellular tumor suppresser pRb and its

related family members, and inactivates cellular cyclin dependent kinase

inhibitors including p21 (Westbrook et al., 2002). As a consequence, there is

increased expression of cyclin E, cyclin A and aberrant CDK2 activity mediated

by constitutively active E2F in infected cells. Since E2F has the ability to

upregulate p14ARF to stabilize p53, the function of high risk E6 protein is also

64
important to cell transformation and carcinogenesis. HPV E6 oncoprotein

induces the rapid degradation of p53 via the ubiquitin proteasome pathway by

interacting with E6-AP, a host cell protein/ ubiquitin ligase (Barbosa et al., 1989;

Patel et al., 1999).

5.2 p53 status of cervical cancers

Most cervical tumor cells that have integrated HPV generally retain wild

type TP53 and RB genes (Denk et al, 2001; Scheffner et al, 1991). As the viral

oncogenes and proteins efficiently and effectively interfere with the functions of

cellular tumor suppressor proteins, p53 and pRB, there is seldom any secondary

mutation within the tumor suppressor or apoptotic genes themselves in HPV-

induced tumors (Crook and Vousden 1994). The wild type status of p53 in HPV-

positive tumors is particularly noteworthy because often the mutation of p53 is

the transforming event in other types of cancer (Vousden 2000).

5.3 Current treatments for HPV-induced cancers of the uterine cervix

Treatment options for HPV positive uterine cervical tumors vary with the

stage of disease at diagnosis, the health of the patient, and desire for bearing

children, but in all cases involves surgical removal of the malignant tumor

(Moore 2006). In early stages (see appendix A), tumors are curable by surgical

65
intervention alone. Procedures such as the loop electrosurgical excision

procedure (LEEP) and trachelectomy are conservative treatments that allow a

patient to bear children in the future (Schlaerth and Abu-Rustum 2006). In more

advanced stages of cervical cancer, hysterectomy is the treatment of protocol,

and in the most advanced stages, radical surgery called pelvic exenteration,

which includes removal of all pelvic organs (bladder, rectum, cervix, and

vagina), is performed (Moore 2006).

More advanced cases and cases of recurrent cervical cancers may require

the use of radiation and/or chemotherapy in addition to surgical treatment. A

variety of chemotherapeutic drugs can be used to treat cervical cancer. The

standard first line treatment for invasive cervical cancer is usually cisplatin

(Platinol) and 5-fluorouracil (5-FU, Adrucil, Efudex) used in combination and in

addition to radiation. Recurrent and late stage cervical cancers are often treated

with a combination of platinum, bleomycin, methotrexate, and 5-FU (DuPont

and Monk 2006). Chemotherapy is administered intravenously, through

injection, or in pill form. Side effects often accompany treatment and may be

severe; they can include nausea, vomiting, diarrhea, and leukopenia (low white

blood cell count). Most of these side effects are a result of drug toxicity to

healthy tissues such as skin, hair follicles, and epithelial cells that line the

66
digestive tract (Stewart and Viswanathan 2006). Patient tumors can develop

resistance to these therapies as well (Muggia et al, 2004). Hence, there is the need

to develop alternate, safer, less toxic, and more specific therapies for UCC.

6. Natural Products as Anticancer Agents

Some of the most successful currently used anticancer drugs are extracts

or derivatives of compounds from plants or other natural products. These

include the breakthrough class of drugs known as taxanes (Paclitaxel, Docetaxel)

that come from the yew tree (Taxus baccata ), and the vinca alkaloids (Vincristine,

Vinblastine, Vinorelbine) which are derivatives from the periwinkle plant

(Catharanthus roseus). Other examples of natural products include the antitumor

antibiotics such as anthracyclines, dactinomycin, bleomycin, adriamycin, and

mithramycin, which are all compounds derived from fungi. These compounds

affect the growth of all rapidly dividing cells, whether normal or malignant, and

like other drugs, are associated with significant toxicity and side effects.

Millions of research dollars each year go to the discovery of new natural

products with limited side effects that may be used as effective antitumor agents.

Some compounds that are currently being investigated are components of

dietary plants (Wilson and Danishefsky 2006; Tan et al., 2006). Examples of such

67
compounds are resveratrol, indole-3 carbinol, quercetin and other catechin

compounds, azaphilones, and flavonoids. Many of these potential drugs are

antioxidants and fall into a class of chemical compounds called polyphenols

(structures shown in figure 18).

Recent studies have identified a class of naturally occurring polyphenol

compounds called lignans that may be potent antiviral and anticancer agents

(Hwu et. al, 1998; Gnabre et. Al, 1996; McDonald et al, 2001). Three such

compounds, nordihydroguaiaretic acid (NDGA), and its methylated derivatives,

3-O-methyl-nordihydroguaiaretic acid (3-O-methyl-NDGA) and tetra-O-

methyl-nordihydroguaiaretic acid (tetra-O-methyl-NDGA) are derived from the

creosote bush, larrea tridentata. The structures of these compounds are depicted

in figure 19. It has been suggested that NDGA or its derivatives may be an

effective treatment for tumors as well as other diseases (Artega et al., 2005).

NDGA is the most studied of the lignans. It has a variety of potential

antitumor properties and is a compound that has been used by Native

Americans throughout history for treating a variety of diseases including cancer.

Some of the potential anticancer properties of the lignan are described as follows.

First, NDGA is a potent inhibitor of 5-lipoxygenase and other lipoxygenase

enzymes (Bhattacherjee et al., 1988). By interfering with the actions of the

68
OH
CH 2OH
HO

N
H
OH

Indole-3-Carbinol
Resveratrol
R5
OH O R4 R6
OH O
R1
R7
OH
HO O R3
OH R2 O

Quercetin Flavonoids

O
O O
O
OH

O OH

Azaphilones

Figure 18. Structures of some naturally occurring polyphenols that have

anticancer properties.

69
HO HO

HO HO

OH OMe
OH OH

Nordihydroguaiaretic Acid 3'-O-Methyl-Nordihydroguaiaretic Acid

MeO

MeO

OMe
OMe

Tetra-O-Methyl-Nordihydroguaiaretic Acid

Figure 19. Chemical structures of lignans derived from the creosote bush

Nordihydroguaiaretic Acid (NDGA), 3-O-Methyl-Nordihydroguaiaretic Acid

(3-OMe-NDGA), and tetra-O-methyl-nordihydroguaiaretic acid (tetra-O-methyl-

NDGA).

70
enzyme, NDGA blocks the synthesis of inflammatory mediators such as

prostaglandins and leukotrienes. Products from the prostaglandin pathway,

such as 5-HETE, are shown to be elevated in cancers and also stimulate tumor

growth (Wallace 2002). At higher concentrations in the micromolar range,

NDGA also inhibits cyclooxygenase (COX) enzymes (Wallace 2002). Second,

NDGA is known to prevent leukocyte infiltration into tissues and to stop the

release of reactive oxygen species (ROS) (Bhattacherjee 1988). NDGA can also

interfere with the platelet derived growth factor receptor and the protein kinase

C intracellular signaling pathway (Gschwendt et al., 1984). These signaling

pathways are known to be important in proliferation and survival of tumors.

Finally, NDGA is a direct inhibitor of the tyrosine kinase IGF-1 and

Her2/neureceptors (Youngren et al., 2005).

Experimental evidence for the success of NDGA as an antitumor drug

includes the observations NDGA induces apoptosis in tumor cells and tumor

xenografts and inhibits the growth of tumors in vitro and in animal models. One

potential benefit of its use as an antitumor drug is that NDGA is fairly nontoxic

to normal cells. Because of these results, there is interest in the compound for

clinical study. However, other studies of NDGA indicate it may have dose

limiting hepatotoxicity in animals and relatively high concentrations of NDGA

71
are required for efficacy. Hence, the discovery of more potent analogues of

NDGA is required (McDonald et al., 2001).

Studies show that the plant lignan, 3-O-methyl-nordihydroguaiaretic acid

(3-O-methyl-NDGA), from the creosote bush (Larrea tridentata) and other

methylated analogs of its parent compound, nordihydroguaiaretic acid (NDGA),

are potent antiviral agents against human immunodeficiency virus (HIV-1),

herpes simplex virus (HSV), and SV-40 (Hwu et al., 1998; Gnabre et al., 1995). 3-

O-methyl-NDGA is also a powerful inhibitor of gene expression from the P97

promoter of high-risk human papillomavirus type 16 (HPV-16) and endogenous

HPV E6/E7 gene expression within HPV DNA containing cervical cancer cells

(Craigo et al., 2000 and unpublished data). Previous work in our lab has

suggested that 3-O-methyl-NDGA may be not only be an effective antiviral

compound but also an anticancer agent against HPV induced cancers and

precancerous lesions of the uterine cervix. Since both viral oncogenes E6 and E7

are critical to malignant transformation and cancer progression, the disruption of

E6 and E7 expression from the HPV P97 promoter by 3-O-methyl-NDGA may

result in the stabilization of wild-type p53 in these cells. There is considerable

evidence establishing that stabilization and accumulation of active p53 protein

within the HPV transformed cervical tumor cell can induce cell cycle arrest or

72
apoptosis (Hietanen et al., 2000). Based on these observations, it is possible that

the lignans that inhibited the episomal P97 promoter would also inhibit E6

protein expression from the integrated P97 promoter in HPV transformed cervical

tumor cells and interfere with oncogene control over cellular tumor suppressor

genes, which would likely result in a return of cellular control over p53 stability.

If functional p53 can be reactivated within these cells then a cascade of events for

either cell cycle arrest or apoptosis would be set in motion meaning that the

compound may be an effective antitumor agent.

7. Specific aims for this study

The possibility that 3-O-methyl-NDGA can interfere with E6 and E7

functions makes it a potential small molecule therapy against HPV-induced

cancers of the uterine cervix. The central hypothesis of this research study is the

following: 3-O-methyl-NDGA stabilizes transcriptionally-active p53 in HPV-

positive cervical carcinoma cells resulting in the activation of pro-apoptotic

genes and apoptosis, and its action is mediated via an interference with the

function of HPV E6. Figure 20 illustrates this hypothesis. If the hypothesis is

the correct, it would represent a novel mechanism of inducing tumor cell death

in HPV-positive cancers. Because 3-O-methyl-NDGA may specifically target

73
HO

HO
3-O-methyl-NDGA
OMe
OH

PPrrooccaassppaassee--99

B
BCCLL--22
BBaaxx
E66//EE77 pp5533
PPUUMMAA
PPrrooccaassppaassee--3

C
Caassppaassee--99

C
Caasspaassee--33

APOPTOSIS

Figure 20. Hypothetical mechanism of action of 3-O-methyl-NDGA. The

lignan interferes with E6/E7 oncogene activity and results in the stabilization of

p53 protein. Stabilized p53 is active in inducing pro-apoptotic bax and caspase

enzymes resulting in programmed cell death.

74
HPV-positive cells, treatment of precancerous and UCC lesions with this lignan

in combination with other currently approved anticancer therapies may result in

a synergistic cytotoxic effect and permit treatment with lower doses of

conventional chemotherapy. This could potentially improve patient response to

the therapy while, at the same time, reducing side effects associated with

standard treatments.

The specific aims described below were investigated to test this

hypothesis.

(1) The ability of the plant lignan to induce cell cycle arrest in a variety of

cell lines was investigated.

(2) The cellular effect the stabilization of p53 by 3-O-Methyl-NDGA

has on HPV-positive cells was determined.

(3) The hypothesis that 3-O-Methyl-NDGA stabilizes transcriptionally

active p53 in HPV-positive cervical cancer cells was tested.

(4) Whether the stabilization of p53 in HPV-positive cancer cells is a

result of a decrease in E6 and E7 protein function was examined.

75
CHAPTER TWO

MATERIALS AND METHODS

1. Reagents

The primary compound used in this study, 3-O-methyl-

nordihydroguaiaretic acid [1-(3,4-dihydroxyphenyl)-4-(3-methoxy-4-

hydroxyphenyl)-2,3-dimethylbutane; 3-O-methyl-NDGA; 3-OMe-NDGA], was

a gift from Dr. R. Huang (Gnabre et. al, 1995, 1996; Hwu et. al, 1998).

Actinomycin D (Dactinomycin), 5-fluorouracil (5-FU), camptothecin (CPT), and

nordihydroguaiaretic acid [1,4-Bis (3,4-dihydroxyphenyl) -2,3-dimethylbutane;

NDGA] were purchased from Sigma. 3-O-methyl-NDGA was dissolved in 40

percent dimethylsulfoxide (DMSO, Sigma) and the stock solution was diluted to

final concentrations in Dulbeccos Modified Eagles Medium (DME) prior to

adding to cell cultures. All other compounds were dissolved in 100 percent

dimethylsulfoxide and stock solutions were diluted to desired concentrations in

Dulbeccos Modified Eagles Medium before addition to cell culture medium.

76
Final DMSO concentrations were 0.2 percent or less. Untreated vehicle controls

contained 0.2 percent DMSO.

2. Cell Culture

Cervical carcinoma cell lines used in this study were purchased from the

American Type Culture Collection (ATCC). C33-A, SiHa, and HeLa cells were

grown in DME, purchased in powder form from Sigma, containing 0.075 percent

sodium bicarbonate, 2 mM glutamine, and supplemented with 5 percent fetal

bovine serum (FBS) and 50 g/mL gentamycin sulfate (Sigma). CaSki cells were

grown in 4.5g/L glucose DME from Gibco supplemented with 10 percent FBS

and 50 g/ml gentamycin sulfate. All cell cultures were maintained at 37

Celsius in a humidified atmosphere of 5 percent CO2 and 95 percent air. The

HPV content and p53 status of these cell lines is outlined in table 6.

3. alamar Blue Assay- to measure cell toxicity

Cells were plated at a density of 7.5 x 103 cells per well in a 96 well plate.

3-O-methyl-NDGA was added to wells in a range of concentrations from

500 nM to 500 M. Cells were incubated for 20 hours after which 20 microliters

of alamar Blue Reagent was added to each well. Cells were incubated with

alamar Blue for an additional 4 hours. Following incubation, the absorbances of

77
Cell Line HPV Status p53 Status tissue

HeLa HPV-18 + wild type cervical carcinoma

CaSki HPV-16 + wild type cervical carcinoma

SiHa HPV-16 + wild type cervical carcinoma

C33-A negative mutant cervical carcinoma

HT-3 HPV-30 + mutant cervical carcinoma

MCF-7 negative wild type breast carcinoma

MRC-5 negative wild type Normal fetal lung fibroblast

Table 6. Cell lines used in this study and their HPV and p53 statuses.

78
each well at 560 and 600 nM were read on a plate reader (Molecular Devices).

Toxicities were calculated as a percentage of the absorbance of DMSO treated

controls and error due to antioxidant activity of the lignan on alamar Blue was

subtracted from drug treated samples.

4. MTT Assay- to measure cell viability

Cells were plated at a density of 7.5 x 103 cells per well in a 96 well plate.

3-O-methyl-NDGA was added to wells in a range of concentrations from

500 nM to 500 M. Cells were incubated for 21 hours after which media

containing lignan was replaced with fresh media. 100 microliters of MTT [3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in serum free medium

was added to each well and the plate was incubated for three hours at 37 C.

Following incubation, MTT/media was removed and replaced with 100

microliters of DMSO to solubilize formazan crystals. The plate was read on a

spectrophotometer (Molecular Devices) at 540 nm and results were reported as

growth percent of control.

5. Trypan Blue Assay- to measure cell death

Cells were cultured and treated with various concentrations of the lignan on a

six well plate. Following treatment, cells were trypsinized and placed in a micro-

79
centrifuge tube and combined with 100 microliters of Trypan Blue dye. Cell

suspension was then placed on a hemacytometer and cells were counted as either

blue or clear using a phase contrast microscope. Data was reported as viability

percent of control.

5. Western Blots

Cells were plated at a density of 1 x 106 cells per 10 cm2 plate, treated with

drug compounds, and cell lysates were prepared using radio immune

precipitation assay (RIPA) lysis buffer (50mM Tris-HCl pH7.4, 1 percent Nonidet

NP-40, 0.25 percent Na-deoxycholate, 150mM NaCl, 1 mM EDTA, 1 mM PMSF, 1

mg/ml aprotinin, 1 mM NaF). Cellular proteins were acetone- precipitated and

the pellet was re-dissolved in RIPA buffer. Protein concentrations in samples

were determined utilizing the Quant-Pro protein assay kit from Sigma. 10-20

micrograms of protein sample was loaded onto a SDS-page gel (Cambrex) and

transferred to a polyvinylidene fluoride (PVDF) (Westran) membrane. Blots

were blocked in blotto (5% Carnation nonfat dry milk in 0.1 percent Tween-20 in

TBS) overnight at 4Celsius. After blocking, blots were incubated with mouse

monoclonal primary antibodies (Santa Cruz) to p53, p21, bax, beta actin, survivin

(Abcam), or, in the case of PUMA, rabbit polyclonal antibodies (Abcam), for one

80
hour at 25 Celsius, followed by incubation with anti-mouse or anti-rabbit

horseradish peroxidase-conjugated secondary antibody (Promega) for one hour

at 25 Celsius. The internal standard used for protein loading was beta actin.

Bands were visualized using the chemiluminescent detection kit (ECL-plus) from

Amersham Biosciences and directions found in the ECL-plus kit.

6. Immunofluorescence Assay for p53- to measure p53 stabilization

CaSki cells were grown at a density of 4x 104 cells per well on an eight

chambered slide (Labtek). Following drug treatment, cells were washed twice

with PBS. Cells were fixed with 4 percent paraformaldehyde for 15 minutes.

Cells were blocked for 20 minutes in blocking buffer (5 percent glycine, 5 percent

FBS, 0.2 percent Triton X-100, 0.1 percent NaN3 in PBS). Cells were incubated

with mouse anti-p53 (Santa Cruz, San F) in blocking buffer for 1 hour at 37

Celsius. Wells were washed with PBS three times for 5 minutes. Cells were then

incubated with goat anti-mouse-FITC secondary antibody (Santa Cruz) for 1

hour at 37Celsius. Following incubation with secondary antibody, wells were

washed again with PBS three times for 5 minutes and allowed to dry. Slides

were viewed with a fluorescence microscope and photographs of the images

were taken.

81
7. Transfection Studies- to measure p53 transcriptional activity

HeLa, CaSki, and SiHa cells were analyzed for the activation of a p53-

luciferase reporter upon treatment with the lignan as an indication of

transcriptional activity of stabilized p53. 5 x 105 cells were seeded on a six well

plate. Eight hours later, the cells were transfected with 1 microgram of the

reporter vector and/or the positive control vector pFC-p53 using the

Lipofectamine method. DNA was removed from cells three hours later and and

replaced with fresh medium or fresh medium containing 15 M 3-O-methyl-

NDGA. Cells were analyzed for luciferase activity the following day using a

scintillation counter (Beckman).

8. Caspase Assays- to measure cell death by apoptosis

Caspase-9 activity in lignan-treated cells was analyzed utilizing the caspase-9

colorimetric assay kit from Calbiochem. Caspase-3 activity was measured using

the CaspACE colorimetric caspase-3 assay kit from Promega. Both assays make

use of a caspase-specific substrate labeled with p-nitroaniline (pNA) (DEVD-

pNA for caspase-3, LEHD-pNA for caspase-9). Cleavage of the substrate by the

specific cellular caspase yields free pNA that can be read by a spectrophotometer

at 405 nm. The caspase-3 or caspase-9 activity in a sample is proportional to the

82
amount of pNA product detected spectrophotometrically. Typically, C33-A,

CaSki, and HeLa cells were seeded at a density of 1 x 106 cells on a 10 cm2 plate.

Cells were allowed to attach overnight and were then treated with drug

compounds for amounts of time ranging from 3-12 hours. Following drug

treatment, cells were washed once with PBS, scraped off the plate, resuspended

in the lysis buffer from the kit and lysed via 3 cycles of freezing/thawing on dry

ice. Equal volumes of cell lysates were then assayed for caspase-3 or caspase-9

activity in a 96 well plate utilizing the corresponding substrates. Caspase activity

was normalized against non-drug-treated non-substrate-treated controls and

non-drug-treated substrate-treated controls. Spectrophotometric readings were

performed on a Spectra Max plate reader (Molecular Devices). Following

caspase assays, the protein concentration for each sample was measured using

the Quant-PRO protein assay from Sigma. Enzyme activity was divided by

protein concentration to provide specific activity.

9. Flow Cytometry- to measure cell death by apoptosis

Cervical carcinoma cells treated with 3-Omethyl-NDGA were assayed for

apoptosis via flow cytometry (Beckman) utilizing the Annexin V-FITC Apoptosis

Detection Kit from Calbiochem. CaSki cells were plated as described above,

83
treated with 30-60 M lignan or 50 nM Actinomycin D for 3-12 hours. Cells were

harvested via trypsinization following drug treatment and apoptosis was

measured by flow cytometry analysis of staining of cells with Annexin V-FITC

and propidium iodide (PI). For each sample, 10,000 events were cataloged on a

fluorescence versus cell number plot and the percentage of Annexin V-FITC

positive cells was determined. Apoptosis was defined as the percentage of cells

that appeared in quadrant A1 (high Annexin V-FITC signal, low PI signal, early

apoptosis) compared to the total number of cells analyzed. Cells that appeared

in quadrant A2 (high Annexin V-FITC signal, low PI signal) were excluded from

the analysis due to the inability to distinguish cell death by apoptosis from cell

death by necrosis in those cells. Throughout the study, common settings for

fluorescence gains, fluorescence threshold, and color compensation were used;

these were determined by analysis of singly dyed and undyed controls. All

apoptosis assays were performed in triplicate utilizing cellular extracts obtained

from separate treatments and cell cultures.

10. TUNEL Assay- to measure in situ apoptosis

Induction of apoptosis in cervical carcinoma cells by 3-O-methyl-NDGA was

confirmed using the TdT-mediated dUTP nick end labeling (TUNEL) assay. The

84
DeadEnd Fluorometric TUNEL System kit used in this study was purchased

from Promega. Cells were seeded at a density of 35,000 cells per well on 8-

chambered slides (Lab Tek). Cells were then treated with 15 M to 80 M lignan

for 18 hours. Control cells were treated with 0.2 percent DMSO only. Following

treatment, cells were fixed on slides via immersion in 4 percent formaldehyde in

PBS for 25 minutes. Cells were permeabilized using 0.2 percent Triton X-100 in

PBS. DNA strand breaks were labeled by enzymatic incorporation of

fluorescein-12-dUTP. The fluorescein-12-dUTP-labled DNA was visualized

directly by fluorescence microscopy and photographs were taken of the images.

Apoptotic cells are reported as a percentage of total cells in the bright field

picture that displayed a green fluorescence under fluorescence microscopy.

11. Assay for inhibition of lipoxygenase activity

A cell-free assay kit for lipoxygenase inhibitors was purchased from

Cayman Chemical. Cell lysates are not compatible for use with this screening

method. A range of concentrations of both the methylated lignan and its parent

compound NDGA, which is known to inhibit lipoxygenase activity, were

dissolved in the assay buffer from the kit in a 96 well plate. 90 L of

lipoxygenase enzyme was added to each well and then the reactions were

85
initiated by the addition of substrate. The reaction was allowed to proceed for 8

minutes after which 100 L of Chromagen was added to stop the reaction. The

absorbance of each well at 495 nm was then read on a plate reader (Molecular

Devices). The activity of the lipoxygenase enzyme is proportional the

absorbance of each well at 495 nm.

86
CHAPTER THREE

RESULTS

1. 3-O-methyl-nordihydroguaiaretic acid (3-O-methyl-NDGA) shows

selective toxicity to cells containing HPV DNA and wild type p53 protein.

Taken together, the CD50 values obtained from the alamar Blue assay,

MTT assay, and Trypan blue assays suggest that the lignan has some selective

toxicity to cells that contain both HPV DNA and wild type p53. While overall

CD50 values varied somewhat with respect to the cell proliferation assay used,

the overall trend of toxicity values shows that the compound is more toxic to

cells that contain HPV DNA and p53 that is wild type. Representative individual

results from the Trypan Blue assay and MTT assay in CaSki cells are shown in

figures 21 and 22. The assays were repeated for a variety of other cell lines (data

not shown). Data obtained from the alamar Blue assay for multiple cell lines is

shown in figure 23. The lignan showed a dose dependent inhibition of cell

87
3'-O-Methyl-NDGA toxicity in Caski cells
at 24 hours as determined by MTT assay

120

100
Growth Percent Control

80

60

40

20

0
0 25 50 100 200 400

[3'-O-methyl-NDGA] in M

Figure 21. Toxicity of 3-O-methyl-NDGA in CaSki cells at 24 hours post

treatment as determined by the MTT assay. (Error bars represent +/- standard

deviation).

88
3'-O-Methyl-NDGA toxicity in Caski cells
at 24 hours as determined by Trypan Blue Assay
120

100
Viability percent control

80

60

40

20

0
0 50 100 150 200

[3'-O-methyl-NDGA] in M

Figure 22. Trypan Blue viability assay of CaSki cells treated with 3-O-methyl-

NDGA and analyzed at 24 hours post treatment. (Error bars represent +/-

standard deviation).

89
Toxicity of 3'-O-Methyl-NDGA in Cancer Cells
at 24 hours determined by alamar Blue assay

100
Growth Percent Control

80

60

40 CaSki Cells
MRC-5 cells
20 C33-A Cells
HT-3 Cells
HeLa Cells
0
0 50 100 150 200
[3'-O-Methyl-NDGA] in M

Figure 23. Toxicity results for 3-O-methyl-NDGA in multiple cancer cell lines

obtained from the alamar Blue assay. Toxicities were calculated as a percentage

of the absorbance of DMSO treated controls and error due to antioxidant activity

of the lignan was subtracted. (Error bars represent +/- standard deviation).

90
growth in all cell lines tested. HPV DNA containing cells appeared to be most

sensitive to lignan treatment and showed the lowest CD50 values. Average CD50

values calculated from data obtained from the three assays used for all cell lines

tested are shown in table 7.

2. The plant lignan, 3-O-methyl nordihydroguaiaretic acid, stabilizes p53

tumor suppressor protein in treated HPV positive cervical carcinoma cell

lines.

In previous studies the plant lignan, 3-O-methyl NDGA, inhibited expression

of a luciferase reporter gene linked to the P97 promoter of high risk human

papillomavirus type 16 (HPV-16) (Craigo, et al 2000). We hypothesized that the

lignan may also decrease E6 expression from the integrated HPV promoters

found in human cervical cancer cell lines. A decrease in viral E6 protein

expression within treated cells should result in the stabilization of p53 protein.

In order to assess whether lignan treatment stabilizes p53 protein steady state

levels within treated cells, we measured p53 protein levels found in the HPV-

containing cervical cell lines HeLa and CaSki with and without treatment with

30 M 3-O-methyl NDGA. Protein extracts from lignan treated and DMSO

treated controls were analyzed for p53 protein amount by Western blot analysis.

91
CELL LINE CD50 M
HeLa (cervical carcinoma, high risk HPV-18 + wt p53) 83
CaSki (cervical carcinoma, high risk HPV-16 + wt p53) 69
SiHa (cervical carcinoma, high risk HPV-16 + wt p53) 64
HT-3 (cervical carcinoma, low risk HPV-30 + mutant p53) 263
C33-A (cervical carcinoma, HPV-negative, mutant p53) 187
MRC-5 (human immortalized fetal lung fibroblasts) >250
HFK (human foreskin keratinocytes) >80

Table 7. Growth arrest induction by of 3-O-methyl-NDGA in various

cell lines. Table lists the corresponding CD 50 values for these cell lines as the

average CD50 values calculated from results of alamar Blue, MTT, and Trypan

Blue assays. Values obtained from each assay were similar (data not shown).

92
The 3-O-methyl NDGA treated cells had increased p53 protein levels from 2 to

12 hours after treatment within CaSki cells (figure 24) and 12 to 18 hours after

treatment in HeLa cells (figure 25). The lignan did not alter the mutant p53

levels in C33-A cells at a concentration of 30 M as shown in figure 26. These

results suggest that the compound is effective in stabilizing wild type p53, but

that mutant p53 may be unaffected by the compound. However, it is also

possible that Western blot is not sensitive enough to detect any changes in

mutant p53 induced by the lignan due to the increased half-life of the mutant

protein and the high levels of p53 protein within the cells tested.

Immunocytochemical staining for p53 protein using a FITC-conjugated

secondary (Santa Cruz) antibody confirmed the increase in p53 protein found in

30 M 3-O-methyl NDGA treated CaSki cells as compared to vehicle treated cell

cultures (figure 27). Immunocytochemical analysis of p53 was performed at

timepoints of 6, 12, and 18 hours post treatment. The optimal time point for

lignan-induced stabilization of p53 protein (brightest signal observed) was found

to be 12 hours after treatment of CaSki cells with 30 M 3-O-methyl-NDGA.

Stabilization of p53 and an increase in its half-life by lignan treatment is apparent

since it is usually not possible to visualize p53 by immunocytochemistry in cells

where the half life of p53 is normal (Bartley and Ross 2002; Moll et al, 1995).

93
CaSki Cells

Control 30 M
3-O-Methyl-NDGA Treated

12h 2h 4h 6h 9h 12h

p53

-Actin

Figure 24. p53 protein is stabilized in CaSki cells treated with 30 M 3-O-

Methyl-NDGA. Western blot analysis shows stabilization of p53 in lignan

treated CaSki cells. Stabilization of p53 is enhanced over time from 2 to 12 hours

following treatment with the lignan.

94
HeLa Cells

Control __Treated__

12h 12h 18h

p53

-Actin

Figure 25. p53 protein is stabilized in HeLa cells treated with 30 M 3-O-

Methyl-NDGA. Western blot shows stabilization of p53 in HeLa cells treated

with 30 M 3-OMe-NDGA for 12 and 18 hours. p53 levels are elevated in lignan

treated cells compared to controls.

95
C33-A Cells

Control ________Treated_
3-OMe-NDGA treated _
30 uM
control
hrs 12h
12 6h
6 9h
9 12h
12

p53

C33A Cells

Figure 26. p53 protein levels do not change in C33-A cells treated with 30 M

3-O-Methyl-NDGA. p53 Western blot shows no change in p53 levels in 30

M lignan treated C33-A cells which contain mutant p53. Hence, the lignan does

not stabilize mutant p53 in these cells.

96
CaSki Cells

p53p53

(-) 3-O-Methyl-NDGA (+) 30 M 3-O-methyl-NDGA

Figure 27. Immunocytochemical staining of p53 in CaSki cells after lignan

treatment confirms that p53 levels are increased in lignan-treated cells.

Results show a larger amount of p53 staining in lignan treated cells compared to

untreated control indicating the lignan stabilizes p53 protein.

97
3. The p53 protein stabilized by 3-O-methyl NDGA treatment is

transcriptionally active.

It is confirmed that the p53 protein stabilized in lignan-treated cells is

transcriptionally active because 15 M 3-O-methyl-NDGA p53 increases the

luciferase activity of HeLa cells transfected with a p53-luciferase responsive

reporter. The p53-luciferase reporter plasmid has p53 response elements linked

to the luciferase gene and the activation of p53 in transfected cells drives

expression of luciferase (figure 28). The p53-luciferase transfected HeLa cells

treated with the 3-O-methyl-NDGA showed a 5 fold increase in luciferase

activity compared to the transfected non-treated cells (figure 29) indicating that

the compound stabilizes transcriptionally-active p53 in treated cells.

HeLa cells were also co-transfected with a pFC- p53 vector (Stratagene)

that has the wild type p53 gene linked to the immediate early CMV promoter as

a positive control. pFC-p53 (positive control plasmid) activates the transcription

of the reporter from the enhancer element-TATA box region. Transfection of

wild type p53 along with the reporter plasmid into HeLa cells shows that the

reporter vector responds to excess production of p53 within the cells as shown in

figure 29. Similar increases were observed when CaSki cells and SiHa cells were

transfected with the p53-luciferase vector indicating that the stabilized p53 in

98
Figure 28. Structure of the p53-luciferase vector used to transfect HeLa

cells with p53. The vector has the wild type p53 responsive gene linked to

the immediate early CMV promoter. The inducible reporter plasmid contains

the luciferase reporter gene driven by a basic promoter (TATA box) plus a cis-

enhancer element. Expression of the luciferase gene is controlled by the

promoter/ enhancer because it contains transcription recognition sequences

for p53 protein. If p53 is stabilized within p53-luciferase transfected cells, a

higher luciferase activity would be expected.

99
p53-luc Transfection of HeLa Cells
50

40
Specific Activity Luciferase
(cpm/ug protein x 10 )
7

30

20

10

0
p53-luciferase p53-luciferase + p53-luciferase + 15
pFC uM lignan

Figure 29. 15 M 3-O-methyl-NDGA stabilizes p53 within HeLa cells

transfected with p53-luciferase. Specific activity was obtained by dividing

sample activity in cpm by the protein concentration. p53-luciferase transfected

cells that were treated with the lignan showed a 5-6 fold increase in luciferase

activity compared to control indicating that the compound stabilizes

transcriptionally-active p53 in treated cells. (Error bars represent +/- standard

deviation. P values for treated vs. control <<.001 at alpha = .05 for 2 tailed

Student T test).

100
these cells is transcriptionally active as well (figures 30 and 31).

4. 3-O-methyl-NDGA fails to activate p21 protein in HPV-positive cervical

cancer cell lines.

The activation of p21 (Cip1/Waf1) protein by p53 is an indication of the

activation of growth arrest pathways. p21 was not induced at 12 or 18 hour time

points in 30 M lignan-treated CaSki or HeLa cells as determined by Western

blot analysis (figures 32 and 33). However, treatment of the same cell lines with

5 M 5-fluorouracil or 5 M camptothecin resulted in the up-regulation of p21

protein (positive control). This indicates treated cells are not undergoing growth

arrest at the times studied.

5. 3-O-methyl NDGA activates caspases within treated HPV-positive

cervical carcinoma cell lines.

The activation of caspases, which are cysteine proteases, is a signal for the

cells commitment to undergoing apoptosis. We examined whether treatment

with 30 M 3-O-methyl NDGA induced caspase activity within C33-A, HeLa,

and CaSki cell lines. The HPV positive cell lines HeLa and CaSki both showed

caspase-3 induction after treatment with 30 M lignan. Caspase-3 activity

increased over time and peaked at 12 hours post-treatment (figure 34). The

101
p53-luc Transfection of CaSki cells

40
35
Specific Activity Luciferase
(cpm/ug protein x 10 )
6

30
25
20
15
10
5
0
p53-luciferase p53-luciferase + p53-luciferase +
15 uM lignan pFC-p53

Figure 30. 15 M 3-O-methyl-NDGA stabilizes p53 within CaSki cells

transfected with p53-luciferase. Specific activity was calculated by dividing

sample activity in cpm by protein concentration in micrograms. p53-luciferase

transfected cells that were treated with the lignan showed a 3 fold increase in

luciferase activity compared to control indicating that the compound stabilizes

transcriptionally-active p53 in treated cells. (Error bars represent +/- standard

deviation. P values for treated vs. control <<.001 at alpha = .05 for 2 tailed

Student T test).

102
p53-luciferase transfection of SiHa cells
14
Specific Activity Luciferase

12
(cpm/ug protein x 106)

10

0
p53-luciferase p53-luciferase p53-luciferase +
+15 uM lignan pFC-p53

Figure 30. 15 M 3-O-methyl-NDGA stabilizes p53 within SiHa cells

transfected with p53-luciferase. Total protein concentration was determined for

each sample and specific activity was calculated by dividing sample activity in

cpm by protein concentration in micrograms. p53-luciferase transfected cells that

were treated with the lignan showed an 8 fold increase in luciferase activity

compared to control indicating that the compound stabilizes transcriptionally-

active p53 in treated cells. (Error bars represent +/- standard deviation. P values

for treated vs. control <<.001 at alpha = .05 for 2 tailed Student T test).

103
CaSki Cells
Control ____Treated_____
12h 12h 18h AD

p21

-Actin

5-FU 12h L Camptothecin

p21

Figure 32. Western blot analysis of p21 induction in CaSki cells by 30 M 3-

OMe-NDGA. Cells were treated with lignan and analyzed for p21 expression via

Western blot. 3-O-Methyl-NDGA failed to induce p21 CaSki cells. However,

p21 is can be induced in CaSki cells by 100 nM Actinomycin D (AD), 5 M 5-

fluorouracil (5-FU) and 5 M camptothecin in CaSki cells (positive controls) as

shown. Beta actin was used as an internal control for protein loading for all blots

depicted above.

104
HeLa Cells
Control _Treated__
12h 12h 18h

p21

-actin

Figure 33. Western blot analysis of p21 induction in HeLa cells by 30 M 3-

OMe-NDGA. Cells were treated with lignan and analyzed for p21 expression via

Western blot. 3-O-Methyl-NDGA failed to induce p21 CaSki cells. Beta actin

was used as an internal control for protein loading for all blots depicted above.

105
Caspase-3 Activity in Cervical Cancer Cells
Treated With 3'-O-Me-NDGA
35
Specific Activity of Caspase-3

30 HeLa Cells *
CaSki Cells
25 *
C33-A Cells
(nmol/h/mg)

20

15

10

5
0
0 3 6 9 12
Time Post Treatment (hours)

Figure 34. 30 M 3-O-methyl-NDGA activates caspase-3 in treated HPV-

containing cervical cancer cells, but not in HPV-negative C33-A cells. Caspase-

3 activity in a sample is proportional to the amount of pNA product detected

spectrophotometrically. 30 M 3-OMe-NDGA induces the effector caspase-3 in

HPV-positive HeLa and CaSki cells. C33-A cells do not show caspase-3

activation when treated the lignan. (Error bars represent +/- standard deviation).

106
caspase-3 levels induced with micromolar amounts of lignan were comparable to

the levels found with 50 nM Actinomycin D treatment (data not shown). HPV

negative C33-A cells, which also contain a mutant p53 gene, did not show

significant caspase-3 activity when cultures where treated with 30 M lignan

over 3 to 12 hours post treatment. C33-A cells contain inducible caspase-3 that

was observed when the cells were treated with 5 M camptothecin, a

topoisomerase inhibitor (figure 35).

The intrinsic mitochondrial apoptotic pathway is dependent upon p53

mediated caspase activation. This includes the activation of the initiator

caspase-9. Caspase-3, on the other hand, is an effector caspase in the apoptotic

pathway. It can be activated by initiator caspase-8 in the extrinsic pathway or by

caspase-9 in the intrinsic pathway. Hence, to gauge which apoptotic pathway

was being activated, we examined caspase-9 activity. Caspase-9 activity was

induced in HPV positive cells after treatment with 30 M lignan (figure 36). The

caspase-9 activity reached a maximum at 6 hours post drug treatment in both

HeLa and CaSki cells, which was about 6 hours before maximal effector caspase-

3 activity was observed. The HPV negative C33-A cells did not shown any

caspase-9 activity when treated with lignan (figure 36).

107
Caspase-3 Activity in C33-A Cells
Treated with Camptothecin
Specific Activity of Caspase-3

80
70
60
(nmol/h/mg)

*
50
40
30
20
10
0
0 3 6 9 12 15 18

Time Post Treatment (hours)

Figure 35. C33-A cells have an intact caspase-3 pathway. Caspase-3 activity in

a sample is proportional to the amount of pNA product detected

spectrophotometrically. C33-A cells do not show caspase-3 activation when

treated the lignan but C33-A cells have an intact caspase-3 pathway as shown

when treated with 5 M camptothecin. (Error bars represent +/- standard

deviation).

108
Caspase-9 Activity in Cervical Cancer Cells
Treated with 3'-O-Me-NDGA
45
Specific Activity of Caspase-9

40 HeLa Cells
35 CaSki Cells
30 C33-A Cells
(nmol/h/mg)

25
20
15
10
5
0
0 3 6 9
Time Post Treatment (hours)

Figure 36. 30 M 3-O-methyl-NDGA activates caspase-9 in treated HPV-

containing cervical cancer cells, but not in HPV-negative C33-A cells. The

caspase-9 activity in a sample is proportional to the amount of pNA product

detected spectrophotometrically. Results demonstrated that 30 M 3-OMe-

NDGA induces caspase-9 activity in HPV positive HeLa and CaSki cells but fails

to induce caspase-9 in HPV-negative C33-A cells which contain mutant p53.

109
6. The caspase-3 inhibitor Z-VAD-FMK rescues lignan treated cells from the

apoptotic pathway.

The effect of 30 M 3-O-methyl-NDGA on HPV-positive CaSki cells is

partially reversible when caspase-3 activity is blocked by the addition of the

caspase inhibitor Z-VAD-fluoromethylketone (Z-VAD-FMK). Z-VAD-FMK is a

potent cell permeable inhibitor of all caspases including caspases-3. Cells treated

with a combination of the lignan and the caspase inhibitor showed lower caspase

activity than cells treated with the lignan alone as shown in figure 37. This data

suggests that lignan treatment is acting directly on the cellular path to apoptosis.

The activation of caspase-3 is a committed step for the induction of apoptosis.

However, the data does not distinguish whether the intrinsic pathway or the

extrinsic pathway is being inhibited, since caspase-3 can be activated by both

pathways to apoptosis.

7. 3-O-methyl NDGA induces apoptosis in HPV-positive cervical

carcinoma cell lines.

The induction of caspase-9 and caspase-3 activity within HPV containing cell

lines after treatment with lignan strongly suggests that the cells are undergoing

p53-induced apoptosis. To confirm that the treated cells were undergoing

110
(3)
Caspase-3 Inhibitor Z-VAD-FMK Protects
CaSki Cells From Lignan Induced Apoptosis
50
Percent Cell Death as determined by TUNEL Assay

45

40

35

30

25

20

15

10

Control Z-VAD-FMK Lignan Lignan + Z-


VAD-FMK

Figure 37. Addition of the caspase-3 inhibitor ZADFMK to cells treated with 30

M lignan inhibits caspase-3 activation in those cells.

111
programmed cell death, lignan treated cells were assayed for apoptosis by flow

cytometry and the TUNEL assay. We were able to confirm that treated cells

were undergoing apoptosis using flow cytometry and Annexin V-FITC and

propidium iodide staining. Cells staining with Annexin V-FITC were found in

non-synchronous lignan treated cell cultures with maximal cell numbers at 6-9

hours after lignan treatment (figure 38). Apoptosis was defined as the

percentage of cells that appeared in quadrant A1 (high Annexin V, low

propidium iodide signal; early apoptosis) compared to the total number of cells

analyzed. Cells appearing in quadrant A2 (high Annexin V, high propidium

iodide signal) were excluded from analysis due to the fact that it cannot be

determined whether the cells were dying via apoptosis or necrosis. Cells treated

with 30 M lignan showed an increased number of cells undergoing apoptosis at

6 and 9 hours post treatment compared to vehicle treated controls (figure 38).

The TUNEL assay confirmed the flow cytometry results. HPV DNA

containing cervical cancer cells had positive TUNEL staining when treated with

the lignan. The TUNEL assay also showed a different sensitivity between the

HPV positive cell lines CaSki and SiHa (figures 39 and 40) as compared to the

HPV negative cervical cancer HT-3 and C33-A cell lines (figures 41 and 42).

HeLa cells were not tested due to difficulty in fixing the cells.

112
Annexin V-FITC/ PI Staining in CaSki Cells
35
*
Percent of Annexin V Only Positive Cells

30

25
*
20

15

10

Control 6h 30 uM 9h 30uM

Figure 38. Lignan treated cells are undergoing apoptosis as assayed using flow

cytometry and Annexin V-FITC and propidium iodide staining. Apoptosis is

defined as the percentage of cells that appeared in quadrant A1 (high Annexin V,

low propidium iodide signal; early apoptosis) compared to the total number of

cells analyzed. Cells treated with 30 M lignan showed an increased number of

cells undergoing apoptosis at 6 and 9 hours post treatment compared to vehicle

treated controls. (Error bars represent +/- standard deviation. * denotes

significance versus control according to 2 tailed Student T test at alpha = .05).

113
CaSki Cells

Control 30 M 3-OMe-NDGA
3% cell death 50% cell death

Figure 39. Analysis of apoptosis in HPV-16 positive CaSki cells by TUNEL

assay showing induction of cell death by 30 M 3-O-methyl-NDGA. HPV-

positive CaSki cells, which contain wild-type p53, showed an increased number

of cells undergoing apoptosis compared to control when treated with 3-OMe-

NDGA. Actinomycin D was used as a positive control (data not shown).

(Significant difference between treated and control according to two tailed

student T test at alpha = 0.05; p < 0.001).

114
SiHa Cells

Control 30 M 3-OMe-NDGA
<5% cell death >50% cell death

Figure 40. Analysis of apoptosis in HPV-16 positive SiHa cells by TUNEL

assay showing induction of cell death by 30 M 3-O-methyl-NDGA. SiHa

cells, which contain wild-type p53, showed an increased in cells undergoing

apoptosis compared to control when treated with 3-OMe-NDGA. (Actinomycin

used as a positive control for comparison). (Significance between treated and

control according to two tailed student T test at alpha = 0.05; p < 0.001).

115
C33-A Cells

Control 30 M 3-OMe-NDGA
<5% cell death 10% cell death

Figure 41. Analysis of apoptosis in HPV-negative C33-A cells by TUNEL

assay. C33-A cells, which contain a mutation in the p53 tumor suppressor, did

not show significant apoptosis and were not nearly as sensitive to lignan

treatment as CaSki and SiHa cells. (No significant difference between treated

and control according to two tailed student T test at alpha = 0.05).

116
HT-3 Cells

Control 30 M 3-OMe-NDGA
4% Cell death 9% Cell Death

Figure 42. Analysis of apoptosis in HT-3 by TUNEL assay showing induction

of cell death by 30 M 3-O-methyl-NDGA primarily in HPV-containing cells

with wild type p53. 30 M lignan does not induce significant apoptosis in HT-3

cells which contain HPV-30 DNA and mutant p53. (No significant difference

between treated and control according to two tailed student T test at alpha =

0.05).

117
8. The Bax and PUMA genes are induced within lignan treated CaSki cells.

Genes that are transactivated by p53 when it becomes stabilized include

Bax and PUMA. Both proteins are pro-apoptotic and are induced via the

intrinsic p53-dependent apoptotic pathway. The result of bax induction is the

activation of caspases from the mitochondria. We analyzed the ability of the

lignan to induce bax and PUMA by examining protein levels by Western blot.

CaSki cells and HeLa cells treated with 30 M 3-O-methyl NDGA showed

increased levels of Bax protein at 12 hours post treatment as analyzed by

Western blot (figures 43 and 44). PUMA protein increased in 30 M lignan

treated CaSki cells 6 to 18 hours post treatment (figure 45). The elevations in

both Bax and PUMA protein levels are consistent with induction of the intrinsic

apoptotic pathway within lignan treated cervical tumor cells.

9. The lignan reduces E7 protein levels within treated CaSki cells.

Western blot analysis shows that CaSki cells treated with 30 M lignan

have a reduction in HPV E7 protein levels over time. E7 protein levels decreased

over time from 9 to 18 hours post treatment (figure 46). This correlates with the

loss of full length E6/E7 mRNA as determined by real time PCR (D. Tschantz and

A. DeLucia, manuscript in preparation). The linkage of protein reduction for E7

118
CaSki Cells

Control Treated
12h 12h

Bax

-Actin

Figure 43. Western blot analysis shows that bax protein is induced by 30 M

3-O-Methyl-NDGA in CaSki cells. Bax was induced in CaSki cells at 12 hours

following treatment with 30 M lignan. Western blot analysis shows that bax

protein levels are higher in lignan-treated cells versus control. The internal

standard used for protein loading was beta actin.

119
HeLa Cells

Control Treated
12h 12h

Bax

-Actin

Figure 44. Western blot analysis shows that bax protein is induced by 30 M

3-O-Methyl-NDGA in HeLa cells. Bax was induced in HeLa cells at 12 hours

following treatment with 30 M lignan. The internal standard used for protein

loading was beta actin.

120
CaSki Cells

Control Treated____________
12h CPT AD 6h 9h 12h 18h

Figure 45. Western blot analysis shows that both PUMA protein is induced by

30 M 3-O-Methyl-NDGA in HPV DNA containing cells. PUMA was induced

in CaSki cells after treatment with the lignan for 9-18 hours and were compared

to 5 M camptothecin (CPT) or 100 M Actinomycin D (AD) treatments used as

positive controls. Blots were checked for accuracy of protein loading by

reprobing the blot for Beta actin (data not shown).

121
CaSki Cells

0.2%DMSO 30 M 3-O-methyl-NDGA

Cont 9h 12h 18h

E7

Figure 46. E7 protein levels decrease when CaSki cells are treated with 30 M

lignan. The decrease in E7 protein correlates with the observed loss of E6/E7

mRNA in CaSki cells treated with the lignan (Tschantz and DeLucia, manuscript

in preparation)

122
with mRNA loss suggests that the lignan may work at the transcriptional level.

By interfering with the transcription of E6, p53 could be stabilized, setting in

motion the events described above.

123
CHAPTER FOUR

DISCUSSION

Previous work indicated that natural plant lignans isolated or derived

from the creosote bush interfered with HPV type 16 early promoter, P97 , activity

(Craigo et al., 2000). This led to the prediction that such lignans could inhibit the

transcription of the HPV genome including the oncogenes E6 and E7. Most

cervical tumor cells that have integrated HPV generally retain wild type p53 and

RB genes (Denk et al., 2001; Scheffner et al., 1991). Viral oncogenes and proteins

efficiently and effectively suppress the functions of cellular tumor suppressor

proteins, p53 and pRB, so there is seldom any accumulation of further mutations

to maintain the transformed state. Since HPV tumors contain wild-type p53, it is

plausible that the protein could be reactivated if the functions of the HPV

oncogenes were blocked. In fact, there is considerable evidence establishing that

stabilization and accumulation of active p53 protein within the HPV transformed

cervical tumor cell can induce cell cycle arrest or apoptosis (Ying and Xiao 2006).

124
Since E6s primary function is to shuttle endogenous p53 to the

proteosome, the delivery of small molecule inhibitors of E6 protein to the cervical

tumor or pre-cancerous cell could reactivate the silenced cellular repair

machinery or activate the apoptotic pathway. Based on these observations, we

tested the natural plant lignan 3-O-methyl-NDGA for the interference with HPV

E6 function in HPV transformed cervical tumor cells. We observed a return of

p53 stability and the stabilized p53 protein was transcriptionally active as shown

by luciferase reporter assay and the induction of p53 responsive genes. We also

observed the induction of apoptosis in lignan treated cells.

Upon treatment of HPV positive cervical cancer cell lines with the lignan,

stabilization and accumulation of transcriptionally active p53 protein was

quickly established within 2 hours post-treatment with lignan. The early and

rapid appearance of increased p53 levels and the lack of concomitant increases in

p53 mRNA (Tschantz and DeLucia, manuscript in preparation) suggest a return

of cellular control to the post-translational regulation of p53 protein. However,

an alternative explanation for the increase in p53 could be an increase in

translation of stabilized p53 mRNA. The increased p53 protein levels observed

in HPV positive cell lines does not infer by itself that the lignan is acting through

an HPV specific factor nor does it establish that p53 is functional. Typical signals

125
that are considered important for p53 protein stabilization work through the

disruption of p53 regulation by MDM2 (Momand et al., 1992; Picksley and Lane

1993). However, MDM2 protein is not considered to be an important player in

p53 regulation in HPV-positive cervical tumor cells due to the presence of E6.

The high levels of p53 stabilization found in lignan treated cells likely depend on

an interruption of the HPV E6 protein. Future studies will distinguish whether it

is the level of E6 protein itself that is being affected or the function of E6 protein

that is being interfered with by the action of the lignan. It is also possible that the

lignan modulates the interaction of E6, E6-AP, and p53 thereby stabilizing p53 in

treated cells. The functional status of p53 stabilized by the lignan was

established by showing increased activity of a p53 luciferase reporter vector in

lignan-treated versus untreated cells, by the induction of p53 responsive genes

PUMA and bax in lignan-treated cells, and indirectly by the activation of the p53-

dependent initiator caspase-9.

HPV positive cervical tumor cells did not show an increase in p21 protein

levels, which might be expected as a result of p53 stabilization. The lignan-

treated cells appear to follow a pathway directed at apoptosis rather than growth

arrest. One possible explanation for the absence of p21 may be that the lignan is

interfering with Sp1 transcription factor levels, and the transcription of p21 is

126
highly Sp1 dependent. It has been suggested previously that the lignan is

capable of interfering with Sp1, and the interference with Sp1 may also explain

the ability of the compound to block gene expression from the P97 promoter,

since P97 also contains sever Sp1 binging sites. It is also possible that we failed to

observe the induction of p21 at the time points tested. p21 is known to be a

substrate for active caspase-3, and the induction of caspase-3 may have

destroyed any p21 that was produced at these time points.

HPV-positive cervical cancer cell lines treated with 30 M lignan were

next examined for induction of apoptosis, since induction of apoptosis can be a

result of p53 stabilization in cancer cells. When compared to both untreated

controls and HPV negative cervical cancer cell lines, lignan-treated HPV positive

tumor cells underwent apoptosis as shown by several methods, including the

activation of caspases, flow cytometry using fluorophore conjugated Annexin V

Propidium Iodine staining, and TUNEL assay.

Caspases are cysteine-containing proteases and are an integral part of the

apoptotic pathway. They are maintained in a quiescent state within cells until

they are activated (Cereghetti and Scorano 2006). The p53 dependent intrinsic

apoptotic pathway begins the cascade of proteolytic destruction within the cell

127
by first activating procaspase-9 which, in turn, cleaves and activates the other

caspases and terminates with the formation of the apoptosome and the activation

of effector caspase-3. Active caspase-9 began increasing 3 hours post-treatment

with the lignan and levels peaked between 6 and 9 hours. The effector caspase-3

activity was observed to be increasing at 6 hours after treatment and peaked at

12 hours. The kinetics of caspase activations provides strong evidence for the

induction of the intrinsic apoptotic pathway by the active p53 at 2-3 hours post-

treatment since caspase activation coincided with the observed increases in p53

protein obtain by Western blot.

This data also shows that induction of cell death by the plant lignan is

likely occurring through the intrinsic apoptotic pathway. That endogenous wild

type p53 was responsible for the caspase activation was further supported by the

lack of caspase-9 and caspase-3 activation within C-33A cells, a cervical tumor

cell line that lacks HPV DNA and also has a mutant p53 gene, but has normal

caspase 3, as shown by activation by the addition of camptothecin. The

induction of apoptosis after lignan treatment and stabilization of p53 protein was

further confirmed by both flow cytometry using Annexin V and the counter stain

propidium iodide, and TUNEL assay. Non-labeled or non-apoptotic cells require

further investigation as they may be either resistant to the treatment or due to

128
asynchrony of the cells under the experimental conditions.

The functionality of the stabilized p53 was also established by the

activation of p53 responsive genes in lignan treated cells. PUMA protein, a

newly identified BH3-only protein involved in apoptosis, has recently been

shown to coordinate apoptotic events when it interacts with cytosolic p53

protein. Increasing p53 protein levels in the nucleus activates expression of the

PUMA gene, which then increases its protein levels after translation of increased

PUMA mRNA. PUMA protein then interacts with a p53-BCL-XL complex in the

cytosol, and results in the release of p53 protein from the grip of BCL-XL,

allowing p53 to interact with the Bax protein stationed at the surface of the

mitochondria. This starts the cascade for the intrinsic apoptotic pathway. The

induction of PUMA protein was observed in lignan treated cells at time points

following the stabilization of p53 protein. Bax protein was also shown to be

accumulated within treated cells. The proteins identified as increasing during

treatment are explained by the cell initiating and carrying out the intrinsic

pathway for programmed cell death.

Other data in our lab shows that lignan treatment induces a significant

reduction in mRNA coding for both E6 and E7 proteins (Tschantz and DeLucia,

manuscript in preparation). Due to the unavailability of suitable E6 antibodies,

129
only E7 protein levels could be measured following lignan treatment. A decrease

in E7 protein levels was observed in lignan treated CaSki cells and this is

supported by the disappearance of E6/E7 mRNA. Hence, the lignan appears to

be interfering with the activity of the endogenous P97 promoter in these cells.

Since most currently used chemotherapy drugs target all rapidly dividing cells,

the lignan may represent a novel, HPV-tumor-specific treatment for cervical

cancer.

The observations found in this work coupled with previous work permit

us to put forward the following model for 3-O-methyl-NDGA mode of action.

Upon entry of the compound into the cell, the lignan overwhelms the viral E6

protein control of wild type p53 within the cell. The loss of E6 function is at a

level that permits the stabilization of substantial amounts of p53 within a short

time. The rapid accumulation of transcriptionally active p53 protein induces key

proteins that function in the apoptotic pathway, and end point assays suggest

that the cell death induced by the lignan follows a p53-dependent pathway.

Lignan treatment of HPV-positive cells once again demonstrates that HPV

induced carcinogenesis is likely initiated by an assault on the p53 mediated

regulatory mechanism of the cervical epithelial cell by E6. There is no known

significant accumulation of secondary pro-carcinogenic mutations even in high

130
passage immortalized cell lines that can interrupt cell death and p53 stabilization

induced by the lignan. This work argues that, as E6 protein is a principle player

in the transformation of cervical epithelium in pre-cancerous and cancerous

lesions, any interference with its function may disrupt the viral induced pathway

to carcinogenesis.

By identifying a naturally occurring small molecule that can significantly

and specifically inhibit a key oncogene, we identified an important parent

compound from which a more efficacious compound may be derived. Natural

products represent a promising start since their metabolism and uptake within

transformed cells may actually stress them more than normal cells. The lignans

may also be starting points to examine if such products can lower the

concentrations of more conventional chemotherapy drugs, hence greatly

increasing the therapeutic index of those drugs.

131
REFERENCES

Akgul B, Cooke JC, Storey A. HPV-associated skin disease. J Pathol. 2006


Jan;208(2):165-75.

Alexander KA. Diagnosis and management of human papillomavirus infections.


Pediatr Infect Dis J. 2005 Nov;24(11):1007-8.

Alirol E and Martinou JC. Mitochondria and cancer: is there a morphological


connection? Oncogene. 2006 Aug 7;25(34):4706-16.

Antonsson A, Forslund O, Ekberg H, Sterner G, Hansson BG. The ubiquity and


impressive genomic diversity of human skin papillomaviruses suggest a
commensalic nature of these viruses. J Virol. 2000 Dec;74(24):11636-41.

Antson AA et al. Structure of the intact transactivation domain of the human


papillomavirus E2 protein. Nature 2000;403:805-9.

Arteaga S, Andrade-Cetto A, Cardenas R. Larrea tridentata (Creosote bush), an


abundant plant of Mexican and US-American deserts and its metabolite
nordihydroguaiaretic acid. J Ethnopharmacol. 2005 Apr 26;98(3):231-9.

Ashrafi GH, et al. E5 protein of human papillomavirus type 16 selectively


downregulates surface HLA class I. Int J Cancer. 2005;113:276-283.

Avrich E, Sulik S, Nashelsky J. What is the appropriate management for a


patient with CIN1 on colposcopy? J Fam Pract. 2006 Feb;55(2):145-6.

Barbosa MS, et al. Papillomavirus polypeptides E6 and E7 are zinc binding


proteins. J Virol. 1989; 63:1404-1407.

Bartley AN and Ross DW. Validation of P53 immunohistochemistry as a


prognostic factor in breast cancer in clinical practice. Archives of Pathology
& Laboratory Medicine. 2002 Apr; 126(4):456-8.

Baseman JG and Koutsky LA. The epidemiology of human papillomavirus


infections. J Clin Virol. 2005 Mar;32 Suppl 1:S16-24.

132
Beausoleil SA, et al. Large-scale characterization of HeLa cell nuclear
phosphoproteins. Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12130-5.
Epub 2004 Aug 9.

Bernard HU. Gene expression of genital human papillomaviruses and


considerations on potential antiviral approaches. Antivir.Ther. 2002; 7:219-
237.

Bhattacherjee P, et al. The effects of a novel series of selective inhibitors of


arachidonate 5-lipoxygenase on anaphylactic and inflammatory
responses. Ann N Y Acad Sci. 1988;524:307-20.

Bouchet BP et al. p53 as a target for anti-cancer drug development. Crit Rev
Oncol Hematol. 2006 Jun;58(3):190-207.

Buck CB, et. al. Maturation of papillomavirus capsids. J Virol 2005; 79:2839-46.

Caldeira S, de Villiers EM, Tommasino M. Human papillomavirus E7 proteins


stimulate proliferation independently of their ability to associate with
retinoblastoma protein. Oncogene. 2000 Feb 10;19(6):821-6.

Cereghetti GM and Scorano L. The many shapes of mitochondrial death.


Oncogene. 2006 Aug 7;25(34):4717-24.

Corden S, et al. The integration of HPV-18 DNA in cervical carcinoma. Mol


Pathol 1999: 52: 275-282.

Cox JT. Human papillomavirus testing in primary cervical screening and


abnormal Papanicolaou management. Obstet Gynecol Surv. 2006 Jun;61(6
Suppl 1):S15-25.

Cox JT. Epidemiology and natural history of HPV. J Fam Pract. 2006 Nov;
Suppl:3-9.

Chiang CM et al. Viral E1 and E2 proteins support replication of homologous


and heterologous papillomaviral origins. Proc Natl Acad Sci USA
1992;89:5799-5803.

133
Craigo J, Callahan M, Huang RC, DeLucia AL. Inhibition of human
papillomavirus type 16 gene expression by nordihydroguaiaretic acid
plant lignan derivatives. Antiviral Res. 2000 Jul;47(1):19-28.

Crook T and Vousden KH. Properties of p53 mutations detected in primary and
secondary cervical cancers suggest mechanisms of metastasis and
involvement of environmental carcinogens. EMBO J. 1992 Nov;11(11):3935-
40.

Crusius K, Auvinen E, Alonso A. Enhancement of EGf- and PMA-mediated


MAP kinase activation in cells expressing human papillomavirus type 16
E5 protein. Oncogene 1997;15:1437-1444.

Crusius K, et al. The human papillomavirus type 16 E5 protein modulates ligand


dependent activation of the EGF receptor family in the human epithelial
cell line HaCaT. Exp Cell Res 1998;241:76-83.

Davy CE, et al. Human papillomavirus type 16 E1-E4-induced G2 arrest is


associated with cytoplasmic retention of active CDK/cyclin B1 complexes.
J Virol 2005;79:3998-4011.

Deng W, et al. mRNA splicing regulates human papillomavirus type 11 E1


protein production and DNA replication. J Virol 2003; 77:10213-10226.

Deng W et al. Cyclin/CDK regulates the nucleocytoplasmic localization of the


human papillomavirus E1 DNA helicase. J Virol 2004;78:13954-65.

Denk C, Butz K, Schneider A, Durst M, Hoppe-Seyler F. p53 mutations are rare


events in recurrent cervical cancer. J Mol Med. 2001 Jun;79(5-6):283-8.

De Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H. Classification
of papillomaviruses. Virology. 2004 Jun 20; 324(1):17-27.

DiMaio D and Liao JB. Human papillomaviruses and cervical cancer. Adv Virus
Res. 2006;66:125-59.

134
Doorbar J, et al. Specific interaction between HPV-16 E1-E4 and cytokeratins
results in collapse of the epithelial cell intermediate filament network.
Nature 1991;352:824-827.

Doorbar J. The papillomavirus life cycle. J Clin Virol. 2005 Mar;32 Suppl 1:S7-
15.
Doorbar J. Molecular biology of human papillomavirus infection and cervical
cancer. Clin Sci (Lond) 2006; 110(5): 525-41.

Dreier B et al.Development of zinc finger domains for recognition of the 5'-ANN-


3' family of DNA sequences and their use in the construction of artificial
transcription factors. J. Biol. Chem. 2001; 276(31): 29466-78.

Dreyer G, Snyman LC, Mouton A, Lindeque BG. Management of recurrent


cervical cancer. Best Pract Res Clin Obstet Gynaecol. 2005 Aug;19(4):631-44.

Dreyer, G. Operative management of cervical cancer. Best Pract Res Clin


Obstet Gynaecol. 2005 Aug;19(4):563-76.

Dunne EF and Markowitz LE. Genital human papillomavirus infection. Clin


Infect Dis. 2006 Sep 1;43(5):624-9.

DuPont NC and Monk BJ. Chemotherapy in the management of cervical


carcinoma. Clin Adv Hematol Oncol. 2006 Apr;4(4):279-86.

Erster S and Moll UM. Stress-induced p53 runs a transcription-independent


death program. Biochem Biophys Res Commun. 2005 Jun 10;331(3):843-50.

Fadeel B. Programmed cell clearance. Cell Mol Life Sci. 2003 Dec;60(12):2575-
85.

Felisani A, Mileo AM, Paggi MG. Retinoblastoma family proteins as key targets
of the small DNA virus oncoproteins. Oncogene. 2006 Aug 28;25(38):5277-
85.

Finnen RL, et al. Interactions between papillomavirus L1 and L2 capsid proteins.


J Virol 2003;77:4818-26.

135
Foijer F and te Riele H. Check, double check: the G2 barrier to cancer. Cell
Cycle. 2006 Apr;5(8):831-6. Epub 2006 Apr 17.

Fox PA and Tung MY. Human papillomavirus: burden of illness and treatment
cost considerations. Am J Clin Dermatol. 2005;6(6):365-81.

Giacinti C and Giordano A. RB and cell cycle progression. Oncogene. 2006 Aug
28;25(38):5220-7.

Glover JN and Harrison SC. Crystal structure of the heterodimeric BZip


transcription factor c-fos / c-jun bound to DNA. Nature. 1995; 373: 257-261.

Gnabre JN, Brady JN, Clanton DJ, Ito Y, Dittmer J, Bates RB, Huang RC.
Inhibition of human immunodeficiency virus type 1 transcription and
replication by DNA sequence-selective plant lignans. Proc Natl Acad Sci U
S A. 1995 Nov 21;92(24):11239-43.

Greer CE, Wheeler CM, Ladner MB, Beutner K, Coyne MY, Liang H, Langenberg
A, Yen TS, Ralston R. Human papillomavirus (HPV) type distribution
and serological response to HPV type 6 virus-like particles in patients
with genital warts. J Clin Microbiol. 1995 Aug;33(8):2058-63.

Gschwendt M et al. Calcium and phospholipid-dependent protein kinase


activity in mouse epidermis cytosol. Stimulation by complete and
incomplete tumor promoters and inhibition by various compounds.
Biochem Biophys Res Commun. 1984 Oct 15;124(1):63-8.

Gu Z and Matlashewski G. Effect of human papillomavirus type 16 oncogenes


on MAP kinase activity. J Virol 1995; 69:8051-56.

Hazard K, Karlsson A, Andersson K, Ekberg H, Dillner J, Forslund O.


Cutaneous Human Papillomaviruses Persist on Healthy Skin. J Invest
Dermatol. 2006 Oct 5.

Hebner CM and Laimins LA. Human papillomaviruses: basic mechanisms of


pathogenesis and oncogenicity. Rev Med Virol. 2006 Mar-Apr;16(2):83-97.

136
Hegde RS et al. Crystal structure at 1.7 A of the bovine papillomavirus-1 E2
DMA binding domain bound to its DNA target. Nature 1992;359:505-12.

Herrington CS. Do HPV-negative cervical carcinomas exist?--revisited. J Pathol.


1999 Sep;189(1):1-3.

Hietanen S, Lain S, Krausz E, Blattner C, Lane DP. Activation of p53 in cervical


carcinoma cells by small molecules. Proc Natl Acad Sci U S A. 2000 Jul
18;97(15):8501-6.

Hughes FJ and Romanos MA. E1 protein of human papillomavirus is a DNA


helicase/ATPase. Nucleic Acids Res. 1993;21(25):5817-23.

Hwu JR, Tseng WN, Gnabre J, Giza P, Huang RC. Antiviral activities of
methylated nordihydroguaiaretic acids. 1. Synthesis, structure
identification, and inhibition of tat-regulated HIV transactivation. J Med
Chem. 1998 Jul 30;41(16):2994-3000.

James MA, Lee JH, and Klingelhutz AJ. Human papillomavirus type 16 E6
activates NF-kappaB, induces cIAP-2 expression, and protects against
apoptosis in a PDZ binding motif-dependent manner. J Virol. 2006
Jun;80(11):5301-7.

Jeon S, Allen-Hoffman B, Lambert P. Integration of human papillomavirus type


16 into the human genome correlates with a selective growth advantage of
cells. J Virol 1995; 69: 2989-2997.

Keen N, Elston R, Crawford L. Interaction of the E6 protein of human


papillomavirus with cellular proteins. Oncogene. 1994 May;9(5):1493-9.
Lindeque, BG. Management of cervical premalignant lesions. Best Pract
Res Clin Obstet Gynaecol. 2005 Aug;19(4):545-61

Kim SS, et al. The structural basis of DNA target discrimination by


papillomavirus E2 proteins. J Biol Chem 2000;275:31245-54.

Kitayner M, et al. Structural basis of DNA recognition by p53 tetramers. Mol


Cell. 2006 Jun 23;22(6):741-53.

137
Lindeque BG. Management of cervical premalignant lesions. Best Pract Res Clin
Obstet Gynaecol. 2005 Aug;19(4):545-61.

Ma T et al. Interaction between cyclin-dependent kinases and human


papillomavirus replication-initiation protein E1 is required for efficient
viral replication. Proc Natl Acad Sci USA 1999; 96:382-87.

Malanchi I, Caldeira S, Krutzfeldt M, Giarre M, Alunni-Fabbroni M, Tommasino


M. Identification of a novel activity of human papillomavirus type 16 E6
protein in deregulating the G1/S transition. Oncogene. 2002 Aug
22;21(37):5665-72.

Manfredi JJ. p53 and apoptosis: its not just in the nucleus anymore. Mol Cell.
2003;11:552-554.

Maruta H and Burgess AW. Regulation of the ras signaling network. Bioessays.
1994 Jul;16(7):489-96.

McDonald RW, et al. Synthesis and anticancer activity of nordihydroguaiaretic


acid (NDGA) and analogues. Anticancer Drug Design. 2001; 16(6):261-70.

Meek DW. The p53 response to DNA damage. DNA Repair. 2004(3):1049-56.

Michalak E, et al. Death squads enlisted by the tumour suppressor p53. Biochem
Biophys Res Commun. 2005 Jun 10;331(3):786-98.

Moll UM, LaQuaglia M, Benard J, and Riou G . Wild-type p53 protein


undergoes cytoplasmic sequestration in undifferentiated neuroblastomas
but not in differentiated tumors. Proc Natl Acad Sci U S A. 1995 May
9;92(10):4407-11.

Momand J, et al. The mdm-2 oncogene product forms a complex with the p53
protein and inhibits p53-mediated transactivation. Cell. 1992 Jun
26;69(7):1237-45.

Moore DH. Cervical cancer. Obstet Gynecol. 2006 May;107(5):1152-61.

138
Muggia FM. Recent updates in the clinical use of platinum compounds for the
treatment of gynecologic cancers. Semin Oncol. 2004 Dec;31(6 Suppl 14):17-
24.

Munger K, et al. Complex formation of human papillomavirus E7 proteins with


the retinoblastoma tumor suppressor gene product. EMBO J. 1989 Dec
20;8(13):4099-105.

Munger K, et al. Interactions of HPV E6 and E7 oncoproteins with tumour


suppressor gene products. Cancer Surv. 1992;12:197-217.

Padilla LA, Leung BS, Carson LF. Evidence of an association between human
papillomavirus and impaired chemotherapy-induced apoptosis in cervical
cancer cells. Gynecol Oncol. 2002 Apr;85(1):59-66.

Patel D, et al. The E6 protein of human papillomavirus type 16 binds to and


inhibits coactivation by CBP and p300. EMBO J. 1999; 18: 5061-72.

Pearson G, et al. Mitogen-activated protein (MAP) kinase pathways: regulation


and physiological functions. Endocr Rev. 2001 Apr;22(2):153-83.

Picksley SM and Lane DP. The p53-mdm2 autoregulatory feedback loop: a


paradigm for the regulation of growth control by p53? Bioessays. 1993
Oct;15(10):689-90.

Pollack M and Leeuwenburgh C. Apoptosis and aging: role of the mitochondria.


J Gerontol A Biol Sci Med Sci. 2001 Nov;56(11):B475-82.

Pray TR and Laimins LA. Differentiation-dependent expression or E1-E4


proteins in cell lines maintaining episomes of human papillomavirus type
31b. Virology 1995;206:679-85.

Ranganathan R and Ross E. PDZ domain proteins: scaffolds for signaling


complexes. Curr Biol 1997; 7 (12): R770-3.

139
Reed J. The survivin saga goes in vivo. J Clin Invest. 2001 Oct;108(7): 965-
969.

Roberts S, et al. Cutaneous and mucosal human papillomavirus E4 proteins


form intermediate filament-like structures in epithelial cells. Virology
1993; 197:176-87.

Roberts S, et al. Mutational analysis of the human papillomavirus type 16 E1-E4


protein shows that the C terminus is dispensable for keratin cytoskeleton
association but is involved in inducing disruption of the keratin filaments.
J Virol 1997;71:3554-3562.

Sapp M, et al. Organization of the major and minor capsid proteins in human
papillomavirus type 33 virus-like particles. J Gen Virol 1995;76:2407-12.

Scheffner M. Ubiquitin, E6-AP, and their role in p53 inactivation. Pharmacol


Ther. 1998 Jun;78(3):129-39.

Scheffner M, Munger K, Byrne JC, Howley PM. The state of the p53 and
retinoblastoma genes in human cervical carcinoma cell lines. Proc Natl
Acad Sci U S A. 1991 Jul 1;88(13):5523-7.

Schiffman M and Castle PE. Human papillomavirus: epidemiology and public


health. Arch Pathol Lab Med. 2003 Aug;127(8):930-4.

Schlaerth AC, and Abu-Rustum NR. Role of minimally invasive surgery in


gynecologic cancers. Oncologist. 2006 Sep;11(8):895-901.

Schmiedeskamp MR, Kockler DR. Human Papillomavirus Vaccines (CE)


(July/August). Ann Pharmacother. 2006 Jul 18 [Epublication ahead of
print].

Sen E, Alam S, Meyers C. Genetic and Biochemical analysis of cis regulatory


elements within the keratinocyte enhancer region of the human
papillomavirus type 31 upstream regulatory region during different
stages of the viral life cycle. J Virol 2004; 78:612-619.

140
Sengupta S and Harris CC. p53: traffic cop at the crossroads of DNA repair and
recombination. Nat Rev Mol Cell Biol. 2005;6:44-55.

Shi Y, et al. Transcriptional repression by YY1, a human GLI-Kruppel-related


protein, and relief of repression by adenovirus E1A protein. Cell. 1991 Oct
18;67(2):377-88.

Stanley MA. Human papillomavirus and cervical carcinogenesis. Best Pract Res
Clin Obstet Gynaecol. 2001 Oct;15(5):663-76.

Stanley M. Immune responses to human papillomavirus. Vaccine. 2006 Mar


30;24 Suppl 1:S16-22.

Steger G and Corbach S. Dose dependent regulation of the early promoter of


human papillomavirus type 18 by the viral E2 protein. J Virol 1997; 71: 50-
58.

Sterling JC, Skeper JN, Stanley MA. Immunoelectron microscopical location of


human papillomavirus type 16 L1 and E4 proteins in cervical
keratinocytes cultured in vivo. J Invest Dermatol 1993;100:154-158.

Stewart AJ and Viswanathan AN. Current controversies in high-dose-rate versus


low-dose-rate brachytherapy for cervical cancer. Cancer. 2006 Sep
1;107(5):908-15.

Straight SW, et al. The E5 oncoprotein of human papillomavirus type 16


transforms fibroblasts and effects the downregulation of the epidermal
growth factor receptor in keratinocytes. J Virol 1993; 67:4521-4532.

Sverdrup F and Khan SA. Replication of human papillomavirus (HPV) Dnas


supported by the HPV type 18 E1 and E2 proteins. J Virol 1994;68:505-509.

Tan G, Gyllenhaal C, Soejarto DD. Biodiversity as a source of anticancer drugs.


Curr Drug Targets. 2006 Mar;7(3):265-77.

141
Tewari KS, Monk BJ. Gynecologic oncology group trials of chemotherapy for
metastatic and recurrent cervical cancer. Curr Oncol Rep. 2005
Nov;7(6):419-34.

Tommasino M and Crawford L. Human papillomavirus E6 and E7: proteins


which deregulate the cell cycle? Bioessays. 1995 Jun;17(6):509-18.

Tomson TT, Roden RB, Wu TC. Human papillomavirus vaccines for the
prevention and treatment of cervical cancer. Curr Opin Investig Drugs.
2004 Dec;5(12):1247-61.

Ustav M and Stenlund A. Transient replication of BPV-1 requires two viral


polypeptides encoded by the E1 and E2 open reading frames. EMBO J
1991;10:449-457.

Veldman T, et al. Human papillomavirus E6 and Myc proteins associate in vivo


and bind to and cooperatively activate the telomerase reverse
transcriptase promoter. Proc Natl Acad Sci USA 2003 Jul 8;100(14):8211-6.

Vousden KH. p53: death star. Cell. 2000 Nov 22;103(5):691-4.

Vousden KH. P53 and PUMA: A deadly duo. Science. 2005 Sept 9; 309:1685-
1686.

Vousden KH and Lu X. Live or let die: the cells response to p53. Nat Rev
Cancer. 2002;2:594-604.

Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV,
Snijders PJ, Peto J, Meijer CJ, Munoz N. Human papillomavirus is a
necessary cause of invasive cervical cancer worldwide. J Pathol. 1999
Sep;189(1):12-9.

Wang Q, et al. Functional analysis of the human papillomavirus type 16 E1=E4


protein provides a mechanism for in vivo and in vitro keratin filament
reorganization. J Virol 2004;78:821-33.

142
Werness BA, Levine AJ, Howley PM. Association of human papillomavirus
types 16 and 18 E6 proteins with p53. Science. 1990 Apr 6;248(4951):76-9.

Westbrook TF, Nguyen DX, Thrash BR, McCance DJ. E7 abolishes raf-induced
arrest via mislocalization of p21(Cip1). Mol Cell Biol. 2002 Oct;22(20):7041-
52.

Wiley D and Masongsong E. Human papillomavirus: the burden of infection.


Obstet Gynecol Surv. 2006 Jun;61(6 Suppl 1):S3-14.

Wilson RM and Danishefsky SJ. Small molecule natural products in the


discovery of therapeutic agents: the synthesis connection. J Org Chem.
2006 Oct 27;71(22):8329-51.

Yee KS and Vousden KH. Complicating the complexity of p53. Carcinogenesis.


2005 Aug;26(8):1317-22.

Ying H and Xiao ZX. Targeting retinoblastoma protein for degradation by


proteasomes. Cell Cycle. 2006 Mar;5(5):506-8.

Youngren JF, et al. Nordihydroguaiaretic acid (NDGA) inhibits the IGF-1 and c-
erbB2/HER2/neu receptors and suppresses growth in breast cancer cells.
Breast Cancer Res Treat. 2005 Nov;94(1):37-46.

Yi S. p53 and its downstream proteins as molecular targets of cancer. Mol


Carcinogenesis. 2006 Jun;45(6):409-15.

Yoon CS, et al. alpha(6) Integrin is the main receptor of human papillomavirus
type 16 VLP. Biochem Biophys Res Commun. 2001 May 11;283(3):668-73.

Yu J and Zhang L. The transcriptional targets of p53 in apoptosis control.


Biochem Biophys Res Commun. 2005 Jun 10;331(3):851-8.

Zheng ZM and Baker CC. Papillomavirus genome structure, expression, and


post-transcriptional regulation. Front Biosci. 2006 Sep 1;11:2286-302.

143
Zimmerman JM and Maher, III, LJ. Solution measurement of DNA curvature in
papillomavirus E2 binding sites. Nucleic Acids Res. 2003 September 1; (17):
51345139.

zur Hausen, H. Papillomaviruses and cancer: from basic studies to clinical


application. Nat Rev Cancer. 2002 May 2(5):342-50.

Zur Hausen, H. Oncogenic DNA viruses. Oncogene. 2001 Nov 26;20(54):7820-


3.

zur Hausen, H. Papillomaviruses causing cancer: evasion from host-cell control


in early events in carcinogenesis. J Natl Cancer Inst. 2000 May 3;92(9):690-
8.

zur Hausen H. Papillomaviruses in human cancers. Proc Assoc Am Physicians.


1999 Nov-Dec;111(6):581-7.

zur Hausen, H. Viruses in human cancers. Eur J Cancer. 1999


Dec;35(14):1878-85.

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