Biology of Aging

Lecture 9

Telomeres and telomerase

continued
2009 Nobel prize in Medicine
Correlation between telomere length
and social status

Divided the SES into nonmanual (white-collar)

workers (I-IIIN) and manual (blue-collar) workers
(IIIM-V)!
 Mean telomere length and standard error by
manual vs. nonmanual social class groupings.
Comparison of TRFL within 17 twin pairs
most discordant for SES.!
Correlation of telomere length
with:
Social status
Depression
Some reports were unable to replicate these
results
Various disease conditions

Conditions associated with low-grade

inflammation lead to higher cell turnover and
telomere shortening
 Why do we need replicative
senescence?
Not to make us mortal!
Senescence is a powerful anticancer
mechanism
 TMM-telomere maintenance mechanism

Normal somatic cells permanently exit from the cell cycle (that is, become senescent) after a limited
number of cell divisions. Cells might escape temporarily from the senescence barrier if they lose the
function of key tumour-suppressor genes, especially TP53 and/or RB, but most will eventually die, at which
stage the cell population is described as being in 'crisis'. Cells might bypass crisis and become immortalized
(capable of unlimited proliferation) if a telomere maintenance mechanism ? telomerase or ALT ? is
activated. Immortalized cells, but not their mortal predecessors, might be susceptible to malignant
transformation by an activated oncogene such as RAS.
 Telomerase paradox

Humans Mice

-Telomerase is repressed -Telomerase is active

in somatic tissues in somatic tissues
-Short telomeres -Long telomeres
Replicative senescence No Replicative senescence
Cancer mortality ~25% Cancer mortality up to 95%
Mouse telomeres
Human Mouse

PD100
PD60
PD74
PD45

PD13
kb

200
150
100

50
33
25
19
15
12
10
8
6
5

2
 Oxygen sensitivity of mouse fibroblasts
110
110

100 100

90

Doublings
90

80

80 Human

70

Population Doublings
70
Human

Human
60

60 Beaver

50

Capybara

Population
50 Porcupine
Beaver

40

Paca
40
Capybara

30

30
3% or 20% O2
Porcupine
20

20 Paca

10

10

0

0

20
40
60
80
100
120
140
160
180
200

00 20 40 60 80 100 120 140 160 180 200

Time, days

Parrinello et al., Nature Cell Biol. 2003

Does telomerase repression
and replicative senescence
coevolve with long life?
 Rodents present wide variety of lifespans
and body sizes

Gerbil
Deer mouse
Mouse
Rat
Chipmunk

53 g / 6 yrs
20 g / 8 yrs
30 g / 4 yrs
400 g / 5 yrs
96 g / 10 yrs

Chinchilla
Guinea pig
Woodchuck
Nutria
Muskrat

642 g / 17 yrs
728 g / 12 yrs
5000 g / 14 yrs
7850 g / 9 yrs
1360 g / 10 yrs

Capybara
Porcupine
Beaver
Squirrel
Naked molerat

55000 g / 15 yrs
8600 g / 23 yrs
20250 g / 24 yrs
530 g / 24 yrs
35 g / 28 yrs

 Rodent phylogeny
Capybara
Guinea pig
N. American porcupine
Chinchilla
Nutria
Naked mole-rat
American beaver
House mouse
Norway rat
Mongolian gerbil
Deer mouse
Golden hamster
Muskrat
Woodchuck
E. American chipmunk
Eastern grey squirrel

Rodents with maximum lifespan greater than 20 years.

 TRAP assay
Tissue Extract (0.5 g)

repeat by telomerase
Addition of telomeric
+
MTS primer

* 1. Elongation with telomerase

* 2. Binding of RPC3g on telomerase products

2 cycles
RPC3g primer
3. DNA synthesis

*
PCR amplification

4. Binding of RP primer

27 cycles
RP primer
MTS primer

* 5. DNA synthesis

*
Telomerase activity

Seluanov et al. 2007, Aging Cell

 Telomerase activity
Telomerase
Maximum Adult body
Common Name activity
lifespan (yr) mass (g)
coefficient

Capybara 15 55,000 1.000.10

Guinea pig 12 728 6.670.27
Chinchilla 17 642 7.000.28
Nutria 9 7,850 4.000.47
Naked mole-rat 28 35 9.500.53
American beaver 24 20,250 1.670.17
House mouse 4 30 11.000.30
Norway rat 5 400 9.000.22
Mongolian gerbil 6 53 10.000.42
Deer mouse 8 20 11.000.50
Golden hamster 4 105 10.500.35
Muskrat 10 1,360 5.500.31
Woodchuck 14 5,000 12.000.34
Chipmunk 10 96 15.000.24
Eastern grey squirrel 24 530 13.330.43

Seluanov et al. 2007, Aging Cell

 Correlation between telomerase activity and
lifespan and body mass
18 18

Telomerase activity coefficient

Telomerase activity coefficient

16 16

14 14

12 12

10 10

8 8

6 6

4 4

2 2

0 0
0.5 0.7 0.9 1.1 1.3 1.5 1 2 3 4 5

Log maximum lifespan (yr) Log body mass(g)

Raw data r2=0.085, P=0.293 Raw data r2=0.534, P=0.002*

Contrasts r2=0.024, P=0.584 Contrasts r2=0.76, P<0.0001*

Seluanov et al. 2007, Aging Cell

 Hypothesis

Evolution of additional tumor

Cancer risk suppressor mechanisms Cancer risk
Replicative senescence

Seluanov et al. 2007, Aging Cell

Large mammals
Rodent species with replicative senescence

110
110

100

100
90

Doublings
90

80

80

70

Human

Population Doublings

70
Human
60

60 Beaver
Capybara
50

Population

50 Porcupine
Beaver

40

Paca
40
Capybara

30

30
Porcupine

20

20 Paca

10

10

0

0

20
40
60
80

00 20 40 60 80






100
100 120
120 140
140 160
160 180
180 200
200
Time, days

Seluanov et al. 2008, Aging Cell

+
PD10
Beaver
PD39

PD11
Capybara
PD23

PD6
Porcupine
PD32

PD11
Paca
fibroblasts

PD21
Telomerase activity in rodent
 Overexpression of p21 and p16 in
replicatively senescent rodent fibroblasts

p16Ink4a p21Waf1

kDa Y S Y S Y S Y S Y S
20 kDa Y S Y S Y S Y S Y S
15 20
p16
12 15 p21
12
10

Actin Actin
 kb

2
3
4
5
6
8
10
12
15
19
25
33
50
100
150
200
PD45
PD74
PD8
PD32
PD7
PD27
PD46
PD6
PD21
PD39
PD11
PD23
PD11
PD21

PD7
PD25
PD10
PD24

PD13
PD60
PD100
PD12
PD28
fibroblasts

PD73
PD11
PD29
PD113
PD8
PD25
PD66
Telomere length in rodent
Rodent species with continuous cell
proliferation

0











20 40 60 80 100 120 140 160 180 200
110
110
Deer Mouse

100

100 Rat

90

90 Hamster

Population Doublings

80

House Mouse

Deer Mouse
80
Guinea Pig

70

Rat
70

Gerbil

Hamster
Population Doublings

60

60 House
Mouse
50

Guinea Pig
50 Naked mole-rat

Gerbil
40

40
Chipmunk

NMR
Muskrat

30

30 Chipmunk
Chinchilla

20

Muskrat
20
Squirrel

Chinchilla

10

10 Squirrel

0

0
0
0
20
40
60
80
100
120
140
160
180
200

20 40 60 80 100 120 140 160 180 200

Time,
Time, days

days

Seluanov et al. 2008, Aging Cell

Species with replicative senescence
(body mass >8,000 g)

Capybara
Guinea pig
Paca
N. American porcupine
Chinchilla
Naked mole-rat
American beaver
House mouse
Norway rat
Mongolian gerbil
Deer mouse
Muskrat
Golden hamster
E. American chipmunk
Eastern grey squirrel
Species that display slow in vitro cell growth
(lifespan >10 years)

Capybara
Guinea pig
Paca
N. American porcupine
Chinchilla
Naked mole-rat
American beaver
House mouse
Norway rat
Mongolian gerbil
Deer mouse
Muskrat
Golden hamster
E. American chipmunk
Eastern grey squirrel
 Model

Cancer risk Cancer risk

Large body mass Long lifespan

Short lifespan

Slow cell growth

in culture

Repression of somatic
Rapid cell growth
telomerase activity
in culture Alternative
(telomere-independent)
Replicative senescence
mechanisms
to control cell proliferation

Seluanov et al. 2008, Aging Cell

Telomerase knockout
 Telomerase

TERT
protein

RNA TR
TERC
Normal human cells do not have telomerase activity
RNA is expressed but TERT is not
 Telomere shortening and tumor formation by mouse cells lacking telomerase RNA.
Blasco MA, Lee HW, Hande MP, Samper E, Lansdorp PM, DePinho RA, Greider CW.
Cell. 1997 Oct 3;91(1):25-34.

KO telomerase RNA component (TR)

Mice were viable for 3-6 generations
(laboratory mice have very long telomeres)
Telomerase shorten 3-5 kb/generation
Late generation mice start to show
abnormalities
 Progressive telomere shortening

Figure 6. Telomere Fluorescence of Metaphase Chromosomes from Embryonic FibroblastsFibroblasts from mTR+/+ (Wt) and indicated generations
of mTR/ null mice (KO, G2G6) were hybridized with a Cy3 labeled (CCCTAA)3 PNA probe. Arrows indicate telomeres without detectable
TTAGGG. Images were obtained using a Zeiss Axioplan fluorescence microscope coupled to a charge-coupled device (CCD) camera. Separate
images of DAPI-stained chromosomes and telomeres hybridized with (CCCTAA)3 PNA probe were merged and assigned pseudocolors using Adobe
Photoshop (Adobe Systems, Mountain View, California).(A) Metaphase from Wt cells showing distinct bright signal intensities for all telomeres.(B) A
KO-G2 cell showing decreased overall fluorescence and occasional absence of telomere fluorescence signal.(C) A KO-G4 metaphase showing
strong heterogeneity in the telomere signal intensities. Many telomeres show little if any fluorescence (arrows), yet bright telomere staining can be
seen on some chromosome ends.(D) Metaphase from a KO-G6 cell. Note the overall weaker telomere fluorescence and the increased frequency of
telomeres without detectable TTAGGG. Two Robertsonian fusions in this metaphase were observed with no detectable TTAGGG at the fusion point
(arrow).
Late generation mice show abnormalities in
highly proliferative tissues: atrophy in skin and
small intestine, hair graying, problems with bone
marrow regeneration, abnormal hematological
profile

Genomic instability: chromosome fusions

 a, Impaired reproductive function in late-
generation mTR-deficient mice. Males aged
612 weeks and females aged 412 weeks of
the same generation of telomerase-deficient
mice were mated over a period of 2 months
or longer; n indicates the number of mating
pairs examined. The P values of each
generation were calculated compared with G
1 (G2, P = 0.063; G3, P = 0.070; G4, P =
0.021; and G5, P = 0.004).
b, Testicular atrophy and testes weights.
Scale bar, 5mm.
c, Uterine horns. Grossly dissected uterine
horns (u) and ovaries (arrows) (scale bar,
5mm) and haematoxylin and eosin (H&E)
staining (magnification 50).
d, Ovarian structure and function. Oestrus-
matched ovaries are shown at 50
magnification (left).
E, endometrium; M, myometrium; GF,
Graafian follicle; CL, corpora lutea. The
graphs show a weight comparison of ovaries,
average egg yields following natural mating
with a wild-type stud male, and the
percentage of in vitro development of pre-
implantation embryos. BC, blastocyst.
 Tumor formation in TR -/- mice

Figure 7. Tumor Formation in Nude Mice by mTR/ Transformed Cells (A) Tumor formation in nude mice from TAg and
rasv12 transformed mTR+/+ and G1 mTR/ cells. Each transformed cell line (5 105 cells) was injected at a total of four
different sites (two sites in each of two mice), collected, and weighed 20 days after injection. Tumor size for each cell line
is plotted as the average of four tumors arising from four different injection sites (two each on two different mice). (B)
Tumor formation in nude mice injected with E1A and rasv12 transformed cells from wild type and mTR/ G1, G2, G3, and
G4 embryos. Transformed cells were selected after infection with the retroviral vector, and 1.5 106 cells were injected
into nude mice at four independent sites. The average size of the tumor 47 days after injection is plotted. (C) A second
experiment was done similar to that described in (B) using wild type, mTR/ G4 cells, and two different isolates of mTR/
G6 cells. A total of 1.5 106 cells was injected at four independent sites, and the size of the tumor is shown 55 days after
injection.
Overexpression of telomerase

2-fold increase in the rate of wound healing

2-fold increase in tumor formation

Mouse telomeres are already too long, this

result indicates that telomerase has additional
role in stimulating tumor growth
Overexpressing mice are short-lived, die of
cancer
 Human conditions related to
telomerase function
Dyskeratosis congenita
The typical symptoms of dyskeratosis congenita involve the skin,
nails, and mucous membranes, as well as bone marrow failure. Die
before age 20.
DKC-mutation in dyskerin-protein that interacts with telomerase RNA.
Mutation reduces the level of telomerase RNA

Pulmonary fibrosis
 Pulmonary fibrosis
Shortness of breath, fatigue, cough
~5 mln affected worldwide

Scarring of lung tissue

Patients have short telomeres
Telomerase in human
development
In germ cells telomere length is
maintained at equilibrium

Telomere length is longer in

Germ cells than in somatic cells
Telomerase activity in oocytes
Telomerase activity in sperm
 Telomerase in development

Schematic overview of the telomerase activity and hTR expression during different stages of human fetal development and in the adult. Telomerase
activity (measured as the catalytic activity in extraction) and hTR expression during fetal and neonatal development (after Ulaner et al., 1998,
Ulaner et al., 2001, Wright et al., 1996, Wright et al., 2001, Yashima et al., 1998a and Yashima et al., 1998b). In humans, telomerase activity is
detected from the blastocyst stage until 20 weeks of gestation in most embryonic tissues (Wright et al., 1996). During fetal development, tissue- and
developmental phase-dependent regulation generates different levels of telomerase activity. Telomerase activity disappears more rapidly during fetal
development in heart, brain, and kidney as compared to liver, lung, testes, and spleen (Ulaner and Giudice, 1997). Whereas hTR expression is
measured in the kidney up to 21 weeks postgestation, no telomerase activity is measured from 17 weeks onwards due to inactivation hTERT via
alternative splicing (Ulaner et al., 1998). Whereas telomerase is absent in most adult tissues, some exceptions exist with expression of telomerase at
different levels, closely linked to dividing cells. Telomerase activity has been detected in lymphocyte stem and progenitor cells, and in peripheral blood
lymphocytes upon proliferation induction (Counter et al., 1995 and Hiyama et al., 1995). High expression was found in primary spermatocytes and
Sertoli cells within the testis, intermediate expression in lymphoid germinal centers, and weak expression was detected in regenerative epithelia
(Yashima et al., 1998a and Yashima et al., 1998b).
 Animal cloning

Animals are produced from somatic cells. Somatic cells have shorter
telomeres.
Dolly the sheep had shorter than usual telomeres.
Cloned calves - two reports of normal telomere length
 Lane 2, donor cow
lanes 36, live cloned calves
lanes 710, age-matched control calves
lanes 1116, cloned calves died shortly after birth
lane 17, uncut DNA.

Figure1.Telomere length analyses.a, A representative TRF assay on the donor cow, clones and age-matched control calves. Lane 1,
Lambda HindIII markers; lane 2, donor cow; lanes 36, live cloned calves; lanes 710, age-matched control calves; lanes 1116,
cloned calves died shortly after birth; lane 17, uncut DNA. The TRF assays were performed using the TeloQuant Telomere Length
Assay kit (Pharmingen). b, Quantified telomere lengths of the donor cow, all clones (live and died) and control calves. c, A
representative TRF assay on donor fibroblast cells at different passages. Lane 1, Lambda BstEII markers; lanes 27, skin fibroblast
cells at passages 1, 5, 10, 20, 30 and 40; lane 8, 2.5 g Telo Hi markers (mean telomere lengths=11.3 kb); lane 9, 7.5 g Telo Lo
markers (mean telomere lengths=3.3 kb).
 Growth of donor cells,
these calls had about
2 PDs remaining before
senescence

Cells from
cloned fetus

Original strain

Extended proliferation
in clones

Figure 3. Ability of nuclear transfer to restore the proliferative life-span of senescent donor cells.
(A) The growth curve of the original BFF cell strain (green) is compared with that of cells derived from the
fetus (ACT99-002) (black) that was cloned from late-passage BFF cells (CL53 cells).
(B) The growth curve of the CL53 donor cells demonstrating that the cultures had about two population
doublings (PDs) remaining.
(C) Late-passage CL53 cells (n = 97) were seeded at clonal density, and the proliferative capacity after 1
month was determined.
(D) Single-cell clones from early-passage BFF cultures (original) and early-passage ACT99-002 (clone)
showed a capacity for extended proliferation.
 Figure 2. Normal heifers cloned from senescent somatic cells. (A) CL53-8, CL53-9, CL53-10,
CL53-11, and CL53-12 (nicknamed Lily, Daffodil, Crocus, Forsythia, and Rose, respectively) at 5
months of age and (B) CL53-1 (Persephone) at 10 months of age.
 Cloned animals have somewhat longer
telomeres
Telomeres are extended during
embryogenesis