III.

Telomeres and Cancer

telomeres

telomeres
centromeres
 Why chromosomes need special
telomeres?
• Since eukaryotic chromosomes are linear DNA,
the ends need to be protected.
• The special telomere structure prevent the ends of
chromosomes to fuse with each other.
• Prevent the ends to be recognized as double
stranded DNA breaks that would activate cell
cycle check points.
• Telomeres provide a solution for end-replication
problem.

End Protection and End-Replication

 What is telomere?
• Telomere is the nucleoprotein structure that terminates or
“cap”a eukaryotic chromosome
• Contain a special region of DNA consisting of tandemly
repeated sequences
- 500 to 1000s of repeat units
TTGGGGTTGGGGTTGGGG… (protozoan repeat)
-…TTGGGGTTGGGG

-…TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG… (human repeat)

•The single stranded region of terminal sequences are

organised in a loop structure that is not recognised by
nucleases.
•Telomeric repeats are synthesised onto the end of the
chromosome from an RNA template by telomerase.
http://www.hgu.mrc.ac.uk/Research/Chrombio/MouseTelo/davidk.htm
 Solution for end-protection
problem: no free end
T-loop

TRF1
D-loop
TRF2

Subtelomeric (TTAGGG)n
Sequence Pot1 de Lange/Griffin

Single stranded 3’ overhang

T­loops protect single stranded ends of DNA 
and prevent end­end fusion and provoke of 
cell cycle check point  
End replication problem:
- DNA polymerase needs an RNA primer to get started: it only starts at
double-stranded DNA
- DNA polymerase builds in a 5’ to 3’ direction to copy existing strands
- When the RNA primer is removed, it leaves a small overhang, which is
a problem at the very end of a chromosome DNA because piece is lost.
- eventually too much of the chromosome would be lost
5’ 3’
3’ 5’
replication
5’ 3’
3’ 5’ primers disappear because
5’ 3’ they are made of RNA
3’ 5’
5’ 3’
3’ 5’
5’ 3’
3’ 5’
 The solution for end-replication
problem: telomere and telomerase
Telomerase: an RNA - containing enzyme which adds
more telomeres to the 5’ end of DNA strands by using
its RNA as template
 How do telomere and telomerase together solve
the end-replication problem(protozoan)?

Adopted from Cancer Letter 194, 139-154

Telomere maintenance
 Telomerase

Dyskerin
Anchor site

Catalytic site

--TTAGGGTTAGGGTTAGGGTTAG

hTR

hTERT

hTERT = human telomerase reverse transcriptase

hTR = human telomerase template RNA
What is the connection of telomeres with aging ?

Most somatic cells do not have detectable telomerase 
activity

In human somatic cells telomeres shorten by 5­20 repeats 
with every cell division

In culture normal human somatic cells exhibit a restricted 
lifespan and enter senescence after a set number of 
division (~50)

Telomere length shortens with the age of a cell and 
eventually cell dies when telomere becomes too short

Telomeres seem to be part of a mechanism that counts 
the lifespan of cells
 Telomere Hypothesis of Aging:
The Human Generational Clock

• Repetitive, 500-1000 copies of non-coding

DNA (TTAGGG)n
• Progressively lost with each cell division
–“End replication problem”
• Senescence (growth arrest) occurs when
telomeres are short
Length of telomeres varies among
different cell types

Germline/ES cells (telomeres maintained)

Pluripotent Stem cells

Telomere Length

(intermediate telomere loss)

Normal cells (greatest telomere loss)

Growth
Arrest

Cell divisions
 Telomeres Shorten With Increased Age

Tissue Source Telomere Length (kb)

Sperm
Placenta
Fetal brain
Fetal kidney

Colon mucosa (30-65 yrs)

Colon mucosa (65-88 yrs)

Blood (20-39 yrs)

Blood (40-59 yrs)
Blood (60-79 yrs)

Hastie et al, Nature, 346: 866 0 4 8 12 16 20 (kb)

 Short Telomeres are Associated
with pre-mature aging

• Werner’s Syndrome: 3’ to 5’ DNA

helicase deficiency - telomeres are lost
at a greater rate
Hutchinson-Gilford Progeria
• Hutchinson-Gilford Progeria: a point

Telomere length
mutation in lamin A, a component of the
inner nuclear membrane
– 1:10,000,000 live births (short stature,
high pitched voice, beaked nose,
myocardial fibrosis, arteriosclerosis,
depletion of vascular smooth muscle,
alopecia, thin skin, lack of sexual
maturation, no neurological deficits)
– Die from stroke and heart disease Birth Middle Age Old Age
– Some tissues have short telomeres
 How short telomeres Induce (M1)
Senescence

Young telomere Silent

(TTAGGG)n
heterochromatin
Cell Division and
Progressive Telomere Mid-life omere Leaky
Shortening

DNA Damage Signal Old re Active

A DNA damage signal from a Telomere position effects

“too short” telomere
hTERT Expression is Sufficient to
Immortalize Normal Human Cells

160

140
hTERT +
Population Doublings

120

100

80

60 hTERT -

40
0 50 100 150 200 250 300

Days
hTERT Expression is Sufficient to
Immortalize Normal Human Cells

Cells without telomerase Cells with telomerase

cell divisions

Senescence
No senescence

Telomere repeat
 Telomere Function and Dysfunction

• Functional telomeres protect chromosome termini from

degradation, recombination and end-joining reactions and
are essential for continuous cellular proliferation

• At least two mechanisms for uncapping: progressive

shortening and change in “state” (e.g. loss of chromosomal
end protection, not merely loss of DNA sequence)

• Uncapped telomeres appear as double-stranded breaks that

lead to rearrangements contributing to genomic instability

Telomere loss can result in chromosomal

instability or senescence, and thus has
consequences in carcinogenesis and aging
How do cancer cells arise and remain
immortal?
1. Activation of Telomerase Activity in
Cancer Cells Stabilizes Telomere Length

er
Ca l
a
rm
nc
nk
Telomere
Telomere shortening promotes

No

Bla
length
genomic instability
Abrogation of p53
Telomere length

M1

M2 Telomere stabilization

No telomerase activity Telomerase activity ITAS

senescence crisis TRAP

Cell divisions
 Telomeres and Cancer
Tumors are caused by mutations, which activate oncogenes
and switch off tumor suppressor genes. However, with few
remarkable exceptions, not many human tumor cells would
make it into a full-blown, clinically relevant tumors without
overcoming telomere-dependent replicative senescence. While
there is a multitude of different, often parallel oncogenic
pathways and tumor suppressor mechanisms, there appears to
be just one major and one minor mechanism to maintain
telomeres indefinitely: telomerase and ALT (alternative
lengthening of telomeres). Telomerase, which is stably
switched down in most human somatic cells except germ line
and stem cells, is activated in 85–90% of human tumors and
thus the single most frequent alteration found with
malignancy.
Reactivation of Telomerase Bypasses
Senescence and Crisis Leading to Cell
Immortality
 Telomerase is Not absolutely Required
For Tumorigenesis: Malignant Neuroblastoma IVS
Have Critically Shortened Telomeres and No
Telomerase Activity

I II III IV IVS
NTT NTT NTT NTT NTT
RNase - - + - - + - - + - - + - - +

TRAP
 2. ALT: Alternative Lengthening
of Telomeres
• Some telomerase-negative tumors (10-15% of cancer)
maintain the length of their telomeres independent of
telomerase activity by ALT.

• ALT is believed to be accomplished as follow: a DNA strand

from one telomere anneals with the complementary strand of
another telomere, thereby priming synthesis of new telomeric
DNA using the complementary strand as a copy template
 ALT is Characterized by Lack of Telomerase
Activity, Long and Heterogeneous Telomeres,
Extrachromosomal Telomeric Repeats and ALT
Associated PML Bodies
g.

g.
s.

s.
T

T
AL

AL
Ne

Ne
Po

Po
Telomerase Serves as a Target for
Cancer Therapy

T-loop
D-loop

Subtelomeric (TTAGGG)n
Sequence deLange
Lange/Griffin
Single stranded 3’ overhang de and Griffith

Telomerase inhibitor

Stasis (premature senescence)

Telomere-based senescence Aging
Apoptosis Death
DNA Damage Signal
Increased genomic instability
(engagement of ALT or telomerase revertants)
 Telomerase Inhibitor Approaches

• Catalytic (hTERT) component

– Reverse transcriptase inhibitors
– hTERT promoter/gene therapy
– Dominant-negative or shRNA hTERT gene therapy
– Small molecule inhibitors
• Template (hTERC) functional RNA component
– Hammerhead ribozymes – hTR template
– Oligonucleotides – telomerase template antagonist
 Telomerase Promoter is Active in Cancer
Cells But Not Normal Cells or Tissues

Ad/CMV-LacZ CMV-LacZ Adenovirus GT

Ad/hTERT-LacZ hTERT-LacZ Adenovirus GT

CMV-GFP CMV-LacZ TERT-LacZ CMV-GFP CMV-LacZ hTERT-LacZ

H1299
Spleen
A549

NHBE
Liver

NHLF
Fang et al, 2002
Selective Killing of Telomerase Expressing
Cancer Cells with Replication Competent
Viruses

hTERT-promoter Adenovirus

Normal cell

Viral replication
Telomerase -
blocked
Viral agent
hTERT adenovirus

Cell destruction
Cancer cell Viral release
Virus spread

Telomerase + Viral
replication
 Inhibition of Telomerase With DN-hTERT
(D869A) Leads to Cancer Cell Death
H1299 RCC23 DU145
l

l
tro

tro
l
tro

T
on

on
DN-hTERT
r

ER
ffe

T
on
rc

rc
d

d
d
ER

ER
Bu
ate

ate

ate

-hT
rc
cto

cto
-hT

-hT
c to
tre

tre
tre
sis

DN
Ve

Ve
DN

DN
Un

Un

Un
Ve
Ly

DN-hTERT
Cre excised

LTR DN-hTERT IRES BLAST LTR

lox site lox site
36 bp Internal
Standard
 Telomerase Inhibitor Approaches

• Catalytic (hTERT) component

– Reverse transcriptase inhibitors
– hTERT promoter/gene therapy
– Immunotherapy
– Dominant-negative or shRNA hTERT gene therapy
– Small molecule inhibitors
• Template (hTR) functional RNA component
– Hammerhead ribozymes – hTR template
– Oligonucleotides – telomerase template antagonist
 The Telomerase RNA (hTR) Template
Should be a Good Target for Inhibition
GRN163: 3 ‘NH2-AACAGATTGGGAT-OH 5'
hTERC: 5‘…UUGUCUAACCCUAAC…3'

hTR/(RNA)

hTERT
Catalytic protein
 GRN163 Competitive Telomerase
Enzyme Inhibitor

Thiophosphoramidate backbone chemistry

• Stable to nucleases
• Forms extremely stable heteroduplex with RNA (Tm13mer >
70oC)
A competitive template antagonist (not classical
antisense)
• Mechanism of action: competitive with telomere binding
• Binding is base pair-dependent
• When bound, prevents telomeric substrate binding
Telomerase Activity and Telomere Length in
Cells Treated with GRN163

GRN163 GRN163

12 µ M

nM
12 nM
1. rol

l
tro

tro
kb

n
nt

.5
25

co

co
5
co

19

7.7
6.2

4.3
3.5

2.7

1.9

1.5
ITAS

1 µm 3x/wk over 2 months HME50 cells

 Telomerase Therapy Conclusions

• Potential as a universal oncology target

• High tumor specificity
• Synergy expected with cytotoxic drugs
• Systemic delivery possible
 The End

Thank you for your attention!