Download Meir Lahav - Telomeres in Cacer

Overview of telomeres &
telomerase biology: Clinical
implications in cancer and aging
Meir Lahav MD
Laboratory for telomere research, Rabin
Medical Center, Beillinson Campus
Felsenstein Medical Research Center
8 March 2010
Historical perspective
•
1908, McClintock & Muller
“Chromosome bore a special
component at their ends that
provided stability”
• Telomere: telos- end, merospart
•
1961, Hayflick & Moorehead
“Normal somatic cells have a
limited life span- a status
that is terminated in M1
stage- replicative
senescence”.
Leonard Hayflick
Biological landmarks
•
1971, Olovnikov:
“Marginotomy”- the end-replication
problem may account for the Hayflick
limit
•
1972, Watson:
DNA polymerase could not replicate
chromosomes to the tip
The end-replication problem
5’
3’
3’
5’
DNA Replication
5’
3’
R
R
R
R
R
5’
3’
RNA primer removal
Fill-in DNA replication
Ligation
5’
3’
3’
5’
Each division 50-100 bp loss
Biological landmarks (cont.)
•
1978, Blackburn
discovered telomeres in
Tetrahymena
(TTGGGG)n
•
1984, Blackburn & Greider
telomerase activity
was detected in
Tetrahymena
Telomeric end of DNA
(TTAGGG)n
(AATCCC)n
Unique
Proxim al
subtelomeric
Genomic DNA
Degenerate
(TTAGGG) n
Distal
subtelomeric
(TTAGGG) n
repeats
Telomere
Molecular structure of the telomere
Functions of telomere [(TTAGGG)n]
•
Protects the chromosomal ends from:
•
•
Enables a complete replication of the DNA
Contributes to the functional organization of
chromosomes in the nucleus
Participates in regulation of gene expression
Serves as “mitotic clock”: shortens with each
cell division
•
•
– Recombination
– End-to-end fusion
– Recognition as damaged DNA
Telomere length in healthy population
Uziel et al. 2002
8000
Telomere length (bp)
7000
6000
5000
4000
3000
2000
y = -27.45x + 6972.5
1000
R 2 = 0.4636
0
0
20
40
Age (years)
60
80
100
Consequences of telomere shortening &
damage
Two-step hypothesis of cellular
senescence and immortalization
Wright & Shay Microbiol Mol Biol Rev 2002
Telomerase
5’ TTAGGGTTAG
CAAUCCCAAUC
telomerase
Telomerase
5’ TTAGGGTTAGGGTTAG
CAAUCCCAAUC
telomerase
hTERT
hTR-CAAUCCCAAUC
5’ TTAGGGTTAGGGTTAG
CAAUCCCAAUC
telomerase
5’ TTAGGGTTAGGGTTAGGGTTAG
CAAUCCCAAUC
telomerase
Elongation of a telomere by telomerase.
Wong L S et al. Cardiovasc Res 2009;81:244-252
Published on behalf of the European Society of Cardiology. All rights reserved. © The Author
2008. For permissions please email: [email protected]
Keeping telomerase in its place
Maser & DePinho Nature Medicine 2002
The telomere model for cellular
transformation
Telomere length
Germ cells: telomerase ON
Somatic cells:
telomerase OFF
Oncogenetically
transformed cells:
bypass senescence,
telomerase OFF
Senescence Crisis
# of cell divisions
Immortal cells:
telomerase ON
TRF measurements
Southern blot
Shapiro, Uziel and Lahav 2000
FISH flow
FISH on paraffin embedded tissues
Clinical applications of telomere research
Cancer
Senescence
Acquired capabilities of cancer
(Hanahan and Weinberg, Cell 100: 57-70, 2000)
Minimal set of genetic alterations
required for conversion of fibroblasts to
cancer cells
Sun et al 2006
•
Malignant conversion:
•
Malignant cells are not
immortal - enter crisis and die
Telomerase expression renders
cell immortal
•
– SV40 large T antigen (p53 and
pRb inactivation)
– Ras activation
Telomerase up-regulation cause or
consequence
•
Human cancer cells have
•
Correlation between anaphase bridges and
telomere length
•
Human colorectal cancers show a peak in
anaphase bridges index in early lesions;
– shorter telomeres then normal
– dysfunctional telomeres (anaphase bridges, ends
fusions etc.,)
Effect of telomerase inhibition on
malignant cells growth
Telomerase inhibition in cancer
Lahav 2010
Chemosensitization by telomeres
Lahav 2009
Comet assay DNA damage
Lahav 2010
DNA damage focci telomere
dysfunction
Lahav 2009
Association of telomerase activity with disease
free survival in non-small cell lung cancer
Gonzalez-Quevedo, R. et al. J Clin Oncol. 2002;20:254-262
Thalidomide downregulates
telomerase promoter gene expression molecular
pharmacology
Druker, Uziel, Lahav et al. 2004 molec pharmacol
[ThD] mg/ml 0
ARH-77
12.5 25
50
100
hTERT
IGFI-R
b actin
CD63
[ThD] mg/ml 0
RPMI 8226
12.5 25
50
[ThD] mg/ml 0
100
12.5 25
50
100
hTERT
hTERT
IGFI-R
IGFI-R
b actin
CD63
U266
CD63
b actin
Gleevec inhibits telomerase activity in
SK-N-MC cells
Uziel and Lahav,2005 BJC
10mM
15mM
R8
Telomerase activity after Gleevec 5 days treatment
Telomerase activity
(%)
0mM
120
100
80
60
40
20
0
10
2
10
315
[Gleevec](uM)
Inhibition range: 70-90%
(% of control)
telomerase activity
Kinetics of telomerase activity during
Gleevec treatment
120
100
80
60
40
20
0
1
2
3
time (days)
4
5
Telomerase cellular localization in
STI571 treated cells
Uziel, Beery et al 2003
Control cells
STI571 treated cells
Telomerase as a drug target
•
Significant difference of telomerase expression
between malignant and normal tissues
•
Possible adverse effects: damage to stem and germ
cells
•
Telomerase inhibitors will be effective only when the
telomeres shorten to critical length
•
Will probably be used as an adjuvant therapy
Potential effects of telomerase inhibition over
time on telomere length and proliferative
capacity
Experts reviews in molecular medicine 2002
Strategies for inhibition of
telomerase activity
•
Telomerase targeting agents:
•
Telomeres targeting agents
•
compounds from random
screening
–
–
The RNA template
Reverse transcriptase
inhibitors
– Modulators of telomerase
regulating proteins
–
Inhibitors that interact
with G4-DNA structures
– Inhibitors against
telomeres associated
proteins
– “Old” DNA -interacting
drugs
Effect of telomerase antisense on
malignant cell culture
Uziel and Lahav, 2004
Antimetastatic effects of GRN163L on
pretreated A549-Luc cells
Dikmen, Z. G. et al. Cancer Res 2005;65:7866-7873
Telomere attrition sensitize SK-N-MC cells to
DNA SS breaks inducing agent, Cisplatinum
proliferation (%)
Uziel and Lahav, 2006
120
100
80
60
40
20
0
Control
+GRN163
0
0.2
0.4
120
120
100
100
proliferation (%)
proliferation (%)
Cisplatinum (ug/ml)
80
60
40
20
0
80
60
40
20
0
0
5
10
Vincristine (ng/ml)
15
0
50
100
Doxorubicin (ng/ml)
150
Telomerase inhibition – future
directions
• New effective inhibitors
• Antitelomerase vaccines
• Antitelomerase adoptive immunotherapy
• Promoter driven therapy
• Development of antitelomerase –
cytotoxic drugs – other biologic
interventions combinations
Telomerase promoter-driven gene
therapy
• hTERT promoter is highly active in
•
•
cancer cells (not active in somatic cells)
Expression of harmful genes under the
control of
hTERT promoter- expression directed
to malignant cells
Genes used
– Proapoptotic genes: caspase 8, caspase 6,
TRAIL, Bax
Adenovirus and telomerase promoter
Telomerase immunotherapy
•
•
•
•
Immunizing patients against tumor antigens to elicit
antibody or cytotoxic T-cells killing of tumor cells
T cells against a short hTERT peptide in vitro and in
mouse models in vivo; Somatic cells are not affected
Prostate or breast cancer patients were vaccinated
with cells expressing tert peptide; 4 responded; No
se.
12 prostate cancer patients were treated as above,
majority responded positively
Aging
Aging
Comparison between a single homologue from one
individual and a single homologue from an unrelated
individual carrying the same genetic marker
Dolly or
failure of resetting the cellular clock
Willmut et al, 1997
Telomere length & survival rate
Trans-differentiation of pluripotent
stem cells
Telomerase effect on cells
Telomere binding defect in progeria
Diabetes control and telomeres
Lahav 2006
Telomere-telomerase and p53 function
De Angelis, A. et al. Circulation 2010;121:276-292
Copyright ©2010 American Heart Association
Histogram showing haemoglobin levels and their association with telomere length.
Wong L S et al. Eur J Heart Fail 2010;12:348-353
Published on behalf of the European Society of Cardiology. All rights reserved. © The Author
2010. For permissions please email: [email protected].
Telomere length in the anaemic vs. non-anaemic group.
Wong L S et al. Eur J Heart Fail 2010;12:348-353
Published on behalf of the European Society of Cardiology. All rights reserved. © The Author
2010. For permissions please email: [email protected].
Absolute and Relative Mean Changes in Telomere Length Over 5 Years by Quartile of Omega-3
Fatty Acid Level, Adjusted for Age and Baseline Telomere Length
Farzaneh-Far, R. et al. JAMA 2010;303:250-257.
Copyright restrictions may apply.
Translational applications ;
• Cancer; Mechanism of malignancy
•
Therapeutic approaches
• Aging;
•
•
Cellular ( stem cells)
Organism ; normal
accelerated aging