overview of telomeres & telomerase biology: clinical implications in cancer and aging meir lahav...
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Overview of telomeres & telomerase biology: Clinical implications in cancer and aging
Meir Lahav MDLaboratory for telomere research, Rabin Medical Center, Beillinson CampusFelsenstein 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, meros- part
• 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
3’ 5’R
RNA primer removal
Fill-in DNA replication
Ligation 5’ 3’
3’ 5’
Each division 50-100 bp loss
Biological landmarks (cont.)
• 1978, Blackburndiscovered telomeres in Tetrahymena (TTGGGG)n
• 1984, Blackburn & Greidertelomerase activity was detected in Tetrahymena
Telomeric end of DNA
Genomic DNA Telomere
Molecular structure of the telomere
Functions of telomere [(TTAGGG)n]
• Protects the chromosomal ends from: – Recombination– End-to-end fusion– Recognition as damaged DNA
• 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
Telomere length in healthy population Uziel et al. 2002
y = -27.45x + 6972.5
R2 = 0.4636
0
1000
2000
3000
4000
5000
6000
7000
8000
0 20 40 60 80 100
Age (years)
Tel
om
ere
len
gth
(b
p)
Consequences of telomere shortening & damage
Two-step hypothesis of cellular senescence and immortalization
Wright & Shay Microbiol Mol Biol Rev 2002
Telomerase
telomerase
5’ TTAGGGTTAG CAAUCCCAAUC
telomerase
5’ TTAGGGTTAGGGTTAG CAAUCCCAAUC
telomerase
5’ TTAGGGTTAGGGTTAGGGTTAG CAAUCCCAAUC
telomerase
5’ TTAGGGTTAGGGTTAG CAAUCCCAAUC
Telomerase
hTERT
hTR-CAAUCCCAAUC
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
Germ cells: telomerase ON
Somatic cells:
telomerase OFF
Immortal cells:
telomerase ONOncogeneticallytransformed cells:bypass senescence,
telomerase OFFSenescence Crisis
# of cell divisions
Telo
mere
len
gth
TRF measurementsShapiro, Uziel and Lahav
2000
Southern blot FISH flow
FISH on paraffin embedded tissues
Clinical applications of telomere research
Senescence
Cancer
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: – SV40 large T antigen (p53
and pRb inactivation) – Ras activation
• Malignant cells are not immortal - enter crisis and die
• Telomerase expression renders cell immortal
Telomerase up-regulation cause or consequence
• Human cancer cells have– shorter telomeres then normal– dysfunctional telomeres (anaphase bridges,
ends fusions etc.,)
• Correlation between anaphase bridges and telomere length
• Human colorectal cancers show a peak in anaphase bridges index in early lesions;
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 cancerGonzalez-Quevedo, R. et al. J Clin Oncol. 2002;20:254-262
actin
hTERT
IGFI-R
CD63
actin
[ThD] g/ml 0 12.5 25 50 100
hTERT
IGFI-R
actin
CD63
[ThD] g/ml 0 12.5 25 50 100
hTERT
IGFI-R
CD63
[ThD] g/ml 0 12.5 25 50 100 RPMI 8226 U266
ARH-77
Thalidomide downregulates telomerase promoter gene expression molecular pharmacologyDruker, Uziel, Lahav et al. 2004 molec pharmacol
0M 10M 15M R8
Inhibition range: 70-90% 0
20406080
100120
1 2 3 4 5
time (days)
telo
mer
ase
acti
vity
(% o
f co
ntro
l)
Kinetics of telomerase activity during Gleevec treatment
Telomerase activity after Gleevec 5 days treatment
0
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60
80
100
120
1 2 3
[Gleevec](uM)
Te
lom
era
se
ac
tiv
ity
(%
)
0 10 15
Gleevec inhibits telomerase activity in SK-N-MC cellsUziel and Lahav,2005 BJC
Control cells STI571 treated cells
Telomerase cellular localization in STI571 treated cells
Uziel, Beery et al 2003
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 capacityExperts reviews in molecular medicine 2002
Strategies for inhibition of telomerase activity• Telomerase targeting
agents:– The RNA template– Reverse transcriptase
inhibitors– Modulators of telomerase
regulating proteins
• Telomeres targeting agents– Inhibitors that interact with
G4-DNA structures– Inhibitors against
telomeres associated proteins
– “Old” DNA -interacting drugs
• compounds from random screening
Effect of telomerase antisense on malignant cell cultureUziel and Lahav, 2004
Antimetastatic effects of GRN163L on pretreated A549-Luc cellsDikmen, Z. G. et al. Cancer Res 2005;65:7866-7873
0
20
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60
80
100
120
0 5 10 15
Vincristine (ng/ml)
pro
life
rati
on
(%
)
0
20
40
60
80
100
120
0 50 100 150
Doxorubicin (ng/ml)
pro
life
rati
on
(%
)
Control
+GRN163
Telomere attrition sensitize SK-N-MC cells to DNA SS breaks inducing agent, CisplatinumUziel and Lahav, 2006
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– Prodrugs– Viral lytic genes: adenoviruses
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
Copyright ©2010 American Heart Association
De Angelis, A. et al. Circulation 2010;121:276-292
Telomere-telomerase and p53 function
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].
Copyright restrictions may apply.
Farzaneh-Far, R. et al. JAMA 2010;303:250-257.
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
Translational applications ;
•Cancer; Mechanism of malignancy
• Therapeutic approaches
•Aging; Cellular ( stem cells)
• Organism ; normal
• accelerated aging