71
Roles of telomeres and telomerase in human health and disease Elizabeth Blackburn, Ph. D. UCSF
Roles of telomeres and telomerase in human health
and disease
Elizabeth Blackburn, Ph. D. UCSF
Blackburn lab
Carol Anderson Josh Cheong Beth Cimini Francesca Gazzaniga Kyle Lapham Shang Li
Jue Lin
Imke Listerman
Jason Lukas
Sveta Makovets Jeff Seidel Sherry Wang Dan Levy Shivani Nautiyal
Current
Recent
Jason Lukas Tet Matsuguchi Dana Smith Brad Stohr Tanya Williams Lifeng Xu
Telomeres cap ends of chromosomes
3’
Telomere Structure
G-rich sequence
5’ 3’ 5’
Telomeres form a protective “cap” that prevents improper repair
Protective proteins
Mammalian: de Lange T. G&D, 2005
3’
Telomere Shortening
G-rich sequence
5’ 3’ 5’
cell
divi
sion
s
After a delay, senescence
Predicted, if DNA replication alone acts on DNA: Loss of DNA from the chromosome end (the DNA ‘end-replication problem’)
Watson, 1972, Olovnikov, 1971
WHAT DOES
TELOMERASE DO?
GGGGTTGGGGTTGGGGTTGGGGTTG CCCCAACCC
The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the
The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and
5' 3' AACCCCAAC
5'
3'
The solution to telomere attrition Telomerase: a telomere-synthesizing reverse
transcriptase
Chromosome Terminus
GGGTTG
Greider and Blackburn, 1985, 1987, 1989
TERT protein
TER RNA
TERT apo-protein TERT with partial RNA-DNA modeled in (from HIV RT co-crystal)
2008
Telomerase RNA base sequence also affects
telomerase enzymatic action
WHAT DOES
TELOMERASE DO
FOR CELLS?
cell
divi
sions
Cells keep dividing
Plenty of telomerase:
homeostasis balanced
cell d
ivisio
ns
Telomeres provide a reservoir ofreplenishable DNA
No Senescence
Telomeres replenished by telomerase
Tetrahymena thermophila Plenty of telomerase
Cells are immortal
Yu et al, Nature 1990
Inactivate telomerase
Telomeres progressively shorten
Tetrahymena ceased divisions They became “mortal”
cell d
ivisio
ns
Telomeres provide a reservoir ofreplenishable DNA
No Senescence
Telomeres replenished by telomerase
Cells are immortal
WHAT ELSE DOES
TELOMERASE DO?
Remove active telomerase: Even well before senescence, catastrophic shortening of
occasional telomeres
Chan and Blackburn, 2003
- even when bulk telomeres still
LONG
uncapping fusions freq. 1 in 103
(Wild type freq. <1 in 105 )
An experiment in yeast
Telomerase protects telomeres from fusing to a DSB
-50
0
50
100
150
200
Real time quantitative PCR assay for fused telomeres
Fusi
ons
per
gen
ome
x 10
5
no telomerase
no telomerase
Chan and Blackburn, 2003
Telomere-DSB fusion occurs in telomerase(-) cells, even with long telomeres.
WT! tel1! tlc1! tel1!tlc1!
0
10
20
30
40
50
Long telos LONGLong telos IIWT parent
Parent diploid expressed CDC13-EST1
Fusi
ons
per H
O c
ut x
100
00
no telomerase
Y’ t
elom
eres
*
“Parent”
Chan and Blackburn, 2003
Telomere-DSB fusion occurs in telomerase(-) cells, even with long telomeres.
WT! tel1! tlc1! tel1!tlc1!
0
10
20
30
40
50
Long telos LONGLong telos IIWT parent
Parent diploid expressed CDC13-EST1
Fusi
ons
per H
O c
ut x
100
00
no telomerase
Y’ t
elom
eres
*
“Parent”
Chan and Blackburn, 2003
Telomere-DSB fusion occurs in telomerase(-) cells, even with long telomeres.
WT! tel1! tlc1! tel1!tlc1!
0
10
20
30
40
50
Long telos LONGLong telos IIWT parent
Parent diploid expressed CDC13-EST1
Fusi
ons
per H
O c
ut x
100
00
no telomerase
*
Y’ t
elom
eres
Evans and Lundblad, 1999 *
“Bulked up” “Parent”
Chan and Blackburn, 2003
Telomere-DSB fusion occurs in telomerase(-) cells, even with long telomeres.
Y’ t
elom
eres
WT! tel1! tlc1! tel1!tlc1!
0
10
20
30
40
50
Long telos LONGLong telos IIWT parent
Parent diploid expressed CDC13-EST1
Fusi
ons
per H
O c
ut x
100
00
no telomerase
* Evans and Lundblad, 1999 *
“Bulked up” “Parent”
Chan and Blackburn, 2003
Telomere-DSB fusion occurs in telomerase(-) cells, even with long telomeres.
Y’ t
elom
eres
WT! tel1! tlc1! tel1!tlc1!
0
10
20
30
40
50
Long telos LONGLong telos IIWT parent
Parent diploid expressed CDC13-EST1
Fusi
ons
per H
O c
ut x
100
00
no telomerase
* Evans and Lundblad, 1999 *
“Bulked up” “Parent”
Chan and Blackburn, 2003
Telomerase protects even lengthened telomeres
from catastrophic shortening and fusing to a
double-stranded break
A Protective Function for Telomerase
Chan and Blackburn, 2003
GFP-hTERT (green) and telomeres (TRF2 =red) in a representative LOX melanoma cell nucleus
- deconvolution images projection
Telomerase appears to be widely distributed in the nucleus, not just at telomeres
The cancer cell nucleus
nucleus
nucleolus
Active telomerase RNP
Inactive telomerase protein complex
chromosomes
PinX1
Tert
Telomerase protein Tert sequestrates with nucleolar PinX1 protein in a distinct complex lacking telomerase RNA
J. Lin and E. H.Blackburn, Genes and Dev. 2004
TER
Collaborating lab (UCSF) Mo Kashani-Sabet
Shang Li
Unexpected effects of telomerase RNA depletion in cancer cells
cell
divi
sion
s Telomeres replenished by telomerase
Cells keep dividing IN HUMANS - Active: stem cells, germ cells - Detectable: many normal adult cell types (regulated activity) - Highly active: ~90% of human tumors
cell
divi
sion
s
Cells keep dividing
Plenty of telomerase:homeostasis balanced
GTTAGGGTTAGGGTTAGGGTTAG CAATCCC
The image cannot be displayed. Your
The image cannot be displayed. Your
5' 3'
Chromosome Terminus
CAAUCCCAAUC
5'
3'
Human Telomerase
Telomerase: a telomere-synthesizing ribonucleoprotein reverse transcriptase
TERT protein
TER RNA
150-200 bp/cell division
cell
divi
sion
s
- only gradual shortening of all telomeres - DELAY before eventual senescence or apoptosis
OLD - Predicted, if inhibit or knock down telomerase:
150-200 bp/cell division
cell
divi
sion
s
- only gradual shortening of all telomeres - DELAY before eventual senescence or apoptosis
OLD - Observed, if inhibit or knock down telomerase enzyme, but keep RNP level high:
Con
trol
GFP
siRN
A
GAPDH
hTER
Ratio: 100% 96.8% 11.9%
MCF-7 breast cancer cells
Li et al. 2004, 2005
Control GFP siRNA+GFP
0 1 2 3 4 5 6 7
100 200
300 400 500 600 700 800 900
(Cel
l num
ber x
104 )
Lentiviral-expressed anti-hTER siRNA (RNAi) RAPIDLY inhibits growth of LOX melanoma cells
0
Vector siRNA siRNA-mut
1 2 3 4 5 6 7 Days Days
(bulk unselected cell populations) Li et al 2005
Vector
siRNA
siRNA-mut
(kb)121110987
LOX melanoma cells
YET - Bulk telomere shortening had NOT yet been induced by the telomerase RNA knock down
Teloblot
Li et al 2005
Integrin alpha V > Cyclin G2 >
Met >
Cdc27 >
WT-
hTE
R
MT-
hTE
R-A
U5
siR
NA
MT-
hTer
-AU
5/si
RN
A
Depleting telomerase RNA rapidly down-regulates a
specific set of genes - includes cell cycle and tumor
progression genes
HCT116 cells
Li et al 2005
Knock-down of telomerase (by ribozyme or RNA-interference) inhibits metastasis in in vivo
mouse melanoma models B16 cells LOX cells
Kashani-Sabet et al, 2004; Nosrati et al 2005; Baghari et al 2006; Li et al, unpublished
Effects of telomerase knock-down on metastasis?
YES
Li et al, Cancer Res 2004; Li et al, 2005; Bagheri et al 2006
Telomerase knock-down in cancer cells�
RAPIDLY inhibits cancer cell growth p53 is not required for this
NO telomere uncapping or DNA damage response
Metastasis is reduced
Li et al, Cancer Res 2004; Li et al, 2005; Bagheri et al 2006
HOW?
RAPIDLY downregulates cell cycle and tumor progression genes
Glucose metabolism downregulated
Cell differentiation program induced?
Telomerase knock-down in cancer cells�Metastasis is reduced
Does high telomerase promote stem cell-ness?
High telomerase levels (conditionally induced TERT subunit over-expression) in mice promoted proliferation specifically in stem cells, even in mice deleted for the telomerase RNA component gene
S. Artandi and coworkers: Sarin et al. Nature 436, 1048-1052 (2005)
A NEW CONCEPT FOR TELOMERASE:
Humble bricklayer …..and also upper level manager of
cell’s programs?
Indra Nooyi: PepsiCo's chief executive
How do we age?
A multi-faceted process?
- increased susceptibility to diseases - how much is
-genetic? - environment/life factors?
Genetics contributes a lot
Environment/ stochastic/life
factors • Residual zero expected if inheritance is unimportant for determining life span
Residual Increasing with parental life span expected if
life span is inherited
Residual: the difference between each daughter’s life span and the cohort mean life span �
Gavrilova NS and Gavrilov LA. (2001) Journal Anti-Aging Med.
Res
idua
l
Atzmon et al Journal of the American Geriatrics Society, 2004. Vol. 52, 274
• Elderly subjects demonstrating exceptional longevity have generally been spared major age-related diseases, such as cardiovascular disease (CVD), diabetes mellitus and cancer, which are diseases that are responsible for most deaths in the elderly.
cardiovascular disease, diabetes and cancer
- how much is -genetic? - environment/life factors?
In humans
In vivo
cell
divi
sion
s Telomeres replenished by telomerase
Cells keep dividing
- Active: stem cells, germ cells - Detectable: many normal adult cell types (highly regulated) - Highly active: ~90% of human tumors
cell
divi
sion
s
Cells keep dividing
Plenty of telomerase:homeostasis balanced
cell
divi
sion
s
Cells keep dividing
Extra telomerase:telomeres will even grow
Slower net loss/cell division
MO
RE
cell
div
isio
ns
senescence comes later
Predicted, if some telomerase: Slower loss of DNA from the chromosome end
Faster net loss/cell division
cell
divi
sion
s
Senescence sooner
Predicted, if less telomerase: Faster loss of DNA from the chromosome end
Enter the twilight zone faster…
- how much is -genetic? - environment/life factors?
A rare inherited condition in humans: Premature death from progressive bone marrow failure * when one gene copy of telomerase RNA is deficient
*death in early adulthood to middle age
- shorter telomeres - immune system becomes exhausted - cancer-prone
The other gene copy is wild type * Affected family members
X
One mutated hTER gene copy
Vulliamy et al, Nature, 2001
A full human lifespan requires both telomerase RNA alleles to be functional: Telomerase RNA gene product quantity
matters
Reported: people aged 60 years or older with shorter blood cell telomeres have higher mortality rates
Shorter telomeres associate with: • 3.2 fold higher mortality rate from heart disease • 8.5 fold higher mortality rate from infectious disease • poorer survival overall from aggregate of all causes
Effects in vivo of common variations in telomere maintenance in humans?
Cawthon et al, Lancet 361: 393-395, 2003
What came first?
Chronic life stress, acceleration of cellular aging, and risks of cardiovascular disease
Blackburn Lab Group
• Jue Lin
• Elissa Epel, UCSF • Richard Cawthon, U. Utah • Nancy Adler, UCSF • Firdaus Dhabhar, Ohio State • Jason Morrow, Vanderbilt University • Frank H. Wilhelm, University of Basel, Basel, Switzerland • Owen Wolkowitz, UCSF • Christyn Dolbier, East Carolina University, Greenville, NC • Wendy Mendes, Harvard University
Collaborators
Chronic stress wears down telomeres
First Questions
Are
• level of perceived stress (both groups) • duration of caregiving (range = 1 - 12 years)
related to markers of cell aging?
Two of the Markers of Cellular Aging • Telomerase activity • Telomere length
Epel et al, 2004, PNAS
Study 1: Caregiver mothers and chronic stress
.33 * • n = 30
.27* • n=54
Oxidative stress index
-.35 * • n = 37
-.24* n=59
Telomerase activity
-.40 ** n = 36
-.31** n = 54
Telomere length
Years of caregiving
Perceived stress
Perceived stress (whole sample) was associated with • shorter telomeres • lower telomerase activity Also years of caregiving (in caregiver group)
**: p< 0.01, *:P < 0.05
Study 1: Caregiver mothers and chronic stress
Epel et al, 2004, PNAS
These relationships also hold after accounting for chronological age
0 0.02 0.04 0.06 0.08 0.1
0.12
High stress Low stress
Telomerase activity is ~ 50% lower in the high stress group
PBM
C te
lom
eras
e ac
tivity
/ 10
,000
cel
ls
M = .053, SE = .016 M = .092, SE = .016 p < .045
Telomerase activity in the lowest and highest stress quartiles of the whole sample are compared.
Epel et al, 2004, PNAS
Study 1: Caregiver mothers and chronic stress
More telomere shortening in high stress group (equiv. 9 - 17 yrs of extra “aging”)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
High stress Low stress
Aver
age
t/s ra
tio
controlling for age and body mass index: F(3,27) = 12.8, p < .001
Telomeres in the lowest and highest stress quartiles of the whole sample are compared.
Study 1: Caregiver mothers and chronic stress
P
BM
C T
elom
ere
leng
th
High stress Low stress
• Stress perception & caregiving duration are quantifiably linked to cell aging markers – Telomerase – Telomere length
• Causal directions? • Possible mechanisms?
Chronic stress - reduces telomere length maintenance
Chronic Stress
Telomerase
Telomere length
maintenance
Reduces ability of cell to replenish itself
A new connection
Signal input
Signal Integration and processing
Disease impact?
In the largest epidemiological study of risk factors for cardiovascular disease, six prominent factors were shown to be:
smoking poor lipid profile high blood pressure diabetes abdominal obesity psychological stress (Yusef et al, Lancet 2004:304).
• smoking • cholesterol/blood lipids • resting cardiovascular activity • fasting insulin and glucose • adiposity • psychological stress
Chronic stress was associated with markers of cellular aging • Lower telomerase activity • Shorter telomere length
Telomere maintenance and risk of cardiovascular disease
*Epel et al, 2004
*
A link in vivo
Links to telomere maintenance in many cohorts …
… many independent studies (mainly with mean telomere length data so far)
Low telomere length linkage to very common disease states
Cancer Vulliamy, T. et al. (2001) Joshua et al., Shen et al (2007)
Cardiovascular disease Brouilette, S. et al. (2003) (plaques, heart attacks, Benetos, A. et al. (2004) calcificoric aortic valve stenosis) Kurz, D. J. et al. (2006)
Starr et al (2007) Brouilette et al (2007)
Vascular dementia von Zglinicki, T. et al. (2000) Degenerative conditions (osteoarthritis, osteoporosis) Zhai, G., et al. (2006)
Valdes, A. M. et al. (2007) Diabetes Valdes, A. M. et al. (2005)
Aviv, A. et al. (2006) General risk factors for chronic disease Gardner, J. P. et al. (2005)
- obesity and insulin resistance Pulmonary fibrosis Armanios, M. et al. (2007)
Telomerase - an upstream determinant of telomere length maintenance
Telomerase
Telomere length
maintenance
Reduces ability of cell to replenish itself
Known genetic defects in telomerase genes cause disease risk in mice and humans
Telomerase
Telomere length
maintenance
Reduces ability of cells to replenish
Disease impact
Telomerase - an upstream determinant of telomere length maintenance
Chronic Stress
Signal input
Disease impact
…VIA LOWER telomerase?
Chronic stress - an upstream determinant of disease risk
Telomerase
Telomere length
maintenance
Decision of cell to replenish itself
Chronic stress - an upstream determinant of disease risk
Chronic Stress
Signal input
Disease impact
?
…VIA LOWER telomerase?
SUMMARY
1. - Forms of chronic psychological stress are associated with low telomerase and shortening of telomeres.
2. - It is emerging that low telomerase and shortening of telomeres in human cells in vivo are associated with (and contribute to?) disease susceptibility and shorter life.
In the largest epidemiological study of risk factors for cardiovascular disease, six prominent factors were shown to be:
smoking poor lipid profile high blood pressure diabetes abdominal obesity psychological stress (Yusef et al, Lancet 2004:304).
• smoking • cholesterol/blood lipids • resting cardiovascular activity • fasting glucose • adiposity • psychological stress
Stress was associated with markers of cellular aging • Lower telomerase activity • Telomere length
Telomere maintenance and risk of cardiovascular disease
*Epel et al, 2006
A link in vivo
