Transcript 4150 Lecture 8

Aging
• 1950’s
– Believed that cultivated cells could grow forever
• If not, then it was a result of a culturing deficiency
– In 1943, a cancer cell was grown in culture
indefinitely
– Leonard Hayflick noticed that human fibroblasts
from embryonic tissue could only grow for several
months
3
Hayflick Phenomenon
• Limited replication potential of somatic cells
• 50-60 population doublings
• Stop cell cycle and enter G0 state
– Senescence
4
Senescent Phenotype
Young
Phenotypic differences
Pre-senescent
Senescent
Gene expression differences
Protein activity differences
Cyclin D1& D2
p21 & p16
c-fos and Egr-1
Cyclins A, B, & H
SRF DNA binding
p53 and Rb activity
DNA-PK
Ras
PKC 
Large Flattened cells
Unresponsive to growth mitogens
Increase in acid β-galactosidase
Increased excretion of extracellular matrix
Remain viable and metabolically active
5
Senescence in tumour suppression
Aging
• Late 1950’s
– Cytogenetics could detect Barr Body
• Thus, distinguish male-donated fibroblasts from
female-donated fibroblasts
– Thus, distinguish cells at various cell doubling
stages
7
Aging
• Fibroblasts taken from young donors had a
greater PDL than older doners
• Frozen cells thawed remembered their place
in the PDL
• Must be some “counter”
8
Eurika!
• Harley et al – 1990
– Telomeres shorten during aging of human
fibroblasts
9
Telomeres
• Telomeres
– 3-20 Kb repeat of …TTAGGG…at each end of every
chromosome
• Several functions
– “cap” the end of chromosomes to project against
fusion with other chromosomes
– Replication
– Positioning
10
Eurika!
• Harley et al – 1990
– Telomeres shorten during aging of human
fibroblasts
11
Telomeres
• Telomerase
• Ribonucleoprotein
• Specialized reversetranscriptase
• Binds to 3’ overhang and
synthesizes telomere repeat
13
Telomeres
Molecular Biology of the Cell, 4th Edition, Garland Science Inc.
14
Telomeres
• Numerous proteins bind to telomere
repeats
– Eg. Telomere repeat binding factor-1 and 2
(TRF1/2)
Blackburn, Cell, 2001
15
Telomeres
• Numerous proteins bind to telomere
repeats
– Eg. Telomere repeat binding factor-1 and 2
(TRF1/2)
• Longer repeats – more TRF1/2 binding
• Eventually inhibits telomerase activity
– Thus, telomere length is restricted
16
Telomere erosion correlates with the
onset of cellular senscence.
Allsopp et al.
Proc. Nati. Acad. Sci. USA
Vol. 89, pp. 10114-10118, November 1992
Telomeres
• In somatic cells, telomerase activity is low
• In stem cells, e.g. germ line, telomerase
activity is high – maintain telomere length
• In Cancer cells, telomerase is also high
18
Telomeres
Molecular Biology of the Cell, 4th Edition, Garland Science Inc.
19
Telomerase knockout mice
Telomeres shorten progressively in
telomerase-null mice
What happens when telomeres get too
short?
• Cell detects short telomere ends and become
senescent or undergo apoptosis
• Biological clock for regulating the number of cell
divisions for a cell
• Genes located near telomeres may be regulated by
length – age-regulated gene expression
21
Telomeres
• Loss of telomerase activity in mice leads to
premature aging
22
Dolly the sheep
• Cloned by nuclear transfer from a 6 year
old sheep.
• Telomere length 80% of normal
• Died from Infection/Cancer at age 6 (life
expectancy Age 11-12)
•Chronic Arthritis at age 5
•Cloned sheep generally have shorter
telomeres, but are reset in their progeny.
23
Telomeres and Human Pathology
• Werner syndrome
• Premature senescence and
damage to various tissues
• Fibroblasts from Werner
patients only divide about 20
times
24
Werner Syndrome
• Causative agent is mutation in WRN gene
which encodes a RecQ helicase
• Mutations in WRN gene cause Werner
syndrome in humans
25
Werner Syndrome
• Where does RecQ do
most of its unwinding?
26
Werner Syndrome
• Forced expression of telomerase counteracts the loss of WRN gene
• Maintenance of telomeres in humans is
critical for providing genomic stability and
replication potential
27
Photograph of a Zmpste24−/− mouse, illustrating a characteristic dragging of the hind limb.
Bergo M O et al. PNAS 2002;99:13049-13054
©2002 by National Academy of Sciences
Western blots of extracts from wild-type, Zmpste24–/–, and Zmpste24–/–Lmna+/– MEFs with a
carboxyl (C)-terminal prelamin A antibody and an amino (N)-terminal lamin A/C antibody.
Fong L G et al. PNAS 2004;101:18111-18116
©2004 by National Academy of Sciences
Growth rates and grip strength in mice.
Fong L G et al. PNAS 2004;101:18111-18116
©2004 by National Academy of Sciences