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3
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
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Hayflick Phenomenon
• Limited replication potential of somatic cells
• 50-60 population doublings
• Stop cell cycle and enter G0 state– Senescence
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Senescent Phenotype
Gene expression differences
Cyclin D1& D2
p21 & p16c-fos and Egr-1 Cyclins A, B, & H
Protein activity differences
SRF DNA bindingp53 and Rb activity
DNA-PKRas
PKC
Young Pre-senescent Senescent
Phenotypic differences
Large Flattened cellsUnresponsive to growth mitogens
Increase in acid β-galactosidaseIncreased excretion of extracellular matrix
Remain viable and metabolically active
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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
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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”
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8
Eurika!
• Harley et al – 1990– Telomeres shorten during aging of human
fibroblasts
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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
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Eurika!
• Harley et al – 1990– Telomeres shorten during aging of human
fibroblasts
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12
Telomeres
• Telomerase• Ribonucleoprotein
• Specialized reverse-transcriptase
• Binds to 3’ overhang and synthesizes telomere repeat
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Telomeres
• Numerous proteins bind to telomere repeats– Eg. Telomere repeat binding factor-1 and 2
(TRF1/2)
Blackburn, Cell, 2001
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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
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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
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Telomeres
Molecular Biology of the Cell, 4th Edition, Garland Science Inc.
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Telomerase knockout mice
Telomeres shorten progressively intelomerase-null mice
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Telomeres
• Loss of telomerase activity in mice leads to premature aging
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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
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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.
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Telomeres and Human Pathology
• Werner syndrome
• Premature senescence and damage to various tissues
• Fibroblasts from Werner patients only divide about 20 times
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Werner Syndrome
• Causative agent is mutation in WRN gene which encodes a RecQ helicase
• Mutations in WRN gene cause Werner syndrome in humans
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Werner Syndrome
• Where does RecQ do most of its unwinding?
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Werner Syndrome
• Forced expression of telomerase counter-acts the loss of WRN gene
• Maintenance of telomeres in humans is critical for providing genomic stability and replication potential
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Hutchinson Gilford Progeria Syndrome
• rare progressive autosomal dominant disorder .
• The most striking feature of the disorder is extremely accelerated aging (progeria).
• In most cases, affected infants appear to develop normally until approximately six months of age.
•In most patients, Hutchinson-Gilford Progeria Syndrome is caused by de novo sporadic mutation in lamin A.
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The Zmpste24 -/- Mouse
6 Months4 Months
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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
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Analysis of nuclear shape in wild-type, Zmpste24–/–, and Zmpste24–/–Lmna+/– MEFs by laser-scanning fluorescence microscopy.
Loren G. Fong et al. PNAS 2004;101:18111-18116
©2004 by National Academy of Sciences
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Growth rates and grip strength in mice.
Fong L G et al. PNAS 2004;101:18111-18116
©2004 by National Academy of Sciences
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Crossing the zmpste24 -/- with p53 -/- leads to partial rescue of the progeria phenotype.
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