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

Hayflick Phenomenon Limited replication potential of somatic cells 50-60 population doublings Stop cell cycle and enter G0 state Senescence

Senescent Phenotype Young Pre-senescent Senescent Phenotypic differences Large Flattened cells Unresponsive to growth mitogens Increase in acid β-galactosidase Increased excretion of extracellular matrix Remain viable and metabolically active Gene expression differences Cyclin D1& D2 p21 & p16 c-fos and Egr-1 Cyclins A, B, & H Protein activity differences SRF DNA binding p53 and Rb activity DNA-PK Ras PKC 

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

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”

Eurika! Harley et al – 1990 Telomeres shorten during aging of human fibroblasts

Telomeres Telomeres Several functions 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

Eurika! Harley et al – 1990 Telomeres shorten during aging of human fibroblasts

Telomeres Telomerase Specialized reverse-transcriptase Ribonucleoprotein Specialized reverse-transcriptase Binds to 3’ overhang and synthesizes telomere repeat

Telomeres Molecular Biology of the Cell, 4th Edition, Garland Science Inc.

Telomeres Numerous proteins bind to telomere repeats Eg. Telomere repeat binding factor-1 and 2 (TRF1/2) Blackburn, Cell, 2001

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

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

Telomeres Molecular Biology of the Cell, 4th Edition, Garland Science Inc.

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

Telomeres Loss of telomerase activity in mice leads to premature aging

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.

Telomeres and Human Pathology Werner syndrome Premature senescence and damage to various tissues Fibroblasts from Werner patients only divide about 20 times

Werner Syndrome Causative agent is mutation in WRN gene which encodes a RecQ helicase Mutations in WRN gene cause Werner syndrome in humans

Werner Syndrome Where does RecQ do most of its unwinding?

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

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. 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. Analyses from two independent experiments, with cells prepared from two different sets of embryos, are shown. Protein loading was assessed with an antibody against β-actin. Densitometry showed a 58.4 ± 4.1% reduction in prelamin A and a 78 ± 4.9% decrease in lamin C in the Zmpste24–/–Lmna+/– cells (n = 4) relative to the Zmpste24–/– cells. Fong L G et al. PNAS 2004;101:18111-18116 ©2004 by National Academy of Sciences

Growth rates and grip strength in mice. Growth rates and grip strength in mice. (a) Female Zmpste24–/– Lmna+/– mice (n = 6) exhibit normal growth, indistinguishable from wild-type mice (n = 5). Similar results were obtained with male mice (data not shown). (b) Muscle strength in 3- to 4-week old and 16- to 17-week-old female wild-type (Lmna+/–, n = 7), Zmpste24–/– (n = 5), and Zmpste24–/–Lmna+/– mice (n = 9), as judged by the length of time that they were able to hang on to an upside-down grid (1) (grip time; P < 0.001). Similar results were observed for male mice. Differences became statistically significant at 8 weeks of age. Fong L G et al. PNAS 2004;101:18111-18116 ©2004 by National Academy of Sciences