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

4 Hayflick Phenomenon Limited replication potential of somatic cells population doublings Stop cell cycle and enter G 0 state – Senescence

5 Senescent Phenotype Gene expression differences Cyclin D 1 & D 2 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  YoungPre-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

6 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 – 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

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

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

14 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

15 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

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

Telomerase knockout mice Telomeres shorten progressively in telomerase-null mice

18 Telomeres Loss of telomerase activity in mice leads to premature aging

19 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

20 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.

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

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

23 Werner Syndrome Where does RecQ do most of its unwinding?

24 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

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.

The Zmpste24 -/- Mouse 6 Months 4 Months

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: ©2004 by National Academy of Sciences

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: ©2004 by National Academy of Sciences

Growth rates and grip strength in mice. Fong L G et al. PNAS 2004;101: ©2004 by National Academy of Sciences

Crossing the zmpste24 -/- with p53 -/- leads to partial rescue of the progeria phenotype.