2 1. 2. 1) 2) 3) 3. 4. 1) 2) 3) Microcell 4) 5) 5. 6. 1.. (organism).,...?,??.,. 1935 McCay 27
. 1960 Hayflick. 10 (cytogerontology)... 2. 1),,,,.,,,.. (lipofuscin) (fluidity),., (microvilli)..,, (heat shock). 2). DNA DNA.. 28
.. DNA DNA. (monooxygenase).. NADPH- P-450 P-450. P-450. 3) in vitro. -, -.. (inclusion bodies). DNA. DNA 9.0 10 4. DNA,. 4), (heat-lability), KM,,,, SH,,. 3. (carbonyl). 29
3. 1881 August Weismann,. 20. 1912 Alexis Carrel.. Carrel 1960 Hayflick. 1961 Hayflick Moorhead (human diploid fibroblast; HDF) 40 60 (population doubling, 2 ).,. HDF (epidermal keratinocytes), (smooth muscle cells), (lens epithelial cells), (glial cells), (endothelial cells), T (T lymphocytes), (adrenocortical cells), - (pancreatic -cells).,.. (generation) in vivo. Hayflick, (cytosome),. 30
. HDF (species) (lifespan)......, ph 6.0 SA- -galactosidase (SA- -gal). (mitogen) DNA (, prb ), CDKI (p21, p16 ).., DNA, G1 G1 G1 S (Fig. 1). G0 G0. (growth factor) DNA. mrna. SV40 G1 (G1 phase block). DNA. Dimri ph 6.0 SA- -gal in vivo.. 31
Fig. 1. PRb and cellular senescence. The block of DNA synthesis in senescent cells correlateswith the failure to phosphorylate the prb cell cycle control protein. The kinases and or phosphatases controlling this phosphorylation are probably important in cellular senescence. 4.. (species) (maximum lifespan potential),.., -, - (senescence-associated genes). 1) (cell fusion). 32
(hybrid cell)...,.. 2),,. Pereira-Smith 4 (complementation group A, B, C D).,,. 40 4,.,,,. SV40 (transformation) 7 6 (A ). 7 T B (D ). 4.. 33
3) Microcell. microcell (microcell-mediated chromosome transfer). colcemid cytochalasin B microcell.. Sugawara (1990) Syrian hamster 10W-2 HDF MRC-5 27 15 12 1. 1 (long arm, 1q) 10W-2, 1q C. B 4, D 7. A 6q,. 11. 2, 3, 6q, 10, 11, 16, 17, 18, X.. 4) (tumor suppressor gene) prb p53. prb 13 (13q14), (p105-rb) DNA. p53 34
17 (short arm) DNA.. prb G1 (phase) (transcription factor) E2F DNA G1, S G2 M (Fig. 1). (cyclin) - (cyclin-dependent kinase;cdk) prb G1 (checkpoint) S. p53 p21 CDK prb G1. SV40 (oncoprotein) T (large T antigen). T prb p53. prb. prb G1/G0 (phase), S G2 M. - (CDC2 CDK2) prb G1 (checkpoint) S. G0 prb prb. prb prb. prb G0. prb. prb CDC2 kinase hamster., DNA?.. Shay SV40 T (large T antigen) HPV-E6/E7 (transfection) p53 35
. p53. HDF p53 p21 G1. P53 CDK p16, p21 p24 (overexpression). (premature aging syndrome) (Werner syndrome) DNA (DNA helicase).. 5) DNA- (telomere). DNA (double strand break),. (5'-TTTAGGG-3')n. 15-20 kb DNA 50-200 bp DNA (the end-replication problem)., (germ cells) (telomerase).. Harley (1991) (mitotic clock) (Telomere hypothesis of cell aging). G1 phase (G1 arrest). DNA p53. mortality phase 1(M1 the Hayflick limit).. mortality phase 2 (M2 crisis). intrgrity 36
. M2. Vaziri Benchimol (1996) /DNA (Telomere loss/dna-damage hypothesis of cell aging) (Fig. 2). (dicentric chromosome) (ring chromosome) DNA. DNA p53 p21 CDK DNA. CDK prb G1 Fig. 2. Schematic drawing of the telomere loss/ DNA damage hypothesis of cell aging. Double (ds) and single strand (ss) DNA break signal can go through either p53 or some other protein independent of p53 (px) and induce p21. 37
. p53 p21 prb CDC2... (telomere binding protein). (ribonucleoprotein). (catalytic subunit, htert) (template) RNA (htr). htert (, BJ ),.,..,.., (Alternative Lengthening of Telomeres, ALT).. ALT (recombination) (copy switching). 5. 38
Table 1. Senescent features observed in H2O2-treated early-passage HDFs. G1 arrest Incapable of replicating when G1 arrest is abolished Cell enlargement Reduced saturation density Unable to activate ornithine decarboxylase Unable to activate thymidine kinase Activation of SA -galactosidase Elevated p21 and p16 proteins Unable to phosphorylate Rb Elevated mrna of collagenase-1 apolipoprotein J fibronectin 1( )-procollagen osteonectin SM-22 SS-9. 1956 Harman (free radical theory of aging)..,,.,, ATP,, AP-1 NF-kB, p53.,. (H2O2). HDF (< 200 M) 2, DNA. 4-7 39
. (ornithine decarboxylase) (thymidine kinase) SA- -gal. HDF 1. in vitro (hyperoxia),, (ceramide) buthionine sulfoximine. 6.., (Human Genome Project)..,. Bree RT, Stenson-Cox C, Grealy M, Byrnes L, Gorman AM and Samali A. Cellular longevity: Role of apoptosis and replicative senescence. Biogerontology, 2002, 3, 195-206. Chen QM. Replicative senescence and oxidant-induced premature senescence: Beyond the control of the cell cycle checkpoint. Ann New York Acad Sci, 2000, 908, 111-125. Harman, D. A theory based on free radical and radiation chemistry. J Gerontol, 1956, 11, 298-300. Hayflick, L. Antecedents of cell aging research. Exp Gerontol, 1989, 24, 355-365. Morris M, Hepburn P and Wynford-Thomas D. Sequential extension of proliferative lifespan in human fibroblasts induced by over-expression of CDK4 or 6 and loss of p53 function. Oncogene, 2002, 21, 4277-4288. 40
Smith, JR and Pereira-Smith, OM. Replicative senescence: Implications for in vivo aging and tumor suppression. Science, 1996, 273, 63-67. Timiras, PS. Advances in cell aging and gerontology. Vol. 1. Greenwich JAI Press, 1996, 1-29. Tominaga, K, Olgun A, Smith, JR and Pereira-Smith, OM. Genetics of cellular senescence. Mech Ageing Dev, 2002, 123, 927-936. Uhrbom, L, Nister, M, Westermark, B. Induction of senescence in human malignant glioma cells by p16ink4a. Oncogene, 1997, 15, 505-514. Vaziri, H, Benchimol, S. From telomerase loss to p53 induction and activation of a DNA-damage pathway at senescence: The telomere loss/ DNA damage model of cell aging. Exp Gerontol, 1996, 31, 295-301. 41