Age-related alterations in human chromosome composition and DNA content in vitro during senescence

Abstract

Different theories of ageing involving somatic mutations, error catastrophe, compensation and repair, and programmed ageing were subjected to analysis for their feasibility from experimental data. Due to the relative difficulty of carrying out longitudinal studies in human subjects in vivo and the long periods involved, most of these experiments dealt with in vitro systems. I. Fibroblast cells in culture were found to be ideal materials for demonstrating senescence for a particular species and at the terminal end the cultures showed certain changes associated with age. II. It appears that the ideas regarding the alterations in DNA content at the terminal stages of the culture or otherwise are related to the tissues concerned and the duration of the cultured condition. III. The importance of DNA content specially related to the concept of stability of the DNA strand supports indirectly the error catastrophe theories. The rate of net DNA synthesis, the size of the replicon and duration of S phase is reported not to change during in vitro ageing. The regulation system of DNA replication may however change, but this alteration does not affect the DNA replication machinery. Following cell fusion studies it has been hypothesized that senescent human diploid fibroblasts contain a diffusible inhibitor which blocks cells at G1 phase. Some immortal cell lines like HeLa and SV40-transformed cells would contain a dominant inducer that could override or inactivate the putative inhibitor, the nature of which is not yet clear. IV. DNA repair competence of fibroblast cultures is reported to decline near the end of in vitro life-span. However, it has been noted that the human skin fibroblasts from both young and old donors are equally proficient in repairing damage by UV light. V. The replication patterns of chromosomes from both senescent embryonic fibroblasts and early-passage adult skin fibroblasts were essentially identical. There were very few differences between the early-passage embryonic and adult skin cells. It was concluded that the terminal replication pattern of fibroblasts changes very little with cellular ageing. VI. A statistically significant increase in sister chromatid exchange frequency has been reported during the terminal part of fibroblast cultures. VII. Electron-microscopic studies have confirmed the increased organization of microfillaments into bundles in senescent cells. The presence of a rigid cytoskeletal structure may contribute in part to the inability of such cells to replicate. VIII. Age-related increase in nuclear proteins was attributed to accumulation of residual acid proteins. Densitometric analysis showed that histone HI was low in late-passage cells and H₄ fraction increased relatively at the terminal phase. IX. In contrast to age-matched controls, fibroblasts cultured from progeria and Werner's syndrome undergo significantly low population doubling. Metaphase plates from these patients demonstrated a much higher frequency of chromosomal abnor malities than normal fibroblasts. Frequency of sister chromatid exchange in cells from Fanconi's anaemia did not show any significant change as compared with control sets. X. Significantly lower Feulgen DNA values have been recorded from lymphocytes of the elderly as compared with younger ones, indicated by hypodiploidy as well as by the individual amounts of euchromatin and heterochromatin. However, later data from flow-cytometric measurements indicated that DNA content was the same for all age groups. XI. The UV-induced unscheduled DNA synthesis in lymphocytes of 80 to 90 year-old individuals was reduced as compared to younger persons. However, the rate of repair of DNA strand breaks is apparently constant in all the age groups. XII. Increase in aneuploidy from lymphocyte cultures of aged individuals has been recorded by many workers. XIII. Significantly lower titres of serine, threonine, histidine, ornithine and lysine have been observed in aged persons, and only the first three decreased equally in both sexes. Some of the amino acids were influenced by sex hormones. XIV. Study of the frequency of spontaneous sister chromatid exchanges showed that neither intra-individual variation between replicate cultures established from the same blood sample nor variation among samples from the same individual initiated at different times was significant. However, sensitivity to induced sister chromatid exchange is reported to increase with age.