UBC Theses and Dissertations
Aging of mammalian cells in vitro Atchison , Brad
The purpose of this study was to examine the phenomenon of aging at the cellular level in vitro. Some of the biological mechanisms underlying the aging process, which may invoke a change at the level of the DNA were studied. Morphological changes were analysed with phase-contrast microscopy and functional changes by the use of tritiated thymidine in combination with autoradiography as well as by cell treatment with colchicine. A method is described for obtaining "aged" or "old" cells in vitro. In the human cells (human embryonic kidney) as well as the rat, mouse, and Syrian hamster cells, morphological changes in vitro are basically the same as aging progresses. These include transformation to a polygonal, epithelial -1ike shape; binucleation; an accumulation of "age pigments" around the nucleus; the appearance of ragged edges of the cell membrane; an increase in the overall cell size; and a loss of a regular (often parallel) orientation to adjacent cells. The mitotic rate and DNA-synthesizing capacity in "young" and "aged" cells were examined using autoradiography and cell treatment with colchicine. Evidence is presented that DNA-synthesizing aged cells are non-proliferating while DNA-synthesizing young cells are mitotically active. The significance of DNA-synthesis in non-dividing "aged" cells is discussed. The number of population doublings (generation times or cell divisions) that it takes hamster and mouse cells to age in vitro was also investigated. Thirteen and six cell generation times were found to cause hamster and mouse cells to age with a loss of proliferative capacity. The effect of various molarities of 4-nitroquinoline-1 oxide (4-NQO), on DNA of aged cells, which results in an unscheduled DNA-repair synthesis, was studied using autoradiography. It appears that an aged cell responds to these concentrations in much the same way as young cells; however, there does seem to be a slightly greater sensitivity to toxic doses of 4-NQO in aged cells. Autoradiographic studies also revealed that the duration of DNA-repair is the same in both aged and young cells, but the former appear to have a decreased capacity to repair damage to the DNA of the pretreated cells caused by the 4-NQO. The significance of this apparent decrease in the DNA-repair capacity of "aged" cells is discussed. Mouse cells, aged in vitro, were exposed to human adenovirus type 12 (strain Huie). Evidence is presented that this external agent stimulated these aged cells to increase DNA-synthesis and also pushed them into mitosis (for at least one cell division). The possibility that this might be an accountable mechanism for the observation of an accumulation of mutations in aged cells is evaluated. Aged cells were examined for frequencies and types of chromosome aberrations after exposure to adenovirus type 12. Among the most common are chromatid breaks and double fragments. As well, old cells exhibited a much higher frequency of chromosome aberrations than young cells after viral exposure. A comparison of this in vitro system of cell aging with an in vivo system is presented. The application of all of the aforementioned results and observations concerning the cellular aging process to the problem of carcinogenesis and neoplasia is emphasized.