The life history of fibroblast and fibroblast-like cells includes an initial stage of outgrowth and establishment in culture; a period of vigorous proliferation which has a variable length, depending on the tissue of origin, age of the donor, etc.; a period of declining proliferative vigor which includes substantial cell death; and finally, the emergence of an (apparently) long-lived population which is unable to proliferate in response to growth factors. During the phase of declining proliferative vigor, the cells acquire characteristics, some of which are similar to the characteristics of cells in older individuals. Eventually the culture completely loses proliferative capacity. A comparable life history has been described for glial cells, keratinocytes, vascular smooth muscle cells, endothelial cells, and lymphocytes which suggests that this life history is characteristic of those cell types that, in vivo, retain the capacity for proliferation throughout the life span. Numerous studies have shown a correlation between the age of the tissue donor and the replicative life span of the cells in culture. In addition, for a small sample of species, there is a direct correlation between fibroblast replicative life span in vitro and maximum life span potential of the species. The period in the life history that is usually referred to as the "senescent phase" is probably more complicated than was originally thought, since studies with life span modulators suggest that there is a "conditionally" senescent state from which cells can be rescued for one or more additional rounds of DNA synthesis. Finally, the cells enter an "obligatory" arrested state in which only SV40 infection can reverse the block to DNA synthesis but not the block to mitosis. The modern era of aging research in tissue culture is just over 30 years old. The inception of the field really began with the recognition by Hayflick and Moorhead (109) that the phenomenon of senescence in vitro paralleled, in some of its characteristics, cell aging in vivo and thus provided a model that could be used to study the cellular mechanisms underlying senescence in controlled environmental conditions. The research in this area began with a detailed characterization and comparison of young versus senescent cell morphology and physiology. These studies provided the basis for a wide variety of subsequent studies that addressed possible mechanisms underlying cell senescence. These included studies on DNA repair, protein synthetic errors, chromatin structure and function, and mechanisms for modulating replicative life span.(ABSTRACT TRUNCATED AT 400 WORDS)

Our studies have led us to conclude that senescent cells respond to growth factors in much the same way, in part, as young cells. The receptor systems are largely unchanged with age, although some subtle modifications do occur. Furthermore, many of the early growth factor initiated events occur in a similar way in both young and old cells. This has led us to theorize that senescent cells are not arrested like mitogen-deprived young cells. Rather, they become blocked at a new arrest point in late G1 just prior to entry into DNA synthesis.

We had established that growth of human diploid WI-38 cells is reversibly inhibited by elevated partial pressures of oxygen (Po~) and we were interested in determining where in the cell cycle growth was delayed. A technique combining cytospectrophotometry and autoradiography was used to determine cell cycle parameters. Confluent cells that were subcultivated and exposed to a Po2 of 365 • 8 mm Hg were delayed primarily after DNA synthesis but before metaphase. At a Po2 of 590 • 35 mm Hg, most cells did not initiate DNA synthesis, and the few that did, failed to complete the process. When exponentially growing cells that had already begun DNA synthesis were exposed to aPo2 of 590 • 35 mm Hg, they accumulated after completing DNA synthesis but before initiating mitosis. The rate at which [3H]thymidine was incorporated into DNA was inversely correlated with oxygen tension (Po2 of 135-590 mm Hg). These results suggest that the process most sensitive to oxygen causes cells to be delayed after DNA synthesis but before metaphase. Slightly higher Po's were needed to inhibit the initiation of DNA synthesis. Further, the rate of DNA synthesis is decreased by elevated oxygen tensions.KEY WORDS oxygen 9 cell cycle aging human diploid cells cell proliferationThe precise nature of oxygen's interaction in cellular metabolism is not understood, although it is clear that oxygen participates in several fundamental cellular processes. In addition to oxygen's performing a central role in respiration, oxygeninduced cellular lesions have been suspected of being involved in cancer, aging, differentiation, and evolution (5,6,12,17,20,23,25).In our previous studies, we showed that elevated oxygen tensions reversibly inhibited the growth of human diploid cells (2-4). Oxygeninduced inhibition of cellular proliferation was not due to a generalized oxidation of sulfhydryl enzymes, because under growth-inhibiting conditions glycolysis was markedly stimulated (4). By systematically investigating oxygen's effect on cell growth and metabolism, we are attempting to elucidate the various aspects of oxygen's function in cellular economy. In this report, we describe the biological basis of oxygen-induced inhibition of cell growth by determining whether oxygeninhibited cells accumulate at random points or at particular stages of the cell cycle. 390J. CELL BIOLOGY 9 The Rockefeller University Press 9 0021-9525/78/0801-039051.00 on

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For logarithmically growing cultures of the human diploid cell lines WI-38 and WI-26, there is an exponential increase in the fraction of cells not incorporating [³H]dT under conditions of continuous labelling. Thymidine uptake, phosphorylation and incorporation into DNA can be correlated with cell proliferation. In addition, determination of the labelling index is reproducible within relatively broad limits of thymidine concentrations and specific activity. The plateauing phenomenon of the curve expressing percent-labelled nuclei versus time, which occurs in populations with less than 100% cycling cells, is largely due to radiation damage to the cells. The results of these studies provide insight into the population dynamics of aging fibroblast-like cell cultures. More importantly, however, the measurement of labelling index as described can be used as a reproducible and quantitative measure of the age of the diploid cell culture.