Irreversibility of cellular aging and neoplastic transformation: a clonal analysis.

Abstract

Prolonged incubation of NIH 3T3 cells under the growth constraint of confluence results in a persistent impairment of proliferation when the cells are subcultured at low density and a greatly increased probability of neoplastic transformation in assays for transformation. These properties, along with the large accumulation of age pigment bodies in the confluent cells, are cardinal cellular characteristics of aging in organisms and validate the system as a model of cellular aging. Two cultures labeled a and 3 were obtained after prolonged confluence; both were dominated by cells that were both slowed in growth at low population density and enhanced in growth capacity at high density, a marker of neoplastic transformation. An experiment was designed to study the reversibility of these age-related properties by serial subculture at low density of the two uncloned cultures and their progeny clones derived from assuredly single cells. Both uncloned cultures had many transformed cells and a reduced growth rate on subculture. Serial subculture resulted in a gradual increase in growth rates of both populations, but a reversal of transformation only in the a! population. The clones originating from both populations varied in the degree of growth impairment and neoplastic transformation. None of the a clones increased in growth rate on low density passage nor did the transformed clones among them revert to normal growth behavior. The fastest growing f3 clone was originally slower than the control clone, but caught up to it after four weekly subcultures. The other 13 clones retained their reduced growth rates. Four of the five (8 clones, including the fastest grower, were transformed, and none reverted on subculture. We conclude that the apparent reversal of impaired growth and transformation in the uncloned parental a population resulted from the selective growth at low density of fast growing nontransformed clones. The reversal of impaired growth in the uncloned parental ,B population was also the result of selective growth of fast growing clones, but in this case they were highly transformed so no apparent reversal of transformation occurred. The clonal results indicate that neither the impaired growth nor the neoplastic transformation found in aging cells is reversible. We discuss the possible contribution of epigenetic and genetic processes to these irreversible changes.A general feature of the cells in aging organisms is a slowdown in the rate of multiplication whether measured in vivo (1-4) or after removal to cell culture (5-7). There is also an increase in the probability of neoplastic transformation (8, 9). These changes are reproduced in NIH 3T3 mouse cells subcultured after several weeks of constraint at confluence (10)(11)(12)). Another cellular characteristic of aging organisms is the accumulation of lipofuscin granules or age pigment bodies that are particularly abundant in postmitotic tissue such as nerve and muscle (13,14). A similar accumulation occurs in long-term confluent cultures of human cell...

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