UNMASKING LARGE AND PERSISTENT REDUCTIONS IN PROLIFERATION RATE OF AGING CELLS

We have reported that nontransformed sublines of NIH 3T3 cells that ar e incubated under the growth constraint of confluence for 10 d or long er exhibit heritable reductions of growth rate upon serial subculture at low density, which simulate the effects of aging in vivo on cell gr owth. There is also a marked increase in the likelihood of neoplastic transformation. After switching to a new batch of calf serum (CS), we found the reduced grow-th rate was no longer produced within the previ ously established timeframe. However, substitution of fetal bovine ser um (FBS) for CS during the period of recovery from confluence or the f ollowing tests of growth rate resulted in profound inhibition of growt h in cells serially subcultured from confluent cultures. In some cases , fewer than one in a thousand cells from subcultures of confluent cul tures formed colonies in FBS although they cloned at relatively high e fficiency in CS. The reduced growth in FBS was retained in the postcon fluent subcultures after many generations of multiplication at low den sity in CS. Generally, similar results with individual variations were obtained with three other batches of FBS. The numbers of cells per 3- d colony initiated from subcultures of confluent cultures were lower t han those of control cultures that had never been confluent. Supplemen tation of FBS-containing medium with CS fully restored the growth of t he postconfluent subcultures to the rate in CS medium, indicating that there is a deficiency of growth factor(s) in FBS rather than the pres ence of an inhibitor. The results show that prolonged incubation at co nfluence induces a populationwide heritable increase in requirement fo r growth factor(s) in short supply in FBS. Because clonal studies have shown that the reduction in growth rate is irreversible and varies in degree from clone to clone, we propose it arises from damage to DNA a t any of many different genetic loci or from chromosome aberrations. S uch genetic damage is also consistent with the increased tendency for neoplastic transformation in subcultures from the long-term confluent cultures.