CELLULAR AGING IS A CRITICAL DETERMINANT OF PRIMARY-CELL RESISTANCE TO V-SRC TRANSFORMATION

Primary cell cultures are in general resistant to the transforming eff ect of a single oncogene, a finding considered consistent with the mul tistage theory of carcinogenesis. In the present studies, we examined whether cellular age, differentiation stage, and/or tissue origin of p rimary cells plays a role in determining their response to v-src trans formation. To study the role of cellular age, rat mammary fibroblasts were isolated from a 50-day-old female rat and infected with a recombi nant retrovirus carrying a v-src gene after 2, 7, 14, 21, and 28 days of continuous growth. To determine whether cellular differentiation is important, fibroblasts were isolated from embryos at 12 and 16 days o f gestation, from newborns, and from a 30-day-old rat and similarly in fected. Finally, the role of primary-cell histogenesis was assessed by infecting primary cultures of fibroblasts isolated from the mammary g land, dermis, and lungs of a mature rat. When compared to 3Y1 cells, a ll preparations of primary cultures exhibited considerable resistance to v-src transformation. However, whereas primary cells isolated from different tissues responded similarly to the transforming effect of th e oncogene, major differences were observed when cells were transduced at different stages of their in vitro life span. v-src was capable of inducing formation of foci and growth in soft agar in early-passage c ells but failed to do so in primary cultures infected after 14 days of continuous passaging. Similarly, both the number of foci and the numb er of colonies in soft agar decreased with tissue donor age. The diffe rential response of young and senescing cells could not be explained b y mutations in v-src provirus, by differences in functional v-src expr ession, or by growth stimulation or suppression via paracrine mechanis ms. Furthermore, v-src cooperated with an immortalizing gene, like sim ian virus 40 large T, polyomavirus large T, E6 and E7 of human papillo mavirus, or an activated p53 mutant, to induce anchorage-independent g rowth of primary cultures but failed to do so with cytoplasmic transfo rming genes, like v-abl, v-ras, or v-raf, which did not confer indefin ite division potential. These studies indicate that cellular aging is a critical determinant of primary-cell resistance to v-src transformat ion. It is suggested that v-src requires a nuclear auxiliary function for transformation which is present in early-passage cells, particular ly when these cells are derived from embryonic tissue, but is lost as cells approach replicative senescence. This auxiliary function is prov ided by nuclear oncogenes but not cytoplasmic transforming genes.