Benzo[a]pyrene-diol-epoxide-induced anchorage-independence in diploid human fibroblasts. Analysis of cellular protooncogenes

Abstract

Treatment of diploid human fibroblasts with stereoisomeric benzo[alpha]pyrene anti and syn diol epoxides has been shown to induce anchorage-independent clones of cells with a dose dependence and frequency [(0.5-12) X 10(-4)] not significantly different from mutations at the hypoxanthine-guanine phosphoribosyltransferase locus [(1-8) X 10(-4)] in these cells. The majority of the anchorage-independent clones that were picked retained their mutagen-induced, anchorage-independent phenotype through at least 20 generations of expansion in monolayer culture. No variant cells showing extended life-span were detected among survivors in any of the mutagen treatment groups (less than 1.6 X 10(-7) frequency). Extensive analysis of a pool of 15 cellular protooncogenes (Ha-ras, Ki-ras, N-ras, mos, fos, fes, myc, abl, sis, myb, erbA, erbB, src, raf, N-myc), using Southern and northern blot analysis, was done to determine whether mutagen-induced rearrangement, amplification or overexpression of any of these genes was responsible for the mutagen-induced, anchorage-independent phenotype. We found no evidence that the genomic arrangement or expression level of any of these genes had been altered, thus indicating that an alternative form of mutation, or an alternative gene not included in this screening was responsible for the mutagen-induced, anchorage-independent phenotype.