Transformation of Diploid Human Fibroblasts by Chemical Carcinogens

Abstract

Using N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and following a protocol modeled after that published by Kakunaga (1978, Proc. Natl. Acad. Sci. U.S.A. 75, pp. 1334–1338) we succeeded in causing diploid human fibroblasts (KD cells) to transform into cells which can form foci on the top of confluent monolayers. Cells derived from such foci were isolated, allowed to multiply into large populations, and their progeny assayed for tuinorigenicity by injection into athymic mice. Nodules formed in some of the animals. Of these, some later regressed, but histological examination by several pathologists of a large nodule which did not regress revealed a fibrosarcoma. Cells from this fibrosarcoma exhibited a human karyotype. Thus, although the frequency of induction of focus-forming cells in the population is very difficult to quantitate, we have confirmed the results reported earlier by Kakunaga. More recently, we also succeeded in inducing transformation of diploid human fibroblasts with MNNG following a protocol modeled after that of Milo and DiPaolo (1978, Nature 275, 130–132). In this protocol, selection for transformed cells among the progeny of carcinogen-treated cells derived from foreskins was applied by growth in medium containing high concentrations of amino acids followed by growth in soft agar. Cells capable of forming colonies in agar were produced with high frequency and cells derived from such colonies were isolated, propagated, and assayed for tumorigenicity in athymic mice. Again, nodules were observed and a tumor analyzed independently by several pathologists was diagnosed as a fibrosarcoma. The karyotype of the cells from the tumor was human. Experiments were carried out to compare the frequency of acquisition of anchorage independence with that of 6-thioguaninc resistance, the genetic marker commonly employed in our mutation assays. Using populations exposed to carcinogens under the same conditions and given similar expression periods, we found the frequency of cells able to grow in agar to be ca. 20-fold higher than that of thioguanine resistant cells. However, the carcinogen-induced frequency of both traits showed a dose- dependent increase. We are presently analyzing these protocols to adapt them for studies of the mechanisms involved in carcinogenesis.