Autocrine factor-independent growth of mammalian fibroblasts established in fully synthetic medium: No V-onc requirement in establishment

Summary In a previous study Chinese hamster fibroblasts carrying a partially deleted v-src were established in a synthetic medium lacking macromolecular supplements and shown to possess a particular serum-free phenotype hereafter designatedsf. In cloning efficiency assays,sf, unlike wild-type, fibroblasts required a threshold cell density to growth from single cells, suggesting autocrine stimulation. In the present study a conditioned medium harvested fromsf cells was added to the samesf cells, and the resulting cloning density was found to markedly diminish rather than increase.Sf cells were found to be unable to grow at cloning density because of trypsin damage;sf cells seeded into trypsin inhibitor-containing medium cloned with no requirement for threshold cells and were therefore independent of autocrine secretion from neighboring cells. Their cloning efficiency reached 7.7%; this value could not be improved by subcloning thesf culture, and it diminished when selenium was not added to the assay medium. To determine whether v-src is involved in thesf phenotype, five clones of the parental Chinese hamster fibroblast line not infected with Rous sarcoma virus were explanted into serum-free cultures with no macromolecular additives as in the case of v-src-containing cells. Each clone gave rise to ansf cell line growing indefinitely in synthetic medium like the v-src-containingsf cells, showing that the v-src gene is not required either for the establishment or maintenance of thesf phenotype. Supported by grants from CNRS, INSERM, contract 862015, Association Claude-Bernard, Association pour la Recherche sur le Cancer, and Fondation pour la Recherche Médicale.