Serum-free media LEP-1 and F12/9F, respectively, were developed for mouse keratinocytes (MK) and rat tracheal epithelial (RTE) cells. Clonal growth assays were used to evaluated the basal nutrients and supplementary hormones and growth factors required for each cell type. Serum induced squamous terminal differentiation in both MK and RTE cells. Serum factors were shown to have either stimulatory (albumin) or inhibitory activity for MK cells (fetuin, crude platelet extract TGF beta). When albumin, 100 ug/ml, was added to LEP-1, MK cells continued to multiply and are now at the 33 rd passage. Chromosomal studies showed an altered karyotype by the 2nd or 3rd passage and most cells were triploid to tetraploid by passage 17. This MK system in serum-free medium is currently used in studies oc chemically induced transformation and gene activation. RTE cells in F12/9F gradually stopped multiplying and underwent squamous differentiation when switched to serum-containing medium. This property o serum is used to select for preneoplastic enhanced growth variants (EG). EG variants arose spontaneously in serum-free RTE cultures. The frequency of variants increased with cell number and time. However, they appeared at a constant rate of less than one variant per one hundred thousand cells per generation. This serum-free system is being used in studies of RTE cell growth control, transformation, and differentiation. The culture life span of a tranformed but non-tumorigenic human prostatic epithelial line (NP-2s/T2) has been extended by transfection of the EJ-ras oncogene. Although the line produces a transforming growth factor, it is not tumorigenic in nude mice. Attempts to transfect normal prostatic epithelial cells (NP-2s) with known oncogenes and with DNA from prostatic carcinoma cells (PC-3) are in progress. The presence of known or new oncogenes in PC-3 is being investigated by calcium phosphate-mediated DNA transfection into NIH/3T3 cells.
