Chronic application of a tumor promoter to confluent nontransformed rat cells induces the irreversible expression of transformed phenotype.

The hypothesis was put foreward that 'immortalisation' of an established nontransformed cell line might represent one type of 'initiation' in the process of malignant transformation. Therefore, a nontransformed rat fibroblast cell line, FR3T3, was investigated as to whether or not it could be irreversibly transformed by exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA was applied in different treatment schedules at varying growth phases of the cells (logarithmic or stationary growth). The cells' acquisition of transformation-associated properties was studied by means of various transformation assays (saturation densities and formation of dense foci both at low and high serum concentrations, as well as growth in agarose and production of plasminogen activator, PA). We found that a phase of stationary growth plus TPA treatment was decisive for the induction of transformed cell clones. Growth in 0.5% serum was used as the selection procedure. Several isolated foci manifested, in vitro, highly transformed phenotypes. However, only 1 of 12 clones produced PA. All cell lines derived from these clones, proved to be tumorigenic in syngeneic animals. Since the nontransformed, but 'immortal' rat cell line is susceptible (at certain growth conditions) to the tumor promoting activity of TPA, we conclude that induction of 'immortalisation' may, from an operational point of view, correspond to 'initiation', at least in this particular cell line.