TUMOR PROMOTERS AND EPIDERMAL GROWTH-FACTOR STIMULATE ANCHORAGE-INDEPENDENT GROWTH OF ADENOVIRUS-TRANSFORMED RAT EMBRYO CELLS

Previous studies indicated that the potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) enhances transformation of rat embryo cells (2.degree. RE) by a mutant of human Ad5 (H5ts125). This study examines the effect of TPA, its structural analogs and epidermal growth factor (EGF) on anchorage-independent growth of a cloned population of H5ts125-transformed 2.degree. RE cells (clone E11). Both TPA and EGF (.apprx. 10-8 M) induced a 3-5 fold-increase in agar cloning efficiency of E11 cells. Macroscopic colonies appeared earlier and were larger and more diffuse. The TPA analogs phorbol-12,13-didecanoate (PDD) and ingenol-3,20-dibenzoate also enhanced growth in agar of E11 cells, whereas phorbol, 4.alpha.PDD and 4-O-meTPA, which are inactive as tumor promoters, failed to enhance agar growth. In contrast to the results obtained with E11 cells, TPA, PDD or ingenol-3,20-dibenzoate failed to induce growth in agar of normal 2.degree. RE cells. Dexamethasone (10-5-10-6 M), trans retinoic acid (10-5-10-6 M) and the protease inhibitors leupeptin, antipain and elastatinol did not inhibit the ability of TPA to enhance the growth of E11 cells in agar. The TPA-enhanced anchorage independence was a stable property, since subclones of E11 cells isolated from TPA-agar plates had a higher agar cloning efficiency than the parental E11 cells when retested in the absence of TPA. This effect of TPA does not appear to reflect simple selection of a subpopulation of cells. When the parental E11 cells were first cloned in monolayer culture in the absence of TPA, all 10 randomly picked clones showed enhanced growth in agar in the presence of TPA. In addition, prior growth of E11 cells did not enhance their subsequent growth in agar. This system provides an example in which TPA appears to enhance the acquisition of a stable cell property and may be a useful model for studying mechanisms of tumor promotion and progression.