Effect of phorbol esters on the activity of purified transforming growth factors

Application of either phorbol esters or transforming growth factors to normal cells in culture may induce the appearance of a malignant phenotype. Since the mechanism of transformation by both chemical tumor promoters and growth factors is unknown, it was of interest to investigate whether phorbol esters might potentiate the activity of transforming growth factors. The combination of beta-transforming growth factor (beta-TGF) and epidermal growth factor (EGF) causes the normally anchorage-dependent cells (NRK-49F) to form colonies in soft agar. In the presence of maximally stimulating concentrations of EGF and suboptimally stimulating concentrations of beta-TGF, phorbol 12-myristate-13-acetate (PMA) enhances the soft agar colony growth of NRK-49F cells in a concentration-dependent manner. However, PMA alone or in combination with either EGF or beta-TGF does not stimulate soft agar growth. In contrast to the stimulation of PMA, 4 alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, does not potentiate the effects of EGF plus beta-TGF on soft agar growth. PMA does not stimulate the growth of NRK-49F cells in monolayer, nor does it further potentiate the monolayer growth induced by EGF with or without beta-TGF. Because the addition to NRK-49F cells of compounds which potentiate beta-TGF activity increases EGF receptor number, the effects of PMA on the EGF receptor were studied. A short exposure to PMA (30 min) induces a 50% decrease in EGF receptors of NRK cells whether or not they have been exposed to beta-TGF. Scatchard analysis shows that this decrease involves primarily high affinity EGF receptors. However, cells treated with PMA for longer periods of time (4, 6, and 24 h) show no change in EGF or beta-TGF receptor binding. PMA therefore must potentiate the activity of purified TGFs without causing an increase in EGF or beta-TGF receptor binding.