Tumor‐promoting phorbol esters stimulate the proliferation of interleukin‐3 dependent cells

To determine whether activation of protein kinase C is involved in the proliferation of interleukin‐3 (IL‐3) ‐dependent cells, we examined the effect of tumor‐promoting phorbol esters on the in vitro proliferation of the IL‐3‐dependent cell lines FD and DA‐1. The viability of FD and DA‐1 cells cultured for 24 hours in 100 nM phorbol myristate acetate (PMA) and 10% FCS was similar to that of cells cultured in 25% WEHI‐3 conditioned medium as a source of IL‐3, and 10% FCS. FD cells failed to proliferate in concentrations of FCS of up to 50%, while DA‐1 cell proliferation was not markedly influenced by FCS. By contrast, PMA promoted the proliferation of FD and DA‐1 cells in the absence of FCS and enhanced their proliferation in the presence of 10% FCS, 60‐ and 20‐fold, respectively. Stimulation of proliferation was achieved with as little as 10 nM PMA and was maximal at 100 nM PMA. Low concentrations (0.05‐0.1%) of WEHI‐3 CM promoted the proliferative response of FD and DA‐1 cells to PMA, but at concentrations of WEHI‐3 CM greater than 0.8%, no further increment in proliferation was obtained with PMA. As little as 1/2 hour of exposure to phorbol esters was sufficient to cause translocation of protein kinase C from the cytosol to the membranes of DA‐1 cells, and 1 hour of exposure to phorbol esters was sufficient to stimulate DNA synthesis. A protein kinase C inhibitor, H‐7, at a concentration of 10 μM inhibited phorbol ester‐induced stimulation of DA‐1 cell proliferation. When DA‐1 cells were exposed to the calcium ionophore A23187 in addition to both a phorbol ester and IL‐3, their proliferation was enhanced over that stimulated by only the phorbol ester and IL‐3. The data indicate that stimulation of proliferation of IL‐3‐dependent cells involves the activation of protein kinase C.