Role of phorbol diesters in in vitro murine megakaryocyte colony formation

In vitro megakaryocyte differentiation is regulated by two activities: a megakaryocyte colony-stimulating activity (Mk-CSA), which is required for proliferation, and an auxiliary factor, megakaryocyte potentiating activity, which plays a role in later differentiation events. Tumor-promoting phorbol esters alter many cellular differentiation-related events. Thus, it was hypothesized that phorbol esters may bring about megakaryocyte differentiation in vitro. 4 beta-Phorbol 12-myristate 13-acetate (PMA), when co-cultured with a source of Mk-CSA, stimulated a threefold increase in colony numbers. Co-culture of PMA and megakaryocyte potentiator activity did not stimulate colony formation, thus eliminating any action of PMA as an Mk-CSA. The direct effect of PMA on the formation of megakaryocyte colonies was established by (a) the function of PMA as a megakaryocyte potentiator in serum-free experiments, (b) the ability of PMA to stimulate megakaryocyte colony formation using bone marrow cells depleted of populations known to produce potentiating activity, (c) the inability of bone marrow adherent cells previously treated with phorbol, 12,13-dibutyrate (PDBu) to augment megakaryocyte colony formation, and (d) the ability of PMA to induce the growth of immature megakaryocytes into large single megakaryocytes. Structure:activity experiments resulted in equivalent activities for PMA and PDBu, whereas the nontumor promoter phorbol 12,13-diacetate and phorbol itself lacked activity. The observations in this study indicate that phorbol esters can bring about megakaryocyte differentiation, and during colony formation, can induce effects identical to those brought about by biological sources of megakaryocyte potentiator activity.