Short-term and long-term-effects of phorbol 12-myristate 13-acetate and different inhibitors on the ability of bone marrow cells to form colonies in vitro.

Abstract

The process of signal transduction responsible for the phorbol 12-myristate 13-acetate mediated increase in the colony-forming potential of murine (CBA) bone marrow cells was studied using known modulators of the mitogenic signal. Pretreatment of cells for 60 minutes with staurosporine (1 mumol/l), an inhibitor of protein kinase C, completely prevented colony formation in the control group of cells and significantly reduced the number of colonies formed in the phorbol ester-treated group. Brief exposure (60 min) of cells to the phospholipase A2 inhibitors, mepacrine (500 mumol/l) and heparin (1 g/l), reduced the number of colonies formed in the control group and completely abolished the increase in the number of colonies formed after treatment of the cells with phorbol ester. When inhibitors of protein kinase C or phospholipase A2 were present during the entire period of the colony forming assay (7 days), no colonies could be scored in either the control or phorbol ester-treated groups of bone marrow cells. Long-term treatment or temporary exposure (60 min) of cells to indomethacin (50 mumol/l), an inhibitor of cyclooxygenase, or nordihydroguaiaretic acid (50 mumol/l), an inhibitor of lipoxygenase, had no effect on colony formation in both groups. Pretreatment of cells for 45 min with calcium ionophore A23187 (10 mumol/l) failed to increase the number of colonies, compared with the control group. Moreover, simultaneous treatment of cells for 45 min with phorbol ester (500 nmol/l) and A23187 (10 mumol) did not produce any further increase in the number of colonies, compared with the phorbol ester-treated group, suggesting that elevation of intracellular calcium is unimportant in the phorbol ester-mediated response. Dibutyryl cyclic adenosine monophosphate (50 mumol/l) in the presence or absence of phorbol ester, failed to stimulate colony formation, indicating that cyclic AMP-dependent protein kinases are not involved in the signalling process. Temporary exposure (75 min) of bone marrow cells to okadaic acid (1 mumol/l), a potent inhibitor of serine/threonine phosphatases, or to tyrphostine AG-115 (20 mumol/l), a tyrosine kinase inhibitor, did not effect colony growth in the control or phorbol ester-treated group. The results indicate that phospholipase A2 activation is involved in the phorbol ester-mediated increase in colony formation, since, of the different agents applied, only staurosporine, an inhibitor of protein kinase C, and mepacrine and heparin, putative inhibitors of phospholipase A2, were capable of abolishing phorbol ester-mediated effects.