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

The process of signal transduction responsible for the phorbol 12-myri state 13-acetate mediated increase in the colony-forming potential of murine (CBA) bone marrow cells was studied using known modulators of t he mitogenic signal. Pretreatment of cells for 60 minutes with stauros porine (1 mu mol/l), an inhibitor of protein kinase C, completely prev ented colony formation in the control group of cells and significantly reduced the number of colonies formed in the phorbol ester-treated gr oup. Brief exposure (60 min) of cells to the phospholipase A(2) inhibi tors, mepacrine (500 mu mol/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 phospholip ase A(2) were present during the entire period of the colony forming a ssay (7 days), no colonies could be scored in either the control or ph orbol ester-treated groups of bone marrow cells. Long-term treatment o r temporary exposure (60 min) of cells to indomethacin (50 mu mol/l), an inhibitor of cyclooxygenase, or nordihydroguaiaretic acid (50 mu mo l/l), an inhibitor of lipoxygenase, had no effect on colony formation in both groups. Pretreatment of cells for 45 min with calcium ionophor e A23187 (10 mu mol/l) failed to increase the number of colonies, comp ared with the control group. Moreover, simultaneous treatment of cells for 45 min with phorbol ester (50 mu mol/l) and A23187 (10 mu mol/l) did not produce any further increase in the number of colonies, compar ed with the phorbol ester-treated group, suggesting that elevation of intracellular calcium is unimportant in the phorbol ester-mediated res ponse. Dibutyryl cyclic adenosine monophosphate (50 mu mol/l) in the p resence or absence of phorbol ester, failed to stimulate colony format ion, indicating that cyclic AMP-dependent protein kinases are not invo lved in the signalling process. Temporary exposure (75 min) of bone ma rrow cells to okadaic acid (1 mu mol/l), a potent inhibitor of serine/ threonine phosphatases, or to tyrphostine AG-115 (20 mu mol/l), a tyro sine kinase inhibitor, did not effect colony growth in the control or phorbol ester-treated group. The results indicate that phospholipase A (2) activation is involved in the phorbol ester-mediated increase in c olony formation, since, of the different agents applied, only staurosp orine, an inhibitor of protein kinase C, and mepacrine and heparin, pu tative inhibitors of phospholipase A(2) were capable of abolishing pho rbol ester-mediated effects.