EFFECT OF BRYOSTATIN-1 ON TAXOL-INDUCED APOPTOSIS AND CYTOTOXICITY INHUMAN LEUKEMIA-CELLS (U937)

We have examined the effects of the macrocyclic lactone protein kinase C (PKC) activator bryostatin 1 on taxol-induced apoptosis and inhibit ion of clonogenicity in the human monocytic leukemia cell line U937. E xposure of cells to bryostatin 1 (10 nM; 15 hr) after (but not before) a 6-hr incubation with 0.5 mu M taxol significantly increased apoptos is and resulted in an approximately 3 log reduction in clonogenicity. Cell cycle analysis revealed that the increase in apoptotic cells foll owing bryostatin 1 treatment occurred primarily in the population unde rgoing taxol-mediated G(2)M arrest. The actions of bryostatin 1 were n ot attributable to potentiation of taxol-induced tubulin stabilization or to a reduction in the intracellular retention of taxol. Following exposure of cells to taxol, the Bcl-2 protein displayed an alteration in mobility that was not modified appreciably by bryostatin 1 treatmen t. The mobility shift in Bcl-2 protein from cells exposed to taxol fol lowed by bryostatin 1 was eliminated by treatment of lysates with the protein phosphatase 2A (PP2A); the latter effect was blocked by okadai c acid. Treatment of cells with taxol followed by bryostatin 1 did not increase the amount of total Bar (compared with treatment with taxol alone), but did increase the amount of free Bar in the supernatant fra ction. Finally, the ability of bryostatin 1 to potentiate taxol-induce d apoptosis in U937 cells was :mimicked closely by 2'-amino-3'-methoxy flavone (PD98059), a specific inhibitor of the mitogen activated prote in kinase (MAPK) kinase (MEK). Collectively, these findings indicate t hat bryostatin 1 increases the susceptibility of U937 cells to taxol-i nduced apoptosis and inhibition of clonogenicity. They also raise the possibility that this phenomenon may-involve functional alterations in Bcl-2 and/or other proteins involved in regulation of the cell death pathway. (C) 1998 Elsevier Science Inc.