AGENTS THAT DOWN-REGULATE OR INHIBIT PROTEIN-KINASE-C CIRCUMVENT RESISTANCE TO 1-BETA-D-ARABINOFURANOSYLCYTOSINE-INDUCED APOPTOSIS IN HUMANLEUKEMIA-CELLS THAT OVEREXPRESS BCL-2

The effects of the non-tumor-promoting protein kinase C (PKC) activato r bryostatin 1 and the PKC inhibitors staurosporine and UCN-01 were ex amined with respect to modulation of 1-[beta-D-arabinofuranosyl]cytosi ne (ara-C)-induced apoptosis in human myeloid leukemia cells (HL-60) o verexpressing the antiapoptotic protein Bcl-2, HL-60/Bcl-2 cells displ ayed a 5-fold increase in Bcl-2 protein compared with empty-vector cou nterparts (HL-6-/pCEP4) but comparable levels of Bax, Mcl-1, and Bcl-x (L). After exposure to an equimolar concentration of ara-C (10 mu M fo r 6 hr), HL-60/BCl-2 cells were significantly less susceptible to apop tosis, DNA fragmentation, and loss of clonogenicity than HL-60/pCEP4 c ells. The protective effect of increased Bcl-2 expression was manifest ed by a failure of ara-C; to induce activation/cleavage of the Yama pr otease (CPP32; caspase-3) and degradation of one of its substrates, po ly(ADP-ribose)polymerase to an 85-kDa cleavage product. When HL-60/Bcl -2 cells were preincubated with bryostatin 1 (10 nM; 24 hr) or coincub ated with either staurosporine (50 nM; 6 hr) or UCN-01 (300 nM; 6 hr) after a 1-hr preincubation, exposures that exerted minimal effects alo ne, ara-C-induced apoptosis and DNA fragmentation were restored to lev els equivalent to, or greater than, those observed in empty-vector con trols. These events were accompanied by restoration of the ability of ara-C to induce CPP32 cleavage and activation, poly(ADP-ribose) polyme rase degradation, and inhibition of colony formation. Western analysis of Bcl-2 protein obtained from overexpressing cells treated with bryo statin 1, staurosporine, or UCN-01 revealed the appearance of a slowly migrating species and a general broadening of the protein band, effec ts that were insensitive to the protein synthesis inhibitor cyclohexim ide. Alterations in Bcl-2 protein mobility on sodium dodecyl sulfate-p olyacrylamide gel electrophoresis were reversed by treatment of lysate s with alkaline phosphatase or protein phosphatase 2A; actions of the latter were blocked by the specific phosphatase inhibitor okadaic acid . In vivo labeling studies of Bcl-2 protein demonstrated increased inc orporation of [(PO4)-P-32]orthophosphate in drug-treated cells. Last, phosphorylated Bcl-2 failed to display decreased binding to the proapo ptotic protein Bar. Collectively, these findings indicate that bryosta tin 1, which down-regulates PKC, and staurosporine and UCN-01, which d irectly inhibit the enzyme, circumvent resistance of Bcl-2-overexpress ing leukemic cells to ara-C-induced apoptosis and activation of the pr otease cascade. They also raise the possibility that modulation of Bcl -2 phosphorylation status contributes to this effect.