MODULATION OF PROTEIN-KINASE-C ACTIVITY AND CALCIUM-SENSITIVE ISOFORMEXPRESSION IN HUMAN MYELOID-LEUKEMIA CELLS BY BRYOSTATIN-1 - RELATIONSHIP TO DIFFERENTIATION AND ARA-C-INDUCED APOPTOSIS

Previous studies have shown that pretreatment of human myeloid leukemi a cells (HL-60) with the protein kinase C (PKC) activator bryostatin 1 potentiates ara-C-induced apoptosis. To test the hypothesis that this capacity stems from down-regulation of PMC activity and/or Ca2+-depen dent (group-I; cPKC) isoform expression, comparisons were made between the effects of this agent and the stage-a tumor promoter mezerein und er conditions favoring either cellular differentiation or drug-induced apoptosis. Twenty-four-hour pretreatment of HL-60 cells with 10 nM br yostatin 1, which does not induce differentiation in this cell line, l ed to a profound reduction in membrane and cytosolic PKC activity, dec reased expression of cPKC isoforms (alpha, beta(I), beta(II), gamma), and a marked increase in ara-C induced apoptosis; In contrast, 10 nM m ezerein, which induces HL-60 cell differentiation, was less effective in down-regulating membrane and cytosolic PKC activity as well as alph a, beta(I), and gamma cPKC isoform expression, and failed to potentiat e ara-C-related apoptosis. The effects of bryostatin 1 were dominant t o those of mezerein, in that the combination resulted in down-regulati on of PRC activity and expression and potentiation of ara-C-induced ap optosis, but not cellular maturation. However, coadministration of the Ca2+ ionophore A23187 (250 nM) restored bryostatin 1's differentiatin g ability while antagonizing its capacity to augment apoptosis, despit e failing to reverse bryostatin 1-induced down-regulation of PKC activ ity and cPKC isoform expression. Furthermore, pretreatment of differen tiation-responsive monocytic leukemia cells (U937) with bryostatin 1 s ubstantially reduced PKC activity and cPKC isoform expression, but exe rted minimal effects on ara-C-related apoptosis. In contrast, exposure of U937 cells to bryostatin 1 after ara-C dramatically increased apop tosis, a phenomenon that did not occur in differentiation-unresponsive HL-60 cells. Collectively, these observations indicate that down-regu lation of total assayable PKC activity and cPKC expression by bryostat in 1 are insufficient, by themselves, to account for potentiation of l eukemic cell apoptosis, at least under conditions in which differentia tion occurs. They also provide further evidence that a reciprocal and highly schedule-dependent relationship exists between leukemic cell di fferentiation and drug-induced apoptosis. (C) 1996 Academic Press, Inc .