The goal of the present study was to determine whether partial restoration of the differentiation-inducing capacity of the PKC activator bryostatin 1 by the calcium ionophore A23187 is accompanied by enhancement of apoptosis in ara-C-pretreated human leukemia cells. When HL-60 cells were exposed to ara-C (10 or 100 microM;6 h) followed by bryostatin 1 alone (10 nM; 24 h), no increase in apoptosis was noted. In contrast, subsequent exposure of ara-C-pretreated cells to A23187 (250 nM; 24 h) increased apoptosis by approximately 100%. When ara-C-pretreated cells were incubated with A23187 and bryostatin 1, no further potentiation of cell death (compared to cells exposed to A23187 alone) was observed. Nevertheless, the combination of bryostatin 1 and A23187 substantially increased inhibition of clonogenicity in cells preincubated with ara-C (e.g., by > or = 2 logs). This effect was associated with morphological and functional evidence (i.e., plastic adherence) of enhanced leukemic cell maturation. The differentiating capacity of the combination of bryostatin 1 and A23187 was significantly weaker than that of the phorbol diester, PMA (10 nM), and unaccompanied (at 24 h) by induction of the cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/CIP1 and p27KIP1. However, the extent of apoptosis was comparable in cells exposed to ara-C followed by PMA or bryostatin 1 + A23187, suggesting that differentiation per se is not solely responsible for enhancement of cell death in ara-C-pretreated cells. Coadministration of bryostatin 1 and the organotellurium compound AS101, which mimics the actions of A23187 in some systems, after ara-C also led to enhanced antiproliferative effects which were unaccompanied by an increase in apoptosis. Finally, exposure of cells to ara-C followed by other differentiation-inducing agents, including dimethylsulfoxide and sodium butyrate also resulted in increases in cell death in this cell line. These findings indicate that the inability of bryostatin 1 to potentiate apoptosis in ara-C-pretreated HL-60 cells may involve factors other than an inadequate differentiation stimulus. They also suggest that loss of leukemic self-renewal capacity following exposure to cytotoxic and differentiation-inducing agents may involve mechanisms other than, or in addition to, potentiation of apoptosis, particularly cellular maturation.

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