MODULATION OF THE EXPRESSION OF BCL-2 AND RELATED PROTEINS IN HUMAN LEUKEMIA-CELLS BY PROTEIN-KINASE-C ACTIVATORS - RELATIONSHIP TO EFFECTSON 1-[BETA-D-ARABINOFURANOSYL]CYTOSINE-INDUCED APOPTOSIS

We have previously reported that pretreatment of HL-60 human promyeloc ytic leukemia cells with the non-tumor-promoting protein kinase C (PKC ) activator bryostatin 1 potentiates induction of apoptosis by the ant imetabolite 1-[beta-D-arabinofuranosyl]cytosine (ara-C) (Biochem Pharm acol 47:839,1994). To determine whether this phenomenon results from a ltered expression of Bcl-2 or related proteins, Northern and Western a nalysis was employed to assess the effects of bryostatin 1 and other P KC activators on steady-state levels of Bcl-2, Bax, Bcl-x, and Mcl-1 m RNA and protein. Pretreatment of cells for 24 h with in nM bryostatin 1, or, to a lesser extent, the stage-1 tumor-promoter phorbol dibutyra te (PDB) significantly potentiated apoptosis induced by ara-C (inn mu M;6 h); in contrast, equivalent exposure to the stage-2 tumor promoter , mezerein (MZN), which, unlike bryostatin 1, is a potent inducer of d ifferentiation in this cell line, failed to modify ara-C-related cell death. Neither bryostatin 1 nor PDB altered expression of bcl-2/Bcl-2 over this time frame. In contrast, MZN downregulated bcl-2 mRNA levels , but this effect was not accompanied by altered expression of Bcl-2 p rotein. None of the PKC activators modified expression of Bar or Bcl-x (L) mRNA or protein; levels of Bcl-x(s) were undetectable in both trea ted and untreated cells. However, expression of Mcl-1 mRNA and protein increased modestly after treatment with either bryostatin 1 or PDB, a nd to a greater extent following exposure to MZN. Combined treatment o f cells with bryostatin 1 and MZN resulted in undiminished potentiatio n of ara-C-med iated apoptosis and by antagonism of cellular maturatio n. These effects were accompanied by unaltered expression of Bcl-2, Ba x, and Bcl-x(L), and by a further increase in Mcl-1 protein levels. Wh en cells were co-incubated with bryostatin 1 and calcium ionophore (A2 3187), an identical pattern of expression of Bcl-2 family members was observed, despite the loss of bryostatin 1's capacity to potentiate ap optosis, and the restoration of its ability to induce differentiation. Finally, treatment of cells with bryostatin 1 +/- ara-C (but not ara- C alone) resulted in a diffuse broadening of the Bcl-2 protein band, w hereas exposure of cells to taxol (250 nM, 6 h) led to the appearance of a distinct Bcl-2 species with reduced mobility, phenomena compatibl e with protein phosphorylation. Together, these findings indicate that the ability of bryostatin 1 to facilitate drug-induced apoptosis in h uman myeloid leukemia cells involves factors other than quantitative c hanges in the expression of Bcl-2 family members, and raise the possib ility that qualitative alterations in the Bcl-2 protein, such as phosp horylation status, may contribute to this capacity. They also suggest that increased expression of Mcl-1 occurs early in the pre-commitment stage of myeloid cell differentiation, and that this event does not pr otect cells from drug-induced apoptosis.