Modulation of the expression of Bcl-2 and related proteins in human leukemia cells by protein kinase C activators: relationship to effects on 1-[β-D-arabinofuranosyl]cytosine-induced apoptosis

Abstract

We have previously reported that pretreatment of HL-60 human promyelocytic leukemia cells with the non-tumor-promoting protein kinase C (PKC) activator bryostatin 1 potentiates induction of apoptosis by the antimetabolite 1-[beta-D-arabinofuranosyl]cytosine (ara-C) (Biochem Pharmacol 47:839,1994). To determine whether this phenomenon results from altered expression of Bcl-2 or related proteins, Northern and Western analysis was employed to assess the effects of bryostatin 1 and other PKC activators on steady-state levels of Bcl-2, Bax, Bcl-x, and Mcl-1 mRNA and protein. Pretreatment of cells for 24 h with 10 nM bryostatin 1, or, to a lesser extent, the stage-1 tumor-promoter phorbol dibutyrate (PDB) significantly potentiated apoptosis induced by ara-C (100 microM; 6 h); in contrast, equivalent exposure to the stage-2 tumor promoter, mezerein (MZN), which, unlike bryostatin 1, is a potent inducer of differentiation 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 down-regulated bcl-2 mRNA levels, but this effect was not accompanied by altered expression of Bcl-2 protein. None of the PKC activators modified expression of Bax or Bcl-x(L) mRNA or protein; levels of Bcl-x(S) were undetectable in both treated and untreated cells. However, expression of Mcl-1 mRNA and protein increased modestly after treatment with either bryostatin 1 or PDB, and to a greater extent following exposure to MZN. Combined treatment of cells with bryostatin 1 and MZN resulted in undiminished potentiation of ara-C-mediated apoptosis and by antagonism of cellular maturation. These effects were accompanied by unaltered expression of Bcl-2, Bax, and Bcl-x(L), and by a further increase in Mcl-1 protein levels. When cells were co-incubated with bryostatin 1 and calcium ionophore (A23187), an identical pattern of expression of Bcl-2 family members was observed, despite the loss of bryostatin 1's capacity to potentiate apoptosis, 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, whereas exposure of cells to taxol (250 nM, 6 h) led to the appearance of a distinct Bcl-2 species with reduced mobility, phenomena compatible with protein phosphorylation. Together, these findings indicate that the ability of bryostatin 1 to facilitate drug-induced apoptosis in human myeloid leukemia cells involves factors other than quantitative changes in the expression of Bcl-2 family members, and raise the possibility that qualitative alterations in the Bcl-2 protein, such as phosphorylation 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 protect cells from drug-induced apoptosis.