Modulation of the expression of Bcl-2 and related proteins in human leukemia cells by protein kinase C activators: relationship to effects on 1-[ -D-arabinofuranosyl]cytosine-induced apoptosis
Tina M. Bartimole1,
Julie A. Vrana1,
Alex J. Freemerman1,
W. David Jarvis1,
John C. Reed4,
Lawrence H. Boise3 & Steven Grant2,5
1Department of Medicine, Medical College of Virginia, Richmond, Virginia
2Department of Pharmacology and Toxicology and Division of Hematology/Oncology, Medical College of Virginia, MCV Station Box 230, Richmond 23298, Virginia
3Department of Microbiology and Immunobiology, University of Miami, Miami, Florida
4The Burnham Institute, Cancer Research Centre, La Jolla, California, USA
5Author for correspondence tel: (804) 828-5211; fax: (804) 828-8079
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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-[-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 µM; 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-xL mRNA or protein; levels of Bcl-xS 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-xL, 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. |
