Agents that down-regulate or inhibit protein kinase C circumvent resistance to 1-beta-D-arabinofuranosylcytosine-induced apoptosis in human leukemia cells that overexpress Bcl-2.
The effects of the non-tumor-promoting protein kinase C (PKC) activator bryostatin 1 and the PKC inhibitors staurosporine and UCN-01 were examined with respect to modulation of 1-[beta-D-arabinofuranosyl]cytosine (ara-C)-induced apoptosis in human myeloid leukemia cells (HL-60) overexpressing the antiapoptotic protein Bcl-2. HL-60/Bcl-2 cells displayed a 5-fold increase in Bcl-2 protein compared with empty-vector counter-parts (HL-60/pCEP4) but comparable levels of Bax, Mcl-1, and Bcl-xL. After exposure to an equimolar concentration of ara-C (10 microM for 6 hr), HL-60/Bcl-2 cells were significantly less susceptible to apoptosis, DNA fragmentation, and loss of clonogenicity than HL-60/pCEP4 cells. The protective effect of increased Bcl-2 expression was manifested by a failure of ara-C to induce activation/cleavage of the Yama protease (CPP32; caspase-3) and degradation of one of its substrates, poly(ADP-ribose)polymerase to an 85-kDa cleavage product. When HL-60/Bcl-2 cells were preincubated with bryostatin 1 (10 nM; 24 hr) or coincubated with either staurosporine (50 nM; 6 hr) or UCN-01 (300 nM; 6 hr) after a 1-hr preincubation, exposures that exerted minimal effects alone, ara-C-induced apoptosis and DNA fragmentation were restored to levels equivalent to, or greater than, those observed in empty-vector controls. These events were accompanied by restoration of the ability of ara-C to induce CPP32 cleavage and activation, poly(ADP-ribose) polymerase degradation, and inhibition of colony formation. Western analysis of Bcl-2 protein obtained from overexpressing cells treated with bryostatin 1, staurosporine, or UCN-01 revealed the appearance of a slowly migrating species and a general broadening of the protein band, effects that were insensitive to the protein synthesis inhibitor cycloheximide. Alterations in Bcl-2 protein mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were reversed by treatment of lysates with alkaline phosphatase or protein phosphatase 2A; actions of the latter were blocked by the specific phosphatase inhibitor okadaic acid. In vivo labeling studies of Bcl-2 protein demonstrated increased incorporation of [32PO4]orthophosphate in drug-treated cells. Last, phosphorylated Bcl-2 failed to display decreased binding to the proapoptotic protein Bax. Collectively, these findings indicate that bryostatin 1, which down-regulates PKC, and staurosporine and UCN-01, which directly inhibit the enzyme, circumvent resistance of Bcl-2-overexpressing leukemic cells to ara-C-induced apoptosis and activation of the protease cascade. They also raise the possibility that modulation of Bcl-2 phosphorylation status contributes to this effect.
Wang
S
S
Department of Medicine, Medical College of Virginia, Richmond 23298, USA.
Vrana
J A
JA
Bartimole
T M
TM
Freemerman
A J
AJ
Jarvis
W D
WD
Kramer
L B
LB
Krystal
G
G
Dent
P
P
Grant
S
S
D000470:Alkaloids / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D054713:Bryostatins; D003561:Cytarabine / Q000494:pharmacology*; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D010450:Endopeptidases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018922:HL-60 Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology / Q000031:analogs & derivatives
D000470:Alkaloids; D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D054713:Bryostatins; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D007783:Lactones; D018942:Macrolides; D019253:Proto-Oncogene Proteins c-bcl-2; D003561:Cytarabine; C046785:bryostatin 1; C054852:7-hydroxystaurosporine; D011493:Protein Kinase C; D010450:Endopeptidases; D019311:Staurosporine