PT  - JOURNAL ARTICLE
AU  - Shujie Wang
AU  - Julie A. Vrana
AU  - Tina M. Bartimole
AU  - Alex J. Freemerman
AU  - W. David Jarvis
AU  - Lora B. Kramer
AU  - Geoffrey Krystal
AU  - Paul Dent
AU  - Steven Grant
TI  - Agents that Down-Regulate or Inhibit Protein Kinase C Circumvent Resistance to 1-β-<span class="sc">D</span>-Arabinofuranosylcytosine-Induced Apoptosis in Human Leukemia Cells that Overexpress Bcl-2
AID  - 10.1124/mol.52.6.1000
DP  - 1997 Dec 01
TA  - Molecular Pharmacology
PG  - 1000--1009
VI  - 52
IP  - 6
4099  - http://molpharm.aspetjournals.org/content/52/6/1000.short
4100  - http://molpharm.aspetjournals.org/content/52/6/1000.full
SO  - Mol Pharmacol1997 Dec 01; 52
AB  - 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-[β-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 counterparts (HL-60/pCEP4) but comparable levels of Bax, Mcl-1, and Bcl-xL. After exposure to an equimolar concentration of ara-C (10 μm 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.