Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C.
The present studies were undertaken to characterize further the potential role of protein kinase C (PKC) in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. The capacity of acute exposure to specific and nonspecific pharmacological inhibitors of PKC to promote apoptotic DNA fragmentation was examined both quantitatively and qualitatively and correlated with effects on cellular differentiation and proliferation. Incubation of HL-60 cells for 6 h with chelerythrine and calphostin C (highly specific inhibitors that act at the regulatory domain) or H7 and gossypol (nonspecific inhibitors that act at the PKC catalytic domain) produced concentration-dependent increases in DNA fragmentation. Induction of DNA fragmentation by chelerythrine, calphostin C, and gossypol was biphasic, resulting in a sharp decline in effect at concentrations above 5 microM, 0.1 microM, and 100 microM, respectively, whereas maximal and more stable effects were observed in response to H7 (100 microM). A 6-h exposure to staurosporine, a nonspecific but potent PKC inhibitor, failed to induce DNA fragmentation at concentrations generally used to achieve maximal inhibition of enzyme activity (e.g., 50 nM) but promoted fragmentation at considerably higher concentrations (e.g., > or = 200 nM). In contrast, 6-h exposures to the nonspecific protein kinase inhibitor hypericin (0.1 to 100 microM) or to the nonspecific inhibitor of protein kinase A, HA1004 (50 microM), were without effect on DNA fragmentation. DNA obtained from cells exposed to chelerythrine (5 microM), calphostin C (100 nM), H7 (50 microM), gossypol (50 microM), and staurosporine (200 nM)--but not hypericin (25 microM)--exhibited clear evidence of internucleosomal DNA cleavage on agarose gel electrophoresis; moreover, these cells exhibited the classical morphological features of apoptosis (cell shrinkage, nuclear condensation, and the formation of apoptotic bodies). All of the PKC inhibitors that induced apoptosis, and one of the inhibitors that did not (hypericin), substantially inhibited HL-60 cell clonogenicity at the concentrations evaluated. None of the agents tested induced cellular maturation as assessed by nonspecific esterase and nitro-blue tetrazolium positivity. DNA fragments obtained from cells exposed to specific and nonspecific PKC inhibitors possessed predominantly 5'-phosphate termini, consistent with the action of a Ca(2+)-/Mg(2+)-dependent endonuclease. Finally, Northern blot analysis revealed that exposure to calphostin C at a concentration that induced apoptosis (100 nM) failed to alter expression of bcl-2, an oncogene known to block apoptosis in both lymphoid and myeloid leukemia cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Jarvis
W D
WD
Department of Medicine, Medical College of Virginia, Richmond 23298-0230.
Turner
A J
AJ
Povirk
L F
LF
Traylor
R S
RS
Grant
S
S
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000633:toxicity; D000873:Anthracenes; D000970:Antineoplastic Agents / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects*; D053119:Benzophenanthridines; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000633:toxicity*; D006072:Gossypol / Q000633:toxicity; D006801:Humans; D007546:Isoquinolines / Q000633:toxicity; D015473:Leukemia, Promyelocytic, Acute; D009281:Naphthalenes; D010569:Perylene / Q000031:analogs & derivatives / Q000633:toxicity; D010617:Phenanthridines / Q000633:toxicity; D010879:Piperazines / Q000633:toxicity; D011083:Polycyclic Compounds / Q000633:toxicity; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D019311:Staurosporine; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D000873:Anthracenes; D000970:Antineoplastic Agents; D053119:Benzophenanthridines; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D009281:Naphthalenes; D010617:Phenanthridines; D010879:Piperazines; D011083:Polycyclic Compounds; D010569:Perylene; C004965:hypericin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C016299:chelerythrine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019311:Staurosporine; C058819:calphostin C; D006072:Gossypol