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 potent ial role of protein kinase C (PKC) in the regulation of apoptosis in H L-60 promyelocytic leukemia cells. The capacity of acute exposure to s pecific and nonspecific pharmacological inhibitors of PKC to promote a poptotic DNA fragmentation was examined both quantitatively and qualit atively and correlated with effects on cellular differentiation and pr oliferation. Incubation of HL-60 cells for 6 h with chelerythrine and calphostin C (highly specific inhibitors that act at the regulatory do main) or H7 and gossypol (nonspecific inhibitors that act at the PKC c atalytic domain) produced concentration-dependent increases in DNA fra gmentation. Induction of DNA fragmentation by chelerythrine, calphosti n C, and gossypol was biphasic, resulting in a sharp decline in effect at concentrations above 5 muM, 0.1 muM, and 100 muM, respectively, wh ereas maximal and more stable effects were observed in response to H7 (100 muM). A 6-h exposure to staurosporine, a nonspecific but potent P KC inhibitor, failed to induce DNA fragmentation at concentrations gen erally used to achieve maximal inhibition of enzyme activity (e.g., 50 nM) but promoted fragmentation at considerably higher concentrations (e.g., greater-than-or-equal-to 200 nM). In contrast, 6-h exposures to the nonspecific protein kinase inhibitor hypericin (0.1 to 100 muM) o r to the nonspecific inhibitor of protein kinase A, HA1004 (50 muM), w ere without effect on DNA fragmentation. DNA obtained from cells expos ed to chelerythrine (5 muM), calphostin C (100 nM), H7 (50 muM), gossy pol (50 muM), and staurosporine (200 nM)-but not hypericin (25 muM)-ex hibited clear evidence of internucleosomal DNA cleavage on agarose gel electrophoresis; moreover, these cells exhibited the classical morpho logical features of apoptosis (cell shrinkage, nuclear condensation, a nd 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 concentration s 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 nonspecifi c PKC inhibitors possessed predominantly, 5'-phosphate termini, consis tent with the action of a Ca2+-/Mg2+-dependent endonuclease. Finally, Northern blot analysis revealed that exposure to calphostin C at a con centration that induced apoptosis (100 nM) failed to alter expression of bcl-2, an oncogene known to block apoptosis in both lymphoid and my eloid leukemia cells. Together, these observations suggest that certai n inhibitors of PKC, administered alone and on a transient basis, are capable of inducing apoptotic DNA fragmentation and cell death in HL-6 0 promyelocytic leukemia cells in a highly concentration-dependent man ner. These findings also suggest a protective involvement of basal PKC activity in the regulation or programmed cell death in myeloid leukem ia cells.