EVIDENCE FOR INVOLVEMENT OF MITOGEN-ACTIVATED PROTEIN-KINASE, RATHER THAN STRESS-ACTIVATED PROTEIN-KINASE, IN POTENTIATION OF 1-BETA-D-ARABINOFURANOSYLCYTOSINE-INDUCED APOPTOSIS BY INTERRUPTION OF PROTEIN-KINASE-C SIGNALING

The stress-activated protein kinase (SAPK) and mitogen-activated prote in kinase (MAPK) cascades mediate cytotoxic and cytoprotective functio ns, respectively, in the regulation of leukemic cell survival. Involve ment of these signaling systems in the cytotoxicity of 1-beta-D-arabin ofuranosylcytosine (ara-C) and modulation of ara-C lethality by protei n kinase C PKC inhibition/down-regulation was examined in HL-60 promye locytic leukemia cells. Exposure to ara-C (10 mu M) for 6 hr promoted extensive apoptotic DNA damage and cell death, as well as activation o f PKC. This response was accompanied by downstream activation of the S APK and MARK cascades. PKC-dependent MARK activity seemed to limit ara -C action in that the toxicity of ara-C was enhanced by pharmacologica l reductions of PKC, MAPK, or both. Thus, ara-C action was (1) partial ly attenuated by diradylglycerols, which stimulated PKC and MAPK, but (2) dramatically amplified by sphingoid bases, which inhibited PKC and MAPK. The cytotoxicity of ara-C also was substantially increased by p harmacological reductions of PKC, including down-regulation of PKC by chronic preexposure to the macrocyclic lactone bryostatin 1 or inhibit ion of PKC by acute coexposure to the dihydrosphingosine analog safing ol. Significantly, both of these manipulations prevented activation of MAPK by ara-C. Moreover, acute disruption of the MAPK module by AMF, a selective inhibitor of MEK1, suppressed both basal and drug-stimulat ed MAPK activity and sharply increased the cytotoxicity of ara-C, sugg esting the direct involvement of MARK as a downstream antiapoptotic ef fector for PKC. None of these chemopotentiating agents enhanced ara-CT P formation. Ceramide-driven SAPK activity did not seem to mediate dru g-induced apoptosis, given that (1) neutralization of endogenous tumor necrosis factor-alpha with monoclonal antibodies or soluble tumor nec rosis factor receptor substantially reduced ceramide generation and SA PK activation by ara-C, whereas the induction of apoptosis was unaffec ted; (2) pharmacological inhibition of sphingomyelinase by 3-O-methoxy sphingomyelin reduced ceramide generation and SAPK activation without limiting the drug's cytotoxicity; and (3) potentiation of ara-C action by bryostatin 1 or safingol was not associated with further stimulati on of SAPK. These observations collectively suggest a primary role for decreased MAPK, rather than increased SAPK, in the potentiation of ar a-C cytotoxicity by interference with PKC-dependent signaling.