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 protein kinase (MAPK) cascades mediate cytotoxic and cytoprotective functions, respectively, in the regulation of leukemic cell survival. Involvement of these signaling systems in the cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) and modulation of ara-C lethality by protein kinase C PKC inhibition/down-regulation was examined in HL-60 promyelocytic leukemia cells. Exposure to ara-C (10 microM) for 6 hr promoted extensive apoptotic DNA damage and cell death, as well as activation of PKC. This response was accompanied by downstream activation of the SAPK and MAPK cascades. PKC-dependent MAPK activity seemed to limit ara-C action in that the toxicity of ara-C was enhanced by pharmacological reductions of PKC, MAPK, or both. Thus, ara-C action was (1) partially 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 pharmacological reductions of PKC, including down-regulation of PKC by chronic preexposure to the macrocyclic lactone bryostatin 1 or inhibition of PKC by acute coexposure to the dihydrosphingosine analog safingol. 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-stimulated MAPK activity and sharply increased the cytotoxicity of ara-C, suggesting the direct involvement of MAPK as a downstream antiapoptotic effector for PKC. None of these chemopotentiating agents enhanced ara-CTP formation. Ceramide-driven SAPK activity did not seem to mediate drug-induced apoptosis, given that (1) neutralization of endogenous tumor necrosis factor-alpha with monoclonal antibodies or soluble tumor necrosis factor receptor substantially reduced ceramide generation and SAPK activation by ara-C, whereas the induction of apoptosis was unaffected; (2) pharmacological inhibition of sphingomyelinase by 3-O-methoxysphingomyelin 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 stimulation of SAPK. These observations collectively suggest a primary role for decreased MAPK, rather than increased SAPK, in the potentiation of ara-C cytotoxicity by interference with PKC-dependent signaling.
Jarvis
W D
WD
Department of Medicine, Medical College of Virginia, Richmond, Virginia 23298, USA.
Fornari
F A
FA
Tombes
R M
RM
Erukulla
R K
RK
Bittman
R
R
Schwartz
G K
GK
Dent
P
P
Grant
S
S
D000964:Antimetabolites, Antineoplastic / Q000378:metabolism / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D054713:Bryostatins; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D003561:Cytarabine / Q000378:metabolism / Q000494:pharmacology*; D004075:Diglycerides / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D018922:HL-60 Cells / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D006801:Humans; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013237:Stereoisomerism
D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D054713:Bryostatins; D004075:Diglycerides; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007783:Lactones; D018942:Macrolides; D047428:Protein Kinase Inhibitors; D003561:Cytarabine; C046785:bryostatin 1; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013110:Sphingosine; C005682:safingol; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one