Apoptosis in 7-hydroxystaurosporine-treated T lymphoblasts correlates with activation of cyclin-dependent kinases 1 and 2.
7-Hydroxystaurosporine (UCN-01) is a potent inhibitor of protein kinase C (PKC) isozymes alpha, beta, and gamma [Seynaeve et al., Mol. Pharmacol, 45: 1207-1214, 1994] that also has antitumor effects in vivo. To determine whether inhibition of PKC can be related to inhibition of cell growth with induction of apoptosis, we compared the effects of UCN-01 to those of the highly selective bisindolylmaleimide PKC antagonist GF 109203X in leukemic T-cell lines. Both compounds potently inhibited PKC activity when added to T-cell membrane preparations and reversed phorbol ester-induced c-fos gene expression in intact cells. However, whereas UCN-01 potently inhibited growth of Jurkat, Molt-3, Molt-4, and Hut-78 cells (IC50 = 20-65 nM, irreversible after 24 h of exposure), GF 109203X had IC50s for cell growth of 3.6-5.0 muM. Less than 3 h after addition, UCN-01 but not GF 109203X-treated cells displayed loss of cells with G2-M DNA content, appearance of a hypodiploid DNA fraction, and evidence of internucleosomal DNA fragmentation. Six h after treatment, cells appeared to accumulate with S-phase DNA content. These effects correlated with selective UCN-01 but not GF 109203X-induced decrease in total and tyrosine phosphorylation of cyclin-dependent kinases (cdks) 1 and 2, and with increases in the histone H1 kinase activities of cdk1 and cdk2. UCN-01 was relatively less potent in inhibition of properly activated cdk1 and cdk2 when added in vitro to H1 kinase assays (IC50 = 1000 and 600 nM, respectively). We conclude that inhibition of PKC alone is not sufficient to account for the actions of UCN-01 and are led to the hypothesis that inappropriate cdk activation either correlates with or actually mediates cell growth inhibition with apoptosis in T lymphoblasts exposed to UCN-01.
Wang
Q
Q
Laboratory of Biological Chemistry, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20892, USA.
Worland
P J
PJ
Clark
J L
JL
Carlson
B A
BA
Sausville
E A
EA
D000470:Alkaloids / Q000494:pharmacology*; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects*; D016203:CDC2 Protein Kinase / Q000819:agonists; D042846:CDC2-CDC28 Kinases; D002455:Cell Division / Q000187:drug effects; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000819:agonists*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007938:Leukemia / Q000097:blood / Q000201:enzymology; D008301:Maleimides / Q000494:pharmacology; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D017346:Protein Serine-Threonine Kinases / Q000819:agonists; D019311:Staurosporine / Q000031:analogs & derivatives; D013234:Stem Cells / Q000187:drug effects; D013601:T-Lymphocytes / Q000187:drug effects*; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; C054852:7-hydroxystaurosporine; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D019311:Staurosporine; C070515:bisindolylmaleimide I