7-Hydroxystaurosporine (UCN-01) induces apoptosis in human colon carcinoma and leukemia cells independently of p53.
7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.
Shao
R G
RG
Laboratory of Molecular Pharmacology, Division of Basic Science, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Shimizu
T
T
Pommier
Y
Y
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D000470:Alkaloids / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D016203:CDC2 Protein Kinase / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology*; D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018922:HL-60 Cells; D019073:HT29 Cells; D006801:Humans; D034921:Lamin Type B; D034882:Lamins; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism / Q000473:pathology*; D009687:Nuclear Proteins / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D019311:Staurosporine / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*
D000470:Alkaloids; C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D011500:Protein Synthesis Inhibitors; D015842:Serine Proteinase Inhibitors; D016159:Tumor Suppressor Protein p53; C054852:7-hydroxystaurosporine; D003513:Cycloheximide; D011065:Poly(ADP-ribose) Polymerases; D011493:Protein Kinase C; D016203:CDC2 Protein Kinase; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D019311:Staurosporine