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 i nhibitor than staurosporine. UCN-01 exhibits antitumor activity in exp erimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carci noma HT29 cells undergo internu cleosomal DNA fragmentation and morpho logical changes characteristic of apoptosis after UCN-01 treatment. Th ese three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cel ls occurred after 1 day of treatment while it took less than 4 h in KL 60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromole cular synthesis is selectively required for apoptotic DNA fragmentatio n in HT29 cells. UCN-01-induced DNA fragmentation was preceded by acti vation 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 cyst eine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, pr otected HL60 cells from UCN-01-induced DNA fragmentation. However, onl y 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 r ole in the execution phase of apoptosis in HL60 cells treated with UCN -01. Z-VAD-FRIK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor age nt, UCN-01 is a potent apoptosis inducer in cell lines that are usuall y resistant to apoptosis and lack p53 and that caspases and probably s erine proteases are activated during UCN-01-induced apoptosis. (C) 199 7 Academic Press.