Abrogation of an S-phase checkpoint and potentiation of camptothecin cytotoxicity by 7-hydroxystaurosporine (UCN-01) in human cancer cell lines, possibly influenced by p53 function.
7-Hydroxystaurosporine (UCN-01) is a selective protein kinase C inhibitor in clinical trial for cancer treatment. In this study, we found that nanomolar concentrations of camptothecin (CPT), a topoisomerase I inhibitor, arrest or delay cell cycle progression during the S and G2 phases in p53 mutant human colon carcinoma HT29 cells and that UCN-01 abrogates the S-phase arrest or delay induced by CPT. Under these conditions, CPT increased cyclin A levels and cyclin A/cyclin-dependent kinase 2 activity. UCN-01 prevented the increase of cyclin A/cyclin-dependent kinase 2 activity induced by CPT and enhanced Cdc2 kinase activity. Replication protein A (RPA2) was hyperphosphorylated after CPT treatment, and this effect was also abrogated by UCN-01. UCN-01 potentiated the cytotoxicity of CPT and reduced by 6-fold the concentration of CPT required to kill 50% of the HT-29 cells, as determined by clonogenic assays. This effect was observed at concentrations of UCN-01 that alone were not cytotoxic and had no detectable effect on cell cycle progression. UCN-01 markedly potentiated the cytotoxicity of CPT also in HCT116/E6 and MCF-7/ADR cells defective for p53 function, whereas significantly less potentiation was observed in p53-wild-type HCT116 and MCF-7 cells. These results suggest the existence of an S-phase checkpoint that delays replication and that may extend the time available for DNA repair. Thus, pharmacological abrogation of CPT-induced S- and G2-phase checkpoints by UCN-01 may provide an effective strategy for enhancing the chemotherapeutic activity of CPT, particularly against p53-defective tumors.
Shao
R G
RG
Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Cao
C X
CX
Shimizu
T
T
O'Connor
P M
PM
Kohn
K W
KW
Pommier
Y
Y
D000470:Alkaloids / Q000008:administration & dosage*; D015153:Blotting, Western; D002166:Camptothecin / Q000008:administration & dosage*; D018797:Cell Cycle Proteins / Q000378:metabolism; D004273:DNA, Neoplasm / Q000096:biosynthesis; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000008:administration & dosage; D016158:Genes, p53; D006801:Humans; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D016196:S Phase / Q000187:drug effects*; D019311:Staurosporine / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*
D000470:Alkaloids; D018797:Cell Cycle Proteins; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D016159:Tumor Suppressor Protein p53; C054852:7-hydroxystaurosporine; D011493:Protein Kinase C; D019311:Staurosporine; D002166:Camptothecin