UCN-01, A POTENT ABROGATOR OF G(2) CHECKPOINT FUNCTION IN CANCER-CELLS WITH DISRUPTED P53

Background: Arrest of the cell cycle in G(2) phase following DNA damag e helps protect cell viability by allowing time for DNA repair before entry into mitosis (M phase), Abrogation of G(2) arrest sensitizes cel ls to the effects of DNA-damaging agents, UCN-01 (7-hydroxystaurospori ne), a protein kinase C inhibitor that may block G(2) checkpoint regul ation, has been reported to enhance the cytotoxicity of mitomycin C, a known DNA-damaging agent, Purpose: We studied the effect of UCN-01 on G(2) checkpoint control in human lymphoma CA46 cells, whose sensitivi ty to various DNA-damaging agents and G(2) response to DNA damage have been characterized, We also assessed the ability of UCN-01 to enhance the cytotoxicity of gamma irradiation in CA46 cells and human colon c arcinoma HT-29 cells, both of which are mutant for p53 function, The i nfluence of p53 function on UCN-01-mediated abrogation of the G(2) che ckpoint and enhancement of DNA-damaging agent cytotoxicity was studied in transfected human breast carcinoma MCF-7 cells that either express ed or did not express the human papillomavirus type-16 E6 protein, MCF -7 cells have normal p53 function, and the E6 protein binds p53 protei n and promotes its destruction. Methods: The effect of UCN-01 on cell cycle arrest induced by gamma irradiation was studied in CA46 cells an d in transfected MCF-7 cells by use of flow cytometry, A histone H1 ph osphorylation assay was employed to measure cyclin B1/Cdc2 kinase acti vity in extracts derived from irradiated and nonirradiated CA46 cells that had been either treated or not treated with UCN-01; the phosphory lation status of Cdc2 kinase protein in the same extracts was determin ed by use of western blotting, The effect of UCN-01 on the cytotoxicit y of gamma irradiation in CA46 and HT-29 cells was determined by use o f MTT (thiazolyl blue) and clonogenic (colony-forming) assays, respect ively; a clonogenic assay was also used to measure the effect of UCN-0 1 on the cytotoxicity of cisplatin in transfected and nontransfected M CF-7 cells, Results: G(2) arrest induced in CA46 cells by gamma irradi ation was inhibited by treatment with UCN-01 in a dose-dependent manne r; arrest in G(2) was completely abrogated by exposure to 300 nM UCN-0 1. Biochemical markers indicative of the G(2)/M transition, including the activation of cyclin B1/Cdc2 kinase and the suppression of Cdc2 th reonine-14 and tyrosine-15 phosphorylation, were detected in irradiate d cells treated with UCN-01, UCN-01 enhanced the cytotoxicity of gamma irradiation in CA46 and HT-29 cells, MCF-7 cells with functional p53 protein were more resistant to G(2) checkpoint abrogation by UCN-01 th an MCF-7 cells with disrupted p53 function, UCN-01 markedly enhanced t he cell-killing activity of cisplatin in MCF-7 cells defective for p53 function, Conclusions and Implications: UCN-01 is a potent abrogator of G(2) checkpoint control in cancer cells with disrupted p53 function , UCN-01 might be capable of enhancing the effectiveness of DNA-damagi ng agents in the treatment of tumors with cells lacking normal p53 fun ction.