UCN-01: a Potent Abrogator of G2 Checkpoint Function in Cancer Cells With Disrupted p53

Abstract

Arrest of the cell cycle in G ₂ phase following DNA damage helps protect cell viability by allowing time for DNA repair before entry into mitosis (M phase). Abrogation of G ₂ arrest sensitizes cells to the effects of DNA-damaging agents. UCN-01 (7-hydroxystaurosporine), a protein kinase C inhibitor that may block G ₂ checkpoint regulation, has been reported to enhance the cytotoxicity of mitomycin C, a known DNA-damaging agent. We studied the effect of UCN-01 on G ₂ checkpoint control in human lymphoma CA46 cells, whose sensitivity 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 γ irradiation in CA46 cells and human colon carcinoma HT-29 cells, both of which are mutant for p53 function. The influence of p53 function on UCN-01-mediated abrogation of the G ₂ checkpoint and enhancement of DNA-damaging agent cytotoxicity was studied in transfected human breast carcinoma MCF-7 cells that either expressed or did not express the human papillomavirus type-16 E6 protein. MCF-7 cells have normal p53 function, and the E6 protein binds p53 protein and promotes its destruction. The effect of UCN-01 on cell cycle arrest induced by γ irradiation was studied in CA46 cells and in transfected MCF-7 cells by use of flow cytometry. A histone H1 phosphorylation assay was employed to measure cyclin B1/Cdc2 kinase activity in extracts derived from irradiated and nonirradiated CA46 cells that had been either treated or not treated with UCN-01; the phosphorylation status of Cdc2 kinase protein in the same extracts was determined by use of western blotting. The effect of UCN-01 on the cytotoxicity of γ irradiation in CA46 and HT-29 cells was determined by use of MTT (thiazolyl blue) and clonogenic (colony-forming) assays, respectively; a clonogenic assay was also used to measure the effect of UCN-01 on the cytotoxicity of cisplatin in transfected and nontransfected MCF-7 cells. G ₂ arrest induced in CA46 cells by γ irradiation was inhibited by treatment with UCN-01 in a dose-dependent manner; arrest in G ₂ was completely abrogated by exposure to 300 n M UCN-01. Biochemical markers indicative of the G ₂ /M transition, including the activation of cyclin B1/Cdc2 kinase and the suppression of Cdc2 threonine-14 and tyrosine-15 phosphorylation, were detected in irradiated cells treated with UCN-01. UCN-01 enhanced the cytotoxicity of γ 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 than MCF-7 cells with disrupted p53 function. UCN-01 markedly enhanced the cell-killing activity of cisplatin in MCF-7 cells defective for p53 function. 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-damaging agents in the treatment of tumors with cells lacking normal p53 function. [J Natl Cancer Inst 1996;88:956–65]