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UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53
1Laboratory of Biological Chemistry, Division of Basic Science, National Cancer Institute, Bethesda, MD, USA.
Background:
Arrest of the cell cycle in G2 phase following DNA damage helps protect cell viability by allowing time for DNA repair before entry into mitosis (M phase). Abrogation of G2 arrest sensitizes cells to the effects of DNA-damaging agents. UCN-01 (7-hydroxystaurosporine), a protein kinase C inhibitor that may block G2 checkpoint regulation, has been reported to enhance the cytotoxicity of mitomycin C, a known DNA-damaging agent.
Purpose:
We studied the effect of UCN-01 on G2 checkpoint control in human lymphoma CA46 cells, whose sensitivity to various DNA-damaging agents and G2 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 carcinoma HT-29 cells, both of which are mutant for p53 function. The influence of p53 function on UCN-01-mediated abrogation of the G2 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.
Methods:
The effect of UCN-01 on cell cycle arrest induced by gamma 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 gamma 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.
Results:
G2 arrest induced in CA46 cells by gamma irradiation was minibited by treatment with UCN-01 in a dose-dependent manner; arrest in G2 was completely abrogated by exposure to 300 nM UCN-01. Biochemical markers indicative of the G2/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 gamma irradiation in CA46 and HT-29 cells. MCF-7 cells with functional p53 protein were more resistant to G2 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.
Conclusions And Implications:
UCN-01 is a potent abrogator of G2 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.
Insights
UCN-01 abrogates the G2 cell cycle checkpoint, enhancing cancer cell sensitivity to DNA-damaging agents like gamma irradiation and cisplatin. This effect is more pronounced in cells with disrupted p53 function, suggesting UCN-01 as a potential therapeutic enhancer.
Area of Science:
- Cell cycle regulation
- DNA damage response
- Cancer therapy
Background:
- Cell cycle arrest in G2 phase after DNA damage allows for repair, protecting cell viability.
- Abrogation of G2 arrest sensitizes cells to DNA-damaging agents.
- UCN-01 (7-hydroxystaurosporine), a protein kinase C inhibitor, may disrupt G2 checkpoint control and enhance DNA-damaging agent cytotoxicity.
Purpose of the Study:
- To investigate UCN-01's effect on G2 checkpoint control in human lymphoma CA46 cells.
- To assess UCN-01's ability to enhance cytotoxicity of gamma irradiation in p53-mutant CA46 and HT-29 cells.
- To determine the influence of p53 function on UCN-01-mediated G2 checkpoint abrogation and cytotoxicity enhancement.
Main Methods:
- Flow cytometry to study UCN-01's effect on gamma irradiation-induced G2 arrest.
- Histone H1 phosphorylation assay and western blotting to measure cyclin B1/Cdc2 kinase activity and Cdc2 phosphorylation status.
- MTT and clonogenic assays to determine UCN-01's effect on cytotoxicity of gamma irradiation and cisplatin.
Main Results:
- UCN-01 dose-dependently abrogated gamma irradiation-induced G2 arrest in CA46 cells.
- Biochemical markers of G2/M transition were detected in UCN-01-treated irradiated cells.
- UCN-01 enhanced cytotoxicity of gamma irradiation and cisplatin, particularly in cells with disrupted p53 function.
Conclusions:
- UCN-01 is a potent abrogator of G2 checkpoint control in cancer cells with compromised p53 function.
- UCN-01 holds potential for enhancing DNA-damaging agent efficacy in treating tumors lacking normal p53.
- Targeting the G2 checkpoint with UCN-01 may improve cancer treatment outcomes.