title: 90-kDa heat shock protein inhibition abrogates the topoisomerase I poison-induced G2/M checkpoint in p53-null tumor cells by depleting Chk1 and Wee1.
abstract: The G(2)/M cell cycle checkpoint is regulated by a multitude of signaling pathways after genotoxic stress. Herein, we report that treatment with the 90-kDa heat shock protein (Hsp90) molecular chaperone inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) selectively abrogates the G(2)/M checkpoint induced by 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan, in p53-null compared with p53-intact HCT116 colon cancer cells. The basis for this selectivity can be explained in part by the lack of p21 induction in p53-null cells. In accord with published results, we could show that treatment with 17AAG resulted in depletion of Chk1, a known Hsp90 client protein. In addition, we observed a time- and dose-dependent decrease in Wee1 kinase level, a negative regulator of mitosis, after 17AAG treatment in gastrointestinal cancer cells. Depletion of Wee1 protein preceded mitotic entry induced by 17AAG, and this decrease could be partially rescued by cotreatment with a proteasome inhibitor. Coimmunoprecipitation experiments showed that Hsp90 and Wee1 interacted in whole cells, and 17AAG treatment decreased the degradative half-life of Wee1, indicating that Wee1 is another Hsp90 client in mammalian cells. Knockdown of Chk1 and Wee1 by short interfering RNA each resulted in abrogation of the G(2)/M checkpoint induced by SN-38. The combination of SN-38 and 17AAG was shown to be synergistic in p53-null but not in parental HCT116 cells by median effect/combination index analysis. Taken together, 17AAG specifically inhibits the G(2)/M checkpoint in p53-defective cells by down-regulation of two critical checkpoint kinases, Chk1 and Wee1.
authors.affiliation: Laboratory of New Drug Development, Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. tsea@mskcc.org
authors.forename: Archie N
authors.initials: AN
authors.lastname: Tse
authors.forename: Tahir N
authors.initials: TN
authors.lastname: Sheikh
authors.forename: Ho
authors.initials: H
authors.lastname: Alan
authors.forename: Ting-Chao
authors.initials: TC
authors.lastname: Chou
authors.forename: Gary K
authors.initials: GK
authors.lastname: Schwartz
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D016227:Benzoquinones / Q000494:pharmacology; D002166:Camptothecin / Q000031:analogs & derivatives / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D004264:DNA Topoisomerases, Type I / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D016195:G2 Phase; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D045325:HCT116 Cells; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D000077146:Irinotecan; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013997:Time Factors; D059004:Topoisomerase I Inhibitors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D016227:Benzoquinones; D018797:Cell Cycle Proteins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D009687:Nuclear Proteins; D059004:Topoisomerase I Inhibitors; D016159:Tumor Suppressor Protein p53; C112765:tanespimycin; D000077146:Irinotecan; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin