title: RNA silencing of checkpoint regulators sensitizes p53-defective prostate cancer cells to chemotherapy while sparing normal cells.
abstract: p53 is frequently mutated in patients with prostate cancer, especially in those with advanced disease. Therefore, the selective elimination of p53 mutant cells will likely have an impact in the treatment of prostate cancer. Because p53 has important roles in cell cycle checkpoints, it has been anticipated that modulation of checkpoint pathways should sensitize p53-defective cells to chemotherapy while sparing normal cells. To test this idea, we knocked down ataxia telangiectasia mutated (ATM) gene by RNA interference in prostate cancer cell lines and in normal human diploid fibroblasts IMR90. ATM knockdown in p53-defective PC3 prostate cancer cells accelerated their cell cycle transition, increased both E2F activity and proliferating cell nuclear antigen expression, and compromised cell cycle checkpoints, which are normally induced by DNA damage. Consequently, PC3 cells were sensitized to the killing effects of the DNA-damaging drug doxorubicin. Combining ATM knockdown with the Chk1 inhibitor UCN-01 further increased doxorubicin sensitivity in these cells. In contrast, the same strategy did not sensitize either IMR90 or LNCaP prostate cancer cells, both of which have normal p53. However, IMR90 and LNCaP cells became more sensitive to doxorubicin or doxorubicin plus UCN-01 when both p53 and ATM functions were suppressed. In addition, knockdown of the G(2) checkpoint regulators ATR and Chk1 also sensitized PC3 cells to doxorubicin and increased the expression of the E2F target gene PCNA. Together, our data support the concept of selective elimination of p53 mutant cells by combining DNA damage with checkpoint inhibitors and suggest a novel mechanistic insight into how such treatment may selectively kill tumor cells.
authors.affiliation: Département de Biochimie, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Utpal K
authors.initials: UK
authors.lastname: Mukhopadhyay
authors.forename: Adrian M
authors.initials: AM
authors.lastname: Senderowicz
authors.forename: Gerardo
authors.initials: G
authors.lastname: Ferbeyre
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D004171:Diploidy; D004317:Doxorubicin / Q000494:pharmacology*; D005347:Fibroblasts / Q000502:physiology; D016195:G2 Phase; D015870:Gene Expression; D020868:Gene Silencing; D018816:Genes, cdc; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D008297:Male; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis / Q000235:genetics; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D034622:RNA Interference; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000903:Antibiotics, Antineoplastic; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; D016337:RNA, Catalytic; D012334:RNA, Neoplasm; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C427204:hammerhead ribozyme; C054852:7-hydroxystaurosporine; D004317:Doxorubicin; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine