title: Decreased expression of hMLH1 correlates with reduced 5-fluorouracil-mediated apoptosis in colon cancer cells.
abstract: Patients with sporadic microsatellite instable colorectal cancers, in most of which the function of the hMLH1 mismatch repair gene is impaired, do not gain a survival benefit from 5-fluorouracil (5-FU)-based chemotherapy. However, the effect of hMLH1 on the cytotoxicity induced by 5-FU has not yet been sufficiently confirmed. In this study, we assessed the effect of hMLH1 on cytotoxicity and apoptosis induced by 5-FU using newly developed cell lines. We constructed two cell lines: SW480 (originally hMLH1-proficient), in which the expression of hMLH1 was reduced using a small interfering RNA (siRNA) technique, and HCT116 (originally hMLH1-deficient), in which the expression of hMLH1 can be regulated by doxycycline. Using these cell lines, a clonogenic survival assay, 4',6-diamidino-2-phenylindole (DAPI) staining and an Annexin-V assay were performed. Moreover, the incorporation of 5-FU into DNA was determined using tritium-labeled 5-FU. In both of our two cell lines, hMLH1-deficient cells exhibited approximately 2.4-fold clonal surviving fraction compared to hMLH1-proficient cells for 10 days after the administration of 5-FU. Additionally, hMLH1-deficient cells treated with 5-FU exhibited 34-45% less apoptosis than hMLH1-proficient cells according to the results of DAPI staining and Annexin-V assay. Furthermore, hMLH1-deficient cells treated with 5-FU exhibited an approximately 2-fold greater incorporation of 5-FU into DNA than control cells, suggesting that the recognition of 5-FU-incorporated DNA is impaired in hMLH1-deficient cells, resulting in reduced apoptosis. Our conclusions were that decreased expression of hMLH1 in colon cancer cells reduced the apoptosis induced by 5-FU, suggesting that hMLH1 is a key determinant of 5-FU chemosensitivity.
authors.affiliation: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Fujita
authors.forename: Jun
authors.initials: J
authors.lastname: Kato
authors.forename: Joichiro
authors.initials: J
authors.lastname: Horii
authors.forename: Keita
authors.initials: K
authors.lastname: Harada
authors.forename: Sakiko
authors.initials: S
authors.lastname: Hiraoka
authors.forename: Hidenori
authors.initials: H
authors.lastname: Shiraha
authors.forename: Kohsaku
authors.initials: K
authors.lastname: Sakaguchi
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Shiratori
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology*; D019175:DNA Methylation; D004260:DNA Repair; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000964:Antimetabolites, Antineoplastic; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; D000070957:MutL Protein Homolog 1; D005472:Fluorouracil