title: Deficient MGMT and proficient hMLH1 expression renders gallbladder carcinoma cells sensitive to alkylating agents through G2-M cell cycle arrest.
abstract: The aim of this study was to assess whether combined evaluation of O6-methylguanine methyltransferase (MGMT) and hMLH1 status determines sensitivity to monofuntional alkylating agents such as N-methyl-N-nitrosourea (MNU) and dacarbazine (DTIC) against gallbladder carcinoma cells. The molecular mechanism behind MGMT and hMLH1 status affecting the cell cycle was also addressed. Using 5 gallbladder cancer carcinoma lines and 1 colon carcinoma cell line (SW48), MGMT and hMLH1 expression was analyzed using RT-PCR and Western blotting. MGMT and hMLH1 status in the 6 cell lines was compared with drug sensitivity to MNU. As a result, cell lines that were MGMT-/hMLH1+ had the highest sensitivity to MNU, compared with MGMT+/hMLH1+ and MGMT-/hMLH1- cells. In flow cytometric analysis, G2-M cell cycle arrest was specifically observed in GB-d1 cells with MGMT-/hMLH1+ and expression of cyclin A and Cdc2 in GB-d1 cells was significantly reduced by MNU treatment, but not observed in KMG-C cells with MGMT+/hMLH1+. Finally, we assessed the in vitro and in vivo effect of the clinically used alkylating agent DTIC in these cells. The highest sensitivity to DTIC was also observed in MGMT-/hMLH1+. In conclusion, MNU suppressed cell proliferation of MGMT-/hMLH1+ gallbladder carcinoma cells by arresting the cell cycle at the G2-M phase, accompanied by down-regulation of cyclin A and Cdc2. These results indicated that expression of MGMT and hMLH1 could be used to select candidates for alkylating agent chemotherapy against gallbladder carcinoma.
authors.affiliation: Department of Surgery, Saga University Faculty of Medicine, Saga 849-8501, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Sato
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Kitajima
authors.forename: Naohiko
authors.initials: N
authors.lastname: Kohya
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyoshi
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Koga
authors.forename: Kohji
authors.initials: K
authors.lastname: Miyazaki
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D002352:Carrier Proteins; D002455:Cell Division / Q000187:drug effects*; D045744:Cell Line, Tumor; D003606:Dacarbazine / Q000494:pharmacology / Q000627:therapeutic use; D005260:Female; D005434:Flow Cytometry; D016195:G2 Phase / Q000187:drug effects*; D005706:Gallbladder Neoplasms / Q000737:chemistry / Q000188:drug therapy* / Q000473:pathology; D006801:Humans; D008770:Methylnitrosourea / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000032:analysis* / Q000235:genetics; D009374:Neoplasms, Experimental / Q000188:drug therapy; D009687:Nuclear Proteins / Q000032:analysis* / Q000235:genetics; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018906:Antineoplastic Agents, Alkylating; D002352:Carrier Proteins; C501486:MLH1 protein, human; C501485:Mlh1 protein, mouse; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D008770:Methylnitrosourea; D003606:Dacarbazine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D000070957:MutL Protein Homolog 1