Deficient MGMT and proficient hMLH1 expression renders gallbladder carcinoma cells sensitive to alkylating agents through G2-M cell cycle arrest

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.