DNA mismatch repair proficiency executing 5-fluorouracil cytotoxicity in colorectal cancer cells

Background: 5-fluorouracil (5-FU)-based chemotherapy is the standard treatment for advanced stage colorectal cancer (CRC) patients. Several groups including ours have reported that stage II-III colorectal cancer patients whose tumors retain DNA Mismatch repair (MMR) function derive a benefit from 5-FU, but patients with tumors that lost MMR function do not. Although, MMR recognition of 5-FU incorporated in DNA has been demonstrated biochemically, it has not been demonstrated within cells to execute 5-FU cytotoxicity. Aim: To establish an efficient construction model for 5-FU within DNA and demonstrate that 5-FU incorporated into DNA can trigger cellular cytotoxicity executed by the DNA MMR system. Methods: We constructed a 5FdU-containing heteroduplex plasmid (5FdU plasmid) and 5FdU-containing linear ds-DNA (5FdU linear DNA), and transfected these into MMR-proficient, hMLH1(-/-) and hMSH6(-/-) cells. We observed cell growth characteristics of both transfectants for 5-FU-induced cytotoxicity. Results: MMR-proficient cells transfected with the 5FdU plasmid but not the 5FdU linear DNA showed reduced cell proliferation by MTS and clonogenic assays, and demonstrated cell morphological change consistent with apoptosis. In MMR-deficient cells, neither the 5FdU plasmid nor 5FdU linear DNA induced cell growth or morphological changes different from controls. Conclusion: 5FdU as heteroduplex DNA in plasmid but not linear form triggered cytotoxicity in a MMR-dependent manner. Thus 5-FU incorporated into DNA, separated from its effects on RNA, can be recognized by DNA MMR to trigger cell death.