Both hMutSα and hMutSβ DNA Mismatch Repair Complexes Participate in 5-Fluorouracil Cytotoxicity

Background: Patients with advanced microsatellite unstable colorectal cancers do not show a survival benefit from 5-fluorouracil (5-FU)-based chemotherapy. We and others have shown that the DNA mismatch repair (MMR) complex hMutS alpha binds 5-FU incorporated into DNA. Although hMutS beta is known to interact with interstrand crosslinks (ICLs) induced by drugs such as cisplatin and psoralen, it has not been demonstrated to interact with 5-FU incorporated into DNA. Our aim was to examine if hMutS beta plays a role in 5-FU recognition. Methods: We compared the normalized growth of 5-FU treated cells containing either or both mismatch repair complexes using MTT and clonogenic assays. We utilized oligonucleotides containing 5-FU and purified baculovirus-synthesized hMutS alpha and hMutS beta in electromobility shift assays (EMSA) and further analyzed binding using surface plasmon resonance. Results: MTT and clonogenic assays after 5-FU treatment demonstrated the most cytotoxicity in cells with both hMutS alpha and hMutS beta, intermediate cytotoxicity in cells with hMutSa alone, and the least cytotoxicity in cells with hMutS beta alone, hMutS beta binds 5-FU-modified DNA, but its relative binding is less than the binding of 5-FU-modified DNA by hMutS alpha. Conclusion: Cytotoxicity induced by 5-FU is dependent on intact DNA MMR, with relative cell death correlating directly with hMutS alpha and/or hMutS beta 5-FU binding ability (hMutS alpha>hMutS beta). The MMR complexes provide a hierarchical chemosensitivity for 5-FU cell death, and may have implications for treatment of patients with certain MMR-deficient tumors.