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

Abstract

BackgroundPatients 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 hMutSα binds 5-FU incorporated into DNA. Although hMutSß 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 hMutSß plays a role in 5-FU recognition.MethodsWe 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 hMutSα and hMutSß in electromobility shift assays (EMSA) and further analyzed binding using surface plasmon resonance.ResultsMTT and clonogenic assays after 5-FU treatment demonstrated the most cytotoxicity in cells with both hMutSα and hMutSß, intermediate cytotoxicity in cells with hMutSα alone, and the least cytotoxicity in cells with hMutSß alone, hMutSß binds 5-FU-modified DNA, but its relative binding is less than the binding of 5-FU-modified DNA by hMutSα.ConclusionCytotoxicity induced by 5-FU is dependent on intact DNA MMR, with relative cell death correlating directly with hMutSα and/or hMutSß 5-FU binding ability (hMutSα>hMutSß). 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.

Microsatellite Instability as a Biomarker for PD-1 Blockade

“…Although MSS patients showed a benefit from 5-FU treatment, MSI patients did not (46), a finding confirmed in smaller follow-up studies (47,48). Laboratory-based investigations have suggested that components of the MMR machinery bind to 5-FU incorporated DNA, raising the possibility that they mediate the observed cytotoxic response (49,50). Further clinical studies have generally substantiated the finding that MSI patients do not benefit from 5-FU therapy, with some exceptions (51).…”

Microsatellite Instability Pathway and EMAST in Colorectal Cancer

“…The MMR recognition complexes recognize and bind to ingested or parenteral 5-fluorouracil (5FU) and are then converted and incorporated into DNA. Both MSH2-MSH6 and MSH2-MSH3 complexes can bind 5FU within DNA and trigger cell death [52–57]. When the MMR recognition complexes are not present, cell death does not occur and cells may proceed with mitosis with 5FU-induced and other novel mutations contained within DNA.…”

GRG Profiles: John M. Carethers

“…We later showed that this had an effect on patient survival from colorectal cancer, as patients with a defective DNA mismatch repair system did not show a survival benefit with adjuvant 5-FU treatment [10]. We worked out the mechanism, showing that individual complexes from DNA mismatch repair had specific binding affinities for 5-FU incorporated into DNA [11, 12] and that isolating the DNA component of 5-FU (from the RNA component) proved that DNA mismatch repair could trigger cell death once it recognizes 5-FU [13]. Talented postdocs Akihiro Tajima, Moriya Iwaizumi, and Yasushi Hamaya [14] were key in figuring out this important process.…”