Both hMutSα and hMutSß DNA mismatch repair complexes participate in 5-fluorouracil cytotoxicity.

Akihiro Tajima,Betty L Cabrera,John M Carethers,Moriya Iwaizumi,Stephanie Tseng-Rogenski,Hassan Ashktorab

doi:10.1371/journal.pone.0028117

Akihiro Tajima, Betty L Cabrera + Show 4 more

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https://doi.org/10.1371/journal.pone.0028117

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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.

Highlights

The fluoropyrimidine 5-fluorouracil (5-FU) is the cornerstone for chemotherapy in patients with advanced stage colorectal cancer [1]
The DNA mismatch repair (MMR) system plays an important role in maintaining DNA fidelity after DNA synthesis for cell replication
Systemic 5-FU therapy leads to incorporation into all forms of RNA, but by its action upon thymidylate synthetase (TS), 5-FU after conversion to a deoxyribonucleic acid serves as a substrate for DNA synthesis with cell depletion of TTPs

Summary

Introduction

The fluoropyrimidine 5-fluorouracil (5-FU) is the cornerstone for chemotherapy in patients with advanced stage colorectal cancer [1]. Patients with Lynch syndrome (germline mutation in MMR gene) or patients with sporadic microsatellite unstable (MSI) cancers (hypermethylation of the MMR gene hMLH1) do not show a survival advantage with systemic 5-FU therapy [3,4,5,6], whereas patients with MMR-proficient tumors improve their survival These observations correlate with 5-FU treatment of MMR-deficient cells in that these cells are resistant to 5-FU [7,8,9], and continue to survive in its presence. Given that 5-FU incorporated into DNA would best simulate a single mispair, we initially predicted that hMutSß would not bind or recognize 5-FU, unlike hMutSa. The presence of hMSH3, the DNA recognition component of hMutSß, is the likely molecule that prevents the occurrence of elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in colorectal cancers, as reduced expression of hMSH3 has been detected among these tumors [25,26]. Our aim was to examine if hMutSß plays a role in 5-FU recognition

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