Gastroenterology

Gastroenterology

Volume 117, Issue 1, July 1999, Pages 123-131
Gastroenterology

Alimentary Tract
Mismatch repair proficiency and in vitro response to 5-fluorouracil,☆☆

https://doi.org/10.1016/S0016-5085(99)70558-5Get rights and content

Abstract

Background & Aims: The DNA mismatch repair (MMR) system recognizes certain DNA adducts caused by alkylation damage in addition to its role in recognizing and directing repair of interstrand nucleotide mismatches and slippage mistakes at microsatellite sequences. Because defects in the MMR system can confer tolerance to acquired DNA damage and, by inference, the toxic effects of certain chemotherapeutic agents, we investigated the effect of 5-fluorouracil (5-FU) on colon cancer cell lines. Methods: We determined growth selection by cell enrichment assay and cloning efficiency after treatment with 5 μmol/L 5-FU, assayed nucleic 3H–5-FU incorporation, and analyzed the cell cycle by flow cytometry. Results: 5-FU treatment provided a growth advantage for MMR-deficient cell lines, indicating a relative degree of tolerance to 5-FU by the MMR-deficient cell lines. Enhanced survival was statistically significant after 5 days of growth, and a 28-fold reduction in survival was noted in the MMR-proficient cells by clonagenic assays after 10 days of growth. Differences in nucleotide uptake of 5-FU did not account for the observed growth differences, and specific cell cycle checkpoint arrest was not detected. Conclusions: Intact DNA MMR seems to recognize 5-FU incorporated into DNA but may do so in a different manner than other types of alkylation damage. Defective DNA MMR might be one mechanism for tumor resistance to 5-FU.

GASTROENTEROLOGY 1999;117:123-131

Section snippets

Reagents

5-FU was obtained from Sigma Chemical Co. (St. Louis, MO) and dissolved in Iscove's modified Dulbecco's medium at a stock concentration of 1 mmol/L and maintained at 4°C. 3H–5-FU was obtained from Sigma and stored according to the manufacturer's instructions.

Cell lines and cultures

The human colon cancer cell lines HCT116, SW480, and LoVo were obtained from American Type Culture Collection (Rockville, MD) and maintained in growth medium containing 10% fetal bovine serum (FBS). The HCT116 cell lines containing

MMR-corrected HCT116+ch3 cells are tumorigenic in nude mice

To assess the tumorigenicity of the chromosome 3–transferred cell lines before the in vitro treatment with 5-FU, HCT116+ch3 cells were injected into nude mice. HCT116+ch3 cells do not exhibit microsatellite instability,34 possess a full-length hMLH1 protein by in vitro transcription/translation assay, and have reduced tolerance to alkylation damage compared with HCT116 cells.23 Despite a prolongation in the in vitro doubling time for both the HCT116+ch2 and HCT116+ch3 cell lines (20.7 and 25.3

Discussion

The DNA MMR system repairs strand-to-strand base mispairs, slippage mistakes at microsatellite sequences,1, 2 and recognizes certain DNA adducts caused by alkylation damage.23, 24, 25 It has been suggested that the MMR system may be the afferent limb for the detection of certain types of DNA damage.44 We have found that the widely used agent 5-FU is selectively more cytotoxic for MMR-proficient cells than for MMR-deficient cells. Cytotoxicity with 5-FU was shown with a cell enrichment assay in

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    Supported by National Institutes of Health grants DK02433 and CA72851, the Robert Wood Johnson Foundation, and the Veterans Affairs Research Service.

    ☆☆

    Address requests for reprints to: John M. Carethers, M.D., Department of Medicine, 0688, 4028 Basic Science Building, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0688. e-mail: [email protected]; fax: (619) 822-0301.

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