Alimentary TractMismatch repair proficiency and in vitro response to 5-fluorouracil☆,☆☆
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.
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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.