Mismatch repair proficiency and in vitro response to 5-fluorouracil

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 micromol/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.

Figure 1

Tumorigenicity of HCT116 (●), HCT116+ch2 (■), and HCT116+ch3 (▲) cell lines. Each cell line was grown in Dulbecco’s modified Eagle medium with 10% FBS in 7% CO₂ and injected into the left flank of 5 BALB/c NCR-NU athymic mice, performed in duplicate. Tumor volume was calculated at each time point by the method in Kwan et al. Each point represents the mean ± SD.

Figure 2

Colony-forming ability of MMR-deficient and -proficient cell lines in response to 5-FU. Cells were plated in media containing 0, 1, 2.5, or 5 μmol/L 5-FU and allowed to form colonies over a 10-day period. The plates were then fixed with methanol and stained with 3% Giemsa, and previously viable colonies were counted. The mean number of colonies obtained after 10 days for untreated cells were (plating factor of 10³): HCT116, 390; HCT116+ch2, 438; HCT116+ch3, 602.5; SW480, 610; LoVo, 176; and 2774, 385. Results are expressed as the mean ± SE of the relative surviving fraction. Data are from 5 independent experiments. MMR-proficient cell lines are noted with the asterisk.

Figure 3

Incorporation of ³H–5-FU into nucleic acids of HCT116, HCT116+ch2, and HCT116+ch3 cell lines. Cells were coincubated with 1 μmol/L ³H–5-FU and 10 μCi/mL of H₃-³²PO₄ for 24 hours. The nucleic acids were separated by a cesium chloride gradient and fractionated. Linear regression was determined by cesium chloride density for each fraction. The ³H–5-FU and H₃-³²PO₄ counts banding in the RNA region (between density 1.62 and 1.68 g/mL) and DNA region (between density 1.42 and 1.48 g/mL) of the gradient were determined and used as a measure of the incorporation of 5-FU and phosphate. The RNA to DNA ratio is the total tritium counts per minute from the RNA fraction divided by the total counts per minute from the DNA fraction. Results are from 2 independent experiments.