Circumvention of 5‐Fluorouracil Resistance in Human Stomach Cancer Cells by Uracil Phosphoribosyltransferase Gene Transduction

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Abstract
A human stomach cancer cell line with acquired resistance to 5‐fluorouracil (5‐FU), NUGC‐3/5FU/L, has been found to possess reduced ability to convert 5‐FU into active metabolites. We attempted in vitro gene therapy for this 5‐FU‐resistant cell line. NUGC‐3 and NUGC‐3/5FU/L cells were infected with recombinant adenovirus (Ad) containing Escherichia coli uracil phosphoribosyltransferase (UPRT) gene driven by CAG promoter (CA), AdCA‐UPRT, and changes in their 5‐FU metabolism and sensitivity were investigated. Activities of orotate phosphoribosyltransferase increased from 10.2 and 1.56 (nmol/mg protein/30 min) in the uninfected cells of NUGC‐3 and NUGC‐3/5FU/L to 216 and 237, respectively, after the transfection of UPRT gene. The 5‐FU nucleotide level in the acid‐insoluble fraction increased from 7.32 to 15.9 (pmol/mg protein) in NUGC‐3 cells on infection with AdCA‐UPRT, and in NUGC‐3/5FU/L cells it increased from 1.91 to 21.4. The 50% growth‐inhibition concentration (IC50) was 12.7 μmol/liter for NUGC‐3 and much higher than 100 μmol/liter for NUGC‐3/5FU/L, indicating over 8‐fold resistance. NUGC‐3/5FU/L transfected with the UPRT gene showed very high sensitivity to 5‐FU with an IC50 of 3.2 μmol/liter. The high resistance in this metabolic activation‐deficient cell line was thus completely reversed by transduction of an exogenous gene coding for a 5‐FU‐anabolizing enzyme.

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