Screening of Differentially Expressed Genes in 5‐Fluorouracil‐resistant Human Gastrointestinal Tumor Cells

Abstract

To identify genes differentially expressed in association with resistance to 5-fluorouracil (5FU), an mRNA differential display (DD) analysis was used to compare transcripts from the NUGC-3 human gastric tumor cell line and the NUGC-3/5FU/L line, which had acquired 208-fold resistance as a consequence of repeated exposure to escalating concentrations of 5FU. The 110 cDNA fragments differentially expressed in the DD analysis of either the NUGC-3 or NUGC-3/5FU/L cells were sequenced and subjected to a homology search, and 29 overexpressed and 22 underexpressed genes were identified in NUGC-3/5FU/L as a result. To confirm whether the changes in the gene expression levels in the NUGC-3/5FU/L cells were shared by other 5FU-resistant cells, 35 genes were analyzed by northern hybridization in 3 pairs of parent / 5FU-resistant human gastrointestinal tumor cell lines. The analysis revealed 20 overexpressed and 10 underexpressed genes in at least one of the three 5FU-resistant cells as compared with those in the parent cells. Among them, P-glycoprotein, equilibrative nucleoside transporter 1, and methylenetetrahydrofolate dehydrogenase were highly expressed in two of the three 5FU-resistant cells and cytidine deaminase and integrin α α α α3 were underexpressed. The acquisition of resistance to 5FU by tumor cells may result from multiple changes in cellular functions. Key words:Resistance to 5-fluorouracil -Profiling of gene expression -Human gastrointestinal tumor cells -mRNA differential display -Northern hybridization 5-Fluorouracil (5FU) is widely used in the treatment of solid tumors, but the inherent or acquired resistance of certain tumors to 5FU therapy is a major clinical problem. Various mechanisms of resistance to 5FU have been proposed: overexpression of the target enzyme thymidylate synthase (TS), depletion of folate cofactors, an increase in the level of the competing substrate deoxyuridine monophosphate, altered drug transport/metabolism, and alterations in DNA repair/cell-cycle control pathways. [1][2][3][4][5] Following repeated exposure to escalating concentrations of 5FU, a 5FU-resistant human gastric tumor cell line we established, NUGC-3/5FU/L, developed a 208-fold increase in resistance to 5FU compared with the parent line. Our previous studies 6) showed a significant decrease in the inhibitory effect of 5FU on intracellular TS activity and the incorporation of 5FU into the RNA of NUGC-3/5FU/L cells compared to parent cells. Assays for several enzymes involved in 5FU metabolism or anabolism revealed that the activities of uridine phosphorylase (UP), uridine kinase (UK), and orotate phosphoribosyltransferase (OPRT) were about 70% lower in NUGC-3/ 5FU/L cells than in the parent line. It is possible that other as yet unknown molecular mechanisms are also involved in 5FU resistance, since there appear to be considerable differences between the degree of resistance and the rates of inhibition of these 5FU-activating enzymes.In this study, an mRNA differential display (DD) were performed to screen for genes...

Predicting 5‐fluorouracil chemosensitivity of liver metastases from colorectal cancer using primary tumor specimens: Three‐gene expression model predicts clinical response

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Germ-line variants in methyl-group metabolism genes and susceptibility to DNA methylation in human breast cancer

“…It is not immediately obvious why this variant would be associated with CpG island methylation frequency, other than through a possible link with the concentration of intracellular folate intermediates. In support of this, in vitro studies in colorectal and gastric cell lines have shown a link between MTHFD1 expression and sensitivity to 5-fluorouracil (19,20). Further research is required, however, to evaluate DNA methylation and intracellular folate levels in relation to the MTHFD1 G1958A polymorphism in normal and tumour tissues.…”

MGr1-Ag is associated with multidrug-resistant phenotype of gastric cancer cells

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