AIM:TO investigate the resistance mechanism of 5-fluorouracil(5-FU)in Bel_(7402)/5-FU cells which was establishedin our lab by in vitrocontinuous stepwise exposure of humanhepatocellular carcinoma(HCC)cell line Bel_(7402)to 5-FU.METHODS:The expression of multidrug resistance-associated protein(MRP)and thymidylate synthase(TS)inBel_(7402)cells was detected by immonocytochemistry.Thefluorescein(FLU)accumulation,an index of MRP functionalactivity,was determined by flow cytometry.The distributionof FLU was observed by confocal laser scanning microscope.The spectrofluorometry was used to show the intracelluarcontent of glutathione(GSH).Cell growth inhibition wasdetermined by MTT assay.The activity of glutathione S-transferases(GSTs)was determined by spectrophotometry.RESULTS:A higher expression of MRP in the Bel_(7402)/5-FUcells was observed by using monoclonal mouse anti-MRPantibody,MRPr-1,in comparison with Bel_(7402)cells.Bel_(7402)/5-FU cells also showed a significant decrease of FLUaccumulation.FLU mainly accumulated in the nucleus witha high nuclear/cytoplasmic ratio in Bel_(7402)cells,whereas therewas no difference of FLU accumulation between the nucleusand cytoplasm in Bel_(7402)/5-FU cells.The intracellular GSHcontent in Bel_(7402)/5-FU cells was almost 3 folds higher thanthat in Bel_(7402)cells.Addition of D,L-buthione-S,R-sulfoximine(BSO)dose-dependently reduced the GSH content in Bel_(7402)/5-FU cells,however,only a weak enhancement on thecytotoxicity of 5-FU and doxorubicin(Dox)to Bel_(7402)/5-FU cellswas observed.Bel_(7402)/5-FU cells also exhibited 29.1% highertotal GSTs activity than Bel_(7402)cells.Immunocytochemicalstaining by using anti-TS monoclonal antibody TS 106 showedthat the level of TS in Bel_(7402)/5-FU cells elevated markedly ascompared with Bel_(7402)cells.CONCLUSION:The continuous exposure of Bel_(7402)cells to5-FU led to overexpression of TS and MRP,as well asincreased intracellular GSH content and total GST activity. AIM: TO investigate the resistance mechanism of 5-fluorouracil (5-FU) in Bel 74042/5-FU cells which was established in our lab by in vitrocontinuous stepwise exposure of human hepatocellular carcinoma (HCC) cell line Bel 7402 to 5 -FU.METHODS: The expression of multidrug resistance-associated protein (MRP) and thymidylate synthase (TS) in Bel 7402 was detected by immonocytochemistry. Fluorescein (FLU) accumulation, an index of MRP functionalactivity, was determined by flow cytometry. The distribution of FLU was observed by confocal laser scanning microscope. The spectrofluorometry was used to show the intracelluartent of glutathione (GSH). Cell growth inhibition was determined by MTT assay. The activity of glutathione S-transferases (GSTs) was determined by spectrophotometry .RESULTS: A higher expression of MRP in the Bel_ (7402) / 5-FU cells was observed by using monoclonal mouse anti-MRP antibody, MRPr-1, in comparison with Bel_ (7402) cells. significant decrease of FLUacc umulation.FLU mainly accumulated in the nucleus witha high nuclear / cytoplasmic ratio in Bel_ (7402) cells, there there no difference of FLU accumulation between the nucleus and cytoplasm in Bel_ (7402) / 5-FU cells.The intracellular GSHcontent in Bel_ (7402) (7402) cells. Addition of D, L-buthione-S, R-sulfoximine (BSO) dose- dependently reduced the GSH content in Bel 7402/5-FU cells was almost 3 folds higher thanthat in Bel 7402 cells. FU cells, however, only a weak enhancement on the cytotoxicity of 5-FU and doxorubicin (Dox) to Bel 7404/5-FU cells was observed. Bel_ (7402) / 5-FU cells also exhibited 29.1% highertotal GSTs activity than Bel_ (7402) cells.Immunocytochemical staining by using anti-TS monoclonal antibody TS 106 showed that the level of TS in Bel_ (7402) / 5-FU cells elevated markedly ascompared with Bel_ (7402) cells.CONCLUSION: The continuous exposure of Bel_ (7402) cells to5-FU led to overexpression of TS and MRP, as well as contributing to intracellular GSH content and total GST activity.