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Purpose: Mutations in the p53 gene are reported in 50~ 90% of gallbladder and bile duct cancer, and have been implicated in chemoresistance. We undertook this study to determine whether the introduction of the wild type p53 gene into GBCE (human gallbladder cancer cell line with a heterozygous p53 mutation) by an adenoviral vector could increase the sensitivity of the cell to 5-FU, a commonly used drug in the treatment of gallbladder cancer. Materials and Methods: GBCE cells were transfected with either Ad/p53 or Ad/E1 in the presence of 5-FU. Gene expression was confirmed by western blotting. Nude mice were injected subcutaneously with GBCE cells. When tumors formed, intratumoral injection of Ad/p53 was performed. Reduction of tumor size was compared in two weeks of Ad/p53 gene transfection. Results: Ad/53 transfection induced a dose-dependent inhibition of tumor growth. Tumor colony formation was more inhibited with p53 gene transfection than with mock transfection in the presence of 5-FU. The reduction in tumor size was more pronounced with p53 transfection than with mock infection. Conclusion: These treatment modalities could be utilized in the treatment of p53 mutant human gallbladder cancers. (Cancer Res Treat. 2003;35:521-527)