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BACKGROUND: It has previously been demonstrated that Ad5CMV-p53 gene transfer, either used alone or delivered concomitantly with ionizing radiation, resulted in cytotoxicity mediated by apoptosis in nasopharyngeal carcinoma (NPC) cell lines. In this study, a novel approach was evaluated of combining Ad5CMV-p53 gene therapy with hyperthermia (HT), in the CNE-1 NPC cell line, which harbours a mutation in codon 249 of the p53 gene. MATERIALS AND METHODS: CNE-1 cells were infected using either Ad5CMV-p53 or Ad5CMV-B-gal, followed, 24 h later, by HT (43 degrees C x 0-2 h). Protein was extracted for Western blot analysis, and apoptosis was evaluated using acridine-orange ethidium bromide staining, followed immediately by fluorescent microscopy examination for the proportion of cells displaying morphologic features of apoptosis. RESULTS: Ad5CMV-p53 gene therapy combined with HT resulted in a dose-dependent cytotoxicity with less than 1% clonogenic survival when 10 pfu/cell of Ad5CMV-p53 was combined with 2 h heating at 43 degrees C. Western blotting demonstrated that treatment with Ad5CMV-p53 resulted in the rapid expression of p53, which was minimally affected by HT. The inducible form of hsp70 was maximally expressed at 48 h post-HT, with minimal effect when cells were additionally treated with Ad5CMV-p53. Clonogenic cytotoxicity was associated with the development of apoptosis, with up to 70% of CNE-1 cells displaying morphologic features of apoptosis after the combination treatments. CONCLUSION: Based on the shapes of the clonogenic survival curves, Ad5CMV-p53 gene therapy and HT appear to interact in an additive manner, suggesting the therapeutic potential of this combined treatment approach for patients with NPC.