Radiotherapy for oral squamous cell carcinomas is limited in its efficacy and in its abitity to improve the survival rate in patients at an advanced stage. A protocol is described here which may elevate the therapeutic efficacy of radiation for these cells. The addition of glycerol to the culture medium prior to irradiation of an oral squamous cell carcinoma cell tine (Ca9-22) bearing a mutant p53 (mp53) gene was found to increase the radiosensitivity of these cells. A colony formation assay was used to evaluate the effect of glycerol on the radiation sensitivity of Ca9-22 cells. Apoptosis was analyzed using Hoechst 33342 staining, Western blotting, and a DNA ladder formation assay. Glycerol, when present in the culture medium, enhanced the radiation sensitivity and extent of apoptosis following X-irradiation in the Ca9-22 cells, although neither X-rays or glycerol atone increased the extent of apoptosis. Bax protein was accumulated after treatment with X-rays plus glycerol, but not after exposure to X-rays or glycerol alone. A get mobility-shift assay showed that glycerol restored the DNA-binding activity of mp53 for a p53-consensus sequence to levels similar to that of wild-type p53. These findings suggest that pre-treatment with glycerol may enhance the effectiveness of radiotherapy against oral squamous cell carcinomas bearing an mp53 gene mutation. (c) 2005 Elsevier Ltd. All rights reserved.