Purpose: We have previously reported that relative intrinsic cellular radiosensitivity in a range of human in-vitro cell lines was related to high endogenous expression of the protein product of the full length c-raf-1 proto-oncogene. Further studies involving PCR and sequencing of cDNA for p53 in 15 of these human cell lines have shown 7 to be wild type (wtp53) and 9 to contain functional mutations (mp53) within the 11 exons of the p53 gene. Cell lines expressing mutations in p53 mRNA were significantly more radioresistant than cell lines expressing wild type p53. The wild type p53 cell lines, however, still demonstrated the full range of radiation sensitivities. We have thus investigated whether high c-raf-1 expression in addition to wtp53 status was required for high intrinsic radiosensitivity in human in vitro cell lines.Materials and Methods: The 16 cell lines, their growth requirements and characteristics and response to radiation as determined by multiple clonogenic assays of each cell line, have been previously described as has their p53 status and RAF protein levels (Warenius et al. 1994, 1996a). In addition G2 delay following 2 Gy of radiation has been measured in 6 of these cell lines by flow cytometric analysis of DNA histograms following propidium iodide staining (Warenius et al 1996b).Results: Cell lines which expressed mp53 were all relatively radioresistant, did not show a wide range of radiosensitivities and exhibited a slight but non-significant relationship between RAF protein and cellular radiosensitivity (r = 0.61, p = 0.08). Cell lines expressing wtp53, however, showed a strong relationship between c-raf-1 protein levels and the radiosensitivity parameter alpha (r = 0.88, p = 0.008). RAF protein was expressed at higher levels in wtp53 cells than in mp53 cells. We also examined the putative relationship between c-raf-1 expression and post-irradiation perturbation of G2 in 6 of the wtp53 human cell lines. Cell lines which expressed high levels of c-raf-1 protein and wtp53 appeared to exit more rapidly from G2 (r = 0.93, p = 0.006) and were also more radiosensitive (r = [minus ]0.91, p = 0.01).Conclusion: These results suggest a possible co-operative role for wtp53 and c-raf-1 proto-oncogene in determining cellular radiosensitivity in human cancer which may involve control of the G2/M checkpoint.