We hypothesize that an X-Ray-Induced Protein (Mr: 269 KDa) product, XIP269, observed in radioresistant human melanoma (U1-Mel) cells functions directly or indirectly in DNA repair. This gene product may be a major determinant in the resistance of human tumors to ionizing radiation. The induction of XIP269 correlated with DNA repair in various human normal and tumor cells after X-irradiation (Boothman et. al., Cancer Res. 49: 2871-2878, 1989) and was down-regulated by caffeine, a drug which prevented repair (Hughes and Boothman, Radiat. Res. 125: 313-317, 1991). The objectives of this study are: a) to clone the gene encoding XIP269; b) to investigate the regulation of induction of XIP269 gene/protein within radioresistant tumor versus radiosensitive normal human cells before and after exposure to ionizing radiation; and c) to elucidate the function of XIP269 in DNA repair processes occurring in radioresistant human cells, and to determine if this gene could be a suitable marker for radioresistance in human tumors in a clinical setting. Positive cDNA clones encoding XIP269 will be isolated by sequentially screening a cDNA expression library from X-irradiated radioresistant U1- Mel cells first using a polyclonal anti-XIP269 antibody, followed by a screen with degenerate oligonucleotide probes based on partial amino acid sequences of XIP269. XIP269 cDNA clones will be sequenced and analyzed for homology (and an open reading frame) to known sequences via GenBank database comparisons. We will then examine the regulation of this gene before and after X-irradiation in cycling or arrested radioresistant as compared to radiosensitive human cells. Survival levels will be determined and compared to the expression of XIP269 at both the gene transcript and protein levels. Correlations between radioresistance, arrest points within the cell cycle (e.g., G1-or G2- arrest determined by flow cytometry), and the expression of XIP269 will be attempted. Finally, we will investigate the function of XIP269 using antisense oligonucleotide gene inactivation experiments with survival and DNA repair endpoints. The future goal of this research will be to use anti-XIP269 antibodies of cDNA probes as cellular prognostic markers to determine whether this gene product is a factor in tumor radiation responsiveness.
