MUTATIONAL LESIONS SPECIFIC FOR IONIZING RADIATION

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During the course of experiments to define the spectrum of X-ray-induced mutations at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus in human lymphoblastoid cells, a complex mutation was identified. This mutation has a six base-pair deletion 3 base-pairs downstream of a base-pair substitution. The same mutation has appeared 3 times independently. Based on the number of mutants examined so far, it has been calculated that this particular mutation may constitute as much as 10% of all X-ray-induced events at hprt. However, this alteration has never been observed in more than 500 other mutants examined that arose either spontaneously or after induction by other agents. Consequently, this specific mutation may be a distinctive marker for radiation exposure. It is proposed to develop the technology for the rapid and facile detection of this mutation and then to validate its use in several systems, including human cells in culture, peripheral lymphocytes from cancer patients treated with radiation, and splenic lymphocytes from irradiated mice. Furthermore, the proposed work will seek to identify other X-ray specific mutations in the in vivo mouse model and in the cancer patients. Specific Aim 1 is to develop molecular techniques for the rapid detection and quantification of this X-ray-specific mutation in populations of mixed hprt- mutants of human cells. Two potential approaches will be evaluated. The first is based on a polymerase chain reaction (PCR) assay utilizing a primer which hybridizes specifically to the mutant region. The second is based on differential migration of mutant sequences in denaturing gradient gels. Aim 2 is to examine the dose-response relationship for the induction of this mutation in human lymphoblast cells treated in vitro with radiation, by use of the techniques developed in Aim 1. The third Specific Aim is to determine whether this mutation is induced in an in vivo mouse mutation assay, recently developed by Dr. Skopek. If the X-ray specific mutant is formed in the mouse hprt gene, the dose-response relationship of its induction will be examined. Also, an approach will be developed to look for other mutations specifically induced in vivo by X-rays, based on analyses of mixed mutant populations by denaturing gradient gel electrophoresis. Finally, Specific Aim 4 is to determine whether the X-ray specific mutation is induced and persists in cancer patients who have been treated with high doses of ionizing radiation. Some Hodgkin's disease patients exhibit persistently elevated mutant frequencies many months after the end of treatment, while others have normal frequencies. These two subsets, as well as normal healthy adults, will be compared. Mutant DNAs from these patients also will be screened for additional X-ray specific mutations, by use of the protocols developed in Aim 3.