The goal of the proposed work is to determine whether the phenomenon known as delayed mutation occurs in human cells exposed, in vitro, to ionizing radiation. Traditional mutation assays evaluate the induction of mutations at a single snapshot in time. If radiation exposure induces a persistent instability in the genome, traditional assays may underestimate the risk of heritable alterations in somatic cells. The hypothesis to be tested is that delayed mutations arise as a result of the induction of genomic instability. Initial studies will be performed using an immortal, near-diploid human B-lymphoblastoid cell line, TK6. Subsequent studies will be performed using normal human lymphocytes. To determine whether delayed lethal mutations arise, serial sampling of the plating efficiency of irradiated cells will be performed. To determine the persistence of this phenotype, colonies arising after the initial post-irradiation plating will be subcloned in order to determine their subcultivation efficiency. We will also determine whether radiation exposure induces a persistent elevation in specific locus mutations in the progeny of irradiated cells. The candidate loci to be tested are the hypoxanthine phosphoribosyltransferase (hprt) locus and, where possible, the thymidine kinase locus (tk) and selected VNTR and microsatellite repeat loci. The spectrum of DNA structural alterations found in clonally derived hprt mutants exhibiting the delayed mutation phenotype will be determined using Southern blotting techniques or PCR-based assays to ascertain whether they resemble spontaneous mutants or whether they have a unique signature associated with the induction of instability. A separate measure of genomic instability, the ability to amplify the CAD gene, will also be performed on the cells chosen for the abovementioned studies. We will carry out somatic cell hybridization experiments to determine whether the delayed mutation phenotype in certain irradiated progeny of Chinese hamster ovary cells can be suppressed by human chromosomes. We also seek to determine whether delayed mutation is a process which is invoked in human cells only when the pathway for programmed cell death is abrogated. The overall goal of these studies is to determine whether radiation exposure induces a persistent instability in the genome of human cells, and to disclose whether a predisposition to instability is required for the delayed mutation phenotype to be evident.
| Cherbonnel-Lasserre, C; Gauny, S; Kronenberg, A (1996) Suppression of apoptosis by Bcl-2 or Bcl-xL promotes susceptibility to mutagenesis. Oncogene 13:1489-97 |
