Genetic factors affecting radiation sensitivity and genomic stability

Abstract

A report of the unexpected radiosensitivity of fibroblasts derived from unaffected parents of children with bilateral retinoblastoma (Fitzek, et ah, Int. J. Cancer 99, 2002) prompted the studies discussed in this dissertation. The Rb1+/+ parents may harbor unidentified mutations in DNA repair, cell cycle control, or genomic stability genes, influencing their radiosensitivity and possibly the increased transmission of mutant Rb1 alleles to their offspring. These studies sought to validate the moderate to hyper-radiosensitivity phenotype documented in the Fitzek report in this laboratory, and determine whether a continuous low dose-rate (LDR) irradiation protocol could amplify the survival differential between radiosensitive Rb family member and apparently normal control fibroblast strains sufficiently for genetic complementation and correction purposes. Measurements of fibroblast survival following acute irradiation and 24-hour potentially lethal damage (PLD) repair and G2 chromosomal radiosensitivity were made. Rb family member strains were ~1.5 to 4-fold more radiosensitive than the CIMR control strains, with high degrees of tetraploidy and endoreduplication observed in some cases in the G2 assay. The ability of fibroblasts to form colonies during continuous LDR (0.5-8.4 cGy/h) irradiation was successful in identifying radiosensitive strains (both Rb family members and some CIMR controls), achieving a > 100-fold survival differential at ~2-3 cGy/h for some strains. Continuous LDR (1-15 cGy/h) colony formation measurements were also made with atm-/-, atm+/- and tp53-/- mouse embryo fibroblasts (MEFs). The atm-heterozygote MEFs were intermediate in survival between the hyper-radiosensitive atm-homozygote MEFs and wild-type controls; the tp53-/- MEFs were the most radioresistant. The frequency of giant cells in tp53-/- MEF cultures was significantly increased by continuous LDR exposures but not by acute exposures. Cytogenetic and immunocytochemical analyses of tp53-/- MEFs revealed micronuclei with γ-H2AX foci and aberrant centrosomes. Telomere analysis by fluorescence in situ hybridization (FISH) revealed a novel phenotype of telomere dysfunction, which I term "telomere fission," characterized by large numbers of extra-chromosomal telomeric repeats and telomeric double-minutes. Massive chromosomal rearrangements were revealed by multiplex FISH (mFISH) analysis. These studies indicate continuous LDR irradiation could provide a powerful genetic screening strategy for studying mechanisms involved in the mutation of crucial tumor suppressors underlying carcinogenesis.