X-Ray-Induced Genomic Instability in Normal Human Cells and DNA Repair-Deficient CHO Cells

摘要

Recent studies have shown that delayed expression of effects of radiation, such as lethal mutation or delayed reproductive death, delayed chromosomal instability, and delayed mutation induction are transmitted through many generations. Although the mechanism(s) by which this genomic instability is induced remains to be elucidated, it has been suggested to be associated with radiation-induced carcinogenesis. As the genome of normal human cells is highly stable, it needs to be determined whether genomic instability can be induced in normal human primary cells. Recently, we reported on the extended life span and chromosome aberrations in normal ,human embryonic cells repeatedly irradiated with very low doses of γ rays. These results present the possibility that low-LET radiation may also cause delayed effects in normal human embryo cells. Although they have a limited life span, the higher growth potential of the embryonic cells made it possible to study X-ray-induced genomic instability in normal human primary cells over several generations. Until now, several mechanisms have been proposed to explain the induction of genomic instability by ionising radiation. Since ionising radiation and DNA strand-breaking agents induced genetic instability, double-strand breaks (DSBs) in the DNA are thought to be one of the initiators of this instability. It has been reported that a CHO mutant, xrs-5, which is defective in the repair of DNA DSBs, does not show a persistently reduced cloning efficiency, suggesting that the process of DNA DSB repair may be involved in the induction of genome instability. We have hypothesised that cells surviving irradiation retain abnormal DNA sequences or an unstable chromatin structure which is introduced during the process of damage repair, and that these potentially fragile regions, transmitted through many generations, are expressed to randomly activate recombination or signal transduction pathways, which might result in a chromosomal instability or expression of a variety of delayed effects. In the present study, we examined whether chromosome breakage and rejoining were involved in genetic instability as manifested by a bridge between chromosomes in dividing normal human cells. Although the resultant dicentric chromosomes are frequently used as a marker for chromosome instability, most studies have examined them using cell populations. It has been shown that the pleiotropic phenotypes of genetic instability are not detected uniformly among the progeny of surviving cells. Therefore, we have established an in situ method by which we can evaluate the incidence of chromosome bridges in each growing colony derived from an individual normal human cell which survived X irradiation. Furthermore, we examined X-ray-induced genomic instability in repair-deficient mutants to determine whether DNA DSB repair and SSB repair are involved in the formation of potentially fragile regions.