RESUMEN
The study of Ataxia-telangiectasia (A-T) has benefited significantly from mouse models with knockout mutations for the Atm (A-T mutation) locus. While these models have proven useful for in vivo studies, cell cultures from Atm null embryos have been reported to grow poorly and then senesce. In this study, we initiated primary cultures from adult ears and kidneys of Atm homozygous mice and found that these cultures immortalized readily without loss of sensitivity to ionizing radiation and other Atm related cell cycle defects. A mutational analysis for loss of expression of an autosomal locus showed that ionizing radiation had a mutagenic effect. Interestingly, some spontaneous mutants exhibited a mutational pattern that is characteristic of oxidative mutagenesis. This result is consistent with chronic oxidative stress in Atm null cells. In total, the results demonstrate that permanent cell lines can be established from the tissues of adult mice homozygous for Atm and that these cell lines will exhibit expected and novel consequences of this deficiency.
Asunto(s)
Ataxia Telangiectasia/genética , Línea Celular Transformada , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/genética , Radiación Ionizante , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular , Supervivencia Celular/efectos de la radiación , Aberraciones Cromosómicas , Proteínas de Unión al ADN , Pérdida de Heterocigocidad/efectos de la radiación , Metafase/efectos de la radiación , Ratones , Ratones Noqueados , Mutagénesis , Mutación , Tolerancia a Radiación , Proteínas Supresoras de TumorRESUMEN
The presence of increased frequencies of blood-derived and solid tumors in ataxia-telangiectasia (A-T) patients, coupled with a role for the ATM (A-T mutation) protein in detecting specific forms of DNA damage, has led to the assumption of a mutator phenotype in A TM-deficient cells. Supporting this assumption are observations of increased rates of chromosomal aberrations and intrachromosomal homologous recombinational events in the cells of A-T patients. We have bred mice with knockout mutations for the selectable Aprt (adenine phosphoribosyltransferase) locus and the Atm locus to examine the frequency of second-step autosomal mutations in Atm-deficient cells. Two solid tissues were examined: (a) the ear, which yields predominately mesenchymal cells; and (b) the kidney, which yields predominately epithelial cells. We report here the lack of a mutator phenotype for inactivating autosomal mutations in solid tissues of the Atm-deficient mice.
Asunto(s)
Adenina Fosforribosiltransferasa/genética , Mutación , Proteínas Serina-Treonina Quinasas , Proteínas/genética , Animales , Ataxia Telangiectasia/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Mapeo Cromosómico , Proteínas de Unión al ADN , Oído , Femenino , Genotipo , Heterocigoto , Homocigoto , Riñón/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Especificidad de Órganos , Proteínas/metabolismo , Proteínas Supresoras de TumorRESUMEN
To determine the types of mutations induced by oxidative damage, a kidney cell line with a heterozygous deficiency for the autosomal Aprt (adenine phosphoribosyltransferase) gene was tested for its mutagenic response to hydrogen peroxide. Aprt-deficient cells were selected and scored for loss of heterozygosity (LOH) for 11 microsatellite loci on mouse chromosome 8. On the basis of the LOH analysis, spontaneous mutants (n = 38) were distributed into four classes: apparent point mutation, mitotic recombination, chromosome loss, and large interstitial deletion. However, 9 of 20 (45%) hydrogen peroxide-induced mutants exhibited a novel class of mutations characterized by "discontinuous LOH" for one or more of the microsatellite loci. Interestingly, mutations resembling discontinuous LOH are commonly observed in a wide variety of human cancers. Our data suggest that discontinuous LOH is a signature mutational pattern for oxidative damage and further suggest that such genetic damage is widespread in cancer.