Autosomal mutants of proton-exposed kidney cells display frequent loss of heterozygosity on nonselected chromosomes.

High-energy protons found in the space environment can induce mutations and cancer, which are inextricably linked. We hypothesized that some mutants isolated from proton-exposed kidneys arose through a genome-wide incident that causes loss of heterozygosity (LOH)-generating mutations on multiple chromosomes (termed here genomic LOH). To test this hypothesis, we examined 11 pairs of nonselected chromosomes for LOH events in mutant cells isolated from the kidneys of mice exposed to 4 or 5 Gy of 1 GeV protons. The mutant kidney cells were selected for loss of expression of the chromosome 8-encoded Aprt gene. Genomic LOH events were also assessed in Aprt mutants isolated from isogenic cultured kidney epithelial cells exposed to 5 Gy of protons in vitro. Control groups were spontaneous Aprt mutants and clones isolated without selection from the proton-exposed kidneys or cultures. The in vivo results showed significant increases in genomic LOH events in the Aprt mutants from proton-exposed kidneys when compared with spontaneous Aprt mutants and when compared with nonmutant (i.e., nonselected) clones from the proton-exposed kidneys. A bias for LOH events affecting chromosome 14 was observed in the proton-induced Aprt mutants, though LOH for this chromosome did not confer increased radiation resistance. Genomic LOH events were observed in Aprt mutants isolated from proton-exposed cultured kidney cells; however the incidence was fivefold lower than in Aprt mutants isolated from exposed intact kidneys, suggesting a more permissive environment in the intact organ and/or the evolution of kidney clones prior to their isolation from the tissue. We conclude that proton exposure creates a subset of viable cells with LOH events on multiple chromosomes, that these cells form and persist in vivo, and that they can be isolated from an intact tissue by selection for a mutation on a single chromosome.

Marked aneuploidy and loss of multiple chromosomes are common in autosomal mutants isolated from normal mouse kidney epithelium.

Marked aneuploidy and loss of multiple chromosomes are hallmarks of cancer, but whether these events are only present in malignant cells is not known. In prior work, we showed that approximately half of spontaneous autosomal mutants isolated directly from normal kidney epithelium arose from loss of a marker chromosome 8 containing the wild type Aprt gene. Chromosome loss was detected by loss of heterozygosity (LOH) for all chromosome 8 polymorphic loci examined. To determine whether loss of chromosome 8 reflected a larger mitotic event, LOH was examined for polymorphic loci on 11 nonselected chromosomes in Aprt mutants that lost the selected chromosome 8 homologue. LOH events were detected for one or more nonselected chromosomes in 38% of these mutants. The additional LOH events also reflected apparent chromosome loss based on the molecular analysis. Metaphase spreads from mutants that lost chromosome 8 were markedly aneuploid, and chromosome painting revealed reduced levels for any chromosome shown to be lost with the LOH analysis. In contrast, LOH on nonselected chromosomes was infrequent in Aprt mutants exhibiting intragenic events or mitotic recombination for chromosome 8, and marked aneuploidy was absent. These observations suggest that the mechanism leading to chromosome loss in somatic mammalian cells is often not a simple nondisjunction event and instead could result from a single catastrophic event. They also suggest that cells with characteristics of malignancy are present in normal appearing tissue.