Overlapping loss of heterozygosity by mitotic recombination on mouse chromosome 7F1-ter in skin carcinogenesis.

Abstract

A significant role for mouse chromosome 7 abnormalities during chemically induced skin carcinogenesis has been advanced based on previous cytogenetic and molecular studies. To determine the frequency of allelic losses at different loci of chromosome 7 in skin tumors induced in the outbred SENCAR mouse stock by a two-stage initiation-promotion protocol, we compared the constitutional and tumor genotypes of premalignant papillomas and squamous cell carcinomas for loss of heterozygosity at different informative loci. In a previous study, these tumors had been analyzed for their allelic composition at the Harvey ras-1 (Ha-ras-1) locus and it was found that 39% of squamous cell carcinomas had lost the normal Ha-ras-1 allele exhibiting 3 or 2 copies of the mutated counterpart or gene amplification. In the present study, by combining Southern blot and polymerase chain reaction fragment length polymorphism analyses, we detected complete loss of heterozygosity at the beta-globin (Hbb) locus, distal to Ha-ras-1, in 15 of 20 (75%) skin carcinomas. In addition, 5 of 5 informative cases attained homozygosity at the int-2 locus, 27 centimorgans distal to Hbb. Polymerase chain reaction analysis of DNA extracted from papillomas devoid of stromal contamination by fluorescence-activated sorting of single cell dispersions immunolabeled with anti-keratin 13 antibody revealed loss of heterozygosity at the Hbb locus, demonstrating that this event occurs during premalignant stages of tumor development. Interestingly, loss of heterozygosity was only detected in late-stage lesions exhibiting a high degree of dysplasia and areas of microinvasion. Analysis of allelic ratios by densitometric scanning of tumors that had become homozygous at Hbb but retained heterozygosis at Ha-ras-1 indicated mitotic recombination as the mechanism underlying loss of heterozygosity on mouse chromosome 7 during chemically induced skin carcinogenesis. These findings are consistent with the presence of a putative tumor suppressor gene linked to the Hbb locus in the 7F1-ter region of mouse chromosome 7, the functional inactivation of which may constitute a critical event in skin tumor progression, possibly during the malignant conversion stage.