2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a mutagenic compound found in the human diet in cooked meat, is a mammary gland carcinogen in female Sprague-Dawley rats. PhIP-induced rat mammary gland carcinomas were examined for mutations in several genes (exons) known to regulate cell growth and apoptosis including p53 (4-8), p21Waf1 (coding region), Apc (14, 15), B-catenin (3), E-cadherin (9,13,15), Bcl-x (coding region), Bax (3), IGFIIR (28), and TGFBIIR (3). DNA from 30 carcinomas were examined by single strand conformation polymorphism analysis but no mutations were detected in these genes/gene regions. DNA from carcinomas and matching normal tissue were further screened for allelic imbalance using a polymerase chain reaction-based approach with primers to known microsatellite regions located throughout the rat genome. Out of 53 markers examined, 12 revealed allelic imbalance. Microsatellite instability (MSI) was detected at two markers, one on chromosome 4 and one on chromosome 6. Sixty-five percent and 96% of all carcinomas examined (N=23) showed MSI at these loci on chromosomes 4 and 6, respectively, supporting the notion that MSI plays a role in PhIP-induced mammary carcinogenesis. Loss of heterozygosity (LOH), an indication of a possible tumor suppressor gene, was observed at ten markers distributed on chromosomes 3, 10, 11, 14, and X. The frequency of LOH at these markers ranged from 75%-94% supporting that the regions of allelic imbalance were largely similar for the PhIP-induced carcinomas examined in this study. When PhIP-induced carcinomas from rats placed on high fat and low fat diet were compared, no unique regions of allelic imbalance nor statistical differences in the frequency of allelic imbalance were observed. The results indicate that the high fat diet, known to be a promoter of PhIP-induced rat mammary carcinogenesis, did not influence allelic imbalance in the carcinomas. Interestingly, DMBA-induced mammary carcinomas did not show allelic imbalance at 11 of the 12 loci which showed allelic imbalance in PhIP-induced carcinomas. These findings suggest that distinct chemical carcinogens induce different patterns of allelic imbalance during rat mammary carcinogenesis. Further studies are needed to determine whether regions of LOH harbor potentially novel tumor suppressor genes involved in breast cancer. Stages of mammary gland cancer development and early genomic changes are currently being examined in this rat model.
