Reduced expression of BRCA1 protein, caused by the hypermethylation of its gene promoter and by other mechanisms, is observed in most sporadic human breast cancers, whereas its somatic mutations are rare. In the present study, we demonstrate that immunoreactivity of Brca1 was reduced in almost all rat mammary carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), whereas hypermethylation of its promoter, mutations in its entire coding region, and decreased mRNA expression were absent. Using two kinds of polyclonal antibodies against human BRCA1 that recognized the N and C termini, respectively, immunoreactivity of Brca1 was found to be reduced in all 17 carcinomas examined and, especially, to be almost completely lost in 8 or 10 carcinomas. The reduction was confirmed by immunoblot analysis of five mammary carcinomas and normal mammary epithelial cells. Sequencing of the Brca1 promoter region after bisulfite modification in 10 PhIP-induced mammary carcinomas showed that the region was completely unmethylated in all of them. No mutations were detected in the entire coding region by direct sequencing of the Brca1 cDNA. Decrease in mRNA levels was not detected by quantitative real-time reverse transcription-polymerase chain reaction. These data suggested that PhIP-induced mammary carcinomas could model human breast cancers that show reduced BRCA1 immunoreactivity without promoter hypermethylation and with normal mRNA expression. As underlying mechanisms, alterations in posttranscriptional regulation or stability of Brca1 protein were suggested.
Copyright 2002 Wiley-Liss, Inc.