2-Amino-1-methyl-6-phenylimidazo 4,5-b pyridine (PhIP) is a suspected human breast carcinogen found in cooked meat that induces mammary gland cancer in rats. By real-time polymerase chain reaction (PCR) analysis, PhIP-induced rat mammary gland carcinomas showed statistically higher expression of the G1/S cyclin D1 (5-fold) and its kinase partner Cdk4 (37-fold) in comparison to normal mammary gland, while cyclin D2, cyclin D3, and Cdk6 were not statistically changed. Amplification of cyclin D1 was observed by real-time PCR in 24% of carcinomas (15 of 63). Only 1 of 47 carcinomas showed Cdk4 amplification. By Western blotting, the level of phospho-Rb was over 2-fold higher in carcinomas than in normal mammary gland. By immunohistochemical analysis, cyclin D1, Cdk4, and phospho-Rb nuclear protein expression was 5.7, 3.9 and 2.3-fold higher in carcinomas than in normal mammary gland, whereas the expression of cyclin D2, cyclin D3 and Cdk6 was similar. Among carcinomas, Cdk4 and phospho-Rb levels were positively correlated with cell proliferation. Previous studies by this laboratory indicated that these carcinomas harbor a high frequency of H-ras mutations. The H-ras pathway is known to be linked to the cell cycle via cyclin D1. The results from the current study implicate cyclin D1/Cdk4, phospho-Rb as a central pathway in PhIP-induced rat mammary gland carcinogenesis.Previous studies using comparative genomic hybridization have indicated that PhIP-induced rat mammary gland carcinomas harbor loss of the centromeric region of 2q, a region known to carry the mammary carcinoma susceptibility 1 gene and other genes potentially important for carcinogenesis. Using a polymerase chain reaction based assay, allelic imbalance, including microsatellite instability and loss of heterozygosity, was evaluated in eleven mammary carcinomas induced by PhIP in Sprague-Dawley x Wistar Furth F1 hybrid rats in 34 microsatellite markers putatively coinciding to 2q11-14. Nine of the 34 markers revealed allelic imbalance, the frequency varying from 10- 100% depending on the specific marker. None of the markers showing allelic imbalance, however, coincided with the MCS1 gene locus, suggesting that alterations in this loci were not associated with PhIP-induced rat mammary gland cancer. The expression of several genes physically mapped to 2q11-14 and potentially involved in carcinogenesis including Cyclin H, Ras-GRF2, RasGAP, Cyclin B1, Pi3kr1, Il6st, was also examined by quantitative real-time PCR and immunohistochemistry (IHC) in a large bank of PhIP-induced Sprague-Dawley rat mammary gland carcinomas. By realtime PCR, the expression of RASGAP, Pi3Kp85, Ccnh, and IL6 in carcinomas was respectively 26-, 20-, 3-, and 3-fold higher in carcinomas than in control mammary gland tissue (p<0.05, Student's t-test). A statistically six-fold lower expression of RasGrf2 was detected in carcinomas, whereas no significant change in CCNB1 expression was observed. The findings from RT-PCR were further confirmed by immunohistochemical staining of mammary gland carcinomas and normal mammary gland using specific antibodies. For each gene examined, the change in expression by RT-PCR was shown to correspond to altered expression as detected by immunohistochemistry. The proliferation index in mammary gland carcinomas was also assessed by PCNA immunostaining and shown to correlate with the over-expression of cyclin H (P<0.05, Spearman Rank Order Correlation). The results indicate that multiple genes and loci residing in the centromeric region of chromosome 2 show alterations in PhIP-induced rat mammary gland carcinomas.
