Aquisition of Genomic Alterations During Mammary Gland Cancer Induction in Rats

2-氨基-1-甲基-6-苯基咪唑[4,5-b]吡啶（PhIP）是一种在人类饮食中发现的致突变化合物，是雌性Sprague-Dawley大鼠的乳腺致癌物。我们检测了phip诱导的大鼠乳腺癌中几种已知调节细胞生长和凋亡的基因（外显子）的突变，包括p53（4-8）、p21Waf1（编码区）、Apc（14,15）、B-catenin(3)、E-cadherin（9,13,15）、Bcl-x（编码区）、Bax(3)、IGFIIR（28）和TGFBIIR(3)。对30例肿瘤的DNA进行单链构象多态性分析，未发现这些基因/基因区域发生突变。利用基于聚合酶链反应的方法，利用位于整个大鼠基因组的已知微卫星区域的引物，进一步筛选来自癌组织和匹配正常组织的DNA是否存在等位基因失衡。在检测的53个标记中，有12个显示等位基因不平衡。在4号染色体和6号染色体上检测到微卫星不稳定性（MSI）。65%和96%的癌（N=23）分别在4号和6号染色体上的这些位点上显示MSI，支持MSI在phip诱导的乳腺癌发生中起作用的观点。在分布于3、10、11、14和x号染色体上的10个标记上观察到杂合性缺失（LOH），这是可能存在肿瘤抑制基因的一个指示。这些标记上LOH的频率在75%-94%之间，这支持了在本研究中检测的phip诱导的癌症中，等位基因失衡的区域在很大程度上是相似的。当比较高脂和低脂饮食大鼠的phip诱导的癌时，没有观察到独特的等位基因不平衡区域，也没有观察到等位基因不平衡频率的统计学差异。结果表明，高脂肪饮食，已知是phip诱导的大鼠乳腺癌的促进剂，不影响癌中的等位基因失衡。有趣的是，dmba诱导的乳腺癌在phip诱导的癌中显示等位基因不平衡的12个位点中的11个没有显示等位基因不平衡。这些发现表明，在大鼠乳腺癌发生过程中，不同的化学致癌物诱导不同模式的等位基因失衡。需要进一步的研究来确定LOH区域是否有潜在的新型肿瘤抑制基因参与乳腺癌。

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.