Kuo M T, Zhao J Y, Teeter L D, Ikeguchi M, Chisari F V
Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Cell Growth Differ. 1992 Aug;3(8):531-40.
The expression of multidrug resistance (mdr) genes was investigated in the livers of transgenic mice that express the human hepatitis B virus large envelope polypeptide under the transcriptional control of a liver-specific promoter. These mice develop a storage disease due to the accumulation of a nonsecretable form of hepatitis B surface antigen in the hepatocyte. Liver cell injury is followed by a hepatocellular proliferative response, dysplasia, microscopic nodular hyperplasia, and finally hepatocellular carcinoma. The expression of mdr1, mdr2, and mdr3 genes was analyzed in livers at different stages of the disease by RNase protection assay, Western blot, and immunohistochemistry. RNase protection assay revealed that mdr3 mRNA expression was moderately increased in tissue with microscopic nodular hyperplasia and significantly overexpressed in hepatocellular carcinoma but undetectable in earlier stages of the disease. Western blot using isoform-specific anti-mdr3 antibody demonstrated that the expression of mdr3 protein reflected the steady-state level of mdr3 mRNA. Immunohistochemical analyses using anti-mdr3 isoform-specific antibody and monoclonal antibody C219, which recognizes all the three mdr isoforms, demonstrated selective overexpression in preneoplastic foci during the stage of microscopic nodular hyperplasia as well as in neoplastic hepatocytes in hepatocellular carcinoma. No consistent activation of mdr1 and mdr2 (but occasional coactivation with mdr1) genes during hepatocarcinogenesis was observed. Our results suggest that the hepatocellular mdr3-specific activation mechanism is associated with the late events of hepatocarcinogenesis in this model. The predictable kinetics of mdr gene expression in this transgenic tumor model suggest that it is suitable for future studies of the mechanism of mdr gene activation and the possible pharmacological consequences for mdr3 gene expression of hepatocellular carcinoma.
在肝脏特异性启动子转录控制下表达人乙肝病毒大表面抗原多肽的转基因小鼠肝脏中，研究了多药耐药（mdr）基因的表达。这些小鼠因肝细胞中积累不可分泌形式的乙肝表面抗原而患上一种贮积病。肝细胞损伤后会出现肝细胞增殖反应、发育异常、显微镜下结节性增生，最终发展为肝细胞癌。通过核糖核酸酶保护试验、蛋白质免疫印迹法和免疫组织化学法，分析了疾病不同阶段肝脏中mdr1、mdr2和mdr3基因的表达。核糖核酸酶保护试验显示，mdr3 mRNA表达在显微镜下结节性增生组织中适度增加，在肝细胞癌中显著过表达，但在疾病早期无法检测到。使用异构体特异性抗mdr3抗体的蛋白质免疫印迹法表明，mdr3蛋白的表达反映了mdr3 mRNA的稳态水平。使用抗mdr3异构体特异性抗体和识别所有三种mdr异构体的单克隆抗体C219进行的免疫组织化学分析表明，在显微镜下结节性增生阶段的癌前病灶以及肝细胞癌的肿瘤肝细胞中存在选择性过表达。在肝癌发生过程中未观察到mdr1和mdr2基因的持续激活（但偶尔与mdr1共同激活）。我们的结果表明，肝细胞mdr3特异性激活机制与该模型中肝癌发生的晚期事件有关。该转基因肿瘤模型中mdr基因表达的可预测动力学表明，它适用于未来对mdr基因激活机制以及肝细胞癌mdr3基因表达可能的药理学后果的研究。
