Kakehashi Anna, Inoue Masayo, Wei Min, Fukushima Shoji, Wanibuchi Hideki
Department of Pathology, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan.
Toxicol Appl Pharmacol. 2009 Jul 1;238(1):71-9. doi: 10.1016/j.taap.2009.04.018. Epub 2009 May 3.
Screening of the proteome of microdissected glutathione S-transferase placental form (GST-P) positive foci and normal-appearing liver on anionic (Q10), and cationic (CM10) surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip arrays demonstrated significant overexpression of cytokeratin 8 (CK8; m/z 54,020), cytokeratin 18 (CK18; m/z 47,760), microsomal cytochrome 5A (m/z 15,224) and histone type 2 H2aa3 (m/z 15,964) in the livers of rats initiated with diethylnitrosamine (DEN) followed by 10 weeks on phenobarbital (PB) at a dose of 500 ppm. Furthermore, formation of CK8 and CK18 complexes due to CK8 phosphorylation at Ser73 and Ser431 was found to be strongly associated with promotion of hepatocarcinogenesis by PB and the development of hepatocellular carcinomas. The data were confirmed by immunohistochemistry and real-time Q-PCR and profound overexpression of CK8 and CK18 (CK8/18) proteins and mRNAs were detected in several large size GST-P positive foci and liver tumors. A strong correlation between CK8/18 positive foci development and multiplicity of hepatocellular carcinomas was further observed. Moreover, elevation of CK8/18 was strongly associated with induction of cell proliferation in GST-P positive foci and tumors. In conclusion, our data imply that CK8/18 overexpression, those two cytokeratins complex formation associated with histone type 2 H2aa3 up-regulation and intermediate filament reorganization may drive neoplastic transformation of GST-P positive foci during rat hepatocarcinogenesis leading to the formation of hepatocellular carcinomas.
在阴离子（Q10）和阳离子（CM10）表面增强激光解吸/电离飞行时间质谱（SELDI-TOF-MS）蛋白质芯片阵列上，对显微切割的谷胱甘肽S-转移酶胎盘型（GST-P）阳性灶和外观正常的肝脏蛋白质组进行筛选，结果显示，用二乙基亚硝胺（DEN）启动、随后以500 ppm剂量给予苯巴比妥（PB）10周的大鼠肝脏中，细胞角蛋白8（CK8；m/z 54,020）、细胞角蛋白18（CK18；m/z 47,760）、微粒体细胞色素5A（m/z 15,224）和组蛋白H2aa3（m/z 15,964）显著过表达。此外，发现由于CK8在Ser73和Ser431处磷酸化而形成的CK8和CK18复合物与PB促进肝癌发生及肝细胞癌的发展密切相关。免疫组织化学和实时定量PCR证实了这些数据，并且在几个大尺寸的GST-P阳性灶和肝肿瘤中检测到CK8和CK18（CK8/18）蛋白及mRNA的大量过表达。进一步观察到CK8/18阳性灶的发展与肝细胞癌的多发性之间存在强相关性。此外，CK8/18的升高与GST-P阳性灶和肿瘤中的细胞增殖诱导密切相关。总之，我们的数据表明，CK8/18过表达、这两种细胞角蛋白复合物的形成与组蛋白H2aa3上调及中间丝重组相关，可能在大鼠肝癌发生过程中驱动GST-P阳性灶的肿瘤转化，导致肝细胞癌的形成。
