Studies on establishment and malignant transformation of cell lines from normal human liver

来自18周大人类胚胎的肝细胞通过转染猿猴病毒40（SV40）T抗原基因而永生化，并建立了细胞系OUMS-22。另一方面，来自正常成人肝脏的细胞也被转染了SV40 T抗原基因，但未能成为永生细胞系。 OUMS-22 细胞呈 SV-40 T 抗原阳性、上皮样细胞，并且具有抗角蛋白 18 单克隆抗体的免疫反应性。细胞中细胞角蛋白的染色模式与分化的人肝细胞癌细胞系相似，但与人胆管细胞癌细胞系不同。 OUMS-22 细胞既不表达甲胎蛋白也不表达白蛋白 mRNA。然而，OUMS-22细胞对黄曲霉毒素B1和苯并[a]芘的细胞毒性敏感，而人胚胎肺成纤维细胞对这些致癌物的细胞毒性不敏感。这些发现表明，OUMS-22细胞可能源自未分化的肝干细胞或在永生化之前失去表达肝脏特异性功能的能力的肝细胞。随着OUMS-22细胞的永生化，谷胱甘肽S-转移酶pi mRNA的表达显着增加。核型分析表明，OUMS-22 细胞中染色体的数量和结构畸变很严重。 OUMS-22细胞可以在软琼脂中生长，但移植到裸鼠体内时不具有致瘤性。然后，用在密码子61内含有点突变的克隆的激活的c-Ha-ras癌基因转染OUMS-22细胞。然而，激活的c-Ha-ras转染的细胞不具有致瘤性。即使在进一步转染突变型 p53 基因（该基因在外显子 8 的密码子 273 处将 CGT 转化为 CAT）后，细胞也未能变成恶性。另一方面，黄曲霉毒素B1和苯巴比妥联合处理后，OUMS-22细胞的克隆效率显着提高，但细胞未形成肿瘤。

The liver cells from the 18-week-old human embryo were immortalized by transfection with the simian virus 40 (SV40) T-antigen gene, and a cell line OUMS-22 was established. On the other hand, the cells from normal adult human liver were also transfected with SV40 T-antigen gene, but failed to become an immortal cell line. OUMS-22 cells were SV-40 T-antigen-positive, epithelial-like, and immunoreactive against an anti-keratin 18 monoclonal antibody. The staining pattern of cytokeratin in the cells was similar to that in the differentiated human hepatocellular carcinoma cell lines but not to that in the human cholangiocellular carcinoma cell lines. OUMS-22 cells expressed neither alpha-fetoprotein nor albumin mRNAs. However, OUMS-22 cells were sensitive to cytotoxicity of aflatoxin B1 and benzo[a]pyrene, whereas human embryo lung fibroblasts were insensitive to the cyntotoxicity of these carcinogens. These findings suggest that OUMS-22 cells may arise from undifferentiated liver stem cells or from hepatocytes that lost their ability to express the liver-specific functions prior to immortalization. The expression of glutathione S-transferase pi mRNA highly increased in OUMS-22 cells with their immortalization. Karyotypic analysis showed that numerical and structural aberrations of the chromosomes were profound in OUMS-22 cells. OUMS-22 cells could grow in soft agar, but they were not tumorigenic when transplanted into nude mice. Then, OUMS-22 cells were transfected with the cloned activated c-Ha-ras oncogene containing a point mutation within codon 61. However, the activated c-Ha-ras transfected cells were not tumorigenic. Even after further transfection with the mutant p53 gene, which has a conversion of CGT into CAT at codon 273 in the exon 8, the cells failed to become malignant. On the other hand, the cloning efficiency of OUMS-22 cells was significantly increased by treatment with a combination of aflatoxin B1 and phenobarbital, but the cells formed no tumors whe