Munir K M, Custer R P, Sorof S
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Cancer Res. 1989 Jan 15;49(2):424-32.
A Mr 14,000 polypeptide (p14), identified as liver fatty acid binding protein, in normal liver cytosol was shown previously to be the principal target of the carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene), early during hepatic carcinogenesis in rats. Immunohistochemical analyses using rabbit antiserum against pure p14/liver fatty acid binding protein revealed marked increases in the levels of the protein in cytoplasm specifically during mitosis in normal and regenerating hepatocytes, and throughout the cell cycle in hyperplastic and malignant hepatocytes brought about by carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene) or 3'-methyl-4-dimethylaminoazobenzene. Present also in normal hepatocytes was a nuclear antigen that was not detected in the hyperplastic hepatocytes, benign hepatocytic adenomas, and hepatocellular carcinomas produced by these carcinogens. The nuclear antigen was discerned to be a Mr 17,000 polypeptide (p17) in extracts of normal liver nuclei and nucleosomes. In the present study, the p17 was purified by high-performance liquid chromatography and identified as being the three variants of histone H3, based on common molecular size, amino acid composition, electrophoretic migration in Triton-acetic acid-urea gels, and Western blot and histochemical reactions using affinity-purified antibodies. The histone H3 of all tested organs reacted specifically with the antiserum in Western blots following sodium dodecyl sulfate gel electrophoresis. In contrast, in a survey of 23 normal rat organs, nuclei of virtually only hepatocytes were reactive immunohistochemically. In view of the exceptional immunohistochemical reactivity of nuclei of normal hepatocytes, attributable to accessible histone H3, and the lack of such reaction in carcinogen-altered hepatocytes, the collected evidence indicates that normal hepatocytes contain uniquely available histone H3 sites that become cryptic during the chemical carcinogenesis.
先前已表明，正常肝细胞溶质中一种分子量为14,000的多肽（p14），被鉴定为肝脂肪酸结合蛋白，是大鼠肝癌发生早期致癌物N - 2 - 芴基乙酰胺（2 - 乙酰氨基芴）的主要作用靶点。使用针对纯p14/肝脂肪酸结合蛋白的兔抗血清进行的免疫组织化学分析显示，在正常和再生肝细胞的有丝分裂期间，细胞质中该蛋白水平显著升高，而在由致癌物N - 2 - 芴基乙酰胺（2 - 乙酰氨基芴）或3'-甲基 - 4 - 二甲基氨基偶氮苯导致的增生性和恶性肝细胞的整个细胞周期中，该蛋白水平也显著升高。正常肝细胞中还存在一种核抗原，在这些致癌物产生的增生性肝细胞、良性肝细胞腺瘤和肝细胞癌中未检测到。在正常肝细胞核和核小体提取物中，该核抗原被识别为一种分子量为17,000的多肽（p17）。在本研究中，通过高效液相色谱法纯化了p17，并根据共同的分子量、氨基酸组成、在Triton - 乙酸 - 尿素凝胶中的电泳迁移以及使用亲和纯化抗体的蛋白质印迹和组织化学反应，将其鉴定为组蛋白H3的三种变体。在十二烷基硫酸钠凝胶电泳后的蛋白质印迹中，所有测试器官的组蛋白H3均与抗血清发生特异性反应。相比之下，在对23个正常大鼠器官的调查中，实际上只有肝细胞核在免疫组织化学上有反应。鉴于正常肝细胞核具有特殊的免疫组织化学反应性（归因于可及的组蛋白H3），而在致癌物诱导改变的肝细胞中缺乏这种反应，所收集的证据表明，正常肝细胞含有独特的可及组蛋白H3位点，这些位点在化学致癌过程中变得隐蔽。
