Huitfeldt H S, Brandtzaeg P, Poirier M C
Institute of Pathology, National Hospital, University of Oslo, Norway.
Proc Natl Acad Sci U S A. 1990 Aug;87(15):5955-8. doi: 10.1073/pnas.87.15.5955.
To investigate early cellular alterations in liver DNA during hepatocarcinogenesis, we have visualized replicating cells and analyzed their DNA adduct content in livers of rats continuously fed a carcinogenic level (0.02%) of 2-acetylaminofluorene for periods up to 4 weeks. One hour prior to sacrifice, cells undergoing DNA synthesis were pulse-labeled with the thymidine analogue 5-bromodeoxyuridine. Replicating cells were visualized immunohistochemically with anti-(5-bromodeoxyuridine), and identification of aminofluorene-DNA adducts in replicating nuclei was achieved by staining with an antiserum specific for N-(deoxyguanosin-8-yl)-2-aminofluorene; both stains were observed simultaneously by two-color immunofluorescence. Data were obtained for all cells, including large hepatocytes (nuclei greater than 6 microns) and small cells (nuclei less than 6 microns), such as hepatocytes sliced asymmetrically, oval cells, Kuppfer cells, and sinusoidal lining cells. Based on the size of their nuclei, the hepatocytes were the only cells that could be identified separately from the total. A distinct increase in the number of cells synthesizing DNA was observed after 25 days of 2-acetylaminofluorene feeding; replicating cells were either scattered randomly throughout the liver or clustered in discrete foci. At times up to 28 days, cells with both large and small nuclei that were synthesizing DNA showed reduced aminofluorene-DNA adduct immunofluorescence compared to nonreplicating cells. The results suggest that liver cells replicating during carcinogen exposure have altered metabolic capacities resulting in reduced aminofluorene-DNA adduct formation. It is possible that such cells constitute the progenitors of preneoplastic foci, which have a replicative advantage as compared to normal liver.
为了研究肝癌发生过程中肝脏DNA早期的细胞变化，我们对持续喂食致癌水平（0.02%）的2-乙酰氨基芴长达4周的大鼠肝脏中的复制细胞进行了可视化，并分析了它们的DNA加合物含量。在处死前1小时，用胸苷类似物5-溴脱氧尿苷对正在进行DNA合成的细胞进行脉冲标记。通过抗（5-溴脱氧尿苷）免疫组织化学方法对复制细胞进行可视化，并用对N-（脱氧鸟苷-8-基）-2-氨基芴特异的抗血清染色，以鉴定复制细胞核中的氨基芴-DNA加合物；通过双色免疫荧光同时观察这两种染色。获得了所有细胞的数据，包括大肝细胞（核大于6微米）和小细胞（核小于6微米），如不对称切片的肝细胞、卵圆细胞、库普弗细胞和窦状隙衬里细胞。根据细胞核大小，肝细胞是唯一能从总体中单独识别的细胞。在喂食2-乙酰氨基芴25天后，观察到合成DNA的细胞数量明显增加；复制细胞要么随机分散在整个肝脏中，要么聚集在离散的病灶中。在长达28天的时间里，与非复制细胞相比，正在合成DNA的大核和小核细胞的氨基芴-DNA加合物免疫荧光降低。结果表明，在致癌物暴露期间进行复制的肝细胞具有改变的代谢能力，导致氨基芴-DNA加合物形成减少。这些细胞有可能构成癌前病灶的祖细胞，与正常肝脏相比，它们具有复制优势。
