Wani G, Wani A A, D'Ambrosio S M
Division of Radiobiology, College of Medicine, Ohio State University, Columbus 43210.
Carcinogenesis. 1993 Apr;14(4):737-41. doi: 10.1093/carcin/14.4.737.
The O6-alkylguanine-DNA alkyltransferase (ATase) is known to overcome the effects of promutagenic, precarcinogenic O6-alkylguanine induced in DNA by exposure to environmental, chemotherapeutic and dietary alkylating agents. Within an organ, the cell type-specific responses to these agents may be attributed, in part, to varying expression of critical DNA repair genes, like ATase. In order to determine the cell-specific expression of the human ATase gene, in situ hybridization was used to map the cellular distribution of ATase mRNA in tissue sections of normal human fetal and adult livers. Tissue sections were hybridized with a digoxigenin-labeled 39 base oligomer, antisense to ATase cDNA. Following immunodetection, using an alkaline phosphatase-conjugated anti-digoxigenin antibody, the ATase-specific mRNA levels were visualized in parallel with liver cell type identification. The specificity of the antisense probe and hybridization to human ATase mRNA was demonstrated by: (i) staining of Mer+ and not Mer- cells by the antisense probe; (ii) faint staining of liver sections when the antisense probe was not used during hybridization; (iii) no hybridization of liver sections by the sense probe; (iv) no staining of sections preincubated with RNase before hybridization; and (v) the retention of cell type-specific staining patterns in tissue sections incubated with DNase prior to hybridization with the antisense probe. The staining patterns appeared similar in adjacent sections of tissues obtained from the same liver and in sections obtained from either adult or fetal livers of different individuals. The expression of the ATase mRNA, as noted by stain intensity, appeared highest in all of the bile ductal cells. There was a heterogenous expression in hepatocytes, which varied from moderate to high stain. Staining in Kupffer cells also appeared to be high. Sinusoidal cells, endothelial cells of the hepatic artery and cells of the connective tissue showed weak hybridization, indicating low levels of ATase mRNA. These data explain, in part, the basis for a differential response of various cell types within the liver to the mutagenic and carcinogenic effects of alkylating agents.
O6-烷基鸟嘌呤-DNA烷基转移酶（ATase）能够消除因接触环境、化疗和饮食中的烷基化剂而在DNA中诱导产生的前诱变、前致癌性O6-烷基鸟嘌呤的影响。在一个器官内，细胞类型对这些试剂的特异性反应可能部分归因于关键DNA修复基因（如ATase）的不同表达。为了确定人类ATase基因的细胞特异性表达，采用原位杂交技术绘制正常人胎儿和成人肝脏组织切片中ATase mRNA的细胞分布。组织切片与地高辛标记的39碱基寡聚物杂交，该寡聚物与ATase cDNA反义。免疫检测后，使用碱性磷酸酶偶联的抗地高辛抗体，在识别肝细胞类型的同时可视化ATase特异性mRNA水平。反义探针与人类ATase mRNA杂交的特异性通过以下方式证明：（i）反义探针使Mer+细胞而非Mer-细胞染色；（ii）杂交过程中不使用反义探针时肝脏切片染色微弱；（iii）正义探针不与肝脏切片杂交；（iv）杂交前用RNase预孵育的切片不染色；（v）与反义探针杂交前用DNase孵育的组织切片中细胞类型特异性染色模式得以保留。在取自同一肝脏的相邻组织切片以及取自不同个体的成人或胎儿肝脏的切片中，染色模式看起来相似。从染色强度来看，ATase mRNA的表达在所有胆管细胞中最高。肝细胞中存在异质性表达，染色程度从中等到高不等。库普弗细胞中的染色似乎也很高。窦状细胞、肝动脉内皮细胞和结缔组织细胞显示出弱杂交信号，表明ATase mRNA水平较低。这些数据部分解释了肝脏内各种细胞类型对烷基化剂的诱变和致癌作用产生不同反应的基础。
