IDENTIFICATION AND CHARACTERIZATION OF NEW HUMAN P-450

化学致癌物被认为是人类癌症的主要原因。 这些化合物通常必须被活化，形成称为终极的形式 致癌物，是能够与细胞结合的亲电性物质 蛋白质、RNA和DNA等大分子。人们相信DNA的突变 负责细胞转化。这些酶能够 激活致癌物质的是细胞色素P-450。多种形式的P-450 存在着由50到200个基因编码的生物。在过去 几年来，啮齿动物和人类拥有 P-450可以说是非常独特的。数量和质量物种 分歧已经被发现。特别是其催化特性。 啮齿动物的P-450可能不同于人类的P-450。这些发现有 在致癌研究中的主要意义，因为啮齿动物经常 作为化学物质诱导肿瘤发生的实验模型 曝光。因此，我们把重点放在人类的特征上 通过直接从肝脏和肺文库中克隆P-450的cDNA。这些 然后插入到表达载体中，用于生产培养的 含有催化活性P-450酶的细胞。现役P-450是 然后测试它们激活化学致癌物的能力 DNA结合的代谢物。已经使用了两种系统：1)牛痘病毒 能够产生高水平直接表达蛋白质的瞬时表达 酶和光谱分析；ii)人淋巴母细胞系 稳定的表达系统，独一无二地适用于致癌物检测。

Chemical carcinogens are believed to be the major cause of human cancer. These compounds generally must be activated to forms, termed ultimate carcinogens, that are electrophiles capable of binding to cellular macromolecules such as proteins, RNA, and DNA. Mutation of DNA is believed to be responsible for cellular transformation. The enzymes capable of activating carcinogens are the cytochrome P-450s. Multiple forms of P-450 exist that are encoded by anywhere from 50 to 200 genes. During the past several years it has become quite evident that rodents and humans possess P-450s that can be quite distinct. Quantitative and qualitative species differences have been uncovered. In particular catalytic specificities of rodent P-450s can differ from those of human P-450s. These findings have major implications in carcinogenesis research since rodents are frequently used as experimental models for tumorigenesis mediated by chemical exposures. We have therefore focused on the characterization of human P-450s by directly cloning their cDNAs from liver and lung libraries. These are then inserted into expression vectors and used to produce cultured cells containing catalytically active P-450 enzymes. The active P-450s are then tested for their abilities to activate chemical carcinogens to DNA-binding metabolites. Two systems have been used: 1) vaccinia virus transient expression capable of producing high levels of protein for direct enzymatic and spectral analysis; ii) a human lymphoblastoid cell line stable expression system uniquely suitable for carcinogen testing.