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活化成称为最终的形式， 致癌物质，是亲电体，能够结合细胞 大分子如蛋白质、RNA和DNA。DNA突变被认为是 负责细胞转化。这些酶能够 激活致癌物是细胞色素P-450。多种形式的P-450 由50到200个基因编码。在过去 几年来，很明显啮齿动物和人类拥有 P-450可以很明显。数量和质量物种 差异已经被发现。特别是催化特异性， 啮齿类动物的P-450可能与人类的P-450不同。这些发现 在致癌研究中的主要影响，因为啮齿动物经常 用作化学物质介导的肿瘤发生的实验模型 暴露。因此，我们重点关注人类的特征 P-450的cDNA克隆。这些 然后插入到表达载体中并用于产生培养的 含有催化活性P-450酶的细胞。现役P-450 然后测试它们激活化学致癌物的能力， DNA结合代谢物。已使用两种系统：1）牛痘病毒 瞬时表达能够产生高水平的蛋白质，用于直接 酶和光谱分析; ii）人淋巴母细胞样细胞系 稳定表达系统，特别适用于致癌物检测。

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.