POLYCYCLIC HYDROCARBON METABOLISM AND CARCINOGENESIS

我们的长期目标是了解抗药性的分子基础 以及对多环烃诱发致癌的易感性。我们 曾假设苯并(A)芘(BaP)和 3-甲基胆蒽(3MC)诱导大鼠细胞色素P450c 与4S胞浆以高亲和力和可饱和的方式络合 蛋白。后者会移位到核中，与 细胞色素P450c基因的特异区，并增加转录 这个基因。我们现在已经纯化了4S结合蛋白，并制定了 以下是我们调查的具体目的。我们将a)生产 抗此纯化蛋白的单抗，将表征 “受体”，将该结合蛋白定位于细胞内，b)分离 通过筛选lambda gtll的表达获得该4S结合蛋白的基因 带有该蛋白抗体或具有寡核苷酸的文库 探针，c)根据dna序列和顺式调控来表征基因。 元件，d)表明4S结合蛋白作为一种 利用截短的细胞色素P450c基因表达转录因子 和体外转录检测系统，e)分离9S TCDD结合 蛋白，制备抗该蛋白的单抗并鉴定其结合 来自4S组分的蛋白质，f)定义 4S和9S结合蛋白与细胞色素的相互作用 P450c基因，和g)转化为“受体阴性”的细胞，如早期 RL-PR-C大鼠肝细胞传代为受体阳性 并演示了该转染法的后续效果 细胞色素P450c的诱导。我们希望通过这些具体的 目的我们将对约束的作用有更好的理解 诱导细胞色素P450c的蛋白质。

Our long-term objective is to understand the molecular basis for resistance and susceptibility to polycyclic hydrocarbon-induced carcinogenesis. We have previously postulated that benzo(a)pyrene (BaP) and 3-methylcholanthrene (3MC) induce cytochrome P450c in the rat after complexing with high affinity and in saturable manner to a 4S cytosolic protein. The latter would translocate into the nucleus, interact with a specific region of the cytochrome P450c gene, and increase transcription of this gene. We have now purified the 4S binding protein and have formulated the following specific aims for our investigations. We will a) produce monoclonal antibody to this purified protein, will characterize the "receptor", localize this binding protein intracellularly, b) isolate the gene for this 4S binding protein by screening a lambda gtll expression library with either the antibody to this protein or with an oligonucleotide probe, c) characterize the gene as to DNA sequence and cis-regulatory elements, d) demonstrate that the 4S binding protein acts as a transcriptional factor through the use of truncated cytochrome P450c gene and in vitro transcriptional assay systems, e) isolate the 9S TCDD-binding protein, prepare monoclonal antibody to it and differentiate this binding protein from the 4S component, f) define the specific regions of interaction of both the 4S and 9S binding proteins with the cytochrome P450c gene, and g) convert a "receptor-negative" cell, such as early passage RL-PR-C rat hepatocytes, to a "receptor-positive" one by transfection and demonstrate the subsequent effects of this transfection on the induction of cytochrome P450c. We hope that through these specific aims we will have a better understanding of the role of the binding proteins in the induction of cytochrome P450c.