POLYCYCLIC HYDROCARBON METABOLISM AND CARCINOGENESIS

我们的长期目标是了解耐药性的分子基础 和对多环烃诱导的致癌作用的易感性。 我们 以前曾假设苯并（a）芘（BaP）和 3-甲基胆蒽（3 MC）诱导大鼠细胞色素P450 c 以高亲和力和可饱和的方式与4S胞质复合 蛋白 后者会转移到细胞核中， 细胞色素P450 c基因的特定区域，并增加 这个基因。 我们现在已经纯化了4S结合蛋白，并配制了 以下是我们调查的具体目的。 A.生产 这种纯化蛋白的单克隆抗体，将表征 B）分离该结合蛋白， 通过筛选λ gtll表达， 用针对该蛋白的抗体或用寡核苷酸 探针，c）根据DNA序列和顺式调控序列表征基因 元素，d）证明4S结合蛋白充当 通过使用截短的细胞色素P450 c基因的转录因子 和体外转录测定系统，e）分离9 S TCDD-结合蛋白， 蛋白质，制备针对其的单克隆抗体并区分该结合 f）定义来自4S组分的蛋白质的特定区域， 4S和9 S结合蛋白与细胞色素的相互作用 P450 c基因，和g）转化“受体阴性”细胞，如早期 传代RL-PR-C大鼠肝细胞，通过 转染，并证明该转染的后续影响， 细胞色素P450 c的诱导。 我们希望通过这些具体的 目的是我们将有一个更好的了解约束力的作用 细胞色素P450 c的诱导蛋白质。

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.