POLYCYCLIC HYDROCARBON METABOLISM AND CARCINOGENESIS

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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.