Modulation of Protooncogene Expression in Rat Aortic Smooth Muscle Cells by Benzo[a]Pyrene

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), cause vascular lesions of atherosclerotic etiology in avian and rodent species. Because the development of these lesions is associated with aberrant proliferation of vascular smooth muscle cells (SMCs), the present study was conducted to evaluate the effects of BaP on DNA synthesis and protooncogene expression in rat aortic SMCs. Subcultured cells were exposed to BaP (0.3-30 μM) for various times and processed for measurements of [3H]thymidine incorporation and c-Ha-ms or c-myc protooncogene expression. Sucrose density gradient analysis and gel mobility shift assays were employed to assess binding of BaP to intracellular proteins in the nuclear fraction and their interaction with a synthetic dioxin responsive element (DRE), respectively. BaP (0.3 and 3 μM) increased DNA synthetic rates in randomly cycling SMCs in a concentration- and time-dependent fashion. Increased DNA synthesis was also observed in synchronized SMCs treated with 3.0 μM BaP. Challenge of quiescent SMCs with 10% fetal bovine serum in the presence of BaP for 8 h enhanced serum-induced c-Ha-ras and c-myc protooncogene expression. Velocity sedimentation analysis of the nuclear fraction from SMCs treated with 0.3 μM [3H]BaP resulted in specifically bound peaks of 4.4 S and 6.5 S. The 6.5 S peak was competitively inhibited in the presence of unlabeled 10 μM 2,3,7,8-tetrachlorodibenzofuran, an aryl hydrocarbon ligand, while both peaks were eliminated in cells cotreated with 10 μM α-naphthoflavone (α-NF), an inhibitor of cytochrome P450 and an antagonist of PAH-protein interactions. α-NF also inhibited the induction of c-Ha-ras mRNA and DNA synthesis by BaP and the binding of BaP-Ah receptor complexes to the DRE. These results show that BaP enhances serum-induced protooncogene expression during the early part of the cell cycle in rat nortic SMCs and suggest that binding of HaP to intracellular proteins may play a role in these responses.