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

D. N. Sadhu, M. Merchant, S. H. Safe, Kenneth Ramos

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)124-131
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume300
Issue number1
DOIs
StatePublished - Jan 1993
Externally publishedYes

Fingerprint

Benzo(a)pyrene
Smooth Muscle Myocytes
Muscle
Rats
Cells
Modulation
Dioxins
DNA
Polycyclic Aromatic Hydrocarbons
Serum
Proteins
Electrophoretic Mobility Shift Assay
Nuclear Proteins
Hydrocarbons
Vascular Smooth Muscle
Sedimentation
Thymidine
Cytochrome P-450 Enzyme System
Blood Vessels
Sucrose

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

Modulation of Protooncogene Expression in Rat Aortic Smooth Muscle Cells by Benzo[a]Pyrene. / Sadhu, D. N.; Merchant, M.; Safe, S. H.; Ramos, Kenneth.

In: Archives of Biochemistry and Biophysics, Vol. 300, No. 1, 01.1993, p. 124-131.

Research output: Contribution to journalArticle

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abstract = "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.",
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