Diminished Carcinogen Detoxification Is a Novel Mechanism for Hypoxia-inducible Factor 1-mediated Genetic Instability

Marten A Schults,Leen Timmermans,Roger W Godschalk,Jan Theys,Bradly G Wouters,Frederik J Van Schooten,Roland K Chiu

doi:10.1074/jbc.m109.076323

Abstract

The hypoxia-inducible factor 1 (HIF-1) pathway is induced in many tumors and associated with poorer outcome. The hypoxia-responsive transcription factor HIF-1alpha dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT), which is also an important binding partner for the aryl hydrocarbon receptor (AhR). AhR is an important mediator in the metabolic activation and detoxification of carcinogens, such as the environmental pollutant benzo[a]pyrene (BaP). We hypothesized that HIF-1alpha activation attenuates BaP-induced AhR-mediated gene expression, which may lead to increased genetic instability and malignant progression. Human lung carcinoma cells (A549) were simultaneously stimulated with CoCl(2), which leads to HIF-1alpha stabilization and varying concentrations of BaP. Both quantitative PCR and immunoblot analysis indicated that induction of the hypoxia response pathway significantly reduced the levels of AhR downstream targets CYP1A1 and CYP1B1 and AhR protein binding to ARNT. We further demonstrate that the BaP-induced hypoxanthine-guanine phosphoribosyltransferase mutation frequency and gamma-H2AX foci were markedly amplified when the HIF-1 pathway was induced. BaP-DNA adducts were only marginally increased, and transient strand breaks were diminished by HIF-1 induction, indicating changes in DNA repair. These data indicate that concurrent exposure of tumor cells to hypoxia and exogenous genotoxins can enhance genetic instability.

Highlights

hypoxia-inducible factor 1 (HIF-1)␣ is regarded as the primary molecular switch to alter gene expression in response to reduced oxygen tension
HIF-1 binds to the (A/G)CGTG consensus sequence in the hypoxia-responsive elements of the promoter/enhancer regions in the DNA [12], where it drives the expression of a wide array of hypoxia-inducible genes, including vascular endothelial growth factor [13], glucose transporter 1 [14], and carbonic anhydrase IX (CA-IX) [15]
BaP Down-regulates in a Dose-dependent Manner the CoCl2mediated Induction of CA-IX—The effect of BaP on the HIF-1 pathway was tested by determining changes in mRNA and protein levels of CA-IX

Summary

Introduction

HIF-1␣ is regarded as the primary molecular switch to alter gene expression in response to reduced oxygen tension. HIF-1 binds to the (A/G)CGTG consensus sequence in the hypoxia-responsive elements of the promoter/enhancer regions in the DNA [12], where it drives the expression of a wide array of hypoxia-inducible genes, including vascular endothelial growth factor [13], glucose transporter 1 [14], and carbonic anhydrase IX (CA-IX) [15]. AhR translocates into the nucleus, dimerizes with ARNT, and activates gene expression by binding to the TNGCGTG consensus sequence in the xenobiotic-responsive elements of target genes [16] This leads to the up-regulation of a multitude of genes, including the cytochrome P450 isoforms CYP1A1 and CYP1B1. We measured BPDE-DNA adduct levels and transient strand breaks to further investigate the relationship between the cross-talk and the changed genetic instability

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