title: Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene.
abstract: The microenvironment of rapidly growing tumors is associated with increased energy demand and diminished vascular supply, resulting in focal areas of prominent hypoxia. A number of hypoxia-responsive genes have been associated with growing tumors, and here we demonstrate that the multidrug resistance (MDR1) gene product P-glycoprotein, a Mr approximately 170,000 transmembrane protein associated with tumor resistance to chemotherapeutics, is induced by ambient hypoxia. Initial studies using quantitative microarray analysis of RNA revealed an approximately 7-fold increase in MDR in epithelial cells exposed to hypoxia (pO(2) 20 torr, 18 h). These findings were further confirmed at the mRNA and protein level. P-Glycoprotein function was studied by analysis of verapamil-inhibitable efflux of digoxin and rhodamine 123 in intact T84 cells and revealed that hypoxia enhances P-glycoprotein function by as much as 7 +/- 0.4-fold over normoxia. Subsequent studies confirmed hypoxia-elicited MDR1 gene induction and increased P-glycoprotein expression in nontransformed, primary cultures of human microvascular endothelial cells, and analysis of multicellular spheroids subjected to hypoxia revealed increased resistance to doxorubicin. Examination of the MDR1 gene identified a binding site for hypoxia inducible factor-1 (HIF-1), and inhibition of HIF-1 expression by antisense oligonucleotides resulted in significant inhibition of hypoxia-inducible MDR1 expression and a nearly complete loss of basal MDR1 expression. Studies using luciferase promoter constructs revealed a significant increase in activity in cells subjected to hypoxia, and such hypoxia inducibility was lost in truncated constructs lacking the HIF-1 site and in HIF-1 binding site mutants. Extensions of these studies also identified a role for Sp1 in this hypoxia response. Taken together, these data indicate that the MDR1 gene is hypoxia responsive, and such results may identify hypoxia-elicited P-glycoprotein expression as a pathway for resistance of some tumors to chemotherapeutics.
authors.affiliation: Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Katrina M
authors.initials: KM
authors.lastname: Comerford
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Wallace
authors.forename: Jörn
authors.initials: J
authors.lastname: Karhausen
authors.forename: Nancy A
authors.initials: NA
authors.lastname: Louis
authors.forename: Michael C
authors.initials: MC
authors.lastname: Montalto
authors.forename: Sean P
authors.initials: SP
authors.lastname: Colgan
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics; D018938:Caco-2 Cells / Q000378:metabolism / Q000502:physiology; D015687:Cell Hypoxia / Q000502:physiology; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000502:physiology*; D004727:Endothelium / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic; D019450:Genes, MDR / Q000235:genetics*; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins / Q000502:physiology*; D014157:Transcription Factors; D015533:Transcriptional Activation
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D014157:Transcription Factors