Virus-based reporter systems for monitoring transcriptional activity of hypoxia-inducible factor 1.
Being key regulator of oxygen homeostasis hypoxia-inducible factor 1 (HIF-1) plays significant roles in cancer progression as well as in cardiovascular diseases. The modulation of HIF-1alpha activity in vivo may represent a valuable therapeutic approach to these disorders. In order to monitor HIF-1 transcriptional activity, we have developed HIF-1alpha-responsive reporter constructs, in which lacZ gene expression is driven by minimal Hsp70 gene promoter or minimal immediate early promoter of cytomegalovirus (CMV) and a combination of hypoxia response elements from regulatory regions of PGK1, ENO1 and LDHA genes. For the efficient delivery to a wide variety of cell types we chose retroviral and lentiviral vectors as carriers of the reporter cassette. We demonstrate that the obtained reporter system i) has a high inducibility in response to treatments leading to HIF-1alpha activation, ii) shows upregulation in response to HIF-1 activation and downregulation following inhibition of HIF-1alpha expression by small interfering RNA, iii) follows the dynamics of endogenous HIF-1 target gene expression. The retrovirus- and lentivirus-based reporters can be used for high-throughput screening of HIF-1alpha modulators and for the study of crosstalk between HIF-1 and different related signal transduction pathways. Potential applications for the reporters are discussed.
Razorenova
O V
OV
Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195, USA.
Ivanov
A V
AV
Budanov
A V
AV
Chumakov
P M
PM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D003676:Deferoxamine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D014780:Viruses / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism
C497442:HIF1A protein, human; D018840:HSP70 Heat-Shock Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D001616:beta-Galactosidase; D003676:Deferoxamine