Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9127140 | Gene | 2005 | 10 Pages |
Abstract
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-1α activity in vivo may represent a valuable therapeutic approach to these disorders [Hofer, T., Desbaillets, I., Hopfl, G., Wenger, R.H., Gassmann, M., 2002. Characterization of HIF-1 alpha overexpressing HeLa cells and implications for gene therapy. Comp. Biochem. Physiol., Toxicol. Pharmacol. 133, 475-481]. In order to monitor HIF-1 transcriptional activity, we have developed HIF-1α-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-1α activation, ii) shows upregulation in response to HIF-1 activation and downregulation following inhibition of HIF-1α 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-1α modulators and for the study of crosstalk between HIF-1 and different related signal transduction pathways. Potential applications for the reporters are discussed.
Keywords
LTRENO1Enolase 1von Hippel Lindau proteinlacZLDHAONPGpVHL5-bromo-4-chloro-3-indolyl β-d-galactopyranosideX-GalMCMVHREDesferrioxamine mesylatedsDNADFOHIF-1o-nitrophenyl β-D-galactopyranosideβgalPGK1double stranded DNASmall interfering RNAsiRNAβ-galactosidasesinLong terminal repeatBase pair(s)hour(s)hypoxia-inducible factor 1hypoxia response elementVascular endothelial growth factorVascular Endothelial Growth Factor (VEGF)Phosphoglycerate kinase 1Lactate dehydrogenase Anucleotide(s)unit(s)HIV-1Human immunodeficiency virus type 1β-galactosidase gene
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Authors
O.V. Razorenova, A.V. Ivanov, A.V. Budanov, P.M. Chumakov,