Virus-based reporter systems for monitoring transcriptional activity of hypoxia-inducible factor 1

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-1a 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][476][477][478][479][480][481]. In order to monitor HIF-1 transcriptional activity, we have developed HIF-1a-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-1a activation, ii) shows upregulation in response to HIF-1 activation and downregulation following inhibition of HIF-1a expression by small interfering RNA, iii) follows the dynamics of endogenous HIF-1 target gene expression. The retrovirus-and lentivirusbased reporters can be used for high-throughput screening of HIF-1a modulators and for the study of crosstalk between HIF-1 and different related signal transduction pathways. Potential applications for the reporters are discussed.

Small-molecule RETRA suppresses mutant p53-bearing cancer cells through a p73-dependent salvage pathway

“…We decided to search for small molecules that reactivate the transcriptional activity of p53 in a mutant p53-bearing cancer cell line. We introduced by lentiviral transfer into epidermal carcinoma cell line A431, which is bearing His-273 p53 mutant, a reporter construct LC5 carrying a cassette for expression of ␤-galactosidase under control of a minimal CMV promoter coupled to multiple p53-binding elements (17) [supporting information (SI) Fig. S1].…”

“…We used human carcinoma cell lines expressing mutant p53 A431, HT29, SW480, SW620 (His-273 mutant p53), MDA-MB-231 (Lys-280 mutant p53), MDA-MB-435 (Glu-266 mutant p53), p53-negative cell lines H1299, PC3, Saos-2, MDA041, wild-type p53 expressing cell lines A549, HeLa and human HEFs (normal fibroblasts from a 12-week human embryo, passage 10 -14) and their derivatives with introduced recombinant lentiviral constructs. We created reporter cell lines expressing lacZ under control of p53-responsive promoter by infection of A431, H1299, A549, PC3, Saos-2, and MDA041 cells with recombinant lentiviral construct LC5 as described (17). We generated cell lines expressing p53 mutant with inhibited expression of p53 or p73 by infection with lentiviral constructs pLSLP-sh-p53 and pLSLPsh-p73 as described (20).…”

“…We used the following regions of p63 and p73 mRNA for designing shRNA oligonucleotides and insertion into pLSLG vector: for p63, 5Ј-ctaggtagaagtgagcaaa and 5Ј-gaaaacaatgcccagactcaa; for p73, 5Ј-gccaagtgactgtgtctgaaa and 5Ј-cgacatcttttggttctggat. We described lentiviral reporter construct pLC5 for expression of ␤-galactosidase under the control of a minimal cytomegalovirus promoter and several copies of 20-bp p53-binding segments from the CDKN1A gene (17). We prepared recombinant lentiviral stocks as described (20).…”

VHL loss in renal cell carcinoma leads to up-regulation of CUB domain-containing protein 1 to stimulate PKCδ-driven migration

“…Lentiviral plasmids pLKO.1shCDCP1 (Open Biosystems) and pLKO.1shGFP, and retroviral plasmids LZRS-myc-PKCδR144/145A (31) and LZRS-linker were used for viral production as described in ref. 42.…”

Improving the Post-Stroke Therapeutic Potency of Mesenchymal Multipotent Stromal Cells by Cocultivation With Cortical Neurons: The Role of Crosstalk Between Cells

“…Lentiviral constructs were introduced by transient transfection of 293T cells, along with lentiviral packaging plasmids pCMV-deltaR8.2 and pCMV-VSV-G using Lipofectamine LTX reagent (Invitrogen, Carlsbad, CA), as previously described [20]. Viral particles were harvested starting 24 hours after transfection and used for infection of target cells.…”