Martinez-Flores F.,Skin And Tissue Bank At The Instituto Nacional Of Rehabilitacion |
Martinez-Flores F.,National Autonomous University of Mexico |
Barrera-Lopez A.,Skin And Tissue Bank At The Instituto Nacional Of Rehabilitacion |
Sandoval-Zamora H.E.,Skin And Tissue Bank At The Instituto Nacional Of Rehabilitacion |
And 6 more authors.
Gene Therapy and Molecular Biology
Transcription factor Egr-1 regulates expression of genes related to differentiation and proliferation of several cell types. The Egr-1 promoter has been described to respond, among others, to serum, osmotic changes, gamma radiation, and UV light. Here we describe the in vitro effect of UV light exposure times (30-120 sec) on cell proliferation and on Egr-1 promoter activity of human primary dermal fibroblasts (HPDF) transduced with the nonreplicative adenoviral vector Ad-Egr-1/Luc7 which we designed and constructed to drive transcription of reporter luciferase gene. A 600 bp fragment of the human Egr-1 promoter and a 1.3 kb DNA fragment containing the open reading frame of firefly luciferase gene were cloned into a pShuttle vector, homologously recombined in E.coli DH10B, and packaged into HEK-293 cells for large scale preparation. After establishing the vector's correct orientation and efficient expression in pAd-Egr-1/Luc-transduced HPDF, short exposure to UV light (30 sec) was shown to potentiate their proliferation rate after incubation for 24 and 72 h, as determined by crystal violet viability assays, as well as their Egr-1 promoter activity which was significantly enhanced compared to UV-unexposed controls in either serum-containing or serum-free culture media, as measured in the luciferase reporter assay. Basal and UVinduced Egr-1 promoter activities were repressed by betamethasone. Ad-CMV-Luctransduced HPDF (promoter control for response to UV light) were unresponsive to serum, UV light or betamethasone. In conclusion, Ad-Egr-1/Luc7-transduced HPDF proved useful for assessment of Egr-1-promoter modulation by UV radiation and by betamethasone, a drug used clinically to temporarily drive gene expression of injured skin. Source