CELL-TYPE-SPECIFIC MODULATION OF PDGF-B REGULATORY ELEMENTS VIA VIRALENHANCER COMPETITION - A CAVEAT FOR THE USE OF REFERENCE PLASMIDS IN TRANSIENT TRANSFECTION ASSAYS

The human platelet-derived growth factor-B (PDGF-B) gene has been show n to display a wide range of levels of mRNA transcription in a variety of cell types. Functional analyses of PDGF-B gene expression have beg un to reveal intricate, cell-type-specific regulatory mechanisms invol ving multiple control elements. We have previously isolated and charac terised several elements involved in the control of human PDGF-B gene expression in the JEG-3 choriocarcinoma cell line and in the breast ca ncer-derived cell line, ZR-75. Assessment of the positive or negative regulatory contributions of these elements was carried out using trans ient transfection assays. Such studies routinely require the inclusion of a reference plasmid in order to determine transfection efficiency. Here we show that competition for regulatory factors occurs in transf ected cells between viral enhancers and elements regulating PDGF-B gen e transcription. A frequently used reference plasmid which utilises th e SV40 promoter and enhancer region to drive expression of a beta-gala ctosidase reporter gene was found to severely repress the activity of a co-transfected reporter construct containing the PDGF-B promoter and its intronic enhancer in JEG-3 cells. This competition was localised to the enhancer region of the SV40 regulatory sequences and surprising ly, the effect was reversed in ZR-75 cells; where increasing the amoun t of reference plasmid strongly stimulated the activity of the PDGF-B construct. These results imply that the same intronic region which fun ctions equally well as an enhancer in two distinct cell-types, may ope rate in response to different transcription factor complements. Furthe rmore, this data demonstrates that the choice of reference plasmid and its quantitative use can be a crucial factor when examining putative regulatory elements by transient transfection methods.