Stephen J. Miller

The sequential activation and repression of the human PDGF-B gene during chronic hypoxia reveals antagonistic roles for the depletion of oxygen and glucose.

Abstract Hypoxia and glucose deprivation, are important during many physiological and pathological processes. Cells respond to these stimuli by activating genes involved in the regulation of metabolism and angiogenesis. Platelet derived growth factor-B (PDGF-B) is involved in the regulation of angiogenesis and tumour progression and is induced by hypoxia. Most known hypoxia-induced genes are activated by the hypoxia inducible factor (HIF-1), via its binding to specific response elements. The mechanism of hypoxic induction and the effect of low glucose on PDGF-B expression have not been characterised. We show that PDGF-B exhibits a novel, biphasic regulation (induction, followed by repression below basal levels) in bladder carcinoma cells cultured under chronic hypoxia. We show that the repression observed after long-term hypoxia is due to glucose-depletion and that this can also abrogate short-term hypoxic induction. This is in contrast to the previous results showing that hypoxiahypogly-caemia elicit the same response. We also show that a putative hypoxia response element in the PDGF-B promoter is not sufficient for hypoxic induction, although it does function as a hypoxia independent enhancer element in hepatocellular carcinoma cells.

Cell-type-specific modulation of PDGF-B regulatory elements via viral enhancer 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 shown to display a wide range of levels of mRNA transcription in a variety of cell types. Functional analyses of PDGF-B gene expression have begun to reveal intricate, cell-type-specific regulatory mechanisms involving multiple control elements. We have previously isolated and characterised several elements involved in the control of human PDGF-B gene expression in the JEG-3 choriocarcinoma cell line and in the breast cancer-derived cell line, ZR-75. Assessment of the positive or negative regulatory contributions of these elements was carried out using transient 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 transfected cells between viral enhancers and elements regulating PDGF-B gene transcription. A frequently used reference plasmid which utilises the SV40 promoter and enhancer region to drive expression of a beta-galactosidase 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 surprisingly, the effect was reversed in ZR-75 cells; where increasing the amount of reference plasmid strongly stimulated the activity of the PDGF-B construct. These results imply that the same intronic region which functions equally well as an enhancer in two distinct cell-types, may operate in response to different transcription factor complements. Furthermore, 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.

RANDOM MONOALLELIC EXPRESSION OF THE IMPRINTED IGF2 AND H19 GENES IN THE ABSENCE OF DISCRIMINATIVE PARENTAL MARKS

Abstract The IGF2 and H19 genes are genomically imprinted and expressed preferentially from the paternal and maternal alleles, respectively, during human prenatal development. The exact role of the parental imprint(s), however, is not known. To explore this issue in some detail, we have examined human androgenetic cells which by definition should be incapable of allelic discrimination given the paternal origin of both genomes. Allele-specific in situ hybridisation analysis of dispermic complete hydatidiform moles shows that IGF2 and H19 can be found to be transcriptionally active in a variegated manner, which results in the generation of random monoallelic expression patterns. This data shows that imprinted genes can be expressed monoallelically in the absence of discriminating parental marks and raises the question whether or not mechanisms underlying monoallelic expression preceded the acquisition of parental imprints during evolution.