However, under hypoxic conditions, HIF-1a or 2a heterodimerize with ARNT forming HIF-1 and HIF-2, which bind to hypoxic response elements within the L-838,417 promoters of target genes to promote transcription. HREs have been found in a remarkably large set of genes that affect many different genetic programs, including proliferation, differentiation, tissue-specific responses, and cell death. During our studies of DDX3 we Necrostatin-7 discovered putative HRE sequences within its promoter. Therefore, we hypothesized that the hypoxic microenvironment within solid tumors activates the expression of DDX3. In this study, we investigated whether expression of DDX3 gene is up-regulated by HIF-1a or HIF-2a in response to hypoxia in human breast epithelial cell lines. Our data provide evidence that hypoxic induction of the DDX3 gene is mediated by transactivation of DDX3 promoter by HIF-1a through a consensus HRE binding site. As shown in Figure 4c, under shHIF-1a conditions the reporter activities shown in Figure 4b were decreased in all cases while the pattern of activities was identical to that observed in Figure 4a. To study the activities of the mutated promoters under HIF-1a stabilized conditions, the reporter constructs were transfected into MCF 7 cells that were then treated with CoCl2. As shown in Figure 4d reporter activities were enhanced following CoCl2 treatment without changing the pattern seen in Figure 4a. To further explore HIF-1 regulation of DDX3 expression we cotransiently transfected MCF 7 cells with the mutated promoterreporter constructs and a constitutive expressing HIF-1a vector construct. Under these conditions all reporter activities roughly tripled and the loss of the HRE-1 site showed qualitatively the same diminishing effect on reporter activities. In total, the overall patterns of activities seen in Figure 4 remained qualitatively very similar regardless of the conditions tested and all these results indicate that loss of HRE-1 alone was sufficient to decrease reporter activity to roughly the same degree as what occurred when HRE-1 + 2 or +3 or HRE-1+2+3 were mutated. On the other hand, loss of either HRE-2 or HRE-3 or both HRE-2 + 3 either caused an increase in reporter activities or had no impact on reporter activity respectively.