Our immunoblot data mirrors the mRNA data in that DDX3 protein levels increased in both cell lines Muscimol following 8 h of either hypoxia or CoCl2 treatment but reflect that the protein stability was higher than the mRNA stability in MCF 10A cells. Recent evidence has shown that HIF-1 and not HIF-2 is the principal HIF involved with the regulation of the genes in response to hypoxia in cancer cell lines including hepatoma, neuroblastoma, and breast cancer. Thus, we used shRNA knockdown of HIF-1a to determine the specificity of hypoxia induced DDX3 expression. The data obtained with the HIF-1a knockdown in MCF 10A and MCF 7 cell lines provide consistent evidence supporting the major involvement of HIF-1 and not HIF-2 in the transcriptional activation of DDX3 by hypoxia and indicated that DDX3 could be considered a new member of the growing family of HIF-1 targeted genes. In order to define the binding sites of HIF-1 in the DDX3 promoter that are contributing to increased Leptomycin A expression under hypoxic conditions, we performed luciferase based reporter assays using 2 kb gene sequence that is immediately up stream of the translation start site. In our luciferase reporter studies with MCF 7 cells we observed that the promoter fragment that extended approximately 1.5 kb upstream from the translation start site showed basal activity that was relatively high in comparison to the other promoter-reporter constructs tested. This is consistent with a similar high basal reporter activity reported during a characterization of the DDX3 promoter in two human embryonic carcinoma cell lines NEC8 and NEC14. This is likely due to the binding of basal levels of transcriptional activators to the upstream region of DDX3 promoter. When we co-transfected a constitutively expressing HIF-1a vector along with the promoterreporter constructs the reporter activity increased by an order of magnitude for all the promoter constructs expect the two that lack the 2320 bp region, i.e., the region closest to the translation start site. This indicates that HIF-1 directly or indirectly can regulate DDX3 expression via a cis-element located within the 2320 bp proximal promoter region that is particularly critical to the modulation of expression by HIF-1. An analysis of the 1.5 kb DDX3 promoter sequence showed that it contains at least 3 core hypoxia response elements, A/GCGTG, i.e., HIF-1 binding sites.