Hypoxia Reporter Element Assay

Background

In our recent publication (van Oosterwijk et al., 2018), we sought to examine the regulation of BMX, a nonreceptor tyrosine kinase, in response to sorafenib treatment. BMX, a Tec kinase family member, is known bind to tyrosine-phosphorylated proteins and mediate membrane targeting by binding to phosphatidylinositol 3,4,5-triphosphate (PIP3; Chen et al., 2013). We showed that direct treatment of sorafenib in acute myeloid leukemia (AML) cells lines with or without stromal cell support did not contribute to the upregulation of BMX. Previous studies have shown that BMX expression can be induced by ischemia and that sorafenib has antiangiogenic activity (He et al., 2006; Davis et al., 2011). Therefore, we hypothesized that the antiangiogenic activity of sorafenib causes a hypoxic environment within the bone marrow, thus contributing to a hypoxia-dependent BMX upregulation in AML. Under hypoxic conditions, we were able to show a significant increase in BMX expression in a number of different cell lines. Further analysis of the BMX promoter identified a putative hypoxia-responsive element (HRE; 5’-ACGTG-3’) at -5005. To test whether this HRE was involved in the hypoxia-induced promoter activation of BMX, we developed a hypoxia element reporter assay.

Reporter assays are extensively used throughout the scientific community to study alterations and modifications to gene expression in response to environmental changes. One type of reporter assay that is gaining in acceptance are the dual-reporter assays. This type of reporter assay allows the simultaneous expression and measurement of two different reporters within a single cell and had been widely proven to improve experimental accuracy by reducing extraneous influences. One such dual assay system is the LightSwitch Dual Assay System. This system utilizes the RenSP luciferase reporter gene, a novel luciferase developed by SwitchGear Genomics and the Cypridina luciferase reporter gene. RenSP and Cypridina employ different substrates, thus eliminating cross-reaction between proteins and their substrates. RenSP is used with your favorite gene as the reporter signal, and Cypridina is utilized with a constitutively active promoter as the control signal.

Here, we used the LightSwitch Dual Assay System to evaluate the involvement of the BMX HRE to its upregulation in response to hypoxic conditions.