Real-time imaging of hypoxia-inducible factor-1 activity in tumor xenografts

Hypoxia-inducible factor-1 (HIF-1) is responsible for various gene expressions related to tumor malignancy, such as metastasis, invasion and angiogenesis. Therefore, monitoring HIF-1 activity in solid tumors is becoming increasingly important in clinical and basic studies. To establish a convenient system for visualizing HIF-1 activity in tumor xenografts, we employed a promoter consisting of five copies of hypoxia response elements (5HRE), whose activity depends on HIF-1, and used a derivative of green fluorescence protein (d2EGFP) as a reporter gene. A human melanoma cell line, Bel 1, which contains the 5HRE-d2EGFP gene, showed fluorescence in response to hypoxia. The fluorescent intensity correlated inversely with the surrounding oxygen tension, and was time-dependent for the hypoxic treatment. Reoxygenation resulted in a rapid decrease in fluorescence due to the signal sequence for protein degradation encoded in d2EGFP, which enabled monitoring of HIF-1 activity in real-time. Heterogeneous fluorescence was observed in the solid tumor of a non-sacrificed tumor-bearing mouse. Immunohistochemical analysis confirmed that d2EGFP-expressing regions overlapped with the ones stained with a hypoxia marker, pimonidazole. These results suggest that the 5HRE-d2EGFP gene is suitable for the real-time imaging of HIF-1-activating cells in vivo, due to the short half-life of the d2EGFP protein as well as the specificity of the 5HRE promoter for HIF-1 activity.