Construction of Mutant TKGFP for Real-Time Imaging of Temporal Dynamics of HIF-1 Signal Transduction Activity Mediated by Hypoxia and Reoxygenation in Tumors in Living Mice

The herpes simplex virus type 1 thymidine kinase (HSV1-tk)/green fluorescent protein (TKGFP) dual-reporter gene and a multimodality imaging approach play a critical role in monitoring therapeutic gene expression, immune cell trafficking, and protein-protein interactions in translational molecular-genetic imaging. However, the cytotoxicity and low temporal resolution of TKGFP limits its application in studies that require a rapid turnover of the reporter. The purpose of this study was to construct a novel mutant TKGFP fusion reporter gene with low cytotoxicity and high temporal resolution for use in the real-time monitoring of temporal dynamics and spatial heterogeneity of hypoxia-inducible factor 1 (HIF-1) signal transduction activity mediated by hypoxia and reoxygenation in vitro and in vivo. Methods: Destabilized TKGFP was produced by inserting the nuclear export signal (NES) sequence at the N terminus and fusing the degradation domain of mouse ornithine decarboxylase (dMODC) at the C terminus. The stability of TKGFP in living NG4TL4 cells was determined by Western blot analysis, HSV1-tk enzyme activity assay, and flow cytometric analysis. The suitability of NESTKGFP: dMODC as a transcription reporter was investigated by linking it to a promoter consisting of 8 copies of hypoxia-responsive elements, whose activities depend on HIF-1. The dynamic transcriptional events mediated by hypoxia and reoxygenation were monitored by NESTKGFP: dMODC or TKGFP and determined by optical imaging and PET. Results: Unlike TKGFP, NESTKGFP: dMODC was unstable in the presence of cycloheximide and showed a short half-life of protein and enzyme activity. Rapid turnover of NESTKGFP: dMODC occurred in a 26S proteasome-dependent manner. Furthermore, NESTKGFP: dMODC showed an upregulated expression and low cytotoxicity in living cells. Studies of hypoxia-responsive TKGFP and NESTKGFP: dMODC expression showed that NESTKGFP: dMODC as a reporter gene had better temporal resolution than did TKGFP for monitoring the dynamic transcriptional events mediated by hypoxia and reoxygenation; the TKGFP expression level was not optimal for the purpose of monitoring. Conclusion: In translational molecular-genetic imaging, NESTKGFP: dMODC as a reporter gene, together with optical imaging and PET, allows the direct monitoring of transcription induction and easy determination of its association with other biochemical changes.