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.


CONCLUSIONS
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.
Hsieh
Chia-Hung
CH
Institute of Medical Science, China Medical University, Taichung, Taiwan.
Kuo
Jung-Wen
JW
Lee
Yi-Jang
YJ
Chang
Chi-Wei
CW
Gelovani
Juri G
JG
Liu
Ren-Shyan
RS
D000818:Animals; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D004795:Enzyme Stability; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000201:enzymology; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D009154:Mutation; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D010100:Oxygen / Q000378:metabolism*; D014176:Protein Biosynthesis; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
D051793:Hypoxia-Inducible Factor 1; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase; D010100:Oxygen