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.

Abstract

UNLABELLED:

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.

• Authors: Chia-Hung Hsieh, Jung-Wen Kuo, Yi-Jang Lee, Chi-Wei Chang, Juri G Gelovani, Ren-Shyan Liu
• Journal: Journal of nuclear medicine : official publication, Society of Nuclear Medicine (J Nucl Med) Vol. 50 Issue 12 Pg. 2049-57 (Dec 2009) ISSN: 1535-5667 [Electronic] United States
• PMID: 19910419 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Hypoxia-Inducible Factor 1
• Recombinant Fusion Proteins
• Green Fluorescent Proteins
• Thymidine Kinase
• Oxygen

Topics

• Animals
• Cell Hypoxia
• Cell Line, Tumor
• Enzyme Stability
• Genes, Reporter (genetics)
• Green Fluorescent Proteins (genetics, metabolism)
• Herpesvirus 1, Human (enzymology)
• Humans
• Hypoxia-Inducible Factor 1 (genetics, metabolism)
• Intracellular Space (metabolism)
• Mice
• Molecular Imaging (methods)
• Mutation
• Neoplasms (genetics, metabolism, pathology)
• Oxygen (metabolism)
• Protein Biosynthesis
• Protein Transport
• Recombinant Fusion Proteins (genetics, metabolism)
• Signal Transduction
• Thymidine Kinase (genetics, metabolism)
• Time Factors
• Transcription, Genetic
• Transfection