Generation of Destabilized Herpes Simplex Virus Type 1 Thymidine Kinase as Transcription Reporter for PET Reporter Systems in Molecular-Genetic Imaging

• Main Authors: Chia-Hung Hsieh, 謝佳宏
• Other Authors: Fu-Du Chen
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/71859681196810291670

博士 === 國立陽明大學 === 放射醫學科學研究所 === 95 === The herpes simplex virus type 1 thymidine kinase (HSV1-TK) reporter system is being used to directly and indirectly monitor the therapeutic gene expression, immune cell trafficking, and protein-protein interactions in various animal systems. However, the issues of HSV1-TK enzyme and protein stability in living mammalian cells, and whether this reporter system is optimal for dynamic studies of gene expression events in the molecular-genetic imaging have not be addressed. The purpose of the present study was to evaluate the application of this reporter system in dynamic studies of transcriptional gene regulation and to generate a destabilized HSV1-TK for use in such studies if HSV1-TK is not optimal for dynamic studies of gene expression events and gene regulations. In this study, two tetracycline-inducible murine sarcoma cell lines, tetracycline-turn-off HSV1-tk-expressing cell line (NG4TL4/tet-off-HSV1-tk) and tetracycline-turn-off Luc-expressing cell line (NG4TL4/tet-off-Luc), were established to create an artificial regulated gene expression model in vitro. The dynamic transcriptional events mediating a series of doxycycline inductions were monitored by HSV1-TK or the firefly luciferase reporter gene using HSV1-TK enzyme activity assay and luciferase assay, respectively. The results of dynamic gene expression studies showed that the luciferase gene is an optimal reporter gene for monitoring short time-scale dynamic transcriptional events mediating a series of doxycycline inductions whereas the HSV1-TK is not optimal to achieve this purpose. Furthermore, western blotting assay demonstrated that HSV1-TK has high protein stability in living cells and its enzyme half-life in NG4TL4 cells is about 35 hours after cycloheximide-induced protein inhibition. On the other hand, the results of an efflux assay of [131I] FIAU and [3H] GCV revealed that the molecular probe phosphorylated by HSV1-TK can be trapped long term within HSV1-tk stably-transformed cells. Therefore, a long half-life radionuclide is not suitable for dynamic gene expression studies. Based on these results, we suggest that the HSV1-TK reporter system is not optimal for monitoring short time-scale dynamic processes such as kinetic gene expression controlled by inducible promoters or a less stable protein with a more rapid turnover, due to the limitations of the half-life of the HSV1-TK enzyme and the cellular retention time of their phosphorylated molecular probes. To overcome high protein or enzyme stability of HSV1-TK limited its application in studies that require rapid reporter turnover; we created a destabilized HSV1-TK for use in such studies by targeting inactivating mutations in the nuclear localization signal (NLS) and fusing the degradation domain of mouse ornithine decaroxylase (MODC) to the C-terminal end of HSV1-TK. This destabilized HSV1-TK, unlike wild-type HSV1-TK, was unstable in the presence of cycloheximide and had a short half-life of protein and enzyme activity using western blotting analysis and HSV1-TK enzyme activity assay. The proteasome inhibition assay also confirmed the destabilized HSV1-TK was degraded through 26S proteasome pathway. Furthermore, in vitro cell proliferation assay demonstrated that overexpression of this destabilized HSV1-TK has no significant cytotoxicity in mammalian cells. The suitability of destabilized HSV1-TK as a transcription reporter was tested by linking it to tetracycline-turn-off-expressing system. The dynamic transcriptional events mediating a series of doxycycline (Dox) inductions were monitored by destabilized HSV1-TK or by wild-type HSV1-TK. The results of dynamic gene expression studies showed that the destabilized HSV1-TK is an optimal reporter gene for monitoring the dynamic changes in short time-scale transcriptional events mediating a series of Dox inductions, whereas the wild-type HSV1-TK is not optimal to achieve this purpose. Therefore, the destabilized HSV1-TK is an optimal reporter for dynamic studies of short time-scale gene expression events or gene regulations. In summary, we addressed the issue whether the HSV1-TK is an optimal reporter system for dynamic studies of short time-scale gene expression events and generated a destabilized herpes simplex virus type 1 thymidine kinase as transcription reporter for position emission tomography (PET) reporter system in the molecular-genetic imaging. These results were filled in the gap for unknown protein or enzyme stability of HSV1-TK in living mammalian cells and provided a novel mutant form of HSV1-TK as a reporter in the molecular-genetic imaging. Using the destabilized HSV1-TK as a transcription reporter together with its complimentary molecular probes which have a short physical and biological half-life, can increase the temporal resolution and allow more directly monitoring of transcription induction and more easily monitoring its coincidence with other biochemical change.