Abstract
Imaging 2 different molecular-genetic events in a single subject by PET is essential in a variety of in vivo applications. Using herpes simplex virus-1 thymidine kinase (HSV1-tk) mutants with narrower substrate specificities in combination with wild-type HSV1-tk (wtHSV1-tk) would enable differential imaging with corresponding radiotracers, namely 2′-deoxy-2′- 18F-fluoro-5- ethyl-1-β-D-arabinofuranosyl-uracil ( 18F-FEAU) and the acyclo- guanosine derivative 9-(4- 18F-fluoro-3-[hydroxymethyl]butyl)- guanine ( 18F-FHBG). In this study, we evaluated wtHSV1-tk and the A168H mutant, which has been reported to exhibit enhanced acycloguanosine substrate catalytic activity and diminished pyrimidine phosphorylating activity, as PET reporter genes. Methods: Computational analysis was performed to assess the binding mode of FHBG and FEAU to wtHSVI -tk and the A168H variant. U87 cells were stably transduced with wtHSVI -tk or HSV1-tk(A168H) fused with green fluorescent protein and sorted to obtain equivalent transgene expression. In vitro uptake studies were performed to determine rates of substrate accumulation and retention. Nude mice bearing tumors expressing HSV1-tk variants were subsequently imaged using 18F-FHBG and 18F-FEAU. Results: Docking results indicate that binding of FHBG to the A168H variant is unaffected whereas the binding of FEAU is hindered because of a steric clash with the bulkier mutant residues. U87 cells expressing HSV1-tk(A168H) accumulated 18F-FHBG in in vitro uptake studies at a 3-fold higher rate than did cells expressing wtHSVI -tk without any detectable accumulation of 3H-FEAU. Furthermore, HSV1-tk(A168H) demonstrated no thymidine phosphorylation activity. In contrast, U87 cells expressing wtHSVI-tk preferentially accumulated 3H- FEAU at an 18-fold higher rate than they did 18F-FHBG. Tumors expressing wtHSVI-tk or HSV1-tk(A168H) were distinctly imaged with 18F-FEAU or 18F-FHBG, respectively. Hence, tumors expressing HSV1-tk(A168H) accumulated 8.4-fold more 18F- FHBG than did tumors expressing wtHSVI-tk. In addition, wtHSVI-tk tumors, compared with HSV1-tk(A168H)- expressing tumors (which retained baseline levels of the radiotracer), preferentially accumulated 18F-FEAU. Conclusion: The FEAU and FHBG substrate discrimination capacity of the wtHSVI-tk and HSV1-tk(A168H) reporter enzymes was validated in vivo by PET of mice with tumor xenografts established from U87 cells expressing these different reporters. Thus, HSV1-tk(A168H) may potentially be used as a second reporter gene in combination with wtHSVI -tk to achieve differential PET. COPYRIGHT
Original language | English (US) |
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Pages (from-to) | 409-416 |
Number of pages | 8 |
Journal | Journal of Nuclear Medicine |
Volume | 50 |
Issue number | 3 |
DOIs | |
State | Published - Mar 1 2009 |
Keywords
- A168H
- F-FEAU
- F-FHBG
- HSV1-tk
ASJC Scopus subject areas
- Radiology Nuclear Medicine and imaging
MD Anderson CCSG core facilities
- Small Animal Imaging Facility
- NMR Facility