A new acycloguanosine-specific supermutant of herpes simplex virus type 1 thymidine kinase suitable for PET imaging and suicide gene therapy for potential use in patients treated with pyrimidine-based cytotoxic drugs Journal Article
| Authors: | Likar, Y.; Dobrenkov, K.; Olszewska, M.; Vider, E.; Shenker, L.; Cai, S.; Pillarsetty, N.; Hricak, H.; Ponomarev, V. |
| Article Title: | A new acycloguanosine-specific supermutant of herpes simplex virus type 1 thymidine kinase suitable for PET imaging and suicide gene therapy for potential use in patients treated with pyrimidine-based cytotoxic drugs |
| Abstract: | The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene is widely used as a suicide gene in combination with ganciclovir (GCV) and as a nuclear imaging reporter gene with an appropriate reporter probe. Wild-type HSV1-tk recognizes a variety of pyrimidine and acycloguanosine nucleoside analogs, including clinically used antiviral drugs. PET of HSV1-tk reporter gene expression will be compromised in patients receiving nucleoside-based antiviral treatment. With the use of an acycloguanosine-specific mutant of the enzyme, PET of HSV1-tk reporter gene expression can be successfully performed with acycloguanosine-based radiotracers without interference from pyrimidine-based antiviral drugs. Methods: The levels of expression of wild-type HSV1-tk and HSV1-A167Ytk, HSV1-sr39tk, and HSV1-A167Ysr39tk mutants fused with green fluorescent protein (GFP) and transduced into U87 cells were normalized to the mean fluorescence of GFP measured by fluorescence-activated cell sorting. The levels of enzymatic activities of wild-type HSV1-tk and its mutants were compared by 2-h in vitro radiotracer uptake assays with 3H-2′- fluoro-2′-deoxy-1-β-D-arabinofuranosyl-5-ethyluracil ( 3H-FEAU), 3H-pencyclovir (3H-PCV), and 3H-GCV and by drug sensitivity assays. PET with 18F-FEAU and 18F-9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ( 18F-FHBG) was performed inmice with established subcutaneous tumors, expressing wild-type HSV1-tk and its mutants, followed by tissue sampling. Results: FEAU accumulation was not detected in HSV1-A167Ysr39tk-expressing cells and xenografts. Lack of conversion of pyrimidine derivatives by the HSV1-A167Ysr39tk supermutant was also confirmed by a drug sensitivity assay, in which the 50% inhibitory concentrations for thymine 1-β-D-arabinofuranoside and bromovinyldeoxyuridine were found to be similar to those in nontransduced cells. In contrast, we found that HSV1-A167Ysr39tk could readily phosphorylate 3H-GCV at levels similar to those of wild-type HSV1-tk and HSV1-A167Ytk but showed enhanced activity with 3H-PCV in vitro and with 18F-FHBG in vivo. Conclusion: We developed a new reporter gene, HSV1-A167Ysr39tk, which exhibits specificity and high phosphorylation activity for acycloguanosine derivatives. The resulting supermutant can be used for PET with 18F-FHBG and suicidal gene therapy protocols with GCV in patients treated with pyrimidine-based cytotoxic drugs. Copyright © 2008 by the Society of Nuclear Medicine, Inc. |
| Keywords: | controlled study; human cell; mutation; cytotoxic agent; nonhuman; positron emission tomography; mouse; animals; mice; animal tissue; gene expression; amino acid substitution; green fluorescent protein; animal experiment; animal model; aciclovir; in vivo study; in vitro study; enzyme activity; molecular imaging; cell line, tumor; pyrimidines; tyrosine; phosphorylation; wild type; xenograft; guanine; substrate specificity; positron-emission tomography; gene therapy; radiopharmaceutical agent; reporter gene; thymidine kinase; genes, reporter; herpesvirus 1, human; alanine; tumor; pyrimidine derivative; pet; drug sensitivity; fluorescence activated cell sorting; herpes simplex virus 1; ganciclovir; mutant; feau; hsv1-tk; 9 [4 fluoro 3 (hydroxymethyl)butyl]guanine f 18; pyrimidine; acyclovir; antiviral agents; enzyme; gene expression regulation, viral; suicide gene therapy; 18f-fhbg; 5 (2 bromovinyl) 2' deoxyuridine; clevudine f 18; clevudine h 3; pencyclovir h 3; cytotoxins |
| Journal Title: | Journal of Nuclear Medicine |
| Volume: | 49 |
| Issue: | 5 |
| ISSN: | 0161-5505 |
| Publisher: | Society of Nuclear Medicine |
| Date Published: | 2008-05-01 |
| Start Page: | 713 |
| End Page: | 720 |
| Language: | English |
| DOI: | 10.2967/jnumed.107.046425 |
| PUBMED: | 18413388 |
| PROVIDER: | scopus |
| PMCID: | PMC2735464 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 7" - "Export Date: 17 November 2011" - "CODEN: JNMEA" - "Source: Scopus" |
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