ABSTRACT
UNLABELLED: 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 (3)H-2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-ethyluracil ((3)H-FEAU), (3)H-pencyclovir ((3)H-PCV), and (3)H-GCV and by drug sensitivity assays. PET with (18)F-FEAU and (18)F-9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ((18)F-FHBG) was performed in mice 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-beta-d-arabinofuranoside and bromovinyldeoxyuridine were found to be similar to those in nontransduced cells. In contrast, we found that HSV1-A167Ysr39tk could readily phosphorylate (3)H-GCV at levels similar to those of wild-type HSV1-tk and HSV1-A167Ytk but showed enhanced activity with (3)H-PCV in vitro and with (18)F-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 (18)F-FHBG and suicidal gene therapy protocols with GCV in patients treated with pyrimidine-based cytotoxic drugs.
Subject(s)
Acyclovir/metabolism , Genetic Therapy/methods , Herpesvirus 1, Human/enzymology , Mutation , Positron-Emission Tomography/methods , Thymidine Kinase/genetics , Thymidine Kinase/metabolism , Acyclovir/pharmacology , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Cell Line, Tumor , Cytotoxins/chemistry , Cytotoxins/metabolism , Cytotoxins/pharmacology , Ganciclovir/analogs & derivatives , Ganciclovir/metabolism , Ganciclovir/pharmacology , Gene Expression Regulation, Viral/drug effects , Genes, Reporter/genetics , Guanine/analogs & derivatives , Herpesvirus 1, Human/genetics , Humans , Mice , Phosphorylation/drug effects , Pyrimidines/chemistry , Pyrimidines/metabolism , Pyrimidines/pharmacology , Substrate SpecificityABSTRACT
Evasion of host immune responses is well documented for viruses and may also occur during tumor immunosurveillance. The mechanisms involve alterations in MHC class I expression, Ag processing and presentation, chemokine and cytokine production, and lymphocyte receptor expression. Epithelial tumors overexpress MHC class I chain-related (MIC) molecules, which are ligands for the activating receptor NKG2D on NK and T cells. We report that NK cells from patients with colorectal cancer lack expression of activating NKG2D and chemokine CXCR1 receptors, both of which are internalized. Serum levels of soluble MIC (sMIC) are elevated and are responsible for down-modulation of NKG2D and CXCR1. In contrast, high serum levels of CXC ligands, IL-8, and epithelial-neutrophil-activating peptide (ENA-78) do not down-modulate CXCR1. In vitro, internalization of NKG2D and CXCR1 occurs within 4 and 24 h, respectively, of incubating normal NK cells with sMIC-containing serum. Furthermore, natural cytotoxicity receptor NKp44 and chemokine receptor CCR7 are also down-modulated in IL-2-activated NK cells cocultured in MIC-containing serum-an effect secondary to the down-modulation of NKG2D and not directly caused by physical association with sMIC. The patients' NK cells up-regulate expression of NKG2D, NKp44, CXCR1, and CCR7 when cultured in normal serum or anti-MIC Ab-treated autologous serum. NKG2D(+) but not NKG2D(-) NK cells are tumoricidal in vitro, and in vivo they selectively traffic to the xenografted carcinoma, form immunological synapse with tumor cells, and significantly retard tumor growth in the SCID mice. These results suggest that circulating sMIC in the cancer patients deactivates NK immunity by down-modulating important activating and chemokine receptors.
