PET imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk reporter gene expression in mice and humans using [18F]FHBG

The herpes simplex virus type 1 thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and to image intracellular molecular events and cell trafficking in living subjects. The expression of these PRGs can be imaged using 18F- or 124I-radiolabeled acycloguanosine or pyrimidine analog PET reporter probes (PRPs). This protocol describes the procedures for imaging HSV1-tk or HSV1-sr39tk PRG expression in living subjects with the acycloguanosine analog 9-4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG). [18F]FHBG is a high-affinity substrate for the HSV1-sr39TK enzyme with relatively low affinity for mammalian TK enzymes, resulting in improved detection sensitivity. Furthermore, [18F]FHBG is approved by the US Food and Drug Administration as an investigational new imaging agent and has been shown to detect HSV1-tk transgene expression in the liver tumors of patients. MicroPET imaging of each small animal can be completed in approximately 1.5 h, and each patient imaging session takes approximately 3 h.     

Studying the biodistribution of positron emission tomography reporter probes in mice

Positron emission tomography (PET) reporter probes (PRPs) are used to detect PET reporter gene (PRG) expression in living subjects. This article details protocols for analyzing the biodistribution of a PRP used to detect herpes simplex virus 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk PRG expression. However, the methods described are generalizable to other beta- or gamma/positron-emitting probes. Accumulation of PRPs in animal tissues can be determined by counting PRP activity of isolated tissues, whereas digital whole-body autoradiography (DWBA) provides high-resolution images of PRP biodistribution in 5- to 45-microm tissue slices of killed research animals at a single time point. Biodistribution assay results may be obtained in less than a week after beginning the assay, and DWBA image acquisitions can take up to 3 months depending on the probe's radioisotope.     

Measuring herpes simplex virus thymidine kinase reporter gene expression in vitro

The herpes simplex 1 virus thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and for imaging intracellular molecular events and cell trafficking in living subjects. Two in vitro methods are available to assay gene expression of HSV1-tk or HSV1-sr39tk in cells or tissues. One method determines the level of HSV1-TK or HSV1-sr39TK enzyme activity in cell or tissue lysates by measuring the amount of the radiolabeled substrates that have been phosphorylated by these enzymes in a fixed amount of cell lysate protein after a fixed incubation time. The other method, called the 'cell-uptake assay', takes into account the natural uptake and efflux characteristics of the radiolabeled substrate by specific cells, in addition to the level of HSV1-TK or HSV1-sr39TK activity. Both of these assays can be used to validate molecular models in cultured cells, prior to studying them in living research subjects. Each of these assays can be completed in one day.     

Second-generation triple reporter for bioluminescence, micro-positron emission tomography, and fluorescence imaging

Bioluminescence, positron emission tomography (PET), and fluorescence modalities are currently available for noninvasive imaging in vivo, each with its own merits. To exploit the combined strengths of each and facilitate multimodality imaging, we engineered a dual-reporter construct in which firefly luciferase (FLuc) and a 12-amino acid nonstructural linker were fused in frame to the N-terminus of a mutant herpes simplex virus thymidine kinase (mNLS-SR39TK) kinetically enhanced for positron emission tomography (PET). Furthermore, a triple-reporter construct was developed in which monster green fluorescent protein (MGFP), a recently available enhanced fluorescent protein, was introduced into the fusion vector downstream of an internal ribosome entry site (IRES) to allow analysis by fluorescence microscopy or flow cytometry without compromising the specific activities of the upstream fusion components. FLuc bioluminescence was measured with a cooled charge-coupled device camera and mNLS-SR39TK activity by 9-[4-[(18)F]fluoro-3-(hydroxymethyl) butyl guanine ((18)F-FHBG) microPET or (3)H-penciclovir net accumulation. Importantly, HeLa cells transiently transfected with the FLuc-mNLS-SR39TK-IRES-MGFP triple reporter retained the same specific activities of the FLuc-mNLS-SR39TK heteroenzyme and the individual unfused enzymes with no change in protein half-lives. The presence of the IRES-MGFP modestly decreased upstream heteroprotein expression. In living mice, somatic gene transfer of a ubiquitin promoter-driven FLuc-mNLS-SR39TK-IRES-MGFP plasmid showed a > 1,000-fold increase in liver photon flux and a > 2-fold increase in liver retention of (18)F-FHBG by microPET compared with mice treated with control plasmid. Multifocal hepatocellular fluorescence was readily observed by standard confocal microscopy. This second-generation triple reporter incorporating enhanced components enables bioluminescence, PET, and fluorescence imaging of cells and living animals.     

Molecular PET imaging of HSV1-tk reporter gene expression using [18F]FEAU

Non-invasive imaging of transgene expression requires the appropriate combination of a reporter gene and a reporter probe. [18F]FEAU positron emission tomography (PET) is used for the assessment of herpes simplex virus type-1 thymidine kinase gene expression. Hybrid AAV phage (termed AAVP) can be adapted to transduce mammalian cells by targeting to a specific receptor. We evaluated a targeted AAVP vector using [18F]FEAU PET. This protocol describes [18F]FEAU production and dosing, micro-PET imaging and image analysis. 2-Deoxy-2-trifluoromethanesulfonyl-1,3,5-tri-O-benzoyl-alpha-D-ribofuranose is radio-fluorinated, converted into its 1-bromo derivative and coupled with protected 5-ethyl uracil. The coupled product is hydrolyzed and purified using HPLC. Tumor-bearing animals targeted with either retroviral or AAVP vectors are anesthetized and injected with [18F]FEAU (0.1 mCi per mouse); this is followed 2 h after injection by imaging on a micro-PET. Production of [18F]FEAU requires approximately 3.5 h from the end of bombardment. PET imaging studies require 2-3 h (depending on the number of animals) after synthesis of [18F]FEAU.     

Mutagenesis of non-conserved active site residues improves the activity and narrows the specificity of human thymidine kinase 2

Human thymidine kinase 2 (TK2) is critical for the nucleotide salvage pathway and phosphorylation of nucleoside analog prodrugs in vivo; however, it remains poorly studied because of difficulties in expressing it heterologously. TK2 is strictly pyrimidine-specific, whereas its phylogenetic relative, the Drosophila melanogaster deoxyribonucleoside kinase (DmdNK), shows higher activity and broader specificity towards both pyrimidines and purines. These differences are counterintuitive, as only two of 29 active site residues differ in the two enzymes: F80 and M118 in DmdNK are L78 and L116 in TK2. In addition to reporting an optimized protocol for the expression and purification of TK2, we have used site-directed mutagenesis to introduce the DmdNK-like amino acids into TK2, and characterized the three resulting enzymes (L78F-TK2, L116M-TK2, and L78F/L116M-TK2). These mutations improve the K(M) for thymidine, increasing the catalytic activity of L78F/L116M-TK2 4.4-fold, yet leaving the activity for deoxycytidine or the purine nucleosides unchanged.     

Synthesis and characterization of a C6 nucleoside analogue for the in vivo imaging of the gene expression of herpes simplex virus type-1 thymidine kinase (HSV1 TK)

The synthesis and biological evaluation of '6-(1,3-dihydroxyisobutyl)thymine' (DHBT; 1), which corresponds to 6-[3-hydroxy-2-(hydroxymethyl)propyl]-5-methylpyrimidine-2,4(1H,3H)-dione, is reported. DHBT (1) was designed as a new substrate for herpes simplex virus type-1 thymidine kinase (HSV1 TK). The compound was found to be exclusively phosphorylated by HSV1 TK, and to exhibit good binding affinity (Ki = 35.3+/-1.3 microM). Cell-proliferation assays with HSV1-TK-transduced human osteosarcoma cells (143B-TK+-HSV1-WT) and with both human-thymidine-kinase-1-negative (143B-TK-) and non-transduced parental (MG-63) cells indicate that 1 is less cytotoxic than the standard drug Ganciclovir. Thus, DHBT (1) represents a promising precursor of a nontoxic reporter probe for the monitoring of HSV1 TK gene expression by means of positron-emission tomography (PET).     

Novel radiosynthesis of PET HSV-tk gene reporter probes [18F]FHPG and [18F]FHBG employing dual Sep-Pak SPE techniques

Positron emission tomography (PET) herpes simplex virus thymidine kinase (HSV-tk) gene reporter probes 9-[(3-[(18)F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([(18)F]FHPG) and 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG) were prepared by nucleophilic substitution of the appropriate tosylated precursors with [(18)F]KF/Kryptofix 2.2.2 followed by a quick deprotection reaction and purification with a simplified dual Silica Sep-Pak solid-phase extraction (SPE) method in 15-30% radiochemical yield.     

18F]-2'-Fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (18F-FMAU) in prostate cancer: initial preclinical observations

We hypothesized that imaging-based assessment of cellular proliferation in prostate cancer may improve tumor characterization. We therefore evaluated the biodistribution and effect of androgen on tumor uptake of the cellular proliferation imaging marker [(18)F]-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil ((18)F-FMAU) in xenograft mouse models of human prostate cancer. Castrated and noncastrated athymic male mice were implanted with androgen-independent PC3 and androgen-sensitive CWR22 human prostate cancer cells. Dynamic micro-positron emission tomography (PET)/computed tomography was performed for 1 hour followed by 10-minute static scans at 2 and 3 hours. Animals were sacrificed after imaging for biodistribution studies and immunohistochemical staining of tumors for androgen receptor and Ki-67/MIB expression. (18)F-FMAU uptake was significantly higher in all major organs of the castrated animals in comparison with noncastrated mice, with the highest uptake in liver and the lowest uptake in muscle and bone. When compared to PC3 tumors, CWR22 xenografts showed significantly higher tumor to muscle (2.56 ± 0.30 vs 1.99 ± 0.30, p = .008) and tumor to liver (1.72 ± 0.12 vs 1.26 ± 0.17, p = .0003) uptake ratios in the noncastrated animal at the 3-hour time point. Androgen receptor and Ki-67/MIB expressions were higher in CWR22 than in PC3 xenografts. Our initial preclinical observations suggest that there may be an association between androgen signaling and thymidine metabolism and that (18)F-FMAU PET may be useful in prostate tumor characterization.     

The regulation of thymidine secretion by macrophages.

The secretion of thymidine by mononuclear phagocytes was correlated with the activity of the enzyme thymidine kinase (TK). Macrophages cultured in regular tissue culture medium released thymidine and did not express TK. However, when macrophages were incubated with medium conditioned by L cells, they expressed TK, incorporated 3H thymidine into trichloroacetic acid precipitable material, and ceased to secrete the nucleotide. Furthermore, replicating P388/D1 cells were induced to secrete thymidine by inhibiting TK with d-glucosamine. These results have demonstrated an inverse relationship between thymidine secretion and the expression of TK. They suggest that thymidine secretion by macrophages may be attributed to their lack of TK activity.     

Utilization of exogenous thymidine by Chlamydia psittaci growing in the thymidine kinase-containing and thymidine kinase-deficient L cells.

The incorporation of [3H]thymidine into the deoxyribonucleic acid (DNA) of Chlamydia psittaci (strain 6BC) growing in thymidine kinase (adenosine 5'-triphosphate-thymidine 5'-phosphotransferase, EC 1.7.1.21)-containing L cells, L(TK+), and thymidine kinase-deficient L cells, LM(TK-), was examined by autoradiography. Label was detected over C. psittaci inclusions in L(TK+) but not LM(TK-) cells. No evidence for a chlamydia-specific thymidine kinase activity in either L(TK+) or LM(TK-) cells was obtained. Entry of [3H]thymidine into the DNA of C. psittaci growing in L(TK+) cells was quantitated by measuring label in purified C. psittaci. It was 265 times less efficient than entry into infected host cell DNA. It is concluded that low levels of exogenous thymidine are incorporated into the DNA of C. psittaci and that this incorporation is dependent on a fully competent host thymidine kinase activity. Evidence also is presented that L cells possess at least two thymidine kinase activities, both of which are capable of supplying thymidylate precursors for nuclear DNA synthesis.     

Mutation of herpesvirus thymidine kinase to generate ganciclovir-specific kinases for use in cancer gene therapies

Understanding the functional and mechanistic properties of the multi-substrate herpes simplex virus type-1 thymidine kinase (HSV-1 TK) remains critical to defining its role as a major pharmacological target in herpesvirus and gene therapies for cancer. An inherent limitation of the activity of HSV-TK is the >70-fold difference in the K(m)s for phosphorylation of thymidine over the pro-drug ganciclovir (GCV). To engineer an HSV-1 TK isoform that is specific for GCV as the preferred substrate, 16 site-specific mutants were generated. The mutations were concentrated at conserved residues involved in nucleoside base binding, Gln125 and near sites 3 and 4 involved in catalysis and substrate binding. The substrate preferences of each mutant enzyme were compared with wild-type HSV-1 TK. One mutant, termed Q7530 TK, had a lower K(m) for GCV than thymidine. Expression of the Q7530 TK in tumor cells indicated comparable metabolism to and improved sensitivity to GCV over wild-type HSV-1 TK, with minimal thymidine phosphorylation activity. A molecular modeling simulation of the different HSV-1 TK active-sites was done for GCV and thymidine binding. It was concluded that mutations at Gln125 and near site 4, especially at Ala168, were responsible for loss of deoxypyrimidine substrate binding.     

Substrate diversity of herpes simplex virus thymidine kinase. Impact Of the kinematics of the enzyme.

Herpes simplex virus type 1 (HSV 1) thymidine kinase (TK) exhibits an extensive substrate diversity for nucleobases and sugar moieties, in contrast to other TKs. This substrate diversity is the crucial molecular basis of selective antiviral and suicide gene therapy. The mechanisms of substrate binding of HSV 1 TK were studied by means of site-directed mutagenesis combined with isothermal calorimetric measurements and guided by theoretical calculations and sequence comparison. The results show the link between the exceptionally broad substrate diversity of HSV 1 TK and the presence of structural features such as the residue triad His-58/Met-128/Tyr-172. The mutation of Met-128 into a Phe and the double mutant M128F/Y172F result in mutants that have lost their activity. However, by exchanging His to form the triple mutant H58L/M128F/Y172F, the enzyme regains activity. Strikingly, this triple mutant becomes resistant toward acyclovir. Furthermore, we give evidence for the importance of Glu-225 of the flexible LID region for the catalytic reaction. The data presented give new insights to understand mechanisms ruling substrate diversity and thus are crucial for both the development of new antiviral drugs and engineering of mutant TKs apt to accept novel substrate analogs for gene therapeutic approaches.     

Mitochondrial thymidine kinase and the enzymatic network regulating thymidine triphosphate pools in cultured human cells

In non-proliferating cells mitochondrial (mt) thymidine kinase (TK2) salvages thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1-R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication. In non-proliferating cells p53R2 substitutes for R2. Catabolic enzymes safeguard the size of the dTTP pool: thymidine phosphorylase by degradation of thymidine and deoxyribonucleotidases by degradation of dTMP. Genetic deficiencies in three of the participants in the network, TK2, p53R2, or thymidine phosphorylase, result in severe mt DNA pathologies. Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of thymidine phosphorylase with 5-bromouracil. In proliferating cells the de novo pathway dominates, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by thymidine phosphorylase, which degrades thymidine in the cytoplasm, thus limiting the availability of thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of thymidine concentrations at the nanomolar level.     

Herpes simplex virus thymidine kinase enzymatic assay in transient transfection experiments using thymidine kinase-deficient cells.

An enzymatic assay for herpes virus simplex type 1 thymidine kinase (HSV-TK) that was sensitive enough to quantitate intracellular levels of enzyme transiently expressed after transfection of HSV-TK vectors into TK-deficient cells using the DNA-calcium phosphate coprecipitation technique is described. TK activity in extracts of transfected cells was determined by binding of [methyl-3H]thymidylate product to thin layers of polyethyleneimine (PEI)-impregnated cellulose. The assay used high-specific-activity [methyl-3H]thymidine as substrate, which required removal of anionic material on a column of PEI-cellulose to enhance the signal-to-noise ratio. The assay was linear over a wide range with respect to the amount of HSV-TK plasmid transfected or content of HSV-TK enzyme in cell extracts. To validate the assay in transient expression experiments, HSV-TK and chloramphenicol acetyltransferase (CAT) plasmids were cotransfected into NIH/3T3 tk- fibroblasts. Transient TK and CAT levels were concordant in cell extracts prepared from replicate plates of transfected cells. Normalizing the transient TK activity for CAT activity from the cotransfected "internal standard" CAT plasmid improved precision significantly, reducing the sample-to-sample coefficient of variation from 41 to 19%. CAT normalization reduced experimental variability mostly by correcting outlying results in transfection efficiency. The HSV-TK reporter gene system based on TK enzymatic assay was thus subject to experimental variation similar to that of the well-established CAT reporter function, demonstrating its utility in transient gene expression analysis.     

Limits of [18F]-FLT PET as a Biomarker of Proliferation in Oncology

Background

Non-invasive imaging biomarkers of cellular proliferation hold great promise for quantifying response to personalized medicine in oncology. An emerging approach to assess tumor proliferation utilizes the positron emission tomography (PET) tracer 3’-deoxy-3’[¹⁸F]-fluorothymidine, [¹⁸F]-FLT. Though several studies have associated serial changes in [¹⁸F]-FLT-PET with elements of therapeutic response, the degree to which [¹⁸F]-FLT-PET quantitatively reflects proliferative index has been continuously debated for more that a decade. The goal of this study was to elucidate quantitative relationships between [¹⁸F]-FLT-PET and cellular metrics of proliferation in treatment naïve human cell line xenografts commonly employed in cancer research.

Methods and Findings

[¹⁸F]-FLT-PET was conducted in human cancer xenograft-bearing mice. Quantitative relationships between PET, thymidine kinase 1 (TK1) protein levels and immunostaining for proliferation markers (Ki67, TK1, PCNA) were evaluated using imaging-matched tumor specimens. Overall, we determined that [¹⁸F]-FLT-PET reflects TK1 protein levels, yet the cell cycle specificity of TK1 expression and the extent to which tumors utilize thymidine salvage for DNA synthesis decouple [¹⁸F]-FLT-PET data from standard estimates of proliferative index.

Conclusions

Our findings illustrate that [¹⁸F]-FLT-PET reflects tumor proliferation as a function of thymidine salvage pathway utilization. Unlike more general proliferation markers, such as Ki67, [¹⁸F]-FLT PET reflects proliferative indices to variable and potentially unreliable extents. [¹⁸F]-FLT-PET cannot discriminate moderately proliferative, thymidine salvage-driven tumors from those of high proliferative index that rely primarily upon de novo thymidine synthesis. Accordingly, the magnitude of [¹⁸F]-FLT uptake should not be considered a surrogate of proliferative index. These data rationalize the diversity of [¹⁸F]-FLT-PET correlative results previously reported and suggest future best-practices when [¹⁸F]-FLT-PET is employed in oncology.     

5-Azacytidine induction of thymidine kinase in a spontaneously enzyme-deficient murine tumor line

During the course of our studies on murine tumor cell metastases, one of our variant lines (called L61-M) was found to be unable to incorporate [methyl-3H]thymidine into DNA, due to a spontaneous deficiency in thymidine kinase (TK) activity. L61-M cells are unable to proliferate in HAT selection medium and are resistant to bromodeoxyuridine (BrdU). TK activity in L61-M cells is 4.2% of that found in the wild-type parental MDAY-D2 cell line. Treatment of L61-M with 5-azacytidine, a known inducer of DNA hypomethylation, resulted in the expression of TK activity. These observations suggest that the TK deficiency in the L61-M cell line was due in part to an alteration in the methylation pattern of DNA, resulting in the diminished expression of the TK gene. These results demonstrate the ability of 5-azacytidine to induce TK activity in a spontaneously enzyme-deficient murine tumor cell line.