FIAU, (1-(2’-deoxy-2’-fluoro-1-β-D-arabinofuranosyl)-5-iodouracil) has been used as a substrate for herpes simplex virus thymidine kinases (HSV-TK and HSV-tk, for protein and gene expression respectively) and other bacterial and viral thymidine kinases for noninvasive imaging applications. Previous studies have reported the formation of a de-iodinated metabolite of 18F-FIAU. This study reports the dynamic tumor uptake, biodistribution and metabolite contribution to the activity of 18F-FIAU seen in HSV-tk gene expressing tumors and compares the distribution properties with its de-iodinated metabolite 18F-FAU.
CD-1 nu/nu mice with subcutaneous MH3924A and MH3924A-stb-tk+ xenografts on opposite flanks were used for the biodistribution and imaging studies. Mice were injected IV with either 18F-FIAU or 18F-FAU. Mice underwent dynamic imaging with each tracer for 65 min followed by additional static imaging up to 150 min post injection for some animals. Animals were sacrificed at 60 or 150 min post injection. Samples of blood and tissue were collected for biodistribution and metabolite analysis. Regions of interest were drawn over the images obtained from both tumors to calculate the time activity curves.
Biodistribution and imaging studies showed the highest uptake of 18F-FIAU in the MH3924A-stb-tk+ tumors. Dynamic imaging studies revealed a continuous accumulation of 18F-FIAU in HSV-TK expressing tumors over 60 min. The mean biodistribution values (SUV±SE) for MH3924A-stb-tk+ were 2.07±0.40, 6.15±1.58, and that of MH3924A tumors were 0.19±0.07, 0.47±0.06 at 60 and 150 min respectively. In 18F-FIAU injected mice, at 60 min nearly 63% of blood activity was present as its metabolite 18F-FAU. Imaging and biodistribution studies with 18F-FAU demonstrated no specific accumulation in MH3924A-stb-tk+ tumors and SUVs for both the tumors were similar to those observed with muscle.
18F-FIAU shows a continuous accumulation of activity in HSV-TK expressing tumors. 18F-FAU does not show any preferential accumulation in HSV-TK expressing tumors. In the 18F-FIAU treated mice, the 18F-FAU contribution to the total uptake seen in HSV-TK positive tumors is minimal.
Fluorine-18; FIAU; HSV-TK; Gene Expression; Metabolism; PET Imaging
2'-Fluoro-2'-deoxy-1β-D-arabinofuranosyl-5-[125I]iodouracil ([125I]FIAU), a substrate for the thymidine kinase (TK) present in most bacteria, has been used as an imaging agent for single photon emission computed tomography (SPECT) in an experimental model of lung infection. Using SPECT-CT we show that [125I]FIAU is specific for bacterial infection rather than sterile inflammation. We report [125I]FIAU lung uptake values of 1.26 ± 0.20 percent injected dose per gram (%ID/g) in normal controls, 1.69 ± 0.32 %ID/g in lung inflammation and up to 7.14 ± 1.09 %ID/g in lung infection in ex vivo biodistribution studies at 24 h after intranasal administration of bacteria. Images of [125I]FIAU signal within lung can be used to estimate the number of bacteria present, with a limit of detection of 109 colony forming units per mL on the X-SPECT scanner. [125I]FIAU-Based bacterial imaging may be useful in preclinical models to facilitate the development of new antibiotics, particularly in cases where a corresponding human trial is planned.
Inflammation; thymidine kinase; nucleoside; SPECT; PET; molecular imaging
Traditional imaging techniques for the localization and monitoring of bacterial infections, although reasonably sensitive, suffer from a lack of specificity. This is particularly true for musculoskeletal infections. Bacteria possess a thymidine kinase (TK) whose substrate specificity is distinct from that of the major human TK. The substrate specificity difference has been exploited to develop a new imaging technique that can detect the presence of viable bacteria.
Eight subjects with suspected musculoskeletal infections and one healthy control were studied by a combination of [124I]FIAU-positron emission tomography and CT ([124I]FIAU-PET/CT). All patients with proven musculoskeletal infections demonstrated positive [124I]FIAU-PET/CT signals in the sites of concern at two hours after radiopharmaceutical administration. No adverse reactions with FIAU were observed.
[124I]FIAU-PET/CT is a promising new method for imaging bacterial infections.
One limitation of HSV1-tk reporter PET imaging with nucleoside analogues is the high background radioactivity in the intestine. We hypothesized that endogenous expression of thymidine kinase in bacterial flora could phosphorylate and trap such radiotracers, contributing to the high radioactivity levels in the bowel and therefore explored different strategies to increase fecal elimination of radiotracer.
Intestinal radioactivity was assessed by in vivo microPET imaging and ex vivo tissue sampling following intravenous injection of 18F-FEAU, 124I-FIAU or 18F-FHBG in a germ-free mouse strain. We also explored the use of an osmotic laxative agent and/or a 100% enzymatically hydrolyzed liquid diet.
No significant differences in intestinal radioactivity were observed between germ-free and normal mice. 18F-FHBG-derived intestinal radioactivity levels were higher than those of 18F-FEAU and 124I-FIAU; the intestine-to-blood ratio was more than 20-fold higher for 18F-FHBG than for 18F-FEAU and 124I-FIAU. The combination of Peptamen and Nulytely lowered intestinal radioactivity levels and increased (2.2-fold) the HSV1-tk transduced xenograft-to-intestine ratio for 18F-FEAU.
Intestinal bacteria in germ-free mice do not contribute to the high intestinal levels of radioactivity following injection of radionucleoside analogs. The combination of Peptamen and Nulytely increased radiotracer elimination by increasing bowel motility without inducing dehydration.
reporter gene imaging; HSV1-tk; PET imaging; bacteria; background radioactivity
Cell-tracking methods with molecular-imaging modality can monitor the biodistribution of cells. In this study, the direct-labeling method with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM), indirect cell-labeling methods with herpes simplex virus type 1-thymidine kinase (HSV1-tk)-mediated 124I-2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil (124I-FIAU) were comparatively investigated in vitro and in vivo for tracking of human chronic myelogenous leukemia cells. K562-TL was established by retroviral transduction of the HSV1-tk and firefly luciferase gene in the K562 cell. K562-TL cells were labeled with 64Cu-PTSM or 124I-FIAU. Cell labeling efficiency, viability, and radiolabels retention were compared in vitro. The biodistribution of radiolabeled K562-TL cells with each radiolabel and small-animal positron emission tomography imaging were performed. Additionally, in vivo and ex vivo bioluminescence imaging (BLI) and tissue reverse transcriptase–polymerase chain reaction (RT-PCR) analysis were used for confirming those results. K562-TL cells were efficiently labeled with both radiolabels. The radiolabel retention (%) of 124I-FIAU (95.2%±1.1%) was fourfold higher than 64Cu-PTSM (23.6%±0.7%) at 24 hours postlabeling. Viability of radiolabeled cells was statistically nonsignificant between 124I-FIAU and 64Cu-PTSM. The radioactivity of each radiolabeled cells was predominantly accumulated in the lungs and liver at 2 hours. Both the radioactivity of 64Cu-PTSM- and 124I-FIAU-labeled cells was highly accumulated in the liver at 24 hours. However, the radioactivity of 124I-FIAU-labeled cells was markedly decreased from the body at 24 hours. The K562-TL cells were dominantly localized in the lungs and liver, which also verified by BLI and RT-PCR analysis at 2 and 24 hours postinjection. The 64Cu-PTSM-labeled cell-tracking method is more efficient than 124I-FIAU-labeled cell tracking, because of markedly decrease of radioactivity and fast efflux of 124I-FIAU in vivo. In spite of a high labeling yield and radiolabel retention of 124I-FIAU in vitro, the in vivo cell-tracking method using 64Cu-PTSM could be a useful method to evaluate the distribution and targeting of various cell types, especially, stem cells and immune cells.
gene transfer; molecular imaging; PET
Purpose: A group of radiolabeled thymidine analogs were developed as radio-tracers for imaging herpes viral thymidine kinase (HSV1-tk) or its variants used as reporter gene. A transgenic mouse model was created to express tk upon liver injury or naturally occurring hepatocellular carcinoma (HCC). The purpose of this study was to use this unique animal model for initial testing with radio-labeled thymidine analogs, mainly a pair of newly emerging nucleoside analogs, D-FMAU and L-FMAU.
Methods: A transgeneic mouse model was created by putting a fused reporter gene system, firefly luciferase (luc) and HSV1-tk, under the control of mouse alpha fetoprotein (Afp) promoter. Initial multimodal imaging, which was consisted of bioluminescent imaging (BLI) and planar gamma scintigraphy with [125I]-FIAU, was used for examining the model creation in the new born and liver injury in the adult mice. Carcinogen diethylnitrosamine (DEN) was then administrated to induce HCC in these knock-in mice such that microPET imaging could be used to track the activity of Afp promoter during tumor development and progression by imaging tk expression first with [18F]-FHBG. Dynamic PET scans with D-[18F]-FMAU and L-[18F]-FMAU were then performed to evaluate this pair of relatively new tracers. Cells were derived from these liver tumors for uptake assays using H-3 labeled version of PET tracers.
Results: The mouse model with dual reporters: HSV1-tk and luc placed under the transcriptional control of an endogenous Afp promoter was used for imaging studies. The expression of the Afp gene was highly specific in proliferative hepatocytes, in regenerative liver, and in developing fetal liver, and thus provided an excellent indicator for liver injury and cancer development in adult mice. Both D-FMAU and L-FMAU showed stable liver tumor uptake where the tk gene was expressed under the Afp promoter. The performance of this pair of tracers was slightly different in terms of signal-to-background ratio as well as tracer clearance.
Conclusion: The newly created knock-in mouse model was used to demonstrate the use of the dual-reporter genes driven by well-characterized cancer-specific transcriptional units in conjunction with in vivo imaging as a paradigm in studying naturally occurring cancer in live animals. While BLI is suitable for small animal imaging with luc expression, PET with L-FMAU seemed be the choice for liver injury or liver cancer imaging with this animal model for future investigations.
deoxynucleoside analogs; reporter gene imaging; hepatocellular carcinoma
Bacteria can be selectively imaged in experimentally-infected animals using exogenously administered 1-(2′deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-[125I]-iodouracil ([125I]-FIAU), a nucleoside analog substrate for bacterial thymidine kinase (TK). Our goal was to use this reporter and develop non-invasive methods to detect and localize Mycobacterium tuberculosis.
We engineered a M. tuberculosis strain with chromosomally integrated bacterial TK under the control of hsp60 - a strong constitutive mycobacterial promoter. [125I]FIAU uptake, antimicrobial susceptibilities and in vivo growth characteristics were evaluated for this strain. Using single photon emission computed tomography (SPECT), M. tuberculosis Phsp60 TK strain was evaluated in experimentally-infected BALB/c and C3HeB/FeJ mice using the thigh inoculation or low-dose aerosol infection models. M. tuberculosis Phsp60 TK strain actively accumulated [125I]FIAU in vitro. Growth characteristics of the TK strain and susceptibility to common anti-tuberculous drugs were similar to the wild-type parent strain. M. tuberculosis Phsp60 TK strain was stable in vivo and SPECT imaging could detect and localize this strain in both animal models tested.
We have developed a novel tool for non-invasive assessment of M. tuberculosis in live experimentally-infected animals. This tool will allow real-time pathogenesis studies in animal models of TB and has the potential to simplify preclinical studies and accelerate TB research.
Fialuridine (FIAU) is a halogen-substituted analog of thymidine that was undergoing clinical investigation as a drug for the treatment of chronic hepatitis B viral infection. However, clinical trials of FIAU were terminated after adverse events occurred following chronic oral administration. Prior to the termination of clinical trials, a sensitive assay was needed for the measurement of FIAU because of the anticipated low dose administered to patients. We therefore undertook the development of a radioimmunoassay (RIA). A specific antiserum was raised in rabbits following immunization with a 5'-O-hemisuccinate analog of FIAU coupled to keyhole limpet hemocyanin. Radiolabeled FIAU was synthesized by a destannylation procedure by using sodium [125I]iodide. We developed a competitive-binding procedure and used precipitation with polyethylene glycol as the method for separating the bound and free forms of FIAU. The RIA is sensitive (0.2 ng/ml), specific (negligible interference from known metabolites and endogenous nucleosides), and reproducible (interassay coefficients of variation range from 5 to 19.7% for serum controls). We used the RIA to assess the pharmacokinetics of FIAU in healthy adult volunteers following administration of a single 5-mg oral dose. The sensitivity of the RIA permitted the detection of a prolonged elimination phase for FIAU in healthy volunteers and dogs, with mean elimination half-lives of 29.3 and 35.3 h, respectively. We conclude the RIA is a valid method for the quantification of FIAU in biological fluids.
A decrease in the in vitro sensitivity to acyclovir (ACV) was observed in successive isolates of herpes simplex virus type 1 from three immunocompromised patients during intravenous therapy with this drug. The ACV-resistant isolate from patient 1 was cross-resistant to dihydroxypropoxymethylguanine and bromovinyldeoxyuridine, but still susceptible to three fluoro-substituted pyrimidines, 2'-fluoro-5-iodo-1-beta-D-arabinofuranosylcytosine (FIAC), 2'-fluoro-5-iodo-1-beta-D-arabinofuranosyluracil (FIAU), and 2'-fluoro-5-iodo-1-beta-D-arabinofuranosylthymine (FMAU). The thymidine kinase (TK) from the resistant isolate showed a 50-fold or greater reduction in affinity for thymidine, FIAU, FMAU, and ACV, but the total enzyme activity was similar to that of the sensitive isolate. The ACV-resistant isolate from patient 2 was also resistant to dihydroxypropoxymethylguanine, bromovinyldeoxyuridine, and the fluoro-substituted compounds; TK activity for this isolate was less than 1% of the patient's pretherapy isolate. An isolate obtained during a subsequent recurrence in patient 2 was susceptible to ACV and the other TK-dependent agents. The ACV-resistant isolate from patient 3 was partially resistant to FIAC and FIAU but still susceptible to FMAU; the viral TK had a 10-fold-lower affinity for ACV, FIAU, and FMAU than did the sensitive pretherapy isolate, while the level of TK activity detected was reduced to 6%. In none of the isolates studied was a change in sensitivity to phosphonoformic acid observed. Compared with the corresponding pretherapy ACV-sensitive isolates, there was a 30-fold decrease in neurovirulence for mice of the two drug-resistant isolates with diminished levels of thymidine-phosphorylating activity and no change in virulence for the third isolate. These findings indicate that mixed patterns of drug-resistance to TK-dependent antiviral compounds can occur in clinical isolates, resulting from changes in either the amount or the affinity of viral TK activity.
We investigated the possibility of using a pharmacologic agent to modulate viral gene expression in order to target radiotherapy to tumor tissue. In a murine xenograft model, we had previously shown targeting of [125I]2'-fluoro-2'-deoxy-beta-D-5-iodouracilarabinofuranoside ([125I]FIAU) to tumors engineered to express the Epstein-Barr virus (EBV)-thymidine kinase (TK). Here we extend those results to targeting of a therapeutic radiopharmaceutical [131I]FIAU to slow or stop tumor growth or to achieve tumor regression. These outcomes were achieved in xenografts with tumors that constitutively expressed the EBV-TK, as well as with naturally-infected EBV tumor cell lines. Burkitt's lymphoma and gastric carcinoma required activation of viral gene expression by pretreatment with bortezomib. Marked changes in tumor growth could also be achieved in naturally-infected Kaposi's sarcoma herpesvirus (KSHV) tumors following bortezomib activation. Bortezomib-induced enzyme-targeted radiation (BETR) therapy illustrates the possibility of pharmacologically modulating tumor gene expression to effect targeted radiotherapy.
Quantum dots (QDs) have many intriguing properties suitable for biomedical imaging applications. The poor tissue penetration of optical imaging in general, including those using QDs, has motivated the development of various QD-based dual-modality imaging agents. In this issue of AJNMMI (http://www.ajnmmi.us), Sun et al. reported the synthesis and in vitro/in vivo characterization of intrinsically radio-labeled QDs (r-QDs), where 109Cd was incorporated into the core/shell of QDs of various compositions. These r-QDs emit in the near-infrared range, have long circulation half-life, are quite stable with low cytotoxicity, exhibit small size and low accumulation in the reticuloendothelial system, and can allow for accurate measurement of their biodistribution in mice. With these desirable features demonstrated in this study, future development and optimization will further enhance the biomedical potential of intrinsically radio-labeled QDs.
Quantum-dots (QDs); nanoparticle; positron emission tomography (PET); single-photon emission computed tomography (SPECT); near-infrared (NIR); optical imaging
Fialuridine (FIAU) is a nucleoside analog with potent activity against hepatitis B virus in vitro and in vivo. In this report, the effect of FIAU on mitochondrial DNA (mtDNA) replication in vitro was investigated. CEM cells, a cell line derived from human T cells, were incubated for 6 days in up to 20 microM FIAU. Total cellular DNA was isolated, normalized for the number of cells, and slot hybridized to a probe specific for mtDNA sequences. Treatment of CEM cells with FIAU did not result in a dose-dependent decrease in the amount of mtDNA. In contrast, dideoxycytidine (ddC) inhibited mtDNA replication by 50% at a concentration of approximately 0.1 microM. After 6 days of incubation, both compounds displayed a 50% toxic dose at a concentration of approximately 2 microM in CEM cells and approximately 34 microM in human hepatoblastoma cells (HepG2). In further experiments, CEM cells were incubated for 15 days in up to 2.5 microM FIAU, and again, no inhibition of mtDNA was observed. Over a 6-day incubation, FIAU, at concentrations of up to 200 microM, also failed to inhibit mtDNA replication in either HepG2 or HepG2 cells which constitutively replicate duck hepatitis B virus. In contrast, ddC inhibited mtDNA replication in these cells with a 50% inhibitory concentration of approximately 0.2 microM over a 6-day incubation. Treatment of cells with either FIAU or ddC resulted in a dose-dependent increase in lactate levels in the cell medium, indicating that any effect of FIAU on mitochondrial function may not be related to inhibition of mtDNA replication on the basis of the in vitro data. Alternative explanations for mitochondrial toxicity are considered.
As measured by plaque and yield reduction assays, several metabolites of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) were highly active against herpes simplex virus types 1 and 2. These metabolites included the 2'-deoxy-2'-fluoroarabinosyl derivatives of 5-iodouracil (FIAU), cytosine (FAC), uracil (FAU), and thymine (FMAU). In mice inoculated intracerebrally with herpes simplex virus type 2, the relative order of potency of these compounds and licensed antiviral drugs was as follows: FMAU much greater than FIAC approximately equal to FIAU greater than acyclovir approximately equal to vidarabine much greater than FAC approximately equal to FAU. One of the main metabolites of FMAU, 2'-fluoro-5-hydroxymethyl-arabinosyluracil, was essentially inactive in vivo. FIAC-, FIAU-, FMAU-, FAC-, and FAU-resistant herpes simplex virus variants prepared in cell culture were found to be (i) devoid of viral thymidine kinase, (ii) cross-resistant to one another and resistant to drugs requiring viral thymidine kinase for activation, and (iii) sensitive to vidarabine or phosphonoformate. These results indicate that FIAC, FIAU, and FMAU require the virally encoded thymidine kinase for activation and suggest that the antiviral activity of FAU and FAC in cell cultures is also mediated by this enzyme. The interaction of the fluoroarabinosyl pyrimidine nucleosides with herpes simplex virus thymidine kinase in a cell-free system is also described.
We have explored the mechanism(s) related to FIAU-induced liver toxicity, particularly focusing on its effect on mitochondrial function in a human hepatoma cell line-HepG2. The potential role of FMAU and FAU, metabolites detected in FIAU-treated patients were also ascertained. FIAU and FMAU inhibited cell growth and were effectively phosphorylated. A substantial increase in lactic acid production in medium of cells incubated with 1-10 microM FIAU or FMAU was consistent with mitochondrial dysfunction. Slot blot analysis demonstrated that a two week exposure to 10 microM FIAU or FMAU was not associated with a decrease in total mitochondrial (mt) DNA content. However, FIAU and FMAU were incorporated into nuclear and mtDNA and relative values suggest that both compounds incorporate at a much higher rate into mtDNA. Electron micrographs of cells incubated with 10 microM FIAU or FMAU revealed the presence of enlarged mitochondria with higher cristae density and lipid vesicles. In conclusion, these data suggest that despite the lack of inhibition of mtDNA content, incorporation of FIAU and FMAU into mtDNA of HepG2 cells leads to marked mitochondrial dysfunction as evidenced by disturbance in cellular energy metabolism and detection of micro- and macrovesicular steatosis.
The thymidine analog, 1-(2-deoxy-2-fluoro-beta-D-arabino-furanosyl)-5-iodouracil (FIAU), is incorporated into DNA in cell culture and in vivo. To investigate the effect of incorporation of FIAU into DNA on the binding of transcription factors, oligonucleotide duplexes which bind specifically to activator protein 1 (AP-1) or to TFIID were synthesized and binding of these oligonucleotides to their respective proteins was studied using gel-shift analysis. When thymidine at position -3, -1, 1 or 7 (relative to the first thymidine of the core binding sequence) was replaced with FIAU, binding to AP-1 was approximately 82, 28, 86 and 51%, respectively, of the binding to the non-substituted oligonucleotide to AP-1. When thymidine at position 3 or 5 (each adjacent to the center of dyad symmetry) was replaced with FIAU, binding to AP-1 was abrogated. Oligonucleotides containing FIAU at positions -1, 3 or 5, were much less able to compete with radiolabeled wild-type oligonucleotides for binding to AP-1. In contrast, the presence of FIAU, depending on its location, resulted in the increased binding of TFIID to its consensus target DNA sequence. These results indicate that incorporation of FIAU into DNA may induce local conformational changes resulting in the altered ability of transcriptional factors to bind to their cognate DNA sequences. Additional studies demonstrated that the presence of FIAU at a position 5' to the cleavage site in the consensus sequence T*TAA (where * is the cleavage site) inhibited restriction of the oligomeric duplex by MseI.
FIAC [1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine], FIAU [1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodouracil], and FMAU [1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil] were evaluated for their efficacies in the treatment of genital infections with herpes simplex virus type 2 in guinea pigs. Intraperitoneal administration of these drugs in daily doses of 100 mg/kg of body weight initiated 24 h after virus inoculation and repeated 2 successive days thereafter inhibited development of genital lesions and reduced shedding of virus without evoking untoward reactions. In a comparative study with this 3-day dosage schedule, the efficacy of daily doses of 50 mg of FMAU per kg was greater than that of the same doses of FIAC and FIAU, in that order; all these were more effective than daily doses of 50, 100, or 200 mg of acyclovir or of 500 mg of phosphonoformic acid per kg. These differences in efficacy were enhanced when treatment was delayed for 2 to 3 days after inoculation.
The capacity of recombinant human cytosolic thymidine kinase (TK1) and bovine mitochondrial thymidine kinase (TK2) to phosphorylate the antiviral analogs 1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) and 1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl)-5-methyluracil (FMAU) has been analyzed. The Vmax/Km ratios for FIAU and FMAU with TK2 are about 30% of that for deoxythymidine, while the corresponding values for TK1 are 2 and 5%, respectively. Thus, these two analogs are more efficient substrates for TK2 than for TK1, which may be part of the explanation for the mitochondrial toxicity associated with FIAU during treatment of hepatitis B infection.
The fluorinated pyrimidines 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU) are highly effective inhibitors of herpesvirus infections in vitro and in vivo. This report is concerned with an evaluation of their activities in African green monkeys (Cercopithecus aethiops) infected with simian varicella virus, a herpesvirus closely related to human varicella-zoster virus. Oral or intravenous administration of FIAU at 50 mg/kg per day as divided doses beginning 48 h after virus inoculation prevented the development of evidences of clinical infection. Oral treatment with FIAU at 30 mg/kg per day deferred as late as 7 days after virus inoculation modified the course of the disease. When treatment was started 48 h after virus inoculation, daily doses of FIAU as small as 1 mg/kg inhibited development of infections; daily doses of 0.2 mg/kg were ineffective. At the latter dose FMAU prevented development of clinical disease, suggesting that it was more active than FIAU. No signs of FIAU toxicity were observed, with the single exception of an early but transitory elevation in aspartate aminotransferase activity in serum.
Hepadnaviruses employ a unique mechanism for the initiation of RNA-directed DNA synthesis. Initially, four bases (5'-GTAA-3') are added to a tyrosine residue of the viral polymerase by reverse transcription of a bulge sequence in epsilon, a stem-loop structure which functions as the packaging signal for pregenomic RNA. This protein-DNA complex acts as the primer for minus-strand elongation from the 3' sequence, DR1. To understand this process in greater detail, we investigated whether the protein-mediated priming of viral DNA synthesis is affected by nucleotide analogs. By using cell-free expression of duck hepatitis B virus (DHBV) reverse transcriptase (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992), the 5'-triphosphate of the thymidine analog fialuridine (FIAU) was shown to inhibit the incorporation of radiolabeled TMP into primer DNA in a dose-dependent manner. Inhibition by the 5'-triphosphate of FIAU (FIAU-TP) was nearly complete at a concentration of 10 microM. The dideoxynucleotide analogs ddGTP, ddTTP, and 3'-azidodeoxythymidine triphosphate, known inhibitors of DHBV endogenous DNA polymerase, did not affect substantially the synthesis of primer DNA. Alternate substrate analysis suggested that FIAU is incorporated efficiently into nascent primer DNA as an analog of thymidine. Using site-directed mutagenesis to construct a mutant RNA template yielding a primer with the sequence 5'-GTAC-3', we demonstrated that FIAU-TP inhibited the incorporation of TMP, had no effect on that of dAMP, and decreased markedly the incorporation of dCMP. These results show that the synthesis of full-length DHBV primer DNA is inhibited by FIAU-TP but not by the dideoxynucleotide analogs that we tested. The significance of these findings as they relate to HBV DNA replication is discussed.
The effects of (2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluridine (FMAU), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouridine (FIAU), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU), and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG or BW B759U) on the replication of Epstein-Barr virus (EBV) in vitro were evaluated and compared with that of acyclovir (ACV). The relative potencies of these drugs, on the basis of anti-EBV activity, were: FIAC = FIAU greater than FMAU greater than DHPG greater than BVdU greater than ACV; on the basis of the therapeutic index they were: BVdU greater than DHPG greater than FIAC greater than ACV greater than FIAU greater than FMAU. Differential inhibition of EBV-associated polypeptides by these drugs was observed.
One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.
Better intraprostatic cancer imaging techniques are needed to guide clinicians in prostate cancer treatment decisions. Because many genes are specifically overexpressed in cancer cells, one strategy to improve prostate cancer detection is to image intraprostatic cancer–specific transcriptional activity. Because of the obstacles of weak cancer- or tissue-specific promoter activity and bladder clearance of many PET tracers, intraprostatic PET of gene transcriptional activity has not been previously reported.
The two-step transcriptional amplification (TSTA) system that amplifies the prostate-specific antigen promoter activity was used for PET imaging of the reporter gene herpes simplex virus type-1 sr39 thymidine kinase (HSV1-sr39tk). The TSTA-sr39tk system was injected directly into prostates or prostatic tumors as a replication-incompetent adenovirus (AdTSTA-sr39tk) and imaged using PET.
AdTSTA-sr39tk was able to image prostate-specific antigen promoter transcriptional activity by 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine PET, in both mouse and canine prostates in vivo. Ex vivo small-animal PET images, scintigraphic counts, and sr39tk expression analysis confirmed the specificity of the observed signal.
Here, by combining the TSTA-amplified signal with a protocol for tracer administration, we show that in vivo PET detection of transcriptional activity is possible in both mouse and immunocompetent canine prostates. These results suggest that imaging applications using transcription-based tumor-specific promoters should be pursued to better visualize cancer foci that escape detection by conventional biopsies.
prostate imaging; localized prostate cancer; positron emission tomography; molecular imaging
We have used a genetically attenuated adenoviral vector which expresses HSVtk to assess the possible additive role of suicidal gene therapy for enhanced oncolytic effect of the virus. Expression of TK was measured using a radiotracer-based molecular counting and imaging system.
Materials and Methods
Replication-competent recombinant adenoviral vector (Ad-ΔE1B19/55) was used in this study, whereas replication-incompetent adenovirus (Ad-ΔE1A) was generated as a control. Both Ad-ΔE1B19/55-TK and Ad-ΔE1A-TK comprise the HSVtk gene inserted into the E3 region of the viruses. YCC-2 cells were infected with the viruses and incubated with 2'-deoxy-2'-fluoro-β-D-arabinofuranosyl-5-iodouracil (I-131 FIAU) to measure amount of radioactivity. The cytotoxicity of the viruses was determined, and gamma ray imaging of HSVtk gene was performed. MTT assay was also performed after GCV treatment.
On gamma counter-analyses, counts/minute (cpm)/µg of protein showed MOIs dependency with ΔE1B19/55-TK infection. On MTT assay, Ad-ΔE1B19/55-TK led to more efficient cell killing than Ad-ΔE1A-TK. On plate imaging by gamma camera, both Ad-ΔE1B19/55-TK and Ad-ΔE1A-TK infected cells showed increased I-131 FIAU uptake in a MOI dependent pattern, and with GCV treatment, cell viability of ΔE1B19/55-TK infection was remarkably reduced compared to that of Ad-ΔE1A-TK infection.
Replicating Ad-ΔE1B19/55-TK showed more efficient TK expression even in the presence of higher-cancer cell killing effects compared to non-replicating Ad-ΔE1A-TK. Therefore, GCV treatment still possessed an additive role to oncolytic effect of Ad-ΔE1B19/55-TK. The expression of TK by oncolytic viruses could rapidly be screened using a radiotracer-based counting and imaging technique.
Oncolysis; adenovirus; thymidine kinase; gene therapy; radiotracer
Because of its high selectivity and specificity for the imaging reporter probe 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine (18F-FHBG), the herpes simplex virus type 1 thymidine kinase (HSV1-tk) variant sr39tk is actively being studied as a PET reporter gene. We recently demonstrated the capability of using a prostate-specific transcriptional amplification PET reporter vector, AdTSTA-sr39tk, to target prostate cancer lymph node metastasis. However, one area that warrants further study is the examination of the sensitivity of PET by determining the minimum percentage of cells expressing the sr39tk transgene needed for detection. Addressing this question could determine the sensitivity of vector-mediated sr39tk PET in cancer-targeting strategies.
DU-145, PC-3, and CWR22Rv.1 prostate cancer cell lines (a total of 1 × 106 cells) were studied, of which 7%, 10%, 25%, 50%, or 70% were transduced with the lentiviral vector constitutively expressing HSV1-sr39tk-IRES–enhanced green fluorescent protein (EGFP). Cells were subcutaneously implanted into the left shoulder of severe combined immunodeficient mice and evaluated. Tumor cells comparably transduced with an EGFP control vector were implanted on the right shoulder. Mice were imaged using PET with 18F-FHBG at 8, 15, and 22 d after tumor implant. On day 23, tumors were isolated and analyzed for sr39tk transgene expression by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), Western blotting, immunohistochemistry, and flow cytometry for EGFP expression.
Results showed a linear relationship between the level of sr39tk expression and the quantity of tracer accrual in DU-145, with the minimal value for PET detection at 10%. The magnitude of tracer retention in sr39tk-expressing cells was amplified over time as the tumor grew. Protein levels in the stepwise titration increased with the percentage of sr39tk-transduced cells.
The stepwise titration of prostate cancer cells transduced with the lenti-CMV-sr39tk-IRES-EGFP determined the minimum number of sr39tk-expressing tumor cells necessary to be detected by PET using the 18F-FHBG reporter probe. Furthermore, PET signal correlated well with traditional methods of protein evaluation such as flow cytometry, quantitative RT-PCR, Western blotting, and immunohistochemistry. Unlike the traditional methods, however, the use of PET is noninvasive and will be more advantageous in clinical situations.
prostate cancer; thymidine kinase; PET; transgene expression; lentivirus
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.
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 in mice with established subcutaneous tumors, expressing wild-type HSV1-tk and its mutants, followed by tissue sampling.
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.
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.
molecular imaging; reporter gene; HSV1-tk; PET; FEAU; 18F-FHBG