Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Soft tissue sarcomas are mesenchymal tumors which respond poorly to systemic therapy. Recent studies suggest a higher response rate with an increased doxorubicin dosage. However, this was parallel with a profound hematotoxicity in 75% of patients. Transfer of the human multidrug resistance 1 (MDR1) gene to normal hematopoietic stem cells and transplantation may significantly reduce the hematotoxicity of anthracyclin-based chemotherapy. To test this concept of supportive gene therapy in advance of a clinical study, we transduced mobilized peripheral blood progenitor cells (PBPC) with the retroviral vector SF91m3 containing the human MDR1 gene, transplanted these cells to immune-deficient mice, allowed 6 weeks for engraftment to occur and treated the animals with MDR1-based chemotherapy. In the MDR1-transduced group the human leukocytes were significantly protected from the toxicity of chemotherapy (p < 0.05). While the gene transfer rate was in the range of 10% and thus comparable to recent clinical trials, the gene expression was 59% of transduced cells and thus significantly higher than previously reported for less-advanced vectors. On the other hand, ifosfamide, a drug which has been used successfully for stem cell mobilization, is active in soft tissue sarcoma. Due to these favorable characteristics sarcoma is an attractive target to test the efficacy of MDR1 gene therapy in a clinical setting. Gene therapeutic strategies may also be used to directly target sarcoma cells, e.g. by transfer of suicide genes. We found that adenoassociated virus 2 (AAV-2) vectors efficiently transduce human HS-1 and HT1080 sarcoma cells (>90%) while other tumor cell lines and primary human PBPC were less susceptible. The thymidine kinase (TK) suicide gene was cloned into an AAV-2 vector and a complete kill of TK-transduced HS-1 and HT1080 cells was observed following exposure to aciclovir or ganciclovir (GCV), while >90% of mock-transduced HS-1 cells survived at these dosages. Transplantation of those sarcoma cells to nonobese diabetic (NOD)/LtSz-severe-combined immunodeficient (scid)/scid (NOD/SCID) mice resulted in a survival of >5 months in the AAV-TK-transduced/GCV-treated group, while the mice in the mock-transduced/GCV-treated group had died after 3 weeks. These data show that soft tissue sarcomas are a particularly suitable model system for the development and clinical testing of new gene therapeutic concepts.
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PMID:Gene therapy for sarcoma. 1242 90

New technologies are needed to characterize the migration, survival, and function of antigen-specific T cells in vivo. Here, we demonstrate that Epstein-Barr virus (EBV)--specific T cells transduced with vectors encoding herpes simplex virus-1 thymidine kinase (HSV-TK) selectively accumulate radiolabeled 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU). After adoptive transfer, HSV-TK+ T cells labeled in vitro or in vivo with [131I]FIAU or [124I]FIAU can be noninvasively tracked in SCID mice bearing human tumor xenografts by serial images obtained by scintigraphy or positron emission tomography (PET), respectively. These T cells selectively accumulate in EBV+ tumors expressing the T cells' restricting HLA allele but not in EBV- or HLA-mismatched tumors. The concentrations of transduced T cells detected in tumors and tissues are closely correlated with the concentrations of label retained at each site. Radiolabeled transduced T cells retain their capacity to eliminate targeted tumors selectively. This technique for imaging the migration of ex vivo-transduced antigen-specific T cells in vivo is informative, nontoxic, and potentially applicable to humans.
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PMID:Serial in vivo imaging of the targeted migration of human HSV-TK-transduced antigen-specific lymphocytes. 1265 11

The Herpes simplex virus 1 (HSV) thymidine kinase (tk) suicide gene together with ganciclovir (GCV) have been successfully used for the in vivo treatment of various solid tumors and for the ablation of unwanted transfused stem cells in recent clinical trials. With the aim of improving this therapeutic system, we compared the potential efficacy of adenoviral (Ad) vectors expressing enhanced tk mutants in vitro and in vivo. The previously created HSV-tk mutants dm30 and sr39, created by random sequence mutagenesis, were inserted into a standard Ad.RSV E1(-)E3(-) backbone using homologous recombination. GCV killing of Ad.HSV-tk, Ad.dm30-tk and Ad.sr39-tk was assessed in various tumor cell lines with a cell proliferation assay. Cells expressing the two TK mutants were two-to-five-fold more sensitive to GCV when compared with Ad.HSV-tk transduced cells in all cell lines tested (five human mesotheliomas, one human lung cancer, a human cervical carcinoma, a mouse fibrosarcoma, and a rat glioma line) at equal TK expression levels. Flank tumor models, including cell-mixing studies, assessed the in vivo efficacy of the engineered viruses in BALB/C and SCID mice. In all animal studies, Ad.dm30-tk and Ad.sr39-tk showed more tumor growth inhibition than Ad.HSV-tk when GCV was administered. The use of adenovirus-mediated gene transfer of both tk mutants dm30-tk and sr39-tk for cancer suicide gene therapy should provide a more effective and safer alternative to wild-type HSV-tk.
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PMID:Adenovirus-mediated gene transfer of enhanced Herpes simplex virus thymidine kinase mutants improves prodrug-mediated tumor cell killing. 1271 5

Here we describe the evaluation of 3'-[(18)F]fluoro-3'-deoxythymidine [[(18)F]-FLT] as a tracer for positron emission tomography (PET) in a murine model of B-cell lymphoma and in human malignant lymphoma. The human B-cell line DoHH2 expressed high levels of active thymidine kinase 1 (TK-1) as the key enzyme of [(18)F]-FLT metabolism. Immunostaining confirmed high levels of TK-1 in DoHH2 derived xenograft tumors in SCID/SCID mice. In vitro studies demonstrated a time-dependent uptake of [(18)F]-FLT, an efficient phosphorylation to the respective monophosphate and the incorporation of [(18)F]-FLT into the perchloric acid insoluble fraction in DoHH2 cells, indicating the incorporation of this tracer into the DNA. After incubation with [(18)F]FLT for 240 min, 12.5% +/- 1.0% of radioactivity applied to the medium was intracellularly trapped in DoHH2 cells. Specific accumulation of [(18)F]-FLT in the malignant cell clone was confirmed in biodistribution studies in SCID/SCID mice bearing DoHH2-derived tumors. The percentage of injected dose of [(18)F]-FLT per gram of tumor tissue correlated with the tumor-proliferation index as evaluated in BrdUrd-labeling experiments. In a pilot study of 11 patients with both indolent and aggressive lymphoma, [(18)F]-FLT was suitable and comparable to [(18)F]-FDG in the ability to detect malignant lesions by PET scan. Furthermore, we found a close correlation (r = 0.95, P < 0.005) of the [(18)F]-FLT standardized uptake values with the Ki67-labeling index of tissue biopsies (n = 10) in these patients. These results suggest that [(18)F]-FLT represents a novel tracer for PET that enables imaging of proliferation in human lymphoma in vivo.
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PMID:3'-[18F]fluoro-3'-deoxythymidine ([18F]-FLT) as positron emission tomography tracer for imaging proliferation in a murine B-Cell lymphoma model and in the human disease. 1275 Feb 97

The herpes simplex virus (HSV) virion host shutoff (vhs) protein, the product of the UL41 (vhs) gene, is an important determinant of HSV virulence. vhs has been implicated in HSV interference with host antiviral immune responses, down-regulating expression of major histocompatibility complex molecules to help HSV evade host adaptive immunity. The severe attenuation of vhs-deficient viruses in vivo could reflect their inability to escape immune detection. To test this hypothesis, BALB/c or congenic SCID mice were infected intravaginally (i.vag.) with the HSV type 2 (HSV-2) vhs null mutant 333d41 or the vhs rescue virus 333d41(R). vhs-deficient virus remained severely attenuated in SCID mice compared with rescue virus, indicating that vhs regulation of adaptive immune responses does not influence HSV pathogenesis during acute infection. Innate antiviral effectors remain intact in SCID mice; prominent among these is alpha/beta interferon (IFN-alpha/beta). The attenuation of HSV-2 vhs mutants could reflect their failure to suppress IFN-alpha/beta-mediated antiviral activity. To test this hypothesis, 129 and congenic IFN-alpha/beta receptor-deficient (IFN-alpha/betaR(-/-)) mice were infected i.vag. with wild-type virus, vhs null mutants 333-vhsB or 333d41, or the vhs rescue virus 333d41(R). Whereas vhs-deficient viruses showed greatly reduced replication in the genital mucosa of 129 mice compared with wild-type or vhs rescue viruses, they were restored to nearly wild-type levels of replication in IFN-alpha/betaR(-/-) mice over the first 2 days postinfection. Only wild-type and vhs rescue viruses caused severe genital disease and hind limb paralysis in 129 mice, but infection of IFN-alpha/betaR(-/-) mice restored the virulence of vhs-deficient viruses. vhs-deficient viruses replicated as vigorously as wild-type and rescue viruses in the nervous systems of IFN-alpha/betaR(-/-) mice. Restoration was specific for the vhs mutation, because thymidine kinase-deficient HSV-2 did not regain virulence or the capacity to replicate in the nervous systems of IFN-alpha/betaR(-/-) mice. Furthermore, the defect in the IFN-alpha/beta response was required for restoration of vhs-deficient virus replication and virulence, but the IFN-alpha/beta-stimulated protein kinase R pathway was not involved. Finally, vhs of HSV-2 has a unique capacity to interfere with the IFN-alpha/beta response in vivo, because an HSV-1 vhs null mutant did not recover replication and virulence after i.vag. inoculation into IFN-alpha/betaR(-/-) mice. These results indicate that vhs plays an important role early in HSV-2 pathogenesis in vivo by interfering with the IFN-alpha/beta-mediated antiviral response.
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PMID:Herpes simplex virus type 2 virion host shutoff protein regulates alpha/beta interferon but not adaptive immune responses during primary infection in vivo. 1291 49

Human marrow-derived mesenchymal stem cells (MSC), which have the potential to differentiate into mesenchymal tissues, such as bone, cartilage, adipose and bone marrow stroma, were transduced with a retroviral vector carrying the simian virus 40 large T antigen, hygromycin-resistant gene and herpes simplex virus thymidine kinase gene, that can be excised by Cre/loxP site-specific recombination. This resulted in establishment of an MSC cell line, HMSC-1, which retained original surface characteristics and differentiation potential, and exhibited a higher proliferative capacity than parental cells. HMSC-1 expressed mRNAs of BMP-4, Jagged-1, and SCF that are known to promote hematopoiesis. Human CB CD34+ hematopoietic progenitor cells (HPC) cultured on a layer of HMSC-1 cells showed high expansion of CD34+CD38- immature HPC, capable of reconstituting human hematopoiesis in non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. This cell line may be of value for developing strategies for ex vivo expansion of human HPC.
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PMID:Immortalization of bone marrow-derived human mesenchymal stem cells by removable simian virus 40T antigen gene: analysis of the ability to support expansion of cord blood hematopoietic progenitor cells. 1296 70

A novel therapy for Epstein-Barr virus (EBV)-positive tumors involves the intentional induction of the lytic form of EBV infection combined with ganciclovir (GCV) treatment. Virally encoded kinases (thymidine kinase and BGLF4) which are expressed only during the lytic form of infection convert GCV (a nucleoside analogue) into its active, cytotoxic form. However, tightly latent EBV infection in B cells has made it difficult to identify drugs that can be used clinically to induce lytic viral infection in B-cell lymphomas. Here we demonstrate that gemcitabine and doxorubicin (but not 5-azacytidine, cis-platinum, or 5-fluorouracil) induce lytic EBV infection in EBV-transformed B cells in vitro and in vivo. Gemcitabine and doxorubicin both activated transcription from the promoters of the two viral immediate-early genes, BZLF1 and BRLF1, in EBV-negative B cells. This effect required the EGR-1 motif in the BRLF1 promoter and the CRE (ZII) and MEF-2D (ZI) binding sites in the BZLF1 promoter. GCV enhanced cell killing by gemcitabine or doxorubicin in lymphoblastoid cells transformed with wild-type EBV, but not in lymphoblastoid cells transformed by a mutant virus (with a deletion in the BZLF1 immediate-early gene) that is unable to enter the lytic form of infection. Most importantly, the combination of gemcitabine or doxorubicin and GCV was significantly more effective for the inhibition of EBV-driven lymphoproliferative disease in SCID mice than chemotherapy alone. In contrast, the combination of zidovudine and gemcitabine was no more effective than gemcitabine alone. These results suggest that the addition of GCV to either gemcitabine- or doxorubicin-containing chemotherapy regimens may enhance the therapeutic efficacy of these drugs for EBV-driven lymphoproliferative disease in patients.
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PMID:Lytic induction therapy for Epstein-Barr virus-positive B-cell lymphomas. 1474 54

Hypoxia-inducible factors, key transcription factors for hypoxia-dependent gene expression, play important roles in angiogenesis and tumor growth. The VHL protein binds to the alpha subunit of (HIF-alpha) for its oxygen-dependent degradation. VHL mutations are found frequently in sporadic RCC. Disruption of VHL results in an abnormal accumulation of HIF-alpha, leading to the upregulation of downstream genes such as the vascular endothelial growth factor gene. We constructed a luciferase reporter vector driven by hypoxia-responsive elements (5HRE/luc) and a therapeutic vector expressing a herpes simplex virus thymidine kinase gene (5HRE/tk). In the transient transfection assay using VHL-deficient 786-O cells, constitutive luciferase expression was detected under both aerobic and hypoxic conditions. In contrast, 786-O cells transfected with a wild-type VHL showed hypoxia-inducible luciferase activity. In in vitro MTS assay, 50% of growth inhibition of 786-O cells stably transfected with 5HRE/tk was achieved with exposure to 0.2 microg/mL of GCV under both aerobic and hypoxic conditions. Xenografts of the stable clone in SCID mice exhibited a marked regression on daily injections of GCV (50 mg/kg) for 10 days. In conclusion, a hypoxia-responsive vector may have therapeutic potential for RCC with VHL mutations.
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PMID:A tumor-specific gene therapy strategy targeting dysregulation of the VHL/HIF pathway in renal cell carcinomas. 1590 70

Live attenuated HIV vaccines offer a means to introduce exogenous sequences into the viral genome to target the virus elimination in vivo. Foreign genes inserted into the nef region of HIV-1 NL4-3 were found to be rapidly deleted following virus infection and/or replication, in a size dependent manner, in the human fetal Thymus/Liver implants of severe combined immunodeficient mouse (SCID-hu) model. When the murine heat stable antigen (HSA) of 283 bp was substituted into HIV-1 nef region, the viral loads in vivo were comparable to the negative control nef attenuated HIV-1, and the reporter HSA gene was not deleted upon infection. However, the murine Thy1.2 gene (505 bp) substituted into the nef attenuated HIV-1, upon infection and replication, deleted 441 bp in vitro and 437 bp in vivo, of the inserted Thy1.2 gene. When the enhanced green fluorescence protein (eGFP) gene (720 bp) was substituted for nef, virus replication was aborted in vivo in the Thy/Liv implants, as seen by the background levels of viral loads, comparable to mock infected implants, and the eGFP gene was deleted. When the herpes simplex virus thymidine kinase gene, HSV-TK (1.15 kbp), or HSA gene, was substituted into the viral vpr gene, TK but not HSA gene was deleted, upon infection in vitro. Moreover, NL-TKI reporter virus with both intact nef and vpr genes shows deletion of TK gene both in vitro and in vivo. Excision of foreign genes occurred within the exogenous segments but not in the viral own regions. These results suggest that larger "suicide" genes introduced via HIV-1 can be deleted upon infection. However, smaller size nucleotide sequences or genes (approximately 300 bp) inserted in place of viral nef or vpr gene may be used to target the virus or its components, for attack and elimination in vivo, and thus have implications for the development of live attenuated HIV vaccines.
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PMID:Rapid size dependent deletion of foreign gene sequences inserted into attenuated HIV-1 upon infection in vivo: implications for vaccine development. 1625 Aug 84

Cancer gene therapy based on tissue-restricted expression of cytotoxic gene should achieve superior therapeutic index over an unrestricted method. This study compared the therapeutic effects of a highly augmented, prostate-specific gene expression method to a strong constitutive promoter-driven approach. Molecular imaging was coupled to gene therapy to ascertain real-time therapeutic activity. The imaging reporter gene (luciferase) and the cytotoxic gene (herpes simplex thymidine kinase) were delivered by adenoviral vectors injected directly into human prostate tumors grafted in SCID mice. Serial bioluminescence imaging, positron emission tomography, and computed tomography revealed restriction of gene expression to the tumors when prostate-specific vector was employed. In contrast, administration of constitutive active vector resulted in strong signals in the liver. Liver serology, tissue histology, and frail condition of animals confirmed liver toxicity suffered by the constitutive active cohorts, whereas the prostate-targeted group was unaffected. The extent of tumor killing was analyzed by apoptotic staining and human prostate marker (prostate-specific antigen). Overall, the augmented prostate-specific expression system was superior to the constitutive approach in safeguarding against systemic toxicity, while achieving effective tumor killing. Integrating noninvasive imaging into cytotoxic gene therapy will provide a useful strategy to monitor gene expression and therapeutic efficacy in future clinical protocols.
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PMID:Micro-PET/CT monitoring of herpes thymidine kinase suicide gene therapy in a prostate cancer xenograft: the advantage of a cell-specific transcriptional targeting approach. 1628 8


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