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)

To investigate the possibility of killing tumor cells by the expression of an exogenously introduced toxic gene, we have constructed a novel retroviral vector (LTRNL) which has the polyA signal deleted herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene. The vector becomes toxic by treating cells expressing HSV1-tk with the antiherpetic drugs acyclovir or ganciclovir (GCV). Cells of the human leukemia lines (K562, MEG-01) were infected with this vector and two transduced cell lines (K562/LTRNL, MEG-01/LTRNL) were established. Southern blot analysis confirmed the integration of the HSV1-tk transgene in these cells and Northern blot analysis exhibited the expression of 4.8-kb viral mRNA containing the HSV1-tk gene. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for the in vitro cytotoxic effects of GCV to these cells demonstrated that concentrations of about 2.5 microM for K562/LTRNL and 1.25 microM for MEG-01/LTRNL cells resulted in 50% inhibition of cell growth after 72 hr. Subcutaneous tumors of MEG-01/LTRNL in KSN nude mice, but not those of uninfected MEG-01 cells, showed durable regressions after exposure of the mice to 40 mg/kg of GCV given subcutaneously once a day for 15 days. This study indicates that the LTRNL-infected human leukemia cells exhibit inducible susceptibility to GCV.
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PMID:Transduction of a drug-sensitive toxic gene into human leukemia cell lines with a novel retroviral vector. 839 Jun 93

One of the recent strategies for gene therapy as a cancer control is the targeted introduction of a drug-sensitivity gene into tumor cells. We investigated the gene transfer of herpes simplex virus type I thymidine kinase (HSV-TK) gene as a drug-sensitivity gene into human lung cancer cell lines. We used a recombinant retroviral vector derived from Moloney murine leukemia virus (MuLV) as one of potential vectors for gene therapy. The amphotropic retroviral vector consisted of the HSV-TK gene and the neomycin-resistant gene under Rous sarcoma virus (RSV) promoter control. The antiherpes drugs, acyclovir (ACV) and ganciclovir (GCV), were chosen for testing the activity of HSV-TK that was transferred into human lung cancer cell lines. ACV and GCV are nucleoside analogs specifically converted by HSV-TK to a toxic form capable of inhibiting DNA synthesis. The cytotoxicity was determined by using a tetrazolium-based colorimetric assay (MTT assay). The results obtained from our experiments demonstrated that the retroviral vector-mediated HSV-TK gene transfer leads to ACV- and GCV-dependent cytotoxicity in human lung cancer cell lines, which were both small cell carcinoma and non-small cell carcinoma established from human specimens. These findings suggest that the gene transfer of HSV-TK gene into tumor cells would be one of the models for the use of gene therapy to control lung cancer.
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PMID:Gene transfer of herpes simplex virus type I thymidine kinase gene as a drug sensitivity gene into human lung cancer cell lines using retroviral vectors. 839 27

5-fluorouracil (5-FU), an inhibitor of thymidylate synthase (EC 2.1.1.45), is clinically used in the treatment of several solid tumors, including colorectal, head and neck, gastric, and pancreatic cancer. The drug effectively inhibits deoxynucleoside triphosphate de novo synthesis. However, this inhibition can be circumvented by increased thymidine kinase (EC 2.7.1.21) activity. In the present study we examined the effects of 5-FU combined with azidothymidine (AZT), a competitive inhibitor of thymidine kinase in human colon tumor cells in vitro, including three 5-FU resistant cell lines. The cells were simultaneously incubated with various concentrations of 5-FU (0.015 to 150 microM) and AZT (20 to 300 microM) for 6 days. 5-FU alone yielded an IC50 of 18 microM in the parental CCL 227 cell line and IC50s of 470 and 1100 microM in the 5-FU resistant cell lines as determined by a MTT chemosensitivity assay. Addition of 100 microM AZT alone, a drug concentration that can be achieved in patients, had no effect on the growth of the cell lines examined. However, when added simultaneously with 5-FU, the IC50s of 5-FU synergistically decreased to 10 microM in the sensitive and to 360 or 760 microM in the resistant cell lines, respectively. Our results demonstrate that the combination of 5-FU with AZT synergistically inhibited the growth of 5-FU resistant cells, suggesting the use of 5-FU in combination with AZT for the treatment of 5-FU sensitive as well as resistant human colon tumors.
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PMID:Modulation of 5-fluorouracil resistance in human colon tumor cell lines by azidothymidine. 888 11

In investigating new methods for the treatment of pancreatic cancer, we have explored the possibility of using a combination of radiation and gene therapies. We demonstrate herein that the early growth response gene 1 promoter (Egr-1) is sufficient to confer selective expression of the luciferase gene (Luc) in a human pancreatic tumor cell line (AsPc-1) when exposed to ionizing radiation. The Egr-1 promoter directed the radioinducible expression of luciferase, and yielded higher levels of Luc activity than that in nonirradiated lines. The radioisotopes Tc-99m, I-131, and Ga-67-citrate were selected as Egr-1 activators for their potential to accumulate in tumors. We studied Ga-67-citrate, a radioisotope employed in tumor scintigraphy, for its suitability for selective gene induction. The plasmid vector pEgr-1-Luc was transfected into AsPc-1 cells and then exposed to radioisotopes. Luciferase activity increased by 100-300 times over control. We also inserted the herpes thymidine kinase gene (TK) downstream of Egr-1 and transfected this construct into AsPc-1 cells. Ga-67-citrate and ganciclovir were added to the cells and cell survival was assessed by MTT assay. The growth of AsPc-1 cells transfected with the pEgr-TK construct was suppressed 2 days after exposure of the cells to Ga-67-citrate. The results indicate that Ga-67-citrate may be useful in combining radiation and gene therapies.
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PMID:Induction of the suicide HSV-TK gene by activation of the Egr-1 promoter with radioisotopes. 914 8

The cytomegalovirus(CMV) promoter is considered one of the strongest positive regulators. In this study toxicity, cell killing efficacy and bystander effect of Rous Sarcoma Virus(RSV) driven herpes simplex thymidine kinase(TK) gene therapy was compared with CMV driven TK gene therapy in three ovarian cancer cell lines with different growth patterns using a 3-(4,5-dimethylthiazol)-2,5-diphenyl tetra-zolium bromide (MTT) based assay. ADV/CMV-TK was shown to be 2 to 10 times more effective in tumor cell killing than ADV/RSV-TK. The difference in cell killing efficacy between ADV/CMV-TK and ADV/RSV-TK was dependent on the individual cell line. A CMV promoter dependent eight to ten fold improvement in cell killing efficacy was observed in the relatively slow growing SKOV3 cell line which is not easily transducible, while only a 2 to 4 fold difference was observed in the easily transducible OV-CA-2774 and OV-CA-1225 cell lines. ADV/CMV-TK also showed a stronger bystander effect than ADV/RSV-TK in all three ovarian cancer cell lines. Our data demonstrated that the efficacy of adenovirus-mediated gene therapy of ovarian cancer can be enhanced by using the CMV promoter without increasing toxicity.
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PMID:The efficacy of adenovirus-mediated gene therapy of ovarian cancer is enhanced by using the cytomegalovirus promoter. 961 11

Adenovirus(ADV) mediated thymidine kinase(TK) gene therapy followed by ganciclovir(GCV) administration is widely used in different types of cancer. ACV shares the same mechanism of selective cell killing in ADV/TK positive cells as GCV and can be used at 4.5 times higher doses in patients without significant side effects. An increased dose of TK substrate is associated with improved bystander effect and more efficient cell killing. Toxicity and cell killing efficacy were assessed using a 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide(MTT) based assay in three ovarian cancer cell lines with different proliferation patterns. At the same concentration, equal or higher cell killing efficacy and bystander effect were observed using ACV rather than GCV. 2.5 and 5 times (25 micrograms/ml and 50 micrograms/ml) higher concentrations of ACV always resulted in more effective cell killing than GCV (10 micrograms/ml, P < 0.01). Our data indicate that replacing GCV with ACV in the ADV-TK gene therapy may increase the treatment effect without increasing toxicity.
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PMID:Improvement of gene therapy for ovarian cancer by using acyclovir instead of ganciclovir in adenovirus mediated thymidine kinase gene therapy. 961 10

The current treatment concept of ovarian cancer consists of radical surgery with subsequent chemotherapy. We have shown that adenovirus (ADV) mediated thymidine kinase (TK) gene transduction of cisplatin-resistant human ovarian cancer xenotransplanted into nude mice followed by ganciclovir (GCV) administration leads to prolongation of survival or cure. In this study the interaction of ADV-TK gene therapy and selected chemotherapeutic agents commonly used for the treatment of ovarian cancer was investigated in three ovarian cancer cell lines with different growth patterns. Toxicity and cell killing efficacy of gene therapy, chemotherapy and their combinations with different concentrations and time intervals were measured by a 3-(4,5- dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT) based assay. A slightly increased resistance to gene therapy was observed in cells pretreated with chemotherapy. Removal of the drugs restored the previous susceptibility of the cells to gene therapy. No antagonism was observed with gene therapy followed by chemotherapy. The concomitant application of gene therapy and chemotherapy resulted in a higher rate of cell death than the interval therapy. A dose dependent synergistic interaction was observed only for the combination of gene therapy and the topoisomerase 1 inhibitor topotecan. This synergistic effect was still seen even if the chemotherapeutic agent was added 72 hours later. Our data demonstrate that in addition to its own therapeutic efficacy, ADV-TK based gene therapy may enhance the effect of subsequent chemotherapy while up-front chemotherapy was disadvantageous.
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PMID:Adenovirus mediated thymidine kinase gene therapy may enhance sensitivity of ovarian cancer cells to chemotherapeutic agents. 985 18

A human immunodeficiency virus type 1 (HIV-1)-based retroviral vector pseudotyped with HIV envelope containing the herpes simplex virus-thymidine kinase (HSV-TK) gene under the control of the HIV LTR promoter (pHXTKN) was constructed and stably transferred into human CD4(+) H9, CEM, and U937 cells. RNase protection assays did not initially detect expression of the HSV-TK gene in HXTKN-transduced CD4(+) cells (HXTKN/CD4), but expression was then efficiently induced by infection with HIV-1. MTT assays showed that after HIV-1 infection, the susceptibility of HXTKN/CD4 cells to ganciclovir (GCV) was 1000-fold higher than prior to infection. This enabled HIV-1-infected cells to be selectively killed by transduction with HXTKN followed by exposure to GCV. Because the HSV-TK gene is specifically transferred into HIV-1-permissive cells and expressed only after HIV-1 infection, the frequency of unwanted cell death should be low. Elimination of the HIV-1-infected cells effectively inhibited further spread of infectious virus. In addition, the integrated HIV vector sequences were repackaged on infection with HIV-1 and transferred to surrounding untransduced cells. These results are indicative of the potential benefits of using HIV vectors in gene therapies for the treatment of HIV-1 infection.
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PMID:Selective killing of human immunodeficiency virus-infected cells by targeted gene transfer and inducible gene expression using a recombinant human immunodeficiency virus vector. 1117 60

A group of unnatural 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluorobenzenes having a variety of C-5 two-carbon substituents [-C...C-X, X = I, Br; -C...CH; (E)-CH=CH-X, X = I, Br; -CH=CH2; -CH2CH3; -CH(N3) CH2Br], designed as nucleoside mimics, were synthesized for evaluation as anticancer and antiviral agents. The 5-substituted (E)-CH=CH-I and -CH2CH3 compounds exhibited negligible cytotoxicity in a MTT assay (CC50 = 10(-3) to 10(-4)M range), relative to thymidine (CC50 = 10(-3) to 10(-5)M range), against a variety of cancer cell lines. In contrast, the C-5 substituted -C...C-I and -CH(N3)CH2Br compounds were more cytotoxic (CC50 = 10(-5) to 10(-6)M range). The -C...C-I and -CH2CH3 compounds exhibited similar cytotoxicity against non-transfected (KBALB, 143B) and HSV-1 TK+ gene transfected (KBALB-STK, 143B-LTK) cancer cell lines expressing the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (TK+). This observation indicates that expression of the viral TK enzyme did not provide a gene therapeutic effect. The parent group of 5-substituted compounds, that were evaluated using a wide variety of antiviral assay systems [HSV-1, HSV-2, varicella-zoster virus (VZV), vaccinia virus, vesicular stomatitis, cytomegalovirus (CMV), and human immunodeficiency (HIV-1, HIV-2) viruses], showed that this class of unnatural C-aryl nucleoside mimics are inactive and/or weakly active antiviral agents.
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PMID:Synthesis of 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-substituted-benzenes: "thymine replacement" analogs of thymidine for evaluation as anticancer and antiviral agents. 1130 62

To study the therapeutic effects of herpes simplex virus thymidine kinase (HSV-TK) gene transferred by the EBV-based expression vector (pDR2) on experimental hepatocellular carcinoma, pDR2-TK gene was delivered into human hepatocellular carcinoma cell line SMMC-7721 by using liposome-mediated transfection technique, and then gene expression was detected by RT-PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model, the antitumor effects of pDR2-TK/GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2-TK/GCV had cytotoxic effect and about 70% SMMC-7721 cells were killed when GCV was at 1000 mumol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2-TK gene was significantly smaller than that in control group (P < 0.01). On the 10th day the tumor in 3 mice (60%) disappeared completely after GCV treatment. It is concluded that the pDR2-TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.
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PMID:Gene therapy of HSV-TK transferred by the EBV based expression vector on experimental hepatocellular carcinoma. 1152 15


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