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)

The cytotoxicity of LY231514 was only partially alleviated by thymidine addition (5 microM) in GC3 human colon carcinoma cells, and complete protection required the addition of both hypoxanthine (100 microM) and thymidine. In contrast, the cytotoxic activity of tomudex (raltitrexed, ZD1694) was completely reversed by thymidine alone. MCF-7 human breast and H630 human colon carcinoma cells selected for resistance to tomudex and 5-fluorouracil, respectively via thymidylate synthase (TS) amplification demonstrated only modest resistance to LY231514 compared to tomudex. LY231514-induced cytotoxicity in these resistant cell lines was completely prevented by the addition of hypoxanthine (100 microM), indicating inhibition of purine de novo biosynthesis as a secondary target for LY231514 action. Thymidine at physiologic levels in mouse plasma (approximately 1 microM) produced only a 2.6-fold shift in the IC50 for LY231514-mediated cytotoxicity in GC3/cl1 cells compared to a 128-fold shift for tomudex. LY231514 treatment (i.p., qd x 10) significantly delayed tumor growth in the GC3 carcinoma xenograft model. However, a thymidine kinase-deficient mutant of this same tumor line demonstrated heightened sensitivity to the in vivo antitumor activity of LY231514 with complete regression of established tumors and a large number of tumor-free survivors after one course of treatment. The data demonstrate that inhibition of thymidylate synthase is a prominent mechanism for antitumor activity by LY231514, but important secondary sites of action exist for this multitargeted molecule.
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PMID:Role of thymidylate synthase in the antitumor activity of the multitargeted antifolate, LY231514. 1022 79

Our recent study demonstrates the feasibility of the thyroglobulin (TG) promoter in transcriptionally targeted gene therapy for thyroid carcinomas expressing TG, albeit less effectively than the constitutive viral promoter. The present study was, therefore, designed to enhance the activity of the TG promoter with the Cre-loxP system. Our data demonstrate that the in vitro cytotoxic effect of herpes simplex virus thymidine kinase/ganciclovir obtained with the TG promoter and the Cre-loxP system is approximately 5-10-fold higher than that with the TG promoter alone. Enhanced tumor growth inhibition was also observed in in vivo tumor models. These data indicate the usefulness of the Cre-loxP system to enhance the activity of a tissue (or tumor)-specific promoter in transcriptionally targeted cancer gene therapy.
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PMID:Enhanced efficacy of transcriptionally targeted suicide gene/prodrug therapy for thyroid carcinoma with the Cre-loxP system. 1039 42

A new strategy in anticancer gene therapy uses stress-responsive cellular promoters that offer the advantage of enhanced gene expression in a variety of tumors. Although the feasibility of their selective expression has been demonstrated, functional evidence of their ability to activate therapeutic agents within the tumor environment leading to tumor eradication has not been established. Glucose deprivation, chronic anoxia, and acidic pH known to persist in poorly vascularized solid tumors strongly induce the transcription of the glucose-regulated protein 78 (grp78) gene, which encodes an Mr 78,000 stress-inducible protein. In this report, we tested directly the efficacy of the grp78 promoter in a retroviral system to drive the expression of the herpes simplex virus-thymidine kinase (HSVtk) suicide gene, using a murine fibrosarcoma model, in the context of their syngeneic, immunocompetent hosts. Our results showed that under glucose starvation conditions, the expression of HSVTK was enhanced in tumor cells where the HSVtk gene was driven by the internal grp78 promoter, in contrast to the Moloney murine leukemia virus long terminal repeat, where suppression was observed. We further demonstrated that in vivo, HSVTK expression was elevated to much higher levels inside tumors when driven by the internal grp78 promoter, resulting in complete eradication of sizable tumor mass, with no recurrence of tumor growth. Our study suggests that the glucose starvation-inducible grp78 promoter could be useful for enhanced expression of a variety of therapeutic agents within the solid tumor environment.
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PMID:Use of the glucose starvation-inducible glucose-regulated protein 78 promoter in suicide gene therapy of murine fibrosarcoma. 1039 51

Inherited medullary thyroid carcinomas (MTC) are aggressive and resistant to conventional chemo- and radiotherapies. We evaluated a novel strategy for treatment of MTC, combining "suicide" and interleukin-2 (IL-2) gene therapies. Tumors were produced in Wag/Rij rats by orthotopic injection of the rMTC 6-23 cell line, and/or derivatives expressing the herpes simplex virus 1 thymidine kinase (HSV1-TK) gene (rMTC-TK). Ganciclovir, a nucleoside analog selectively transformed to a toxic metabolite by HSV1-TK, totally eradicated rMTC-TK tumors in 60% of the animals. 1:1 rMTC and rMTC-TK mixed tumors were also strongly inhibited by ganciclovir (P < 0.05), indicating the occurrence of an efficient "bystander" effect in vivo. Double labelling of rMTC cell membranes and apoptotic nuclei revealed that, as with the TK+ cells, some TK- cells died by apoptosis. A 1:1 mixture of rMTC and rMTC-TK cells was administered to produce established tumors and then rMTC cells, transfected to express the IL-2 gene (rMTC-IL2), were inoculated. Combined ganciclovir and IL-2 treatment improved the inhibition of tumor growth compared to that following ganciclovir alone (86% compared to 54%, P < 0.05). This treatment also significantly enhanced macrophage activation and tumor infiltration by CD8+ and CD4+ T lymphocytes. These results open an avenue for combining suicide and immunoregulatory gene therapies for MTC management in man.
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PMID:Treatment of medullary thyroid carcinoma by combined expression of suicide and interleukin-2 genes. 1041 62

Because many tumors have mutated p53, one potential strategy proposed for cancer gene therapy is the introduction of the wild-type p53 gene into tumor cells. One puzzling aspect of this approach is that currently available gene transfer protocols result in a small percentage of tumor cells being transduced in vivo, thus implicating a "bystander effect" to achieve therapeutic efficacy. Because bystander effects in the context of p53-mediated gene therapy have not been well characterized, we evaluated the role of in vitro and in vivo bystander effects of adenovirally delivered p53 (AdWTp53). Using human tumor cell lines that did not express p53 protein but were infectible with adenovirus and showed sensitivity to p53-mediated apoptosis, we were unable to demonstrate an AdWTp53-mediated in vitro bystander effect, despite seeing strong bystander effects when cells were infected with an adenovirus containing the suicide gene herpes simplex virus thymidine kinase and treated with ganciclovir. In contrast, in vivo flank mixing studies using one of these cell lines showed a weak but significant p53-mediated bystander effect (a 40% inhibition of tumor growth). This bystander effect translated into a small survival advantage in an established intraperitoneal tumor model when tumor burden was low at the time of viral instillation. The survival advantage was lost, however, when tumor burden was increased. This study indicates that treatment of human tumors using AdWTp53 may be possible; however, because of the weak bystander effect in vivo, effective treatment will likely require a large percentage of tumor cells to be transduced.
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PMID:The evaluation of adenoviral p53-mediated bystander effect in gene therapy of cancer. 1041 47

Initial studies have demonstrated the therapeutic efficacy for cancer treatment of in vivo transfer of the herpes simplex virus thymidine kinase gene followed by ganciclovir (GCV) treatment. However, recent studies have questioned the validity of this approach. Using retroviral vector-producing cells (VPC) as a source for in vivo gene transfer, we evaluated the efficacy of in vivo transduction of malignant cells using three different tumor cell models: B16 murine and IIB-MEL-LES human melanomas and a C6 rat glioblastoma. In vitro studies showed a bystander effect only in C6 cells. In vivo studies showed an inhibition of tumor growth in the two melanoma models when tumor cells were coinjected with VPC-producing retroviral vectors carrying the herpes simplex virus thymidine kinase gene, followed by GCV treatment; however, 100% of mice developed tumors in both models. Under similar experimental conditions, 70% (7 of 10) of syngeneic rats completely rejected stereotactically transferred C6 tumor cells; most of them (5 of 10) showed a prolonged survival. Treating established C6 tumors with VPC-producing retroviral vectors carrying the herpes simplex virus thymidine kinase gene and GCV led to the cure of 33% (4 of 12) of the animals. Rats that rejected tumor growth developed an antitumor immune memory, leading to a rejection of a stereotactic contralateral challenge with parental cells. The immune infiltrate, which showed the presence of T lymphocytes, macrophages, and polymorphonuclear cells at the site of the first injection and mainly T lymphocytes and macrophages at the site of tumor challenge, strengthened the importance of the immune system in achieving complete tumor rejection.
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PMID:Different efficacy of in vivo herpes simplex virus thymidine kinase gene transduction and ganciclovir treatment on the inhibition of tumor growth of murine and human melanoma cells and rat glioblastoma cells. 1041 54

Multimodal therapy is generally more effective than single-agent treatment for cancer. rRp450 is an engineered herpes simplex viral mutant that replicates in and kills tumor cells in a relatively selective fashion. It also expresses, in infected cells, the cyclophosphamide (CPA)-sensitive rat cytochrome P450 2B1 (CYP2B1) and the ganciclovir (GCV)-sensitive herpes simplex virus thymidine kinase (HSV-TK) transgenes. We show that cultured rat 9L and human U87deltaEGFR glioma cells, infected and lysed by rRp450, also exhibit supra-additive sensitivity to both CPA and GCV, as determined by Chou-Talalay synergy analysis. DNA cross-linking, assayed by ethidium bromide fluorescence, was significantly inhibited in the presence of GCV, suggesting that interactions between the CPA/CYP2B1 and GCV/HSV-TK gene therapies occurred at the level of DNA repair. In vivo, regression of 9L s.c. tumor volumes in athymic mice was achieved only by the multimodal treatment allowed by rRp450 viral oncolysis combined with CPA/CYP2B1 and GCV/HSV-TK gene therapies, whereas all other treatment combinations produced only tumor growth retardation.
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PMID:Multimodal cancer treatment mediated by a replicating oncolytic virus that delivers the oxazaphosphorine/rat cytochrome P450 2B1 and ganciclovir/herpes simplex virus thymidine kinase gene therapies. 1046 70

With the advent of gene therapy, herpes simplex virus type I (HSV-1) thymidine kinase (TK) has garnered much interest as a suicide gene for cancer ablation. As a means to improve the overall efficacy of the prodrug-gene activation approach, as well as to reduce ganciclovir-mediated toxicity, a large library of mutant thymidine kinases was generated and screened for the ability to enhance in vitro cell sensitivity to the prodrugs, ganciclovir (GCV) and acyclovir (ACV). Enzyme kinetics of one thymidine kinase mutant from this library that contains six amino acid substitutions at or near the active site reveals a distinct mechanism for providing enhanced prodrug-mediated killing in mammalian cells. In in vitro rat C6 cell prodrug sensitivity assays the TK mutant (mutant 30) achieves nanomolar IC50 values with GCV and ACV, in contrast to IC50values of 30 microM and >100 microM, respectively, for wild-type TK. In a mouse xenograft tumor model, growth of mutant 30 expressing tumors is restricted by ganciclovir at a dose at least 10- fold lower than one that impedes growth of wild-type TK-expressing tumors. Furthermore, in the presence of GCV a substantial bystander effect is observable when only 20% of the tumor cells express mutant 30 whereas no restriction in tumor growth is seen in tumors bearing the wild-type TK under the same conditions. The enhanced sensitization to prodrugs conferred by mutant 30 is apparently due to a 35-fold increase in thymidine Km which results in reduced competition between prodrug and thymidine at the active site. This provides mutant 30 a substantial kinetic advantage despite very high Kms for both ganciclovir and acyclovir. Molecular modeling of the mutations within the active site suggests that a tyrosine substitution at alanine 168 (A168) alters thymidine and prodrug interactions by causing catalytically important residues to move. The use of mutant 30 in place of the wild-type TK should provide a more effective gene therapy of cancer.
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PMID:Enhancement of tumor ablation by a selected HSV-1 thymidine kinase mutant. 1046 66

The therapeutic effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system on hepatocellular carcinoma was studied in this experiment. The tk-containing retroviral recombinants were used to infect hepatoma cells (BEL-7402) and the cells were treated with ganciclovir (0-1000 microg/ml). The results showed that HSV-tk gene could be efficiently transferred in vitro into hepatoma cells and stably expressed. The growth potential of the tk-containing cells was significantly inhibited by GCV (P<0.01) as compared to the non-tk-containing cells. The antitumor effect of HSV-tk/GCV system was also produced ex vivo in tk-containing tumor of nude mice as characterized by a marked decrease in tumor growth after GCV treatment contrary to a progressive enlargement of non-tk-containing tumors. Although the histological examination demonstrated that the efficiency of the gene transfer was less than 30%, the killing effect of HSV-tk/GCV system on hepatocellular carcinoma was still significantly generated. The proper mechanism of HSV-tk gene therapy on hepatic tumor referred as "bystander effect" in therapeutic approach has not been found in this study and required to be explored further.
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PMID:Retrovirus-mediated herpes simplex virus thymidine kinase gene therapy approach for hepatocellular carcinoma. 1052 Jun 5

We report successful electro-gene therapy (EGT) by using plasmid DNA for tumor-bearing mice. Subcutaneously inoculated CT26 tumor was subjected to EGT, which consists of intratumoral injection of a naked plasmid encoding a marker gene or a therapeutic gene, followed by in vivo electroporation (EP). When this treatment modality is carried out with the plasmid DNA for the green fluorescent protein gene, followed by in vivo EP with the optimized pulse parameters, numerous intensely bright green fluorescent signals appeared within the tumor. EGT, by using the "A" fragment of the diphtheria toxin gene significantly inhibited the growth of tumors, by about 30%, on the flank of mice. With the herpes simplex virus thymidine kinase gene, followed by systemic injection of ganciclovir, EGT was far more effective in retarding tumor growth, varying between 50% and 90%, compared with the other controls. Based on these results, it appears that EGT can be used successfully for treating murine solid tumors.
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PMID:Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene. 1061 22


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