Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nucleoside kinases from several species are investigated as "suicide genes" for treatment of malignant tumors by combined gene/chemotherapy. We have recently cloned a multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK), and we have shown that the enzyme phosphorylates cytotoxic pyrimidine and purine nucleoside analogs. The broad substrate specificity of the enzyme, as well as its very high catalytic rate, makes it a unique member of the nucleoside kinase enzyme family. In the present study, we evaluated Dm-dNK as a suicide gene by constructing a replication-deficient retroviral vector that expresses the enzyme. The human pancreatic adenocarcinoma cell line MIA PaCa-2 and a thymidine kinase-deficient osteosarcoma cell line were transduced with the recombinant virus. We showed that Dm-dNK can be expressed in human cells, that the enzyme retained its enzymatic activity, and that it is localized in the cell nuclei due to a nuclear localization signal in its C-terminal region. The cells expressing Dm-dNK exhibited increased sensitivity to several cytotoxic nucleoside analogs, such as 1-beta-d-arabinofuranosylcytosine, 1-beta-d-arabinofuranosylthymine, (E)-5-(2-bromovinyl)-2'-deoxyuridine, 2-chloro-2'-deoxyadenosine, and 2',2'-difluorodeoxycytidine. These findings suggest that Dm-dNK may be used as a suicide gene in combined gene/chemotherapy of cancer.
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PMID:Retroviral transduction of cancer cell lines with the gene encoding Drosophila melanogaster multisubstrate deoxyribonucleoside kinase. 1099 93

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) can be expressed in human cells with retained enzymatic activity. The cells expressing Dm-dNK exhibit increased sensitivity to several cytotoxic nucleoside analogs. In this study, we further evaluated Dm-dNK as a potential novel suicide gene in combination with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as the prodrug. We used two human cancer cell lines transduced with a retrovirus encoding the Dm-dNK cDNA and investigated whether the cells expressing the enzyme can induce cell death of untransduced cells, a phenomenon known as the "bystander effect". A bystander effect was observed in a thymidine kinase-deficient human osteosarcoma cell line but not in the MIA PaCa-2 human pancreatic adenocarcinoma cell line. The cytotoxicity of BVDU increased in both cell lines when the compound was used in combination with subtoxic concentrations of hydroxyurea. Hydroxyurea also enhanced the bystander effect in the osteosarcoma cells, but not in the MIA PaCa-2 cells, treated with BVDU. These findings indicate that BVDU phosphorylated by Dm-dNK in transduced cancer cells may also induce bystander cell death in certain cell lines.
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PMID:Bystander effects of cancer cell lines transduced with the multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster and synergistic enhancement by hydroxyurea. 1145 12

Pancreatic ductal adenocarcinomas (PDACs) overexpress various cell-surface tyrosine kinase receptors, including the type I high-affinity fibroblast growth factor receptor (FGFR-1). The purpose of this study was to determine whether FGFR-targeted gene therapy is feasible in this disorder. Accordingly, the effects of a conjugate consisting of fibroblast growth factor (FGF)-2 linked to a Fab' fragment against the adenovirus knob region were evaluated in human pancreatic cancer cell lines treated with an adenoviral vector containing the herpes simplex virus thymidine kinase (AdTK) gene. An adenoviral vector containing the firefly luciferase reporter gene (AdLuc) served to assess infection efficiency, and was initially tested in L6 rat myoblasts. In parental L6 cells that express exceedingly low levels of high-affinity FGFRs, transduction with AdLuc was enhanced 7- to 10-fold with the FGF2-Fab' conjugate, whereas in L6 cells transfected to express FGFR-1, it was enhanced 39- to 52-fold. The pancreatic cancer cell lines expressed variable levels of the four high-affinity FGF receptors, and exhibited 2- to 34-fold increases in gene transduction in the presence of the FGF2-Fab' conjugate. In the absence of FGF2-Fab' there was no correlation between surface binding of FGF2 and AdLuc transduction efficiency, whereas in the presence of FGF2-Fab', enhanced AdLuc transduction efficiency correlated with greater surface binding of FGF2. In the absence of AdTK, all the cell lines were insensitive to ganciclovir, whereas after AdTK transduction, only ASPC-1 and PANC-1 cells were resistant to ganciclovir even in the presence of FGF2-Fab'. Ganciclovir-mediated inhibition was dependent on the conjugate in CAPAN-1 and COLO-357 cells, but was independent of the conjugate in T3M4 and MIA-PaCa-2 cells. Real-time quantitative PCR of laser-captured cancer cells revealed high levels of various FGFR mRNA species in six of seven PDAC tumor samples. These findings indicate that transduction efficiency with FGF2-Fab' in pancreatic cancer cells is independent of native adenoviral transduction efficiency and is greatest in cells that exhibit concomitant expression of various high-affinity FGFRs. In view of the overexpression of high-affinity FGFRs in the cancer cells in PDAC, our findings also suggest that the combined use of AdTK, ganciclovir, and FGF2-Fab' may ultimately be a promising therapeutic approach in a subgroup of patients with PDAC.
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PMID:Targeting of suicide gene delivery in pancreatic cancer cells via FGF receptors. 1203 63

Selective killing of tumors can be achieved by targeting the transcription of suicide genes via specific DNA control elements to malignant cells. Three different enhancer-promoter systems were constructed and evaluated for their capability to direct gene expression to melanoma. Two tissue-specific (tyrosine and MIA) promoters and one weak viral promoter were fused to multiple tandem copies of a melanocyte-specific enhancer element. Reporter gene assays revealed a maximum increase in transcription by combining each promoter with 3-4 copies of the enhancer and demonstrated that all enhancer-promoter combinations exhibited tissue-specific activity. Though this activity was still significantly less than that of the strong but unspecific cytomegalovirus (CMV) promoter. In contrast, when those combinations were employed to drive the expression of two suicide genes, encoding the diptheria toxin A chain (DT-A) and the prodrug-activating herpes simplex virus thymidine kinase (TK), respectively, only those constructs in which transcription was under control of tissue-specific promoter elements mediated selective killing of melanoma cells. This killing was in the range of cell death induced by CMV promoter activity. Our data indicate that the enhancer/tyrosinase and enhancer/MIA promoter constructs but not the viral promoter constructs can provide a valuable tool for selective suicide gene expression in melanoma.
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PMID:Evaluation of combined gene regulatory elements for transcriptional targeting of suicide gene expression to malignant melanoma. 1471 61

The clinical efficacy of conditionally replicative oncolytic adenoviruses (CRAd) is still limited by the inefficient infection of the tumor mass. Since tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells, targeting both cell compartments may profoundly influence viral efficacy. Therefore, we developed SPARC promoter-based CRAds since the SPARC gene is expressed both in malignant cells and in tumor-associated stromal cells. These CRAds, expressing or not the Herpes Simplex thymidine kinase gene (Ad-F512 and Ad(I)-F512-TK, respectively) exerted a lytic effect on a panel of human melanoma cells expressing SPARC; but they were completely attenuated in normal cells of different origins, including fresh melanocytes, regardless of whether cells expressed or not SPARC. Interestingly, both CRAds displayed cytotoxic activity on SPARC positive-transformed human microendothelial HMEC-1 cells and WI-38 fetal fibroblasts. Both CRAds were therapeutically effective on SPARC positive-human melanoma tumors growing in nude mice but exhibited restricted efficacy in the presence of co-administered HMEC-1 or WI-38 cells. Conversely, co-administration of HMEC-1 cells enhanced the oncolytic efficacy of Ad(I)-F512-TK on SPARC-negative MIA PaCa-2 pancreatic cancer cells in vivo. Moreover, conditioned media produced by stromal cells pre-infected with the CRAds enhanced the in vitro viral oncolytic activity on pancreatic cancer cells, but not on melanoma cells. The whole data indicate that stromal cells might play an important role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses.
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PMID:Tumor associated stromal cells play a critical role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses. 1933 91