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)

RMP-7, a bradykinin analog, has been shown to selectively open the blood-tumor barrier for the delivery of chemotherapeutic drugs to brain tumors. In contrast to bradykinin, RMP-7 has no hypotensive effects and has been approved for human use. This study was initiated to determine whether RMP-7 would open the blood-tumor barrier to virus vectors encoding tumor-killing genes in an experimental model. The herpes virus vector used, hrR3, which encodes virus thymidine kinase gene and the lacZ reporter gene, is defective in a gene encoding ribonucleotide reductase, replicates selectively in dividing tumor cells and not in postmitotic neural cells. It was determined that an optimum dose of RMP-7 (1.5-3.0 microg/kg over 10-15 minutes) enhanced viral delivery to brain tumors in rats bearing intracranial 9 L gliosarcomas when infused through the carotid artery immediately prior to virus vector application. Maximum expression of the lacZ reporter gene occurred at 3 days after intracarotid infusion. By 8 days, transgene expression was largely confined to tumor foci away from the main tumor mass. Viral delivery was essentially specific to tumor cells, with little transgene expression elsewhere in the brain. Minimal uptake and pathology was noted in the kidney, spleen, and liver. These findings indicate that intracarotid delivery of RMP-7 can augment the selective delivery of virus vectors to brain tumors in an experimental rat model, with the potential for application to human brain tumors.
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PMID:Selective delivery of herpes virus vectors to experimental brain tumors using RMP-7. 1007 59

Continuing our studies on ribonucleotide reductase (RNR) mechanism-based inhibitors, we have now prepared the diphosphates (DP) of 2'-O-allyl-1-beta-D-arabinofuranosyl-uracil and -cytosine and 2'-O-allyl-9-beta-D-arabinofuranosyl-adenine and evaluated their inhibitory activity against recombinant murine RNR. 2'-O-Allyl-araUDP proved to be inhibitory to RNR at an IC(50) of 100 microM, whereas 2'-O-allyl-araCDP was only marginally active (IC(50) 1 mM) and 2'-O-allyl-araADP was completely inactive. The susceptibility of the parent nucleosides to phosphorylation by thymidine kinase and 2'-deoxycytidine kinase was also investigated, and all nucleosides proved to be poor substrates for the above-cited kinases. Moreover, prodrugs of 2'-O-allyl-araU and -araC monophosphates, namely 2'-O-allyl-5'-(phenylethoxy-L-alanyl phosphate)-araU and -araC, were prepared and tested against tumor cell proliferation but proved to be inactive. A molecular modeling study has been conducted in order to explain our results. The data confirm that for both the natural and analogue nucleoside diphosphates, the principal determinant interaction with the active site of RNR is with the diphosphate group, which forms strong hydrogen bonds with Glu623, Thr624, Ser625, and Thr209. Our findings indicate that the poor phosphorylation may represent an explanation for the lack of marked in vitro cytostatic activity of the test compounds.
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PMID:5'-Phosphoramidates and 5'-diphosphates of 2'-O-allyl-beta-D-arabinofuranosyluracil, -cytosine, and -adenine: inhibition of ribonucleotide reductase. 1046 11

A protein of 10,425 Da was purified from the edible mushroom Rozites caperata and shown to inhibit herpes simplex virus types 1 and 2 replication with an IC50 value of < or = 5 microM. The protein designated RC-183 also significantly reduced the severity of HSV-1 induced ocular disease in a murine model of keratitis, indicating in vivo efficacy. HSV mutants lacking ribonucleotide reductase and thymidine kinase were also inhibited, suggesting the mechanism does not involve these viral enzymes. Antiviral activity was also seen against varicella zoster virus, influenza A virus, and respiratory syncytial virus, but not against adenovirus type VI, coxsackie viruses A9 and B5, or human immunodeficiency virus. Characterization of RC-183 by mass spectroscopy, sequencing, and other methods suggests it is composed of a peptide (12 or 13 mer) coupled to ubiquitin via an isopeptide bond between the c-terminal glycine of ubiquitin and the epsilon amino group of a lysine residue in the peptide. The peptide sequence did not match any known sequence. Thus, RC-183 is a novel antiviral that may have clinical utility or serve as a lead compound for further development. Determining the mechanism of action may lead to identification of novel steps in viral replication.
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PMID:Isolation and partial characterization of an antiviral, RC-183, from the edible mushroom Rozites caperata. 1051 9

We have previously demonstrated (L. Z. Rubsam et al., Cancer Res., 59: 669-675, 1999) that low ganciclovir (GCV) triphosphate (TP) levels similar to cellular deoxynucleotide concentrations can induce multilog killing in cells stably expressing herpes simplex virus thymidine kinase (HSV-TK). In this study, we evaluated whether reducing the endogenous competitor of GCV-TP, dGTP, enhanced GCV-mediated cytotoxicity. In SW620 human colon carcinoma cells stably expressing HSV-TK, the addition of the ribonucleotide reductase inhibitor, hydroxyurea (HU), decreased cellular dGTP pools and simultaneously increased the accumulation of GCV-TP levels. The amount of GCV nucleotide transfer from HSV-TK-expressing to nonexpressing (bystander) cells was quantitated in physically separated pHook-expressing bystander cells. Elevation of the GCV-TP:dGTP ratio by HU resulted in increased levels of GCV nucleotides transferred from HSV-TK-expressing to bystander cells during a 24 h drug incubation and enhanced GCV monophosphate incorporation into DNA after drug removal. Isobologram analysis demonstrated that the combination of GCV and HU was additive in 100% HSV-TK cultures and synergistic in HSV-TK/bystander mixtures. IC50 values for GCV in 1:1 cocultures of HSV-TK-expressing and nonexpressing SW620 cells were reduced from 1.5 microM to 0.07 microM with 2 mM HU. A similar reduction was also observed with HT-29 cells and U251 cells. With 2 mM HU, IC50 values for GCV in 10:90, 5:95, and 1:99 SW620 HSV-TK-expressing and nonexpressing cocultures were reduced from 55 microM to 0.3 microM, 71 microM to 0.8 microM, and 118 microM to 7 microM, respectively. These results demonstrate the ability to pharmacologically enhance HSV-TK/GCV-mediated bystander killing and may have an important therapeutic impact.
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PMID:Synergistic enhancement of herpes simplex virus thymidine kinase/ganciclovir-mediated cytoxicity by hydroxyurea. 1074 33

Virulent alpha-herpesvirus genes, though not essential for virus replication in cell culture, play important roles in virus replication in vivo. In this paper, I classify the virulent genes and discuss the relationship between gene function and virulence. The products of the virulent genes of herpes simplex virus, described in this paper, are enzymes (thymidine kinase, ribonucleotide reductase, deoxyuridine triphosphatase, DNA polymerase, and two protein kinases), glycoproteins (gC, gE), immediate early gene product (ICP47) and gamma 34.5. To identify the virulent genes of varicellazoster virus, mutation in the Oka vaccine strain was studied. The low levels of gV expression and mutation found in the immediate early gene were predicted as the cause of the attenuation of the Oka vaccine strain.
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PMID:[The relationship between gene function and virulence in alpha-herpesviruses]. 1077 98

Herpes simplex virus type 1 (HSV-1) replication within tumors can mediate tumor regression (oncolysis). The genetically engineered, HSV-1 mutant rRp450 does not express viral ribonucleotide reductase and is therefore replication conditional. During the course of infection, rRp450 expresses the cytochrome P450 transgene and HSV-1 thymidine kinase gene, thereby enabling it to bioactivate the prodrugs cyclophosphamide and ganciclovir, respectively. rRp450 replication in hepatocellular carcinoma (HCC) cells is cytotoxic and liberates progeny virion that infect adjacent tumor cells. rRp450-mediated oncolysis is enhanced in the presence of cyclophosphamide, whereas it is inhibited in the presence of ganciclovir. As a consequence of defective viral ribonucleotide reductase expression, the yield of rRp450 progeny virions from infection of HCC cells is 3 to 4 log orders greater than that from infection of normal hepatocytes. This is associated with dramatic tumor reduction of diffuse HCC after a single intravascular administration of rRp450. rRp450 holds the promise of the dual therapeutic benefit of selective oncolysis and P450 transgene delivery.
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PMID:Oncolysis of diffuse hepatocellular carcinoma by intravascular administration of a replication-competent, genetically engineered herpesvirus. 1085 Apr 15

Cellular delivery of a replication-conditional herpes simplex virus type 1 (HSV-1) vector provides a means for gene therapy of invasive tumor cells. LacZ-bearing neural precursor cells, which can migrate and differentiate in the brain, were infected with a ribonucleotide reductase-deficient HSV-1 mutant virus (rRp450) that replicates only in dividing cells. Replication of rRp450 in neural precursor cells was blocked prior to implantation into the tumor by growth arrest in late G1 phase through treatment with mimosine. Viral titers in the medium of mimosine-treated, rRp450-infected neural precursor cells were below detection levels 3 days after infection. In culture, after removal of mimosine and passaging, cells resumed growth and replication of rRp450 so that, 7 days later, virus was present in the medium and cell death was evident. Mimosine-treated neural precursor cells injected into established intracerebral CNS-1 gliomas in nude mice migrated extensively throughout the tumor and into the surrounding parenchyma beyond the tumor over 3 days. Mimosine-treated neural precursor cells, infected with rRp450 and injected into intracerebral CNS-1 tumors, also migrated within the tumor with the appearance of foci of HSV-thymidine kinase-positive (TK+) cells, presumably including tumor cells, distributed throughout the tumor and in the surrounding parenchyma over a similar period. This migratory cell delivery method has the potential to expand the range of delivery of HSV-1 vectors to tumor cells in the brain.
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PMID:Neural precursor cells for delivery of replication-conditional HSV-1 vectors to intracerebral gliomas. 1093 53

A human tumour sub-line resistant to 5-fluorouracil (5-FU) was established by once a day and every 5, with at least 50 administrations of 5-FU to KM12C human colorectal xenografts in nude mice. KM12C tumours treated with 5-FU showed less sensitivity to 5-FU with an inhibition rate (IR) of 7.9%, while non-treated tumours were highly sensitive to 5-FU with an IR of 81.8%. To clarify the mechanism of 5-FU-resistance, the activities of various enzymes and gene expressions involved in the metabolism of 5-FU in both parental and 5-FU-treated KM12C tumours were measured. A 2- to 3-fold increase in thymidylate synthase (TS) activity and 4- to 5-fold decrease in ribonucleotide reductase (RNR) activity were observed in 5-FU-resistant KM12C tumours, while the activities of orotate phosphoribosyltransferase (OPRT) thymidine and uridine phosphorylases (TP,UP) and thymidine kinase (TK) were not markedly changed as a consequence of repeated treatment of KM12C tumours with 5-FU. The expression of TS mRNA was also amplified in accordance with the increased TS activity in a 5-FU-treated tumour sub-line (KM12C/5-FU) compared with that in parental tumours, but changed expressions of both RNR-R1 and RNA-R2 mRNA could not be detected in the 5-FU-resistant tumour sub-line compared with the parental tumours, suggesting possible post-transcriptional regulation of RNR. Moreover, RNR, in addition to TS and OPRT, seemed to be related to the inherent insensitivity to 5-FU in human cancer xenografts. From these results, it may be concluded that RNR activity is one of the acquired or inherent resistant factors, including TS, to 5-FU in human cancer xenografts in vivo.
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PMID:Thymidylate synthase (TS) and ribonucleotide reductase (RNR) may be involved in acquired resistance to 5-fluorouracil (5-FU) in human cancer xenografts in vivo. 1152 96

The effects of 2-chloro-2'-deoxyadenosine (CdA, cladribine), an adenosine deaminase-resistant analogue toxic for both proliferating and resting lymphoid cells, were investigated in the human leukemia cell line EHEB, which was derived from a patient with B-cell chronic lymphocytic leukemia. These cells were found to be less sensitive to CdA than B-cell chronic lymphocytic leukemia lymphocytes (approximately 25-fold) and other human lymphoblastic cell lines (10-1000-fold). Phosphorylation of CdA by deoxycytidine kinase and intracellular accumulation of 2-chloro-2'-deoxyadenosine triphosphate (CdATP) were similar in EHEB cells and in other CdA-sensitive cell lines. In contrast, the inhibitory effect of CdA on ribonucleotide reductase activity, which was investigated in situ by the conversion of cytidine into deoxyribonucleotides and its incorporation into DNA, was much less pronounced in EHEB cells than in other human lymphoblastic cells. Accordingly, concentrations of deoxynucleoside triphosphates did not decrease and even tended to rise. Unexpectedly, incorporation of thymidine and deoxycytidine into DNA was increased severalfold after a 24-h incubation with CdA. CdA also increased the activities of deoxycytidine kinase and thymidine kinase approximately 4-fold. Analysis of the cell cycle by flow cytometry showed that after 24 h, CdA provoked an increase in the proportion of cells in S phase, synthesizing DNA. We conclude that the EHEB cell line is resistant to the cytotoxic action of CdA not only because of a lack of inhibition of ribonucleotide reduction but also because CdA, in contrast with its known effects, provokes in this cell line an increase in the proportion of cells replicating their DNA. Unraveling of the mechanism of this effect may shed light on clinical resistance to CdA.
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PMID:Resistance to 2-chloro-2'-deoxyadenosine of the human B-cell leukemia cell line EHEB. 1170 77

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.
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PMID:Profiles of pyrimidine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers. 1224 48


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