brivudine and thymine-arabinoside

Topics: Acyclovir; Adenine; Animals; Antiviral Agents; Arabinonucleosides; Bromodeoxyuridine; Chemical Phenomena; Chemistry; Cytarabine; Deoxyuridine; Guanine; Herpesviridae Infections; Humans; Idoxuridine; Nucleosides; Ribavirin; Thymidine; Virus Diseases; Viruses

Keloid scarring is a type of fibroproliferative disease with a high recurrence rate. However, no effective treatment is currently available. Combined therapy with recombinant lentivirus‑mediated Drosophila melanogaster deoxyribonucleoside kinase (Dm‑dNK) and prodrug has been widely studied and used for cancer treatment. Due to the similarities between keloid scars and tumors, the aim of the present study was to investigate the efficacy of a Dm‑dNK/nucleoside analog system for the treatment of keloid scars. Recombinant lentivirus expression of the Dm‑dNK suicide gene was assessed. Western blotting was used to examine the protein expression of lentivirus mediated Dm‑dNK in keloid fibroblasts. Enzyme activity assays were conducted using [3H]‑labeled substrates. Furthermore, cytotoxicity and bystander effects were evaluated using MTT assays. The expression of green fluorescent protein was observed using fluorescence microscope and results indicated that there was no notable difference in lentivirus infectivity between the multiplicity of infection (MOI) of 1 and 10 in cells. Notably, western blotting revealed that Dm‑dNK was stably expressed in keloid fibroblasts and the enzymatic activity assays revealed that the enzyme was activated following introduction into the keloid fibroblasts via the lentivirus. The cytotoxicity and bystander effects of Dm‑dNK combined with cytotoxic nucleoside analogs were both observed in Dm‑dNK+ keloid fibroblasts. These results demonstrated that the lentivirus‑mediated Dm‑dNK therapy may be effective in treating keloid fibroblasts, which provides some evidence for the use of Dm‑dNK/prodrug therapy for keloid treatment in vivo in the future.

Topics: Adult; Animals; Apoptosis; Arabinonucleosides; Bromodeoxyuridine; Bystander Effect; Cell Proliferation; Combined Modality Therapy; Drosophila melanogaster; Enzyme Assays; Female; Fibroblasts; Genes, Reporter; Genetic Vectors; Green Fluorescent Proteins; Humans; Insect Proteins; Keloid; Lentivirus; Male; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Primary Cell Culture; Thymidine; Transgenes

Cellular models of mitochondrial thymidine kinase (TK2) deficiency require a reliable method to measure TK2 activity in whole cell extracts containing two interfering deoxyribonucleoside kinases, thymidine kinase 1 (TK1) and deoxycytidine kinase. We tested the value of the thymidine analog (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as a TK2-specific substrate. With extracts of OSTTK1- cells containing TK2 as the only thymidine kinase and a highly specific TK2 inhibitor we established conditions to detect the low TK2 activity commonly present in cells. With extracts of TK1-proficient osteosarcoma cells and normal human fibroblasts we showed that BVDU, but not 1-(beta-d-arabinofuranosyl)thymine (Ara-T), discriminates TK2 activity even in the presence of 100-fold excess TK1. A comparison with current procedures based on TK2 inhibition demonstrated the better performance of the new TK2 assay. When cultured human fibroblasts passed from proliferation to quiescence TK2 activity increased by 3-fold, stressing the importance of TK2 function in the absence of TK1.

Topics: Arabinonucleosides; Biological Assay; Bromodeoxyuridine; Cell Extracts; Fibroblasts; Humans; Mitochondria; Phosphorylation; Protein Kinase Inhibitors; Substrate Specificity; Thymidine; Thymidine Kinase; Tumor Cells, Cultured

Nucleoside kinases from several species are investigated as suicide genes for treatment of malignant tumors by combined gene/chemotherapy. In the present study, we have investigated a novel strategy where nucleoside kinase proteins are directly delivered to cells without delivery of genetic material. We used a mix of a trifluoroacetylated lipopolyamine and dioleoyl phosphatidylethanolamine (BioPorter) to form protein-lipid complexes containing either recombinant herpes simplex virus type-1 thymidine kinase or Drosophila melanogaster multisubstrate deoxyribonucleoside kinase. We showed that the nucleoside kinase containing protein-lipid complexes was imported into human osteosarcoma and Chinese hamster ovary cell lines by endocytosis and that the enzymes were delivered to the cytosol and nucleus. The nucleoside kinases imported into the cell lines retained enzymatic activity, and the cells treated with the enzyme-lipid complexes showed increased sensitivity to nucleoside analogues, such as ganciclovir, (E)-5-(2-bromovinyl)-2'-deoxyuridine, and 1-beta-D-arabinofuranosylthymine. Our results show that direct delivery of suicide gene proteins to cells may be an alternative approach to conventional suicide gene therapy strategies.

Topics: Animals; Arabinonucleosides; Bone Neoplasms; Bromodeoxyuridine; Cell Line, Tumor; CHO Cells; Cricetinae; Drosophila melanogaster; Endocytosis; Ganciclovir; Herpesvirus 1, Human; Humans; Liposomes; Osteosarcoma; Phosphatidylethanolamines; Phosphotransferases (Alcohol Group Acceptor); Recombinant Proteins; Thymidine; Thymidine Kinase

We have recently shown that the overexpression of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) in cancer cell lines increases the cells' sensitivity to several cytotoxic nucleoside analogs and the enzyme may accordingly be used as a suicide gene in combined gene/chemotherapy treatment of cancer. To further characterize the enzyme for possible use as a suicide gene, we constructed a replication-deficient retroviral vector that expressed either the wild-type enzyme that localizes to the cell nucleus or a mutant (arg247ser) that localizes to the cytosol. A thymidine kinase-deficient osteosarcoma cell line was transduced with the recombinant virus and we compared the sensitivity and bystander cell killing when the cell lines were incubated with the pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)-2'-deoxyuridine and 1-beta-D-arabinofuranosylthymine. In summary, we showed that the cells' sensitivity and the efficiency of bystander cell killing were not dependent on whether Dm-dNK was located in the nucleus or cytosol.

Topics: Amino Acid Sequence; Animals; Arabinonucleosides; Bromodeoxyuridine; Cell Death; Cell Nucleus; Cytosol; Drosophila melanogaster; Gene Expression; Genes, Insect; Genetic Therapy; Green Fluorescent Proteins; Humans; Luminescent Proteins; Osteosarcoma; Phosphotransferases (Alcohol Group Acceptor); Point Mutation; Recombinant Fusion Proteins; Retroviridae; Thymidine; Transduction, Genetic; Tumor Cells, Cultured

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.

Topics: Adenocarcinoma; Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antiviral Agents; Arabinonucleosides; Bromodeoxyuridine; Catalysis; Cell Division; Cell Nucleus; Cladribine; Cytarabine; Deoxycytidine; Drosophila melanogaster; Gemcitabine; Humans; Inhibitory Concentration 50; Kinetics; Osteosarcoma; Pancreatic Neoplasms; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Promoter Regions, Genetic; Retroviridae; Substrate Specificity; Thymidine; Thymidine Kinase; Transduction, Genetic; Tumor Cells, Cultured

We tested the in vitro sensitivity of Macropodid Herpesvirus 2 to eight commonly used anti-herpetic compounds using plaque reduction tests, March and April, 1995. The virus was most susceptible to inhibition by (E)-5-(2'-bromovinyl)-2'-deoxyuridine and adenine 9-beta-D-arabino-furanoside. Both compounds have been used for anti-herpetic therapy in humans and may prove useful in the treatment of macropodoids in captivity.

Topics: Acyclovir; Animals; Antiviral Agents; Arabinonucleosides; Bromodeoxycytidine; Bromodeoxyuridine; Cell Line; Cytarabine; Deoxycytidine; Herpesviridae; Herpesviridae Infections; Idoxuridine; Macropodidae; Microbial Sensitivity Tests; Neutralization Tests; Thymidine; Trifluridine; Vidarabine

Seven herpes simplex virus mutants which have been previously shown to be resistant to arabinosyladenine were examined for their sensitivities to four types of antiviral drugs. These drugs were a pyrophosphate analog, four nucleoside analogs altered in their sugar moieties, two nucleoside analogs altered in their base moieties, and one altered in both. The seven mutants exhibited five distinct phenotypes based on their sensitivities to the drugs relative to wild-type strain KOS. All mutants exhibited resistance to acyclovir and arabinosylthymine, as well as marginal resistance to iododeoxyuridine, whereas all but one exhibited resistance to phosphonoformic acid. The mutants exhibited either sensitivity or hypersensitivity to other drugs tested--2'-nor-deoxyguanosine, 5-methyl-2'-fluoroarauracil, 5-iodo-2'-fluoroarauracil, and bromovinyldeoxyuridine--some of which differed only slightly from drugs to which the mutants were resistant. These results suggest ways to detect and treat arabinosyladenine-resistant isolates in the clinic. Antiviral hypersensitivity was a common phenotype. Mutations conferring hypersensitivity to 2'-nor-deoxyguanosine in mutant PAAr5 and to bromovinyldeoxyridine in mutant tsD9 were mapped to nonoverlapping regions of 1.1 and 0.8 kilobase pairs, respectively, within the herpes simplex virus DNA polymerase locus. Thus, viral DNA polymerase mediates sensitivity to these two drugs. However, we could not confirm reports of mutations in the DNA polymerase locus conferring resistance to these two drugs. All of the mutants exhibited altered sensitivity to two or more types of drugs, suggesting that single mutations affect recognition of the base, sugar, and triphosphate moieties of nucleoside triphosphates by viral polymerase.

Topics: Acyclovir; Antiviral Agents; Arabinofuranosyluracil; Arabinonucleosides; Bromodeoxyuridine; DNA-Directed DNA Polymerase; Drug Resistance, Microbial; Foscarnet; Ganciclovir; Genes, Viral; Idoxuridine; Mutation; Phosphonoacetic Acid; Simplexvirus; Thymidine; Vidarabine

The effect of several antiviral drugs on the reactivation of herpes simplex virus type 1 in explant cultures of latently infected mouse trigeminal ganglia was investigated. Phosphonoacetate and phosphonoformate, which act directly on the virus-induced DNA polymerase, require a drug concentration of 400 micrograms/ml for the inhibition of virus reactivation in latently infected ganglia. Arabinosyladenine and arabinosyladenine monophosphate, which are phosphorylated to triphosphates by cellular enzymes and inhibit virus synthesis either by blocking the DNA polymerase or by incorporation into viral DNA, require a concentration of only 100 micrograms/ml for the inhibition of the reactivation process. Drugs that are phosphorylated by the virus-induced thymidine kinase, such as acyclovir, arabinosylthymine, bromovinyldeoxyuridine, and three fluorinated pyrimidine nucleosides require the lowest drug concentrations for complete inhibition of virus reactivation in latently infected ganglia explant cultures. Our data suggest that the inhibition of virus reactivation is dependent not only on drug concentration, but also on the number of latently infected neurons in the ganglia.

Topics: Acyclovir; Animals; Antibodies, Viral; Antiviral Agents; Arabinofuranosyluracil; Arabinonucleosides; Bromodeoxyuridine; Culture Media; Culture Techniques; Cytarabine; Foscarnet; Mice; Mice, Hairless; Neutralization Tests; Phosphonoacetic Acid; Simplexvirus; Thymidine; Trigeminal Nerve; Vidarabine Phosphate; Virus Activation

The selective and potent anti-herpesvirus drug, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU), has been examined for its inhibitory effects on several parameters of Epstein-Barr virus (EBV) infection in the lymphoblastoid cell lines Raji, P3HR-1, B-95-8 and P3 hybrid cells (a human embryo oropharyngeal cell line fused with a nasopharyngeal carcinoma cell line). At a dosage of 0.03 to 0.1 mM, BVdU caused a marked inhibition of (i) spontaneous viral capsid antigen (VCA) expression in B-95-8 and P3 hybrid cells, (ii) VCA expression and DNA synthesis in B-95-8 cells induced with croton oil and n-butyrate, (iii) early antigen (EA) expression and DNA synthesis in Raji cells superinfected with EBV, and (iv) VCA expression and DNA synthesis in B-95-8 cells superinfected with EBV. In its inhibitory effects on these various parameters of EBV infection, BVdU appears to be comparable to acyclovir [9-(2-hydroxyethoxymethyl)guanine], another selective anti-herpesvirus drug which has been previously recognized as an effective inhibitor of EBV replication.

Topics: Acyclovir; Animals; Antiviral Agents; Arabinonucleosides; Bromodeoxyuridine; Burkitt Lymphoma; Callitrichinae; Cell Line; Drug Evaluation, Preclinical; Herpesviridae Infections; Herpesvirus 4, Human; Humans; Hybrid Cells; In Vitro Techniques; Thymidine; Virus Cultivation; Virus Replication

The deoxythymidine kinase (dTK) activity of a 5-methoxymethyldeoxyuridine-resistant mutant (MMdU(r)-20) of herpes simplex virus type 1 was compared with that of the parental wild-type (WT) virus. The dTK activity induced by the mutant was consistently less than that induced by the WT virus, was inhibited by antibody specific for herpes simplex virus dTK, and was more thermostable than the WT dTK. Further, it was inhibited to a lesser degree than the WT dTK by the nucleoside analogs MMdU and arabinosylthymine (araT), which suggests that one of the effects of the mutation was a selective alteration in substrate recognition by the dTK. The loss of ability to inhibit the mutant dTK by E-(2)-5-bromovinyldeoxyuridine was not as great as that seen with araT and MMdU. This agrees well with our previous observation that the MMdU(r)-20 mutant of herpes simplex virus is only partially resistant to this analog, as compared with araT and MMdU (V. Veerisetty and G. A. Gentry, Virology 114:576-579, 1981). [2-(14)C]araT was used to explore further the resistance to araT. Extracts of cells infected with the mutant, although producing a small amount of [(14)C]araTMP, were unable to produce [(14)C]araTTP, in contrast to extracts of cells infected with the WT virus. Both extracts, however, produced [(14)C]dTTP from [(14)C]deoxyribosylthymine. Finally, the ability of the extracts to phosphorylate [(14)C]dTMP was examined. It was found that this activity was greatly reduced relative to dTK activity in the case of the mutant. These findings suggest that a mutation in the dTK polypeptide has affected recognition not only of nucleoside substrates but of the nucleotide substrate dTMP as well, which agrees with the suggestion of Chen et al. that both activities are located on the same polypeptide (M. S. Chen and W. H. Prusoff, J. Biol. Chem. 253:1325-1327, 1978; M. S. Chen, J. Walker, and W. H. Prusoff, J. Biol. Chem. 254:10747-10753, 1979; M. S. Chen, W. P. Summers, J. Walker, W. C. Summers, and W. H. Prusoff, J. Virol. 30:942-945, 1979).

Topics: Arabinonucleosides; Blood; Bromodeoxyuridine; Deoxyuridine; Drug Resistance, Microbial; Enzyme Induction; Hot Temperature; Mutation; Phosphorylation; Simplexvirus; Substrate Specificity; Thymidine; Thymidine Kinase

Three acyclovir (ACV)-resistant mutants derived from a strain of herpes simplex virus (HSV) type 1 were studied to determine the range of their resistance to nine drugs active against HSV. Two of the mutants were thymidine kinase-deficient (TK-) and were resistant to drugs that are usually phosphorylated by HSV TK. The other mutant induced normal levels of TK; it was of special interest since TK+ viruses appear more likely to multiply well in vivo. This mutant was inhibited by "TK-mediated" drugs: idoxuridine, which is already in use, and two drugs with promising clinical potential, 1-beta-arabinofuranosylthymine and E-5-(2-bromovinyl)-2'-deoxyuridine. All of the mutants were sensitive to trifluorothymidine and 9-beta-D-arabinofuranosyladenine. These results suggest that the study of cross-resistance of HSV strains in vitro will aid in the investigation of alternative drugs for use in effective chemotherapy.

Topics: Acyclovir; Antiviral Agents; Arabinonucleosides; Bromodeoxyuridine; Drug Resistance, Microbial; Foscarnet; Guanine; Microbial Sensitivity Tests; Mutation; Phosphonoacetic Acid; Simplexvirus; Thymidine; Trifluridine; Vidarabine

Topics: Acyclovir; Animals; Arabinonucleosides; Bromodeoxyuridine; Cell Line; Deoxyuridine; Drug Resistance, Microbial; Enzyme Induction; Guanine; Mice; Mutation; Phosphonoacetic Acid; Simplexvirus; Thymidine; Thymidine Kinase

The therapeutic efficacy of three new antiviral agents-5-iodo-3',5'-diacetyl-2'-deoxyuridine (diacetylidoxuridine, 1% Ac2IDU), E-5-(2-bromovinyl)-2'-deoxyuridine (0.25% BVDU), and 3% thymine arabinoside-is compared with available antivirals in an experimental model of herpes simplex virus type 1 (HSV-1) keratitis in New Zealand white rabbits. Compared with placebo, Ac2IDU significantly reduced ulcerative keratitis on days 4 through 8 after inoculation with virus and iritis on day 8 after inoculation. Compared with placebo, thymine arabinoside reduced ulcerative keratitis but not significantly. Thymine arabinoside caused significant iritis in all eyes. The epithelial disease in BVDU-treated eyes was significantly less than that in placebo-treated eyes on days 5 through 8 after inoculation. The results indicate that 1% Ac2IDU and 0.25% BVDU were effective in our ocular model of HSV-1 keratitis, whereas thymine arabinoside was not.

Topics: Animals; Antiviral Agents; Arabinonucleosides; Bromodeoxyuridine; Drug Evaluation, Preclinical; Idoxuridine; Keratitis, Dendritic; Male; Petrolatum; Rabbits; Sodium Chloride; Thymidine; Vidarabine