bromodeoxycytidine and ibacitabine

The efficacy of 3% Ara-A ophthalmic ointment (Vira A) has been evaluated on 100 epithelial herpetic keratitis; the poor intra-ocular penetration of Ara-A explains the exclusion of stromal keratitis and kerato-unveitis. Patients were treated 5 times a day until complete epithelial healing of ulcers, then twice a day during 7 days. Healing was obtained within 10.6 days for 87% of the patients, who have been treated by Ara-A at first (n = 77) or after failure of IDU or of IDC (n = 23). The healing rate was higher for the 52 first ocular episodes (92%) than for the 48 recurrences (81%); it decreases to 77% for recurrences after failure of IDU or IDC. Geographic ulcers heal in 76% of cases only. Their length has no influence on their healing. The longest healing time, 10.6 days, can be explained by the long period of time before beginning to apply Ara-A, 12.8 days: significative correlation between both periods of time is highlighted and shows the advantage of an early treatment. The need for a local corticotherapy (n = 8) does not hinder healing in 15.5 days. Two weeks after discontinuation of the treatment, 3 patients presented a relapse, sensitive to a 2nd Ara-A course; a maintenance treatment, superior to 7 days, is necessary. Tolerance to Vira A ointment is good. Indications of Ara-A during ocular herpes are superficial keratitis, especially those resistant to IDU or, from experimental data, to ACV, and their prevention by a possible long term treatment.

Topics: Adult; Antiviral Agents; Bromodeoxycytidine; Corneal Ulcer; Deoxycytidine; Drug Resistance; Female; Humans; Idoxuridine; Keratitis, Dendritic; Male; Middle Aged; Ointments; Recurrence; Vidarabine

Topics: Acyclovir; Adolescent; Adult; Aged; Bromodeoxycytidine; Child; Clinical Trials as Topic; Deoxycytidine; Double-Blind Method; Female; Humans; Keratitis, Dendritic; Male; Middle Aged

The mammalian central nervous system (CNS) undergoes significant expansion postnatally, producing astrocytes, oligodendrocytes and inhibitory neurons to modulate the activity of neural circuits. This is coincident in humans with the emergence of pediatric epilepsy, a condition commonly treated with valproate/valproic acid (VPA), a potent inhibitor of histone deacetylases (HDACs). The sequential activity of specific HDACs, however, may be essential for the differentiation of distinct subpopulations of neurons and glia. Here, we show that different subsets of CNS neural stem cells (NSCs) and progenitors switch expression of HDAC1 and HDAC2 as they commit to a neurogenic lineage in the subventricular zone (SVZ) and dentate gyrus (DG). The administration of VPA for only one week from P7-P14, combined with sequential injections of thymidine analogs reveals that VPA stimulates a significant and differential decrease in the production and differentiation of progeny of NSCs in the DG, rostral migratory stream (RMS), and olfactory bulb (OB). Cross-fostering VPA-treated mice revealed, however, that a postnatal failure to thrive induced by VPA treatment had a greater effect on DG neurogenesis than VPA action directly. By one month after VPA, OB interneuron genesis was significantly and differentially reduced in both periglomerular and granule neurons. Using neurosphere assays to test if VPA directly regulates NSC activity, we found that short term treatment with VPA in vivo reduced neurosphere numbers and size, a phenotype that was also obtained in neurospheres from control mice treated with VPA and an alternative HDAC inhibitor, Trichostatin A (TSA) at 0 and 3 days in vitro (DIV). Collectively, these data show that clinically used HDAC inhibitors like VPA and TSA can perturb postnatal neurogenesis; and their use should be carefully considered, especially in individuals whose brains are actively undergoing key postnatal time windows of development.

Topics: Age Factors; Animals; Animals, Newborn; Brain; Bromodeoxycytidine; Cell Differentiation; Cells, Cultured; Deoxycytidine; Deoxyuridine; Dose-Response Relationship, Drug; Gene Expression Regulation, Developmental; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Mice; Nerve Tissue Proteins; Neural Stem Cells; Neurogenesis; Proliferating Cell Nuclear Antigen; SOXB1 Transcription Factors; Valproic Acid

This study evaluated a radioiodinated deoxycytidine analog, (131)I-5-iodo-2'-deoxycytidine ([(131)I]ICdR), as a novel proliferation probe and compared it with (131)I-5-iodo-2'-deoxyuridine ([(131)I]IUdR) in a NG4TL4 sarcoma-bearing mouse model. As an imaging agent, the biological characteristics of [(123)I]IUdR is not satisfactory due to its metabolic instability and short biological half-life in vivo. With [(123)I]ICdR/SPECT it was possible to clearly delineate the tumor lesion at 1h post-injection (tumor-to-muscle ratio 7.74) in tumor-bearing mice. The results of biodistribution were consistent with those observed in scintigraphic imaging. This study demonstrated that [(131)I]ICdR is a more promising SPECT probe than [(131)I]IUdR for imaging proliferation.

Topics: Animals; Bromodeoxycytidine; Cell Line, Tumor; Deoxycytidine; Deoxyuridine; Female; Iodine Radioisotopes; Isotope Labeling; Metabolic Clearance Rate; Mice; Molecular Probe Techniques; Organ Specificity; Radionuclide Imaging; Radiopharmaceuticals; Sarcoma; Tissue Distribution

This study aims to demonstrate that 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) positron emission tomography (PET) is a promising modality for noninvasively monitoring the therapeutic efficacy of Doxisome(®) in a subcutaneous hepatoma mouse model.. Male BALB/c nu/nu mice were inoculated with HepG2 hepatoma xenograft in the right flank. Doxisome(®) (5 mg/kg, three times a week for 2 weeks) was intravenously administrated for treatment. (18)F-FLT-microPET, biodistribution studies, and immunohistochemistry of Ki-67 were performed.. A significant difference (p < 0.05) in tumor volume was observed on day 5 between treated and control groups. The tumor-to-muscle ratio derived from (18)F-FLT-PET and (123)I-ICdR-microSPECT images of Doxisome(®)-treated mice dropped from 12.55 ± 0.76 to 3.81 ± 0.31 and from 2.48 ± 0.42 to 1.59 ± 0.08 after a three-dose treatment, respectively, while that of the control group remained steady. The retarded proliferation rate of treated xenograft was confirmed by Ki-67 immunohistochemistry staining.. This study clearly demonstrated that Doxisome(®) is an effective anti-cancer drug against the growth of HepG2 hepatoma and that (18)F-FLT-PET could provide early information of tumor response during treatment.

Topics: Animals; Bromodeoxycytidine; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Death; Deoxycytidine; Dideoxynucleosides; Disease Models, Animal; Doxorubicin; Endocytosis; Hep G2 Cells; Humans; Immunohistochemistry; Ki-67 Antigen; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Multimodal Imaging; Polyethylene Glycols; Positron-Emission Tomography; Tissue Distribution; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; Treatment Outcome; Xenograft Model Antitumor Assays

Suicide gene therapy of malignant melanoma essentially requires efficient gene transfer and highly selective therapeutic gene expression. To achieve this, recombinant adeno-associated virus (rAAV) particles were constructed containing the tissue-specific promoter of the human melanoma inhibitory activity (hMIA) gene combined with four copies of the enhancer element of the murine tyrosinase gene. Three melanoma and one cervix carcinoma cell line were infected with rAAV particles carrying a reporter gene under control of the enhancer/hMIA promoter in order to determine transcriptional activity and specificity of this system. Viral particles containing the enhancer/hMIA promoter mediated reporter gene activity only in melanoma cells, whereas infection with a cytomegalovirus (CMV)-based promoter construct induced unspecific gene expression. Correspondingly, transient transduction with viral particles bearing the HSVtk gene under the control of the enhancer/MIA promoter elements followed by treatment with ganciclovir (GCV) resulted in growth inhibition only in melanoma cells, whereas the CMV promoter-based construct induced unspecific cytotoxicity. In vivo experiments in nude mice demonstrated that tumors originating from human melanoma cells disappeared after stable, but not transient transduction with vectors bearing the HSVtk gene under the control of the enhancer/hMIA promoter in response to GCV application. In face of higher transduction efficiency, these rAAV particles might therefore be a useful tool for suicide gene therapy of malignant melanoma.

Topics: Animals; Antiviral Agents; Bromodeoxycytidine; Cell Line, Tumor; Cell Separation; Cloning, Molecular; Deoxycytidine; Dependovirus; Enhancer Elements, Genetic; Extracellular Matrix Proteins; Female; Flow Cytometry; Ganciclovir; Gene Transfer Techniques; Genes, Reporter; Genetic Therapy; Humans; Immunosuppressive Agents; Melanoma; Mice; Mice, Nude; Models, Genetic; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Transplantation; Plasmids; Promoter Regions, Genetic; Proteins; Simplexvirus; Thymidine Kinase; Time Factors; Tissue Distribution

DNA fragments containing a sequence d(GCGAAAGC) are known to be highly thermostable. To investigate the structural basis for such a specific property, crystallographic studies of the DNA octamer and a nanomer d(GXGAAAGCT) (X = 2'-deoxy-5-iodocytidine) have been performed. The present higher resolution X-ray analyses have shown that both DNA oligomers are stabilized respectively to form a zipper-type duplex homodimer. PAGE of these oligomers, however, indicates that they are monomeric even when their crystals were dissolved at room temperature.

Topics: Base Sequence; Bromodeoxycytidine; Crystallography, X-Ray; Deoxycytidine; Dimerization; DNA; Models, Molecular; Nucleic Acid Conformation

Telomerase is a ribonucleoprotein enzyme that adds telomeric sequence repeats to the ends of linear chromosomes. In vitro, telomerase has been observed to add repeats to a DNA oligonucleotide primer in a processive manner, leading to the postulation of a DNA anchor site separate from the catalytic site of the enzyme. We have substituted photoreactive 5-iododeoxypyrimidines into the DNA oligonucleotide primer d(T4G4T4G4T4G2) and, upon irradiation, obtained cross-links with the anchor site of telomerase from Euplotes aediculatus nuclear extract. No cross-linking occurred with a primer having the same 5' end and a nontelomeric 3' end. These cross-links were shown to be between the DNA primer and (i) a protein moiety of approximately 130 kDa and (ii) U51-U52 of the telomerase RNA. The cross-linked primer could be extended by telomerase in the presence of [alpha-32P]dGTP, thus indicating that the 3' end was bound in the enzyme active site. The locations of the cross-links within the single-stranded primers were 20 to 22 nucleotides upstream of the 3' end, providing a measure of the length of DNA required to span the telomerase active and anchor sites. When the single-stranded primers are aligned with the G-rich strand of a Euplotes telomere, the cross-linked nucleotides correspond to the duplex region. Consistent with this finding, a cross-link to telomerase was obtained by substitution of 5-iododeoxycytidine into the CA strand of the duplex region of telomere analogs. We conclude that the anchor site in the approximately 130-kDa protein can bind duplex as well as single-stranded DNA, which may be critical for its function at chromosome ends. Quantitation of the processivity with single-stranded DNA primers and double-stranded primers with 3' tails showed that only 60% of the primer remains bound after each repeat addition.

Topics: Animals; Binding Sites; Bromodeoxycytidine; Cross-Linking Reagents; Deoxycytidine; DNA; DNA Primers; Euplotes; Idoxuridine; Models, Genetic; Molecular Weight; Nucleic Acid Conformation; RNA; Telomerase; Ultraviolet Rays

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

We have generated a chimeric gene transfer vector that combines the simplicity of plasmids with the infectivity and long-term expression of retroviruses. We replaced the env gene of a Moloney murine leukemia virus-derived provirus by a foreign gene, generating a plasmid that upon transfer to tumor cells generates noninfectious retroviral particles carrying the transgene. We added to this plasmid an independent expression cassette comprising a cytomegalovirus promoter, an amphotropic retroviral envelope, and a polyadenylylation signal from simian virus 40. These constructs were designed to minimize the risk of recombination generating replication-competent retroviruses. Their only region of homology is a 157-bp sequence with 53% identity. We show that the sole transfection of this plasmid in various cell lines generates infectious but defective retroviral particles capable of efficiently infecting and expressing the transgene. The formation of infectious particles allows the transgene propagation in vitro. Eight days after transfection in vitro, the proportion of cells expressing the transgene is increased by 10-60 times. There was no evidence of replication-competent retrovirus generation in these experiments. The intratumoral injection of this plasmid, but not of the control vector lacking the env gene, led to foci of transgene-expressing cells, suggesting that the transgene had propagated in situ. Altogether, these "plasmoviruses" combine advantages of viral and non-viral vectors. They should be easy to produce in large quantity as clinical grade materials and should allow efficient and safe in situ targeting of tumor cells.

Topics: 3T3 Cells; Animals; Antiviral Agents; beta-Galactosidase; Bromodeoxycytidine; Cell Line; Cell Transformation, Neoplastic; Deoxycytidine; DNA Replication; Ganciclovir; Genes, env; Genetic Therapy; Genetic Vectors; Male; Mice; Mice, Inbred BALB C; Moloney murine leukemia virus; Neoplasms, Experimental; Plasmids; Proviruses; Recombinant Proteins; Retroviridae; Simplexvirus; Thymidine Kinase; Transfection; Virus Replication

In experiments using yeast, without addition of an external metabolic activation system, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was co-mutagenic and showed an insignificant anti-recombinogenic effect in combination with triethylene melamine (TEM). In the presence of activating S9-mix, the anti-recombinogenicity and co-mutagenicity could clearly be seen. At higher concentrations the co-mutagenic effect was converted into anti-mutagenicity. The other three 5-substituted pyrimidine nucleoside analogs were tested only in the presence of activating S9-mix and showed similar effects. As TEM is a direct alkylating agent that is inactivated by liver microsomes, the higher activity in presence of S9-mix can be interpreted as resulting from metabolic activation of the 5-substituted pyrimidine nucleoside analogs. In previous experiments using yeast bacteria, Drosophila or mice, tumor promoters were co-recombinogenic/anti-mutagenic, and co-carcinogens were co-mutagenic/anti-recombinogenic. Thus, there is not only an operational difference between tumor promoters and co-carcinogens but a real difference in respect to their genetic effectiveness. As up to now only co-carcinogens have shown co-mutagenic and anti-recombinogenic effects, it is perhaps possible that, within a certain concentration range, 5-substituted pyrimidine nucleoside analogs may have co-carcinogenic activity in carcinogenicity tests. At higher concentrations the co-carcinogenic effect may be converted into an anti-carcinogenic one.

Topics: Animals; Bromodeoxycytidine; Bromodeoxyuridine; Deoxycytidine; Dose-Response Relationship, Drug; Genes, Fungal; Heterozygote; Idoxuridine; Liver; Male; Mutagens; Mutation; Rats; Recombination, Genetic; Saccharomyces cerevisiae; Triethylenemelamine; Trifluridine

In this work, spermhead survival in mouse testis was used to investigate the radiotoxicity of several intratesticularly localized radioiodinated pharmaceuticals. Radioiodines that decay by electron capture and/or internal conversion (123I, 125I) as well as by beta- decay (131I) were coupled to pharmaceuticals that selectively localize in different cell compartments. Dose response curves yield D37 values of 62 cGy, 75 cGy, 61 cGy and 7.7 cGy for 123IMP (N-isopropyl-p-iodoamphetamine), 131IdU (iododeoxyuridine), H131IPDM (N,N,N'-trimethyl-N'-(2-hydroxyl-3-methyl-5-iodobenzyl)-1,3-propanediami ne) and 125IdC (iododeoxycytidine), respectively. At 37% survival, the relative biological effectiveness (RBE) of these radiochemicals, when compared to the pure gamma-emitting radiochemical 7Be-chloride (D37 = 65 cGy), are 1.0, 0.89, 1.1 and 8.4, respectively. Intratesticular 7Be, with an effective half-life of 430 hr in the organ, was used as the source of reference radiation to determine the RBE values because it solely emits 477 keV gamma rays, and the dose to the testis is delivered chronically, as in the case of the other radiocompounds. Subcellular distribution studies show that all of the cellular activity is localized in the cytoplasm in the cases of 123IMP and H131IPDM, while virtually all of 131IdU and 125IdC were bound to DNA in the cell nucleus. In agreement with our earlier in vivo studies, these data show that subcellular distribution plays a key role in the radiotoxicity of Auger electron emitters such as 123I and 125I, and has no role for beta emitters such as 131I. These findings may have implications in the design of radiopharmaceuticals for both diagnosis (localize Auger emitter in cytoplasm of cell) and therapy (localize Auger emitter in cell nucleus).

Topics: Amphetamines; Animals; Bromodeoxycytidine; Cell Survival; Deoxycytidine; Dose-Response Relationship, Radiation; Drug Design; Idoxuridine; Injections; Iodine Radioisotopes; Iodobenzenes; Iofetamine; Male; Mice; Sperm Head; Subcellular Fractions; Testis

The problem of determining RBE values for Auger emitters incorporated into proliferating mammalian cells is examined. In general, the reference radiation plays a key role in obtaining experimental RBE values. Using survival of cultured Chinese hamster V79 cells as the experimental model, new data are provided regarding selection of a reference radiation for internal Auger emitters. These data show that gamma rays delivered acutely (137Cs) are more than twice as lethal as gamma rays delivered chronically with an exponentially decreasing dose rate (99mTc). The results confirm that the reference radiation should be delivered chronically in a manner consistent with the extended exposure received by the cells in the case of incorporated radionuclides. Through a direct comparison of the radiotoxicity of Auger emitters and alpha emitters, the high RBE values reported for DNA-bound Auger emitters are confirmed. These studies reveal that the DNA binding compound [125I]iododeoxyuridine (125IdU) is about 1.6 times more effective in killing V79 cells than 5.3 MeV alpha particles from intracellularly localized 210Po-citrate. In addition, toxicity studies with the radiochemicals 125IdU and [125]-iododeoxycytidine (125IdC) establish the equivalence of the radiosensitivity of thymine and cytosine base sites in the DNA. In view of these results, and information already available, the question of establishing quality factors for Auger emitters is considered. Finally, a method for calculation of the dose equivalent for internal Auger emitters is advanced.

Topics: Animals; Bromodeoxycytidine; Cell Division; Cell Line; Cell Survival; Cesium Radioisotopes; Cricetinae; Cricetulus; Deoxycytidine; Electrons; Gamma Rays; Idoxuridine; Iodine Radioisotopes; Polonium; Radioisotopes; Relative Biological Effectiveness; Technetium

A new assay method for the measurement of thymidine kinase (TK) is described. Cytosols were prepared from TK- and TK+ cells and evaluated for TK activity using an assay which is based on the phosphorylation of [125I]-iododeoxyuridine, [125I]-iododeoxycytidine, or [3H]thymidine and the precipitation of the monophosphates of these nucleosides by lanthanum chloride. The specificity, reproducibility, sensitivity, and convenience of this assay are demonstrated.

Topics: Bromodeoxycytidine; Cells, Cultured; Chemical Precipitation; Cytosol; Deoxycytidine; Idoxuridine; Lanthanum; Nucleotides; Thymidine; Thymidine Kinase

Topics: Adult; Antiviral Agents; Bromodeoxycytidine; Cross Reactions; Deoxycytidine; Drug Eruptions; Drug Hypersensitivity; Female; Humans; Idoxuridine; Male; Patch Tests

Two Mannich-base prodrugs of 5-iodo-2'-deoxycytidine (5-IDC) have been synthesized. The prodrugs exhibit increased lipid solubility compared to 5-IDC and rapidly revert to 5-IDC in buffer. One of the prodrugs delivered about twice as much 5-IDC from isopropyl myristate (IPM) through hairless mouse skin in diffusion-cell experiments as did 5-IDC from IPM. Subsequent applications of theophylline/propylene glycol onto the diffusion cells to determine the effect of prodrug/IPM, 5-IDC/IPM, or IPM on the resistance of the skins to subsequent applications showed that the prodrug/IPM had no more effect than IPM itself.

Topics: Administration, Topical; Amines; Animals; Antiviral Agents; Bromodeoxycytidine; Deoxycytidine; Diffusion; Half-Life; Mannich Bases; Mice; Mice, Hairless; Prodrugs; Solubility

Carbocyclic analogues of 5-halocytosine nucleosides were prepared by direct halogenation of the carbocyclic analogues of cytidine, 2'-deoxycytidine, 3'-deoxycytidine, or ara-C. The 5-chloro and 5-bromo derivatives of the cytidine (carbodine) and of the 2'-deoxycytidine analogues and the 5-iodo derivatives of all four of the cytosine nucleoside analogues were prepared. All of the C-5-halocytosine nucleosides, as well as the parent C-cytosine nucleosides, were tested against a strain of herpes simplex virus type 1 (HSV-1) that induces thymidine kinase in host cells. Carbodine, 5-bromocarbodine, C-2'-deoxycytidine, C-5-bromo-2'-deoxycytidine, the four C-5-iodocytosine nucleosides, and C-ara-C inhibited replication of this strain of HSV-1 in cultured cells. Most of these compounds were tested also against the type 2 virus (HSV-2) in vitro and were active. The greatest activity observed was exerted by C-5-iodo-2'-deoxycytidine in inhibiting replication of HSV-1 in L929 cells. In tests against these DNA viruses, carbodine, a ribofuranoside analogue that had been shown previously to be highly active against human influenza A virus in vitro, was the most active compound against HSV-2 and one of the most active compounds against HSV-1 in Vero cells. 5-Bromocarbodine was active against influenza virus, but it was less active than carbodine.

Topics: Bromodeoxycytidine; Chemical Phenomena; Chemistry; Cytarabine; Cytidine; Deoxycytidine; Halogens; Simplexvirus; Virus Replication

A novel radioimmunoassay of 5MedCyd is described. The assay, employing a highly specific antiserum raised in rabbits against BSA-conjugated 5MeCyd, used 5-125iodo-2'-deoxycytidine as the tracer. The measuring range for the assay was found to be 1-1000 pmol per assay of 5MedCyd. When the methods were applied to the measurement of methylation in DNA samples a good correlation between the results obtained with the radioimmunoassay and HPLC was demonstrated. The method has several advantages over the more laborious and sophisticated techniques previously available: high sensitivity, large assay range, rapidity and potential for large number of simultaneous assays, simplicity, and low cost provided that the laboratory has equipment for gamma counting.

Topics: 5-Methylcytosine; Animals; Antibody Specificity; Bromodeoxycytidine; Cattle; Chromatography, High Pressure Liquid; Cross Reactions; Cytosine; Deoxycytidine; DNA; Fishes; Methylation; Rabbits; Radioimmunoassay

A rapid screening test for resistance to acyclovir, mediated by a lack of thymidine kinase (TK) activity in herpes simplex virus (HSV), was developed by utilizing the uptake of [125I]iododeoxycytidine (IdC) by infected Vero cells. Cells infected with TK+ virus demonstrate uptake of IdC within 3 hr of infection. The assay can detect as few as 690 pfu of virus. Cells infected with TK virus have an uptake of IdC similar to that of uninfected cells, whereas cells infected with TK+ generally have an uptake greater than 10 times that of uninfected cells if tested when obvious viral cytopathic effect is present. All 19 clinical HSV isolates tested were correctly identified as TK+. Of 17 blinded HSV isolates tested, all six TK- isolates were correctly identified. A single strain with an ID50 of 3.4 micrograms/ml and an altered TK substrate specificity was incorrectly classified as TK+. The assay is a useful, rapid screening test for viral TK activity.

Topics: Acyclovir; Animals; Bromodeoxycytidine; Cell Line; Chlorocebus aethiops; Deoxycytidine; Drug Resistance, Microbial; Herpes Simplex; Humans; Kidney; Microbial Sensitivity Tests; Simplexvirus; Thymidine Kinase

A plaque autoradiography assay to detect and quantitate thymidine kinase (TK) mutants of herpes simplex virus type 1 (HSV-1) and HSV-2 in clinical samples is described. This method utilizes the selective incorporation of [125I]iododeoxycytidine, a pyrimidine analog selectively phosphorylated by the HSV TK. Only cells infected with TK-competent virus will efficiently incorporate iododeoxycytidine and are the only cells detected by autoradiography. Furthermore, this assay discriminates between TK+ virus (TK competent) and TKA virus (TK altered or reduced). This ability to differentiate TK+ from TKA virus is enhanced when infected cells are labeled with [14C]thymidine in tandem with iododeoxycytidine labeling. Reconstruction experiments with mixtures of TK+ (HSV-1 Patton) virus and TK-deficient (TK-) (B2006) or TKA (IUDRr) mutants were performed to determine the limits of detection of this technique. Ten percent TK- or TKA virus was the lower limit for the detection of TK mutants in a mixed population, whereas 1 in 1,000 TK+ virus revertants could be detected in a TK- virus population. In reconstructed populations and 45 clinical samples, a good correlation existed between the increase in 50% inhibitory dose for acyclovir and the percent TK mutant virus present. Similarly, the results of this technique correlated well with the acyclovir phosphorylating activity of extracts from cells infected with isolates or reconstructed mixtures. Plaque autoradiography with [125I]iododeoxycytidine was able to distinguish mixed populations of TK+ and TK- virus and homogeneous populations of TKA virus. The tandem use of [125I]iododeoxycytidine and [14C]thymidine readily identified TKA virus, which appeared as TK+ virus when labeled with [14C]thymidine alone. This technique provides a sensitive screen for antiviral resistance due to alterations in the viral TK and can be used to analyze clinical samples.

Topics: Acyclovir; Autoradiography; Bromodeoxycytidine; Deoxycytidine; Herpes Simplex; Humans; Mutation; Simplexvirus; Thymidine; Thymidine Kinase; Viral Plaque Assay

Topics: Acyclovir; Amantadine; Antiviral Agents; Biguanides; Bromodeoxycytidine; Cytarabine; Deoxycytidine; Humans; Idoxuridine; Methisazone; Morpholines; Trifluridine; Vidarabine

Doubly labeled [U-14C, 5-125I]iododeoxycytidine (IdC) triphosphate was synthesized and incorporated enzymatically into defined positions of the plasmid pBR322. After storage under various conditions, the stable end products were analyzed using radio-GC, radio-HPLC, and electron microscopy. In addition, solutions of 14C-IdC-labeled DNA containing Na125I as an internal radiation source were studied to investigate the influence of internal radiolysis. Transmutation of the covalently bound 125I leads to complete destruction of the labeled nucleotide, giving rise to 14CO2 and 14CO as major products. Fragmentation of the pyrimidine base is independent of solvent and DNA configuration. Internal radiolysis caused by Na125I leads to only minor damage. Electron microscopy studies reveal that decay-induced double strand breaks (dsb) occur both at the site of decay and in areas as far as hundreds of base pairs apart from that site. Number and distribution of the breaks is strongly dependent on solvent and DNA configuration. A direct correlation exists between the extent of fragmentation of the nucleotide and the mean number of dsb.

Topics: Bromodeoxycytidine; Deoxycytidine; DNA; Iodine Radioisotopes; Microscopy, Electron; Plasmids; Radiation Genetics; Radioactivity

The study of the permeability of the cornea to 5-iodo-2'-deoxycytidine (IDC), an antiherpetic agent was performed in the rabbit. In a first experiment, using 125I-IDC eye-drops and a sustained contact between the drug and the cornea, we showed that the penetration of IDC in the aqueous humor was important. In a second experiment, using a HPLC method, we studied comparative ocular penetration and metabolism of IDC and 5-iodo-2'-deoxyuridine (IDU).

Topics: Animals; Antiviral Agents; Bromodeoxycytidine; Chromatography, High Pressure Liquid; Cornea; Deoxycytidine; Herpesviridae; Idoxuridine; Male; Ophthalmic Solutions; Permeability; Rabbits; Radioisotopes

This communication describes the use of 6 different halogenated pyrimidine analogues, bromodeoxyuridine (BrdUrd), chlorodeoxyuridine (CldUrd), iododeoxyuridine (IdUrd), bromodeoxycytidine (BrdCyd), chlorodeoxycytidine (CldCyd), and iododeoxycytidine (IdCyd), to achieve sister chromatid differentiation (SCD) and evaluate sister chromatid exchange (SCE) formation in mitogen-stimulated human lymphocytes. Also included are a description of an in vivo experiment with BrdUrd, CldUrd, and IdUrd; a discussion of pyrimidine metabolism effects on SCEs; and the presentation of an update on the "conformation hypothesis" for SCE formation. This hypothesis revision includes a model that centers on the idea that the sum of the conformational alterations of the DNA polymerase-DNA template complex at replication is the controlling factor in SCE formation.

Topics: Animals; Bone Marrow; Bromodeoxycytidine; Bromodeoxyuridine; Deoxycytidine; Deoxyuridine; Humans; Idoxuridine; Lymphocytes; Mice; Models, Genetic; Pyrimidines; Rabbits; Sister Chromatid Exchange

[125I]Iododeoxycytidine incorporation was used to measure herpes virus (HSV-1) DNA synthesis following specific DNA damage. Xeroderma pigmentosum fibroblasts were less able to replicate UV-irradiated viral DNA than were normal fibroblasts, indicating the necessity for excision repair for the survival of UV-irradiated virus. Because of its rapidity and ease of quantitation, this assay had advantages over standard viral mediated assays of DNA excision repair. It was possible to monitor viral replication as a function of the cellular cell cycle. Other genetic defects which have been proposed to reflect deficiencies in DNA-repair capacity were not detected by this assay. DNA-repair inhibitors, caffeine and 3-aminobenzamide, also did not show synergistic lethal effects on the replication of damaged viral DNA.

Topics: Ataxia Telangiectasia; Bloom Syndrome; Bromodeoxycytidine; Caffeine; Cell Cycle; Cells, Cultured; Deoxycytidine; DNA Repair; Humans; Huntington Disease; Simplexvirus; Ultraviolet Rays; Virus Replication

The incorporation into DNA of 5-bromocytosine and 5-iodocytosine, derived from their respective administered deoxyribonucleoside analogs, has been demonstrated in studies with cells infected with herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and in cells transformed with the thymidine kinase gene of HSV-1. No significant incorporation of iodocytosine or iodouracil occurred in the DNA of uninfected or nontransformed cells when the deaminating enzymes were inhibited, in accord with past studies in our laboratory with 5-bromodeoxycytidine and tetrahydrouridine. When 2'-deoxytetrahydrouridine, a potent inhibitor of cytidine deaminase and dCMP deaminase, was utilized, all the counts in DNA that were derived from [(125)I]iododeoxycytidine appeared as iodocytosine in HSV-infected cells. In the absence of a deaminase inhibitor, 32 to 45% of the counts associated with DNA pyrimidines appeared as iodocytosine, and 55 to 68% appeared as iodouracil in HSV-infected cells. Substantial incorporation of iodocytosine (16%) occurred in cells transformed with the HSV thymidine kinase gene, suggesting the importance of the specificity of cellular nucleoside kinases and the activity of the deaminases in presenting unmodified bases to an undiscriminating polymerase. Incorporation into DNA of bromocytosine derived from [(3)H]bromodeoxycytidine was demonstrated in HSV-2 infected cells; very little incorporation of bromocytosine compared with bromouracil could be demonstrated in these cells in the absence of inhibition of the deaminases (19% of the total counts associated with pyrimidines with deaminase inhibition and 1.5% without). Limited studies with 5-methyl[5-(3)H]deoxycytidine indicated essentially no (or very little) incorporation of this analog as such in the DNA of HSV-1- and HSV-2-infected and -transformed cells. This suggests an exclusion or repair mechanism preventing inappropriate methylcytosine incorporation in DNA. The addition of nucleoside and deoxyribonucleoside deaminase inhibitors, which leads to the incorporation of 5-halogenated analogs of deoxycytidine into DNA as such, does not impair their antiviral activity. We infer from studies with 4-N-alkyl (ethyl and isopropyl)-substituted analogs of iododeoxycytidine that they are incorporated as such into DNA without deamination and effectively inhibit the virus at concentrations that are marginally toxic. Among the several reasons presented for the heightened potential efficacy of analogs of deoxycytidine

Topics: Animals; Antiviral Agents; Bromodeoxycytidine; Cell Transformation, Viral; Cytidine Deaminase; Deamination; Deoxycytidine; DNA, Viral; Kinetics; Simplexvirus; Structure-Activity Relationship; Thymidine Kinase; Tritium

Infection of the cornea due to herpes simplex virus continues to be a problem for ophthalmologists despite treatment of the disease with antiviral drugs. These drugs are known to produce some toxic effects and prolonged administration is sometimes necessary. Using tensile strength measurements to assess tissue repair, healing of a 5 mm perforating corneal stromal incision was measured after treatment with different antiviral drugs four times a day for eighteen postoperative days. Results suggest that 3% adenine arabinoside, 3% acycloguanosine, 1% trifluorothymidine, and 1% iododesoxycytidine ointments do not delay (p greater than 0.05) normal stromal healing.

Topics: Acyclovir; Animals; Antiviral Agents; Bromodeoxycytidine; Cicatrix; Deoxycytidine; Keratitis, Dendritic; Rabbits; Tensile Strength; Trifluridine; Vidarabine; Wound Healing

Topics: Adenosine; Bromodeoxycytidine; Cells, Cultured; Deoxycytidine; DNA, Viral; Herpes Simplex; Humans; Lesch-Nyhan Syndrome; Orotic Acid; Thioguanine; Ultraviolet Rays; Virus Replication; Xeroderma Pigmentosum

Topics: Acyclovir; Antiviral Agents; Bromodeoxycytidine; Deoxycytidine; Deoxyuracil Nucleotides; Eye Diseases; Humans; Idoxuridine; Ophthalmic Solutions; Trifluridine; Vidarabine; Virus Diseases

Topics: Bromodeoxycytidine; Cell Line; Deoxycytidine; Deoxycytidine Kinase; Herpes Simplex; Humans; Phosphorylation; Phosphotransferases

Five clinically-used antiviral drugs (3% adenine arabinoside ointment; 3% acycloguanosine ointment; 0.24% idoxuridine ointment; 1% trifluorothymidine drops) were compared with a control (petrolatum base) to determine their toxic effects on rabbit corneal epithelium after injury by iodine vapors: --Only trifluorothymidine significantly retarded healing of epithelial erosions. --Histopathologic examination after seven-day treatment showed that all five drugs, except vidarabine and to a lesser degree acycloguanosine, caused toxic changes in the regenerating epithelium.

Topics: Acyclovir; Animals; Antiviral Agents; Bromodeoxycytidine; Cornea; Deoxycytidine; Epithelium; Guanine; Idoxuridine; Rabbits; Trifluridine; Vidarabine; Wound Healing

Topics: Animals; Bromodeoxycytidine; Cattle; Deoxycytidine; DNA; Energy Transfer; Photochemistry; Polynucleotides

Topics: Animals; Bromodeoxycytidine; Deoxycytidine; Deoxyuridine; Humans; Idoxuridine; Keratitis, Dendritic; Rabbits

Topics: Adenocarcinoma; Bromodeoxycytidine; Carcinoma; Carcinoma, Squamous Cell; Deoxycytidine; Hodgkin Disease; Humans; Idoxuridine; Leukemia; Nucleosides

Topics: Aminohydrolases; Animals; Bromodeoxycytidine; Deoxycytidine; In Vitro Techniques; Kidney; Mice; Nucleoside Deaminases; Nucleosides

Topics: Antineoplastic Agents; Bromine; Bromodeoxycytidine; Bromodeoxyuridine; Deoxycytidine; Humans; Iodine Isotopes; Neoplasms; Radioisotopes; Radiometry; Radiotherapy

Topics: Antineoplastic Agents; Bromodeoxycytidine; Deoxycytidine; In Vitro Techniques; Nucleosides; Nucleotides

Topics: Animals; Bromodeoxycytidine; Deoxycytidine; DNA; Mice; Nucleosides; Rats

Topics: Bromodeoxycytidine; Deoxycytidine; Nucleosides; Nucleotides