Comparison of the Selectivity of Anti-Varicella-Zoster Virus Nucleoside Analogues

We compared the selectivity of six anti-varicella-zoster virus (VZV) drugs, which are clinically available or of which clinical efficacy for the treatment of VZV infections has been reported. Sorivudine (BV-araU) had the most potent anti-VZV effect in the plaque inhibition assay, followed by brivudine (BVDU) and 5-propynyl-arabinofuranosyluracil (Pry-araU). All test compounds, except vidarabine (AraA), had only a very weak effect on human embryonic lung cell growth. The selectivity indexes (ID₅₀ for cell growth/ED₅₀ for VZV plaque inhibition) of BV-araU, BVDU, and Pry-araU were > 1,000,000, 20,000, and > 10,000, respectively, while those of acyclovir and penciclovir ranged from 600 to 800. AraA was much less selective than any of the other drugs tested. We measured the amount of pH] thymidine incorporated into the acid-insoluble fraction of VZV-infected cells to determine the ability of these drugs to selectively inhibit viral DNA synthesis. [³H]Thymidine incorporation was markedly inhibited by all anti-VZV compounds, except BVDU. Treatment of infected cells with drugs from 32 to 38 hr after infection inhibited the DNA synthesis to the same extent as VZV plaque formation, except that AraA inhibited the DNA synthesis at a lower dose than for VZV plaque formation. DNA synthesis in non-infected growing cells was inhibited to the same extent as cell growth. A particularly high selectivity index for the inhibition of DNA synthesis was noted for BV-araU, which was defined as the ratio of inhibitions of DNA synthesis in VZV-infected and non-infected. The highest selectivity indexes were recorded for BV-araU > Pry-araU > acyclovir ≥ penciclovir > AraA.     

Herpes Simplex Virus Type 2 Functions Expressed During Stimulation of Human Cell DNA Synthesis

Experiments were designed to identify herpes simplex virus type 2 (HSV-2)-specific functions expressed during stimulation of human embryo fibroblast DNA synthesis. Cultures were partially arrested in DNA synthesis by pretreatment with 5-fluorouracil and maintenance in low-serum (0.2%) medium during virus infection. Results showed that continuous [methyl-(3)H]thymidine uptake into cellular DNA was ninefold greater in HSV-2-infected than in mock-infected cultures measured after 24 h of incubation at 42 degrees C. Shifting mock-infected cultures from low- to high-serum (10%) medium also caused some stimulation, but [methyl-(3)H]thymidine uptake was only twofold greater than in cells maintained with low serum. Plating efficiencies of both HSV-2-infected and mock-infected cells at 42 degrees C were essentially the same and ranged from 37 to 76% between zero time and 72 h of incubation. De novo RNA and protein syntheses were continuously required for HSV-2 stimulation of cellular DNA synthesis. HSV-2 infection markedly enhanced transport, phosphorylation, and rate of incorporation of [methyl-(3)H]thymidine into cellular DNA, starting at 3 h and reaching a maximum by 12 h; after 12 h, these processes gradually declined to low levels. In mock-infected cells these processes remained at low levels throughout the observation period. Pretreatment of cells with interferon or addition of arabinofuranosylthymine at the time of virus infection inhibited stimulation caused by HSV-2. 5-Bromodeoxyuridine density-labeled experiments revealed that HSV-2 stimulates predominantly semiconservative DNA replication and some DNA repair. Stimulation of [methyl-(3)H]thymidine into cellular DNA correlated with detection of virus-specific thymidine kinase activity. In conclusion, HSV-2 stimulation of cellular DNA synthesis appeared to involve at least four virus-specific functions: induction of thymidine transport, HSV-2 thymidine kinase activity, semiconservative replication, and repair of cellular DNA.     

Metabolic Fate of [3H]1-β-d-Arabinofuranosyl-5-[(E)-2-bromovinyl]uracil in Herpes Simplex Virus Type 1-Infected Cells

The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [³H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [³H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [³H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [³H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [³H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P₁ digestion reduced 99% of the [³H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [³H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [³H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [³H]BV-araU is incorporated inside viral DNA strands in infected cells.     

Effect of BV-araU and Acyclovir on Varicella-Zoster Virus Replication with Various Length and Timing of Drug Exposure

We studied antiviral effects of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) and acyclovir against varicella-zoster virus (VZV) multiplication varying the length or timing of drug exposure. First, residual anti-VZV effect of drugs, exposed to cells for various periods followed by incubation in drug-free medium, was determined by the plaque inhibition assay. None of the drugs showed activity when removed within 24 hr of incubation. Weakened efficacy of BV-araU was seen in 2 days of treatment. When it was removed after 3 or 4 days, the ED₅₀ was as low as that for cultures in which the drug was not removed. Still, plaque inhibition was not complete even at high concentrations. Acyclovir inhibited plaque formation only by 50% or less in 2 days of treatment. It gave a much higher ED₅₀ in 3 days of treatment than that observed without drug removal. In the experiments, in which BV-araU was added to VZV-infected cells 1 day after infection, BV-araU immediately suppressed increase in the number of infective centers at a concentration of 0.001 μg/ml, and reduced it at concentrations of 0.01 μg/ml or higher. The reduction of infective centers was seen with a dose-dependent manner when added 2 or 3 days after infection. BV-araU stimulated the decrease in the number of infective centers when added 4 days after infection. This inhibitory effect of acyclovir was very weak. Microscopic observations supported the above results. BV-araU was still much superior to acyclovir in the anti-VZV effect when the length and timing of drug exposure were varied.     

Effect of N-trifluoroacetyladriamycin-14-valerate on [3H]thymidine uptake and DNA synthesis of human lymphoma cells

The effects of N-trifluoroacetyladriamycin-14-valerate on the uptake of [3H]thymidine and its incorporation into DNA of human P3HR-1 lymphoma cells were studied. In the absence of the drug, at 0 degrees C, [3H]thymidine was transported into the cells but not incorporated into DNA, as determined by both the trichloroacetic acid-soluble and -precipitable counts obtained with the cells. At 37 degrees C, [3H]thymidine was readily transported into the cells and incorporated into DNA. In the presence of the drug, both [3H]thymidine uptake (as shown by acid-soluble counts) and the amount of its incorporation into acid-precipitable materials were markedly reduced. However, the uptake of [3H]thymidine at 0 degrees C was found to be equally sensitive to drug inhibition as at 37 degrees C. The incorporation at 37 degrees C of [3H]thymidine into acid-precipitable materials of the cells, which had been prelabeled at 0 degrees C with [3H]thymidine, was found to be insensitive to inhibition by the drug. The in vitro activities of DNA polymerases alpha and beta purified from human P3HR-1 cells were also found not to be susceptible to inhibition. Nuclei purified from cells pretreated with the drug continued to synthesize DNA. The cytofluorograms of the cells treated with the drug indicated that the treated cells accumulated at the G2/M phase, whereas the S phase of the cells was not arrested. These results suggest that N-trifluoroacetyladriamycin-14-valerate inhibits [3H]thymidine uptake but not cellular DNA synthesis in human P3HR-1 lymphoma cells.     

Cytofluorometry of lymphocytes infected with Epstein-Barr virus: effect of phosphonoacetic acid on nucleic acid.

DNA synthesis in Epstein-Barr virus (EBV)-infected lymphocytes was inhibited by phosphonoacetic acid (PAA) as measured by [3H]thymidine incorporation. PAA, at a concentration of 200 microgram/ml, inhibited [3H]thymidine incorporation by human umbilical cord lymphocytes infected with EBV strain P94 but had little effect on DNA synthesis in mitogen-stimulated cells. Transformed cell lines did not develop from infected cord cell cultures treated with 100 microgram of PAA per ml. Cytofluorometric analysis showed marked increases in cellular nucleic acid content (RNA plus DNA) as early as 9 days after infection of cord cells in the absence of PAA and before significant enhancement of [3H]thymidine incorporation became apparent. Moreover, EBV led to increases in cellular nucleic acid even when 200 microgram of PAA per ml was added to cell cultures before infection. The apparent discrepancy between results obtained by [3H]thymidine incorporation and cytofluorometry is explained either by significant inhibition of cellular DNA polymerases by PAA or by a block at the G2 + M phase of the cell cycle. The data suggest that EBV initiates alterations in cellular nucleic acid synthesis or cell division without prior replication of viral DNA by virus-induced DNA polymerases.     

Replicatin of the resident marek''s disease virus genome in synchronized nonproducer MKT-1 cells

MKT-1, a virus nonproducer lymphoblastoid cell line established from a Marek's disease tumor, was synchronized by double thymidine block to determine the sequence of events in the synthesis of cellular and latent marek's disease virus DNA. Cellular DNA synthesis was measured by incorporation of [3H]thymidine, whereas viral DNA synthesis was determined by DNA-DNA reassociation kinetics. The results of these studies indicate that the resident Marek's disease viral DNA in MKT-1 cells replicates during the early S phase of the cell cycle, before the onset of active cellular DNA synthesis. This observation is similar to that seen in the replication of resident Epstein-Barr virus DNA in synchronized Raji cells.     

EFFECT OF PHOSPHONIC ACID ANALOGS OF ACYCLOVIR AND GANCICLOVIR ON IN VITRO CYTOMEGALOVIRUS INFECTIONS

Abstract

Eight phosphonic acid analogs of acyclovir (ACV) or ganciclovir (DHPG) inhibited human cytomegalovirus in vitro. Therapeutic indices were: phosphonate diacid of DHPG: 500; DHPG: 500; phosphonate monoethylester of DHPG: 258; phosphonate monoethylester of ACV: 94; cyclic phosphonate of DHPG: 64; ACV: 60; phosphonate monobutylester of ACV: 1.5; phosphonate monoethylester of deoxy DHPG: 4.6; 8-bromo ACV phosphonate monoethylester: >2; phosphonate monoethylester heptyl of ACV: 1. Types 1 and 2 herpesvirus (HSV-1, HSV-2) and varicella zoster virus (VZV) were poorly inhibited by these new compounds, suggesting highly specific anti-HCMV activity. None exhibited significant cytotoxic effects as measured by uptake of [³H]thymidine, [³H]uridine and [³H]leucine.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

Differential effect of arabinofuranosylthymine of the replication of human herpesviruses.

The thymidine analog 1-beta-arabinofuranosylthymine (ara-T) has previously been found to selectively inhibit herpes simplex virus replication. At a relatively nontoxic conentration (50 microgram/ml), ara-T reduced herpes simplex virus yields by 4 to 5 log10. Ara-T was also effective in inhibiting the replication of varicellazoster virus (VZV) in vitro in human embryo fibroblasts, completely preventing VZV-specific cytopathic effects. The inhibition of VZV was reversible upon drug removal at 48 h after addition but was not reversible after 5 days of treatment. ara-T also reduced cell-free virus infectivity and the plaque-forming cell yield of VZV. Compared with the untreated controls, which demonstrated a 1-log10 increase over input plaque-forming cells at 24 h after infection, 50 microgram of ara-T per ml resulted in a 1-log10 decrease. In contrast to herpes simplex virus and VZV, cytomegalovirus replication was relatively resistant to ara-T. Neither cytopathic effects nor the incorporation of [3H]thymidine into acid-insoluble material in cytomegalovirus-infected cells was markedly affected. Analysis of the newly synthesized labeled DNA by CsCl buoyant density determinations indicated that the same relative proportions of cell and virus DNA were synthesized with or without added drug. Interpretation of these results with regard to virus-induced deoxypyrimidine kinase is discussed.     

Detection of varicella-zoster virus DNA by field-inversion gel electrophoresis

A new method for detection of varicella-zoster virus (VZV) DNA using field-inversion gel electrophoresis (FIGE) was devised. VZV-genomic DNA could be differentiated from the host cell DNA of human embryonic lung (HEL) fibroblasts infected with VZV under electrophoretic conditions allowing resolution of linear and double-stranded DNAs in the 49-230 kilobase pairs (Kb) range. The detection of VZV-genomic DNA from infected HEL cells was successful regardless of whether the VZV was a laboratory strain, live vaccine strain, or fresh isolate. Under the same electrophoretic conditions, DNA of VZV-infected HEL cells could be clearly differentiated from DNA obtained from HEL cells infected with herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), or human cytomegalovirus (HCMV). Furthermore, VZV genomic DNA could be detected from as small a sample as 1.9 x 10(4) VZV-infected HEL cells. Finally, we could detect VZV genomic DNA from 10 samples of vesicle tissue (blister lids, each about 1-4 mm2) and one sample of vesicle fluid (about 5 microliters) obtained from patients diagnosed as having herpes-zoster. The results of this study indicate that FIGE is a simple and promising method for the detection of VZV from clinical materials as well as infected in vitro cultured cells.     

Inhibitory effects of orotate on precursor incorporation into nucleic acids.

The influence of orotic acid on the incorporation of precursors into nucleic acids was studied in mice and rats and in isolated cells. In vivo, orotate levels were modified by two diets which are known to increase the rate of pyrimidine nucleotide synthesis in rat liver. Of these diets, a 1% orotate diet had greater inhibitory effects than an arginine-deficient diet on the incorporation of [3H]orotate into RNA of mouse kidney than mouse liver. This contrasted with the situation in the rat where there was a greater effect in the liver than the kidney. The situation in the rat was more readily interpreted than in the mouse in terms of previously established effects of these diets on ribonucleotide pool sizes. However, studies using [3H]adenosine as a precursor for incorporation into RNA suggested that even in the mouse the effects of orotate were on pool sizes rather than an inhibitory effect on RNA synthesis. The incorporation of [3H]thymidine into DNA was inhibited by orotate to a similar degree in cultured HTC hepatoma cells and a line of rat liver epithelial cells. An effect on DNA synthesis rather than solely on pool sizes was suggested by the observation that the pool size of dTTP was not increased by 5 mM orotate under conditions in which there was a four-fold increase in the level of UTP in HTC cells. An inhibitory effect of orotate on DNA synthesis was further supported by an observation of decreased incorporation of [3H]deoxyadenosine into DNA and a lower rate of cellular proliferation.     

Analysis of toxic and mutagenic activities of antiherpesvirus nucleosides against HeLa cells and herpes simplex virus type 1.

The toxic and mutagenic activities of five antiherpesvirus agents to HeLa cells and herpes simplex virus type 1 (HSV-1) were investigated. 5-Iodo-2'-deoxyuridine (IDU) and 9-beta-D-arabinofuranosyl-adenine (araA) showed very potent inhibitory effects on cell growth and the cloning efficiency of HeLa cells, whereas 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU), E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 9-(2-hydroxyethoxymethyl)guanine (ACV) showed less inhibitory effect. 50% inhibitory doses of BV-araU and BVDU for cell growth were 657 and 253 micrograms/ml, respectively. Although the growth inhibitory activity of BVDU was very weak, as above, the mutagenic activity of this drug to the cells, estimated by induction of colchicine-resistant mutants, was observed to be 4 micrograms/ml, which was a markedly smaller dose than the inhibitory dose for cell growth, and the highest frequency of mutation of the cells was shown at 100 micrograms/ml of BVDU. This activity was more potent than that of IDU. No mutagenic activity of BV-araU, araA and ACV to cells was observed within the concentration range of 1-800 micrograms/ml. IDU showed high mutagenic activity to HSV-1 growing in human embryo lung fibroblasts, and IDU-resistant mutants were induced at a high frequency. BVDU also induced a small amount of BVDU-resistant mutant virus, although this drug induced many mutant cells. No mutagenic activity of BV-araU, araA and ACV to HSV-1 was observed.     

Metabolism of the carbocyclic analogue of (E)-5-(2-iodovinyl)-2'-deoxyuridine in herpes simplex virus-infected cells. Incorporation of C-IVDU into DNA

The carbocyclic analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), in which the sugar moiety is replaced by a cyclopentane ring and which have been designated as C-BVDU and C-IVDU, respectively, are, like their parent compounds BVDU and IVDU, potent and selective inhibitors of herpes simplex virus type 1 (HSV-1) and, to a lesser extent, herpes simplex virus type 2 (HSV-2) replication. We have now synthesized the radiolabeled C-IVDU analogue, C-[125I]IVDU, and determined its metabolism by HSV-infected and mock-infected Vero cells. C-[125I]IVDU was effectively phosphorylated by HSV-1-infected cells and, to a lesser extent, HSV-2-infected cells. C-[125I]IVDU was not phosphorylated to an appreciable extent by either mock-infected cells or cells that had been infected with a thymidine kinase-deficient mutant of HSV-1. Furthermore, C-[125I]IVDU was incorporated into both viral and cellular DNA of HSV-1-infected Vero cells. This finding represents the first demonstration of the incorporation of a cyclopentylpyrimidine into DNA.     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Cellular uptake of [3H]thymidine [( 3H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 X 10(-18) mol/cell. The pool activity of G1 cells was unmeasurable but rose to maximum values at the border of the G1-S phase. It decreased again during G2. The [3H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10% X min-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.     

Differential effects of ovarian steroids and triphenylethylene compounds on macromolecular uptake and thymidine incorporation in the mouse uterus

In the rodent uterus, estrogen elicits a biphasic response i.e. an early phase (Phase I) and a late phase (Phase II). Estradiol-17 beta (E2) and estriol (E3), as well as triphenylethylene (TPE) compounds, CI-628 and clomiphene citrate (CC), were used to characterize Phase I and Phase II responses in uterine preparation for implantation in the mouse. While uterine macromolecular uptake (vascular permeability), a Phase I response, was studied in progesterone (P4)-primed animals, uterine [3H]thymidine incorporation (DNA synthesis), a Phase II response, was investigated with and without P4-priming. In the P4-primed uterus, all compounds, except CC, significantly increased uterine macromolecular uptake as determined by interstitial tissue accumulation of [125I]bovine serum albumin [( 125I]BSA). DNA synthesis as determined by cellular incorporation of [3H]thymidine was modulated by P4, estrogens and TPE compounds in a cell-type specific and temporal manner. As a single injection and in the absence of P4, E2 induced [3H]thymidine incorporation in the luminal and glandular epithelium at 18 and 24 h. E3 was inferior to E2 in this response. On the other hand, treatment with P4 for 1 day or 4 days induced [3H]thymidine incorporation primarily in stromal cells. However, stromal cell incorporation was potentiated when P4 treatment was combined with estrogens or TPE compounds. These results reveal the relative importance of Phase I and cell-type specific Phase II responses in uterine preparation for implantation.     

Induction of human cell DNA synthesis by herpes simplex virus type 2.

The effect of herpes simplex virus type 2 (HSV-2) infection on the synthesis of DNA in human embryonic fibroblast cells was determined at temperatures permissive (37 C) and nonpermissive (42 C) for virus multiplication. During incubation of HSV-2 infected cultures at 42 C for 2 to 4 days or after shift-down from 42 to 37 C, incorporation of (3H)TdR into total DNA was increased 2-to 30-fold as compared with mock-infected cultures. Analysis of the (3H)DNA suggested that host cell DNA synthesis was induced by HSV-2 infection. Induction of host cell DNA synthesis by HSV-2 also occurred in cells arrested in DNA replication by low serum concentration. The three strains of HSV-2 tested were capable of stimulating cellular DNA synthesis. Virus inactivated by UV irradiation, heat, or neutral red dye and light did not induce cellular DNA synthesis, suggesting that an active viral genome is necessary for induction.     

Antiviral activity of the 3''-amino derivative of (E)-5-(2-bromovinyl)-2''-deoxyuridine.

3'-NH2-BV-dUrd, the 3'-amino derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, was found to be a potent and selective inhibitor of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) replication. 3'-NH2-BV-dUrd was about 4-12 times less potent but equally selective in its anti-herpes activity as BV-dUrd. Akin to BV-dUrd, 3'-NH2-BV-dUrd was much less inhibitory to herpes simplex virus type 2 than type 1. It was totally inactive against a thymidine kinase-deficient mutant of HSV-1. The 5'-triphosphate of 3'-NH2-BV-dUrd (3'-NH2-BV-dUTP) was evaluated for its inhibitory effects on purified herpes viral and cellular DNA polymerases. Among the DNA polymerases tested, HSV-1 DNA polymerase and DNA polymerase alpha were the most sensitive to inhibition by 3'-NH2-BV-dUTP (Ki values 0.13 and 0.10 microM, respectively). The Km/Ki ratio for DNA polymerase alpha was 47, as compared with 4.6 for HSV-1 DNA polymerase. Thus, the selectivity of 3'-NH2-BV-dUrd as an anti-herpes agent cannot be ascribed to a discriminative effect of its 5'-triphosphate at the DNA polymerase level. This selectivity most probably resides at the thymidine kinase level. 3'-NH2-BV-dUrd would be phosphorylated preferentially by the HSV-1-induced thymidine kinase (Ki 1.9 microM, as compared with greater than 200 microM for the cellular thymidine kinase), and this preferential phosphorylation would confine the further action of the compound to the virus-infected cell.     

Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3':5'-monophosphate.

A single injection of either isoproternol or N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [3H]thymidine. Inhibition of [3H]thymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2, 02-Dibutyryl guanosine 3':5'-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilibrium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [3H]thymidine incorporation into DNA that is replicating semiconservatively. Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [3H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in vivo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E1) also inhibit [3H]thymidine incorporation into DNA or cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect. Isoproterenol administered together with theophylline to neonatal rats signficantly stimulates the in corporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [3H]phenylalanine. DNA synthesis decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.     

Bovine thyrotropin inhibits DNA synthesis inversely with stimulation of cyclic AMP production in cultured porcine thyroid follicles

The effects of bovine thyrotropin (TSH) on DNA synthesis and cyclic AMP production were studied in porcine thyroid follicles using suspension culture. During the early 72 hours incubation, the time-dependent uptake of [3H]thymidine by the follicles was observed. In the presence of 10 mU/ml TSH, the uptake of [3H] thymidine was significantly depressed at 72 hours incubation. TSH inhibition of [3H] thymidine incorporation was related to its concentration and the 50% inhibition was observed by using 1.0 mU/ml TSH. Under the same conditions, cyclic AMP production was stimulated by TSH and the stimulation was observed to be related to TSH concentration. In these experiments, the incubation time was 30 min. Dibutyryl cyclic AMP, an analogue of cyclic AMP, inhibited the [3H] thymidine uptake at 72 hours incubation. From these results, it is suggested that TSH inhibits DNA synthesis, and that the inhibition may be mediated by cyclic AMP that is produced by TSH stimulation.