Synthesis, molecular conformation and activity against herpes simplex virus of (E)-5-(2-bromovinyl)-2'-deoxycytidine analogs.

Analogs of (E)-5-(2-bromovinyl)-2'-deoxycytidine (BrVdCyd) (1) by substitution at N(4) were synthesized to impart resistance against deamination. The anti-HSV-1 activity and solution conformation of these analogs were determined. N(4)-Acetyl-BrVdCyd (2) was a potent inhibitor of HSV-1 replication whereas N(4)-propanoyl-BrVdCyd (3) had good activity and N(4)-Butanoyl-BrVdCyd (4) had only low activity against HSV-1 replication. N(4)-Methyl-BrVdCyd (5) was devoid of activity against HSV-1.

Effects of the genotoxic agent 5-bromo-2'-deoxyuridine with or without pre-pubertal gonadectomy on brain monoamines and their metabolites in female rats.

A nucleotide analog 5-bromo-2'-deoxyuridine (BrdU) is a genotoxic compound. Previous studies have demonstrated that prenatal treatment of rodents with BrdU affects the development of cortical neurons, reduces dopamine levels, and elevates serotonin (5-HT) levels in the striatum in adult male offspring from BrdU-treated dams. Moreover, prenatal BrdU-treated rats show locomotor hyperactivity in both males and females. This study investigated sexual dimorphism in the effect of prenatal BrdU on monoamine metabolism. Sprague-Dawley rats were treated with BrdU on gestational days 9-15 (50mg/kg, i.p.) and monoamine metabolism was examined in female rats at 10 weeks of age. The influence of pre-pubertal gonadectomy on the effects of BrdU was also investigated. BrdU-treated females showed elevations of dopamine and 5-HT levels in the striatum; reductions in dopamine, dihydroxyphenylacetic acid, or homovanillic acid (HVA) in the hypothalamus or the midbrain; and elevated HVA and 5-HT in the hippocampus. Pre-pubertal gonadectomy had a suppressive effect on striatal dopamine levels in prenatal BrdU-treated females. The present data indicate sexual dimorphic effects of prenatal BrdU-treatment in striatal dopamine metabolism but not in serotonergic metabolism and suggest a contribution of the increasing gonadal hormones that accompany puberty to this sex difference.

Comparative cytostatic activity of different antiherpetic drugs against herpes simplex virus thymidine kinase gene-transfected tumor cells.

A series of selective antiherpetic compounds were found to exert pronounced cytostatic activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) thymidine kinase (TK) gene-transfected mammary carcinoma FM3A cells. Based on their potency and mechanism of cytostatic action, the antiherpetic compounds could be divided into two different classes. The first class encompasses (E)-5-(2-bromovinyl)-2'-deoxyuridine and structurally related analogues thereof [i.e., the cytosine derivative (E)-5-(2-bromovinyl)-2'-deoxycytidine and the 4'-thio derivative (E)-5-(2-bromovinyl)-2'-deoxy-4'-thiouridine]. These compounds are exquisitely cytostatic against FM3A/TK-/HSV-1 TK+ and FM3A/TK-/HSV-2 TK+ cells (50% inhibitory concentrations ranging from 0.047 to 0.001 microM) and inhibit tumor cell proliferation by inhibiting cellular thymidylate synthase. The second class consists of the acyclic guanosine derivatives penciclovir, buciclovir, and ganciclovir. These compounds are also more inhibitory to the HSV-1 TK or HSV-2 TK gene-transfected FM3A cells than to FM3A/0 or FM3A/TK- cells, but at concentrations that are higher than the concentrations at which the (E)-5-(2-bromovinyl)-2'-deoxyuridine derivatives proved to be inhibitory. These acyclic guanosine analogues appear to be targeted at the cellular DNA polymerase. From this study, (E)-5-(2-bromovinyl)-2'-deoxy-4'-thiouridine emerged as a promising candidate compound for the treatment of HSV-1 TK gene-transfected tumors in vivo, due to its metabolic stability (i.e., resistance to hydrolysis by thymidine phosphorylase).

[A mathematical model of the proliferative kinetics of cells labelled with brominated DNA precursors]. / Matematicheskaia model' proliferativnoi kinetiki kletok, mechennykh bromirovannymi predshestvennikami DNK.

A mathematical model of cell division is presented. It permits analyzing cell proliferation obtained by using BrdU. Numerical experiments show that this model adequately describes experimental data. Application of this model permits decreasing experimental data bulk.

[The frequency of sister chromatid exchanges in cultured cells of mouse blood, bone marrow and spleen]. / Chastota sestrinskikh khromatidnykh obmenov v kul'tiviruemykh kletkakh krovi, kostnogo mozga i selezenki myshei.

Analysis of factors leading to the increase of SCE in vitro is presented. Frequency of SCE is the same in the blood, spleen, marrow cell culture and does not influence upon the time of culture and BDU or BDC concentration. The authors consider that procedure preparation of the culture leads to an increase of SCE in vitro as compared to in vivo.

Selective radiosensitization and cytotoxicity of human melanoma cells using halogenated deoxycytidines and tetrahydrouridine.

The halogenated pyrimidines 5-chloro-2'-deoxycytidine (CldCyd) and 5-bromo-2'-deoxycytidine (BrdCyd) can act as radiosensitizers and cytotoxic agents. It was hypothesized that tumor cells and normal cells might use different metabolic pathways to incorporate these halogenated deoxycytidines into DNA. This difference could potentially be exploited to produce selective radiosensitization and cytotoxicity of human tumor cells compared to normal human fibroblasts. This hypothesis was tested using two human melanoma cell lines and two normal fibroblast cell lines. Either CldCyd or BrdCyd alone caused both cytotoxicity and radiosensitization of tumor and normal cells. The addition of the cytidine deaminase inhibitor tetrahydrouridine (H4U) significantly protected the normal cells but had relatively little effect on the tumor cells. These data indicate that it may be possible to exploit differences between the pyrimidine metabolism of normal cells and melanoma cells to improve the therapeutic index of halogenated pyrimidines both as radiosensitizers and as cytotoxic agents.

[The duration of cell cycle phases studied using 5-bromodeoxycytidine]. / Izuchenie prodolzhitel'nosti faz kletochnogo tsikla s pomoshch'iu 5-bromdezoksitsitidina.

The quantitative method is suggested to estimate cell cycle phase durations and dispersions of progress through the phases for population of cells. The method is based on the analysis of frequency of cells with different staining of sister chromatids by means of 5-bromodeoxycytidine. The process of cell population progress is described by the Gauss probability integral. The durations of the cell cycle phases are determined for cell culture of Chinese hamster.

Synthesis and antiviral properties of (E)-5-(2-bromovinyl)-2'-deoxycytidine-related compounds.

Treatment of 3',5'-di-O-acetyl-(E)-5-(2-bromovinyl)-2'-deoxyuridine (2) with p-chlorophenyl phosphorodichloridate and 1,2,4-triazole gave 1-(3,5-di-O-acetyl-2-deoxy-beta-D-erythro-pentofuranosyl)-(E)-5-(2-br o movinyl)- 4-(1,2,4-triazol-1-yl)pyrimidin-2(1H)-one (3). Reaction of 3 with ammonia gave (E)-5-(2-bromovinyl)-2'-deoxycytidine (1), the overall yield from 2 being 60%. A similar 4-(1,2,4-triazol-1-yl) derivative (4) was obtained from 3',5'-di-O-acetyl-thymidine by the use of phosphoryl chloride as the condensing agent. Treatment of thymidine with trimethylsilyl chloride and then with phosphoryl chloride and 1,2,4-triazole gave upon workup 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-5-methyl-4(1,2,4-triazol -1-yl) pyrimidin-2(1H)-one (5). (E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU) when similarly treated gave the corresponding (E)-5-(2-bromovinyl) compound 7. A minor product formed in both cases was a 4-(1,2,4-triazol-1-yl) derivative in which the nucleoside 5'-hydroxyl group had been replaced by chlorine (6 and 8). Whereas compounds 4-6 and 8 did not exhibit a selective antiviral effect, compounds 1-3 and 7 proved almost as active as the reference compound BVDU. In particular, compound 7, the 4-triazolyl derivative of BVDU, would seem worth pursuing for its potential as an inhibitor of herpes simplex virus type 1 and varicella-zoster virus.

Incorporation of 5-bromodeoxycytidine in the adenovirus 2 replication origin interferes with nuclear factor 1 binding.

We have studied the binding of nuclear factor 1 (NFI), a human sequence-specific DNA-binding protein, to a DNA fragment substituted in vitro with 5-bromodeoxycytidine (5-BrdC). Even at low substitution grades binding of NFI to its recognition sequence was considerably lower than with the unsubstituted control fragment. We developed a procedure to cleave substituted DNA specifically at a BrdC residue and searched for contacts between NFI and 5-BrdC residues by an interference assay. Surprisingly, no specific contacts were found in or near the recognition sequence. It appeared instead that interference was inversely related to the distance of a 5-BrdC residue from the NFI binding site. Models to explain these results, including a possible sliding mechanism, are discussed.

Murine mammary FM3A carcinoma cells transformed with the herpes simplex virus type 1 thymidine kinase gene are highly sensitive to the growth-inhibitory properties of (E)-5-(2-bromovinyl)-2'-deoxyuridine and related compounds.

Murine mammary carcinoma (FM3A TK-/HSV-1 TK+) cells, which are thymidine kinase (TK)-deficient but have been transformed with the herpes simplex virus type 1 (HSV-1) TK gene are inhibited in their growth by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC) at 0.5, 0.5 and 0.8 ng/ml, respectively; i.e., a concentration 5000 to 20 000-fold lower than that required to inhibit the growth of the corresponding wild-type FM3A/0 cells. Hence, transformation of tumor cells with the HSV-1 TK gene makes them particularly sensitive to the cytostatic action of BVDU and related compounds.

Heritable fragile sites on human chromosomes. XI. Factors affecting expression of fragile sites at 10q25, 16q22, and 17p12.

The fragile sites at 10q25, 16q22, and 17p12 can all be induced in lymphocyte culture by BrdU or BrdC added 6-12 hrs prior to harvest. Without induction, fra(10)(q25) is rarely expressed spontaneously, whereas fra(16)(q22) is frequently expressed spontaneously. Fra(17)(p12) is frequently expressed spontaneously but is probably expressed only after induction in some individuals. Distamycin A, netropsin, and Hoechst 33258 induced high levels of expression of fra(16)(q22) and fra(17)(p12) but did not enhance expression of fra(10)(q25). The mechanisms of induction of fra(16)(q22) by BrdU and distamycin A appear to be different, since the time of induction by BrdU reaches a maximum about 12 hrs prior to harvest whereas induction by distamycin A requires much longer exposure. The fragile sites at 10q25 and 16q22 were both induced in fibroblast culture by BrdU. Fra(17)(p12) is accepted as a fragile site because preliminary studies show that it behaves similarly in lymphocyte culture to fra(16)(q22); however, there is only limited evidence for fragility at 17p12.

Probing sister chromatid exchange formation with halogenated pyrimidines.

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.

Use of 5-BrdUrd/313 nm light technique for identification of cells in initial S-phase within GM-CFCc compartment.

Long-term, low-level, BrdUrd infusion identifies two subpopulations of GM-CFCc with quite dissimilar sensitivities to 313 nm light. The responses of these two GM-CFCc subpopulations to hydroxyurea indicate that both are rapidly proliferating at the time of the assay. However, the absolute UV-light sensitivity of the S-phase components and the effects of increasing BrdUrd concentration indicate that the two GM-CFCc subpopulations passed through the previous cell cycle at widely disparate rates. Further, those GM-CFCc originating from a parental cell with a slow turnover are associated with a lower buoyant density than those GM-CFCc that have been in rapid cycle for at least two generations. These results indicate that the resistance to 313 nm-light irradiation, shown by S-phase cells in the first cell cycle of the BrdUrd labeling, may provide evidence of the proliferative history of the cell being assayed.

[Analysis of sister chromatid exchanges in the 1st, 2d and 3d mitoses using 5-bromodeoxyuridine and 5-bromodeoxycytidine]. / Analiz sestrinskikh khromat dnykh obmenov v pervom, vtorom i tret'em mitozakh s pomoshch'iu 5-bromdezoksiuridina i 5-bromdezoksitsitidina.

The cultures of Chinese hamster cells were treated with different concentrations (2.5, 5.0, 10.0, 20.0, 80.0 and 160.0 microgram/ml) 5-BrdU and 5-BrdC during 12 hours. The cultures were fixed at the 24-th hour. The linear increase of sister chromatid exchanges (SCE) was discovered with the increase of BrdU concentration. No change of SCE frequency was observed at different BrdC concentrations. The reasons for these differences in a concentration effect are discussed.

Hemimethylated duplex DNAs prepared from 5-azacytidine-treated cells.

Duplex heavy-light (HL) DNAs synthesized in the presence of brdUrd and methylation inhibitors were separated from bulk cellular DNA by CsCl density gradient centrifugation and analysed for 5-methylcytosine (5mC) contents by HPLC. DNAs synthesized in the presence of 5 mM ethionine or 2 mg/ml cycloleucine were not detectably hypomethylated, was undermethylated with respect to control DNA. The heavy, or H-strand, in which up to 5% of the cytosine residues were replaced by intact 5-azacytosine, was undermethylated and the HL duplex DNA was therefore strand asymmetrically methylated. This duplex DNA served as an efficient substrate for a crude DNA methyltransferase preparation which transferred the methyl group from S-adenosylmethionine specifically into cytosine residues within the hypomethylated H strand. Increasing levels of incorporated 5-azacytosine inhibited the action of the methyltransferase suggesting that incorporation of 5-azacytosine into DNA may be responsible for the inhibitory effect of 5-azacytidine on DNA methylation.

5-Bromodeoxycytidine in the study of sister chromatid exchanges in human lymphocytes.

When [3H]dC was added with a high dose (4 x 10(-1) mM) of dT to human blood lymphocyte cultures, much heavier labeling of interphase nuclei and metaphase chromosomes was observed compared with that in cultures treated with [3H]dC alone. This observation indicates that in the presence of excess dT, exogenous dC is included into cytosine bases of DNA, releasing the cells from the thymidine block. BrdC 5 x 10(-2) mM added with a high dose of dT (4 x 10(-1) to 1.0 mM) to the cultues did not relieve the thymidine block as determined from the percentage of metaphases of the first to third divisions. It is concluded that BrdC, in contrast to dC, is not utilized as a cytosine DNA precursor even in the presence of high concentrations of dT. The frequency of SCEs per cell was the same when studied with the aid of BrdC and BrdU used under similar conditions. The distribution of SCEs among chromosomes was also identical for both analogues: The number of SCEs was significantly higher than expected in chromosomes of group B and lower than expected in chromosomes of groups E, F, and G.

[Sister chromatid exchanges following incorporation of bromine into cytosine DNA nucleotides. I. Combined use of 5-bromdeoxycytidine and thymidine for the purpose of incorporating bromine into DNA cytosine]. / Sestrinskie khromatidnye obmeny pri vkliuchenii broma v tsitozinovye nukleotidy DNK. I. Sovmestnoe primenenie 5-bromdezoksitsitidina i timidina s tseil'iu vkliucheniia broma v tsitozin DNK.

In the cultured in vitro human blood lymphocytes treated with 5-bromodeoxycytidine (BrdC) plus thymidine (dT) in concentrations of 0.05 mM and 0.4 mM, respectively, metaphases of the second division, containing chromosomes with differentially stained sister chromatids appear after a 30 hours treatment, attaining 85 per cent at 36 hours. Under the treatment with BrdC or BrdU alone, the similar percentage of metaphases is observed at 28 hours. Similar results are obtained if, at 16--18 hours, the culture medium, containing BrdC plus dT is either replaced with a medium free of both the precursors, or supplied once more vith (0.05 mM) or dC(0.05 mM). The The observed slowing movement of cells through out the cell cycle, due to the treatment with BrdC plus dT, is explained by the inhibitory effect of dT on DNA synthesis which is manifested as BrdC catabolism. It is concluded that BrdC administered together with the cell cycle-slowing doses of thymidine is predominantly incorporated into cytosine of DNA.

Recovery of cells from induced, potentially lethal damage.

The recovery of mammalian cells after a variety of treatments is, in part, governed by the cells' ability to deal with repairable, but potentially lethal, lessions. Kinetics of such recovery show a T1/2 of 10-20 hours after ultraviolet (UV) irradiation and 1.5-2.5 hours after X-irradiation. Recovery after exposure to mechlorethamine and bleomycin (BLM) is similar to X-ray recovery; after methylmethane sulfonate, recovery has components similar to X-ray and UV recovery. The sequential treatments of cells with 43 degrees C hyperthermia and X-rays (or reverse order) modify both the immediate survival after treatments as well as the subsequent recovery kinetics. Very similar results are found after BLM and hyperthermia treatments, suggesting strongly that after exposure to that drug a real repair system is operative. However, although recovery after X-irradiation is similar in vitro and in vivo, after BLM the site of treatment and of recovery strongly influences the magnitude and kinetics of recovery.

Enzymatic basis for the selective inhibition of varicella-zoster virus by 5-halogenated analogues of deoxycytidine.

5-Bromodeoxycytidine (BrdC) and 5-iododeoxycytidine, at a concentration of 100 mug/ml, effectively inhibit the replication of varicella-zoster (VZ) virus in tissue culture. No toxicity could be demonstrated in uninfected cells under the same conditions. Studies on the enzymatic basis for this selective inhibition were undertaken. Infection of human embryonic lung cell monolayers with VZ virus-infected cells results in the induction of thymidine (dT), deoxycytidine (dC), and BrdC kinase activities (which are increased 10-, 40-, and 60-fold, respectively) and in a 70-fold stimulation in the incorporation of 3H nucleotide (5-bromodeoxyuridylate) derived from BrdC into DNA. The thermal stability of the VZ virus-induced activities differs significantly from the activities induced by herpes simplex virus type 1 and herpes simplex virus type 2 and those present in uninfected human embryonic lung cells. The VZ virus-induced dT, dC, and BrdC kinase are similarly affected by temperature and cofractionate upon Sephadex gel filtration, findings consistent with the hypothesis that these activities are the function of a single enzyme: a pyrimidine deoxyribonucleoside kinase. The molecular weight, calculated on the basis of the elution pattern on Sephadex G-150, is 70,000. Kinetic studies, demonstrating that dT and dC competively inhibit the phosphorylation of BrdC, are consistent with the phosphorylation of these substrates at a common active site. Kinetic parameters include: KidT = 0.6 MUM; KidC = 60 muM; KmBrdC = 8.5 muM. In contrast to its relatively high affinity for the VZ virus-induced kinase, BrdC is a relatively poor substrate for the host kinases. Therefore, the basis for the selective inhibition of VZ virus by 5-halogenated analogues of dC is reflected in the induction of a pyrimidine deoxyribonucleoside kinase with a high affinity for BrdC.