ß-l-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (l-BHDU) prevents varicella-zoster virus replication in a SCID-Hu mouse model and does not interfere with 5-fluorouracil catabolism.

The alphaherpesvirus varicella-zoster virus (VZV) causes chickenpox and shingles. Current treatments are acyclovir (ACV) and its derivatives, foscarnet and brivudine (BVdU). Additional antiviral compounds with increased potency and specificity are needed to treat VZV, especially to treat post-herpetic neuralgia. We evaluated ß-l-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (l-BHDU, 1) and 5'-O-valyl-l-BHDU (2) in three models of VZV replication: primary human foreskin fibroblasts (HFFs), skin organ culture (SOC) and in SCID-Hu mice with skin xenografts. The efficacy of l-BHDU in vivo and its drug-drug interactions were previously not known. In HFFs, 200µM l-BHDU was noncytotoxic over 3days, and l-BHDU treatment reduced VZV genome copy number and cell to cell spread. The EC50 in HFFs for l-BHDU and valyl-l-BHDU were 0.22 and 0.03µM, respectively. However, l-BHDU antagonized the activity of ACV, BVdU and foscarnet in cultured cells. Given its similar structure to BVdU, we asked if l-BHDU, like BVdU, inhibits 5-fluorouracil catabolism. BALB/c mice were treated with 5-FU alone or in combination with l-BHDU or BVdU. l-BHDU did not interfere with 5-FU catabolism. In SCID-Hu mice implanted with human skin xenografts, l-BHDU and valyl-l-BHDU were superior to ACV and valacyclovir. The maximum concentration (Cmax) levels of l-BHDU were determined in mouse and human tissues at 2h after dosing, and comparison of concentration ratios of tissue to plasma indicated saturation of uptake at the highest dose. For the first time, an l-nucleoside analog, l-BHDU, was found to be effective and well tolerated in mice.

Endothelins reciprocally regulate VEGF-A and angiopoietin-1 production in cultured rat astrocytes: implications on astrocytic proliferation.

Vascular endothelial growth factors (VEGFs) and angiopoietins (ANGs) are involved in pathophysiological responses in damaged nerve tissues. Astrocytes produce VEGFs and ANGs upon brain ischemia and traumatic injury. To clarify the extracellular signals regulating VEGF and ANG production, effects of endothelins (ETs), a family of endothelium-derived peptides, were examined in cultured rat astrocytes. ET-1 (100 nM) and Ala(1,3,11,15)-ET-1 (100 nM), an ET(B) receptor agonist, increased VEGF-A mRNA levels in cultured astrocytes, while ANG-1 mRNA was decreased by ETs. ET-1 did not affect astrocytic VEGF-B, placental growth factor (PLGF), and ANG-2 mRNA levels. The effects of ET-1 on VEGF-A and ANG-1 mRNAs were inhibited by BQ788, an ET(B) antagonist. Release of VEGF-A proteins from cultured astrocytes was increased by ET-1. In contrast, ET-1 reduced release of astrocytic ANG-1. Exogenous ET-1 (100 nM) and VEGF(165) (100 ng/mL), an isopeptide of VEGF-A, stimulated bromodeoxyuridine (BrdU) incorporation into cultured astrocytes. Treatment with ET-1 and VEGF(165) increased the numbers of cyclin D1-positive astrocytes. Exogenous ANG-1 (250 ng/mL) did not stimulate the BrdU incorporation. Increases in BrdU incorporation by ET-1 and VEGF(165) were not affected by ANG-1. In 60-70% confluent cultures, SU4312 (10 µM), a VEGF receptor tyrosine kinase inhibitor, partially reduced the effects of ET-1 on BrdU incorporation and cyclin D1 expression. ET-induced BrdU incorporation and cyclin D1 expression were reduced by a neutralizing antibody against VEGF-A. Our findings suggest that ET-1 is a factor regulating astrocytic VEGF-A and ANG-1, and that increased VEGF-A production potentiates ET-induced astrocytic proliferation by an autocrine mechanism.

Molecular mechanism of green microalgae, Dunaliella salina, involved in attenuating balloon injury-induced neointimal formation.

The pathological mechanism of restenosis is primarily attributed to excessive proliferation of vascular smooth muscle cells (VSMC). The preventive effects of ethanol extract of Dunaliella salina (EDS) on balloon injury-induced neointimal formation were investigated. To explore its molecular mechanism in regulating cell proliferation, we first showed that EDS markedly reduced the human aortic smooth muscle cell proliferation via the inhibition of 5'-bromo-2'-deoxyuridine (BrdU) incorporation at 40 and 80 microg/ml. This was further supported by the G0/G1-phase arrest using a flow cytometric analysis. In an in vivo study, EDS at 40 and 80 microg/ml was previously administered to the Sprague-Dawley rats and found that the thickness of neointima, and the ratio of neointima:media were also reduced. EDS inhibited VSMC proliferation in a dose-dependent manner following stimulation of VSMC cultures with 15 % fetal bovine serum (FBS). Suppressed by EDS were 15 % FBS-stimulated intracellular Raf, phosphorylated extracellular signal-regulated kinases (p-Erk) involved in cell-cycle arrest and proliferating cell nuclear antigen. Phosphorylated focal adhesion kinase (p-FAK) was also suppressed by EDS. Also active caspase-9, caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) protein expression levels were increased by administration with EDS; the apoptotic pathway may play an important role in the regulatory effects of EDS on cell growth. These observations provide a mechanism of EDS in attenuating cell proliferation, thus as a potential intervention for restenosis.

Overexpression of HAM1 gene detoxifies 5-bromodeoxyuridine in the yeast Saccharomyces cerevisiae.

5-Bromodeoxyuridine (BrdU) is known to modulate expression of particular genes, and eventually arrest cell division in mammalian and yeast cells. To study a molecular basis for these phenomena, we adopted a genetic approach with a yeast cell system. We screened multicopy suppressor genes that confer resistance to BrdU with a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae. One of such genes was found to encode Ham1 protein, which was originally identified as a possible triphosphatase for N-6-hydroxylaminopurine triphosphate. Consistent with this, overexpression of the HAM1 gene reversed growth arrest caused by BrdU, and blocked incorporation of BrdU into genomic DNA. On the contrary, disruption of the gene sensitized cells to BrdU. A crude extract from Ham1-overproducing cells showed a high activity to hydrolyze BrdUTP to BrdUMP and pyrophosphate in addition to abnormal purine nucleotides. Purified recombinant Ham1 protein showed the same activity. These results demonstrate that Ham1 protein detoxifies abnormal pyrimidine as well as purine nucleotides.

Angiotensin-converting enzyme inhibitor enhances liver regeneration following partial hepatectomy: involvement of bradykinin B2 and angiotensin AT1 receptors.

Angiotensin-converting enzyme (ACE) inhibitor enhances the liver regeneration in rats after partial hepatectomy (PH), though the precise mechanisms are unknown. To determine the roles of bradykinin and angiotensin II in the ACE inhibitor-induced enhancement of liver regeneration, we investigated effects of lisinopril (ACE inhibitor), candesartan and losartan (angiotensin II type 1 (AT1) receptor antagonists) and icatibant (bradykinin B2 receptor antagonist) on the hepatic regenerative response to 70% PH in the rat. The liver regeneration was evaluated by measuring the frequency of 5-bromo-2'-deoxyuridine (BrdU) incorporation into hepatocyte nuclei 48 h after PH. We found that administration of candesartan or losartan, as well as lisinopril, enhanced BrdU incorporation after PH, and the lisinopril-induced enhancement was inhibited in part (40%) by icatibant. PH induced the expression of hepatocyte growth factor (HGF) mRNA in remnant liver, and this PH-induced up-regulation of HGF mRNA was further enhanced not only by lisinopril but also by candesartan and losartan. Administration of icatibant inhibited up to 40% of the lisinopril-induced up-regulation of HGF mRNA. These results suggest that the blockade of the renin-angiotensin system by either ACE inhibitor or AT1 receptor antagonist enhances the hepatic regenerative response to PH, probably through an augmentation of hepatic HGF production. In addition to this mechanism, the activation of B2 receptors may also be involved in the ACE inhibitor-induced enhancement of hepatic regenerative response.

Identification of potent 5-pyrimidinyl-2-aminothiazole CDK4, 6 inhibitors with significant selectivity over CDK1, 2, 5, 7, and 9.

5-Pyrimidinyl-2-aminothiazole 1 was identified as an inhibitor of cyclin-dependent kinases (CDKs) by a screening of the Merck sample repository. The introduction of a methyl group at the C-5 or C-6 position on the pyrimidine ring, directed toward the gate keeper residue of CDK4 (Phe93), led to significant enhancement of selectivity for CDK4 over other CDKs. Compound 3 exhibited more than 300-fold selectivity for CDK4 over CDK1, 2, 5, 7, and 9. Subsequent improvements in aqueous solubility afforded compound 4, which is available for further in vivo studies and this compound inhibited pRb phosphorylation and BrdU incorporation in tumor models.

Cyclic AMP-mediated regulation of transcription factor Lot1 expression in cerebellar granule cells.

Lot1, a zinc finger transcription factor acting as a tumor suppressor gene on tumoral cells, is highly expressed during brain development. In developing rat cerebellum, Lot1 expression is high in cerebellar granule cells (CGC), a neuronal population undergoing postnatal neurogenesis. The time course of Lot1 cerebellar expression closely matches the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors coupled to adenylyl cyclase. The aim of this study was to ascertain whether Lot1 expression is regulated by cAMP-dependent pathways and to identify mechanisms of Lot1 activation in CGC cultures. Our results show that Lot1 expression in CGC is cAMP-dependent, as treatments with either forskolin or PACAP-38 induced an increase in its expression at both the mRNA and protein levels. This effect on Lot1 expression was mimicked by dibutyryl cAMP and suppressed by protein kinase A and MEK inhibitors. In parallel, we found that treatments with forskolin and PACAP-38 in precursor CGC inhibited bromodeoxyuridine incorporation by 25 and 35%, respectively, indicating a negative effect on neuronal precursor proliferation. Luciferase reporter analysis and mutagenesis of the Lot1 promoter region indicated a crucial role of the AP1-binding site (located at -268 bp) in cAMP-induced Lot1 transcription. In addition, cotransfection experiments indicated that the c-Fos/c-Jun heterodimer is responsible for cAMP-dependent Lot1 transcriptional activation. In conclusion, our data demonstrate that, in CGC, Lot1 is under the transcriptional control of cAMP through an AP1 site regulated by the c-Fos/c-Jun heterodimer and suggest that this gene may be an important element of the cAMP-mediated pathway that regulates neuronal proliferation through the protein kinase A-MEK signaling cascade.

A novel approach to thymidylate synthase as a target for cancer chemotherapy.

Tumor cell resistance to fluoropyrimidines and other inhibitors of thymidylate synthase (TS) is a serious problem often associated with increased intracellular TS. Clinically, another problem that arises from the use of TS inhibitors is toxicity, which develops, in part, because normal cells may be adversely affected by doses of inhibitor that do not impact tumor cells. To circumvent this problem, we have devised a new strategy called enzyme-catalyzed therapeutic activation (ECTA), which takes advantage of overexpressed TS to enzymatically generate cytotoxic moieties preferentially in tumor cells. We show herein that tumor cells expressing elevated levels of TS are preferentially sensitive to NB1011, a phosphoramidate derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine. We find support for the proposed mechanism of NB1011 in the following results: 1) positive relationship between TS protein level and sensitivity to NB1011 in engineered HT1080 tumor cells, designed to express defined levels of TS protein; 2) NB1011 activity is enhanced on tumor cells which express endogenous elevated TS; 3) cytotoxicity of NB1011 is blocked by raltitrexed (Tomudex); 4) NB1011 selection of TS-overexpressing MCF7TDX tumor cells results in recovery of cell populations and clones with diminished TS levels (and restored sensitivity to raltitrexed). A preliminary comparison of TS mRNA levels in multiple normal tissues versus colon tumor samples suggests that selective tumor cytotoxicity of NB1011 may be possible in the clinical setting. Because NB1011 cytotoxicity is dependent upon activation by TS, its proposed mechanism of action is distinct from current TS-targeted drugs, which require inhibition of TS to be effective.

Enumeration of S-phase cells in normal rat liver by immunohistochemistry using bromodeoxyuridine-antibromodeoxyuridine system.

The visualization of incorporation sites of the thymidine analog bromodeoxyuridine (BrdU) into DNA, detected by immunocytochemistry, has been proposed as an index of the percentage of S-phase cells in a variety of tissues and as an easy, less expensive alternative to autoradiography. This technique has not yet been applied to the study of physiological cell renewal in the normal liver. In the present study, results obtained with this method in the liver of normal young adult rats is reported. BrdU was administered in vivo and subsequent incorporation was detected by the PAP technique using monoclonal anti-BrdU antibodies. The nuclei exhibiting a positive reaction within the liver were few and accounted for about 0.45% of all hepatocytes. Positive cells were located preferentially in zone 1, which contained 82.7% of the labeled cells. Zone 2 contained 15.4%, while only 1.9% of the labeled cells were found in zone 3. Positive-staining Kupffer cell nuclei were rare (about 0.5% of all Kupffer cells) and were distributed randomly in the hepatic lobule. These findings provide quantitative data about hepatocyte renewal in the normal liver in the absence of a growth stimulus. The simplicity and the reproducibility of this technique suggests that further application of this method in situations assessing hepatic regeneration are indicated.

Inhibition of bromodeoxyuridine-associated sister chromatid exchanges in Bloom's syndrome cells with cycloheximide.

Effects of cycloheximide (CH) and deoxycytidine (dC) on the frequency of sister chromatid exchanges (SCEs) in normal and Bloom's syndrome (BS) cells labeled with bromodeoxyuridine (BrdU) during first, second, and third cell cycles were evaluated using endomitotic and three-way differentiation analyses. When CH at 0.2 and 2.0 ng/ml was added to normal and BS cultures of BrdU-labeled endomitoses, the rate of single SCEs was significantly decreased in BS cells, though the rate of reduction in single SCEs was slight in normal cells. No significant change was detected in the twin SCE rate. In BS cells, treatment with CH at 0.2 and 2.0 ng/ml produced significant reductions in SCEs in both the second (SCE2) and third (SCE3) cell cycles, sometimes reaching the normal level. Treatment with dC at 13 and 26 micrograms/ml resulted in almost no significant changes in rates of SCE during first, second, and third cell cycles. When CH was added to BrdU-labeled normal and BS cell cultures, the cell growth rates improved from 35% to 70% over the control level in the BS cells, though in normal cells, the addition of CH resulted in a close-dependent lower cell growth rate. Deoxycytidine did not noticeably affect the cell growth rates in BrdU-labeled normal and BS cultures. The finding that the reduction of BrdU-induced SCEs in BS is paralleled by cell growth improvement is of special interest.

[Fusion of cultured Djungarian hamster tumor cells with the host cells in vivo and the characteristics of the hybrids obtained]. / Sliianie kul'tiviruemykh opukholevykh kletok dzhungarskogo khomiachka s kletkami khoziaina in vivio i kharakateristika poluchennykh gibridov.

Tumor Djungarian hamster cells resistant to 5-bromodeoxyuridine (5-BrdU) were inoculated to newborn hamsters. Tumors occurred in animals and were seeded into HAT medium in vitro. This procedure permitted to select hybrids between tumor and normal cells established in vivo. Hybrid nature of cell cultures was confirmed by karyological analysis. Hybrid cells were tested for their ability to grow progressively in newborn Djungarian hamsters and to form colonies in soft agar. The hybrid cells were less malignant than 5-BrdU-resistant tumor cells, but they could grow in soft agar with the efficiency of the parental tumor cells. Chromosomal constitution of the hybrid tumors indicate that as a rule, the expression of malignancy correlates with elimination of the morphologically normal chromosome pairs No 1, 4, 6, 8. Our data suggest that at least two genes located on different chromosomes of the normal cell are needed for suppression of malignancy in somatic cell hybrids.

The BrdU content of DNA is decreased during reversal of inhibition of myogenesis by deoxycytidine.

The mechanism by which 5-bromodeoxyuridine (BrdU) inhibits cell differentiation is unresolved. The ability of deoxycytidine to reverse the inhibition of myogenesis produced by BrdU has been cited as evidence that the inhibition is not a direct result of the incorporation of BrdU into cellular DNA. In contrast to previous work, the present study demonstrates a direct correlation between the effects of deoxycytidine on myogenic cells and a reduction in the substitution of BrdU for thymidine in the DNA. Further-more, the reversal occurs at the same degree of BrdU substitution (20-30%) as is required to inhibit myogenesis when cells are grown in BrdU alone or with deoxycytidine in a medium that prevents the conversion of deoxycytidine to thymidine. The effects of deoxycytidine thus do not support a mechanism of action of BrdU in myogenic cells independent of its effects on DNA.

Inhibition of protein synthesis antagonizes induction of sister-chromatid exchanges by exogenous agents.

Cycloheximide (CH) and puromycin (PM)strongly antagonize induction of sister-chromatid exchanges (SCEs) by exogenous agents regardless of the mechanism for initiating damage. 5-Bromodeoxyuridine (BUdR) substitution was used to monitor SCEs, but the background level of BUdR-induced SCEs was unaffected by the presence of protein inhibitors. Antagonism between DNA-damaging agents and protein inhibitors was strongest in euchromatic regions. Possible relationships between SCE formation and the mechanism of antagonism by protein inhibitors are discussed.

The effects of 5-bromodeoxyuridine on the growth and morphology of transformed rat liver cells.

The effects of bromodeoxyuridine (BrdUrd) on the growth, morphology, and tumorigenicity of the spontaneously transformed rat liver cell line R72/3 were studied. These cells grow either in suspension or in a monolayer and are tumorigenic. In monolayer cultures, cells treated with low concentrations (2.5 micrograms/ml) of BrdUrd were larger, more spread out, and more firmly attached to the substratum than were untreated controls. Treated cells failed to grow in suspension or on confluent monolayers of 3T3 cells and did not form colonies in soft agar. Scanning electron microscopy revealed extensive flattening of treated cells and a dramatic reduction in the number of microvilli on the cell surface. Transmission electron microscopy showed an increase in polyribosomes and rough endoplasmic reticulum, as well as an enlargement of endoplasmic reticulum cisternae and a complete absence of the bundles of intermediate size filaments that were conspicuous in untreated cells. The persistence of these changes required the continuous presence of BrdUrd in the medium. The effects of BrdUrd were readily reversed by withdrawal of BrdUrd and were not expressed in the presence of excess thymidine.

Induction of sister chromatid exchanges by BUdR is largely independent of the BUdR content of DNA.

The halogenated thymidine (dT) analogue, 5-bromodeoxy-uridine (BUdR), has a variety of effects on mammalian cells, including toxicity, suppression of differentiation, and mutagenesis. Although it is generally assumed that the effects of BUdR are due primarily to its presence in DNA, results from our laboratory have raised doubts about such assumptions. We have shown, for example, that BUdR mutagenesis in mammalian cells is determined by the concentration of BUdR in the medium rather than in DNA, and that mutagenesis can be suppressed by deoxycytidine (dC) without changing the amount of BUdR in DNA. BUdR has also been shown to induce sister chromatid exchanges (SCEs) in mammalian cells. Initial results suggested that the relationship between BUdR and SCEs might not be explained by a single factor, and various correlations between BUdR and SCEs have been proposed. However, the results to date have been inconclusive, because the experiments did not resolve as independent variables the concentration of BUdR in the medium and the amount of BUdR incorporated into nuclear DNA. We have now carried out experiments to resolve these two factors; the results indicate that the major factor in determining the frequency of SCEs is the concentration of BUdR in the medium.

Differentiation of 3T3-L2 fibroblasts into adipose cells in bromodeoxyuridine-suppressed cultures.

Growth of 3T3-L2 fibroblasts in medium containing 5-bromo-2'-deoxyuridine prevents the spontaneous cytodifferentiation of these cells into adipose cells. Treatment of bromodeoxyuridine-suppressed cultures at confluence with 1-methyl-3-isobutylxanthine, a promoter of the adipose conversion, results in differentiation of the fibroblasts into adipose cells but the extent of the conversion is less than that seen in cultures not treated with bromodeoxyuridine. 1-Methyl-3-isobutylxanthine appears to function without altering the amount of bromodeoxyuridine present in DNA.

[Production and characteristics of a Djzungarian hamster cell line (DX-TK-) resistant to 5-bromodeoxyuridine]. / Poluchenie i kharakteristika linii kletok dzhungarskogo khomiachka (DKh-TK-), rezistentnoi k 5-bromdezoksiuridinu.

New biochemically marked Djungarian hamster cell line (DX-TK-) was established. These cells are resistant to 5-bromodeoxyuridine (25 mkg/ml) and deficient in thymidine kinase activity (TK-). Due to this biochemical defect they have lost the ability to grow in HAT medium. DX-TK- cells are malignant. They grow as tumours after the inoculation to newborn Djungarian hamsters. Tumorigenecity of DX-TK- cells was decreased as compared with the parent TK+ cell line. DX-TK- cell line is a hypodiploid cell culture (26 chromosomes) with 7 chromosome markers easily identified by means of G-band staining. This line is a new model for somatic cell genetic experiments, particularly for somatic cell hybridization.

Biological and biochemical effects of bromodeoxyuridine and deoxycytidine on Syrian hamster melanoma cells.

The addition of deoxycytidine (dCyd) to the growth medium of cultured Syrian hamster melanoma cells causes a reversal of the toxic effects of 5-bromodeoxyuridine (BrdU) and a decrease in the extent of incorporation of BrdU into nuclear DNA. These effects of dCyd can be accounted for, in part, by the intracellular conversion of the exogenously supplied dCyd to thymidine (dThd) nucleotides which can compete with BrdU nucleotides for incorporation into DNA. To some extent, the conversion of dCyd to dThd nucleotides can be inhibited by increasing the concentration of BrdU in the growth medium. The conversion of dCyd to dThd nucleotides is inhibited completely by aminopterin (Apt), and Apt also prevents dCyd from reversing BrdU toxicity and from decreasing the level of BrdU incorporation into nuclear DNA. In a clone of Syrian hamster melanoma cells, increasing the concentration of dCyd in the growth medium from 1 micron to 1000 micron resulted in a progressive increase in the percentage of dThd residues in nuclear DNA being derived from the exogenous dCyd, until more than 90% of the dThd residues came from the exogenous dCyd. However, despite the increasing amount of dThd derived from exogenous dCyd, there was a plateau in the decrease in BrdU incorporation into nuclear DNA at concentrations of dCyd above 8 micron.