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At least 775 records · Page 43Linked to original sources
PubMed1986

1-B-D arabinofuranosyl cytosine and all-trans retinoic acid in combination accelerates and increases monocyte differentiation of myeloid leukemic cells.

The effect of the combination of two different agents on myeloid differentiation was studied. Human promyelocytic cells (HL-60) and monoblast-like cells (U-937) were treated with 1-B-D arabinofuranosyl cytosine (Ara-C) alone, or in combination with retinoic acid (trans-RA). The known dual potentiality of HL-60 promyelocytes was confirmed by their ability to mature into granulocytes following induction by retinoic acid and into monocyte-like cells following treatment with 1-B-D arabinofuranosyl cytosine. The U-937 cell line differentiated to monocyte-like cells with either of the two drugs. The differentiation induced by Ara-C involved an irreversible step after 24-h incubation with the drug, was concentration dependent, and far more superior on U-937 cells than on HL-60 cells. The outcome of these two cell lines after treatment with both Ara-C and trans-RA was also different: this combination maintained the monocyte differentiation in the HL-60 cell line, and resulted in a higher sensitivity of U-937 cells to Ara-C, as indicated by a cell response to low Ara-C concentrations and a more rapid expression of monocyte specific properties.

Antibodies, Monoclonal↗
PubMed1987

In-vitro studies on phosphorylation and dephosphorylation of cytosine arabinoside in human leukemic cells.

The cytotoxic effect of cytosine arabinoside (ara-C) depends on the capacity of cells to form and retain intracellularly the phosphorylated metabolite cytosine arabinoside triphosphate (ara-CTP). In this study accumulation and cellular retention of ara-CTP have been measured in vitro in the bone marrow cells of 69 patients with acute leukemia. Cells were incubated with 3H-ara-C and the amount of ara-CTP formed was determined after separation of the nucleotides by thin-layer chromatography. Phosphorylation of ara-C to ara-CTP appeared to be a saturable process. The Km-equivalents varied between 1.1 and 16.2 microM ara-C. Maximal ara-CTP formation ranged from 12 to 125 pmol ara-CTP/10(6) cells in 30 min. The phosphorylation activity did not correlate with the percentage of S-phase cells. The intracellular half-life time of ara-CTP measured in vitro ranged from 53 to 210 min. Phosphorylation of ara-C was comparable in patients with acute myeloid leukemia (n = 51) and in patients with acute lymphoblastic leukemia (n = 18). Ara-CTP elimination appeared slower in lymphoblasts than in myeloblasts. The average intracellular ara-CTP level in relapsed patients (n = 34) appeared higher than in previously untreated patients (n = 52). The less favourable outcome of second remission induction therapy with conventional doses of ara-C compared to the first remission induction treatment is not explained by an alteration in the intracellular metabolism of ara-C.

Bone Marrow↗
PubMed1990

Saturation of intracellular cytosine arabinoside triphosphate accumulation in human leukemic blast cells.

Accumulation of cytosine arabinoside triphosphate (araCTP) from a range of cytosine arabinoside (araC) concentrations (1-50 microM) was measured during incubations of leukemic cells freshly isolated from patients with acute leukemia. In all but one patient, increments in extracellular araC above 10 microM did not increase intracellular araCTP levels. This maximal level of araCTP accumulation ranged from 254 to 1607 pmol/10(7) cells attained after 1 h incubation and did not correlate with either the number of nucleoside transporters on the cell membrane or the Vmax of araC phosphorylation in cell free extracts. Extremely low araCTP accumulation (103 pmol/10(7) cells/h at 50 microM araC) was observed in an AML patient with the unusual finding of micromyeloblasts. These cells also had very low numbers of nucleoside transport sites (less than 500 sites/cell) and were mitotically inactive. The unique feature of the myeloblasts from this patient was that intracellular araCTP accumulation showed a linear dependence on extracellular araC up to 50 microM with no evidence of saturation.

Acute Disease↗
PubMed1991

Modulation of cytosine arabinoside toxicity by 3-deazauridine in a murine leukemia model.

3-Deazauridine (DAUrd), a competitive inhibitor of CTP synthetase, inhibits both RNA and DNA synthesis. Murine leukemia cells resistant to cytosine arabinoside (ara-C) due to a deletion of deoxycytidine kinase are collaterally sensitive to DAUrd, which inhibits the de novo production of CTP and hence results in dCTP depletion. We evaluated DAUrd in combination with the palmitate derivative of ara-C (palmO-ara-C) in mice bearing L1210 leukemia cells with a subpopulation resistant to ara-C. Both simultaneous administration and a sequential schedule of palmO-ara-C at its maximally tolerated dose (MTD), followed by DAUrd treatment, failed to produce a therapeutic gain. We also studied whether non-toxic doses of DAUrd (15-250 mg/kg i.p. at h 0 and 6 on days 4 and 8) could modulate the antileukemic activity of palmO-ara-C (7.5-120 mg/kg i.p. at h 3 on days 4 and 8). The addition of DAUrd produced a modest (but statistically significant) prolongation of life span and a further 2-log10 reduction in tumor burden compared to the same dose of palmO-ara-C alone, and resulted in long-term survivors in five of 30 treated animals. Two-dimensional dose-response analysis of the survival data indicated a positive drug interaction (p less than or equal to 0.01) when the dosage of DAUrd was modeled to reflect an apparent threshold effect. Cyclopentenyl cytosine (CPE-C; 0.625-2.5 mg/kg i.p. at h 0 and 6 on days 4 and 8), a more potent inhibitor of CTP synthetase, was also given with palmO-ara-C. This combination resulted in an additional 2-6 log10 units of cell kill and occasional long-term survivors at palmO-ara-C dosages that alone resulted in no more than 2 log10 units of cell kill and no long-term survivors. However, DAUrd and CPE-C given with palmO-ara-C increased host toxicity, compromising the tolerable dose of palmO-ara-C. Single-agent palmO-ara-C given at its MTD produced a similar reduction in tumor burden and increase in life span compared to the highest palmO-ara-C dose that could be given in combination with either modulator.

3-Deazauridine↗
PubMed1996

Modulation of metabolism and cytotoxicity of cytosine arabinoside with N-(phosphon)-acetyl-L-aspartate in human leukemic blast cells and cell lines.

Cytosine arabinoside (Ara-C) activation to cytosine arabinoside triphosphate (Ara-CTP) and subsequent incorporation into DNA is regulated by the pyrimidine nucleotides UTP, CTP and dCTP. Inhibition of the de novo synthesis of these pyrimidine nucleotides by N-(phosphon)-acetyl-L-aspartate (PALA) may enhance the cytotoxicity of Ara-C. We therefore studied the effect of PALA on Ara-C cytotoxicity and on Ara-CTP accumulation and incorporation into DNA on cell lines and patient samples. Fifty micromolar PALA increased the growth inhibitory effect of Ara-C in U937 cells several fold both with pre- and coincubation. Ara-C cytotoxicity was not potentiated by PALA in Hl60 cells. However, coincubation with PALA did not enhance Ara-CTP accumulation both in HL60 and U937 cells, nor affect Ara-C incorporation into DNA. Ara-C cytotoxicity to leukemic blast cells from 11 untreated patients with different types of leukemia was only modulated to a small extent by high PALA concentrations in only two cases. Ara-CTP accumulation in leukemic blast cells varied from non-detectable levels to 200 pmol/10(6) cells. Fifty micromolar PALA enhanced the accumulation of Ara-CTP significantly in only one patient with no apparent effect on UTP and CTP levels. Raising PALA to 500 microM decreased UTP and CTP levels to 50% but had no effect on Ara-CTP levels. In conclusion, modulation by PALA of Ara-C cytotoxicity and metabolism is limited in leukemic cells, both in culture and from patients. This suggests the possibility for selective modulation of other agents by PALA on non-hematological cells.

Antimetabolites, Antineoplastic↗
PubMed1982

An ultrastructural study of cortical remodeling in cytosine arabinoside induced granuloprival cerebellum in tissue culture.

Mouse-derived cerebellar explants were exposed for 5 days to cytosine arabinoside, an inhibitor of deoxyribonucleic acid synthesis. They were then maintained in normal nutrient medium until fixation for electron microscopy at 15-20 days in vitro. The cerebellar cortex lacked granule cells, but Purkinje cells, Golgi neurons and a few basket and stellate cells survived. Astrocytes and oligodendrocytes were diminished in number and myelination was absent. Purkinje cell recurrent axon collaterals increased in number and formed synapses with the surviving cortical neurons and their processes. The ultrastructural alterations that occurred in the cytosine arabinoside-treated cultures were consistent with an interpretation of cortical remodeling in which Purkinje cell axon collaterals were the dominant inhibitory elements.

Animals↗
PubMed1985

Simultaneous determination of cytosine arabinoside, its nucleotides and metabolites by ion-pair high-performance liquid chromatography.

Currently available high-performance liquid chromatographic assays for cytosine arabinoside (ara-C) and its metabolites suffer from two major shortcomings: inability to resolve both ara-C and its nucleotides in a single chromatographic step and/or inadequate sensitivity to allow quantitation of intracellular cytosine arabinofuranoside-5'-triphosphate (ara-CTP) without the use of radiolabelled drug. In this paper, we describe a new ion-pairing high-performance liquid chromatographic assay for ara-C in biological samples that can separate ara-C from its nucleotides, metabolites, and naturally occurring ribonucleotides in a single chromatographic step with a lower limit of quantitation of 5 pmol for ara-C and 10 pmol for ara-CTP. Examples of the utility of this assay are shown in studies of intracellular pharmacokinetics of ara-C in cultured human breast cancer cells and in analysis of plasma nucleoside levels in patients receiving high-dose thymidine chemotherapy. We conclude that this assay provides a rapid and versatile system that can be applied to the study of both cellular and plasma nucleoside pharmacokinetics.

Animals↗
PubMed1975

Photoamidation of unsaturated carbohydrates. Synthesis of 1-(3-C-carbamoyl-3-deoxy-beta-D-glucopyranosyl)-thymine and -cytosine.

The acetone-initiated photochemical addition of formamide to 1,2,4,6-tetra-O-acetyl-3-deoxy-alpha-D-erythro-hex-2-enopyranose (1) afforded 1,2,4,6-tetra-O-acetyl-3-C-carbamoyl-3-deoxy-alpha-D-glucopyransoe (2), 1,2,4,6-tetra-O-acetyl-3-C-carbamoyl-3-deoxy-alpha-D-allopyranose (3), 1,2,4,6-tetra-O-acetyl-3-C-carbamoyl-3-deoxy-alpha-D-altropyranose (4), and 1,2,4,6-tetra-O-acetyl-3-deoxy-3-C-(1-hydroxy-1-methylethyl)-alpha-D-mannopyranose (5), in 46, 13, 1, and 7% yields, respectively. Condensation of 2,4,6-tri-O-acetyl-3-C-carbamoyl-3-deoxy-alpha-D-glucopyranosyl bromide (produced from 2 with hydrogen bromide) with 2,4-bis(trimethylsilyl)thymine afforded 1-(2,4,6-tri-O-acetyl-3-C-carbamoyl-3-deoxy-beta-D-glucopyranosyl)thymine (6) in 40% yield. Deacetylation of 6 with sodium methoxide in methanol yielded the pure unprotected nucleoside 7. Similar condensation of the bromide from 2 with 2,4-bis(trimethylsilyl)-N4-acetylcytosine afforded the protected cytosine nucleoside 8 in 55% yield, which was converted into 1-(3-C-carbamoyl-3-deoxy-beta-D-glucopyranosyl)cytosine (9).

Anti-Bacterial Agents↗
PubMed2002

Retinoic acid reduces the cytotoxicity of cyclopentenyl cytosine in neuroblastoma cells.

In this paper, it is demonstrated that all-trans, 9-cis and 13-cis retinoic acid (RA) decreased the sensitivity of SK-N-BE(2)c neuroblastoma cells towards the chemotherapeutic agent cyclopentenyl cytosine (CPEC), a potent inhibitor of cytosine-5'-triphosphate synthetase. Retinoic acid attenuated CPEC-induced apoptosis as reflected by a decreased caspase-3 induction. Retinoic acid decreased the accumulation of CPEC, whereas the salvage of cytidine was strongly increased. Metabolic labeling studies using [(3)H]uridine showed a strongly decreased biosynthesis of CTP via CTP synthetase. Retinoic acid likely confers resistance of neuroblastoma cells to CPEC in part by slowing down proliferation, and in part by shifting the synthesis of CTP towards the salvage of cytidine, thereby bypassing CTP synthetase.

Antineoplastic Agents↗
PubMed2001

Cytosine methylation of plastid genome in higher plants. Fact or artefact?

DNA methylation of chloroplast genome has been studied in a large variety of angiosperm species using restriction enzyme analysis of three genomic loci (totally encompassing about 10% of chloroplast genome) and bisulfite genomic sequencing of tobacco ribulose bisphosphate carboxylase/oxygenase (large subunit) gene (rbcL). Except for CCWGG (W=A or T) sites that were partially refractory to the cleavage with methylation sensitive EcoRII in all loci, no cytosine methylation was found at the CCGG (MspI/HpaII) and several other restriction sites tested. However, EcoRII was unable to completely digest an unmethylated CCWGG site in the cloned rbcL gene on plasmid. Further a bisulfite genomic sequencing performed on EcoRII-restricted DNA failed to show any 5-methylcytosine either within or outside inspected EcoRII sites along the 3' end of rbcL coding region. In conclusion our results do not support evidence for methylated cytosine residues in plant chloroplast genomes and we suggest that results obtained with EcoRII should be interpreted with great care especially when small differences in methylation levels are analysed.

Journal Article↗
PubMed1998

Antitumor activity of a novel orally effective nucleoside, 1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine.

The antitumor activity of a novel 4'-thionucleoside, 1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine (4'-thio-FAC), was evaluated. 4'-Thio-FAC inhibited the in vitro growth of various human cancer cell lines, particularly the growth of cell lines established from gastric and colorectal carcinomas, while its oxy-type congener 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)cytosine (FAC) showed little or no activity against such solid cancer cell lines. 4'-Thio-FAC showed a remarkable antitumor effect against human tumors subcutaneously implanted into nude mice and was highly effective even by oral administration. 4'-Thio-FAC was less susceptible to deamination by cytidine deaminase than FAC and 2'-deoxy-2',2'-difluorocytidine (gemcitabine) and therefore is a promising drug candidate for cancer chemotherapy.

Administration, Oral↗
PubMed1999

Gene amplification of human cytidine deaminase proviral cDNA and increased levels of its mRNA produces enhanced drug resistance to cytosine arabinoside in retroviral-transduced murine fibroblasts.

Hematopoietic toxicity is one of the major problems that limits the effectiveness of many antineoplastic drugs. One approach to overcome this problem is to confer chemoresistance to the hematopoietic cells by gene transfer of drug resistance genes. Human cytidine deaminase (CD) inactivates the cytosine nucleoside analogues, such as cytosine arabinoside (ARA-C), by deamination. We have reported previously that retroviral-mediated gene transfer of CD conferred drug resistance to ARA-C in murine cells. One of the major problems in the use of these vectors is to obtain adequate and prolonged expression of the transferred gene to produce a therapeutic effect in the transduced cells. The objective of this investigation was to determine if it is possible to increase the expression of CD proviral DNA in transduced murine fibroblast cells. We observed that by the use of continuous exposure to increasing concentrations of ARA-C it was possible to enhance drug resistance in the transduced cells. This drug resistance was found to be associated with increases in CD enzyme activity and CD proviral mRNA and by amplification of the proviral CD gene.

3T3 Cells↗
PubMed1982

Two simple high-performance liquid chromatographic methods for simultaneous determination of 2'-deoxycytidine 5'-triphosphate and cytosine arabinoside 5'-triphosphate concentrations in biological samples.

Cytosine arabinoside (ara-C) has been used in the treatment of leukemia, but its exact mechanism of cytotoxicity is not yet known. One of the proposed mechanisms for the effectiveness of this drug in treating leukemias suggests that a metabolite of ara-C, i.e., 2'-deoxycytidine 5'-triphosphate (araCTP), competes with cytosine arabinoside 5'-triphosphate (dCTP) for binding to DNA polymerase. The ratio of the drug metabolite to the endogenous nucleotide (araCTP/dCTP) may, therefore, be important in determining the effectiveness of ara-C therapy. This ratio may also play a role in drug resistance. Previously published methods have focused on either araCTP or dCTP, along with metabolites and analogues of one of these compounds. The methods presented here provide two simple, sensitive ways to measure dCTP and araCTP in the same biological sample.

Arabinofuranosylcytosine Triphosphate↗
PubMed1984

Neuroanatomical study of somatomotor cortex in microcephalic mice induced by cytosine arabinoside.

Somatomotor cortex of mice with microcephaly induced by DNA polymerase inhibitor cytosine arabinoside (Ara-C), has been studied with a modified Golgi-Cox staining and a HRP retrograde tracing method. Microcephalic mice were prepared by prenatal injections of cytosine arabinoside on days 13.5 and 14.5 of pregnancy. Cytoarchitectonically, the cerebral cortices of adult microcephalic mice are characterized by atypical pyramidal cells with abnormal dendrites and irregular patterns of cellular lamination. Semiquantitative analyses of the abnormality of dendrites in Golgi-Cox preparation indicate that both the degree and direction of ramification are severely affected in Ara-C treated mice. In adult control cerebrum, original neurons of corticospinal tract labeled after HRP injection into the lumbar cord were situated in layer V. In the microcephalic brains, however, HRP labeled neurons, some of which had abnormal polarity, were scattered throughout all layers. This HRP study for corticospinal tract neurons also confirms the irregular pattern of the cortex in which only three layers are recognized.

Afferent Pathways↗
PubMed2000

Cyclopentenyl cytosine inhibits cytidine triphosphate synthetase in paediatric acute non-lymphocytic leukaemia: a promising target for chemotherapy.

Cytidine triphosphate (CTP) synthetase is a key enzyme in the anabolic pathways of cytosine and uracil ribonucleotide metabolism. The enzyme catalyses the conversion of uridine triphosphate (UTP) into CTP, and has a high activity in various malignancies, which has led to the development of inhibitors of CTP synthetase for therapeutic purposes. We studied both CTP synthetase activity and ribonucleotide concentrations in leukaemic cells of 12 children suffering from acute non-lymphocytic leukaemia (ANLL), and performed incubation experiments with cyclopentenyl cytosine (CPEC), a nucleoside analogue that is capable of inhibiting CTP synthetase. The CTP synthetase activity in ANLL cells (5.1+/-2.3 nmol CTP/mg/h) was significantly higher compared with granulocytes of healthy controls (0.6+/-0.4 nmol CTP/mg/h, P=0.0002), but was not different from the CTP synthetase activity in non-malignant CD34+ bone marrow cells (5. 6+/-2.4 nmol CTP/mg/h). Major shifts were observed in the various ribonucleotide concentrations in ANLL cells compared with granulocytes: the absolute amount of ribonucleotides was increased with a substantial rise of the CTP (2.4 versus 0.4 pmol/microg protein, P=0.0007) and UTP (8.7 versus 1.6 pmol/microg protein, P=0. 0007) concentrations in ANLL cells compared with granulocytes. Treatment of ANLL cells in vitro with CPEC induced a major depletion (77% with 2.5 microM of CPEC) in the concentration of CTP, whilst the concentrations of the other ribonucleotides remained unchanged. Therefore, the high activity of CTP synthetase in acute non-lymphocytic leukaemic cells can be inhibited by CPEC, which provides a key to a new approach for the treatment of ANLL.

Adolescent↗
PubMed2001

Strand displacement recognition of mixed adenine-cytosine sequences in double stranded DNA by thymine-guanine PNA (peptide nucleic acid).

Mixed pyrimidine-purine peptide nucleic acids (PNAs) composed of thymines and guanines are shown to form a PNA(2)-DNA triplex with Watson-Crick complementary adenine-cytosine oligonucleotides and to bind complementary adenine-cytosine targets in double stranded DNA by helix invasion. These results for the first time demonstrate binding of an unmodified PNA oligomer to a mixed pyrimidine-purine target in double stranded DNA and illustrate a novel binding mode of PNA.

Base Composition↗
PubMed2003

Expression of DNA (5-cytosin)-methyltransferases (DNMTs) in hepatocellular carcinomas.

Down-regulation of tumor suppressor genes by hypermethylation of 5'-CpGs is one of the important mechanisms involved in tumor development. DNA (5-cytosine)-methyltransferases (DNMTs) are enzymes that methylate the cytosine residue of CpGs, and four types have been identified (DNMT1, 2, 3a and 3b). To examine the involvement of DNMTs in hepatocellular carcinogenesis, we measured DNMT mRNAs in hepatocellular carcinomas (HCCs). mRNAs of DNMT1, 2, 3a and 3b were detected by reverse transcription-PCR analysis and quantified by a real-time PCR method in surgically resected HCCs and adjacent non-tumorous liver tissue. DNMT1 was expressed in all tissues and at a significantly higher level in HCCs than in non-tumorous liver tissue (P=0.01). DNMT2 was expressed at a low level in all tissues. DNMT3a and DNMT3b mRNA were undetectable in normal liver. DNMT3a was expressed in all HCCs and was expressed at similar levels in 60% of the non-tumorous liver tissues. DNMT3b mRNA was detected at a significantly higher level (P=0.002) in HCCs than in non-tumorous liver tissues. The amount of DNMT1, 3a and 3b mRNA was not different between HCCs with or without hypermethylation of the CDH1 promoter. These data suggest that overexpression of DNMT1 and DNMT3b contributes to hepatocellular carcinogenesis.

Journal Article↗
PubMed1988

Methylation of cytosine in the 5-position alters the structural and energetic properties of the supercoil-induced Z-helix and of B-Z junctions.

The structural and energetic consequences of cytosine methylation in the 5-position on the supercoil-dependent B-Z equilibrium in alternating dC-dG sequences cloned into recombinant plasmids were investigated. The helical parameters determined with the band shift method for right-handed [10.7 base pairs (bp)/turn] and left-handed (12.8 bp/turn) 5MedC-dG inserts were different from the helical repeat values for unmethylated dC-dG inserts (10.5 bp/turn in the right-handed and 11.5 bp/turn in the left-handed form). We analyzed the thermodynamic parameters delta GBZ (free energy difference per base pair between right-handed and left-handed helix structure), delta Gjx (free energy for formation of one B-Z junction), and b (helix unwinding at a junction region) for varying lengths of dC-dG inserts by two-dimensional gel electrophoresis and application of a statistical mechanics model. A comparison of plasmids fully methylated in vitro with HhaI methylase and their unmethylated counterparts revealed that delta Gjx is not significantly changed by cytosine methylation. However, this base modification results in an approximate 3-fold decrease of delta GBZ and an approximate 2-fold decrease of the unwinding b at B-Z junction regions. Analysis of a pair of related plasmids, each containing two dC-dG blocks, revealed qualitatively different transition behaviors. When the two dC-dG blocks were separated by 95 bp of a mixed sequence, they underwent independent B to Z transitions with separate nucleation events and junction formations. When the two blocks were separated by only a 4 bp GATC sequence, only one nucleation event was necessary, and the Z-helix spread across the nonalternating GATC region.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Topoisomerases, Type I↗