Detection of cytosine arabinoside resistant cells at low frequency using the bromodeoxyuridine/DNA assay

This report describes a rapid and sensitive procedure for detection of cytosine arabinoside- (Ara-C) resistant mouse leukemia cells (L1210) in a predominantly Ara-C-sensitive population. L1210 cell lines sensitive or resistant to Ara-C were grown and treated with Ara-C in vitro or in vivo. Ara-C-resistant cells were detected as those cells with S-phase DNA content retaining the ability to incorporate bromodeoxyuridine (BrdUrd) after treatment with Ara-C. The BrdUrd incorporation ability of the S-phase cells was assessed by simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated BrdUrd. The proportion of Ara-C-resistant cells was accurately estimated at frequencies approaching 10(-3).     

Determination of 1-β- -arabinofuranosylcytosine and 1-β- -arabinofuranosyluracil in human plasma by high-performance liquid chromatography

A method is described for the determination of 1-β- -arabinofuranosylcytosine (Ara-C) and its metabolite 1-β- -arabinofuranosyluracil (Ara-U) in human plasma. After deproteinization of the plasma sample, separation is performed by reversed-phase liquid chromatography. For Ara-C concentrations exceeding 0.05 mg/l and for Ara-U concentrations exceeding 1 mg/l, injection volumes of 100 μl are applied. For lower concentrations an injection volume of 500 μl is used. Ara-C is detected at 280 nm with a lowest detection limit of 0.002 mg/l in plasma. Ara-U is detected at 264 nm with a lowest detection limit varying from 0.01 to 0.1 mg/l in plasma. This variation is caused by an unknown substance with the same elution properties as Ara-U and which appears to be present in plasma in variable concentrations. The coefficient of variation of the whole procedure is about 6% for Ara-C concentrations above 0.005 mg/l and for Ara-U concentrations above 0.1 mg/l. For lower concentrations the coefficient of variation is about 14%.     

Determination of 1-β-d-arabinofuranosylcytosine and 1-β-d-arabinofuranosyluracil in human plasma by high-performance liquid chromatography

A method is described for the determination of 1-β-d-arabinofuranosylcytosine (Ara-C) and its metabolite 1-β-d-arabinofuranosyluracil (Ara-U) in human plasma. After deproteinization of the plasma sample, separation is performed by reversed-phase liquid chromatography. For Ara-C concentrations exceeding 0.05 mg/l and for Ara-U concentrations exceeding 1 mg/l, injection volumes of 100 μl are applied. For lower concentrations an injection volume of 500 μl is used. Ara-C is detected at 280 nm with a lowest detection limit of 0.002 mg/l in plasma. Ara-U is detected at 264 nm with a lowest detection limit varying from 0.01 to 0.1 mg/l in plasma. This variation is caused by an unknown substance with the same elution properties as Ara-U and which appears to be present in plasma in variable concentrations. The coefficient of variation of the whole procedure is about 6% for Ara-C concentrations above 0.005 mg/l and for Ara-U concentrations above 0.1 mg/l. For lower concentrations the coefficient of variation is about 14%.     

Effect of high dose cytosine arabinoside on quantitative EEG in patients with acute myeloid leukemia

Background EEG activity is considered an index of functional state of brain. Chemotherapy (CT), used for non-central nervous system (CNS) cancer, can cross the blood brain barrier and contribute to changes in the functional state of brain that can alter background EEG activity. Quantitative EEG (qEEG) is superior to conventional EEG in the detection of subtle alterations of EEG background activity and for this reason, the use of qEEG might assist the clinician in evaluating the possible effect of CT on the CNS. The nucleoside analog cytosine arabinoside (Ara-C) is one of the milestone chemotherapeutic agents used for treatment of acute myeloid leukemia (AML). Our observational study evaluates the possible effect of Ara-C on the qEEG of patients with AML, without CNS involvement. We conducted an observational study on newly diagnosed AML patients without CNS involvement, undergoing treatment with Ara-C to analyze the possible effect of Ara-C high doses on EEG background activity using qEEG analyses. A total of nine AML patients, 5 with Ara-C i.v. high dose (≥3 g/m² die), 4 with standard dose (100 mg/m² die) underwent qEEG (at rest, during hyperpnoea, mental arithmetic task and blocking reaction). We compared the EEG background activity of the two groups at baseline and after 6 months. Statistical analysis showed no significant differences between the two groups in mean relative power for all frequency bands, at rest and during hyperpnoea, mental arithmetic task and blocking reaction. Our data indicate that high dose Ara-C i.v. did not induce significant changes on EEG background activity in our patients. Future research in this area could include prospective studies that would combine qEEG and neuropsychological testing to assess the impact of CT on brain functions.     

Induction of endoreduplication in Chinese hamsters V79 cells by cytosine arabinoside

Endoreduplication (ER) could be induced very effectively in Chinese hamster V79 cells exposed to cytosine arabinoside (1-β-D-arabinofuranosylcytosine; Ara-C). Cells were cultured for 48 hours in Ara-C containing medium. ER frequency increases rapidly after Ara-C release. About 60% of metaphase cells were endoreduplicated at 8–10 hours after release from Ara-C (5 μg/ml). Induction of ER also depends on Ara-C concentrations.     

Cytokinetic properties of asynchronous and cytosine arabinoside perturbed murine tumors measured by simultaneous bromodeoxyuridine/DNA analyses

This paper describes the determination of cytokinetic properties of asynchronous and cytosine arabinoside- (Ara-C) treated KHT tumors growing in vivo using the bromodeoxyuridine (BrdUrd)/DNA analysis technique. The cytokinetic properties of asynchronously growing tumors were estimated by computer analysis of sequential BrdUrd/DNA distributions measured at 2- to 3-h intervals after administration of a single i.p. injection of BrdUrd. The cytokinetic properties of the Ara-C-treated tumors were estimated by computer analysis of BrdUrd/DNA distributions measured at 2- to 3-h intervals after Ara-C treatment. BrdUrd was injected 30 min prior to tumor harvest. The cytokinetic properties of the cells rendered nonclonogenic by Ara-C were followed in BrdUrd/DNA distributions measured at 2- to 3-h intervals after Ara-C treatment of tumors that were labeled with BrdUrd 30 min prior to Ara C injection. The G1-, S-, and G2M-phase durations were estimated to be 7.6, 10.9, and 2.0 h prior to Ara-C; decreasing to 1.2, 4.1, and 1.4 after Ara-C. The growth fraction was estimated to be 0.8 prior to Ara-C. Complete recruitment of the normally noncycling subpopulation was observed after Ara-C treatment. Ara-C-killed cells were removed from the tumor within 24 h following Ara-C injection. These cytokinetic properties were similar to those reported in other studies.     

Effects of cytosine arabinoside on differential gene expression in embryonic neural retina. II. Immunochemical studies on the accumulation of glutamine synthetase

Cytosine arabinoside (Ara-C) elicits a significant increase in the level of the enzyme glutamine synthetase (GS) while it markedly reduces overall RNA and protein synthesis in cultures of embryonic chick neural retina. This increase was analyzed by radioimmunochemical procedures and compared with the induction of GS by hydrocortisone (HC). Accumulation of GS in Ara-C-treated retinas was found to be due to de novo synthesis of the enzyme; however, unlike the induction of GS by HC, Ara-C caused no measurable increase in the rate of GS synthesis. The results indicate that Ara-C facilitates GS accumulation largely by preventing degradation of the enzyme. Even though Ara-C inhibits the bulk of RNA synthesis in the retina, it does not stop the formation of GS-specific RNA templates. However, the progressive accumulation of these templates does not result in an increased rate of GS synthesis unless Ara-C is withdrawn from such cultures under suitable experimental conditions. Thus, it is suggested that the continuous presence of Ara-C imposes a reversible hindrance at the translational level which limits the rate of GS synthesis. The results demonstrate that the increase in retinal GS elicited by Ara-C is achieved through mechanisms which are quite different from those involved in the hydrocortisone-mediated induction of this enzyme.     

Enhancing effects of cytosine arabinoside on ethyl methanesulfonate-induced 6-thioguanine resistance mutations in Chinese hamster V79 cells

We studied the synergistic enhancement effects of two chemicals which are different in their mechanism of action on DNA in cells. The test chemicals used were ethyl methanesulfonate (EMS) as an alkylating agent and cytosine arabinoside (Ara-C) as an analogue of cytidine. For determination of mutagenesis we measured the induction of resistance to 6-thioguanine (6-TG) in Chinese hamster V79 cells. EMS had a strong mutagenic effect on V79 cells, but for Ara-C the results were less clear. In this study, Ara-C had no detectable effect in inducing mutation up to a concentration of 5 X 10(-4) M. The mutation frequency of combined treatment with EMS and Ara-C was significantly higher than that obtained with EMS alone. These results indicate that Ara-C had an enhancing effect on mutations induced by EMS.     

In vivo enrichment of cytidine deaminase gene-modified hematopoietic cells by prolonged cytosine-arabinoside application

Background aimsDrug-resistance genes have been explored as powerful in vivo selection markers in hematopoietic cell gene therapy, and cytidine deaminase (CDD) represents a particularly attractive candidate given the virtual absence of non-hematopoietic side-effects after low/intermediate dose application of the associated drug cytosine-arabinoside (Ara-C).MethodsWe investigated the in vivo selection potential of CDD overexpression and prolonged low/intermediate-dose Ara-C application in a murine model. Furthermore, non-transplanted mice were utilized to study Ara-C toxicity in different hematopoietic cell compartments.ResultsSignificant protection of myelo- and thrombopoiesis and up to 6-fold in vivo enrichment of CDD-transduced hematopoietic cells was observed. Enrichment was most robust early after Ara-C application and was correlated with dosage and duration of chemotherapy. Enrichment remained significant for several weeks, indicating selection at the level of a progenitor population. This notion was supported by Ara-C toxicity studies, demonstrating profound hematotoxicity and a marked delay in hematopoietic recovery, specifically in the progenitor/stem cell compartment after low/intermediate-dose Ara-C. Conclusions. These data support the concept of CDD/Ara-C as a clinically applicable in vivo selection system in hematopoietic gene therapy. The data also demonstrate marked differences in hematotoxicity between alternative Ara-C dosing schemes and suggest thorough in vivo toxicity studies to optimize further Ara-C dosing en route to safe and stable enrichment of gene-corrected hematopoiesis.     

Effects of naringin on cytosine arabinoside (Ara-C)-induced cytotoxicity and apoptosis in P388 cells

Naringin (NG), a flavonoid in grapefruit and citrus, has been reported to exhibit antioxidant effects and pharmacological actions. Recently, we have reported that NG suppressed the cytotoxicity and apoptosis induced by H(2)O(2), a typical pro-oxidant, in mouse leukemia P388 cells. Cytosine arabinoside (1-beta-d-arabinofuranosylcytosine; Ara-C) is the most important antimetabolite chemotherapeutic drug used for acute leukemia. It has been suggested that Ara-C-induced cytotoxicity is caused by apoptosis, which is mediated by reactive oxygen species (ROS). In this study, we examined the effect of NG on the cytotoxicity and apoptosis in mouse leukemia P388 cells treated with Ara-C. Ara-C caused cytotoxicity in a concentration and time-dependent manner in the cells. N-Acetyl-L-cysteine (NAC), cystamine (CysA) or a reduced form of glutathione (GSH), typical antioxidants significantly blocked Ara-C-induced cytotoxicity. Similarly, Ara-C-induced cell death was completely prevented by NG. NG strongly reduced ROS production caused by Ara-C in the cells. NG slightly increased the activities of antioxidant enzymes, catalase and glutathione peroxidase. Ara-C caused apoptosis with nuclear morphological change and DNA fragmentation. NG remarkably attenuated the Ara-C-induced apoptosis. NG completely blocked the DNA damage caused by Ara-C treatment at 6 h using the Comet assay. Our data suggest that NG reduces Ara-C-induced oxidative stress through both an inhibition of the generation of ROS production and an increase in antioxidant enzyme activities. Consequently, NG blocked apoptosis caused by Ara-C-induced oxidative stress, resulting in the inhibition of the cytotoxicity of Ara-C.     

Mechanism of interaction of the antileukemic drug cytosine arabinoside with aromatic peptides: role of sugar conformation and peptide backbone

Interaction of the antileukemic drugs, cytosine-arabinoside (Ara-C) and adenosine-arabinoside (Ara-A) and a structural analogue, cytidine, with aromatic dipeptides has been studied by fluorescence and NMR spectroscopy. Ara-C and cytidine bind tryptophanyl and histidyl dipeptides but not tyrosyl dipeptides, while Ara-A does not bind to any of them. Both studies indicate association involving stacking of aromatic moieties. NMR spectra also indicate a protonation of the histidine moiety by Ara-C. In case of cytidine, the chemical shifts observed on binding to His-Phe imply that the backbone protons of the dipeptide participate in the binding. The conformation of the sugar and the base seem to play a very important role in the binding phenomenon as three similar molecules, Ara-C, Ara-A and cytidine bind in totally different ways.     

Mechanism of the antiviral action of 1-beta-D-arabinofuranosylcytosine on Borna disease virus

Borna disease virus (BDV) is a nonsegmented, negative-stranded RNA virus that causes neurological diseases in a variety of warm-blooded animal species. Recently, we showed that the nucleoside analog 1-beta-D-arabinofuranosylcytosine (Ara-C) was a potent inhibitor of BDV. This finding was surprising for an RNA virus, since Ara-C is a DNA polymerase inhibitor. Thus, we sought to better define the mechanism of action of Ara-C on BDV. Here, we show that (i) this effect is specific for an arabinoside ring carrying a cytosine base, (ii) it requires phosphorylation of the nucleotide, and (iii) it can be reversed by an excess of cytidine. Using the recently described minigenome assay for BDV, we provide evidence suggesting that Ara-C may act as a competitive inhibitor of the BDV replication complex.     

HES4抑制急性T淋巴细胞白血病细胞系Jurkat对阿糖胞苷的敏感性

摘要 目的：研究hes家族bHLH转录因子4（HES4）与急性T淋巴细胞白血病（T-ALL）细胞系Jurkat对化疗药物阿糖胞苷（Ara-C）的敏感性作用。方法：构建Lenti-pCDH-HES4-puro质粒,包装慢病毒感染Jurkat细胞,感染空载体Lenti-pCDH-puro为对照组,puromycin筛选阳性细胞,Realtime-PCR检测转录水平HES4过表达情况,CCK8法检测Jurkat对Ara-C敏感性,Ara-C终浓度为0.1、1、10、100、1000、10000 nmol?L^-1;设计3对针对于HES4的特异sgRNA序列,构建LentiGuide-sgRNA1-puro、LentiGuide-sgRNA2-Puro、LentiGuide-sgRNA3-puro质粒,LentiGuide-puro空载体为对照组,包装慢病毒感染Jurkat细胞,puromycin筛选,提取基因组DNA,PCR扩增sgRNA的靶序列,一代测序检测HES4的敲除效率,CCK8法检测敲除HES4后Jurkat对Ara-C敏感性,Ara-C终浓度为0.1、1、10、100、1000、10000 nmol?L^-1。结果：与对照组相比,HES4在Jurkat中过表达（2.37 ± 0.09）倍（P < 0.001）,在Ara-C为1 nmol?L-1、100 nmol?L^-1 浓度下,过表达HES4使Jurkat对Ara-C的药物敏感性降低（P值分别小于0.01、0.05）; sgRNA1、sgRNA 2、sgRNA 3敲除分值分别为83、71、63,其中sgRNA1的敲除效果最佳,在Ara-C为1000 nmol?L^-1浓度下,敲除HES4促进Jurkat对Ara-C的敏感性（P < 0.05）,3条sgRNAs之间无明显区别（P > 0.05）。结论：HES4抑制T-ALL细胞系Jurkat对Ara-C的敏感性,为T-ALL化疗耐受的机制研究提供指导。     

Exploring the possible mitoprotective and neuroprotective potency of thymoquinone,betanin, and vitamin D against cytarabine-induced mitochondrial impairment and neurotoxicity in rats' brain

It has been suggested that cytarabine (Ara-C) induces toxicity via mitochondrial dysfunction and oxidative stress. Therefore, we hypothesized that mitochondrial protective agents and antioxidants can reduce cytarabine-induced neurotoxicity. For this purpose, 48 male Wistar rats were assigned into eight equal groups include control group, Ara-C (70 mg/kg, i.p.) group, Ara-C plus betanin (25 mg/kg, i.p.) group, Ara-C plus vitamin D (500 U/kg, i.p.) group, Ara-C plus thymoquinone (0.5 mg/kg, i.p.) group, betanin group, vitamin group, and thymoquinone group. The activity of acetylcholinesterase (AChE), and butyrylcholinesterase (BChE), the concentrations of antioxidants (reduced glutathione and oxidized glutathione), oxidative stress (malondialdehyde) biomarkers, mitochondrial toxicity parameters as well as histopathological alteration in brain tissues were measured. Our results demonstrated that Ara-C exposure significantly declines the brain enzymes activity (AChE and BChE), levels of antioxidant biomarkers (GSH), and mitochondrial functions, but markedly elevate the levels of oxidative stress biomarkers (MDA) and mitochondrial toxicity. Almost all of the previously mentioned parameters (especially mitochondrial toxicity) were retrieved by betanin, vitamin D, and thymoquinone compared to Ara-C group. These findings conclusively indicate that betanin, vitamin D, and thymoquinone administration provide adequate protection against Ara-C-induced neurotoxicity through modulations of oxidative, antioxidant activities, and mitochondrial protective (mitoprotective) effects.     

1-beta-D-arabinofuranosylcytosine inhibits borna disease virus replication and spread

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that causes neurological diseases in a variety of warm-blooded animal species. There is general consensus that BDV can also infect humans, being a possible zoonosis. Although the clinical consequences of human BDV infection are still controversial, experimental BDV infection is a well-described model for human neuropsychiatric diseases. To date, there is no effective treatment against BDV. In this paper, we demonstrate that the nucleoside analog 1-beta-D-arabinofuranosylcytosine (Ara-C), a known inhibitor of DNA polymerases, inhibits BDV replication. Ara-C treatment inhibited BDV RNA and protein synthesis and prevented BDV cell-to-cell spread in vitro. Replication of other negative-strand RNA viruses such as influenza virus or measles virus was not inhibited by Ara-C, underscoring the particularity of the replication machinery of BDV. Strikingly, Ara-C treatment induced nuclear retention of viral ribonucleoparticles. These findings could not be attributed to known effects of Ara-C on the host cell, suggesting that Ara-C directly inhibits the BDV polymerase. Finally, we show that Ara-C inhibits BDV replication in vivo in the brain of infected rats, preventing persistent infection of the central nervous system as well as the development of clinical disease. These findings open the way to the development of effective antiviral therapy against BDV.     

Structural impact of the leukemia drug 1-beta-D-arabinofuranosylcytosine (Ara-C) on the covalent human topoisomerase I-DNA complex

1-beta-d-Arabinofuranosylcytosine (Ara-C) is a potent antineoplastic drug used in the treatment of acute leukemia. Previous biochemical studies indicated the incorporation of Ara-C into DNA reduced the catalytic activity of human topoisomerase I by decreasing the rate of single DNA strand religation by the enzyme by 2-3-fold. We present the 3.1 A crystal structure of human topoisomerase I in covalent complex with an oligonucleotide containing Ara-C at the +1 position of the non-scissile DNA strand. The structure reveals that a hydrogen bond formed between the 2'-hydroxyl of Ara-C and the O4' of the adjacent -1 base 5' to the damage site stabilizes a C3'-endo pucker in the Ara-C arabinose ring. The structural distortions at the site of damage are translated across the DNA double helix to the active site of human topoisomerase I. The free sulfhydryl at the 5'-end of the nicked DNA strand in this trapped covalent complex is shifted out of alignment with the 3'-phosphotyrosine linkage at the catalytic tyrosine 723 residue, producing a geometry not optimal for religation. The subtle structural changes caused by the presence of Ara-C in the DNA duplex may contribute to the cytotoxicity of this leukemia drug by prolonging the lifetime of the covalent human topoisomerase I-DNA complex.     

Recycling of resting cells in the JB-1 ascites tumour after treatment with 1-beta-D-arabinofuranosylcytosine (ara-C).

Resting cells in tumours present a major problem in cancer chemotherapy. In the plateau phase of grwoth of the murine JB-1 ascites tumour (i.e. 10 days after 2-5 X 10(6) cells i.p.) large fractions of non-cycling cells with G1 and G2 DNA content (Q1 and Q2 cells) are present, and the fate of these resting cells was investigated after treatment with 1-beta-D-arabinofuranosylcytosine (Ara-C).The experimental work of growth curves, percentage of labelled mitoses curves after continuous labelling with 3H-TdR, and cytophotometric determination of single-cell DNA content in unlabelled tumour cells. Treatment with an i.p. single injection of Ara-C 200 mg/kg in the plateau JB-1 tumour resulted in a significant reduction in the number of tumour cells 1 and 2 days later as compared with untreated controls, while no difference in the number of tumour cells was observed after 3 days. In tumours prelabelled with 3H-TdR 24 hr before Ara-C treatment, a significant decrease in the percentage of labelled mitoses was observed 6-8 hr later followed by a return to the initial value after 12 hr, and a new pronounced fall from 20 hr after Ara-C. The second fall in the percentage of labelled mitoses disappeared when the labelling with 3H-TdR was continued also after Ara-C treatment. Cytophotometry of unlabelled tumour cells prelabelled for 24 hr with 3H-TdR before Ara-C treatment showed 20 hr after Ara-C a pronounced decrease in the fraction of Q1 cells paralleled by an increase in the fraction of unlabelled cells with S DNA content. The results indicate recycling of resting cells first with G2 and later with G1 DNA content, which contribute to the regrowth of the tumours.     

Low-dose Ara-C in the treatment of acute leukemia. Cytotoxicity or differentiation induction?

The differentiation inducing effect of low-dose Ara-C on human myeloid leukemic cells was studied in two patients with subacute myelocytic and subacute myelomonocytic leukemia in vivo and in vitro. By continuous i.v. administration of 10 mg Ara-C/m2 over 12 h daily for 12 or 20 days complete remissions were obtained in both patients with normalization of the incidence of the committed progenitor cells BFU-E and CFU-C in the marrow while the incidence of pluripotent CFU-GEMM remained subnormal. Parallel cultures of the patients' bone marrow cells in diffusion chambers (DC) implanted in mice demonstrated a clear cytotoxic effect of low-dose Ara-C. The greater increase of granulopoietic cells within DC in the Ara-C exposed group than in control mice after the end of drug administration is, in addition, an indication for differentiation induction by this kind of Ara-C therapy.