Detection of single nucleotide insertion of BCR/ABL region in imatinib-resistant human myelogenous leukemia SR-1 cells

Imatinib mesylate is a selective Bcr/Abl kinase inhibitor and an effective anticancer agent for Bcr/Abl-positive chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Mutations within the BCR/ABL kinase domain are the most commonly identified mechanism associated with relapse. To overcome the imatinib resistance in CML, many investigators have tried to clarify molecular mechanism for imatinib resistance in cells of patients who failed to respond to imatinib. Our aim was to invesitigate underlying mechanism for imatinib resistance in SR-1 cells, which were derived from a CML patient in blast crisis. We detected the new mutation of BCR/ABL, resulting in premature termination and loss of BCR/ABL fusion protein expression, which might be possible mechanism for the resistance to imatinib in SR-1 cells.     

Determination of imatinib and its active metabolite N-desmethyl imatinib in human plasma by liquid chromatography/tandem mass spectrometry

Imatinib is a first-line treatment for chronic myelogenous leukaemia (CML). The pharmacokinetics of imatinib in patients with CML are characterised by large interpatient variability. Concentration monitoring of imatinib and its active metabolite N-desmethyl imatinib (DMI) is considered necessary to enhance the safe and effective use of imatinib. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry assay was developed for the simultaneous determination of imatinib and its metabolite DMI in human plasma. After proteins were precipitated with acetonitrile, imatinib, DMI and the internal standard D8-imatinib were resolved on a Gemini-NX 3?μm C18 column using gradient elution of 0.05?% formic acid and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves of imatinib and DMI were adequately fitted by quadratic equations (r?>?0.999) over the concentration range of 10 to 2,000?ng/mL which encompasses clinical concentrations. Bias was ≤±8.3?%, intra- and inter-day coefficients of variation (imprecision) were ≤8.0?% and the limit of quantification was 10?ng/mL for both imatinib and DMI. The assay is being used successfully in clinical practice to enhance the safe and effective use of imatinib.     

Quantification of Intracellular Thiols by HPLC-Fluorescence Detection

Biothiols, such as cysteine and glutathione, play important roles in various intracellular reactions represented by the redox equilibrium against oxidative stress. In this study, a method for intracellular thiol quantification using HPLC-fluorescence detection was developed. Thiols were derivatized with a thiol-specific fluorescence derivatization reagent, viz. ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), followed by reversed-phase separation on an InertSustain AQ-C18 column. Six different SBD-thiols (homocysteine, cysteine, cysteinylglycine, γ-glutamylcysteine, glutathione, and N-acetylcysteine as an internal standard) were separated within 30 min using a citric buffer (pH 3.0)/MeOH mobile phase. The calibration curves of all the SBD-thiols had strong linearity (R² > 0.999). Using this developed method, the thiol concentrations of human chronic myelogenous leukemia K562 cell samples were found to be 5.5–153 pmol/1 × 10⁶ cells. The time-dependent effect of a thiol scavenger, viz. N-ethyl maleimide, on intracellular thiol concentrations was also quantified. This method is useful for elucidating the role of intracellular sulfur metabolism.     

Zinc, copper and aluminium in the corpuscular components of peripheral blood in patients with pre-terminal and terminal renal failure

Summary Trace elements play an essential role in the activation and regulation of metabolic processes. By using the flameless graphite furnace-AAS, accurate results can be obtained. In order to verify blood cells as more representative than plasma to reflect the whole body content of trace elements at uremic state, two patient groups, one with preterminal renal failure (n=40) and the second with chronic renal failure undergoing hemodialysis (n=20) were examined. The concentration levels of zinc, copper and aluminium were measured in different matrices, i.e. whole blood, plasma and corpuscular blood components. The analytical technique is eligible to isolate mono- and polymorphnuclear leucocytes for the determination of intracellular contents. In agreement with other reports, zinc and copper were decreased and aluminium was increased in the plasma of both patient groups. Different results could be found for intracellular contents with mean zinc concentration of erythrocytes, thrombocytes and leucocytes between mean and higher levels, though staying within normal values. Copper was decreased in erythrocytes and increased in thrombocytes, but not detectable in leucocytes. The results of this study point out the differences between the plasmatic and intracellular alteration of trace element contents in uremic patients. However, the concentration levels of all cellular compartments show high standard deviation, which shows the necessity of measuring additional matrices, e.g. plasma. Uremic symptoms relate to the disorders of the trace element homeostasis. Regarding the increased fragility of erythrocytes with shortened life duration, decreased copper contents could play an essential role. Increased aluminium concentration levels might also be one of the interfering factors in the uremic thrombocytopathy. Yet the measurement of the plasma content is inevitable to diagnose and reflect the whole body content of trace elements at uremic state. By keeping analysis control, important information about progression and influence of therapy can be obtained.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday     

Single-cell sphingosine kinase activity measurements in primary leukemia

Sphingosine kinase (SK) is a promising therapeutic target in a number of cancers, including leukemia. Traditionally, SK has been measured in bulk cell lysates, but this technique obscures the cellular heterogeneity present in this pathway. For this reason, SK activity was measured in single cells loaded with a fluorescent sphingosine reporter. An automated capillary electrophoresis (CE) system enabled rapid separation and quantification of the phosphorylated and nonphosphorylated sphingosine reporter in single cells. SK activity was measured in tissue-cultured cells derived from chronic myelogenous leukemia (K562), primary peripheral blood mononuclear cells (PBMCs) from three patients with different forms of leukemia, and enriched leukemic blasts from a patient with acute myeloid leukemia (AML). Significant intercellular heterogeneity existed in terms of the degree of reporter phosphorylation (as much as an order of magnitude difference), the amount of reporter uptake, and the metabolites formed. In K562 cells, the average amount of reporter converted to the phosphorylated form was 39?±?26 % per cell. Of the primary PBMCs analyzed, the average amount of phosphorylated reporter was 16?±?25 %, 11?±?26 %, and 13?±?23 % in a chronic myelogenous leukemia (CML) patient, an AML patient, and a B-cell acute lymphocytic leukemia (B-ALL) patient, respectively. These experiments demonstrated the challenge of studying samples comprised of multiple cell types, with tumor blasts present at 5 to 87 % of the cell population. When the leukemic blasts from a fourth patient with AML were enriched to 99 % of the cell population, 19?±?36 % of the loaded sphingosine was phosphorylated. Thus, the diversity in SK activity remained even in a nearly pure tumor sample. These enriched AML blasts loaded significantly less reporter (0.12?±?0.2 amol) relative to that loaded into the PBMCs in the other samples (≥1 amol). The variability in SK signaling may have important implications for SK inhibitors as therapeutics for leukemia and demonstrates the value of single-cell analysis in characterizing the nature of oncogenic signaling in cancer. Figure

Phosphorylation of a fluorescent sphingosine kinase reporter was used to measure single-cell SK activity in primary cells from leukemic patients. Peripheral blood mononuclear cells as well as enriched leukemic blasts were analyzed.     

Simple methodology for the therapeutic drug monitoring of the tyrosine kinase inhibitors dasatinib and imatinib

A simple HPLC method has been developed to measure imatinib and N‐desmethylimatinib (norimatinib) in plasma or serum at concentrations attained during therapy. Adaptation of this method to LC‐MS/MS also allows dasatinib assay. A small sample volume (100 μL HPLC‐UV, 50 μL LC‐MS/MS) is required and analysis time is <5 min in each case. Detection was by UV (270 nm) or selective reaction monitoring (two transitions per analyte) tandem mass spectrometry. Assay calibration was linear (0.05–10 mg/L imatinib, 0.01–2.0 mg/L norimatinib and 1–200 µg/L dasatinib), with acceptable accuracy (86–114%) and precision (<14% RSD) for both methods. A comparison between whole blood and plasma confirmed that plasma is the preferred sample for imatinib and norimatinib assay. For dasatinib, although whole blood concentrations were slightly higher, plasma is still the preferred sample. Despite considerable variation in the (median, range) plasma imatinib and norimatinib concentrations in patient samples [1.66 (0.02–4.96) and 0.32 (0.01–0.99) mg/L, respectively, N = 104], plasma imatinib was >1 mg/L (suggested target for response) in all but one sample from patients achieving complete molecular response. As to dasatinib, the median (range) plasma dasatinib concentration was 13 (2‐143) µg/L (N = 33). More observations are needed to properly assess the potential role of therapeutic drug monitoring in guiding treatment with dasatinib. Copyright © 2012 John Wiley & Sons, Ltd.     

Meta-analysis of microarray data: The case of imatinib resistance in chronic myelogenous leukemia

With the proliferation of related microarray studies by independent groups, a natural approach to analysis would be to combine the results across studies. In this article, we address a meta-analysis of the gene expression data on imatinib resistance in chronic myelogenous leukemia. First, an analysis of the overlapping among 6 published studies revealed that only 3 genes were coincident between 2 studies. A later reprocessing using different methods on 4 publicly available datasets revealed that 2 extra genes were overlapped between two sets. Both poor overlappings may be due to large differences in the sample source, the microarray platforms used, and a small difference in gene expression between the imatinib non-responder and responder patients. A search of common genes inside 4 public datasets afforded 404 well defined genes. Nevertheless, this necessary condition for meta-analysis caused the loss of many genes of possible interest. The expression signals of the common genes in the four datasets were reanalyzed using three summary statistical methods for combining quantitative information: Fisher, Stouffer and effect-size. Taking the three methods together and using an FDR < 0.10 threshold, a gene-list with 33 differentially expressed genes was found. Considering all the reanalysis approaches used in this work, a final gene-list with 38 differentially expressed genes is reported. Despite the important limitations to this microarray meta-analysis, the presented procedures and integrated gene-list may have some potential value as regards imatinib resistance in CML patients since it is the first attempt to integrate evidence about gene-lists in this area.     

A non-radioactive assay for precise determination of intracellular levels of imatinib and its main metabolite in Bcr-Abl positive cells

Multidrug resistance (MDR) is often associated with overexpression of the P-glycoprotein (P-gp, ABCB1). It was demonstrated that the P-gp mediated efflux decreases the drug concentration in cancer cells which results in the failure of chemotherapy. However, the MDR phenotype in cancer cells obviously involves various mechanisms. Therefore, if we want to estimate a contribution of the P-gp expression to the MDR phenotype, a clear quantitative relationship between the intracellular drug level and cell sensitivity must be established. To achieve this goal, a sensitive and non-radioactive assay for precise determination of intracellular levels of imatinib and its main metabolite N-desmethyl imatinib (CGP 74588) has been developed. The assay is based on an optimised extraction of cells with 4% formic acid after their separation from the growth medium by centrifugation through a layer of silicone oil. Cell extracts are subsequently analyzed by LC/MS/MS. Calibration curves were linear from 1 to 500 nmol/l for imatinib and from 2 to 500 nmol/l for CGP 74588, with correlation coefficients (r²) better than 0.998 and 0.996, respectively. The limit of quantitation (LOQ) was 1 nmol/l for imatinib and 2 nmol/l for CGP 74588. Our method has been successfully applied to the determination of intracellular levels of imatinib in sensitive K562 and their resistant variant, K562/Dox cells.     

Uranium concentration in blood samples of Southern Iraqi leukemia patients using CR-39 track detector

The simple and effective technique of fission track etch has been applied to determine trace concentration of uranium in human blood samples taken from two groups of male and female participants: leukemia patients and healthy subjects group. The blood samples of leukemia patients and healthy subjects were collected from three key southern governorates namely, Basrah, Muthanna and Dhi-Qar. These governorates were the centers of intensive military activities during the 1991 and 2003 Gulf wars, and the discarded weapons are still lying around in these regions. CR-39 track detector was used for registration of induced fission tracks. The results show that the highest recorded uranium concentration in the blood samples of leukemia patients was 4.71 ppb (female, 45 years old, from Basrah) and the minimum concentration was 1.91 ppb (male, 3 years old, from Muthanna). For healthy group, the maximum uranium concentration was 2.15 ppb (female, 55 years old, from Basrah) and the minimum concentration was 0.86 ppb (male, 5 years old, from Dhi-Qar). It has been found that the uranium concentrations in human blood samples of leukemia patients are higher than those of the healthy group. These uranium concentrations in the leukemia patients group were significantly different (P < 0.001) from those in the healthy group.     

Design and Synthesis of New Imatinib Analogs Containing Thiazolyl Moiety

A series of thiazolyl moiety‐containing pyrimidine derivatives were synthesized and evaluated for their cytotoxicity against chronic myelogenous leukemia cell line K562 and human breast cancer cell line MCF‐7. Most compounds showed good antiproliferation activity.     

Bone marrow scintigraphy with 111In-chloride--a clinical value for the hematological diseases

Bone marrow scintigraphy with indium chloride (111In) was performed in fifty-one patients with the hematological diseases. The results of the investigation were that 1. in all patients, as well as in patients with aplastic anemia, no correlation was there between the degree of the indium chloride accumulation and peripheral blood counts, 2. in patients with aplastic anemia and pure red cell aplasia (PRCA) a tendency to reduction in uptake of indium chloride in bone marrow, 3. in patients with these two good correlation between the degree of indium chloride accumulation and histology of the erythroid bone marrow, but in patients with chronic myelocytic leukemia (CML) and atypical leukemia no correlation between the two, so it seemed unlikely that indium chloride should reflect the effective production of erythrocytes, 4. four patients with leukemia were studied with indium chloride bone marrow imaging two times to evaluate their responses to chemotherapy, and peripheral expansion was no change or reduced in two patients with acute myelocytic leukemia (AML) and one patient with acute lymphocytic leukemia (ALL) who obtained complete remission, but on the other hand, it enlarged in one patient with acute myelocytic leukemia who obtained partial remission, and 5. in two patients with chronic myelocytic leukemia it enlarged up to the ankle joints, which was considerably specific.     

3-Isopropyloxy-6-morpholino-2-phenylphenalen-1-one as Lipophilic Fluorescent Probe for Lymphocyte Investigations

Fluorescent properties of two naphthalimides and a phenalenone derivative in organic solvents and when they bind to human peripheral blood lymphocytes were investigated. Different spectral characteristics were observed using lymphocytes of healthy donors and patients with nonmalignant (chronic myeloid leukemia) and malignant (B-cell lymphoid leukemia) diseases. It was found that spectral properties of the used fluorophores in cell suspension qualitatively characterize its structural and functional alterations during pathological phenomena. The intensity of fluorescence increased in samples from patients with B-cell lymphoid leukemia, and the fluorescence maximum shifted to the long-wavelength region by 20 nm compared with normal lymphocytes. It is concluded that 3-isopropyloxy-6-morpholino-2-phenylphenalen-1-one as most promising probe may be applied to the study of malignant diseases.     

Trace and major elements in serum of patients with chronic myelogenous leukemia

Summary Major and trace elements were determined in serum of patients with chronic myelogenous leukemia (CML) using total reflection X-ray fluorescence induced by synchrotron radiation (SRTXRF). CML affects 1 to 2 people per 100,000 and accounts for 7-20% cases of leukemia. It was possible to determine the concentrations of the following elements: P, S, Cl, K, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Br and Rb. Using analysis of variance (ANOVA) it was observed that the contents of the P, S, Ca, Cr, Mn, Fe, Cu and Rb elements differed significantly at a = 0.05 between groups of healthy subjects and CML patients and also genders (males and females).     

SPR imaging biosensor for the 20S proteasome: sensor development and application to measurement of proteasomes in human blood plasma

The 20S proteasome is a multicatalytic enzyme complex responsible for intracellular protein degradation in mammalian cells. Its antigen level or enzymatic activity in blood plasma are potentially useful markers for various malignant and nonmalignant diseases. We have developed a method for highly selective determination of the 20S proteasome using a Surface Plasmon Resonance Imaging (SPRI) technique. It is based on the highly selective interaction between the proteasome’s catalytic β5 subunit and immobilized inhibitors (the synthetic peptide PSI and epoxomicin). Inhibitor concentration and pH were optimized. Analytical responses, linear ranges, accuracy, precision and interferences were investigated. Biosensors based on either PSI and epoxomicin were found to be suitable for quantitative determination of the proteasome, with a precision of ±10% for each, and recoveries of 102% and 113%, respectively, and with little interference by albumin, trypsin, chymotrypsin, cathepsin B and papain. The proteasome also was determined in plasma of healthy subjects and of patients suffering from acute leukemia. Both biosensors gave comparable results (2860 ng·mL-1 on average for control, and 42300 ng·mL-1 on average for leukemia patients).

Development of a plate-based assay for high-content analysis of individual untethered non-adherent cells

Research involving non-adherent cell lines, primary cells and blood cells is definitely important, but its application in image-based assays, especially in high-content systems, is highly limited. Accordingly, efficient high-content methods to study non-adherent cells are needed not only to improve diagnostics but also for early screening of targeted drugs. A plate-based assay using adhesion reagents for multiparametric measurement with single non-adherent and non-anchored cells in a large cell population in high-content cytometry was developed and optimized. The cells preserved their identity even during extensive biomanipulations. The proposed method is highly robust for better imaging and can be used in various assays in different cellular backgrounds. Furthermore, as exemplary experiments, novel optimized assay protocols were used to study extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity after cell inhibition with imatinib in chronic myelocytic leukemia K562 cells, revealing the phosphorylation kinetics of ERK MAPK. The results showed that the proposed assay detects kinase phosphorylation with good sensitivity and may be used in rapid drug screening.     

Binding Mechanism and Molecular Design of Benzimidazole/Benzothiazole Derivatives as Potent Abl T315I Mutant Inhibitors

Despite the efficacy of imatinib therapy in chronic myelogenous leukemia, the development of drug-resistant Abl mutants, especially the most difficult overcoming T315I mutant, makes the search for new Abl T315I inhibitors a very interesting challenge in medicinal chemistry. In this work, a multistep computational framework combining the three dimensional quantitative structure-activity relationship (3D-QSAR), molecular docking, molecular dynamics (MD) simulation and binding free energy calculation, was performed to explore the structural requirements for the Abl T315I activities of benzimidazole/benzothiazole derivatives and the binding mechanism between the inhibitors and Abl T315I. The established 3D-QSAR models exhibited satisfactory internal and external predictability. Docking study elucidated the comformations of compounds and the key amino acid residues at the binding pocket, which were confirmed by MD simulation. The binding free energies correlated well with the experimental activities. The MM-GBSA energy decomposition revealed that the van der Waals interaction was the major driving force for the interaction between the ligands and Abl T315I. The hydrogen bond interactions between the inhibitors and Met318 also played an important role in stablizing the binding of compounds to Abl T315I. Finally, four new compounds with rather high Abl T315I activities were designed and presented to experimenters for reference.     

利用原子力显微镜探测人正常与慢性淋巴白血病外周血B淋巴细胞

采用Ficoll密度梯度离心法得到人外周血单个核细胞(PBMC),并结合磁珠分选的方法进一步纯化得到正常B淋巴细胞,探索了正常和肿瘤B淋巴细胞之间的差异。通过应用具有高分辨率的原子力显微镜(AFM)对正常人和慢性淋巴白血病人外周血B淋巴细胞进行成像,并对这两种B淋巴细胞的高度、直径、体积及膜表面的颗粒平均高度、平均粗糙度和颗粒分布进行测量,对比观察两组细胞膜表面宏观和纳米结构的变化。结果表明,慢性淋巴白血病B淋巴细胞比正常的B淋巴细胞高大,细胞膜表面颗粒更大且细胞膜粗糙。此外,对这两组淋巴细胞进行了机械性质方面的测量和统计,结果发现慢性淋巴白血病B淋巴细胞粘附力(524.1±160.0)pN比正常B淋巴细胞粘附力(1091±260)pN约小1倍,且癌变的B淋巴细胞硬度明显比正常的小。当正常细胞癌变时,细胞的形貌、超微结构及骨架会发生一定的改变。实验证明应用AFM可在形态学和机械性质上明显区别正常和慢性淋巴白血病B淋巴细胞,为临床诊断慢性淋巴白血病提供新的技术手段。     

Betulin,a Newly Characterized Compound in Acacia auriculiformis Bark,Is a Multi-Target Protein Kinase Inhibitor

The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/β (GSK-3 α/β), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.     

DNA detection of chronic myelogenous leukemia by magnetic nanoparticles

A novel tool for the detection of BCR/ABL fusion gene in chronic myelogenous leukemia (CML) was developed by a magneto-polymerase chain reaction (PCR)-enzyme linked gene technique. The forward primers covalently bound to the surface of magnetic nanoparticles allowed a convenient separation of PCR products with high sensitivity (0.5 pg ml(-1)) and high specificity using K562 cell line and CML patients. The results were obtained when the biotinylated-reverse primer bound to streptavidin-horseradish peroxidase (HRP) and hydrolysed the substrate. This novel readout system was approximately 1000-fold more sensitive than the conventional agarose gel electrophoresis. The present technique is practical and useful for following up CML patients and for providing appropriate treatment, particularly to patients in remote areas.