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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a rapid and convenient method for the quantitative assessment of nuclear translocation of glucocorticoid-receptor complexes in lymphoid cells. In this assay, cells are incubated with [3H]dexamethasone at 4 C or 21 C, treated for 15 min at 4 C with the sulfhydryl blocking reagent sodium tetrathionate or methyl methanethiolsulfonate, and then ruptured with the neutral detergent Triton X-100 to separate the nuclei from the cytoplasmic constituents. When cells are incubated with [3H]dexamethasone at 21 C, an average of 45-60% of the specifically bound cellular radiolabel remains with the nuclei. In contrast, less than 15% of the hormone remains with the nuclei when cells are labeled at 4 C. In rat thymocytes, quantitatively similar results were obtained using this new method and a conventional method of isolating nuclei (homogenization of cells in a hypotonic buffer). For both methods, preincubation of the labeled cells with the sulfhydryl blocking reagents was essential to prevent release of the nuclear hormone (or hormone-receptor complex) by a sulfhydryl-dependent labilizing activity apparently located in the cytoplasm. The new method has been successfully used to quantitate uptake of nuclear [3H]dexamethasone-receptor complexes in rat thymocytes, human lymphocytes, and human leukemia cells of lymphoid and myeloid origin. It should prove successful for separating nuclear from cytoplasmic glucocorticoid-receptor complexes in a wide variety of cells, many of which resist disruption by more conventional techniques.
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PMID:An alternative approach to the quantitation of glucocorticoid-receptor complexes in the nuclei of lymphoid cells. 705 27

Protein-protein interaction of Moloney murine leukaemia virus was studied by an assay where one protein preparation was coupled covalently to Sepharose, and binding of radiolabelled proteins to the protein-Sepharose was examined. It was found that the virus proteins gp70, p30, p15E and p15 in solution could associate weakly to disrupted virus particles and to p30. However, when the disrupted virus particles and p30 were coupled to Sepharose in the presence of Triton X-100, stronger binding of the four proteins was observed. Only low or no binding of p12 and p10 was observed to these protein-Sepharoses. The results are discussed with respect to the assembly and structure of the virus particle.
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PMID:Association of moloney murine leukaemia virus proteins: an assay for hydrophobic protein-protein interactions. 714 70

Roughly 10% of surface glycoproteins in the envelope of mature Friend murine leukemia virus are coupled to membrane polypeptides by disulfide bridges. The remaining 90% of these glycoproteins are associated noncovalently. However, they could also be linked to membrane polypeptides by the treatment of purified Friend murine leukemia virus with 2,2'dithiobis(m-nitropyridine). These amphiphilic heterodimer polypeptides, gp84/86, were recovered almost quantitatively in the form of aggregates, termed rosettes, when prepared by solubilization of the viral membrane with Triton X-100 and subsequent velocity sedimentation. gp69/71 and p12(E)/15(E) were purified from these protein micelles after reduction of the disulfide bonds by gel chromatography. Electron micrographs of rosettes, as well as of purified p12(E)/15(E), showed structures different from native viral knobs. Isolated gp84/86 could be reassociated and then displayed more similarity to these viral surface projections. As shown by peptide mapping, the primary structures of the glycoproteins gp69/71 are highly related as are those of the membrane polypeptides p12(E) and p15(E). Furthermore, it was shown by two-dimensional polyacrylamide gel electrophoresis and re-electrophoresis of purified gp84/86 that the larger component, gp86, was composed of gp71 associated with p15(E) and p12(E), whereas the smaller component, gp84, was formed by gp69 bound only to p12(E).
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PMID:Envelope polypeptides of Friend leukemia virus: purification and structural analysis. 741 88

In an attempt to define the role of cell adhesion molecules (CAMs) within the bone marrow (BM) microenvironment in normal hematopoiesis and in leukemia development, a novel cell-blotting technique that involved cell adhesion to protein bands after separation by lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane has been developed. Human BM stromal cell membrane fractions have been prepared from Dexter-type cultures after cell lysis by sonification and differential centrifugations of the sonification contents. The 20,000 g pellets representing membrane fractions have been solubilized by 2% Triton X-100, 0.575% LDS, and 8 mol/L urea in sequential order. The protein extracts are fractionated by LDS-PAGE and screened for CAMs by the new cell-blotting technique. This led to identification of nine protein bands in lanes containing LDS extracts showing adhesion of KG1a (CD34+ progenitor myeloid) cells. Evidence that the BM proteins exhibiting KG1a cell adhesion are novel CAMs is based on the observations that these proteins, in comparison with known CAMs, specifically VCAM-1, CD54, and CD44, show (1) contrasting detergent-solubility properties, (2) different temperature requirement for mediating cell adhesion function, and (3) markedly distinct electrophoretic mobilities. The various cell types tested, notably KG1a, NALM-6, WIL-2, Ramos, HS-Sultan, K562, JY B lymphoblastoid cells, and T lymphoblasts, showed distinctive patterns of binding to different subsets of BM CAMs. These results demonstrate a new approach to studies of molecular mechanisms that may determine specificity of hematopoietic cellular localization within BM microenvironment and may play an important role in controlling hematopoiesis.
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PMID:Discovery of novel hematopoietic cell adhesion molecules from human bone marrow stromal cell membrane protein extracts by a new cell-blotting technique. 751 96

A disulfide-linked heterodimeric antigen from human B leukemia cells was detected by radioimmunoprecipitation and Western blot analysis using a monoclonal antibody (mAb). The mAb was generated against a cell membrane antigen preparation from human B prolymphocytic leukemia cells and found to define an extracellular epitope of the smaller component (beta chain) of a heterodimeric antigen on human B leukemia cells. The antigen from BALL-1, a human B leukemia cell line, and fresh (uncultured) B prolymphocytic leukemia cells was found to consist of a 44-49 kDa (alpha chain) and a 36-40 kDa (beta chain) component. An additional minor component of 34 kDa was detected in the reduced antigen from BALL-1. For chemical identification of the antigen, we isolated the antigen from a Triton X-100 lysate of BALL-1 by immunoaffinity chromatography using the mAb. Determination of the amino-terminal amino acid sequences of the alpha and beta chains unequivocally identified them as the human mb-1 and B29 proteins, respectively. The sequence analyses indicate the molecular heterogeneity of the mb-1 protein and also perhaps the heterogeneity of the B29 protein. We detected three forms of the mb-1 protein which share an identical amino-terminal amino acid sequence and probably two forms of the B29 protein which share an identical amino-terminal sequence. Our sequence data allowed us to establish the authentic amino-terminal amino acid sequence of the human B29 protein which is different from those proposed by others based on cDNA sequence analyses. Comparison of the amino-terminal sequences of the human mb-1 and B29 proteins with those of the mouse mb-1 and B29 proteins showed that the majority of the conserved amino acids in the mb-1 proteins are hydrophobic amino acids. Such conservation of hydrophobic amino acids is not observed in the amino termini of the human and mouse B29 proteins. A beta-tubulin-like protein was found to be co-purified with the mb-1-B29 antigen in the present study. In addition, we found that there is a strong amino acid sequence homology between the microtubule-binding domains of certain human microtubule-associated proteins and an intracellular segment of the human mb-1 protein.
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PMID:Isolation and chemical characterization of the human B29 and mb-1 proteins of the B cell antigen receptor complex. 751 67

PHA-stimulated human lymphocytes or myelogenous leukemia cells (strain K-562) were pulse labeled with 3H-thymidine and submitted to various fixation-permeabilization procedures. They were then immunostained with the 19A2, 19F4 or PC10 monoclonal antibody against the proliferating cell nuclear antigen (PCNA). The preparations were finally scored for the proportion of unlabeled, double-labeled and single PCNA or 3H-thymidine-labeled nuclei. Unstimulated lymphocytes were immunonegative in all the conditions tested, as also were stimulated lymphocytes checked with an isotype of the primary antibody. A specificity (Sp) and a sensitivity (Se) score was calculated to evaluate the recognition by PCNA staining of the S-phase cells, as defined by the 3H-labeling. The data show that in most instances the three antibodies recognized the 3H-labeled cells with high sensitivity, ie with few false negative, but with low specificity, ie with PCNA positivity extending to variable proportions of non-S-phase cells. By contrast, methanol fixation followed by a brief treatment with the detergent Triton X-100 and immunostaining with either 19F4 or PC10 (but not with 19A2) combined a high sensitivity and specificity scores of the recognition of the 3H-thymidine-labeled cells: PC10 gave a more intense and, hence, more readable reaction. PHA-stimulated lymphocytes that had been preserved at -20 degrees C as cytocentrifuged smears failed to show any immunopositivity for PCNA if not submitted to further fixation prior to the immunocytochemical assay. When methanol-Triton was used for this step, only PC10 gave positive immunoreaction, yet with a lower specificity score (Sp = 76%) than in cells submitted to this fixation-permeabilization procedure without prior cryopreservation (Sp = 91.7%). The PCNA index was measured in cryopreserved, methanol-fixed smears of lymphocytes from patients with various hematological diseases and was compared to the Ki-67 index established independently on a serial sample. A good correlation was found between the two indices (r = 0.79; P < 0.0001) with the PCNA index generally lower than or close to the Ki-67 index. This warrants a note of caution about the use of total (ie stable and labile) PCNA immunostaining to measure the growth fraction (GF), classically defined as the proportion of proliferating cells in a population. However, in the absence of an absolute reference marker for G0 cells, there is no reason to assume that the PCNA index would necessarily be a worse estimate of GF than the Ki-67 index.(ABSTRACT TRUNCATED AT 400 WORDS)
Leukemia 1995 Jun
PMID:PCNA immunopositivity index as a substitute to 3H-thymidine pulse-labeling index (TLI) in methanol-fixed human lymphocytes. 759 73

We evaluated the detection of terminal deoxynucleotidyltransferase by flow cytometry using two commonly reported methods of permeabilization and compared them with immunofluorescence microscopy. Thirty-four consecutive cases referred for leukemia workup were studied prospectively. We found decreased viability of cells with both flow cytometry methods in association with shifts in forward and side scatter patterns. Both procedures demanded careful manipulation of cells to avoid total lysis. The Triton X-100 method had a sensitivity of 100% and a specificity of 93%, while the methanol method had a sensitivity of 54% and a specificity of 100%. Therefore, the Triton X-100 method correlated better with immunofluorescence microscopy than did the methanol method. Despite technical difficulties, the advantages of increased objectivity, increased diagnostic capability associated with multicolor analysis, and the increased sensitivity in detecting minimal residual disease make flow cytometry a more attractive and advantageous procedure.
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PMID:Flow cytometric terminal deoxynucleotidyltransferase analysis. Evaluation of Triton X-100 and methanol permeabilization methods compared with immunofluorescence microscopy. 797 98

The experiments have been undertaken whether DNA contents could be measured using whole blood lysis method by FACScan. Cell population in the phases of G1, S and G2 + M were well analyzed, when we used 3 x 10(6) cells lysed with 0.1% Triton X-100 in 1 ml of phosphate buffered saline, staining with 30 micrograms/ml of propidium iodide (PI) within 30 min after staining with PI. We have further developed cell cycle analysis for cells bearing lineage specific antigens recognized with FITC-conjugated monoclonal antibodies using two color analysis. When we fixed cells with 50% ice-cold ethanol after staining cells with FITC-conjugated antibodies, positive population ratio in these cells have been unchanged before and after fixing for CD3, CD4, CD5, CD8. CD10, CD19, CD14, CD33, and HLA-DR, but CD7 positive cells were markedly decreased after fixing. Using this method, CD41 positive leukemia cells have 3.4% in S phase and 6.8% in G2 + M phase, while CD41 negative cells have 1.8% in S phase and 2.0% in G2 + M phase in a patient with AML: M7, resulting leukemia cells were rich in S phase and G2 + M phase. The similar results were obtained in patients with AML:M2 using CD33 antibodies. During the clinical course, the changes of the blast numbers were well-correlated with changes of S-phase proportion in the patient with AML:M2. Among 47 patients with hematological malignancies in our hospital tested here, only 2 cases with 4.3% of total patients showed to have aneuploidy in malignant cells. One is a patient with non-Hodgkin lymphoma, the other is myelodysplastic syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Analysis of DNA contents in hematological malignant cells using whole blood lysis method]. 799 13

Inside-out membrane vesicles prepared from multidrug resistant human leukemic cells (CEM/VBL1000), but not from sensitive cells, transported [3H]-labelled vinblastine (VBL) in an ATP-dependent manner, reaching a plateau level by 15 min. The transport occurred with an apparent Km of 60 +/- 20nM. Verapamil (10 microM), and taxol (IC50 = 1 microM) prevented VBL uptake and evoked VBL diffusion from vesicles when added after VBL uptake had reached steady state. The channel forming agent alamethicin prevented net uptake of VBL and addition of alamethicin to the vesicles after the steady-state had been reached resulted in the rapid efflux of [3H]VBL. Very low concentrations of Triton X-100 (0.01 % v/v) also prevented net uptake of VBL, whilst addition of Triton X-100 and making the medium hypo-osmotic after the steady state had been reached caused the [3H]VBL to rapidly diffuse out of the vesicles. These observations indicate that VBL is actively transported into the lumen of inside-out vesicles from multidrug resistant leukaemia cells.
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PMID:Vinblastine transport by membrane vesicles from human multidrug-resistant CCRF-CEM leukaemia cells: inhibition by taxol and membrane permeabilising agents. 810 21

A murine monoclonal antibody (mAb i3A; IgG1, kappa light chain) was obtained using human red blood cells as immunogen. The antibody showed Fy6 specificity since it agglutinated all but Fy(a-b-)-untreated red cells and failed to agglutinate chymotrypsin-treated cells. An erythrocyte membrane protein of 42-46 kD was revealed as the major component recognized by the antibody on immunoblots. The antibody also bound to 92- to 95- and 200-kD proteins, tentatively identified as oligomers of the 42- to 46-kD monomeric form. The affinity-purified Fy6-active protein was converted to a sharp band of 35 kD after N-glycanase treatment. The molecule appeared as a slightly broadly band after neuraminidase treatment but was not further altered by O-glycanase. The i3A mAb bound to 6,000 +/- 1,000 receptor sites on either Fy(a-b+), Fy (a+b+) and Fy(a+b-) red cells with an affinity constant in the range of 3-6 x 10(8) M-1. No binding was observed to other blood cells nor to several cells (B, T, myelomonocytic and erythro-leukemia cell lines). Also, the bulk of i3A-Fy6 immune complexes could be dissociated from the red cell membrane with as low as 0.2% Triton X-100, showing that the Fy6-active glycoprotein is not tightly associated with the membrane skeleton. Our data obtained with a new monoclonal antibody directed to the Fy6 antigen demonstrate that the blood group Duffy-active component is a red cell-specific glycoprotein carrying one or more N-linked oligosaccharides.
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PMID:Production of a new murine monoclonal antibody with Fy6 specificity and characterization of the immunopurified N-glycosylated Duffy-active molecule. 814 85


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