Leukemic Leukocytes Research Articles

The detection of membrane-associated complement components (C 3 and C 4) on circulating human normal and leukemic leukocytes and on cultured cells with monkey erythrocytes.

Monkey erythrocyte (Mk) rosette formation is described as an exquisitely sensitive method for the detection of complement (C) components on the membrane of human leukocytes. Blocking of the immune adherence receptor on Mk blocked subsequent rosette formation as did pretreatment of leukocytes with antiserum to the C components C 3 and C 4. In vitro C deposition by immune complex formation with normal human lymphocytes enhanced Mk rosette formation, and this could be inhibited with antiserum to C 3. The use of Mk rosette formation revealed that cells from a wide variety of human lymphoid and myeloid leukemias carry membrane-bound C. It was also shown that several lymphoblastoid cell lines, including a T cell line, probably synthesize both C 3 and C 4. Mk rosette formation is not dependent on metabolic activity of the rosetting leukocyte, and it is suggested that this technique will be of value in detecting C deposition in a variety of situations.

Inosine 5?-phosphate dehydrogenase activity in normal and leukemic blood cells

The enzyme inosinic acid dehydrogenase (EC 1.2.1 [14]) was measured and partially purified (10- to 15-fold) from normal and leukemic leukocytes. From the normal blood cells, the highest activities could be detected in lymphocytes and bone marrow cells. Dependent on the blast cell count, the leukemic IMP dehydrogenase had a higher mean specific activity than the enzymes of fractionated, immature bone marrow cells, or normal granulocytes. The partially purified enzymes from the various blood cells were apparently identical; they exhibited hyperbolic substrate saturation kinetics and were inhibited by a number of purine nucleotides. For the leukemic blast cell enzyme, the Km values for the substrates, IMP and NAD+, were 28 +/- 11; 227 +/- 98 microM, and 34 +/- 10; 240 +/- 67 microM for the partially purified enzyme from normal, immature bone marrow cells. The hypoxanthine-guanine and adenine phosphoribosyltransferase activities increased in the leukemic cells when compared with mature granulocytes, but nearly always showed similar activities when compared with fractionated bone marrow cells. Only one of the 30 investigated leukemic patients exhibited a marked decrease in hypoxanthine phosphoribosyltransferase activity of 0.5 nmol/mg/h. The phosphoribosyltransferase-specific activities of the leukemic cells are more variable than for the normal ones and no correlation of enzyme activities and blast cell count was apparent.

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection. The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemic leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE 2, PGD 2, PGF 2α, TXB 2 and 6-keto-PGF 1α. In all types studied PGE 2 and TXB 2 were the major products formed. The identification of PGE 2 and TXB 2 was confirmed by GC/MS with multiple ion monitoring. The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.

Comparative studies on phenol-soluble nonhistone chromatin proteins in normal and leukaemic human leukocytes

Phenol-soluble chromatin proteins which may be involved in gene control mechanisms have been isolated from citric acid nuclei of normal and leukaemic human leukocytes derived from freshly obtained venous blood. They were compared by one-dimensional gel electrophoresis and by comparative labelling with14C-leucine or32P-orthophosphate. In addition, the influence of intercalating agents such as adriamycin and daunomycin was studied.

Isolation and sequence organization of human ribosomal DNA

The genes coding for 28 S and 18 S ribosomal RNA have been purified from leukemic leukocytes of one human individual by density gradient centrifugation. The purified ribosomal DNA was analyzed by restriction endonuclease digestion and electron microscopy. The location of cleavage sites for the restriction endonuclease EcoRI was established by R-loop mapping of restriction fragments by electron microscopy. The results are in agreement with gel analysis and gel transfer hybridization. One type of ribosomal DNA repeating unit contains four cleavage sites for EcoRI. Two of these cuts are located in the genes coding for 28 S and 18 S rRNA, while the other two are in the non-transcribed spacer. Thus, one of the restriction fragments generated contains non-transcribed spacer sequences only and is not detected by gel transfer hybridization if labeled rRNA is used as the hybridization probe. A second type of repeating unit lacks one of the EcoRI cleavage sites within the non-transcribed spacer. This indicates that sequence heterogeneity exists in human rDNA spacers. R-loop mapping of high molecular weight rDNA in the electron microscope reveals that the majority of repeats are rather uniform in length. The average size of 22 repeats was 43.65(±1.27) kb. Two repeats were found with lengths of 28.6 and 53.9 kb, respectively. This, and additional evidence from gels, indicates that some length heterogeneity does exist in the non-transcribed spacer. The structure of the human rDNA repeat is summarized in Figure 10.

Normal, Transformed and Leukemic Leukocytes.

Normal, Transformed and Leukemic Leukocytes.

Normal, Transformed and Leukemic Leukocytes

Normal, Transformed and Leukemic Leukocytes

Specific and non-specific folate binding protein in normal and malignant human tissues.

Binding of tritiated folic acid by supernatants prepared from extracts of normal and leukaemic leucocytes, normal mucosa, and malignant tumours from different parts of the gastrointestinal tract has been measured using Sephadex-gel filtration and albumin-coated charcoal techniques. Non-specific binding (measured by Sephadex G-75 gel filtration) was almost invariably greater than specific binding measured by albumin-coated charcoal separation of bound and unbound folate. In nine normal leucocyte extracts, binding measured by Sephadex G-75 filtration ranged from 1.3 to 18.2 (mean 8.2) pg/mg protein and by albumin-coated charcoal from 1.0 to 14.8 (mean 6.7) pg/mg protein. Raised specific binding was found in the extracts from leucocytes of eight of 14 patients with chronic granulocytic leukaemia, in four substantially so (389, 121, 108, 59.7 pg/mg protein), but was only marginally increased in one of eight cases of acute myeloid leukaemia and in two of five cases of chronic lymphocytic leukaemia. Binding was normal in the extracts of all three cases of acute lymphoblastic leukaemia tested. Among the tissues of the gastrointestinal tract binding was greatest by the duodenal mucosa and liver. Extracts of carcinoma of the stomach and colon bound greater amounts of (3)H-folic acid than the corresponding normal mucosal extracts but the differences were not large. Sephadex G-200 gel chromatography showed more than one binding peak in the extracts of liver and duodenum but only one peak in the other tissues of the gastrointestinal tract, and only one peak, of molecular weight either about 50 000 or over 200 000, in the leucocyte extracts.

I and i antigens on normal and leukemic leukocytes

Human leukocyte I and i antigens were quantitated using 125I labelled purified antibodies. Binding of these antibodies to leukocytes was dependent on reduced temperature. No significant difference in antigen content was observed between normal and leukemic myeloid leukocytes. B lymphocytes bound much greater amounts of both I and i antibodies than did T lymphocytes. Neoplastic lymphoid cells bound widely divergent amounts of both antibodies with chronic lymphocytic leukemia and lymphosarcoma cell leukemia cells binding much decreased amounts compared to normal lymphocytes. Cells from patients with hairy cell leukemia bound very large quantities of these antibodies in a cold dependent fashion. These elevated levels of binding were not due to nonspecific binding of IgM.

Merocyanine 540 as a fluorescent probe of membranes: Selective staining of leukemic and immature hemopoietic cells

We have reported ( Easton, Valinsky and Reich, 1978) that merocyanine 540 (MC 540) specifically stains a variety of living excitable cells, but not nonexcitable cells. This paper describes the exceptional permeability to MC 540 of leukemic leukocytes and immature hemopoietic precursor cells. We have used fluorescence microscopy and uptake of radioactive dye to study MC 540 staining of peripheral blood leukocytes from 80 leukemic and 34 normal individuals; leukemic leukocytes stain, whereas normal leukcytes do not. The leukocyte staining reaction differs from that previously described for excitable cells since it is independent of the ionic composition of the staining medium, kinetically complex, enhanced by light, enhanced by oxygen and essentially irreversible. Virtually all circulating nucleated cells from leukemic individuals are stained to approximately the same extent, and there is no qualitative or quantitative distinction between the various forms of leukemia. We have also found that MC 540 interacts with granulopoietic colony-forming cells (CFU-C) and with spleen colony-forming cells derived from mouse bone marrow (CFU-S). We cannot as yet identify a specific property of leukocyte plasma membranes that determines MC 540 permeability; since changes in MC 540 uptake appear to be correlated with cellular maturation during normal hemopoiesis, the retention of staining by leukemic cells, some of which appear morphologically normal, may indicate a failure in membrane maturation during leukemic blood cell development.

Glycogen phosphorylase from human normal and leukemic leucocytes: Activities and interconversions between active and inactive forms

1. 1. Leukemic leucocyte extracts show a lower glycogen phosphorylase and amylo (1,6) glucosidase activities than normal leucocytes. 2. 2. Two forms of phosphorylase, interconvertibles by phosphorylation-dephosphorylation reactions, are present in all types of leucocytes isolated. 3. 3. Total phosphorylase activity in leucocyte extracts can be measured with 1 mM AMP −0.4 M Na 2SO 4. 4. 4. Lymphocyte and leukemic leucocyte phosphorylase is related to the liver type. 5. 5. Glucose, glucose-6-phosphate and ATP appear to be important metabolites in regulating phosphorylase activity.

Quantitative estimation of cationic leukocyte antigen (cla) and lysozyme in leukaemic leukocytes (author's transl)

Cationic lysosomal proteins such as cationic leukocyte antigen (CLA) and lysozyme were estimated in leukocyte lysates of acute and chronic leukaemics using quantitative enzymatic and immunoprecipitation techniques. The studies demonstrate that both lysosomal conponents are markers of non-lymphatic leukaemias. Therefore the determination of CLA and lysozyme is valuable for the differential diagnosis of acute leukaemias. The intracellular cationic protein contents showed characteristic kinetic variations as showing during X-irradiation of the spleen in cases of chronic myelocytic leukaemia. White cells in leukocytosis could be distinguished from normals by remarkably low cationic protein contents in leukocyte lysates. The results are interpreted in the light of current results revealed by immunofluorescent analyses.

Trimethoprim-induced elevation of dihydrofolate reductase activity in human leukocytes.

The administration of trimethoprim (TMP)--a diamino benzylpyrimidine compound which binds very tightly the bacterial dihydrofolate reductase--was accompanied by the appearance of measurable levels of dihydrofolate reductase in peripheral leukocytes from patients with nonhematological diseases. In all instances, enzyme activity rose rapidly between the fourth and eighth day after TMP. The time course of the rise and fall of dihydrofolate activity approaches cellular life span and is similar to that obtained after methotrexate or triamterene administration. Dihydrofolate reductases, partially purified from leukocytes of patients treated with TMP, bone marrow and leukemic leukocytes, had simila molecular weights, pH optima, Ki of inhibitor (methotrexate); they were stimulated to the same degree by KCl and urea. Electrophoresis of the enzyme on cellulose acetate strip resulted in the separation of two enzymatically active protein components. No differences in the electrophoretic behavior of the three blood cell enzymes were noted. The findings noted above are consistent with the suggestion that the observed rise in dihydrofolate reductase activity is a quantitative one. Moreover, the effect of TMP in vivo is discussed in comparison with the currently held hypothesis for methotrexate action (stabilization by the drug of a previously synthetized enzyme).

Structure analysis of small proteins by electron microscopy: Valinomycin, bacitracin and low molecular weight cell growth stimulators

Dark field electron microscopy was combined with optical filtering to study at high resolution the structure of the cyclopeptide antibiotics, bacitracin and valinomycin, and two proteins of unknown structure, LMW-CSA N and B, low molecular weight granulocyte colony stimulating activity isolated from medium conditioned with normal or leukemic leukocytes. For bacitracin and valinomycin the images faithfully represented the known structural features at a resolution of 0.5 nm or better, depicting a two-ring structure for bacitracin, as well as the position of the potassium ion in valinomycin. Both proteins of unknown structrue had at least one cyclic peptide portion. LMW-CSA N had a size of 2.0 nm, LMW-CSA B of 2.4 nm. A potential site of the calcium ionophoric activity in the latter protein was found to be in the larger of the two ring portions constituting the molecule.

DNA repair in human leukemic leucocytes treated with neocarzinostatin.

Neocarzinostatin, an antineoplastic agent which is effective against human leukemia, induced unscheduled DNA synthesis in human leukemic leucocytes. This indicated the existence of repair process against at least a part of DNA damage caused by the agent. The extent of unscheduled DNA synthesis increased in parallel with the concentration of Neocarzinostatin up to 5 microgram/ml, followed by a decline at more than 5 microgram/ml. Leucocytes from patients with chronic myelogenous leukemia had higher repair synthesis after Neocarzinostatin treatment compared to those from patients with acute leukemia. The amount of repair synthesis correlated well with the proportion of immature leucocytes among chronic myelogenous leukemia samples, but such correlation was not found among acute leukemia samples.

Catalase in salivary gland striated and excretory duct cells. II. phi body: an ellipsoidal peroxisomal organelle with crystalloid axial projections.

A new distinctive and unique peroxisomal organelle with a spindle shape has been observed in luminal epithelial cells of striated and excretory ducts of mouse salivary glands. Light microscopic studies indicate it has an ellipsoidal centre from which catalase-positive filamentous or rod-like processes protrude along its major axis; hence, it is called a phi body. A role for this specialized peroxisome in the formation of nearby free filaments and rods is suggested by the frequent observation of segmentation of its axial processes. Complementary ultrastructural studies of osmium-fixed preparations show that the deformation to an oval shape results from the pressure of the extruding crystalloid coincident with the major axis of the ellipsoidal body. The size range and conformation of phi body axial processes are comparable to those of free catalase-positive rods and filaments observed in the same cells. The periodic substructure of the crystalloid in the phi body core is identical with that of nearby cytoplasmic rods. These observations are consistent with the view that the rods and filaments observed free in the cytoplasm are formed by extrusion from the crystalloid core of the phi body. phi Bodies could also be responsible for the Aver rods of leukemic leukocytes.

Immunologic detection of intracellular and cell-surface lysozyme with human and experimental leukemic leukocytes

Intracellular lysozyme (muramidase) was detected by both indirect and direct immunofluorescent microscopy in human and rat leukocytes by labeling methanol-fixed cells with the globulin fraction of immune rabbit serum prepared against the homologous lysozyme. Normal human polymorphonuclear leukocytes (PMNLs), monocytes, and cells from patients with chronic myelogenous leukemia, acute myelogenous leukemia, and acute monomyelogenous leukemia were lysozyme positive, while lymphocytes, basophils, eosinophils, and cells from patients with acute lymphatic leukemia were negative. Similarly, normal rat PMNLs, monocytes, and cells from three transplantable rat myelogenous leukemias were positive in the rat lysozyme assay, while normal rat lymphocytes and spontaneous acute mononuclear cell leukemias of two inbred strains were negative. By labeling viable cells with the purified F(ab 1) 2 fragment of anti-human lysozyme immunoglobulin, lysozyme could be detected on the surface of cells and surface-lysozyme-positive cells could be separated from other human bone marrow leukocytes using a fluorescence activated cell sorter. Lysozyme appears to be a potentially valuable cell-surface marker for identification and separation of myeloid, monocytic, and histiocytic cells.

Low-molecular-weight peptide inhibits RNA synthesis in human leukemic and phytohemagglutinin-stimulated leukocytes and globin mRNA transcription in differentiating Friend cells.

The RNA synthesis of human leukemic leukocytes and phytohemagglutinin-stimulated lymphocytes is markedly reduced by administration of a low-molecular-weight nonhistone peptide factor from calf thymus. Treatment with the factor strongly inhibits hemoglobin production and globin mRNA transcription in dimethyl sulfoxide-stimulated Friend cells without appreciably modifying the rate of cell growth. Evidence for specificity of these effects is provided by the lack of action of the factor on both growth rate and RNA synthesis of a number of nondifferentiating cell lines from various animal species. After removal of the compound, both human lymphocytes and Friend cells can be stimulated by phytohemagglutinin and by dimethyl sulfoxide, respectively, ruling out any toxic effect.

Phi Bodies: Peroxidatic Particles that Produce Crystalloidal Cellular Inclusions

Unique, spindle-shaped particles (phi bodies) and rods with peroxidatic activity are found in certain epithelial cells of normal mice, clofibrate-fed rats, and in leukemic leukocytes. The ellipsoidal shape of phi bodies apparently results from the deformation of spherical granules by extrusion of axial crystalloid that subsequently fragments into rods.

Filamin inhibits actin activation of heavy meromyosin ATPase

Filamin is a high molecular weight (240 000) actin-binding protein. It has been isolated from alveolar macrophages [l] , leukemic leukocytes [2] and smooth muscle [3,4] and identified immunochemically in rat and mouse fibroblasts [3,5]. Although the protein is present in substantial amounts in some tissues (l-2% of the protein of macrophage and smooth muscle cells), little is known of its function in the cell. Stossel and Hartwig [ 1,6,7] have shown that when macrophage filamin and actin are mixed together at 25°C they form a gel. Shizuta et al. [4] also observed gel formation when chicken-gizzard filarnin and actin were combined. Although little information is available on what effect filamin might have on actin function and what factors may regulate the interaction of filamin and actin, a known function of actin is activation of the Mg-ATPase activity of myosin or heavy meromyosin (HMM) (for review see ref. [S] ), We have studied the effects of filamin on F-actin activation of HMM ATPase and myosin ATPase and have found that filamin markedly reduced actin activation of these activities.