Deoxycytidylate deaminase activity in non-stimulated and phytohemagglutinin-stimulated human lymphocytes,and in leukemic cells

Deoxycytidylate deaminase isolated from normal human lymphocytes and from mononuclear leucocytes from patients with acute lymphoblastic leukemia, chronic lymphocytic leukemia and acute monocytic leukemia has been characterized in regard to the substrate, dAMP and the allosteric regulators dCTP and dTTP. The enzymes exhibited sigmoidal initial velocity versus dCMP concentration whereas in the presence of the activator, dCTP, Michaelis-Menten kinetics were obtained.At saturating substrate concentrations dTTP acted as an allosteric inhibitor of the enzyme isolated from non-stimulated as well as from stimulated lymphocytes. However, the enzymes isolated from the leukemic cells had lost the allosteric regulation by dTTP.At low substrate concentrations the competitive inhibitor, dAMP, activated all the enzymes. This activation was abolished in the presence of dCTP which indicates that dAMP might be involved in the regulation of dCMP deaminase activity and thus influence the dCTP and dTTP pools under physiological conditions.Abbreviations dCMP deaminase deoxycytidylate deaminase - PHA Phytohemagglutinin - ALL acute lymphoblastic leukemia - CLL chronic lymphocytic leukemia - AMOL acute monocytic leukemia - WBC white blood cells     

Differentiation of myeloid cells in liquid culture: 2. Acute myelocytic leukemia cells

In a companion paper we demonstrated that normal peripheral blood granulocytic precursor cells differentiate after 2-3 weeks in suspension culture. In the studies described here leukemic blast cells obtained from 14 patients with acute myelocytic leukemia (AML) and two patients with chronic myelocytic leukemia in blastic crisis were cultured in McCoy's 5A medium containing 15 per cent fetal bovine serum for 2-3 weeks at 37 degrees C in an atmosphere of 5 per cent CO2-95 per cent room air. 'Spontaneous' myeloid differentiation (20 x 10(4) viable mature myeloid cells ml-1) occurred in the cultures of cells obtained from 8 pts. The differentiation was granulocytic in three cases, monocytic in four cases and of mixed type in one case. Differentiation was independent of the growth of the cells in culture and occurred in four cases after the first week. Monocytic differentiation was seen only in AML of the FAB M4 type whereas granulocytic or mixed differentiation were seen only in AML of the FAB M1 or M2 types. When PHA leucocyte conditioned medium (PHA-LCM) was added to the cultures monocytic/macrophage differentiation was favoured. Inducers of the differentiation of the HL-60 cell line (N-methylacetamide, cytosine arabinoside, or retinoic acid) had no consistent effect on the differentiation and were at times inhibitory. Three patients received therapy with low dose cytosine arabinoside and no correlation was observed between the outcome of the treatment and leukemic cell differentiation in culture in the presence of the drug.     

Detection of terminal deoxynucleotidyl transferase (TdT) by flow cytometry in leukemic disorders

We applied a new technique to the detection of intracellular TdT in 26 leukemic patients, including 16 non-T acute lymphoblastic leukemia (ALL), four T-ALL, one T-lymphoblastic lymphoma in leukemia phase, one undifferentiated leukemia, one de novo lymphoblastic phase of chronic myeloid leukemia, and three acute monocytic leukemias (AMOL). Mononuclear cell suspensions were incubated in saponin to permeabilize the cell membrane. The cells were then stained by indirect immunofluorescence (IF) using anti-human TdT monoclonal antibodies and were analyzed by flow cytometry (FCM). The TdT results were compared with those obtained by biochemical TdT assay (26 cases), immunoperoxidase determination (PAP) (12 cases), and fluorescence microscopy (seven cases). The results obtained by PAP and fluorescence microscopy were 100% concordant with those obtained by FCM and biochemical assay. TdT determination by FCM allows the analysis of large numbers of cells in a fast, objective, and reliable manner, as compared with biochemical assay, PAP, and fluorescence microscopy determinations.     

Recombinant human colony stimulating factor-granulocyte/macrophage and -granulocyte, but not macrophage induce the development of a respiratory burst in primary human myeloid leukemic cells in vitro

Respiratory burst develops in myeloid blast cells if they differentiate functionally along the monocytic or granulocytic lineage. Using the nitroblue tetrazolium (NBT) assay we studied the effects of recombinant human granulocyte/macrophage colony stimulating factor (rhuGM-CSF), rhuG-CSF and rhuM-CSF on development of respiratory burst activity in primary blast cells from patients with myeloid leukemia. Assessing suspension cultures containing cells from patients with acute myeloid leukemia (AML, n = 13) or myeloid-blast crisis (myBC) of chronic myeloid leukemia (CML, n = 5) it was found that the percentage of NBT positive cells was increased by at least 20% as compared to control cultures by rhuGM-CSF in 6/17 cases, by rhuG-CSF in 7/17 cases and by rhuM-CSF in 0/16 cases, representing in 'responders' a mean increase of 267% and 270% in the absolute number of NBT positive cells by rhuGM-CSF and rhuG-CSF, respectively. Morphological examination of cultured cells from 'responders', as compared to controls, showed decreased blast cell content but generally no evidence of terminal differentiation. The demonstration of Auer rods in NBT positive cells indicates that respiratory burst developed in a leukemic clone. These findings may be of physiological, pathophysiological and clinical relevance.     

Further evidence for the B-cell nature of hairy cells. A study using immunostaining of splenic tissue with a wide panel of monoclonal antibodies

Phenotypic characterization of neoplastic cells from 5 patients with hairy cell leukemia (HCL) was performed with 29 monoclonal and 6 polyclonal (anti-Ig) antibodies using immunoperoxidase staining of fresh frozen splenic tissue. Monotypic Ig was expressed in 4 cases, one case was non-expressive. Strong staining was obtained in all cases by monoclonal antibodies (MAs) specific for 3 pan-B-lymphocyte antigens (by anti-B 1, To 15, anti-Leu 12). Five other B-cell related antigens detectable with appropriate MAs (BA-1, anti-B2, DAKO-C3 b R, Tü 1, 38.13) were absent in all cases. The stainings with 13 T-cell associated MAs (OKT 3, OKT 4, anti-Leu 3 a, OKT 6, OKT 8, Tü 68, OKT 10, anti-Lyt 2, Tü 71, OKT 11, anti-Lyt 3, Tü 14, Tü 33) were all negative. Stainings with 4 MAs recognizing myelocytic and/or monocytic antigens (OKM 1, anti-Mo 1, anti-Mo 2, 3 C4) were also negative. We included 14 frozen biopsies with B-type chronic lymphatic leukemia (B-CLL) into our immunohistological study in order to establish phenotypic differences between HCL and B-CLL. Five MAs (Tü 1, anti-Lyt 2, Tü 71, BA-1 and anti-B2) gave consistently negative staining in HCL cases but positive staining in most or all B-CLL cases. The study provides significant evidence for the B-cell nature of HCL and also establishes important phenotypic differences between HCL and B-CLL.     

Membrane protein hMYADM preferentially expressed in myeloid cells is up-regulated during differentiation of stem cells and myeloid leukemia cells

We report here the molecular cloning and characterization of a novel human gene (hMYADM) derived from a human bone marrow stromal cell (BMSC) cDNA library, which shares high homology with mouse myeloid-associated differentiation marker (MYADM). hMYADM is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved MYADM-like family. hMYADM with 322-residue protein contains eight putative transmembrane segments and confocal microscopic analysis confirmed its membrane localization by using anti-hMYADM monoclonal antibody. hMYADM mRNA was selectively expressed in human monocytes, dendritic cells, promyeloid or monocytic leukemia cell lines, but not in CD4+, CD8+, CD19+ cells, nor in T cell leukemia or lymphocytic leukemia cell lines. hMYADM expression was also found in normal human bone marrow enriched for CD34+ stem cells, and the expression was up-regulated when these cells were induced to differentiate toward myeloid cells. The mRNA expression level of hMYADM significantly increased in acute promyelocytic leukemia HL-60 and chronic myelogenous leukemia K562 cell line after phorbol myristate acetate (PMA)-induced differentiation. Our study suggests that hMYADM is selectively expressed in myeloid cells, and involved in the myeloid differentiation process, indicating that hMYADM may be one useful membrane marker to monitor stem cell differentiation or myeloid leukemia differentiation.     

Microangiopathic hemolytic anemia in patients with diseases of the hematopoietic and lymphatic systems

Microangiopathic haemolytic anaemia was diagnosed in the course of haematopoietic and lymphatic disorders such as chronic granulocytic leukemia, chronic myelofibrosis, chronic lymphatic leukemia, Osler's disease, chronic monocytic leukemia, and lymphoplasmocytic lymphoma, in 11 patients (6 women and 5 men) aged between 33 and 81 years (mean age 58.8 years) treated at the Haematological Out-Patient Clinic of the Postgraduate Medical Education Centre within 1977-1987. The following laboratory tests were carried out: 1) morphology of the peripheral blood and bone marrow, especially some haematological parameters concerning erythrocytes and blood platelets; 2) biochemical tests reflecting erythrocytes disintegration; 3) haemostasis. All examined patients suffered from haemolytic anaemia of various degree with characteristic changes in erythrocyte shape (helmets, tear-drops etc.). Haemolytic origin of anaemia was confirmed by the increased LDH activity. In the majority of patients no compensative stimulation of haematopoiesis (reticulocytosis, red blood cells hyperproliferation in bone marrow) was seen. Clinical symptoms of haemostatic disorders such as haemorrhagic diathesis and vein thrombosis were diagnosed in 50% of the patients. Blood platelet counts ranged from markedly decreased to significantly increased. Bone marrow smears did not show increased number of megacariocytes. Bleeding time was prolonged in the majority of examined patients while prothrombin index--decreased). Abnormal fibrinogen levels (decreased or increased) were found in the majority of patients with fibrin degradation products. Microangiopathic haemolytic anaemia in these patients differ from the typical Moschowitz's disease clinically probably due to the lack of compensative stimulation of erythropoiesis and lower thrombocytopenia.     

Maturation inducer activity and the process of human myeloid leukemia cell differentiation

The process and mechanism of human myeloid leukemia cell differentiation induced by T-cell lymphokine maturation inducer activity was investigated. The maturation inducer activity was purified from conditioned medium of normal peripheral blood lymphocytes and shown to be a 50,000 M.W. protein. The degree of maturation of myeloid cell cultures was directly related to the dosage of the inducer. The interaction of the leukemia cells with the inducer led to initiation of terminal differentiation to monocytic cells. Proliferation cessation of the leukemia cells and the expressions of mature monocytic cells indicated a continuous and multistaged process.     

Myelogenous leukemia and electric blanket use

In a case-control study of adult acute and chronic myelogenous leukemia in Los Angeles County, we tested the hypothesis that excess exposure to electromagnetic fields from electric blankets was associated with risk of leukemia. We did this by studying 116 cases of acute myelogenous leukemia (AML) and 108 cases of chronic myelogenous leukemia (CML) along with matched neighborhood controls. The cases and controls were queried as to electric blanket use and the risks computed. For AML the risk was 0.9 (95% CI 0.5-1.6) and for CML the risk was 0.8 (95% CI 0.4-1.6). Cases did not differ from controls by duration of use, year of first regular use, year since last use, or socioeconomic status. Our best estimates of exposure indicate that electric blanket use increases overall exposure to electric fields by less than 50% and magnetic fields by less than 100%. We conclude that there is no major leukemogenic risk associated with electric blanket use in Los Angeles County.     

Detection of trisomy 12 by fluorescence in situ hybridization on archival cytopathologic material in chronic lymphocytic leukemia/small lymphocytic lymphoma

OBJECTIVE: To evaluate fluorescence in situ hybridization (FISH) for the detection of trisomy 12 in archival cytologic specimens of chronic lymphocytic leukemia/small lymphocytic lymphoma. STUDY DESIGN: The cytopathology database was searched for all cases of chronic lymphocytic leukemia/small lymphocytic lymphoma. Six cases of chronic lymphocytic leukemia/small lymphocytic lymphoma obtained by fine needle aspiration and one case of small lymphocytic lymphoma with plasmacytoid features were analyzed for trisomy 12 by FISH. These cases had been archived between 1 week to 16 months prior to analysis. RESULTS: We detected trisomy 12 in four of the six cases of small lymphocytic lymphoma/chronic lymphocytic leukemia. The case of small lymphocytic lymphoma with plasmacytoid features was negative for trisomy 12. CONCLUSION: Detection of trisomy 12 by FISH can be effectively performed on routinely prepared, stained and coverslipped archival cytologic material.     

Reactivity patterns of monoclonal antibodies positive on myelomonocytic leukemia cells as defined by esterase isoenzyme analysis

Summary The reactivity with monoclonal antibodies (MoAbs) specific for myelomonocytic cells and the expression of a particular esterase isoenzyme were analyzed in 159 cases of acute myeloid leukemias. The incidence of positivity of 16 MoAbs (MCS-2, MCS-1, OKM1, My-1, Leu-M1, Leu-M3, CA-2-38, MY4, MY7, MY8, MY9, VIM-D2, VIM-D5, Mo1, Mo2, 63D3) was studied using the indirect immunofluorescence technique. A carboxylic esterase isoenzyme which can be inhibited completely and selectively by sodium fluoride (NaF) was demonstrated by isoelectric focusing on horizontal polyacrylamide gels. This NaF-sensitive isoenzyme indicated the monocytic origin of the blast cells as it is specific for this cell lineage. Prior to the immunological-isoenzymatic analysis all cases were categorized into two subtypes according to morphological criteria of the FAB classification system: 147 cases of AML (FAB M1-3) and 12 cases of AMMoL/AMoL (FAB M4/5). However, 15 out of 147 cases of AML expressed the NaF-sensitive isoenzyme and were therefore assigned to the group AMMoL/AMoL. Likewise, 1 case, diagnosed morphologically as AMMoL, was negative for this marker isoenzyme and was assigned to the other leukemia subtype. The incidence of reactivity varied widely for the MoAbs tested regarding the overall results on all cases and the positivity on cases of either AML or AMMoL/AMoL. The MoAbs were grouped into four classes depending on the pattern of reactivity with myeloblastic or monoblastic or both subtypes of acute myeloid leukemia. The MoAbs MCS-2, MY7, Leu-M1, and MY9 detected the vast majority of cases with either myelocytic or monocytic involvement (group-I: pan-myelomonocytic reactivity). The MoAbs MCS-1, OKM1, VIM-D5, and Mo1 showed a predominance in their staining pattern for monocytic variants, but were also positive on a substantial percentage of nonmonocytic cases (group-II: predominantly reactive with monocytic, but also myelocytic cases). The MoAbs Leu-M3, MY4, VIM-D2, Mo2, and MY8 reacted with the large majority of AMMoL/AMoL cases and with a small number of AML cases (group-III: monocyte-specific reactivity). The MoAbs of group-I are useful in differentiating acute lymphoid from acute myeloid leukemias. The MoAbs of group-III, and to a lower extent those of group-II, will be of considerable value in the subtyping of acute myeloid leukemias. The results show that (1) accuracy of leukemia classification might not always be achieved by morphology alone, but that immunological and biochemical aspects should be included as well, and (2) several MoAbs are very useful tools for classification and subtyping of acute myeloid leukemias.     

Active cell membrane mechanisms involved in the exclusion of RH 123 allow distinction between normal and tumoral cells

Human cell lines derived from three epithelial carcinomas (CaSki, HeLa, SiHa), one B lymphoma (BL60), one promyelocytic (HL60), one monocytic (U937) leukemia, one chronic myelogenous leukemia (sensitive K562S; multichemoresistant K562R) and normal human skin fibroblasts were compared for their capacity of staining with rhodamine 123 (Rh 123) and their kinetics of dye exclusion. Cells were exposed for 30 min to 10 g/ml of Rh 123 in culture medium; fluorescence intensity was measured by flow cytometry immediately or 1, 2, 3 and 4 h after staining. The highest fluorescence intensity was observed in carcinoma cell lines; there was no incorporation in multichemoresistant K562R cells. Exclusion of Rh 123 was evaluated from 0 to 4 h, both by flow cytometry and by fluorimetry. Fluorescence intensity measured by flow cytometry decreased slightly in carcinoma and leukemia cells and rapidly in fibroblasts. In all cell lines Rh 123 exclusion was inhibited by 40 mol/L verapamil and 5 mmol/L probenecid. Thus, incorporation and exclusion of Rh 123 allows distinction between normal and tumoral cells; moreover, inhibition of exclusion by verapamil and probenecid favors the involvement of active cell membrane mechanisms in the exclusion process.Abbreviations PBS phosphate-buffered saline - Rh 123 rhodamine 123     

NSE/αNAE positivity in B-lineage acute lymphoblastic leukemia: revisiting a potential cytochemical diagnostic pitfall

Cytochemical staining for leukemia typing is declining in hematology laboratories, but the use of flow cytometry may not be possible in some settings. Aberrant cytochemical nonspecific esterase/α-naphthyl acetate esterase (NSE/αNAE) positive B-lymphoblasts can cause confusion with monoblasts, a potentially dangerous pitfall. This unusual cytochemical NSE/αNAE positivity had been associated with relatively poorer outcome of acute lymphoblastic leukemia (ALL) in the era prior to the advent of routine multicolor flow cytometric immunophenotyping. We reviewed morphological, cytochemical and flow-cytometric data from five cases of B-lineage ALL that showed NSE/αNAE positivity and were diagnosed definitively using multi-parametric flow cytometric immunophenotypic analysis. Diffuse or dot-like (localized) strong cytochemical NSE/αNAE activity was detected in all cases and all showed one or more features of high risk disease. The number of NSE/αNAE positive blasts in the marrow varied from 10 to 75%. The morphological differential diagnoses included T-lymphoid lineage ALL and acute monoblastic leukemia (AML-M5). Flow cytometric data revealed B-lineage antigens and the absence of monocytic or other myeloid markers resolved the diagnosis. These cases underscore the importance of immunophenotyping in all cases of suspected ALL regardless of the cytochemical findings. Although the numbers are small, the association with high risk disease observed in all five of our cases may corroborate the previously reported poor prognostic value of such aberrant cytochemical staining.     

Affinity chromatographic purification of human lysozyme, with special reference to human leukemia lysozyme.

Lysozyme [EC 3.2.1.17] was purified from human tears, serum, and urine of acute monocytic leukemia patients, renal disease patients, and residents in cadmium-polluted areas of Tsushima Island using an affinity adsorbent containing lysozyme-lysate of Micrococcus lysodeikticus cell walls as the ligand. By means of this procedure, leukemia lysozyme was purified 100- to 200-fold with an activity recovery of 80%. It was crystallized at pH 10. This purified preparation appeared homogeneous in disc electrophoresis and showed a specific activity 2.5-fold higher than that of crystalline lysozyme from hen egg-white. Tear lysozyme was also purified to a nearly homogeneous state while the enzymes from normal serum and urine of a nephrosis patient and of residents in cadmium-polluted area were still disc electrophoretically heterogeneous and showed low specific activity as compared with purified leukemia lysozyme.     

Expression of ��1,3-N-acetylglucosaminyltransferases during differentiation of human acute myeloid leukemia cells

The expressions of β1,3-N-acetylglucosamonyltransferase-2 and -8 (β3GnT-2, β3GnT-8),-the two main glycosyltransferases responsible for the synthesis of poly-N-acetyllactosamine (polyLacNAc) in glycans, and β3GnT-5 participating in the syntheses of sphingoglycolipids were studied in leukemia cell lines during differentiation using RT-PCR method. β3GnT-2 and β3GnT-8 distribute widely in six myeloid and monocytoid leukemia cell lines with different abundances, while β3GnT-4 was only present in NB4 cells. ATRA (all-trans retinoic acid) and dimethylsulfoxide (DMSO), which induce the differentiation of HL-60 and NB4 (two human acute myeloid leukemia cell lines) to myelocytic lineage, up-regulated these two enzymes with various degrees at 2 and 72 h of treatment. In HL-60 cells treated with ATRA, the increase of β3GnT-8 was more than β3GnT-2, while in NB4 cells treated with DMSO, the increase of β3GnT-2 was more than β3GnT-8. However, when HL-60 and NB4 were differentiated to monocytic lineage induced by phorbol 12-myristate 13-acetate the expressions of β3GnT-2 and β3GnT-8 showed no alterations or the increase of expressions was far less than those in myelocytic differentiation. By means of FITC-labeled tomato lectin affinity staining and flow-cytometry, it was found that the product of β3GnT-2 and -8, polyLacNAc was also increased on the cell surface of HL-60 and NB4 treated with ATRA or DMSO, but unchanged when treated with PMA. These results were in accordance with the up-regulation of the mRNAs of β3GnT-2 and -8. The expression of β3GnT-5, however, was not changed both in myelocytic and monocytic differentiations. The difference in the up-regulation of β3GnT-2 and -8, especially their products may become a useful index to discriminate the myelocytic and monocytic differentiation of leukemia cells.     

N-acetyl-beta-glucosaminidase activity in normal and malignant leukocytes.

An improved cytochemical method demonstrating N-acetyl-beta-glucosaminidase in peripheral blood and bone marrow leukocytes is described. A significant elevation in enzyme activity in circulating monocytes from patients with solid tumor malignancies was observed. In a large series of cases of acute nonlymphocytic leukemia, elevated levels were found in the vast majority of those leukemias that had a predominant monocytic component identified either morphologically or by standard cytochemical methods. This reaction would appear to be useful as a monocyte marker.