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Expressions of Four Major Protein Ser/Thr Phosphatases in Human

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Expressions of Four Major Protein Ser/Thr Phosphatases in Human Leukemia (1999) 13, 595–600 1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu Expressions of four major protein Ser/Thr phosphatases in human primary leukemic cells M Yamamoto1, Y Suzuki1, H Kihira1, H Miwa1, K Kita1, M Nagao2, S Tamura3, H Shiku1 and M Nishikawa1 1The 2nd Department of Internal Medicine, Mie University School of Medicine, Edobashi, Tsu, Mie; 2Biochemistry Division, National Cancer Center Research Institute, Chuo-ku, Tokyo; and 3Institute of Development, Aging and Cancer, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan Activity and expression of four major protein serine/threonine ted with cessation of cell proliferation.5 We reported that oka- (Ser/Thr) phosphatases, protein phosphatase type 1 (PP1), pro- daic acid (OKA) and calyculin-A, potent inhibitors of PP1 and tein phosphatase type 2A (PP2A), protein phosphatase type 2B (PP2B) and protein phosphatase type 2C (PP2C) were evalu- PP2A, augmented either ATRA-induced granulocytic or ated in normal peripheral leukocytes, and in various leukemic 1,25(OH)2D3-induced monocytic differentiation of HL-60 cells from patients with acute myelogenous leukemia (AML), cells.6 ATRA induces downregulation of the PP2A catalytic common acute lymphocytic leukemia (cALL), or chronic lym- subunit in HL-60 cells, whereas PP1 expression is phocytic leukemia (CLL). PP1 was the most abundant phospha- unchanged.7–9 We also found that PP1 catalytic subunit iso- tase in blood cells, and relative abundance of each phospha- Ͼ Ͼ Լ zymes have a differential distribution and translocation during tase was: PP1 PP2A PP2B PP2C. PP1 activity and its 10 expressions were higher in blasts of AML-M4 and -M5 than in 1,25(OH)2D3-induced monocytic differentiation. 1,25 cells of AML-M1, cALL and CLL. PP2A activity and its (OH)2D3 induces upregulation of both PP2B and PP2C in HL- expression were higher in blasts of AML-M3, -M4 and -M5 than 60 cells.11,12 ATRA also induces upregulation of PP2B in HL- in cells of AML-M1, cALL and CLL. Activity and expression of 60 cells, while the PP2C expression is unaltered.13 Thus, both PP1 and PP2A in normal monocytes were highest, and levels of both activity and expression of each protein phos- PP2A activity in normal neutrophils was lowest among normal phatase either increase or decrease during HL-60 cell leukocytes. PP2B activity and its expression were higher in blasts of AML-M2, -M3 and normal lymphocytes. PP2C activity differentiation. and its expression were relatively constant in various leukemic The role and expression of each protein phosphatase have cell types. Activities of PP1 and PP2A of AML blasts correlated yet to be clarified with regard to blast cells from patients with positively with the expression of CD11b, whereas activities of various types of primary leukemia, and in normal mature leu- PP1 and PP2B correlated negatively with the expression of kocytes. Leukemic cells likely arise from a clonal expansion CD7. Thus, each phosphatase was ubiquitously but differently of cells after transformation and arrest at a certain stage of expressed in various leukemic cell types and in normal leuko- 14 cytes. These data also suggest that expressions of PP1, PP2A differentiation. We analyzed activities and enzyme and PP2B are relatively low in leukemic blasts arresting at the expressions of protein Ser/Thr phosphatases in adult human stage of early pluripotent stem cells, and are differently modu- primary leukemic cells, the objective being to evaluate cell lated during the course of myelomonocytic commitment and type- or lineage-specific expression. maturation. Keywords: protein phosphatase type 1; protein phosphatase type 2A; protein phosphatase type 2B (calcineurin); protein phosphatase type 2C; human primary leukemic cells Materials and methods Cells Introduction Leukemic cells from 85 adult Japanese patients (65, 8 and 12 cases of AML, cALL and B-CLL, respectively) referred to our Protein phosphatases, which functionally counteract protein laboratory between 1985 and 1997 were evaluated in this kinases, control a wide range of events such as cell division, study. Informed consent was obtained for these studies from cell signalling, cell motility, gene transcription, apoptosis, cell all patients. AML and cALL diagnoses were made by morpho- proliferation, differentiation and carcinogenesis.1–3 The major logical and cytochemical analyses according to conventions protein Ser/Thr phosphatase catalytic subunits of mammalian of the FAB Cooperative Group.14,15 CLL diagnosis was based cells comprise four forms which have been designated PP1, on established hematological criteria and was confirmed by PP2A, PP2B (calcineurin), and PP2C, based on their substrate an immuno-phenotypic analysis.14 Heparinized venous blood specificity, dependence on metal ions and sensitivity to inhibi- was obtained from these patients prior to the start of chemo- tory agents.1 PP1 and PP2A seem to have no absolute require- + therapy, and mononuclear cells were separated using Ficoll– ment for divalent cations, whereas PP2B and PP2C are Ca2 - + Hypaque density gradient centrifugation. Selection of patient and Mg2 -dependent, respectively. Protein phosphatases may samples was based on the availability of large cell numbers be involved in the processes of differentiation and prolifer- (Ͼ2 × 108 cells) needed to measure the activity and protein ation of human myeloid leukemia cell line HL-60 cells, in expression of protein phosphatase. Samples containing more addition to protein kinases.4 HL-60 cells can be induced to than 95% leukemic cells were frozen in liquid nitrogen and differentiate into monocytes by 1␣,25-dihydroxyvitamin D 3 stored at −80°C until measurements of phosphatase activity. (1,25(OH) D ), or into granulocytes by all-trans retinoic acid 2 3 Normal neutrophils were separated from buffy coats of 10 nor- (ATRA) and Me SO; terminal differentiation is usually associa- 2 mal healthy donors by Ficoll–Hypaque density gradient centri- fugation, and residual erythrocytes were removed by lysis in hypotonic medium. Peripheral blood lymphocytes and mono- Correspondence: M Mishikawa, 2nd Department of Internal Medi- cine, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie cytes were isolated by Ficoll–Hypaque sedimentation pro- 514-8507, Japan; Fax: 81 59 231 5200 duced leukocyte preparations from buffy coats of 10 normal Received 16 September 1998; accepted 17 December 1998 healthy donors. Lymphocyte purification was achieved by Protein phosphatase in human primary leukemic cells M Yamamoto et al 596 removing monocytes by adherence to glass from the elutri- after immediately photographing the visualized band.7–13 The ation fraction. Monocytes were isolated with CD14 mono- signal was then compared with that of known amounts of clonal antibody and magnetic Dynabeads (Dynal, Oslo, each purified recombinant phosphatase. Routinely, at least Norway). The purities of these normal leukocytes were Ͼ90%, three different amounts of each phosphatase were included as as determined by May–Giemsa staining. Surface markers of standards to quantify the level of phosphatase. The amount of AML blasts were assessed by cytofluorometry in an immuno- each phosphatase was expressed as ng per 108 cells. fluorescence-activated cell sorter (Cytron; Ortho Diagnostic System, Tokyo, Japan) using monoclonal antibodies, including CD11b (OKM1), CD11c (LeuM5), CD13 (MCS2), CD14 Statistical analysis (My4), CD33 (My9), CD34 (My10) as myeloid markers, CD7 (WT1 or TP40) as a T cell marker, and CD19 (B4 or Leu12) Differences between groups were evaluated by Student’s as a B cell marker. At least 5000 cells were examined for sur- non-parametric t-test, and P Ͻ 0.05 indicated a significant face marker analysis.16 The specimen was considered positive difference. for a monoclonal antibody if over 15% of cells showed specific labeling above the control. Results and discussion Preparation of cytosolic fractions Figure 1 shows differences in the level of each phosphatase activity in AML (65 cases), cALL (eight cases), and B-CLL (12 Leukemic cells (2 × 108 cells) disrupted in 500 ␮l of homogen- cases) and in normal mature leukocytes. AML cases were ization buffer consisting of 250 mM sucrose and buffer A classified into following subtypes according to French–Amer- (20 mM Tris-HCl, pH 7.5, 2 mM dithiothreitol, 5 mM EDTA, ican–British (FAB) classification:15 12 M1, 20 M2, 12 M3, 12 5mM EGTA, and protease inhibitors, including 75 mg/l phe- M4, 9 M5. Because of the comparatively small number of nylmethyl-sulfonylfluoride, 10 mg/l leupeptin, 10 mg/l N-tos- cases, the AML-M5a and -M5b subclasses were pooled yllysine chloromethyl ketone, and 5 mg/l N-tosyl phenylala- together. PP1 is the most abundant phosphatase among four nine chloromethyl ketone), using a glass to glass Potter– major Ser/Thr phosphatases in blood cells, and relative abun- Elvehjem homogenizer at 4°C.10,12 The supernatant was cen- dance of each phosphatase was PP1ϾPP2AϾPP2BԼPP2C. trifuged at 100 000 g for 1 h to obtain the cytosolic fraction. Although there were considerable variations in the level of PP1 activity, even from patients with a similar clinical diag- nosis, the level of PP1 activity in AML-M1 and lymphoid leu- Measurement of phosphatase activity kemic cells from ALL and B-CLL appeared to be lower than that in other types of AML blasts. The distribution of PP2A Activities of PP1, PP2A, PP2B and PP2C were determined activity among various types of leukemic blasts was similar to using 32P-phosphorylated myosin light chain (MLC) of chicken that of PP1 activity. Cases of AML with myelomonocytic fea- gizzard as a substrate. PP1 and PP2A activities were measured tures (AML-M4 and -M5) exhibited significantly higher activi- in a reaction mixture (50 ␮l) containing 20 mM Tris-HCl, pH ties of both PP1 and PP2A than the less differentiated cases 7.4, 50 mM NaCl, 0.1 mM EGTA, ± 10 nM OKA and ± a heat (AML-M1).
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