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). PP1 activity of normal monocytes was highest stable phosphatase inhibitor-2.6–9 PP1 can be taken as the among normal leukocytes and was approximately three times activity sensitive to the inhibitor-2 and PP2A can be taken as higher than that of normal lymphocytes. PP2A activity was the activity blocked by 10 nM OKA. PP2B and PP2C activities significantly lower in the immature form (AML-M1) of the were determined as the activity in the presence of 0.5 mM granulocytic leukemia than in the more differentiated subtypes

CaCl2,5mm MgCl2, 500 nM OKA, 0.1 mg/ml bovine serum (AML-M2 and -M3). PP2A activity of neutrophils was signifi- albumin, 1 ␮M calmodulin and ± 0.15 mM trifluoperazine.11,12 cantly lower than that of monocytes or lymphocytes. The dis- PP1 and PP2A activities were completely inhibited at 500 nM tribution of PP2B activity among various types of leukemic OKA, therefore this activity was taken as the total activity of blasts differed from that of PP1. PP2B activity was significantly PP2B and PP2C. PP2B activity was determined by subtracting higher in AML-M2 and -M3 blasts than that in AML-M1 or PP2C (the activity in the presence of 0.15 mM trifluoperazine) -M5 blasts. PP2B activity of lymphocytes was highest among from the total activity. Under these conditions, the rates of leukocytes, although this difference did not achieve statistical phosphorylation were linear with respect to time and enzyme significance between lymphocytes and monocytes. Lymphoid dilution. Assays were done in duplicate, and the c.p.m. meas- leukemic cells from cALL and CLL tended to display relatively ured in blank assays lacking cell sample was subtracted from higher PP2B activity, compared to AML-M1 blasts. PP2C the total c.p.m. The phosphatase activity was indicated as activity was relatively constant among various leukemic cell nanomoles released 32Pi per min in 108 cells. types, although PP2C activity in monocytic subtype AML-M5 blasts tended to be higher. PP2C activity of monocytes showed the highest activity among normal leukocytes. Immunoblot analysis Activities of protein phosphatases can be modulated by changes in the levels of activators or inhibitors for a prolonged Immunoblot analysis was carried out using polyclonal anti- period or by changes in the levels of functional phosphatases bodies specific against PP1 catalytic subunits (PP1␣, PP1␥, in the cells in response to growth factors and differentiation PP1␦), PP2A catalytic subunit, PP2B catalytic subunits (PP2B- factors.1–3 We next examined amounts of PP1 catalytic sub- A␣ and PP2B-A␤), and PP2C catalytic subunits (PP2C␣ and unit isozymes (PP1␣, PP1␥, and PP1␦), PP2A catalytic subunit, PP2C␤), as described.7–13 The specificity of all immunoreactive PP2B catalytic subunit isozymes (PP2B-A␣, PP2B-A␤), and proteins has been established by immunoblot analysis.7–13 PP2C catalytic subunit isozymes (PP2C␣, PP2C␤) using a Quantitative estimation of the level of each phosphatase was quantitative immunoblot method and respective polyclonal done densitometrically by scanning the immunoreactive band antibodies,7–13 as shown in Figure 2. Our data indicated that Protein phosphatase in human primary leukemic cells M Yamamoto et al 597

Figure 1 Activities of four major protein phosphatases in various primary leukemic cells and normal leukocytes. Activities of PP1, PP2A, PP2B and PP2C were assayed in AML blasts subgrouped according to FAB classification (M1–M5), ALL blasts, CLL cells, normal neutrophils (N), normal lymphocytes (L), and normal monocytes (M). Number of cases is indicated in parenthesis under the cell types. Each value represents mean (columns) ± s.e. (bars). Student’s t-test was used for statistical analysis. *P Ͻ 0.05, **P Ͻ 0.01, ***P Ͻ 0.001.

Figure 2 Expressions of four major phosphatase catalytic subunits in various primary leukemic cells and normal leukocytes. Amounts of PP1, PP2A, PP2B and PP2C catalytic subunits in AML blasts subgrouped according to FAB classification (M1–M5), ALL blasts, CLL cells, normal neutrophils (N), normal lymphocytes (L), normal monocytes (M) were analyzed by immunoblot analysis, using antisera specific for PP1␣ ({), PP1␥ (`), PP1␦ (a), PP2A ({), PP2B-A␣ ({), PP2B-A␤ (`), PP2C␣ (࡯) and PP2C␤ (`) as described in Materials and methods. Number of cases is indicated in parenthesis under the cell types. Values are expressed as means (columns) ± s.e. (bars). Student’s t-test. *P Ͻ 0.05, **P Ͻ 0.01, ***P Ͻ 0.001. the total amount of each phosphatase catalytic subunit AML-M4 blasts and in normal monocytes. PP1␦ was highly appeared to correlate with the level of its phosphatase activity expressed in AML-M4, -M5 blasts and in normal monocytes. in various leukemic cell types (compare Figures 1 and 2). All PP2A enzyme expressions in AML-M4 blasts and in normal PP1 catalytic subunit isozymes (PP1␣, PP1␥ and PP1␦) were monocytes were more abundant than in other cell types. In ubiquitously and comparably expressed in most leukemic cell AML-M1, ALL, and CLL cells, the expression of PP2A was low. types, whereas the expression of PP1␣ was significantly higher The pattern of PP2B-A␣ expression differed from that of PP2B- among three PP1 isozymes in normal neutrophils and lympho- A␤ expression. In AML-M1, -M2, -M3, CLL cells and normal cytes. Expressions of PP1␣ were prominently increased in neutrophils, expression of PP2B-A␣ was more abundant than Protein phosphatase in human primary leukemic cells M Yamamoto et al 598 Table 1 Relationship between activities of four major protein phosphatases and immunophenotypes of AML blasts

Phosphatase activity (nmol/min/108 cells)

PP1 PP2A PP2B PP2C

CD34 ± ± ± ± negative (25) 0.485 0.095P = 0.625 0.205 0.036P = 0.457 0.094 0.016P = 0.456 0.118 0.020 P = 0.722 positive (24) 0.354 ± 0.066 0.252 ± 0.051 0.115 ± 0.023 0.132 ± 0.032

CD11b ± ± ± ± negative (32) 0.359 0.070]* P = 0.048 0.156 0.078]* P = 0.018 0.115 0.020P = 0.212 0.114 0.025 P = 0.392 positive (16) 0.629 ± 0.110 0.363 ± 0.182 0.081 ± 0.020 0.146 ± 0.028

CD11c ± ± ± ± negative (17) 0.392 0.095P = 0.966 0.246 0.056P = 0.217 0.126 0.029P = 0.077 0.115 0.024 P = 0.276 positive (11) 0.400 ± 0.154 0.166 ± 0.030 0.062 ± 0.020 0.194 ± 0.065

CD13 ± ± ± ± negative (6) 0.707 0.258P = 0.332 0.196 0.034P = 0.313 0.074 0.028P = 0.268 0.104 0.045 P = 0.587 positive (40) 0.425 ± 0.062 0.247 ± 0.036 0.112 ± 0.016 0.132 ± 0.022

CD14 ± ± ± ± negative (34) 0.402 0.068P = 0.227 0.163 0.018]* P = 0.048 0.128 0.027P = 0.801 0.107 0.021 P = 0.179 positive (12) 0.606 ± 0.147 0.377 ± 0.096 0.118 ± 0.031 0.181 ± 0.049

CD33 ± ± ± ± negative (5) 0.348 0.100P = 0.355 0.320 0.061P = 0.226 0.111 0.022P = 0.841 0.208 0.134 P = 0.544 positive (42) 0.467 ± 0.069 0.227 ± 0.034 0.105 ± 0.016 0.119 ± 0.016

CD7 ± ± ± ± negative (38) 0.494 0.073]* P = 0.030 0.250 0.037]* P = 0.027 0.119 0.016]** P = 0.003 0.116 0.016 P = 0.515 positive (10) 0.280 ± 0.061 0.142 ± 0.029 0.047 ± 0.015 0.162 ± 0.067

CD19 ± ± ± ± negative (41) 0.478 0.070P = 0.187 0.248 0.036P = 0.072 0.094 0.014P = 0.136 0.127 0.039 P = 0.764 positive (6) 0.291 ± 0.111 0.166 ± 0.026 0.189 ± 0.053 0.140 ± 0.039

Results are expressed as the mean ± s.e. * P Ͻ 0.05; **P Ͻ 0.01.

that of PP2B-A␤. In ALL blasts and normal lymphocytes, the entiation antigens, thereby reflecting commitment to the amount of PP2B-A␣ expression was similar to that of PP2B- myeloid lineage, as well as level of maturation.14 Immuno- A␤. The lower PP2B activity in neutrophils appeared to be phenotyping has made feasible elucidation of the relationship mostly due to the significant lower expression of PP2B-A␤. between the stage of myeloid differentiation and expression The amount of PP2B-A␤ enzyme expression in AML-M4, -M5 of each phosphatase in AML blasts.14,17 The analysis was blasts, and normal monocytes tended to be more abundant restricted to blast population by gating on forward and side than that of PP2-BA␣. In most leukemic cell types and normal light scatter parameters and excluding the majority of con- leukocytes, PP2C␣ expression was higher than that of PP2C␤. taminating mature leukocytes. Among the myeloid lineage Thus, PP1, PP2A, PP2B and PP2C were ubiquitously but dif- antibodies, panmyeloid-associated antigens, CD13 and/or ferently expressed in primary leukemic blasts and in normal CD33 were detected on most AML blasts and did not correlate leukocytes, suggesting that these phosphatases have essential with FAB classification. Expression of CD34, a marker for roles in cellular proliferation, differentiation of hematopoietic myeloid progenitor cells, including stem cells, was high in cells, and in specific functions of different terminal myeloid AML-M1 (four cases positive/seven cases tested), -M2 (12/18) cells. Although distribution of the total amount of each phos- and -M4 (5/9) subtypes and low in AML-M3 (2/8) and -M5 phatase catalytic subunit appeared to parallel that of its (1/7). The presence of CD34 antigen was more frequently activity in various leukemic cell types, our study does not observed in immature FAB subtypes, AML-M1 and -M2. exclude the possibility that other factors may influence the net CD11b and CD11c are of a different integrin ␣ subunit that phosphatase activity. PP1, PP2A and PP2B isolated in various covalently associates with the common ␤2 integrin (CD18) to ␣ ␤ ␣ ␤ forms from mammalian tissues are composed of catalytic and form heterodimers termed Mac-1 ( M 2) and p150,90 ( x 2), regulatory subunits.1–3 The activity or substrate specificity of respectively.18 CD11b/CD18 and CD11c/CD18 are critical to these phosphatases may be altered by modification or inter- monocyte and granulocyte adhesion-dependent functions.5 action with regulatory proteins, without change in the level While expression of these antigens begins only at the myelo- of catalytic subunit expression. Further investigations on these cyte stage of granulocyte maturation with highest levels found regulatory subunits in various leukemic cells are expected to in mature granulocytes, the antigens are markers of immature shed light on the roles of these phosphatases in hemato- monocytes (monoblasts), appearing earlier in monocytic poietic cells. development than the prototype antigen CD14.5,18 CD11b Leukemic myeloblasts express a variety of leukocyte differ- was highly positive in AML-M5 (7/7) and -M4 (7/8), reactions Protein phosphatase in human primary leukemic cells M Yamamoto et al 599

Figure 3 Correlation of phosphatase activity with cell surface markers in AML blasts. in myeloid subtypes AML-M1 (0/7), -M2 (2/18) and promyelo- CD34-positive AML blasts tended to be lower than that of cytic leukemia AML-M3 (0/8) were rarely expressed. CD11c CD34-negative blasts, but the level of PP1 activity of AML was highly positive in AML-M5 (4/4), -M1 (4/5) and -M4 (2/5); blasts showed a negative correlation with CD34 positivity (r expression was negative in AML-M2 (1/12) and AML-M3 (0/2). =−0.588, P Ͻ 0.05). There was no significant difference in The monocyte/macrophage marker, CD14 was frequently each phosphatase activity between CD19-positive and -nega- expressed in monocytic variant AML-M5 (6/6) and -M4 (3/9), tive AML blasts. Interestingly, in AML-M2 blasts associated whereas the antigen was rarely expressed in AML-M1 (1/6), - with B-lymphoid leukemic antigen (CD19), PP2B activity M2 (2/18) and -M3 (0/7) cases. T cell antigen (CD7) was posi- tended to be higher than that observed in AML blasts lacking tive in 20% of cases (10/49), this antigen being mainly CD19, and was similar to that in lymphoid leukemic cells expressed in AML-M1 (4/7), -M2 (3/18) and -M4 (3/9). B cell such as ALL and CLL. The CD7-positive AML blasts had sig- antigen (CD19) was only found in six of 18 cases (33%) of nificantly lower activities of PP1, PP2A and PP2B than those AML-M2 blasts. Four major phosphatase activities in relation of blasts without such surface antigen. These findings suggest to various phenotypical markers are shown in Table 1. Figure that T cell antigen-positive and B cell antigen-positive AML 3 summarizes data on the significant correlation between the blasts might have different biologic features. The correlation level of phosphatase activity and positivity of the cell surface coefficients between CD7 positivity of AML blasts and activity marker. CD11b-positive AML cells showed significantly of PP1, PP2A or PP2B were −0.661, −0.276, or −0.475 (P Ͻ higher activities of both PP1 and PP2A than those of CD11b- 0.05), respectively. Such CD7-positive AML blasts may arise negative AML cells. Correlation coefficients between CD11b from malignant transformation of early progenitor cells cap- positivity and PP1 or PP2A activity were 0.704 and 0.529 (P able of differentiating into more than one lineage.16,19 These Ͻ 0.05), respectively. No significant difference in each phos- findings suggest that the levels of PP1, PP2A and PP2B activi- phatase activity was demonstrated between CD11c-positive ties are relatively low in leukemic cells which are considered and -negative cells, or between CD13-positive and -negative to be morphologically and immunophenotypically immature cells. The CD14-positive AML cells showed significantly and that each phosphatase activity is differently modulated higher PP2A activity than did CD14-negative cells. The corre- during the course of myelomonocytic cell differentiation. lation coefficient between CD14 positivity and PP2A activity Our earlier studies revealed that expression of each protein was 0.412 (P Ͻ 0.05). PP1 activity of the stem cell marker phosphatase, including PP1, PP2A, PP2B and PP2C, is differ- Protein phosphatase in human primary leukemic cells M Yamamoto et al 600 ently regulated during differentiation of HL-60 cells,6–13 and HL-60 leukemic cells by serine/threonine protein phosphatase this present study is the first that provides a detailed analysis inhibitors. FEBS Lett 1992; 314: 340–344. of four major protein phosphatases in primary human leu- 7 Tawara I, Nishikawa M, Morita K, Kobayashi K, Toyoda H, Omay SB, Shima H, Nagao M, Kuno T, Tanaka C, Shirakawa S. Down- kemic cells and in normal peripheral leukocytes. Our study regulation by retinoic acid of the catalytic subunit of protein phos- indicates that four major phosphatases are ubiquitously but phatase type 2A during granulocytic differentiation of HL-60 cells. differently expressed in various types of leukemic cells and in FEBS Lett 1993; 321: 224–228. normal mature leukocytes. Although the exact mechanism of 8 Nishikawa M, Omay SB, Toyoda H, Tawara I, Shima H, Nagao different expression of each phosphatase is unknown, there M, Hemmings BA, Mumby MC, Deguchi K. Expression of the cata- is evidence that the regulation may be at the level of RNA lytic and regulatory subunits of protein phosphatase type 2A may be differentially modulated during retinoic acid-induced granulo- transcription, rather than mRNA stabilization or protein turn- cytic differentiation of HL-60 cells. Cancer Res 1994; 54: 4879– over. Our reported data demonstrated that alterations in phos- 4885. phatase expression during HL-60 cell differentiation correlate 9 Omay SB, Nishikawa M, Morita K, Toyoda H, Nakai K, Shima H, with the level of phosphatase mRNA.7–9,11,12 Expressions of Nagao M, Kitagawa S, Saito M, Shiku H. Decreased expression of r PP1, PP2A and PP2B appear to be relatively low in leukemic protein phosphatase type 2A in HL-60 variant (HL-60RA ) cells blasts arresting at the stage of early pluripotent stem cells, and resistant to induction of cell differentiation by all-trans retinoic acid. Exp Hematol 1995; 23: 244–251. each phosphatase may be differently modulated during the 10 Omay SB, Ogasawara H, Toyoda H, Nakai K, Shima H, Nagao course of myelomonocytic commitment and maturation. M, Mumby MC, Shiku H, Nishikawa M. 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