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[CANCERRESEARCH54, 4879-4884, September15. 19941 Expression of the Catalytic and Regulatory Subunits of Protein Phosphatase Type 2A May Be Differentially Modulated during Retinoic Acid-induced Granulocytic Differentiation of HL-60 Cells1 Masakatsu Nishikawa,2 Serdar B. Omay, Hideki Toyoda, Isao Tawara, Hiroshi Shima, Minako Nagao, Brian A. Hemmings, Marc C. Mumby, and KatsUmi Deguchi The 2nd Department of internal Medicine. Mie University School of Medicine, E4obashi, Tsu, Mie 514 Japan (M. N.. S. B. 0., H. T., i. T., K. DI; Carcinogenesis Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, 104 Japan (H. S., M. N.]; Friedrich Miescher-institut, CH-41X12, Base!, Switzerland (B. A. H.]; and Department of Pharmaco!ogy, University of Texas. Southwestern Medica! Center, Da!!as, Texas 75235-9041 (M. C. M.] ABSTRACT To elucidate the regulation of protein phosphatases types 1 (PP1) and 2A (PP2A) during all-trans retinoic acid (ATRA)-induced granulocytic differentiation of HL-60 cells, the phosphatase activity, proteins, and gene expressions ofPPl and PP2A were examined. Treatment with 1 pM ATRA caused an 85% decrease in the PP2A activity In extracts from HL-60 cells, while the PP1 activity was constant. This reduction in PP2A activity appeared to parallel phenotypic and functional changes of HL-60 cells Induced by A1'RA. Western blot analysis showed that the level of PP2A catalytic subunit (PP2A-C) decreased during the course of ATRA-induced differentiation, whereas expressions of A and B (Mr 55,000) regulatory subunits subunit of PP2A were relatively isozymes (PP1a, Unaltered. Expressions of PP1 catalytic PP1'y, and PP18) were not significantly affected by ATRA treatment. Northern blot analysisrevealedthat mRNA levelsof PP2A-C@and Aa regulatory subunits were decreased following treatment with ATRA, while levels of PP2A-Ca and B (Mr 55,000) a regulatory subunit transcripts were relatively constant. Selective down regulation of PP2A-Cf3 preceded the granulocytic maturation induced by ATRA. Ex pressions of PP2A-C isoforms and A and B regulatory subunits may be differentially modulated during ATRA-induced granulocytic differentia don ofHL-60cells. INTRODUCTION ATRA3 induces granulocytic differentiation in cultured leukemic HL-60 cells (1, 2) and is clinically effective as the differentiation therapy in inducing high remission rates in patients with acute pro myelocytic leukemia (3, 4). The biological effects of ATRA appear to be mediated through a number of closely related nuclear retinoic acid receptors that possess discrete DNA-binding and retinoic acid-binding domains (5). Although the exact mechanism of the ATRA-induced granulocytic differentiation remains to be elucidated, protein phos phorylation/dephosphorylation is also thought to be a regulatory de vice eminently suited for the control of differentiation processes (6). The levels of both protein kinase C activity and the expression of its isoforms have been shown to increase during HL-60 cell differentia tion induced by dimethyl sulfoxide and ATRA (7). However, little is known concerning protein phosphatases responsible for reversing the actions of protein kinase-catalyzed phosphorylation reactions in HL-60 cell differentiation. The protein serine/threonine phosphatase catalytic subunits of mammalian cells comprise four forms which have been designated type 1 (PP1), type 2A (PP2A), type 2B (calcineurin), and type 2C (8, 9). They differ in metal ion requirements and sensitivities to two heat-stable protein inhibitors, inhibitor-i and inhibitor-2 (8, 9). Sev eral isoforms of PP1 catalytic subunit, PP1a, PP1'y, and PP1@ have been cloned from rat cDNAs (10). PP1s consist of multimeric struc tures composed of a catalytic subunit complexed to several regulatory subunits (8, 9). Formation of heteromeric complexes is thought to play an important role in regulating the activity of PP1 catalytic subunit, as yet not defined in HL-60 cells. The PP2A catalytic subunit (PP2A-C, Mr 36,000) Received 3/16/94; accepted 7/11/94. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported in part by grants for research from The Ministry of Education, Science, and Culture, Japan. 2 To 3 The whom requests abbreviations for used reprints are: ATRA, MATERIALS AND METHODS superoxide, as monitored by the reduction of NBT (Sigma) and surface antigen analysis. The ability of NBT reduction was evaluated as described elsewhere Surface antigens were assessed by cytofluorometry in a fluorescence activated cell sorter scan (Becton Dickinson, Mountain View, CA), using be addressed. alh-trans-retinoic forming a heterotrimer Cells, Culture Conditions, and Evaluation of Differentiation. Proce dures for the maintenance of HL-60 cells and determination of variable cell counts have been described previously (19). Cells in the logarithmic growth phase were used for experiments. The extent of differentiation by ATRA (Sigma Chemical Co., St Louis, Mo) was assayed by the ability to produce (20). should is mainly present as a holoenzyme with A (Mr 65,000) regulatory and different B regulatory subunits; the basic form is the PP2A-C/A complex, and the B subunit is associated with the dimer through A subunit (9, 11, 12). The A and B regulatory subunits were shown to modulate the phosphatase activity of the PP2A-C in vitro. cDNA clones for the two respective isoforms of PP2A-C and A subunit, PP2A-Ca and PP2A-C(3 and Aa and A(3, respectively, have been isolated from several animal species (13—15). Based on biochemical evidence, the B subunit of PP2A holoenzyme is comprised of several distinct families of proteins of B (M1 55,000), B' (Mr 54,000), B― (Mr 74,000), and Mr 72,000 (9). Three cDNAs for isoforms of the B (Mr 55,000) subunit (a, @3, ‘y) have been cloned from human, rabbit, rat, and yeast libraries (9, 16). A, B, and PP2A-C subunits show a high degree of sequence conservation among species (9). Although the effect of the regulatory subunits of PP2A on the substrate specificity has clearly been demonstrated in vitro (8,9), the roles of individual regulatory proteins in the regulation of metabolism, growth, differentiation, and development remain largely unknown. We reported that OKA and calyculin-A, both potent and specific inhibitors of PP1 and PP2A, augment the granulocytic differentiation of HL-60 cells induced by ATRA but not the monocytic differentia tion induced by phorbol diester (17). In addition, PP2A-C is down regulated during ATRA-induced granulocytic differentiation of HL-60 cells, whereas the PP1 catalytic subunit is unchanged (18). To determine the effects of ATRA treatment on regulation of cellular PP2A holoenzyme, we analyzed PP2A activity and expressions of the A and B regulatory subunits in addition to that of PP2A-C, following ATRA treatment. acid; PP1, protein phosphatase type 1; PP2A, protein phosphatase type 2A; PP2A-C, the catalytic subunit of PP2A; MLC, myosin light chain; OK.A,okadaic acid; NBT, nitrobluetetrazolium;cDNA, complementary DNA, SDS, sodium dodecyl sulfate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. monochonal antibodies, including CD! lb (0KM!; Ortho Diagnostic System Inc., Raritan, NJ), CDllc (LeuM5; Becton Dickinson), and CD54 (anti-ICAM; Cosmo Bio Co., Tokyo, Japan). 4879 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research. PROTEIN PHOSPHATASE 2A IN GRANULOCYI1C DIFFERENTIATION Preparation of CytosohicFractions of HL-60 Cells. HL-60 cells (2 X l0@ cells each) were washed saline three times with ice-cold and then disrupted Tris-HC1-buffered in 1 ml of homogenization buffer consisting ATRA-induced Granulocytic Differentiation in HL-60 Cells. of 250 mM sucrose and buffer A (20 mM Tris-HC1, pH 7.5, 2 mM dithiothreitol, 2 mM EDTA, 2 mMethylene glycol bis-(13-aminoethyhether)-N-N'-tetraacetic acid, and protease inhibitors, including 75 mg/liter phenylmethyh-sulfonylfluoride, 10 mg/liter heupeptin, 10 mg/liter N-tosyl hysinechloromethyl ketone, and 5 mg/liter N-tosyhphenylalanine chhoromethylketone) by a glass-glass Potter Ehvehjem homogenizer 1000 at 4°C(19). The sample was then centrifuged X g for 10 mm. The resulting fraction. The supernatant pellet was centrifuged was used at 100,000 as the crude at nuclear X g for 1 h to separate the cytosol and membrane fractions. The membrane and nuclear fractions were rinsed with homogenization nization buffer buffer and subsequently containing 1% Nonidet resuspended P-40. Following in homoge a 1-h incubation on ice, these fractions were sonicated and further centrifuged at 100,000 X g for 1 h to obtain solubihized fractions. In preliminary experiments, we have found that Nonidet P-40 is most suitable for solubilization does not inhibit the activity of phosphatase of phosphatase at concentrations because it as high as 1%. Assay of Protein Phosphatases. Activities of PP1 and PP2A were assayed in the absence of divahent cations using 32P-phosphorylated isolated MLC of chicken gizzard as a substrate, because it is known to be a good substrate for both mammalian PP1 and PP2A (21). Phosphatase activity was determined by the liberation of inorganic 32P from the substrate (32P-labehed Mr 20,000 at 30°C according phatase activity preparation to the method of Pato and Kerc (22). Inhibition by OKA was determined by adding OKA MLC) of phos to the enzyme 10 mm prior to adding the substrate. The extent of dephosphory lation was restricted to <10%. Under these conditions, the rates of phospho ryhationwere linear with respect to time and enzyme dilution. Immunobhot Analysis. Antisera against PP1 catalytic subunit, PP2A-C, and A, B (Mr 55,000), by immunizing @ B' (Mr 54,000), and Mr 72,000 rabbits with synthesized (23, 24), A (25) regulatory, fragments B (Mr 55,000), subunits were obtained of PPla, PPl@y, and PP1& and B (M, 55,000) (3 (12), B' (M, 54,000) (26), or Mr 72,000 (27) regulatory subunits. HL-60 cells were incu bated with 1 ,iM ATRA, trichloroacetic acid-lO and the reaction mM dithiothreitoh-2 ethyl ether)-N,N'-tetraacetic twice with acetone-lO was terminated with cold mM ethyheneglycol 10% bis-(f3-amino acid solution. The resulting cell pellet was washed mM dithiothreitol and then dried. The dried acetone powder was dissolved with Laemmhi sample buffer (28). The sample was subjected to 0.1% SDS-12.5% electrophoretical transfer polyacrylamide to a nitrocehlulose gel electrophoresis membrane RESULTS (pH 7.4) followed by Treatment of HL-60 cells with 1 ,.LMATRA for 4 days led to acquisition of a mature phenotype resembling that of a granulocyte (17). The mature phenotype in HL-60 cells was confirmed by flow cytometric assessments with various monoclonal antibodies and an increased NBT reduction. HL-60 cell proliferation was inhibited after treatment with 1 ,.LMATRA. As shown in Fig. 1, treatment of HL-60 cells with ATRA resulted in a time-dependent increase in NBT reduction and increase in reactivities of the cells with CD1 lb. CDS4, and CD1 ic, although the kinetics of each differentiation marker acquisition were not necessarily identical. The reactivity of HL-60 cells with CD54 reached a plateau faster than that of CD1 lb or CD11c. Alterations of Activity and Expression of PP1 and PP2A during ATRA-induced Differentiation. The activity of protein serine/thre onine protein-phosphatase in HL-60 cells was assayed in the absence of divalent cations with 32P-phosphorylated MLC as substrate (21). The activities of cytosol, membrane, and nuclear fractions in HL-60 cells were 17.6 ±3.1, 4.90 ±0.65, and 5.84 ±0.58 nmol/mg/min (mean ±SE; n = 3), respectively. In untreated HL-60 cells, approx imately 60-70% of the total activity of MLC phosphatase was present in the cytosol fraction, with the remaining activity located in the membrane and nuclear fractions. Incubation of HL-60 cells with 1 @M ATRA resulted in a 66% decrease in MLC phosphatase activity in the cytosol fraction, as shown in Fig. 2, but the phosphatase activity of the membrane or nuclear fraction was unaltered (data not shown), thereby suggesting that the translocation of phosphatase from the cytosol fraction to membrane or nuclear fractions did not occur. PP2A is inhibited completely by S n@tOKA, while PP1 is hardly affected at this concentration and complete inhibition requires 500 n@i(21). The varying concentrations producing 50% inhibition for different phos phatases mean that OKA can be used to distinguish between these activities in cell-free systems. Therefore, we examined effects of OKA (5 and 500 ni@i)on MLC phosphatase activities of HL-60 cells during ATRA-induced differentiation to estimate the relative amounts (29). The membrane was reacted with the avidin-biotin peroxide complex method (Vectastain; Vector Laboratories, Burhingame, CA). Prestained SDS-polyacrylamide gel ehectrophoresis standards (Bio-Rad, Richmond, CA) were used as molecular 100 weight standards. Quantitative estimation of the level of PP1 and PP2A was carried out densitometrically with a Bio-Rad videodensitometer by scanning the immunoreactive band after immediately photographing the band. The area of an individual peak was measured tracings and expressed as absorbence above background 80 in densitometric X mm (29). RNA Isolation and Northern Blot Analysis. Total cellular RNA was extracted by the guanidinium isothiocyanatecesium chloride method (30). Total RNA (20 @g) of HL-60 cells was electrophoresed through 0.8% agarose with 18% formaldehyde and transferred to Nytran membranes (Schleicher >1 > & Schuell, Dassel, Germany). After baking at 95°Cfor 2 h under vacuum, the blots were hybridized at 42°Cin 50% formamide, 5X Denhardt's solution, SX 0 0. SSPE (1 x SSPE is 0.15 MNaCh-lOmMNaH2PO4-0.lmr@i EDTA), 0.5% SDS, 200 @g/mhof denatured salmon @40 20 sperm DNA, and the 32P-habeled cDNA probe. The probes used were as follows: PsrI-EcoRI fragment of rat PP2A-Ca cDNA and synthetic ohigomer (3'-noncoding region) of rat PP2A-C@3 cDNA (31), 0. EcoRl fragments of rat Aa and A@3regulatory subunit cDNAs (32), Hind!! fragment of rat B (M1 55,000) a and AccI fragment of rat B (Mr 55,000) 13 regulatory fragment subunit cDNAs (32), EcoRI of rat PP1 y, and EcoRI fragment fragment of rat PPI1a, 0 EcoRV-PstI of rat PP1@ (10). These probes were labeled with [a-32P]dCFP by a multiprime labeling system kit (Amersham, Buckinghamshire, England). Hybridized blots were finally washed with 0.1 x standard saline citrate (1X is 0.15 MNaCl-15 mMtrisodium citrate) and 0.5% SDS at room temperatureand autoradiographed.Quantificationof hy bridization was determined by scanning densitometry (18). 24 48 72 96 Time ( hr) Fig. 1. Induction of markers of cell differentiation in HL-60 cells after treatment with ATRA. HL-60 cells were incubated with 1 @M ATRA. Percentages of NBT-positive cells (•) and surface markers using CD11b (0),CD11c (L.@), and CD54(0)were analyzed by flow cytometry, as described in “Materials and Methods.― Points, means of three separate experiments expressed as percentages of positive cells. 4880 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research. PROTEIN PHOSPHATASE 2A IN GRANULOCYI1C DIFFERENTIATION @ @I 20 Expressions of mRNAs of PP2A during ATRA-induced cell differentiation of HL-60 cells. To determine whether the ATRA induced down regulation of PP2A-C in HL-60 cells was reflected at the level of RNA transcripts, Northern blot analyses using 32P-labeled cDNA probes for PP2A catalytic subunits (PP2A-Ca and PP2A-C@3) and regulatory subunits (Aa, A(3, B (Mr 55,000) a and B (Mr 55,000) I 15 f3subunits) wereperformed on totalcellularRNAsobtained from 10 5 @A @ A A A A __________________________________________ , ______________________________________________ ,— 0 0 24 48 Time ( hr ) 72 96 Fig. 2. Time course of protein phosphatase activity during ATRA-induced differenti ation of HL-6O cells. HL-60 cells (2 x 108 cells) were treated with 1 @LM ATRA, and cytosolic fractions were prepared at the indicated times. Protein phosphatase activity was measured in the absence (•)or presence of 5 nsi OKA (A) and 500 nsi OKA (I), as described in “Materials and Methods.―Point, mean from three separate experiments. SD, <10%. of PP1 and PP2A (Fig. 2). OKA (5 flM) inhibited about 68% of the MLC phosphatase activity in the cytosol of untreated HL-60 cells, and this OKA -sensitive phosphatase portion (5 nM) was decreased by 85% during ATRA-induced differentiation of HL-60 cells. Because the MLC phosphatase activity of HL-60 cells in the presence of 5 nM OKA was relatively constant for 4 days after the addition of ATRA, @ the decreased MLC phosphatase activity of HL-60 cells after treat ment with ATRA is probably mainly due to decreased PP2A activity, even though the PP1 activity is relatively constant. Decrease in PP2A activity appeared to coincide with an increase in NBT reduction (Figs. 1 and 2). We then examined the effects of ATRA on the regulation of PP2A activity, protein expressions of PP2A-C, A, and B regulatory subunits in whole cell extracts by Western blot analysis using poly clonal antibodies specific for PP2A-C, A regulatory, and various B regulatory subunits [B (Mr 55,000), B' (Mr 54,000), and 72,000J of PP2A. Because precise titers of these antibodies against PP2A-C and A and B (Mr 55,000) regulatory subunits await purification of these subunits to homogeneity from HL-60 cells, it is impossible, at present, to estimate the accurate molar ratio for each subunit, but one can compare change in the amount of individual PP2A subunits during ATRA-induced differentiation. As shown in Fig. 3, the major immu noreactive species of PP2A-C, A, and B (Mr 55,000) subunits were bands of Mr 35,000, 65,000, and 55,000, respectively. Antibodies against the B (Mr 55,000)13, B' (Mr 54,000), or Mr 72,000 regulatory subunit detected no protein in HL-60 cells. The time-dependent and dramatic decrease in immunoreactive PP2A-C was evident in HL-60 cells after the addition of ATRA, whereas expressions of both A and B (M@55,000) a subunits were relatively constant (Fig. 3B). In contrast, the expression of each isozyme of PP1 catalytic subunit (PP1a, PP1y1, PP16) appeared to be unchanged in HL-60 cells after ATRA treatment (Fig. 3A). Quantitative immunoblot analysis using purified recombinant proteins as standard revealed that PP2A-C, HL-60 cells following treatment with ATRA (Fig. 4). HL-60 cells expressed mRNAs of PP2A-Ca, PP2A-C@, and Aa and B (Mr 55,000) a subunits. The 2.0-kilobase mRNA was a major transcript of HL-60 cells after hybridization to the PP2A-C@3 probe. The down regulation of PP2A-C(3 mRNA was evident within 3 h after the addition of 1 p.M ATRA. The level of PP2A-C@3mRNA was further decreased to approximately one-third of the basal level of untreated HL-60 cells within 6 h, when normalized to GAPDH mRNA expres sion. Rehybridization of these blots with GAPDH cDNA probe con firmed that relatively equal amounts of RNA were loaded in each lane. The PP2A-Ca probe also detected a major band of 2.0 kilobase and the expression of PP2A-Ca 2.0-kilobase transcript was relatively unaltered by treatment with ATRA. Thus, our data suggest that two isotypes of PP2A could be differently regulated during ATRA-in duced differentiation. The Aa and B (M@55,000) a genes were also expressed in HL-60 cells, and A a and B (M1 55,000) a transcripts were approximately 3.1 and 2.6 kilobases, respectively. The levels of Aa transcript gradually decreased by treatment of HL-60 cells with ATRA, although expression of the Aa regulatory protein did not fluctuate. The levels of B (Mr 55,000) a mRNA remained relatively constant after ATRA treatment. Transcripts of the A(3 and B (M1 55,000) 3 subunits in HL-60 cells were not detectable. The mRNA expressions of PP1a, PP1y, and PPl@ were unaltered during ATRA induced differentiation of HL-60 cells (data not shown). DISCUSSION Our present results suggest that PP1 enzyme is not altered during ATRA-induced granulocytic differentiation of HL-60 cells, while PP2A activity is down regulated during ATRA-induced differentia tion. Subunit composition and specific complex formation play im portant roles in regulating the activity and specificity of PP2A-C (9). Cellular PP2A is composed of a common PP2A-C/A complex that interacts with different families of endogenous and exogenous regu latory proteins including B (Mr 55,000), B' (Mr 54,000), B― (Mr 74,000) and Mr 72,000 proteins and tumor antigen (9). We found that the expres sion of PP2A-C was markedly decreased during the course of granulo cytic differentiation, whereas the levels of A and B (Mr 55,000) a regulatory subunits were relatively constant. Other different B regulatory subunits, including B (Mr 55,000) (3' B' (M1 54,000), and M1 72,000 regulatory proteins were not expressed in HL-60 cells before and after treatment with ATRA. Although the molar ratio for the PP2A-C and A and B (M@55,000) subunits in the cells is unknown, the selective down regulation of PP2A-C may change the ratio ofdimeric and trimeric PP2A holoenzymes and, therefore, may alter specific catalytic properties such as substrate specificities or response to effectors. HL-60 cells expressed RNA transcripts of PP2A-Ca, PP2A-C@3,and Aa and B (Mr 55,000) a regulatory subunits. Treatment with ATRA led to a dramatic reduction in mRNA expression of PP2A-C@3,whereas the level of a isozyme mRNA was unchanged after exposure to ATRA. The down regulation of PP2A-C@3 RNA transcript clearly preceded the ATRA-induced granulocytic differentiation. The amino acid sequences of PP2A-Ca and PP2A-C(3 proteins, deduced from cDNA sequences, are PP1a, PP1'y, and PP1@ accounted for 5.0, 4.2, 3.9, and 1.4 @tg/mg 97% identical (8, 9). Although mRNAs encoding both isoforms have protein (means of three determinations), respectively, of the total been detected in mammalian cells and tissues (14, 33), the steady-state cytosol fractions in untreated HL-60 cells. PP2A-Ca mRNA is more abundant than PP2A-C13 mRNA in various cell 4881 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research. PROTEIN PHOSPHATASE2A IN GRANULOCYTIC DIFFERENTIATION A ATRA(1#) B 49.5 @ 32.5 @- • . HL-60 — —‘PPla @ ATRA(1j@) 80.0'— 49.5-'—0 @ .@ 49.5 PP2A-C fl5.O ; Z5—@@---------—---—-‘PP17 @ -* @ @ @ @ @lO6.0 @. 80.0-'—'-― —‘A 49.5..- Fig. 3. Time course of changes in immunoreac tive PP1 and PP2A during ATRA-induced differ entiation of HL-60 cells. HL-6O cells (1 X 10@ 49.5 cells) were treated with 1 p@ A'I'RA, and the re action was stopped by the addition of ice-cold 32.5 —‘PPlo 80.0 49.5 trichloroacetic acid at the times indicated (abscis sas). The acid-precipitated proteins were analyzed by immunoblot analysis, using polyclonal antibod ies specific for PP2A and PP1 isozymes, as de scribed in “Materialsand Methods.―A, blots incu 0 24 C batedwithPP1a,PP1y,andPP1&antisera.B,blots incubated with PP2A-C and A and B (M, 55,000) a regulatory subunit antisera. All antibodies were used at a 1:100 dilution. Ordinate, molecular weight (MW.) standard markers (Bio-Rad). These data are from one representative experiment, but similar results were obtained in two other experi ments. C, graphic display of PP2A-C (•),A (U), 48 72(1w) -—B @. 0 24 48 72 96 ( hr) 6 5 E and B (M, 55,000)a (A) regulatory subunits shown 4 in B, quantitated by densitometry. C 3 2 -e I 0 0 24 48 TIme(hr) 72 96 lines other than hematopoietic cells, and this differential expression is thought to be due to different promoter activities (14). PP2A-Ca and PP2A-C13 are encoded by diStinCtgenes whose expression appears to be differentially regulated (14, 31). Therefore, it is conceivable that the may be resistant to proteolysis and, therefore, has a long life span in the cell. The ATRA-induced granulocytic differentiation of HL-60 cells appears to be mediated through the nuclear retinoic acid receptor expression superfamily of transcription factors and possesses discrete retinoic of PP2A-Ca and PP2A-C13 is separately modulated in the (34).Theretinoicacidreceptor isa member of thethyroidhormone process of ATRA-induced granulocytic differentiation. We did not dem onstrate directly the differential expression of two PP2A-C isoforms at protein levels, because a specific antibody against each isoform was not available. The expression ratio of PP2A-Ca to PP2A-C(3 proteins may possibly be increased in the PP2A activity of ATRA-induced mature granulocytes. The mRNA level of Aa regulatory subunit was also decreased following treatment with ATRA, while that of B (Mr 55,000) a regulatory subunit was relatively constant. These data suggest that mRNA expressions of PP2A-C and Aa subunits are coordinately regulated in HL-60 cells following treatment with ATRA. In spite of the early decrease in mRNA expression of Aa regulatory subunit, the level of expression of the A subunit protein did not fluctuate through out ATRA-induced differentiation. While the reason for this discrep acid-binding (ligand-binding) and DNA-binding domains that regu late transcription of certain target genes (5). ATRA can influence PP2A gene expression either directly, by activating transcription of the PP2A gene, or indirectly, by altering expression of genes encoding transcription factors. In addition to the modulation of protein phosphatases, the levels of both activity of protein kinase C and expression of its isoforms (a, @, and y) of protein kinase C was shown to increase during granulocytic differentiation of HL-60 cells induced by ATRA and dimethyl sulf oxide (7). 1,25-Dihydroxyvitamin D3-induced monocytic differentia tion of HL-60 cells is also associated with an increase in both activity of protein kinase C and expression of protein kinase C-a and -@, as well as steady-state levels of mRNA of protein kinase C-a and -@(35, ancy be involved in the regulation of HL-60 cell differentiation by TPA, remains unknown, we speculate that the A regulatory subunit 36).The activationof cAMP-dependentproteinkinasealsoappearsto 4882 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research. PROTEIN PHOSPHATASE 2A IN GRANULOCYTIC DIFFERENTIATION @ A @ 3 5 12 24(1w) PP2A-C@ — @ @ 0 .@ - 2.0kb 5 12 24(1w) Act— . 3.1kb GAPDH— 01 3 5 1224(1w) @ @ 3 Bar— ,@ Fig. 4. Time course of mRNA expressions of PP2A-Cf3, PP2A-Ca, Aa and B (M, 55,000) a subunits in HL-60 cells stimulated with ATRA. PP2A@C@— r@ , , -26kb —2.0kb HL-60cells(2 x iO@ cells)weretreatedwith1 ps, ATRA, and total RNA (20 pg) was isolated from @ the cells at the indicated times (abscissas). A, Northern blots of total RNA were hybridized with GAPDH - 32Plabeled cDNA probes for PP2A-Ca, PP2A-C13, Aa, and B (M, 55,000) a subunits. The same RNA blots were hybridized to GAPDH cDNA probe for assessment of RNA quantities in each lane. B, graphic representation of relative intensity of hy GAPDH bridization signals of PP2A-Ca (0), PP2A-C@ (•), Aa(U), andB (M,55,000) a (A)obtained B from densitometric scanning of the autoradiogram pictured in A. The ratio of these signals to the GAPDH signal was determined for each time point following ATRA addition and the resulting value is 120 expressed relative to control untreated HL-60 cells. Similar results were obtained in two independent experiments. 0 5- 100 C 0 C-) C 0 4) Cl) 0) 80 60 5- 40 w 0) > 20 0) 0@ 1@_ 0 6 12 18 24 Time(hr) ATRA or dimethyl formamide (37, 38). Although the monocytic and granulocytic inducers share protein kinases as target molecules (6), it is also true that activation and/or modulation of protein kinases may be solely insufficient to explain the mechanisms for bringing about terminal differentiation into granulocytic or monocytic phenotypes. Thus, the modulation of protein phosphatases may play important roles in regulating the net phosphorylation of critical substrate(s) that subsequently mediate the differentiation of HL-60 cells into either phenotype. PP2A is thought to regulate multiple functions in vivo, including several metabolic pathways, protein synthesis, DNA repli cation, and the cell cycle (8, 9). Differentiation of HL-60 cells by chemical agents, such as ATRA, 1,25-dihydroxyvitamin D3, and phorbol diester is accompanied by withdrawal from the cell cycle (1, 2). All of these data taken together suggest that down regulation of PP2A may be associated with ATRA-induced differentiation HL-60 cells, although the modulation of protein phosphatases only one of several parallel events which mediate the in is various effects of ATRA. Further studies of HL-60 cells utilizing antisense oligodeoxynucleotides directed against PP2A-C and variant cell lines (39) arrested at discrete points of differentiation may provide insights into the mechanisms by which PP2A influences myeloid differentiation. ACKNOWLEDGMENTS We thank M. 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Human myelogenous leukemia cell line HL-60 cells resistant to differentiation induction by retinoic acid. J. Biol. Chem., 264: 16149—16154, 1989. 4884 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research. Expression of the Catalytic and Regulatory Subunits of Protein Phosphatase Type 2A May Be Differentially Modulated during Retinoic Acid-induced Granulocytic Differentiation of HL-60 Cells Masakatsu Nishikawa, Serdar B. Omay, Hideki Toyoda, et al. Cancer Res 1994;54:4879-4884. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/54/18/4879 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1994 American Association for Cancer Research.