Differentiation-induced Changes in Protein-Tyrosine ... · [CANCER RESEARCH 52. 4027-4035, July 15, 1992) Differentiation-induced Changes in Protein-Tyrosine Phosphatase Activity

[CANCER RESEARCH 52. 4027-4035, July 15, 1992)

Differentiation-induced Changes in Protein-Tyrosine Phosphatase Activity andCommensurate Expression of CD45 in Human Leukemia Cell Lines1

Marina Buzzi,2- 3 Li Lu,3 Anthony J. Lombardi, Jr., Marshall R. Posner,4 David L. Brautigan, Lauren D. Fast,and A. Raymond Frackelton, Jr.5

Department of Medicine, Roger Williams Cancer Center [M. B., L. L, A. J. L., M. R. P., A. K. F.], and Department of BioMedical Sciences, Brown University[D. L. B., A. R. F.], and Division of Clinical Hematology, Rhode Island Hospital [L. D. F./, Providence. Rhode Island 02908

ABSTRACT

Pharmacologie differentiation of the promyelocytic leukemia HL60 isassociated with an increase in cellular tyrosine phosphatase activity. Weasked (a) if this increase might, at least in part, be due to changes in atransmembranous protein-tyrosine phosphatase, CD45; and (b) it ( 1)45changes similarly in other differentiating leukemias. Differentiation ofHL60, several chronic myelogenous leukemias, a monocytic leukemia(TIIP-l), and a monoblastoid leukemia (U-937) could be induced byphorbol ester, 1,25-dihydroxy vitamin 1),, dimethyl sulfoxide, or cyclicAMP analogues. This differentiation was associated with a markedincrease in (a) total cellular tyrosine phosphatase activity (2-4-fold asmeasured by the ability to dephosphorylate a tyrosine-phosphorylatedpeptide); (b) CD45-specific tyrosine phosphatase activity (2-4-fold); (c)CD45 cell surface expression by flow cytometry (2-5-fold); (d) synthesisof both exon B-dependent M, 205,000 and exon ABC M, 185,000CD45 proteins, as revealed by immunoprecipitation with antisera specific for CD45 isoforms. Both isoforms have enhanced electrophoreticmobility when isolated from the differentiated cells. This enhancedmobility did not appear to be due to decreased stoichiometry of CD45phosphorylation on serine/threonine residues. Interestingly, 12-0-tetradecanoylphorbol-13-acetate transiently reduced CD45 protein-tyrosine phosphatase activity in the chronic myelogenous leukemia cellRWLeu4 without altering the CD45 amount (as measured by cell surface immunofluorescence).

Modulation of CD45 tyrosine phosphatase activity (and proteinlevels) may play a role in differentiation or in maintaining cells ina nonproliferative state or may represent a phenotypic marker of differentiation.

INTRODUCTION

Studies of many transforming retroviruses and normal mammalian growth factor receptors have demonstrated that proliferation of both normal and neoplastic cells is dependent on thephosphorylation of certain proteins on tyrosine residues (for areview, see Ref. l). For example, the binding of polypeptidegrowth factors such as epidermal growth factor, platelet-derivedgrowth factor, fibroblast growth factors, insulin-like growth factor I, insulin, and colony-stimulating factor I to their respectivereceptors causes activation of the intrinsic tyrosine kinases oftheir receptors, typically resulting in the tyrosine phosphorylation of several important cellular proteins, including the rafserine kinase, phospholipase C 7, a M, 85,000 subunit of the

Received 2/6/92; accepted 5/7/92.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.

1Supported in part by National Cancer Institute Cancer Center Grant P30-CA13943-18. by National Cancer Institute Grant RO1-CA39235 (A. R. F.) and bya grant from the Italian Association of Cancer Research (M. B.).

2 Present address: Institute of Hematology, University of Bologna. Bologna.Italy.

1 M. B. and L. L. contributed equally to this work.4 Present address: Department of Medicine. New England Deaconess Hospital.

Boston. MA.5 To whom requests for reprints should be addressed, at Department of Medi

cine. Roger Williams General Hospital. Providence. RI 02908.

3'-phosphatidyl inositol kinase, the ras-GTPase activating pro

tein, and a A/r 62,000 protein associated with the ras-GTPaseactivating protein (1, 2). Mutated receptors which either lacktyrosine kinase activity or fail to associate with and phospho-rylate certain of these cellular proteins fail to transduce growthfactor signals (1-3). Similarly, many neoplastic cells rely onabnormally expressed tyrosine kinases, growth-factor receptors, and growth factors to demonstrate their transformed phe-

notype.The extent to which a protein is tyrosine phosphorylated is

the result of a dynamic equilibrium between two opposing reactions: phosphorylation by protein-tyrosine kinases and de-phosphorylation by tyrosine phosphatases (4-7). Altering thisequilibrium can have profound effects on cells. For example,vanadate, a potent inhibitor of tyrosine phosphatases, can causenormal cells to take on a transformed phenotype (8). In contrast, tyrosine phosphatase activity increases markedly duringpharmacologically induced terminal differentiation of HL-60promyelocytic leukemia cells (9-10). The potential role playedby the changes in tyrosine phosphatase activity during differentiation is particularly interesting.

The leukocyte common antigen CD45 has recently been de-montrated to be a protein-tyrosine phosphatase (6, 7, 11-13).CD45 is a family of structurally related, high-molecular-weight,membrane-spanning molecules found in all hematopoietic cellsexcept mature red cells, platelets, and their immediate progenitors. Here we sought to determine if the increase in tyrosinephosphatase activity during the differentiation of HL60 mightbe due, at least in part, to changes in CD45 expression andactivity, and to extend our observations to other leukemiacells.

MATERIALS AND METHODS

Reagents. 1,25-Dihydroxy vitamin D, was kindly provided by R.Uskokovic (Hoffman-LaRoche, Inc., Nutley, NJ). Stock solutions wereprepared at 0.2 niM in ethanol, protected from direct light, and storedat -20Â°C. PMA6 (Sigma Chemical Co., St. Louis, MO) was dissolved

in acetone at 10 Â¿ig/ml(17 MM)-protected from direct light, and storedat -20'C. DMSO (cell culture grade). Nonidet-P40, phenylmethylsul-

fonyl fluoride, bovine serum albumin (radioimmunoassay grade), leu-peptin, aprotinin, and pepstatin were all obtained from Sigma. Raytideand the p43v ahl tyrosine kinase were obtained from Oncogene Science(Unionville, NY). Affinity matrices (wheat germ agglutinin bound toSepharose and protein A bound to Sepharose) were obtained fromPharmacia (Piscataway, NJ). RPMI-1640 was purchased from Gibco

(Grand Island, NY).Monoclonal Antibodies. Monoclonal antibodies to CD45 included

13.4, GAP 8.3, CMRF.l 1(806), UCHL 1(826), and PD-7/26/l6(816).

6 The abbreviations used are: VDi 1.25-dihydroxyvitamin D}; PMA, 12-0-tetradecanoylphorbol-13-acetate; DMSO, dimethyl sulfoxide; HÃˆPES, /V-(2-hy-droxyethyl)piperazine-,V-(2-ethanesulfonic acid); PTPase. protein-tyrosine phosphatase: CML, chronic myelogenous leukemia: FCS. fetal calf serum; PBS.phosphate-buffered saline; PKC, protein kinase C.

4027

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CD45 AND PTPase DURING LEUKEMIC DIFFERENTIATION

Monoclonal antibodies 13.4 and GAP 8.3 (raised from the corresponding hybridomas obtained from the American Type Culture Collection)are specific for all members of the CD45 family (14). Antibodies 806,816, and 826 were obtained from the Third Leukocyte Typing workshop (15). Antibody 806 is dependent on the presence of the CD45 Aexon, antibody 816 is dependent on the presence of the B exon, andantibody 826 requires the absence of the A, B, and C exons and therefore recognizes the smallest, M, 180,000 CD45 isoform. Monoclonalantibody OKT8, which is specific for CDS, normal mouse IgG, or amouse IgGlk anti-dinitrophenyl monoclonal antibody (gift of H. Eisen,Massachusetts Institute of Technology) served as negative controls forflow cytometry and immunoprecipitations. Antibody to /32-microglob-ulin served as a positive control for changes in cell size (16). Monoclonal antibodies to the macrophage-specific antigens, CD1 Ib (Mol)and CD 14 (Mo2), were obtained from Coulter Corporation (Hialeah,FL), and monoclonal antibodies to the macrophage activation antigen,Mo3, were from R Todd (Ann Arbor, MI). Monoclonal antibody to thegranulocyte antigen, CD15, was obtained from Dako (Carpiteria, CA).

Cells Lines and Cell Culture. HL-60 human promyelocytic leukemiacells (17) (obtained from the American Type Culture Collection, Rock-ville, MD); human chronic myelogenous leukemia cell lines BV173(obtained from J. Ritz) (18), EM2 and EM3 (19) (obtained from A.Keating), RWLeu4 (16, 20, 21), and K562 (22) (obtained from theAmerican Type Culture Collection); and the monocytic leukemia-celllines U-937 and THP-I (obtained from H. Lazarus) were maintained inlogarithmic growth in RPMI 1640 supplemented with 10% PCS, non-essential amino acids, 2 HIML-glutamine. 100 units/ml penicillin, and100 Mg/ml streptomycin at 37Â°Cin a humidified 95% air/5% CO2

atmosphere.Induction of Differentiation. Cells growing logarithmically were sed-

imented for 5 min at 400 x g and resuspended in culture media containing various concentrations of maturational agents (typically 17 n\iPMA, 50 n%tVD.,, or 1.2% DMSO) at 2 x IO5cells/ml. The cells werethen typically cultured in 6-well tissue culture plates (Costar, Cambridge, MA) containing 5 ml of media/well for 0 to 5 days. Differentiation along the monocytic pathway was monitored by characteristicchanges in cellular and nuclear morphology (Wright-Giemsa staining),development of adherance to plastic substratum, the appearance ofcharacteristic macrophage antigens [CDllb (Mol), CD14 (Mo2),Mo3, monitored by flow cytometry': see belowj (23-25), and the abilityto reduce nitrobluetetrazolium (performed as detailed in kit instructions from Sigma). Differentiation along the myeloid pathway wasmonitored by changes in cellular and nuclear morphology and the appearance of cytoplasmic granules (May Grundwaldt and Wright-Giemsa staining) and the level of the granulocyte antigen CD 15(26-27).

Flow Cytometry. Cells were collected, sedimented at 400 x g,washed with PBS (0.14 M NaCl, 0.01 M sodium phosphate, pH 7.4,containing 1% PCS), resuspended at 1 x 10" in 200 n\ of PBS containing 2% FCS, and chilled on ice for 5 min. Then, 5-10 M' of primaryantibody (e.g., anti-CD45 or anti-CDl Ib) were added and incubated onice for 30-45 min. The cells were washed once in PBS at 4Â°C,and if the

first antibody added was not fluorescently labeled, the cells were thenresuspended in 100 p\ of PBS containing 5 M!of fluorescein isothiocy-anate goat anti-mouse IgG (Tago, Inc.. Burlingame, CA) and incubatedon ice for 30 min. The cells were washed 3 times with 4 ml of PBS andfixed with 1% formaldehyde in 1 ml of PBS. If the first antibody wasfluorescently labeled, the cells were fixed in the 1% formaldehyde solution after the two washes with PBS. The samples were stored at 4Â°C

and analyzed within 3 days by flow cytometry using an EPICS model Cflow cytometer (Coulter).

Cell Fractionation. Adherent cells were removed from tissue cultureplates after a 15-min incubation in 0.14 M NaCl, 0.01 Msodium phosphate. 0.1% EDTA, pH 7.4, on ice by dislodging with gentle triturationusing a Pasteur pipet and centrifuged at 400 x g for 5 min at 4Â°C.For

the assay of PTPase activity associated with the paniculate fraction ofcells, cell pellets were resuspended in buffer [10 imi HEPES, 5 IHMEDTA, (pH 7.0), 15 HIM2-mercaptoethanol, 0.14 imi phenylmethyl-

sulfonyl fluoride, and 10 Mg/ml of leupeptin, aprotinin, and pepstatin]at 1 x IO7cells/ml. The cells were disrupted by 10 strokes in a Douncehomogenizer (B pestle) or by sonication (three to six 5-s bursts at powersetting 5 with a microprobe-equipped sonicator; model W140D; Ultrasonics, Plainview, NY) and centrifuged at 8000 x g for 30 min at 4Â°C

to sediment particulate material. The pellet was resuspended in 25 HIMHEPES, l mM EDTA (pH 7.0), 50 HIM2-mercaptoethanol, 1 mg/mlbovine serum albumin, and 1% Triton X-100 and centrifuged for 10min at 4Â°C.The resulting detergent-soluble portion of the pellet wasaliquoted and stored at â€”¿�70Â°C.Alternatively, for PTPase assays of

immunoprecipitated CD45, cells were harvested as described above andthen extracted at 2 x IO6 cells/ml with lysis buffer (1% Triton X-100,0.15 Msodium chloride, 20 m\i Tris, 1 mM EDTA, 1 mm phenylmeth-ylsulfonyl fluoride, 0.04% bovine serum albumin. 0.04% Ficoll, and0.04% polyvinylpyrrolidone, pH 8.0) for 10 min on ice. The extract wasclarified by centrifugation (400 x g for 5 min) and reacted with 25 n\ ofprotein-A Sepharose beads which had been precoated with rabbit antibody to mouse IgG (Gateway) and the GAP 8.3 monoclonal antibody toCD45. After rotating overnight at 4Â°C,the CD45-immunosorbent com

plex was washed three times with 1 ml of the lysis buffer and three timeswith 0.14 Msodium chloride, 0.01 Msodium phosphate, pH 7.4. ThisCD45-immunosorbent complex was then directly assayed for its abilityto hydrolyze -12P-Tyr-Raytide as described below.

PTPase Assays. Tyrosine-phosphorylated Raytide was prepared using the tyrosine-containing peptide, Raytide, [-y-32P]ATP, and thep4jv-abi tyrosine kinase, as directed by packaging instructions (Oncogene Science). Typically, 10-15% of the 12P radioactivity was incorpo

rated into the peptide; by phosphoamino acid analysis, all of the labelwas in the tyrosine (data not shown). Nonincorporated [32P]ATP was

removed by multiple precipitations of the labeled peptide using 10%trichloroacetic acid and, finally, acetone. The labeled peptide was thenredissolved in 0.2 MTris, pH 8.0, and stored at -20'C until used. The

phosphatase reaction was performed essentially as described by Streuliet al. (28). Briefly, 5 n\ of reaction buffer (250 HIMHEPES, 50 mMEDTA, 100 mM dithiothreitol, pH 7.3), 15 M!of water, and 25 M!of thesample to be assayed (e.g., CD45-immunosorbent complex describedabove) were combined with 5 M!of 32P-Raytide (20,000 cpm) and incubated at 30Â°Cfor 20 min (reaction was still linear at 30 min at low

PTPase concentrations). Reactions were terminated by adding 750 M!of4% Norit A charcoal in 0.9 MHCI, 90 mMsodium pyrophosphate, 2 HIMmonobasic sodium phosphate, and unhydrolyzed 32P-Raytide was sedimented at 8000 x g for 10 min at 4Â°C.A 400-n\ aliquot of the supernatant was taken for measurement of hydrolyzed 32PO4 by scintillation

spectrometry. Dilutions of PTPase were utilized that gave between 5%and 20% hydrolysis of the 12P-Raytide. The release of 4000 cpm cor

responded to the hydrolysis of 2.4 pmol of PO4 .Data are expressed as the percentage of substrate hydrolyzed; where

indicated, this has been normalized to the activity extractable from thesame cell lines but which had not been exposed to maturational agents,to facilitate comparison of different cell lines.

Immunoprecipitation of Metabolically Labeled CD45. For metaboliclabeling of CD45 with [35S]methionine, cells were washed twice inincili ion ine tree media, resuspended at 10'' cells/ml in the same mediacontaining 0.25 mCi L-35S-Translabel/ml (ICN, Irvine, CA), and incubated at 37Â°Cfor 4 h (or as indicated) in a humidified 95% air/5% CO2atmosphere. For metabolic labeling of CD45 with 32PÂ¡,cells werewashed twice in phosphate-free media, resuspended at IO6 cells/ml inthe same media containing 1 mCi 32PÂ¡/ml(ICN), and incubated at 37Â°C

for 3 h as for methionine labeling. Proteins were then extracted fromthe cells with detergent (1% Triton X-100, 10 nui Tris, 5 IHMEDTA, 50HIMNaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 100MMsodium orthovanadate, 0.1% bovine serum albumin, 1 mM phenyl-methylsulfonyl fluoride, pH 7.6, at 4'C), and cellular debris was removed by centrifugation (8000 x g for 15 min at 4"C). To partially

purify the glycosylated, mature CD45 proteins, clarified protein extractwas rotated for l h at 4"( ' with 25 M!of wheat germ agglutinin linked to

Sepharose beads, after which the beads were washed three times with 1ml of the extraction buffer, and glycoproteins were specifically eluted

4028




using 40 n\ of 0.3 M/V-acetyl glucosamine prepared in extraction buffer.CD45 proteins in this eluate were reacted overnight at 4Â°Cwith the

indicated anti-CD45 monoclonal antibody (e.g., 13.4, GAP 8.3, etc.)and then coprecipitated using protein A-Sepharose immunosorbentwhich had been precoated overnight with rabbit antibody to mouseimmunoglobulin G. The CD45-immunosorbent complex was washedtwice with 1-ml aliquots of the extraction buffer (lacking bovine serumalbumin) and finally eluted by boiling for 5 min in Laemmli samplebuffer. CD45 proteins were resolved by electrophoresis on sodiumdodecyl sulfate polyacrylamide gels (5-7.5% acrylamide) and visualizedby fluorography (for -15S)or by autoradiography using preexposedKodak XAR-5 film and Lightning Plus intensifying screens, exposingat â€”¿�70Â°Cfor 1-2 days. Densitometric analysis was performed on the

autoradiograms using a GS300 scanning densitometer and GS370Macintosh software (Hoefer, San Fransisco, CA). The CD45 peak wasintegrated by Gaussian curve fitting of the densitometric differencebetween the anti-CD45 lane and the anti-dinitrophenyl nonspecific control lane.

Cell Proliferation. Cells were seeded into wells of 24-well tissueculture plates at 1 x IO4 cells/well (~20% confluence) in 1 ml culture

media containing the indicated concentrations of maturational agents.After 24, 48, and 72 h, cells were harvested by treatment with trypsin-EDTA and enumerated using a Coulter counter (model Channelyzer,ZM).

RESULTS

Pharmacological differentiation of the promyelocytic leukemia HL60 is associated with an increase in cellular tyrosinephosphatase activity (9, 10, 29). We were interested in whethersimilar changes would be observed during pharmacologicallyinduced differentiation of other leukemia cell lines, in particularcell lines derived from chronic myelogenous leukemias. Onerecently derived CM L cell line, RWLeu4, appears to be arrestedat a developmental stage very similar to that of HL60. TheRWLeu4 cells clearly lie on the myelomonocytic pathway: theylack Fc receptors, have a basophilic cytoplasm with prominentcytoplasmic granules, have a high nuclearcytoplasmic ratio,bear/32-microglobulin and CD1 Ib antigen on their surface, lack

immunoglobulin, and display histocytochemically both perox-

idase and esterase activity (16, 20, 21). Furthermore, like HL60(30-34), these cells can be induced to differentiate along the

monocytic pathway into macrophages by VD, (15) and byPMA (Figs. 1-3). Exposure of RWLeu4 cells to 10 HMPMA

caused them to adhere to the plastic tissue culture substratumand to develop lobulated nuclei and irregular cellular morphology with pseudopodia (Fig. 1, A and B). These morphologicalchanges were accompanied by striking increases in the myelo/monocyte-specific antigen CDllb (Fig. 2A) but a decrease inthe granulocyte-specific antigen CD 15.

Also like HL60 (35, 36), RWLeu4 can be differentiated alongthe myeloid pathway into granulocytes by polar solvents such asDMSO (Figs. 1 and 2). DMSO causes RWLeu4 cells to becomeadherent and to exhibit morphological changes similar to butless extreme than those described for PMA-induced differenti

ation (Fig. 1C). Furthermore, unlike PMA, DMSO caused nosignificant decrease in the granulocyte-specific antigen CD 15

(Fig. 2B).Both PMA and DMSO showed a marked, dose-dependent

inhibition of RWLeu4 proliferation (Fig. 3), with 50% inhibition doses of <1 n\i and 1.2%, respectively. RWLeu4 viabilitywas >95% after 3 days' exposure to 10 n\i PMA and >60%after 3 days' exposure to 1.5% DMSO, as measured by trypan

blue exclusion.PTPase Activity during RWLeu4 Differentiation. We sought

first to determine the effect of PMA-induced differentiation onPTPase activity. PTPase activity was assayed using tyrosine-phosphorylated Raytide, an excellent substrate, compared toseveral other artificial substrates, for the CD45 families of tyrosine phosphatases (28, 37, 38). Preliminary experiments indicated that most PTPase activity was associated with the par-ticulate fraction of the HL-60 and RWLeu4 cells, irrespective

of their exposure to maturational agent, and therefore all subsequent data are reported on that fraction. Raytide PTPaseactivity in HL60 cells increased by 90% (P < 0.01) after 1 day

Fig. 1. PMA and DMSO induce morphological differentiation of RWLeu4 CML cells. R\VLeu4 cells were seeded at 2 x 10s cells/well in 6-well tissue culture dishesand cultured for 3 days in RPMI 1640, 10% FCS. as indicated in "Materials and Methods." in the absence of maturational agents (.â€¢/),in the presence of 35 nin PMA(B), or in the presence of 1.2% DMSO (for 3 days (C). Cellular morphology is shown through phase-contrast optics. Bar. 10 Â¡Â¿m.

4029




100 .

05Ãœ

:â€¢=so .<no

CL

0123 0123

Exposure (days)

Fig. 2. PMA and DMSO cause the appearance of differentiation antigens onR\VLeu4 cells. RWLeu4 cells were exposed to 16 HMPMA or 1.2% DMSO as inFig. 1 for the indicated times, harvested by chilling and gentle trituration with apipet and analyzed for the presence and relative amount of antigenic markers ofmonocytic and granulocytic differentiation, using flow cytometry (see "Materialsand Methods"). Data are expressed as percentage positive cells, where each datapoint is the average Â±SE, typically of 4-6 replicates from two or more experiments. G. CD1 Ib; â€¢¿�CD14: O. Mo3; â€¢¿�.CDI5.

0 1 2 301

Exposure (days)

Fig. 3. PMA and DMSO inhibit proliferation of RWLeu4 CML cells.R\VLeu4 cells were seeded into culture dishes as in Fig. I and cultured in thepresence of various concentrations of PMA (A) or DMSO (A). At the indicatedtimes cell numbers were determined with a Coulter cell counter. Cell numbersexpressed arc the means of at least six separate determinations in three experiments. Bars, SE. typically <5% of the mean. Inhibition of cell growth by PMAwas statistically significant (P < 0.01 by Student's t test) at all times and concen

trations of PMA, and inhibition by DMSO was statistically significant at DMSOconcentrations >1%.

of PMA exposure and then remained essentially unchanged(Fig. 4). RWLeu4 responded somewhat differently to PMA,however. Most strikingly, Raytide PTPase activity was transiently inhibited, showing 30% inhibition after a 30-min exposure (P < 0.05) and partial recovery by 2 h (22% inhibition).Like HL60 cells, RWLeu4 showed maximal Raytide PTPase(70% increase) after 24 h of exposure to PMA.

Effects of PMA on CD45 Expression. CD45 is a major,transmembranous PTPase found on nearly all cells in the he-matopoetic lineage. Therefore we asked whether CD45 expression changed in response to PMA-induced differentiation andwhether any changes correlated with the changes in cellularPTPase observed above. Various leukemia cell lines were exposed to PMA for 6, 24, 48, and 72 h, harvested, and examinedfor CD45 expression by flow cytometry (Table 1 and data notshown). As expected, nearly all lines showed >95% of theircells to be positive for CD45 expression. However, there weremarked differences in the amount of CD45 expressed per cell,with untreated RWLeu4 cells displaying several times moreCD45 than HL60 cells or K562 cells. Exposure of RWLeu4and HL60 cells to PMA caused an increase in cell surface

expression of CD45, first detectable in HL60 cells after 6 h (nochange detected after a 30-min PMA exposure) and maximalafter 24 h in RWLeu4 and 48 h in HL60 (Table 1). Interestingly, CD45 increased to nearly the same final amount onHL60 and on RWLeu4, even though this represents a 200%increase for HL60 and only a 60% increase for RWLeu4 (Table1). Of the eight cell lines examined, only K562 failed to showsignificantly increased CD45 in response to PMA (Table 1).K562 is unique in its position on the erythroid lineage and isinduced by PMA along a differentiation pathway to megakary-ocytes (39).

To test whether the increase in CD45 was specific for maturation induced by PMA, we examined the effects of three othermaturational agents: DMSO, 1,25-dihydroxyvitamin D3, and8-chloro-cAMP. As mentioned above, DMSO differentiatesHL60 and RWLeu4 along a granulocytic pathway, 1,25-dihydroxyvitamin D3 along a monocyte/macrophage pathway, and8-chloro-cAMP along a monocytic pathway (40). Each of theseagents induced marked increases in the cell surface expressionof CD45, with DMSO and VD3 inducing CD45 expressionlevels in RWLeu4 surpassing those induced by PMA (Table 2).

Effects of PMA on CD45 Synthesis and Electrophoretic Mobility. The increased level of cell surface CD45 could haveresulted from increased CD45 synthesis, decreased degradation, or transfer of already synthesized CD45 from an intrac-ellular compartment to the cell surface. To begin to addressthese possibilities, we examined the effects of PMA on CD45synthesis in RWLeu4. RWLeu4 cells were metabolically labeled with [35S]methionine, and mature, glycosylated CD45was partially purified from cell extracts by lectin-affinity chro-matography and specific immunoprecipitation. Fluorographyof the immunoprecipitated CD45 revealed a broad band, frequently resolved as a pronounced doublet, corresponding inmigration to A/r ~210,000-230,000 proteins (Fig. 5, Lanes 0h). Exposure to PMA caused a dramatic increase in CD45synthesis: nearly 3-fold after 24 h, 5-fold after 48 h, and still2-fold after 72 h by densitometric analysis (Fig. 5, Lanes 14, 48,and 72 h). Increased synthesis of CD45 could be detected asearly as 1 h after PMA exposure (data not shown). Similarpronounced increases in CD45 synthesis were observed inHL60, U937, EM2, and EM3 cells (data not shown).

2 20 40

PMA Exposure (hr)

60

Fig. 4. Protein-tyrosine phosphatase activity during PMA-induced differentiation of HL60 (D) and RWLeu4 (A) leukemia cells. Cells were seeded in tissueculture dishes and exposed to 16 n\i PMA or carrier solvent for O min, 30 min, 2h, 24 h, 48 h, or 72 h. Cell extracts were prepared and assayed for tyrosinephosphatase activity as described in "Materials and Methods," using 12P-Tyr-

Raytide as substrate. Phosphatase activity is expressed relative to the activity in anequal number of untreated cells. Data points (Â±SE)are the average of 4-5 experiments. In each experiment, 4-10 replicate serial dilutions of cell extract wereused to determine the enzymatic activity.

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Table 1 Changes in cell surface CD45 during PMA-induced differentiation

CelllineHL60RWLeu4BV173K562EM2EM3U937THP-1CelltypePromylCMLCMLCMLCMLCMLMono.Mono.Days

ofexposure01001001001001001001001001162*160*104122ND'121NDNDÂ±SE139341292282*152*218*124ND118148*NDtoPMA-Â±SE397711423309*157*222*120256*188*206*216*Â±SE4189111936915

"Cells were exposed for the indicated times to 16 nw PMA (except BV173.which required 160 n\i PMA to induce differentiation) and then examined for cellsurface CD45 by flow cytometry as detailed in "Materials and Methods." Numbers,

CD45 mean fluorescence intensity compared to untreated cells. Each measurementhas been repeated typically 8 to 10 times.

* CD45 fluorescence was significantly greater (P < 0.01 ) than in cells cultured inmedia or exposed to carrier solvent alone.

CND, not determined.

(Table 1) might have been responsible for at least a part of theincrease in PTPase activity seen after PMA exposure (Fig. 4),but this would have been possible only if the CD45 from cellsexposed to PMA is fully active. To test this, CD45 was isolatedfrom untreated and PMA-treated RWLeu4 cells and thentested for its ability to catalyze the release of 32P from 32P-Tyr-

Raytide. Interestingly, PMA acutely and transiently inhibitedCD45 PTPase activity (50% inhibited, P < 0.01, after a 30-minexposure) (Fig. SA). By 2 h of exposure, cellular CD45 activityhad begun to climb, and activity peaked near 24 h at approximately 3-4 times its initial activity. One can obtain a crude

measure of CD45 relative specific activity by dividing CD45PTPase activity (Fig. 8/4) by the relative amount of CD45 determined by flow cytometry (Table 1 and data not shown).Examining this measure of specific activity as a function ofPMA exposure (Fig. SÃ„), CD45 specific activity decreased

The electrophoretic mobility of CD45 increased slightly, butvery consistently, after 24 h of PMA exposure in RWLeu4(Fig. 5) (and in all other cells examined, including HL60, U937,EM2, and EM3, and in response to 1,25-dihydroxyvitamin D3(data not shown). The increased mobility persisted in the differentiated cells (Fig. 5 and data not shown). Because CD45exists in several splicing isoforms (41-43), and because isoformusage has been reported to change during differentiation(28, 44, 45), we asked whether the increased mobility was dueto changes in splicing isoforms. To address this question,RWLeu4 cells were exposed to PMA for various times, meta-bolically labeled, extracted with detergent, and subjected to lec-tin-affinity chromatography as before. Immunoprecipitation ofCD45 with isoform-specific antibodies revealed that the CD45doublet seen with the non-isoform-specific CD45 antibody(13.4; see Figs. 5 and 6) consisted of a M, ~215,000 exonB-dependent isoform and a Mr ~ 190,000 isoform lacking theA, B, and C exons (Fig. 6). After exposure to PMA, synthesis ofboth isoforms increased severalfold, but they each now migrated slightly faster, with apparent A/r of ~ 205,000 and~ 180,000. Exon A-containing CD45 isoforms were not de

tected.Another possible explanation for the increased electro

phoretic mobility was a PMA-induced hypophosphorylation:less phosphorylated species migrate faster than more highlyphosphorylated species (46). Therefore, we examined the effects of PMA on the extent of CD45 phosphorylation. RWLeu4cells were exposed to PMA for various times as before, meta-bolically labeled with 32Pi, and then extracted with detergent.

CD45 was immunoprecipitated from the detergent extracts,resolved by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis, and visualized by autoradiography (Fig. 7). Althoughlittle change in CD45 phosphorylation was noticeable after 30min, CD45 phosphorylation increased markedly after 24 and48 h of PMA exposure, at least commensurately with the 70%increase in cell surface CD45 (Table 1) and consistently withthe severalfold increase in CD45 synthesis (Figs. 5 and 6).These data seem inconsistent with the hypothesis that decreasesin overall CD45 phosphorylation are responsible for shifts in itsgel mobility.

Effects of PMA on CD45 PTPase Activity. Mechanisms regulating CD45 PTPase activity are not well understood but mayinvolve posttranslational modifications such as changes inserine or tyrosine phosphorylation or the addition of a lipidmoiety (47-49). The PMA-induced increase in CD45 protein

Table 2 Effects of other maturational agents on CD45Cells were cultured for 3 days in media with 1.2% DMSO, 50 nui VD3, or 308-chloro-cAMP.

CelllineHL60RWLeu4AgentNoneDMSO1,25-Dihydroxy

D,8-Chloro-cAMPNoneDMSO1,25-Dihydroxy

D,8-Chloro-cAMPFl.

Inten"100185*202Â»148Â»100255Â»281Â»198Â»Â±SE148433312

" Mean fluorescence intensity of CD45 measured by flow cytometry and normalized to cells cultured in media alone (carrier solvents did not affect CD45expression).

* Significantly greater (P < 0.01) than cells cultured in media alone.

PMA (hrs)

205 kD >

Fig. 5. Effects of PMA on CD45 synthesis and electrophoretic mobility inRWLeu4 CML cells. RWLeu4 cells were exposed to 16 nw PMA for the indicatedtimes and were metabolically labeled with [-15S]methionine during the final 3 h ofPMA exposure as described in "Materials and Methods." CD45 was partially

purified from detergent extracts of equal numbers of these cells by sequentialwheat germ affinity chromatography and specific immunoprecipitation using amonoclonal antibody broadly reactive with CD45 isoforms (monoclonal 13.4; see"Materials and Methods"). The partially purified CD45 proteins were analyzed by-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7.5% acrylamide, reducing conditions) and visualized by fluorography (20 h) at -70"C using pre-flashed XAR-5 film. C, monoclonal mouse anti-dinitrophenylate was used as anegative control. Ab, antibody 13.4 specific for CD45.

4031



CD45 AND PTPase DURING LEl'KEMIC DIFFERENTIATION

PMA (hrs) 24 48[c Ab A B Nile Ab A B N Ile Ab A B N l

205 kD Â»~

Fig. 6. Effects of PMA on synthesis of CD45 isoforms. RWLeu4 cells wereexposed to 16 nM PMA for the indicated times, metabolically labeled with |-"S]-mcthionine. and extracted with detergent as in Fig. 5. CD45 was partially purifiedfrom the detergent extracts of equal numbers of cells by affinity chromatographyon wheat germ agglutinin and then immunoprecipitated with monoclonal anti-dinitrophenylate as negative control (c); monoclonal antibody 1.1.4 (Un. whichreacts with all CD45 isoforms; monoclonal antibody 806 (A), which reacts withCD45 isoforms containing exon A; monoclonal antibody 816 (B). which reactswith CD45 isoforms containing exon B; or monoclonal antibody 826 (N), whichreacts with a CD45 isoform lacking the A. B. and C exons. Fluorography was for20 h.

markedly and transiently within 30 min of PMA exposure (P<0.01) but then displayed a trend toward increased activity after1 day of exposure and remained at approximately that specificactivity thereafter.

DISCUSSION

Mounting evidence has pointed to pivotal roles for tyrosinephosphorylation of cellular proteins in growth factor signaltransduction, immune recognition (T- and B-cell responses toantigen), and progression through the cell cycle. The steady-state level of tyrosine phosphorylation of any particular proteinis the result of a dynamic balance between tyrosine phosphorylation and tyrosine dephosphorylation. Although much isknown about the dozens of tyrosine kinases and the roles theyplay in cellular processes, comparatively little is known aboutthe counterbalancing PTPases. One PTPase, CD45, is found inlarge amounts on virtually all cells of hematopoetic lineages(except cells late in the erythroid lineage). CD45 appears to playa critical role in the antigen activation of T-cells by removing anegative regulatory phosphate from tyrosine at position 505 oflek, a src family tyrosine kinase which, when activated, appearsto phosphorylate the f subunit of the T-cell receptor; CD45appears to play a similar role in the antigen activation of B-cells(50-57). The function of CD45 in other cells is not known,however.

One interesting question is whether PTPases play a role innormal or in pharmacologically induced differentiation. In thisregard, Frank et al. (9, 10) have reported severalfold increasesin PTPase activity during pharmacologically induced differentiation of the promyelocytic leukemia cell line HL60.

Here we asked whether changes in PTPase activity were moregenerally observed during the pharmacological maturation of

leukemia cell lines and whether changes in PTPase activitymight be due, at least in part, to changes in the number of CD45molecules per cell or in the specific activity of CD45 PTPase inthese cells. We chose to focus most of our studies on a CML cellline, RWLeu4, which is rather similar to HL60 in that it ispositioned along the myeloid lineage (Refs. 16, 20, 21, andFigs. 1-3), can be induced to differentiate along a monocyte/macrophage pathway by exposure to 1,25-dihydroxy vitaminD, (16, 21) and phorbol esters (Figs. 1-3), and along a granu-locytic pathway by exposure to DMSO (Figs. 1-3). However,differentiation induced by PMA is not completely equivalent todifferentiation induced by VD,. Whereas both PMA and VD,caused similar morphological changes (Fig. 1), increases in theearly macrophage-specific antigen CD1 Ib (Fig. 3) and inhibi

tion of proliferation (Fig. 2), VD, but not PMA increased thelate macrophage differentiation antigen CD 14, and PMA butnot VD, increased the macrophage activation antigen Mo3(Fig. 3). By contrast, both PMA and VD, increased CD1 Ib andCD14 on HL60. Another difference between RWLeu4 andHL60 is seen with the myeloid marker CD 15. CD 15 is highlyexpressed in RWLeu4, reduced during PMA and VD, mono-cytic differentiation, but maintained at high levels duringDMSO-induced granulocytic differentiation (Fig. 3). In HL60,CD 15 is expressed at low levels and induced by DMSO but notby PMA or VD,,.

Clearly, PMA-induced differentiation of RWLeu4, as well asof HL60, is associated with a general increase in PTPase activity (Fig. 4). Using a substrate (12P-Tyr-Raytide) that the CD45

PTPase family hydrolyzes well, the maximal increase inPTPase activity was observed after HL60 cells had been exposed to PMA for only 1 day. This would suggest that the majorincrease in PTPase seen after 1 day of PMA exposure was dueto CD45 (or a CD45 family member). However, this is notcompletely consistent with the flow cytometric evaluation ofCD45 following PMA treatment of HL60 (Table 1 and data notshown). Although nearly all HL60 cells expressed CD45, theydid so at a relatively low level per cell; exposure to PMA caused

PMA(h) ro-,r-0.5-i|-24-ir48-,Ab C AD c AD AD

CD45Â»-

_ .Fig. 7. Effects of PMA on CD45 phosphorylation. RWLeu4 cells were exposed

to 16 nM PMA for the indicated times and were metabolically labeled with "PÂ¡during the final 3 h of PMA exposure as described in "Materials and Methods."

CD45 was partially purified from detergent extracts of these cells as in Fig. 6. C,monoclonal anti-DNP asa negative control: Ab. antibody 13.4 specific forCD45.Arrow, position of CD45. Autoradiograph is from a 20-h exposure at -70'C witha Lightning Plus (Kodak) intensifying screen and preflashcd XAR-5 film.

4032




"- oÃ 3

Â§ Â£o â€”¿�Ã¼ 2

$ tj 3

Ã s-u- W 0in > 2^ "^^Q J5Ã¼

1

B

O 20 40 60

PMA Exposure (h)Fig. 8. CD45 tyrosine phosphatase activity during PMA-induced differentia

tion of RWLeu4 CML cells. A. RWLeu4 cells treated with 16 niw PMA for theindicated times and then harvested and fractionated as detailed in "Materials andMethods." The paniculate fraction was solubilized with detergent, and CD45 waspartially purified by sequential lectin affinity chromatography and anti-CD45immunoprecipitation. The partially purified CD45 was tested for its ability tohydrolyze 32P from "P-Tyr-Raytide, as detailed in "Materials and Methods."

PTPase activity is expressed relative to activity in an equal number of untreatedor carrier-solvent treated controls. B, CD45 specific activity estimated by the ratioof CD45 PTPase activity in A to the amount of CD45 determined by flow cy-tometry in Table 1. Ordinale, the same ratio, normalized to 1.0 for cells notexposed to PMA. Points, average (Â±SE)of 3-5 experiments. In each experiment,6-10 replicate serial dilutions of CD4S were used to determine its enzymaticactivity.

a marked increase in CD45 expression, but this increase did notpeak until at least 2 days of PMA exposure: CD45 increased byonly 60% after 1 day, 180% after 2 days, and 210% after 3 daysof exposure to PMA (Table 1). This suggests that in HL60,PMA maximally induces within 1 day some PTPase other thanCD45 having a preference for hydrolyzing "P-Tyr-Raytide.

Alternatively, the specific enzymatic activity of CD45 may begreatest after 1 day of PMA exposure.

Frank and Sartorelli (9, 10) reported previously that PMAcauses a 2-fold increase in HL60 PTPase activity after 1 day'sexposure and 8-fold increase after 2 or 3 days' exposure. They

used a copolymer of glutamine, alanine, and tyrosine, phospho-rylated on tyrosine residues, as a substrate for the cellular PT-Pases. Because similar polyanionic copolymers of glutamineand tyrosine actually inhibit some PTPases (particularlyPTPase 1A, IB, and PTP5) (38, 58), it is difficult to comparethe kinetics and magnitude of their PTPase changes with ourresults obtained with "P-Raytide as a PTPase substrate.

The overall time course of changes in RWLeu4 PTPase wassimilar to that seen in HL60 cells. However, in RWLeu4, incontrast to HL60, PMA caused a commensurate and coincidentincrease in total particulate Raytide PTPase (Fig. 4) and inCD45 Raytide PTPase per cell (Fig. 8/1). This suggests that

CD45 constitutes a large fraction of RWLeu4 PTPase activity.Consistent with this interpretation, CD45 was present at muchhigher levels on untreated RWLeu4 cells than on untreatedHL60 cells (data not shown).

In RWLeu4 (but not reproducibly in HL60 or in U937; datanot shown), PMA caused a transient, marked inhibition of Ray-tide PTPase. Interestingly, the time course of this inhibitioncorresponded to the time course of PMA-induced decrease in

CD45 total PTPase activity (Fig. SA) and specific PTPase activity (Fig. 8Ã„).The mechanism whereby CD45 PTPase activityis inhibited is unclear. We did not detect gross changes in thephosphorylation of CD45 that might be responsible for thedecrease in CD45 PTPase activity, although subtle changes inthe phosphorylation of individual sites might well have occurred. (Alternatively, CD45 function may be inhibited by someother posttranslational modification or by complexing with aninhibitory protein.) CD45 may well be the target of regulatoryprotein kinases other than PKC: CD45 is highly phosphory-lated in RWLeu4 cells under conditions where PKC is >90%down-regulated (Fig. 7; data not shown).

In some human T-cells PKC increases the serine phosphorylation of CD45, and this correlates with a transient decrease inCD45 PTPase activity in human T-cells (59, 60). In contrast,however, direct phosphorylation of CD45 by PKC in vitro doesnot alter CD45 PTPase activity (37). Furthermore, Ostergaardand Trowbridge (61) have recently reported that ionomycin, butnot PMA, reduces the serine phosphorylation of a specific siteon CD45 in mouse thymocytes. Coincident with this decrease,they observed a decrease in CD45 PTPase activity.

The biological significance of the rapid decrease in CD45specific activity is unclear. Inasmuch as we have detected this inresponse to PMA-induced differentiation of RWLeu4 but notHL60 or U937, this transient decrease in CD45 PTPase activity may not play a general role in PMA-induced differentiation.This would also be consistent with the severalfold variation inCD45 levels expressed by different myelomonocytic leukemiccell lines (Table 1; data not shown) that differentiate in response to PMA. Nevertheless, rapid increases in CD45-medi-ated dephosphorylation of tyrosine 505 of the lek protein arebelieved to be important in T-cell activation (55). T-cell activation by antigen results in the activation of PKC (see Ref. 62): wecould speculate that one result of this might be to feedback-down-regulate CD45 activity, tightly regulating responses toantigen.

In addition to the general increase in the amount of CD45that occurs during PMA-induced differentiation of most leukemia cell lines, we also observed a clear increase in the electro-phoretic mobility of CD45 (Figs. 5-7). Because of reports ofchanges in CD45 isotype usage during T-cell differentiation(44, 45), we initially speculated that analogous changes wereoccuring in RWLeu4 (and other leukemia cell lines). Thisclearly was not the case, however (Fig. 6); one isoform wedetected contained the B exon but lacked the A exon, while theother isoform lacked the A, B, and C exons. PMA caused nochange in the usage of these exons.

It is also well known that decreases in the stoichiometry ofphosphorylation can increase electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (46).We had already determined that PMA down-regulated PKC inRWLeu4 cells (data not shown). We speculated that, in theabsence of PKC, CD45 might be serine phosphorylated to amuch lower extent. Two findings argue against this possibility.

4033



CD45 AND PTPasc DURING LEUKEMIC DIFFERENTIATION

First, allowing cells to recover to normal levels of PKC (bywashing away phorbol ester and then culturing cells an additional 24 h in its absence) does not result in return of CD45 toits slower electrophoretic mobility (data not shown). Second,PMA exposure caused commensurate increases in CD45 (byflow cytometry; Table 1) and in CD45 phosphorylation (bydensitometric analysis of Fig. 7). We are left, then, with fourpossible explanations. PMA may change the usage of someother CD45 exon or may cause a posttranslational modificationin polypeptide length (proteolysis), carbohydrate content, orlipid moieties. In this regard, PKC is known to activate a protease which cleaves the colony-stimulating factor I receptor

(63).Our data also suggest that CD45 specific activity (Fig. 8Ã„)

increases concurrently with increases in CD45 mobility (Figs.5-7). Our observation of a trend to increased CD45 specific

activity is limited in that we determined cellular CD45 usingflow cytometry. Our antibodies do not immunoblot, and concurrent changes in the rate of CD45 synthesis and protein half-life complicated attempts to compare CD45 in undifferentiatedand differentiated cells by isotopie labeling. This raises thepossibility that the faster-migrating CD45 may be a more en-

zymatically active form of CD45. Alternatively, the apparentincreased specific activity of CD45 may reflect the down-regulation of PKC. This could be easily tested by washing away thephorbol ester and allowing PKC to return to normal levels.Although this tactic does not alter CD45 migration (Fig. 7), wedo not know whether this would return CD45 apparent specificactivity to levels found normally in RWLeu4 cells.

The generality of increases in CD45 during the pharmacologically induced differentiation of leukemia cells was seen inthe effects of PMA on several leukemia cells lines (Table 1) andin the effects of other maturational agents on RWLeu4 andHL60 (Table 2). This raises the question of whether the increased expression of CD45 (and CD45 PTPase activity) playsa role in the differentiation process itself or, rather, plays a rolein the function of the differentiated cell. Our data do not distinguish between these possibilities. Although one CM L cellline, K562, showed no changes in CD45 during PMA-induceddifferentiation, PMA causes K562 to differentiate along amegakaryocytic pathway, and mature megakaryocytes wouldnot be expected to have high levels of CD45, inasmuch asplatelets, produced by megakaryocytes, lack CD45 (64). Thusthe failure of CD45 to increase during the differentiation ofthese cells is not surprising. However, other PTPases may substitute for increased CD45 during differentiation of these cells.Consistent with this possibility, Butler et al. (65) have recentlyreported increases in a M, 40,000 cytosolic PTPase duringPMA-induced differentiation of K562.

Our results with HL60 cells are also generally consistent witha recent report of increases in CD45 expression and CD45PTPase activity during PMA-induced monocytic and DMSO-induced granulocytic differentiation (66). Our results appear todiffer from theirs regarding U937 cells, however. We detect a100% increase in the amount of CD45 on U937 cells duringPMA-induced differentiation (Table 1), whereas Taetle et al.(66) mention that PMA does not increase CD45 immunofluo-

rescence on U937 cells.Coordinate expression of CD45 and various antigenic mark

ers of monocytic and myeloid differentiation has been examined in human bone marrow by flow cytometry (64). In normalmonocytic differentiation, CD45 is expressed at high levels on

monocytic precursors and increases further during monocyticdifferentiation. In contrast, CD45 is present at low levels onmyeloid precursors and remains low and constant duringgranulocytic differentiation. Thus while our data (in contrastto Taetle's) are consistent with normal monocytic differentia

tion (monocytic differentiation induced by PMA and VD,caused increased expression of CD45 in RWLeu4, HL60,BV173, EM2, U937, and THP-1), our (and Taeltle's HL60)

observations on DMSO-induced granulocytic differentiation(CD45 increases) do not appear consistent with normal myeloiddifferentiation.

What might be the function of the large amounts of CD45 inthe differentiated monocytic cells? Given that CD45 appears toplay a role in transducing antigen recognition signals in bothT-cells and B-cells, it is tempting to speculate about a rolefor CD45 in signal transduction in mature monocytes/macrophages, perhaps transducing signals from Fc or other cellsurface receptors.

ACKNOWLEDGMENT

We thank Akiko Takeda for helpful discussions and for critical reading of the manuscript.

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inPhosphatase Activity and Commensurate Expression of CD45 Differentiation-induced Changes in Protein-Tyrosine

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Differentiation-induced Changes in Protein-Tyrosine ... · [CANCER RESEARCH 52. 4027-4035, July 15, 1992) Differentiation-induced Changes in Protein-Tyrosine Phosphatase Activity