[CANCER RESEARCH57. 4141-4147, september 15, l997J

second meningioma locus on chromosome 22q (6). Other candidatemeningioma genes on chromosome 22q include a member of thehuman /3-adaptin family (7) and a gene termed MNJ that is disruptedby a balanced translocation in a meningioma cell line (8).

Gain-of-function genetic changes in meningioma, such as amplification of cellular proto-oncogenes, have been investigated but are yetto be described (9). One gain-of-function regulatory change, thecoordinate expression of PDGF B chain and of PDGF-j3 receptorsubunits, is a common feature of human meningiomas (10—13).Multiple studies have shown that the vast majority of meningioma tissuesamples express both PDGF B mRNA and protein (10—12, 14, 15).The coexpression of PDGF-BB and PDGF-(3 receptors suggests thepresence of autocrine stimulatory loops in these tumors. However, afunctional role for PDGF autocrine loops in human meningioma hasnot been established as it has for another common brain neoplasm,malignant astrocytoma (16, 17). Meningiomas are not tumors thatcommonly progress from lower to higher grades. Thus, there is nobasis for correlating the expression of PDGF ligands and their receptors with tumor malignancy, as can be done with astrocytoma (18, 19).The scarcity of established meningioma cell lines makes it difficult toassess the role of PDGF autocrine loops in maintaining the transformed phenotype of these tumors. In fact, there is no evidence thatPDGF receptors within meningioma are exposed to sufficient levels ofPDGF ligand in vivo to trigger receptor activation, and in turn,transduce a mitogenic response.

The detection of PDGF receptor activation in primary samples ofbrain tumor tissue requires especially sensitive immunochemical reagents, because these tumors contain significant amounts of normalstroma. Several years ago, we and others showed that synthetictyrosine phosphopeptides, corresponding to functional autophosphorylation sites in growth factor receptors, could be used as immunogensto raise antisera which only interact with the receptor in its activeconfiguration (20, 21). In contrast to generic, commercially availableantibodies to pY, these “pY-directed―antibodies recognize pY onlywithin the context of a defined sequence of flanking amino acids.pY-directed antibodies can be used as immunohistochemical reagentsto detect the activation of specific growth factor receptors at the singlecell level.6 Using somewhat different methodology, comparable phosphoserine/threonine-directed antibodies to signal generating proteinshave also been developed (22—27).

We describe here the development and characterization of a pYdirected antibody to the PDGF-(3 receptor. Our antibody is targeted toY-75l of the human PDGF-j3 receptor, which, in its phosphorylatedstate, forms part of the recognition motif for the p85:P13 kinasesignal-generating complex (28). The p85:P13 kinase recognition motifof PDGF-j3 receptors plays a pivotal role in PDGF-induced mitogenesis, chemotaxis, and membrane ruffling (29—32). Using both bio

6 A. Bhattacharyya,F. L. Watson,T. A. Bradlee,S. L. Pomeroy,C. D. Stiles, andR. A.Segal.unpublishedobservations.

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Detection of Activated Platelet-derived Growth Factor Receptors inHuman Memngioma'

Steven M. Shamah,2 John A. Alberta, William V. Giannobile, Abhi.jit Guha, Yunhee K. Kwon,3 Rona S. Carroll,Peter M. Black, and Charles D. Stiles4Department of Microbiology and Molecular Genetics, Hareard Medical School, Boston. Massachusetts 02115 [S. M. S., J. A. A., W. V. G., Y. K. K., C. D. 5.1: The Dana-FarberCancer institute, Boston, Massachusetts 02115 [S. M. S., J. A. A., W. V. G., 1'.K. K., P. M. B., C. D. S.]: Department of Periodontology, Harvard School of Dental Medicine.Boston, Massachusetts 02115 (W V. G.J; Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research institute, Mt. Sinai Hospital. Toronto, Ontario. Canada M5T-258 [A. G.J; Division of Neurosurgery, The Toronto Hospital, Toronto, Ontario, Canada M5T-2S8 (A. G.J; and Neurosurgical Laboratories and Brain Tumor Center, Brighamand Women's Hospital. Boston, Massachusetts 02115 (R. S. C., P. M. B.], Department of Surgery, Children's Hospital. Boston, Massachusetts 02115 (R. S. C., P. M. B.J, andDepartment ofSurgery, Harvard Medical School, Boston. Massachusetts 02115 (R. S. C., P. M. B.]

ABSTRACT

The @3receptor subunit of platelet-derived growth factor (PDGF) andIts corresponding Ilgand (PDGF-B8) are coordinately expressed in freshsurgical Isolates of human menlagloma. These observations Imply thatPDGF autocrine loops are engaged in human meningioma and suggestthat activated PDGF-@3receptors might contribute to the pathology of thiscommon brain neoplasm. The study of PDGF autocrine loops and humanmeningloma has been slowed by the scarcity of meningioma cell culturemodel systems@ Furthermore, in meningioma tumor tissue, the activation

state of PDGF receptors is difficult to assess with conventional reagents,because the tumor Is intermixed with normal stroma. In fact, there is noevidence that PDGF receptors within the tumor are activated by ligand.We used a synthetic tyrosine phosphopeptide to raise an antibody thatreports the phosphorylation state of tyrosine 751 in the human PDGF-fireceptor. Phosphorylated tyrosine 751 is a recognition site for phosphatidylinositol 3'-kinase, a cytoplasmic effector ofPDGF-induced mitogenesis,chemotaxis, and membrane niffling. Immunoblotting and immunostaining analyses with this antibody show that the PDGF4J receptor isconstitutlvely phosphorylated at tyrosine 751 within multiple fresh surgical IsOlates of human meningioma. These findings are consistent with arole for activated PDGF receptors in the proliferation of human meningiomas.

INTRODUCTION

Meningiomas are common, primary intracranial tumors which areslow-growing and mostly benign (1). The genetic aberrations thatestablish memngioma and contribute to its growth have not been wellcharacterized. Approximately 50% of meningiomas have deletions inchromosome 22, suggesting that loss-of-function mutations in tumor

suppressor genes at this locus may be involved in meningioma etiology (2—4).The increased incidence of meningiomas in NF-25 patientsand the location of the NF-2 gene on chromosome 22q makes NF-2 acandidate tumor suppressor gene for both sporadic and NF-2-associated meningiomas. In those sporadic meningiomas with loss of heterozygosity on chromosome 22q, approximately 50—60% of the tumors have mutations in the NF-2 gene, with loss or reduction ofexpression of the encoded protein (5). However, examination ofpatients and families with multiple meningiomas have not showngerm-line mutations in the NF-2 gene, suggesting the presence of a

Received 3/27/97; accepted 7/17/97.Thecostsof publicationof thisarticleweredefrayedin partby the payrnentof page

charges. This article must therefore be hereby marked advertisement in accordance with18U.S.C.Section1734solelyto indicatethisfact.

I This work, to which the first two authors contributed equally, was supported by NIH

Grant SF01 HD24926.2 Present address: Division of Neuroscience, Department of Neurology, Children's

Hospital, Boston, Massachusetts 02115.3 Present address: Department of Biology, College of Arts and Sciences, Kyunghee

University, Seoul, South Korea.4 To whom requests for reprints should be addressed, at Division of Cellular and

Molecular Biology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115.5 The abbreviations used are: NF-2, neurofibromatosis type 2; Y-75l, tyrosine 751;

pY, phosphotyrosine; P13, phosphatidylinositol 3; TBST, Tris-buffered saline withTween 20.

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ACTIVATED PDGF RECEFFORS IN HUMAN MENINGIOMA

chemical and immunohistochemical methods, we show that PDGFreceptors are constitutively phosphorylated at Y-75l in fresh surgicalspecimens of human meningioma.

MATERIALS AND METHODS

Antibody Design and Purification. The @3isoform of PDGF receptor,

rather than the a isoform, is most commonly expressed in meningioma (10—14,33). Therefore, we synthesized a peptide, CKDESVD-pY-VPMLDMK, where

pY refers to phosphotyrosine, corresponding to amino acids 745—758of thehuman PDGF-(3 receptor (Self-Y-P). The corresponding unphosphorylatedpeptide (Self-Y) was also synthesized. Both peptides were generated using

tertiary-butyloxycarbonyl (TBOC) chemistry on an Applied Biosystems, Inc.430A peptide synthesizer at the Core Molecular Biology Facility, Dana-FarberCancer Institute (Boston, MA). The NH,-terminal cysteine residue was addedto facilitate coupling to maleimide-activated keyhole limpet hemocyanin, aswas accomplished using the IMJECT system from Pierce Chemical Co. (Rock

ford, IL) according to the manufacturer's instructions. Immune serum (antipY-751) was generated in a New Zealand white rabbit at HRP, Inc. (Denver,

PA) by injecting 0.5 mg of coupled peptide in Freund's complete adjuvant(primary injection) and 0.25 mg/injection in Freund's incomplete adjuvant(subsequent injections). Immunoglobulin was isolated using protein A-Sepha

rose column chromatography, with elution by 100 mM glycine (pH 3.0).Protein containing fractions as determined by the Bio-Rad (Melville, NY)

microprotein assay were pooled, and 5 mg of this material were passed

repeatedly through a peptide-affinity column overnight. This column wasgenerated using Affi-Gel 10 (Bio-Rad) following the instructions for anhydrous peptide coupling with DMSO as a solvent. The unbound material fromthat column was then applied to a phosphopeptide column (generated in thesame manner) overnight. The column was then washed extensively and consecutively with 100 mM Tris, 10 nmi Iris, 0.5 M NaC1, 10 nmi Iris, 100 mM

glycine (pH 3.0), and 10 mt@i Iris (Iris pH was 8.0 in all cases). Active

material was eluted with 100 mM triethylamine (pH 11.5). Fractions werecollected in tubes containing 2 M Iris (pH 8.0) to neutralize the base andassayed for specific reactivity toward the activated PDOF receptor using

immunoblots containing 40 p@gIlane of protein extracts generated from quies

cent or PDGF-BB-stimulated (30 ng/ml for 10 mm) BALB/c 3T3 cells.

Activation state-specific fractions were pooled and concentrated using a Cen

triprep-10 concentrator in the presence of I mg/ml BSA (final). Concentratedpools were stored in aliquots at —20°C.

Cell Culture and Harvesting. BALB/c 3T3 cells were maintainedinDMEM (Life lechnologies, Inc.) supplemented with 10% bovine calf serum.Cells were grown to confluence on tissue culture dishes 150 mm in diameter.The medium was changed at confluence to 5% platelet-poor plasma for anadditional 18 h. Cells were incubated in the absence or presence of 30 ng/ml

of PDGF-BB (Upstate Biotechnology Inc., Lake Placid, NY). Cells wererinsed three times with Iris-buffered saline [10 mrviIris (pH 8.0) and 157 msi

sodium chloridel and then lysed in 0.5 ml of NP4Olysis buffer [1% NP4O,20mM HEPES (pH 8), 10% glycerol, 2.5 mM EGIA, 2.5 msi EDIA, 10 @tg/mlleupeptin, 2 @.tg/mlaprotinin, 1m@iphenylmethylsulfonyl fluoride, and 0.5 msisodium vanadatej. Cell lysates were scraped, transferred to an Eppendorf tube,

and rocked for 10 mm at 4°C.Cellular debris was removed by microcentrifugation, and the supernatants were stored at —70°C.

Immunoblotting. Cell lysates were reconstituted in Laemmli samplebuffer (34), boiled for 10 mm, and separated on SDS-7.5% polyacrylamidegels. Proteins were transferred to Immobilon-P (Millipore) in 250 mMIris, 200mM glycine, and 20% methanol for 1.5 h. The blots were blocked in 2%

BSA-IBSI (10 mMIris, 150 mMsodium chloride, and 0.1% Iween 20) for2 h at room temperature. Primary antibody incubations were performed in

TBST for I.5 h at room temperature at the following dilutions: anti-pY-751 at1:1,500,anti-PDGF-j3receptor (pan-a) (Upstate Biotechnology,Inc.) at1:5,000, and 4GlO (a gift from Dr. Thomas Roberts, Dana-Farber Cancer

Institute, Boston, MA) at I:10,000. For peptide competition experiments,anti-pY-75 I at 1: 1500 was incubated for 2 h at 4°C in the presence of 10 nM

immunizing (Self-Y-P) peptide, the corresponding unphosphorylated peptide(Self-Y), or the following unrelated phosphopeptides: CAENPE-pY-LGLDVPV (NPXY), CSDGHE-pY-I-pY-VDPVG(pY-579/-58l ; Src-bindingpeptide sequence), CDIES PS-pY-MAPYDN (pY-77 I ; GAP-binding peptide se

quence), CSSVL-pY-IAVQP (pY-857; SHP2-binding peptide sequence), and

CDESDND-pY-IIP (pY-102I ; PLC--y-bindingpeptide sequence). The blot wasthen probed overnight at 4°Cwith this material. The Immobilon blots wererinsed four times for 5 mm each and then incubated with goat antirabbit or goatantimouse horseradish peroxidase-coupled secondary antibody (Bio-Rad) at1:4000 in IBSI for 45 mm at room temperature. The blots were rinsed as

above and then developed using Amersham enhanced chemiluminescencedetection reagents.

Harvesting of Tumor Samples. Meningioma tissue specimens were collected at the time of craniotomy for tumor resection and were immediatelysnap frozen in liquid nitrogen in the operating room and subsequently stored inliquid nitrogen. For the purposes of comparison, nonneoplastic tissue wasobtained from patients undergoing temporal lobectomy for uncontrollableseizures. Each sample was taken from a specimen that was used by the

neuropathologist for diagnosis. Tissue samples were Dounce homogenized onice in 1 ml of NP4O lysis buffer (described above) per 100 mg of tissue. The

homogenate was clarified by centrifugation at 10,000 X g for 30 mm at 4°Cand stored at —80°C.

Immunocytochemistry. For cell culture staining experiments, quiescentBALB/c 3T3 cells were treated for 10 mm with 30 ng/ml of PDGF-BB or notreatment as indicated. The cells were fixed with 2% paraformaldehyde contaming 1 mM orthovanadate, permeabilized with 0.5% NP4O, and blocked with

2% normal goat serum. For tissue section staining experiments, control brainand meningioma samples were harvested by tissue excision at the time ofsurgery. Specimens were placed directly into ice-cold 4% paraformaldehyde

fixative for 2—8h. The fixed biopsies were embedded subsequently in paraffinand cut into 5-,.zm sagittal tissue sections. The tissue specimens were next

exposed to a series of graded xylene and ethanol rinses followed by hydration

of the sections. This pretreatment was followed by incubation with the antipY-75l or pan-PDGF-@3 receptor antibodies. Following treatment with primary

antibody (I :50 dilution), the cultures were processed for peroxidase stainingusing a commercial avidin-biotin complex method kit (Vector Laboratories,

Burlingame, CA) and True Blue (KPL Laboratories, Gaithersburg, MD) substrate. In some cases, anti-pY-751 was incubated, prior to cell staining, for 30mm at room temperature with its phosphopeptide immunogen (Self-Y-P), thecorresponding unphosphorylated peptide (Self-Y), or an unrelated phosphopeptide (NPXY). In tissue-staining experiments, sections were also processed for H&E staining to obtain descriptive histology (magnification, X40;

phase contrast; H&E).

RESULTS

Generation of a pY-directed Antibody to the PDGF-fi Receptor. A peptide encompassing residues 745—758of the human PDGF-(3receptor was synthesized with a pY at position 751 (Y-75l) and used asan immunizing antigen. Affinity-purified rabbit antiserum was tested for

its capacity to recognize PDGF receptors in a receptor-specific andactivation-specific fashion by immunoblot analysis. Fig. lA shows that

anti-pY-75 1 detected PDGF receptors in PDGF-treated BALB/c 3T3fibroblasts but not in untreated, quiescent cells. Preincubation of anti-pY

751 with an excess of the immunizing phosphopeptide (Self-Y-P) dimmated the reactivity with tyrosine phosphorylated PDGF receptor,whereas preincubation with the unphosphorylated peptide (Self-Y) failedto compete (Fig. 1A). In immunoblotting experiments (but not in cellstaining experiments; see below), anti-pY-75l also detected the activation of PDGF-a receptors in cells treated with PDGF-AA. Thus, antipY-75l can be used as an activation-specific and PDGF receptor-specificantibody for immunoblot analysis.

Positional Specificity of the pY-751 Antibody. Peptide competition experiments were performed in which the pY-75 I antibodywas preincubated with phosphopeptides corresponding to otherSH2 recognition motifs in the PDGF-@ receptor. Phosphopeptidescorresponding to the SH2 recognition motifs for Src (pY-579/

-58 1), GTPase-activating protein (pY-77 1), SHP-2 (pY- 1009), andphospholipase C-y (pY-l021) failed to compete (Fig. 2). In addition, neither the PDGF-/3 receptor major autophosphorylation site

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ACTIVATED PDGF RECEPTORS IN HUMAN MENINGIOMA

@d@

Competitor: /@

PDGF: —÷ -+ -+ AA

200kD—@

@ a

98kD

64kD

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Fig. 1. The pY-751 antiserum specifically detects PDGF receptors in anactivated,tyrosine-phosphorylatedstate.QuiescentBALB/c3T3fibroblastswere incubated in the absence (—)or presence (+) of 30 nglml PDGF-BB or30ng/mlPDGF-AA(An)for 10mmat 37°C.CelllysateswereseparatedbySDS-PAGE on a 7.5% polyacrylamide gel, transferred to Immobilon-P (Millipore), and analyzed by immunoblotting with either anti-pY-75l or theanti-pYantibody4010. Peptidecompetitionexperimentswereperformedbypreincubaling the pY-75l antibody with a 10-fold molar excess of thephosphopeptide antigen (Self-Y-P) or the corresponding nonphosphorylatedpeptide (Self-Y)prior to blotting.

phosphopeptide (pY-857) nor an Shc recognition phosphopeptidederived from the erbB2 receptor tyrosine kinase (pY-l248) competed for binding of the pY-75l antibody. The possibility ofanti-pY-75l recognizing other untested pY motifs on PDGF receptors or other proteins cannot be dismissed. Nevertheless, these datademonstrate a clear preference of anti-pY-75 I for pY-75 1 relativeto six other PDGF-@ receptor pYs.

Anti-pY-751 Detects Activated PDGF Receptors as an Immunostaining Reagent. Quiescent BALB/c 3T3 cells were stimulatedwith PDGF or left untreated, and they were immunostained withanti-pY-75 1 (Fig. 3A). The pY-75 1 antibody failed to stain quiescentcells, despite the presence of moderately high levels of PDGF-@3receptor in the cultures (Fig. 3A, first two panels). In contrast, antipY-75l readily stained PDGF-BB-treated cells, a pattern that wascompeted by the Self-Y-P peptide but not by the unphosphorylatedpeptide (Self-Y) or an unrelated peptide (NPXY; Fig. 3A). The staining appeared uniform throughout the cell bodies, with some perinuclear concentration.

As a test of receptor specificity, we exposed 3T3 cells to PDGF-AA,which activates PDGF-ct receptors rather than PDGF-(3 receptors. AntipY-751 did not stain cells that were exposed to PDGF-AA (Fig. 3B). Asa positive control to show that activated PDGF-a receptors can bedetected with this methodology, we stained PDGF-AA-treated 3T3 cellswith a different pY-directed antibody that we raised to the positionalequivalent ofY-751 in the PDGF-a receptor(tyrosine-742). As expected,the pY-directed antibody to tyrosine-742 (anti-pY-742) in the PDGF-csreceptor stained 3T3 cells that had been exposed to either PDGF-AA orPDGF-BB. The PDGF-a receptor-directed anti-pY-742 antibody was not

characterized further for this study.The Anti-pY-751 PDGF-fi Receptor Antibody Detects

PDGF-BB (c-sis) Autocrine Loops. We initially tested the capacityof anti-pY-75l to detect PDGF autocrine loops in PDGF-transformed

BALB/c 3T3 fibroblasts. This well-defined cell culture model systemdisplays constitutive PDGF receptor kinase activity and hallmarkmorphologicalchangesassociatedwith cellular transformation(16,17, 35). Anti-pY-751 stains c-sis-transformed 3T3 cells (16) whengrown in PDGF-free medium, demonstrating the presence of PDGFautocrine loops (Fig. 3C). In contrast, neither v-src nor Ha-rastransformed fibroblasts grown under the same conditions stainedpositive for activated PDGF receptors with the pY-75l antibody. Allthree transformed fibroblast cell lines expressed comparable levels ofPDGF-f3 receptor, as detected by staining with a pan-j3 receptorantibody (Fig. 3C). These results demonstrate the utility of the pY-75lantibody as an immunocytochemical reagent capable of detectingPDGF autocrine loops in situ.

Activated PDGF Receptor Detected in Human MeningiomaTissue with Anti-pY-751. Immunoblot analysis with anti-pY-75l revealed a signal, of varying intensities, in the majority of meningiomatumors investigated (Fig. 4). In most cases, the pY-75l antibody preferentially recognized a protein band that comigrates with the lower of twobands detected with pan-a, a conventional antibody targeted specificallyto human PDGF-(3 receptors. The lower band recognized by anti-pY-75lis likely to represent partially glycosylated PDGF receptor that is acti

vated by PDGF from within the meningioma cells (see “Discussion―).Immunocytochemical analysis of human meningioma tumor specimensrevealed positive staining with the anti-pY-751 antibody (Fig. 5). Forcomparison, nonneoplastic tissue obtained from seizure patients undergoing temporal lobectomy consistently stained negative with anti-pY751. Staining of these tissue sections with the pan-a receptor antibodyranged from nondetectable/low for control brain to moderatelhigh formeningioma. There are currently no useful molecular or histochemicalmarkers for meningioma. Although the solubility of the peroxidase staining substrate used to display anti-pY-75l immunoreactivity prevented usfrom obtaining histological counterstains, anti-pY-75l consistently

Fig. 2. The pY-751 antibody preferentially recognizes PDGF receptorstyrosine phosphorylated at position Y751. Quiescent BALB/c 3T3 fibroblasts were incubated in the absence (—)or presence (+) of 30 ng/mlPDGF-BB. Cell lysates were fractionated by SDS gel electrophoresis andimmunoblotted with anti-pY-75l . Prior to immunoblotting, the pY-75lantibody was incubated with the indicated peptides (see “MaterialsandMethods―for exact sequences) at a concentration of 100 nr@ifor I h at roomtemperature.

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ACTIVATED PDGF RECEPTORS IN HUMAN MENINGIOMA

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Fig. 3. A, detection of activated PDGF receptors on cultured cells in situ. Quiescent BALB/c 3T3 were treated for 10 mm with 30 ng/ml of PDGF-BB (+) or no treatment (—)asindicated. The indicated cells were fixed with 2% paraformaldehyde containing 1 msi orthovanadate, permeabilized with 0.5% NP4O,and blocked with 2% normal goat serum. Thispretreatment was followed by incubation with anti-pY-751 or a pan-PDGF-f3 receptor that is not sensitive to the phosphorylation status of the receptor (Pan-a). Following treatmentwith primary antibody (1:50 dilution), the cultures were processed for peroxidase staining using a commercial avidin-biotin complex method kit (Vector Laboratories) and True Blue(KPL Laboratories) substrate. In some cases, anti-pY-75l was incubated prior to cell staining for 30 mm at room temperature with its phosphopeptide immunogen (Self.Y-P), thecorresponding unphosphorylated peptide (Self- Y), or a generic phosphopeptide (NPXY). B, activation of the PDGF-a and/or@ receptors detected at the single-cell level withphosphorylationstate-specificantibodies.QuiescentBALB/c313 cellsweregrownon fibronectin-coatedcoverslipsandtreatedfor7 mmwith30 ng/mlPDGF-AAor PDGF-BBatthe same concentration or were not treated (nt) as indicated. Cells were fixed with parafonnaldehyde, permeabilized with NP4O,and blocked with normal goat serum. Left, the primaryantibody used in each panel. Anti-pY742 is directed against the corresponding pY in the PDGF-a receptor. C, the pY-75l antibody detects PDGF autocrine stimulatory loops inPDGF-transforrnedcells.3T3fibroblaststransformedbyPDGF-BB(c-sis),v-src,orHa-msweregrowninPDGF-freemediumfor3—4daysandthenfixedandstainedwithanti-pY-75lor pan-@3as described in A. Magnification, X63, phase contrast.

stained tumor sections positive and failed to stain control brain sections. DISCUSSIONIn immunoblotting experiments, the anti-pY-75l antibody also repeatedly detected activated PDGF receptors in tumor cell lysates (Fig. 4) butnot in control brain tissue lysates (data not shown). Because stromal cellsare present in both tumor and benign tissue specimens, it is likely that theimmunoreactivity of anti-pY-751 in the meningioma samples reflects thepresence of activated PDGF receptors in tumor cells. These resultsconfirm the presence of activated PDGF-@ receptors in human meningioma tissue.

Coordinate and elevated expression of PDGF-BB and the PDGF-(3receptor is a common feature of meningioma (10—13).The PDGF-f3receptors expressed in memngiomas are functional in that they stimulate transcriptional induction of the immediate-early gene c-los (12)and induce a mitogenic effect when stimulated with PDGF-BB (12,13, 15). Conditioned medium from meningioma cell cultures alsostimulates the proliferation of meningioma in vitro, and this activity is

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PDGF-AA

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ACTIVATED PDOF RECEPTORS IN HUMAN MENINGIOMA

Fig. 4. Anti-pY-75l detects constitutively active PDGF receptors in human meningioma protein lysates. Human meningioma tissue samples were collected and frozen in liquidnitrogen immediately following tumor resection. Protein lysates were obtained by homogenization in an NP4O lysis buffer containing protease and phosphatase inhibitors, separatedby SDS-PAGE, and analyzed by immunoblotting for activated PDGF receptors (anti-pY-751) and total PDGF-(3 receptors (pan-a). The arrows correspond to fully processed (top arrow)and partially processed (bottom arrow) PDGF receptors. Protein lysates from c-sis-transformed BALB/c 3T3 fibroblasts (515) were also analyzed for anti-pY-751 and pan-f3immunoreactivity as a positive control for displaying PDGF autocrine loops.

neutralized by anti-PDGF-BB antibodies (15, 36, 37) as well as by PDGF-BB in vivo to actually trigger receptor activation. We describesuramin and trapidil, two nonspecific growth factor antagonists (37, here a pY-directed antibody to the p85:PI3 kinase recognition motif of

38).Cumulatively,thesefindingsdemonstratethepotentialforPDGF activatedPDGF-(3receptors.In 3T3 fibroblastmodelsystems,theautocrine stimulation as a hyperproliferative signal in human mean- pY-751 antibody detects receptors that have been activated by exoggioma. The one important issue that has been unresolved to date is enous PDGF. The antibody also reacts with PDGF receptors in 3T3whether PDGF-j3 receptors are exposed to sufficient levels of cells that have been transformed with PDGF-BB (c-sis) by an auto

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ACFLVATED PDGF RECEFFORS IN HUMAN MENINGIOMA

crine loop mechanism. However, it does not react with receptors in313 cells transformed by other activated oncogenes (e.g., v-src andHa-ras). In both immunoblot assays and immunohistochemical experiments, the pY-75 I antibody detects phosphorylated PDGF receptors

in the majority of human meningioma specimens studied.Can phosphorylation at Y-75 1 be equated with a gain-of-function

mitogenic signal? Y-75 I serves as a recognition motif for the p85

subunit of P13 kinase (28). The p85:P13 kinase recognition motif plays

a critical role in the mitogenic response to PDGF (29, 3 1, 39). Forthese reasons, it is reasonable to speculate that constitutively activatedPDGF receptors provide a gratuitous and chronic proliferative cue thatcontributes to the transformed phenotype of sporadic meningioma.However, our data, strictly speaking, show only that PDGF receptorsare phosphorylated at a significant signal-generating position. They donot show that the tumor cells are dividing as a consequence ofreceptor phosphorylation or that the phosphorylated receptors areeven engaged with signal-generating proteins. It is notable that freshsurgical isolates of meningioma express two isoforms of PDGF re

ceptor with apparent M,s of 180,000 and 160,000. These receptorisoforms are comparable to mature, fully glycosylated and immature,

partially glycosylated PDGF-j3 receptors detected in sis-transformed3T3 cells (40). In meningiomas, the Mr 160,000 receptor isoform ispreferentially tyrosine phosphorylated. In contrast, the Mr 180,000receptor isoform appears to be primarily accountable for mitogenicsignaling in sis-transformed 3T3 cells (40). This discrepancy mayreflect cellular differences in receptor processing or autocrine loopmechanisms or an absence of PDGF autocrine proliferative signalingin these tumors. It is also possible that the Mr 160,000 band corre

sponds to activated PDGF-a receptor because anti-pY-75 I detectsPDGF-a receptors activated by PDGF-AA in 3T3 fibroblasts (see Fig.I ). However, it has been widely reported that meningioma tumortissue expresses abundant levels of PDGF-@3 receptor (10—14, 33), but

little, if any, PDGF-ca receptor ( I2—14).Thus, the tyrosine phosphorylated Mr 160,000 protein detected by anti-pY-75l in human meningioma is more likely to be a differentially processed form of thePDGF-@3 receptor. Additional studies involving dominant-negativemutations of PDGF and/or its receptors will be required to specificallyaddress the functional consequence of activated PDGF receptors inmeningioma. Reverse genetic approaches will be technically challenging for meningiomas due to the scarcity of established meningioma

cell lines. This approach has been successful in determining thatPDGF autocrine loops contribute to the transformed phenotype ofmalignant astrocytoma (16, 17).

Another unresolved issue is how the expression of PDGF ligandsand receptors is up-regulated to establish the coordinate and elevatedpatterns that are observed in human meningioma. A deletion in thefifth intron of the PDGF-B chain has been detected in a population ofmeningioma patients (I 3, 4 1). However, there was no correlation

between cytogenetic analysis and PDGF-B expression, and an intron5 deletion has been shown to have no effect on PDGF-B chain genepromoter activity or PDGF-BB expression (13, 42). Although othershave observed increased PDGF-B immunoreactivity with highergrades of meningioma ( 13), we did not observe a correlation betweentumor grade and degree of PDGF-j3 receptor expression or autophosphorylation (data not shown). Nonetheless, it is possible that PDGFautocrine loops contribute to the tumorigenicity of meningiomas orthe transition to higher grades.

In summary, we have detected activated PDGF-f3 receptors inhuman meningiomas using an antibody that exclusively recognizestyrosine phosphorylated PDGF receptors. These data extend previous

observations on the coordinate expression of PDGF ligand and receptor in two ways: (a) they show that receptors are exposed to sufficientlevels of ligand in vivo to trigger receptor dimerization and autophos

phorylation and (b) they show that receptors are activated withintumor cells as opposed to endothelium or normal stroma. Thesestudies also demonstrate the utility of activation state-dependent an

tibodies for the study of growth factor receptor activation in complexanimal tissues such as brain. Their use as immunohistochemicalreagents to localize the activation state of specific receptors or signalgenerators in situ provides a significant advantage over existingreagents. The prevalence of ligand/receptor coexpression patterns in amyriad of tumor types suggests that, in some cases, autocrine stimulation as detected with phosphospecific antibodies might serve as auseful marker for the onset or progression of tumorigenesis.

ACKNOWLEDGMENTS

The meningioma tumor sections were reviewed by Dr. Matthew Frosch(Brigham and Women's Hospital, Boston, MA).

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