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Expression and Distribution of Id Helix-Loop-Helix Proteins in Human Astrocytic Tumors DMITRI A.A. VANDEPUTTE, 1,2 DIRK TROOST, 1 SIEGER LEENSTRA, 2 HELEN IJLST-KEIZERS, 1 MARJA RAMKEMA, 1 D. ANDRIES BOSCH, 2 FRANK BAAS, 3 NAB K. DAS, 1 AND ELEONORA ARONICA 1 * 1 Department of (Neuro)Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 2 Department of Neurosurgery, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 3 Department of Neurozintuigen, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands KEY WORDS astrocytoma; glioblastoma; inhibitors of DNA binding; tumor blood vessels immunocytochemistry ABSTRACT The Id family of helix-loop-helix proteins is involved in a variety of processes, such as development, proliferation, and angiogenesis. In this study, we investigated the expression pattern of Id1, Id2, and Id3 in surgical specimens of human glial tumors. Western blot analysis demonstrated that all three Id proteins were ex- pressed in astrocytic tumors. Expression levels in high-grade tumors were higher than in low-grade tumors. Immunohistochemical analysis confirmed that many of the tumor astrocytes exhibited strong Id1-3 IR. In contrast, in adult human normal brain, Id expression was low both in resting astrocytes and in endothelial cells. In tumor cells, Id proteins displayed cytoplasmic as well as nuclear localization. Id1-3 IR scores in tumor cells were positively correlated with proliferation indices. Moreover, Id1-3 IR was de- tected in endothelial cells of the astrocytic tumor blood vessels. The vascular Id1-3 expression correlated positively with tumor vascularity and grade. These results support the role of the Id gene family in the enhanced proliferative potential of tumor astrocytes. The evidence also supports the involvement of the Id gene family in tumor angiogenesis, a process that critically influences the malignant behavior of glial tumors. GLIA 38: 329 –338, 2002. © 2002 Wiley-Liss, Inc. INTRODUCTION An important feature of glial cells is represented by their ability to divide postnatally (Gensert and Gold- man, 2001). Glial proliferation characterizes the brain response to injury (reactive gliosis), as well as the ma- lignant transformation of glial cells (Dirks and Rutka, 1997; Ridet et al., 1997). Astrocytes have been shown to have the highest predisposition to malignant transfor- mation compared with other central nervous system (CNS) cell types. Accordingly, astrocyte-derived tumors represent the most common type of primary brain tu- mors (Kleihues and Cavanee, 2000). Tumor cells in astrocytoma share some common features with reac- tive astrocytes, including expression of proteins that are normally produced at different developmental stages in the astrocytic differentiation pathway (Kristt et al., 1993; Kaluza et al., 1994; Krishna et al., 1995; Ridet et al., 1997; Godbout et al., 1998; Aronica et al., 2000). These observations may reflect the existence of Grant sponsor: National Epilepsy Fund Power of the Small and Hersenstich- ting Nederland; Grant number: NEF 02-10; Grant sponsor: Stichting AZUA. *Correspondence to: E. Aronica, Department of (Neuro)pathology, H2, Aca- demic Medical Center,Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: [email protected] Received 5 November 2001; Accepted 20 February 2002 DOI 10.1002/glia.10076 Published online 00 Month 2002 in Wiley InterScience (www.interscience. wiley.com). GLIA 38:329 –338 (2002) © 2002 Wiley-Liss, Inc.

Expression and distribution of id helix-loop-helix proteins in human astrocytic tumors

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Page 1: Expression and distribution of id helix-loop-helix proteins in human astrocytic tumors

Expression and Distribution ofId Helix-Loop-Helix Proteins in

Human Astrocytic TumorsDMITRI A.A. VANDEPUTTE,1,2 DIRK TROOST,1 SIEGER LEENSTRA,2HELEN IJLST-KEIZERS,1 MARJA RAMKEMA,1 D. ANDRIES BOSCH,2

FRANK BAAS,3 NAB K. DAS,1 AND ELEONORA ARONICA1*1Department of (Neuro)Pathology, Academic Medical Center, University of Amsterdam,

Amsterdam, The Netherlands2Department of Neurosurgery, Academic Medical Center, University of Amsterdam,

Amsterdam, The Netherlands3Department of Neurozintuigen, Academic Medical Center, University of Amsterdam,

Amsterdam, The Netherlands

KEY WORDS astrocytoma; glioblastoma; inhibitors of DNA binding; tumor bloodvessels immunocytochemistry

ABSTRACT The Id family of helix-loop-helix proteins is involved in a variety ofprocesses, such as development, proliferation, and angiogenesis. In this study, weinvestigated the expression pattern of Id1, Id2, and Id3 in surgical specimens of humanglial tumors. Western blot analysis demonstrated that all three Id proteins were ex-pressed in astrocytic tumors. Expression levels in high-grade tumors were higher thanin low-grade tumors. Immunohistochemical analysis confirmed that many of the tumorastrocytes exhibited strong Id1-3 IR. In contrast, in adult human normal brain, Idexpression was low both in resting astrocytes and in endothelial cells. In tumor cells, Idproteins displayed cytoplasmic as well as nuclear localization. Id1-3 IR scores in tumorcells were positively correlated with proliferation indices. Moreover, Id1-3 IR was de-tected in endothelial cells of the astrocytic tumor blood vessels. The vascular Id1-3expression correlated positively with tumor vascularity and grade. These results supportthe role of the Id gene family in the enhanced proliferative potential of tumor astrocytes.The evidence also supports the involvement of the Id gene family in tumor angiogenesis,a process that critically influences the malignant behavior of glial tumors. GLIA 38:329–338, 2002. © 2002 Wiley-Liss, Inc.

INTRODUCTION

An important feature of glial cells is represented bytheir ability to divide postnatally (Gensert and Gold-man, 2001). Glial proliferation characterizes the brainresponse to injury (reactive gliosis), as well as the ma-lignant transformation of glial cells (Dirks and Rutka,1997; Ridet et al., 1997). Astrocytes have been shown tohave the highest predisposition to malignant transfor-mation compared with other central nervous system(CNS) cell types. Accordingly, astrocyte-derived tumorsrepresent the most common type of primary brain tu-mors (Kleihues and Cavanee, 2000). Tumor cells inastrocytoma share some common features with reac-tive astrocytes, including expression of proteins that

are normally produced at different developmentalstages in the astrocytic differentiation pathway (Kristtet al., 1993; Kaluza et al., 1994; Krishna et al., 1995;Ridet et al., 1997; Godbout et al., 1998; Aronica et al.,2000). These observations may reflect the existence of

Grant sponsor: National Epilepsy Fund Power of the Small and Hersenstich-ting Nederland; Grant number: NEF 02-10; Grant sponsor: Stichting AZUA.

*Correspondence to: E. Aronica, Department of (Neuro)pathology, H2, Aca-demic Medical Center,Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.E-mail: [email protected]

Received 5 November 2001; Accepted 20 February 2002

DOI 10.1002/glia.10076

Published online 00 Month 2002 in Wiley InterScience (www.interscience.wiley.com).

GLIA 38:329–338 (2002)

© 2002 Wiley-Liss, Inc.

Page 2: Expression and distribution of id helix-loop-helix proteins in human astrocytic tumors

an “active” state shared by astrocytes in both reactiveand neoplastic processes. Although significant progresshas been made in understanding glial pathology, themolecular mechanisms governing glial cell activationremain unclear. Activation and malignant transforma-tion of astrocytes may involve the regulation of severalgenes that are also typical of cancer cells outside theCNS (Collins, 1995).

Using serial analysis of gene expression (SAGE) inanaplastic astrocytoma (Vandeputte et al., 2001), werecently identified Id genes expressed at significantlyhigher levels in tumor cells compared with controlwhite matter astrocytes (D.A.A. Vandeputte, unpub-lished observations). Id genes encode a family of fourhelix-loop-helix (HLH) proteins that lack the basicDNA binding domain (Benezra et al., 1990). Theseproteins heterodimerize with other basic HLH (bHLH)proteins, preventing them from binding to DNA andinhibiting transcription of differentiation-associatedgenes (Norton et al., 1998). Several studies support therole of HLH proteins as key mediators of cell growth,differentiation, and tumorigenesis (reviewed in Israelet al., 1999; Norton, 2000). Introduction of Id genes intodifferent cell lines leads to a block in differentiationand to an increase in proliferation. Tumor xenograftsimplanted in mice lacking copies of the Id1 and Id3genes are unable to grow because of impaired angio-genesis (Lyden et al., 1999). Tumor cell proliferation,as well as tumor neovascularization, are critical for thegrowth and progression of malignant gliomas (Burgerand Vogel, 1985; Puduvalli and Sawaya, 2000).

In the present study, we report the immunohisto-chemical expression pattern of Id1-3 proteins in humanastrocytic tumors. Id protein expression has not beenpreviously studied in primary human glial tumors.Previous reports that have investigated glial cell-de-rived tumors have largely focused on well-establishedhuman tumor cell lines or rat astrocytes in culture(Andres-Barquin et al., 1997; Tzeng and de Vellis,1997). In an effort to detect changes in Id protein ex-pression and/or localization in relation to the progres-sion of the tumor, both high-grade as well as low-gradeglial tumors have been included in our study.

MATERIALS AND METHODSSubjects

Tissue samples and pathology reports were retrievedfrom the files of the department of neuropathology ofthe Academic Medical Center (University of Amster-dam). Informed consent was obtained for the use ofbrain tissue. Tumor samples from 58 patients withprimary glial tumors who underwent surgical resectionwere included in the study for immunocytochemicalanalysis of Id1-3 proteins: 6 pilocytic astrocytomas, 14astrocytomas grade II, 6 anaplastic astrocytomas, 16glioblastoma multiforme (GBM), 3 oligodendrogliomas,10 anaplastic oligodendrogliomas, and 3 oligoastrocy-tomas (Table 1). All cases were reviewed independently

by two neuropathologists; the diagnosis was confirmedaccording to the revised World Health Organization(WHO) classification of tumors of the nervous system(Kleihues and Cavanee, 2000). Control brain tissue(including normal cortex and white matter from thetemporal, frontal, and parietal regions and from thecerebellum) was obtained from 8 age-matched patientswho died as a result of non-neurological disease. Fro-zen tissues (stored at �80°C) from astrocytoma gradeII (n � 6), anaplastic astrocytoma (n � 6), glioblastomamultiforme (n � 6), and white matter (n � 2) were usedfor Western blot analysis.

Cell lines U-118 and U-87 were obtained from theAmerican Type Culture Collection (ATCC, Rockville,MD). Human glioblastoma cell lines Gly-6 and Gly-8were obtained from the Brain Tumor Research Bank,University of Amsterdam, The Netherlands, main-tained as described previously (Fehlauer et al., 2000).Loss of heterozygosity (LOH) for chromosome arm 10qand LOH for the tumor suppressor gene p53 have beenfound in Gly-6 tumor cells (T.J. Hulsebos, unpublishedobservations). Cell lines were cultured in DMEM sup-plemented with 50 U/ml penicillin, 50 �g/ml strepto-mycin, and 10% fetal calf serum (FCS), except for U-87,which was cultured in MEM supplemented with 10%FCS.

Tissue Preparation

All specimens used in the study for immunocyto-chemistry were fixed in 10% buffered formalin andwere embedded in paraffin. Paraffin-embedded tissuewas sectioned at 5 �m on a sliding microtome andmounted on organosilane (3-aminopropylethoxysilane;Sigma)-coated slides. Representative sections of allspecimens were processed for hematoxylin & eosin(H&E) staining, as well as for immunocytochemicalreactions, using a number of glial markers describedbelow.

Antibody Characterization

Antbodies (Ab) against glial fibrillary acidic protein(GFAP; polyclonal rabbit; Dako, Denmark; 1: 2,000)and vimentin (mouse clone V9; Dako; 1:25) were used

TABLE 1. Histopathological Features of Glial Tumors

Specific diagnosis No. Location

Pilocytic astrocytoma 6 Frontal, 2; parietal, 2;cerebellum, 2

Astrocytoma II 14 Frontal, 6; parietal, 4;temporal, 4

Anaplastic astrocytoma 6 Frontal, 4; parietal, 2Glioblastoma multiforme 16 Frontal, 6; parietal, 6;

temporal, 4Oligodendroglioma 3 Frontal, 2; parietal, 1Anaplastic oligodendroglioma 10 Frontal, 5; parietal, 2;

temporal, 3Anaplastic oligoastrocytoma 3 Frontal

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in the routine immunocytochemical analysis of glio-mas. In studying tumor proliferation characteristics,we performed labeling with Ab against the proliferat-ing nuclear cellular antigen Ki67 (clone MIB-1; Diag-nostic Products Corporation (DPC), The Netherlands;1:100) (Sallinen et al., 2000). To detect Id proteins, weused specific rabbit anti-human Id1, Id2, and Id3 poly-clonal antibodies (Santa Cruz Biotechnology, CA).These Abs specifically react with Id1, Id2, and Id3,respectively, of human origin, as previously reported(Maruyama et al., 1999). The specificity of these Abswas tested by preincubating each Ab with 100-foldexcess of the corresponding antigenic peptide (SantaCruz Biotechnology) and by demonstration of specificbands on Western blots of the total homogenates ofhuman astrocytomas and glioma cell lines (see Figs. 1and 4D–F). Sections incubated without the primary Abor with preimmune sera were essentially blank.

Immunoblot Analysis

For immunoblot analysis, human normal brain(white matter), tumor samples, and four glioma celllines (U-118, U-87, Gly-6, and Gly-8) were homoge-nized in lysis buffer containing 10 mM Tris (pH 8.0),150 mM NaCl, 10% glycerol, 1% Nonidet P-40 (NP-40),5 mM ethylenediamine tetraacetic acid, and proteaseinhibitor cocktail (Boehringer-Mannheim, Germany).Protein content was determined using the bicincho-ninic acid method (Smith et al., 1985). For electro-phoresis, equal amounts of proteins (30 �g/lane) weresubjected to sodium dodecyl sulfate-polyacrylamide gelelectrophoretic (SDS-PAGE) analysis. Separated pro-teins were transferred to nitrocellulose paper for 1 h,using a semidry electroblotting system (Bio-Rad,Transblot SD, Hercules, CA), and incubated in TTBS(50 mM Tris-HCl, 0.1% Tween-20, 154 mM NaCl, pH7.5), containing 5% nonfat dry milk and 1% bovineserum albumin (BSA) for 1 h. Samples were then in-cubated overnight in TTBS/3% BSA/0.1% sodiumazide, containing the primary antibody (anti- Id1, -Id2,or -Id3, 1: 1,000; anti-actin, monoclonal mouse; Sigma,St. Louis, MO, 1:1,000). After several washes in TTBS,the membranes were incubated in TTBS/5% nonfat drymilk/1% BSA, containing the goat anti-rabbit coupledto horseradish peroxidase (HRP; 1:1,500; Dako, Den-mark) for 2 h. After several washes in TTBS, immuno-reactive bands were visualized using an enhancedchemiluminescence (ECL) kit (Amersham, Bucking-hamshire, UK). The levels of Id1-3 protein were eval-uated by measuring optical densities of the proteinbands, using Scion Image for Windows (beta 4.02) im-age-analysis software. To compare the relative increasein expression of the respective Id proteins in high-grade tumors, the same astrocytoma II samples wereused to compare astrocytoma II/anaplastic astrocytomaand astrocytoma II/GBM. The data were compared byanalysis of variance (ANOVA) with Fisher protectedleast significant difference post hoc analysis. Expres-

sion of �-actin (as the reference protein) in the sameprotein extracts did not change (Fig. 1A).

Immunohistochemistry

The sections were deparaffinated in xylene; afterbeing rinsed in ethanol (100% and 95%), they wereincubated with 1% H2O2 diluted in methanol for 20min. Slides were then washed with phosphate-bufferedsaline (PBS; 10 mM, pH 7.4). To detect Id1-3 proteins,the slides were placed into sodium citrate buffer (0.01M, pH 6.0) and heated in a microwave oven at 650 Wfor 10 min. The slides were allowed to cool for 20 min inthe same solution at room temperature (RT) and werethen washed in PBS. At this point, they were incubatedin PBS containing 10% normal goat serum (NGS) for 15min before incubation with the primary Ab. Incubationwith Id1 (1:150), Id2 (1: 250), or Id3 (1:200) was per-formed in PBS at RT for 30 min and at 4°C for 16 h.Sections were then washed thoroughly with PBS andincubated at RT for 1 h with the appropriate biotinyl-ated secondary Ab diluted in PBS (1:400 goat-anti rab-bit, for Id1-3 and GFAP; 1:200 goat-anti-mouse, forvimentin and MIB-1; Dako). Single-label immunocyto-chemistry was carried out using the avidin-biotin per-oxidase method (Vector Elite) and 3,3�-diaminobenzi-dine (DAB) as the chromogen. Sections werecounterstained with hematoxylin, dehydrated in alco-hol and xylene, and coverslipped. Sections incubatedwithout the primary Ab or with pre-immune sera wereessentially blank.

Serial sections of four astrocytomas were used todetect in the same sample the expression of Id3 andE47 (class A bHLH protein). As both antibodies to Id3and E47 (1:100; Santa Cruz Biotechnology) are poly-clonal rabbit antibodies, this combination could not beexamined directly with double-labeling procedures.

Evaluation of Immunostaining

Using a light microscope, all labeled sections wereexamined independently by two observers with respectto the presence or absence of various histopathologicalparameters and specific immunoreactivity (IR) for thedifferent markers in tumor cells and tumor vessels. Aspreviously proposed by Wilson et al. (2001), the stain-ing was semiquantitatively evaluated by assigning ascore for the intensity of the immunohistochemical re-action and for the proportion of Id1-3 cells stained. Theproduct of these two values was taken to give theoverall IR score (total score). The intensity of the im-munohistochemical reaction (intensity score) wasstratified into to four categories: 0, no IR; 1, weak IR; 2,moderate IR; and 3, strong IR. The proportion of posi-tive cells (cell score) was also stratified into fourgroups: 0, no tumor cells exhibiting IR; 0.33, �33% ofthe tumor cells exhibiting IR; 0.67, 33–67% of the

331ID PROTEINS IN ASTROCYTIC TUMORS

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tumor cells exhibiting IR; and 1, �67–100% of thetumor cells exhibiting IR. In mixed gliomas (oligoastro-cytomas), the astrocytic component was differentiatedfrom the oligogendroglial component on the basis ofmorphology and by staining of a serial section withGFAP.

MIB-1 staining was examined using an ocular gridand counting 1,000 cells from representative fields ofthe tumor, as previously described (Aronica et al.,2001b). The section border, necrotic and hemorrhagicareas were omitted. The result was recorded as theKi-67 (MIB-1) labeling index (LI), expressing the per-centage of the immunostained nuclei (number of la-beled cells per total number of cells).

We also evaluated the degree of vascular endothelialproliferation (VEP score) using the criteria previouslydescribed by Christov et al. (1998). The VEP score arestratified into three groups: (1) mild cell nuclei in-creased in number; (2) moderate conspicuous crowdingof the nuclei in thickened vessels; and (3) marked mul-

tilayered proliferation (glomeruloid hyperplasia). Thescores of the individual vessels were pooled to calculatethe mean specimen score.

The data were compared using a nonparametricKruskal-Wallis test followed by a Mann-Whitney testto assess the difference between groups; P � 0.05 wastaken as the level of significance. Correlation betweenId immunostaining and other variables (MIB-1 LI andVEP score) were assessed using the Spearman’s rankcorrelation test.

RESULTSCase Material and Histological Features

A total of 58 surgical specimens from patients withglial tumors were examined. The histopathological fea-tures of these cases are summarized in Table 1. Bothlow-grade and high-grade tumors were included.

Fig. 1. Expression of Id1-3 proteins in astrocytic tumors and glio-blastoma cell lines. A: Representative immunoblots of Id1, Id2, andId3 in total homogenates from astrocytomas grade II (1, 2, 3), ana-plastic astrocytomas (4, 5), glioblastoma multiforme (GBM), glioblas-toma cell lines, and white matter (Wm). Proteins (30 �g/lane) weresubjected to Western blot analysis with specific antibodies, as de-scribed in the Materials and Methods section. Expression (as refer-ence protein) of �-actin (actin; 42 kDa) is shown in the same tumor

protein extracts. B: Densitometric analysis of Western blots. Values(optical density units) are mean �SD of 6 astrocytomas II (A II), 6anaplastic astrocytomas (AA), and 6 glioblastoma multiforme (GBM).By comparison with low-grade astrocytomas (A II), both anaplasticastrocytomas and GBM exhibited a significant increase in Id1-3 pro-tein expression (P � 0.05; ANOVA with Fisher protected least signif-icant difference post hoc analysis).

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Id1-3 Protein Expression Levelsin Astrocytic Tumors

Specific anti-human Id1, Id2, and Id3 polyclonal Abswere used to study the expression of Id proteins inprimary human glial tumors and human glioblastomacell lines. In control white matter, Id1-3 protein expres-sion by Western blot analysis was not detectable. Incontrast, all three Id proteins were present at varyinglevels in four glioblastoma cell lines and in tumor sam-ples from low-grade (astrocytoma II) and high-grade(anaplastic astrocytoma and GBM) glial tumors (Fig.1A). Id1 and Id3 were detectable by immunoblot asbands of 14 kDa. Immunoblot with the Id2 Ab showedtwo bands at 16 and 18 kDa. As previously suggested(Barone et al., 1994; Maruyama et al., 1999), it ispossible that the two Id2 immunoreactive bands mayrepresent separate translation products of the Id2 gene(Maruyama et al., 1999). All immunoreactive bandsdisappeared after preadsorption with the correspond-ing peptide (data not shown).

As compared with astrocytomas grade II, both ana-plastic astrocytomas and GBM exhibited significant

increase in Id1-3 protein expression (P � 0.05). Theoptical density measurements of Id1-3 protein expres-sion levels in each tumor group are represented inFigure 1B.

Id1-3 Immunoreactivity in Glial Tumor Cells

Immunocytochemistry was performed to study thecellular distribution of Id1, Id2, and Id3 proteins inboth normal and tumor astrocytes. This was particu-larly important for high-grade tumors in which thedetection of Id proteins by blot analysis could reflectexpression in hyperplastic vascular cells (Lyden et al.,1999).

In adult normal brain, only a few astrocytic processesshowed weak to moderate intracytoplasmic Id1, Id2,and Id3 staining (in particular, in the white matter),whereas oligodendrocytes did not exhibit detectableamounts of Id proteins (Fig. 2A,D,G). A panel of 42astrocytic tumors was screened by immunocytochemis-try with each of the three Id antibodies. All the Idproteins were expressed at higher levels in tumor as-

Fig. 2. Expression of Id1-3 immunoreactivity (IR) in astrocytic tu-mors. Representative photomicrographs of Id1 (A–C), Id2 (D–F), andId3 (G–I) IR in normal adult white matter (A,D,G), astrocytoma gradeII (B,E,H), and glioblastoma multiforme (C,F,I). Sections were coun-terstained with hematoxylin. In normal adult white matter, there wasonly weak Id1-3 IR in resting astrocytes (arrows); Id1-3 IR in endo-thelial cells was not detectable (arrowheads). Id1-3 IR was clearly

detectable in astrocytoma (B,E,H; arrows) and glioblastoma tumorcells (C,F,I). Id1- and Id2-positive cells displayed mainly cytoplasmicstaining, whereas prominent nuclear localization was observed for Id3protein in both low-grade (H; arrows) and in high-grade tumors (ar-rows; detail in I). Astrocytoma II showed only weak Id1-3 IR inendothelial cells (arrowheads). Scale bar � 120 �m in A (for A,D,G);80 �m in B and C (for B,E,H and C,F,I, respectively).

333ID PROTEINS IN ASTROCYTIC TUMORS

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trocytes versus astrocytes from normal brain tissue.Typical Id1-3 staining is shown in a representativeastrocytoma grade II (Fig. 2B,E,H) and in GBM (Fig.2C,F,I). Id1-positive and Id2-positive cells displayedmainly cytoplasmic staining, whereas prominent nu-clear localization was observed for Id3 protein (Fig.2H,I). E47 (target of Id3) (Deed et al., 1996) also dis-played nuclear localization in tumor astrocytes (datanot shown).

Despite the overall increase in tumor-associated IdIR, there was heterogeneity in both the intensity ofstaining and the proportion of positive tumor cells,which was taken into account in the further evaluationof the data. Figure 3A shows the distribution of Id1 IR(total) scores in the patient series. High-grade tumors(anaplastic astrocytomas and GBM) showed consis-tently elevated levels of immunoreactivity for Id1, aswell as for Id2 and Id3 (not shown). Table 2 summa-rizes the average IR scores for all three Id proteins inastrocytic tumors, with a different degree of malig-nancy. Id1, Id2, and Id3 IR scores in tumor cells weresignificantly higher in high-grade (anaplastic astrocy-toma and GBM) compared with low-grade tumors (pi-locytic astrocytoma and astrocytoma II; P � 0.05). Inmixed gliomas (oligoastrocytomas), Id1-3 expression

was observed mainly in the astroglial component of thetumor (Fig. 3G; Table 3). Table 3 summarizes the im-munohistochemical data concerning the oligodendro-gliomas, showing low IR scores in both low-grade aswell as high-grade tumors.

MIB-1 Labeling Index and Correlation With Id1-3 Expression in Astrocytic Tumors

The proliferation indices of the astrocytic tumorsincluded in this study were investigated by evaluationof the immunostaining performed with the MIB-1 an-tibody against the Ki-67 proliferation antigen. Inagreement with previous studies (Sallinen et al., 2000;Kim et al., 2000), the MIB-1 labeling index (LI) showeda significant increase in tumors with higher his-topathological malignancy (P � 0.05; Table 2). In as-trocytic tumors MIB-1 LI was well correlated with theId1-3 expression in tumor cells (r � 0.733 for Id1; r �0.781 for Id2; r � 0.728 for Id3; P � 0.05). Figure 3Billustrates this significant correlation with MIB-1 LIfor the Id1 protein expression.

Fig. 3. Id1 immunoreactivity (IR) scores in astrocytic tumors. A:Scatter plot showing the distribution of Id1 IR in tumor cells andvessels of specimens with different degree of histological malignancy.B: Scatter plot showing the significant correlation between MIB-1 LI(labeling index) and Id1 expression in tumor cells. IR score represents

the total score, which was taken as the product of the intensity scoreand the cell score (proportion of positive cells within the specimen).PyA, pilocytic astrocytoma; AA, anaplastic astrocytoma; GBM, glio-blastoma multiforme.

TABLE 2. Id1, Id2, and Id3 Immunoreactivity Scores and MIB-1 Labeling Index in Astrocytic Tumors*

Astrocytic tumors

Id1 Id2 Id3 MIB-1

Tumor cells Vessels Tumor cells Vessels Tumor cells Vessels Labeling Index

Pilocytic astrocytoma (n � 6) 0.5 � 0.1 0.2 � 0.1 0.6 � 0.1 0.2 � 0.1 0.6 � 0.1 0.2 � 0.1 2.7 � 3Astrocytoma II (n � 14) 0.9 � 0.1 0.1 � 0.1 0.6 � 0.1 0.2 � 0.1 0.6 � 0.1 0.2 � 0.1 8.0 � 6.0Anaplastic astrocytoma (n � 6) 2.5 � 0.2a 1.5 � 0.2a 1.7 � 0.1a 1.8 � 0.3a 2.7 � 0.2a 2.7 � 0.2a 20 � 11a

Glioblastoma multiforme (n � 16) 2.3 � 0.2a 2.1 � 0.2a 2.4 � 0.2a 1.6 � 0.2a 2.7 � 0.1a 2.5 � 0.2a 27.7 � 9.6a

*Scoring of the histological specimens was performed as described in the Materials and Methods section. Values represent the mean �SEM of the number of samplesindicated in parentheses.aSubgroups with significantly elevated Id expression relative to other subgroups: Id1-3 score for anaplastic astrocytoma and glioblastoma multiforme greater thanId1-3 score for pilocytic astrocytoma and astrocytoma II (P � 0.05); MIB-1 LI (labelling index) for anaplastic astrocytoma and glioblastoma multiforme greater thanMIB-1 LI for pilocytic astrocytoma and astrocytoma II (p � 0.05). Mann-Whitney U-test.

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Id1-3 Immunoreactivity in Tumor Vessels

In adult normal brain, endothelial cells did not ex-hibit detectable amounts of Id proteins (Fig. 2A,D,G).Only faint Id IR was present in the endothelial cells ofsome low-grade astrocytic tumors (Fig. 2B,E,H). In con-trast, strong Id IR in tumor vessels was observed inspecimens from high-grade tumors (anaplastic astrocy-toma and GBM) (Fig. 4A–C). Preabsorption of the re-spective Ab with the blocking peptide specific for Id1,Id2, and Id3 completely abolished IR in both tumor andvascular cells (Fig. 4D–F). The data concerning the Id

IR score in tumor vessels for the different tumor groupsare summarized in Table 2. The vascular expression ofId protein was greater in high-grade (anaplastic astro-cytoma and GBM) than in low-grade (pilocytic astrocy-toma and astrocytoma II) astrocytic tumors, showing astrong association with the degree of histological ma-lignancy (P � 0.05; Table 2; Fig. 3A). Moreover, theId1-3 expression in tumor vessels was correlated withthe vascular endothelial proliferation score (r � 0.646for Id1; r � 0.579 for Id2; r � 0.572 for Id3; P � 0.05).In contrast to the astrocytic tumors, low Id IR scoreswere found in the tumor vessels of oligodendrocytic

TABLE 3. Id1, Id2, and Id3 Immunoreactivity Scores in Oligodendrocytic Tumors*

Oligodendrocytic tumors

Id1 Id2 Id3

Tumor cells Vessels Tumor cells Vessels Tumor cells Vessels

Oligogendroglioma (n � 3) 0.4 � 0.1 0.5 � 0.1 0.4 � 0.1 0.2 � 0.1 0.5 � 0.1 0.3 � 0.2Anaplastic oligodendroglioma

(n � 10)0.5 � 0.1 0.3 � 0.1 0.5 � 0.2 0.3 � 0.1 0.6 � 0.1 0.5 � 0.2

Oligoastrocytoma (n � 3) 0.3 � 0.2a 0.4 � 0.2 0.4 � 0.2a 0.3 � 0.2 0.4 � 0.1a 0.6 � 0.31.3 � 0.4b — 1.1 � 0.2b — 1.1 � 0.1b —

*Scoring of the histological specimens was performed as described in the Materials and Methods section. Values represent the mean �SEM of the number of samplesindicated in parentheses.aOligodendrocytes.bAstrocytes.

Fig. 4. Expression of Id1-3 immunoreactivity (IR) in blood vessels ofhigh-grade glial tumors. Representative photomicrographs of Id1 (A),Id2 (B), and Id3 (C) IR in glioblastoma multiforme with strong Idexpression in the tumor vessels (arrows). Preabsorption of the respec-tive Ab with the blocking peptide specific for Id1 (D), Id2 (E), and Id3(F), completely abolished IR in both tumor and endothelial cells. G:

Representative photomicrograph of Id1 IR in oligoastrocytoma, show-ing IR only in the astrocytic component of the tumor (arrows). H–J:Representative photomicrographs of Id1 (H), Id2 (I), and Id3 (J) inanaplastic oligodendroglioma showing weak to moderate Id1-3 IR intumor oligodendrocytes, but no detectable IR in the tumor bloodvessels (arrows). Scale bars � 100 �m in A (for A–F); 95 �m in G andJ (for G and H–J, respectively).

335ID PROTEINS IN ASTROCYTIC TUMORS

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tumors, including anaplastic oligodendrogliomas (Ta-ble 3; Fig. 4H–J).

DISCUSSION

The present study describes the expression patternand the cellular distribution of Id proteins in primaryhuman glial tumors. Several observations were made:(1) in adult human normal brain, Id1-3 expression islow in resting astrocytes and endothelial cells; (2) pri-mary brain astrocytic tumors show high Id1-3 proteinexpression levels; and (3) Id1-3 expression in tumorcells and tumor blood vessels is significantly higher inhigh-grade compared with low-grade astrocytic tumorsand correlates with proliferation indices. The signifi-cance of these findings in relation with activation, ma-lignant transformation of astrocytes and glial tumorprogression is discussed below.

Id1-3 Proteins Are Expressed in Primary BrainGlial Tumors

Previous reports have shown expression of one ormore Id mRNAs in different glioblastoma cell lines(Zhu et al., 1995; Andres-Barquin et al., 1997). How-ever, the expression pattern and subcellular localiza-tion of Id proteins have not been previously investi-gated in primary human glial tumors. In the presentstudy, we showed by Western blot analysis that astro-cytic tumor samples expressed Id1-3 proteins at levelsthat appeared to be enhanced in tumors with higherhistopathological malignancy. Immunohistochemicalanalysis confirmed that many of the tumor cells exhib-ited abundant Id1-3 IR, whereas in adult normal brainonly a few astrocytic processes displayed low Id1-3expression. Low Id protein expression in resting astro-cytes has also been reported in adult rat brain (Tzenget al., 1999; Aronica et al., 2001a).

Given the oncogenic properties of Id genes and theirability to promote proliferation in different cell types invitro (Norton, 2000), the high expression of Id proteinsobserved in primary astrocytic tumors supports theirinvolvement in the malignant transformation of hu-man astrocytes in vivo.

A role for Id proteins in the control of oligodendrocytedevelopment was recently suggested (Tzeng and deVellis, 1998). In particular, Id2 overexpression hasbeen found to powerfully inhibit oligodendrocytic dif-ferentiation in vitro (Wang et al., 2001). In our study,all three Id proteins were detected within the tumormass of oligodendroglioma samples. However, oligo-dendrocytic tumor cells, including cells of high-gradetumors, exhibited low IR scores. The different levels ofId protein expression that we observed may reflect thedifferent origin and biology of specific glial-derived tu-mor cells.

Because all three Id proteins are expressed in glia-derived tumor cells, it is likely that these proteins have

some redundant functions in these cells. The precisemechanism by which Id proteins exert their function isonly partially understood. The ability to antagonizebHLH transcription factors is suggested as a primarymechanism of Id protein function (Norton, 2000). Inparticular, the heterodimeric interaction with class AbHLH proteins (E12, E47, E2-2, HEB) appears to reg-ulate the function of Id proteins both critically anddifferentially.

Heterodimerization of Id3 with E47 extends the half-life of Id3, whereas the E47 protein is more rapidlydegraded (Deed et al., 1996). Heterodimerization regu-lates also the subcellular distribution, leading to a se-questration of the Id protein in the nucleus (Deed et al.,1996). Id proteins do not normally possess the nuclearlocalization found for many bHLH proteins (Norton,2000). Accordingly, cytoplasmic localization of all threeId proteins is found in adult resting glial cells, as wellas in rat or human reactive glial cells (Tzeng et al.,1999; Aronica et al., 2001a). It is possible that an activemechanism in these cells is involved in sequesteringId1-3 from E-proteins in the cytoplasm and preventingtheir nuclear translocation. A nuclear localization of Idproteins, however, was observed in tumor astrocytes.This observation indicates a deregulation of Id expres-sion in glial tumors that involves a perturbation oftheir subcellular location. The fact that the nuclearlocalization was prominent for Id3, and only occasion-ally observed for Id1 and Id2, suggests the existence ofadditional regulatory mechanisms for the different Idproteins. Accordingly, in cultured astrocytes, Id2 andId3, but not Id1, are translocated from the cytoplasm tothe nucleus in response to serum (Tzeng and de Vellis,1997). Nuclear localization of Id3 was recently reportedin squamous cell carcinoma, cervical cancer, and colo-rectal adenocarcinoma (Langlands et al., 2000; Schindlet al., 2001; Wilson et al., 2001). The physiologicalrelevance of this transit remains to be established. Arecent report, however, points to a critical role for thenuclear translocation of Id proteins in the control ofglial cell differentiation (Wang et al., 2001).

The present study does not directly address themechanisms through which Id genes are regulated inglial tumor cells. However, previous observations indi-cate that the expression and the function of glial Idgenes are plastic and can be regulated by differentextracellular environmental signals (Andres-Barquinet al., 1997; Tzeng et al., 1999).

Overexpression of Id1-3 Proteins in TumorCells and Blood Vessels of High-Grade

Astrocytic Tumors

Recent reports suggest a link between Id expressionand tumor progression (Desprez et al., 1998; Langlandset al., 2000; Lin et al., 2000; Schindl et al., 2001).Accordingly, we show that the Id1-3 expression in as-trocytic tumors is related to the degree of histologicalmalignancy. The highest Id IR scores in tumor cells

336 VANDEPUTTE ET AL.

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were seen in high-grade tumors. Such tumors tend tohave much less differentiated features than low-gradetumors and a higher proliferation index (Russell andRubinstein, 1989). The correlation between prolifera-tion index and expression of Id1-3 proteins may reflectthe ability of id proteins to promote cell proliferationwhen experimentally overexpressed into different celltypes (Israel et al., 1999; Norton, 2000). Furthermore,Tzeng and Vellis (1997) showed that abrogation of Idfunction by antisense oligonucleotide blockade resultsin an inhibition of glial cells proliferation.

Recently, attention has been focused on the expres-sion of Id proteins in endothelial cells of tumor bloodvessels. Lyden et al. (1999) showed that the Id proteinsare required for the proliferative and invasive pheno-type of endothelial cells during tumor angiogenesis.Expression of Id1 and Id3 mRNAs has been found inblood vessels of some high-grade human tumors of neu-ral origin (Lyden et al., 1999). Neovascularization isindeed an important feature of CNS tumors, includingglial-derived tumors. Astrocytomas are among themost angiogenic tumors and the presence of vascularproliferation is significantly associated with malig-nancy (Folkerth, 2000; Brat and Van Meir, 2001). Ourstudy demonstrates that, in contrast to the undetect-able levels found in endothelial cells of adult normalbrain, Id1-3 proteins were detectable in blood vessels ofastrocytic tumors. In particular, the highest Id IR wasobserved in vessels of high-grade tumors and was cor-related with the severity of vascular endothelial prolif-eration. This observation supports the possible role ofId proteins in the creation of a permissive environmentfor tumor angiogenesis. In contrast to high-grade as-trocytomas, anaplastic oligodendrogliomas did not ex-hibit strong Id protein expression in tumor vessels.This finding was unexpected, as the presence of a richcapillary network with endothelial proliferation is ahallmark of these glial tumors. It is possible that fac-tors other than Id molecules regulate tumor angiogen-esis in oligodendroglial tumors. A similar differencebetween astrocytic and oligodendrocytic tumors hasbeen reported for other angiogenic factors (Stan et al.,1994; Pietsch et al., 1997). The functional role of Idproteins in endothelial cells (through interaction withendothelial-specific bHLH proteins) (Quertermous etal., 1994), as well as the mechanisms of regulation of Idexpression in different brain tumors, require furtherinvestigation.

In conclusion, these results support the role of the Idgene family in the enhanced proliferative potential oftumor astrocytes as well as its involvement in tumorangiogenesis, a process that critically influences themalignant behavior of glial tumors.

ACKNOWLEDGMENTS

The authors are grateful to Dr. J. Aten for help withthe statistical analysis and Dr. J.A. Gorter for criticallyreviewing of the manuscript. We thank W.P. Meun for

expert photography. E.M.A. Aronica was supported bythe National Epilepsy Fund Power of the Small andHersenstichting Nederland (NEF 02-10) and by fund-ing from Stichting AZUA.

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