Oncogene (2002) 21, 239 ± 247 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc

MGEA6 is tumor-speci®c overexpressed and frequently recognized by patient-serum antibodies

Nicole Comtesse1, Isolde Niedermayer2, Brenda Glass1, Dirk Heckel1, Esther Maldener1, Wolfgang Nastainczyk3, Wolfgang Feiden2 and Eckart Meese*,1

1Institut fuÈr Humangenetik, UniversitaÈtskliniken des Saarlandes, 66421 Homburg/Saar, Germany; 2Abteilung fuÈr Neuropathologie, UniversitaÈtskliniken des Saarlandes, 66421 Homburg/Saar, Germany; 3Institut fuÈr Medizinische Biochemie, UniversitaÈtskliniken des Saarlandes, 66421 Homburg/Saar, Germany

Tumorigenesis of meningioma has been associated with 40% of the meningiomas show no mutation in the NF2 chromosome 22, most notably the NF2 gene, but gene, indicating that other genes are involved in the additional genes have also been implicated in meningioma tumorigenesis (Ruttledge et al., 1994; Akagi et al., development. Previously, we have cloned the cDNAs for 1995). the meningioma expressed antigen 6 (MGEA6) and its A variety of tumors are known to elicit immune splice variant MGEA11. Here, we show that antibodies responses in patients bearing the tumor. Immunogenic against recombinantly expressed MGEA6/11 are found in tumor-associated antigens have been described pri- 41.7% (10/24) of the sera from meningioma patients and marily for malignant tumors including melanoma, in 2/8 sera of glioblastoma patients, whereas no response breast cancer, colon cancer, ovarian cancer (Real et was seen in 12 sera from healthy persons. Western-blot al., 1988; Ben-Mahrez et al., 1990; Gallagher et al., analyses using generated polyclonal antibodies, revealed 1991; Disis et al., 1994; Disis and Cheever, 1996; overexpression in meningioma and glioma tumor samples Boon and Lloyd, 1997). However, di€erent studies compared to normal brain. Immunohistochemical staining have indicated that meningiomas are also capable of of tissue sections con®rms reactivity in meningioma tumor inducing an immune response in the host. In 1976, cells and tumor cells of glial origin. We found no Pees and Seidel already described a cellular immune reactivity to normal astrocytes and only faint reactivity response against autologous and heterologous menin- to normal leptomeninges. Sequence analysis predicted gioma cells in the serum of patients (Pees and Seidel, membranic localization of MGEA6/11, that was con- 1976). Another study concerning a cytotoxic immune ®rmed by cell fractionation. The immune response to response presents data suggesting that meningiomas MGEA6/11 is frequent in both meningioma and glioma can induce complex immunological responses in the patients and may likely be attributed to overexpression of host (Pees, 1979). The presence of IgG in meningioma the MGEA6/11 protein in the tumor cells. was shown by Tabuchi et al. (1979). Expression of Oncogene (2002) 21, 239 ± 247. DOI: 10.1038/sj/onc/ MHC class II and MHC class I molecules have also 1205005 been detected in meningioma tissue (Becker and Roggendorf, 1989). Keywords: tumor antigen; overexpression; central- Previously, we reported cloning of immunoreactive nervous-system neoplasm; meningioma; glioma antigens expressed in meningioma, providing the ®rst evidence for the expression of immunogenic antigens in this tumor type (Heckel et al., 1997, 1998; Comtesse et Introduction al., 1999). The meningioma expressed antigens 6/11 (MGEA6/11; previously called MEA6/11) induced an Meningiomas are neoplasms of the leptomeninges immune response in the patient bearing the meningioma covering the brain and the spinal cord. With *20% (Heckel et al., 1997). Expression analysis on the mRNA of all primary intracranial tumors, they are among the level revealed constitutive expression of the MGEA6/11 most common tumors of the human nervous system in all human tissues tested. MGEA6 belongs to a (Kleihues and Cavenee, 2000). Although there is a multigene family with an active locus on chromosome large amount of evidence for involvement of the 14q and at least nine pseudogenes on di€erent neuro®bromatosis type 2 (NF2) gene in tumor growth, chromosomes. The smaller MGEA11 is generated by alternative splicing of exon 19 of the MGEA6 gene (Comtesse et al., 2001). In this study we report that the immune response *Correspondence: E Meese, Department of Human Genetics, against MGEA6/11 is found in 41.7% (10/24) of University of Saarland, Building 60, 66421 Homburg/Saar, meningioma and in 2/8 of glioblastoma patients, with Germany; E-mail: [email protected] Received 19 June 2001; revised 28 September 2001; accepted 1 no antibodies present in 12 sera of healthy persons, October 2001 indicating that MGEA6/11 probably acts as a tumor MGEA6 is overexpressed and immunogenic N Comtesse et al 240 antigen in these tumor types. We report that on the protein level, MGEA6/11 is overexpressed in menin- gioma and glioma tumor cells compared to the normal tissue. Immunohistochemical staining of tissue sections revealed reactivity of meningioma cells and tumor cells of glial origin, whereas no reactivity was found in the cellular elements of the normal control brain and only faint reactivity in normal leptomeninges. No expression was found in tumor cells of cerebral carcinoma metastases. Sequence analysis purported a membranic localization of the protein, which was con®rmed by Western-blot analysis of cellular fractions of MGEA6/ 11 expressing cells. Our ®ndings suggest that the immune response in glioma and meningioma patients may likely be attributed to overexpression of this gene at the protein level.

Results

Recombinant expression of MGEA6/11 in a eukaryotic expression system To further characterize the meningioma expressed antigens 6 and 11 (MGEA6/11), we expressed the two variants in a recombinant eukaryotic expression system. As shown previously, cDNA for MGEA6 shows an open reading frame (ORF) of 2415 bp and cDNA for MGEA11 an ORF of 2286 bp, with the coding regions di€ering only in a 129 bp sequence (Heckel et al., 1997). As most recently described, Figure 1 Western-blot analyses using recombinantly expressed mRNA for MGEA11 probably results from alternative MGEA6/11. (a) Serum of meningioma patient H4 detects a 110 kDa and a 105 kDa protein corresponding to MGEA6 and splicing of exon 19 of the MGEA6 gene on chromo- MGEA11. (b) Detection of MGEA6 and MGEA11 with serum some 14q (Comtesse et al., 2001). Sequence analysis of from glioblastoma patient H57. (c, left) Western blot with three the cDNA clones revealed a high number of codons pooled normal sera. Reactivity of the sera was shown using a rarely used in bacteria. Therefore, we used the positive control (data not shown). (Right) Same blot as left, stripped and incubated with meningioma serum H4 detecting baculovirus expression system to facilitate complete MGEA6 and 11. NR: not recombinant clone translation of the clones. After recombinant expression, the proteins were visualized by Western blot analysis using the serum of the autologous patient for detection. We determined molecular masses for MGEA6 of subtype of meningiomas (Tables 1 and 2). In detail, we 110 kDa and for MGEA11 of 105 kDa (Figure 1a). found antibodies against MGEA6/11 in 4/10 sera from Deducting the fusion portion of 34AA (about 4 kDa), WHO grade I meningiomas including arachnothelial, results in a molecular mass of 106 kDa for MGEA6 transitional and secretory subtypes, in 5/12 sera from and of 101 kDa for MGEA11. This is in good atypical meningiomas, and in one of two sera from accordance with the predicted theoretical molecular anaplastic meningioma. masses of 91 kDa for MGEA6 and 89 kDa for We further tested sera from patients with primary MGEA11. The minimal mass di€erences can be brain tumors of glial origin (Table 2). We detected attributed to post-translational modi®cation. antibodies against MGEA6 in 2/8 sera from glioblas- toma (WHO grade IV) patients (Figure 1b) and in one of two patients with WHO grade II astrocytomas. Humural immune response against MGEA6/11 is found in Sera derived from two patients with WHO grade III meningioma and glioma patients, not in healthy persons astrocytomas and from three patients with pilocytic We also used the baculovirus-expressed recombinant astrocytomas did not react with MGEA6/11. When proteins to examine the immune response against using 12 sera from healthy, tumor-free persons, in no MGEA6/11 in other patients bearing meningiomas. case was an antibody response detected against We tested 24 sera derived from patients with MGEA6/11 (Figure 1c). Additionally, for testing meningioma of di€erent grading and histological tumors derived from other embryonic origin, we tested subtype. An antibody reaction was detected with 10 six sera of patients with squamous cell lung of 24 (41.7%) meningioma sera. The immune response carcinoma. In no case were antibodies against was not correlated with tumor grading or histological MGEA6/11 found.

Oncogene MGEA6 is overexpressed and immunogenic N Comtesse et al 241 Table 1 Summary of sera from meningioma-patients examined for C-terminal region is characterized by a high percentage antibody-response against MGEA6 (22%) of proline residues including several polyproline Meningioma Immune stretches. Notably, the insertion found in MGEA6 also No WHO grading Subtype response possesses a high amount of proline residues and shows H 4 WHO I Arachnothelial + high hydrophobicity. H 19 WHO I Transitional + H 28 WHO I Arachnothelial 7 H 52 WHO I Secretory 7 Generation of polyclonal antibodies against MGEA6/11 H 53 WHO I Arachnothelial 7 via peptide immunization H 63 WHO I Transitional, regressive + H 64 WHO I Angiomatous, regressive 7 To investigate the MGEA6/11 expression at the protein H 71 WHO I Secretory 7 level, we generated polyclonal rabbit antibodies against H 78 WHO I Fibroblastic 7 MGEA6/11. We synthesized a peptide from the C- H 81 WHO I Secretory + terminus of MGEA6/11. For immunization, the H 15 WHO II Atypical 7 peptide was coupled with an additional N-terminal H 60 WHO II Atypical + cysteine residue to keyhole limpet hemocyanine. One H 62 WHO II Atypical + week after each immunization, the serum was tested for H 72 WHO II Atypical + antibodies against MGEA6/11 by Western blot H 73 WHO II Atypical 7 H 82 WHO II Atypical 7 analysis using the recombinant proteins from the H 99 WHO II Atypical + baculovirus-expressing clones. Positive serum was H 103 WHO II Atypical + enriched for peptide-speci®c antibodies by anity H 105 WHO II Atypical 7 puri®cation using a peptide-coupled sepharose column. H 106 WHO II Atypical 7 H 122 WHO II Atypical 7 Speci®city of the peptide-enriched serum was shown by H 138 WHO II Atypical 7 Western blot analysis (Figure 3a).

H 108 WHO III Anaplastic 7 H 46 WHO III Anaplastic + Western blot analysis shows overexpression of MGEA6/11 in meningioma and glioblastoma +: positive reaction; 7: negative reaction compared to normal brain The peptide-puri®ed, polyclonal antibody was used to examine MGEA6/11 expression in di€erent tissue Table 2 Summary of anti-MGEA6 antibody response lysates by Western blot analysis. We examined the Antibody Percentage MGEA6/11 expression of 19 human meningiomas of Sera reaction (%) di€erent WHO grading, a pilocytic astrocytoma (WHO Meningiomas 10/24 41.7 I), an oligoastrocytoma (WHO II), three glioblastomas common type meningiomas (WHO I) 4/10 40 (WHO IV), and an anaplastic oligodendroglioma arachnothelial 1/3 (WHO III). Additionally we tested three normal brain transitional 2/2 samples, two derived from an autopsy case (cerebellum fibroblastic 0/1 and frontal lobe) and one derived from the neurochir- secretory 1/3 angiomatous 0/1 urgical resection of meningioma H 560. Table 3 atypical meningiomas (WHO II) 5/12 41.7 summarizes the protein expression analysis. As shown anaplastic meningiomas (WHO III) 1/2 50 in Figure 3b, a *106 kDa protein was detected in all Pilocytic astrocytomas (WHO I) 0/3 0 tested meningiomas and brain tumors. In most samples Diffuse astrocytomas (WHO II) 1/2 50 Diffuse anaplastic astrocytomas (WHO III) 0/2 0 a second lighter signal, about 3 ± 5 kDa smaller, was Glioblastomas (WHO IV) 2/8 25 detected. This protein corresponds to the smaller Squamous cell lung carcinomas 0/3 0 variant MGEA11. Notably, the expression level of Healthy persons 0/12 0 the larger MGEA6 protein is higher than that of the smaller variant in normal brain and meningioma grades I and II. Only the grade III meningiomas show an equivalent expression of both variants. The level of protein expression in normal human brain samples is Prediction of MGEA6/11 protein structure indicates a signi®cantly lower than in the examined brain tumor membrane protein with several domains tissues. To obtain information about the protein structure of When the peptide used to generate the MGEA6/11 MGEA6/11, we analysed the amino acid sequences antibody is incubated with the anity-puri®ed serum, with several computer programs. The predicted domain it e€ectively blocks the binding demonstrating the structure is shown in Figure 2. The amino acid speci®city of the puri®ed MGEA6/11 polyclonal anti- sequence possesses a transmembrane domain (AA body. To con®rm that the signals are not generated by 39 ± 59) close to the N-terminus. Additionally, the a non-speci®c interaction with the secondary antibody, amino acid sequence shows two coiled coil domains we incubated the Western blot with the anti-rabbit (AA 115 ± 261; 322 ± 496). Within the ®rst one is secondary antibody only. No signal was revealed with located a heptad repeat of four leucine residues. The the anti-rabbit secondary antibody (data not shown).

Oncogene MGEA6 is overexpressed and immunogenic N Comtesse et al 242 patchy reactivity. Also positive for MGEA6/11 expres- Immunohistochemical detection of MGEA6/11 in sion were brain tumors of glial origin. We tested a frozen tissue sections circumscribed glioma (pilocytic astrocytoma), further We also used the peptide-puri®ed, polyclonal rabbit diverse di€use gliomas and an atypical teratoid serum to investigate MGEA6/11 immunohistochemi- rhabdoid tumor. In the tumor cells of two examined cally on di€erent frozen tumor sections (Figure 4). Table primary cerebral non-Hodgkin's lymphomas, no 1 summarizes the immunohistochemical staining results. MGEA6/11 expression was detected. Furthermore, All meningiomas demonstrated, independent of the brain metastases of di€erent carcinomas were investi- WHO grading, reactivity to the MGEA6/11 polyclonal gated. In contrast to tumors derived from the antibodies. While in some meningiomas all tumor cells epithelium, which lacked any MGEA6/11-reactivity, a were positive for MGEA6/11, others showed rather brain metastasis of a malignant melanoma, showed focal

Figure 2 Schematic representation of the predicted protein domains of MGEA6/11. T: transmembrane domain, L: leucine zipper- motif, CC: coiled-coil domain, cross-hatched line: insertion of MGEA6

Figure 3 (a) Veri®cation of the speci®city of the peptide-puri®ed anti-MGEA6/11 polyclonal serum using recombinantly expressed MGEA6 and 11 and tumor tissue lysate H313. NR: non-recombinant clone. (b) Western blot analyses of di€erent tissue lysates using the peptide-puri®ed anti-MGEA6/11 polyclonal serum. Hybridization with anti-tubulin antibodies was performed as the control. The tumor grading according to the WHO classi®cation is indicated by roman numerals. Arabic numbers refer to tumor samples. NB: normal brain (derived from tumor patient H560), FL: frontal lobe, CB: cerebellum, PA: pilocytic astrocytoma, OA: oligoastrocytoma; OD: anaplastic oligodendroglioma

Oncogene MGEA6 is overexpressed and immunogenic N Comtesse et al 243 staining within the tumor cells. Notably, in the reactive gen. In some samples, there seemed to be a globular brain tissue surrounding the tumors, MGEA6/11 was perinuclear staining, comparable with the Golgi-®eld. detected within the cytoplasm of activated astrocytes. Normal astrocytes, in contrast, showed no MGEA6/11 Verification of membranic localization of MGEA6/11 reactivity. Leptomeninges of two autopsy cases showed faint reactivity of the arachnoid cells (Figure 4). To determine subcellular localization of MGEA6/11, In all cases where immunoreactivity was observed, we prepared cellular fractions from the glioblastoma MGEA6/11 was localized in the cytoplasm. No nuclear cell line TX3868, which expresses the MGEA6/11 staining was detected throughout. Also, there was protein. Nucleus-, cytosol- and membrane fractions partially enhanced staining of the cytoplasmic mem- were analysed by Western-blot using the anti-MGEA6/ brane, indicating membranic localization of this anti- 11 antibody (Figure 5). Speci®city of fractions was

Table 3 Western blot analyses and immunohistochemical staining with peptide-puri®ed anti-MGEA6/11 polyclonal serum on di€erent tissue samples Expression analysis Numbering WHO grading Western blot Immunohistochemistry

Intracranial meningioma H53* WHO I (ara) positive nd H28* WHO I (ara) positive nd H274 WHO I (ara) positive positive H487 WHO I (trans) positive positive (1359/99) WHO I (ara) nd positive H514 WHO I (trans) nd positive H72* WHO II positive nd H73* WHO II positive nd H99* WHO II positive nd H60* WHO II positive nd H127 WHO II positive positive H313 WHO II positive positive H350 WHO II positive positive H391 WHO II nd positive H493 WHO II positive positive H560 WHO II positive nd H566 WHO II nd positive H583 WHO II positive positive (749/99) WHO II nd positive H5 WHO III positive nd H46 a WHO III positive nd H46 b* WHO III positive nd H108* WHO III positive nd H588 WHO III positive positive

Primary brain tumors Circumscribed glioma pilocytic astrocytoma H585 WHO I positive positive Diffuse glioma oligoastrocytoma H593 WHO II nd positive H494 WHO II positive nd anaplastic astocytoma H619 WHO III nd positive glioblastoma H104* WHO IV positive nd H322* WHO IV positive nd H324* WHO IV positive positive H600 WHO IV nd positive

Atypical teratoid rhabdoid tumor WHO IV nd positive

Primary cerebral Non-Hodgkin's lymphoma nd negative

Brain metastases of mamma carcinoma nd negative lung carcinoma nd negative colorectal cancer nd negative malignant melanoma (from neural crest) nd patchy positivity

Normal tissues brain faintly positive negative fetal leptomeninges nd faintly positive adult leptomeninges nd faintly positive nd: not done; ara: arachnothelial; trans: transitional; *: serum used (see Table 1)

Oncogene MGEA6 is overexpressed and immunogenic N Comtesse et al 244 con®rmed using anti-Ki67 and anti-N-cadherin anti- Discussion bodies. The Western-blot analysis clearly con®rmed the localization of MGEA6/11 to the membranic fraction Previously, the meningioma expressed antigens 6 and as postulated based on the predicted transmembranic 11 (MGEA6/11) were identi®ed by immunoscreening a domain (Figure 2). meningioma cDNA expression library with autologous

Figure 4 (a) Immunohistochemistry for MGEA6/11 in a common-type meningioma (WHO grade I) with uniform reactivity con®ned to the cytoplasm of the tumor cells, in cases pronounced at the cell membranes (Objective, 106). (b) MGEA6/11 staining of a di€usely manifested anaplastic astrocytoma (WHO grade III) displays cytoplasmic immunoreactivity of part of the tumor cells only, in areas with clustering of decorated cells around blood vessels. No staining was observed in the pre-existing brain tissue (Objective, 406). (c) Normal brain tissue completely devoid of MGEA6/11 reactivity in the neuropil and the cellular elements, among them some cortical neurons with lipofuscin granula in their cytoplasm (Objective, 406). (d) Normal leptomeninges showing faint granular immunoreactivity con®ned to the cytoplasm of arachnoid cells (Objective, 406)

Oncogene MGEA6 is overexpressed and immunogenic N Comtesse et al 245 serum (Heckel et al., 1997). Here, we report that antibodies against MGEA6/11 are found in 41.7% of meningioma patients, without correlation to tumor grade or histological subtype. Furthermore, we showed that 2/8 glioblastoma patients and 1/2 of patients with astrocytoma grade II had antibodies against MGEA6/ 11. There was no antibody response in two sera from patients with anaplastic astrocytomas (WHO grade III), in three patients with pilocytic astrocytoma and in 12 sera from healthy persons. Our data show that MGEA6/11 can induce antibody response in menin- gioma patients and in patients with di€use gliomas. In addition, our Western blot studies revealed overexpression of MGEA6/11 in meningioma and glioma tissues compared to normal brain. Notably, the increased expression was detected in all examined meningiomas, independent of the WHO grading and seems not to be correlated with the grade of malignancy. Our immunohistochemical studies of di€erent frozen tumor sections support the Western blot data. Reactivity against MGEA6/11 was shown in meningioma tumor cells and in tumor cells of brain tumors with glial origin, whereas no reactivity was Figure 5 Cellular localization of MGEA6/11 by Western-blot analysis of cell fractions of MGEA6/11 expressing glioblastoma found in normal astrocytes and faint reactivity in cell line TX3868. The speci®city of the cell fractions was normal leptomeninges. The faint expression found in con®rmed by Ki-67, known to be localized in the nucleus and normal brain by Western blotting is likely attributable N-cadherin, known to be localized at the cytoplasmic membrane. to higher sensitivity of Western blot in comparison to Incubation with anti-MGEA6/11 serum con®rms the localization immunohistochemical staining. In brain metastasis of of MGEA6/11 to the membranic fraction tumors derived from the epithelium, MGEA6/11 was not detected. In neural crest derivatives, however, such as the brain metastasis of a malignant melanoma, frequently associated with an antibody response. patchy reactivity was noted. These results are con- However, various studies propose alternative mechan- sistent with the idea that overexpression of MGEA6/11 isms, causing the induction of immune responses (Disis is restricted to tumors of neuroectodermal origin. and Cheever, 1996; Boon and Lloyd, 1997). There is a long standing concept that brain is an A question is what biological function MGEA6/11 immunologically privileged site (Barker and Billingham, may have during tumor development. Prediction of the 1977). However, meningiomas as tumors derived from protein structure of MGEA6/11 shows an N-terminal leptomeninges covering the brain and the spinal cord, transmembrane domain. Membranic localization was are not protected by the blood-brain barrier. As for con®rmed by Western-blot analysis of cellular fractions gliomas, the astrocytes as the essential part of the of a MGEA6/11 expressing cell line. Additionally, from blood-brain barrier are a€ected by tumorigenesis. Our the protein structure prediction, some other domains data suggest that antibody-response against MGEA6/11 were identi®ed which could be involved in protein ± is not patient-speci®c and that MGEA6/11 frequently protein interactions (Figure 2). For example, interac- elicits an immune response in patients with primary tions mediated by proline-rich regions are described for intracranial tumors. We postulate that the observed WW domains found in proteins of signal transduction overexpression of MGEA6/11 in the tumor cells may pathways (Marcias et al., 1996). Coiled-coil domains cause the immune response detected in meningioma and are known to be present in structure-forming proteins glioma patients. Interestingly, overexpressed oncogenic and also in protein-binding regions. These data suggest proteins are known to represent potential tumor that MGEA6/11 could have some binding partners. antigens. C-erbB-2, for example, is overexpressed in Together with the membranic localization, this makes human tumors, especially in breast carcinoma, and MGEA6/11 probably involved in signaling pathways. antibodies against this overexpressed protein have been detected in several patient sera (Pupa et al., 1996). Autoantibodies against the L-myc oncogene, which is known to be overexpressed in tumors with neuroendo- Materials and methods crine characteristics, have been identi®ed in sera from patients with small cell lung cancer (Yamamoto et al., Recombinant protein expression in 1996). However, these studies have not established a Baculovirus Expression system correlation between overexpression and induction of Expression of MGEA6 and MGEA11 was performed using immune response for the same tumor. Here we showed Xpress System Baculovirus Protein Expression system that the observed overexpression of MGEA6/11 is (Invitrogen). In brief, open reading frames of MGEA6 and

Oncogene MGEA6 is overexpressed and immunogenic N Comtesse et al 246 MGEA11 were cloned into the pBlueBacHis2A expression immediately after surgery and then stored at 7708C. Serum vector (Invitrogen). Transformation into SF158 cells was was taken from patients at the day of surgery and stored at performed using the Bac-N-Blue Transfection Kit (Invitro- 7708C. gen). Plaque assay for puri®cation of recombinant virus was performed twice. High titer virus stocks were generated and Tissue lysates used for SF158 cell infection. Cells were harvested 46 h post- infection and stored at 7758C. Cell pellets were resuspended Frozen tissues were homogenized in sample bu€er (6% SDS; in lysis bu€er (20 mM Sodium phosphate, pH 7.0; 100 mM 125 mM Tris pH 6.8; 10% (v/v) mercaptopropanediol; 10% NaCl; 2% NP-40; 1% Complete, EDTA-free Protease (v/v) glycerol; 1% Complete, EDTA-free Protease Inhibitor Inhibitor Cocktail (Boehringer Mannheim)). Lysis was Cocktail (Boehringer Mannheim)) using an ultra-turrax. performed for 2 h on ice. Cell debris was collected (20 min, Samples were incubated 10 min at room temperature. Cell 13 500 r.p.m., in a Hermle Z 323 K centrifuge at 48C). Lysate debris was separated (20 min, 3000 r.p.m. in a Hermle Z 323 was mixed with Roati-Load1 (Roth) and stored at 7758C. K centrifuge at 48C), and the lysate was soni®ed (two times, 10 s). The debris was separated (20 min, 13 500 r.p.m.) again, and the supernatant was stored at 7758C. The protein Protein structure prediction concentration of the tissue lysates was examined with the Protein structure prediction was performed with the Bio-Rad protein assay. SMART-Simple Molecular Architecture Research Tool (http://smart.embl.heidelberg.de), TMHMM 2.0 (http:// Western blot analysis www.cbs.dtu.dk/services), TMpred (http://wwwch.embnet. org/software) and the algorithms PROSITE and BEAUTY. Proteins were separated by electrophoresis on a 10% SDS- polyacrylamide gel at 25 mA for 3 h. Proteins were transferred to Hybond-P membrane (Amersham Pharmacia Peptide synthesis Biotech) by electroblotting at 500 mA for 3 h. Membranes A peptide from the C-terminal amino acids 776 to 795 of were blocked in 16PBS; 0.5% dry-milk for 1 h at room MGEA6/11 was synthesized by the Fmoc method of solid temperature. Peptide-speci®c, anity-puri®ed rabbit serum phase peptide chemistry (Merri®eld, 1963) on a Milligen/ was diluted 1:50, human preabsorbed patient serum 1:100 ± Biosearch 9050 Pep-synthesizer. Cleavage of the peptides 1:500 in 16PBS; 0.5% dry-milk and incubated overnight at from the resin and removal of the protecting groups were 48C. After washing three times for 10 min each in 16PBS, achieved by treating the peptide with 90% tri¯uoroacetic the membrane was hybridized with the secondary antibody acid, 2.5% phenol, 2.5% 1,2-ethanedithiol, 1% triisopropyl- (goat anti-rabbit or goat anti-human IgG Fc-speci®c anti- silane (all from Sigma) and 5% water. body conjugated to peroxidase (Dianova)) 1 : 5000 in 16PBS, 5% dry-milk for 1 h at room temperature. Detection was carried out using the ECL detection reagent (Amersham Polyclonal antibodies Pharmacia Biotech) according to the manufacturer's instruc- Antibodies were generated against the peptide plus an tions. additional amino-terminal cysteine-residue. The peptide was coupled to keyhole limpet hemocyanine that had been Tissue preparation for immunohistochemistry activated with N-succinimidyl-3-maleinimido-propionate (Fulka). To obtain antibodies, rabbits (bastards) were Fresh tissue samples were snap frozen in nitrogen cooled immunized by subcutaneous injections of 300 mg of coupled isopentane and protected from lyophilization by coating them peptide emulsi®ed in Freund's complete adjuvant (Sigma) with aqueous 2% Tragant (Merck, Germany). For any case (Harlow and Lane, 1998). The immunization was repeated included in the study, at least one hematoxylin-eosin-stained two times at 2-week intervals with the same amount of 8 mm thick section plus serial unstained sections were coupled peptide in Freund's incomplete adjuvant. The rabbits available. After cutting, they were allowed to air dry at were bled 8 ± 10 days after the immunizations. room temperature for 5 min, were ®xed by immersion in 48C cold Zamboni's ®xative for 10 min (PBS with 15% (v:v) twice ®ltered saturated picric acid and 2% (w:v) paraformaldehyde, Affinity purification of the polyclonal antibodies ®nal pH 7.3), thereafter thoroughly washed under running The peptide-speci®c polyclonal antibodies were anity tap water for 10 min, and again air dried. They were kept puri®ed using the HiTrap NHS-activated anity columns frozen at 7808C until use. from Amersham Pharmacia Biotech according to instruction manual. Ten milligrams of peptide were coupled to the NHS- Immunohistochemistry activated Sepharose column, and puri®cation was performed according to Harlow and Lane (1988). In brief, 5 ml of the Endogenous peroxidase activity was quenched by immersing rabbit serum were diluted 1 : 10 with 10 mM Tris, pH 7.5, and the sections in 3% hydrogen peroxide in methanol for the diluted serum was applied two times to the equilibrated 30 min. For immunohistochemistry, the optimal dilution of column. Antibodies were eluted with 100 mM glycine, pH 2.5. the peptide-speci®c, anity puri®ed polyclonal antibody to Fractions were immediately neutralized with 1 M Tris, pH 8. MGEA6/11 was 1 : 5. Both the background reducing anti- Finally, bu€er exchange against PBS pH 7.5 was performed body diluent (S 3022) and the components of the detection using NAP-25 Columns (Amersham Pharmacia Biotech). systems, including color substrate, were purchased from Final volume was 14 ml. DAKO, Hamburg, Germany. Incubation of the sections with the primary antibody was done in a moist chamber at 378C for 30 min. Optimal detection was achieved with biotin Tumor tissues and sera labeled swine F(ab')2 fragments to rabbit immunoglobulins The tumor tissues and sera were used with consent by the (E 0431) (1 : 200, 378C, 10 min) followed by a second patients. Tissue samples were frozen in liquid nitrogen incubation step with horseradish peroxidase (HRP) con-

Oncogene MGEA6 is overexpressed and immunogenic N Comtesse et al 247 jugated streptavidin (P 0397) (1 : 300, 378C, 20 min). After 0.1 mM PMSF, 0.1 mM NaNO4,10mM NaF, 50 mM each incubation step, the sections were thoroughly rinsed in glycerolphosphate, 1% Triton X-100, 1% NP40) and phosphate bu€ered saline (PBS). Reaction products were centrifuged at 38 000 r.p.m. for 1 h at 48C. The supernatant visualized with diaminobenzidine as the color substrate (DAB was referred to as membrane fraction. The nuclear pellet (see Plus substrate solution, 1 : 50; K 3468). Counterstaining of above) was washed with bu€er A (10 mM HEPES, 10 mM the nuclei was done with Harris' hematoxylin followed by KCl, 1.5 mM MgCl2,5mM DTT, 0.5 mM PMSF) and covering the slides with an organic mounting medium and resuspended in three volumes bu€er B (20 mM HEPES, cover slips after dehydration with alcohol and xylol. 450 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 5 mM DTT, 0.5 mM PMSF, 20% Glycerol), incubated for 30 min on ice and centrifuged for 20 min at 15 000 r.p.m. and 48C. The Preparation of cellular fractions supernatant was referred to as nuclear extract. Cells were rinsed twice with cold PBS and collected (10 min; 1200 r.p.m.). The cell pellet was washed and then lysed in 2 volumes of lysis bu€er (10 mM HEPES (pH 7.9), 10 mM KCl, Acknowledgments 3 mg/ml Aprotinin) 5 min at 48C. The lysate was homo- The authors thank Prof Dr WI Steudel and the sta€ of the genized in a Dounce homogenizer (20 strokes) and Neurosurgical Clinic for providing fresh tumor samples. centrifuged at 1500 r.p.m. for 15 min at 48C to obtain a We thank Mr Marc Voss, Department of Virology, for the nuclear pellet. The supernatant was collected, adjusted with excellent help preparing the cell fractions. The skilled 5 M NaCl to a concentration of 0.15 M and centrifuged for technical assistance of Mr W FoÈ rderer, Mr P KruÈ ck, Mrs S 1 h at 38 000 r.p.m. and 48C (Centrikon T-2060, Kontron). Leyes, Mrs B Simon and Mr B Stock, Department of The supernatant contained the cytosolic fraction. The pellet Neuropathology, is greatly appreciated. This work was was resuspended with NTENT-bu€er (150 mM NaCl, 10 mM supported by a grant from the Deutsche Forschungsge- Tris/Cl (pH 8), 1 mM EDTA (pH 8), 3 mg/ml Aprotinin, meinschaft (SFB 399, A4).

References

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