Proteomic Studies on Low- and High-Grade Human Brain Astrocytomas

Human brain astrocytomas range from the indolent low-grade to the highly infiltrating and aggressive high-grade form, also known as glioblastoma multi...
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Proteomic Studies on Low- and High-Grade Human Brain Astrocytomas Federico Odreman,† Marco Vindigni,‡ Marlen Lujardo Gonzales,† Benedetta Niccolini,† Giovanni Candiano,§ Bruno Zanotti,‡ Miran Skrap,‡ Stefano Pizzolitto,| Giorgio Stanta,† and Alessandro Vindigni*,† International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy, Neurosurgery Unit, Hospital “S.M. della Misericordia” 15, I-33100 Udine, Italy, Laboratory of Pathophysiology of Uremia, G. Gaslini Children’s Hospital, Genova, Italy, and Pathology Unit, Hospital “S.M. della Misericordia” 15, I-33100 Udine, Italy Received October 7, 2004

Human brain astrocytomas range from the indolent low-grade to the highly infiltrating and aggressive high-grade form, also known as glioblastoma multiforme. The extensive heterogeneity of astrocytic tumors complicates their pathological classification. In this study, we compared the protein pattern of low-grade fibrillary astrocytomas to that of glioblastoma multiforme by 2D electrophoresis. The level of most proteins remains unchanged between the different grade tumors and only few differences are reproducibly observable. Fifteen differentially expressed proteins, as well as seventy conserved spots, were identified by mass spectrometry. Western and immnunohistochemical analysis confirmed the differential expression of the identified proteins. These data provide an initial reference map for brain gliomas. Among the proteins more highly expressed in glioblastoma multiforme, we found peroxiredoxin 1 and 6, the transcription factor BTF3, and R-B-crystallin, whereas protein disulfide isomerase A3, the catalytic subunit of the cAMP-dependent protein kinase, and the glial fibrillary acidic protein are increased in low-grade astrocytomas. Our findings contribute to deepening our knowledge of the factors that characterize this class of tumors and, at the same time, can be applied toward the development of novel molecular biomakers potentially useful for an accurate classification of the grade of astrocytomas. Keywords: astrocytomas • gliomas • proteomics • 2D-gel electrophoresis • mass spectrometry

Introduction The brain is composed of both nerve cells (neurons) and glial cells.1 Glial cells are more numerous than neurons, and unlike neurons, do not generate electrical signals. Instead, these cells serve to support, nourish, and insulate neurons.2 Glial cells include ependymal cells, oligodendrocytes, astrocytes, and microgliocytes.2 Tumors involving glial cells are known as gliomas and are more common than tumors involving neurons. In fact, gliomas account for about 45% of all primary brain tumors, and their incidence is around five to 10 per 100 000 general population.3 Most of brain gliomas arise from astrocytes, the most abundant type of glial cells, and are known as astrocytomas. Astrocytomas are divided into four grades according to their histological characteristics and following the classification guidelines given by the World Health Organization (WHO).4 The * To whom correspondence should be addressed. Tel: +39-040-3757369. Fax: +39-040-226555. E-mail: [email protected]. † International Centre for Genetic Engineering and Biotechnology. ‡ Neurosurgery Unit, Hospital “S.M. della Misericordia”. § Laboratory of Pathophysiology of Uremia, G. Gaslini Children’s Hospital. | Pathology Unit, Hospital “S.M. della Misericordia”.

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Journal of Proteome Research 2005, 4, 698-708

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most aggressive type of astrocytoma, glioblastoma multiforme (WHO grade IV), is an infiltrating and highly vascularized tumor with a very poor prognosis.5 Unfortunately, there are situations in which the WHO four-grade classification system is problematic, primarily because pathological diagnosis remains subjective. Given these difficulties, several hospitals have adopted a simplified classification system, dividing the astrocytomas into low- and high-grade. The majority of human gliomas have a set of genetic alterations that cause the loss of protein regulation.6,7 Studies based on DNA microarray analyses indicated that glioblastomas overexpress various gene families related to cell adhesion, motility, invasion, and angiogenesis.8 A down-regulation of cell cycle-regulating genes was also observed.8 Protease profiling studies indicated that the expression levels of several proteases such as urokinase-type plasminogen activator (uPA) and its receptor (uPAR), the cysteine cathepsin B, as well as the matrix metalloproteases MMP2 and MMP9 are more abundant in high-grade astrocytomas.9 Despite the information already emerged from these and other studies, additional investigation, especially at the protein level, is indispensable to uncover new molecules that characterize the different grades of astrocytomas 10.1021/pr0498180 CCC: $30.25

 2005 American Chemical Society

Proteomics of Human Brain Astrocytomas

and that are associated with the elevated malignancy of glioblastoma multiforme. In the present work, a proteomic approach has been used to compare the protein expression profiles of low- and highgrade astrocytomas. Particular care was taken to only select gliomas originating from astrocytes and not from other glial cells to make sure that differences between 2D gels would be exclusively related to the different grade of the tumor. Proteins extracted from tissues were separated on 16 × 18 cm twodimensional (2D) polyacrylamide gels. The majority of protein spots were conserved among the gliomas of different grades and only few differences were reproducibly visible. All the differentially expressed proteins as well as several of the conserved ones were identified by mass spectrometry analysis. Western and immunohistochemical analysis were utilized to validate our findings. The results presented here contribute to deepening our understanding on the differences between lowgrade fibrillary astrocytomas and glioblastoma multiforme at the protein level and provide an initial description of proteins that are consistently present in human brain gliomas.

Materials and Methods Materials. CHAPS, IPG buffer, immobiline DryStrips, and cover fluid were purchased from Amersham Pharmacia Biotech AB (Uppsala, Sweden). DTT, urea, thiourea, TrisHCl, iodoacetamide, glycine, agarose, SDS, were from Sigma (St. Louis, MO). Acrylamide, and broad range molecular mass markers were from Bio-Rad (Munich, Germany). Silver nitrate, glycerol, TEMED, ammonium persulfate, and ammonium bicarbonate were bought from Fluka (Buchs, Switzerland). Methanol, ethanol, and acetic acid were from Merck (Darmstadt, Germany). Protease inhibitors cocktail was from Roche (Mannheim, Germany) and sequencing grade trypsin and Taq polymerase from Promega (Madison, WI). The rabbit polyclonal antibodies against the catalytic subunit of the cAMP-dependent protein kinase and peroxiredoxin 1, and the mouse monoclonal antibodies against R-tubulin and protein disulfide isomerase were from Abcam (Cambridge, UK). The goat polyconal antibody against ubiquitin-protein hydrolase was from from Santa Cruz (Santa Cruz, CA), while the rabbit polyclonal antibodies against R-internexin, transcription factor BTF3, and the glial fibrillary acidic protein were from Zymed Lab. (San Francisco, CA), EnCor Biotech. (Alaschua, FL), and Ventana (Tucson, AZ), respectively. The mouse monoclonal antibody against peroxiredoxin 6 was from LabFrontier (Wiltshire, UK). Preparation of Tissue Proteins. Frozen surgical tumor samples were provided by the Neurosurgical unit of the hospital “Santa Maria della Misericordia” of Udine, Italy. The tissues of low- and high-grade astrocytomas were obtained by surgical resection. Perilesional tissues that immediately surround the lesion and that did not show any tumor infiltration at the microscopic examination were also provided. Samples were immediately frozen in liquid nitrogen and stored at -80 °C. A portion of the tumor was fixed in 10% formaldehyde and embedded in paraffin for histopathological diagnosis. A total of 20 astrocytomas were analyzed: 10 fibrillary astrocytomas (grade II) and 10 glioblastoma multiforme (grade IV). Tissues were homogenized in 700 µL of lysis buffer consisting of 7 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 0.5 mM EDTA, and 1X protease inhibitors cocktail. Successively, the suspension was sonicated for 30 s to facilitate protein solubilization and incubated for 1 h at room temperature. The samples were then centrifuged at 16 000 g for 30 min. The protein concentra-

research articles tion in the supernatant was estimated by SDS-PAGE gels stained with coomassie brilliant blue R-250 and with the RC DC protein assay from Bio-Rad. This colorimetric assay allows the determination of protein concentration in the presence of reducing agents and detergents. BSA was used as a standard. Samples were either directly used for 2-DE analysis or stored at -80 °C. 2D Electrophoresis. Immobilized pH gradient (IPG) strips (pH 3-10 NL and pH 4-7) of 18 cm were rehydrated passively overnight in a strip holder with 350 µL of rehydration buffer containing 8 M urea, 1.5 M thiourea, 2% CHAPS, 1% DTT, 0.5% v/v Pharmalyte pH 3-10 NL, and ∼100 µg of protein extract. Isoelectro focusing was carried out under the following conditions: 500 V for 1 h, 1000 V for 1 h, and finally 8000 V hold for 48 000 Vh. The strips were kept at 200 V until loaded on the second dimension. Before starting the second dimension the strips were equilibrated in 6 M urea, 30% glycerol, 2% CHAPS, 50 mM Tris pH 6.8, and 1% DTT for 15 min. Afterward, they were briefly rinsed in double distilled water (ddwater) and equilibrated in 6 M urea, 30% glycerol, 2% CHAPS, 50 mM Tris pH 7.8, and 2.5% iodoacetamide for an additional 15 min. The second dimension was carried out using 10% or 14% polyacrylamide gels and a current of 30 mA per gel. Protein Visualization. Proteins were visualized using the modified silver staining protocol developed by Mortz and coworkers.10 Briefly, after the second dimension, the gels were fixed overnight at room temperature in 50% methanol, 12% acetic acid, and 0.05% formalin. The day after, the gels were washed 3 times in 35% ethanol for 20 min, and then sensitized in a 0.02% solution of Na2S2O3 for 2 min. Next, the gels were washed 3 times in ddwater for 5 min and placed into a solution containing 0.2% silver nitrate and 0.076% formalin for 30 min. Successively, the gels were washed twice with ddwater for 1 min and developed in a solution containing 6% Na2CO3, 0.05% formalin, and 0.0004% Na2S2O3. Finally, the gels were placed in a solution containing 50% methanol, 12% acetic acid for 5 min to stop the reaction. The stained gels were stored in 1% acetic acid. Computer Analysis of 2D Gels. The silver-stained gels were scanned with a VersaDoc imaging system (Model 3000 from Bio-Rad). The data were analyzed using the PDQuest software from Bio-Rad. Protein Digestion. Selected spots were excised manually from the gel with a scalpel. Individual gel samples were placed in 1.5 mL microcentrifuge tubes and washed twice for 10 min with ddwater. To remove silver, samples were placed in a 1:1 mix of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate until the gel pieces turned clear. This solution was then discarded and each gel sample was washed with ddwater for 15 min. Successively, 300 µL of acetonitrile were added to the solution, and the gel pieces were incubated for additional 15 min. After discarding the solution, the bands were washed with 100 mM of ammonium bicarbonate/acetonitrile (50:50 v/v) for 15 min. This solution was removed and gel samples were crushed with a Teflon stick, after which 100 µL of acetonitrile were added for dehydration. After 5 min, the acetonitrile was removed and the bands were dried in a Speed Vac for 5 min. Samples were resuspended in 50 µL of 10 mM DTT in 100 mM ammonium bicarbonate and incubated for 1 h at 56 °C. After this time, DTT was removed and samples were placed in 50 µL of 50 mM iodoacetamide in 100 mM ammonium bicarbonate at room temperature for 30 min in the Journal of Proteome Research • Vol. 4, No. 3, 2005 699

research articles dark. The solution was removed and gel samples were washed with 300 µL of 100 mM ammonium bicarbonate for 15 min. Samples were then placed in 300 µL of 20 mM ammonium bicarbonate/acetonitrile (50:50 v/v) for 15 min. The previous solution was replaced with 100 µL of acetonitrile and samples were left at room temperature for 5 min. The samples were then dried in a Speed Vac for 10 min and resuspended with 5 µL of trypsin (0.1 µg/µL) in 100 mM ammonium bicarbonate pH 8.0. After 10 min, 100 µL of 100 mM ammonium bicarbonate were added and the digestion was carried out overnight at 37 °C. The supernatant was collected in a second microcentrifuge tube. The gel pieces were washed once with 100 µL ddwater and twice with 100 µL of 60% acetonitrile/1% TFA. The washes were pooled and added to the previously collected supernatant. The volume of the solution was reduced to 5-10 µL in a Speed Vac and the samples were stored at 4 °C until analyzed with the mass spectrometer. Mass Spectrometry Analysis. Preparative gels were stained using the silver staining protocol described above. Spots of interest were excised from the gels and digested with sequencing grade trypsin. LC-ESI-MS/MS was performed on a LCQ Deca ion trap (ThermoFinnigan, San Jose, CA) coupled with an LC-PAL autosampler (CTC Analytics, Zwingen, Switzerland) and a Surveyor micro HPLC pump (ThermoQuest-Finningan). For each experiment, 20 µL of sample in 0.1% formic acid was injected on a C8 reverse-phase column (1 × 150 mm) from Thermo-Hypersil. Peptides were eluted from the column using a linear gradient of acetonitrile (from 0 to 85% CH3CN in 70 min) in the presence of 0.1% formic acid. When smaller amounts of material were available, 5 µL of sample were loaded onto a PicoFrit column (0.075 × 50 mm) from New Objective and eluted with a CH3CN gradient (from 0 to 80% CH3CN) at a flow rate of ∼200 nL/min. Helium was used as collision gas and the collision energy was set at 35% of the maximum. Data analysis was performed either using TurboSequest 2.0 or MASCOT 2.0. For MASCOT analysis, the spectra were converted to DTA files and regrouped using in-house software. The combined Swiss-Prot and TrEMBL database was searched without species restriction. Western Blot Analysis. Brain tissues were homogenized in lysis buffer containing 50 mM TrisHCl pH 8.0, 100 mM NaCl, 1% NP-40 and protein inhibitors. The solutions were sonicated on ice (three times for 30 s). The cell homogenates were centrifuged at 1000 rpm for 5 min at 4 °C and the supernatants were stored at -80 °C. Protein extracts (80 µg) were loaded on 12% polyacrylamide gels that were run at 130 V for 2h. SDSPAGE gels were transferred onto PVDF membranes using a current of 200 mA for 2 h. Nonspecific binding was prevented by blocking the membrane with 5% of nonfat dried milk, 0.1% v/v Tween 20 in TBS. Next, membranes were washed 5 times, 5 min each, with 0.1% v/v Tween 20 in TBS. Monoclonal antibodies raised against protein disulfide isomerase, the catalytic subunit of the cAMP-dependent protein kinase, peroxiredoxin 1, and peroxiredoxin 6 were used in a 1:1000 dilution in 0.5% nonfat dried milk, 0.1% v/v Tween 20 in TBS. Ubiquitin carboxyl-terminal hydrolase isozyme L1, R-internexin, and transcription factor BTF3 were instead detected using rabbit generated polyclonal antibodies in a 1:1000 dilution. The immunocomplexes were detected with 1:10 000 dilution of antimouse IgG, anti-rabbit IgG, or anti-goat IgG (Pierce). Immunoblots were developed using the ECL detection system. Immunohistochemical Analysis. Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded 700

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tissues obtained from the Pathology unit of the hospital “Santa Maria della Misericordia” of Udine, Italy. After deparaffinization and hydration, the sections were treated with a 0.4% pepsin solution for 30 min at 37 °C to unmask the antigens. Then endogenous peroxidases activity was blocked with 0.6% H2O2 in methanol for 30 min at room temperature. After washing the slides in cold tap water for 10 min, fixed tissues section were incubated with 3% BSA in PBS and normal goat serum (1:100 dilution) for 45 min. Slides were then incubated with primary antibodies for glial fibrillary acidic protein (rabbit polyclonal, 1:500 dilution), the catalytic subunit of the cAMPdependent protein kinase (rabbit polyclonal antibody, 1:400 dilution), protein disulfide isomerase (mouse monoclonal antibody, 1:100 dilution), peroxiredoxin 1 (rabbit polyclonal antibody, 1:200 dilution), R-internexin (rabbit polyclonal antibody, 1:200 dilution) for 1 h at room temperature. After three serial washes with PBS, the avidin-biotin peroxidase method was utilized to visualize the bound antibodies (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA). Diaminobenzidine was used as chromagen and counterstained with hematoxylin for 60 s, then dehydrated and mounted using Eukit Mounting medium (Fluka).

Results Protein extracts from fibrillary astrocytomas (low-grade) and glioblastoma multiforme (high-grade) were analyzed by 16 × 18 cm 2D gel electrophoresis. The first dimension analysis was performed either with broad (pH 3-10NL) or narrow (pH 4-7) pH range IPG strips. For the second dimension, we used 10% and 14% polyacrylamide gels. Lower percentage polyacrylamide gels were used to better visualize proteins of high molecular mass, whereas proteins of low molecular mass were resolved on 14% gels. Thus, we were able to detect proteins with molecular masses ranging from 15 to 150 kDa. Typical 2-D gel images of low-grade astrocytomas obtained with the broad range pH strips (pH 3-10 NL) are shown in Figure 1. Following computational analysis, we detected approximately 700-800 protein spots on each gel (16 × 18 cm) using a silver- stained based protocol optimized for sensitivity and compatible with mass spectrometry analysis.10 Most of the protein spots were distributed in a molecular mass range from 20 to 120 kDa. Fewer protein spots were detected using the IPG strips with pH range 4-7 (data not shown). A total of 20 astrocytomas from different patients were analyzed with this technique: 10 fibrillary astrocytomas (grade II) and 10 glioblastoma multiforme (grade IV). The 2D electrophoresis profiles and relative spot intensities obtained for most of the samples were perfectly reproducible when performed in duplicate or triplicate. In addition, analysis of 2D gels of gliomas of the same grade coming from different individuals yielded matching means of 82 ( 7% for low-grade astrocytomas and 79 ( 9% for glioblastoma multiforme. The differences observed between tumors of the same grade were due to the heterogeneity present between gliomas of the same histological category and to the varying levels of protein degradation resulting from tissue necrosis. Comparison of the 2D gels of low- and high-grade astrocytomas showed that most of the protein spots reproducibly present in all low-grade astrocytomas were also conserved in all glioblastoma multifome and only few differences were clearly visible. Proteins were identified using an ion trap mass spectrometer equipped with a micro-high pressure liquid chromatography system and a nano-electrospray source (see Materials and

Proteomics of Human Brain Astrocytomas

research articles 6 spots that showed a constant increase and 9 spots that were significantly less intense in the 2D gels of low-grade astrocytomas relative to the 2D gels of glioblastoma multiforme (Figures 2 and 3). After mass spectrometry analysis, we found that ubiquitin-protein hydrolase, protein disulfide isomerase A3, dihydropteridine reductase, the catalytic subunit of the cAMP-dependent protein kinase, glial fibrillary acidic protein, and T-complex protein 1 epsilon subunit were more highly expressed in low-grade astrocytomas, whereas R-internexin, β-actin, fibrinogen fragment D, apolipoprotein A-I, peroxiredoxin 1 and 6, annexin A5, transcription factor BTF3 homologue 1, and R-B-crystallin were increased in high-grade tumors.

Figure 1. 2D protein expression profiles of low-grade astrocytomas. 2D gels were generated, stained, and analyzed as described in Materials and Methods. The amount of protein loaded was approximately 100 µg. Top: 10% polyacrylamide gel. Bottom: lower region of 14% polyacrylamide gel. All the 2D gels images are representative of at least three independent experiments. Spot numbers (from 1 to 70) indicate all the proteins identified by mass spectrometry and refer to the numbers reported in Table 1.

Methods). We initially identified 70 proteins that were consistently present in all the low- and high-grade astrocytomas analyzed so far (Table 1). Among these conserved proteins we found 27 metabolism related proteins, 7 structural proteins, 5 brain proteins, 11 stress related proteins, 2 oncoproteins, and 18 proteins involved in other cellular functions (Figure 4). A cluster of proteins was detected in the basic and low molecular mass region of the gels (Figure 1). After mass spectrometry analysis, we established that this protein cluster was mainly formed by hemoglobin. Hemoglobins were more abundant in high-grade astrocytomas consistent with the major grade of vascularization of these tissues compared to low-grade tumors. For the same reason, a group of spots containing albumin was visible in the upper right region of the 2D gels and was more represented in glioblastoma multifome (Figure 1). To identify proteins that were significantly correlated to the histological grading, we selected the 15 proteins that were consistently increased or decreased between all the low- and high-grade astrocytomas (Table 2). In particular, we detected

The results obtained from the 2D gel analysis were validated by Western and immunohistochemical analysis. In particular, Western analysis was performed using specific antibodies raised against 7 out of the 15 differentially expressed proteins previously identified by mass spectrometry (Figure 5). In addition, perilesional tissues were utilized to compare the expression level of the six proteins in the low- and high-grade tumors with their expression in the tissues that immediately surround the lesion. The western results confirmed our previous findings and showed that protein disulfide isomerase A3 and ubiquitinprotein hydrolase were equally expressed in perilesional tissues and low-grade astroctyomas, whereas they were basically absent in the high-grade tumors. Similarly, the catalytic subunit of the cAMP-dependent protein kinase was not detectable in glioblastoma multiforme although its expression level was already reduced in low-grade astrocytomas relative to the perilesional tissues. Among the proteins previously found as more highly expressed in glioblastoma multiforme, peroxiredoxin 1 and 6, transcription factor BTF3, and R-internexin, were clearly more represented in the high-grade tumors not only relative to the low-grade astrocytomas, but also to the perilesional tissues. Immunohistochemical analysis was performed on paraffin-embedded tissues for 5 out of the 15 differentially expressed proteins providing additional information on the localization of these proteins in the cells (Figure 6). High levels of glial fibrillary acidic protein and the catalytic subunit of the cAMP-dependent protein kinase were clearly visible in the cytoplasm of the low-grade tumor cells, while the same proteins were significantly reduced in the high-grade tumors. Elevated levels of protein disulfide isomerase A3 were observed mainly in the nuclei of the fibrillary astrocytomas, whereas the glioblastoma multiforme were hardly stained with the antibody. Among the proteins increased in the high-grade tumors, our immunohistochemical analysis confirmed that R-internexin and peroxiredoxin 1 were more abundant in glioblastoma multiforme compared to the lower grade astrocytomas and are primarily localized in the cytoplasm. Remarkably, earlier studies had already demonstrated that some of the identified proteins were differentially expressed between low- and high-grade astrocytomas. In particular, R-B-crystallin, a member of the small heat shock protein (HSP20) family, was also found by Aoyama and co-workers to be overexpressed in high-grade astrocytomas.11 Similarly, previous studies suggested that the protein disulfide isomerase A3, the catalytic subunit of the cAMP-dependent protein kinase, and the glial fibrillary acidic protein are more represented in low-grade tumors.12-14 In addition, the higher abundance of apolipoprotein A-I and fibrinogen fragment D in high-grade astrocytomas is also in agreement with previous findings and is probably related to the higher degree of vascularization of these tissues.15 To our knowledge, the other proteins identified by mass spectrometry Journal of Proteome Research • Vol. 4, No. 3, 2005 701

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Table 1. Proteins Conserved in Low- and High-Grade Astrocytomas

no.

protein

Swiss-Prot or NCBI entry

theoretical Mr(kDa)/pI

sequence coverage (%)

P07195

Metabolism 36.5/5.72

24

Phosphoglycerate kinase 1

P00558

44.5/8.30

18

3

Glutamate dehydrogenase 1, mitochondrial [Precursor]

P00367

61.3/7.66

23

4

Methylmalonate-semialdehyde dehydrogenase Triosephosphate isomerase

Q02252

57.8/8.72

20

P00938

26.5/6.51

71

Glyceraldehyde-3-phosphate dehydrogenase Acetyl-CoA acetyltransferase Pyruvate kinase, M2 isozyme 3-phosphoglycerate dehydrogenase Succinyl CoA:3-oxoacid CoA-transferase Aconitate hydratase, mitochondrial Alpha enolase Protein-L-isoaspartate(D-aspartate) O-methyltransferase Heterogeneous nuclear ribonucleoprotein H′ 3-Hydroxyacyl-CoA dehydrogenase type II

P00354

36.2/8.26

33

P24752 P14786 O43175 P55809 Q99798 P06733 P22061

45.2/8.98 58.0/7.95 57/6.29 56/7.13 86/7.36 47/7.01 25/6.70

40 26 14 25 18 23 30

P55795

50/5.89

19

Q99714

27/7.66

54

ATP synthase R chain, mitochondrial Carbonic anhydrase II

P25705

40/8.94

15

P00921

29/6.63

28

P13804

33/6.95

51

19 20 21

Electron-transfer flavoprotein R-subunit Fructose-bisphosphate aldolase A Pyruvate kinase, M1 isozyme Aldose reductase

P04075 P14618 P15121

40/8.39 59/7.95 36/6.56

42 22 16

22

Hexokinase, type I

P19367

102/6.44

10

23

Cyclophilin A

P05092

17.8/7.8

53

24

Carbonyl reductase

P16152

31/8.55

60

25

Glucose-6-phosphate isomerase

P06744

63/8.42

36

26 27

Glycerol-3-phosphate dehydrogenase Aflatoxin B1 aldehyde reductase 1

P21695 O43488

38/5.81 39/6.52

33 22

28

Cofilin, nonmuscle isoform

P23528

Structural 19/8.2

34

29 30

Tubulin R-1 chain F-actin capping protein R-1 subunit

P05209 P52907

51/5.02 33/5.45

16 17

31

PDZ and LIM domain protein 1

O00151

37/6.56

12

32

Histone H1.3

P16402

22/11.02

14

33 34

Tubulin β-1 chain Profilin II

P07437 P35080

50.0/4.75 15/6.78

24 21

35

Fatty acid-binding protein, brain

O15540

Brain 15/5.41

35

702

Journal of Proteome Research • Vol. 4, No. 3, 2005

1

L-lactate

2

5 6 7 8 9 10 11 12 13 14 15

16 17 18

dehydrogenase B chain

function

Belongs to the LDH family; involved in the final step of the anaerobic glycolysis pathway Glycolytic enzyme that might also act as a polymerase R cofactor protein Belongs to the Glu/Leu/Phe/Val dehydrogenases family; catalyses the reaction of L-glutamate metabolism Involved in valine and pyrimidine metabolism Plays an important role in several metabolic pathways Homotetramer involved in the second phase of glycoslysis Plays a major role in ketone body metabolism Homotetramer involved in the glycolysis pathway Involved in the serine biosynthesis Key enzyme for ketone body catabolism Involved in nucleic acid synthesis and processing Involved in the glycolysis pathway Plays a role in the repair and/or degradation of damaged proteins Nucleic acid synthesis and processing Binds intracellular amyloid-β and it may contribute to the neuronal dysfunction associated with Alzheimer disease Produces ATP from ADP in the presence of a proton gradient across the membrane Catalyses the reversible hydration of carbon dioxide Specific electron acceptor for several dehydrogenases Involved in the glycolysis pathway Involved in the final step of the glycolysis pathway Catalyzes the NADPH-dependent reduction of a wide variety of carbonyl-containing compounds to their corresponding alcohols Allosteric enzyme involved in the final step of several metabolic pathways Accelerates the folding of proteins; catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides Catalyzes the reduction of a wide variety of carbonyl compounds including the antitumor anthracycline antibiotics Involved in glycolysis and in gluconeogenesis; is a neurotrophic factor for spinal and sensory neurons Converts NAD+ in NADH Can reduce the dialdehyde protein-binding form of aflatoxin B1 to the nonbinding AFB1 dialcohol Controls reversibly actin polymerization and depolymerization in a pH-sensitive manner Constituent of microtubules Binds in a Ca(2+)-independent manner to the fast growing ends of actin filaments Protein that may act as an adapter that brings other proteins to the cytoskeleton Histones H1 are necessary for the condensation of nucleosome chains into higher order structures Tubulin is the major constituent of microtubules Binds to actin and affects the structure of the cytoskeleton Could be involved in the transport of a so far unknown hydrophobic ligand with potential morphogenic activity during CNS development

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Proteomics of Human Brain Astrocytomas Table 1 (Continued) Swiss-Prot or NCBI entry

theoretical Mr(kDa)/pI

sequence coverage (%)

no.

protein

36 37 38 39 40

Neuropolypeptide h3 5′-TG-3′ interacting factor Fructose-bisphosphate aldolase C Dihydropyrimidinase related protein-1 Glutathione transferase omega 1

P30086 Q15583 P09972 Q14194 P78417

Stress 21/7.4 29.7/8.24 39.3/6.46 62/6.55 27.6/6.24

23 12 37 26 38

41

Macrophage migration inhibitory factor (MIF)

P14174

12/8.2

46

42

Superoxide dismutase [Cu-Zn]

P00441

15.8/5.7

24

43

Superoxide dismutase [Mn

P04179

24.7/8.35

56.8

44 45

Stress-induced-phosphoprotein 1 60 kDa heat shock protein

P31948 P10809

62.6/6.4 61.0/5.70

25 34

46

O94760

31.0/5.53

42

P38646

74/5.97

16

48

NG,NG-dimethylarginine dimethylaminohydrolase 1 Stress-70 protein, mitochondrial [Precursor] Glutathione S-transferase P

P09211

23.2/5.44

48.8

49 50

Peroxiredoxin 2 Heat shock 27 kDa protein

P32119 P04792

22/5.66 23/5.98

15 29

51

Zinc finger protein 397

Q8NF99

Oncoproteins 56/7.36 32

52

Q99497

20/6.33

49

53

HDJ-1 Other proteins Phosphatidylethanolamine-binding protein

P30086

20.9/7.43

22

54

Lambda-crystallin homolog

Q9Y2S2

34.3/5.95

15

55 56

Ankyrin (brank-1) Serotransferrin [Precursor]

29489* P02787

70.0/9.08 77/6.81

10 8

57 58

YF13H12 protein Proteasome R 2 subunit

AAH08250* P25787

28/6.35 26/7.12

28 28

59

T-complex protein 1, β subunit

P78371

58/6.01

17

60 61 62 63 64 65

Inorganic pyrophosphatase IgG κ chain Ig κ chain V-III region B6 Selenium-binding protein 1 Adenylate cyclase Vimentin

Q15181 BAA37169* P01619 Q13228 P00936 P08670

33/5.54 24/6.92 12/9.30 53/5.93 24/5.43 54/5.06

12 41 42 13 10

66

Elongation factor Tu, mitochondrial [Precursor]

P49411

50/7.26

33

67 68

Pirin Poly(rC)-binding protein 1

O00625 Q15365

32/6.42 38/6.66

14 20

69 70

Hemoglobin Serum albumin

P01922 P02768

15/8.73 69/5.92

25 13

47

have not been previously reported as differentially expressed between low- and high-grade gliomas adding novel information

function

Binds ATP, opioids and phosphatidylethanolamine Nucleic acid synthesis and processing Energy and metabolism Cellular communication and signal transduction Exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities The expression of MIF at sites of inflammation suggest a role for the mediator in regulating the function of macrophage in host defense Destroys radicals which are normally produced within the cells and which are toxic to biological systems Destroys radicals which are normally produced within the cells and which are toxic to biological systems Unclassified Implicated in mitochondrial protein import and macromolecular assembly Has a role in nitric oxide generation Implicated in the control of cell proliferation and cellular aging. May also act as a chaperone Conjugation of reduced glutathione to a wide number of exogenous and endogenous hydrophobic electrophiles Involved in redox regulation of the cell Involved in stress resistance and actin organization Isoform 3 acts as a DNA-dependent transcriptional repressor Protect against polyglutamine toxicity HCNP may be involved in the function of the presynaptic cholinergic neurons of the central nervous system Belongs to the 3-hydroxyacyl-CoA dehydrogenase family Binds integral membrane proteins It is responsible for the transport of iron from sites of absorption and heme degradation to those of storage and utilization Function unknown The proteasome cleaves peptides with Arg, Phe, Tyr, Leu, and Glu adjacent to the leaving group at neutral or slightly basic pH Molecular chaperone; assist the folding of proteins upon ATP hydrolysis. Known to play a role, in vitro, in the folding of actin and tubulin Belongs to the PPase family This is a Bence-Jones protein This is a Bence-Jones protein Function unknown; bind selenium Cellular communication and signal transduction Vimentins are class-III intermediate filaments found in various nonepithelial cells, especially mesenchymal cells This protein promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis Nucleic acid synthesis and processing Single-stranded nucleic acid binding protein that binds preferentially to oligo dC Involved in oxygen transport The main protein of plasma

on the differences present at the protein level between astrocytomas of different grade. Journal of Proteome Research • Vol. 4, No. 3, 2005 703

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Figure 2. Enlarged images of the 2D gels highlighting the proteins increased in low-grade astrocytomas. Right: 2D polyacrylamide gels of low-grade astrocytomas. Left: 2D polyacrylamide gels of high-grade astrocytomas. The arrows indicate the position of the differentially expressed proteins. Spot numbers (from L1 to L6) indicate all the proteins identified by mass spectrometry and refer to the numbers reported in Table 2.

Conclusions Human brain astrocytomas are heterogeneous neoplasms originating from astrocytes, the most abundant of all glial cells, and represent the most common type of brain tumors.3,16 Given the extensive heterogeneity of astrocytic tumors, an accurate distinction between the malignant high-grade and the indolent low-grade astrocytomas on the basis of their microscopic appearance can be difficult, thus affecting prognosis estimation and therapeutic decisions. The proteomic study presented here describes the protein expression profiles of different grade astrocytomas. This analysis allowed the identification of differentially expressed proteins that could eventually serve as novel molecular makers for this class of tumors. Previous studies based on DNA microarray techniques highlighted a number of differentially expressed genes between normal brain and gliomas of different grades.8,12,17,18 In particular, Fuller and co-workers showed that the insulin-like growth factor binding protein 2 (IGFBP2) is consistently overexpressed in high-grade astrocytomas.17 Since this gene is normally expressed in fetal cells and is turned off in adult cells, this discovery suggested that the progression of gliomas could result from a block in differentiation. This finding was confirmed in later studies showing that additional genes involved in cell adhesion, motility, invasion, and angiogenesis are overexpressed in glioblastomas.8 Moreover, Rickman and coworkers found that genes involved in cell proliferation, migration, and inhibition of apoptosis are overexpressed in highgrade gliomas.12 The same authors discovered that some members of the cytoskeletal-associated proteins and the winged-helix family of transcription factors, such as FOXG1B and FOXM1, are also highly expressed in high-grade tumors. In the first proteomic study related to brain gliomas, Zhang and co-workers compared the protein expression profiles of 704

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human fetal astrocytes and human glioblastoma cell lines.13 They found four proteins highly expressed in glioblastoma cells, namely Hsp27, a major vault protein, brain-specific transglutaminase (TTG), and cystatin B. Western blot, ELISA, and RT-PCR analysis using brain tissue confirmed that these proteins were highly expressed in high-grade astrocytomas compared to low-grade tumors. Successively, Hiratsuka and coworkers compared the protein expression profiles of gliomas of different grade and nontumor tissues.15 In this work, the authors highlighted 11-up-regulated and 4-down-regulated proteins in gliomas relative to nontumor samples. On the other hand, information on the type of glioma was not provided in the text raising the question of whether some of the observed changes might be due to additional factors related to cell type variation. Among the down-regulated proteins, particular attention was dedicated to the cytoskeleton-related protein SIRT2 that is poorly expressed in gliomas. Ectopic expression of this protein in glioma cells leads to the perturbation of the microtubule network suggesting that SIRT2 may operate as a tumor suppressor gene through the regulation of this network.15 A more recent study highlighted 25 proteins that were differentially expressed between astrocytomas of different grade.14 Interestingly, eight of the proteins increased in high-grade gliomas were small G-proteins suggesting that aberrant Gprotein signaling might be involved in the malignant transformation of gliomas. Notwithstanding the important information that already emerged from these studies, much further investigation is still required to reach a more comprehensive knowledge of the proteins that are selectively up- or downregulated in gliomas of different grades. In the present work, we compared for the first time the protein expression profiles of low- and high-grade astrocytomas using 16 × 18 cm 2D-gels. The only previous comparative study

Proteomics of Human Brain Astrocytomas

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Figure 5. Western blot analysis. Western blot analysis of surgically removed perilesional tissues, fibrillary astrocytomas, and glioblastoma multiforme were performed using rabbit polyclonal antibodies against the catalytic subunit of the cAMP-dependent protein kinase (PKA), peroxiredoxin 1 (PRX1), R-internexn, and the transcription factor BTF3; mouse monoclonal antibodies against protein disulfide isomerase A3 (PDI), peroxiredoxin 6, and R-tubulin, and goat polyclonal antibody against ubiquitin carboxyl-terminal hydrolase (UCHL1). R-Tubulin was used as an internal control to guarantee that the amount of proteins loaded on each well was the same.

Figure 3. Enlarged images of the 2D gels highlighting the proteins increased in high-grade astrocytomas. Right: 2D polyacrylamide gels of low-grade astrocytomas. Left: 2D polyacrylamide gels of high-grade astrocytomas. The arrows indicate the position of the differentially expressed proteins. Spot numbers (from H1 to H9) indicate all the proteins identified by mass spectrometry and refer to the numbers reported in Table 2.

Figure 4. Distribution of all 70 identified proteins conserved in low- and high-grade astrocytomas according to their general function.

on low- and high-grade gliomas was performed with small 2D gels (7 × 8 cm) raising the possibility that some of the differences in protein pattern might be lost.14 It is important to remark that, on the basis of their microscopic appearance, we only selected astrocytomas of different grade and not gliomas originating from other glial cells, such as oligodendriomas. In addition, among the low- and high-grade astrocytomas we only selected those that were classified, respectively, as fibrillary astrocytomas (grade II) and glioblastoma

multiforme (grade IV) on the basis of the histopathological analysis. Thus, eventual variations in protein expression should be entirely related to the different grade of the tumor. Our results show that most protein spots are conserved among the different grade tumors and only a limited number of differences are clearly visible. All the differentially expressed proteins as well as 70 of the conserved spots were identified by mass spectrometry (Table 1). Among the conserved proteins, we found metabolism related proteins (38%), structural proteins (10%), brain specific proteins (7%), stress related proteins (16%), oncoproteins (3%), and proteins involved in other cellular functions (26%) (Figure 4). This information provides a preliminary description of the proteins consistently present in the human brain astrocytomas and represents the first step toward the creation of a glioma protein database. Among the differentially expressed proteins, we identified 6 proteins that are more represented in low-grade astrocytomas and 9 in glioblastoma multiforme (Table 2) (Figure 7). Peroxiredoxin 1 and 6 are two proteins whose corresponding spots are significantly stronger in the 2D gels of the high-grade tumors. The higher expression of peroxiredoxin 1 in glioblastoma multiforme relative to low-grade astrocytomas was confirmed by Western and immunohistochemical analysis. Moreover, Western analysis showed that also peroxiredoxin 6 is overexpressed in glioblastoma multiforme and that both peroxiredoxin’s variants are barely detectable in perilesional tissues indicating that these proteins are up-regulated in the high-grade tumors compared to normal tissues. Peroxiredoxins are a recently characterized family of proteins that regulate the intracellular concentration of peroxides, which mediate signal transduction in mammalian cells.19 These two proteins have not been previously described as overexpressed in glioblastoma multifome, although earlier studies have already suggested their potential role in cancer development.20-22 In particular, recent studies indicated that peroxiredoxin 1 and 6 are overexpressed in lung cancer, breast carcinoma and malignant mesothelioma.23-26 In addition, Yanagawa and co-workers suggested that peroxiredoxin 1 might serve as a novel tumor marker to Journal of Proteome Research • Vol. 4, No. 3, 2005 705

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Table 2. Proteins Differentially Expressed between Low- and High-Grade Astrocytomas

no.

protein

Swiss-Prot or NCBI entry

Mr(kDa)/pI

sequence coverage (%)

L1

Glial fibrillary acidic protein, astrocyte

P14136

Low-grade 50/5.42

44

L2

P48643

60/5.45

32

L3

T-complex protein 1,  subunit Protein disulfide isomerase A3

P30101

57/5.98

19

L4

cAMP-dependent protein kinase

P17612

40.6/8.84

15

L5

Dihydropteridine reductase

P09417

25.6/6.90

23

L6

Ubiquitin carboxyl-terminal hydrolase isozyme L1

P09936

24.8/5.43

36

H1

R-internexin

Q16352

High-grade 55.5/5.34

26

H2

Actin, cytoplasmic 1

P02570

42/5.22

15

H3

Fibrinogen fragment D

2781208

38.0/5.84

11

H4 H5

Peroxiredoxin 1 Apolipoprotein A-I

Q06830 P02647

22/8.27 30.7/5.56

34 40

H6 H7

Peroxiredoxin 6 Annexin A5

P30041 P08758

24.9/6.02 38.5/6.64

58 22

H8

Transcription factor BTF3 homologue 1 R-B-crystallin

Q13890

12.5/9.7

12

P02511

20/6.8

34

H9

discriminate between tissue types of tumors on the basis of the observation that this protein is overexpressed in follicular neoplasms, while its expression level remains normal in papillary carcinomas.27 Our results add more evidences to the proposed relationship between peroxiredoxins and tumor progression, and indicate that these proteins could be used as novel markers for glioblastoma multifome. The transcription factor BTF3 is another interesting protein that we can only detect in the high-grade tumors. Previous studies have shown that the expression level of BTF3 is significantly decreased in apoptotic human Burkitt lymphoma cells relative to nonapoptotic cells suggesting that this factor is a potential candidate for the control of apoptosis in B-lymphocytes.28 The overexpression of apoptotic inhibitors in glioblastoma multiforme, such as thioredoxin and surviving (apoptotic inhibitor 4), has already been reported in the literature indicating that these proteins are essential factors for the hyperproliferation of glioblastoma cells as previously observed for other type of cancers.12 Among the remaining proteins that are more abundant in glioblastoma multiforme, R-B-crystallin has already been described by other groups as up-regulated in the highgrade astrocytomas. R-B-crystallin is heat-shock protein, previ706

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function

During the development of the central nervous system, distinguishes astrocytes from other glial cells Molecular chaperone; assist the folding of proteins upon ATP hydrolysis Protein synthesis processing and protein fate Phosphorylates a large number of substrates in the cytoplasm and the nucleus The product of this enzyme, tetrahydrobiopterin (BH-4), is an essential cofactor for phenylalanine, yrosine, and tryptophan hydroxylases Ubiquitin-protein hydrolase is involved both in the processing of ubiquitin precursors and of ubiquinated proteins It is involved in the morphogenesis of neurons Are involved in various types of cell motility and are ubiquitously expressed in all eukaryotic cells Fibrinogen has a double function: yielding monomers that polymerize into fibrin and acting as a cofactor in platelet aggregation Involved in redox regulation of the cell Participates in the reverse transport of cholesterol Involved in redox regulation of the cell Acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade Nucleic acid synthesis and processing Member of the small heat shock protein family. Acts as molecular chaperones and possess autokinase activity

ously shown to be overexpressed in individuals suffering from Alexander and Parkinson’s disease.29,30 In agreement with our results, Western blot analysis showed that R-B-crystallin is highly expressed in astrocytic tumors and is preferentially found in the most aggressive stages of gliomas.11 Similarly, three of the six proteins that are overexpressed in low-grade astrocytomas relative to glioblastoma multiforme were already discovered by other groups: protein disulfide isomerase A3, the catalytic subunit of the cAMP-dependent protein kinase, and the glial fibrillary acidic protein. The first protein is a molecular chaperone that catalyzes disulfide formation and that was found by Iwadate and co-workers to be more highly expressed in low-grade astrocytomas relative to glioblastoma multiforme.14 The catalytic subunit of the cAMP-dependent protein kinase is responsible for the phosphorylation of a large number of proteins in the cytoplasm and in the nucleus. Rickman and co-workers identified the gene encoding for the catalytic subunit of the cAMP-dependent protein kinase among those expressed at higher levels in low-grade gliomas compared to grade IV gliomas suggesting that, in agreement with our findings, this protein is overexpressed in the low-grade tumors.12 The third protein, the glial fibrillary acidic protein

Proteomics of Human Brain Astrocytomas

Figure 6. Immunohystochemical analysis of low- and high-grade tumors. Expression of glial fibrillary acidic protein (GFAP), protein disulfide isomerase (PDI), the catalytic subunit of the cAMPdependent protein kinase (PKA), peroxiredoxin 1 (PRX1), and R-internexin in the paraffin-embedded section of 10 tumors samples: 5 high-grade glioblastoma multiforme and 5 low-grade fibrillary astrocytomas. (A) GFAP was mainly detected in the cytoplasm of low-grade tumors, while it was weakly expressesed in high-grade tumors. PDI protein was detected mainly in the nuclei of low-grade tumors and was barely detected in highgrade tumors. PKA was mainly detected in low-grade astrocytomas, while we observed a modest level of expression in highgrade tumors. (B) PRX1 and R-internexin were expressed in the cytoplasm of high-grade tumors but were scarcely detected in low-grade tumors.

(GFAP), is a cell-specific marker that, during the development of the central nervous system, distinguishes astrocytes from other glial cells. Mutations in the gene encoding for GFAP are associated with the Alexander disease, a rare disorder of the central nervous system.31 Several studies reported a decreased expression level of GFAP in high-grade gliomas compared to the low-grade astrocytomas indicating that this protein could be a potential specific marker for the low-grade tumors.13,14,32-34 This conclusion is supported by our immunohistochemical results that show a marked difference between the paraffinembedded tissues of the low- and high-grade astrocytomas stained with the GFAP antibody. The other three proteins more highly expressed in the low-grade astrocytomas are the Tcomplex polypeptide 1, another molecular chaperone, the ubiquitin carboxyl-terminal hydrolase, an enzyme responsible

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Figure 7. Magnetic Resonance Imaging (MRI) of low- and highgrade astrocytomas. Post-contrast T1-weighted MR images in sagittal planes of brains showing the localization of low-grade (bottom) and high-grade (top) astrocytomas. The 9 proteins more abundant in glioblastoma multiforme and the 6 proteins more represented in low-grade astrocytomas are indicated.

for the processing of ubiquitin precursors and of ubiquinated proteins, and dihydropteridine reductase, an enzyme involved in the production of tetrahydrobiopterin that is an essential cofactor for phenylalanine, tyrosine, and tryptophan hydroxylases. These proteins have not been previously described as differentially expressed between the gliomas of different grade and thus provide novel information on the differences existing between low- and high-grade astrocytomas. In summary, our work offers an initial description of the proteins conserved in low- and high-grade astrocytomas as well as novel information on proteins that are differentially expressed between the gliomas of diverse histological grade. The information on the identity of the fifteen differentially expressed proteins can be used for the development of novel markers that are highly required for an appropriate classification of astrocytomas. Further studies will be focused on the systematic Journal of Proteome Research • Vol. 4, No. 3, 2005 707

research articles analysis of astrocytomas of the same grade to highlight differences at the protein level that could be related to the different outcome of the patients during the follow-up period providing crucial information for a correct prediction of prognosis and treatment response of patients.

Acknowledgment. We thank Youhna Mary Ayala for valuable discussion and for her comments on the manuscript. Federico Moretti and Milena Sinigaglia are acknowledged for help with the immunohistochemical analysis. The work was supported by a grant from the Human Frontier Science Program, by a FIRB Grant of MIUR (Ministero dell’Istruzione dell’Universita’ e della Ricerca), and by Grant No. 02.00648.ST97 of Consiglio Nazionale delle Ricerche, Rome. References (1) Burt, A. M. Textbook of Neuroanatomy, 1st ed.; Elsevier Science: New York, 1993. (2) Bear, M. F.; Connors, B. W.; Paradiso, M. A. Neuroscience: Exploring the Brain; Lippincott Williams & Wilkins: Baltimore, 2003. (3) Legler, J. M.; Gloeckler Ries, L. A.; Smith, M. A.; Warren, J. L.; Heineman, E. F.; Kaplan, R. S.; Linet, M. S. J. Natl. Cancer. Inst. 1999, 91, 2050A-22051. (4) Kleihues, P.; Louis, D. N.; Scheithauer, B. W.; Rorke, L. B.; Reifenberger, G.; Burger, P. C.; Cavenee, W. K. J. Neuropathol. Exp. Neurol. 2002, 61, 215-225; discussion 226-219. (5) Maher, E. A.; Furnari, F. B.; Bachoo, R. M.; Rowitch, D. H.; Louis, D. N.; Cavenee, W. K.; DePinho, R. A. Genes Dev. 2001, 15, 13111333. (6) Benjamin, R.; Capparella, J.; Brown, A. Cancer J. 2003, 9, 82-90. (7) Kitange, G. J.; Templeton, K. L.; Jenkins, R. B. Curr. Opin. Oncol. 2003, 15, 197-203. (8) Sallinen, S. L.; Sallinen, P. K.; Haapasalo, H. K.; Helin, H. J.; Helen, P. T.; Schraml, P.; Kallioniemi, O. P.; Kononen, J. Cancer Res. 2000, 60, 6617-6622. (9) Rao, J. S. Nat. Rev. Cancer 2003, 3, 489-501. (10) Mortz, E.; Krogh, T. N.; Vorum, H.; Gorg, A. Proteomics 2001, 1, 1359-1363. (11) Aoyama, A.; Steiger, R. H.; Frohli, E.; Schafer, R.; von Deimling, A.; Wiestler, O. D.; Klemenz, R. Int. J. Cancer 1993, 55, 760-764. (12) Rickman, D. S.; Bobek, M. P.; Misek, D. E.; Kuick, R.; Blaivas, M.; Kurnit, D. M.; Taylor, J.; Hanash, S. M. Cancer Res. 2001, 61, 6885-6891. (13) Zhang, R.; Tremblay, T. L.; McDermid, A.; Thibault, P.; Stanimirovic, D. Glia 2003, 42, 194-208.

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Odreman et al. (14) Iwadate, Y.; Sakaida, T.; Hiwasa, T.; Nagai, Y.; Ishikura, H.; Takiguchi, M.; Yamaura, A. Cancer Res. 2004, 64, 2496-2501. (15) Hiratsuka, M.; Inoue, T.; Toda, T.; Kimura, N.; Shirayoshi, Y.; Kamitani, H.; Watanabe, T.; Ohama, E.; Tahimic, C. G.; Kurimasa, A.; Oshimura, M. Biochem. Biophys. Res. Commun. 2003, 309, 558-566. (16) Behin, A.; Hoang-Xuan, K.; Carpentier, A. F.; Delattre, J. Y. Lancet 2003, 361, 323-331. (17) Fuller, G. N.; Rhee, C. H.; Hess, K. R.; Caskey, L. S.; Wang, R.; Bruner, J. M.; Yung, W. K.; Zhang, W. Cancer Res. 1999, 59, 42284232. (18) Huang, H.; Colella, S.; Kurrer, M.; Yonekawa, Y.; Kleihues, P.; Ohgaki, H. Cancer Res. 2000, 60, 6868-6874. (19) Wood, Z. A.; Schroder, E.; Robin Harris, J.; Poole, L. B. Trends Biochem. Sci. 2003, 28, 32-40. (20) Chen, W. C.; McBride, W. H.; Iwamoto, K. S.; Barber, C. L.; Wang, C. C.; Oh, Y. T.; Liao, Y. P.; Hong, J. H.; de Vellis, J.; Shau, H. J. Neurosci. Res. 2002, 70, 794-798. (21) Kim, H. J.; Chae, H. Z.; Kim, Y. J.; Kim, Y. H.; Hwangs, T. S.; Park, E. M.; Park, Y. M. Cell Biol. Toxicol. 2003, 19, 285-298. (22) Kinnula, V. L.; Paakko, P.; Soini, Y. FEBS Lett. 2004, 569, 1-6. (23) Lehtonen, S. T.; Svensk, A. M.; Soini, Y.; Paakko, P.; Hirvikoski, P.; Kang, S. W.; Saily, M.; Kinnula, V. L. Int. J. Cancer 2004, 111, 514-521. (24) Karihtala, P.; Mantyniemi, A.; Kang, S. W.; Kinnula, V. L.; Soini, Y. Clin Cancer Res. 2003, 9, 3418-3424. (25) Noh, D. Y.; Ahn, S. J.; Lee, R. A.; Kim, S. W.; Park, I. A.; Chae, H. Z. Anticancer Res. 2001, 21, 2085-2090. (26) Kinnula, V. L.; Lehtonen, S.; Sormunen, R.; Kaarteenaho-Wiik, R.; Kang, S. W.; Rhee, S. G.; Soini, Y. J. Pathol. 2002, 196, 316323. (27) Yanagawa, T.; Ishikawa, T.; Ishii, T.; Tabuchi, K.; Iwasa, S.; Bannai, S.; Omura, K.; Suzuki, H.; Yoshida, H. Cancer Lett. 1999, 145, 127132. (28) Brockstedt, E.; Otto, A.; Rickers, A.; Bommert, K.; WittmannLiebold, B. J. Protein Chem. 1999, 18, 225-231. (29) Head, M. W.; Corbin, E.; Goldman, J. E. Am. J. Pathol. 1993, 143, 1743-1753. (30) Renkawek, K.; Stege, G. J.; Bosman, G. J. Neuroreport 1999, 10, 2273-2276. (31) Brenner, M.; Johnson, A. B.; Boespflug-Tanguy, O.; Rodriguez, D.; Goldman, J. E.; Messing, A. Nat. Genet. 2001, 27, 117-120. (32) Rutka, J. T.; Murakami, M.; Dirks, P. B.; Hubbard, S. L.; Becker, L. E.; Fukuyama, K.; Jung, S.; Tsugu, A.; Matsuzawa, K. J. Neurosurg. 1997, 87, 420-430. (33) Toda, M.; Shirao, T.; Uyemura, K. Brain Res. Dev. Brain Res. 1999, 114, 193-200. (34) Peraud, A.; Mondal, S.; Hawkins, C.; Mastronardi, M.; Bailey, K.; Rutka, J. T. Brain Tumor Pathol. 2003, 20, 53-58.

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