Overexpression and Nucleolar Localization of F

J Neuropathol Exp Neurol Copyright ! 2015 by the American Association of Neuropathologists, Inc.

Vol. 74, No. 7 July 2015 pp. 723Y742

ORIGINAL ARTICLE

Overexpression and Nucleolar Localization of F-Tubulin Small Complex Proteins GCP2 and GCP3 in Glioblastoma Eduarda Dra´berova´, PhD, Luca D_Agostino, PhD, Valentina Caracciolo, PhD, Vladimı´ra Sla´dkova´, MSc, Tetyana Sulimenko, MSc, Vadym Sulimenko, PhD, Margaryta Sobol, PhD, Nicoletta F. Maounis, MD, PhD, Elias Tzelepis, MD, Eleni Mahera, MD, PhD, Leox Kren, MD, PhD, Agustin Legido, MD, PhD, Antonio Giordano, MD, PhD, Sverre Mo¨rk, MD, PhD, Pavel Hoza´k, DSc, Pavel Dra´ber, PhD, and Christos D. Katsetos, MD, PhD, FRCPath, FRCP Edin

Abstract The expression, cellular distribution, and subcellular sorting of the microtubule (MT)-nucleating F-tubulin small complex (FTuSC) proteins, GCP2 and GCP3, were studied in human glioblastoma cell lines and in clinical tissue samples representing all histologic grades of adult diffuse astrocytic gliomas (n = 54). Quantitative real-time polymerase chain reaction revealed a significant increase in the expression of GCP2 and GCP3 transcripts in glioblastoma

From the Departments of Biology of Cytoskeleton (ED, VS, TS, VS, PD) and Biology of the Nucleus (MS, PH), Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Department of Pediatrics (LD, ET, AL, CDK), Drexel University College of Medicine, Section of Pediatric Neurology and Neurooncology Program (AL, CDK), St. Christopher’s Hospital for Children, and Department of Pathology and Laboratory Medicine (NFM, CDK) Drexel University College of Medicine, Philadelphia, Pennsylvania; Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania (VC, AG); Department of Clinical Cytology, Sismanoglion General Hospital, Athens, Greece (NFM); Department of Pathology, KAT General Hospital, Athens, Greece (EM); Department of Pathology, Faculty Hospital Brno, Brno, Czech Republic (LK); and Gades Institute, Department of Pathology, University of Bergen, Haukeland Hospital, Bergen, Norway (SM). Send correspondence and reprint requests to: Christos D. Katsetos, MD, PhD, FRCPath, FRCP Edin, Department of Pediatrics, Section of Neurology, St. Christopher’s Hospital for Children,160 East Erie Ave., Philadelphia, PA 19134; E-mail: [email protected]; or Pavel Dra´ber, PhD, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vı´defska´ 1083, 14220 Prague 4, Czech Republic; E-mail: [email protected] Eduarda Dra´berova´ and Christos D. Katsetos contributed equally to this work. This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant no LH12050 to PD); the Ministry of Health of the Czech Republic (grant no NT14467 to LK, VS, PD); the Grant Agency of the Czech Republic (grant no P302/12/1673 to VS, TS, PD); the Academy of Sciences of the Czech Republic (grant no M200521203PIPP to PD); the Technology Agency of the Czech Republic (grant no TE01020118 to MS, PH); the Ministry of Industry and Trade of the Czech Republic (grant no FR-TI3/588 to PH); Institutional Research Support (grant no RVO 68378050 to PD, PH); and the Philadelphia Health Education Corporation (PHEC) and St. Christopher’s Hospital for Children Reunified Endowment (grant no 323256 to CDK). Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.jneuropath.com).

cells versus normal human astrocytes; these were associated with higher amounts of both FTuSC proteins. GCP2 and GCP3 were concentrated in the centrosomes in interphase glioblastoma cells, but punctate and diffuse localizations were also detected in the cytosol and nuclei/nucleoli. Nucleolar localization was fixation dependent. GCP2 and GCP3 formed complexes with F-tubulin in the nucleoli as confirmed by reciprocal immunoprecipitation experiments and immunoelectron microscopy. GCP2 and GCP3 depletion caused accumulation of cells in G2/M and mitotic delay but did not affect nucleolar integrity. Overexpression of GCP2 antagonized the inhibitory effect of the CDK5 regulatory subunit-associated tumor suppressor protein 3 (C53) on DNA damage G2/M checkpoint activity. Tumor cell GCP2 and GCP3 immunoreactivity was significantly increased over that in normal brains in glioblastoma samples; it was also associated with microvascular proliferation. These findings suggest that FTuSC protein dysregulation in glioblastomas may be linked to altered transcriptional checkpoint activity or interaction with signaling pathways associated with a malignant phenotype. Key Words: Gamma-tubulin, Gamma-tubulin complex proteins, GCP2, GCP3, Glioma, Glioblastoma, Nucleolus.

INTRODUCTION Gliomas account for the majority of brain tumors in children and adults. Histologically, these tumors are broadly divided into low-grade (World Health Organization [WHO] grades I and II) and high-grade gliomas (WHO grades III and IV), including anaplastic astrocytomas (WHO grade III) and glioblastomas (WHO grade IV). Diffuse gliomas (WHO grades IIYIV) are characterized by infiltrative patterns of growth and spread within the CNS parenchyma. A subset of these tumors may start as ‘‘low-grade’’ tumors (WHO grade II) with potential for anaplastic change (WHO grades III/IV), whereas others may arise as glioblastomas de novo (1). A plethora of studies have focused on stratifying brain tumors by molecular subtype depending on the presence of specific mutations (2, 3). This approach has gained clinical significance both in gliomas and medulloblastomas (3). However, despite significant strides in the genomics and epigenomics of brain tumors, there is no efficacious treatment for diffuse gliomas, especially for glioblastoma.

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There is currently urgent need for new treatment strategies to combat the dire prognosis of these tumors. Microtubules (MTs), assembled from >A-tubulin heterodimers, play a decisive role in many cellular functions such as intracellular transport, cell organization, and cell division (4). Tubulin is the target of some of the most widely used and time-honored anticancer drugs, including vinca alkaloids, colchicine, taxanes, and epothilones (5Y7). Although abnormalities in the organization of MTs constitute a common mechanism of genetic instability in cancer cells, the molecular mechanisms underlying such changes remain unknown. Our approach to MT dysregulation in cancer cells takes into consideration potential changes involving a host of MT regulatory proteins, including MT-nucleating and severing proteins (4). One of the key components required for MT nucleation and stabilization is F-tubulin (8), a minor member of the tubulin superfamily, concentrated in interphase cells at the pericentriolar material of centrosomes, the conventional MT-organizing centers (MTOC) (9). Nucleation-competent F-tubulin forms complexes with other proteins, which are embedded in the MTOC matrix. The human F-tubulin small complex (FTuSC) comprises 2 molecules of F-tubulin and 1 molecule each of F-tubulin complex proteins GCP 2 and GCP 3 (10). Large F-tubulin ring complexes (FTuRCs) are formed by FTuSCs together with other cytoplasmic complex proteins, including GCPs 4, 5, and 6 (11). In addition to MTOC-derived MT nucleation, FTuRCs are also involved in the regulation of MT minus-end dynamics (12) and in spindle assembly checkpoint signaling (13). In previous studies, we and others have demonstrated that F-tubulin is associated with cellular membranes (14, 15), where it can participate in noncentrosomal MT nucleation (14, 16, 17). Aside from MTOCs, cell membranes, and cytosol, F-tubulin is also present in the nucleus and nucleolus where it modulates the activity of the cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated tumor suppressor protein 3 (C53) (18). Moreover, nuclear F-tubulin affects E2F transcriptional activity (19). In the context of brain tumors, we and others have previously demonstrated that F-tubulin is overexpressed in glioblastomas (20Y24) and in medulloblastomas (25). A recent study has suggested that F-tubulin may be a prognostic indicator in patients with astrocytic gliomas as determined by a significant relationship between high levels of expression of this protein in surgically excised tumor specimens and high tumor grade, poor patient performance status after resection, and a poorer overall survival status (24). To gain a deeper insight into the expression and function of proteins involved in MT nucleation in glioma cells, we examined the expression, cellular distribution, and subcellular sorting of FTuSC proteins, GCP2 and GCP3, in several human glioblastoma cell lines and in surgically excised clinical samples of diffuse astrocytic gliomas corresponding to all histologic grades (WHO grades IIYIV). We report, for the first time, evidence of increased expression of GCP2 and GCP3 coupled with nuclear and nucleolar compartmentalization of these proteins in glioblastoma cells. Our observations lend credence to the hypothesis that proteins involved in MT nucleation are dysregulated in neoplastic glial cells and also shed a new light on divergent roles of FTuSC proteins in cancer cells.

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MATERIALS AND METHODS Cell Cultures Human glioblastoma cell lines T98G, U87MG, U118MG, and U138MG; human osteogenic sarcoma cell line U2OS; and human neuroblastoma cell line SH-SY5Y were obtained from the American Type Culture Collection (Manassas, VA). Pediatric glioma cell line KNS-42 was obtained from JCRB Cell Bank (Ibaraki, Japan) (26). Human kidney embryonal cells HEK293FT (HEK) were from Promega Biotec (Madison, WI). Mouse bone marrowYderived mast cell line (BMMCL) was kindly provided by M. Hibbs (Ludwig Institute for Cancer Research, Melbourne, Australia) (27), and immortalized human retinal pigment epithelial cells hTERT-RPE1 (RPE1) were from Dr M. Bonhivers (Universite´ Bordeaux, Bordeaux, France). Preparation of U2OS cells stably expressing C53-enhanced green fluorescent protein (EGFP) was performed as previously described (18). All cells, except BMMCL, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (0.1 mg/mL). In the case of RPE1 cells, medium was supplemented with hygromycin B (0.01 mg/mL). BMMCL were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with serum, antibiotics, and interleukin 3, as previously described (28). Cells were grown at 37-C in 5% CO2 in air and passaged every 2 or 3 days using 0.25% trypsin/0.01% ethylenediaminetetraacetic acid (EDTA) in phosphate buffered saline (PBS), pH 7.5. Proliferating nonimmortalized, nontransformed normal human fetal astrocytes (NHA; Clonetics Astrocyte Cell Systems), were from Cambrex Bio Science (Walkersville, MD) and were maintained as described (29). To induce genotoxic stress, cells were incubated for 3 hours in 20 Kmol/L etoposide (Sigma-Aldrich, St. Louis, MO) in serum-free medium, and after washing in PBS, cells were incubated for 17 hours in medium with serum.

Antibodies For the detection of GCP2, a rabbit antibody from GeneTex (Irvine, CA; Catalog No. GTX102281), and mouse monoclonal antibodies (mAbs) GCP2-01 and GCP2-02 were used (see below under ‘‘Results’’ for preparation and characterization of anti-GCP2 mAbs). For the detection of GCP3, rabbit antibody from GeneTex (Catalog. No. GTX87444), rabbit antibody from Proteintech (Manchester, UK; Catalog No. 15719-1-AP), and mAb C-3 (IgG1) from Santa Cruz Biotechnology (Santa Cruz, CA; Catalog No. sc-373758) were used. MAb TU-01 (IgG1) (30, 31) and rabbit antibody (GeneTex; Catalog No. GTX15246) were used for the detection of >-tubulin. Mouse mAbs TU-31 (IgG2b), TU-32 (IgG1) (32), and GTU88 (IgG1) from Sigma-Aldrich (Catalog No. T5326) and rabbit antibody DQ-19 (Sigma-Aldrich; Catalog No. T3195) were used for the detection of F-tubulin. Mouse mAb to nucleophosmin (IgG1; Catalog No. WH0004869M1) and rabbit antibodies to actin (Catalog No. A2066), nucleolin (Catalog No. 2662), green fluorescent protein (GFP, Catalog No. G1544), and FLAG (Catalog No. F7425) were obtained from SigmaAldrich. Rabbit antibody to cyclophilin A was obtained from Upstate Biotechnology (Lake Placid, NY; Catalog No. 07Y313), rabbit antibody to cyclin B1 from Cell Signaling (Danvers, MA; ! 2015 American Association of Neuropathologists, Inc.



Catalog No. 4138), and mAb to Cdk1-p-Y15 from Santa Cruz (IgG1; Catalog No. sc-136014). Mouse mAb to growth factor receptor-bound protein 2 (Grb2) was obtained from BD Transduction Laboratories (Lexington, KY; IgG1; Catalog No. 610112). Rabbit antibody to ATM-p-S1981 (Catalog. No. ab8192) and mAb to H2AX-p-S139 (IgG2b; Catalog No. ab18311) were obtained from Abcam (Cambridge, UK). Mouse mAb NF-09 (IgG2a) to neurofilament protein NF-M (33) and mAb HTF14 (IgG1) to human transferrin (34) were used as negative controls. The DY549-conjugated anti-mouse (Catalog No. 115505-146) and DyLight 549-conjugated anti-mouse (Catalog No. DI-2549) antibodies were obtained from Jackson Immunoresearch Laboratories (West Grove, PA) and Vector Laboratories, Inc. (Burlingame, CA), respectively. DY488-conjugated antirabbit (Catalog. No. 111-485-144) and DyLight 488Yconjugated anti-rabbit (Catalog No. DI-1488) antibodies were obtained from Jackson Immunoresearch Laboratories and Vector Laboratories, Inc., respectively. Horseradish peroxidase-conjugated anti-mouse (Catalog. No. W402B) and anti-rabbit (Catalog. No. W401B) antibodies were obtained from Promega. Twelvenanometer gold-conjugated anti-rabbit (Catalog No. 111-205-144) and 6-nm gold-conjugated anti-mouse (Catalog. No. 115-195068) antibodies were obtained from Jackson Immunoresearch Laboratories. For the preparation of mAbs specific for GCP2, glutathione transferase (GST)Ytagged fragment coding mouse GCP2 (SwissProt accession No. Q921G8) polypeptide (a.a. 1-194) was used as immunogen. GST-tagged fragment was prepared as described (17). Briefly, the fragment encoding mouse GCP2 (GenBank nucleotide sequence database accession number NM_133755) was amplified by polymerase chain reaction (PCR) using forward (5¶-AGTCGGATCCAGCGAATTTCGGATTCAC-3¶) (BamHI restriction site is underlined) and reverse (5¶-AAGC GTCGACTCAGCCAATCAGGAAATCTC-3¶) (Sal restriction site is underlined) primers, with total cDNA from mouse brain used as template. The isolated fragment was ligated into pGEX-6P-1 (Amersham Biosciences, Uppsala, Sweden) for preparation of GST-tagged fusion protein. The procedures pertaining to immunization of F1(B10A " BALBc) mice, fusion with mouse myeloma cells Sp2/0, screening by ELISA and immunofluorescence, as well as cloning have been described (30, 35). Class and subclass of mAbs were determined using IsoStrip (Roche Diagnostics, Basel, Switzerland).

DNA Constructs C-terminally FLAG-tagged human F-tubulin and Cterminally FLAG-tagged mouse protein tyrosine kinase p59Fyn (Fyn) constructs were previously described (18). C-terminally FLAG-tagged human GCP2 construct was commercially obtained from Origene Technologies (Rockville, MD; Catalog No. RC210833). Cells were transfected with DNA using Lipofectamine LTX (Thermo Fisher Scientific, Waltham, MA), as described (18).

Reverse Transcription Quantitative Real-Time PCR Total cellular RNA was extracted in 3 independent isolations from human astrocytes and from U87MG, T98G, U118MG, U138MG, and KNS-42 human glioblastoma cell lines using RNeasy Mini kit (Qiagen, Valencia, CA), according

F-Tubulin complex proteins in glioblastoma

to the manufacturer’s protocol. The quality of RNA was checked on 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). All RNA samples were of good quantitative PCR (qPCR) quality (RNA integrity number Q9.0 for all samples). Purified RNA was stored at j70-C. RNA from each sample was converted to cDNA using the SuperScript VILO cDNA Synthesis Kit with random primers (Thermo Fisher Scientific) according to the manufacturer’s protocol; each reaction sample (20 KL) contained 1 Kg RNA. Quantitative PCR was performed with gene-specific primers for human F-tubulin complex-associated protein 2 (TUBGCP2, NCBI Ref. Seq.: NM_001256617.1, NM_001256618.1, NM_006659.3; primers anneal to all transcript variants), Ftubulin complex-associated protein 3 (TUBGCP3, NCBI Ref. Seq.: NM_006322.4), for human F-tubulin 1 (TUBG1, NCBI Ref. Seq.: NM_001070.4) and A-actin (ACTB, NCBI Ref. Seq.: NM_001101.3). All primers were tested in silico by NCBI BLAST to amplify specific targets. Primer sequences are summarized in Table, Supplemental Digital Content 1, http://links.lww.com/NEN/A752. Oligonucleotides were from Sigma-Aldrich. Quantitative PCRs were carried out on LightCycler 480 System (Roche). Each reaction (5 KL) consisted of 2.5 KL of LightCycler 480 SYBR Green I Master (Roche), 0.5 KL of mixed gene-specific forward and reverse primers (5 Kmol/L of each), and 2 KL of diluted cDNA. cDNA samples were diluted 1:40. Calibration curves for tested genes were made by serial dilutions (dilution factor 3) of brain cDNA. Each sample was run in triplicate. Thermocycling parameters are described in Text, Supplemental Digital Content 2, http://links.lww.com/NEN/A753. The identity of PCR products was verified by sequencing. Statistical analysis was performed with the Student 2-tailed unpaired t-test using Microsoft Excel.

Cell Fractionation Nucleoli were prepared from T98G and U87MG cells at 4-C according to the procedure originally described by Andersen et al (36) and modified by Horejxı´ et al (18). Briefly, washed cells were resuspended in buffer A (36), supplemented with protease and phosphatase inhibitors, disrupted in Dounce homogenizer, and centrifuged at 228 g for 5 minutes. Supernatant represented the cytosolic fraction. The pellet was resuspended in 10 mmol/L of MgCl2 in 0.25 mol/L of sucrose and layered over 0.35 mol/L of sucrose containing 0.5 mmol/L of MgCl2. After centrifugation at 1430 g for 5 minutes, the pellet (nuclear fraction) was resuspended in 0.35 mol/L of sucrose containing 0.5 mmol/L of MgCl2 and sonicated. The sonicated sample was layered over 0.88 mol/L of sucrose containing 0.5 mmol/L of MgCl2 and centrifuged at 2800 g for 10 minutes. Supernatant represents the nucleoplasmic fraction. The pellet was resuspended in 0.5 mL of 0.35 mol/L sucrose containing 0.5 mmol/L of MgCl2 and centrifuged at 2000 g for 2 minutes to obtain highly purified nucleoli. For immunoprecipitation experiments or gel filtration chromatography, nucleoli were extracted by radio-immunoprecipitation assay (RIPA) buffer (50 mmol/L of Tris pH 8.0, 150 mmol/L of NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with protease and phosphatase inhibitors.

! 2015 American Association of Neuropathologists, Inc.


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Whole-cell extracts for SDS-PAGE were prepared by rinsing the cells twice in Hepes buffer (50 mmol/L of Hepes adjusted to pH 7.6 with NaOH, 75 mmol/L of NaCl, 1 mmol/L of MgCl2, and 1 mmol/L of ethylene glycol tetraacetic acid), scraping them into Hepes buffer supplemented with protease (Roche; Complete EDTA-free protease mixture) and phosphatase (1 mmol/L of Na3VO4 and 1 mmol/L of NaF) inhibitors, and solubilizing in hot SDS-sample buffer (37) without bromphenol blue and boiling for 5 minutes. Low-speed supernatant of brain extracts were prepared as previously described (38). Protein quantifications in lysates and SDS-PAGE samples were performed, respectively, with Pierce BCA Proteins assay kit (Pierce, Rockford, IL) and silver dot assay (39), using bovine serum albumin as standard.

RNA Silencing T98G or U87MG glioblastoma cells in 24-well plates were transfected with siRNAs (final concentration 10 nmol/L) using Lipofectamine RNAi MAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Two siRNAs (Applied Biosystems/Ambion, Prague, Czech Republic) that target regions present in all human GCP2 isoforms (Cat. No. S21286, siRNA#1, 5¶-GGCUUGACUUCAAUGGUUU-3¶ Cat. No. S21285, siRNA#2, 5¶-GAAAGAUGCUUCGAGACAA-3¶) and 2 siRNAs (Ambion) that target regions present in all human GCP3 isoforms (Cat. No. S20394, siRNA#1, 5¶-GAGUUGGGAUG GUUGCAUA-3¶ Cat. No. S20395, siRNA#2, 5¶-GGACUUG CUAAAACCAGAA-3¶) were used. The siRNA (Ambion) that efficiently targets the region present in human F-tubulin 1 (5¶-CGCA UCUCUUU CUCAUAUA-3¶, siRNA ID #120194) was previously described (40). dTdT overhangs were added to the 3¶ of the oligomers. Cells were harvested 48 hours after transfection with siRNA. Negative control siRNA was from Ambion (Silencer Negative Control 1 siRNA). To evaluate cell cycle changes after GCP2 and GCP3 depletion, cells in serum-free media were incubated in Hoechst 33342 at concentration 10 Kg/mL for 30 minutes and analyzed using the BD Influx cell sorter (BD Bioscience). Emission was triggered by 355-nm laser, and fluorescence was detected with a 460/50 band-pass filter.

Gel Filtration Chromatography Gel filtration was performed using fast protein liquid chromatography (AKTA-FPLC system, Amersham) on Superose 6 10/300 GL column (Amersham). Column equilibration and chromatography was performed in RIPA buffer. Column was eluted at 30 mL/h, and 0.5-mL aliquots were collected. Samples for SDS-PAGE were prepared by mixing with 5" concentrated SDS sample buffer. The following molecular mass standards were used: immunoglobulin IgM (900 kDa), thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), and bovine serum albumin (66 kDa).

Immunoprecipitation, Gel Electrophoresis, and Immunoblotting Immunoprecipitation was performed as described (41). Cell extracts were incubated with beads of protein A (Pierce, Rockford, IL) saturated with (i) mAb GCP2-01 (IgG2b) to

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GCP2, (ii) mAb TU-31 (IgG2b) to F-tubulin, mAb NF-09 (IgG2a; negative control), or with immobilized protein A alone. Alternatively, cell extracts were incubated with beads of protein G (Pierce) saturated with (i) mAb to C-3 (IgG1) to GCP3, (ii) mAb HTF-14 (IgG1; negative control), or with immobilized protein G alone. One-dimensional electrophoresis (SDS-PAGE) and immunoblotting were performed using standard protocols (31). For immunoblotting mAbs to GCP2 (GCP2-01 and GCP202), >-tubulin (TU-01) and F-tubulin (TU-32), in the form of hybridoma spent culture media, were diluted 1:5. MAb to GCP3, Cdk1-p-Y15, nucleophosmin, Grb2 and H2AX-p-S139 were diluted, 1:300, 1:1000, 1:2000, 1:5000, and 1:5000, respectively. Rabbit antibodies to cyclin B1, ATM-p-S1981, actin, GCP2, GCP3 (Proteintech), FLAG, GFP, and cyclophilin A were diluted, 1:1000, 1:1000, 1:2000, 1:2000, 1:3000, 1:3000, 1:5000, and 1:10,000, respectively. Peroxidase-conjugated antimouse and anti-rabbit antibodies were diluted 1:10,000. Bound antibodies were detected by SuperSignal WestPico Chemiluminescent reagents (Pierce) and quantified using the‘LAS 3000 imaging system (Fujifilm, Tokyo, Japan) and AIDA image analyzer (ver. 4) software (Raytest, Straubenhardt, Germany).

Cell Synchronization To obtain a synchronous population of cells, confluent plates of T98G and U87 MG cells were incubated with DMEM containing only 0.1% FBS for 72 hours. The medium was then changed with DMEM containing 10% FBS and 2 mmol/L of hydroxyurea (Sigma). The treatment with hydroxyurea lasted 21 hours. The cells were then replated in new plates (at a lower density), and samples of cells were collected each hour for fluorescence-activated cell sorting analysis on Accuri C6 flow cytometer (Accuri BD Biosciences, Franklin Lakes, NJ). The performance of the instrument was validated according to the manufacturer’s instructions and using 6 and 8 peak fluorescent bead mixtures (Accuri) to identify the best time point for G0/G1, S, and G2/M phases of the cell cycle, and the corresponding pellets were used for immunoblot analysis as previously described (25).

Immunofluorescence on Cell Lines Immunofluorescence microscopy was performed as previously described (42). Briefly, cells on glass coverslips were rinsed with PBS and fixed in methanol for 20 minutes at j20-C. Air-dried preparations were washed 3 " 5 minutes in PBS before staining. Alternatively, cells were washed in MTstabilizing buffer (42) fixed at 37-C for 30 minutes in 3% formaldehyde in MT-stabilizing buffer before extraction for 4 minutes with 0.5% Triton X-100 in MT-stabilizing buffer at 37-C. To expose epitopes in compact nucleoli, methanolfixed cells were stored up to 1 month in PBS at 4-C before staining. mAbs GCP2-01 and GCP2-02, in the form of hybridoma spent culture media, were diluted 1:5. MAb to GCP3 was diluted 1:50. Rabbit antibodies to GCP2, >-tubulin, and F-tubulin were diluted 1:50, 1:200, and 1:500, respectively. The rabbit antibody to nucleolin was diluted 1:1000. DY549conjugated anti-mouse antibody was diluted 1:500 and DY488conjugated anti-rabbit antibody was diluted 1:200. ! 2015 American Association of Neuropathologists, Inc.



For double-label immunofluorescence, coverslips were incubated separately with the primary antibodies, and simultaneously with the secondary conjugated antibodies. The preparations were mounted in MOWIOL 4Y88 (Calbiochem, San Diego, CA), supplemented with 4,6-diamidino-2-phenylindole ([DAPI], Sigma) to label nuclei, and examined on Olympus AX-70 Provis (Olympus Optical Co., Hamburg, Germany) equipped with 60"/1.0 NA water objectives. Conjugates alone rendered no significant staining.

Immunoelectron Microscopy Cells, grown on coverslips coated with gelatin, were fixed for 30 minutes in 3% paraformaldehyde, 0.1% glutaraldehyde in So¨rensen buffer (SB; 0.1 mol/L of Na/K phosphate buffer, pH 7.3). They were washed (2 " 10 minutes) in SB and then incubated in 0.02 mol/L glycine in SB for 20 minutes. Samples were then dehydrated in ethanol. Ethanol was replaced in 2 steps with LR White resin (Sigma), and the resin was polymerized by ultraviolet light (48 hours, 4-C). Alternatively, cells were fixed, washed in ice-cold PBS and dehydrated in ethanol followed by Lowicryl HM20 resin (Electron Microscopy Sciences, Hatfield, PA) infiltration by the progressive lowering of temperature method. Polymerization was effected by ultraviolet light during 24 hours at j35-C, then during 72 hours at 20-C. After cutting 80-nm sections, samples were treated with 0.5% SDS in water during 12 minutes for antigen retrieval of GCP2. Nonspecific labeling was blocked with a mixture of 10% normal goat serum (Life Technologies, Prague, Czech Republic), 1% bovine serum albumin, and 0.1% Tween 20 in PBS for 5 minutes at room temperature. The sections were then coincubated with rabbit anti-F-tubulin antibody (diluted 1:75) and anti-GCP2 (GCP2-01, undiluted supernatant) or anti-GCP3 (C-3, diluted 1:10). The samples were washed 3 times in PBS containing Tween 20 (PBT) (0.005% Tween 20 in PBS), incubated with 12-nm gold-conjugated anti-rabbit antibody and 6 nm gold-conjugated anti-mouse antibody, both diluted 1:30, washed twice in PBT and twice in bidistilled water, and then air dried. Finally, sections were contrasted with a saturated solution of uranyl acetate in water (4 minutes) and inspected in Morgagni 268 transmission electron microscope (FEI, Brno, Czech Republic) equipped with Mega View III CCD camera. Negative control incubations without primary antibodies proved that the signals were specific.

Time-lapse Imaging Time-lapse imaging was performed as described (43). Shortly, T98G cells were transfected with either GCP2-specific, GCP3-specific, or negative control siRNA as previously described (43). Seventy-two hours after transfection, cells were detached, counted, and diluted in culture media. A total of 2 " 104 transfected cells were plated in a single well of a 6-well tissue culture dish and allowed to adhere for 24 hours. Cells were subsequently incubated in a medium containing Hoechst 33342 (Sigma-Aldrich) at a final concentration of 0.5 Kg/mL and were imaged on a microscope (IX-81; Olympus) equipped with a platform for live cell imaging. Images were obtained


in a bright-field channel to visualize whole cells and in a fluorescence channel (excitation: 350 nm; emission: 460 nm) to visualize nuclei. The total imaging time was 10 hours with 10-minute intervals.

Clinical Tumor Samples and Brain Tissues Formaldehyde-fixed, paraffin-embedded biopsy/resection samples from adults (n = 54) representative of all WHO grades of astrocytic gliomas (WHO grades IYIV) were collected retrospectively from the KAT General Hospital (Athens, Greece), Gades Institute, Department of Pathology, University of Bergen, Haukeland Hospital (Bergen, Norway), and the Department of Pathology, University of Patras Hospital (Rion, Patras, Greece), under the approval of an institutional review board exempt review. All specimens were deidentified. A number of tumor specimens used in this study were also used in previous studies (20, 43). Histologic classification was according to the recommendations of the 2007 WHO classification of tumors of the CNS (44). Tumor specimens from adult patients included low-grade diffuse astrocytomas (WHO grade II; n = 14), anaplastic astrocytomas (WHO grade III; n = 5) and glioblastoma (WHO grade IV; n = 35). A panel of 4 senior pathologists (NFM, EM, SM, CDK), including 2 senior neuropathologists (SM, CDK), independently evaluated the histology of tumor samples. Participants were blinded to the original diagnosis and with regard to each others’ readings. Microtome sections from archived formalin-fixed, paraffin-embedded tissue blocks were cut at 5-Km thickness, placed on electromagnetically charged slides, and stained with hematoxylin and eosin for morphologic evaluation. Adjacent, serially cut, and sequentially numbered sections were processed for immunohistochemistry. Control tissues included brain biopsy and autopsy tissue samples from cases without tumor (n = 10). Nonneoplastic brain autopsy tissue samples (n = 5) derived from 5 adults (21, 49, 54, 59, and 63 years old) who had died of nonneurologic conditions and whose postmortem CNS examination was without pathologic findings were examined. Some of these tissues were used in previous studies (20, 43). The use of existing autopsy tissue specimens was approved by an institutional review board exempt review protocol. No patient identifiers were used. For immunoblot analysis, fresh human brain specimens were obtained during autopsy, within a 6-hour postmortem interval, from patients who died from causes unrelated to an underlying neurologic disorder or terminal hypoxic/ischemic brain damage. Before tissue procurement for immunoblotting, gross examination of the freshly sliced brain was carried out by a senior pathologist (LK) in order to confirm that there was absence of pathologic findings. Brain tissues corresponding to cerebral cortex and white matter were flash frozen in liquid nitrogen and stored at j80-C. All tissues were obtained after informed consents and the approval of an institutional review board (University Hospital Brno, Czech Republic). Mouse (Mus musculus), chicken (Gallus domesticus), African clawed frog (Xenopus laevis), and zebrafish (Danio rerio) brains were obtained from animals kept in the Animal Facility of the Institute of Molecular Genetics in Prague.



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Immunohistochemistry on Clinical Tumor Samples Immunohistochemistry was performed with the avidin biotin complex (ABC) peroxidase method using Rabbit and Mouse IgG ABC Elite detection kits (Vector Laboratories), as previously described (20, 21). Before the performance of the immunohistochemical procedure, 5-Km-thick histologic sections from paraffin-embedded tissue blocks were subjected to nonenzymatic antigen unmasking in 0.01 mol/L of sodium citrate buffer (pH 6.0) for 10 minutes in a pressure cooker at medium power setting such that the temperature ranged between 90-F and 100-F. The rabbit antibody to GCP2 was used at a 1:500 dilution; the rabbit antibody to GCP3 (GeneTex) was used at a dilution of 1:250. mAb GCP2-01 in the form of hybridoma spent culture media was used undiluted. Methanol/ hydrogen peroxide treatment of the slides was for 30 minutes at room temperature to suppress endogenous peroxidase activity. The concentration of serum in blocking and antibody dilution buffers was increased 2 times when compared with the manufacturer’s recommendation. Incubations with primary antibodies were performed overnight at 4-C, whereas incubations with the corresponding biotinylated secondary antibodies were performed at room temperature for 1 hour. Hematoxylin nuclear counterstaining was performed using Hematoxylin QS (Vector Laboratories). Negative controls included omission of primary antibody. For the evaluation of immunoperoxidase staining, an Eclipse E400 brightfield microscope (Nikon, Tokyo, Japan) equipped with a digital BFC 280 camera (Leica Microsystems, Buffalo Grove, IL) was used.


Laboratories; Catalog No. H-1200) then sealed with nail polish and allowed to air dry in the dark under a chemical hood. The tissue slides were then stored at 4-C in the dark until ready for fluorescent microscopy.

Immunofluorescence on Clinical Tumor Samples Immunohistochemical hydration of the tissue slides was performed as previously described (20, 21). The tissue samples were treated with chilled sodium borohydride (1 mg/mL) in PBS for 50 minutes. The rabbit antibody to GCP2 was used at a 1:250 dilution; the rabbit antibody to GCP3 was used at a dilution of 1:125; the mouse mAb to F-tubulin (clone GTU88) was used at a 1:150 dilution; and purified mAb GCP2-01 to GCP2 at concentration 1 mg/mL was used at a 1:62 dilution. DyLight 549 anti-mouse antibody was used at a 1:400 dilution; DyLight 488 anti-rabbit antibody was used at a 1:800 dilution. Normal goat serum (Vector Laboratories; Catalog. No. S-1000) and normal horse serum (Vector Laboratories; Catalog No. S-2000) were used in blocking and antibody dilution buffers when rabbit and mouse primary antibodies, respectively, were applied. Concentration of serum in blocking and antibody dilution buffers was increased 2 times when compared with manufacturer’s recommendation. All washes were done in PBS containing 0.5% of blocking buffer. Incubations with primary antibodies were performed overnight at 4-C, whereas incubations with the corresponding fluorescent dye conjugated secondary antibodies were performed at room temperature for 1 hour. Final treatment of autofluorescence was done by incubating the tissue slides in 0.2% Sudan black in 70% ethanol for 10 minutes at room temperature after which tissue slides were washed copiously in double-distilled water. The tissue slides were then mounted with Vectashield with DAPI (Vector

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FIGURE 1. Characterization of mAb GCP2-02 to F-tubulin complex protein 2 (GCP2). (A) Immunofluorescence staining of interphase and mitotic U2OS cells with mouse mAb GCP202. Double-label staining with GCP2-02 (a) and polyclonal antibody to F-tubulin (b). Double-label staining with GCP2-02 (c) and polyclonal antibody to >-tubulin (d). Fixation, methanol. Scale bar for aYd = 20 Km. (B) Immunoblot analysis of total cell lysates from human and mouse cell lines of different cellular origin with mAb GCP2-02. Human kidney embryonal cells (HEK), human retinal pigment epithelial cells (RPE1), human neuroblastoma cells (SH-SY5Y), human osteogenic sarcoma cells (U2OS) and mouse BMMCL. (C) Immunoblot analysis of brain extracts from different species with mAb GCP2-02. Bars on the left margins in (B) and (C) indicate positions of molecular mass markers in kilodalton. ! 2015 American Association of Neuropathologists, Inc.




Cell Counting Immunohistochemical preparations were evaluated by 2 observers (CDK, LD). Representative images were captured by a digital BFC 280 camera (Leica Microsystems). Manual cell counting of GCP2- and GCP3-labeled tumor cells was performed on the digital images. Between 683 and 1120 tumor cells were evaluated per case, in 20 nonoverlapping high-power (40") fields; a labeling index was determined for each case. The labeling index was expressed as the percentage (%) of GCP2, GCP3, or F-tubulin-labeled cells out of the total number of tumor cells counted in each case and for each antibody, as previously described (20, 43). The median labeling index (MLI) and the interquartile range (IQR), delimited by the 25th and 75th percentiles, were determined for the set of cases in each histologic grade using 1-way analysis of variance in SigmaStat (Systat Software, San Jose, CA). The statistical significance of differences in labeling indices between WHO histologic grades were examined with nonparametric statistical techniques using Kruskal-Wallis analysis of variance tests. A p value of less than 0.05 was considered significant.

RESULTS Characterization of New mAbs to GCP2 The practice of using different mAbs recognizing distinct epitopes on the same molecule rests on the rationale that it is likely to enhance the fidelity of immunochemical detection of a target protein of interest. Toward this goal, we prepared and characterized 2 mouse mAbs against the N-terminal region of mouse GCP2 (a.a. 1-194). From a panel of 6 hybridomas producing mAbs reacting in ELISA with GSTGCP2 fusion protein used for immunization but not with GST, 2 mAbs named GCP2-01 (IgG2bJ) and GCP2-02 (IgG1J) were characterized using immunofluorescence, immunoblotting, and immunoprecipitation. Although GCP2-01 is already commercially available, its detailed characterization has not been hitherto described in a published work. Both mAbs GCP2-01 and GCP2-02 labeled centrosomes in which the presence of GCP2 was expected. Figure 1A shows double-label staining of GCP2-02 with an anti-F-tubulin antibody (Fig. 1Aa, b), or with an anti->-tubulin antibody FIGURE 2. Comparison of F-tubulin complex protein 2 and 3 (GCP2 and GCP3) expression in NHA and glioblastoma cell lines. (A) Immunofluorescence staining of NHA (a, c) and T98G cells (b, d) with mAb GCP2-02 to GCP2 (a, b) and mAb to GCP3 (c, d). Fixation, methanol. Images were collected and processed in exactly the same manner. Scale bar = 20 Km. (B) Immunoblotting analysis of total cell lysates from NHA and glioblastoma cell lines U87MG (U87), T98G (T98), U118MG (U118), U138MG (U138), and KNS-42 (KNS). Staining with mAbs to GCP2 (GCP2-02), GCP3, and >-tubulin (>-Tub). Bars on the left margins indicate positions of molecular mass markers in kilodalton. (C) Transcription of genes for GCP2 and GCP3 in glioblastoma cell lines relative to the level in NHA. Data are presented as the mean fold change T SE obtained from 2 independent experiments with triplicate samples. **p G 0.01; ***p G 0.001. ! 2015 American Association of Neuropathologists, Inc.


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(Fig. 1Ac, d). mAb GCP2-02 stained centrosomes within interphase, duplicated centrosomes within prophase, spindle poles and parts of 2 half-spindles within metaphase (inserts in Fig. 1Aa, c). The mAb did not label interphase MTs or short aster MTs at the spindle poles. Similar structures were detected with the mAb GCP2-01 (Figure, Supplemental Digital Content 3, part A, http://links.lww.com/NEN/A754). Labeling was extinguished by preabsorption of antibodies with GSTtagged GCP2 fragment used for immunization but not with GST (not shown). The antibodies reacted with centrosomes in all tested cell lines derived from divergent cell types in different species. Cells from the following cell lines were immunostained: human U2OS, RPE1, SH-SY5Y, and HEK and mouse BMMCL. The mAbs differed in their reactivity under various fixation conditions. Both antibodies robustly decorated centrosomes in methanol-fixed samples. In formaldehyde-fixed/Triton X100-extracted cells, centrosomes were stained only with mAb GCP2-01. On immunoblots of total cell lysates, the antibodies interacted with proteins with relative molecular weight corresponding to GCP2 as illustrated using mAb GCP2-02 in Figure 1B and mAb GCP2-01 in Figure, Supplemental Digital Content 3, part B, http://links.lww.com/NEN/A754. In immunoblots of brain tissues from various species, namely, human, mouse, chicken, African clawed frog, and fish, mAb GCP2-02 recognized the corresponding GCP2 epitope (Fig. 1C). Conversely, when a simultaneously prepared blot was probed with GCP2-01, the mAb reacted with GCP2 in human, mouse, and chicken brains but not in lysates from African clawed frog or fish brains (Figure, Supplemental Digital Content 3, part C, http://links.lww.com/NEN/A754). No cross reactivity was detected with the other GCPs (GCP3-GCP6) on immunoblots (not shown). As documented under the ‘‘Results’’ heading below, mAb GCP2-01 is also suitable for immunohistochemical probing on formaldehyde-fixed, paraffin-embedded histologic sections. Both antibodies are directed against phylogenetically conserved epitopes on GCP2 and can be used for detection of this protein in ELISA, immunofluorescence, immunoblotting, and immunoprecipitation. The differential reactivity of these antibodies in immunoblots and in fixed cells suggests that mAbs GCP2-01 and GCP2-02 recognize nonidentical epitopes. The findings of this study suggest that mAb GCP2-01 is more suitable for immunoprecipitation and immunohistochemistry, whereas mAb GCP2-02 is more appropriate for immunoblotting. Together, mAbs GCP2-01 and GCP2-02 represent specific and standard immunoprobes for the detection of GCP2.

GCP2 and GCP3 are Overexpressed in Glioblastoma Cell Lines The subcellular distribution of GCP2 and GCP3 was compared by immunofluorescence microscopy in nonimmortalized, nontransformed NHA and human glioblastoma cell line T98G. In interphase NHA, both proteins were localized mainly in centrosomes (Fig. 2Aa, c). By comparison, in T98G cells immunostaining was found both in centrosomes and cytoplasm, where robust and variously dense localization of GCP2 and GCP3 was detected diffusely throughout the cytoplasm, occasionally extending into the cell periphery

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(Fig. 2Ab, d). In T98G cells, colocalization of F-tubulin with both GCP2 (Figure, Supplemental Digital Content 4, parts a, b, http://links.lww.com/NEN/A755) and GCP3 (Figure, Supplemental Digital Content 4, parts c, d, http://links.lww.com/NEN/A755) was demonstrated. The association of GCP2 with F-tubulin in the mitotic spindle is shown in Figure, Supplemental Digital Content 4, parts a, b, arrows, http://links.lww.com/NEN/A755). The spatial distribution of MTs in NHA and T98G cells was similar and the increased levels of GCP2 and GCP3 in the cytosol of T98G glioblastoma cells did not affect the organization of MT network (not shown). Prominent punctate cytoplasmic localizations of GCP2 and GCP3 were found in all 5 tested glioblastoma cell lines (U87MG, T98G, U118MG, U138MG, and KNS-42). Overall, a similar staining pattern was obtained with mAbs GCP2-01 and GCP2-02 compared to the polyclonal antibody to GCP2 (not shown). To compare and contrast the expression of GCP2 and GCP3 in NHA and glioblastoma cell lines further, blots of total cell lysates were probed with antibodies to GCP2, GCP3, and >-tubulin. Higher amounts of GCP2 and GCP3 were detected in all glioblastoma cell lines, while no significant differences were observed in the expression level of >-tubulin (Fig. 2B). Densitometric measurements of immunoblots showed that the amount of GCP2 and GCP3 was more then 3-fold in U87MG and T98G glioblastoma cell lines as compared with NHA (p G 0.005, n = 3). To evaluate differences of expression in NHA and glioblastoma cell lines at the transcriptional level, mRNAs for GCP2 and GCP3 were isolated from NHA and tested glioblastoma cell lines; the levels of transcript expression were determined by reverse transcription quantitative real-time PCR (RT-qPCR). The expression of both GCP2 and GCP3 was significantly higher in all 5 glioblastoma cell lines when compared with NHA (U87MG and T98G, p G 0.001; U118MG, U138MG, and KNS-42, p G 0.01) (Fig. 2C). Collectively, these data demonstrate that the significant increase in transcripts for GCP2 and GCP3 in glioblastoma cell lines, as compared with NHA, is associated with higher amounts of GCP2 and GCP3 proteins, as determined by both immunoblotting and immunofluorescence. In order to follow the distribution of GCP2 throughout the different phases of the cell cycle, synchronization experiments were performed in T98G cells. Cell cycle characteristics were determined by flow cytometric analysis of asynchronous and synchronous cell populations. The distribution of the percentage of cells in the different phases of the cell cycle is shown in Figure, Supplemental Digital Content 5, http://links.lww.com/NEN/A756. Immunoblot analysis of synchronized T98G cells revealed no substantial changes in the amount of GCP2 in G0/G1, S, and G2/M phases of the cell cycle (Figure, Supplemental Digital Content 5, http://links.lww.com/NEN/A756).

GCP2 and GCP3 Associate with Nuclei and Nucleoli in Glioblastoma Cells In previous studies, we have reported overexpression of F-tubulin in T98G cells (20), and an association of F-tubulin with nucleoli in these cells (18). To detect nucleolar F-tubulin by immunofluorescence, it was essential to unmask F-tubulin ! 2015 American Association of Neuropathologists, Inc.




FIGURE 3. Nucleolar localization of F-tubulin complex proteins 2 and 3 (GCP2 and GCP3) in T98G glioblastoma cells. (A) Nucleolar localization of GCP2 and GCP3 in immunofluorescence. Methanol fixed cells stained with mAb GCP2-01 to GCP2 (a) or with mAb to GCP3 (b). Scale bar for aYb = 10 Km. (B) Immunoblot analysis of subcellular fractions. Cytosolic, nuclear, and nucleolar fractions of cells were probed with mAbs to nucleophosmin (Nucl.), >-tubulin (>-Tub), GCP2 (GCP2-02), GCP3, F-tubulin (F-Tub), and rabbit antibody to cyclophilin A (Cycl.) The same amount of proteins was loaded into each lane. Bars on the left margins indicate positions of molecular mass markers in kilodalton. (C, D) Ultrastructural localization of GCP2 (C) and GCP3 (D) in nucleolus. Double-label staining with mAb GCP2-01 to GCP2 or mAb to GCP3 (detection with 6-nm gold-conjugated anti-mouse antibody) and rabbit antibody to F-tubulin (detection with 12-nm gold-conjugated anti-rabbit antibody). White arrows indicate position of 6-nm gold particles. DFC, dense fibrillar component; FC, fibrillar center; GC, granular component; N, nucleoplasma. Scale bar for a and b = 100 nm. (E) Size distribution of nucleolar proteins. Proteins extracted from nucleoli were fractionated on Superose 6. Blots of collected fractions were probed with mAbs to F-tubulin, GCP2 (GCP2-02), and GCP3. Calibration standards (in kilodalton) are indicated on the top. Numbers at the bottom denote individual fractions. (F) Coprecipitation of F-tubulin with GCP2 or GCP3. Nucleolar extracts were immunoprecipitated with mAbs to GCP2 (GCP2-01), GCP3, or F-tubulin. Blots were probed with mAbs to GCP2 (GCP2-02), GCP3, or F-tubulin (F-Tub). Load (1), immobilized mAb not incubated with extract (2), immunoprecipitated proteins (3), antibody carrier without mAb incubated with extract (4). ! 2015 American Association of Neuropathologists, Inc.


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epitopes within the compact structure of the nucleolus. Nucleolar F-tubulin was clearly detected after prolonged washing of methanol-fixed preparations, as previously detailed (18). Therefore, we used the same approach to evaluate the possible association of GCP2 and GCP3 with nucleoli in T98G cells. Both mAbs to GCP2 labeled discrete nuclear regions in methanol-fixed preparations as illustrated using mAb GCP201 (Fig. 3Aa). A similar nuclear staining pattern was observed using a mAb to GCP3 (Fig. 3Ab). Expected localization of GCP2 and GCP3 was detected in centrosomes (arrows in Fig. 3Aa, b). Double labeling using the nucleolar marker, nucleolin, revealed that both GCP2 and GCP3 localized in nucleoli (as shown for GCP3 in Fig. 4Ca, b). Diffuse GCP2 and GCP3 localizations were also observed in the nucleoplasm. Omission of primary antibodies (application of secondary antibodies alone) or the use of a mAb to transferrin, both serving as negative controls, did not render nucleolar labeling. To corroborate independently the presence of GCP2 and GCP3 in nucleoli of T98G cells, immunoblots were performed using purified cytosolic, nuclear, and nucleolar extracts. The purity of isolated nucleolar preparations was checked by phase contrast microscopy. When blots were probed with an antibody to nucleophosmin, a protein that shuttles between the nucleus and cytoplasm, but predominantly resides in nucleoli, immunoreactivity was substantially higher in the nucleolar fraction (Fig. 3B, Nucl.). No signal in nucleolar fraction was detected with an antibody to cytosolic cyclophilin A (Fig. 3B, Cycl.), indicating that the nucleolar fraction was devoid of cytosolic contaminants. Similarly, when an antibody to >-tubulin (Fig. 3B, >-Tub) was applied, no staining was detected in the nucleolar fraction. Conversely, GCP2, GCP3, and F-tubulin (Fig. 3B) were all detected in the nucleolar fraction. Both mAb to GCP2 (GCP2-02) and rabbit antibody to GCP2, as well as both mAb to GCP3 and rabbit antibody to GCP3 (Proteintech) detected corresponding proteins in the nucleolar fraction. The nucleolus consists of 3 different regions: fibrillar centers, dense fibrillar components, and granular components. To obtain data on subnucleolar localization of GCP2 and GCP3, postembedding immunoelectron microscopy was carried out on 80-nm sections of T98G cells. Electron microscopy examination of formaldehyde-fixed preparations disclosed that GCP2 in interphase T98G cells was located in granular components or on the boundary between dense fibrillar components and granular components (Fig. 3C, white arrows). A similar distribution was observed for GCP3 (Fig. 3D, white arrows). Neither GCP2 nor GCP3 accumulated in fibrillar centers where transcription of ribosomal DNA takes place. Nucleolar GCP2 and GCP3 were visualized both as single gold particles and small or large clusters. Double-labeling with antiF-tubulin antibody disclosed that occasionally GCP2 and GCP3 were in close proximity to F-tubulin as demonstrated for GCP3 (insert in Fig. 3D). To determine whether nucleolar GCP2, GCP3, and F-tubulin exist in the form of complexes, nucleolar extracts were subjected to gel filtration chromatography on Superose 6 column. As reported previously (18), F-tubulin was distributed through a large zone in complexes of various sizes. Large F-tubulin complexes of È2 MD were not detected, and GCP2 and GCP3 only partially overlapped with F-tubulin (Fig. 3E).

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To ascertain whether nucleolar GCP2 and/or GCP3 associate with F-tubulin, immunoprecipitation experiments were performed from nucleolar extracts. Immunoblot analysis revealed that a mAb GCP2-01 to GCP2 coprecipitated both GCP3 and F-tubulin (Fig. 3F, left part, lane 3), whereas a mAb to GCP3 coprecipitated both GCP2 and F-tubulin (Fig. 3F, middle part, lane 3). In addition, the reciprocal precipitation with a mAb to F-tubulin confirmed an interaction of GCP2 and GCP3 with F-tubulin (Fig. 3F, right part, lane 3). In contrast, negative control mAbs did not coprecipitate GCP2, GCP3, or F-tubulin. Altogether, these data strongly suggest that GCP2 and GCP3 associate with nucleoli of T98G cells where they are capable of forming complexes with F-tubulin. Both immunofluorescence microscopy (Figure, Supplemental Digital Content 6, part A, http://links.lww.com/NEN/A757) and subcellular fractionation (Figure, Supplemental Digital Content 6, part B, http://links.lww.com/NEN/A757) revealed that nucleolar GCP2 and GCP3 were also present in U87MG glioblastoma cells. Similarly, as in the case of T98G glioblastoma cells, nucleolar F-tubulin formed complexes with GCP2 and GCP3 in U87MG glioblastoma cells (Figure, Supplemental Digital Content 6, part C, http://links.lww.com/NEN/A757). To determine whether GCP2 or GCP3 depletion is essential for nucleolar integrity, both proteins were depleted in T98G cells by siRNA. For this, 2 different siRNAs were used. As demonstrated in RT-qPCR (Fig. 4A) and immunoblotting (Fig. 4B) experiments, 2 siRNAs for GCP2 as well as 2 siRNAs for GCP3 effectively depleted corresponding proteins. Because GCP2 was more efficiently depleted by siRNA#1 (denoted KD1 in Fig. 4A, TUBGCP2) and similarly, GCP3 was more efficiently depleted by siRNA#1 (denoted KD1 in Fig. 4A, TUBGCP3), they were used in further experiments. Specific depletion of GCP2 or GCP3 effectively decreased immunofluorescence signals both in the cytosol and nuclei/nucleoli. An illustrative example in this regard is shown for GCP3 (Fig. 4Ca, c). No overt impairment of nucleolar integrity was detectable in cells with depleted level of GCP3 (Fig. 4Cb, d) or GCP2 (not shown) on the basis of immunostaining with an antibody to nucleolin. The most prominent phenotypic feature of GCP2 or GCP3 depletion was accumulation of cells in G2/M phase of the cell cycle as documented by flow cytometry and staining of mitotic spindles with an antibody to >-tubulin (Fig. 4D). At variance with the depletion of F-tubulin (Figure, Supplemental Digital Content 7, part A, B, http://links.lww.com/NEN/A758), which was characterized by profound mitotic spindle defects (Figure, Supplemental Digital Content 7, part C, http://links.lww.com/NEN/A758), depletion of GCP2 and GCP3 did not result in severe mitotic spindle abnormalities. Moreover, live cell imaging revealed that, in sharp contrast to Ftubulin depletion, which led to complete mitotic arrest in T98G glioblastoma cells and other cell types (40, 45), approximately 20% of cells passed with delay through mitosis in GCP2depleted or GCP3-depleted T98G glioblastoma cells. Depletion of GCP2 and GCP3 cells in U87MG glioblastoma cells resulted in the accumulation of cells in G2/M phase of the cell cycle, albeit at a lower level as compared with T98G glioblastoma cells (Figure, Supplemental Digital Content 8, part A-C, http://links.lww.com/NEN/A759). Similar to T98G ! 2015 American Association of Neuropathologists, Inc.




FIGURE 4. Effect of F-tubulin complex protein 2 or 3 (GCP2 or GCP3) depletion on nucleolar integrity and cell cycle in T98G glioblastoma cells. (A) Transcription of GCP2 and GCP3 genes in cells transfected with specific siRNAs relative to the level in cells transfected with negative control siRNA (Control). Both GCP2 and GCP3 were depleted with 2 different siRNAs (siRNA#1 is denoted KD1; siRNA#2 is denoted KD2). Data are represented as the fold change T SE. ***p G 0.005. (B) Immunoblot analysis of whole cell extracts from cells transfected with negative control (Control), GCP2 (GCP2-KD1; GCP2-KD2), or GCP3 (GCP3-KD1; GCP3-KD2) specific siRNAs. Staining with mAbs to GCP2 (GCP2-02), GCP3, and rabbit antibody to actin. Bars on the left margins indicate positions of molecular mass markers in kilodalton. (C) Comparison of GCP3 distribution in cells transfected with negative control siRNA (Control) and with GCP3-specific siRNA (GCP3-KD1). Cells were double label stained with antibodies to GCP3 and nucleolin. Methanol fixation. Fluorescence images of cells stained for GCP3 were captured under identical conditions and processed in exactly the same manner. Scale bar for aYd = 10 Km. (D) T98G cells with depleted level of GCP2 (GCP2-KD1), GCP3 (GCP3-KD1), negative control cells (RNAi control), or untreated cells (T98G) were stained with antibody to >-tubulin. Scale bar for all images = 10 Km. Note the accumulation of mitotic cells in GCP2- or GCP3-depleted cells when compared with control cells. The flow cytometry analysis of corresponding cell cultures is shown under the immunofluorescence images. ! 2015 American Association of Neuropathologists, Inc.


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glioblastoma cells, depletion of GCP2 and GCP3 in U87MG cells did not result in severe mitotic spindle defects.

GCP2 Overexpression Antagonizes the Inhibitory Effect of Tumor Suppressor C53 on DNA Damage G2/M Activation Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry. In response to DNA damage, G2/M checkpoint system (ATM/ATR and checkpoint kinases) inactivates phosphatase Cdc25C by phosphorylation, which in turn leads to accumulation of Cdk1 phosphorylated at Y15 and inactivation of Cdk1 (46). It has previously been demonstrated that overexpression of CDK5 regulatory subunitassociated protein 3 (C53; gene CDK5RAP3) overrides the G2/M DNA damage checkpoint induced by genotoxic agents as etoposide (47). We have previously reported that F-tubulin forms complexes with C53 and overexpression of F-tubulin in U2OS human osteosarcoma cells stably expressing C53 (U2OS/C53-EGFP) antagonizes C53 action and restores accumulation of Cdk1-p-Y15 and cyclin B1 (18). To evaluate whether GCP2 plays role in the modulation of C53 activity, we used the same experimental setup and overexpressed GCP2 in U2OS/C53-EGFP cells. Treatment of U2OS cells with etoposide caused accumulation of Cdk1-p-Y15, and the C53 overexpression (U2OS/C53-EGFP cells) attenuated the phosphorylation of Cdk1 (Fig. 5, Cdk1-p-Y15) and accumulation of cyclin B1 (Fig. 5, cyclin B1). Intriguingly, both the overexpression of F-tubulin (+F-Tb) and GCP2 (+GCP2) in U2OS/C53-EGFP antagonized the C53 action and restored


the accumulation of Cdk1-p-Y15 and cyclin B1. Overexpression of control Fyn (+Fyn) had a much lower effect. No obvious changes in the amount of endogenous GCP2 were detected in etoposide-treated U2OS cells (data not shown). This finding suggests, albeit indirectly, that the activation of Cdk1/cyclin B1 by C53 overexpression is diminished by virtue of an excess of F-tubulin and GCP2. Phosphorylation of ATM at S1981 and histone H2AX at S139 were used as indicators of ATM/ ATR activity responsible for phosphorylation of checkpoint kinases (Fig. 5, ATM-p-S1981 and H2AX-p-S139). The expression of C53-EGFP and FLAG-tagged proteins was determined using antibodies to GFP (Fig. 5, GFP) and FLAG (Fig. 5, FLAG), respectively. Collectively taken, the data indicate that C53 activity during G2/M DNA damage checkpoint is subject to modulation by FTuSC proteins.

GCP2 and GCP3 Are Overexpressed in Clinical Samples of Glioblastoma Compared to Low-grade Diffuse Gliomas In the normal adult CNS, diffuse cytoplasmic GCP2 and GCP3 staining, beyond the expected centrosomal localization, was detected predominantly in neuronal perikarya and proximal neurites (dendrites and axon hillocks) (Figure, Supplemental Digital Content 9, http://links.lww.com/NEN/A760). In occasional neurons, GCP2 staining was also detected in the nucleus either in the form of diffuse staining in the nucleoplasm (Figure, Supplemental Digital Content 9, part a, http://links.lww.com/NEN/A760), or infrequently, as single,

FIGURE 5. Overexpression of F-tubulin complex protein 2 (GCP2) antagonizes the inhibitory effect of CDK5 regulatory subunitassociated tumor suppressor protein 3 (C53) on DNA damage G2/M checkpoint activation. U2OS cells expressing EGFP-C53 were transfected with vectors encoding FLAG-tagged GCP2 (+GCP2), F-tubulin (+F-Tb; positive control), or Fyn (+Fyn; negative control). After 48 hours, the cells were treated with 20 Kmol/L of etoposide for 3 hours. U2OS cells served as control. Immunoblot analysis of whole cell extracts with Abs to Cdk1-p-Y15, cyclin B1, ATM-p-S1981, H2AX-p-S139, GFP, and FLAG. Numbers under the blots indicate relative amounts of proteins normalized to cells not treated with etoposide. Means T SD were calculated from 3 experiments.

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sharply delineated GCP2-labeled inclusion-like particles adjacent to the nucleolus (arrow in Figure, Supplemental Digital Content 9, part b, http://links.lww.com/NEN/A760). For the most part, quiescent glial cells did not exhibit significant noncentrosomal GCP2/GCP3 staining (arrows in Figure, Supplemental Digital Content 9, part a, c-e, http://links.lww.com/NEN/A760). Scant GCP2 and GCP3 labeling was also detected, albeit to a lesser degree, in the cytoplasm of subpopulations of astrocytes and brain endothelial cells (not shown). As compared with normal brain tissues, GCP2 and GCP3 immunoreactivity was significantly increased in glial tumor cell (gliomatous) phenotypes of diffuse astrocytic gliomas of all histologic grades (Figs. 6, 7). Typical GCP2 and GCP3 tumor cell labeling in areas of tumor invasion (in a case of diffuse astrocytic glioma with anaplastic change) compared with the nearby normal-appearing brain, whereby sparse labeling is detected in neuronal cell bodies and blood vessels (Fig. 6). A trend toward higher GCP2 and GCP3 expression and more widespread cellular distribution was observed in line with an ascending scale of histologic malignancy (Figure, Supplemental Digital Content 10, http://links.lww.com/NEN/A761). Increased GCP2 and GCP3 median labeling indices were present in high-grade diffuse astrocytic gliomas (anaplastic astrocytoma/WHO grade III and glioblastoma/WHO grade IV) compared with low-grade diffuse astrocytic gliomas (WHO grade II) (p G 0.05). In low-grade diffuse astrocytic gliomas (WHO grade II), GCP2 and GCP3 tumor cell labeling was variable within individual tumor specimens, and was also characterized by a wide range of labeling counts of an uneven distribution among tumors of the same histologic grade (GCP2: IQR 2%Y42%; MLI 16%. GCP3: IQR 5%Y34%, MLI 23%). Immunoreactivity was significantly more prominent and widespread in high-grade gliomas (WHO grades III/IV),


notwithstanding the presence of intratumoral staining variability (GCP2: IQR 24%Y76%, MLI 44%; GCP3: IQR 35%Y88%, MLI 57%) (p G 0.05). No significant differences in median labeling indices were found between WHO grade III and WHO grade IV tumors. A linear relationship was also determined between the percentages of F-tubulin and GCP2/ GCP3-labeled tumor cells (p G 0.05). Three overlapping patterns of abnormal GCP2/GCP3 tumor cell localization were identified in clinical tumor samples: (i) diffuse cytoplasmic (Fig. 7AaYd); (ii) granular/ punctate cytoplasmic (Fig. 7BaYd); and (iii) nuclear, either in the form of diffuse GCP2 staining (Fig. 7Ab) or seldom, as isolated GCP2-labeled inclusion-like particles (arrow in Fig. 7Ad) akin to those described above in the nuclei of nonneoplastic neurons (arrow in Figure, Supplemental Digital Content 9, part b, http://links.lww.com/NEN/A757). A variously dense, diffuse, and homogeneous cytoplasmic labeling was mainly obtained with both monoclonal and polyclonal antibodies to GCP2 (Fig. 7AaYd). Nuclear localization in paraffin sections was clearly observed with mAb GCP2-01 (Fig. 7Ab). A dot-like, multipunctate pattern of cytoplasmic tumor cell labeling was rendered with the polyclonal antibody to GCP3 (Fig. 7BaYd). In large tumor cells with plump cytoplasm, with morphologic features reminiscent of gemistocytic or giant cell morphologic phenotypes, partial GCP3 staining of a granular/particulate quality was detected in discrete portions of the cytoplasm (Fig. 7Bc), occasionally exhibiting a proclivity for the periphery of tumor cells (Fig. 7Bd). By comparison, GCP3 labeling was diffuse and homogeneous in small (anaplastic) tumor cells (Fig. 7Bb). Double labeling revealed partial colocalization of GCP2 and F-tubulin (Fig. 8a), GCP3 and F-tubulin (Fig. 8b) as well as GCP2 and GCP3 in a large proportion of tumor cells (Fig. 8c, d). In double-labeled

FIGURE 6. Comparison of F-tubulin complex protein 2 and 3 (GCP2 and GCP3) expression in tumor cells versus non-neoplastic brain. GCP2 (a) and GCP3 (b) labeling using rabbit anti-GCP2 and anti-GCP3 antibodies in areas of tumor compatible with diffuse astrocytoma with anaplastic change (anaplastic astrocytoma/World Health Organization grade III) compared with the nearby infiltrated normal-appearing cerebral cortex. ABC peroxidase with hematoxylin counterstain. Scale bars for a and b = 100 Km. ! 2015 American Association of Neuropathologists, Inc.


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FIGURE 8. Colocalization of F-tubulin, F-tubulin complex protein 2 and 3 (GCP2 and GCP3) in glioblastoma cells. Double immunofluorescence labeling performed on paraffin sections from a clinical tumor sample of glioblastoma using mAb GTU-88 (Sigma) to F-tubulin (green) and rabbit antibodies to GCP2 (a) and GCP3 (red) (b). Panels c and d illustrate partial colocalization of GCP2 and GCP3 by double labeling using mAb GCP2-01 to GCP2 (green) and a rabbit antibody to GCP3 (red). Arrows in b and d depict, respectively, localization of F-tubulin and GCP3 in the periphery of tumor cells. Scale bars for aYd = 10 Km.

preparations, there was a tendency for F-tubulin and GCP3 to accumulate in the periphery of occasional tumor cells (Fig. 8b, d, arrows). In addition to the detection of FTuSC protein immunoreactivity in neoplastic glial cells, variously intense GCP2

(Fig. 9aYc) and GCP3 (Fig. 9c, d) labeling was also observed in areas of microvascular proliferation in which partial colocalization was demonstrated by double-label immunofluorescence (Fig. 9c). Localizations were demonstrated in morphologically overt endothelial cells of tumor microvessels (Fig. 9a),

FIGURE 7. Immunoreactivity profiles of F-tubulin complex protein 2 and 3 (GCP2 and GCP3) in clinical samples of glioblastoma. (A) Immunohistochemical reactivity profiles of GCP2 in neoplastic cells from paraffin sections of clinical tumor samples. Diffuse cytoplasmic (aYd) and nuclear localizations, the latter either in the form of diffuse staining (b) or seldom, as labeling of single, discrete inclusion-like particles (arrow in d). Immunostaining in panel b is rendered using mAb GCP2-01; labeling shown in panels a, c, and d is obtained using a rabbit anti-GCP2 antibody. Scale bars for aYd = 20 Km. (B) Immunohistochemical staining for GCP3 using rabbit antibody. Granular/punctate cytoplasmic (aYd) labeling. Note the sharp, punctate GCP3 staining in portions of the cytoplasm of large tumor cells (c) occasionally exhibiting a proclivity for the periphery of tumor cells (d). Compare granular/ punctate GCP3 localization in large tumor cells with plump cytoplasm to the diffuse homogeneous GCP3 staining in small (anaplastic) tumor cells (b). ABC peroxidase with hematoxylin counterstain. Scale bar for aYd = 20 Km. ! 2015 American Association of Neuropathologists, Inc.


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FIGURE 9. F-tubulin complex protein 2 and 3 (GCP2 and GCP3) in tumor blood vessels of glioblastoma. (a, b) Panels a and b show localization of GCP2 using rabbit antibody and mAb GCP2-01, respectively, in endothelial cells of microvessels (a, b), and in vasculocentric/perivascular tumor-like cells merging imperceptibly with vascular endothelial-like cells bordering the vascular lumen in a focus of microvascular proliferation that are difficult to tell apart from comingled glioma cells (b). The vascular lumens are denoted by asterisks. (c) Partial colocalization of GCP2 (green, mAb GCP2-01) and GCP3 (red, rabbit antibody) by double label immunofluorescence microscopy in cells bordering the lumen of a blood vessel (asterisk). (d) Granular GCP3 labeling using a rabbit antibody in hypertrophic vascular-like cells of a glomeruloid structure that are morphologically indistinguishable from neoplastic glial cells. The capillary-like lumens are demarcated by asterisks. Avidin-biotin complex peroxidase with hematoxylin counterstain. Scale bars for a, b, and d = 20 Km; scale bar for c = 10 Km.

in vasculocentric/perivascular tumor-like cells indistinguishable from atypical vascular-like cells/pericytes (Fig. 9b), as well as in hypertrophic cells of glomeruloid structures that were difficult to tell apart from neoplastic glial cells (Fig. 9d).

DISCUSSION To our knowledge, this is the first study to demonstrate overexpression of FTuSC proteins, GCP2 and GCP3, both in surgically resected glioblastoma tissue specimens and in

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human glioblastoma cell lines. A significant relationship between level of expression of GCP2 or GCP3 and histologic malignancy (WHO grade II vs WHO grade III/IV) was clearly detected. Divergent subcellular sorting was found in the cytosol, nuclei, and nucleoli of 2 widely used human glioblastoma cell lines, T98G and U87MG. To that end, to our knowledge, this is also the first study to demonstrate nuclear and nucleolar localizations of GCP2 and GCP3 in human cells. The findings reported here complement and extend the data derived from previous reports concerning overexpression of F-tubulin in ! 2015 American Association of Neuropathologists, Inc.



diffuse astrocytic gliomas, according to an ascending histologic gradient of malignancy (20, 21, 23, 24, 48), and of a differential compartmentalization of F-tubulin in cell nuclei and nucleoli (18). However, unlike F-tubulin, depletion in GCP2 and GCP3 does not lead to severe mitotic spindle abnormalities and a subpopulation of T98G cells passes, albeit with delay, through the M-phase of the cell cycle. The present study has also unraveled that GCP2 is capable of modulating the inhibitory effect of CDK5 regulatory subunit-associated protein 3 (C53) on DNA damage G2/M checkpoint activation, in a manner similar to F-tubulin (18). Collectively, these findings may have broad relevance to the biology and therapeutic targeting of glioblastomas, particularly in light of a renewed interest in the mechanisms and signaling networks involved in centrosome amplification in cancer (49). The present study brings attention to the fact that increased expression of FTuSC proteins in glioblastoma may be linked to pro-oncogenic events and cellular dysfunction(s) at the transcriptional level through interactions with tumor suppressor genes and/or signaling pathways, which fall outside the conventional role of these proteins in MT nucleation.

Accumulation of FTuSC Proteins in Cytosol The multisubunit FTuRC is vital for MT nucleation in eukaryotic cells, but the elucidation of mechanisms by which FTuRC is activated either at conventional MTOCs or other potential sites of MT nucleation in normal cell types or in the context of centrosome abnormalities in cancer cells is still evolving (50). Although a large number of proteins bind to cytoplasmic FTuRCs in both interphase and mitotic cells (51), only some of them, with FTuRC-mediated nucleation activator (FTuNA) motif (52), stimulate MT-nucleating activity. Regulation of FTuRC activity may also be accomplished via conformational modulation of GCP3 (50). The increased coexpression of F-tubulin and FTuSC proteins, GCP2 and GCP3, in glioma/glioblastoma cells raises the question of activation of ectopic MT nucleation in tumor cells. Such increased production of newly formed MTs might serve to meet the increased demands of tumor cells for abnormal multipolar spindles, but this may also increase the numbers of MTs for altered cellular architecture and control of focal adhesion dynamics in the context of force generation for directional cell migration and tumor invasion (49, 53). However, no evidence of ectopic, noncentrosomal, MT nucleation was observed in T98G or U87MG glioblastoma cells as compared with normal human astrocytes (P. Dra´ber and E. Dra´berova´, unpublished data). Moreover, unlike the MTsevering protein spastin (43), depletion of GCP2 or F-tubulin had no effect on cell migration of T98G glioblastoma cells grown in monolayer cultures (E. Dra´berova´, unpublished data). Interestingly, depletion of GCP2 or GCP3 in T98G and U87MG glioblastoma cells did not result in complete mitotic arrest and generation of mitotic defects, contrasting the arrest of mitotic cell cycle progression caused by F-tubulin depletion in T98G glioblastoma cells and other cell types (40, 45). GCP2 and GCP3 are essential proteins of FTuSCs that are core structural components of MT-nucleating FTuRCs. During mitotic spindle assembly, MTs are newly nucleated in


large amount from FTuRCs on centrosomes (54). In addition, noncentrosomal FTuRCs are important for the generation and maintenance of a robust mitotic spindle (55). Partial depletion of GCP2 or GCP3 can decrease the amount of nucleation competent FTuRCs resulting in accumulation of cells in G2/M and delay in mitosis. Furthermore, mitotic delay may reflect the presence of an underlying substitution mechanism whereby other GCP proteins might step in and partially take over the role of depleted GCP2 or GCP3. Studies focusing on the elucidation of the crystal structure of human GCP4 have unraveled that GCP4 fits remarkably well into FTuSC cryo-electron microscopic structure in the positions of GCP2 and GCP3 (56). It has been suggested that GCP3, GCP4, and GCP6 can act as FTuSC-like complexes (50). An alternative, but not necessarily mutually exclusive, explanation with regard to the overexpression and abnormal cytoplasmic accumulation and distribution of F-tubulin and GCP2/GCP3 in glioblastoma cells goes beyond the traditionally assigned role of these proteins in MT nucleation. It is possible that FTuSC proteins interact with tumor-activated signaling pathways and/or other cellular organelles such as mitochondria, endoplasmic reticulum, and Golgi complexes, which are also activated in cancer (57). Regarding the postulated interaction of FTuSC proteins with activated signaling pathways, it is thought that tyrosine kinases may serve as regulators of F-tubulin function and thus play an important role in signal transduction as well as in the regulation of MT protein interactions (58Y60). It is well established that F-tubulin forms complexes with a host of kinases such as 1phosphatidylinositol 4-kinase (Plk1), Wee1, MT affinityregulating kinase 4 (MARK4), Syk tyrosine kinase, Src family tyrosine kinases (Lyn, Src, Fyn), BubR1 kinase, and phosphoinositide 3-kinase (PI3K) (61). Direct interaction of F-tubulin with the C-terminal Src homology 2 domain of p85> (regulatory p85> subunit of PI3K) was described, and it was suggested that signaling-cascade proteins such as tyrosine kinases and PI3K could modulate noncentrosomal MT nucleation by membrane-associated F-tubulin (17). A noteworthy observation in this study is the detection of GCP2 and GCP3 labeling in areas of microvascular proliferation in clinical specimens, which mirrors that of F-tubulin in glioblastomas (20). The extent to which centrosome abnormalities in endothelial cells (62) may underlie neoangiogenesis in glioblastomas remains to be determined in future studies. An alternative interpretation relates to the emerging concept of vasculogenic mimicry according to which glioma phenotypes are capable of undergoing transdifferentiation and forming microvascular channels in glioblastoma independent of endothelial cell angiogenesis (63, 64).

Localization of FTuSC Proteins in Nucleus/Nucleolus Contrary to the view that GCP2 and GCP3 are solely cytosolic proteins in mammalian cells, data generated in the present study show that these proteins are also located in nuclei and nucleoli of T98G and U87MG glioblastoma cells. Several lines of evidence support this conclusion. First, proteins were detected by double-labeling immunofluorescence



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microscopy in nucleoli using an antibody to the nucleolar marker nucleolin. Second, the immunofluorescence staining was extinguished after depletion of GCP2 or GCP3. Third, nucleolar GCP2 or GCP3 localizations were visualized by immunoelectron microscopy in the form of either single molecules or variously sized clusters (Fig. 3C, D). Neither F-tubulin nor GCP2 or GCP3 were localized in fibrillar centers, suggesting that FTuSC proteins are not involved in the process of ribosomal transcription. Fourth, immunoblot analysis of isolated nucleoli confirmed that they contain GCP2 and GCP3 but not cytosolic proteins. Fifth, both FTuSC proteins coprecipitated with Ftubulin in nucleolar fractions. Although no known nuclear localization sequences have been to date unraveled in human GCP2 or GCP3 (https:// www.predictprotein.org), nuclear localization signal has been identified in F-tubulin (19), suggesting that GCP2 or GCP3 might be transported to the nucleus via hitchhiking on the Ftubulin molecule. Coprecipitation of GCP2 with F-tubulin in the nuclear membrane fraction of 3T3 mouse fibroblast and U2OS human osteosarcoma cell extracts, but not from chromatin fraction and without concomitant GCP2 localization in the nucleus by immunofluorescence microscopy, has been recently reported (65). There are several explanations that may account for the differences in the results presented in this study compared with the study by Eklund et al (65). First, different cell types were used for analysis. Eklund et al used U2OS and 3T3 cells. A comparison between U2OS cells and T98G glioblastoma cells by RT-qPCR in the present study revealed approximately a 2.5-fold increase in GCP2 transcripts in T98G cells compared with U2OS cells (V. Sla´dkova´ and P. Dra´ber, unpublished data). Second, there were differences in the methodology of protein sample preparation for immunoprecipitation experiments. In our study, we isolated nucleoli and then prepared concentrated protein extracts, whereas Eklund et al (65) used differential extraction to prepare nuclear membrane and chromatin fractions. Importantly, in the present study, we did not detect signal in the nucleolar fraction with antibodies to cytosolic cyclophilin A or >-tubulin, indicating that the isolated nucleolar fraction was devoid of cytosolic contaminants. Third, different anti-F-tubulin antibodies were used for immunoprecipitation, and different antibodies were also used for the detection of GCP2 in the 2 studies. Our data indicate that both monoclonal and polyclonal antibodies to GCP2 are capable of detecting this protein in the nucleolar fraction by immunoblotting. Similarly, we have detected GCP3 in immunoblots with polyclonal as well as mAbs. And fourth, both the fixation method and also the antibodies used to visualize GCP2 in the nucleoli by immunofluorescence microscopy in the present study were different from the methods employed by Eklund et al (65). In an earlier study, we have shown that in order to detect nucleolar F-tubulin by immunofluorescence, it is necessary to unmask F-tubulin epitopes within the compact structure of the nucleolus (18). The findings presented herein indicate that same holds true for GCP2 and GCP3. Previous studies have shown that F-tubulin may have nuclear-/nucleolar-specific functions beyond its conventionally assigned role in MT nucleation and spindle formation. These functions include (a) modulation of transcriptional activity of E2 promoter binding factor (E2F) (19, 66); (b) complementary

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interaction with retinoblastoma protein (Rb1) in the regulation of E2F activity in the context of a F-tubulin/Rb1 signaling network (19, 66); (c) a regulatory role in the anaphase-promoting complex/cyclosome, which is a key mitotic and cell cycle regulatory complex (67); and (d) modulation of the activity of tumor suppressor protein C53 (18). Interestingly, suppression of F-tubulin protein levels in tumors with nonfunctional Rb1 has a proapoptotic effect (66). The C53 protein, which is a caspase substrate, plays a role in the regulation of apoptosis induced by genotoxic stress through modulating Cdk1-cyclin B1 function (47). C53 deficiency confers partial resistance to genotoxic agents such as etoposide and ionizing irradiation, whereas ectopic expression of C53 makes cells susceptible to various genotoxins that typically trigger G2/M arrest (47). Cdk5 is considered to be a multifunctional regulator of cytoskeletal organization, cell adhesion, and apoptosis in human glioblastoma cells in vitro (68). In particular, Cdk5 has been found in the detergent-insoluble cytoskeletal fraction of the U373 human glioblastoma cell line (68). Our results regarding potential involvement of GCP2 in the regulation of DNA damage G2/M checkpoint protein C53 suggest that GCP2 could have nuclear-/nucleolar-specific function(s). Immunoelectron microscopy has revealed that GCP2 and GCP3 proteins can be found occasionally in close proximity to F-tubulin. Thus, we postulate that GCP2 in complex with F-tubulin may participate, either directly or indirectly, in the modulation of C53 function in DNA damage G2/M checkpoint. Collectively, in the present study, we have demonstrated for the first time evidence of nucleolar localization of GCP2 and GCP3 and have functionally elucidated the effect of depletion of these FTuSC proteins on cell cycle progression in 2 widely used human glioblastoma cell lines, T98G and U87MG. However, these findings should be interpreted with the caveat that neither the genomic alterations nor the intratumoral cellular heterogeneity of primary patient tumors (gliomas in situ) are faithfully reproduced in adherent serumgrowing cultures of established human glioblastoma cell lines (69, 70). This pitfall is not limited to cell lines as it can also be encountered in primary cultures derived from explanted tumors (70). It is thought that phenotypic and transcriptomic alterations, clonal selection, and genetic drifts represent adaptive changes of tumor cells to the conditions of monolayer cultures (69, 70). At the same time, it should be recognized that phenotypic shifts can also occur in vivo where they may be swayed by factors related to tumor microenvironment (70Y72). Compared with serum-growing monolayer cultures of established human glioblastoma cell lines, culture conditions that are suitable for the isolation of neural progenitors and cancer stem cells, such as neurosphere cultures or biopsy-derived spheroid cultures (70), are more likely to reflect the phenotypic and genotypic profiles of original tumors (73). Future studies are needed to determine possible differences in the behavior of FTuSC proteins within homogenous populations of glioma stem cellYlike phenotypes.

Therapeutic Targeting of F-Tubulin and FTuSC Proteins in Glioblastoma One of the principal and time-honored strategies used in cancer therapeutics has been to disrupt the integrity of MTs in ! 2015 American Association of Neuropathologists, Inc.



order to prevent or curtail mitotic division. Because MTs are nucleated during spindle assembly from FTuRCs, the development of anticancer drugs targeting MT-nucleating complexes emerges as a rational approach in cancer therapy (61). Previous studies have shown that depletion of F-tubulin or FTuRC proteins induces changes in MT dynamics and spindle defects that resemble the effects of MT drugs (45, 74). Glioblastomas are endowed with a high concentration of F-tubulin and FTuSC proteins, GCP2 and GCP3. The development of specific drugs targeting MT-nucleating proteins, that is, F-tubulin and/or GCPs, is currently underway. Friesen et al (75) have shown that colchicine and combretastatin A-4 are the first compounds known to bind and interact with F-tubulin, which makes them potentially promising small molecule inhibitors in the therapeutic targeting of glioblastomas. Another recent study has demonstrated in silico the existence of a binding pocket at the interface between GCP4 and F-tubulin in FTuRC (76). This opens up new possibilities for drug development against proteins of FTuRC (76). Up to now, no compounds that specifically interact only with F-tubulin or GCPs have been identified. In summary, this study presents novel data on the overexpression and differential subcellular sorting (nucleolar compartmentalization) of GCP2 and GCP3 in glioblastoma and unravels divergent functional roles of FTuSC proteins in the regulation of C53 during DNA damage G2/M activation. In addition, it raises awareness about the potential of F-tubulin and FTuSC proteins as molecular targets in brain cancer therapy. ACKNOWLEDGMENTS Dedicated to the memory of Dr. Nicholas K. Gonatas. The authors thank Dr Theodoros Maraziotis (Department of Neurosurgery, University of Patras Hospital, Rion, Patras, Greece) for providing sections from existing paraffin blocks of clinical specimens used in this study. We also thank Dr M. Hibbs (Ludwig Institute for Cancer Research, Melbourne, Australia) for the gift of BMMCL cells and Dr M. Bonhivers (Universite´ Bordeaux, Bordeaux, France) for the gift of RPE1 cells. Parts of this work were presented in the 2nd International Medical Olympiad (Thessaloniki, Greece, October 18Y20, 2013). Published abstract: Katsetos CD, Dra´berova E, D’Agostino L, Caracciolo V, Sla´dkova´ V, Sulimenko T, Maounis NF, Tzelepis EG, Mahera E, Legido A, Giordano A, Dra´ber P. Overexpression of human FTuSC proteins GCP2 and GCP3 in glioblastomas and human glioblastoma cell lines. Hell J Nucl Med 2014;17 (Supplement): 82. REFERENCES 1. Ohgaki H, Kleihues P. The definition of primary and secondary glioblastoma. Clin Cancer Res 2013;19:764Y72 2. McDonald KL, Aw G, Kleihues P. Role of biomarkers in the clinical management of glioblastomas: what are the barriers and how can we overcome them? Front Neurol 2013;3:e188 3. Louis DN, Perry A, Burger P, et al. International Society of NeuropathologyHaarlem consensus guidelines for nervous system tumor classification and grading. Brain Pathol 2014;24:429Y35 4. Dra´ber P, Dra´berova´ E. Microtubules. In: Kavallaris M, ed. Cytoskeleton and Human Disease. New York: NY: Humana Press; 2012:29Y54

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Text S1 Thermocycling parameters at quantitative PCR Data presented in Figs. 2 and 4 were obtained in the LightCycler® 480 (Roche) using the following thermocycling parameters: initial denaturation - 95°C/5 min; cycling - 45 cycles. Target temperature 95°C 60°C 72°C

Fluorescence acquisition mode None None Single

Hold

Ramp Rate

10 s 15 s 15 s

4.8°C/s 2.5°C/s 4.8°C/s

Melting curve Target temperature 95°C 55°C 96°C 40°C

Fluorescence acquisition mode None None Continuous None

Hold

Ramp Rate

Acquisitions #

5s 60 s X 30 s

4.8°C/s 2.5°C/s 0.06°C/s 2.5°C/s

X X 10/1°C X

Cp values of all samples were determined in LightCycler® 480 Software, release 1.5.0, by the module “Abs quant/2nd Derivative Max”. Melting curves were analyzed in the module ”Melting curve genotyping”. Only samples with the correct melting and amplification curves were further evaluated. PCR efficiencies (E) for probed genes were calculated from calibration curves by the LightCycler® 480 Software. Calculation of the normalized relative quantity (NRQ) of evaluated transcripts was based on the following formula (1): Cp,goi E goi

NRQ 

f

f

E

Cp,ref0 ref0

0

1.

Hellemans J, Mortier G, De Paepe A, et al. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data.  Genome Biol 2007;8:R19

Table S1.

Sequences of primers used for RT-qPCR analysis of human genes

Name

Sequence

Amplicon length

___________________________________________________________________________

TUBGCP2, fwd

5’- CTTCCTTCGAGTACGGGCAG-3’

TUBGCP2, rev

5’- GGCTGGCTGGATGTAGAACC-3’

TUBGCP3, fwd

5’- TGCTACGTTATTTGCTCAGGC-3’

TUBGCP3, rev

5’- TGGGCACTCCGATCTTGGTA-3’

TUBG1, fwd

5’- CCCTCATCTGCCTTACTGGTTG-3’

TUBG1, rev

5’- AGGTCCCTGATCTGTGCTCTGA-3’

ACTB, fwd

5’- ATGTGGCCGAGGACTTTGATT-3’

ACTB, rev

5’- AGTGGGGTGGCTTTTAGGATG-3’

151 bp

104 bp

72 bp

106 bp

___________________________________________________________________________

FIGURE S1. Characterization of monoclonal antibody GCP2-01 to GCP2. (A) Double-label immunofluorescence staining of interphase and mitotic U2OS cells with mAb GCP2-01 (a) and polyclonal antibody to α-tubulin (b). Fixation methanol. Scale bar, 20 μm. (B) Immunoblot analysis of total cell lysates from human and mouse cell lines of different origin with mAb GCP201. Human kidney embryonal cells (HEK), human retinal pigment epithelial cells (RPE1), human neuroblastoma cells (SH-SY5Y), human osteogenic sarcoma cells (U2OS) and mouse bone marrow-derived mast cells (BMMCL). (C) Immunoblot analysis of brain extracts from different species with mAb GCP2-01. Bars on the left margins in (B) and (C) indicate positions of molecular mass markers in kDa.

FIGURE S2. Codistribution of GCP2 and GCP3 with γ-tubulin in T98G glioblastoma cells. Double-label immunofluorescence staining of glioblastoma cells with mAb GCP2-02 to GCP2 (a), a mAb to GCP3 (c) and a polyclonal antibody to γ-tubulin (b, d). The same microscopic fields are shown in (a-b) and (c-d). Arrows indicate the position of mitotic spindles. Fixation in methanol. Scale bar, 20 μm.

FIGURE S3. Expression of GCP2 in synchronized T98G glioblastoma cells. (A) The flow cytometry analysis. Asynchronous/actively growing cells (AG); cells grown for 72 hours in a serum deprived medium, under which conditions cells go into arrest in the G0/G1 phase (72h SD); starting from G0/G1 synchronized cells, cells were re-plated and a incubated for 23 hours for the S phase (23 h Rel); starting from G0/G1 synchronized cells, cells were re-plated and a incubated for 27 hours for the G2/M phase (27 h Rel). (B) The distribution of the percentage of cells in the different phases of the cell cycle. (C) Immunoblot analysis of total cell lysates from asynchronous cells and after cell synchronization. Blots were probed with mAbs to GCP2 (GCP2-02) and Grb2.

FIGURE S4. Nucleolar localization of GCP2 and GCP3 in U87MG glioblastoma cells. (A) Nucleolar localization of GCP2 and GCP3 in immunofluorescence. Methanol fixed cells stained with mAb GCP2-01 to GCP2 (a) or with mAb to GCP3 (b). Scale bar for a-b, 10 μm. (B) Immunoblot analysis of subcellular fractions. Cytosolic, nuclear and nucleolar fractions of cells were probed with antibodies to nucleophosmin (Nucl.), cyclophilin A (Cycl.), α-tubulin (α-Tub), GCP2 (GCP2-02), GCP3 (Santa Cruz) and γ-tubulin (γ-Tub). The same amount of proteins was loaded into each lane. Bars on the left margins indicate positions of molecular mass markers in kDa. (C) Co-precipitation of γ-tubulin with GCP2 or GCP3. Nucleolar extracts were immunoprecipitated with mAbs to GCP2 (GCP2-01), GCP3 or γ-tubulin. Blots were probed with mAb to GCP2 (GCP2-02), rabbit antibody to GCP3 (Proteintech) or mAb to γ-tubulin (γ-Tub). Load (1), immobilized mAb not incubated with extract (2), immunoprecipitated proteins (3), antibody carrier without mAb incubated with extract (4).

FIGURE S5. Depletion of human γ-tubulin 1 leads to mitotic spindle defects and metaphase arrest in T98G glioblastoma cells. (A) Transcription of TUBG1 gene in cells transfected with specific siRNA (KD) relative to the level in cells transfected with negative control siRNA (Control). Data are represented as the fold change ± SE. ∗∗∗, p