BAG3 Protein Is Overexpressed in Human Glioblastoma and Is a Potential Target for Therapy
2011; Elsevier BV; Volume: 178; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2011.02.002
ISSN1525-2191
AutoresMichelina Festa, Luis Del Valle, Kamel Khalili, Renato Franco, Giosuè Scognamiglio, Vincenzo Graziano, Vincenzo De Laurenzi, Maria Caterina Turco, Alessandra Rosati,
Tópico(s)Force Microscopy Techniques and Applications
ResumoGlioblastoma multiforme, which represents 80% of malignant gliomas, is characterized by aggressiveness and high recurrence rates. Despite therapeutic advances, patients with glioblastoma multiforme show a poor survival, and identification of novel markers and molecular targets for therapy is needed. A role for BAG3, a member of the BAG family of HSC/HSP70 co-chaperones, in promoting tumor cell growth in vivo has recently been described. We analyzed BAG3 levels by IHC in specimens from patients affected by brain tumors and we found that BAG3, although negative in normal brain tissues, was highly expressed in astrocytic tumors and increasingly expressed in more aggressive types of cancer; it was particularly high in glioblastomas. Down-regulating BAG3 both in vitro and in vivo in a rat glioblastoma model resulted in increased sensitivity to apoptosis, suggesting that BAG3 is a potential target for novel therapies. Finally, we determined that the underlying molecular mechanism requires the formation of a complex of BAG3, HSP70, and BAX that prevents BAX translocation to mitochondria, thus protecting tumor cells from apoptosis. Our data identify BAG3 as a potential marker of glial brain tumor sensitivity to therapy and thus also an attractive candidate for new molecular therapies. Glioblastoma multiforme, which represents 80% of malignant gliomas, is characterized by aggressiveness and high recurrence rates. Despite therapeutic advances, patients with glioblastoma multiforme show a poor survival, and identification of novel markers and molecular targets for therapy is needed. A role for BAG3, a member of the BAG family of HSC/HSP70 co-chaperones, in promoting tumor cell growth in vivo has recently been described. We analyzed BAG3 levels by IHC in specimens from patients affected by brain tumors and we found that BAG3, although negative in normal brain tissues, was highly expressed in astrocytic tumors and increasingly expressed in more aggressive types of cancer; it was particularly high in glioblastomas. Down-regulating BAG3 both in vitro and in vivo in a rat glioblastoma model resulted in increased sensitivity to apoptosis, suggesting that BAG3 is a potential target for novel therapies. Finally, we determined that the underlying molecular mechanism requires the formation of a complex of BAG3, HSP70, and BAX that prevents BAX translocation to mitochondria, thus protecting tumor cells from apoptosis. Our data identify BAG3 as a potential marker of glial brain tumor sensitivity to therapy and thus also an attractive candidate for new molecular therapies. Glioblastoma multiforme (GBM) is the most aggressive and most common tumor of the brain, accounting for approximately 25% of all brain tumors, 50% to 60% of all astrocytic tumors, and 80% of all malignant gliomas.1Central Brain Tumor Registry of the United States (CBTRUS)2009–2010 CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2004–2006. CBTRUS, Hinsdale, IL2010Google Scholar, 2Ohgaki H. Kleihues P. Epidemiology and etiology of gliomas.Acta Neuropathol. 2005; 109: 93-108Crossref PubMed Scopus (941) Google Scholar Poorly circumscribed margins, microinvasion, and the infiltrating nature of astrocytes are contributing factors for the notorious aggressiveness and high rates of recurrence of glioblastomas; moreover, the severe neurological dysfunctions that accompany this tumor compromise both quality of life and survival. Despite significant advances in neurosurgical techniques, including the introduction of gamma knife surgery, and aggressive multimodal treatments, the median survival time for GBM is approximately 56 weeks.3Grossman S.A. Ye X. Piantadosi S. Desideri S. Nabors L.B. Rosenfeld M. Fisher J. NABTT CNS ConsortiumSurvival of patients with newly diagnosed glioblastoma treated with radiation and temozolomide in research studies in the United States.Clin Cancer Res. 2010; 16: 2443-2449Crossref PubMed Scopus (350) Google Scholar, 4Clarke J. Butowski N. Chang S. Recent advances in therapy for glioblastoma.Arch Neurol. 2010; 67: 279-283Crossref PubMed Scopus (219) Google Scholar Recent studies elucidating some of the molecular abnormalities underlying the pathogenesis of glioblastomas are contributing to the development of novel therapeutic approaches.5Zheng H. Ying H. Wiedemeyer R. Yan H. Quayle S.N. Ivanova E.V. Paik J.H. Zhang H. Xiao Y. Perry S.R. Hu J. Vinjamoori A. Gan B. Sahin E. 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An evolutionarily conserved family of Hsp70/Hsc70 molecular chaperone regulators.J Biol Chem. 1999; 274: 781-786Crossref PubMed Scopus (406) Google Scholar In addition, BAG3 contains a WW domain and a proline-rich repeat (PXXP), through which it interacts with other proteins.16Doong H. Price J. Kim Y.S. Gasbarre C. Probst J. Liotta L.A. Blanchette J. Rizzo K. Kohn E. CAIR-1/BAG-3 forms an EGF-regulated ternary complex with phospholipase c-gamma and hsp70/hsc70.Oncogene. 2000; 19: 4385-4395Crossref PubMed Scopus (132) Google Scholar BAG3 expression is induced in leukocytes and other normal cell types in response to stress.17Pagliuca M.G. Lerose R. Cigliano S. Leone A. Regulation by heavy metals and temperature of the human BAG-3 gene, a modulator of Hsp70 activity.FEBS Lett. 2003; 541: 11-15Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 18Bonelli P. Petrella A. Rosati A. Romano M.F. Lerose R. Pagliuca M.G. Amelio T. Festa M. Martire G. Venuta S. Turco M.C. 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The anti-apoptotic protein BAG-3 is overexpressed in pancreatic cancer and induced by heat stress in pancreatic cancer cell lines.FEBS Lett. 2001; 503: 151-157Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 21Chiappetta G. Ammirante M. Basile A. Rosati A. Festa M. Monaco M. Vuttariello E. Pasquinelli R. Arra C. Zerilli M. Todaro M. Stassi G. Pezzullo L. Gentilella A. Tosco A. Pascale M. Marzullo L. Belisario M.A. Turco M.C. Leone A. The antiapoptotic protein BAG3 is expressed in thyroid carcinomas and modulates apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand.J Clin Endocrinol Metab. 2007; 92: 1159-1163Crossref PubMed Scopus (96) Google Scholar, 22Romano M.F. Festa M. Pagliuca G. Lerose R. Bisogni R. Chiurazzi F. Storti G. Volpe S. Venuta S. Turco M.C. Leone A. BAG3 protein controls B-chronic lymphocytic leukemia cell apoptosis.Cell Death Differ. 2003; 10: 383-385Crossref PubMed Scopus (96) Google Scholar, 23Romano M.F. Festa M. Petrella A. Rosati A. Pascale M. Bisogni R. Poggi V. Kohn E.C. Venuta S. Turco M.C. Leone A. BAG3 protein regulates cell survival in childhood acute lymphoblastic leukemia cells.Cancer Biol Ther. 2003; 2: 508-510Crossref PubMed Scopus (64) Google Scholar Several lines of evidence indicate that BAG3 plays a role in tumor cell survival. Indeed down-regulation of BAG3 in primary samples of B-cell chronic lymphocytic leukemia and acute lymphoblastic leukemia results in increased basal as well as drug-induced apoptosis.22Romano M.F. Festa M. Pagliuca G. Lerose R. Bisogni R. Chiurazzi F. Storti G. Volpe S. Venuta S. Turco M.C. Leone A. BAG3 protein controls B-chronic lymphocytic leukemia cell apoptosis.Cell Death Differ. 2003; 10: 383-385Crossref PubMed Scopus (96) Google Scholar, 23Romano M.F. Festa M. Petrella A. Rosati A. Pascale M. Bisogni R. Poggi V. Kohn E.C. Venuta S. Turco M.C. Leone A. BAG3 protein regulates cell survival in childhood acute lymphoblastic leukemia cells.Cancer Biol Ther. 2003; 2: 508-510Crossref PubMed Scopus (64) Google Scholar Furthermore, in thyroid carcinomas, BAG3 down-regulation sensitizes cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent apoptosis.21Chiappetta G. Ammirante M. Basile A. Rosati A. Festa M. Monaco M. Vuttariello E. Pasquinelli R. Arra C. Zerilli M. Todaro M. Stassi G. Pezzullo L. Gentilella A. Tosco A. Pascale M. Marzullo L. Belisario M.A. Turco M.C. Leone A. The antiapoptotic protein BAG3 is expressed in thyroid carcinomas and modulates apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand.J Clin Endocrinol Metab. 2007; 92: 1159-1163Crossref PubMed Scopus (96) Google Scholar The role of BAG3 in cell survival is, at least in part, mediated by the regulation of HSC70/HSP70 function.11Rosati A. Ammirante M. Gentilella A. Basile A. Festa M. Pascale M. Marzullo L. Belisario M.A. Tosco A. Franceschelli S. Moltedo O. Pagliuca G. Lerose R. Turco M.C. Apoptosis inhibition in cancer cells: a novel molecular pathway that involves BAG3 protein.Int J Biochem Cell Biol. 2007; 39: 1337-1342Crossref PubMed Scopus (116) Google Scholar We have recently shown that BAG3 sustains melanoma cell survival by interfering with the binding of HSP70 to the IKK-γ subunit of the NF-κB-activating IKK complex, thus favoring IKK complex formation and preventing the proteasomal degradation of IKK-γ and finally enhancing NF-κB activation.12Ammirante M. Rosati A. Arra C. Basile A. Falco A. Festa M. Pascale M. d'Avenia M. Marzullo L. Belisario M.A. De Marco M. Barbieri A. Giudice A. Chiappetta G. Vuttariello E. Monaco M. Bonelli P. Salvatore G. Di Benedetto M. Deshmane S.L. Khalili K. Turco M.C. Leone A. IKK{gamma} protein is a target of BAG3 regulatory activity in human tumor growth.Proc Natl Acad Sci USA. 2010; 107: 7497-7502Crossref PubMed Scopus (92) Google Scholar It is likely that in different tumors BAG3 can interfere with the binding of HSP70 with other partners known to sustain cell survival, preventing their degradation.Here we show that BAG3 is robustly expressed in a large proportion of astrocytomas and glioblastomas and that its expression increases with tumor grade. Furthermore, we demonstrate that BAG3 promotes the binding of HSC/HSP70 to the proapoptotic BCL2 family member BAX, preventing its translocation to mitochondria and protecting glioblastoma cells from apoptosis, and that silencing of BAG3 results in a dramatic decrease in cell proliferation in vitro and in vivo.Materials and MethodsAntibodiesAntibodies used to detect BAG3 protein were a mouse monoclonal antibody (AC-1) and a rabbit polyclonal antibody (TOS-2) distributed by Enzo Life Sciences (Lausen, Switzerland; Farmingdale, NY) and a mouse monoclonal antibody clone AC-2 produced in our laboratories. Polyclonal antibodies recognizing BAX, cleaved caspase-3 (Asp175), and BAD were obtained from Cell Signaling Technology (Danvers, MA). An anti-α-tubulin monoclonal antibody was obtained from Sigma-Aldrich (St. Louis, MO). Secondary antibodies were obtained from Pierce (Thermo Fisher Scientific, Rockford, IL). Anti-GAPDH and -HSP60 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-HSC/HSP70 polyclonal antibody was obtained from Stressgen (Victoria, BC, Canada; now Enzo Life Sciences).Cell Cultures and ReagentsThe rat malignant glial tumor cell line C6 and the human glioblastoma cell lines A172, T98G, and DBTRG-05MG were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The C6 and A172 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS); the T98G cells were grown in Eagle's minimal essential medium supplemented with 10% FBS, 1% Na-pyruvate, and 1% nonessential amino acids; the DBTRG-05MG cells were grown in Roswell Park Memorial Institute growth medium (RPMI-1640) supplemented with 10% FBS. All of the cell lines were maintained in 5% CO2 at 37°C in a humidified incubator. All of the growth media and supplements were obtained from Gibco-Invitrogen (Carlsbad, CA). Cisplatin [cis-diamminedichloroplatinum(II) (CDDP)] was obtained from Sigma-Aldrich.Cell Viability, Apoptosis Assay, and Mitochondrial Membrane Potential MeasurementCell viability was measured by Trypan Blue exclusion using a Bürker counting chamber. Apoptosis was analyzed by propidium iodide incorporation in permeabilized cells and flow cytometry was performed as described previously.12Ammirante M. Rosati A. Arra C. Basile A. Falco A. Festa M. Pascale M. d'Avenia M. Marzullo L. Belisario M.A. De Marco M. Barbieri A. Giudice A. Chiappetta G. Vuttariello E. Monaco M. Bonelli P. Salvatore G. Di Benedetto M. Deshmane S.L. Khalili K. Turco M.C. Leone A. IKK{gamma} protein is a target of BAG3 regulatory activity in human tumor growth.Proc Natl Acad Sci USA. 2010; 107: 7497-7502Crossref PubMed Scopus (92) Google Scholar Mitochondrial membrane potential was assessed by flow cytometry using tetramethylrhodamine ethyl ester as described previously.24Rosati A. Quaranta E. Ammirante M. Turco M.C. Leone A. De Feo V. Quassinoids can induce mitochondrial membrane depolarisation and caspase 3 activation in human cells.Cell Death Differ. 2004; 11: S216-S218Crossref PubMed Scopus (32) Google Scholar Briefly, cells were exposed to tetramethylrhodamine ethyl ester (Molecular Probes, Eugene, OR) for 1 hour at 37°C. Changes in dye fluorescence were analyzed with a FACScan flow cytometer (BD Biosciences, San Jose, CA).Co-ImmunoprecipitationFor co-immunoprecipitation assays, cells were lysed in HNT buffer (HEPES 20 mmol/L pH 7.5, NaCl 150 mmol/L, Triton 0.1%) supplemented with a protease inhibitor cocktail (Sigma-Aldrich) on ice for 20 minutes. After 5 cycles of freeze and thaw, cell membranes were centrifuged at 15,000 × g. Next, 500 μg of soluble proteins were subjected to immunoprecipitation with 1 μg of polyclonal anti-BAX antibody or 1 μg of rabbit IgG as control in HNTG buffer (HNT buffer, 10% glycerol), overnight at 4°C. Protein A-Sepharose was then added (45 minutes at 4°C), and immunocomplexes were precipitated by centrifugation at 15,000 × g. Next, immunoprecipitated proteins were washed with a low-salt buffer (0.1% SDS, 1% Triton, 20 mmol/L EDTA, 20 mmol/L Tris pH 8.0, 150 mmol/L NaCl), a high-salt buffer (0.1% SDS, 1% Triton, 20 mmol/L EDTA, 20 mmol/L Tris pH 8.0, 500 mmol/L NaCl), and a LiCl buffer (0.25 mol/L LiCl, 1% NP-40, 1% deoxycholate, 1 mmol/L EDTA, 10 mmol/L Tris pH 8.0), followed by two final washes with HNT buffer. Obtained proteins were then loaded onto SDS-PAGE gels and were analyzed for presence of HSC/HSP70 and BAG3 protein by Western blotting.DensitometryScanning densitometry of the bands was performed with image scanning software (SnapScan 1212; Agfa-Gevaert, Mortsel, Belgium). The area under the curve related to each band was determined using Gimp 2 software version 2.6 (available at http://www.gimp.org). Background was subtracted from the calculated values. Results are expressed as means of at least three separate experiments.IHCA brain tumor microarray was obtained from US Biomax (Rockville, MD). The TMA represented 151 cases: 13 grade I glial tumors, 85 low-grade diffuse astrocytomas (grade II), 17 anaplastic astrocytomas (grade III), and 36 glioblastomas (grade IV), as well as 8 samples of normal brain adjacent to a brain tumor and 8 normal brain tissues, along with pathology diagnosis and tumor grade data. Immunohistochemistry (IHC) was performed using the avidin-biotin-peroxidase system, according to the manufacturer's instructions (Leica Microsystems, Bannockburn, IL). Our modified protocol included deparaffination in xylene, rehydration through descending degrees of alcohol up to water, nonenzymatic antigen retrieval in citrate buffer, pH 6.0, for 30 minutes at 95°C, and endogenous peroxidase quenching with H2O2 in methanol for 20 minutes. After rinsing with PBS, the samples were blocked with 5% normal horse serum in 0.1% PBS/bovine serum albumin.To detect BAG3, samples were incubated for 1 hour at room temperature with the monoclonal antibody AC-1 (1:100 dilution, Enzo Life Sciences). After a thorough washing with PBS, sections were incubated with a biotinylated secondary anti-mouse IgG for 20 minutes, then were rinsed, incubated with avidin-biotin-peroxidase (ABC) complexes, and developed with diaminobenzidine (Sigma-Aldrich). Finally, the sections were counterstained with hematoxylin, dehydrated in alcohol, cleared in xylene, and mounted with Permount (Thermo Fisher Scientific, Waltham, MA). Ten nonoverlapping high-power fields (×400) were evaluated, and a labeling index of tumor cells was calculated for each specimen. The labeling index was defined as the percentage of positive cells out of the total number of cells counted in all fields. Only neoplastic cells were counted; to avoid nontumoral cells, margins and areas of infiltration into the brain were excluded. The number of positive cells were divided into three groups: BAG3 negative ( 40% positive cells). The results from the IHC experiments were evaluated separately by two observers (R.F. and L.D.V.), blinded to the histological diagnosis and grading of the tumors.siRNAs and TransfectionsA specific small interfering RNA (siRNA) (5′-AAGGUUCAGACCAUCUUGGAA-3′) targeting bag3 mRNA and a control, nontargeted (NT) RNA (5′-CAGUCGCGUUUGCGACUGG-3′) were obtained from Dharmacon (Thermo Fisher Scientific, La Fayette, CO). Glioma cell lines were transfected with siRNAs at final concentration of 100 nmol/L using TransIT-TKO reagent (Mirus Bio, Madison, WI). Cells were harvested at indicated time points.Statistical AnalysisResults are expressed as means ± SD or ± SE. Data were analyzed by Student's t-test or χ2 test using GraphPad Prism statistical software version 4.01 (La Jolla, CA). P values from 0.01 to 0.05 were considered significant, P values from 0.001 to <0.01 were considered very significant, and P values of <0.001 were considered highly significant.Stereotactic Surgeries, C6 Cell Implants, and siRNA TreatmentsRats were housed in an animal facility and were maintained in a temperature-controlled and light-controlled environment with an alternating 12-hour light/dark cycle. All protocols were approved by the local Ethical Committee (DiFarma). For the surgical procedures, all instruments were sterilized beforehand and sterile small-animal surgical techniques were used. The rats were allowed to feed and drink freely until the time of the surgery. Animals were anesthetized by intraperitoneal injection with a ketamine/xylazine solution (200 mg ketamine and 20 mg xylazine in 10 mL of saline solution) at a dosage of 0.15 mg per 10 g body weight. Once the rats were anesthetized, the head of the animals was shaved and positioned in a stereotactic frame with a rat teeth adaptor (World Precision Instruments, Sarasota, FL). The skin was prepared with povidone-iodine 10% and alcohol and a 2- to 3-mm incision was made at the midline and anterior to the interaural line, for clear identification of the bregma and lambda sutures. A burr hole was drilled in the skull at 1.4 mm anterior to bregma and 2.5 mm lateral to the midsagittal suture (the coordinates for the caudate putamen), as described previously.25Lal S. Lacroix M. Tofilon P. Fuller G.N. Sawaya R. Lang F.F. An implantable guide-screw system for brain tumor studies in small animals.J Neurosurg. 2000; 92: 326-333Crossref PubMed Scopus (189) Google Scholar A guide screw with an 0.5-mm channel (Plastics One, Roanoke, VA) was inserted into the drilled hole. The top of the screw was approximately 1 mm above the skull surface, and its shaft protruded through the dura and into the brain surface. For glioma formation, 1 × 106 C6 cells suspended in 10 μL of PBS were placed in a 26-gauge Hamilton syringe and inoculated through the guide screw, 4.5 mm down from the surface, to reach the caudate putamen, with an automated microinfuser pump (World Precision Instruments). Finally, a cross-shaped stylet was placed in the central hole of the guide screw, to prevent neoplastic cells from growing into the guide screw hole.After 2 weeks, the animals were separated into two groups for treatment, with one group receiving BAG3 siRNA and the second group a nontargeted (NT) siRNA as control. The animals were again anesthetized and placed into the stereotactic frame as described above. After removal of the stylet, siRNA was delivered in a Hamilton syringe through the hole in the screw. The guide screw ensured that the tip of the needle penetrated the glioma cells implant and that, as a result, the siRNAs were delivered directly into the tumor. Treatments were performed three times per week for 3 weeks, after which the animals were euthanized. Rat brains were removed from the cranial cavity, bisected coronally at the injection site, fixed in 10% formalin for 3 days, and embedded in paraffin; sections (4 μm thick) were stained with H&E for routine histological evaluation. The maximum cross-sectional area of the intracranial glioblastomas was used to determine tumor area by computer-assisted image analysis (Olympus cellSens version 1.4).Subcellular Fractionation and Western Blot AnalysisCells were harvested and washed twice with PBS 1× solution (Mediatech, Herndon, VA). Total proteins were extracted in Tris 250 mmol/L, pH 7.6, supplemented with a protease inhibitor cocktail (Sigma-Aldrich), using five cycles of freeze and thaw. The lysates were centrifuged at 15,000 × g at 4°C and the soluble fractions were collected. Protein concentrations were measured using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of total protein (30 μg) from each sample were separated electrophoretically in a 12% SDS-PAGE and were blotted on a nitrocellulose membrane (Hybond; Amersham Life Sciences, St Louis, MO). Immunodetection was performed using an enzymatic chemiluminescence kit (ECL Plus; Amersham Biosciences, Piscataway, NJ) according to the protocol provided by the manufacturer. Cell cytosolic and mitochondrial fractions were generated by a digitonin-based subcellular fractionation technique as described previously.26Sun X.M. Bratton S.B. Butterworth M. MacFarlane M. Cohen G.M. Bcl-2 and Bcl-XL inhibit CD95-mediated apoptosis by preventing mitochondrial release of Smac/DIABLO and subsequent inactivation of X-linked inhibitor-of-apoptosis protein.J Biol Chem. 2002; 277: 11345-11351Crossref PubMed Scopus (203) Google ScholarTUNEL AssayLabeling of apoptotic cells from the tumor glioma cells implanted in the rat brain was performed using a TUNEL ApopTag peroxidase in situ apoptosis detection kit following the manufacturer's instructions (Intergen, Purchase, NY). Briefly, deparaffinized sections were pretreated with 30 μg/mL of proteinase K (Roche Diagnostics, Indianapolis, IN). After endogenous peroxidase quenching, sections were treated with digoxigenin nucleotide-containing reaction buffer, terminal deoxynucleotidyl transferase, and the modified nucleotides were then detected by immunolabeling with anti-digoxigenin antibody. Finally, sections were developed with diaminobenzidine (Dako, Carpinteria, CA) and counterstained with hematoxylin. A breast carcinoma sample provided by the manufacturer was used as a positive control for apoptosis. Incubation of the sections with reaction buffer, but without terminal deoxynucleotidyl transferase, was performed as a negative control.ResultsUsing a BAG3-specific monoclonal antibody, we investigated BAG3 expression in patients affected by gliomas of various grades, taking advantage of a TMA representing 151 cases of tumors (13 grade I astrocytomas, 85 grade II astrocytomas, 17 malignant astrocytomas, and 36 glioblastomas), along with 8 cancer-adjacent normal tissue samples and 8 normal brain tissues. Positivity to BAG3 staining increased in more aggressive tumors, whereas normal brain samples were completely BAG3 negative (Figure 1A). The number of BAG3 positive cells within the tumor samples increased with tumor aggressiveness, and a statistically significant difference was observed when comparing glioblastomas to low grade astrocytomas (P = 0.0003, versus grade I; P = 0.017, versus grade II) (Figure 1B). We defined a scoring system to analyze biopsy samples based on the percentage of positive cells: BAG3 negative ( 40% positive cells). Distribution of BAG3 positivity based on these criteria is summarized in Table 1. Over all tumor samples studied, 81% were BAG3 positive; the percentage of positive cells increased in more aggressive tumors, reaching 63.9% of high-positive biopsy samples in the GBM group (Table 1). A χ2 test confirmed significance of the differences among tumor groups.Table 1Clinicodemographic and Pathology Data, with Distribution of BAG3 PositivityAge (years ± SD)M/F (no.)Samples, no.BAG3 negative, no. (%)BAG3 positive, no. (%)BAG3+low BAG3+high BAG3+Tissue type NBT31.6 ± 16.31/788 (100)0 (0) NBT adjacent to brain tumor45.5 ± 11.26/288 (100)0 (0) Brain tumor tissue (combined N = 151)46.2 ± 10.477/7415114 (9)137 (91) Astrocytoma grade I133 (23)6 (46)4 (31) Astrocytoma grade II856 (7)43 (50.6)36 (42.4) Anaplastic astrocytoma (grade III)171 (5.9)6 (35.3)10 (58.8) Glioblastoma multiforme364 (11.1)9 (25)23 (63.9)⁎P = 0.013. 3×3 contingency table for BAG3 levels distribution between Glioblastoma Multiforme, Anaplastic astrocytoma, and Astrocytoma grade II groups.BAG3 negative, 40% positive cells.NBT, normal brain tissue. P = 0.013. 3×3 contingency table for BAG3 levels distribution between Glioblastoma Multiforme, Anaplastic astrocytoma, and Astrocytoma grade II groups. Open table in a new tab Because BAG3 has a well-established antiapo
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