Primary Glioblastoma Multiforme Cell Lines Research Articles

Dual blockade of EGFR and PI3K signaling pathways offers a therapeutic strategy for glioblastoma

BackgroundGlioblastoma multiforme (GBM) is a devastating disease that lacks effective drugs for targeted therapy. Previously, we found that the third-generation epidermal growth factor receptor (EGFR) inhibitor AZD-9291 persistently blocked the activation of the ERK pathway but had no inhibitory effect on the phosphoinositide 3-kinase (PI3K)/Akt pathway. Given that the PI3K inhibitor GDC-0084 is being evaluated in phase I/II clinical trials of GBM treatment, we hypothesized that combined inhibition of the EGFR/ERK and PI3K/Akt pathways may have a synergistic effect in the treatment of GBM.MethodsThe synergistic effects of cotreatment with AZD-9291 and GDC-0084 were validated using cell viability assays in GBM and primary GBM cell lines. Moreover, the underlying inhibitory mechanisms were assessed through colony formation, EdU proliferation, and cell cycle assays, as well as RNA-seq analyses and western blot. The therapeutic effects of the drug combination on tumor growth and survival were investigated in mice bearing tumors using subcutaneously or intracranially injected LN229 xenografts.ResultsCombined treatment with AZD-9291 and GDC-0084 synergistically inhibited the proliferation and clonogenic survival, as well as induced cell cycle arrest of GBM cells and primary GBM cells, compared to monotherapy. Moreover, AZD-9291 plus GDC-0084 combination therapy significantly inhibited the growth of subcutaneous tumors and orthotopic brain tumor xenografts, thus prolonging the survival of tumor-bearing mice. More importantly, the combination of AZD-9291 and GDC-0084 simultaneously blocked the activation of the EGFR/MEK/ERK and PI3K/AKT/mTOR signaling pathways, thereby exerting significant antitumor activity.ConclusionOur findings demonstrate that the combined blockade of the EGFR/MEK/ERK and PI3K/AKT/mTOR pathways is more effective against GBM than inhibition of each pathway alone, both in vitro and in vivo. Our results suggest that AZD-9291 combined with GDC-0084 may be considered as a potential treatment strategy in future clinical trials.396yfhm1sdcH9_KhVkr-McVideo

Temozolomide-induced increase of tumorigenicity can be diminished by targeting of mitochondria in in vitro models of patient individual glioblastoma

Glioblastoma multiforme (GBM) is a highly heterogeneous and aggressive brain tumor with a dismal prognosis. Development of resistance towards cytostatic drugs like the GBM standard drug temozolomide is a severe problem in GBM treatment. One potential source of GBM relapse could be so called cancer stem like cells (CSCs). These represent an undifferentiated subpopulation of cells with high potential for tumor initiation. Furthermore, it has been shown that differentiated GBM cells can regain CSC properties when exposed to continuous temozolomide treatment in vitro. In this study, treatment of several primary GBM cell lines with clinically relevant doses of temozolomide increased their tumorigenicity as determined by colony formation assays in soft agar. Increased tumorigenicity is a known property of CSCs. Hence, therapy options that specifically target CSCs are under investigation. CSCs appear to be particularly dependent on mitochondria biogenesis which may represent a useful target for CSC elimination. Toxicity towards mitochondria is a known side effect of several antibiotics. Thus, addition of antibiotics like doxycycline may represent a useful tool to inhibit CSCs in GBM. Here, we show that combining temozolomide treatment of primary GBM cells with doxycycline could counteract the increase of tumorigenicity induced by temozolomide treatment.

Abstract 1959: Global microRNA expression analysis of Sox-2 positive and negative glioblastoma cells

Abstract Introduction: Glioblastoma multiforme (GBM) is the most frequent intracranial malignity of astrocytic origin in adults. This tumor is characterized by infaust prognosis, which is primarily caused by resistance to the therapy and early relapses relate to the presence of glioblastoma stem cells (GSCs). These cells form neurospheres in vitro and express markers of stemness such as Sox-2, Oct-4, and nestin. Targeting of GSCs could be a novel promising therapeutic approach leading to the overcome of therapy resistance and better prognosis of GBM patients. One of the approaches how to successfully regulate GSC is a targeted regulation of microRNAs (miRNAs). These small non-coding RNA molecules post-transcriptionally regulate an expression of more than 2/3 of all human genes that are also involved in stem cell associated signaling pathways. Moreover, deregulated expression of some miRNAs has been observed in many cancers including GBM. Cell lines and Methods: We have prepared eleven Sox-2 positive and negative paired primary GBM cell lines, which have been cultured under DMEM/F12 medium supplemented with bFGF, EGF, and B12 supplement and DMEM medium supplemented with 10% FBS, respectively. The global miRNA expression analysis was performed using GeneChip miRNA 4.0 Array (Affymetrix). Targeted regulation of miRNA levels have been carried out by the transient transfection of specific anti-miRNAs or miRNA mimics in GSC cell lines NCH 601 acquired from Interdisciplinary Center For Neurosciences (Heidelberg, Germany). The sphere formation assay was analyzed using IncuCyte ZOOM instrument (Essen BioScience). Sox-2 and nestin expressions were analyzed on both protein and transcriptional levels using combination of PAGE with Western blotting and real-time PCR, respectively. Results: Analysis of Sox-2 positive and negative paired GBM cell lines revealed 29 differentially expressed miRNAs, from which miR-93-3p, miR-95-5p, miR-106b-5p, miR-22-3p, and miR-3195 showed high significance (adjust. P value < 0.05) and association with both Sox-2 (Spearman R; p < 0.002) and nestin (Spearman R; p < 0.02) expressions. MiR-22-3p and miR-3195 showed lower expression whereas other miRNAs higher expression in Sox-2 positive GBM cells. The in vitro analyses also suggest that miR-22-3p and miR-106b-5p affect the sphere formation potential and Sox-2 expression in GSC cells. Conclusion: Taken together, our data suggest that miR-22-3p and miR-106b-5p are probably involved in GSC biology and, thus, should be promising molecular targets to overcome GBM therapeutic resistance. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A and 15-34678A. All rights reserved. Citation Format: Jiri Sana, Marek Vecera, Romana Butova, Pavel Fadrus, Leos Kren, Jaroslav Juracek, Robert Illiev, Jitka Mlcochova, Zuzana Ozanova, Petra Vychytilova, Ondrej Slaby. Global microRNA expression analysis of Sox-2 positive and negative glioblastoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1959.

A combination of tyrosine kinase inhibitors, crizotinib and dasatinib for the treatment of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Despite the advances in surgery, radiotherapy and chemotherapy, patient survival averages only 14.6 months. In most GBM tumors, tyrosine kinases show increased activity and/or expression and actively contribute to the development, recurrence and onset of treatment resistance; making their inhibition an appealing therapeutic strategy. We compared the cytotoxicity of 12 tyrosine kinase inhibitors in vitro. A combination of crizotinib and dasatinib emerged as the most cytotoxic across established and primary human GBM cell lines. The combination treatment induced apoptotic cell death and polyploidy. Furthermore, the combination treatment led to the altered expression and localization of several tyrosine kinase receptors such as Met and EGFR and downstream effectors as such as SRC. Furthermore, the combination treatment reduced the migration and invasion of GBM cells and prevented endothelial cell tube formation in vitro. Overall, our study demonstrated the broad specificity of a combination of crizotinib and dasatinib across multiple GBM cell lines. These findings provide insight into the development of alternative therapy for the treatment of GBM.

Abstract 3317: Radiation induced multinucleated giant cells: A novel therapeutic target to prevent survival and relapse of glioblastoma

Abstract In this study, we address the fundamental issue of radiation resistance in Glioblastoma Multiforme (GBM) a highly malignant form of brain tumor. However, because of the unavailability of multiple biopsies of GBM patients after treatment, we are limited in our understanding of resistance mechanisms. To circumvent this problem we recapitulated clinical scenario of GBM resistance in a cellular model developed from fresh naive primary GBM patient samples and cell lines. Using our model we show that upon lethal dose of radiation, small percentage of GBM cells survive, are non-apoptotic and transiently become non- proliferative. They are arrested in G2/M phase of cell cycle mediated by inhibitory phosphorylation of Cdk1(Y15) and p21. Surprisingly, we find although non -proliferative, these cells are highly motile and undergo homotypic cell-cell fusions at high frequency to form multinucleated and giant cells (MNGCs). Cell fusion leads to the induction of senescence as seen by β-galactosidase staining and accordingly high expression of SASPs (senescence associated secretory proteins like GM-CSF, SCF, IL-6 and IL-8) is also observed. SASPs provide survival and proliferation signals to the cells in an autocrine manner. Accordingly, we find enhanced expression of anti-apoptotic genes BIRC3 and Bcl-xL and unaltered levels of Bax (pro-apoptotic protein) mRNA in the resistant cells. MNGCs also activate AKT, which is a survival signal and a central convergence node of signals downstream to SASPs. AKT activation along with Bcl-xL is reported to regulate apoptosis synergistically. Additionally, resistant cells also undergo active DNA damage repair mediated by sensory kinases ATM and ATR. Interestingly, irrespective of the phase of cell cycle, they adopt Non Homologous End Joining (NHEJ) as their pathway of choice to repair their double strand breaks in DNA. Since we find increased presence of H3K36me2 marks known to modulate NHEJ, a possible link between NHEJ and this modification in GBM is worth exploring. Finally, unlike previous reports we demonstrate that MNGCs do not die of mitotic catastrophe instead escape senescence, undergo normal cell division and proliferation giving rise to mono-nucleated relapse cells with unaltered ploidy compared to parent cells. Importantly, we also show the potential ability to prevent the relapse in Glioma by disrupting the non-proliferative state of radiation resistant cells using mitotic inducer (MK1775) and selectively ablating growth of MNGCs by cytokinesis inhibitor. In summary, our data provides a mechanistic explanation for clinical observations and novel insights into an unexplored multi-step process of radiation survival and relapse in Glioblastoma We identified homotypic cell fusions of resistant Glioma cells as a novel non-genetic mechanism to sustain survival and relapse and generate rationale for novel combinatorial targeted therapies. Citation Format: Ekjot Kaur, Jacinth Rajendra, Sanket Shah, Jyothi Nair, Ankit Khushwaha, Aliasgar Moiyadi, Shilpee Dutt. Radiation induced multinucleated giant cells: A novel therapeutic target to prevent survival and relapse of glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3317. doi:10.1158/1538-7445.AM2015-3317

Hypoxia upregulates aldehyde dehydrogenase isoform 1 (ALDH1) expression and induces functional stem cell characteristics in human glioblastoma cells

Aldehyde dehydrogenase 1 (ALDH1) has been used to isolate tumorigenic stem-like cells in a large number of tumors, including glioblastoma multiforme (GBM). We recently showed that human glioblastoma cells with high ALDH1 (ALDH1(high)) activity contain stem-cell-like characteristics. In the study reported here, we isolated established and primary human glioblastoma cells based on their ALDH1 expression. When tested for asymmetric division, only cells with ALDH1(high) expression were able to restore heterogeneous populations after a few days, whereas cells with ALDH1(low) levels could not. Most interestingly, the capacity of cells with ALDH1(low) levels to divide asymmetrically into cells with either ALDH1(high) or ALDH1(low) expression could be restored after exposure to hypoxic culture conditions. Consequently, we found neurosphere formation reinstated in posthypoxic, formerly ALDH1(low), cells. The direct involvement of ALDH1 could be confirmed by ALDH1 small hairpin ribonucleic acid (shRNA) knockdown, suggesting ALDH1 as an intracellular marker for the identification and isolation of stem-like glioblastoma cells. In summary, we show that ALDH1 expression correlates well with asymmetric division capacity and tumor sphere formation. Furthermore, we demonstrated that hypoxic culture conditions induce and/or upregulate ALDH1 expression in established and primary GBM cell lines.

FoxM1 Inhibition Sensitizes Resistant Glioblastoma Cells to Temozolomide by Downregulating the Expression of DNA-Repair Gene Rad51

Recurrent glioblastoma multiforme (GBM) is characterized by resistance to radiotherapy and chemotherapy and a poor clinical prognosis. In this study, we investigated the role of the oncogenic transcription factor FoxM1 in GBM cells' resistance to alkylator temozolomide (TMZ) and its potential molecular mechanism. FoxM1 expression levels were measured by immunohistochemical analysis in 38 pairs of primary and recurrent GBM tumor samples. Expression levels were also measured in primary recurrent GBM cell lines, and their responses to TMZ were characterized. In a mechanistic study, an siRNA array was used to identify downstream genes, and a chromatin immunoprecipitation assay was used to confirm transcriptional regulation. Recurrent tumors that were TMZ resistant expressed higher levels of FoxM1 than did primary tumors. Recurrent GBM cell lines expressed higher levels of FoxM1 and the DNA damage repair gene Rad51 and were resistant to TMZ. TMZ treatment led to increased FoxM1 and Rad51 expression. FoxM1 knockdown inhibited Rad51 expression and sensitized recurrent GBM cells to TMZ cytotoxicity. FoxM1 directly regulated Rad51 expression through 2 FoxM1-specific binding sites in its promoter. Rad51 reexpression partially rescued TMZ resistance in FoxM1-knockdown recurrent GBM cells. A direct correlation between FoxM1 expression and Rad51 expression was evident in recurrent GBM tumor samples. Targeting the FoxM1-Rad51 axis may be an effective method to reverse TMZ resistance in recurrent GBM.

Therapeutic stem cells expressing variants of EGFR-specific nanobodies have antitumor effects.

The deregulation of the epidermal growth factor receptor (EGFR) has a significant role in the progression of tumors. Despite the development of a number of EGFR-targeting agents that can arrest tumor growth, their success in the clinic is limited in several tumor types, particularly in the highly malignant glioblastoma multiforme (GBM). In this study, we generated and characterized EGFR-specific nanobodies (ENb) and imageable and proapoptotic ENb immunoconjugates released from stem cells (SC) to ultimately develop a unique EGFR-targeted therapy for GBM. We show that ENbs released from SCs specifically localize to tumors, inhibit EGFR signaling resulting in reduced GBM growth and invasiveness in vitro and in vivo in both established and primary GBM cell lines. We also show that ENb primes GBM cells for proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Furthermore, SC-delivered immunoconjugates of ENb and TRAIL target a wide spectrum of GBM cell types with varying degrees of TRAIL resistance and significantly reduce GBM growth and invasion in both established and primary invasive GBM in mice. This study demonstrates the efficacy of SC-based EGFR targeted therapy in GBMs and provides a unique approach with clinical implications.

Enhanced stability and activity of temozolomide in primary glioblastoma multiforme cells with cucurbit[n]uril

Temozolomide (TMZ) is the primary chemotherapeutic agent for treatment of glioblastoma multiforme (GBM) yet it has a fast rate of degradation under physiological conditions to the 'active' MTIC, which has poor penetration of the blood-brain barrier and cellular absorption. Herein we have demonstrated binding of TMZ within the cavity of nano-container cucurbit[7]uril, resulting in a decreased rate of drug degradation. Prolonging the lifetime of the TMZ under physiological conditions through encapsulation dramatically improved the drug's activity against primary GBM cell lines as more TMZ could be absorbed by the cells before degradation. This work can potentially lead to increases in the drug's propensity for crossing the blood-brain barrier and absorption into the GBM cells, thereby increasing the efficacy of this chemotherapy.

Alkylpurine–DNA–N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients

Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). O⁶-methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O⁶-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG), which repairs the cytotoxic lesions N³-methyladenine and N⁷-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.

Abstract 2484: Aurora B silencing attenuates radiation resistance through PARP and identification of a missense mutation in aurora B as a determinant of radiosensitization in gliomas

Abstract Purpose: Aurora B (Aur B), a mitotic protein located on chromosome 17p13.1, is critical for proper chromosome segregation and changes in chromosome 17p are quite common in glioblastoma multiforme (GBM). Upregulation of Aur B induces multicellularity and fosters polyploidy. Aur B reaches maximum levels later in mitosis and regulates the chromosomal passenger complex progression. It has been reported that Aur B expression markedly correlates with short survival of GBM patients and may be a prognostic factor. Targeting Aur B is an ideal strategy, since it is active during mitosis and non-proliferating cells would not be adversely affected. Materials and Methods: The cell lines used for this study include both established and primary GBM cell lines. Radiation experiments were conducted using gammacell irradiator. DNA damage was estimated using comet assay. Apoptosis, cell cycle and multinucleated polyploid cells were estimated using flow cytometry. Gene silencing and overexpression were carried out using established protocols. Results and Discussion: Aur B expression increases after radiation in GBM cells and not in normal human astrocytes as determined both at transcriptional and translational levels. The radiosensitivity of cells lines was estimated by clonogenic survival assay. Through sequencing Aur B, we discovered mutations at the 885(C) and 893(T) residues in the GBM cell lines. The 885(C) was a silent mutation and 893(T) leads to an amino acid change from methionine (M) to threonine (T). This missense mutation leads to a new phospho site. The GBM cell lines tested had heterozygous and homozygous deletions. The radiosensitivity assay conducted on these cell lines shows a differential response with regards to the nature of mutation. The cell lines which had homozygous deletions on both sites 885(C) and 893(T) exhibit an increased radioresistance under in vitro conditions when compared to one of them being a heterozygote. Further, we show that Aur B silencing inhibits cytokinesis leading to 4N or 8N DNA content in p53 functional cell lines. The endoreduplicated cells due to Aur-B inhibition leads to the loss of viability in p53 mutant/null cell lines. Overexpression of Aur B increases the DNA repair enzyme PARP and scaffolding protein XRCC1 after radiation, which are critical for the activation and recruitment of Base Excision Repair. Whereas, silencing Aur B decreases the PARP expression levels. Conclusion: In GBM cell lines, we report that Aur B knockdown sensitizes cells to radiation and over expression of Aur B leads to radioresistance. Thus far, one of the prime targets in mediating this effect was found to be PARP, since silencing and forced induction of Aur B correlates with expression levels of PARP. For the first time, we have shown that the mutational status of Aur B alters the radiation sensitivity. Further in vivo studies are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2484. doi:10.1158/1538-7445.AM2011-2484

Attenuated AMPA Receptor Expression Allows Glioblastoma Cell Survival in Glutamate-Rich Environment

BackgroundGlioblastoma multiforme (GBM) cells secrete large amounts of glutamate that can trigger AMPA-type glutamate receptors (AMPARs). This commonly results in Na+ and Ca2+-permeability and thereby in excitotoxic cell death of the surrounding neurons. Here we investigated how the GBM cells themselves survive in a glutamate-rich environment.Methods and Findings In silico analysis of published reports shows down-regulation of all ionotropic glutamate receptors in GBM as compared to normal brain. In vitro, in all GBM samples tested, mRNA expression of AMPAR subunit GluR1, 2 and 4 was relatively low compared to adult and fetal total brain mRNA and adult cerebellum mRNA. These findings were in line with primary GBM samples, in which protein expression patterns were down-regulated as compared to the normal tissue. Furthermore, mislocalized expression of these receptors was found. Sequence analysis of GluR2 RNA in primary and established GBM cell lines showed that the GluR2 subunit was found to be partly unedited.ConclusionsTogether with the lack of functional effect of AMPAR inhibition by NBQX our results suggest that down-regulation and afunctionality of AMPARs, enable GBM cells to survive in a high glutamate environment without going into excitotoxic cell death themselves. It can be speculated that specific AMPA receptor inhibitors may protect normal neurons against the high glutamate microenvironment of GBM tumors.

Radiation Enhances the Invasive Potential of Primary Glioblastoma Cells via Activation of the Rho Signaling Pathway

Glioblastoma multiforme (GBM) is among the most treatment-refractory of all human tumors. Radiation is effective at prolonging survival of GBM patients; however, the vast majority of GBM patients demonstrate progression at or near the site of original treatment. We have identified primary GBM cell lines that demonstrate increased invasive potential upon radiation exposure. As this represents a novel mechanism by which radiation-treated GBMs can fail therapy, we further investigated the identity of downstream signaling molecules that enhance the invasive phenotype of irradiated GBMs. Matrigel matrices were used to compare the extent of invasion of irradiated vs. non-irradiated GBM cell lines UN3 and GM2. The in vitro invasive potential of these irradiated cells were characterized in the presence of both pharmacologic and dominant negative inhibitors of extracellular matrix and cell signaling molecules including MMP, uPA, IGFR, EGFR, PI-3K, AKT, and Rho kinase. The effect of radiation on the expression of these signaling molecules was determined with Western blot assays. Ultimately, the in vitro tumor invasion results were confirmed using an in vivo 9L GBM model in rats. Using the primary GBM cell lines UN3 and GM2, we found that radiation enhances the invasive potential of these cells via activation of EGFR and IGFR1. Our findings suggest that activation of Rho signaling via PI-3K is required for radiation-induced invasion, although not required for invasion under physiologic conditions. This report clearly demonstrates that radiation-mediated invasion is fundamentally distinct from invasion under normal cellular physiology and identifies potential therapeutic targets to overcome this phenomenon.

Proteins and Protein Pattern Differences between Glioma Cell Lines and Glioblastoma Multiforme

Research into the pathogenesis, molecular signaling, and treatment of glioblastoma multiforme (GBM) has traditionally been conducted using cell lines derived from malignant gliomas. We compared protein expression patterns between solid primary GBMs and GBM cell lines to identify proteins whose expression may be altered in cell culture. We cultured cell lines U87, U118, U251, and A172 and used tissue-selective microdissection of eight primary GBMs to obtain pure populations of tumor cells, which we studied using two-dimensional gel electrophoresis (2DGE) and examined using differential expression software. Select protein targets expressed differentially between GBM tumors and GBM cell lines were sequenced using tandem mass spectrometry. Analysis of the primary GBM tumor samples (n = 8) and the GBM cell lines revealed reproducibly similar proteomic patterns for each group, which distinguished tumors from the cell lines. Gels contained up to 500 proteins that were consistently identified in the pH 4 to 7 range. Comparison of proteins identified in the GBM tumors and in the cell lines showed approximately 160 proteins that were gained and 60 proteins that were lost on culture. Using normalized intensity patterns from the 2DGE images, ANOVA tests were done and statistically significant spots were identified. Seven proteins found in the cell lines were significantly increased when compared with the GBM tumors (P < 0.05), whereas 10 proteins were significantly decreased from cell lines compared with the GBM tumors. Proteins identified included transcription factors, tumor suppressor genes, cytoskeletal proteins, and cellular metabolic proteins. Global protein and proteomic differences were identified between primary GBM tumor samples and GBM cell lines. The proteins identified by 2DGE analysis elucidate some of the selection pressures of in vitro culture, help accentuate the advantages and limitations of cell culture, and may aid comprehension of gliomagenesis and enhance development of new therapeutics.

Changes in intracellular Ca2+ and pH in response to thapsigargin in human glioblastoma cells and normal astrocytes

Despite extensive work in the field of glioblastoma research no significant increase in survival rates for this devastating disease has been achieved. It is known that disturbance of intracellular Ca(2+) ([Ca(2+)](i)) and intracellular pH (pH(i)) regulation could be involved in tumor formation. The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is a major regulator of [Ca(2+)](i). We have investigated the effect of inhibition of SERCA by thapsigargin (TG) on [Ca(2+)](i) and pH(i) in human primary glioblastoma multiforme (GBM) cells and GBM cell lines, compared with normal human astrocytes, using the fluorescent indicators fura-2 and BCECF, respectively. Basal [Ca(2+)](i) was higher in SK-MG-1 and U87 MG but not in human primary GBM cells compared with normal astrocytes. However, in tumor cells, TG evoked a much larger and faster [Ca(2+)](i) increase than in normal astrocytes. This increase was prevented in nominally Ca(2+)-free buffer and by 2-APB, an inhibitor of store-operated Ca(2+) channels. In addition, TG-activated Ca(2+) influx, which was sensitive to 2-APB, was higher in all tumor cell lines and primary GBM cells compared with normal astrocytes. The pH(i) was also elevated in tumor cells compared with normal astrocytes. TG caused acidification of both normal and all GBM cells, but in the tumor cells, this acidification was followed by an amiloride- and 5-(N,N-hexamethylene)-amiloride-sensitive recovery, indicating involvement of a Na(+)/H(+) exchanger. In summary, inhibition of SERCA function revealed a significant divergence in intracellular Ca(2+) homeostasis and pH regulation in tumor cells compared with normal human astrocytes.

796 Proteins and Protein Pattern Differences between Glioma Cell Lines and Glioblastoma Multiforme

INTRODUCTION: Research into the pathogenesis, molecular signaling, and treatment of glioblastoma multiforme (GBM) has traditionally been conducted using cell lines derived from malignant gliomas. To identify proteins whose expression may be altered in cell culture, we compared protein expression patterns between solid primary GBMs and GBM cell lines. METHODS: Cultured human glioma cell lines U87, U118, U251, and A172 and tissue from selective microdissection of eight primary GBMs to obtain pure populations of tumor cells were studied using two-dimensional gel electrophoresis (2DGE) and differential expression software. Select protein targets expressed differentially between GBM tumors and GBM cell lines were sequenced using tandem mass spectrometry (MS/MS). RESULTS: Analysis of primary GBM (n = 8) and GBM cell lines revealed reproducibly similar and distinct proteomic patterns for each group, which distinguished tumors from the cell lines. Gels contained up to 500 proteins that were consistently identified in the pH 4 to 7 range. Comparison of proteins identified in the tumors and cell lines demonstrated approximately 160 proteins that were gained and 60 proteins that were lost on culture. Using normalized intensity patterns from the 2DGE images, analysis of variance tests were performed and statistically significant spots were identified. Seven proteins from the cell lines were increased compared with the tumors (P < 0.05), whereas 10 proteins were significantly decreased from cell lines compared with the tumors. Proteins identified included transcription factors, tumor suppressor genes, cytoskeletal proteins, and cellular metabolic proteins. CONCLUSION: Global protein and proteomic differences were identified between primary GBM tumor samples and human GBM cell lines. The proteins identified elucidate some of the selection pressures of in vitro culture, expose certain advantages and limitations of cell culture, and may enhance our understanding of gliomagenesis and spur development of new therapeutics.

AIM-2: a novel tumor antigen is expressed and presented by human glioma cells.

Antigen isolated from Immunoselected Melanoma-2 (AIM-2) was recently identified using melanoma-reactive CD8 T cells. AIM-2 antigen is expressed in a wide variety of tumor types, including neuroectodermal tumors, as well as breast, ovarian and colon carcinomas. In this study, we analyzed AIM-2 expression in glioblastoma multiforme (GBM) in primary cultured cells and established GBM cell lines. We found that the primary GBM cell lines expressed 88.4% and 93.0% of non-spliced and spliced AIM-2, respectively. Five out of seven of the established GBM cell lines expressed both non-spliced and spliced AIM-2. Furthermore, the C9 CTL clone, which is specific for AIM-2 peptide (RSDSGQQARY), efficiently recognized GBM tumor cells in an antigen-specific and HLA-A1 restricted manner. IFN-gamma treatment of the GBM tumor cells dramatically increased HLA-A1 expression levels and, consequently, increased CTL recognition of the treated tumor cells. More importantly, seven out of 12 HLA-A1 and AIM-2 positive patients from our dendritic cell clinical trial generated AIM-2 specific CTL activity in their PBMC after vaccinations. These data indicate that AIM-2 could be used as a tumor antigen target for monitoring vaccine trials or to develop antigen specific active immunotherapy for glioma patients.

Malignant Astrocytomas with Homozygous CDKN2/p16 Gene Deletions Have Higher Ki-67 Proliferation Indices

p16 is involved in a cell-cycle regulatory cascade that includes cyclin-dependent kinase 4 (cdk4), cyclin D1 and pRb. Alterations of each of these components have been described in primary human glioblastoma multiforme (GBM) or GBM cell lines, and alterations of the individual components of this pathway appear inversely correlated with one another. While this suggests that disruption of any individual component has similar oncogenic effects, homozygous deletions of the CDKN2/p16 gene are the most common genetic alteration. We investigated the relationship between homozygous CDKN2/ p16 deletions and cellular proliferation in 50 primary astrocytomas (2 WHO grade I pilocytic astrocytoma, 15 grade II astrocytomas, 20 grade III anaplastic astrocytomas and 13 grade IV GBMs). Using a comparative multiplex PCR assay, homozygous deletions of the CDKN2/p16 gene were detected in 5 anaplastic astrocytomas (25%) and 6 GBMs (46%), but in none of the lower-grade tumors. Ki-67 immunohistochemistry was used to assess the number of proliferating cells in the same samples used for molecular genetic analysis. In both anaplastic astrocytomas and GBMs, Ki-67 proliferation indices were significantly higher in tumors with CDKN2/p16 deletions (20%) than in those without deletions (10%; p = 0.0001). These results suggest that homozygous CDKN2/p16 deletions in high-grade astrocytomas may have a more deleterious effect on cell cycle control than the other aberrations in the p16-cdk4-cyclin D1-pRb pathway, and may provide one explanation for why homozygous CDKN2/p16 deletions are more common genetic events in high-grade astrocytomas than RB mutations or CDK4 amplification.