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Roles of the P2X7 receptor in C6 astroglioma: in vitro and in vivo studies Wei, Wei 2007

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Roles of the P2X7 receptor in C6 astroglioma: in vitro and in vivo studies by Wei Wei B.Sc., Tsinghua University, (P.R.China) 2005 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Pharmacology) THE UNIVERSITY OF BRITISH COLUMBIA December 2007 0 Wei Wei, 2007 Abstract The purinergic P2X7 receptor (P2X7R) is an ionotropic adenosine triphosphate (ATP) receptor which is closely linked with pathological conditions in the central nervous system (CNS). Gliomas are the most common primary brain tumors with presently no cures. The roles of the P2X7R in these diseases have not been previously studied and in this work, I have used the rat C6 glioma as an experimental model system to investigate expression and function of the P2X7R in vitro and in vivo. The in vitro study has examined expression of the P2X7R in C6 cells and the involvement of this receptor in mediating cell functional responses. C6 glioma cells were found to express the P2X7R at both mRNA and protein levels. The P2X 7R agonist, 2', 3 '-(benzoy1-4-benzoy1)-ATP (BzATP) induced an increase in intracellular Ca2+ concentration, an effect which was largely inhibited by periodate-oxidized ATP (OxATP), an irreversible P2X7R antagonist. BzATP treatment of C6 cells also resulted in ethidium bromide dye uptake indicating pore formation was induced by P2X7R activation. Chronic exposure of C6 cells to BzATP showed up-regulation of several pro-inflammatory factors including the chemokines monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) and the angiogenic factor vascular endothelial growth factor (VEGF) suggesting the P2X7R in C6 cells is involved in mediating inflammation in tumors. In addition, BzATP treatment was found to enhance wound-induced cell migration, an effect which was inhibited in the presence of OxATP, or another P2X7R antagonist, Brilliant Blue G (BBG). The in vivo study examined whether pharmacological modulation of P2X7R with BBG altered tumor growth. C6 glioma cells were implanted into the striatum of rat brain and in situ P2X7R expression was shown to be associated with glioma cells and resident microglia. Preliminary results have indicated that inhibition of P2X7R leads to a reduced volume of brain tumors formed by transplanted C6 cells. The overall results from this study demonstrate the novel finding that C6 glioma cells express functional P2X7R and suggest pharmacological modulation of the P2X7R could serve as an effective strategy to inhibit the development and progression of brain tumors. Ill Table of Contents Abstract^ ii Table of Contents^ iv List of Figures vi List of Abbreviations vii Acknowledgements^ x 1. Introduction  1 1.1. P2 receptors: general^  1 1.2. P2X7 receptors 2 1.2.1. The structure of the P2X7R^ 2 1.2.2. The pharmacological properties of the P2X7R^ 5 1.2.3. P2X7 receptors in brain: expression and functions  6 Expression of P2X7 receptors in different cell types in brain ^ 6 Functional roles of P2X7 receptors in glia^ 8 The pathological roles of the P2X7R in brain 11 1.3. Glioma brain tumors^  13 1.3.1. Glioma brain tumors: general^  13 1.3.2. C6 glioma: the experimental model system^  14 1.3.3. Microglia in brain tumors  15 1.4. Research hypotheses^ 20 2. Materials and Methods 21 2.1. In vitro studies 21 2.1.1. Cell Culture^ 21 2.1.2. Calcium-Sensitive Spectrofluorescence^ 21 2.1.3. Reverse-transcriptase polymerase chain reaction (RT-PCR)^ 22 2.1.4. Western blot analysis^  23 2.1.5. Immunocytochemistry of C6 glioma cells^ 24 2.1.6. Ethidium bromide (EtBr) uptake assay 25 2.1.7. Scratch-wound migration assay^ 25 2.1.8. Statistical analysis^ 27 2.2. In vivo studies^ 27 2.2.1. Intracerebral implantation of C6 cells^  27 2.2.2. Immunohistochemical analysis 28 2.2.3. Double immunofluorescence staining 29 2.2.4. BBG administration^  30 2.2.5. Quantification of tumor volumes^ 30 3. Results^  31 3.1 In vitro studies^  31 3.1.1. Intracellular calcium levels are altered in response to P2X7R agonist BzATP 31 3.1.2. P2X7 receptors are expressed in C6 cells and regulate expression of pro-inflammatory factors  33 iv 3.1.3. P2X7R protein is present in C6 cells and is up-regulated by BzATP stimulation^  35 3.1.4. P2X7R activation induces pore formation in C6 cells^ 38 3.1.5. P2X7 receptors in C6 glioma cells regulate cell mobility  40 3.2. In vivo studies^ 42 3.2.1. Tumor progression in the rat brain^ 42 3.2.2. Co-localization of P2X7R with C6/LacZ cells^ 44 3.2.3. Co-localization of P2X7R with microglia/macrophages and astrocytes ^ 46 3.2.4. Administration of BBG reduced the tumor size^ 49 4. Discussion 51 4.1. In vitro studies^  51 4.2. In vivo studies  55 5. Conclusions of thesis research and future directions^ 58 Bibliography^ 59 v List of Figures Figure 1-1. Structure of the P2X7R: membrane topology^ .3 Figure 1-2. Possible interactions between neoplastic cells, microglia, and macrophages in brain tumors^ 17 Figure 3-1. [Ca21i responses induced by the P2X7 agonist BzATP^32 Figure 3-2. Effects of BzATP stimulation of C6 glioma cells on expression of P2X7R and pro-inflammatory factors ^ 34 Figure 3-3. BzATP-induced up-regulation of P2X7R protein in C6 glioma ^ ..36 Figure 3-4. BzATP-induced uptake of ethidium bromide ^ ..39 Figure 3-5. BzATP effects on C6 repopulation of the wound area^ 41 Figure 3-6. Progression of C6 glioma^ 43 Figure 3-7. Co-localization of P2X7R with C6/LacZ cells ^ 45 Figure 3-8. Microglia and P2X7R stain throughout tumor 47 Figure 3-9. Astrocytes and P2X7R stain throughout tumor^ 48 Figure 3-10. Administration of BBG reduced the tumor size ^ 50 vi List of Abbreviations AD^ Alzheimer's disease Af3 amyloid-beta 2-AG^ 2-arachidonoylglycerol AP-1 activator protein-1 ATP^ adenosine triphosphate BBG Brilliant Blue G f3-gal^ beta-galactosidase BzATP 2', 3' -(benzoy1-4-benzoy1)-ATP [Ca21i^ intracellular calcium concentration CNS central nervous system CNTF^ ciliary neurotropic factor COX-2 cyclooxygenase-2 CT-1^ cardiotrophin-1 EGF epidermal growth factor ER^ endoplasmic reticulum ERK extracellular signal-regulated kinase EtBr^ ethidium bromide F12K Kaighn's modification of Ham's F12 medium GBM^ glioblastoma multiforme GFAP glial fibrillary acidic protein IL-1 /3^ interleulcin-113 vi i IL-6^ interleukin-6 IL-8 interleukin-8 IL-10^ interleukin-1 0 iNOS inducible nitric oxide synthase 1P3^ inositol triphosphate LIF leukemia inhibitory factor LPS^ lipopolysaccharide M-MLV moloney murine leukemia virus MCAO^ middle cerebral arterial occlusion MCP-1 monocyte chemoattractant protein-1 MG^ microglia MMP-9 matrix metalloprotease-9 MP^ macrophage NF-K13 nuclear factor-KB NO^ nitric oxide OSM oncostatin-M OxATP^ periodate-oxidized ATP P2XR P2X receptor P2YR^ P2Y receptor PGE2 prostaglandin E2 ROI^ reactive oxygen intermediates RT-PCR reverse-transcriptase polymerase chain reaction SEM^ standard error of mean VIII SOC^ store-operated channels SP substance P TGF-13^ transforming growth factor-f3 TNF-a tumor necrosis factor-a VEGF^ vascular endothelial growth factor ix Acknowledgements First and foremost I would like to express my sincerest gratitude to my supervisor, Dr. James G McLamon who has supported me throughout my graduate study with his patience and knowledge. Without his guidance, this work would never have been completed. I am also grateful to my committee members, Dr. Kenneth G Baimbridge, Dr. David V. Godin and Dr. William Jia for their valuable insights into this work. I would like to acknowledge my colleague Jae K. Rye and our past lab member Dr. Hyun B. Choi for their precious suggestions and technical assistance. Finally, I would like to thank my parents for always being there for me. I can feel your love and support although you are thousands of miles away from me. 1. Introduction 1.1. P2 receptors: general Adenosine triphosphate (ATP) is not only a cellular energy source but also a purinergic ligand with important signaling roles as a neurotransmitter and as a modulator of cell functions. ATP produces its effects via the activation of P2 receptors comprising the metabotropic P2Y receptor (P2YR) family and the ionotropic P2X receptor (P2XR) family (Erb, 2006; Illes, 2004). The P2Y receptors are G-protein-coupled and linked to inositol triphosphate (IP3)-mediated release of calcium (Ca 2+) from intracellular endoplasmic reticulum (ER) stores. Depletion of Ca2+ from ER stores can subsequently trigger entry of Ca2+ through store-operated channels (SOC) located in plasma membrane (McLarnon, 2005). To date, eight P2Y receptor subtypes have been cloned in mammals: P2Y1, P2Y2, P2Ya, P2Y6, P2Y1 1, P2Y12, P2Y13 and P2Y14 (Fries, 2006; White and Burnstock, 2006). The ionotropic P2XR family is coupled to non-selective cationic channels allowing influx of Na+ and Ca2+ and efflux of K. To date, seven distinct P2XR subtypes have been identified at the molecular level and named P2X1R-P2X7R. An important consequence of activation of P2XR is cell depolarization, which can serve as a modulatory factor in shaping cellular responses (North, 2002). A particularly unique member of the P2XR family is the subtype P2X7 receptor (P2X7R), which is described in considerable detail in section 1.2. 1.2. P2X7 receptors 1.2.1. The structure of the P2X7R The P2X7R was first described by Buisman et al. (1988) and labeled P2Z receptor (Di Virgilio, 1995), until its molecular identification. The full length cDNAs of the P2X7R were first cloned from a rat brain cDNA library. The P2X7 subunit is 595 amino acids long, having 35-40% homology with the other six members of the P2XR family (Surprenant et al., 1996). Its structure is basically similar to the residual P2X receptor subunits, having two transmembrane domains (M1, M2), a large extracellular domain and N- and C-terminal residues both on the cytoplasmic side (Surprenant et al., 1996). The extracellular loop contains 10 cysteine residues that can form disulfide bonds and a sequence stretch (aa 170-330) likely containing six-stranded 13-pleated sheets (Freist et al., 1998). This region has been proposed to host the ATP binding site (Gu et al., 2004). The main structural distinctive feature of the P2X7R is that its intracellular carboxy terminal domain is much longer (239 amino acids) than those of other P2XR subunits (27-129 amino acids). It is now believed that the long C-terminal tail harbors multiple potential protein and lipid interaction motifs (Denlinger et al., 2001). The representative membrane topology of the P2X7R subunit is presented in Figure 1-1. 2 Figure removed due to copyright reasons. Figure 1 -1. Structure of the P2X7R: membrane topology The full-length and truncated (\C) splice variant of the P2X 7R are shown. The truncated form lacks almost the entire COOH tail (249aa, green, red and short black traits) but bears an extra 18aa (light blue trait). TNFRI homology domain (green) and putative LPS-binding region (red) are shown. Cysteine residues forming putative disulfide bridges are also shown (Ferrari et al., 2006). 3 Following the cloning and expression of the rat P2X 7R, the genes encoding the human (Rssendren et al., 1997), mouse (Chessell et al., 1998) and Xenopus laevis (Paukert et al., 2002) P2X7R were also identified, having 80%, 85% and 50% amino acid sequence homology with the rat receptor, respectively. It is now also clear, that unlike other P2XR subunits, the P2X7 subtype does not hetero-oligomerize and functions only in homo-oligomeric form (Torres et al., 1999), most likely as a homo-trimer. The activation of P2X7R-gated ion channels leads to two distinct responses depending on the exposure time of the agonist. Following a single brief ATP application, a non-selective inward cationic current can be recorded which is similar to inward currents caused by the activation of homo- or hetero-oligomeric assemblies of other P2X receptor subunits, although with different deactivation kinetics. Upon repeated or prolonged application, the opening of a membrane pore can be detected which allows permeability to high molecular weight molecules and ions up to the size of 800 Da (Surprenant et al., 1996). The pore formation is characterized by the uptake of high molecular weight fluorescent dyes such as ethidium bromide, Lucifer yellow or YO-PRO. Pore activation is followed by cytoskeletal rearrangement such as membrane blebbing (Virginio et al., 1997, 1999a), which can eventually lead to apoptosis in immune cells. The long intracellular C-terminal domain has been shown to be instrumental for the pore-forming property of the P2X7R (Adriouch et al., 2002; Surprenant et al., 1996). Moreover, a lipopolysaccharide (LPS)-binding site has also been identified close to the carboxy terminus of the receptor (Denlinger et al., 2001), whereby the receptor could translate inflammatory signals to signal transduction 4 events. 1.2.2. The pharmacological properties of the P2X7R The pharmacological phenotype of the P2X7R is relatively well established and distinguishable from other members of the P2XR family. The main properties of the P2X7R are summarized below (Anderson and Nedergaard, 2006; Duan and Neary, 2006; North, 2002; Sperlagh et al., 2006). 1) A relatively low affinity of the P2X7R for ATP. The P2X7R generally requires higher concentrations of ATP for activation, in excess of 100 ii.M, compared to that required by other members of the P2 receptor family. 2) 2', 3'-(benzoyl-4-benzoyl)-ATP (BzATP), the most commonly used P2X7R agonist, is 10-30 times potent than ATP. Although BzATP also activates other P2X receptors, a higher potency of BzATP than that of ATP is often taken as evidence of P2X7R involvement. 3) The effect of ATP and BzATP is potentiated by reducing the concentration of extracellular divalent cations (Ca2÷ or Mg2+). This property indicates that ATP4" may be the active ligand. Since most of extracellular ATP is complexed with Mg 2+ or Ca2+ in normal physiological media, the proportion of ATP 4- in total added ATP is low and relatively high levels of ATP are required to activate the P2X7R. It is also possible that the divalent ions bind on the receptor and exert allosteric inhibition. 5 4) Relatively specific antagonists that can be used for identifying the P2X 7R include periodate-oxidized ATP (OxATP), Brilliant Blue G (BBG) and KN-62. OxATP, a commonly used antagonist that irreversibly blocks P2X7 receptors, is used at concentrations of 0.1-0.3 mM. OxATP is a slowly equilibrating antagonist so pre-treatment is required to allow enough time for blockade (Murgia et al., 1993). Another compound, BBG is a potent, noncompetitive antagonist displaying remarkable selectivity towards P2X7 receptors in the nanomolar range, having IC50 values of about 10 nM at the rat P2X7R (Jiang et al., 2000). A third antagonist which may be useful for the pharmacological identification of P2X7 receptors is KN-62, a selective antagonist at the human, but not the rat, P2X7R (Gargett and Wiley, 1997). 1.2.3. P2X7 receptors in brain: expression and functions Expression of P2X7 receptors in different cell types in brain Initial studies of the P2X7R distribution in brain concluded that P2X7 immunoreactivity was localized only to immune cells and reactive glial populations in infarct-surrounding regions following middle cerebral arterial occlusion (MCAO) (Collo et al., 1997). This outlook began to change in the late 1990s when several reports of P2X7R expression in neurons were published (Sperlagh et al, 2006). However, substantial controversy over the use of available antibodies has prompted some to conclude that neurons are actually devoid of the P2X7R (Anderson and Nedergaard, 2006). Therefore, it appears to be premature to draw final conclusions as 6 to the neuron-specific localization of P2X 7R. As noted above, glial cells have been thought to be the major cell types expressing P2X7R in the brain. According to current view, the function of glia is far more than a simple support for the neuronal network. Glial cells play an active role in information processing, controlling the homeostasis of the neuronal microenvironment, the development and differentiation of neural cells and the host-defense response. The main glial cell types of the nervous system are astrocytes, oligodendrocytes, and microglia. To fulfill their diverse roles, glial cells have to communicate with each other and with neurons. For this purpose, they utilize their own signaling systems and the P2X7R seems to have a pivotal role in these processes. Astrocytes express mRNA for the P2X 7R (Sperlagh et al., 2006). The P2X 7R immunoreactivity is present on the cell surface of human fetal astrocytes (Narcisse et al., 2005) and is co-localized with astrocytic markers in acutely isolated and cultured astrocytes (Panenka et al., 2001). Microglia originate from monocyte/macrophage precursors and are regarded as the major immunocompetent cell type in the brain. It has been known for a considerable time that microglia express both ionotropic and metabotropic receptors (Norenberg et al., 1994), and the presence of the "pore-forming" ATP receptor was also described before the molecular identification of the P2X7R (Ferrari et al., 1996). The expression P2X7R transcripts and of the corresponding protein has been confirmed later both in cultured microglia (Collo et al., 1997; Ferrari et al., 1997a) and in microglia activated following middle cerebral artery occlusion (Collo et al., 1997). 7 Functional roles of P2X7 receptors in glia Astrocytes: The astroglial P2X7R participates in a wide array of normal and pathological functions in brain. The primary intracellular signal following the activation of the P2X7R is a sustained elevation of intracellular Ca-")+ caused by Ca2+ influx from the extracellular space through the receptor-ion channel complex (Ballerini et al., 1996). This Ca2+ signal is then translated to various signal transduction pathways conveying diverse functions. However, it is noteworthy that some of the pathways are not dependent on Ca2+ . Thus, P2X7R activation seems to be a common trigger for astrocytic transmitters and modulators to enter the extracellular space either by Ca2+-dependent or by Ca2+-independent signaling pathways. It has been well known that astrocytes participate in neuroinflainmation underlying a variety of CNS insults including trauma, ischemia and neurodegeneration. Inflammatory stimuli rapidly induce the proliferation and hypertrophy of glial cells, a process called reactive gliosis, and respond with the expression and production of inflammatory cytokines, chemokines and other mediators. P2X7 receptors are involved in these regulatory pathways. For example, activation of the P2X7R promotes the expression of monocyte chemoattractant protein-1 (MCP-1), a critical factor in the early monocyte infiltration during the neuroinflammatory process (Panenka et al., 2001). Furthermore, phosphorylation of ERK1, ERK2 and p38 MAP kinase is increased; these have determinant roles in the commitment of the cells to apoptosis (Panenka et al., Wang et al., 2003). The phosphorylation of ERK1/2 is mediated through a cellular pathway that is dependent 8 on both Ca2+ and the expression of other intracellular signaling proteins, i.e., Pyk2, c-Scr, IP3K and MEK1/2, respectively (Gendron et al., 2003). Other signaling proteins regulated by the P2X7R include phospolipase D (PLD) (Sun et al., 1999), transforming growth factor-(31 (TGF-(31) (Wang et al., 2003) and protein kinase B (Akt) (Jacques-Silva et al., 2004). P2X7 receptors have been suggested to mediate the effect of ATP to potentiate interleukin-1 f3 (IL-1(3)-induced expression of nuclear factor-KB (NF-KB) and to promote activator protein-1 (AP-1) protein expression (John et al., 2001). In addition, a recent study revealed that the activation of the P2X7R attenuates LPS-induced tumor necrosis factor-a (TNF-a) release from primary cortical astrocytes (Kucher and Neary, 2005); this contrasts with corresponding data in microglia, where P2X7 receptors stimulate the production of this pro-inflammatory cytokine (Suzuki et al., 2004). Microglia: Microglia are the resident immune cells in the brain and they help maintain brain homeostatic mechanisms and detect changes in the brain microenvironment. In these functions, microglia can be considered as surveillance cells of the brain (Kreutzberg, 1996). Microglia can be rapidly activated in response to pathological signals such as ischemia and undergo morphological changes transforming the resting ramified cells to an ameboid form with phagocytic activity, proliferation and production of a wide array of inflammatory mediators. Microglia express P2X 7 receptors which convey functions analogous to those of other cells of the monocyte/macrophage lineage upon inflammatory stimuli. Primary microglial cultures and immortalized microglial cell lines respond to ATP and BzATP stimulation with an inward current (Chessell et al., 9 1997; Haas et al., 1996; Visentin et al., 1999), membrane depolarization, a sustained increase in intracellular Ca 2+ levels ([Ca21i) (Ferrari et al., 1996), and the uptake of dyes (Chessell et al., 1997; Ferrari et al., 1996). Activation of the P2X7R in microglia stimulates a host of intracellular signaling pathways and modulates cellular responses such as production of inflammatory and neurotoxic compounds. In microglia, P2X7R is considered as a key player in IL-1 processing and release (Ferrari et al., 2006). The mechanism underlying ATP-dependent IL-1(3 maturation and release involves an outwardly directed IC conductance and the activation of caspase-1 responsible for the cleavage of pro-IL-113 to the mature, 17kDa form (Sanz and Di Virgilio, 2000). ATP is known to be a full stimulus to induce TNF-a production via a Ca2+ -dependent, ERK/JNK/p38 signaling pathway (Hide et al., 2000; Suzuki et al., 2004). In addition, high concentrations of ATP induce inducible nitric oxide synthase (iNOS) mRNA expression and increases nitric oxide (NO) production from rat microglia (Ohtani et al., 2000), an effect likely mediated by the P2X7R. ATP and BzATP also promote generation of reactive oxygen intermediates (ROI), in particular superoxide radicals, dependent on extracellular Ca 2+ ; both OxATP and BBG prevent this effect. The actions of ATP are mediated by the p38MAPK pathway (Parvathenani et al., 2003). 2-arachidonoylglycerol (2-AG) is one of the most abundant endogenous cannabinoid ligands (endocannabinoids) and plays a critical role in neuroinflammation. The activation of the P2X7R using high concentration of ATP or BzATP markedly increases 2-AG production in primary microglial cultures (Witting et al., 2004). Finally, activated microglia can also provide neuroprotection by releasing trophic factors such as plasminogen (Inoue et al., 1998). This neurotrophic I 0 factor is known to promote the development of mesencephalic dopaminergic neurons and neurite outgrowth from explants of neo-cortical tissue. The pathological roles of the P2X7R in brain As mentioned in section 1.2.2, P2X7R generally require higher concentration of ATP for activation and this characteristic could indicate that activation of the P2X7R is associated with elevated levels of extracellular ATP. A number of pathological events are known to stimulate ATP release. Such pathological signals include hypotonic (Wang et al., 1996) and mechanical stimuli (Coco et al., 2003; Verderio and Matteoli, 2001), energy deprivation (Juranyi et al., 1999) and inflammatory signals such as bacterial lipopolysaccharide (LPS) (Ferrari et al., 1997b), concanvavalin-A (Filippini et al., 1990), or IL-10 (Sperlagh et al., 2004). In addition, taking into account that the free ATP concentration within living cells under the normal metabolic rate is in the millimolar range, cellular damage is hypothesized to result in very high regional levels of ATP. The up-regulation of the P2X7R has been observed in a number of pathological models including energy deprivation (Cavaliere et al., 2004), in vivo ischemia (Franke et al., 2004), epilepsy (Vianna et al., 2002), mechanical injury (Franke et al., 2001), transgenic models of Alzheimer's disease (AD) (Parathenani et al., 2003), as well as in human brain tissue samples from patients with sclerosis multiplex (Narcisse et al., 2005) and AD (Mclarnon et al., 2006). The first study showing an up-regulation of the P2X7R in brain was that of Collo 1 1 et al. (1997) who found increased immunostaining for the P2X7R in activated microglia of the zona penumbra in the MCAO model. The microglial up-regulation of the P2X7R in response to oxygen deprivation has also been documented in retinal microglia (Morigiwa et al., 2000). In a more recent study, an early (1 day after MCAO occlusion) up-regulation of the P2X7R protein was observed in microglial cells, and later (4-7 days after MCAO occlusion) the receptor was also over-expressed in neurons and astrocytes of the pre-infarct area in spontaneously hypersensitive rats (Franke et al., 2004). P2X7R immunoreactivity appears to be up-regulated around amyloid plaques in activated microglia and astrocytes in Tg2576 transgenic AD mice having mutant amyloid precursor protein (APP) (Parvathenani et al., 2003). Expression of the P2X7R is also up-regulated in microglia obtained from AD individuals compared with levels in nondemented (ND) brain (McLarnon et al., 2006). In addition, the P2X7R was markedly up-regulated in amyloid-f3 (A13)142-injected, compared with PBS- or reverse peptide A(342.1-injected, rat hippocampus (McLarnon et al., 2006). P2X7R immunostaining has also been localized in reactive astrocytes around lesions in autopsy brain sections of multiple sclerosis patients (Narcisse et al., 2005). Altogether these data indicate that the expression of the P2X7R is strongly activity-dependent during pathological conditions, suggesting that the receptor is a potential therapeutic target in neurodegenerative disorders. This has led to testing of protective roles of P2X7R antagonism in functional disease models. The neuroprotective action of non-selective P2 receptor antagonists is well documented in in vitro models of glutamate- (Volonte et al., 1999; Volonte and Merlo, 1996) and 12 kainate- (Zona et al., 2000) mediated excitotoxicity, glucose deprivation (Cavaliere et al., 2001b; Geng et al., 1997) and chemical hypoxia (Cavaliere et al., 2001a). At present, however, inhibition of the P2X 7R as a strategy to protect neurons has not been well studied. 1.3. Glioma brain tumors 13.1. Glioma brain tumors: general Gliomas are the most common brain tumors which originate from glial cells — astrocytes, oligodendrocytes, Schwann cells or their precursors. Astrocytomas are the most common, accounting for 4-5% of all cancer related deaths in North America (Kleihues and Caenee, 2000), and responsible for more than 40% of all central nervous system neoplasms (Kleihues et al., 1996). The World Health Organization (WHO) grades astrocytomas histopathologcially into four grades with increasing malignancy: Grade I and II astrocytomas can be considered as low-grade astrocytomas, while grade III and grade IV astrocytomas are highly malignant. Grade IV astrocytomas (glioblastoma multiforme; GBM) are composed of poorly differentiated cells with high mitotic activity and are specified by rapid growth and high vascularization accompanied by cell necrosis in less capillarized regions. Unfortunately, they are the most common primary brain tumors with mean survival time of only 5-12 months following diagnosis. Traditional treatment options for malignant gliomas include surgery, radiation therapy, and chemotherapy. While therapies for high-grade gliomas are helpful, at 13 present, these treatments cannot cure these tumors. The two major reasons are that tumor cells infiltrate into surrounding brain and thus cannot be completely removed by surgery and that most glioma cells are at least partially resistant to radiation and chemotherapy. Therefore, high-grade glioma cells almost always start to grow again at some point in time. Since these lethal brain tumors often outsmart traditional cancer treatment, new methods of therapy are required. In this regard a better understanding of malignant gliomas may help in the discovery of novel strategies to block their development and progression. In our study, rat C6 glioma has been used as an experimental model system. 1.3.2. C6 glioma: the experimental model system The C6 glioma cell line is a widely used cell line in neurobiological research and has been used as an experimental model system to elucidate the mechanisms underlying the aggressive nature of glioma brain tumors. Compared to primary neural cells, the C6 cell line offers some major advantages, such as easy accessibility and culture as well as availability of high cell numbers. The rapidly proliferating rat C6 glioma cell line was originally induced in random-bred Wistar-Furth rats by exposure to N,N'-nitroso-methylurea (Benda et al. 1968). When transplanted into rat brains, C6 cells form tumors with many characteristics of malignant glioma and the tumor formed by C6 cells was histopathologically classified as astrocytoma (Grobben et al., 2002). 14 The C6 glioma has been and will continue to be useful for a variety of studies related to brain tumor biology including studies on tumor growth, invasion and migration, capillary permeability, blood-brain barrier disruption, neovascularization, growth factor regulation and production and biomedical studies. It has also been used to evaluate the therapeutic efficacy of cancer treatments such as chemo-, radiation-, immuno- and gene therapies (Barth, 1998). In the C6 glioma model, a number of factors have been shown to participate in tumor growth, invasion and angiogenesis (extensively reviewed by Grobben et al., 2002). In addition, recent evidence suggests that ATP signaling may also be involved in glioma development and that P2 receptors might provide novel therapeutic targets in the treatment of brain cancer (Morrone et al., 2005; Morrone et al., 2006). Several purinergic receptors of the P2YR family are expressed in C6 cells including P2Y1, P2Y2 and P2Y12 (Czajkowski et al., 2002; Sabala et al., 2001). P2YR have been reported to mediate changes in [Ca21i following ATP application (Sabala et al, 2001) and to regulate glioma cell proliferation (Czajkowski et al., 2004). At present, however, no studies have reported involvement of P2XR in the progression of brain tumors. In this work, we document expression and functional responses of the subtype P2X7R in C6 glioma cells. 1.3.3. Microglia in brain tumors As noted above, microglia play important roles in inflammatory diseases of the CNS. These cells have been identified in brain neoplasms as well. The presence of 15 microglia in brain tumors was first reported by Rio-Hortega and Asua in 1921 (Rio-Hortega and Asua, 1921) and Penfield in 1925 (Penfield, 1925). In the 1980s, expanding interest in immunotherapy of brain tumors prompted the use of immunohistochemistry to characterize the inflammatory cell response to brain tumors (Rossi et al., 1988, 1987, 1989). These initial findings were confirmed later in both human and animal brain tumors, where a significant macrophage and microglia infiltration was demonstrated (Roggendorf et al., 1996; Shinonaga et al., 1988; Streit, 1994; Wierzba-Bobrowicz et al., 1994). Some studies also suggested a direct correlation between the grade of gliomas and levels of resident tumor microglia/macrophage (MG/MP) cells, indicating that gliomas may play an active role in MG/MP recruitment (Roggendorf et al., 1996). An interesting question is if the MG/MP response is due to local tissue damage or active chemoattraction by tumor cells. However, it has been known that a variety of chemokines and growth factors can be released directly from tumor cells themselves or as a result of local tissue injury due to tumor growth. This can result in recruitment of microglia and macrophages from two main sources: resident brain microglia or perivascular macrophages that can become activated and migrate toward brain tumors (Figure 1-2). The specific cytokins/chemokines involved in glioma recruitment of microglia remain poorly understood. However, MCP-1 is believed to be a major contributor to MG/MP recruitment to gliomas. MCP-1 is expressed at both mRNA and protein levels in gliomas (Leung et al., 1997) and it has been shown to functionally recruit microglia to gliomas in vivo, while promoting their proliferation (Platten et al., 2003). 16 Figure removed due to copyright reasons Figure 1-2. Possible interactions between neoplastic cells, microglia, and macrophages in brain tumors (Watters et al., 2005) 17 At present, the biological role of microglia and macrophages in gliomas remains unknown. It is also not known if the presence of microglia in and around tumors is an attempt by the immune response to combat the tumor or if microglia are recruited by the tumor to promote tumor growth and proliferation. It has become increasingly clear that the defense functions of immune cells such as lymphocytes and microglia are compromised and the well-known immunosuppressive, intratumoral milieu of gliomas is partially due to the action of cytokines and important regulatory proteins (Schneider et al., 1992). Gliomas produce several immunsuppressive cytokines and factors to inhibit cytotoxic T-cell activation as well as microglial activation. All interleukin (IL)-6 type (strongly immunosuppressive) cytokines IL-1, IL-11, ciliary neurotropic factor (CNTF), cardiotrophin (CT)-1, leukemia inhibitory factor (LIF) and oncostatin-M (OSM)) have been demonstrated at the protein and mRNA levels in glioma cell lines (Goswami et al., 1998; Halfter et al., 1998a, b; Hao et al., 2002; Murphy et al., 1995), as have all tumor growth factor (TGF)-f3 cytokine family members (Constam et al., 1992; Hao et al., 2002; Olofsson et al., 1992). TGF-f3 inhibits the proliferation of microglia as well as their production of cytokines in vitro (Suzumura et al., 1993), whereas IL-6 and IL-10 have been postulated to promote glioma cell line proliferation (Goswami et al., 1998; Huettner et al., 1997). Glioma cells secrete IL-6 (Goswami et al., 1998) and infiltrating MG/MP are the main source of IL-10 (Huettner et al., 1997; Wagner et al., 1999). The immunosuppressive cytokine IL-10 not only promotes glioma cell proliferation but also enhances their ability to migrate in vitro (Huettner et al., 1997). Prostaglandin E2 (PGE2) is another important immunosuppressant in 18 gliomas. Cyclooxygenase-2 (COX-2) is one of the major enzymes responsible for the arachidonic acid conversion to prostaglandins and it has been demonstrated that in the C6 tumor model, glioma-infiltrating microglia, not the glioma cells themselves, are the major source of COX-2 activity (Badie et al., 2003). On the other hand, the presence of MG/MP in and around gliomas suggests that they may play a functional role in glioma survival. Many substances produced by microglia may contribute to glioma proliferation, migration and angiogenesis. The proliferation factor epidermal growth factor (EGF) can be produced by activated microglia in vitro (Briers et al., 1994) and microglia express receptors for EGF that enable them to proliferate in response to local release of this growth factor (Nolte et al., 1997). Both microglia and gliomas secrete vascular endothelial growth factor (VEGF) (Lafuente et al., 1999; Tsai et al., 1995), known to be a critical angiogenic factor. In vitro, VEGF can also induce proliferation and migration of microglia (Forstreuter et al., 2002). VEGF production may thus facilitate glioma survival and promote the recruitment and proliferation of microglia whose production of tumor survival factors can also contribute to glioma growth and malignancy. Substance P (SP) and its receptor NK-1 are expressed in microglia (Lai et al., 2000; Rasley et al., 2002) and in human gliomas (Palma and Maggi, 2000; Palma et al., 1999). SP induces microglial release of cytokines involved in angiogenesis and tumor cell proliferation, including IL-113 and TNF-a (Luber-Narod et al., 1994; Martin et al., 1993). IL-113 has been shown to be involved in the invasiveness and angiogenesis of several tumor types in vivo (Saijo et al., 2002; Sunderkotter et al., 1994; Voronov et al., 2003), and it increases the expression and activity of Matrix metalloprotease-9 19 (MMP-9) in vitro (Esteve et al., 1998, 2002; Nakano et al., 1995), which is thought to be critical for local invasion of brain tissue by tumor cells. Microglia also highly express MMP-9 in vitro in response to chemokines such as MCP-1 (Cross and Woodroofe, 1999). Therefore, microglia may be attracted to gliomas and stimulated to produce factors permitting glioma invasion and metastases. TNF-a is produced by microglia and can have very potent effects on glioma biology. According to previous studies, it is likely that the major source of this factor within gliomas is microglial cells (Hao et al., 2002). TNF-a induces the migration of leukocytes into gliomas (Tada et al., 1993) while increasing the expression of factors involved in tumor cell migration and angiogenesis in glioma cells. The factors include VEGF (Ryuto et al., 1996), EGF receptor (Adachi et al., 1992), and MMP-9 (Esteve et al., 2002). 1.4. Research hypotheses 1) I postulate that P2X7 receptors are functionally expressed in C6 glioma cells. Furthermore, activation of P2X7R is predicted to mediate changes in intracellular calcium levels and are coupled to cell functional responses. 2) Pharmacological modulations of the P2X7R will alter tumor development in vivo. In this study, calcium imaging and molecular biology methods have been applied to examine P2X7R-mediated responses in vitro in cultured C6 cells and the rat C6 glioma model has been used to investigate the properties of the P2X7R in vivo 20 2. Materials and Methods 2.1. In vitro studies 2.1.1. Cell Culture Glioma C6 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells from passage number 39 to 59 were used in this work. Cells were cultured in Kaighn's modification of Ham's F 12 medium (F 12K) with 2 mM L-glutamine modified by ATCC to contain 1.5g/L sodium bicarbonate (Javitt et al., 2005) The medium was then supplemented with 15% horse serum, 2.5% fetal bovine serum, 0.5 µg/ml fungizone (Invitrogen: GIBCO, Grand Island, NY) and 0.02 mg/ml gentamicin (Invitrogen: GIBCO). Cells were maintained in 100 mm culture dishes (SARSTEDT, Newton, NC) at 37 °C in a humidified 5% CO2 air atmosphere. 2.1.2. Calcium-Sensitive Spectrofluorescence The methods for preparation of cells for calcium imaging followed published procedures (McLamon et al., 2005). Briefly, cultured C6 glioma cells were loaded with fura-2 acetoxymethyl ester (fura-2AM at 1 gM; Molecular Probes, Eugene, OR) and pluronic acid (at 1 !AM) in normal physiological saline solution (PSS) for 20 min at room temperature (20-22 °C). In some experiments, cells were incubated with the P2X7R antagonist periodate-oxidized ATP (OxATP, 300 AM for 2 hr; Sigma, St. Louis, MO) prior to dye loading. Cells were then washed for 10 min in PSS solution containing (in mM): 126 NaCl, 5 KC1, 1 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 21 D-glucose (pH 7.4). In several studies, Ca t '-free PSS was used; this solution had the same composition as PSS with the exception that EGTA was added (at 1 mM) with no CaC12. Coverslips were placed in a perfusion chamber mounted on an inverted microscope (Zeiss, Jena, Germany) and fluorescence was measured through a 40x quartz objective lens. Alternating wavelengths (340/380 nm) of ultraviolet light were applied at 6-sec intervals for excitation and fluorescence signals were measured at 510 nm of emission light. Signals were acquired from a digital camera (DVC-1310, DVC Co. Austin, TX) and were processed using an imaging system (Empix, Mississauga, ON, Canada) to determine ratios of the 340 and 380 nm intensities which were used as quantitative measures of fluorescence levels in this work. All studies were done at room temperature. 2.1.3. Reverse-transcriptase polymerase chain reaction (RT-PCR) Total RNA was isolated using TRIzol (GIBCO-BRL, Gathersburg, MD) and then processed for the first strand complementary DNA (cDNA) synthesis using Moloney murine leukemia virus (M-MLV) reverse transcriptase (GIBCO-BRL). The cDNA products were then amplified by PCR using a GeneAmp thermal cycler (Applied Biosystems, Foster City, CA). Specific sense and antisense primers with the expected product size were: rat P2X7R sense 5'-AGGAGCCCCTTATCAGCTCT-3' and rat P2X7R antisense 5'-CATTGGTGTACTTGTCGTCC-3' (692 bp); rat monocyte chemoattractant protein-I (MCP-1) sense 5' -CCTGTTGTTCACAGTTGCTGCC-3 ' and rat MCP-1 antisense 5'-TCTACAGAAGTGCTTGAGGTGGTTG-3' (396 bp); 22 rat interleukin-8 (IL-8) sense 5'-GAAGAT AGATTGCACCGATG-3' and rat IL-8 antisense 5'-CATAGCCTCTCACACATTTC-3' (365 bp); rat vascular endothelial growth factor (VEGF) sense 5'-GCTCTCTTGGGTGCACTGGA-3' and rat VEGF antisense 5'-CACCGCCTTGGCTTGTCACA-3' (644 bp); rat 13—actin sense 5-GTGGGGCGCCCCAGGCACCA-3' and rat f3—actin antisense 5'-GTCCTTAATGTCACGCACGATTTC-3' (526 bp). PCR conditions were as follows: initial denaturation at 95 °C for 6 min followed by a 25- to 30- cycle amplification program consisting of denaturation at 95 °C for 45 sec, annealing at 55-60°C for 1 min and extention at 72 °C for 1 min. A final extention was carried out at 72°C for 10 min. (3-actin was used as a reaction standard. The amplified PCR products were identified using 1.5% agarose gels containing ethidium bromide (final concentration 0.5 µg/m1) and visualized under ultraviolet light. The intensities of each band were measured by densitometry using NIH ImageJ 1.37b software (National Institute of Health, Bethesda, MD) and expressed as relative mRNA levels (mRNA levels normalized to (3-actin). 2.1.4. Western blot analysis Total proteins from the C6 glioma cell line or rat brain tissues were prepared as described previously (Hu et al., 2003). Protein samples were analyzed by SDS-PAGE and Western blot analysis (Ryu et al., 2006). The primary antibodies used were P2X7R (1: 1000, Alomone Labs, Jerusalem, Israel) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1: 800, Lake Placid, NY). The bound primary antibody was 23 detected by the relevant horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000, Amersham-Pharmacia Biotech, NJ) and an enhanced chemiluminescence kit (ECL, Amersham-Pharmacia Biotech). The signals were captured by Fluor-S MultiImager and analyzed using Quantity One software (B io-Rad). 2.1.5. Immunocytochemistry of C6 glioma cells For immunocytochemical staining, C6 glioma cells grown on poly-L-lysine-coated Aclar plastic coverslips were incubated in the standard medium with BzATP (300 1,M; Sigma) in the presence or absence of OxATP (300 uM) for 8 hr. In some experiments, cells were pre-treated with OxATP for 2 hr. After incubation, cells were rinsed in 0.1 M PBS and fixed in 4% paraformaldehyde for 10 min and placed in PBS containing 1% bovine serum albumin, 10% normal goat serum and 0.2% Triton X-100 for 30 min. The cells were then incubated with rabbit anti-P2X7R antibody (1:500, LifeSpan Biosciences, Seattle, WA) for 48 hr at 4°C, followed by biotinylated anti-rabbit IgG (1:1000; Vector, Burlingame, CA) for 1 hr and avidin-biotin complex (1:1000; Vector) for 1 hr at room temperature. The reaction product was visualized with 0.05% 3,3 '-diaminobenzidine (Sigma) and 0.003% H202. After a PBS wash, coverslips were mounted onto glass slides using gelvatol and examined under a microscope. For negative staining controls, primary antibody was omitted during the staining procedure. For quantitative analysis, four fields in each coverslip were selected (magnification of 40X) and images were digitized using a Zeiss Axioplan-2 light microscope equipped with a DVC camera (Diagnostic Instruments, Sterling Heights, MI). Digitized images were then analyzed using Northern Eclipse software (Empix Imaging). The staining was quantified by measurement of the pixel intensity 24 above a predetermined threshold level of staining intensity. Quantification was done in a blinded manner. 2.1.6. Ethidium bromide (EtBr) uptake assay C6 cells were seeded in 6-well plates and were allowed to grow to confluency in standard culture media. In some experiments, cells were treated with 300 KM OxATP for 2 hr at 37°C prior to performing the EtBr uptake assay. Cells were then centrifuged, collected and suspended with PBS at 1 X 10 6 cells/ml in 1.5 ml Eppendorf tubes and then incubated for 5 min at room temeperture with EtBr (25 iiM), with or without treatment (BzATP alone, BzATP plus OxATP or OxATP alone). After incubation, cells were centrifuged and supernatant was discarded prior to adding distilled water to rupture plasma membrane. An Ultrospec 2100 pro UV/Visible spectrophotometer (GE Healthcare, Uppsala, Sweden) was used to measure the absorbance of the samples at 488 nm (the excitation wavelength of EtBr). The reference cuvette contained the lysate from cells that were not incubated with EtBr or any other compound. Four independent experiments were performed and the results were normalized relative to the untreated control and presented as mean percentage. Quantification was done in a blinded manner. 2.1.7. Scratch-wound migration assay The methods for the scratch-wound migration assay followed published procedures (Faber-Elman et al., 1996; Lind et al., 2006) with some modifications. C6 cells were plated onto coverslips and were allowed to grow to confluency in the same 25 medium as for cell culture. Once cells were confluent, the medium was switched to serum-free F12K for 24 hr to minimize the influence of growth factors. After the period of serum starvation, the confluent monolayers were scratched using a 200 III sterile plastic pipette tip. In some experiments, cells were incubated with 300 prM OxATP for 2 hr or 11.1M BBG (Sigma) for 15 min prior to performing the scratch wound. The coverslips were then washed three times with the serum-free F 12K, and treatment was applied. BzATP was added alone or with OxATP (or BBG) in the serum-free medium. After 24 hr of incubation (37 °C, 5% CO2), the cells were fixed for DAPI staining Cells were washed in PBS twice and then fixed with 4% paraformaldehyde in 0.1 M PBS (10 min at room temperature). After wash in PBS, cells were incubated in DAPI (Invitrogen) at 1 mg/m1 in PBS to visualize nuclei and determine cell numbers in the field of view. Cells were then washed in water and mounted onto glass slides using gelvatol. The extent of the repopulation of the wound area by DAPI-stained cells was assessed in digitalized images, using the NIH Image J 1.37b software (National Institute of Health). For quantification of data, overall areas of the wound were first examined. Consistent patterns of uniform migration throughout the experimental region were observed. We then quantified the number of cells in a representative area of 0.5 x 0.5 mm2 Four experiments were performed in triplicate. The results were normalized relative to the number of cells counted in equal areas in the control untreated cultures and are presented as mean percentage. Quantification was done in a blinded manner. 26 2.1.8. Statistical analysis Data are presented as mean ± standard error of mean (SEM). Statistical significance (p < 0.05) was evaluated using Student t-test or one-way analysis of variance followed by Student-Newman-Keuls multiple comparison test when applicable (GraphPad Prism 3.0; San Diego, CA). 2.2. In vivo studies 2.2.1. Intracerebral implantation of C6 cells All animal procedures were carried out according to protocol approved by the UBC Animal Care Ethics Committee, adhering to guidelines of the Canadian Council on Animal Care. Male Sprague-Dawley rats (250-300 g; Charles River Laboratories, St. Constant, PQ) were anesthetized with intraperitoneal (i.p.) injection of ketamine hydrochloride (72 mg/kg; Bimeda-MTC, Cambridge, ON) and xylazine hydrochloride (9 mg/kg; Bayer Inc., Etobicoke, ON) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). For implantation of glioma cells, C6 cells were harvested, centrifuged and suspended in HEPES-buffered physiological salt solution (HPSS) and 3 gl of cell suspension (1x10 5 cells/µl) was injected into the striatum of each animal. Injection coordinates for the striatum were as follows: anteriorposterior (AP): 1.0 mm; mediallateral (ML): -3.0 mm; dorsoventral (DV): -5.0 mm, from bregma. A midline skin incision was made in the scalp to expose the skull and stereotaxic unilateral transplantation of C6 glioma cells was performed as previously described (Ryu et al., 2004). In some experiments, C6/LacZ cells (ATCC, 27 Manassas, VA) were used. They are modified C6 cells that constitutively express the lacZ reporter gene product, E. coli-derived beta galactosidase 03-gal). The cells were maintained under the same conditions as C6 glioma cells (see 2.1.1.). C6 cells were slowly injected (0.2 ill/min) into the striatum using a 10 Hamilton syringe attached to a 26-gauge needle. The injection syringe was left in place for an additional 3 min and the needle was slowly withdrawn. After removing the needle, the skin was sutured and the animals were allowed to recover and then returned to their cages. 2.2.2. Immunohistochemical analysis Animals were transcardially perfused with heparinized cold saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (0.1 M PB, pH 7.4) under ketamine/xylazine anesthesia. Brains were removed from the skull, post-fixed in the same fixative overnight and then placed in 30% sucrose for cryoprotection. The brains were then rapidly frozen in powdered dry ice and stored at -70°C. Coronal sections (40 gm) were cut on a cryostat at the interval of 0 8mm throughout the striatum and hippocampus and stored in cryoprotectant solution. Free-floating sections were processed for single immunohistochemistry, as described previously (Ryu and McLamon, 2006). Briefly, sections were permeabilized by incubation in PBS containing 1% bovine serum albumin (BSA; Sigma), 10% normal goat serum (NGS) and 0.2% Triton X-100 for 1 hr. Sections were then incubated overnight at 4°C with the following primary antibodies: anti-CR3R (OX-42, a marker for microglia/macrophages, 1:500; Serotec, Oxford, UK), anti-glial fibrillary acidic 28 protein (GFAP, a marker for astrocytes, 1:1000; Sigma), or anti-neuronal nuclei (NeuN, a marker for neurons, 1:1000; Chemicon, Temecula, CA). Sections were rinsed in PBS with 0.5% BSA and incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Molecular Probes) or Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200; Molecular Probes) at room temperature for 2 hr in the dark. Sections were then washed in 0.1M PBS, mounted on Superfrost/Plus microscope slides (Fisher Scientific; Pittsburgh, PA), and mounted in DPX Mountant (Fluka, Toronto, ON, Canada). For the negative control, the primary antibody was omitted from the staining procedures. 2.2.3. Double immunofluorescence staining Double immunofluorescence staining was performed as described previously (Ryu and McLarnon, 2006). Free-floating sections were incubated overnight at 4°C with a mixture of two primary antibodies: anti-P2X7R (1:200; Alomone Labs, Jerusalem, Israel) in combination with either anti-13-gal (1:200; Abcam, Cambridge, MA), anti-OX-42 (1:500; S erotec), anti-GFAP (1:1000; Sigma) or anti-NeuN (1:500; Chemicon). Sections were then incubated in a mixture of Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:100; Molecular Probes, Eugene, OR) and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:100; Molecular Probes) at room temperature for 2 hr in the dark 29 2.2.4. BBG administration In some experiments, drug administration was started right after C6 implantation. Conscious rats (n = 3) were injected i.p. with BBG (50 mg/kg, dissolved in PBS) twice per day and control animals (n = 3) received an equivalent volume of pyrogen-free saline. Two weeks after tumor transplantation, all animals were sacrificed and coronal sections were taken as described in 2.2.2. Nissl staining was then performed to determine tumor volumes. 2.2.5. Quantification of tumor volumes Tumor volumes were determined from coronal striatal sections (40 at the interval of 0 8 mm) stained for Nissl substance. First, we measured the absolute tumor area using the computer program NIH ImageJ 1.37b (National Institute of Health). The absolute tumor volume was then calculated after integration of the area measurements from every section and the relative tumor volume was expressed as the percentage of the tumor volume to the volume of the intact hemisphere. 30 3. Results 3.1 In vitro studies 3.1.1. Intracellular calcium levels are altered in response to P2X7R agonist BzATP Initial experiments examined effects of the specific P2X7 agonist, BzATP, to increase mobilization of [Ca 2±]i in C6 glioma cells. A representative response elicited by BzATP (300 NM) is presented in Figure 3-1A and shows the ligand induces a rapid rise in [Ca21i (measured as ratio F340/380) followed by a slowly decreasing component (mean response from n = 38 cells). The [Ca 21i increase induced by BzATP was largely inhibited in the presence of the P2X7R antagonist OxATP (300 p.M) (Figure 3-1B; n = 30 cells). Quantification for the amplitudes of responses for BzATP, in the absence and presence of OxATP, is presented in Figure 3-1C; responses were diminished by 67% with application of OxATP (N = 5 experiments with a total of 132 cells for BzATP treatment and 151 cells for BzATP treatment in the presence of OxATP). 31 0.4 M Li- 02 0 C A 1 - 0.8 - 0.6 - 0.4 - 0.2 - 0 ^ 0 BzATP 300 u M 100^200 Time (sec) 300 B 1- as - esse 2 0.6 - .0. 0.4 - Mu. 0.2 - 0 ^  OxATP 300 u M BzATP 300 u M 0 300100^200 Time (sec) BzATP^BzATP + OxATP Figure 3-1. [Ca2li responses induced by the P2X7 agonist BzATP Representative traces show Ca2+ responses induced by BzATP alone (Fig. 1(A); n = 38 cells) or (B) in the presence of OxATP added 2 hr before BzATP application (n = 30 cells). (C) Amplitudes of responses for different treatments are shown (N = 5 independent experiments for each group). *p < 0.05. 32 3.1.2. P2X7 receptors are expressed in C6 cells and regulate expression of pro-inflammatory factors Expression of P2X7R and several inflammatory cytokines was examined using RT-PCR with C6 cells exposed to control (standard medium alone) or 300 11M BzATP solution (treatments for 8 hr). Representative results are presented in Figure 3-2A and show in control low levels of basal expression of P2X7R, the chemokines MCP-1 and IL-8, and the angiogenic factor VEGF. Treatment with BzATP caused marked increases in expression of P2X7R and levels of the inflammatory factors (Figure 3-2A). Semi-quantitative results (Figure 3-2B; from N = 4 independent experiments) showed that stimulation with the P2X7R ligand significantly increased expression of P2X7R and all three factors (relative to control) as follows: P2X7R (by 116%), MCP-1 (by 549%), IL-8 (by 140%), and VEGF (by 62%). Surprisingly, OxATP was ineffective in blocking the BzATP-induced increase in expression of the pro-inflammatory factors (data not shown); this point is discussed below. 33 ti Goy 4:7 1111=P2X7R MCP-1 IL-8 VEGF == 13 -actin A B 1 0.11 0.6 E 0.4 a 0.2 0 ■ Control BzATP * * P2X7R MCP-1^VEGF Figure 3-2. Effects of BzATP stimulation of C6 glioma cells on expression of P2X7R and pro-inflammatory factors (A) Representative RT-PCR results for P2X7R, MCP-1, IL-8 and VEGF for exposure of C6 to control (standard medium) and BzATP (300 1.1M for 8 hr). 13-actin served as a reaction standard. (B) Overall results from N = 4 independent experiments. *p < 0.05. 34 3.1.3. P2X7R protein is present in C6 cells and is up-regulated by BzATP stimulation Western blotting and immunocytochemical staining were used to determine whether the P2X7R protein is produced by C6 cells and if production of the P2X7R is modulated with cell exposure to BzATP. Western blot analysis using antibodies against P2X7R demonstrated the presence of P2X7R protein in C6 cells (Figure 3-3A) and cell lysates from rat brain tissues were used as positive controls. This result was further corroborated by immunocytochemical staining. Representative results show that under unstimulated conditions, immunoreactivity for P2X7R was low (Figure 3-3B, upper left panel). BzATP markedly enhanced P2X7R protein levels (Figure 3-3B upper right panel) which were reduced if OxATP was included with BzATP (Figure 3-3B lower left panel). OxATP alone did not alter immunoreactivity for P2X7R protein levels (Figure 3-3B lower right panel). Quantification of the results (from N = 3 independent experiments) is presented in Figure 3-3C and shows significant increases in P2X7R protein levels with BzATP (by 34-fold relative to the untreated control) and marked inhibition in the presence of OxATP (by 61% relative to BzATP alone). 35 Figure 3-3. BzATP-induced up-regulation of P2X7R protein in C6 glioma (A) Expression of P2X7R protein in C6 cells was analyzed by Western blotting in cell lysates using specific P2X7R antibodies. Lysates from rat brain tissues were used as positive controls. (B) Representative immunocytochemical staining for control (upper left panel), BzATP (upper right panel), BzATP+OxATP (lower left panel) and OxATP alone (lower right panel); Scale bar = 10 Am. (C) P2X7R protein expression in C6 cells for the different treatments (N = 3 independent experiments); * p<0.05 for BzATP vs control and ** for BzATP+OxATP vs BzATP alone. 36 A KDa 97 — 66 — ^ P2X7R B CONT^1111  BzATP + OxATP OxATP C ** <•^4^4^400 NI- v-1 .0+ 0+ 4iky- o 37 3.1.4. P2X7R activation induces pore formation in C6 cells An ethidium bromide (EtBr) uptake assay was used to examine P2X7R-induced pore formation. The overall results (Figure 3-4, N = 4 for each group) show incubation with BzATP (300 ttIVI, 5 min) increased absorbance (measured at 488 nm) by more than two-fold compared with untreated controls indicating pore-formation was induced with P2X7R activation. Treatment of OxATP with BzATP significantly inhibited dye uptake (by 58%) compared to BzATP application alone. OxATP applied separately did not alter absorbance relative to control. Interestingly, although P2X7R activation leads to formation of large pores, exposure of C6 cells to a maintained stimulation of a high level of BzATP (at 500 iiM for 72 hr) had no effect to diminish cellular viability (data not shown). Thus, chronic stimulation of P2X7R was not linked to activation of apoptosis or necrosis pathways in C6 cells. 38 300 *3^250co 4-e. 200 03 0 150 t 100 e 50 0 VO ^ ‹rz Figure 3-4. BzATP-induced uptake of ethidium bromide N = 4 independent experiments for each treatment; * p<0.05 for BzATP vs control and ** for BzATP+OxATP vs BzATP alone. 39 3.1.5. P2X7 receptors in C6 glioma cells regulate cell mobility The mobility of C6 glioma was also investigated in this work using a scratch-wound assay. As shown in Figure 3-5A (top left panel), little or no migration of C6 was observed in control serum-free condition. However, in the presence of BzATP (3001.1M, 24 h incubation) C6 cells showed considerable mobility (Figure 3-5A, top right panel) which was largely inhibited in the presence of OxATP (300RM; lower left panel). No mobility of C6 was observed with OxATP alone (Figure 3-5A, lower right panel). Quantification of the results (from N = 4 independent experiments) is presented in Figure 3-5B and shows a significant increase in C6 mobility with exposure to BzATP (by 290% relative to the untreated control) and marked inhibition in the presence of OxATP (by 91% relative to BzATP alone). Interestingly, levels of C6 mobility were higher in control compared with OxATP suggesting that basal expressions of P2X7R were sufficient to permit C6 mobility in unstimulated cells. Another P2X7R agonist, BBG, showed similar effects in blocking cell mobility induced by BzATP (Figure 3-5C; by 74% relative to BzATP alone) while treatment with BBG alone did not affect C6 cell migration. 40 •• A •."1- • • Untreated BzATP • • • • • :^ • •vlertt..., •^- - ^ it",i'y • • :•••^• it" • . • . 71•711a. *. • ot: IV" • •^• BzATP + OxATP ^ OxATP B^ C C 500 .42 as a) 400 -.. 300 200 0 0 ae 100 0 * c 500 * as in 400 eL o 300 E3 t; 0 200 ** ** 0 ae 100 --3111.• 0 06 ,KIZ 4 4^,,etzfr^P ,c(Z^ %)‘ 4r1' Figure 3-5. BzATP effects on C6 repopulation of the wound area (A) Representative results for treatments with control (upper left panel), BzATP (upper right panel), BzATP+OxATP (lower left panel) and OxATP alone (lower right panel). Scale bar = 100 gm. Experiments with BBG showed similar results. (B) Quantification for wound repopulation with OxATP as the P2X7R antagonist from four independent experiments performed in triplicate. (C) Quantification for wound repopulation with BBG as the P2X7R antagonist from four independent experiments performed in triplicate; * p<0.05 for BzATP vs untreated control and ** for BzATP+OxATP or BzATP+BBG vs BzATP alone. • 41 3.2. In vivo studies 3.2.1. Tumor progression in the rat brain Rodent C6 tumor model was used to investigate P2X7R expression and function in vivo. Initial experiments used immunohistochemical methods to determine tumor progression at two and four weeks following C6 injection into rat striatum. As shown in Figure 3-6A, Nissl staining indicates tumor development at two weeks (2 W) and four weeks (4 W) after transplantation. Quantification (from n = 3 animals per group) is provided in Figure 3-6B and the relative tumor area is expressed as the percentage of the area occupied by the tumor to the area of the contralateral side. 42 A2 Weeks 4 Weeks B Relative tumor areas 100 7-S2 80 60 40 0 20 0 2W^4W Figure 3-6. Progression of C6 glioma (A) Nissl staining shows development of tumor at 2 and 4 weeks after C6 transplantation. No Nissl staining of C6 glioma was evident in the contralateral side. (B) Quantification of tumor areas (n = 3 animals per group). 43 3.2.2. Co-localization of P2X7R with C6/LacZ cells In order to label injected C6 cells in situ, we used C6/LacZ cells. The cells constitutively express the lacZ reporter gene product, E. coli-derived beta galactosidase ((3-gal), as revealed on tissue sections by histochemical stain, and single tumor cells can be identified. At 2W after injection, expression of P2X7R associated with C6 cells was examined. Tumor development at 2W following C6/LacZ injection was shown by Nissl staining (left panel, Figure 3-7A) and single C6 cells were identified by j3-gal immunoreactivity (ir) (right panel Figure 3-7A). A high degree of co-localization of P2X7R and C6 was observed (top panel, Figure 3-7B), indicating that C6 cells express P2X7R in situ. Intratumoral regions show marked 0-gal and P2X7R staining (bottom left panel, Figure 3-7B) while boundary regions show 0-gal ir in intratumoral, but not peritumoral regions (bottom right, Figure 3-7B). 44 C6-IacZ^Beta- • alactosidase Boundary region B A Figure 3-7. Co-localization of P2X7R with C6/LacZ cells (A) Nissl staining shows development of C6 glioma at 2 weeks after C6/LacZ transplantation (left panel) and C6 cells were also identified by 13-gal it (right panel). (B) Co-localization of P2X7R (red) with C6/LacZ cells (green). 45 3.2.3. Co-localization of P2X 7R with microglia/macrophages and astrocytes We then examined expression of P2X7R associated with microglia/macrophages (Figure 3-8) and astrocytes (Figure 3-9) (microglia/macrophages; OX-42 marker; astrocytes; GFAP marker). Considerable OX-42 and P2X7R ir was observed in striatum (top panel, Figure 3-8) with little ir evident in contralateral regions (second panel, Figure 3-8). Intratumoral areas show high levels of staining for OX-42 and P2X7R (third panel, Figure 3-8). Boundary regions show co-localization of P2X7R and OX-42 in intratumoral, but not peritumoral (P2X7R ir in peritumoral region is likely astrocytic). Astrocyte and P2X7R immunostaining is presented in Figure 3-9 (top panel) with only low levels of ir observed in contralateral striatum (second panel, Figure 3-9). GFAP ir was minimal in intratumoral regions (third panel, Figure 3-9). Co-localization of GFAP positive astrocytes with P2X7R was evident in the peritumoral portion of the boundary region (bottom panel, Figure 3-9). No P2X7R ir was observed with neurons (NeuN marker, data not shown) 46 Figure 3-8. Microglia and P2X7R stain throughout tumor A high degree of co-localization of microglia and P2X7R is found within intratumoral regions. For boundary region, both OX-42 and P2X7R show it in intratumoral, but not peritumoral areas. T is standing for tumor. 47 Figure 3-9. Astrocytes and P2X7R stain throughout tumor GFAP staining is minimal in intratumoral regions which contain high levels of P2X7R. Astrocytes are co-localized with P2X7R in peritumoral regions. 48 3.2.4. Administration of BBG reduced the tumor size The P2X7R antagonist BBG was used in our study to inhibit P2X7R expressed in the tumor brain. Figure 3-10A shows serial coronal sections from tumor bearing animals injected only with saline (C6) or with BBG (C6 + BBG). Administration of BBG markedly reduced the size of brain tumors formed by transplanted C6 cells compared with saline injection. Quantification of the preliminary results (from n = 3 animals per group) is presented in Figure 3-10B and shows the tumor volume was reduced by about 65 % with BBG administration. 49 A "-^^A PAY 06 • C6 + BBG B Relative tumor volumes a• 120 -TO (1) 80 231 di O 40 O 0 C6^C6 + BBG Figure 3-10. Administration of BBG reduced the tumor size (A) Serial coronal sections from tumor bearing animals injected only with saline (C6) or with BBG (C6 + BBG). In both animals, the left hemisphere did not receive any surgery and served as the histological control. Nissl staining indicates tumor progression. A: anterior; P: posterior. (B) Quantification of tumor volumes. The relative tumor volume is expressed as the percentage of the absolute tumor volume to the volume of the intact hemisphere. n = 3 animals per group. 50 A 4. Discussion 4.1. In vitro studies Our in vitro studies provide the first report for expression, at both mRNA and protein levels, of the purinergic subtype receptor, P2X7R, in C6 glioma. Activation of this receptor using the specific ligand BzATP induced a rapid transient increase in [Ca21i which was sensitive to pretreatment of cells with the P2X7R antagonist, OxATP. Functional responses mediated by P2X7R in C6 cells were also demonstrated in this work. These included increased expression of pro-inflammatory factors, enhanced migration of cells and pore formation in the cell membrane; these responses are considered in more detail below. In calcium imaging, BzATP induced a rapid increase in intracellular levels of Ca2+ followed by a slowly decreasing phase to basal level. This increase in [Ca 21i following application of BzATP was partially, but not completely, antagonized by OxATP. Interestingly, we also observed a small increase in [Ca 21i when BzATP was applied in a Ca2+-free solution (data not shown). These findings indicate additional actions for BzATP in C6 glioma other than these mediated by P2X7R. One possibility is that this agent also activated purinergic receptors of the metabotropic P2YR type. Some evidence is available indicating expression of P2Y2 receptors (P2Y2R) in C6 cells (Sabala et al., 2001) and our RT-PCR results also showed expression of this subtype P2YR in the present work (data not shown). Furthermore, BzATP has been shown as an activator of cloned human and rat P2Y2 receptors (Erb et al., 1993; Wildman et al., 2003). 51 Previous studies have reported that P2X 7R expression in cells other than C6 are up-regulated under pathological conditions and can mediate inflammatory responses (Franke et al., 2004; Lister et al., 2007; McLarnon et al., 2006; Yiangou et al., 2006). The present data suggest that activation of P2X 7R in C6 glioma is associated with enhanced cellular expression of several pro-inflammatory factors including MCP-1, IL-8 and VEGF. Although the up-regulation of these factors in C6 cells was examined for a single duration of BzATP stimulation (8 hr), it is likely each would exhibit its own specific pattern of time changes with P2X 7R activation. The factors could also act on differential targets. For example, expression of MCP-1 in malignant gliomas is closely associated with microglial and macrophage infiltration (Kielian et al., 2002; Platten et al., 2003). Although the functional responses of infiltrating immune cells are not well-understood, there is some evidence suggesting that these cells can contribute to the overall growth and invasion of brain tumors (Graeber et al., 2002; Wattere et al., 2005). Both VEGF and IL-8 are potent activators of angiogenic activity for vascular cells (Brat et al., 2005; Grobben et al., 2002) and these agents have reported actions to promote tumor neovascularity and progression (Brat et al., 2005; Grobben et al., 2002). Overall, our results are consistent with the idea that P2X7R in C6 cells could enhance tumor development by increasing tumor-associated inflammation and angiogenesis. Activation of P2X7R was also linked to other functional responses including enhancement of cell migration in a scratch-wound assay and pore induction in a dye loading protocol. In the former case, increased mobility of C6 cells could aid migration of cells in a tumor environment. Recent work has reported extracellular 52 signal-regulated kinase (ERK) is activated downstream of P2X7R (Panenka et al., 2001) and is involved in migration of C6 glioma cells (Lind et al., 2006). Although pore formation is considered as a common trigger of cell death (Bulanova et al., 2005; Virginio et al., 1999a), we found no evidence for any cell damage with BzATP stimulation of C6 cells. Recent work using human neuroblastoma cell lines has reported that the two events are not necessarily linked (Raffaghello et al., 2006). P2X7R activation in neuroblastoma cells led to uptake of the YO-PRO dye without causing apoptosis or necrosis; indeed P2X7R acted as a growth-promoting factor in these cells. It is possible that in some cell types negative (death inducing) responses linked to P2X7 activation have been modified to fit other functions; in C6, for example, it could link purinergic activation with pro-inflammatory responses. In addition, it should be noted that P2X2 and P2X4 receptors can also form large pores with longer term application of ATP (10-60 seconds) (Virginio et al., 1999b). However, the present results (Fig. 4) show that the P2X7R antagonist OxATP effectively blocked BzATP-induced dye uptake. This result suggests that P2X2 or P2X4 receptors do not participate in pore formation induced by BzATP. Our work did not address the specific intracellular pathways linking activation of P2X7R with the specific cellular functional responses. It should be noted that although OxATP inhibited the increased [Ca21i induced by BzATP and also reduced migration of C6 cells it does not constitute definitive proof these processes are linked. Interestingly, OxATP treatment (300 AM for 8 hr) was ineffective in blocking the BzATP-induced increase in expression of the pro-inflammatory factors MCP-1, IL-8 and VEGF. A similar result was obtained with different application times of OxATP 53 (data not shown), suggesting that time of exposure of the P2X 7R antagonist was not a factor involved. Our results could indicate that expression of the pro-inflammatory factors is mediated by Ca2+-independent pathways coupled to activation of P2X7R. However, as noted above, OxATP did not completely block [Ca 2li induced by BzATP. Purinergic receptors other than P2X7R could also contribute to C6 responses in this study. As discussed above, P2Y 2R can be expressed in C6 cells and could account for the residual calcium response induced by BzATP in the presence of the P2X7R antagonist, OxATP. In addition, BzATP has been reported as an agonist for several P2X receptor subtypes including P2X 1 R (Duan et al., 2003). Although our data report P2X7R-mediated signaling in C6 cells, it is possible that other P2XR family members could be expressed in these cells. In addition, it should be noted that in the migration study, C6 cells were cultured in media devoid of serum for 48 hours. This procedure was employed to minimize the effects of growth factors on cell mobility. However, it is possible that serum-free treatment could also alter the biological behavior of the cells. It has been reported that long-term serum starvation (up to 96 hrs) had effects on cell properties such as morphology and the adhesion pattern and expression of purinergic P2Y receptors in C6 glioma cells (Krzeminski et al., 2007). Finally, it is important to recognize that no currently available animal tumor model would be expected to simulate human gliomas and species differences could affect expression and functions of the P2X7R in rat and human glioma cells. In addition, the properties of glioma cells derived from different tumors vary from each other so that data derived from a nitrosurea-induced tumor may not be valid for all gliomas. 54 Despite these limitations, the C6 glioma model has provided a wealth of information on in vitro and in vivo biochemical and biological properties of brain tumors (Barth, 1998). Overall, our in vitro work has found C6 glioma cells express P2X7 receptors which mediate Ca2+ mobilization and formation of large pores. Activation of the P2X7R is linked with cell expression of pro-inflammatory factors and cell migration which could contribute to tumor development. 4.2. In vivo studies The preliminary in vivo studies showed a majority of P2X7R were associated with C6 glioma cells and microglia/macrophages (referred to only as microglia below). We also found pharmacological modulation of P2X7R with administration of the receptor antagonist Brilliant Blue G (BBG) markedly reduced tumor size. BBG was used in the in vivo studies due to the following reasons: (1) It is a potent and selective P2X7R antagonist having 1050 values of about 10 nM at the rat P2X7R (Jiang et al., 2000). (2) We have demonstrated that it can cross the blood-brain barrier (BBB; data not shown), which makes it easy to be applied to animals (i.p. injection). However, since BBG is a blue dye it can interfere with signals, the dye is not suitable for in vitro assays such as Ca2+ imaging experiments. In the in vitro studies, we have found that activation of the P2X7R is linked with cell expression of pro-inflammatory factors and cell migration which could contribute to tumor development. Since the P2X7R is not only expressed by C6 glioma cells in 55 vitro but also in vivo, it could have functional roles in regulating tumor growth and migration in the rat brain. One important link between in vitro and in vivo studies is that BBG inhibits C6 cell migration induced by BzATP (see 3.1.5.). Therefore, BBG might reduce tumor size at least partially by inhibiting cell migration associated with P2X7R activation. It was also clearly demonstrated that P2X7R were expressed by tumor-associated microglia in intratumoral regions. The presence of macrophages and microglia was reported in both human and rat gliomas (Morioka et al., 1992; Roggendorf et al., 1996; Shinonaga et al., 1988; Streit, 1994; Wierzba-Bobrowicz et al., 1994) and we also observed marked microglial infiltration within tumors. Astrocytes appeared primarily to be localized to boundary regions (data not shown). Although microglia are thought to play a significant role in neuronal injury in CNS inflammatory diseases, their function in brain tumors is poorly understood. A number of substances produced by microglia may contribute to glioma proliferation, migration and angiogenesis and the P2X7R is functionally involved in release of several of these factors such as IL-113 and TNF-a (see and 1.3.3.). Hence, overall tumor progression and invasion could be reduced by blockade of P2X7R-mediated activities of microglia. In addition, it is important to note that BBG is also a commonly used dye to stain protein. Although there is no report on toxicity of BBG to cells, there is the possibility that the compound may have some non-specific effects since it can bind to protein. According to our present data, we suggest that BBG inhibits tumor growth by acting on P2X7R in both C6 glioma cells and microglia. However, further investigation is required to confirm the effects of BBG administration on tumor 56 development and to study the specific roles of P2X7R in brain tumors. 57 5. Conclusions of thesis research and future directions Our work has found C6 glioma cells express P2X 7 receptors which mediate Ca 2+ mobilization and formation of large pores. Activation of the P2X 7R is linked with cell expression of pro-inflammatory factors and cell migration which could contribute to tumor development. In addition, preliminary in vivo studies showed that C6 glioma cells and tumor-associated macrophages/microglia are major contributors to P2X7R expression in tumor-bearing rat brains Importantly, BBG administration inhibited tumor growth. The results summarized above demonstrate that the P2X 7 receptor is an important modulatory factor in C6 glioma. Furthermore, pharmacological modulation of P2X7R in vivo alters tumor development. Understanding the roles of P2X 7R in glioma brain tumors may lead to a rational testing of novel therapeutic strategies to inhibit tumor growth. Further research needs to be carried out to confirm the effects of BBG administration on tumor development. The role of P2X 7R in tumor-bearing brains could be further investigated by examining tumor-associated pathological processes such as angiogenesis and inflammation. 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