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1.
Genes Immun ; 8(3): 262-74, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17344888

ABSTRACT

Sparc-null mice have been used as models to assess tumor-host immune cell interactions. However, it is not known if they have a competent immune system. In this study, the immune systems of Sparc wild-type and null mice were compared. Mice were assessed for differences in total body weight, spleen weight and spleen-to-body weight ratios. Spleens were compared with respect to morphology, and Sparc, Ki-67, MOMA-1 and IgM expression. Immune cells in blood, bone marrow and spleen were assessed by blood smears, automated blood panel, and flow cytometry. Additionally, the ability of Sparc-null mice to respond to immune challenge was evaluated using a footpad model. The morphological and immunohistochemical results indicated that Sparc-null spleens had more white pulp, hyperproliferative B cells in the germinal centers, and decreased marginal zones. Sparc-null spleens lacked normal Sparc expression in red and white pulp, marginal zones, endothelial and sinusoidal cells. By flow analysis, B cells were decreased and T cells were increased in the bone marrow. Finally, Sparc-null mice were unable to mount an immune response following footpad lipopolysaccharide challenge. These data confirm that Sparc-null mice have an impaired immune system.


Subject(s)
Osteonectin/deficiency , Osteonectin/immunology , Spleen/immunology , Animals , B-Lymphocytes/cytology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Base Sequence , Body Weight , DNA Primers/genetics , Flow Cytometry , Gene Expression , Immune Tolerance , Lymphocytes/cytology , Lymphocytes/immunology , Lymphocytes/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Organ Size , Osteonectin/genetics , Osteonectin/metabolism , Spleen/anatomy & histology , Spleen/metabolism
2.
Oncogene ; 26(34): 4918-27, 2007 Jul 26.
Article in English | MEDLINE | ID: mdl-17297440

ABSTRACT

PIKE-A (phosphoinositide 3-kinases (PI 3)-kinase enhancer) is a ubiquitously expressed GTPase, which binds to and enhances protein kinase B (Akt) kinase activity in a guanine nucleotide-dependent manner. PIKE-A is one of the components of the CDK4 amplicon that is amplified in numerous human cancers. However, whether PIKE-A itself can mediate cell transformation, proliferation and migration remains unknown. Here, we show that PIKE-A is overexpressed in various human cancer samples, escalates U87MG glioblastoma invasion and provokes NIH3T3 cell transformation. Overexpression of wild-type (WT) PIKE-A enhances NIH3T3 and U87MG cell growth, which is further increased by cancer cell-derived PIKE-A active mutants. In contrast, both the dominant-negative mutant and the phosphoinositide lipids interaction-defective mutant antagonize cell proliferation. Moreover, PIKE-A and its active and inactive mutants similarly enhance or antagonize U87MG cell survival and invasion, and their ability to do so is coupled with the catalytic effect they have on Akt activation. Furthermore, PIKE-A WT and its active mutants significantly elicit NIH3T3 cell transformation. Thus, our findings support the concept that PIKE-A acts as a proto-oncogene, promoting cell transformation through Akt activation.


Subject(s)
Cell Transformation, Neoplastic , GTP-Binding Proteins/physiology , GTPase-Activating Proteins/physiology , Neoplasm Invasiveness , Neoplasms/enzymology , Proto-Oncogenes , Animals , Apoptosis , Cell Line, Tumor , Cell Proliferation , GTP-Binding Proteins/genetics , GTPase-Activating Proteins/genetics , Humans , Mice , Mutation , NIH 3T3 Cells , Neoplasms/pathology , Proto-Oncogene Mas , Proto-Oncogene Proteins c-akt/metabolism
5.
J Neurooncol ; 53(2): 149-60, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11716067

ABSTRACT

We have identified secreted protein acidic and rich in cysteine (SPARC) as a potential glioma invasion-promoting gene. To determine whether SPARC alters the growth, attachment, or migration of gliomas, we have used U87T2 and doxycycline-regulatable SPARC-transfected clones to examine the effects of SPARC on (1) cell growth, (2) cell cycle progression, (3) cell attachment, and (4) cell migration, using growth curves, flow cytometry, attachment, and migration analyses on different brain ECMs, including collagen IV, laminin, fibronectin, vitronectin, hyaluronic acid, and tenascin. Our data indicate that SPARC delays tumor cell growth in the log phase of the growth curve. The clones secreted different levels of SPARC. The clone secreting the lowest level of SPARC was associated with a higher percentage of cells in G2M, whereas the clones secreting the higher levels of SPARC were associated with a greater percentage of cells in G0/G1. In comparison to the parental U87T2 clone, the SPARC-transfected clones demonstrated increased attachment to collagen, laminin, hyaluronic acid, and tenascin, but not to vitronectin or fibronectin. SPARC-transfected clones also demonstrated altered migration on the different extracellular matrix proteins. The modulation of migration, either positive or negative, was associated with changes in the level of secreted SPARC. These data suggest that SPARC may modulate glioma proliferation and invasion by modulating both the growth and migration of glioma cells.


Subject(s)
Brain Neoplasms/pathology , Extracellular Matrix Proteins/metabolism , Glioma/pathology , Neoplasm Proteins/physiology , Osteonectin/physiology , Brain Neoplasms/metabolism , Cell Adhesion , Cell Division , Cell Movement , Collagen/metabolism , Fibronectins/metabolism , G1 Phase , Glioma/metabolism , Humans , Hyaluronic Acid/metabolism , Laminin/metabolism , Osteonectin/genetics , Recombinant Fusion Proteins/physiology , Resting Phase, Cell Cycle , Tenascin/metabolism , Transfection , Tumor Cells, Cultured/metabolism , Tumor Cells, Cultured/pathology , Vitronectin/metabolism
6.
Hematol Oncol Clin North Am ; 15(6): 979-1006, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11770303

ABSTRACT

Many genetic alterations that contribute to CNS tumorigenesis and progression have been identified. One goal of such studies is to identify loci that would serve as diagnostic prognostic markers or both. A significant advance is the observation that chromosome 1p loss identified anaplastic oligodendroglioma and a subset of high-grade glioma patients who responded to chemotherapy and had longer survival times. Combined 1p and 19q loss was a predictor of prolonged survival of patients having pure oligodendrogliomas. Such markers eventually may be used to identify patients upfront who would benefit from treatment, while sparing patients who would not benefit. Although many molecular participants involved in the biologic pathways that promote proliferation, angiogenesis, and invasion have been elucidated, there are still many gaps in clinicians' knowledge. It is expected that the use of the human genome project information and databases such as SAGEmap, in combination with techniques such as cDNA arrays and proteomics, will facilitate greatly the identification of novel genes that contribute to CNS tumors. cDNA arrays and tissue arrays will permit the construction of CNS-specific screening tools that will permit the identification of tumor-specific mutations and alterations so that patient-specific therapies can be designed.


Subject(s)
Central Nervous System Neoplasms/metabolism , Central Nervous System Neoplasms/pathology , Cell Division , Central Nervous System Neoplasms/blood supply , Central Nervous System Neoplasms/genetics , Genes, Tumor Suppressor , Growth Substances/metabolism , Humans , Neoplasm Invasiveness , Neoplasm Proteins/metabolism , Neovascularization, Pathologic , Oncogenes , Receptors, Growth Factor/metabolism , Signal Transduction
7.
Int J Oncol ; 17(4): 683-93, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10995878

ABSTRACT

SPARC is a secreted glycoprotein that interacts with extracellular matrix (ECM) proteins to promote de-adhesion of cells from the matrix, thereby inducing a biological state conducive to cell migration. We have demonstrated that SPARC is highly expressed in gliomas (grades II-IV) and promotes glioma invasion in vitro. Therefore, the protein itself or its mechanisms of action might become therapeutic targets to arrest glioma invasion. Vitronectin is an ECM protein found in the blood vessel basement membranes and may promote glioma invasion along these structures. It binds to SPARC in vitro. However, it is not known whether SPARC and vitronectin colocalize and/or interact to contribute to brain tumor cell migration in vivo. In this study, we immuno-histochemically determined if the grade I juvenile pilocytic astrocytomas (JPAs) also express SPARC, if vitronectin is expressed in grades I, II, and IV astrocytomas, and if the proteins colocalize in brain tumors in vivo. We performed western blot analyses to determine if different grades of tumors had different intracellular and/or secreted levels of SPARC and vitronectin. We performed migration assays to determine whether vitronectin is a permissive substrate for glioma migration, and whether the extent of migration correlates with the level of secreted SPARC. Our data demonstrated that JPAs expressed SPARC and secreted significantly higher levels than glioblastomas multiforme (GBMs). Vitronectin was absent from well-preserved tumor but present in areas of disrupted tumor, such as degeneration and/or necrosis. SPARC and vitronectin colocalized only in regions of angiogenesis. We observed that the extent of migration on vitronectin inversely correlated with the level of secreted SPARC: the higher the level, the lesser the migration. These data suggest that the outcome of SPARC - ECM interactions may depend on local SPARC concentrations. The high levels of SPARC secreted by the JPAs, paradoxically, may be more prohibitive for migration on vitronectin than the lower levels secreted by the GBMs. This may account, in part, for the lack of JPA invasion into brain tissue along blood vessel membranes.


Subject(s)
Brain Neoplasms/pathology , Glioma/pathology , Osteonectin/biosynthesis , Adult , Blotting, Western , Brain Neoplasms/metabolism , Cell Movement/drug effects , Child , Dose-Response Relationship, Drug , Glioma/metabolism , Humans , Immunohistochemistry , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Osteonectin/pharmacology , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism
8.
Hum Mol Genet ; 9(10): 1495-500, 2000 Jun 12.
Article in English | MEDLINE | ID: mdl-10888600

ABSTRACT

Meningiomas are common nervous system tumors, whose molecular pathogenesis is poorly understood. To date, the most frequent genetic alteration detected in these tumors is loss of heterozygosity (LOH) on chromosome 22q. This finding led to the identification of the neurofibromatosis 2 (NF2) tumor suppressor gene on 22q12, which is inactivated in 40% of sporadic meningiomas. The NF2 gene product, merlin (or schwannomin), is a member of the protein 4.1 family of membrane-associated proteins, which also includes ezrin, radixin and moesin. Recently, we identified another protein 4.1 gene, DAL-1 (differentially expressed in adenocarcinoma of the lung) located on chromosome 18p11.3, which is lost in approximately 60% of non-small cell lung carcinomas, and exhibits growth-suppressing properties in lung cancer cell lines. Given the homology between DAL-1 and NF2 and the identification of significant LOH in the region of DAL-1 in lung, breast and brain tumors, we investigated the possibility that loss of expression of DAL-1 was important for meningioma development. In this report, we demonstrate DAL-1 loss in 60% of sporadic meningiomas using LOH, RT-PCR, western blot and immunohistochemistry analyses. Analogous to merlin, we show that DAL-1 loss is an early event in meningioma tumorigenesis, suggesting that these two protein 4.1 family members are critical growth regulators in the pathogenesis of meningiomas. Furthermore, our work supports the emerging notion that membrane-associated alterations are important in the early stages of neoplastic transformation and the study of such alterations may elucidate the mechanism of tumorigenesis shared by other tumor types.


Subject(s)
Cytoskeletal Proteins , Genes, Tumor Suppressor/physiology , Membrane Proteins/biosynthesis , Membrane Proteins/metabolism , Membrane Proteins/physiology , Meningeal Neoplasms/genetics , Meningioma/genetics , Neuropeptides , Tumor Suppressor Proteins , Animals , Blotting, Western , Chromosomes, Human, Pair 18 , Humans , Immunohistochemistry , Loss of Heterozygosity , Membrane Proteins/chemistry , Membrane Proteins/genetics , Microfilament Proteins , Neurilemmoma/genetics , Neurilemmoma/metabolism , Neurofibromin 2 , Polymorphism, Single-Stranded Conformational , Protein Structure, Tertiary , Rats , Reverse Transcriptase Polymerase Chain Reaction , Schwann Cells/metabolism , Tumor Cells, Cultured
9.
Int J Cancer ; 87(2): 261-8, 2000 Jul 15.
Article in English | MEDLINE | ID: mdl-10861485

ABSTRACT

The solid growth of high-grade glioma appears to be critically dependent on tumor angiogenesis. It remains unknown, however, whether the diffuse infiltration of glioma cells into healthy adjacent tissue is also dependent on the formation of new tumor vessels. Here, we analyze the relationship between tumor angiogenesis and tumor cell infiltration in an experimental glioma model. C6 cells were implanted into the dorsal skinfold chamber of nude mice, and tumor angiogenesis was monitored by intravital fluorescence videomicroscopy. Glioma infiltration was assessed by the extent of tumor cell invasion into the adjacent chamber tissue and by expression of SPARC, a cellular marker of glioma invasiveness. To test the hypothesis that glioma angiogenesis and glioma infiltration are codependent, we assessed tumor infiltration in both the presence and the absence of the angiogenesis inhibitor SU5416. SU5416 is a selective inhibitor of the VEGF/Flk-1 signal-transduction pathway, a critical pathway implicated in angiogenesis. Control tumors demonstrated both high angiogenic activity and tumor cell invasion accompanied by strong expression of SPARC in invading tumor cells at the tumor-host tissue border. SU5416-treated tumors demonstrated reduced vascular density and vascular surface in the tumor periphery accompanied by marked inhibition of glioma invasion and decreased SPARC expression. A direct effect of SU5416 on glioma cell motility and invasiveness was excluded by in vitro migration and invasion assays. These results suggest a crucial role for glioma-induced angiogenesis as a prerequisite for diffuse tumor invasion and a possible therapeutic role for anti-angiogenic compounds as inhibitors of both solid and diffuse infiltrative tumor growth.


Subject(s)
Glioma/pathology , Neovascularization, Pathologic , Osteonectin/biosynthesis , Angiogenesis Inhibitors/pharmacology , Animals , Biomarkers, Tumor/biosynthesis , Cell Division/drug effects , Cell Movement/drug effects , Dimethyl Sulfoxide/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Glioma/blood supply , Glioma/metabolism , Immunohistochemistry , Indoles/pharmacology , Male , Mice , Mice, Nude , Microscopy, Fluorescence , Microscopy, Video , Neoplasm Invasiveness , Neoplasm Transplantation , Pyrroles/pharmacology , Rats , Solvents/pharmacology , Time Factors , Tumor Cells, Cultured
10.
Clin Cancer Res ; 6(4): 1248-54, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10778947

ABSTRACT

Carboxyamido-triazole (CAI), an inhibitor of non-voltage-gated calcium channels, has been studied in Phase I/II clinical trials following the identification of its inhibitory effects on tumor cell invasion and motility. It has also been reported to inhibit human endothelial cell proliferation, migration, and adhesion to the basement membrane. In glioma, biological assays have shown CAI to be active in inhibiting the phenotypes of invasion and angiogenesis. The exact mechanism of action is not clearly understood, although it appears to work via inhibition of calcium influx in several signal transduction pathways that inhibit cell cycle progression. Recent evidence implicates apoptosis as a contributing mechanism of chemotherapy-induced tumor cytotoxicity. Therefore, we studied the effects of CAI on apoptosis in bovine aortic endothelial cells and a human glioma cell line (U251N) using a variety of methods, including: (a) cell morphology; (b) terminal deoxynucleotidyl transferase-mediated nick end labeling analysis of in situ DNA strand breaks; (c) agarose gel electrophoresis to visualize DNA fragmentation; and (d) flow cytometry. Here we report that the kinetics of CAI-induced apoptosis in bovine aortic endothelial cells and glioma cells was determined to be both dose and time dependent in micromolar concentrations achievable in brain tissue in vivo.


Subject(s)
Apoptosis/drug effects , Calcium Channel Blockers/pharmacology , Endothelium, Vascular/drug effects , Glioma/drug therapy , Triazoles/pharmacology , Animals , Aorta/cytology , Cattle , Cells, Cultured , DNA Fragmentation/drug effects , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Flow Cytometry , Glioma/genetics , Glioma/pathology , Humans , In Situ Nick-End Labeling , Time Factors , Tumor Cells, Cultured
11.
Clin Cancer Res ; 6(1): 102-11, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10656438

ABSTRACT

Glioblastoma multiforme (GBM) tumors display extensive histomorphological heterogeneity, with great variability in the extent of invasiveness, angiogenesis, and necrosis. The identification of genes associated with these phenotypes should further the molecular characterization, permitting better definition of glioma subsets that may ultimately lead to better treatment strategies. Therefore, we performed a differential mRNA display analysis comparing six GBM-derived primary cell cultures from patients having tumors with varied histomorphological features. We identified stromal cell-derived factor 1 (SDF1) as a gene with varied expression. SDF1 (cytokine) and CXC chemokine receptor 4 (CXCR4) interactions are implicated in modulating cell migration. They are also implicated in modulating the immune response in AIDS patients by macrophage-mediated T-cell apoptosis. GBM patients also fail to mount an immune response, although their tumors are seemingly exposed to immune cells in regions of angiogenesis, where the blood-brain barrier is absent, or in areas of necrosis. To determine whether the expression and localization of SDF1 and CXCR4 are consistent with such a role in these brain tumors, immunohistochemical analyses of these proteins were performed on normal brain and astrocytomas (grades II-IV). In normal brain tissue, low levels of SDF1 (0.5+) were observed in astrocytic processes, in neurons, and in the occasional phagocytic cells around vessels. CXCR4 expression was negative in brain tissue but was observed in phagocytic cells within the vessel lumen. In tumors, SDF1 and CXCR4 expression was colocalized when both were expressed, and SDF1 and CXCR4 expression increased with increasing tumor grade (from 0.5+ to 6+). Additionally, CXCR4 was expressed in neovessel endothelial cells. The proteins were expressed in regions of angiogenesis and degenerative, necrotic, and microcystic changes. Those tumors displaying greater amounts of these features had greater staining intensity of the proteins. The expression of SDF1 and CXCR4 did not colocalize with the proliferation marker MIB-1. Thus, our data suggest that SDF1 and CXCR4 expressions: (a) increase with increasing grade; (b) colocalize to regions within these tumors where their interaction may contribute to angiogenesis and/or modulation of the immune response; and (c) may serve to characterize subsets of GBMs.


Subject(s)
Brain Neoplasms/blood supply , Brain Neoplasms/pathology , Chemokines, CXC/analysis , Glioblastoma/blood supply , Glioblastoma/pathology , Neovascularization, Pathologic , Receptors, CXCR4/analysis , Astrocytoma/blood supply , Astrocytoma/genetics , Astrocytoma/immunology , Astrocytoma/pathology , Brain Neoplasms/genetics , Brain Neoplasms/immunology , Chemokine CXCL12 , Chemokines, CXC/genetics , Glioblastoma/genetics , Glioblastoma/immunology , Humans , Immunohistochemistry , Necrosis , Receptors, CXCR4/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
12.
Neuro Oncol ; 2(1): 1-5, 2000 01.
Article in English | MEDLINE | ID: mdl-11302248

ABSTRACT

The prognosis for most patients with astrocytic glioma is poor, and postoperative life expectancy has not significantly improved in the last decade despite advances in diagnosis, surgery, and adjuvant therapy. Progress has been made, however, in cataloging the genetic alterations that occur in these tumors. Studying the allelic changes using loss of heterozygosity analysis has proven to be a reliable and rapid way of identifying genetic alterations fundamental to the pathology of this disease. In this study, we used a series of fluorescent-labeled markers and a new horizontal ultrathin gel electrophoresis technology (HUGE; GeneSys Technologies, Inc.) to analyze loss of heterozygosity on 11p15 in a series of 24 matched normal/tumor glioma pairs that included both anaplastic astrocytomas and glioblastomas. These studies significantly narrowed the region harboring a putative 11p15.5 glioma-associated gene and further suggest that a second gene involved in the pathogenesis of brain tumors may exist, centromeric, in bands 11p15.5-p15.4.


Subject(s)
Alleles , Astrocytoma/genetics , Brain Neoplasms/genetics , Chromosomes, Human, Pair 11/genetics , Electrophoresis, Polyacrylamide Gel/methods , Gene Deletion , Glioblastoma/genetics , DNA Fragmentation , Humans , Loss of Heterozygosity
13.
Int J Dev Neurosci ; 17(5-6): 463-72, 1999.
Article in English | MEDLINE | ID: mdl-10571408

ABSTRACT

Our recent studies have focused on identifying invasion-promoting genes that are expressed early in brain tumor progression. We identified and characterized SPARC (secreted protein acidic and rich in cysteine) as a potential candidate. To determine whether increased SPARC expression functionally promotes brain tumor invasion, SPARC was transfected into U87MG glioblastoma cells using the tetracycline-off gene expression system. The parental cell line (U87MG), the parental transactivator-transfected clone (U87T2) and three selected U87T2-SPARC-transfected clones (A2bi, A2b2 and C2a4) were characterized for endogenous and transfected SPARC expression. In comparison to the parental or U87T2 cell lines, the SPARC-transfected clones demonstrated: (1) morphological changes, (2) increased SPARC transcript and protein abundances that were down-regulated by the tetracycline analog doxycycline, (3) perinuclear localization of the transfected SPARC (consistent with reported localization of SPARC in normal cells in culture) and (4) altered adhesion and increased invasion as assessed by the spheroid confrontation assay. These data indicate that increased SPARC expression contributes to U87 glioblastoma tumor invasion in vitro and that these cell lines will serve as useful reagents to investigate the mechanism(s) by which SPARC promotes this phenotype in vitro and in vivo.


Subject(s)
Cysteine/analysis , Gene Expression Regulation, Neoplastic/physiology , Neoplasm Proteins/metabolism , Animals , Neoplasm Invasiveness , Rats , Rats, Sprague-Dawley , Tumor Cells, Cultured
14.
Clin Cancer Res ; 5(2): 237-41, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10037170

ABSTRACT

SPARC, a secreted, extracellular matrix-associated protein implicated in the modulation of cell adhesion and migration, was evaluated as a marker for invasive meningiomas. Although the majority of meningiomas are clinically and morphologically benign, approximately 10% progress into atypical and malignant tumors, according to the standard criteria. However, a subset of meningiomas presents as histomorphologically benign tumors (WHO grade I), but they are clinically invasive. It has been suggested that these tumors should be classified as malignant, and that the patients may require adjuvant therapy and closer follow up. Unfortunately, a significant number of these tumors may not be recognized because the surgical specimen used to assess the grade of a tumor lacks the infiltrative interface with the brain, which is currently necessary to determine its invasive character. Therefore, a marker of heightened invasiveness would greatly facilitate the identification of this subset of patients. In this study, the immunohistochemical expression of SPARC in benign, noninvasive primary meningiomas was compared with its expression in invasive, aggressive, primary and recurrent meningiomas. SPARC was not expressed in the 9 benign, noninvasive tumors, but was highly expressed in the 20 invasive tumors, regardless of the grade. The findings suggest that SPARC is a potential diagnostic marker of invasive meningiomas and is capable of distinguishing the histomorphologically benign noninvasive from the histomorphologically benign but invasive meningiomas, in the absence of the infiltrative interface.


Subject(s)
Biomarkers, Tumor/biosynthesis , Meningeal Neoplasms/metabolism , Meningioma/metabolism , Osteonectin/biosynthesis , Humans , Immunohistochemistry , Meningeal Neoplasms/chemistry , Meningeal Neoplasms/secondary , Meningioma/chemistry , Meningioma/secondary , Neoplasm Invasiveness
15.
J Neuropathol Exp Neurol ; 57(12): 1112-21, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9862633

ABSTRACT

In an attempt to identify genetic alterations occurring early in astrocytoma progression, we performed subtractive hybridization between astrocytoma and glioblastoma cDNA libraries. We identified secreted protein acidic and rich in cysteine (SPARC), a protein implicated in cell-matrix interactions, as a gene overexpressed early in progression. Northern blot and immunohistochemical analyses indicated that transcript and protein were both elevated in all tumor specimens (grades II-IV) examined when compared with levels in normal brain. The level of SPARC expression was found to be tumor-dependent rather than grade-related. Immunohistochemically, SPARC protein was found to be overexpressed in 1) cells in the less cellularly dense regions within the tumor mass, 2) histomorphologically neoplastic-looking cells in adjacent normal brain at the tumor/brain interface, 3) neovessel endothelial cells in both the tumor and adjacent normal brain, and 4) reactive astrocytes in normal brain adjacent to tumor. Using a combination of DNA in situ hybridization and protein immunohistochemical analyses of the human/rat xenograft, SPARC expression was observed in the human glioma cells within the tumor mass, and in cells that invaded along vascular basement membranes and individually into the rat brain parenchyma, suggesting it may be an invasion-related gene. While it remains to be determined whether SPARC functionally contributes to tumor cell invasion, these data suggest that the early onset of increased SPARC expression, though complex, may serve as a signal indicative of neoplastic astrocytic transformation and reactive response to tumor-induced stress.


Subject(s)
Astrocytes/physiology , Brain Neoplasms/pathology , Gene Expression Regulation, Neoplastic/physiology , Glioma/pathology , Osteonectin/genetics , Signal Transduction/physiology , Animals , Astrocytoma/genetics , Cell Transformation, Neoplastic , Cloning, Molecular , Glioblastoma/pathology , Humans , Neoplasm Invasiveness , Neoplasm Transplantation , Rats , Transplantation, Heterologous
16.
Curr Opin Oncol ; 10(3): 179-85, 1998 May.
Article in English | MEDLINE | ID: mdl-9619352

ABSTRACT

Cell growth, cell migration, and angiogenesis are normal biologic processes hijacked by tumor cells to promote tumor proliferation, invasion, and neovascularity. Great strides have been made in characterizing the altered gene expression and acquired genetic mutations in central nervous system tumors. This review focuses on the known or implicated genetic alterations and their contribution to these biologic processes, with an emphasis on recent contributions to the field.


Subject(s)
Central Nervous System Neoplasms/genetics , Cell Cycle Proteins/physiology , Cell Division , Central Nervous System Neoplasms/metabolism , Central Nervous System Neoplasms/pathology , Extracellular Matrix/physiology , Growth Substances/physiology , Humans , Metalloendopeptidases/physiology , Neoplasm Invasiveness/genetics , Neoplasm Proteins/physiology , Neovascularization, Pathologic/genetics , Receptors, Growth Factor/physiology , Remission Induction
17.
Neurol Med Chir (Tokyo) ; 38 Suppl: 161-4, 1998.
Article in English | MEDLINE | ID: mdl-10235000

ABSTRACT

The factors responsible for the development of cerebral arteriovenous malformations (AVMs) are not well known. Patients with hereditary hemorrhagic telangiectasia (HHT) have cutaneous vascular dysplasia and a high propensity to develop systemic and cerebral AVMs. Transforming growth factor-beta (TGF-beta) complex has been implicated in HHT. The aim of this study was to evaluate the expression of TGF-beta 1, TGF-beta 2, TGF-beta 3, and their two receptors (R1 and R2) in AVMs and in normal brain vessels. Formalin-fixed, paraffin-embedded tissues from 20 patients with cerebral AVMs (including two patients with HHT) were sequentially sectioned into 6 microns sections. Similar sections from normal brain tissue were obtained from five patients without AVMs and no intracranial pathology, who had died from unrelated causes. The normal tissue sections included large intracranial arteries, small arteries, venous sinuses, cortical veins, and brain tissue containing arterioles, capillaries, and venules. All specimens underwent immunohistochemical analyses with polyclonal antibodies to the following antigens: TGF-beta 1, TGF-beta 2, TGF-beta 3, and R1 and R2. The immunoreactivity, when present, was consistently noted in endothelial cells and in the medial smooth muscle. The intensity of vessel wall immunostaining was graded on a scale from 0 to 3. The mean staining grades of normal vessels for TGF-beta 1, TGF-beta 2, TGF-beta 3, R1, and R2 were 0.6 (range 0-1), 3, 2.8 (range 2-3), 1.6 (range 0-2), and 3, respectively, whereas the mean staining grades of AVM vessels were 0.3 (range 0-1), 0.8 (range 0-1), 0.6 (range 0-1), 1.4 (range 0-2), and 0.9 (range 0-1), respectively. The study thus demonstrated that normal brain vessels (arteries, veins, small vessels) have strong (range 2.8-3) immunostaining for TGF-beta 2, TGF-beta 3, and R2, and that the AVM nidus vessels have a paucity (range 0.8-0.9) of staining for these factors. In AVM vessels that had zero immunoreactivity to the above three factors, the vessel wall was fibrocollagenous rather than muscular. Further studies to examine the TGF-beta complex behavior in AVMs are needed.


Subject(s)
Arteriovenous Malformations/metabolism , Transforming Growth Factor beta/biosynthesis , Antibodies/immunology , Arteriovenous Malformations/genetics , Arteriovenous Malformations/immunology , Gene Expression Regulation, Developmental/genetics , Humans , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/immunology
18.
Mol Chem Neuropathol ; 35(1-3): 23-37, 1998.
Article in English | MEDLINE | ID: mdl-10343969

ABSTRACT

The messenger ribonucleic acid (mRNA) of gap junction protein connexin 43 was quantified in the tetanus toxin rat model of focal epilepsy following injection of toxin into the left amygdala. Animals were monitored electrographically at weekly intervals with bilateral amygdala electrodes. Cohorts of 3 rats were sacrificed at weeks 1, 2, 3, 4, 6, 8, and 10, and bilateral regions containing the amygdala and posterior cerebral cortex were sampled, frozen, and later pooled for northern blot analysis. Spike generation was manifest in all animals during the first 4 wk followed by variable attenuation and cessation by 10 wk. Electrode implantation alone was shown by regression analysis to cause significant (p < 0.05) elevation of connexin mRNA in weeks 1-4. Injection of toxin diminished connexin mRNA expression in the amygdala when compared to electrode implantation alone. No trend in connexin mRNA expression was established over time in either amygdala or cerebral cortex in the acute epileptic or chronic postepileptic phase. No association between connexin 43 mRNA expression and the development of epileptogenicity was found in the context of a self-limiting animal model of focal epilepsy.


Subject(s)
Connexin 43/genetics , Gene Expression Regulation , Seizures/metabolism , Tetanus Toxin/toxicity , Transcription, Genetic , Amygdala/metabolism , Amygdala/physiopathology , Animals , Cerebral Cortex/metabolism , Electroencephalography , Functional Laterality , Male , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Seizures/chemically induced , Seizures/physiopathology , Time Factors , Transcription, Genetic/drug effects
19.
Exp Neurol ; 145(1): 154-64, 1997 May.
Article in English | MEDLINE | ID: mdl-9184118

ABSTRACT

An increase in the cellular production of gap junction proteins and increased numbers of gap junctions in the neuronoglial syncytium of an epileptic focus have been proposed as a possible mechanism underlying synchronization of discharge. To study this issue, both Northern and Western blot analyses of the gap junction protein connexin 43 mRNA and protein abundance were performed on hippocampal tissue resected from patients presenting with a complex partial seizure disorder arising from the medial temporal area and the hippocampus in particular. Samples from 15 patients with medically intractable seizures were compared to those from 5 nonepileptic patients requiring temporal lobectomy in life-threatening situations. Six of the 15 epileptic patients underwent noninvasive electrographic recording, whereas the remaining 9 patients required intracerebral electrodes for extraoperative recording and therefore showed a more discrete focality than the noninvasive recordings. A decline in the mean levels of connexin 43 mRNA expressed predominantly in astrocytes was noted in the epileptic patient groups, particularly for those cases requiring intracranial electrode placement where ictal onset was more clearly established to be intrahippocampal. Quantitation of connexin 43 protein in both epileptogenic and nonepileptogenic hippocampal tissues showed no significant differences in expression. Although mean values for mRNA showed a decline, clinical outcomes postoperatively showed no correlation with either mRNA or protein expression individually in our epileptic population. The findings indicate that there is effectively no upregulation of mRNA and no increased production of connexin 43 protein in response to the development of epileptogenicity. Rather it appears the influence of gap junctions as a substrate of epileptogenicity in any mechanism(s) underlying synchrony or electrical propagation may be a function of the dynamic state (open versus closed) of the membrane-bound gap junction.


Subject(s)
Connexin 43/genetics , Epilepsy, Complex Partial/metabolism , Hippocampus/metabolism , Adult , Blotting, Northern , Blotting, Western , Connexin 43/analysis , Electroencephalography , Epilepsy, Complex Partial/diagnosis , Epilepsy, Complex Partial/genetics , Female , Gene Expression/physiology , Hippocampus/chemistry , Hippocampus/physiopathology , Humans , Magnetic Resonance Imaging , Male , Middle Aged , RNA, Messenger/metabolism , Temporal Lobe/physiopathology , Temporal Lobe/surgery
20.
Mol Chem Neuropathol ; 32(1-3): 75-88, 1997.
Article in English | MEDLINE | ID: mdl-9437659

ABSTRACT

The expression of mRNA for connexin 43, a gap junction protein putatively found in astrocytes, is studied in two experimental models of epilepsy: the electrically kindled rat and the tetanus-toxin-injected rat. Rats were kindled by electrical stimulation of the amygdala to Racine class 5 seizures and divided into cohorts of three to undergo 3, 6, or 10 such events, respectively. Another two cohorts of rats received injections of tetanus toxin at strengths of 3 and 9 MLD50, respectively, into the amygdala. Features of epileptogenicity were identified electrographically in both cohorts during the first 4 wk following toxin injection with spontaneous ictal events recorded in the latter cohort. All rats were sacrificed 4 wk after electrode or cannula implantation, except for two toxin-injected cohorts that were sacrificed at wk 8 or 10. The epileptogeonic area in the region of the amygdala was harvested and pooled by cohort for Northern blot analysis. These were compared with control nonimplanted tissues. In the tetanus-toxin-injected animals, at time-points of 4, 8, and 10 wk, connexin 43 mRNA expression in epileptogenic tissues is found to be decreased or unchanged relative to control cases. Kindled rats demonstrated reductions of connexin mRNA with a trend toward normalizing levels with increasing numbers of stimulations when compared to control animals. Connexin 43 immunostained sections of the basolateral amygdala showed a similar trend in protein expression. Both experimental models of epilepsy show no connexin 43 mRNA upregulation despite varying degrees of epileptogenicity. This study therefore does not support the hypothesis that an increase in transcription is the basis for any proposed increase in gap junction communication involving connexin 43 in the context of epileptogenicity or as a reaction to increased neuronal excitability.


Subject(s)
Connexin 43/biosynthesis , Epilepsy/metabolism , RNA, Messenger/biosynthesis , Animals , Blotting, Northern , Electric Stimulation , Electroencephalography , Epilepsy/chemically induced , Immunohistochemistry , Kindling, Neurologic/physiology , Male , Rats , Rats, Sprague-Dawley , Tetanus Toxin
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