ECF-Type ATP-Binding Cassette Transporters.

Energy-coupling factor (ECF)-type ATP-binding cassette (ABC) transporters catalyze membrane transport of micronutrients in prokaryotes. Crystal structures and biochemical characterization have revealed that ECF transporters are mechanistically distinct from other ABC transport systems. Notably, ECF transporters make use of small integral membrane subunits (S-components) that are predicted to topple over in the membrane when carrying the bound substrate from the extracellular side of the bilayer to the cytosol. Here, we review the phylogenetic diversity of ECF transporters as well as recent structural and biochemical advancements that have led to the postulation of conceptually different mechanistic models. These models can be described as power stroke and thermal ratchet. Structural data indicate that the lipid composition and bilayer structure are likely to have great impact on the transport function. We argue that study of ECF transporters could lead to generic insight into membrane protein structure, dynamics, and interaction.

Cysteine-mediated decyanation of vitamin B12 by the predicted membrane transporter BtuM.

Uptake of vitamin B12 is essential for many prokaryotes, but in most cases the membrane proteins involved are yet to be identified. We present the biochemical characterization and high-resolution crystal structure of BtuM, a predicted bacterial vitamin B12 uptake system. BtuM binds vitamin B12 in its base-off conformation, with a cysteine residue as axial ligand of the corrin cobalt ion. Spectroscopic analysis indicates that the unusual thiolate coordination allows for decyanation of vitamin B12. Chemical modification of the substrate is a property other characterized vitamin B12-transport proteins do not exhibit.

Correlation of perfusion parameters with genes related to angiogenesis regulation in glioblastoma: a feasibility study.

BACKGROUND AND PURPOSE: Integration of imaging and genomic data is critical for a better understanding of gliomas, particularly considering the increasing focus on the use of imaging biomarkers for patient survival and treatment response. The purpose of this study was to correlate CBV and PS measured by using PCT with the genes regulating angiogenesis in GBM. MATERIALS AND METHODS: Eighteen patients with WHO grade IV gliomas underwent pretreatment PCT and measurement of CBV and PS values from enhancing tumor. Tumor specimens were analyzed by TCGA by using Human Gene Expression Microarrays and were interrogated for correlation between CBV and PS estimates across the genome. We used the GO biologic process pathways for angiogenesis regulation to select genes of interest. RESULTS: We observed expression levels for 92 angiogenesis-associated genes (332 probes), 19 of which had significant correlation with PS and 9 of which had significant correlation with CBV (P < .05). Proangiogenic genes such as TNFRSF1A (PS = 0.53, P = .024), HIF1A (PS = 0.62, P = .0065), KDR (CBV = 0.60, P = .0084; PS = 0.59, P = .0097), TIE1 (CBV = 0.54, P = .022; PS = 0.49, P = .039), and TIE2/TEK (CBV = 0.58, P = .012) showed a significant positive correlation; whereas antiangiogenic genes such as VASH2 (PS = -0.72, P = .00011) showed a significant inverse correlation. CONCLUSIONS: Our findings are provocative, with some of the proangiogenic genes showing a positive correlation and some of the antiangiogenic genes showing an inverse correlation with tumor perfusion parameters, suggesting a molecular basis for these imaging biomarkers; however, this should be confirmed in a larger patient population.

Splenic and immune alterations of the Sparc-null mouse accompany a lack of immune response.

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.

PIKE-A is a proto-oncogene promoting cell growth, transformation and invasion.

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.

SPARC modulates cell growth, attachment and migration of U87 glioma cells on brain extracellular matrix proteins.

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.

Molecular biology of nervous system tumors.

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.

A study of SPARC and vitronectin localization and expression in pediatric and adult gliomas: high SPARC secretion correlates with decreased migration on vitronectin.

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.

Loss of DAL-1, a protein 4.1-related tumor suppressor, is an important early event in the pathogenesis of meningiomas.

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.

Targeting angiogenesis inhibits tumor infiltration and expression of the pro-invasive protein SPARC.

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.

Carboxyamido-triazole induces apoptosis in bovine aortic endothelial and human glioma cells.

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.

Identification and localization of the cytokine SDF1 and its receptor, CXC chemokine receptor 4, to regions of necrosis and angiogenesis in human glioblastoma.

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.

Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis.

Modified viruses are used as gene transfer vectors because of their ability to transfer genetic material efficiently to the nucleus of a target cell. To better understand intracellular translocation of adenovirus serotype 5 (Ad), fluorophores were covalently conjugated to Ad capsids, and movement of fluorescent Ad within the cytoplasm was observed during the first hour of infection of a human lung epithelial carcinoma cell line (A549). Ad translocation was characterized with respect to its ability to achieve nuclear envelope localization as well as directed movement in the cytoplasm. Whereas Ad achieved efficient nuclear localization 60 min after infection of A549 cells under control conditions, depolymerization of the microtubule cytoskeleton by addition of 25 microM nocodazole reversibly inhibited development of nuclear localization. In contrast, depolymerization of microfilaments by addition of 1 microM cytochalasin D had no effect on nuclear localization. Direct video observation of Ad motility showed that nocodazole, but not cytochalasin D, caused a reversible decrease in rapid linear translocations of Ad in the cytoplasm of A549 cells. Microinjection of function-blocking antibodies against the microtubule-dependent motor protein, cytoplasmic dynein, but not kinesin, blocked nuclear localization of Ad, consistent with net minus end-directed motility indicated by accumulation of Ad at mitotic spindles. Fluorescence ratio imaging revealed a neutral pH in the environment of translocating Ad, leading to a model in which the interaction of Ad with an intact microtubule cytoskeleton and functional cytoplasmic dynein occurs after escape from endosomes and is a necessary prerequisite to nuclear localization of adenovirus serotype 5.

Use of horizontal ultrathin gel electrophoresis to analyze allelic deletions in chromosome band 11p15.5 in gliomas.

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.

Increased SPARC expression promotes U87 glioblastoma invasion in vitro.

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.

SPARC: a potential diagnostic marker of invasive meningiomas.

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.

SPARC: a signal of astrocytic neoplastic transformation and reactive response in human primary and xenograft gliomas.

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.