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Bozena Kaminska
Professor, head of laboratory, Nencki Institute of Experimental Biology
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Journal of neurochemistry 2010 Apr; 113(4)
Prolonged activation of ERK triggers glutamate-induced apoptosis of astrocytes: neuroprotective effect of FK506.
Although, astrocytes are more resistant than neurons to ischemic injury, astrocyte death has been demonstrated in animal models of brain ischemia. Astrocytes death after ischemia/reperfusion may strongly affect neuronal survival because of the absenc... expand abstracte of their trophic and metabolic support to neurons, and astrocytic glutamate uptake. Early signals involved in astrocytes death are poorly understood. We demonstrated enhanced and mostly cytoplasmic activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) during glutamate-induced apoptosis of cultured astrocytes. Treatment with UO126, inhibitor of MEK1, threo-beta-benzyloxyaspartic acid, glutamate transporter inhibitor, and FK506, a cytoprotective drug prevented ERK activation and glutamate-induced apoptosis. Over-expression of ERK dual specificity phosphatases 5 and 6 reduced apoptosis in transfected astrocytes. Prolonged ERK1/2 activation was observed in ischemic brain: in the nucleus and cytoplasm of astrocytes in the cerebral cortex, and exclusively in the cytoplasm of astrocytes in the striatum. Global gene expression profiling in the cortex revealed that FK506 blocks middle cerebral artery occlusion-induced expression of numerous genes associated with ERK signaling pathway and apoptosis. The results demonstrate a pro-apoptotic role of sustained activation of ERK1/2 signaling in glutamate-induced death of astrocytes and the ability of FK506 to block both ERK activation and astrocytic cell death in vitro and in ischemic brains. collapse abstract
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The American journal of pathology 2010 Mar; 176(4)
Novel proteins regulated by mTOR in subependymal giant cell astrocytomas of patients with tuberous sclerosis complex and new therapeutic implications.
Subependymal giant cell astrocytomas (SEGAs) are rare brain tumors associated with tuberous sclerosis complex (TSC), a disease caused by mutations in TSC1 or TSC2, resulting in enhancement of mammalian target of rapamycin (mTOR) activity, dysregulati... expand abstracton of cell growth, and tumorigenesis. Signaling via mTOR plays a role in multifaceted genomic responses, but its effectors in the brain are largely unknown. Therefore, gene expression profiling on four SEGAs was performed with Affymetrix Human Genome arrays. Of the genes differentially expressed in TSC, 11 were validated by real-time PCR on independent tumor samples and 3 SEGA-derived cultures. Expression of several proteins was confirmed by immunohistochemistry. The differentially-regulated proteins were mainly involved in tumorigenesis and nervous system development. ANXA1, GPNMB, LTF, RND3, S100A11, SFRP4, and NPTX1 genes were likely to be mTOR effector genes in SEGA, as their expression was modulated by an mTOR inhibitor, rapamycin, in SEGA-derived cells. Inhibition of mTOR signaling affected size of cultured SEGA cells but had no influence on their proliferation, morphology, or migration, whereas inhibition of both mTOR and extracellular signal-regulated kinase signaling pathways led to significant alterations of these processes. For the first time, we identified genes related to the occurrence of SEGA and regulated by mTOR and demonstrated an effective modulation of SEGA growth by pharmacological inhibition of both mTOR and extracellular signal-regulated kinase signaling pathways, which could represent a novel therapeutic approach. collapse abstract
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Anatomical record (Hoboken, N.J. : 2007) 2009 Nov; 292(12)
MAPK signal transduction underlying brain inflammation and gliosis as therapeutic target.
A majority, if not all, acute and progressive neurodegenerative diseases are accompanied by local microglia-mediated inflammation, astrogliosis, infiltration of immune cells, and activation of the adaptive immunity. These processes progress by the ex... expand abstractpression of cytokines, adhesion molecules, proteases, and other inflammation mediators. In response to brain injury or infection, intracellular signaling pathways are activated in microglia, which turn on inflammatory and antigen-presenting cell functions. Different extrinsic signals shape microglial activation toward neuroprotective or neurotoxic phenotype under pathological conditions. This review discusses recent advances regarding molecular mechanisms of inflammatory signal transduction in neurological disorders and in in vitro models of inflammation/gliosis. Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine protein kinases responsible for most cellular responses to cytokines and external stress signals and crucial for regulation of the production of inflammation mediators. Increased activity of MAPKs in activated microglia and astrocytes, and their regulatory role in the synthesis of inflammatory cytokines mediators, make them potential targets for novel therapeutics. MAPK inhibitors emerge as attractive anti-inflammatory drugs, because they are capable of reducing both the synthesis of inflammation mediators at multiple levels and are effective in blocking inflammatory cytokine signaling. Small molecule inhibitors targeting of p38 MAPK and JNK pathways have been developed and offer a great potential as potent modulators of brain inflammation and gliosis in neurological disorders, where cytokine overproduction contributes to disease progression. Many of the pharmacological MAPK inhibitors can be administered orally and initial results show therapeutic benefits in preclinical animal models. collapse abstract
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International journal of oncology 2009 Oct; 35(5)
Efficacy and mechanism of anti-tumor action of new potential CK2 inhibitors toward glioblastoma cells.
Malignant gliomas are highly resistant to current therapeutic approaches due to genetic alterations rendering them resistant to cell death. CK2, a ubiquitous and constitutively active serine/threonine kinase, frequently elevated in tumors, contribute... expand abstracts to enhanced cell proliferation and resistance to apoptosis. Inhibition of CK2 expression or treatment with inhibitors of CK2 affected survival or induced apoptosis in various cancer cells. Here we compared cytotoxic effects of well-known and new CK2 inhibitors: 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), 4,5,6,7-tetrabromo-1H-benzimidazole (TBI), 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), the related 3-(4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)propan-1-ol (MB001), 3-(4,5,6,7-tetrabromo-1H-1,2,3-benzotriazol-1-yl) propan-1-ol (MB002), 3-(4,5,6,7-tetrabromo-2H-1,2,3-benzotriazol-2-yl)propan-1-ol (MB003) and also structurally similar to above compounds pentabromobenzylisothiourea (ZKK1) and its derivatives (ZKK2-8) on cultured malignant glioma cells. TBI, ZKK1 and MB001-3 were more effective than TBB in inducing growth arrest and cell death in glioma cells. TBI and ZKK1 strongly induced apoptotic death involving caspase 3 and 7 activation followed by PARP cleavage. DMAT strongly upregulated the expression of cytotoxic ligand and its receptor Fas. Structural modifications of ZKK1 largely affected its efficacy: exchange of Br- to Cl- or F-substituents on the pentabromophenyl ring and inclusion of the bulky N-phenyl substituent in thiourea fragment of ZKK1 diminished cytotoxic activity, while N-substitution with short alkyl groups or an allyl group had opposite effects. Interestingly, TBI at moderate dose did not affect viability of non-transformed astrocytes, suggesting some specificity toward tumor cells in cytotoxic action. TBI, DMAT and ZKK1-induced apoptosis associated with caspase cascade activation in human malignant glioblastoma cells with mutated PT53 and PTEN genes. The reported data demonstrate that suitably modified polybromobenzene molecules exhibit a significant cytotoxic potential towards malignant glioblastoma cells. collapse abstract
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Cellular signalling 2010 Jan; 22(2)
Non-apoptotic Fas signaling regulates invasiveness of glioma cells and modulates MMP-2 activity via NFkappaB-TIMP-2 pathway.
Fas (CD95/APO-1) is a cell surface "death receptor" that mediates apoptosis upon engagement by its ligand, FasL. Paradoxically, Fas/FasL can also promote cell invasion among non-apoptotic cells; here, we show that Fas/FasL signaling can promote tumor... expand abstract invasion when apoptosis is compromised. We have developed a recombinant FasL Interfering Protein (FIP) to interfere with Fas signaling in C6 glioma cells expressing both Fas receptor and its ligand. FIP administration did not affect cell viability but impaired cell motility and invasiveness of glioma cells. Blockade of Fas signaling reduced MMP-2 activity in glioma cells, that was associated with down-regulation of MAPK signaling, and AP-1 and NFkappaB-driven transcription. FIP treatment did not affect mmp-2 and mt1-mmp expression but significantly attenuated timp-2 expression and TIMP-2 amount in the culture medium. Studies with pharmacological inhibitors of JNK/c-Jun (SP600125) and NFkappaB (BAY11-7082) signaling pathways demonstrated that timp-2 expression is regulated by NFkappaB transcription factor. Our findings show that non-apoptotic Fas signaling activated in the autocrine manner or through microenvironment derived factors can regulate invasiveness of glioma cells via modulation of MMP-2 activation, likely by controlling TIMP-2 expression. collapse abstract
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Methods in molecular biology (Clifton, N.J.) 512
Molecular characterization of inflammation-induced JNKc-Jun signaling pathway in connection with tumorigenesis.
Tumor cells recruit inflammatory cells to the tumor site and transform them into tumor-supportive cells which in turn release numerous cytokines, including Transforming Growth Factor-beta that enhances tumor proliferation, invasion, angiogenesis and ... expand abstractinduces immune paralysis. Activation of JNK/c-Jun signaling pathway by various stimuli often leads to a formation of the AP-1 transcriptional complex, which is a critical regulator of a complex program of gene expression that defines the invasive phenotype. Recent studies on JNK/c-Jun phosphorylation have been carried out using phospho-specific antibodies, which have greatly facilitated analysis of signal transduction. The electrophoretic mobility shift assay (EMSA, gel shift) helps in determining the transcription factor activation and is based on the observation that complexes of protein and DNA migrate through a non-denaturing polyacrylamide gel more slowly than free DNA fragments or double-stranded oligonucleotides. The specificity of the DNA-binding protein is established by competition experiments and the protein composition of DNA binding activity can be analyzed with specific antibodies in a supershift assay. EMSA provides a sensitive and quantitative measure of a particular DNA binding activity under various experimental conditions. collapse abstract
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Methods in molecular biology (Clifton, N.J.) 512
Molecular characterization of STAT signaling in inflammation and tumorigenesis.
The Janus kinases (JAK) and signal transducer and activator of transcription (STAT) signaling are strongly activated in many tumors. STAT proteins are activated by phosphorylation at the tyrosine residue, then dimerize, translocate to the nucleus and... expand abstract bind DNA, initiating the transcription of target genes. Activation of JAK-STAT pathway is implicated in the regulation of cell growth, differentiation, survival and cross-talk between cancer and immune cells. The activation of STATs depends on phosphorylation on a single tyrosine residue (e.g., Tyr705 in STAT3 and Tyr694 in STAT5) in the C-terminal domain. Commercially available antibodies discriminate between total and specifically phosphorylated (active) forms of different STATs, which allows to measure directly STATs activation in crude cell extracts. Nuclear translocation and transcriptional activity of STATs can be measured in transfected cells using STAT dependent promoter driving reporter luciferase gene. STAT signaling pathway and STAT-dependent gene expression in cells can be specifically modulated using oligodeoxynucleotide (ODN) STAT decoy which is a double-stranded fragment of DNA containing an overlapping ISRE/GAS binding site. collapse abstract
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Proceedings of the National Academy of Sciences of the United States of America 2009 Jul; 106(30)
Gliomas induce and exploit microglial MT1-MMP expression for tumor expansion.
Diffuse infiltration of glioma cells into normal brain tissue is considered to be a main reason for the unfavorable outcomes of patients with malignant gliomas. Invasion of glioma cells into the brain parenchyma is facilitated by metalloprotease-medi... expand abstractated degradation of the extracellular matrix. Metalloproteases are released as inactive pro-forms and get activated upon cleavage by membrane bound metalloproteases. Here, we show that membrane type 1 metalloprotease (MT1-MMP) is up-regulated in glioma-associated microglia, but not in the glioma cells. Overexpression of MT1-MMP is even lethal for glioma cells. Glioma-released factors trigger the expression and activity of MT1-MMP via microglial toll-like receptors and the p38 MAPK pathway, as deletion of the toll-like receptor adapter protein MyD88 or p38 inhibition prevented MT1-MMP expression and activity in cultured microglial cells. Microglial MT1-MMP in turn activates glioma-derived pro-MMP-2 and promotes glioma expansion, as shown in an ex vivo model using MT1-MMP-deficient brain tissue and a microglia depletion paradigm. Finally, MyD88 deficiency or microglia depletion largely attenuated glioma expansion in 2 independent in vivo models. collapse abstract
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Cellular and molecular neurobiology 2009 Aug; 29(6-7)
Molecular mechanisms leading to neuroprotectionischemic tolerance: effect of preconditioning on the stress reaction of endoplasmic reticulum.
Ischemic tolerance can be developed by prior ischemic non-injurious stimulus preconditioning. The molecular mechanisms underlying ischemic tolerance are not yet fully understood. The purpose of this study is to evaluate the effect of preconditioning/... expand abstractpreischemia on ischemic brain injury. We examined the endoplasmic reticulum stress response (unfolded protein response (UPR)) by measuring the mRNA and protein levels of specific genes such as ATF6, GRP78, and XBP1 after 15 min 4-VO ischemia and different times of reperfusion (1, 3, and 24 h). The data from the group of naïve ischemic rats were compared with data from the group of preconditioned animals. The results of the experiments showed significant changes in the gene expression at the mRNA level in the all ischemic/reperfusion phases. The influence of preischemia on protein level of XBP was significant in later ischemic times and at 3 h, the reperfusion reached 230% of the controls. The protein levels of GRP78 in preischemic animals showed a significant increase in ischemic and reperfusion times. They exceeded to 50% levels of corresponding naïve ischemic/reperfusion groups. Preconditioning also induced remarkable changes in the levels of ATF6 protein in the ischemic phase (about 170%). The levels of ATF6 remained elevated in earlier reperfusion times (37 and 62%, respectively) and persisted significantly elevated after 24 h of reperfusion. This data suggest that preconditioning paradigm (preischemia) underlies its neuroprotective effect by the attenuation of ER stress response after acute ischemic/reperfusion insult. collapse abstract
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Journal of molecular medicine (Berlin, Germany) 2009 Jan; 87(2)
Transferrin-receptor-mediated iron accumulation controls proliferation and glutamate release in glioma cells.
Transferrin receptors (TfR) are overexpressed in brain tumors, but the pathological relevance has not been fully explored. Here, we show that TfR is an important downstream effector of ets transcription factors that promotes glioma proliferation and ... expand abstractincreases glioma-evoked neuronal death. TfR mediates iron accumulation and reactive oxygen formation and thereby enhanced proliferation in clonal human glioma lines, as shown by the following experiments: (1) downregulating TfR expression reduced proliferation in vitro and in vivo; (2) forced TfR expression in low-grade glioma accelerated proliferation to the level of high-grade glioma; (3) iron and oxidant chelators attenuated tumor proliferation in vitro and tumor size in vivo. TfR-induced oxidant accumulation modified cellular signaling by inactivating a protein tyrosine phosphatase (low-molecular-weight protein tyrosine phosphatase), activating mitogen-activated protein kinase and Akt and by inactivating p21/cdkn1a and pRB. Inactivation of these cell cycle regulators facilitated S-phase entry. Besides its effect on proliferation, TfR also boosted glutamate release, which caused N-methyl-D-aspartate-receptor-mediated reduction of neuron cell mass. Our results indicate that TfR promotes glioma progression by two mechanisms, an increase in proliferation rate and glutamate production, the latter mechanism providing space for the progressing tumor mass. collapse abstract
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Cellular and molecular neurobiology 2009 Feb; 29(2)
Molecular analysis of endoplasmic reticulum stress response after global forebrain ischemiareperfusion in rats: effect of neuroprotectant simvastatin.
Simvastatin is a cholesterol-lowering agent whose functional significance and neuroprotective mechanism in ischemic brain injury is not yet solved. The purpose of this study is to evaluate the effect of simvastatin on ischemic brain injury. We examin... expand abstracted the endoplasmic reticulum stress response (UPR/unfolded protein response), by measuring the mRNA and protein levels of specific genes such as ATF6, GRP78, and XBP1 after 15 min 4-VO ischemia and different times of reperfusion (1, 3, and 24 h). The results from the group of naïve ischemic rats were compared with results from the group of pre-treated animals with simvastatin. The results of the experiments showed significant increase in all genes at the mRNA level in ischemic phase (about 43% for XBP1, 58% for GRP78, and 39% for ATF6 more than control). The protein level of XBP1 was decreased in pre-treated animals at ischemic phase and first hour of reperfusion (about 15% less), and did not reach control levels. The protein levels of GRP78 were maximal at third hour of reperfusion in statin group with a small decrease at 24 h of reperfusion in both groups. The levels of ATF6 mRNA in statin-treated animals was higher in comparison to non-statin animals at the ischemic phase and the third hour of reperfusion (about 35% higher), which was also translated into the higher protein level. This could indicate that one of the main proteins targeted to enhance neuroprotective effect to ER during the first two hours of reperfusion was ATF6 protein, the levels of which were 60% higher than in non-treated animals. These data suggest that simvastatin, in addition to the proposed neuroprotective effect, exerts a neuroprotective role in the attenuation of ER stress response after acute ischemic/reperfusion insult. collapse abstract
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Stem cells (Dayton, Ohio) 2008 Oct; 26(11)
The antitumorigenic response of neural precursors depends on subventricular proliferation and age.
Glioblastomas, the most aggressive primary brain tumors, occur almost exclusively in adult patients. Neural precursor cells (NPCs) are antitumorigenic in mice, as they can migrate to glioblastomas and induce tumor cell death. Here, we show that the a... expand abstractntitumor effect of NPCs is age-dependently controlled by cell proliferation in the subventricular zone (SVZ) and that NPCs accumulating at a glioblastoma are diverted from their normal migratory path to the olfactory bulb. Experimentally induced cortical glioblastomas resulted in decreased subventricular proliferation in adult (postnatal day 90) but not in young (postnatal day 30) mice. Adult mice supplied fewer NPCs to glioblastomas and had larger tumors than young mice. Apart from the difference in proliferation, there was neither a change in cell number and death rate in the SVZ nor a change in angiogenesis and immune cell density in the tumors. The ability to kill glioblastomas was similar in NPCs isolated from young and adult mice. The proliferative response of NPCs to glioblastomas depended on the expression of D-type cyclins. In young mice, NPCs express the cyclins D1 and D2, but the expression of cyclin D1 is lost during aging, and in adult NPCs only cyclin D2 remains. In young and adult cyclin D2-deficient mice we observed a reduced supply of NPCs to glioblastomas and the generation of larger tumors compared with wild-type mice. We conclude that cyclin D1 and D2 are nonredundant for the antitumor response of subventricular NPCs. Loss of a single D-type cyclin results in a smaller pool of proliferating NPCs, lower number of NPCs migrating to the tumor, and reduced antitumor activity. Disclosure of potential conflicts of interest is found at the end of this article. collapse abstract
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Acta biochimica Polonica 55(1)
Cloning and purification of functionally active Fas ligand interfering protein (FIP) expressed in Escherichia coli.
This report presents purification and characterization of the extracellular domain of rat Fas protein, called FIP (FasL interfering protein), expressed as inclusion bodies in Escherichia coli. FIP was extracted from the inclusion bodies, solubilized ... expand abstractwith 8 M urea, purified by a single-step immobilized metal ion (Ni(2+)) affinity chromatography and refolded. SDS/PAGE and mass spectrometry analysis of the purified protein verified its purity. Fluorescence spectrum analysis showed that the refolding procedure caused structural changes which presumably might have led to oligomerization. The purified FIP has biological activities: it binds specifically soluble Fas ligand and protects human Jurkat lymphocytes against FasL-dependent apoptosis. This efficient procedure of FIP expression in E. coli and renaturation may be useful for production of therapeutically important proteins. collapse abstract
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Oncogene 2008 Feb; 27(7)
Microglia-derived TGF-beta as an important regulator of glioblastoma invasion--an inhibition of TGF-beta-dependent effects by shRNA against human TGF-beta type II receptor.
The invasion of tumor cells into brain tissue is a pathologic hallmark of malignant gliomas and contributes to treatment failures. Diffuse glioblastomas contain numerous microglial cells, which enhance the progression of gliomas; however, factors res... expand abstractponsible for invasion-promoting role of microglia are unknown. Transforming growth factor-beta (TGF-beta) can enhance tumor growth, invasion, angiogenesis and immunosuppression. Antagonizing TGF-beta activity has been shown to inhibit tumor invasion in vitro and tumorigenicity, but a systemic inhibition or lack of TGF-beta signaling results in acute inflammation and disruption of immune system homeostasis. We developed plasmid-transcribed small hairpin RNAs (shRNAs) to downregulate the TGF-beta type II receptor (TbetaIIR) expression, which effectively inhibited cytokine-induced signaling pathways and transcriptional responses in transiently transfected human glioblastoma cells. Silencing of TbetaIIR abolished TGF-beta-induced glioblastoma invasiveness and migratory responses in vitro. Moreover, tumorigenicity of glioblastoma cells stably expressing TbetaIIR shRNAs in nude mice was reduced by 50%. Microglia strongly enhanced glioma invasiveness in the co-culture system, but this invasion-promoting activity was lost in glioma cells stably expressing shTbetaRII, indicating a crucial role of microglia-derived TGF-beta in tumor-host interactions. Our results demonstrate a successful targeting of TGF-beta-dependent invasiveness and tumorigenicity of glioblastoma cells by RNAi-mediated gene silencing. collapse abstract
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Journal of molecular biology 2007 Aug; 371(3)
Dimer composition and promoter context contribute to functional cooperation between AP-1 and NFAT.
The transcription factors activator protein 1 (AP-1) and nuclear factor of activated T-cells (NFAT) cooperate to induce the expression of cytokines during the immune response. While much is known about the signaling pathways and physical interactions... expand abstract between NFAT and AP-1 dimers following lymphocyte activation, few studies have addressed the role of AP-1 composition in modulating NFAT:AP-1-dependent transcription. We examined the function of specific AP-1 complexes using "tethered" AP-1 dimers with defined composition. We found that NFAT can functionally cooperate with all AP-1 dimers tested. Noteworthy, Jun approximately Jun-containing dimers, which are relatively inactive when tested on an AP-1-dependent promoter, are effective co-activators of an NFAT:AP-1-dependent promoter. Interestingly, specific AP-1 dimer combinations behave differently when tested on interleukin 2 (IL2) and interleukin 4 (IL4) gene regulatory regions. Moreover, the requirement for NFAT to activate each of the promoters is different. Our results suggest that higher NFAT levels are necessary to activate the IL4 promoter. Hence changes in AP-1 composition and the level of participating NFAT proteins can differentially influence cytokine gene expression, resulting in biological consequences for the modulation and dynamics of the immune response. collapse abstract
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Biochemical and biophysical research communications 2007 Mar; 354(4)
Cross-talk between Smad and p38 MAPK signalling in transforming growth factor beta signal transduction in human glioblastoma cells.
Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine involved in the regulation of cell proliferation, differentiation, and survival. Malignant tumour cells often do not respond to TGF-beta by growth inhibition, but retain respons... expand abstractiveness to cytokine in regulating extracellular matrix deposition, cell adhesion, and migration. We demonstrated that TGF-beta1 does not affect viability or proliferation of human glioblastoma T98G, but increases transcriptional responses exemplified by induction of MMP-9 expression. TGF-beta receptors were functional in T98G glioblastoma cells leading to SMAD3/SMAD4 nuclear translocation and activation of SMAD-dependent promoter. In parallel, a selective activation of p38 MAPK, and phosphorylation of its substrates: ATF2 and c-Jun proteins were followed by a transient activation of AP-1 transcription factor. Surprisingly, an inhibition of p38 MAPK with a specific inhibitor, SB202190, abolished TGF-inducible activation of Smad-dependent promoter and decreased Smad2 phosphorylation. It suggests an unexpected interaction between Smad and p38 MAPK pathways in TGF-beta1-induced signalling. collapse abstract
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Cellular signalling 2007 May; 19(6)
Alternative pathway of transcriptional induction of p21WAF1Cip1 by cyclosporine A in p53-deficient human glioblastoma cells.
The cyclin-dependent kinase inhibitor p21WAF1/CIP1, a critical regulator of the cell cycle, is mainly regulated by p53 tumour suppressor at the transcriptional level. Restoration of p21WAF1/Cip1 expression in p53-deficient malignant cells suppress tu... expand abstractmour growth. Cyclosporine A (CsA) affects proliferation and survival of cultured malignant glioma cells and impairs growth of experimental gliomas. CsA induced p21WAF1/Cip1 expression de novo in human glioblastoma cells with p53 deficiency. We demonstrate that transcriptional activation of p21WAF1/Cip1 expression correlated with induction of ERK1/2 and c-Jun phosphorylation in CsA-treated glioblastoma cells. Pre-treatment with ERK pathway inhibitors or overexpression of dominant-negative mutants MKK1, ERK2 and c-Jun reduced activation of the p21WAF1/Cip1 promoter. Overexpression of tethered AP-1 dimers containing c-Jun was sufficient to activate the truncated -200 bp p21WAF1/Cip1 promoter, which does not contain p53 binding sites. Chromatin immunoprecipitation revealed that P-c-Jun is bound to the proximal part of p21WAF1/Cip1 promoter in CsA-treated glioblastoma cells. It suggests that CsA activates p53-independent, transcriptional activation p21WAF1/Cip1 expression, mediated by ERK/c-Jun/AP-1 signaling pathway. collapse abstract
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Brain research 2007 Mar; 1137(1)
Distinctive pattern of cannabinoid receptor type II (CB2) expression in adult and pediatric brain tumors.
The efficacy of cannabinoids against high-grade glioma in animal models, mediated by two specific receptors, CB1 and CB2, raised promises for targeted treatment of the most frequent and malignant primary brain tumors. Unlike the abundantly expressed ... expand abstractCB1, the CB2 receptor shows a restricted distribution in normal brain. Although brain tumors constitute the second most common malignancy in children and the prevalence of histological types of brain tumors vary significantly between the adult and pediatric populations, cannabinoid receptor expression in pediatric tumors remains unknown. In the present study, we compared the expression of the CB2 receptor in paraffin-embedded sections from primary brain tumors of adult and pediatric patients. Most glioblastomas expressed very high levels of CB2 receptors and the expression correlated with tumor grade. Interestingly, some benign pediatric astrocytic tumors, such as subependymal giant cell astrocytoma (SEGA), which may occasionally cause mortality owing to progressive growth, also displayed high CB2 immunoreactivity. The high levels of CB2 expression would predestine those tumors to be vulnerable to cannabinoid treatment. In contrast, all examined cases of embryonal tumors (medulloblastoma and S-PNET), the most frequently diagnosed malignant brain tumors in childhood, showed no or trace CB2 immunoreactivity. Our results suggest that the CB2 receptor expression depends primarily on the histopathological origin of the brain tumor cells and differentiation state, reflecting the tumor grade. collapse abstract
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Brain : a journal of neurology 2007 Jan; 130(Pt 2)
The invasion promoting effect of microglia on glioblastoma cells is inhibited by cyclosporin A.
The invasion of tumour cells into brain tissue is a pathologic hallmark of WHO grades II-IV gliomas and contributes significantly to the failure of current therapeutic treatments. Activated microglial cells are abundant in brain tumours and may suppo... expand abstractrt tumour invasiveness. We have previously demonstrated that cyclosporin A (CsA) can affect growth of glioma cells in vitro by inhibiting signalling pathways, which are essential for tumour proliferation and invasiveness. In this work, we demonstrate that migration of EGFP-transfected glioblastoma cells in organotypic brain slices was significantly inhibited by treatment with CsA. On average 77% of untreated cells migrated beyond 500 mum, while only 28-33% cells migrated as far in the brain slices treated with CsA (P < 0.001). This inhibitory effect on glioblastoma invasion was lost when glioblastoma cells were injected into microglia-depleted brain slices. Moreover, CsA significantly inhibits intracranial glioma growth in vivo. We demonstrate that microglia-derived factors increase glioma invasiveness in Matrigel assay in vitro and this is associated with activation of the PI-3K/Akt signalling pathway. The invasion promoting effect of microglia is abolished in the presence of CsA. Furthermore, glioma-derived soluble factors induce morphological transformation of microglia and activate MAPK signalling, although production of pro-inflammatory factors was not observed. Our findings that CsA interferes at clinically relevant concentrations with the tumour-promoting role of microglia and impairs invasive growth of glioma cells in vivo may provide a novel therapeutic strategy against gliomas. collapse abstract
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Journal of neurochemistry 2006 Oct; 99(3)
Neuroprotectant FK506 inhibits glutamate-induced apoptosis of astrocytes in vitro and in vivo.
Neuron-astrocyte interactions are critical for signalling, energy metabolism, extracellular ion and glutamate homeostasis, volume regulation and neuroprotection in the CNS. Glutamate uptake by astrocytes may prevent excitotoxic glutamate elevation an... expand abstractd determine neuronal survival. However, an excess of glutamate can cause the death of astrocytes. FK506, an inhibitor of calcineurin, and an immunosuppressive drug, is neuroprotective in animal models of neurologic diseases, including focal and global ischaemia. In the present work, we demonstrate that a single injection of FK506 60 min after a transient middle cerebral artery occlusion (MCAo) significantly decreases the number of terminal deoxynucleotidyl transferase nick-end labelling (TUNEL)-positive cells in the ischaemic cortex and striatum. Using 3-D confocal microscopy we found that, 24 h after MCAo, many TUNEL-positive cells in the ischaemic striatum and cortex are astrocytes. Furthermore, we demonstrate that exposure of cultured cortical astrocytes to 50-100 mM Glu for 24 h induces apoptotic alterations in nuclear morphology, DNA fragmentation, dissipation of mitochondrial transmembrane potential (DeltaPsi) and caspase activation. FK506 (1 muM) efficiently inhibits Glu-induced apoptosis of cultured astrocytes, DNA fragmentation and changes in mitochondrial DeltaPsi. Our findings suggest that modulation of glutamate-induced astrocyte death early after reperfusion may be a novel mechanism of FK506-mediated neuroprotection in ischaemia. collapse abstract
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European journal of pharmacology 2007 Jan; 554(1)
Neuroprotective activity of selective mGlu1 and mGlu5 antagonists in vitro and in vivo.
The neuroprotective potential of allosteric mGlu5 and mGlu1 antagonists such as 6-methyl-2-(phenylethynyl)-pyridin (MPEP)/[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) and (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methan... expand abstractesulfonate (EMQMCM), was tested in vitro in organotypic hippocampal cultures and in the middle cerebral artery occlusion model of stroke in vivo. Both classes of agent have high selectivity toward mGlu sub-types and are active in animal models of various diseases indicating satisfactory CNS penetration. In organotypic hippocampal cultures MPEP showed high neuroprotective potency against sub-chronic (12 days) insult produced by 3-NP with an IC50 of c.a. 70 nM. In contrast, although the mGlu1 antagonist EMQMCM was also protective, it seems to be weaker yielding an IC50 of c.a. 1 microM. Similarly, in the transient (90 min) middle cerebral artery occlusion model of ischaemia in rats, MTEP seems to be more effective than EMQMCM. MTEP, at 2.5 mg/kg and at 5 mg/kg provided 50 and 70% neuroprotection if injected 2 h after the onset of ischaemia. At a dose of 5 mg/kg, significant (50%) neuroprotection was also seen if the treatment was delayed by 4 h. EMQMCM was not protective at 5 mg/kg (given 2 h after occlusion) but at 10 mg/kg 50% of neuroprotection was observed. The present data support stronger neuroprotective potential of mGlu5 than mGlu1 antagonists. collapse abstract
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BMC bioinformatics 7
Prediction of a key role of motifs binding E2F and NR2F in down-regulation of numerous genes during the development of the mouse hippocampus.
BACKGROUND: We previously demonstrated that gene expression profiles during neuronal differentiation in vitro and hippocampal development in vivo were very similar, due to a conservation of the important second singular value decomposition (SVD) mode... expand abstract (Mode 2) of expression. The conservation of Mode 2 suggests that it reflects a regulatory mechanism conserved between the two systems. In either dataset, the expression vectors of all the genes form two large clusters that differ in the sign of the contribution of Mode 2, which for the majority of them reflects the difference between down- or up-regulation. RESULTS: In the current work, we used a novel approach of analyzing cis-regulation of gene expression in a subspace of a single SVD mode of temporal expression profiles. In the putative upstream regulatory sequences identified by mouse-human homology for all the genes represented in either dataset, we searched for simple features (motifs and pairs of motifs) associated with either sign of the loading of Mode 2. Using a cross-system training-test set approach, we identified E2F binding sites as predictors of down-regulation of gene expression during hippocampal development. NR2F binding sites, for the transcription factors Nr2f/COUP and Hnf4, and also NR2F_SP1 pairs of binding sites, were predictors of down-regulation of expression both during hippocampal development and neuronal differentiation. Analysis of another dataset, from gene profiling of myoblast differentiation in vitro, shows that the conservation of Mode 2 extends to the differentiation of mesenchymal cells. This permitted the identification of two more pairs of motifs, one of which included the CDE/CHR tandem element, as features associated with down-regulation both in the differentiating myoblasts and in the developing hippocampus. Of the features we identified, the E2F and CDE/CHR motifs may be associated with the cycling progenitor cell status, while NR2F may be related to the entry into differentiation along the neuronal pathway. CONCLUSION: Our results constitute the first prediction of an expression pattern from the genomic sequence for the developing mammalian brain, and demonstrate a potential for the analysis of gene regulation in a subspace of a single SVD mode of expression. collapse abstract
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Journal of neurochemistry 2006 Mar; 97 Suppl 1
Identification of conserved modes of expression profiles during hippocampal development and neuronal differentiation in vitro.
Gene expression profiles can be regarded as sums of simpler modes, analogous to the modes of a vibrating violin string. Decomposition of temporal gene expression profiles into modes by singular value decomposition (SVD) was reported before, but the q... expand abstractuestion as to what degree the SVD modes can be interpreted in terms of biology remains open. We report and compare the results of SVD of published datasets from hippocampal development, neuronal differentiation in vitro, and a control time-series hippocampal dataset. We demonstrate that the first SVD mode reflects the magnitude of expression, interpretable on the Affymetrix platform. In the datasets from gene profiling of hippocampal development and neuronal differentiation, the second mode reflects a monotonous change in expression, either up- or down-regulation, in the time course of experiment. We demonstrate that the top two SVD modes are conserved between datasets and therefore, likely reflect properties of the underlying system (gene expression in hippocampus) rather than of a particular experiment or dataset. Our results also indicate that the magnitude of expression, and the direction of change in expression during hippocampal development, are uncorrelated, suggesting that they are regulated by largely independent mechanisms. collapse abstract
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Neurochemistry international 2005 Oct; 47(6)
Cyclosporine a induces growth arrest or programmed cell death of human glioma cells.
Human malignant gliomas are highly resistant to current therapeutic approaches. We previously demonstrated that cyclosporine A (CsA) induces an apoptotic cell death in rat C6 glioma cells. In the present study, we found the induction of growth arrest... expand abstract or cell death of human malignant glioma cells exposed to CsA. In studied glioma cells, an accumulation of p21Cip1/Waf1 protein, a cell cycle inhibitor, was observed following CsA treatment, even in the absence of functional p53 tumour suppressor. CsA induced a senescence-associated growth arrest, in U87-MG glioma cells with functional p53, while in U373 and T98G glioma cells with mutated p53, CsA treatment triggered cell death associated with alterations of cell morphology, cytoplasm vacuolation, and condensation of chromatin. In T98G cells this effect was completely abolished by simultaneous treatment with an inhibitor of protein synthesis, cycloheximide (CHX). Moreover, CsA-induced cell death was accompanied by activation of executory caspases followed by PARP cleavage. CsA treatment did not elevate fasL expression and had no effect on mitochondrial membrane potential. We conclude that CsA triggers either growth arrest or non-apoptotic, programmed cell death in human malignant glioma cells. Moreover, CsA employs mechanisms different to those in the action of radio- and chemotherapeutics, and operating even in cells resistant to conventional treatments. Thus, CsA or related drugs may be an effective novel strategy to treat drug-resistant gliomas or complement apoptosis-based therapies. collapse abstract
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International journal of cancer. Journal international du cancer 2005 Oct; 117(1)
Cyclosporine A and its non-immunosuppressive derivative NIM811 induce apoptosis of malignant melanoma cells in in vitro and in vivo studies.
Advanced melanoma is a highly malignant tumor with an increasing incidence that has a poor prognosis due to resistance to common therapeutic strategies. We have demonstrated previously that cyclosporine A (CsA) induces apoptosis of rat glioma cells, ... expand abstractreactive astrocytes, and fibroblasts. In our present study, we investigated effects of CsA and its nonimmunosuppressive derivative NIM811 on survival of human and murine melanoma cells. We demonstrated that CsA and NIM811 affect survival of human and murine melanoma cells and induce morphological changes, alterations in nuclear morphology and an internucleosomal DNA fragmentation, consistent with an apoptotic type of death. Western blot analysis showed an activation of caspases 9, 7, 3 and PARP cleavage detectable at 24 hr after exposure of human melanoma cells to the drugs. CsA and NIM811 induced a significant increase in subG1 population of murine B16F10 melanoma cells indicative of apoptotic DNA fragmentation. Studies in murine model of melanoma showed that NIM811, but not CsA, retards tumor progression and significantly decreases tumor volume after intratumoral application. Our findings indicate that CsA and its derivatives may be new candidates for the treatment of melanoma patients. collapse abstract
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