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Nuclear Accumulation (nuclear + accumulation)
Selected AbstractsDeterminants of the nucleocytoplasmic shuttling of muscle glycogen synthaseFEBS JOURNAL, Issue 12 2005Emili Cid Muscle glycogen synthase (MGS) presents a nuclear speckled pattern in primary cultured human muscle and in 3T3-L1 cells deprived of glucose and with depleted glycogen reserves. Nuclear accumulation of the enzyme correlates inversely with cellular glycogen content. Although the glucose-induced export of MGS from the nucleus to the cytoplasm is blocked by leptomycin B, and therefore mediated by CRM1, no nuclear export signal was identified in the sequence of the protein. Deletion analysis shows that the region comprising amino acids 555,633 of human MGS, which encompasses an Arg-rich cluster involved in the allosteric activation of the enzyme by Glc6P, is crucial for its nuclear concentration and aggregation. Mutation of these Arg residues, which desensitizes the enzyme towards Glc6P, interferes with its nuclear accumulation. In contrast, the known phosphorylation sites of MGS that regulate its activity are not involved in the control of its subcellular distribution. Nuclear human MGS colocalizes with the promyelocytic leukaemia oncoprotein and p80-coilin, a marker of Cajal bodies. The subnuclear distribution of MGS is altered by incubation with transcription inhibitors. These observations suggest that, in addition to its metabolic function, MGS may participate in nuclear processes. [source] Nuclear accumulation of glycogen synthase kinase-3 during replicative senescence of human fibroblastsAGING CELL, Issue 5 2004Jaroslaw W. Zmijewski Summary Activation of the tumor suppressor protein p53 contributes to cellular senescence. As glycogen synthase kinase-3 (GSK3) was recently found to interact with p53 and contribute to the actions of p53, this study examined whether GSK3 accumulated in the nucleus and associated with p53 in senescent cells. Compared with young and middle-aged human WI-38 fibroblasts, senescent cells were found to contain increased nuclear levels of GSK3,, and also tended to accumulate in the nucleus the other isoform of GSK3, GSK3,. Co-immunoprecipitation experiments demonstrated that GSK3, and p53 formed a complex in the nucleus. Further experiments tested whether inhibition of GSK3 altered the development of senescence using long-term treatment with the selective GSK3 inhibitor lithium. Lithium treatment reduced the senescence-associated accumulation of p53 and caused cells to enter a reversible quiescent state. These results indicate that a portion of the p53 that is activated in senescent cells is modulated by its association with GSK3, in the nucleus, an association that is known to facilitate the actions of p53 and that may contribute to senescence. [source] Nuclear accumulation of the ankyrin repeat protein ANK1 enhances the auxin-mediated transcription accomplished by the bZIP transcription factors BZI-1 and BZI-2THE PLANT JOURNAL, Issue 6 2009Stefan Böttner Summary The tobacco (Nicotiana tabacum) basic leucine zipper (bZIP) transcription factor BZI-1 has been implicated in auxin-mediated gene regulation. Yeast two-hybrid analysis has led to the identification of two BZI-1 protein interaction partners: the heterodimerizing bZIP factor BZI-2 and an ankyrin repeat domain protein, ANK1. Analysis in transgenic plants confirms that low levels of functional BZI-1, BZI-2 and ANK1 result in reduced auxin responses. This finding indicates that the three proteins act in the same functional context. The in vivo interaction of ANK1 and BZI-1 has been confirmed by protoplast two-hybrid analysis, as well as by bimolecular fluorescence complementation (BiFC) studies. Whereas YFP-BZI-1 has been found to be localized in the nucleus, YFP-ANK1 resides in the cytosol. Nevertheless, the inhibition of nuclear export with the inhibitor leptomycin B (LMB) and the co-expression with BZI-1, as well as treatment with auxin, results in the accumulation of YFP-ANK1 in the nucleus. Whereas BZI-1 is a weak activator, BZI-1/BZI-2 heterodimers efficiently support transcription. Importantly, conditions that lead to the accumulation of ANK1 in the nucleus, such as the expression of an ANK1 protein fused to a nuclear localization sequence (NLS) or auxin treatment, lead to a significant enhancement of BZI-1/BZI-2-mediated transcription. We therefore propose a mechanism in which the nuclear accumulation of ANK1 enhances BZI-1/BZI-2-mediated transcription in an auxin-dependent manner, presumably facilitated by protein,protein interaction. In summary, this study defines novel components in auxin-dependent signalling and transcriptional control. [source] Vanadium-induced apoptosis of HaCaT cells is mediated by c-fos and involves nuclear accumulation of clusterinFEBS JOURNAL, Issue 14 2009Soultana Markopoulou Vanadium exerts a variety of biological effects, including antiproliferative responses through activation of the respective signaling pathways and the generation of reactive oxygen species. As epidermal cells are exposed to environmental insults, human keratinocytes (HaCaT) were used to investigate the mechanism of the antiproliferative effects of vanadyl(IV) sulfate (VOSO4). Treatment of HaCaT cells with VOSO4 inhibited proliferation and induced apoptosis in a dose-dependent manner. Inhibition of proliferation was associated with downregulation of cyclins D1 and E, E2F1, and the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p27Kip1. Induction of apoptosis correlated with upregulation of the c-fos oncoprotein, changes in the expression of clusterin (CLU), an altered ratio of antiapoptotic to proapoptotic Bcl-2 protein family members, and poly(ADP-ribose) polymerase-1 cleavage. Forced overexpression of c-fos induced apoptosis in HaCaT cells that correlated with secretory CLU downregulation and upregulation of nuclear CLU (nCLU), a pro-death protein. Overexpression of Bcl-2 protected HaCaT cells from vanadium-induced apoptosis, whereas secretory CLU overexpression offered no cytoprotection. In contrast, nCLU sensitized HaCaT cells to apoptosis. Our data suggest that vanadium-mediated apoptosis was promoted by c-fos, leading to alterations in CLU isoform processing and induction of the pro-death nCLU protein. [source] Determinants of the nucleocytoplasmic shuttling of muscle glycogen synthaseFEBS JOURNAL, Issue 12 2005Emili Cid Muscle glycogen synthase (MGS) presents a nuclear speckled pattern in primary cultured human muscle and in 3T3-L1 cells deprived of glucose and with depleted glycogen reserves. Nuclear accumulation of the enzyme correlates inversely with cellular glycogen content. Although the glucose-induced export of MGS from the nucleus to the cytoplasm is blocked by leptomycin B, and therefore mediated by CRM1, no nuclear export signal was identified in the sequence of the protein. Deletion analysis shows that the region comprising amino acids 555,633 of human MGS, which encompasses an Arg-rich cluster involved in the allosteric activation of the enzyme by Glc6P, is crucial for its nuclear concentration and aggregation. Mutation of these Arg residues, which desensitizes the enzyme towards Glc6P, interferes with its nuclear accumulation. In contrast, the known phosphorylation sites of MGS that regulate its activity are not involved in the control of its subcellular distribution. Nuclear human MGS colocalizes with the promyelocytic leukaemia oncoprotein and p80-coilin, a marker of Cajal bodies. The subnuclear distribution of MGS is altered by incubation with transcription inhibitors. These observations suggest that, in addition to its metabolic function, MGS may participate in nuclear processes. [source] Transcriptional activity of interferon regulatory factor (IRF)-3 depends on multiple protein,protein interactionsFEBS JOURNAL, Issue 24 2002Hongmei Yang Virus infection results in the activation of a set of cellular genes involved in host antiviral defense. IRF-3 has been identified as a critical transcription factor in this process. The activation mechanism of IRF-3 is not fully elucidated, yet it involves a conformational change triggered by the virus-dependent phosphorylation of its C-terminus. This conformational change leads to nuclear accumulation, DNA binding and transcriptional transactivation. Here we show that two distinct sets of Ser/Thr residues of IRF-3, on phosphorylation, synergize functionally to achieve maximal activation. Remarkably, we find that activated IRF-3 lacks transcriptional activity, but activates transcription entirely through the recruitment of the p300/CBP coactivators. Moreover, we show that two separate domains of IRF-3 interact with several distinct regions of p300/CBP. Interference with any of these interactions leads to a complete loss of transcriptional activity, suggesting that a bivalent interaction is essential for coactivator recruitment by IRF-3. [source] Smad3-dependent signaling underlies the TGF-,1-mediated enhancement in astrocytic iNOS expressionGLIA, Issue 11 2010Mary E. Hamby Abstract We previously demonstrated that transforming growth factor-,1 (TGF-,1), while having no effect alone, enhances nitric oxide (NO) production in primary, purified mouse astrocytes induced by lipopolysaccharide (LPS) plus interferon-, (IFN-,), by recruiting a latent population of astrocytes to respond, thereby enhancing the total number of cells that express Nos2. In this investigation, we evaluated the molecular signaling pathway by which this occurs. We found that purified murine primary astrocytes express mRNA for TGF,RII as well as the TGF,RI subunit activin-like kinase 5 (ALK5), but not ALK1. Immunofluorescence microscopy confirmed the expression of TGF,RII and ALK5 protein in astrocytes. Consistent with ALK5 signaling, Smad3 accumulated in the nucleus of astrocytes as early as 30 min after TGF-,1 (3 ng/mL) treatment and persisted upto 32 hr after TGF-,1 administration. Addition of ALK5 inhibitors prevented TGF-,1-mediated Smad3 nuclear accumulation and NO production when given prior to the Nos2 induction stimuli, but not after. Finally, astrocyte cultures derived from Smad3 null mutant mice did not exhibit a TGF-,1-mediated increase in iNOS expression. Overall, this data suggests that ALK5 signaling and Smad3 nuclear accumulation is required for optimal enhancement of LPS plus IFN,-induced NO production in astrocytes by TGF-,1. © 2010 Wiley-Liss, Inc. [source] Prostaglandin E2 promotes cell proliferation via protein kinase C/extracellular signal regulated kinase pathway-dependent induction of c-Myc expression in human esophageal squamous cell carcinoma cellsINTERNATIONAL JOURNAL OF CANCER, Issue 11 2009Le Yu Abstract Overexpression of cyclooxygenase-2 (COX-2) and elevation of its derivative prostaglandin E2 (PGE2) are implicated in human esophageal squamous cell carcinoma. The expression of c-Myc, an oncogenic transcription factor, is also upregulated in this malignant disease. This study sought to elucidate whether a functional connection exists between COX-2/PGE2 and c-Myc in esophageal squamous cell carcinoma. Results showed that PGE2 substantially increased the proliferation of cultured esophageal squamous cell carcinoma cells. In this regard, PGE2 substantially increased the mRNA and protein expression of c-Myc and its association with the binding partner Max. Knockdown of c-Myc by RNA interference also significantly attenuated PGE2 -induced cell proliferation. Further, mechanistic study revealed that PGE2 increased the protein stability and nuclear accumulation of c-Myc via phosphorylation on serine 62 in an extracellular signal regulated kinase (ERK)-dependent manner. To this end, ERK activation by PGE2 was completely abolished by protein kinase C (PKC) inhibitors. Moreover, the effect of PGE2 on c-Myc expression was mimicked by EP2 receptor agonist. In addition, knockdown of EP2 receptor by EP2 siRNA attenuated PGE2 -induced c-Myc expression. Collectively, our findings suggest that PGE2 upregulates c-Myc via the EP2/PKC/ERK pathway. This study sheds new light on the carcinogenic mechanism of PGE2 in esophageal squamous cell carcinoma. © 2009 UICC [source] Ribosome-inactivating proteins isolated from dietary bitter melon induce apoptosis and inhibit histone deacetylase-1 selectively in premalignant and malignant prostate cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 4 2009Su Dao Xiong Abstract Epidemiologic evidence suggests that a diet rich in fruits and vegetables is associated with a reduced risk of prostate cancer (PCa) development. Although several dietary compounds have been tested in preclinical PCa prevention models, no agents have been identified that either prevent the progression of premalignant lesions or treat advanced disease. Momordica charantia, known as bitter melon in English, is a plant that grows in tropical areas worldwide and is both eaten as a vegetable and used for medicinal purposes. We have isolated a protein, designated as MCP30, from bitter melon seeds. The purified fraction was verified by SDS-PAGE and mass spectrometry to contain only 2 highly related single chain Type I ribosome-inactivating proteins (RIPs), ,-momorcharin and ,-momorcharin. MCP30 induces apoptosis in PIN and PCa cell lines in vitro and suppresses PC-3 growth in vivo with no effect on normal prostate cells. Mechanistically, MCP30 inhibits histone deacetylase-1 (HDAC-1) activity and promotes histone-3 and -4 protein acetylation. Treatment with MCP30 induces PTEN expression in a prostatic intraepithelial neoplasia (PIN) and PCa cell lines resulting in inhibition of Akt phosphorylation. In addition, MCP30 inhibits Wnt signaling activity through reduction of nuclear accumulation of ,-catenin and decreased levels of c- Myc and Cyclin-D1. Our data indicate that MCP30 selectively induces PIN and PCa apoptosis and inhibits HDAC-1 activity. These results suggest that Type I RIPs derived from plants are HDAC inhibitors that can be utilized in the prevention and treatment of prostate cancer. © 2009 UICC [source] Somatic mutations of adenomatous polyposis coli gene and nuclear b-catenin accumulation have prognostic significance in invasive urothelial carcinomas: Evidence for Wnt pathway implicationINTERNATIONAL JOURNAL OF CANCER, Issue 1 2009Efstathios Kastritis Abstract Wnt pathway signaling is crucial in many cancers and data indicate crosstalk with other key cancer pathways, however in urothelial carcinogenesis it has not been extensively studied. We searched for mutations in adenomatous polyposis coli (APC), a key regulator of the pathway, and studied b-catenin expression and interactions with the expression of other markers of apoptosis, angiogenesis, and proliferation in patients with invasive urothelial cancer. The mutation cluster region of APC was directly sequenced in 70 patients with muscle invasive disease who were treated with surgery and adjuvant chemotherapy. COX-2, p53, Ki67, and b-catenin were studied immunohistochemically and micro vessel density was quantified by CD105 expression. Single somatic amino-acid substitutions (missense) were found in 9 (13%) and frameshift deletions in 2 (3%) tumors, all located in regions adjacent to b-catenin binding sites. Patients having either APC missense mutations or b-catenin nuclear accumulation had less frequent COX-2 overexpression (24% vs. 76%, p = 0.043) and more frequent lymph node involvement (75% vs. 38%, p = 0.023). Patients with either APC mutations or b-catenin accumulation had shorter disease-free interval (13.4 vs. 28 months, p = 0.07), whereas in multivariate analysis they had shorter disease-specific survival (60.5 vs. 20.6 months, p = 0.048). Somatic APC missense mutations are not rare in advanced urothelial neoplasms. Either APC mutations and/or aberrant expression of b-catenin are associated with worse outcome. Further study of the role of the Wnt pathway, potential crosstalk with other pathways and potential candidate therapeutic targets in urothelial cancer is needed. © 2008 Wiley-Liss, Inc. [source] Mutations of the Wnt antagonist AXIN2 (Conductin) result in TCF-dependent transcription in medulloblastomasINTERNATIONAL JOURNAL OF CANCER, Issue 2 2007Arend Koch Abstract Medulloblastomas (MBs) represent the most common malignant brain tumors in children. Most MBs develop sporadically in the cerebellum, but their incidence is highly elevated in patients with familial adenomatous polyposis coli. These patients carry germline mutations in the APC tumor suppressor gene. APC is part of a multiprotein complex involved in the Wnt signaling pathway that controls the stability of ,-catenin, the central effector in this cascade. Previous genetic studies in MBs have identified mutations in genes coding for ,-catenin and its partners, APC and AXIN1, which cause activation of Wnt signaling. The pathway is negatively controlled by the tumor suppressor AXIN2 (Conductin), a scaffold protein of this signaling complex. To investigate whether alterations in AXIN2 may also be involved in the pathogenesis of sporadic MBs, we performed a mutational screening of the AXIN2 gene in 116 MB biopsy samples and 11 MB cell lines using single-strand conformation polymorphism and sequencing analysis. One MB displayed a somatic, tumor-specific 2 bp insertion in exon 5, leading to carboxy-terminal truncation of the AXIN2 protein. This tumor biopsy showed nuclear accumulation of ,-catenin protein, indicating an activation of Wnt signaling. In 2 further MB biopsies, mutations were identified in exon 5 (Glu408Lys) and exon 8 (Ser738Phe) of the AXIN2 gene, which are due to predicted germline mutations and rare polymorphisms. mRNA expression analysis in 22 MBs revealed reduced expression of AXIN2 mRNA compared to 8 fetal cerebellar tissues. Promoter hypermethylation could be ruled out as a major cause for transcriptional silencing by bisulfite sequencing. To study the functional role of AXIN2 in MBs, wild-type AXIN2 was overexpressed in MB cell lines in which the Wnt signaling pathway was activated by Wnt-3a. In this assay, AXIN2 inhibited Wnt signaling demonstrated in luciferase reporter assays. In contrast, overexpression of mutated AXIN2 with a deleted C-terminal DIX-domain resulted in an activation of the Wnt signaling pathway. These findings indicate that mutations of AXIN2 can lead to an oncogenic activation of the Wnt pathway in MBs. © 2007 Wiley-Liss, Inc. [source] Use of [125I]4,-iodoflavone as a tool to characterize ligand-dependent differences in Ah receptor behaviorJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2002Hollie I. Swanson Abstract We have synthesized [125I]4,-iodoflavone to study Ah receptor (AhR),ligand interactions by a class of AhR ligands distinct from the prototypic ligand 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). This radioligand allows the comparison of AhR,ligand interactions using a ligand that differs in AhR affinity, and yet has the same radiospecific activity as [125I]2-iodo-7,8-dibromodibenzo- p -dioxin. Specific binding of [125I]4,-iodoflavone with the AhR was detected as a single radioactive peak (,9.7 S) following density sucrose gradient analysis. Cytosolic extracts from both Hepa 1 and HeLa cells were used as the source of mouse and human AhR, respectively. A ,6.7 S form of radioligand-bound Ah receptor was detected in the high salt nuclear extracts of both cell lines. In HeLa cells approximately twofold more [125I]4,-iodoflavone,AhR 6 S complex, compared with [125I]2-iodo-7,8-dibromodibenzo- p -dioxin, was recovered in nuclear extracts. A comparison of the ability of 4,-iodoflavone and TCDD to cause time-dependent translocation of AhR-yellow fluorescent protein revealed that 4,-iodoflavone was more efficient at enhancing nuclear accumulation of the receptor. These results suggest that [125I]4,-iodoflavone is a particularly useful and easily synthesized ligand for studying the AhR. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:298,310, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10053 [source] Cyclo(His-Pro) promotes cytoprotection by activating Nrf2-mediated up-regulation of antioxidant defenceJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6 2009Alba Minelli Abstract Hystidyl-proline [cyclo(His-Pro)] is an endogenous cyclic dipeptide produced by the cleavage of thyrotropin releasing hormone. Previous studies have shown that cyclo(His-Pro) protects against oxidative stress, although the underlying mechanism has remained elusive. Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells. Cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion caused by glutamate, rotenone, paraquat and ,-amyloid treatment. Moreover, real-time PCR analyses revealed that cyclo(His-Pro) induced the expression of a number of ARE-related genes and protected cells against hydrogen peroxide-mediated apoptotic death. Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown. Finally, pharmacological inhibition of p-38 MAPK partially prevented both cyclo(His-Pro)-mediated Nrf2 activation and cellular protection. These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence. [source] miR-20b modulates VEGF expression by targeting HIF-1, and STAT3 in MCF-7 breast cancer cells,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2010Sandra Cascio MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of different genes, including genes involved in cancer progression. A functional link between hypoxia, a key feature of the tumor microenvironment, and miRNA expression has been documented. We investigated whether and how miR-20b can regulate the expression of vascular endothelial growth factor (VEGF) in MCF-7 breast cancer cells under normoxic and hypoxia-mimicking conditions (CoCl2 exposure). Using immunoblotting, ELISA, and quantitative real-time PCR, we demonstrated that miR-20b decreased VEGF protein levels at 4 and 24,h following CoCl2 treatment, and VEGF mRNA at 4,h of treatment. In addition, miR-20b reduced VEGF protein expression in untreated cells. Next, we investigated the molecular mechanism by which pre-miR-20b can affect VEGF transcription, focusing on hypoxia inducible factor 1 (HIF-1) and signal transducer and activator of transcription 3 (STAT3), transcriptional inducers of VEGF and putative targets of miR-20b. Downregulation of VEGF mRNA by miR-20b under a 4,h of CoCl2 treatment was associated with reduced levels of nuclear HIF-1, subunit and STAT3. Chromatin immunoprecipitation (ChIP) assays revealed that HIF-1,, but not STAT3, was recruited to the VEGF promoter following the 4,h of CoCl2 treatment. This effect was inhibited by transfection of cells with pre-miR-20b. In addition, using siRNA knockdown, we demonstrated that the presence of STAT3 is necessary for CoCl2 -mediated HIF-1, nuclear accumulation and recruitment on VEGF promoter. In summary, this report demonstrates, for the first time, that the VEGF expression in breast cancer cells is mediated by HIF-1 and STAT3 in a miR-20b-dependent manner. J. Cell. Physiol. 224:242,249, 2010 © 2010 Wiley-Liss, Inc. [source] cAMP-induced differentiation of human neuronal progenitor cells is mediated by nuclear fibroblast growth factor receptor-1 (FGFR1)JOURNAL OF NEUROCHEMISTRY, Issue 6 2003E. K. Stachowiak Abstract Activation of cAMP signaling pathway and its transcriptional factor cyclic AMP response element binding protein (CREB) and coactivator are key determinants of neuronal differentiation and plasticity. We show that nuclear fibroblast growth factor receptor-1 (FGFR1) mediates cAMP-induced neuronal differentiation and regulates CREB and CREB binding protein (CBP) function in ,-internexin-expressing human neuronal progenitor cells (HNPC). In proliferating HNPC, FGFR1 was associated with the cytoplasm and plasma membrane. Treatment with dB-cAMP induced nuclear accumulation of FGFR1 and caused neuronal differentiation, accompanied by outgrowth of neurites expressing MAP2 and neuron-specific neurofilament-L protein and enolase. HNPC transfected with nuclear/cytoplasmic FGFR1 or non-membrane FGFR1(SP-/NLS), engineered to accumulate exclusively in the cell nucleus, underwent neuronal differentiation in the absence of cAMP stimulation. In contrast, FGFR1/R4, with highly hydrophobic transmembrane domain of FGFR4, was membrane associated, did not enter the nucleus and failed to induce neuronal differentiation. Transfection of tyrosine kinase-deleted dominant negative receptor mutants, cytoplasmic/nuclear FGFR1(TK-) or nuclear FGFR1(SP-/NLS)(TK-), prevented cAMP-induced neurite outgrowth. Nuclear FGFR1 localized in speckle-like domains rich in phosphorylated histone 3 and splicing factors, regions known for active RNA transcription and processing, and activated the neurofilament-L gene promoter. FGFR1(SP-/NLS) transactivated CRE, up-regulated phosphorylation and transcriptional activity of CREB and stimulated the activity of CBP several-fold. Thus, cAMP-induced nuclear accumulation of FGFR1 provides a signal that triggers molecular events leading to neuronal differentiation. [source] Reversal of multidrug resistance-associated protein-mediated daunorubicin resistance by camptothecinJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 8 2002David Chauvier Abstract The multidrug-resistance (MR) status of camptothecin (CPT) was investigated in colon adenocarcinoma HT29 cells, leukemia K562, and breast carcinoma MCF7 cells expressing P-glycoprotein (Pgp) and/or MR-associated protein (MRP1). The concentration that induced 50% growth inhibition (IC50) against CPT was 0.14 and 0.20 ,M in parental K562/WT and MCF7/WT cells, respectively. The drug resistant subline KH30 and MCF7/VP cells, which both overexpress MRP1, presented IC50 values of 0.63 and 3.10 ,M, respectively. The resulting resistance indexes were 3.80 and 12.50, respectively. However, in KH300 cells, a cell line that preferentially overexpresses Pgp, the IC50 of CPT was 0.08 ,M and thus did not exhibit resistance against CPT. In MCF7/DoX cells, preferentially overexpressing Pgp, but also a significant level of MRP1, the IC50 of CPT was 0.64 ,M and thus presented a resistance index of 3.26 against CPT. The cytotoxic effect of CPT was modulated in cells expressing MRP1 (MCF7/VP, HT29 cells) by the specific MRP1 modulators, probenecid and MK571. These results led us to consider CPT as a substrate for MRP1 and a potential modulator of MRP1 activity. To test this hypothesis, we examined the ability of nontoxic concentrations of CPT to sensitize MRP1-overexpressing cells to daunorubicin (DNR). In MCF7/VP and KH30 cells, nontoxic concentrations of CPT were able to enhance cytotoxicity of DNR and its nuclear accumulation. Sequential and simultaneous associations of CPT (100 nM) and DNR provided complete reversal of resistance, thus showing a synergistic effect in KH30 cells. However, simultaneous association (with 10 or 20 nM CPT) had an additive effect in MCF7/VP. These data suggest that CPT could be proposed as a candidate for the reversal of the MRP1 phenotype at clinically achievable concentrations. © 2002 Wiley-Liss Inc. and the American Pharmaceutical Association J Pharm Sci 91:1765,1775, 2002 [source] Intracellular uptake and trafficking of Pluronic micelles in drug-sensitive and MDR cells: Effect on the intracellular drug localizationJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 1 2002Natalya Rapoport Abstract The intracellular uptake and localization of a fluorescently labeled Pluronic P-105 in HL-60 leukemia cells and in A2780 drug-sensitive and A2780/ADR MDR ovarian carcinoma cells were characterized by flow cytometry and fluorescence microscopy. Pluronic P-105 molecules were labeled with a pH-sensitive fluorescent label, 5-(and 6-)carboxy-2,7,-dichlorofluorescein. The fluorescence intensity of labeled Pluronic was about twofold higher at pH 7.4 than at pH 5.5. At Pluronic concentrations exceeding the critical micelle concentration (cmc), flow cytometry histograms manifested bimodal distribution of cell fluorescence for all types of cells. Cell population characterized by higher fluorescence intensity presumably resulted from Pluronic transfer from the acidic environment of cytoplasmic vesicles (endosomes or lysosomes) into the neutral environment of the cytoplasm and cell nuclei, which suggested the permeabilization of the membranes of acidic vesicle by Pluronic molecules. For the MDR cells, the bimodal distribution of cell fluorescence was already observed at very low Pluronic concentrations in the incubation medium (i.e., below the cmc). The data suggest that the membranes of acidic vesicles of MDR cells are more susceptible to the action of polymeric surfactants than those of drug-sensitive cells. Permeabilization of acidic vesicles had a dramatic effect on the intracellular trafficking of drugs: when delivered in PBS, the anthracyclin drug ruboxyl (Rb) sequestered in cytoplasmic vesicles and was excluded from cell nuclei; however, when delivered in Pluronic micelles, drug accumulated in cell nuclei. Drug uptake from/with Pluronic micelles was substantially enhanced by ultrasound. These findings suggest that the nuclear accumulation of drugs internalized via fluid-phase endocytosis can be enhanced by the application of Pluronic micelles and can be further augmented by ultrasonic irradiation. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:157,170, 2002 [source] Loss of E-cadherin and ,-catenin is correlated with poor prognosis of ampullary neoplasmsJOURNAL OF SURGICAL ONCOLOGY, Issue 5 2010Hui-Ping Hsu MD Abstract Background and Objectives Distant metastasis resulting from carcinoma cell detachment from the primary tumor involves modification of adhesion molecules. This study was conducted to examine the correlation of E-cadherin/,-catenin expression with survival and recurrence in ampullary neoplasms. Methods Patients with diagnoses of ampullary neoplasms were enrolled in the study. Demographics, operative findings, and histopathological data were collected by retrospective chart review. Expression of E-cadherin and ,-catenin were detected by immunohistochemistry. Results A total of 110 patients were enrolled in the study. Preservation of membranous staining of E-cadherin was noted in 41 (37%) patients, aberrant cytoplasmic staining in 48 (44%) patients, and complete loss in 21 (19%) patients. Loss of E-cadherin was associated with pancreatic invasion, recurrence, and poor prognosis. Membranous staining of ,-catenin was noted in 65 (59%) patients, cytoplasmic or nuclear accumulation in 16 (15%) patients, and complete loss in 29 (26%) patients. Loss of ,-catenin expression was associated with tumor markers, ulcerative type, liver metastases, and poor prognosis. Pancreatic invasion, lymph node involvement, and loss of ,-catenin expression were predictors of disease recurrence. Conclusions Loss of the E-cadherin/,-catenin complex is related to poor prognosis in ampullary cancer. Loss of ,-catenin is predictor of recurrence in multivariate analysis. J. Surg. Oncol. 2010; 101:356,362. © 2010 Wiley-Liss, Inc. [source] Activation of the redox sensor Pap1 by hydrogen peroxide requires modulation of the intracellular oxidant concentrationMOLECULAR MICROBIOLOGY, Issue 5 2004Ana P. Vivancos Summary The transcription factor Pap1 and the MAP kinase Sty1 are key regulators of hydrogen peroxide-induced responses in Schizosaccharomyces pombe. Pap1 can be activated quickly at low, but not high, hydrogen peroxide concentrations. The MAP kinase Sty1 has been reported to participate in Pap1 activation by the oxidant. Here, we provide biochemical and genetic evidence for the in vivo formation of a hydrogen peroxide-induced disulphide bond in Pap1, which precedes the rapid and reversible nuclear accumulation of the transcription factor. We show that activation of the Sty1 cascade before the oxidative insult, or overexpression of the Sty1-regulated genes ctt1 (encoding catalase) or gpx1 (encoding glutathione peroxidase), can accelerate Pap1 entry even at high doses of hydrogen peroxide. In fact, the lack of Sty1 impedes Pap1 nuclear localization, but only at high doses of the oxidant. We propose that, whereas low doses of hydrogen peroxide lead directly to Pap1 oxidation-activation, high concentrations of the oxidant initially activate the Sty1 pathway, with the consequent increase in scavenging enzymes, which in turn helps to decompose the excess of hydrogen peroxide and achieve an appropriate concentration for the subsequent activation of Pap1. Our results also suggest that activation of Sty1 at high doses of hydrogen peroxide may also be required to trigger other antioxidant activities such as those reverting the overoxidation of cysteine residues at the Pap1 pathway. [source] Pathogenesis and molecular targeted therapy of spinal and bulbar muscular atrophyNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 2 2007H. Adachi Spinal and bulbar muscular atrophy (SBMA) or Kennedy's disease is a motor neurone disease characterized by muscle atrophy, weakness, contraction fasciculations and bulbar involvement. SBMA mainly affects males, while females are usually asymptomatic. SBMA is caused by expansion of a polyglutamine (polyQ)-encoding CAG trinucleotide repeat in the androgen receptor (AR) gene. AR belongs to the heat shock protein 90 (Hsp90) client protein family. The histopathologic hallmarks of SBMA are diffuse nuclear accumulation and nuclear inclusions of the mutant AR with expanded polyQ in residual motor neurones in the brainstem and spinal cord as well as in some other visceral organs. There is increasing evidence that the ligand of AR and molecular chaperones play a crucial role in the pathogenesis of SBMA. The success of androgen deprivation therapy in SBMA mouse models has been translated into clinical trials. In addition, elucidation of its pathophysiology using animal models has led to the development of disease-modifying drugs, that is, Hsp90 inhibitor and Hsp inducer, which inhibit the pathogenic process of neuronal degeneration. SBMA is a slowly progressive disease by nature. The degree of nuclear accumulation of mutant AR in scrotal skin epithelial cells was correlated with that in spinal motor neurones in autopsy specimens; therefore, the results of scrotal skin biopsy may be used to assess the efficacy of therapeutic trials. Clinical and pathological parameters that reflect the pathogenic process of SBMA should be extensively investigated. [source] Non-cystic solid-pseudopapillary tumor of the pancreas showing nuclear accumulation and activating gene mutation of ,-cateninPATHOLOGY INTERNATIONAL, Issue 11 2006Isao Nishimori Solid-pseudopapillary tumor (SPT) is an unusual pancreatic neoplasm that is characterized by a mixture of solid and cystic components and a fibrous capsule. Recently, the tumorigenesis of SPT has been reported to be associated with gene mutations of ,-catenin, which is a molecule participating in the Wnt signaling pathway. Reported herein is the case of a 53-year-old woman with SPT. The tumor, approximately 3 cm in diameter in the pancreas body, had a clear margin and central calcification but had neither a cystic component nor fibrous capsule. Several lines of pathological findings in the surgically resected specimen indicated SPT: (i) pseudopapillary proliferation of eosinophilic polygonal cells with oval nuclei; (ii) positive expression of several marker molecules indicating differentiation into acinar and endocrine cells; and (iii) zymogen granule-like structures in the cytoplasm on electron microscopy. Further, the tumor cells had intense nuclear accumulation of ,-catenin and an activating mutation, 34Gly(GGA) to Arg(AGA), in exon 3 of the ,-catenin gene, as previously reported in most SPT. These findings suggest that association of the ,-catenin phenotype with development of the rare phenotype of SPT, a non-cystic and unencapsulated tumor, is unlikely. [source] Nrf2-mediated induction of detoxifying enzymes by alantolactone present in Inula heleniumPHYTOTHERAPY RESEARCH, Issue 11 2008Ji Yeon Seo Abstract Our previous study showed that a methanol extract of Inula helenium had the potential to induce detoxifying enzymes such as quinone reductase (QR) and glutathione S -transferase (GST) activity. In this study the methanol extract was further fractionated using silica gel chromatography and vacuum liquid chromatography, to yield pure compounds alantolactone and isoalantolactone as QR inducers. Alantolactone caused a dose-dependent induction of antioxidant enzymes including QR, GST, , -glutamylcysteine synthase, glutathione reductase, and heme oxygenase 1 in hepa1c1c7 mouse hepatoma cells. The compound increased the luciferase activity of HepG2-C8 cells, transfectants carrying antioxidant response element (ARE)-luciferase gene, in a dose-dependent manner, suggesting ARE-mediated transcriptional activation of antioxidant enzymes. Alantolactone also stimulated the nuclear accumulation of Nrf2 that was inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitors. In conclusion, alantolactone appears to induce detoxifying enzymes via activation of PI3K and JNK signaling pathways, leading to translocation of Nrf2, and subsequent interaction between Nrf2 and ARE in the encoding genes. Copyright © 2008 John Wiley & Sons, Ltd. [source] Beta-catenin accumulation in the progression of human hepatocarcinogenesis correlates with loss of E-cadherin and accumulation of p53, but not with expression of conventional WNT-1 target genesTHE JOURNAL OF PATHOLOGY, Issue 2 2003Wilhelm Prange Abstract Beta-catenin integrates intracellular WNT signalling and the intercellular E-cadherin,catenin adhesion system. To date, little is known about the role of ,-catenin activation and nuclear accumulation in hepatocarcinogenesis. This study has analysed ,-catenin expression patterns in human dysplastic nodules (DNs), as well as in hepatocellular carcinomas (HCCs) in comparison with proliferation, expression of WNT-1 target genes, E-cadherin, and p53. One hundred and seventy HCCs and 25 DNs were categorized according to established criteria and analysed for the expression pattern of ,-catenin. Analysis of the proliferative activity and expression of E-cadherin, cyclin D1, MMP-7, c-myc, and p53 was performed on a representative subgroup of cases. All DNs lacked nuclear ,-catenin, while 36% of all HCCs were positive, with the number of nuclear stained cells ranging from less than 1% to more than 90%. Increasing nuclear accumulation of ,-catenin correlated with reduced membranous E-cadherin expression and nuclear p53 but not with proliferation. Cyclin D1, MMP-7, and c-myc expression was detected in 54%, 26%, and 65% of HCCs, respectively, but did not correlate with nuclear ,-catenin, proliferation, or grading. Sequence analysis of the ,-catenin gene revealed no detectable mutations in DNs, but mutations in the GSK-3, binding site were present in 14.3% of the HCCs. In conclusion, this study has demonstrated that nuclear accumulation of ,-catenin is a frequent progression event in human hepatocarcinogenesis which correlates with nuclear p53 accumulation and loss of membranous E-cadherin, but not with the expression pattern of established WNT-1 target genes. It is hypothesized that the role of ,-catenin in human HCC differs significantly from its established function in colon carcinogenesis. Copyright © 2003 John Wiley & Sons, Ltd. [source] Nuclear accumulation of the ankyrin repeat protein ANK1 enhances the auxin-mediated transcription accomplished by the bZIP transcription factors BZI-1 and BZI-2THE PLANT JOURNAL, Issue 6 2009Stefan Böttner Summary The tobacco (Nicotiana tabacum) basic leucine zipper (bZIP) transcription factor BZI-1 has been implicated in auxin-mediated gene regulation. Yeast two-hybrid analysis has led to the identification of two BZI-1 protein interaction partners: the heterodimerizing bZIP factor BZI-2 and an ankyrin repeat domain protein, ANK1. Analysis in transgenic plants confirms that low levels of functional BZI-1, BZI-2 and ANK1 result in reduced auxin responses. This finding indicates that the three proteins act in the same functional context. The in vivo interaction of ANK1 and BZI-1 has been confirmed by protoplast two-hybrid analysis, as well as by bimolecular fluorescence complementation (BiFC) studies. Whereas YFP-BZI-1 has been found to be localized in the nucleus, YFP-ANK1 resides in the cytosol. Nevertheless, the inhibition of nuclear export with the inhibitor leptomycin B (LMB) and the co-expression with BZI-1, as well as treatment with auxin, results in the accumulation of YFP-ANK1 in the nucleus. Whereas BZI-1 is a weak activator, BZI-1/BZI-2 heterodimers efficiently support transcription. Importantly, conditions that lead to the accumulation of ANK1 in the nucleus, such as the expression of an ANK1 protein fused to a nuclear localization sequence (NLS) or auxin treatment, lead to a significant enhancement of BZI-1/BZI-2-mediated transcription. We therefore propose a mechanism in which the nuclear accumulation of ANK1 enhances BZI-1/BZI-2-mediated transcription in an auxin-dependent manner, presumably facilitated by protein,protein interaction. In summary, this study defines novel components in auxin-dependent signalling and transcriptional control. [source] Phase 2 trial of leuprorelin in patients with spinal and bulbar muscular atrophy,ANNALS OF NEUROLOGY, Issue 2 2009Haruhiko Banno MD Objective Spinal and bulbar muscular atrophy (SBMA) is a hereditary motor neuron disease caused by the expansion of a polyglutamine tract in the androgen receptor (AR). Animal studies have shown that the pathogenesis of SBMA is dependent on serum testosterone level. This study is aimed at evaluating the efficacy and safety of androgen deprivation by leuprorelin acetate in patients with SBMA. Methods Fifty SBMA patients underwent subcutaneous injections of leuprorelin acetate or placebo in a randomized, placebo-controlled trial for 48 weeks, followed by an open-label trial for an additional 96 weeks, in which 19 patients of the leuprorelin group and 15 of the placebo group received leuprorelin acetate. The patients who did not participate in the open-label trial were also followed up for the 96-week period (UMIN000000474). Results Leuprorelin acetate significantly extended the duration of cricopharyngeal opening in videofluorography and decreased mutant AR accumulation in scrotal skin biopsy. The patients treated with leuprorelin acetate for 144 weeks exhibited significantly greater functional scores and better swallowing parameters than those who received placebo. Autopsy of one patient who received leuprorelin acetate for 118 weeks suggested that androgen deprivation inhibits the nuclear accumulation or stabilization, or both, of mutant AR in the motor neurons of the spinal cord and brainstem. Interpretation These observations suggest that administration of leuprorelin acetate suppresses the deterioration of neuromuscular impairment in SBMA by inhibiting the toxic accumulation of mutant AR. The results of this phase 2 trial support the start of large-scale clinical trials of androgen deprivation for SBMA. Ann Neurol 2009;65:140,150 [source] Rho kinase,dependent activation of SOX9 in chondrocytesARTHRITIS & RHEUMATISM, Issue 1 2010Dominik R. Haudenschild Objective The transcription factor SOX9 directly regulates the expression of the major proteoglycans and collagens comprising the cartilage extracellular matrix. The DNA binding activity and cellular localization of SOX9 is controlled through posttranslational modifications, including phosphorylation. The activity of Rho kinase (ROCK) has profound effects on the actin cytoskeleton, and these effects are instrumental in determining the phenotype and differentiation of chondrocytes. However, the mechanisms linking ROCK to altered chondrocyte gene expression remain unknown. The purpose of the present study was to test for a direct interaction between ROCK and SOX9. Methods Human SW1353 chondrosarcoma cells were transfected with constructs coding for RhoA, ROCK, Lim kinase, and SOX9. The interaction between ROCK and SOX9 was tested on purified proteins, and was verified within a cellular context using induced overexpression and activation of the Rho pathway. The effects of SOX9 transcriptional activation were quantified with a luciferase reporter plasmid containing SOX9 binding sites from the COL2A1 enhancer element. Results SOX9 was found to contain a consensus phosphorylation site for ROCK. In vitro, ROCK directly phosphorylated SOX9 at Ser181, and the overexpression of ROCK or the activation of the RhoA pathway in SW1353 chondrosarcoma cells increased SOX9Ser181 phosphorylation. ROCK caused a dose-dependent increase in the transcription of a SOX9-luciferase reporter construct, and increased phosphorylation and nuclear accumulation of SOX9 protein in response to transforming growth factor , treatment and mechanical compression. Conclusion These results demonstrate a new interaction that directly links ROCK to increased cartilage matrix production via activation of SOX9 in response to mechanical and growth factor stimulation. [source] Stimulation of DNA synthesis, activation of mitogen-activated protein kinase ERK2 and nuclear accumulation of c-fos in human aortic smooth muscle cells by ketamineCELL PROLIFERATION, Issue 3 2002V. Boulom Proliferation of vascular smooth muscle cells is known to be regulated by autocrine and paracrine stimuli, including extracellular matrix, reactive oxygen species, lipids, and biomechanical forces. The effect of many pharmacological agents to which smooth muscle cells may be exposed, however, is widely unexplored. Ketamine, an intravenous anaesthetic and a phencyclidine derivative, regulates diverse intracellular signalling pathways in smooth muscle cells, several of which are known to affect cell proliferation. The effect of ketamine on proliferative response of smooth muscle cells, however, is not determined. We tested the hypothesis that ketamine may regulate proliferation of smooth muscle cells, and investigated the effects of pharmacological doses of ketamine on their proliferative capacity by measuring DNA synthesis and activation of mitogen-activated protein (MAP) kinase signalling pathway in human aortic smooth muscle cells. DNA synthesis, as determined by incorporation of 3H-thymidine into DNA, was enhanced by 73% (P < 0.0001) and 130% (P < 0.0001) with 10 and 100 µm ketamine, respectively. Ketamine-induced DNA synthesis was dependent on de novo protein synthesis, as it was abolished by an inhibitor of protein synthesis, cycloheximide. A synthetic inhibitor of MAP kinase pathway, PD98059, decreased 50% (P < 0.0001) of ketamine-induced DNA synthesis, suggesting that the activation of MAP kinase pathway was partially responsible for ketamine-induced effects. Consistently, in-gel kinase assay and in vitro kinase assay of cell lysates showed ketamine-induced MAP kinase activation and expression of ERK2 (extracellular signal-regulated kinase) in smooth muscle cells. This effect of ketamine was not dependent on de novo protein synthesis. Immunofluorescent light microscopy showed ketamine-induced nuclear accumulation of c-fos, a downstream effect of MAP kinase activation, in smooth muscle cells. In conclusion, these data support the hypothesis of the study and demonstrate that ketamine, by stimulating DNA synthesis in human aortic smooth muscle cells, may have an impact on proliferative capacity of these cells. The present results also demonstrate that ketamine induces the activation of MAP kinase pathway and nuclear accumulation of transcription factor c-fos in smooth muscle cells. They further demonstrate that the activation of MAP kinases is partially responsible for ketamine-induced DNA synthesis in human aortic smooth muscle cells. Together, these findings suggest that ketamine may play a role as a pharmacological regulator of mechanisms involved in proliferation of smooth muscle cells. [source] | |||||||||||||||||||||||||