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Ectopic Expression (ectopic + expression)
Selected AbstractsEnhancement of Immunogenicity of Jeg3 Cells by Ectopic Expression of HLA-A*0201 and CD80AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2003Serpil Koc Problem: The choriocarcinoma cell line Jeg3 suppresses immunity in vitro by secretion of soluble factors like leukemia inhibitory factor suppressing leukocyte activation. The cells lack expression of classical human leukocyte antigen (HLA)-A and -B alleles but express some HLA-C, and non-classical HLA-G and -E. Upon binding to killing inhibitory receptor on natural killer (NK) cells, HLA-G prevents activation of cytolytic activity. We investigated whether Jeg3 cells are capable of immune stimulation after complementation with classical HLA and T cell costimulatory signal CD80. Method of study: Jeg3 cells were transduced to express HLA-A*0201 and/or CD80. Parental Jeg3 or transfectants Jeg3-A2, Jeg3-CD80 or Jeg3-CD80-A2 were used to stimulate allogeneic resting and activated peripheral blood lymphocytes (PBL). The different cell lines were loaded with a HLA-A2-restricted Epstein-Barr virus (EBV) recall antigen peptide epitope and antigen presenting ability was examined. T cell lines specific for Jeg3 and transfectants were generated from HLA-A2 matched and nonmatched donors and compared for expansion, phenotypes and cytolytic activity. Results: While all Jeg3 cell lines induced only marginal proliferation of resting T cells, phytohemagglutinin (PHA)-activated T cells were stimulated by CD80 or CD80-A2 expressing Jeg3. Only the transfectant Jeg3-CD80-A2 was capable of specific T cell stimulation by EBV recall antigen presentation. T cell lines of HLA-A2 non-matched donors stimulated with the Jeg3 transfectants showed significant expansion only when HLA-A2 and the costimulus CD80 were present. T cells from HLA-A2 positive donors did not expand significantly or differentially. No NK cells grew under any condition. In Jeg3-CD80-A2 stimulated T cells lines CD8+ cells expanded preferentially. These T cells exerted cytolytic activity toward all Jeg3 cell lines. Conclusion: Our data suggest that, in spite of immunosuppressive mechanisms, proliferative and cytolytic T cell responses are induced by Jeg3 cells when classical HLA- and/or costimulatory signals are present on the cells. [source] Cloning a novel developmental regulating gene, Xotx5: Its potential role in anterior formation in Xenopus laevisDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2000Hiroki Kuroda The vertebrate Otx gene family is related to otd, a gene contributing to head development in Drosophila. In Xenopus, Xotx1, Xotx2, and Xotx4 have already been isolated and analyzed. Here the cloning, developmental expression and functions of the additional Otx Xenopus gene, Xotx5 are reported. This latter gene shows a greater degree of homology to Xotx2 than Xotx1 and Xotx4. Xotx5 was initially expressed in Spemann's organizer and later in the anterior region. Ectopic expression of Xotx5 had similar effects to other Xotx genes in impairing trunk and tail development, and especially similar effects to Xotx2 in causing secondary cement glands. Taken together, these findings suggest that Xotx5 stimulates the formation of the anterior regions and represses the formation of posterior structures similar to Xotx2. [source] Rab23 GTPase is expressed asymmetrically in Hensen's node and plays a role in the dorsoventral patterning of the chick neural tubeDEVELOPMENTAL DYNAMICS, Issue 11 2007Naixin Li Abstract The mouse Rab23 protein, a Ras-like GTPase, inhibits signaling through the Sonic hedgehog pathway and thus exerts a role in the dorsoventral patterning of the spinal cord. Rab23 mouse mutant embryos lack dorsal spinal cord cell types. We cloned the chicken Rab23 gene and studied its expression in the developing nervous system. Chick Rab23 mRNA is initially expressed in the entire neural tube but retracts to the dorsal alar plate. Unlike in mouse, we find Rab23 in chick already expressed asymmetrically during gastrulation. Ectopic expression of Rab23 in ventral midbrain induced dorsal genes (Pax3, Pax7) ectopically and reduced ventral genes (Nkx2.2 and Nkx6) without influencing cell proliferation or neurogenesis. Thus, in the developing brain of chick embryos Rab23 acts in the same manner as described for the caudal spinal cord in mouse. These data indicate that Rab23 plays an important role in patterning the dorso-ventral axis by dorsalizing the neural tube. Developmental Dynamics 236:2993,3006, 2007. © 2007 Wiley-Liss, Inc. [source] Cloning and functional characterization of a novel connexin expressed in somites of Xenopus laevisDEVELOPMENTAL DYNAMICS, Issue 3 2005Teun P. De Boer Abstract Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (,62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals. Developmental Dynamics 233:864,871, 2005. © 2005 Wiley-Liss, Inc. [source] Functional analysis in Drosophila indicates that the NBCCS/PTCH1 mutation G509V results in activation of smoothened through a dominant-negative mechanismDEVELOPMENTAL DYNAMICS, Issue 4 2004Gary R. Hime Abstract Mutations in the human homolog of the patched gene are associated with the developmental (and cancer predisposition) condition Nevoid Basal Cell Carcinoma Syndrome (NBCCS), as well as with sporadic basal cell carcinomas. Most mutations that have been identified in the germline of NBCCS patients are truncating or frameshift mutations, with amino acid substitutions rarely found. We show that a missense mutation in the sterol-sensing domain G509V acts as a dominant negative when assayed in vivo in Drosophila. Ectopic expression of a Drosophila patched transgene, carrying the analogous mutation to G509V, causes ectopic activation of Hedgehog target genes and ectopic membrane stabilisation of Smoothened. The G509V transgene behaves in a manner similar, except in its subcellular distribution, to a C-terminal truncation that has been characterised previously as a dominant-negative protein. G509V exhibits vesicular localisation identical to the wild-type protein, but the C-terminal truncated Patched molecule is localised predominantly to the plasma membrane. This finding suggests that dominant-negative function can be conferred by interruption of different aspects of Patched protein behaviour. Another mutation at the same residue, G509R, did not exhibit dominant-negative activity, suggesting that simple removal of the glycine at 509 is not sufficient to impart dominant-negative function. Developmental Dynamics 229:780,790, 2004. © 2004 Wiley-Liss, Inc. [source] B-cell co-receptor CD72 is expressed on NK cells and inhibits IFN-, production but not cytotoxicityEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2009Valeria L. Alcón Abstract NK cells have two main functions, namely cell-mediated cytotoxicity and production of cytokines. Multiple inhibitory receptors that regulate NK-cell cytotoxicity have been characterized whereas little is known about receptors regulating cytokine production. Here we report that CD72, which is considered to be an important co-receptor regulating B-cell activation, is also expressed on mouse NK cells. NK cells expressing high levels of CD72, upon stimulation with IL-12 and IL-18 or target cells, produce significantly less IFN-, than those expressing low levels of CD72, whereas both subsets are equally cytotoxic. Ectopic expression of CD72 in the murine NK-cell line KY2 inhibits cytokine-induced IFN-, production, and the inhibitory effect is diminished by mutations in the inhibitory motifs in the intracellular domain or replacement of the extracellular domain of CD72. Thus, CD72 is an inhibitory receptor on NK cells regulating cytokine production. [source] Direct role of NF-,B activation in Toll-like receptor-triggered HLA-DRA expressionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2006Keun-Wook Lee Abstract Microbial components, such as DNA containing immunostimulatory CpG motifs (CpG-DNA) and lipopolysaccharides (LPS), elicit the cell surface expression of MHC class II (MHC-II) through Toll-like receptor (TLR)/IL-1R. Here, we show that CpG-DNA and LPS induce expression of the HLA-DRA in the human B cell line, RPMI 8226. Ectopic expression of the dominant negative mutant of CIITA and RNA interference targeting the CIITA gene indicate that CIITA activation is not enough for the maximal MHC-II expression induced by CpG-DNA and LPS. Additionally, nuclear factor (NF)-,B activation is required for the CpG-DNA-activated and LPS-activated HLA-DRA expression, whereas IFN-,-induced MHC-II expression depends on CIITA rather than on NF-,B. Comprehensive mutant analyses, electrophoretic mobility shift assays and chromatin immunoprecipitation assays, reveal that the functional interaction of NF-,B with the promoter element is necessary for the TLR-mediated HLA-DRA induction by CpG-DNA and LPS. This novel mechanism provides the regulation of MHC-II gene expression with complexity and functional diversity. [source] PU.1 is required for transcriptional activation of the Stat6 response element in the Ig, promoterEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2003Marko Pesu Abstract Signal transducer and activator of transcription 6 (Stat6) has a crucial role in regulation of IL-4-induced gene responses. Stat6-binding sites are present in the promoters of both ubiquitously and cell-type-specifically expressed genes. The promoter regions of IL-4-inducible genes contain cis -acting elements for several transcription factors that act in concert with Stat6 and are also likely to modulate lineage-specific gene expression. We have observed that the Stat6 response element from the B-cell-specific Ig, promoter is readily activated upon IL-4 stimulation in B cells but not in non-hematopoietic cells. A minimal low-affinity PU.1-core-binding sequence (5,-AGAA-3,) was identified within the Stat6 DNA-binding site in the Ig, promoter. Ectopic expression of the myeloid- and B-cell-specific transcription factor PU.1 restored the IL-4-inducibility of the Ig,-Stat6 response element in HepG2 cells, and the induction required an intact PU.1-binding sequence. Both the transactivation and the DNA-binding domains of PU.1 were required for induction of Stat6-mediated transcription. The co-operation between PU.1 and Stat6 in transactivation of the Ig, gene represents a molecular mechanism for the fine-tuning of cell-type-restricted expression of IL-4-induced gene responses. [source] The myeloid leukemia factor interacts with COP9 signalosome subunit 3 in Drosophila melanogasterFEBS JOURNAL, Issue 3 2008Wakana Sugano The human myeloid leukemia factor 1 (hMLF1) gene was first identified as an NPM,hMLF1 fusion gene produced by chromosomal translocation. In Drosophila, dMLF has been identified as a protein homologous to hMLF1 and hMLF2, which interacts with various factors involved in transcriptional regulation. However, the precise cellular function of dMLF remains unclear. To generate further insights, we first examined the behavior of dMLF protein using an antibody specific to dMLF. Immunostaining analyses showed that dMLF localizes in the nucleus in early embryos and cultured cells. Ectopic expression of dMLF in the developing eye imaginal disc using eyeless-GAL4 driver resulted in a small-eye phenotype and co-expression of cyclin E rescued the small-eye phenotype, suggesting the involvement of dMLF in cell-cycle regulation. We therefore analyzed the molecular mechanism of interactions between dMLF and a dMLF-interacting protein, dCSN3, a subunit of the COP9 signalosome, which regulates multiple signaling and cell-cycle pathways. Biochemical and genetic analyses revealed that dMLF interacts with dCSN3 in vivo and glutathione S -transferase pull-down assays revealed that the PCI domain of the dCSN3 protein is sufficient for this to occur, possibly functioning as a structural scaffold for assembly of the COP9 signalosome complex. From these data we propose the possibility that dMLF plays a negative role in assembly of the COP9 signalosome complex. [source] In vivo potentiation of human oestrogen receptor , by Cdk7-mediated phosphorylationGENES TO CELLS, Issue 10 2004Saya Ito Phosphorylation of the Ser118 residue in the N-terminal A/B domain of the human oestrogen receptor , (hER,) by mitogen-activated protein kinase (MAPK), stimulated via growth factor signalling pathways, is known to potentiate ER, ligand-induced transactivation function. Besides MAPK, cyclin dependent kinase 7 (Cdk7) in the TFIIH complex has also been found to potentiate hER, transactivation in vitro through Ser118 phosphorylation. To investigate an impact of Cdk7 on hER, transactivation in vivo, we assessed activity of hER, in a wild-type and cdk7 inactive mutant Drosophila that ectopically expressed hER, in the eye disc. Ectopic expression of the wild-type or mutant receptors, together with a green fluorescent protein (GFP) reporter gene, allowed us to demonstrate that hER, expressed in the fly tissues was transcriptionally functional and adequately responded to hER, ligands in the patterns similar to those observed in mammalian cells. Replacement of Ser118 with alanine in hER, (S118A mutant) significantly reduced the ligand-induced hER, transactivation function. Importantly, while in cdk7 inactive mutant Drosophila the wild-type hER, exhibited reduced response to the ligand; levels of transactivation by the hER, S118A mutant were not affected in these inactive cdk7 mutant flies. Furthermore, phosphorylation of hER, at Ser118 has been observed in vitro by both human and Drosophila Cdk7. Our findings demonstrate that Cdk7 is involved in regulation of the ligand-induced transactivation function of hER,in vivo via Ser118 phosphorylation. [source] Decreased expression and promoter methylation of the menin tumor suppressor in pancreatic ductal adenocarcinomaGENES, CHROMOSOMES AND CANCER, Issue 5 2009Ilaria Cavallari Loss of menin, a tumor suppressor coded by the MEN1 gene, is a key factor in the pathogenesis of multiple endocrine neoplasia type I and in a percentage of sporadic endocrine tumors of the pancreas and parathyroid glands. This study investigated expression of the menin protein in the normal exocrine pancreas and in pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic tumor. Immunofluorescence (IF) analyses showed that menin is expressed at high levels in normal acinar and duct cells. Examination of 24 clinical samples of PDAC revealed a pronounced decrease in menin expression in all tumors examined. To identify alterations underlying this defect, we searched for disruption and epigenetic silencing of the MEN1 gene. Analysis of nine laser-microdissected tumors revealed loss of heterozygosity of intragenic (one tumor) or adjacent (three tumors) MEN1 microsatellite markers. Methylation of CpG sites in the MEN1 promoter was documented in five of 24 tumors. IF analyses also revealed low to undetectable menin expression in the PDAC cell lines MiaPaCa-2 and Panc-1. Ectopic expression of menin in these cells resulted in a marked alteration of the cell cycle, with an increase in the G1/S+G2 ratio. These findings represent the first evidence that the MEN1 gene is a target of mutation and methylation in PDAC and that menin influences the cell cycle profile of duct cells. © 2009 Wiley-Liss,Inc. [source] Inactivation of the cystatin E/M tumor suppressor gene in cervical cancerGENES, CHROMOSOMES AND CANCER, Issue 9 2008Mysore S. Veena We have previously localized a cervical cancer tumor suppressor gene to a 300 kb interval of 11q13. Analysis of candidate genes revealed loss of expression of cystatin E/M, a lysosomal cysteine protease inhibitor, in 6 cervical cancer cell lines and 9 of 11 primary cervical tumors. Examination of the three exons in four cervical cancer cell lines, 19 primary tumors, and 21 normal controls revealed homozygous deletion of exon 1 sequences in one tumor. Point mutations were observed in six other tumors. Two tumors contained mutations at the consensus binding sites for cathepsin L, a lysosomal protease overexpressed in cervical cancer. Introduction of these two point mutations using site directed mutagenesis resulted in reduced binding of mutated cystatin E/M to cathepsin L. Although mutations were not observed in any cell lines, four cell lines and 12 of 18 tumors contained promoter hypermethylation. Reexpression of cystatin E/M was observed after 5,aza 2-deoxycytidiene and/or Trichostatin A treatment of cervical cancer cell lines, HeLa and SiHa, confirming promoter hypermethylation. Ectopic expression of cystatin E/M in these two cell lines resulted in growth suppression. There was also suppression of soft agar colony formation by HeLa cells expressing the cystatin E/M gene. Reexpression of cystatin E/M resulted in decreased intracellular and extracellular expression of cathepsin L. Overexpression of cathepsin L resulted in increased cell growth which was inhibited by the reintroduction of cystatin E/M. We conclude, therefore, that cystatin E/M is a cervical cancer suppressor gene and that the gene is inactivated by somatic mutations and promoter hypermethylation. © 2008 Wiley-Liss, Inc. [source] Thirty-kilodalton Tat-interacting protein suppresses tumor metastasis by inhibition of osteopontin transcription in human hepatocellular carcinoma,HEPATOLOGY, Issue 1 2008Jian Zhao It has been previously demonstrated that the 30-kDa Tat-interacting protein (TIP30) plays an important role in the suppression of hepatocarcinogenesis by acting as a tumor suppressor. Here we report that TIP30 suppresses metastasis of hepatocellular carcinoma (HCC) through inhibiting the transcription of osteopontin (OPN), a key molecule in the development of tumor metastasis. The expression of TIP30 messenger RNA was reverse to that of OPN messenger RNA in HCC cell lines. Ectopic expression of TIP30 greatly suppressed OPN expression, inhibited invasion of HCC cells through extracellular matrix (ECM) and adhesion with fibronectin in vitro, whereas down-regulation of TIP30 by RNA-mediated interference enhanced OPN expression and promoted metastatic abilities of HCC cells in vitro. Moreover, overexpression of TIP30 significantly inhibited the growth and lung metastases of HCC cells in nude mice. In contrast, down-regulation of TIP30 greatly promoted tumor cell growth and metastases in vivo. TIP30 repressed OPN transcription through interaction with Ets-1 and suppressed the transcriptional activity of Ets-1 and synergistic actions of Ets-1 and alkaline phosphatase-1. Thus, TIP30 may act as an Ets-1 modulator and inhibit tumor metastasis through abrogating Ets-1,dependent transcription. Moreover, expression of TIP30 was inversely associated with OPN expression in HCC tissue samples as detected by immunohistochemistry assay. Conclusion: Our results reveal a novel pathway by which OPN and possibly other Ets-1 target genes involved in tumor metastasis are regulated by TIP30 and elucidate a mechanism for metastasis promoted by TIP30 deficiency. (HEPATOLOGY 2008.) [source] Inhibition of CK2 Activity by TGF-,1 Promotes I,B-, Protein Stabilization and Apoptosis of Immortalized HepatocytesHEPATOLOGY, Issue 6 2003Lakita G. Cavin Nuclear factor ,B (NF-,B) is an antiapoptotic factor involved in development, regeneration, and neoplastic progression of the liver. Previously, we have shown that stabilization of inhibitor ,B (I,B)-, protein following treatment of hepatocytes with transforming growth factor (TGF)-,1 promoted NF-,B repression, which then permitted induction of AP-1/SMAD-mediated liver cell death. Because basal I,B-, protein turnover is regulated by protein kinase CK2, here we have elucidated the regulation of CK2 kinase activity and its role in control of NF-,B levels following treatment with TGF-,1. We show that both messenger RNA (mRNA) and protein levels of the CK2, catalytic subunit are down-regulated following TGF-,1 stimulation in murine hepatocyte cells. The ensuing inhibition of CK2 kinase activity promotes stabilization of I,B protein, which is followed by the shutoff of constitutive NF-,B activity and induction of apoptosis. Ectopic expression of CK2, inhibits TGF-,1-induced apoptosis through sustained activation of NF-,B. Conversely, expression of a kinase-dead mutant of CK2, potentiates TGF-,1 cell killing. Importantly, we show that hepatocellular carcinomas (HCCs) derived from TGF-,1 transgenic mice and human HCC cell lines display enhanced CK2 I,B kinase activity that contributes in part to an elevated NF-,B activity in vivo. In conclusion, inhibition of CK2 expression levels by TGF-,1 is crucial for the induction of apoptosis of hepatocytes. Circumvention of this process by up-regulation of CK2 activity in transformed cells may contribute to the promotion of TGF-,1-induced liver carcinogenesis. [source] Expression and role of Bcl-xL in human hepatocellular carcinomasHEPATOLOGY, Issue 1 2001Tetsuo Takehara Transformed hepatocytes survive various apoptotic insults during their growth in vivo. However, molecular mechanisms that inhibit apoptosis and support their survival are not well understood. In this study, we investigated the expression and role of Bcl-xL, an antiapoptotic member of the Bcl-2 family, in human hepatocellular carcinoma (HCC). The Bcl-xL protein was expressed in HepG2, Hep3B, and Huh7 human hepatoma cell lines at high levels, but none of these cells expressed Bcl-2. Down-modulation of Bcl-xL by antisense oligonucleotide activated apoptosis in HepG2 cells in response to cellular stresses induced by staurosporine treatment or by serum starvation. Ectopic expression of transcriptionally active p53 alone was not sufficient for the activation of apoptosis in p53 -null Hep3B cells, but apoptosis was induced when endogenous Bcl-xL was simultaneously inhibited by antisense oligonucleotide in these cells. Bcl-xL was expressed in all 20 surgically resected human HCC tissues when examined by Western blot analysis and immunohistochemistry, and levels of its expression were higher in a subset of HCC tissues than those of adjacent nontumor liver tissues or normal livers. We conclude that Bcl-xL expressed in human HCC cells inhibits apoptosis produced by various cellular stresses, such as staurosporine treatment, serum starvation, and p53 activation, and may play an important role in their survival. [source] Antitumor activity of ALK1 in pancreatic carcinoma cellsINTERNATIONAL JOURNAL OF CANCER, Issue 8 2007Hendrik Ungefroren Abstract In this study, the authors investigated the expression of activin receptor-like kinase 1 (ALK1) in pancreatic carcinoma and evaluated its potential role as a tumor suppressor in vitro and in vivo. Endogenous ALK1 expression was demonstrated by immunohistochemistry in both pancreatic tumor tissue and peritumoral normal tissue from 6 patients and by RT-PCR in 8/12 established pancreatic cancer cell lines. Ectopic expression of a constitutively active (ca) ALK1 mutant in TGF-, sensitive PANC-1 and COLO-357 cells augmented transcriptional activation of a Smad2/3 responsive reporter, and slowed down basal growth in vitro. Both effects were further enhanced by TGF-,/ALK5 stimulation, suggesting largely independent nuclear Smad signaling by both type I receptors. Upon orthotopic transplantation of PANC-1-caALK1 into immunodeficient mice, tumor size was strongly reduced and was associated with a lower microvessel density in the PANC-1-caALK1-derived tumors. In vitro, this mutant efficiently blocked TGF-,-induced epithelial-to-mesenchymal transdifferentiation and suppressed TGF-,/ALK5-mediated activation of the p38 MAPK pathway. Mechanistically, caALK1 silenced MyD118, an immediate TGF-, target gene whose protein product, GADD45,, couples Smad signaling to p38 activation. These results show that ALK1 activation in pancreatic tumor cells is antioncogenic by inducing ALK5-independent growth inhibition and by blocking TGF-,/ALK5-mediated epithelial-to-mesenchymal transdifferentiation and, possibly, invasion and metastatic progression. © 2007 Wiley-Liss, Inc. [source] Inactivation of RASSF2A by promoter methylation correlates with lymph node metastasis in nasopharyngeal carcinomaINTERNATIONAL JOURNAL OF CANCER, Issue 1 2007Zhe Zhang Abstract RASSF2 can bind directly to K-Ras and function as a negative effector of Ras protein. RASSF2A is the only isoform of RASSF2 that contains CpG islands in its promoter and it has been reported to be inactivated by its promoter methylation in several human cancers. In the present study, we investigated the correlation of RASSF2A expression with its promoter methylation in nasopharyngeal carcinoma (NPC). Expression of RASSF2A was down-regulated in 80% (4/5) of NPC cell lines. Decreased RASSF2A expression was also observed in NPC primary tumors compared with normal nasopharyngeal epithelia. Promoter methylation of RASSF2A could be detected in all the RASSF2A -silenced cell lines (4/5) of the NPC cell lines and 50.9% (27/53) of primary tumors, but not in any of the normal epithelia. RASSF2A -methylated cases showed a significantly lower level of RASSF2A expression than unmethylated cases. Loss of RASSF2A expression can be greatly restored by the methyltransferase inhibitor 5-aza-dC in NPC cell lines. In addition, patients with methylated RASSF2A presented a higher frequency of lymph node metastasis (p < 0.05). Ectopic expression of RASSF2A in RASSF2A -silenced and -methylated NPC cell line CNE2 shows that RASSF2A could inhibit cell cycle progression, colony formation and cell migration, which provided further evidence that RASSF2A is a candidate tumor suppressor gene. In conclusion, RASSF2A, a candidate tumor suppressor gene (TSG), is frequently inactivated by its promoter methylation and this aberrant methylation correlates with lymph node metastasis in NPC. © 2006 Wiley-Liss, Inc. [source] Overexpression of Smurf2 Stimulates Endochondral Ossification Through Upregulation of ,-Catenin,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2008Qiuqian Wu MD Abstract Ectopic expression of Smurf2 in chondrocytes and perichondrial cells accelerated endochondral ossification by stimulating chondrocyte maturation and osteoblast development through upregulation of ,-catenin in Col2a1-Smurf2 embryos. The mechanism underlying Smurf2-mediated morphological changes during embryonic development may provide new mechanistic insights and potential targets for prevention and treatment of human osteoarthritis. Introduction: Our recent finding that adult Col2a1-Smurf2 mice have an osteoarthritis-like phenotype in knee joints prompted us to examine the role of Smurf2 in the regulation of chondrocyte maturation and osteoblast differentiation during embryonic endochondral ossification. Materials and Methods: We analyzed gene expression and morphological changes in developing limbs by immunofluorescence, immunohistochemistry, Western blot, skeletal preparation, and histology. A series of markers for chondrocyte maturation and osteoblast differentiation in developing limbs were examined by in situ hybridization. Results: Ectopic overexpression of Smurf2 driven by the Col2a1 promoter was detected in chondrocytes and in the perichondrium/periosteum of 16.5 dpc transgenic limbs. Ectopic Smurf2 expression in cells of the chondrogenic lineage inhibited chondrocyte differentiation and stimulated maturation; ectopic Smurf2 in cells of the osteoblastic lineage stimulated osteoblast differentiation. Mechanistically, this could be caused by a dramatic increase in the expression of ,-catenin protein levels in the chondrocytes and perichondrial/periosteal cells of the Col2a1-Smurf2 limbs. Conclusions: Ectopic expression of Smurf2 driven by the Col2a1 promoter accelerated the process of endochondral ossification including chondrocyte maturation and osteoblast differentiation through upregulation of ,-catenin, suggesting a possible mechanism for development of osteoarthritis seen in these mice. [source] E2F1 represses ,-catenin/TCF activity by direct up-regulation of Siah1JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8b 2009Wei Xie Abstract Transcription factor E2F1 is a key regulator of cell proliferation and apoptosis. Its activity is strictly controlled by the pRB/E2F pathway. In the majority of cancer cells, however, this pathway is frequently found deregulated, and the underlying mechanism involving transcriptional control by E2F1 has not yet been fully elucidated. Here we report the identification of two putative E2F1-binding sites located upstream from Siah1 transcription start site (+1). Chromatin immunoprecipitation assay reveals that transcription factor E2F1 is capable of binding to the putative sites, and luciferase reporter assay shows that E2F1 can activate transcription from the Siah1 promoter. Ectopic expression of E2F1 elevates the Siah1 level, hence suppressing the ,-catenin/TCF activity. Consistently, knock-down of endogenous E2F1 by a shRNA strategy results in reduced expression of Siah1. Moreover, repression of ,-catenin/TCF activity by E2F1 can be attenuated by shRNA-based repression of endogenous Siah1, implying that Siah1 is a bona fide E2F1 target gene, which at least partly, mediates the suppression of ,-catenin/TCF signalling pathway. [source] Overexpression of Par-4 enhances thapsigargin-induced apoptosis via down-regulation of XIAP and inactivation of Akt in human renal cancer cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Tae-Jin Lee Abstract The prostate-apoptosis-response-gene-4 (Par-4) protein has been shown to function as an effector of cell death in response to various apoptotic stimuli that trigger mitochondria and membrane receptor-mediated cell death pathways. We found that overexpressing Par-4 by stable transfection sensitizes Caki cells to induction of apoptosis by TRAIL and drugs that induce endoplasmic reticulum (ER) stress [thapsigargin (TG), tunicamycin (TU) and etoposide]. Ectopic expression of Par-4 is associated with decreased levels of XIAP protein in TG-treated cells, caused in part by XIAP protein instability and caspase activation. Levels of phospho-Akt are decreased in Caki/Par-4 cells to a significantly greater extent than in Caki/Vector cells by treatment with TG, and this is in turn associated with decreased levels of phospho-PDK1, the kinase upstream of Akt. In conclusion, we provide evidence that ectopic expression of Par-4 sensitizes Caki cells to TG and that XIAP protein instability and inactivation of Akt are important in cellular pathways affected by Par-4. J. Cell. Biochem. 103: 358,368, 2008. © 2007 Wiley-Liss, Inc. [source] Cell-cycle regulation and mammalian gametogenesis: A lesson from the unexpectedMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 8 2006Abraham L. Kierszenbaum Abstract The progression of mammalian gametogenesis requires a precise balance between cell-cycle activities and elimination of defective gametogenic cells to ensure the perpetuation of species. Both spermatogonia and oogonia are stem cell populations committed to meiosis with the aim of generating haploid gametes for fertilization. At puberty, mitotically dividing spermatogonial cell cohorts maintain the ability of cell renewal and occupy niches in the seminiferous tubule. In contrast, mitotically dividing oogonial cell cohorts produced in the fetal ovary, are exclusively committed to meiosis and produce primordial follicles housing a primary oocyte surrounded by somatic follicular cells. A consistent physiological event during mammalian gametogenesis is the disposal of spermatogenic cells by apoptosis and ovarian follicles by atresia. Cyclin-dependent kinases (Cdks) and their cyclin partners coordinate the activities of the cell cycle. An additional cell-cycle regulatory component is the centrosome. The centrosome harbors regulatory proteins controlling the normal progression of the cell cycle. Changes in individual centrosome proteins can lead to cell-cycle arrest and a decrease in the genomic protective function of p53 that promotes apoptosis. Disruption of cyclin A1, Cdk2, and Cdk4 expression in transgenic mice results in infertility and gonadal atrophy. Cdk,cyclin complexes interact with regulatory proteins, which may fine-tune the activities of the complex. One of the many regulatory proteins is p12, a 115 amino acid growth suppressor polypeptide designated p12CDK2AP1, partner of Cdk2 and with binding affinity to DNA polymerase ,/primase. Overexpression of p12 is associated with testicular and ovarian atrophy without affecting fertility. Ectopic expression of p12 was driven by the keratin 14 promoter. Keratin 14 is the pairing partner of keratin 5 and both keratins are expressed in testis. The efficiency of keratin promoters in driving ectopic gonadal gene expression, the association of gonadal atrophy with the ectopic expression of a Cdk2 regulatory protein and the centrosome, as a reservoir of cell-cycle regulatory proteins, open new experimental opportunities to address still lingering questions concerning cell differentiation and division during mammalian gametogenesis. Mol. Reprod. Dev. 939,942, 2006. © 2006 Wiley-Liss, Inc. [source] Ligand-independent Regulation of the hairless Promoter by Vitamin D Receptor,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2008Andrew Engelhard The characteristic alopecia associated with mutations in the hairless (hr) and vitamin D receptor (VDR) genes defines the resulting genetic disorders, known as atrichia and VDRRIIa rickets, as phenocopies. In both cases, the separation of the dermal papilla from the regressing hair follicle at the onset of the first catagen phase of the hair cycle and the development of dermal cysts and utricules subsequent to mutation of either gene suggests that their activities affect the same regulatory pathways. VDR functions as a hormonally activated transcription factor, and a role in transcription has been postulated for Hr due in part to its nuclear localization and homology with the GATA-1 zinc-finger domain. Therefore, we examined the hypothesis that VDR and Hr have a direct regulatory effect on each other via a transcriptional mechanism. Ectopic expression of the VDR repressed hr promoter activity in HaCaT cells and primary human keratinocytes (PHKs). While this repression occurs in the absence of 1,25 dihydroxyvitamin D3 (D3), the addition of ligand greatly augments the effect. However, we also demonstrate the rare phenomenon of ligand-independent promoter transactivation by VDR. We show that the full-length promoter is transactivated by VDR in a ligand-independent and cell type-specific manner, suggesting that direct transcriptional regulation of hr by the VDR accounts in part for the phenotypic overlap between atrichia and VDRRIIa rickets. [source] Galectin-9, a Novel Prognostic Factor with Antimetastatic Potential in Breast CancerTHE BREAST JOURNAL, Issue 2006Akira Yamauchi MD Abstract: Galectin-9, a member of the ,-galactoside-binding animal lectin family, is involved in various cellular biological events, including aggregation and apoptosis, adhesion of cancer cells, and dendritic cell maturation. We recently reported the relationship between galectin-9 expression in tumor tissue and distant metastasis in breast cancer. Tumors in 42 of the 84 patients were galectin-9-positive, and tumors in 19 of the 21 patients with distant metastasis were galectin-9-negative, assessed by immunohistochemistry. The cumulative distant metastasis-free survival ratio for galectin-9-positive patients was better than for the galectin-9-negative group (p < 0.0001). Multivariate analysis revealed that galectin-9 status influenced distant metastasis independent of and much more than lymph node metastasis. MCF-7 subclones with a high level of galectin-9 expression formed tight clusters during proliferation in vitro, whereas a subclone (K10) with the lowest level of galectin-9 expression did not. However, K10 cells stably transfected with a galectin-9 expression vector aggregated in nude mice as well as in culture. Ectopic expression of galectin-9 also reduced MCF-7 cell adhesion to extracellular matrix proteins., [source] The Arabidopsis ARGOS-LIKE gene regulates cell expansion during organ growthTHE PLANT JOURNAL, Issue 1 2006Yuxin Hu Summary Cell expansion, and its coordination with cell division, plays a critical role in the growth and development of plant organs. However, the genes controlling cell expansion during organogenesis are largely unknown. Here, we demonstrate that a novel Arabidopsis gene, ARGOS-LIKE (ARL), which has some sequence homology to the ARGOS gene, is involved in this process. Reduced expression or overexpression of ARL in Arabidopsis results in smaller or larger cotyledons and leaves as well as other lateral organs, respectively. Anatomical examination of cotyledons and leaves in ARL transgenic plants demonstrates that the alteration in size can be attributed to changes in cell size rather than cell number, indicating that ARL plays a role in cell expansion-dependent organ growth. ARL is upregulated by brassinosteroid (BR) and this induction is impaired in the BR-insensitive mutant bri1, but not in the BR-deficient mutant det2. Ectopic expression of ARL in bri1,119 partially restores cell growth in cotyledons and leaves. Our results suggest that ARL acts downstream of BRI1 and partially mediates BR-related cell expansion signals during organ growth. [source] Phytochromes confer the photoperiodic control of flowering in rice (a short-day plant)THE PLANT JOURNAL, Issue 5 2000Takeshi Izawa Summary The photoperiodic sensitivity 5 (se5) mutant of rice, a short-day plant, has a very early flowering phenotype and is completely deficient in photoperiodic response. We have cloned the SE5 gene by candidate cloning and demonstrated that it encodes a putative heme oxygenase. Lack of responses of coleoptile elongation by light pulses and photoreversible phytochromes in crude extracts of se5 indicate that SE5 may function in phytochrome chromophore biosynthesis. Ectopic expression of SE5 cDNA by the CaMV 35S promoter restored the photoperiodic response in the se5 mutant. Our results indicate that phytochromes confer the photoperiodic control of flowering in rice. Comparison of se5 with hy1, a counterpart mutant of Arabidopsis, suggests distinct roles of phytochromes in the photoperiodic control of flowering in these two species. [source] Cell-free production of transducible transcription factors for nuclear reprogramming,BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009William C. Yang Abstract Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli -based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target. Biotechnol. Bioeng. 2009; 104: 1047,1058. © 2009 Wiley Periodicals, Inc. [source] Genetic link between p53 and genes required for formation of the zonula adherens junctionCANCER SCIENCE, Issue 5 2004Masamitsu Yamaguchi Ectopic expression of human p53 in Drosophila eye imaginal disc cells induces apoptosis and results in a rough eye phenotype in the adult flies. We have screened Drosophila stocks to identify mutations that enhance or suppress the p53-induced rough eye phenotype. One of the dominant enhancers of the p53-induced rough eye phenotype corresponds to a loss-of-function mutation of the crumbs gene, which is essential for the biogenesis of the zonula adherens junction and the establishment of apical polarity in epithelial cells. Enhancement of p53-induced apoptosis in the eye imaginal discs by a half-reduction of the crumbs gene dose was confirmed by a TUNEL method. Furthermore, mutations of genes for Shotgun (Drosophila E-cadherin) and Armadillo (Drosophila,-catenin), the two main components of the adherens junction, also strongly enhanced the p53-induced rough eye phenotype. These results suggest that human p53 senses subtle abnormality at the adherens junction or in signals derived from the junction, and consequently induces apoptosis to remove abnormal cells from tissue. Thus p53 likely plays a role as a guardian of the tissue not only by sensing the damaged DNA, but also by sensing signals from the adherens junction. [source] Stem cells and gastric cancer: Role of gastric and intestinal mixed intestinal metaplasiaCANCER SCIENCE, Issue 2 2003Masae Tatematsu All of the different types of stomach epithelial cells are known to be derived from a single progenitor cell in each gland. Similarly, cancers develop from single cells, based on data from clonality analysis in C3H/HeN , BALB/c chimeric mice. Using gastric and intestinal epithelial cell markers, intestinal metaplasia (IM) can be divided into two major types: a gastric and intestinal (GI) mixed type, and a solely intestinal (I) type. Ectopic expression of Cdx genes and down-regulation of Sox2 in isolated single GI mixed IM glands suggests abnormal differentiation of stem cells that can produce both gastric (G) and I type cells. Similarly, phenotypic expression of gastric cancer cells of each histological type can be clearly classified into G and I type epithelial cells. The heterogeneity of phenotypic expression of gastric cancer cells in individual cancers is assumed to reflect this intrinsic potential for differentiation in two directions. Gastric cancers at early stages, independent of the histological type, mainly consist of G type cells, and phenotypic shift from G to I type expression is clearly observed with progression. The data thus suggest IM may not be a preneoplastic change in gastric carcinoma, but rather that cells of the I type may appear independently in the gastric mucosa in IM and in gastric cancers. Intestinalization of gastric mucosa and cancer cells may represent a kind of homeotic transformation. Whether disturbance of the regulation of Sox2 and Cdx genes may be of importance to the biological behavior of gastric cancers should therefore be clarified in future studies. (Cancer Sci 2003; 94: 135,141) [source] CDK2 regulation through PI3K and CDK4 is necessary for cell cycle progression of primary rat hepatocytesCELL PROLIFERATION, Issue 4 2007L. Wierød In response to mitogenic stimuli, CDK4 and CDK2 form complexes with cyclins D and E, respectively, and translocate to the nucleus in the late G1 phase. It is an on-going discussion whether mammalian cells need both CDK4 and CDK2 kinase activities for induction of S phase. Methods and results: In this study, we have explored the role of CDK4 activity during G1 progression of primary rat hepatocytes. We found that CDK4 activity was restricted by either inhibiting growth factor induced cyclin D1-induction with the PI3K inhibitor LY294002, or by transient transfection with a dominant negative CDK4 mutant. In both cases, we observed reduced CDK2 nuclear translocation and reduced CDK2-Thr160 phosphorylation. Furthermore, reduced pRb hyperphosphorylation and reduced cellular proliferation were observed. Ectopic expression of cyclin D1 alone was not sufficient to induce CDK4 nuclear translocation, CDK2 activity or cell proliferation. Conclusions: Thus, epidermal growth factor-induced CDK4 activity was necessary for CDK2 activation and for hepatocyte proliferation. These results also suggest that, in addition to regulating cyclin D1 expression, PI3K is involved in regulation of nuclear shuttling of cyclin-CDK complexes in G1 phase. [source] Serine-71 phosphorylation of Rac1/Cdc42 diminishes the pathogenic effect of Clostridium difficile toxin ACELLULAR MICROBIOLOGY, Issue 12 2009Janett Schoentaube Summary Clostridium difficile toxin A and B (TcdA/TcdB) are glucosyltransferases that glucosylate GTPases of the Rho family. The epidermal growth factor (EGF) positively modulates C. difficile toxin-induced disturbance of the intestinal barrier function by an unknown mechanism. We found that EGF-treated CaCo-2 monolayers were less susceptible to TcdA-catalysed glucosylation of Rac1 but not of RhoA, which correlated with phosphorylation of Rac1 at Ser-71. Phospho-Rac1/phospho-Cdc42 (Ser-71) still bound to the PAK-CRIB domain indicating an active state. A more detailed characterization of phospho-Rac1 was performed using the phosphomimetic mutant Rac1 S71E. Ectopic expression of Rac1 S71E induced a specific phenotype of cells showing an increase in filopodial structures that were also induced by EGF. Rac1 S71E (and Cdc42 S71E) but not Rac1 S71A was at least fivefold weaker substrate for TcdA-catalysed glucosylation compared with wild type Rac1. The protective effect was checked in transfection experiments where Rac1 S71E and, to a lesser extent, Cdc42 S71E reduced the TcdA-induced cytopathic effect. Thus, Ser-71 phosphorylation of Rac1 might be interesting for modulation of microbial pathogenesis where Rho GTPases, especially Rac1 and Cdc42, are involved. In addition, this is the first description of a specific functional outcome of Rac1 phosphorylation at Ser-71. [source] | ||||||||||||||||||||||||||||||||||