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Oliver Müller - One of the best experts on this subject based on the ideXlab platform.
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APC promoter methylation and Protein expression in hepatocellular carcinoma
Journal of Cancer Research and Clinical Oncology, 2007Co-Authors: Antal Csepregi, Oliver Müller, Christoph Röcken, Nadine Kutzner, Juliane Hoffmann, Stephan Saliger, Regine Schneider-stock, Albert Roessner, Peter MalfertheinerAbstract:Purpose We investigated the impact of promoter methylation on APC Protein expression in patients with hepatocellular carcinoma (HCC).
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Molecular analysis of APC promoter methylation and Protein expression in colorectal cancer metastasis.
Carcinogenesis, 2005Co-Authors: Jie Chen, Oliver Müller, Christoph Röcken, Catherine Lofton-day, Hans-ulrich Schulz, Nadine Kutzner, Peter Malfertheiner, Matthias P. A. EbertAbstract:APC (adenomatous polyposis coli) promoter methylation has been linked to the early development of colorectal cancers. However, the role of APC methylation and its effect on Protein expression in colon cancer metastasis is largely unknown. In this study, we investigated APC promoter methylation by Methylight analysis and analysed the APC Protein levels by immunohistochemistry and western blot analysis in 24 liver metastasis and 39 primary colorectal cancers. Promoter methylation of the APC gene was found to be a frequent event in liver metastasis (10/24) and significantly more frequent compared with primary colorectal cancer (7/39, P = 0.047). APC methylation was not found in 14 matched normal colon tissues. APC Protein was detected in the cytoplasm of primary and metastatic cancer cells and non-tumorous colon epithelium. By western blot analysis, APC Protein levels were found to be decreased in primary tumour tissues compared with the normal colon mucosa. In contrast, APC Protein levels were not decreased in the cancer cells that had metastasized to the liver. APC Protein levels were independent of the presence of APC promoter methylation or gene mutations. In summary, APC promoter methylation is a frequent epigenetic alteration in colorectal cancer metastasis. However, we observed no significant association between APC promoter methylation or gene mutation and APC Protein expression in colorectal metastasis. Therefore, metastatic cancer cells seem to harbour a heterogenous genetic and epigenetic background, in which cancer cells may exhibit APC promoter methylation that is independent of APC expression.
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Differences Between the Interaction of β-Catenin with Non-phosphorylated and Single-mimicked Phosphorylated 20-amino acid Residue Repeats of the APC Protein
Journal of Molecular Biology, 2003Co-Authors: Lara Tickenbrock, Katja Kössmeier, Holger Rehmann, Christian Herrmann, Oliver MüllerAbstract:The tumour suppressor Protein adenomatous polyposis coli (APC) regulates the level and the intracellular localisation of the proto-oncoProtein β-catenin. There are indications that a region comprising seven homologous 20-amino acid residue repeats within the APC Protein is responsible for the interaction with β-catenin and that the phosphorylation of conserved serine residues within these repeats increases the affinity for β-catenin. We used biophysical methods to analyse the β-catenin binding of single repeats or repeat combinations as non-phosphorylated or phosphorylated recombinant Proteins. The non-phosphorylated repeats showed similar affinities, no matter whether they were tested as single recombinant repeats or in combination with neighbouring repeats. This result makes a cooperative influence between the repetitive motifs unlikely. The phosphorylation of the APC Protein was mimicked by specific serine/aspartate mutations, which align to serine residues in the cytoplasmic β-catenin binding domain of E-cadherin. Remarkably, the mimicked phosphorylation of a serine, which is not involved in β-catenin interaction in the E-cadherin/β-catenin complex, led to a significant increase in the APC affinity for β-catenin. These results indicate structural differences between the E-cadherin/β-catenin and the APC/β-catenin complexes and provide quantitative evidence for the importance of the APC phosphorylation for its interaction with β-catenin.
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The Adenomatous Polyposis Coli-Protein (APC) interacts with the Protein tyrosine phosphatase PTP-BL via an alternatively spliced PDZ domain.
Oncogene, 2000Co-Authors: Kai S. Erdmann, Oliver Müller, Jürgen Kuhlmann, Volkmar Lessmann, Lutz Herrmann, Volker Eulenburg, Rolf HeumannAbstract:Mutations of the tumor suppressor Protein APC (Adenomatous Polyposis Coli) are linked to familiar and sporadic human colon cancer. Here we describe a novel interaction between the APC Protein and the Protein tyrosine phosphatase PTP-BL carrying five PDZ Protein–Protein interaction domains. Exclusively, the second PDZ domain (PDZ2) of PTP-BL is binding to the extreme C-terminus of the APC Protein, as determined by yeast two-hybrid studies. Using surface plasmon resonance analysis we established a dissociation constant (KD) of 8.1×10−9 M. We find that a naturally occurring splice insertion of five amino acids (PDZ2b) abolishes its binding affinity to the APC Protein. The in vivo interaction between PTP-BL and the APC Protein was shown by coprecipitation experiments in transfected COS cells. Furthermore, in cultured epithelial Madine Carnine Kidney cells the subcellular colocalization was demonstrated for the nucleus and also for the tips of cellular extensions. The interaction of the APC Protein with a Protein tyrosine phosphatase may indirectly modulate the steady state levels of tyrosine phosphorylations of associated Proteins, such as β-catenin playing a major role in the regulation of cell division, migration and cell adhesion.
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The APC Protein binds to A/T rich DNA sequences.
Oncogene, 1999Co-Authors: J. Deka, Peter Herter, Markus Sprenger-haussels, S. Koosch, D. Franz, K. M. Muller, C. Kuhnen, Ingrid Hoffmann, Oliver MüllerAbstract:The tumor suppressor Protein APC (Adenomatous Polyposis Coli) is localized in the cytosol and in the nucleus. In this study, we demonstrate that the nuclear APC Protein level is high in cells in the basal crypt region of the normal colorectal epithelium. Strikingly, the APC Protein staining resembles the staining pattern of a nuclear proliferation marker. As a first step towards a possible role of the nuclear APC Protein, we provide data showing the direct interaction of the nuclear APC Protein with DNA. A nuclear APC isoform precipitates with matrix-immobilized DNA. Vice versa, the immunoprecipitation of APC from nuclear lysates results in co-precipitation of genomic DNA. Using recombinant APC fragments we mapped three DNA binding domains: one within the β-catenin binding and regulatory domain, and two in the carboxyterminal third of the APC Protein. All these three domains contain clusters of repetitive S(T)PXX sequence motifs that were described to mediate the DNA interaction of many other DNA binding Proteins. In analogy to S(T)PXX Proteins, the APC Protein binds preferentially to A/T rich DNA sequences rather than to a single DNA sequence motif.
Tetsu Akiyama - One of the best experts on this subject based on the ideXlab platform.
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APC Protein is required for initiation of neuronal differentiation in rat pheochromocytoma PC12 cells.
Biochemical and Biophysical Research Communications, 2000Co-Authors: Yoh Dobashi, Kazuhiro Katayama, Masataka Kawai, Tetsu Akiyama, Toru KameyaAbstract:The adenomatous polyposis (APC) gene product is highly expressed in the central nervous system. To elucidate the contribution of the APC Protein to neuronal differentiation, we used an inducible antisense mRNA vector to suppress APC Protein expression and examined neuronal differentiation of PC12 cells induced by nerve growth factor (NGF). When antisense mRNA was induced, APC Protein expression was suppressed to 20% of the noninduced level. In those cells, neurite extension induced by NGF and expression of microtubule-associated Protein 2 (MAP2) was completely inhibited. However, once cells had differentiated, antisense APC mRNA expression and subsequent suppression of APC Protein expression had no effect on either cell morphology or MAP2 Protein expression. These results suggest that the wild type APC is critically involved only in the initiation of neuronal differentiation, but not in the maintenance of the differentiated phenotype, or that the neuronal phenotype could be maintained at lower level of APC Protein.
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The Tumor Suppressor Protein APC Colocalizes with β-Catenin in the Colon Epithelial Cells
Biochemical and Biophysical Research Communications, 1996Co-Authors: Takao Senda, Isao Miyashiro, Akihiko Matsumine, Gyeong Hun Baeg, S. Kobayashi, Morito Monden, Takushi Monden, Kumao Toyoshima, Tetsu AkiyamaAbstract:Abstract The APC gene is mutated in familial adenomatous polyposis and sporadic colorectal tumors. The product of this gene is a 300 kDa cytoplasmic Protein associated with catenin. In the present study, we examined the subcellular localization of the APC Protein and β-catenin in the mouse colon by double-labeling immunocytochemistry. While the APC Protein was localized in the lateral and apical cytoplasm and in microvilli of the epithelial cells, β-catenin was present exclusively in the lateral cytoplasm. Double-labeling-immunoelectron microscopy demonstrated precise colocalization of the APC Protein and β-catenin along the lateral plasma membrane. These results suggest that the APC Protein functions in cooperation with β-catenin in the lateral cytoplasm but has other functions independent of β-catenin in the apical cytoplasm and in microvilli.
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The Tumor Suppressor Gene Product APC Is Hyperphosphorylated during the M Phase
Biochemical and Biophysical Research Communications, 1996Co-Authors: Rabindra N. Bhattacharjee, Kumao Toyoshima, Fumihiko Hamada, Tetsu AkiyamaAbstract:Abstract The APC gene is mutated in familial adenomatous polyposis and sporadic colorectal tumors. The product of this gene is a 300 kDa cytoplasmic Protein and its overexpression results in the block of cell cycle progression from the G0/G1 to the S phase. In the present study, we studied the expression and phosphorylation of the APC Protein through the cell cycle. The APC Protein was found to be constantly expressed and phosphorylated at serine and threonine residues. Moreover, the APC Protein immunoprecipitated from cells arrested in the M phase by nocodazole treatment migrated in SDS–PAGE more slowly than those from the G1 and S phases. Phosphatase treatment abolished this M phase-specific retarded migration, suggesting that APC is transiently hyperphosphorylated in the M phase.
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Upregulation of theAPCGene Product during Neuronal Differentiation of Rat Pheochromocytoma PC12 Cells
Biochemical and Biophysical Research Communications, 1996Co-Authors: Yoh Dobashi, Kumao Toyoshima, Rabindra N. Bhattacharjee, Tetsu AkiyamaAbstract:The adenomatous polyposis coli (APC) gene, the mutation of which is responsible for familial adenomatous polyposis and sporadic colorectal tumors, is highly expressed in the central nervous system. To elucidate the contribution of the APC Protein to neuronal differentiation, changes in APC expression were examined during nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma PC12 cells. The expression of APC gradually increased throughout the time course, in particular it increased markedly after 7 days of exposure to NGF. However, forced expression of APC did not induce neuronal differentiation of PC12 cells. These results suggest that the APC Protein itself does not have the potential to induce neuronal differentiation, but rather is upregulated secondary to the differentiation of PC12 cells.
Inke S. Näthke - One of the best experts on this subject based on the ideXlab platform.
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Interactions and functions of the adenomatous polyposis coli (APC) Protein at a glance.
Journal of Cell Science, 2013Co-Authors: Scott M. Nelson, Inke S. NäthkeAbstract:Since its discovery as the major tumour suppressor in colorectal cancer, the adenomatous polyposis coli (APC) Protein has emerged as a multi-functional Protein that directly or indirectly regulates the cellular processes that govern epithelial tissues ([McCartney and Nathke, 2008][1]). Today, the
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microtubule assembly by the APC Protein is regulated by importin β rangtp
Journal of Cell Science, 2010Co-Authors: Dina Dikovskaya, Ian P. Newton, Iain Davidson, James R. A. Hutchins, Petr Kalab, Paul R. Clarke, Inke S. NäthkeAbstract:Mutations in the tumour suppressor Adenomatous polyposis coli (APC) initiate most sporadic colorectal cancers. APC is implicated in regulating microtubule (MT) dynamics in interphase and mitosis. However, little is known about the underlying mechanism or regulation of this APC function. We identified importin-β as a binding partner of APC that regulates its effect on MTs. APC binds importin-β in vitro and in Xenopus egg extracts, and RanGTP inhibits this interaction. The armadillo-like repeat domain of importin-β binds to the middle of APC, where it can compete with β-catenin. In addition, two independent sites in the C terminus of APC bind the N-terminal region of importin-β. Binding to importin-β reduces the ability of APC to assemble and bundle MTs in vitro and to promote assembly of microtubule asters in Xenopus egg extracts, but does not affect the binding of APC to MTs or to EB1. Depletion of APC decreases the formation of cold-stable spindles in Xenopus egg extracts. Importantly, the ability of purified APC to rescue this phenotype was reduced when it was constitutively bound to importin-β. Thus, importin-β binds to APC and negatively regulates the MT-assembly and spindle-promoting activity of APC in a Ran-regulatable manner.
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Microtubule assembly by the APC Protein is regulated by importin-β—RanGTP
Journal of Cell Science, 2010Co-Authors: Dina Dikovskaya, Ian P. Newton, Iain Davidson, James R. A. Hutchins, Petr Kalab, Paul R. Clarke, Inke S. NäthkeAbstract:Mutations in the tumour suppressor Adenomatous polyposis coli (APC) initiate most sporadic colorectal cancers. APC is implicated in regulating microtubule (MT) dynamics in interphase and mitosis. However, little is known about the underlying mechanism or regulation of this APC function. We identified importin-β as a binding partner of APC that regulates its effect on MTs. APC binds importin-β in vitro and in Xenopus egg extracts, and RanGTP inhibits this interaction. The armadillo-like repeat domain of importin-β binds to the middle of APC, where it can compete with β-catenin. In addition, two independent sites in the C terminus of APC bind the N-terminal region of importin-β. Binding to importin-β reduces the ability of APC to assemble and bundle MTs in vitro and to promote assembly of microtubule asters in Xenopus egg extracts, but does not affect the binding of APC to MTs or to EB1. Depletion of APC decreases the formation of cold-stable spindles in Xenopus egg extracts. Importantly, the ability of purified APC to rescue this phenotype was reduced when it was constitutively bound to importin-β. Thus, importin-β binds to APC and negatively regulates the MT-assembly and spindle-promoting activity of APC in a Ran-regulatable manner.
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Microtubule assembly by the APC Protein is regulated by importin-beta--RanGTP.
Journal of cell science, 2010Co-Authors: Dina Dikovskaya, Ian P. Newton, Iain Davidson, James R. A. Hutchins, Petr Kalab, Paul R. Clarke, Inke S. NäthkeAbstract:Mutations in the tumour suppressor Adenomatous polyposis coli (APC) initiate most sporadic colorectal cancers. APC is implicated in regulating microtubule (MT) dynamics in interphase and mitosis. However, little is known about the underlying mechanism or regulation of this APC function. We identified importin-beta as a binding partner of APC that regulates its effect on MTs. APC binds importin-beta in vitro and in Xenopus egg extracts, and RanGTP inhibits this interaction. The armadillo-like repeat domain of importin-beta binds to the middle of APC, where it can compete with beta-catenin. In addition, two independent sites in the C terminus of APC bind the N-terminal region of importin-beta. Binding to importin-beta reduces the ability of APC to assemble and bundle MTs in vitro and to promote assembly of microtubule asters in Xenopus egg extracts, but does not affect the binding of APC to MTs or to EB1. Depletion of APC decreases the formation of cold-stable spindles in Xenopus egg extracts. Importantly, the ability of purified APC to rescue this phenotype was reduced when it was constitutively bound to importin-beta. Thus, importin-beta binds to APC and negatively regulates the MT-assembly and spindle-promoting activity of APC in a Ran-regulatable manner.
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Cell regulation by the APC Protein: APC as master regulator of epithelia
Current Opinion in Cell Biology, 2008Co-Authors: Brooke M. Mccartney, Inke S. NäthkeAbstract:The adenomatous polyposis coli (APC) Protein participates in many of the fundamental cellular processes that govern epithelial tissues: APC is directly involved in regulating the availability of beta-catenin for transcriptional de-repression of Tcf/LEF transcription factors, it contributes to the stability of microtubules in interphase and mitosis, and has an impact on the dynamics of F-actin. Thus APC contributes directly and/or indirectly to proliferation, differentiation, migration, and apoptosis. This particular multifunctionality can explain why disruption of APC is especially detrimental for the epithelium of the gut, where APC mutations are common in most cancers. We summarise recent data that shed light on the molecular mechanisms involved in the different functions of APC.
Alan Hall - One of the best experts on this subject based on the ideXlab platform.
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Neuronal polarity is regulated by glycogen synthase kinase-3 (GSK-3β) independently of Akt/PKB serine phosphorylation
Journal of Cell Science, 2006Co-Authors: Annette Gärtner, Xu Huang, Alan HallAbstract:An essential step during the development of hippocampal neurons is the polarised outgrowth of a single axon. Recently, it has been suggested that inhibition of glycogen synthase kinase-3β (GSK-3β) via Akt/PKB-dependent phosphorylation of Ser9, specifically at the tip of the presumptive axon, is required for selective axonal outgrowth. We now report that, by using neurons from double knock-in mice in which Ser9 and Ser21 of the two GSK-3β isoforms have been replaced by Ala, polarity develops independently of phosphorylation at these sites. Nevertheless, global inhibition of GSK-3β disturbs polarity development by leading to the formation of multiple axon-like processes in both control and knock-in neurons. This unpolarised outgrowth is accompanied by the symmetric delivery of membrane components to all neurites. Finally, the adenomatous polyposis coli (APC) Protein accumulates at the tip of one neurite before and during axon elongation, but global inhibition of GSK-3β leads to APC Protein accumulation in all neurites. We conclude that GSK-3β inhibition promotes the development of neuronal polarity, but that this is not mediated by Akt/PKB-dependent phosphorylation.
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Neuronal polarity is regulated by glycogen synthase kinase-3 (GSK-3beta) independently of Akt/PKB serine phosphorylation.
Journal of cell science, 2006Co-Authors: Annette Gärtner, Xu Huang, Alan HallAbstract:An essential step during the development of hippocampal neurons is the polarised outgrowth of a single axon. Recently, it has been suggested that inhibition of glycogen synthase kinase-3beta (GSK-3beta) via Akt/PKB-dependent phosphorylation of Ser9, specifically at the tip of the presumptive axon, is required for selective axonal outgrowth. We now report that, by using neurons from double knock-in mice in which Ser9 and Ser21 of the two GSK-3beta isoforms have been replaced by Ala, polarity develops independently of phosphorylation at these sites. Nevertheless, global inhibition of GSK-3beta disturbs polarity development by leading to the formation of multiple axon-like processes in both control and knock-in neurons. This unpolarised outgrowth is accompanied by the symmetric delivery of membrane components to all neurites. Finally, the adenomatous polyposis coli (APC) Protein accumulates at the tip of one neurite before and during axon elongation, but global inhibition of GSK-3beta leads to APC Protein accumulation in all neurites. We conclude that GSK-3beta inhibition promotes the development of neuronal polarity, but that this is not mediated by Akt/PKB-dependent phosphorylation.
Joanna Groden - One of the best experts on this subject based on the ideXlab platform.
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Chain-terminating mutations in the APC gene lead to alterations in APC RNA and Protein concentration.
Genes Chromosomes and Cancer, 1998Co-Authors: Viviana Gismondi, Paola Stagnaro, Simona Pedemonte, Roberta Biticchi, Silvano Presciuttini, Paola Grammatico, Paola Sala, Lucio Bertario, Joanna Groden, Liliana VarescoAbstract:APC mutations introduce premature stop codons into the open reading frame of the gene, leading to the formation of truncated tumor suppressor Proteins. Both RNA and Protein levels are likely to be profoundly altered by such nonsense mutations. To test this hypothesis, Western blotting and RT-PCR strategies were used to characterize mutant and normal APC Protein and APC RNA concentrations in lymphoblastoid cell lines from 22 unrelated polyposis patients carrying different APC mutations. Variable levels of truncated APC peptides were observed in 14 of 14 cell lines with APC mutations within exon 15. No truncated APC Protein was detected in six of eight cell lines with APC mutations located 5′ of exon 15. Mutations located in exon 15 showed mutant RNA underrepresentation in four of eight cell lines, whereas mutations located 5′ of exon 15 showed RNA reduction in five of six cell lines. These findings indicate that a two- to threefold decrease in RNA concentration is common when APC alleles carry chain-terminating mutations. They also suggest that the severe decrease of truncated APC Protein observed in some cell lines is due to mechanisms acting at the Protein level. Genes Chromosomes Cancer 22:278–286, 1998. © 1998 Wiley-Liss, Inc.
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Phosphorylation of the tumor suppressor adenomatous polyposis coli (APC) by the cyclin-dependent kinase p34.
Journal of Biological Chemistry, 1997Co-Authors: Chris Trzepacz, Andrew M. Lowy, Jennifer J. Kordich, Joanna GrodenAbstract:Mutations in the tumor suppressor gene APC invariably lead to the development of colorectal cancer. The vast majority of these mutations are nonsense or frameshifts resulting in nonfunctional, truncated APC Protein products. Eleven cyclin-dependent kinase (CDK) consensus phosphorylation sites have been identified in the frequently deleted carboxyl-terminal region of APC; loss of these phosphorylation sites by mutation could therefore compromise the ability of APC to inhibit cell growth. This report demonstrates that immunoprecipitates of full-length, but not truncated, APC Protein include a mitosis-specific kinase activity in vivo. Biochemical and Western analysis of these immunoprecipitates confirms the presence of the CDK p34(cdc2). We also show that APC is a substrate for recombinant human p34(cdc2)-cyclin B1. Modification of APC by p34(cdc2) implicates phosphorylation as a mechanism for regulating APC function via a link to the cell cycle.
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Microsatellite Instability in Colorectal Adenocarcinoma Cell Lines That Have Full-Length Adenomatous Polyposis Coli Protein
Cancer research, 1995Co-Authors: Christopher D. Heinen, Diane Richardson, Joanna GrodenAbstract:Almost 20% of colon cancers are characterized by genomic instability at simple repeated sequences. This instability is the result of a deficient DNA mismatch repair system. Sporadic, as well as hereditary carcinomas of the proximal colon display this effect. In this study, we examined colorectal adenocarcinoma cell lines, with or without wild-type adenomatous polyposis coli (APC) Protein, for the presence of microsatellite instability. The three cell lines that maintained full-length APC Protein also displayed the highest level of instability, suggesting a negative correlation between APC mutations and microsatellite instability. This data, in addition to other studies that show a negative correlation between microsatellite instability and mutations in p53 and K-ras, support the idea of a second pathway for colorectal cancer development.