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Sample records for identified apc germline

  1. APC germline mutations in families with familial adenomatous polyposis.

    PubMed

    De Queiroz Rossanese, Lillian Barbosa; De Lima Marson, Fernando Augusto; Ribeiro, José Dirceu; Coy, Claudio Saddy Rodrigues; Bertuzzo, Carmen Silvia

    2013-11-01

    Adenomatous polyposis coli (APC) germline mutations are responsible for the occurrence of familial adenomatous polyposis (FAP). Somatic mutations lead to malignant transformation of adenomas. In this context, considering the significance of APC germline mutations in FAP, we aimed to identify APC germline mutations. In the present study, 20 FAP patients were enrolled. The determination of APC germline mutations was performed using sequencing, and the mutations were compared with clinical markers (gender, age at diagnosis, smoking habits, TNM stage, Astler‑Coller stage, degree of differentiation of adenocarcinoma). The data were compared using the SPSS program, with the Fisher's exact test and χ2 test, considering α=0.05. According to the main results in our sample, 16 alleles with deleterious mutations (80% of the patients) were identified while 7 (35%) patients had no deleterious mutations. There was a predominance of nonsense (45% of the patients) and frameshift (20% of the patients) mutations. There was no statistical significance between the APC germline mutations identified and the clinical variables considered in our study. Only TNM stage was associated with the presence of deleterious mutations. Patients with deleterious mutations had an OR, 0.086 (IC=0.001-0.984); TNM stage I+II in comparison with III+IV, when compared with the patients with no deleterious mutations identified. In this context, as a conclusion, we demonstrated the molecular heterogeneity of APC germline mutations in FAP and the difficulty to perform molecular diagnostics in a Brazilian population, considering the admixed population analyzed. PMID:23970361

  2. Germ-line mutations of the APC gene in 53 familial adenomatous polyposis patients.

    PubMed Central

    Miyoshi, Y; Ando, H; Nagase, H; Nishisho, I; Horii, A; Miki, Y; Mori, T; Utsunomiya, J; Baba, S; Petersen, G

    1992-01-01

    We searched for germ-line mutations of the APC gene in 79 unrelated patients with familial adenomatous polyposis using a ribonuclease protection analysis coupled with polymerase chain reaction amplifications of genomic DNA. Mutations were found in 53 patients (67%); 28 of the mutations were small deletions and 2 were 1- to 2-base-pair insertions; 19 were point mutations resulting in stop codons and only 4 were missense point mutations. Thus, 92% of the mutations were predicted to result in truncations of the APC protein. More than two-thirds (68%) of the mutations were clustered in the 5' half of the last exon, and nearly two-fifths of the total mutations occurred at one of five positions. This information has significant implications for understanding the role of APC mutation in inherited forms of colorectal neoplasia and for designing effective methods for genetic counseling and presymptomatic diagnosis. Images PMID:1316610

  3. Exome Sequencing Identifies Biallelic MSH3 Germline Mutations as a Recessive Subtype of Colorectal Adenomatous Polyposis.

    PubMed

    Adam, Ronja; Spier, Isabel; Zhao, Bixiao; Kloth, Michael; Marquez, Jonathan; Hinrichsen, Inga; Kirfel, Jutta; Tafazzoli, Aylar; Horpaopan, Sukanya; Uhlhaas, Siegfried; Stienen, Dietlinde; Friedrichs, Nicolaus; Altmüller, Janine; Laner, Andreas; Holzapfel, Stefanie; Peters, Sophia; Kayser, Katrin; Thiele, Holger; Holinski-Feder, Elke; Marra, Giancarlo; Kristiansen, Glen; Nöthen, Markus M; Büttner, Reinhard; Möslein, Gabriela; Betz, Regina C; Brieger, Angela; Lifton, Richard P; Aretz, Stefan

    2016-08-01

    In ∼30% of families affected by colorectal adenomatous polyposis, no germline mutations have been identified in the previously implicated genes APC, MUTYH, POLE, POLD1, and NTHL1, although a hereditary etiology is likely. To uncover further genes with high-penetrance causative mutations, we performed exome sequencing of leukocyte DNA from 102 unrelated individuals with unexplained adenomatous polyposis. We identified two unrelated individuals with differing compound-heterozygous loss-of-function (LoF) germline mutations in the mismatch-repair gene MSH3. The impact of the MSH3 mutations (c.1148delA, c.2319-1G>A, c.2760delC, and c.3001-2A>C) was indicated at the RNA and protein levels. Analysis of the diseased individuals' tumor tissue demonstrated high microsatellite instability of di- and tetranucleotides (EMAST), and immunohistochemical staining illustrated a complete loss of nuclear MSH3 in normal and tumor tissue, confirming the LoF effect and causal relevance of the mutations. The pedigrees, genotypes, and frequency of MSH3 mutations in the general population are consistent with an autosomal-recessive mode of inheritance. Both index persons have an affected sibling carrying the same mutations. The tumor spectrum in these four persons comprised colorectal and duodenal adenomas, colorectal cancer, gastric cancer, and an early-onset astrocytoma. Additionally, we detected one unrelated individual with biallelic PMS2 germline mutations, representing constitutional mismatch-repair deficiency. Potentially causative variants in 14 more candidate genes identified in 26 other individuals require further workup. In the present study, we identified biallelic germline MSH3 mutations in individuals with a suspected hereditary tumor syndrome. Our data suggest that MSH3 mutations represent an additional recessive subtype of colorectal adenomatous polyposis. PMID:27476653

  4. Putative direct and indirect Wnt targets identified through consistent gene expression changes in APC-mutant intestinal adenomas from humans and mice

    PubMed Central

    Segditsas, Stefania; Sieber, Oliver; Deheragoda, Maesha; East, Phil; Rowan, Andrew; Jeffery, Rosemary; Nye, Emma; Clark, Susan; Spencer-Dene, Bradley; Stamp, Gordon; Poulsom, Richard; Suraweera, Nirosha; Silver, Andrew; Ilyas, Mohammad; Tomlinson, Ian

    2008-01-01

    In order to identify new genes with differential expression in early intestinal tumours, we performed mRNA (messenger ribonucleic acid) expression profiling of 16 human and 63 mouse adenomas. All individuals had germline APC mutations to ensure that tumorigenesis was driven by ‘second hits’ at APC. Using stringent filtering to identify changes consistent between humans and mice, we identified 60 genes up-regulated and 151 down-regulated in tumours. For 22 selected genes—including known Wnt targets—expression differences were confirmed by qRT–PCR (quantitative reverse transcription polymerase chain reaction). Most, but not all, differences were also present in colorectal carcinomas. In situ analysis showed a complex picture. Expression of up-regulated genes in adenomas was usually uniform/diffuse (e.g. ITGA6) or prominent in the tumour core (e.g. LGR5); in normal tissue, these genes were expressed at crypt bases or the transit amplifying zone. Down-regulated genes were often undetectable in adenomas, but in normal tissue were expressed in mesenchyme (e.g. GREM1/2) or differentiated cells towards crypt tops (e.g. SGK1). In silico analysis of TCF4-binding motifs showed that some of our genes were probably direct Wnt targets. Previous studies, mostly focused on human tumours, showed partial overlap with our ‘expression signature’, but 37 genes were unique to our study, including TACSTD2, SEMA3F, HOXA9 and IER3 (up-regulated), and TAGLN, GREM1, GREM2, MAB21L2 and RARRES2 (down-regulated). Combined analysis of our and published human data identified additional genes differentially expressed in adenomas, including decreased BMPs (bone morphogenetic proteins) and increased BUB1/BUB1B. Several of the newly identified, differentially expressed genes represent potential diagnostic or therapeutic targets for intestinal tumours. PMID:18782851

  5. Screens for piwi suppressors in Drosophila identify dosage-dependent regulators of germline stem cell division.

    PubMed Central

    Smulders-Srinivasan, Tora K; Lin, Haifan

    2003-01-01

    The Drosophila piwi gene is the founding member of the only known family of genes whose function in stem cell maintenance is highly conserved in both animal and plant kingdoms. piwi mutants fail to maintain germline stem cells in both male and female gonads. The identification of piwi-interacting genes is essential for understanding how stem cell divisions are regulated by piwi-mediated mechanisms. To search for such genes, we screened the Drosophila third chromosome ( approximately 36% of the euchromatic genome) for suppressor mutations of piwi2 and identified six strong and three weak piwi suppressor genes/sequences. These genes/sequences interact negatively with piwi in a dosage-sensitive manner. Two of the strong suppressors represent known genes--serendipity-delta and similar, both encoding transcription factors. These findings reveal that the genetic regulation of germline stem cell division involves dosage-sensitive mechanisms and that such mechanisms exist at the transcriptional level. In addition, we identified three other types of piwi interactors. The first type consists of deficiencies that dominantly interact with piwi2 to cause male sterility, implying that dosage-sensitive regulation also exists in the male germline. The other two types are deficiencies that cause lethality and female-specific lethality in a piwi2 mutant background, revealing the zygotic function of piwi in somatic development. PMID:14704180

  6. Urinary Bladder Paragangliomas: How Immunohistochemistry Can Assist to Identify Patients With SDHB Germline and Somatic Mutations.

    PubMed

    Giubellino, Alessio; Lara, Karlena; Martucci, Victoria; Huynh, Than; Agarwal, Piyush; Pacak, Karel; Merino, Maria J

    2015-11-01

    Urinary bladder paraganglioma (paraganglioma) is a rare tumor of chromaffin cells of the sympathetic system of the urinary bladder wall. We studied 14 cases of this entity and investigated the usefulness of SDHB protein staining by immunohistochemistry (IHC) as a diagnostic tool to identify patients with bladder paragangliomas that could be associated with SDHB gene mutations, as these patients have a more aggressive disease. Eleven tumors from these patients were stained by IHC. Six of 11 tumors were negative for SDHB staining by IHC with no cytoplasmic staining in tumor cells when compared with normal tissues. Five of these 6 negative cases were confirmed to be positive for germline SDHB mutations. One case showed negative staining and no germline SDHB mutation; however, further investigation of the tumor revealed a somatic SDHB gene deletion. The remaining 5 cases showed strong cytoplasmic staining, but they were negative for the presence of SDHB mutation. They were found to be either sporadic tumors or part of von Hippel-Lindau syndrome. Staining for SDHA was positive in all cases. Our study confirms that there is very good correlation between the presence of an SDHB mutation, whether germline or sporadic, and negative SDHB IHC staining in urinary bladder paragangliomas, and this is the first study to demonstrate that somatic mutations can be recognized by IHC staining. PMID:26457353

  7. Nuclear APC.

    PubMed

    Neufeld, Kristi L

    2009-01-01

    Mutational inactivation of the tumor suppressor gene APC (Adenomatous polyposis coli) is thought to be an initiating step in the progression of the vast majority ofcolorectal cancers. Attempts to understand APC function have revealed more than a dozen binding partners as well as several subcellular localizations including at cell-cell junctions, associated with microtubules at the leading edge of migrating cells, at the apical membrane, in the cytoplasm and in the nucleus. The present chapter focuses on APC localization and functions in the nucleus. APC contains two classical nuclear localization signals, with a third domain that can enhance nuclear import. Along with two sets of nuclear export signals, the nuclear localization signals enable the large APC protein to shuttle between the nucleus and cytoplasm. Nuclear APC can oppose beta-catenin-mediated transcription. This down-regulation of nuclear beta-catenin activity by APC most likely involves nuclear sequestration of beta-catenin from the transcription complex as well as interaction of APC with transcription corepressor CtBP. Additional nuclear binding partners for APC include transcription factor activator protein AP-2alpha, nuclear export factor Crm1, protein tyrosine phosphatase PTP-BL and perhaps DNA itself. Interaction of APC with polymerase beta and PCNA, suggests a role for APC in DNA repair. The observation that increases in the cytoplasmic distribution of APC correlate with colon cancer progression suggests that disruption of these nuclear functions of APC plays an important role in cancer progression. APC prevalence in the cytoplasm of quiescent cells points to a potential function for nuclear APC in control of cell proliferation. Clear definition of APC's nuclear function(s) will expand the possibilities for early colorectal cancer diagnostics and therapeutics targeted to APC. PMID:19928349

  8. Reduced expression of APC-1B but not APC-1A by the deletion of promoter 1B is responsible for familial adenomatous polyposis.

    PubMed

    Yamaguchi, Kiyoshi; Nagayama, Satoshi; Shimizu, Eigo; Komura, Mitsuhiro; Yamaguchi, Rui; Shibuya, Tetsuo; Arai, Masami; Hatakeyama, Seira; Ikenoue, Tsuneo; Ueno, Masashi; Miyano, Satoru; Imoto, Seiya; Furukawa, Yoichi

    2016-01-01

    Germline mutations in the tumor suppressor gene APC are associated with familial adenomatous polyposis (FAP). Here we applied whole-genome sequencing (WGS) to the DNA of a sporadic FAP patient in which we did not find any pathological APC mutations by direct sequencing. WGS identified a promoter deletion of approximately 10 kb encompassing promoter 1B and exon1B of APC. Additional allele-specific expression analysis by deep cDNA sequencing revealed that the deletion reduced the expression of the mutated APC allele to as low as 11.2% in the total APC transcripts, suggesting that the residual mutant transcripts were driven by other promoter(s). Furthermore, cap analysis of gene expression (CAGE) demonstrated that the deleted promoter 1B region is responsible for the great majority of APC transcription in many tissues except the brain. The deletion decreased the transcripts of APC-1B to 39-45% in the patient compared to the healthy controls, but it did not decrease those of APC-1A. Different deletions including promoter 1B have been reported in FAP patients. Taken together, our results strengthen the evidence that analysis of structural variations in promoter 1B should be considered for the FAP patients whose pathological mutations are not identified by conventional direct sequencing. PMID:27217144

  9. Reduced expression of APC-1B but not APC-1A by the deletion of promoter 1B is responsible for familial adenomatous polyposis

    PubMed Central

    Yamaguchi, Kiyoshi; Nagayama, Satoshi; Shimizu, Eigo; Komura, Mitsuhiro; Yamaguchi, Rui; Shibuya, Tetsuo; Arai, Masami; Hatakeyama, Seira; Ikenoue, Tsuneo; Ueno, Masashi; Miyano, Satoru; Imoto, Seiya; Furukawa, Yoichi

    2016-01-01

    Germline mutations in the tumor suppressor gene APC are associated with familial adenomatous polyposis (FAP). Here we applied whole-genome sequencing (WGS) to the DNA of a sporadic FAP patient in which we did not find any pathological APC mutations by direct sequencing. WGS identified a promoter deletion of approximately 10 kb encompassing promoter 1B and exon1B of APC. Additional allele-specific expression analysis by deep cDNA sequencing revealed that the deletion reduced the expression of the mutated APC allele to as low as 11.2% in the total APC transcripts, suggesting that the residual mutant transcripts were driven by other promoter(s). Furthermore, cap analysis of gene expression (CAGE) demonstrated that the deleted promoter 1B region is responsible for the great majority of APC transcription in many tissues except the brain. The deletion decreased the transcripts of APC-1B to 39–45% in the patient compared to the healthy controls, but it did not decrease those of APC-1A. Different deletions including promoter 1B have been reported in FAP patients. Taken together, our results strengthen the evidence that analysis of structural variations in promoter 1B should be considered for the FAP patients whose pathological mutations are not identified by conventional direct sequencing. PMID:27217144

  10. Preferential Allele Expression Analysis Identifies Shared Germline and Somatic Driver Genes in Advanced Ovarian Cancer

    PubMed Central

    Halabi, Najeeb M.; Martinez, Alejandra; Al-Farsi, Halema; Mery, Eliane; Puydenus, Laurence; Pujol, Pascal; Khalak, Hanif G.; McLurcan, Cameron; Ferron, Gwenael; Querleu, Denis; Al-Azwani, Iman; Al-Dous, Eman; Mohamoud, Yasmin A.; Malek, Joel A.; Rafii, Arash

    2016-01-01

    Identifying genes where a variant allele is preferentially expressed in tumors could lead to a better understanding of cancer biology and optimization of targeted therapy. However, tumor sample heterogeneity complicates standard approaches for detecting preferential allele expression. We therefore developed a novel approach combining genome and transcriptome sequencing data from the same sample that corrects for sample heterogeneity and identifies significant preferentially expressed alleles. We applied this analysis to epithelial ovarian cancer samples consisting of matched primary ovary and peritoneum and lymph node metastasis. We find that preferentially expressed variant alleles include germline and somatic variants, are shared at a relatively high frequency between patients, and are in gene networks known to be involved in cancer processes. Analysis at a patient level identifies patient-specific preferentially expressed alleles in genes that are targets for known drugs. Analysis at a site level identifies patterns of site specific preferential allele expression with similar pathways being impacted in the primary and metastasis sites. We conclude that genes with preferentially expressed variant alleles can act as cancer drivers and that targeting those genes could lead to new therapeutic strategies. PMID:26735499

  11. Preferential Allele Expression Analysis Identifies Shared Germline and Somatic Driver Genes in Advanced Ovarian Cancer.

    PubMed

    Halabi, Najeeb M; Martinez, Alejandra; Al-Farsi, Halema; Mery, Eliane; Puydenus, Laurence; Pujol, Pascal; Khalak, Hanif G; McLurcan, Cameron; Ferron, Gwenael; Querleu, Denis; Al-Azwani, Iman; Al-Dous, Eman; Mohamoud, Yasmin A; Malek, Joel A; Rafii, Arash

    2016-01-01

    Identifying genes where a variant allele is preferentially expressed in tumors could lead to a better understanding of cancer biology and optimization of targeted therapy. However, tumor sample heterogeneity complicates standard approaches for detecting preferential allele expression. We therefore developed a novel approach combining genome and transcriptome sequencing data from the same sample that corrects for sample heterogeneity and identifies significant preferentially expressed alleles. We applied this analysis to epithelial ovarian cancer samples consisting of matched primary ovary and peritoneum and lymph node metastasis. We find that preferentially expressed variant alleles include germline and somatic variants, are shared at a relatively high frequency between patients, and are in gene networks known to be involved in cancer processes. Analysis at a patient level identifies patient-specific preferentially expressed alleles in genes that are targets for known drugs. Analysis at a site level identifies patterns of site specific preferential allele expression with similar pathways being impacted in the primary and metastasis sites. We conclude that genes with preferentially expressed variant alleles can act as cancer drivers and that targeting those genes could lead to new therapeutic strategies. PMID:26735499

  12. Bacterial cytoplasmic display platform Retained Display (ReD) identifies stable human germline antibody frameworks.

    PubMed

    Beasley, Matthew D; Niven, Keith P; Winnall, Wendy R; Kiefel, Ben R

    2015-05-01

    Conventional antibody surface display requires fusion protein export through at least one cellular membrane, constraining the yield and occasioning difficulties in achieving scaled production. To circumvent this limitation, we developed a novel cytoplasmic display platform, Retained Display (ReD), and used it to screen for human scFv frameworks that are highly soluble and stable in the bacterial cytoplasm. ReD, based on the retention of high-molecular weight complexes within detergent-permeabilized Escherichia coli, enabled presentation of exogenous targets to antibodies that were expressed and folded in the cytoplasm. All human λ and κ light chain family genes were expressed as IGHV3-23 fusions. Members of the λ subfamilies 1, 3 and 6 were soluble cytoplasmic partners of IGHV3-23. Contrary to previous in vivo screens for soluble reduced scFvs, the pairings identified by ReD were identical to the human germline sequences for the framework, CDR1 and CDR2 regions. Using the most soluble scFv scaffold identified, we demonstrated tolerance to CDR3 diversification and isolated a binding scFv to an exogenous protein target. This screening system has the potential to rapidly produce antibodies to target threats such as emerging infectious diseases and bioterror agents. PMID:25712138

  13. Detection of APC mosaicism by next-generation sequencing in an FAP patient.

    PubMed

    Yamaguchi, Kiyoshi; Komura, Mitsuhiro; Yamaguchi, Rui; Imoto, Seiya; Shimizu, Eigo; Kasuya, Shinichi; Shibuya, Tetsuo; Hatakeyama, Seira; Takahashi, Norihiko; Ikenoue, Tsuneo; Hata, Keisuke; Tsurita, Giichiro; Shinozaki, Masaru; Suzuki, Yutaka; Sugano, Sumio; Miyano, Satoru; Furukawa, Yoichi

    2015-05-01

    Familial adenomatous polyposis (FAP) of the colon is characterized by multiple polyps in the intestine and extra-colonic manifestations. Most FAP cases are caused by a germline mutation in the tumor-suppressor gene APC, but some cases of adenomatous polyposis result from germline mutations in MUTYH, POLD1 or POLE. Although sequence analysis of APC by the Sanger method is routinely performed for genetic testing, there remain cases whose mutations are not detected by the analysis. Next-generation sequencing has enabled us to analyze the comprehensive human genome, improving the chance of identifying disease causative variants. In this study, we conducted whole-genome sequencing of a sporadic FAP patient in which we did not find any pathogenic APC mutations by the conventional Sanger sequencing. Whole-genome sequencing and subsequent deep sequencing identified a mosaic mutation of c.3175G>T, p.E1059X in ~12% of his peripheral leukocytes. Additional deep sequencing of his buccal mucosa, hair follicles, non-cancerous mucosa of the stomach and colon disclosed that these tissues harbored the APC mutation at different frequencies. Our data implied that genetic analysis by next-generation sequencing is an effective strategy to identify genetic mosaicism in hereditary diseases. PMID:25716913

  14. Whole-genome sequencing in autism identifies hot spots for de novo germline mutation.

    PubMed

    Michaelson, Jacob J; Shi, Yujian; Gujral, Madhusudan; Zheng, Hancheng; Malhotra, Dheeraj; Jin, Xin; Jian, Minghan; Liu, Guangming; Greer, Douglas; Bhandari, Abhishek; Wu, Wenting; Corominas, Roser; Peoples, Aine; Koren, Amnon; Gore, Athurva; Kang, Shuli; Lin, Guan Ning; Estabillo, Jasper; Gadomski, Therese; Singh, Balvindar; Zhang, Kun; Akshoomoff, Natacha; Corsello, Christina; McCarroll, Steven; Iakoucheva, Lilia M; Li, Yingrui; Wang, Jun; Sebat, Jonathan

    2012-12-21

    De novo mutation plays an important role in autism spectrum disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes and may also include nucleotide-substitution hot spots. We investigated global patterns of germline mutation by whole-genome sequencing of monozygotic twins concordant for ASD and their parents. Mutation rates varied widely throughout the genome (by 100-fold) and could be explained by intrinsic characteristics of DNA sequence and chromatin structure. Dense clusters of mutations within individual genomes were attributable to compound mutation or gene conversion. Hypermutability was a characteristic of genes involved in ASD and other diseases. In addition, genes impacted by mutations in this study were associated with ASD in independent exome-sequencing data sets. Our findings suggest that regional hypermutation is a significant factor shaping patterns of genetic variation and disease risk in humans. PMID:23260136

  15. A novel germline mutation in SDHA identified in a rare case of gastrointestinal stromal tumor complicated with renal cell carcinoma.

    PubMed

    Jiang, Quan; Zhang, Yong; Zhou, Yu-Hong; Hou, Ying-Yong; Wang, Jiong-Yuan; Li, Jing-Lei; Li, Ming; Tong, Han-Xing; Lu, Wei-Qi

    2015-01-01

    Succinate dehydrogenase (SDH), which is located on the mitochondrial inner membrane, is essential to the Krebs cycle. Mutations of the SDH gene are associated with many tumors, such as renal cell carcinoma, wild type gastrointestinal stromal tumors (WT GISTs) and hereditary paragangliomas/pheochromocytomas. Herein we present a rare case diagnosed as a WT GIST complicated with a renal chromophobe cell tumor and detected a novel germline heterozygous mutation (c.2T>C: p.M1T) in the initiation codon of the SDHA gene. We also conduct a preliminary exploration for the mechanism of reduced expression of SDHB without mutation of SDHB gene. Our case enriches the mutation spectrum of the SDH gene. After reviewing previous studies, we found it to be the first case diagnosed as a WT GIST complicated with a synchronous renal chromophobe cell tumor and identified a novel germline heterozygous mutation. It was also the second reported case of a renal cell carcinoma associated with an SDHA mutation. PMID:26722403

  16. Whole-animal genome-wide RNAi screen identifies networks regulating male germline stem cells in Drosophila

    PubMed Central

    Liu, Ying; Ge, Qinglan; Chan, Brian; Liu, Hanhan; Singh, Shree Ram; Manley, Jacob; Lee, Jae; Weideman, Ann Marie; Hou, Gerald; Hou, Steven X.

    2016-01-01

    Stem cells are regulated both intrinsically and externally, including by signals from the local environment and distant organs. To identify genes and pathways that regulate stem-cell fates in the whole organism, we perform a genome-wide transgenic RNAi screen through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). Here we identify 530 genes that regulate GSC maintenance and differentiation. Of these, we further knock down 113 selected genes using cell-type-specific Gal4s and find that more than half were external regulators, that is, from the local microenvironment or more distal sources. Some genes, for example, versatile (vers), encoding a heterochromatin protein, regulates GSC fates differentially in different cell types and through multiple pathways. We also find that mitosis/cytokinesis proteins are especially important for male GSC maintenance. Our findings provide valuable insights and resources for studying stem cell regulation at the organismal level. PMID:27484291

  17. Whole-animal genome-wide RNAi screen identifies networks regulating male germline stem cells in Drosophila.

    PubMed

    Liu, Ying; Ge, Qinglan; Chan, Brian; Liu, Hanhan; Singh, Shree Ram; Manley, Jacob; Lee, Jae; Weideman, Ann Marie; Hou, Gerald; Hou, Steven X

    2016-01-01

    Stem cells are regulated both intrinsically and externally, including by signals from the local environment and distant organs. To identify genes and pathways that regulate stem-cell fates in the whole organism, we perform a genome-wide transgenic RNAi screen through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). Here we identify 530 genes that regulate GSC maintenance and differentiation. Of these, we further knock down 113 selected genes using cell-type-specific Gal4s and find that more than half were external regulators, that is, from the local microenvironment or more distal sources. Some genes, for example, versatile (vers), encoding a heterochromatin protein, regulates GSC fates differentially in different cell types and through multiple pathways. We also find that mitosis/cytokinesis proteins are especially important for male GSC maintenance. Our findings provide valuable insights and resources for studying stem cell regulation at the organismal level. PMID:27484291

  18. Whole-exome identifies RXRG and TH germline variants in familial isolated prolactinoma.

    PubMed

    Melo, Flavia M; Couto, Patrícia P; Bale, Allen E; Bastos-Rodrigues, Luciana; Passos, Flavia M; Lisboa, Raony G C; Ng, Jessica M Y; Curran, Tom; Dias, Eduardo P; Friedman, Eitan; De Marco, Luiz

    2016-06-01

    Familial isolated pituitary adenoma (FIPA) is a rare genetic disorder. In a subset of FIPA families AIP germline mutations have been reported, but in most FIPA cases the exact genetic defect remains unknown. The present study aimed to determine the genetic basis of FIPA in a Brazilian family. Three siblings presented with isolated prolactin genes. Further mutation screening was performed using whole-exome sequencing and all likely causative mutations were validated by Sanger sequencing. In silico analysis and secreting pituitary adenoma diagnosed through clinical, biochemical and imaging testing. Sanger sequencing was used to genotype candidate prolactinoma-mutated additional predictive algorithms were applied to prioritize likely pathogenic variants. No mutations in the coding and flanking intronic regions in the MEN1, AIP and PRLR genes were detected. Whole-exome sequencing of three affected siblings revealed novel, predicted damaging, heterozygous variants in three different genes: RXRG, REXO4 and TH. In conclusion, the RXRG and TH possibly pathogenic variants may be associated with isolated prolactinoma in the studied family. The possible contribution of these genes to additional FIPA families should be explored. PMID:27245436

  19. Genome-Wide Association Study of Golden Retrievers Identifies Germ-Line Risk Factors Predisposing to Mast Cell Tumours

    PubMed Central

    Arendt, Maja L.; Melin, Malin; Tonomura, Noriko; Koltookian, Michele; Courtay-Cahen, Celine; Flindall, Netty; Bass, Joyce; Boerkamp, Kim; Megquir, Katherine; Youell, Lisa; Murphy, Sue; McCarthy, Colleen; London, Cheryl; Rutteman, Gerard R.; Starkey, Mike; Lindblad-Toh, Kerstin

    2015-01-01

    Canine mast cell tumours (CMCT) are one of the most common skin tumours in dogs with a major impact on canine health. Certain breeds have a higher risk of developing mast cell tumours, suggesting that underlying predisposing germ-line genetic factors play a role in the development of this disease. The genetic risk factors are largely unknown, although somatic mutations in the oncogene C-KIT have been detected in a proportion of CMCT, making CMCT a comparative model for mastocytosis in humans where C-KIT mutations are frequent. We have performed a genome wide association study in golden retrievers from two continents and identified separate regions in the genome associated with risk of CMCT in the two populations. Sequence capture of associated regions and subsequent fine mapping in a larger cohort of dogs identified a SNP associated with development of CMCT in the GNAI2 gene (p = 2.2x10-16), introducing an alternative splice form of this gene resulting in a truncated protein. In addition, disease associated haplotypes harbouring the hyaluronidase genes HYAL1, HYAL2 and HYAL3 on cfa20 and HYAL4, SPAM1 and HYALP1 on cfa14 were identified as separate risk factors in European and US golden retrievers, respectively, suggesting that turnover of hyaluronan plays an important role in the development of CMCT. PMID:26588071

  20. Allele-Specific Deletions in Mouse Tumors Identify Fbxw7 as Germline Modifier of Tumor Susceptibility

    PubMed Central

    Perez-Losada, Jesus; Wu, Di; DelRosario, Reyno; Balmain, Allan; Mao, Jian-Hua

    2012-01-01

    Genome-wide association studies (GWAS) have been successful in finding associations between specific genetic variants and cancer susceptibility in human populations. These studies have identified a range of highly statistically significant associations between single nucleotide polymorphisms (SNPs) and susceptibility to development of a range of human tumors. However, the effect of each SNP in isolation is very small, and all of the SNPs combined only account for a relatively minor proportion of the total genetic risk (5–10%). There is therefore a major requirement for alternative routes to the discovery of genetic risk factors for cancer. We have previously shown using mouse models that chromosomal regions harboring susceptibility genes identified by linkage analysis frequently exhibit allele-specific genetic alterations in tumors. We demonstrate here that the Fbxw7 gene, a commonly mutated gene in a wide range of mouse and human cancers, shows allele-specific deletions in mouse lymphomas and skin tumors. Lymphomas from three different F1 hybrids show 100% allele-specificity in the patterns of allelic loss. Parental alleles from 129/Sv or Spretus/Gla mice are lost in tumors from F1 hybrids with C57BL/6 animals, due to the presence of a specific non-synonymous coding sequence polymorphism at the N-terminal portion of the gene. A specific genetic test of association between this SNP and lymphoma susceptibility in interspecific backcross mice showed a significant linkage (p = 0.001), but only in animals with a functional p53 gene. These data therefore identify Fbxw7 as a p53-dependent tumor susceptibility gene. Increased p53-dependent tumor susceptibility and allele-specific losses were also seen in a mouse skin model of skin tumor development. We propose that analysis of preferential allelic imbalances in tumors may provide an efficient means of uncovering genetic variants that affect mouse and human tumor susceptibility. PMID:22348067

  1. Ectopic Activation of Germline and Placental Genes Identifies Aggressive Metastasis-Prone Lung Cancers

    PubMed Central

    Rousseaux, Sophie; Debernardi, Alexandra; Jacquiau, Baptiste; Vitte, Anne-Laure; Vesin, Aurélien; Nagy-Mignotte, Hélène; Moro-Sibilot, Denis; Brichon, Pierre-Yves; Lantuejoul, Sylvie; Hainaut, Pierre; Laffaire, Julien; de Reyniès, Aurélien; Beer, David G.; Timsit, Jean-François; Brambilla, Christian; Brambilla, Elisabeth; Khochbin, Saadi

    2016-01-01

    Activation of normally silent tissue-specific genes and the resulting cell “identity crisis” are the unexplored consequences of malignant epigenetic reprogramming. We designed a strategy for investigating this reprogramming, which consisted of identifying a large number of tissue-restricted genes that are epigenetically silenced in normal somatic cells and then detecting their expression in cancer. This approach led to the demonstration that large-scale “off-context” gene activations systematically occur in a variety of cancer types. In our series of 293 lung tumors, we identified an ectopic gene expression signature associated with a subset of highly aggressive tumors, which predicted poor prognosis independently of the TNM (tumor size, node positivity, and metastasis) stage or histological subtype. The ability to isolate these tumors allowed us to reveal their common molecular features characterized by the acquisition of embryonic stem cell/germ cell gene expression profiles and the down-regulation of immune response genes. The methodical recognition of ectopic gene activations in cancer cells could serve as a basis for gene signature–guided tumor stratification, as well as for the discovery of oncogenic mechanisms, and expand the understanding of the biology of very aggressive tumors. PMID:23698379

  2. Genome-Wide Analysis Identifies Germ-Line Risk Factors Associated with Canine Mammary Tumours

    PubMed Central

    Melin, Malin; Murén, Eva; Gustafson, Ulla; Starkey, Mike; Borge, Kaja Sverdrup; Lingaas, Frode; Saellström, Sara; Rönnberg, Henrik; Lindblad-Toh, Kerstin

    2016-01-01

    Canine mammary tumours (CMT) are the most common neoplasia in unspayed female dogs. CMTs are suitable naturally occurring models for human breast cancer and share many characteristics, indicating that the genetic causes could also be shared. We have performed a genome-wide association study (GWAS) in English Springer Spaniel dogs and identified a genome-wide significant locus on chromosome 11 (praw = 5.6x10-7, pperm = 0.019). The most associated haplotype spans a 446 kb region overlapping the CDK5RAP2 gene. The CDK5RAP2 protein has a function in cell cycle regulation and could potentially have an impact on response to chemotherapy treatment. Two additional loci, both on chromosome 27, were nominally associated (praw = 1.97x10-5 and praw = 8.30x10-6). The three loci explain 28.1±10.0% of the phenotypic variation seen in the cohort, whereas the top ten associated regions account for 38.2±10.8% of the risk. Furthermore, the ten GWAS loci and regions with reduced genetic variability are significantly enriched for snoRNAs and tumour-associated antigen genes, suggesting a role for these genes in CMT development. We have identified several candidate genes associated with canine mammary tumours, including CDK5RAP2. Our findings enable further comparative studies to investigate the genes and pathways in human breast cancer patients. PMID:27158822

  3. Genome-Wide Analysis Identifies Germ-Line Risk Factors Associated with Canine Mammary Tumours.

    PubMed

    Melin, Malin; Rivera, Patricio; Arendt, Maja; Elvers, Ingegerd; Murén, Eva; Gustafson, Ulla; Starkey, Mike; Borge, Kaja Sverdrup; Lingaas, Frode; Häggström, Jens; Saellström, Sara; Rönnberg, Henrik; Lindblad-Toh, Kerstin

    2016-05-01

    Canine mammary tumours (CMT) are the most common neoplasia in unspayed female dogs. CMTs are suitable naturally occurring models for human breast cancer and share many characteristics, indicating that the genetic causes could also be shared. We have performed a genome-wide association study (GWAS) in English Springer Spaniel dogs and identified a genome-wide significant locus on chromosome 11 (praw = 5.6x10-7, pperm = 0.019). The most associated haplotype spans a 446 kb region overlapping the CDK5RAP2 gene. The CDK5RAP2 protein has a function in cell cycle regulation and could potentially have an impact on response to chemotherapy treatment. Two additional loci, both on chromosome 27, were nominally associated (praw = 1.97x10-5 and praw = 8.30x10-6). The three loci explain 28.1±10.0% of the phenotypic variation seen in the cohort, whereas the top ten associated regions account for 38.2±10.8% of the risk. Furthermore, the ten GWAS loci and regions with reduced genetic variability are significantly enriched for snoRNAs and tumour-associated antigen genes, suggesting a role for these genes in CMT development. We have identified several candidate genes associated with canine mammary tumours, including CDK5RAP2. Our findings enable further comparative studies to investigate the genes and pathways in human breast cancer patients. PMID:27158822

  4. Mechanism of APC/CCDC20 activation by mitotic phosphorylation

    PubMed Central

    Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G.; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A.; Brunner, Michael R.; Davidson, Iain F.; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A.; Peters, Jan-Michael

    2016-01-01

    Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/CCDC20 activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/CCDC20 activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/CCDC20 activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis. PMID:27114510

  5. Mechanism of APC/CCDC20 activation by mitotic phosphorylation.

    PubMed

    Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A; Brunner, Michael R; Davidson, Iain F; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A; Peters, Jan-Michael

    2016-05-10

    Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/C(CDC20) activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/C(CDC20) activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/C(CDC20) activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis. PMID:27114510

  6. APC functions at the centrosome to stimulate microtubule growth.

    PubMed

    Lui, Christina; Ashton, Cahora; Sharma, Manisha; Brocardo, Mariana G; Henderson, Beric R

    2016-01-01

    The adenomatous polyposis coli (APC) tumor suppressor is multi-functional. APC is known to localize at the centrosome, and in mitotic cells contributes to formation of the mitotic spindle. To test whether APC contributes to nascent microtubule (MT) growth at interphase centrosomes, we employed MT regrowth assays in U2OS cells to measure MT assembly before and after nocodazole treatment and release. We showed that siRNA knockdown of full-length APC delayed both initial MT aster formation and MT elongation/regrowth. In contrast, APC-mutant SW480 cancer cells displayed a defect in MT regrowth that was unaffected by APC knockdown, but which was rescued by reconstitution of full-length APC. Our findings identify APC as a positive regulator of centrosome MT initial assembly and suggest that this process is disrupted by cancer mutations. We confirmed that full-length APC associates with the MT-nucleation factor γ-tubulin, and found that the APC cancer-truncated form (1-1309) also bound to γ-tubulin through APC amino acids 1-453. While binding to γ-tubulin may help target APC to the site of MT nucleation complexes, additional C-terminal sequences of APC are required to stimulate and stabilize MT growth. PMID:26556314

  7. APC16 is a conserved subunit of the anaphase-promoting complex/cyclosome

    PubMed Central

    Kops, Geert J. P. L.; van der Voet, Monique; Manak, Michael S.; van Osch, Maria H. J.; Naini, Said M.; Brear, Andrea; McLeod, Ian X.; Hentschel, Dirk M.; Yates, John R.; van den Heuvel, Sander; Shah, Jagesh V.

    2010-01-01

    Error-free chromosome segregation depends on timely activation of the multi-subunit E3 ubiquitin ligase APC/C. Activation of the APC/C initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for destruction. The APC/C is the principle target of the mitotic checkpoint, which prevents segregation while chromosomes are unattached to spindle microtubules. We now report the identification and characterization of APC16, a conserved subunit of the APC/C. APC16 was found in association with tandem-affinity-purified mitotic checkpoint complex protein complexes. APC16 is a bona fide subunit of human APC/C: it is present in APC/C complexes throughout the cell cycle, the phenotype of APC16-depleted cells copies depletion of other APC/C subunits, and APC16 is important for APC/C activity towards mitotic substrates. APC16 sequence homologues can be identified in metazoans, but not fungi, by four conserved primary sequence stretches. We provide evidence that the C. elegans gene K10D2.4 and the D. rerio gene zgc:110659 are functional equivalents of human APC16. Our findings show that APC/C is composed of previously undescribed subunits, and raise the question of why metazoan APC/C is molecularly different from unicellular APC/C. PMID:20392738

  8. Identification of five novel modifier loci of ApcMin harbored in the BXH14 recombinant inbred strain

    PubMed Central

    Siracusa, Linda D.

    2012-01-01

    Every year thousands of people in the USA are diagnosed with small intestine and colorectal cancers (CRC). Although environmental factors affect disease etiology, uncovering underlying genetic factors is imperative for risk assessment and developing preventative therapies. Familial adenomatous polyposis is a heritable genetic disorder in which individuals carry germ-line mutations in the adenomatous polyposis coli (APC) gene that predisposes them to CRC. The Apc Min mouse model carries a point mutation in the Apc gene and develops polyps along the intestinal tract. Inbred strain background influences polyp phenotypes in Apc Min mice. Several Modifier of Min (Mom) loci that alter tumor phenotypes associated with the Apc Min mutation have been identified to date. We screened BXH recombinant inbred (RI) strains by crossing BXH RI females with C57BL/6J (B6) Apc Min males and quantitating tumor phenotypes in backcross progeny. We found that the BXH14 RI strain harbors five modifier loci that decrease polyp multiplicity. Furthermore, we show that resistance is determined by varying combinations of these modifier loci. Gene interaction network analysis shows that there are multiple networks with proven gene–gene interactions, which contain genes from all five modifier loci. We discuss the implications of this result for studies that define susceptibility loci, namely that multiple networks may be acting concurrently to alter tumor phenotypes. Thus, the significance of this work resides not only with the modifier loci we identified but also with the combinations of loci needed to get maximal protection against polyposis and the impact of this finding on human disease studies. Abbreviations:APCadenomatous polyposis coliGWASgenome-wide association studiesQTLquantitative trait lociSNPsingle-nucleotide polymorphism. PMID:22637734

  9. Industrial strength lithography APC

    NASA Astrophysics Data System (ADS)

    Ausschnitt, Christopher P.; Barker, Brian; Muth, William A.; Postiglione, Marc; Walentosky, Thomas

    2003-06-01

    Fully automated semiconductor manufacturing, becoming a reality with the ramping of 300mm fabricators throughout the world, demands the integration of advanced process control (APC). APC is particularly critical for the lithography sector, whose performance correlates to yield and whose productivity often gates the line. We describe the implementation of a comprehensive lithography APC system at the IBM Center for Nanoelectronics, a 300mm manufacturing and development facility. The base lithography APC function encompasses closed-loop run-to-run control of exposure tool inputs to sustain the overlay and critical dimension outputs consistent with product specifications. Automation demands that no decision regarding the appropriate exposure tool run-time settings be left to human judgment. For each lot, the APC system provides optimum settings based on existing data derived from pertinent process streams. In the case where insufficient prior data exists, the APC system either invokes the appropriate combination of send ahead processing and/or pre-determined defaults. We give specific examples of the application of APC to stitched field and dose control, and quantify its technical benefits. Field matching < 0.1 ppm and critical dimension control < 2.5% is achieved among multiple exposure tools and masks.

  10. Genome-wide analysis identifies 16q deletion associated with survival, molecular subtypes, mRNA expression, and germline haplotypes in breast cancer patients.

    PubMed

    Nordgard, Silje H; Johansen, Fredrik E; Alnaes, Grethe I G; Bucher, Elmar; Syvänen, Ann-Christine; Naume, Bjørn; Børresen-Dale, Anne-Lise; Kristensen, Vessela N

    2008-08-01

    Breast carcinomas are characterized by DNA copy number alterations (CNAs) with biological and clinical significance. This explorative study integrated CNA, expression, and germline genotype data of 112 early-stage breast cancer patients. Recurrent CNAs differed substantially between tumor subtypes classified according to expression pattern. Deletion of 16q was overrepresented in Luminal A, and a predictor of good prognosis, both overall and for the nonluminal A subgroups. The deleted region most significantly associated with survival mapped to 16q22.2, harboring the genes TXNL4B and DXH38, whose expression was strongly correlated with the deletion. The area most frequently deleted resided on 16q23.1, 3.5 MB downstream of the area most significantly associated with survival, and included the tumor suppressor gene ADAMTS18 and the cell recognition gene CNTNAP4. Whole-genome association analysis identified germline single nucleotide polymorphisms (SNPs) and their corresponding haplotypes, residing on several different chromosomes, to be associated with deletion of 16q. The genes where these SNPs reside encode proteins involved in the extracellular matrix (CHST3 and SPOCK2), in regulation of the cell cycle (JMY, PTPRN2, and Cwf19L2) and chromosome stability (KPNB1). PMID:18398821

  11. HectD1 E3 ligase modifies adenomatous polyposis coli (APC) with polyubiquitin to promote the APC-axin interaction.

    PubMed

    Tran, Hoanh; Bustos, Daisy; Yeh, Ronald; Rubinfeld, Bonnee; Lam, Cynthia; Shriver, Stephanie; Zilberleyb, Inna; Lee, Michelle W; Phu, Lilian; Sarkar, Anjali A; Zohn, Irene E; Wertz, Ingrid E; Kirkpatrick, Donald S; Polakis, Paul

    2013-02-01

    The adenomatous polyposis coli (APC) protein functions as a negative regulator of the Wnt signaling pathway. In this capacity, APC forms a "destruction complex" with Axin, CK1α, and GSK3β to foster phosphorylation of the Wnt effector β-catenin earmarking it for Lys-48-linked polyubiquitylation and proteasomal degradation. APC is conjugated with Lys-63-linked ubiquitin chains when it is bound to Axin, but it is unclear whether this modification promotes the APC-Axin interaction or confers upon APC an alternative function in the destruction complex. Here we identify HectD1 as a candidate E3 ubiquitin ligase that modifies APC with Lys-63 polyubiquitin. Knockdown of HectD1 diminished APC ubiquitylation, disrupted the APC-Axin interaction, and augmented Wnt3a-induced β-catenin stabilization and signaling. These results indicate that HectD1 promotes the APC-Axin interaction to negatively regulate Wnt signaling. PMID:23277359

  12. ApcMin, A Mutation in the Murine Apc Gene, Predisposes to Mammary Carcinomas and Focal Alveolar Hyperplasias

    NASA Astrophysics Data System (ADS)

    Moser, Amy Rapaich; Mattes, Ellen M.; Dove, William F.; Lindstrom, Mary J.; Haag, Jill D.; Gould, Michael N.

    1993-10-01

    ApcMin (Min, multiple intestinal neoplasia) is a point mutation in the murine homolog of the APC gene. Min/+ mice develop multiple intestinal adenomas, as do humans carrying germ-line mutations in APC. Female mice carrying Min are also prone to develop mammary tumors. Min/+ mammary glands are more sensitive to chemical carcinogenesis than are +/+ mammary glands. Transplantation of mammary cells from Min/+ or +/+ donors into +/+ hosts demonstrates that the propensity to develop mammary tumors is intrinsic to the Min/+ mammary cells. Long-term grafts of Min/+ mammary glands also gave rise to focal alveolar hyperplasias, indicating that the presence of the Min mutation also has a role in the development of these lesions.

  13. Intestinal flora of FAP patients containing APC-like sequences.

    PubMed

    Hainova, K; Adamcikova, Z; Ciernikova, S; Stevurkova, V; Tyciakova, S; Zajac, V

    2014-01-01

    Colorectal cancer mortality is one of the most common cause of cancer-related mortality. A multiple risk factors are associated with colorectal cancer, including hereditary, enviromental and inflammatory syndromes affecting the gastrointestinal tract. Familial adenomatous polyposis (FAP) is characterized by the emergence of hundreds to thousands of colorectal adenomatous polyps and FAP syndrome is caused by mutations within the adenomatous polyposis coli (APC) tumor suppressor gene. We analyzed 21 rectal bacterial subclones isolated from FAP patient 41-1 with confirmed 5bp ACAAA deletion within codons 1060-1063 for the presence of APC-like sequences in longest exon 15. The studied section was defined by primers 15Efor-15Erev, what correlates with mutation cluster region (MCR) in which the 75% of all APC germline mutations were detected. More than 90% homology was showed by sequencing and subsequent software comparison. The expression of APC-like sequences was demostrated by Western blot analysis using monoclonal and polyclonal antibodies against APC protein. To study missing link between the DNA analysis (PCR, DNA sequencing) and protein expresion experiments (Western blotting) we analyzed bacterial transcripts containing the 15Efor-15Erev sequence of APC gene by reverse transcription-PCR, what indicated that an APC gene derived fragment may be produced. We observed 97-100 % homology after computer comparison of cDNA PCR products. Our results suggest that presence of APC-like sequences in intestinal/rectal bacteria is enrichment of bacterial genetic information in which horizontal gene transfer between humans and microflora play an important role. PMID:24824929

  14. Next-generation sequencing identifies germline MRE11A variants as markers of radiotherapy outcomes in muscle-invasive bladder cancer

    PubMed Central

    Teo, M. T. W.; Dyrskjøt, L.; Nsengimana, J.; Buchwald, C.; Snowden, H.; Morgan, J.; Jensen, J. B.; Knowles, M. A.; Taylor, G.; Barrett, J. H.; Borre, M.; Ørntoft, T. F.; Bishop, D. T.; Kiltie, A. E.

    2014-01-01

    Background Muscle-invasive bladder cancer (MIBC) can be cured by radical radiotherapy (RT). We previously found tumour MRE11 expression to be predictive of survival following RT in MIBC, and this was independently validated in a separate institute. Here, we investigated germline MRE11A variants as possible predictors of RT outcomes in MIBC, using next-generation sequencing (NGS). Patients and methods The MRE11A gene was amplified in germline DNA from 186 prospectively recruited MIBC patients treated with RT and sequenced using bar-coded multiplexed NGS. Germline variants were analysed for associations with cancer-specific survival (CSS). For validation as a prognostic or predictive marker, rs1805363 was then genotyped in a cystectomy-treated MIBC cohort of 256 individuals. MRE11A mRNA isoform expression was measured in bladder cancer cell lines and primary tumour samples. Results Carriage of at least one of six (five novel) rare variants was associated with the worse RT outcome (hazard ratio [HR] 4.04, 95% confidence interval [95% CI] 1.42–11.51, P = 0.009). The single-nucleotide polymorphism (SNP), rs1805363 (minor allele frequency 11%), was also associated with worse CSS (per-allele HR 2.10, 95% CI 1.34–3.28, Ptrend = 0.001) following RT in MIBC, with a gene-dosage effect observed, but no effect seen on CSS in the cystectomy cohort (Ptrend = 0.89). Furthermore, rs1805363 influenced relative MRE11A isoform expression, with increased isoform 2 expression with carriage of the rs1805363 minor A allele. Conclusions Germline MRE11A SNP rs1805363 was predictive of RT, but not of cystectomy outcome in MIBC. If successfully validated in an independent RT-treated cohort, this SNP could be a useful clinical tool for selecting patients for bladder-conserving treatment. PMID:24623370

  15. The Anaphase-Promoting Complex (APC) ubiquitin ligase affects chemosensory behavior in C. elegans

    PubMed Central

    Wang, Julia; Jennings, Alexandra K.

    2016-01-01

    The regulation of fundamental aspects of neurobiological function has been linked to the ubiquitin signaling system (USS), which regulates the degradation and activity of proteins and is catalyzed by E1, E2, and E3 enzymes. The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that controls diverse developmental and signaling processes in post-mitotic neurons; however, potential roles for the APC in sensory function have yet to be explored. In this study, we examined the effect of the APC ubiquitin ligase on chemosensation in Caenorhabditis elegans by testing chemotaxis to the volatile odorants, diacetyl, pyrazine, and isoamyl alcohol, to which wild-type worms are attracted. Animals with loss of function mutations in either of two alleles (g48 and ye143) of the gene encoding the APC subunit EMB-27 APC6 showed increased chemotaxis towards diacetyl and pyrazine, odorants sensed by AWA neurons, but exhibited normal chemotaxis to isoamyl alcohol, which is sensed by AWC neurons. The statistically significant increase in chemotaxis in the emb-27 APC6 mutants suggests that the APC inhibits AWA-mediated chemosensation in C. elegans. Increased chemotaxis to pyrazine was also seen with mutants lacking another essential APC subunit, MAT-2 APC1; however, mat-2 APC1 mutants exhibited wild type responses to diacetyl. The difference in responsiveness of these two APC subunit mutants may be due to differential strength of these hypomorphic alleles or may indicate the presence of functional sub-complexes of the APC at work in this process. These findings are the first evidence for APC-mediated regulation of chemosensation and lay the groundwork for further studies aimed at identifying the expression levels, function, and targets of the APC in specific sensory neurons. Because of the similarity between human and C. elegans nervous systems, the role of the APC in sensory neurons may also advance our understanding of human sensory function and disease. PMID

  16. The Anaphase-Promoting Complex (APC) ubiquitin ligase affects chemosensory behavior in C. elegans.

    PubMed

    Wang, Julia; Jennings, Alexandra K; Kowalski, Jennifer R

    2016-01-01

    The regulation of fundamental aspects of neurobiological function has been linked to the ubiquitin signaling system (USS), which regulates the degradation and activity of proteins and is catalyzed by E1, E2, and E3 enzymes. The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that controls diverse developmental and signaling processes in post-mitotic neurons; however, potential roles for the APC in sensory function have yet to be explored. In this study, we examined the effect of the APC ubiquitin ligase on chemosensation in Caenorhabditis elegans by testing chemotaxis to the volatile odorants, diacetyl, pyrazine, and isoamyl alcohol, to which wild-type worms are attracted. Animals with loss of function mutations in either of two alleles (g48 and ye143) of the gene encoding the APC subunit EMB-27 APC6 showed increased chemotaxis towards diacetyl and pyrazine, odorants sensed by AWA neurons, but exhibited normal chemotaxis to isoamyl alcohol, which is sensed by AWC neurons. The statistically significant increase in chemotaxis in the emb-27 APC6 mutants suggests that the APC inhibits AWA-mediated chemosensation in C. elegans. Increased chemotaxis to pyrazine was also seen with mutants lacking another essential APC subunit, MAT-2 APC1; however, mat-2 APC1 mutants exhibited wild type responses to diacetyl. The difference in responsiveness of these two APC subunit mutants may be due to differential strength of these hypomorphic alleles or may indicate the presence of functional sub-complexes of the APC at work in this process. These findings are the first evidence for APC-mediated regulation of chemosensation and lay the groundwork for further studies aimed at identifying the expression levels, function, and targets of the APC in specific sensory neurons. Because of the similarity between human and C. elegans nervous systems, the role of the APC in sensory neurons may also advance our understanding of human sensory function and disease. PMID

  17. A multigene mutation classification of 468 colorectal cancers reveals a prognostic role for APC

    PubMed Central

    Schell, Michael J.; Yang, Mingli; Teer, Jamie K.; Lo, Fang Yin; Madan, Anup; Coppola, Domenico; Monteiro, Alvaro N. A.; Nebozhyn, Michael V.; Yue, Binglin; Loboda, Andrey; Bien-Willner, Gabriel A.; Greenawalt, Danielle M.; Yeatman, Timothy J.

    2016-01-01

    Colorectal cancer (CRC) is a highly heterogeneous disease, for which prognosis has been relegated to clinicopathologic staging for decades. There is a need to stratify subpopulations of CRC on a molecular basis to better predict outcome and assign therapies. Here we report targeted exome-sequencing of 1,321 cancer-related genes on 468 tumour specimens, which identified a subset of 17 genes that best classify CRC, with APC playing a central role in predicting overall survival. APC may assume 0, 1 or 2 truncating mutations, each with a striking differential impact on survival. Tumours lacking any APC mutation carry a worse prognosis than single APC mutation tumours; however, two APC mutation tumours with mutant KRAS and TP53 confer the poorest survival among all the subgroups examined. Our study demonstrates a prognostic role for APC and suggests that sequencing of APC may have clinical utility in the routine staging and potential therapeutic assignment for CRC. PMID:27302369

  18. A multigene mutation classification of 468 colorectal cancers reveals a prognostic role for APC.

    PubMed

    Schell, Michael J; Yang, Mingli; Teer, Jamie K; Lo, Fang Yin; Madan, Anup; Coppola, Domenico; Monteiro, Alvaro N A; Nebozhyn, Michael V; Yue, Binglin; Loboda, Andrey; Bien-Willner, Gabriel A; Greenawalt, Danielle M; Yeatman, Timothy J

    2016-01-01

    Colorectal cancer (CRC) is a highly heterogeneous disease, for which prognosis has been relegated to clinicopathologic staging for decades. There is a need to stratify subpopulations of CRC on a molecular basis to better predict outcome and assign therapies. Here we report targeted exome-sequencing of 1,321 cancer-related genes on 468 tumour specimens, which identified a subset of 17 genes that best classify CRC, with APC playing a central role in predicting overall survival. APC may assume 0, 1 or 2 truncating mutations, each with a striking differential impact on survival. Tumours lacking any APC mutation carry a worse prognosis than single APC mutation tumours; however, two APC mutation tumours with mutant KRAS and TP53 confer the poorest survival among all the subgroups examined. Our study demonstrates a prognostic role for APC and suggests that sequencing of APC may have clinical utility in the routine staging and potential therapeutic assignment for CRC. PMID:27302369

  19. The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators

    PubMed Central

    Hagting, Anja; Izawa, Daisuke; Mansfeld, Jörg; Gibson, Toby J.; Pines, Jonathon

    2016-01-01

    The APC/C is the ubiquitin ligase that regulates mitosis by targeting specific proteins for degradation at specific times under the control of the Spindle Assembly Checkpoint (SAC). How the APC/C recognises its different substrates is a key problem in the control of cell division. Here, we have identified the ABBA motif in Cyclin A, BUBR1, BUB1 and Acm1, and show that it binds to the APC/C co-activator CDC20. The ABBA motif in Cyclin A is required for its proper degradation in prometaphase through competing with BUBR1 for the same site on CDC20. Moreover, the ABBA motifs in BUBR1 and BUB1 are necessary for the SAC to work at full strength and to recruit CDC20 to kinetochores. Thus, we have identified a conserved motif integral to the proper control of mitosis that connects APC/C substrate recognition with the SAC. PMID:25669885

  20. The APC tumor suppressor is required for epithelial cell polarization and three-dimensional morphogenesis.

    PubMed

    Lesko, Alyssa C; Goss, Kathleen H; Yang, Frank F; Schwertner, Adam; Hulur, Imge; Onel, Kenan; Prosperi, Jenifer R

    2015-03-01

    The Adenomatous Polyposis Coli (APC) tumor suppressor has been previously implicated in the control of apical-basal polarity; yet, the consequence of APC loss-of-function in epithelial polarization and morphogenesis has not been characterized. To test the hypothesis that APC is required for the establishment of normal epithelial polarity and morphogenesis programs, we generated APC-knockdown epithelial cell lines. APC depletion resulted in loss of polarity and multi-layering on permeable supports, and enlarged, filled spheroids with disrupted polarity in 3D culture. Importantly, these effects of APC knockdown were independent of Wnt/β-catenin signaling, but were rescued with either full-length or a carboxy (c)-terminal segment of APC. Moreover, we identified a gene expression signature associated with APC knockdown that points to several candidates known to regulate cell-cell and cell-matrix communication. Analysis of epithelial tissues from mice and humans carrying heterozygous APC mutations further supports the importance of APC as a regulator of epithelial behavior and tissue architecture. These data also suggest that the initiation of epithelial-derived tumors as a result of APC mutation or gene silencing may be driven by loss of polarity and dysmorphogenesis. PMID:25578398

  1. Integrated tumor and germline whole-exome sequencing identifies mutations in MAPK and PI3K pathway genes in an adolescent with rosette-forming glioneuronal tumor of the fourth ventricle

    PubMed Central

    Lin, Frank Y.; Bergstrom, Katie; Person, Richard; Bavle, Abhishek; Ballester, Leomar Y.; Scollon, Sarah; Raesz-Martinez, Robin; Jea, Andrew; Birchansky, Sherri; Wheeler, David A.; Berg, Stacey L.; Chintagumpala, Murali M.; Adesina, Adekunle M.; Eng, Christine; Roy, Angshumoy; Plon, Sharon E.; Parsons, D. Williams

    2016-01-01

    The integration of genome-scale studies such as whole-exome sequencing (WES) into the clinical care of children with cancer has the potential to provide insight into the genetic basis of an individual's cancer with implications for clinical management. This report describes the results of clinical tumor and germline WES for a patient with a rare tumor diagnosis, rosette-forming glioneuronal tumor of the fourth ventricle (RGNT). Three pathogenic gene alterations with implications for clinical care were identified: somatic activating hotspot mutations in FGFR1 (p.N546K) and PIK3CA (p.H1047R) and a germline pathogenic variant in PTPN11 (p.N308S) diagnostic for Noonan syndrome. The molecular landscape of RGNT is not well-described, but these data are consistent with prior observations regarding the importance of the interconnected MAPK and PI3K/AKT/mTOR signaling pathways in this rare tumor. The co-occurrence of FGFR1, PIK3CA, and PTPN11 alterations provides further evidence for consideration of RGNT as a distinct molecular entity from pediatric low-grade gliomas and suggests potential therapeutic strategies for this patient in the event of tumor recurrence as novel agents targeting these pathways enter pediatric clinical trials. Although RGNT has not been definitively linked with cancer predisposition syndromes, two prior cases have been reported in patients with RASopathies (Noonan syndrome and neurofibromatosis type 1 [NF1]), providing an additional link between these tumors and the mitogen-activated protein kinase (MAPK) signaling pathway. In summary, this case provides an example of the potential for genome-scale sequencing technologies to provide insight into the biology of rare tumors and yield both tumor and germline results of potential relevance to patient care. PMID:27626068

  2. Integrated tumor and germline whole-exome sequencing identifies mutations in MAPK and PI3K pathway genes in an adolescent with rosette-forming glioneuronal tumor of the fourth ventricle.

    PubMed

    Lin, Frank Y; Bergstrom, Katie; Person, Richard; Bavle, Abhishek; Ballester, Leomar Y; Scollon, Sarah; Raesz-Martinez, Robin; Jea, Andrew; Birchansky, Sherri; Wheeler, David A; Berg, Stacey L; Chintagumpala, Murali M; Adesina, Adekunle M; Eng, Christine; Roy, Angshumoy; Plon, Sharon E; Parsons, D Williams

    2016-09-01

    The integration of genome-scale studies such as whole-exome sequencing (WES) into the clinical care of children with cancer has the potential to provide insight into the genetic basis of an individual's cancer with implications for clinical management. This report describes the results of clinical tumor and germline WES for a patient with a rare tumor diagnosis, rosette-forming glioneuronal tumor of the fourth ventricle (RGNT). Three pathogenic gene alterations with implications for clinical care were identified: somatic activating hotspot mutations in FGFR1 (p.N546K) and PIK3CA (p.H1047R) and a germline pathogenic variant in PTPN11 (p.N308S) diagnostic for Noonan syndrome. The molecular landscape of RGNT is not well-described, but these data are consistent with prior observations regarding the importance of the interconnected MAPK and PI3K/AKT/mTOR signaling pathways in this rare tumor. The co-occurrence of FGFR1, PIK3CA, and PTPN11 alterations provides further evidence for consideration of RGNT as a distinct molecular entity from pediatric low-grade gliomas and suggests potential therapeutic strategies for this patient in the event of tumor recurrence as novel agents targeting these pathways enter pediatric clinical trials. Although RGNT has not been definitively linked with cancer predisposition syndromes, two prior cases have been reported in patients with RASopathies (Noonan syndrome and neurofibromatosis type 1 [NF1]), providing an additional link between these tumors and the mitogen-activated protein kinase (MAPK) signaling pathway. In summary, this case provides an example of the potential for genome-scale sequencing technologies to provide insight into the biology of rare tumors and yield both tumor and germline results of potential relevance to patient care. PMID:27626068

  3. Identification of APC mutations and evaluation of their expression level using a functional screening assay

    SciTech Connect

    Varesco, L.; Gismondi, V.; Bafico, A.

    1994-09-01

    A functional screen for chain-terminating mutations in the APC gene recently has been developed. It is based on the PCR and cloning of a segment of the gene in-frame with a colorimetric marker gene (lacz) followed by screening for the level of activity of the marker polypeptide (beta-galactosidase). This method scores colony number with different blue colors that are produced by bacteria containing normal and mutant APC segments. In the present work this method was used to screen the entire APC coding region by using eight primer pairs. DNA segments with known APC mutations at different positions in the gene were used as controls and were clearly identifiable with this assay. In addition, the entire APC coding region has been examined in 21 APC patients in whom PCR-SSCP did not identify an APC mutation. Novel mutations (n=14) were identified by the blue/white assay and were all confirmed by sequence analysis. This method also was used to quantitate the expression of paternal and maternal APC alleles taking advantage of an RsaI site polymorphism at position 1458 in a small number of informative individuals. Differential expression of some known mutant APC mRNAs was observed.

  4. Characteristics of Germline and Non-germline Retinoblastomas

    PubMed Central

    Ghassemi, Fariba; Chams, Hormoz; Sabour, Siamak; Karkhaneh, Reza; Farzbod, Farzad; Khodaparast, Mehdi; Vosough, Parvaneh

    2014-01-01

    Purpose To discuss the clinical characteristics, treatment and outcomes of germline and non-germline retinoblastoma tumors. Methods A retrospective study was performed on retinoblastoma cases from 1979 to 2007. General characteristics of the patients, treatment modalities, histopathological findings and survival were compared in germline versus non-germline cases. Results We analyzed 557 cases of retinoblastoma with mean age of 32.2±22.0 months including 177 and 380 patients with germline and non-germline tumors, respectively. Germline cases were significantly different from non-germline counterparts in terms of mean age (24.7±17.7 vs 35.7±23.0 months), symptoms (leukocoria in 49.4% vs 62.9%), and outcomes (death in 40.1% vs 13.9%), respectively (P<0.001). In the germline group 66.5% and in non-germline group over 97% of patients had stage Va or higher (ICRB D-E disease). Disease-free survival was 48.6% for germlines cases versus 80.9% for non-germline patients (with mean follow up of 61.9 months, P<0.001). Histopathologically, more invasions to intraocular and extraocular tissues were seen with non-germline tumors of (66% vs 39.8%). Mortality rates in germline cases and non-germline were 40.1% and 13.9%, respectively (P<0.001). Conclusion Despite higher tumor staging in nongermline cases at the time of diagnosis and therefore more aggressive behavior of the tumor, germline cases had a higher rate of mortality during the follow up period. PMID:25279120

  5. Integrated analysis of germline and somatic variants in ovarian cancer.

    PubMed

    Kanchi, Krishna L; Johnson, Kimberly J; Lu, Charles; McLellan, Michael D; Leiserson, Mark D M; Wendl, Michael C; Zhang, Qunyuan; Koboldt, Daniel C; Xie, Mingchao; Kandoth, Cyriac; McMichael, Joshua F; Wyczalkowski, Matthew A; Larson, David E; Schmidt, Heather K; Miller, Christopher A; Fulton, Robert S; Spellman, Paul T; Mardis, Elaine R; Druley, Todd E; Graubert, Timothy A; Goodfellow, Paul J; Raphael, Benjamin J; Wilson, Richard K; Ding, Li

    2014-01-01

    We report the first large-scale exome-wide analysis of the combined germline-somatic landscape in ovarian cancer. Here we analyse germline and somatic alterations in 429 ovarian carcinoma cases and 557 controls. We identify 3,635 high confidence, rare truncation and 22,953 missense variants with predicted functional impact. We find germline truncation variants and large deletions across Fanconi pathway genes in 20% of cases. Enrichment of rare truncations is shown in BRCA1, BRCA2 and PALB2. In addition, we observe germline truncation variants in genes not previously associated with ovarian cancer susceptibility (NF1, MAP3K4, CDKN2B and MLL3). Evidence for loss of heterozygosity was found in 100 and 76% of cases with germline BRCA1 and BRCA2 truncations, respectively. Germline-somatic interaction analysis combined with extensive bioinformatics annotation identifies 222 candidate functional germline truncation and missense variants, including two pathogenic BRCA1 and 1 TP53 deleterious variants. Finally, integrated analyses of germline and somatic variants identify significantly altered pathways, including the Fanconi, MAPK and MLL pathways. PMID:24448499

  6. Integrated Analysis of Germline and Somatic Variants in Ovarian Cancer

    PubMed Central

    Kanchi, Krishna L.; Johnson, Kimberly J.; Lu, Charles; McLellan, Michael D.; Leiserson, Mark D.M.; Wendl, Michael C.; Zhang, Qunyuan; Koboldt, Daniel C.; Xie, Mingchao; Kandoth, Cyriac; McMichael, Joshua F.; Wyczalkowski, Matthew A.; Larson, David E.; Schmidt, Heather K.; Miller, Christopher A.; Fulton, Robert S.; Spellman, Paul T.; Mardis, Elaine R.; Druley, Todd E.; Graubert, Timothy A.; Goodfellow, Paul J.; Raphael, Benjamin J.; Wilson, Richard K.; Ding, Li

    2014-01-01

    We report the first large-scale exome-wide analysis of the combined germline-somatic landscape in ovarian cancer. Here we analyze germline and somatic alterations in 429 ovarian carcinoma cases and 557 controls. We identify 3,635 high confidence, rare truncation and 22,953 missense variants with predicted functional impact. We find germline truncation variants and large deletions across Fanconi pathway genes in 20% of cases. Enrichment of rare truncations is shown in BRCA1, BRCA2, and PALB2. Additionally, we observe germline truncation variants in genes not previously associated with ovarian cancer susceptibility (NF1, MAP3K4, CDKN2B, and MLL3). Evidence for loss of heterozygosity was found in 100% and 76% of cases with germline BRCA1 and BRCA2 truncations respectively. Germline-somatic interaction analysis combined with extensive bioinformatics annotation identifies 237 candidate functional germline truncation and missense variants, including 2 pathogenic BRCA1 and 1 TP53 deleterious variants. Finally, integrated analyses of germline and somatic variants identify significantly altered pathways, including the Fanconi, MAPK, and MLL pathways. PMID:24448499

  7. Germline Mutations in Predisposition Genes in Pediatric Cancer

    PubMed Central

    Edmonson, Michael N.; Gruber, Tanja A.; Easton, John; Hedges, Dale; Ma, Xiaotu; Zhou, Xin; Yergeau, Donald A.; Wilkinson, Mark R.; Vadodaria, Bhavin; Chen, Xiang; McGee, Rose B.; Hines-Dowell, Stacy; Nuccio, Regina; Quinn, Emily; Shurtleff, Sheila A.; Rusch, Michael; Patel, Aman; Becksfort, Jared B.; Wang, Shuoguo; Weaver, Meaghann S.; Ding, Li; Mardis, Elaine R.; Wilson, Richard K.; Gajjar, Amar; Ellison, David W.; Pappo, Alberto S.; Pui, Ching-Hon; Downing, James R.

    2016-01-01

    BACKGROUND The prevalence and spectrum of predisposing mutations among children and adolescents with cancer are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis, direct patient care, and enable genetic counseling of patients and families. METHODS In 1120 patients younger than 20 years of age, we sequenced the whole genomes (in 595 patients), whole exomes (in 456), or both (in 69). We analyzed the DNA sequences of 565 genes, including 60 that have been associated with autosomal dominant cancer-predisposition syndromes, for the presence of germline mutations. The pathogenicity of the mutations was determined by a panel of medical experts with the use of cancer-specific and locus-specific genetic databases, the medical literature, computational predictions, and second hits identified in the tumor genome. The same approach was used to analyze data from 966 persons who did not have known cancer in the 1000 Genomes Project, and a similar approach was used to analyze data from an autism study (from 515 persons with autism and 208 persons without autism). RESULTS Mutations that were deemed to be pathogenic or probably pathogenic were identified in 95 patients with cancer (8.5%), as compared with 1.1% of the persons in the 1000 Genomes Project and 0.6% of the participants in the autism study. The most commonly mutated genes in the affected patients were TP53 (in 50 patients), APC (in 6), BRCA2 (in 6), NF1 (in 4), PMS2 (in 4), RB1 (in 3), and RUNX1 (in 3). A total of 18 additional patients had protein-truncating mutations in tumor-suppressor genes. Of the 58 patients with a predisposing mutation and available information on family history, 23 (40%) had a family history of cancer. CONCLUSIONS Germline mutations in cancer-predisposing genes were identified in 8.5% of the children and adolescents with cancer. Family history did not predict the presence of an underlying predisposition syndrome in most patients. (Funded by the American

  8. Atomic-Resolution Structures of the APC/C Subunits Apc4 and the Apc5 N-Terminal Domain

    PubMed Central

    Cronin, Nora B.; Yang, Jing; Zhang, Ziguo; Kulkarni, Kiran; Chang, Leifu; Yamano, Hiroyuki; Barford, David

    2015-01-01

    Many essential biological processes are mediated by complex molecular machines comprising multiple subunits. Knowledge on the architecture of individual subunits and their positions within the overall multimeric complex is key to understanding the molecular mechanisms of macromolecular assemblies. The anaphase-promoting complex/cyclosome (APC/C) is a large multisubunit complex that regulates cell cycle progression by ubiquitinating cell cycle proteins for proteolysis by the proteasome. The holo-complex is composed of 15 different proteins that assemble to generate a complex of 20 subunits. Here, we describe the crystal structures of Apc4 and the N-terminal domain of Apc5 (Apc5N). Apc4 comprises a WD40 domain split by a long α-helical domain, whereas Apc5N has an α-helical fold. In a separate study, we had fitted these atomic models to a 3.6-Å-resolution cryo-electron microscopy map of the APC/C. We describe how, in the context of the APC/C, regions of Apc4 disordered in the crystal assume order through contacts to Apc5, whereas Apc5N shows small conformational changes relative to its crystal structure. We discuss the complementary approaches of high-resolution electron microscopy and protein crystallography to the structure determination of subunits of multimeric complexes. PMID:26343760

  9. Extensive metabolic disorders are present in APC(min) tumorigenesis mice.

    PubMed

    Liu, Zhenzhen; Xiao, Yi; Zhou, Zhengxiang; Mao, Xiaoxiao; Cai, Jinxing; Xiong, Lu; Liao, Chaonan; Huang, Fulian; Liu, Zehao; Ali Sheikh, Md Sayed; Plutzky, Jorge; Huang, He; Yang, Tianlun; Duan, Qiong

    2016-05-15

    Wnt signaling plays essential role in mesenchymal stem cell (MSC) differentiation. Activation of Wnt signaling suppresses adipogenesis, but promotes osteogenesis in MSC. Adenomatous polyposis coli (APC) is a negative regulator of β-catenin and Wnt signaling activity. The mutation of APC gene leads to the activation of Wnt signaling and is responsible for tumorigenesis in APC(min) mouse; however, very few studies focused on its metabolic abnormalities. The present study reports a widespread metabolic disorder phenotype in APC(min) mice. The old APC(min) mice have decreased body weight and impaired adipogenesis, but severe hyperlipidemia, which mimic the phenotypes of Familial Adenomatous Polyposis (FAP), an inherited disease also caused by APC gene mutation in human. We found that the expression of lipid metabolism and free fat acids (FA) use genes in the white adipose tissue (WAT) of the APC(min) mice is much lower than those of control. The changed gene expression pattern may lead to the disability of circulatory lipid transportation and storage at WAT. Moreover, the APC(min) mice could not maintain the core body temperature in cold condition. PET-CT determination revealed that the BAT of APC(min) mice has significantly impaired ability to take up (18)FDG from the blood. Morphological studies identified that the brown adipocytes of APC(min) mice were filled with lipid droplets but fewer mitochondria. These results matched with the findings of impaired BAT function in APC(min) mice. Collectively, our study explores a new mechanism that explains abnormal metabolism in APC(min) mice and provides insights into studying the metabolic disorders of FAP patients. PMID:26948948

  10. A conserved germline multipotency program

    PubMed Central

    Juliano, Celina E.; Swartz, S. Zachary; Wessel, Gary M.

    2010-01-01

    The germline of multicellular animals is segregated from somatic tissues, which is an essential developmental process for the next generation. Although certain ecdysozoans and chordates segregate their germline during embryogenesis, animals from other taxa segregate their germline after embryogenesis from multipotent progenitor cells. An overlapping set of genes, including vasa, nanos and piwi, operate in both multipotent precursors and in the germline. As we propose here, this conservation implies the existence of an underlying germline multipotency program in these cell types that has a previously underappreciated and conserved function in maintaining multipotency. PMID:21098563

  11. Disruption of the APC gene by t(5;7) translocation in a Turcot family.

    PubMed

    Sahnane, Nora; Bernasconi, Barbara; Carnevali, Ileana; Furlan, Daniela; Viel, Alessandra; Sessa, Fausto; Tibiletti, Maria Grazia

    2016-03-01

    Turcot syndrome (TS) refers to the combination of colorectal polyps and primary tumours of the central nervous system. TS is a heterogeneous genetic condition due to APC and/or mismatch repair germline mutations. When APC is involved the vast majority of mutations are truncating, but in approximately 20%-30% of patients with familial polyposis no germline mutation can be found. A 30-year-old Caucasian woman with a positive pedigree for TS was referred to our Genetic Counselling Service. She was negative for APC and MUTYH but showed a reciprocal balanced translocation t(5;7)(q22;p15) at chromosome analysis. FISH analysis using specific BAC probes demonstrated that 5q22 breakpoint disrupted the APC gene. Transcript analysis by MLPA and digital PCR revealed that the cytogenetic rearrangement involving the 3' end of the APC gene caused a defective expression of a truncated transcript. This result allowed cytogenetic analysis to be offered to all the other family members and segregation analysis clearly demonstrated that all the carriers were affected, whereas non-carriers did not have the polyposis. A cytogenetic approach permitted the identification of the mutation-causing disease in this family, and the segregation analysis together with the transcript study supported the pathogenetic role of this mutation. Karyotype analysis was used as a predictive test in all members of this family. This family suggests that clinically positive TS and FAP cases, which test negative with standard molecular analysis, could be easily and cost-effectively resolved by a classical and molecular cytogenetic approach. PMID:26797314

  12. APC15 mediates CDC20 auto-ubiquitylation by APC/CMCC and MCC disassembly

    PubMed Central

    Uzunova, Kristina; Dye, Billy T.; Schutz, Hannelore; Ladurner, Rene; Petzold, Georg; Toyoda, Yusuke; Jarvis, Marc A.; Brown, Nicholas G.; Poser, Ina; Novatchkova, Maria; Mechtler, Karl; Hyman, Anthony A.; Stark, Holger; Schulman, Brenda A.; Peters, Jan-Michael

    2012-01-01

    The anaphase-promoting complex/cyclosome bound to CDC20 (APC/CCDC20) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) which inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which has been proposed to require CDC20 auto-ubiquitylation. Here we characterized APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C’s MCC binding site, is required for APC/CMCC-dependent CDC20 auto-ubiquitylation and degradation, and for timely anaphase initiation, but is dispensable for substrate ubiquitylation by APC/CCDC20 and APC/CCDH1. Our results support the view that MCC is continuously assembled and disassembled to enable rapid activation of APC/CCDC20 and that CDC20 auto-ubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators, and report the first generation of recombinant human APC/C. PMID:23007861

  13. Structure of an APC3-APC16 complex: Insights into assembly of the Anaphase Promoting Complex/Cyclosome

    PubMed Central

    Yamaguchi, Masaya; Yu, Shanshan; Qiao, Renping; Weissmann, Florian; Miller, Darcie J.; VanderLinden, Ryan; Brown, Nicholas G.; Frye, Jeremiah J.; Peters, Jan-Michael; Schulman, Brenda A.

    2015-01-01

    The Anaphase Promoting Complex/Cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the “Platform” centers around a cullin-RING-like E3 ligase catalytic core; the “Arc Lamp” is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-twofold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3’s TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, via their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7, and contains a >200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3’s loop and IR-tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer, and together with biochemistry and prior data explain how APC16 recruits APC7 to APC3, show how APC3’s C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR-tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C. PMID:25490258

  14. High performance APCS conceptual design and evaluation scoping study

    SciTech Connect

    Soelberg, N.; Liekhus, K.; Chambers, A.; Anderson, G.

    1998-02-01

    This Air Pollution Control System (APCS) Conceptual Design and Evaluation study was conducted to evaluate a high-performance (APC) system for minimizing air emissions from mixed waste thermal treatment systems. Seven variations of high-performance APCS designs were conceptualized using several design objectives. One of the system designs was selected for detailed process simulation using ASPEN PLUS to determine material and energy balances and evaluate performance. Installed system capital costs were also estimated. Sensitivity studies were conducted to evaluate the incremental cost and benefit of added carbon adsorber beds for mercury control, specific catalytic reduction for NO{sub x} control, and offgas retention tanks for holding the offgas until sample analysis is conducted to verify that the offgas meets emission limits. Results show that the high-performance dry-wet APCS can easily meet all expected emission limits except for possibly mercury. The capability to achieve high levels of mercury control (potentially necessary for thermally treating some DOE mixed streams) could not be validated using current performance data for mercury control technologies. The engineering approach and ASPEN PLUS modeling tool developed and used in this study identified APC equipment and system performance, size, cost, and other issues that are not yet resolved. These issues need to be addressed in feasibility studies and conceptual designs for new facilities or for determining how to modify existing facilities to meet expected emission limits. The ASPEN PLUS process simulation with current and refined input assumptions and calculations can be used to provide system performance information for decision-making, identifying best options, estimating costs, reducing the potential for emission violations, providing information needed for waste flow analysis, incorporating new APCS technologies in existing designs, or performing facility design and permitting activities.

  15. APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane.

    PubMed

    Mills, Kate M; Brocardo, Mariana G; Henderson, Beric R

    2016-02-01

    Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations. PMID:26658612

  16. APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane

    PubMed Central

    Mills, Kate M.; Brocardo, Mariana G.; Henderson, Beric R.

    2016-01-01

    Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations. PMID:26658612

  17. Screening for germline BRCA1, BRCA2, TP53 and CHEK2 mutations in families at-risk for hereditary breast cancer identified in a population-based study from Southern Brazil

    PubMed Central

    Palmero, Edenir Inêz; Alemar, Bárbara; Schüler-Faccini, Lavínia; Hainaut, Pierre; Moreira-Filho, Carlos Alberto; Ewald, Ingrid Petroni; dos Santos, Patricia Koehler; Ribeiro, Patricia Lisbôa Izetti; de Oliveira, Cristina Brinkmann; Kelm, Florence Le Calvez; Tavtigian, Sean; Cossio, Silvia Liliana; Giugliani, Roberto; Caleffi, Maira; Ashton-Prolla, Patricia

    2016-01-01

    Abstract In Brazil, breast cancer is a public health care problem due to its high incidence and mortality rates. In this study, we investigated the prevalence of hereditary breast cancer syndromes (HBCS) in a population-based cohort in Brazils southernmost capital, Porto Alegre. All participants answered a questionnaire about family history (FH) of breast, ovarian and colorectal cancer and those with a positive FH were invited for genetic cancer risk assessment (GCRA). If pedigree analysis was suggestive of HBCS, genetic testing of the BRCA1, BRCA2, TP53, and CHEK2 genes was offered. Of 902 women submitted to GCRA, 214 had pedigrees suggestive of HBCS. Fifty of them underwent genetic testing: 18 and 40 for BRCA1/BRCA2 and TP53 mutation screening, respectively, and 7 for CHEK2 1100delC testing. A deleterious BRCA2 mutation was identified in one of the HBOC probands and the CHEK2 1100delC mutation occurred in one of the HBCC families. No deleterious germline alterations were identified in BRCA1 or TP53. Although strict inclusion criteria and a comprehensive testing approach were used, the suspected genetic risk in these families remains unexplained. Further studies in a larger cohort are necessary to better understand the genetic component of hereditary breast cancer in Southern Brazil. PMID:27223485

  18. Screening for germline BRCA1, BRCA2, TP53 and CHEK2 mutations in families at-risk for hereditary breast cancer identified in a population-based study from Southern Brazil.

    PubMed

    Palmero, Edenir Inêz; Alemar, Bárbara; Schüler-Faccini, Lavínia; Hainaut, Pierre; Moreira-Filho, Carlos Alberto; Ewald, Ingrid Petroni; Santos, Patricia Koehler Dos; Ribeiro, Patricia Lisbôa Izetti; Oliveira, Cristina Brinkmann de Netto; Kelm, Florence Le Calvez; Tavtigian, Sean; Cossio, Silvia Liliana; Giugliani, Roberto; Caleffi, Maira; Ashton-Prolla, Patricia

    2016-05-24

    In Brazil, breast cancer is a public health care problem due to its high incidence and mortality rates. In this study, we investigated the prevalence of hereditary breast cancer syndromes (HBCS) in a population-based cohort in Brazils southernmost capital, Porto Alegre. All participants answered a questionnaire about family history (FH) of breast, ovarian and colorectal cancer and those with a positive FH were invited for genetic cancer risk assessment (GCRA). If pedigree analysis was suggestive of HBCS, genetic testing of the BRCA1, BRCA2, TP53, and CHEK2 genes was offered. Of 902 women submitted to GCRA, 214 had pedigrees suggestive of HBCS. Fifty of them underwent genetic testing: 18 and 40 for BRCA1/BRCA2 and TP53 mutation screening, respectively, and 7 for CHEK2 1100delC testing. A deleterious BRCA2 mutation was identified in one of the HBOC probands and the CHEK2 1100delC mutation occurred in one of the HBCC families. No deleterious germline alterations were identified in BRCA1 or TP53. Although strict inclusion criteria and a comprehensive testing approach were used, the suspected genetic risk in these families remains unexplained. Further studies in a larger cohort are necessary to better understand the genetic component of hereditary breast cancer in Southern Brazil. PMID:27223485

  19. Prevalence of Germline Mutations in Cancer Predisposition Genes in Patients with Pancreatic Cancer

    PubMed Central

    Grant, Robert C.; Selander, Iris; Connor, Ashton A.; Selvarajah, Shamini; Borgida, Ayelet; Briollais, Laurent; Petersen, Gloria M.; Lerner-Ellis, Jordan; Holter, Spring; Gallinger, Steven

    2015-01-01

    Background & Aims We investigated the prevalence of germline mutations in APC, ATM, BRCA1, BRCA2, CDKN2A, MLH1, MSH2, MSH6, PALB2, PMS2, PRSS1, STK11, and TP53 in patients with pancreatic cancer. Methods The Ontario Pancreas Cancer Study enrolls consenting participants with pancreatic cancer from a province-wide electronic pathology database; 708 probands were enrolled from April 2003 through August 2012. To improve precision of BRCA2 prevalence estimates, 290 probands were randomly selected from 3 strata, based on family history of breast and/or ovarian cancer, pancreatic cancer, or neither. Germline DNA was analyzed by next-generation sequencing using a custom multiple-gene panel. Mutation prevalence estimates were calculated from the sample for the entire cohort. Results Eleven pathogenic mutations were identified: 3 in ATM, 1 in BRCA1, 2 in BRCA2, 1 in MLH1, 2 in MSH2, 1 in MSH6, and 1 in TP53. The prevalence of mutations in all 13 genes was 3.8% (95% confidence interval, 2.1%–5.6%). Carrier status was significantly associated with breast cancer in the proband or first-degree relative (P<.01), and colorectal cancer in the proband or first-degree relative (P<.01), but not family history of pancreatic cancer, age of diagnosis, or stage at diagnosis. Of patients with a personal or family history of breast and colorectal cancer, 10.7% (4.4%–17.0%) and 11.1% (3.0%–19.1%) carried pathogenic mutations, respectively. Conclusions A small but clinically important proportion of pancreatic cancer is associated with mutations in known predisposition genes. The heterogeneity of mutations identified in this study demonstrates the value of using a multiple-gene panel in pancreatic cancer. PMID:25479140

  20. The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators.

    PubMed

    Di Fiore, Barbara; Davey, Norman E; Hagting, Anja; Izawa, Daisuke; Mansfeld, Jörg; Gibson, Toby J; Pines, Jonathon

    2015-02-01

    The anaphase-promoting complex or cyclosome (APC/C) is the ubiquitin ligase that regulates mitosis by targeting specific proteins for degradation at specific times under the control of the spindle assembly checkpoint (SAC). How the APC/C recognizes its different substrates is a key problem in the control of cell division. Here, we have identified the ABBA motif in cyclin A, BUBR1, BUB1, and Acm1, and we show that it binds to the APC/C coactivator CDC20. The ABBA motif in cyclin A is required for its proper degradation in prometaphase through competing with BUBR1 for the same site on CDC20. Moreover, the ABBA motifs in BUBR1 and BUB1 are necessary for the SAC to work at full strength and to recruit CDC20 to kinetochores. Thus, we have identified a conserved motif integral to the proper control of mitosis that connects APC/C substrate recognition with the SAC. PMID:25669885

  1. Phf7 controls male sex determination in the Drosophila germline

    PubMed Central

    Yang, Shu Yuan; Baxter, Ellen M.; Van Doren, Mark

    2013-01-01

    Summary Establishment of germline sexual identity is critical for production of male and female germline stem cells, and sperm vs. eggs. Here we identify PHD Finger Protein 7 (PHF7) as an important factor for male germline sexual identity in Drosophila. PHF7 exhibits male-specific expression in early germ cells, germline stem cells and spermatogonia. It is required for germline stem cell maintenance and gametogenesis in males, whereas ectopic expression in female germ cells ablates the germline. Strikingly, expression of PHF7 promotes spermatogenesis in XX germ cells when they are present in a male soma. PHF7 homologs are also specifically expressed in the mammalian testis, and human PHF7 rescues Drosophila Phf7 mutants. PHF7 associates with chromatin and both the human and fly proteins bind histone H3 N-terminal tails with a preference for dimethyl lysine 4 (H3K4me2). We propose that PHF7 acts as a conserved epigenetic “reader” that activates the male germline sexual program. PMID:22595675

  2. Germ-line variants identified by next generation sequencing in a panel of estrogen and cancer associated genes correlate with poor clinical outcome in Lynch syndrome patients

    PubMed Central

    Jóri, Balazs; Delvoux, Bert; Blok, Marinus J.; Van de Vijver, Koen K.; de Koning, Bart; Oei, Felicia Trups; Tops, Carli M.; Speel, Ernst J. M.; Kruitwagen, Roy F.; Gomez-Garcia, Encarna B.; Romano, Andrea

    2015-01-01

    Background The risk to develop colorectal and endometrial cancers among subjects testing positive for a pathogenic Lynch syndrome mutation varies, making the risk prediction difficult. Genetic risk modifiers alter the risk conferred by inherited Lynch syndrome mutations, and their identification can improve genetic counseling. We aimed at identifying rare genetic modifiers of the risk of Lynch syndrome endometrial cancer. Methods A family based approach was used to assess the presence of genetic risk modifiers among 35 Lynch syndrome mutation carriers having either a poor clinical phenotype (early age of endometrial cancer diagnosis or multiple cancers) or a neutral clinical phenotype. Putative genetic risk modifiers were identified by Next Generation Sequencing among a panel of 154 genes involved in endometrial physiology and carcinogenesis. Results A simple pipeline, based on an allele frequency lower than 0.001 and on predicted non-conservative amino-acid substitutions returned 54 variants that were considered putative risk modifiers. The presence of two or more risk modifying variants in women carrying a pathogenic Lynch syndrome mutation was associated with a poor clinical phenotype. Conclusion A gene-panel is proposed that comprehends genes that can carry variants with putative modifying effects on the risk of Lynch syndrome endometrial cancer. Validation in further studies is warranted before considering the possible use of this tool in genetic counseling. PMID:26517685

  3. A targeted RNAi screen for genes involved in chromosome morphogenesis and nuclear organization in the Caenorhabditis elegans germline.

    PubMed Central

    Colaiácovo, M P; Stanfield, G M; Reddy, K C; Reinke, V; Kim, S K; Villeneuve, A M

    2002-01-01

    We have implemented a functional genomics strategy to identify genes involved in chromosome morphogenesis and nuclear organization during meiotic prophase in the Caenorhabditis elegans germline. This approach took advantage of a gene-expression survey that used DNA microarray technology to identify genes preferentially expressed in the germline. We defined a subset of 192 germline-enriched genes whose expression profiles were similar to those of previously identified meiosis genes and designed a screen to identify genes for which inhibition by RNA interference (RNAi) elicited defects in function or development of the germline. We obtained strong germline phenotypes for 27% of the genes tested, indicating that this targeted approach greatly enriched for genes that function in the germline. In addition to genes involved in key meiotic prophase events, we identified genes involved in meiotic progression, germline proliferation, and chromosome organization and/or segregation during mitotic growth. PMID:12242227

  4. Deconstructing the ßcatenin destruction complex: mechanistic roles for the tumor suppressor APC in regulating Wnt signaling.

    PubMed

    Roberts, David M; Pronobis, Mira I; Poulton, John S; Waldmann, Jon D; Stephenson, Elise M; Hanna, Shahnaz; Peifer, Mark

    2011-06-01

    Negatively regulating signaling by targeting key effectors for ubiquitination/destruction is essential for development and oncogenesis. The tumor suppressor adenomatous polyposis coli (APC), an essential negative regulator of Wnt signaling, provides a paradigm. APC mutations occur in most colon cancers. Acting in the "destruction complex" with Axin, glycogen synthase kinase 3, and casein kinase, APC targets ßcatenin (ßcat) for phosphorylation and recognition by an E3 ubiquitin-ligase. Despite 20 years of work, the internal workings of the destruction complex and APC's role remain largely mysterious. We use both Drosophila and colon cancer cells to test hypotheses for APC's mechanism of action. Our data are inconsistent with current models suggesting that high-affinity ßcat-binding sites on APC play key roles. Instead, they suggest that multiple ßcat-binding sites act additively to fine-tune signaling via cytoplasmic retention. We identify essential roles for two putative binding sites for new partners--20-amino-acid repeat 2 and conserved sequence B--in destruction complex action. Finally, we demonstrate that APC interacts with Axin by two different modes and provide evidence that conserved sequence B helps ensure release of APC from Axin, with disassembly critical in regulating ßcat levels. Using these data, we suggest a new model for destruction complex action in development, which also provides new insights into functions of truncated APC proteins in cancer. PMID:21471006

  5. Xenopus polo-like kinase Plx1 regulates XErp1, a novel inhibitor of APC/C activity

    PubMed Central

    Schmidt, Andreas; Duncan, Peter I.; Rauh, Nadine R.; Sauer, Guido; Fry, Andrew M.; Nigg, Erich A.; Mayer, Thomas U.

    2005-01-01

    Metaphase-to-anaphase transition is a fundamental step in cell cycle progression where duplicated sister-chromatids segregate to the future daughter cells. The anaphase-promoting complex/cyclosome (APC/C) is a highly regulated ubiquitin-ligase that triggers anaphase onset and mitotic exit by targeting securin and mitotic cyclins for destruction. It was previously shown that the Xenopus polo-like kinase Plx1 is essential to activate APC/C upon release from cytostatic factor (CSF) arrest in Xenopus egg extract. Although the mechanism by which Plx1 regulates APC/C activation remained unclear, the existence of a putative APC/C inhibitor was postulated whose activity would be neutralized by Plx1 upon CSF release. Here we identify XErp1, a novel Plx1-regulated inhibitor of APC/C activity, and we demonstrate that XErp1 is required to prevent anaphase onset in CSF-arrested Xenopus egg extract. Inactivation of XErp1 leads to premature APC/C activation. Conversely, addition of excess XErp1 to Xenopus egg extract prevents APC/C activation. Plx1 phosphorylates XErp1 in vitro at a site that targets XErp1 for degradation upon CSF release. Thus, our data lead to a model of APC/C activation in Xenopus egg extract in which Plx1 targets the APC/C inhibitor XErp1 for degradation. PMID:15713843

  6. Germ-line and somatic DICER1 mutations in pineoblastoma.

    PubMed

    de Kock, Leanne; Sabbaghian, Nelly; Druker, Harriet; Weber, Evan; Hamel, Nancy; Miller, Suzanne; Choong, Catherine S; Gottardo, Nicholas G; Kees, Ursula R; Rednam, Surya P; van Hest, Liselotte P; Jongmans, Marjolijn C; Jhangiani, Shalini; Lupski, James R; Zacharin, Margaret; Bouron-Dal Soglio, Dorothée; Huang, Annie; Priest, John R; Perry, Arie; Mueller, Sabine; Albrecht, Steffen; Malkin, David; Grundy, Richard G; Foulkes, William D

    2014-10-01

    Germ-line RB-1 mutations predispose to pineoblastoma (PinB), but other predisposing genetic factors are not well established. We recently identified a germ-line DICER1 mutation in a child with a PinB. This was accompanied by loss of heterozygosity (LOH) of the wild-type allele within the tumour. We set out to establish the prevalence of DICER1 mutations in an opportunistically ascertained series of PinBs. Twenty-one PinB cases were studied: Eighteen cases had not undergone previous testing for DICER1 mutations; three patients were known carriers of germ-line DICER1 mutations. The eighteen PinBs were sequenced by Sanger and/or Fluidigm-based next-generation sequencing to identify DICER1 mutations in blood gDNA and/or tumour gDNA. Testing for somatic DICER1 mutations was also conducted on one case with a known germ-line DICER1 mutation. From the eighteen PinBs, we identified four deleterious DICER1 mutations, three of which were germ line in origin, and one for which a germ line versus somatic origin could not be determined; in all four, the second allele was also inactivated leading to complete loss of DICER1 protein. No somatic DICER1 RNase IIIb mutations were identified. One PinB arising in a germ-line DICER1 mutation carrier was found to have LOH. This study suggests that germ-line DICER1 mutations make a clinically significant contribution to PinB, establishing DICER1 as an important susceptibility gene for PinB and demonstrates PinB to be a manifestation of a germ-line DICER1 mutation. The means by which the second allele is inactivated may differ from other DICER1-related tumours. PMID:25022261

  7. Targeting the DNA replication checkpoint by pharmacologic inhibition of Chk1 kinase: a strategy to sensitize APC mutant colon cancer cells to 5-fluorouracil chemotherapy

    PubMed Central

    Martino-Echarri, Estefania

    2014-01-01

    5-fluorouracil (5-FU) is the first line component used in colorectal cancer (CRC) therapy however even in combination with other chemotherapeutic drugs recurrence is common. Mutations of the adenomatous polyposis coli (APC) gene are considered as the initiating step of transformation in familial and sporadic CRCs. We have previously shown that APC regulates the cellular response to DNA replication stress and recently hypothesized that APC mutations might therefore influence 5-FU resistance. To test this, we compared CRC cell lines and show that those expressing truncated APC exhibit a limited response to 5-FU and arrest in G1/S-phase without undergoing lethal damage, unlike cells expressing wild-type APC. In SW480 APC-mutant CRC cells, 5-FU-dependent apoptosis was restored after transient expression of full length APC, indicating a direct link between APC and drug response. Furthermore, we could increase sensitivity of APC truncated cells to 5-FU by inactivating the Chk1 kinase using drug treatment or siRNA-mediated knockdown. Our findings identify mutant APC as a potential tumor biomarker of resistance to 5-FU, and importantly we show that APC-mutant CRC cells can be made more sensitive to 5-FU by use of Chk1 inhibitors. PMID:25301724

  8. Intestinal Peyer's patches prevent tumorigenesis in Apc (Min/+) mice.

    PubMed

    Fujimoto, Kyoko; Fujii, Gen; Sakurai, Hitomi; Yoshitome, Hiroko; Mutoh, Michihiro; Wada, Morimasa

    2015-01-01

    Peyer's patches are nodules that play a central role in intestinal immunity. Few studies demonstrate the relationship between the number of Peyer's patches and intestinal polyps. Here we identify a statistically significant inverse correlation between the quantity of Peyer's patches and of the development of intestinal polyps in Apc (Min/+) mice, which are a useful model to clarify the role of Peyer's patches in intestinal tumorigenesis. Using this model, we increased the number of Peyer's patches using 0.1% and 1% corn husk arabinoxylan through feed. Intestinal polyp formation significantly decreased, concomitant with an increase in Peyer's patches development (n = 12/group). In Aly (-/-) Apc (Min/+) mice (negative control; no Peyer's patches) there was no change in the amount of intestinal polyps (n = 10/group). Immune reaction following corn husk arabinoxylan treatment was measured by cytokine array. Increasing the number of Peyer's patches decreased interleukin-17 production, which showed a dose dependent correlation with transcription factor/lymphoid enhancer-binding factor. This study identified a relationship between levels of Peyer's patches and intestinal polyp formation, partly explained by the involvement of interleukin-17 production and β-catenin signaling in Apc (Min/+) mice. PMID:25678750

  9. Proteomic identification of germline proteins in Caenorhabditis elegans

    PubMed Central

    Turner, B Elizabeth; Basecke, Sophia M; Bazan, Grace C; Dodge, Eric S; Haire, Cassy M; Heussman, Dylan J; Johnson, Chelsey L; Mukai, Chelsea K; Naccarati, Adrianna M; Norton, Sunny-June; Sato, Jennifer R; Talavera, Chihara O; Wade, Michael V; Hillers, Kenneth J

    2015-01-01

    Sexual reproduction involves fusion of 2 haploid gametes to form diploid offspring with genetic contributions from both parents. Gamete formation represents a unique developmental program involving the action of numerous germline-specific proteins. In an attempt to identify novel proteins involved in reproduction and embryonic development, we have carried out a proteomic characterization of the process in Caenorhabditis elegans. To identify candidate proteins, we used 2D gel electrophoresis (2DGE) to compare protein abundance in nucleus-enriched extracts from wild-type C. elegans, and in extracts from mutant worms with greatly reduced gonads (glp-4(bn2) worms reared at 25°C); 84 proteins whose abundance correlated with germline presence were identified. To validate candidates, we used feeding RNAi to deplete candidate proteins, and looked for reduction in fertility and/or germline cytological defects. Of 20 candidates so screened for involvement in fertility, depletion of 13 (65%) caused a significant reduction in fertility, and 6 (30%) resulted in sterility (<5 % of wild-type fertility). Five of the 13 proteins with demonstrated roles in fertility have not previously been implicated in germline function. The high frequency of defects observed after RNAi depletion of candidate proteins suggests that this approach is effective at identifying germline proteins, thus contributing to our understanding of this complex organ. PMID:26435885

  10. CDKN2A Germline Mutations in Familial Pancreatic Cancer

    PubMed Central

    Bartsch, Detlef K.; Sina-Frey, Mercedes; Lang, Sven; Wild, Anja; Gerdes, Berthold; Barth, Peter; Kress, Ralf; Grützmann, Robert; Colombo-Benkmann, Mario; Ziegler, Andreas; Hahn, Stephan A.; Rothmund, Matthias; Rieder, Harald

    2002-01-01

    Objective To evaluate the prevalence of mutations in the CDKN2A gene encoding p16INK4a and p14ARF in familial pancreatic cancer (FPC). Summary Background Data The genetic basis of FPC is still widely unknown. Recently, it has been shown that germline mutations in the p16INK4a tumor suppressor gene can predispose to pancreatic cancer. The presence of p14ARF germline mutations has yet not been determined in this setting. Methods Eighteen families with at least two first-degree relatives with histologically confirmed pancreatic cancer and five families with at least one patient with pancreatic cancer and another first-degree relative with malignant melanoma of the German National Case Collection for Familial Pancreatic Cancer were analyzed for CDKN2A germline mutations including p16INK4a and p14ARF by direct DNA sequencing. All participating family members were genetically counseled and evaluated by a three-generation pedigree. Results None of 18 FPC families without malignant melanoma revealed p16INK4a mutations, compared to 2 of 5 families with pancreatic cancer and melanoma. Truncating p16INK4a germline mutations Q50X and E119X were identified in the affected patients of pancreatic cancer plus melanoma families. None of the 23 families revealed p14ARF germline mutations. Conclusions CDKN2A germline mutations are rare in FPC families. However, these data provide further evidence for a pancreatic cancer–melanoma syndrome associated with CDKN2A germline mutations affecting p16INK4a. Thus, all members of families with combined occurrence of pancreatic cancer and melanoma should be counseled and offered screening for p16INK4a mutations to identify high-risk family members who should be enrolled in a clinical screening program. PMID:12454511

  11. Multiloop photolithography control using hierarchical context information for APC models

    NASA Astrophysics Data System (ADS)

    Stuber, John D.

    2003-06-01

    Automated process control loops running in semiconductor manufacturing facilities must be able to compensate for machine variations as well as identify differences between products. With a number of exposure tools, pattern levels, and active devices manufactured in a typical ASIC fab, for photo APC system must maintain thousands of control loops. Control loop context information in TI's Semiconductor Manufacturing System (SMS) is defined in a hierarchal fashion which allows default values in a manufacturing specification to be overridden for particular products or lots. ProcessWORKS APC software automatically adds new control loops for new devices and pattern levels to a defined model structure. This system scales well and supports hundreds of thousands of control loops from a single database server in TI fabs. Application of product specific control systems for alignment and exposure control has provided increased exposure capacity due to decreased reworks and setup time, a substantial reduction in engineering maintenance and improved process capability. The APC system has evolved into a requirement for leading edge photolithography processes in Texas Instruments.

  12. Synergistic Blockade of Mitotic Exit by Two Chemical Inhibitors of the APC/C

    PubMed Central

    Sackton, Katharine L.; Dimova, Nevena; Zeng, Xing; Tian, Wei; Zhang, Mengmeng; Sackton, Timothy B.; Meaders, Johnathan; Pfaff, Kathleen L.; Sigoillot, Frederic; Yu, Hongtao; Luo, Xuelian; King, Randall W.

    2014-01-01

    Summary Protein machines are multi-subunit protein complexes that orchestrate highly regulated biochemical tasks. An example is the Anaphase-Promoting Complex/Cyclosome (APC/C), a thirteen-subunit ubiquitin ligase that initiates the metaphase-anaphase transition and mitotic exit by targeting proteins such as securin and cyclin B1 for ubiquitin-dependent destruction by the proteasome1,2. Because blocking mitotic exit is an effective approach for inducing tumor cell death3,4, the APC/C represents a potential novel target for cancer therapy. APC/C activation in mitosis requires binding of Cdc205, which forms a co-receptor with the APC/C to recognize substrates containing a Destruction box (D-box)6-14. Here we demonstrate that we can synergistically inhibit APC/C-dependent proteolysis and mitotic exit by simultaneously disrupting two protein-protein interactions within the APC/C-Cdc20-substrate ternary complex. We identified a small molecule, called apcin (APC inhibitor), which binds to Cdc20 and competitively inhibits the ubiquitylation of D-box-containing substrates. Analysis of the crystal structure of the apcin-Cdc20 complex suggests that apcin occupies the D-box-binding pocket on the side face of the WD40-domain. The ability of apcin to block mitotic exit is synergistically amplified by co-addition of tosyl-L-arginine methyl ester (TAME), a small molecule that blocks the APC/C-Cdc20 interaction15,16. This work suggests that simultaneous disruption of multiple, weak protein-protein interactions is an effective approach for inactivating a protein machine. PMID:25156254

  13. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1.

    PubMed

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-07-15

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1-3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4-9 did not influence the cell cycle-regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4-9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1. PMID:27226481

  14. Structure of an APC3–APC16 Complex: Insights into Assembly of the Anaphase-Promoting Complex/Cyclosome

    SciTech Connect

    Yamaguchi, Masaya; Yu, Shanshan; Qiao, Renping; Weissmann, Florian; Miller, Darcie J.; VanderLinden, Ryan; Brown, Nicholas G.; Frye, Jeremiah J.; Peters, Jan-Michael; Schulman, Brenda A.

    2015-08-21

    The anaphase-promoting complex/cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the “Platform” centers around a cullin-RING-like E3 ligase catalytic core; the “Arc Lamp” is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-2-fold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3's TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, via their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7 and contains a > 200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3's loop and IR tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer and, together with biochemistry and prior data, explain how APC16 recruits APC7 to APC3, show how APC3's C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C.

  15. Oncogenic mutations in adenomatous polyposis coli (Apc) activate mechanistic target of rapamycin complex 1 (mTORC1) in mice and zebrafish

    PubMed Central

    Valvezan, Alexander J.; Huang, Jian; Lengner, Christopher J.; Pack, Michael; Klein, Peter S.

    2014-01-01

    Truncating mutations in adenomatous polyposis coli (APC) are strongly linked to colorectal cancers. APC is a negative regulator of the Wnt pathway and constitutive Wnt activation mediated by enhanced Wnt–β-catenin target gene activation is believed to be the predominant mechanism responsible for APC mutant phenotypes. However, recent evidence suggests that additional downstream effectors contribute to APC mutant phenotypes. We previously identified a mechanism in cultured human cells by which APC, acting through glycogen synthase kinase-3 (GSK-3), suppresses mTORC1, a nutrient sensor that regulates cell growth and proliferation. We hypothesized that truncating Apc mutations should activate mTORC1 in vivo and that mTORC1 plays an important role in Apc mutant phenotypes. We find that mTORC1 is strongly activated in apc mutant zebrafish and in intestinal polyps in Apc mutant mice. Furthermore, mTORC1 activation is essential downstream of APC as mTORC1 inhibition partially rescues Apc mutant phenotypes including early lethality, reduced circulation and liver hyperplasia. Importantly, combining mTORC1 and Wnt inhibition rescues defects in morphogenesis of the anterior-posterior axis that are not rescued by inhibition of either pathway alone. These data establish mTORC1 as a crucial, β-catenin independent effector of oncogenic Apc mutations and highlight the importance of mTORC1 regulation by APC during embryonic development. Our findings also suggest a new model of colorectal cancer pathogenesis in which mTORC1 is activated in parallel with Wnt/β-catenin signaling. PMID:24092877

  16. Male Germline Stem Cells: From Mice to Men

    PubMed Central

    Brinster, Ralph L.

    2016-01-01

    The production of functional male gametes is dependent on the continuous activity of germline stem cells. The availability of a transplantation assay system to unequivocally identify male germline stem cells has allowed their in vitro culture, cryopreservation, and genetic modification. Moreover, the system has enabled the identification of conditions and factors involved in stem cell self-renewal, the foundation of spermatogenesis, and the production of spermatozoa. The increased knowledge about these cells is also of great potential practical value, for example, for the possible cryopreservation of stem cells from boys undergoing treatment for cancer to safeguard their germ line. PMID:17446391

  17. Lessons for Inductive Germline Determination

    PubMed Central

    Seervai, Riyad N.H.; Wessel, Gary M.

    2015-01-01

    SUMMARY Formation of the germline in an embryo marks a fresh round of reproductive potential, yet the developmental stage and location within the embryo where the primordial germ cells (PGCs) form differs wildly among species. In most animals, the germline is formed either by an inherited mechanism, in which maternal provisions within the oocyte drive localized germ-cell fate once acquired in the embryo, or an inductive mechanism that involves signaling between cells that directs germ-cell fate. The inherited mechanism has been widely studied in model organisms such as Drosophila melanogaster, Caenorhabditis elegans, Xenopus laevis, and Danio rerio. Given the rapid generation time and the effective adaptation for laboratory research of these organisms, it is not coincidental that research on these organisms has led the field in elucidating mechanisms for germline specification. The inductive mechanism, however, is less well understood and is studied primarily in the mouse (Mus musculus). In this review, we compare and contrast these two fundamental mechanisms for germline determination, beginning with the key molecular determinants that play a role in the formation of germ cells across all animal taxa. We next explore the current understanding of the inductive mechanism of germ-cell determination in mice, and evaluate the hypotheses for selective pressures on these contrasting mechanisms. We then discuss the hypothesis that the transition between these determination mechanisms, which has happened many times in phylogeny, is more of a continuum than a binary change. Finally, we propose an analogy between germline determination and sex determination in vertebrates—two of the milestones of reproduction and development—in which animals use contrasting strategies to activate similar pathways. PMID:23450642

  18. Co-regulation proteomics reveals substrates and mechanisms of APC/C-dependent degradation

    PubMed Central

    Singh, Sasha A; Winter, Dominic; Kirchner, Marc; Chauhan, Ruchi; Ahmed, Saima; Ozlu, Nurhan; Tzur, Amit; Steen, Judith A; Steen, Hanno

    2014-01-01

    Using multiplexed quantitative proteomics, we analyzed cell cycle-dependent changes of the human proteome. We identified >4,400 proteins, each with a six-point abundance profile across the cell cycle. Hypothesizing that proteins with similar abundance profiles are co-regulated, we clustered the proteins with abundance profiles most similar to known Anaphase-Promoting Complex/Cyclosome (APC/C) substrates to identify additional putative APC/C substrates. This protein profile similarity screening (PPSS) analysis resulted in a shortlist enriched in kinases and kinesins. Biochemical studies on the kinesins confirmed KIFC1, KIF18A, KIF2C, and KIF4A as APC/C substrates. Furthermore, we showed that the APC/CCDH1-dependent degradation of KIFC1 regulates the bipolar spindle formation and proper cell division. A targeted quantitative proteomics experiment showed that KIFC1 degradation is modulated by a stabilizing CDK1-dependent phosphorylation site within the degradation motif of KIFC1. The regulation of KIFC1 (de-)phosphorylation and degradation provides insights into the fidelity and proper ordering of substrate degradation by the APC/C during mitosis. PMID:24510915

  19. Targeting of Fzr/Cdh1 for timely activation of the APC/C at the centrosome during mitotic exit

    PubMed Central

    Meghini, Francesco; Martins, Torcato; Tait, Xavier; Fujimitsu, Kazuyuki; Yamano, Hiroyuki; Glover, David M.; Kimata, Yuu

    2016-01-01

    A multi-subunit ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), regulates critical cellular processes including the cell cycle. To accomplish its diverse functions, APC/C activity must be precisely regulated in time and space. The interphase APC/C activator Fizzy-related (Fzr or Cdh1) is localized at centrosomes in animal cells. However, neither the mechanism of its localization nor its importance is clear. Here we identify the centrosome component Spd2 as a major partner of Fzr in Drosophila. The localization of Fzr to the centriole during interphase depends on direct interaction with Spd2. By generating Spd2 mutants unable to bind Fzr, we show that centrosomal localization of Fzr is essential for optimal APC/C activation towards its centrosomal substrate Aurora A. Finally, we show that Spd2 is also a novel APC/CFzr substrate. Our study is the first to demonstrate the critical importance of distinct subcellular pools of APC/C activators in the spatiotemporal control of APC/C activity. PMID:27558644

  20. The actin-binding protein profilin is required for germline stem cell maintenance and germ cell enclosure by somatic cyst cells

    PubMed Central

    Shields, Alicia R.; Spence, Allyson C.; Yamashita, Yukiko M.; Davies, Erin L.; Fuller, Margaret T.

    2014-01-01

    Specialized microenvironments, or niches, provide signaling cues that regulate stem cell behavior. In the Drosophila testis, the JAK-STAT signaling pathway regulates germline stem cell (GSC) attachment to the apical hub and somatic cyst stem cell (CySC) identity. Here, we demonstrate that chickadee, the Drosophila gene that encodes profilin, is required cell autonomously to maintain GSCs, possibly facilitating localization or maintenance of E-cadherin to the GSC-hub cell interface. Germline specific overexpression of Adenomatous Polyposis Coli 2 (APC2) rescued GSC loss in chic hypomorphs, suggesting an additive role of APC2 and F-actin in maintaining the adherens junctions that anchor GSCs to the niche. In addition, loss of chic function in the soma resulted in failure of somatic cyst cells to maintain germ cell enclosure and overproliferation of transit-amplifying spermatogonia. PMID:24346697

  1. Attenuated APC alleles produce functional protein from internal translation initiation

    PubMed Central

    Heppner Goss, Kathleen; Trzepacz, Chris; Tuohy, Thérèse M. F.; Groden, Joanna

    2002-01-01

    Some truncating mutations of the APC tumor suppressor gene are associated with an attenuated phenotype of familial adenomatous polyposis coli (AAPC). This work demonstrates that APC alleles with 5′ mutations produce APC protein that down-regulates β-catenin, inhibits β-catenin/T cell factor-mediated transactivation, and induces cell-cycle arrest. Transfection studies demonstrate that cap-independent translation is initiated internally at an AUG at codon 184 of APC. Furthermore, APC coding sequence between AAPC mutations and AUG 184 permits internal ribosome entry in a bicistronic vector. These data suggest that AAPC alleles in vivo may produce functional APC by internal initiation and establish a functional correlation between 5′ APC mutations and their associated clinical phenotype. PMID:12034871

  2. Integrated metrology: an enabler for advanced process control (APC)

    NASA Astrophysics Data System (ADS)

    Schneider, Claus; Pfitzner, Lothar; Ryssel, Heiner

    2001-04-01

    Advanced process control (APC) techniques become more and more important as short innovation cycles in microelectronics and a highly competitive market requires cost-effective solutions in semiconductor manufacturing. APC marks a paradigm shift from statistically based techniques (SPC) using monitor wafers for sampling measurement data towards product wafer control. The APC functionalities including run-to-run control, fault detection, and fault analysis allow to detect process drifts and excursions at an early stage and to minimize the number of misprocessed wafers. APC is being established as part of factory control systems through the definition of an APC framework. A precondition for APC is the availability of sensors and measurement methods providing the necessary wafer data. This paper discusses integrated metrology as an enabler for APC and demonstrates practical implementations in semiconductor manufacturing.

  3. Physiological Control of Germline Development

    PubMed Central

    Hubbard, E. Jane Albert; Korta, Dorota Z.; Dalfó, Diana

    2013-01-01

    The intersection between developmental programs and environmental conditions that alter physiology is a growing area of research interest. The C. elegans germ line is emerging as a particularly sensitive and powerful model for these studies. The germ line is subject to environmentally regulated diapause points that allow worms to withstand harsh conditions both prior to and after reproduction commences. It also responds to more subtle changes in physiological conditions. Recent studies demonstrate that different aspects of germ line development are sensitive to environmental and physiological changes and that conserved signaling pathways such as the AMPK, Insulin/IGF, TGFβ, and TOR-S6K, and nuclear hormone receptor pathways mediate this sensitivity. Some of these pathways genetically interact with but appear distinct from previously characterized mechanisms of germline cell fate control such as Notch signaling. Here, we review several aspects of hermaphrodite germline development in the context of “feasting,” “food-limited,” and “fasting” conditions. We also consider connections between lifespan, metabolism and the germ line, and we comment on special considerations for examining germline development under altered environmental and physiological conditions. Finally, we summarize the major outstanding questions in the field. PMID:22872476

  4. Hereditary desmoid disease due to a frameshift mutation at codon 1924 of the APC gene.

    PubMed Central

    Eccles, D. M.; van der Luijt, R.; Breukel, C.; Bullman, H.; Bunyan, D.; Fisher, A.; Barber, J.; du Boulay, C.; Primrose, J.; Burn, J.; Fodde, R.

    1996-01-01

    Desmoid tumors are slowly growing fibrous tumors highly resistant to therapy and often fatal. Here, we report hereditary desmoid disease (HDD), a novel autosomal dominant trait with 100% penetrance affecting a three-generation kindred. Desmoid tumors are usually a complication of familial adenomatous polyposis, a predisposition to the early development of premalignant adenomatous polyps in the colorectum due to chain-terminating mutations of the APC gene. In general, one or more members in approximately 10% of the FAP families manifest desmoid tumors. Affected individuals from the HDD kindred are characterized by multifocal fibromatosis of the paraspinal muscles, breast, occiput, arms, lower ribs, abdominal wall, and mesentery. Osteomas, epidermal cysts, and other congenital features were also observed. We show that HDD segregates with an unusual germ-line chain-terminating mutation at the 3' end of the APC gene (codon 1924) with somatic loss of the wild-type allele leading to tumor development. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:8940264

  5. Attenuated familial adenomatous polyposis with desmoids caused by an APC mutation.

    PubMed

    Ikenoue, Tsuneo; Yamaguchi, Kiyoshi; Komura, Mitsuhiro; Imoto, Seiya; Yamaguchi, Rui; Shimizu, Eigo; Kasuya, Shinichi; Shibuya, Tetsuo; Hatakeyama, Seira; Miyano, Satoru; Furukawa, Yoichi

    2015-01-01

    We present here a case of attenuated familial adenomatous polyposis (AFAP) with a family history of desmoids and thyroid tumors. This patient had no colonic polyps but did have multiple desmoids. Genetic analysis identified a 4-bp deletion in codon 2644 (c.7932_7935delTTAT: p.Tyr2645LysfsX14) of the adenomatous polyposis coli (APC) gene. In cases with limited numbers of colonic polyps and desmoids, AFAP may be caused by a mutation in the 3' region of APC. PMID:27081525

  6. Attenuated familial adenomatous polyposis with desmoids caused by an APC mutation

    PubMed Central

    Ikenoue, Tsuneo; Yamaguchi, Kiyoshi; Komura, Mitsuhiro; Imoto, Seiya; Yamaguchi, Rui; Shimizu, Eigo; Kasuya, Shinichi; Shibuya, Tetsuo; Hatakeyama, Seira; Miyano, Satoru; Furukawa, Yoichi

    2015-01-01

    We present here a case of attenuated familial adenomatous polyposis (AFAP) with a family history of desmoids and thyroid tumors. This patient had no colonic polyps but did have multiple desmoids. Genetic analysis identified a 4-bp deletion in codon 2644 (c.7932_7935delTTAT: p.Tyr2645LysfsX14) of the adenomatous polyposis coli (APC) gene. In cases with limited numbers of colonic polyps and desmoids, AFAP may be caused by a mutation in the 3′ region of APC. PMID:27081525

  7. Molecular analysis of the APC gene in 71 Israeli families: 17 novel mutations.

    PubMed

    Gavert, Nancy; Yaron, Yuval; Naiman, Tova; Bercovich, Dani; Rozen, Paul; Shomrat, Ruth; Legum, Cyril; Orr-Urtreger, Avi

    2002-06-01

    Familial adenomatous polyposis (FAP) is caused by germline mutations in the APC gene. This study included 71 Israeli families referred for molecular analysis of the APC gene. Analysis was performed by the protein truncation test (PTT) of exon 15, and if negative, by direct sequencing of exon 1 to 14. Mutations were found in 36 (50.7%) probands. Mutation detection rates depended on the pattern of referral, such that among the 40 probands referred from the Service for Hereditary Cancer the mutation detection rate was 70%, whereas among the 31 probands referred by other gastroenterologists detection rate was significantly lower (25.8%). Of the 36 mutations detected, 21 were within exon 15, 13 within exons 1 to 14 and 2 were newly-described splicing mutations in introns 9 and 14. A relatively high proportion of the mutations was detected in exon 9 (6/36), five of them newly described. Altogether, we describe here 17 new mutations. Within the two major ethnic groups in Israel, patients of Ashkenazi and non-Ashkenazi origin, there was no significant differences in the mutation detection rate or the distribution of mutations within the APC gene. No founder mutation was detected in any of these populations. Our data confirm that higher detection rates may be expected in patients referred by clinical services specializing in hereditary colon cancer. These results further underscore the importance of complete analysis of all exons and exon/intron boundaries, in order to achieve maximal detection rate in patients suspected of FAP. PMID:12007223

  8. Apc-driven colon carcinogenesis in Pirc rat is strongly reduced by polyethylene glycol.

    PubMed

    Femia, Angelo Pietro; Becherucci, Caterina; Crucitta, Stefania; Caderni, Giovanna

    2015-11-01

    Polyethylene glycol (PEG) is one of the most powerful agents in reducing chemically induced carcinogenesis in rat colon. However, contrasting results in Min mice dampened the enthusiasm on this potentially strong and virtually safe, cancer chemopreventing agent. Pirc (F344/NTac-Apc (am1137) ) rats carrying a germline heterozygous mutation in the Apc gene, spontaneously develop multiple tumours in the colon thus modelling both familial adenomatous polyposis (FAP) and sporadic colorectal cancer (CRC). Given this similarity, we thought that these rats could be appropriate to test the efficacy of PEG 8000 in reducing carcinogenesis. Pirc male rats aged one month were treated with 5% PEG in drinking water for 2 or 6 months. Precancerous lesions were dramatically reduced after 2 months of PEG treatment (Mucin depleted foci (MDF)/colon were 99 ± 17 and 12 ± 8 in Controls and PEG-treated rats, respectively; p < 0.001; mean ± SD). Similarly, colon tumors were significantly reduced after 6 months of treatment (tumors/rat were 8.1 ± 2.3 and 3.6 ± 2.2 in Controls and PEG-treated rats, respectively; p < 0.05; mean ± SD). Colon proliferation, a parameter correlated to cancer risk, was also significantly lower in PEG-treated rats than in Controls, while apoptosis was not significantly affected. In conclusion, PEG markedly reduces colon carcinogenesis in Pirc rats mutated in Apc; we thus suggest that PEG may be used as chemopreventive agent to reduce cancer risk in FAP and CRC patients. PMID:25912754

  9. Molecular mechanism of APC/C activation by mitotic phosphorylation.

    PubMed

    Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

    2016-05-12

    In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

  10. APC/C-Mediated Degradation of dsRNA-Binding Protein 4 (DRB4) Involved in RNA Silencing

    PubMed Central

    Marrocco, Katia; Criqui, Marie-Claire; Zervudacki, Jérôme; Schott, Gregory; Eisler, Herfried; Parnet, Aude; Dunoyer, Patrice; Genschik, Pascal

    2012-01-01

    Background Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized. Methodology/Principal Findings In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed. Conclusions/Significance Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular

  11. APC is an RNA-Binding Protein and its Interactome Provides a Link to Neural Development and Microtubule Assembly

    PubMed Central

    Preitner, Nicolas; Quan, Jie; Nowakowski, Dan W.; Hancock, Melissa L.; Shi, Jianhua; Tcherkezian, Joseph; Young-Pearse, Tracy L.; Flanagan, John G.

    2014-01-01

    SUMMARY Adenomatous polyposis coli (APC) is a microtubule plus-end scaffolding protein important in biology and disease. APC is implicated in RNA localization, although the mechanisms and functional significance remain unclear. We show that APC is an RNA-binding protein, and identify an RNA interactome by HITS-CLIP. Targets were highly enriched for APC-related functions, including microtubule organization, cell motility, cancer and neurologic disease. Among the targets is β2B-tubulin, known to be required in human neuron and axon migration. We show β2B-tubulin is synthesized in axons and localizes preferentially to dynamic microtubules in the growth cone periphery. APC binds the β2B-tubulin 3'UTR; treatments interfering with this interaction reduced β2B-tubulin mRNA axonal localization and expression, depleted dynamic microtubules and the growth cone periphery, and impaired neuron migration. These results identify APC as a platform binding functionally-related protein and RNA networks, and suggest a self-organizing model for the microtubule to localize synthesis of its own subunits. PMID:25036633

  12. Analysis of APC mutation in human ameloblastoma and clinical significance.

    PubMed

    Li, Ning; Liu, Bing; Sui, Chengguang; Jiang, Youhong

    2016-01-01

    As a highly conserved signaling pathway, Wnt/β-catenin signal transduction pathway plays an important role in many processes. Either in the occurrence or development of tumor, activation of this pathway takes an important place. APC inhibits Wnt/β-catenin pathway to regulate cell proliferation and differentiation. This study aimed to investigate the function of cancer suppressor gene. PCR amplification and sequencing method was used to analyze APC mutations of human clinical specimens. The pathological specimens were collected for PCR and clear electrophoretic bands were obtained after electrophoresis. The gene sequence obtained after purification and sequencing analysis was compared with the known APC gene sequence (NM_000038.5). Base mutations at APC 1543 (T → C), APC-4564 (G → A), APC-5353 (T → G), APC-5550 (T → A) and APC-5969 (G → A) locus existed in 22 (27.5 %), 12 (15 %), 5 (6.25 %), 13 (16.25 %) and 12 patients (15 %), respectively. Gene mutations existed in ameloblastoma, and the mutation loci were 1543 locus (T → C), 4564 locus (G → A), 5353 locus (T → G), 5550 locus (T → A) and 5969 locus (G → A) 15 %, respectively. APC mutation plays a certain role in monitoring the tumor malignant degree as it may indicate the transition process of ameloblastoma malignant phenotype. PMID:27065015

  13. Molecular genetic analysis of exons 1 to 6 of the APC gene in non-polyposis familial colorectal cancer.

    PubMed

    Joyce, J A; Froggatt, N J; Davies, R; Evans, D G; Trembath, R; Barton, D E; Maher, E R

    1995-12-01

    Familial adenomatous polyposis coli is caused by constitutional mutations in the APC gene. The hallmark of familial adenomatous polyposis coli is the presence of numerous (> 100) colorectal polyps, but mutations in the 5' end of the APC gene have been associated with familial colorectal cancer without florid polyposis. Although familial adenomatous polyposis coli accounts for only a minority of familial colorectal cancer cases, we hypothesised that APC mutations which were not associated with florid polyposis might make a significant contribution to nonpolyposis familial colorectal cancer. To investigate this possibility, we analysed 40 unrelated patients with familial colorectal cancer without classical familial adenomatous polyposis coli for mutations in exons 1 to 6 (codons 1 to 243) of the APC gene. No mutations were detected, but a C-->T polymorphism at nucleotide 333 (Arg-->Trp at codon 99) was identified. No 5' APC mutations were detected in two patients with desmoid tumours and a family history of colorectal cancer and polyps. We conclude that mutations in exons 1 to 6 of the APC gene are infrequent in patients with familial colorectal cancer who do not have many colorectal polyps. PMID:8835324

  14. Germline mosaicism at the fragile X locus

    SciTech Connect

    Papp, A.C.; Snyder, P.J.; Sedra, M.S.

    1994-09-01

    The fragile X full mutation, which is associated with the phenotypic expression of the disorder, is characterized by an expansion of CGG repeat and hypermethylation of the CpG island adjacent to the FMR1 gene. New mutations leading to amplification of the CGG repeat have not been reported. We have identified a fragile X syndrome pedigree where the disorder is associated with a molecular deletion. The deletion was present in the DNA of two affected sons but was absent in the mother`s somatic cell (lymphocyte) DNA. This was confirmed by dosage analysis of the Southern blot using StB12-3 and an additional probe against the dystrophin gene and by PCR analysis of DXS548 alleles. The results are consistent with the deletion arising as a postzygotic event in the mother, who therefore is germinally mosaic. The case reported here clearly demonstrates that FMR1 deletions, unlike the expansions, are not always inherited and the finding of heterozygosity or normal dosage from lymphocyte DNA in the mother of a deletion case does not necessarily rule out the possibility of having a second affected child. The deletion of FMR1 gene may be responsible for a small but significant number of fragile X cases. Therefore, it is imperative that those involved in genetic counseling recognize this diagnostic pitfall. Since it depends upon the size of the mutant clone in the mosaic mother, the exact recurrence risk in germline carriers is unknown. However, prenatal and carrier testing should be performed independently of the outcome of the mother. Furthermore, it is possible that the deletion may not be restricted to the germline, and therefore the mother may actually be a somatic mosaic.

  15. Inducible loss of one Apc allele in Lrig1-expressing progenitor cells results in multiple distal colonic tumors with features of familial adenomatous polyposis

    PubMed Central

    Powell, Anne E.; Vlacich, Gregory; Zhao, Zhen-Yang; McKinley, Eliot T.; Washington, M. Kay; Manning, H. Charles

    2014-01-01

    Individuals with familial adenomatous polyposis (FAP) harbor a germline mutation in adenomatous polyposis coli (APC). The major clinical manifestation is development of multiple colonic tumors at a young age due to stochastic loss of the remaining APC allele. Extracolonic features, including periampullary tumors, gastric abnormalities, and congenital hypertrophy of the retinal pigment epithelium, may occur. The objective of this study was to develop a mouse model that simulates these features of FAP. We combined our Lrig1-CreERT2/+ mice with Apcfl/+ mice, eliminated one copy of Apc in leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1)-positive (Lrig1+) progenitor cells with tamoxifen injection, and monitored tumor formation in the colon by colonoscopy and PET. Initial loss of one Apc allele in Lrig1+ cells results in a predictable pattern of preneoplastic changes, culminating in multiple distal colonic tumors within 50 days of induction, as well as the extracolonic manifestations of FAP mentioned above. We show that tumor formation can be monitored by noninvasive PET imaging. This inducible stem cell-driven model recapitulates features of FAP and offers a tractable platform on which therapeutic interventions can be monitored over time by colonoscopy and noninvasive imaging. PMID:24833705

  16. GPCRs Direct Germline Development and Somatic Gonad Function in Planarians

    PubMed Central

    Saberi, Amir; Beets, Isabel; Schoofs, Liliane; Newmark, Phillip A.

    2016-01-01

    Planarians display remarkable plasticity in maintenance of their germline, with the ability to develop or dismantle reproductive tissues in response to systemic and environmental cues. Here, we investigated the role of G protein-coupled receptors (GPCRs) in this dynamic germline regulation. By genome-enabled receptor mining, we identified 566 putative planarian GPCRs and classified them into conserved and phylum-specific subfamilies. We performed a functional screen to identify NPYR-1 as the cognate receptor for NPY-8, a neuropeptide required for sexual maturation and germ cell differentiation. Similar to NPY-8, knockdown of this receptor results in loss of differentiated germ cells and sexual maturity. NPYR-1 is expressed in neuroendocrine cells of the central nervous system and can be activated specifically by NPY-8 in cell-based assays. Additionally, we screened the complement of GPCRs with expression enriched in sexually reproducing planarians, and identified an orphan chemoreceptor family member, ophis, that controls differentiation of germline stem cells (GSCs). ophis is expressed in somatic cells of male and female gonads, as well as in accessory reproductive tissues. We have previously shown that somatic gonadal cells are required for male GSC specification and maintenance in planarians. However, ophis is not essential for GSC specification or maintenance and, therefore, defines a secondary role for planarian gonadal niche cells in promoting GSC differentiation. Our studies uncover the complement of planarian GPCRs and reveal previously unappreciated roles for these receptors in systemic and local (i.e., niche) regulation of germ cell development. PMID:27163480

  17. GPCRs Direct Germline Development and Somatic Gonad Function in Planarians.

    PubMed

    Saberi, Amir; Jamal, Ayana; Beets, Isabel; Schoofs, Liliane; Newmark, Phillip A

    2016-05-01

    Planarians display remarkable plasticity in maintenance of their germline, with the ability to develop or dismantle reproductive tissues in response to systemic and environmental cues. Here, we investigated the role of G protein-coupled receptors (GPCRs) in this dynamic germline regulation. By genome-enabled receptor mining, we identified 566 putative planarian GPCRs and classified them into conserved and phylum-specific subfamilies. We performed a functional screen to identify NPYR-1 as the cognate receptor for NPY-8, a neuropeptide required for sexual maturation and germ cell differentiation. Similar to NPY-8, knockdown of this receptor results in loss of differentiated germ cells and sexual maturity. NPYR-1 is expressed in neuroendocrine cells of the central nervous system and can be activated specifically by NPY-8 in cell-based assays. Additionally, we screened the complement of GPCRs with expression enriched in sexually reproducing planarians, and identified an orphan chemoreceptor family member, ophis, that controls differentiation of germline stem cells (GSCs). ophis is expressed in somatic cells of male and female gonads, as well as in accessory reproductive tissues. We have previously shown that somatic gonadal cells are required for male GSC specification and maintenance in planarians. However, ophis is not essential for GSC specification or maintenance and, therefore, defines a secondary role for planarian gonadal niche cells in promoting GSC differentiation. Our studies uncover the complement of planarian GPCRs and reveal previously unappreciated roles for these receptors in systemic and local (i.e., niche) regulation of germ cell development. PMID:27163480

  18. Investigating polymorphisms by bioinformatics is a potential cost-effective method to screen for germline mutations in Chinese familial adenomatous polyposis patients

    PubMed Central

    YANG, JUN; LIU, WEI QING; LI, WEN LIANG; CHEN, CHENG; ZHU, ZHU; HONG, MIN; WANG, ZHI QIANG; DONG, JIAN

    2016-01-01

    The aim of this study was to investigate germline mutations of the APC, MUTYH and AXIN2 genes in Chinese patients with familial adenomatous polyposis (FAP), and further assess the value of bioinformatics in screening the pathogenic changes predisposing to FAP. APC genes from 11 unrelated FAP patients in Yunnan province in China were firstly examined by exon-specific DNA sequencing. For samples without already known pathogenic changes predisposing to FAP in the APC gene, whole-gene sequencing of MUTYH and AXIN2 was performed. Mutational analysis of each gene was performed by bioinformatics. Eleven different types of APC polymorphisms were observed in the cohort of families analyzed. Of these polymorphisms, four were missense substitutions (V1822D, V1173G, P1760H and K2057), one was a nonsense substitution (S1196X), and six were silent substitutions (Y486Y, T449T, T1493T, G1678G, S1756S and P1960P). One missense mutation (Q335H) and two intronic substitutions (c.264+11G>A and c.420+35A>G) were detected in the MUTYH gene, and four synonymous mutations (I144I, P455P, P462P and L688L) and three intonic mutations (c.1060–77G>T, c.1060–287A>G and c.1060–282 A>G) of the AXIN2 gene were observed. In addition to the already reported pathogenic mutations, by using function assessment tools and databases, the synonymous substitutions observed in the APC gene of our samples were predicted to affect splicing regulation in the translation of mRNA, while the missense mutations observed in the APC gene and MUTYH gene were predicted to be disease-related polymorphisms; however, no functional effect of the mutations was observed in the AXIN2 gene. Comprehensive screening for germline mutations in APC, MUTYH and AXIN2 genes followed by prediction of pathogenicity using bioinformatic tools contributes to a cost-effective way of screening germline mutations in Chinese familial adenomatous polyposis patients. PMID:27347161

  19. The postsynaptic adenomatous polyposis coli (APC) multiprotein complex is required for localizing neuroligin and neurexin to neuronal nicotinic synapses in vivo.

    PubMed

    Rosenberg, Madelaine M; Yang, Fang; Mohn, Jesse L; Storer, Elizabeth K; Jacob, Michele H

    2010-08-18

    Synaptic efficacy requires that presynaptic and postsynaptic specializations align precisely and mature coordinately. The underlying mechanisms are poorly understood, however. We propose that adenomatous polyposis coli protein (APC) is a key coordinator of presynaptic and postsynaptic maturation. APC organizes a multiprotein complex that directs nicotinic acetylcholine receptor (nAChR) localization at postsynaptic sites in avian ciliary ganglion neurons in vivo. We hypothesize that the APC complex also provides retrograde signals that direct presynaptic active zones to develop in register with postsynaptic nAChR clusters. In our model, the APC complex provides retrograde signals via postsynaptic neuroligin that interacts extracellularly with presynaptic neurexin. S-SCAM (synaptic cell adhesion molecule) and PSD-93 (postsynaptic density-93) are scaffold proteins that bind to neuroligin. We identify S-SCAM as a novel component of neuronal nicotinic synapses. We show that S-SCAM, PSD-93, neuroligin and neurexin are enriched at alpha3*-nAChR synapses. PSD-93 and S-SCAM bind to APC and its binding partner beta-catenin, respectively. Blockade of selected APC and beta-catenin interactions, in vivo, leads to decreased postsynaptic accumulation of S-SCAM, but not PSD-93. Importantly, neuroligin synaptic clusters are also decreased. On the presynaptic side, there are decreases in neurexin and active zone proteins. Further, presynaptic terminals are less mature structurally and functionally. We define a novel neural role for APC by showing that the postsynaptic APC multiprotein complex is required for anchoring neuroligin and neurexin at neuronal synapses in vivo. APC human gene mutations correlate with autism spectrum disorders, providing strong support for the importance of the association, demonstrated here, between APC, neuroligin and neurexin. PMID:20720115

  20. Adenomatous polyposis coli (APC) membrane recruitment 3, a member of the APC membrane recruitment family of APC-binding proteins, is a positive regulator of Wnt-β-catenin signalling.

    PubMed

    Brauburger, Katharina; Akyildiz, Senem; Ruppert, Jan G; Graeb, Michael; Bernkopf, Dominic B; Hadjihannas, Michel V; Behrens, Jürgen

    2014-02-01

    The adenomatous polyposis coli (APC) membrane recruitment (Amer) family proteins Amer1/Wilms tumour gene on the X chromosome and Amer2 are binding partners of the APC tumour suppressor protein, and act as negative regulators in the Wnt signalling cascade. So far, nothing has been known about the third member of the family, Amer3. Here we show that Amer3 binds to the armadillo repeat domain of APC, similarly to Amer1 and Amer2. Amer3 also binds to the Wnt pathway regulator conductin/axin2. Furthermore, we identified Amer1 as binding partner of Amer3. Whereas Amer1 and Amer2 are linked to the plasma membrane by an N-terminal membrane localization domain, Amer3 lacks this domain. Amer3 localizes to the cytoplasm and nucleus of epithelial cells, and this is dependent on specific nuclear import and export sequences. Functionally, exogenous Amer3 enhances the expression of a β-catenin/T-cell factor-dependent reporter gene, and knockdown of endogenous Amer3 reduces Wnt target gene expression in colorectal cancer cells. Thus, Amer3 acts as an activator of Wnt signalling, in contrast to Amer1 and Amer2, which are inhibitors, suggesting a nonredundant role of Amer proteins in the regulation of this pathway. Our data, together with those of previous studies, provide a comprehensive picture of similarities and differences within the Amer protein family. PMID:24251807

  1. Interaction between APC and Fen1 during breast carcinogenesis.

    PubMed

    Narayan, Satya; Jaiswal, Aruna S; Law, Brian K; Kamal, Mohammad A; Sharma, Arun K; Hromas, Robert A

    2016-05-01

    Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER - adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) - promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and

  2. Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests

    PubMed Central

    Syred, Heather M.; van der Sar, Sjaak; Patel, Hitesh; Moresco, James J.; Sarkeshik, Ali; Yates, John R.; Rappsilber, Juri; Hardwick, Kevin G.

    2016-01-01

    The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1Mph1 kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1Mph1 kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors in vitro, demonstrating that Mad3p modification can directly influence Cdc20Slp1-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1Mph1 kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests. PMID:26882497

  3. Sex-lethal, master and slave: a hierarchy of germ-line sex determination in Drosophila.

    PubMed

    Oliver, B; Kim, Y J; Baker, B S

    1993-11-01

    Female sex determination in the germ line of Drosophila melanogaster is regulated by genes functioning in the soma as well as genes that function within the germ line. Genes known or suspected to be involved in germ-line sex determination in Drosophila melanogaster have been examined to determine if they are required upstream or downstream of Sex-lethal+, a known germ-line sex determination gene. Seven genes required for female-specific splicing of germ-line Sex-lethal+ pre-mRNA are identified. These results together with information about the tissues in which these genes function and whether they control sex determination and viability or just sex determination in the germ line have been used to deduce the genetic hierarchy regulating female germ-line sex determination. This hierarchy includes the somatic sex determination genes transformer+, transformer-2+ and doublesex+ (and by inference Sex-lethal+), which control a somatic signal required for female germ-line sex determination, and the germ-line ovarian tumor genes fused+, ovarian tumor+, ovo+, sans fille+, and Sex-lethal+, which are involved in either the reception or interpretation of this somatic sex determination signal. The fused+, ovarian tumor+, ovo+ and sans fille+ genes function upstream of Sex-lethal+ in the germ line. PMID:8187645

  4. SCML2 Establishes the Male Germline Epigenome through Regulation of Histone H2A Ubiquitination

    PubMed Central

    Hasegawa, Kazuteru; Sin, Ho-Su; Maezawa, So; Broering, Tyler J.; Kartashov, Andrey V.; Alavattam, Kris G.; Ichijima, Yosuke; Zhang, Fan; Bacon, W. Clark; Greis, Kenneth D.; Andreassen, Paul R.; Barski, Artem; Namekawa, Satoshi H.

    2015-01-01

    SUMMARY Gametogenesis is dependent on the expression of germline-specific genes. However, it remains unknown how the germline epigenome is distinctly established from that of somatic lineages. Here we show that genes commonly expressed in somatic lineages and spermatogenesis-progenitor cells undergo repression in a genome-wide manner in late stages of the male germline and identify underlying mechanisms. SCML2, a germline-specific subunit of a Polycomb repressive complex 1 (PRC1), establishes the unique epigenome of the male germline through two distinct antithetical mechanisms. SCML2 works with PRC1 and promotes RNF2-dependent ubiquitination of H2A, thereby marking somatic/progenitor genes on autosomes for repression. Paradoxically, SCML2 also prevents RNF2-dependent ubiquitination of H2A on sex chromosomes during meiosis, thereby enabling unique epigenetic programming of sex chromosomes for male reproduction. Our results reveal divergent mechanisms involving a shared regulator by which the male germline epigenome is distinguished from that of the soma and progenitor cells. PMID:25703348

  5. Germline modification of domestic animals

    PubMed Central

    Tang, L.; González, R.; Dobrinski, I.

    2016-01-01

    Genetically-modified domestic animal models are of increasing significance in biomedical research and agriculture. As authentic ES cells derived from domestic animals are not yet available, the prevailing approaches for engineering genetic modifications in those animals are pronuclear microinjection and somatic cell nuclear transfer (SCNT, also known as cloning). Both pronuclear microinjection and SCNT are inefficient, costly, and time-consuming. In animals produced by pronuclear microinjection, the exogenous transgene is usually inserted randomly into the genome, which results in highly variable expression patterns and levels in different founders. Therefore, significant efforts are required to generate and screen multiple founders to obtain animals with optimal transgene expression. For SCNT, specific genetic modifications (both gain-of-function and loss-of-function) can be engineered and carefully selected in the somatic cell nucleus before nuclear transfer. SCNT has been used to generate a variety of genetically modified animals such as goats, pigs, sheep and cattle; however, animals resulting from SCNT frequently suffer from developmental abnormalities associated with incomplete nuclear reprogramming. Other strategies to generate genetically-modified animals rely on the use of the spermatozoon as a natural vector to introduce genetic material into the female gamete. This sperm mediated DNA transfer (SMGT) combined with intracytoplasmatic sperm injection (ICSI) has relatively high efficiency and allows the insertion of large DNA fragments, which, in turn, enhance proper gene expression. An approach currently being developed to complement SCNT for producing genetically modified animals is germ cell transplantation using genetically modified male germline stem cells (GSCs). This approach relies on the ability of GSCs that are genetically modified in vitro to colonize the recipient testis and produce donor derived sperm upon transplantation. As the genetic change

  6. Assigning and visualizing germline genes in antibody repertoires

    PubMed Central

    Frost, Simon D. W.; Murrell, Ben; Hossain, A. S. Md. Mukarram; Silverman, Gregg J.; Pond, Sergei L. Kosakovsky

    2015-01-01

    Identifying the germline genes involved in immunoglobulin rearrangements is an essential first step in the analysis of antibody repertoires. Based on our prior work in analysing diverse recombinant viruses, we present IgSCUEAL (Immunoglobulin Subtype Classification Using Evolutionary ALgorithms), a phylogenetic approach to assign V and J regions of immunoglobulin sequences to their corresponding germline alleles, with D regions assigned using a simple pairwise alignment algorithm. We also develop an interactive web application for viewing the results, allowing the user to explore the frequency distribution of sequence assignments and CDR3 region length statistics, which is useful for summarizing repertoires, as well as a detailed viewer of rearrangements and region alignments for individual query sequences. We demonstrate the accuracy and utility of our method compared with sequence similarity-based approaches and other non-phylogenetic model-based approaches, using both simulated data and a set of evaluation datasets of human immunoglobulin heavy chain sequences. IgSCUEAL demonstrates the highest accuracy of V and J assignment amongst existing approaches, even when the reassorted sequence is highly mutated, and can successfully cluster sequences on the basis of shared V/J germline alleles. PMID:26194754

  7. Assigning and visualizing germline genes in antibody repertoires.

    PubMed

    Frost, Simon D W; Murrell, Ben; Hossain, A S Md Mukarram; Silverman, Gregg J; Pond, Sergei L Kosakovsky

    2015-09-01

    Identifying the germline genes involved in immunoglobulin rearrangements is an essential first step in the analysis of antibody repertoires. Based on our prior work in analysing diverse recombinant viruses, we present IgSCUEAL (Immunoglobulin Subtype Classification Using Evolutionary ALgorithms), a phylogenetic approach to assign V and J regions of immunoglobulin sequences to their corresponding germline alleles, with D regions assigned using a simple pairwise alignment algorithm. We also develop an interactive web application for viewing the results, allowing the user to explore the frequency distribution of sequence assignments and CDR3 region length statistics, which is useful for summarizing repertoires, as well as a detailed viewer of rearrangements and region alignments for individual query sequences. We demonstrate the accuracy and utility of our method compared with sequence similarity-based approaches and other non-phylogenetic model-based approaches, using both simulated data and a set of evaluation datasets of human immunoglobulin heavy chain sequences. IgSCUEAL demonstrates the highest accuracy of V and J assignment amongst existing approaches, even when the reassorted sequence is highly mutated, and can successfully cluster sequences on the basis of shared V/J germline alleles. PMID:26194754

  8. Functional Comparison of Human Adenomatous Polyposis Coli (APC) and APC-Like in Targeting Beta-Catenin for Degradation

    PubMed Central

    Schneikert, Jean; Vijaya Chandra, Shree Harsha; Ruppert, Jan Gustav; Ray, Suparna; Wenzel, Eva Maria; Behrens, Jürgen

    2013-01-01

    Truncating mutations affect the adenomatous polyposis coli (APC) gene in most cases of colon cancer, resulting in the stabilization of β-catenin and uncontrolled cell proliferation. We show here that colon cancer cell lines express also the paralog APC-like (APCL or APC2). RNA interference revealed that it controls the level and/or the activity of β-catenin, but it is less efficient and binds less well to β-catenin than APC, thereby providing one explanation as to why the gene is not mutated in colon cancer. A further comparison indicates that APCL down-regulates the β-catenin level despite the lack of the 15R region known to be important in APC. To understand this discrepancy, we performed immunoprecipitation experiments that revealed that phosphorylated β-catenin displays a preference for binding to the 15 amino acid repeats (15R) rather than the first 20 amino acid repeat of APC. This suggests that the 15R region constitutes a gate connecting the steps of β-catenin phosphorylation and subsequent ubiquitination/degradation. Using RNA interference and domain swapping experiments, we show that APCL benefits from the 15R of truncated APC to target β-catenin for degradation, in a process likely involving heterodimerization of the two partners. Our data suggest that the functional complementation of APCL by APC constitutes a substantial facet of tumour development, because the truncating mutations of APC in colorectal tumours from familial adenomatous polyposis (FAP) patients are almost always selected for the retention of at least one 15R. PMID:23840886

  9. Germline BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers

    PubMed Central

    Abdel-Rahman, Mohamed H; Pilarski, Robert; Cebulla, Colleen M; Massengill, James B; Christopher, Benjamin N; Boru, Getachew; Hovland, Peter; Davidorf, Frederick H

    2013-01-01

    Objective To investigate the potential contribution of germline sequence alterations in the BAP1 gene in uveal melanoma (UM) patients with possible predisposition to hereditary cancer. Design A total of 53 unrelated UM patients with high risk for hereditary cancer and five additional family members of one proband were studied. Mutational screening was carried out by direct sequencing. Results Of the 53 UM patients studied, a single patient was identified with a germline BAP1 truncating mutation, c. 799 C→T (p.Q267X), which segregated in several family members and was associated with UM and other cancers. Biallelic inactivation of BAP1 and decreased BAP1 expression were identified in the UM, lung adenocarcinoma and meningioma tumours from three family members with this germline BAP1 mutation. Germline BAP1 variants of uncertain significance, likely non-pathogenic, were also identified in two additional UM patients. Conclusion This study reports a novel hereditary cancer syndrome caused by a germline BAP1 mutation that predisposes patients to UM, lung carcinoma, meningioma, and possibly other cancers. The results indicate that BAP1 is the candidate gene in only a small subset of hereditary UM, suggesting the contribution of other candidate genes. PMID:21941004

  10. Regulation of the expression of human C[epsilon] germline transcript

    SciTech Connect

    Ichiki, T.; Takahashi, W.; Watanabe, T. )

    1993-06-15

    Transcriptional regulation for Ig H chain germline transcripts induced by cytokines is a topic of recent interest for the understanding of the mechanism of class switch recombination. Among human B cell lines examined, the authors have found that a human IgM-producing B cell line, DND39 (EBV negative) expressed germ-line transcripts of [epsilon] constant gene (C[epsilon]) when stimulated with lL-4. In this study, the regulatory element responsible for the expression of lL-4-induced human C[epsilon] germ-line transcript was determined using DND39 cells. To identify the lL-4 responsive promotor/enhancer element, deletion analysis of the upstream region of the germ-line exon (l[epsilon]) of the C[epsilon] germ-line transcript which is located 5' to the switch region, was performed by using a luciferase gene as a reporter. Deletion analysis showed that a DNA fragment which lies between [minus]215 and [minus]154 bp upstream from the most 3' transcriptional initiation site of human l[epsilon] gene is fully responsible for the induction of germ-line transcripts by IL-4. According to a mutational analysis, the DNA fragment between [minus]163 and [minus]152 bp was identified to be a novel IL-4 responsive element in a human C[epsilon] gene. Electrophoretic gel mobility shift assay showed the presence of IL-4-induced nuclear factor that specifically bound to this IL-4 responsive element. This novel IL-4 responsive element and an IL-4-induced DNA binding protein may play an important role for the induction of C[epsilon] germ-line transcript as well as class switching to IgE. 54 refs., 7 figs.

  11. Antigen targeting to APC: from mice to veterinary species.

    PubMed

    Alvarez, B; Poderoso, T; Alonso, F; Ezquerra, A; Domínguez, J; Revilla, C

    2013-10-01

    Antigen delivery to receptors expressed on antigen presenting cells (APC) has shown to improve immunogenicity of vaccines in mice. An enhancement of cytotoxic T lymphocyte (CTL), helper T cell or humoral responses was obtained depending on the type of APC and the surface molecule targeted. Although this strategy is being also evaluated in livestock animals with promising results, some discrepancies have been found between species and pathogens. The genetic diversity of livestock animals, the different pattern of expression of some receptors among species, the use of different markers to characterize APC in large animals and sometimes the lack of reagents make difficult to compare results obtained in different species. In this review, we summarize the data available regarding antigen targeting to APC receptors in cattle, sheep and pig and discuss the results found in these animals in the context of what has been obtained in mice. PMID:23648645

  12. Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2

    PubMed Central

    Ruane, Peter T.; Gumy, Laura F.; Bola, Becky; Anderson, Beverley; Wozniak, Marcin J.; Hoogenraad, Casper C.; Allan, Victoria J.

    2016-01-01

    Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons. PMID:27272132

  13. Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2.

    PubMed

    Ruane, Peter T; Gumy, Laura F; Bola, Becky; Anderson, Beverley; Wozniak, Marcin J; Hoogenraad, Casper C; Allan, Victoria J

    2016-01-01

    Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons. PMID:27272132

  14. Proteomic profiling of a mouse model of acute intestinal Apc deletion leads to identification of potential novel biomarkers of human colorectal cancer (CRC).

    PubMed

    Hammoudi, Abeer; Song, Fei; Reed, Karen R; Jenkins, Rosalind E; Meniel, Valerie S; Watson, Alastair J M; Pritchard, D Mark; Clarke, Alan R; Jenkins, John R

    2013-10-25

    Colorectal cancer (CRC) is the fourth most common cause of cancer-related death worldwide. Accurate non-invasive screening for CRC would greatly enhance a population's health. Adenomatous polyposis coli (Apc) gene mutations commonly occur in human colorectal adenomas and carcinomas, leading to Wnt signalling pathway activation. Acute conditional transgenic deletion of Apc in murine intestinal epithelium (AhCre(+)Apc(fl)(/)(fl)) causes phenotypic changes similar to those found during colorectal tumourigenesis. This study comprised a proteomic analysis of murine small intestinal epithelial cells following acute Apc deletion to identify proteins that show altered expression during human colorectal carcinogenesis, thus identifying proteins that may prove clinically useful as blood/serum biomarkers of colorectal neoplasia. Eighty-one proteins showed significantly increased expression following iTRAQ analysis, and validation of nine of these by Ingenuity Pathaway Analysis showed they could be detected in blood or serum. Expression was assessed in AhCre(+)Apc(fl)(/)(fl) small intestinal epithelium by immunohistochemistry, western blot and quantitative real-time PCR; increased nucelolin concentrations were also detected in the serum of AhCre(+)Apc(fl)(/)(fl) and Apc(Min)(/)(+) mice by ELISA. Six proteins; heat shock 60kDa protein 1, Nucleolin, Prohibitin, Cytokeratin 18, Ribosomal protein L6 and DEAD (Asp-Glu-Ala-Asp) box polypeptide 5,were selected for further investigation. Increased expression of 4 of these was confirmed in human CRC by qPCR. In conclusion, several novel candidate biomarkers have been identified from analysis of transgenic mice in which the Apc gene was deleted in the intestinal epithelium that also showed increased expression in human CRC. Some of these warrant further investigation as potential serum-based biomarkers of human CRC. PMID:23998936

  15. Achieving immortality in the C. elegans germline.

    PubMed

    Smelick, Chris; Ahmed, Shawn

    2005-01-01

    Germline immortality is a topic that has intrigued theoretical biologists interested in aging for over a century. The germ cell lineage can be passed from one generation to the next, indefinitely. In contrast, somatic cells are typically only needed for a single generation and are then discarded. Germ cells may, therefore, harbor rejuvenation mechanisms that enable them to proliferate for eons. Such processes are thought to be either absent from or down-regulated in somatic cells, although cell non-autonomous forms of rejuvenation are formally possible. A thorough description of mechanisms that foster eternal youth in germ cells is lacking. The mysteries of germline immortality are being addressed in the nematode Caenorhabditis elegans by studying mutants that reproduce normally for several generations but eventually become sterile. The mortal germline mutants probably become sterile as a consequence of accumulating various forms of heritable cellular damage. Such mutants are abundant, indicating that several different biochemical pathways are required to rejuvenate the germline. Thus, forward genetics should help to define mechanisms that enable the germline to achieve immortality. PMID:15619471

  16. Spontaneous generation of germline characteristics in mouse fibrosarcoma cells

    NASA Astrophysics Data System (ADS)

    Ma, Zhan; Hu, Yao; Jiang, Guoying; Hou, Jun; Liu, Ruilai; Lu, Yuan; Liu, Chunfang

    2012-10-01

    Germline/embryonic-specific genes have been found to be activated in somatic tumors. In this study, we further showed that cells functioning as germline could be present in mouse fibrosarcoma cells (L929 cell line). Early germline-like cells spontaneously appeared in L929 cells and further differentiated into oocyte-like cells. These germline-like cells can, in turn, develop into blastocyst-like structures in vitro and cause teratocarcinomas in vivo, which is consistent with natural germ cells in function. Generation of germline-like cells from somatic tumors might provide a novel way to understand why somatic cancer cells have strong features of embryonic/germline development. It is thought that the germline traits of tumors are associated with the central characteristics of malignancy, such as immortalization, invasion, migration and immune evasion. Therefore, germline-like cells in tumors might provide potential targets to tumor biology, diagnosis and therapy.

  17. Germline Modification and Engineering in Avian Species.

    PubMed

    Lee, Hong Jo; Lee, Hyung Chul; Han, Jae Yong

    2015-09-01

    Production of genome-edited animals using germline-competent cells and genetic modification tools has provided opportunities for investigation of biological mechanisms in various organisms. The recently reported programmed genome editing technology that can induce gene modification at a target locus in an efficient and precise manner facilitates establishment of animal models. In this regard, the demand for genome-edited avian species, which are some of the most suitable model animals due to their unique embryonic development, has also increased. Furthermore, germline chimera production through long-term culture of chicken primordial germ cells (PGCs) has facilitated research on production of genome-edited chickens. Thus, use of avian germline modification is promising for development of novel avian models for research of disease control and various biological mechanisms. Here, we discuss recent progress in genome modification technology in avian species and its applications and future strategies. PMID:26333275

  18. Germline Modification and Engineering in Avian Species

    PubMed Central

    Lee, Hong Jo; Lee, Hyung Chul; Han, Jae Yong

    2015-01-01

    Production of genome-edited animals using germline-competent cells and genetic modification tools has provided opportunities for investigation of biological mechanisms in various organisms. The recently reported programmed genome editing technology that can induce gene modification at a target locus in an efficient and precise manner facilitates establishment of animal models. In this regard, the demand for genome-edited avian species, which are some of the most suitable model animals due to their unique embryonic development, has also increased. Furthermore, germline chimera production through long-term culture of chicken primordial germ cells (PGCs) has facilitated research on production of genome-edited chickens. Thus, use of avian germline modification is promising for development of novel avian models for research of disease control and various biological mechanisms. Here, we discuss recent progress in genome modification technology in avian species and its applications and future strategies. PMID:26333275

  19. Germline mutation analysis of MLH1 and MSH2 in Malaysian Lynch syndrome patients

    PubMed Central

    Zahary, Mohd Nizam; Kaur, Gurjeet; Abu Hassan, Muhammad Radzi; Singh, Harjinder; Naik, Venkatesh R; Ankathil, Ravindran

    2012-01-01

    AIM: To investigate the protein expression profile of mismatch repair (MMR) genes in suspected cases of Lynch syndrome and to characterize the associated germline mutations. METHODS: Immunohistochemical analysis of tumor samples was performed to determine the protein expression profile of MMR protein. Germline mutation screening was carried out on peripheral blood samples. The entire exon regions of MLH1 and MSH2 genes were amplified by polymerase chain reaction, screened by denaturing high performance liquid chromatography (dHPLC) and analyzed by DNA sequencing to characterize the germline mutations. RESULTS: Three out of 34 tissue samples (8.8%) and four out of 34 tissue samples (11.8%) showed loss of nuclear staining by immunohistochemistry, indicating the absence of MLH1 and MSH2 protein expression in carcinoma cells, respectively. dHPLC analysis followed by DNA sequencing showed these samples to have germline mutations of MSH2 gene. However, no deleterious mutations were identified in any of the 19 exons or coding regions of MLH1 gene, but we were able to identify MLH1 promoter polymorphism, -93G > A (rs1800734), in 21 out of 34 patients (61.8%). We identified one novel mutation, transversion mutation c.2005G > C, which resulted in a missense mutation (Gly669Arg), a transversion mutation in exon 1, c.142G > T, which resulted in a nonsense mutation (Glu48Stop) and splice-site mutation, c.2006-6T > C, which was adjacent to exon 13 of MSH2 gene. CONCLUSION: Germline mutations were identified in four Malaysian Lynch syndrome patients. Immunohistochemical analysis of tumor tissue proved to be a good pre-screening test before proceeding to germline mutation analysis of DNA MMR genes. PMID:22371642

  20. Association of APC, GSTP1 and SOCS1 promoter methylation with the risk of hepatocellular carcinoma: a meta-analysis.

    PubMed

    Liu, Meng; Cui, Lian-Hua; Li, Cheng-Cheng; Zhang, Li

    2015-11-01

    Studies of the relationships of adenomatous polyposis coli (APC), glutathione-S-transferase P1 (GSTP1) and suppressor of the cytokine signalling 1 (SOCS1) promoter region methylation with the risk of hepatocellular carcinoma (HCC) have yielded inconsistent results. We carried out the current meta-analysis to comprehensively assess the associations between APC, GSTP1 and SOCS1 promoter methylation frequency and the risk of HCC. All relevant reports were identified by searching the PubMed, Embase, Web of Science, CNKI and the Chinese BioMedical Literature databases before 1 March 2014, with restriction to articles published in the Chinese and English languages. Pooled odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated to investigate the rates of APC, GSTP1 and SOCS1 promoter methylation and the risk of HCC. Our meta-analysis identified relationships of APC (12 studies with 592 HCC tumour tissues), GSTP1 (14 studies including 646 HCC tumour tissues) and SOCS1 (11 studies with 512 HCC tumour tissues) promoter methylation with the risk of HCC. Compared with paracancerous tissues, the pooled ORs of APC, GSTP1 and SOCS1 promoter region methylation in HCC cancer tissues were 5.32 (95% CI=2.96-9.56), 5.65, (95% CI=3.41-9.35) and 2.73 (95% CI=1.37-5.44), respectively. Compared with normal liver tissues as controls, the pooled ORs of APC, GSTP1 and SOCS1 promoter region methylation in HCC cancer tissues were 20.43 (95% CI=5.56-75.08), 18.78 (95% CI=5.76-61.19) and 13.00 (95% CI=5.20-32.47), respectively. Subgroup analysis by ethnicity showed that APC, GSTP1 and SOCS1 promoter methylation was associated significantly with the risk of HCC in both Asian and White populations (all P<0.05). Our meta-analysis suggested strong associations between APC, GSTP1 and SOCS1 gene promoter methylation and the risk of HCC, suggesting these to be promising biomarkers for HCC. PMID:25853848

  1. Identification of Mom12 and Mom13, two novel modifier loci of Apc (Min) -mediated intestinal tumorigenesis.

    PubMed

    Crist, Richard C; Roth, Jacquelyn J; Lisanti, Michael P; Siracusa, Linda D; Buchberg, Arthur M

    2011-04-01

    Colorectal cancer is a heterogeneous disease resulting from a combination of genetic and environmental factors. The C57BL/6J (B6) Apc (Min/+) mouse develops polyps throughout the gastrointestinal tract and has been a valuable model for understanding the genetic basis of intestinal tumorigenesis. Apc (Min/+) mice have been used to study known oncogenes and tumor suppressor genes on a controlled genetic background. These studies often utilize congenic knockout alleles, which can carry an unknown amount of residual donor DNA. The Apc (Min) model has also been used to identify modifer loci, known as Modifier of Min (Mom) loci, which alter Apc (Min) -mediated intestinal tumorigenesis. B6 mice carrying a knockout allele generated in WW6 embryonic stem cells were crossed to B6 Apc (Min/+) mice to determine the effect on polyp multiplicity. The newly generated colony developed significantly more intestinal polyps than Apc (Min/+) controls. Polyp multiplicity did not correlate with inheritance of the knockout allele, suggesting the presence of one or more modifier loci segregating in the colony. Genotyping of simple sequence length polymorphism (SSLP) markers revealed residual 129X1/SvJ genomic DNA within the congenic region of the parental knockout line. An analysis of polyp multiplicity data and SSLP genotyping indicated the presence of two Mom loci in the colony: 1) Mom12, a dominant modifier linked to the congenic region on chromosome 6, and 2) Mom13, which is unlinked to the congenic region and whose effect is masked by Mom12. The identification of Mom12 and Mom13 demonstrates the potential problems resulting from residual heterozygosity present in congenic lines. PMID:21386660

  2. Distinct germline polymorphisms underlie glioma morphologic heterogeneity

    PubMed Central

    Jenkins, Robert B.; Wrensch, Margaret R.; Johnson, Derek; Fridley, Brooke L.; Decker, Paul A.; Xiao, Yuanyuan; Kollmeyer, Thomas M.; Rynearson, Amanda L.; Fink, Stephanie; Rice, Terri; McCoy, Lucie S.; Halder, Chandralekha; Kosel, Matthew L.; Giannini, Caterina; Tihan, Tarik; O’Neill, Brian P.; Lachance, Daniel H.; Yang, Ping; Wiemels, Joseph; Wiencke, John K.

    2010-01-01

    Two recent genome-wide association studies reported that single nucleotide polymorphisms (SNPs) in (or near) TERT (5p15), CCDC26 (8q24), CDKN2A/B (9p21), PHLDB1 (11q23), and RTEL1 (20q13) are associated with infiltrating glioma. From these reports it was not clear if the SNP associations predispose to glioma in general or whether they are specific to certain glioma grades or morphologic subtypes. To identify hypothesized associations between susceptibility loci and tumor subtype, we genotyped two case/control groups composed of the spectrum of infiltrating glioma subtypes, and stratified the analyses by type. We report that specific germline polymorphisms are associated with different glioma subtypes. CCDC26 (8q24) region polymorphisms are strongly associated with oligodendroglial tumor risk (rs4295627, OR=2.05, p=8.3*10−11), but not glioblastoma risk. The opposite is true of RTEL (20q13) region polymorphisms which are significantly associated with glioblastoma (rs2297440, OR = 0.56, p= 4.6*10−10) but not oligodendroglial tumor. The SNPs in or near CCDC26 (8q24) are associated with oligodendroglial tumors regardless of combined 1p and 19q deletion status; however, the association is greatest for those with combined deletion (rs4295627, OR=2.77, p=2.6*10-9). These observations generate hypotheses concerning the possible mechanisms by which specific SNPs (or alterations in linkage disequilibrium with such SNPs) are associated with glioma development. PMID:21356187

  3. Cell-Type Specific Expression of Apc in Lung Development, Injury and Repair

    PubMed Central

    Li, Aimin; Xing, Yiming; Chan, Belinda; Heisterkamp, Nora; Groffen, John; Borok, Zea; Minoo, Parviz; Li, Changgong

    2010-01-01

    Adenomatous polyposis coli (Apc) is critical for Wnt signaling and cell migration. The current study examined Apc expression during lung development, injury and repair. Apc was first detectable in smooth muscle layers in early lung morphogenesis, and was highly expressed in ciliated and neuroendocrine cells in the advanced stages. No Apc immunoreactivity was detected in Clara or basal cells, which function as stem/progenitor cell in adult lung. In ciliated cells, Apc is associated mainly with apical cytoplasmic domain. In response to naphthalene induced injury, Apcpositive cells underwent squamous metaplasia, accompanied by changes in Apc subcellular distribution. In conclusion, both spatial and temporal expression of Apc is dynamically regulated during lung development and injury repair. Differential expression of Apc in progenitor vs. non-progenitor cells suggests a functional role in cell type specification. Subcellular localization changes of Apc in response to naphthalene injury suggest a role in cell shape and cell migration. PMID:20658693

  4. Liver fatty acid binding protein (L-Fabp) modifies intestinal fatty acid composition and adenoma formation in ApcMin/+ mice

    PubMed Central

    Dharmarajan, Sekhar; Newberry, Elizabeth P.; Montenegro, Grace; Nalbantoglu, ILKe; Davis, Victoria R.; Clanahan, Michael J.; Blanc, Valerie; Xie, Yan; Luo, Jianyang; Fleshman, James W.; Kennedy, Susan; Davidson, Nicholas O.

    2013-01-01

    Evidence suggests a relationship between dietary fat intake, obesity and colorectal cancer, implying a role for fatty acid (FA) metabolism in intestinal tumorigenesis that is incompletely understood. Liver fatty acid binding protein (L-Fabp), a dominant intestinal FA binding protein, regulates intestinal FA trafficking and metabolism and L-Fabp deletion attenuates diet-induced obesity. Here we examined whether changes in intestinal FA metabolism following L-Fabp deletion modify adenoma development in ApcMin/+ mice. Compound L-Fabp−/−ApcMin/+ mice were generated and fed a 10% fat diet balanced equally between saturated, monounsaturated and polyunsaturated fat. L-Fabp−/−ApcMin/+ mice displayed significant reductions in adenoma number and total polyp area compared to ApcMin/+controls, reflecting a significant shift in distribution toward smaller polyps. Adenomas from L-Fabp−/−ApcMin/+ mice exhibited reductions in cellular proliferation, high-grade dysplasia and nuclear β-catenin translocation. Intestinal FA content was increased in L-Fabp−/−ApcMin/+ mice and lipidomic profiling of intestinal mucosa revealed significant shifts to polyunsaturated FA species with reduced saturated FA species. L-Fabp−/−ApcMin/+mice also demonstrated corresponding changes in mRNA expression of enzymes involved in FA elongation and desaturation. Furthermore, adenomas from L-Fabp−/−ApcMin/+mice displayed significant reductions in mRNA abundance of nuclear hormone receptors involved in cellular proliferation and in enzymes involved in lipogenesis. These findings collectively implicate L-Fabp as an important genetic modifier of intestinal tumorigenesis and identify FA trafficking and metabolic compartmentalization as an important pathway linking dietary fat intake, obesity and intestinal tumor formation. PMID:23921281

  5. NUP98 fusion oncoproteins interact with the APC/C(Cdc20) as a pseudosubstrate and prevent mitotic checkpoint complex binding.

    PubMed

    Salsi, Valentina; Fantini, Sebastian; Zappavigna, Vincenzo

    2016-09-01

    NUP98 is a recurrent partner gene in translocations causing acute myeloid leukemias and myelodisplastic syndrome. The expression of NUP98 fusion oncoproteins has been shown to induce mitotic spindle defects and chromosome missegregation, which correlate with the capability of NUP98 fusions to cause mitotic checkpoint attenuation. We show that NUP98 oncoproteins physically interact with the APC/C(Cdc20) in the absence of the NUP98 partner protein RAE1, and prevent the binding of the mitotic checkpoint complex to the APC/C(Cdc20). NUP98 oncoproteins require the GLEBS-like domain present in their NUP98 moiety to bind the APC/C(Cdc20). We found that NUP98 wild-type is a substrate of APC/C(Cdc20) prior to mitotic entry, and that its binding to APC/C(Cdc20) is controlled via phosphorylation of a PEST sequence located within its C-terminal portion. We identify S606, within the PEST sequence, as a key target site, whose phosphorylation modulates the capability of NUP98 to interact with APC/C(Cdc20). We finally provide evidence for an involvement of the peptidyl-prolyl isomerase PIN1 in modulating the possible conformational changes within NUP98 that lead to its dissociation from the APC/C(Cdc20) during mitosis. Our results provide novel insight into the mechanisms underlying the aberrant capability of NUP98 oncoproteins to interact with APC/C(Cdc20) and to interfere with its function. PMID:27097363

  6. Mosaic parental germline mutations causing recurrent forms of malformations of cortical development.

    PubMed

    Zillhardt, Julia Lauer; Poirier, Karine; Broix, Loïc; Lebrun, Nicolas; Elmorjani, Adrienne; Martinovic, Jelena; Saillour, Yoann; Muraca, Giuseppe; Nectoux, Juliette; Bessieres, Bettina; Fallet-Bianco, Catherine; Lyonnet, Stanislas; Dulac, Olivier; Odent, Sylvie; Rejeb, Imen; Jemaa, Lamia Ben; Rivier, Francois; Pinson, Lucile; Geneviève, David; Musizzano, Yuri; Bigi, Nicole; Leboucq, Nicolas; Giuliano, Fabienne; Philip, Nicole; Vilain, Catheline; Van Bogaert, Patrick; Maurey, Hélène; Beldjord, Cherif; Artiguenave, François; Boland, Anne; Olaso, Robert; Masson, Cécile; Nitschké, Patrick; Deleuze, Jean-François; Bahi-Buisson, Nadia; Chelly, Jamel

    2016-04-01

    To unravel missing genetic causes underlying monogenic disorders with recurrence in sibling, we explored the hypothesis of parental germline mosaic mutations in familial forms of malformation of cortical development (MCD). Interestingly, four families with parental germline variants, out of 18, were identified by whole-exome sequencing (WES), including a variant in a new candidate gene, syntaxin 7. In view of this high frequency, revision of diagnostic strategies and reoccurrence risk should be considered not only for the recurrent forms, but also for the sporadic cases of MCD. PMID:26395554

  7. Germline Transformation of Caenorhabditis elegans by Injection

    NASA Astrophysics Data System (ADS)

    Kadandale, Pavan; Chatterjee, Indrani; Singson, Andrew

    Microinjection is a commonly used technique for DNA transformation in Caenorhabditis elegans. It is a powerful tool that links genetic and molecular analysis to phenotypic analysis. In this chapter we shall provide an overview of microinjection for germline transformation in worms. Our discussion will emphasize C. elegans reproductive biology, applications and protocols for carrying out microinjection in order to successfully obtain transgenic worms.

  8. Autophagy SEPArates germline and somatic cells.

    PubMed

    Baehrecke, Eric H

    2009-01-23

    Cellular determinants of the germline selectively accumulate in germ cell precursors and influence cell fate during early development in many organisms. Zhang et al. (2009) now report that targeted autophagy mediated by the SEPA-1 protein depletes germplasm proteins from somatic cells during early development of the nematode. PMID:19167322

  9. Germline Stem Cell Transplantation and Transgenesis

    PubMed Central

    Brinster, Ralph L.

    2016-01-01

    The recently developed testis cell transplantation method provides a powerful approach to studying the biology of the male germline stem cell and its microenvironment, the stem cell niche. The technique also is being used to examine spermatogenic defects, correct male infertility, and generate transgenic animals. PMID:12077400

  10. Germline BRCA1 mutations increase prostate cancer risk

    PubMed Central

    Leongamornlert, D; Mahmud, N; Tymrakiewicz, M; Saunders, E; Dadaev, T; Castro, E; Goh, C; Govindasami, K; Guy, M; O'Brien, L; Sawyer, E; Hall, A; Wilkinson, R; Easton, D; Goldgar, D; Eeles, R; Kote-Jarai, Z

    2012-01-01

    Background: Prostate cancer (PrCa) is one of the most common cancers affecting men but its aetiology is poorly understood. Family history of PrCa, particularly at a young age, is a strong risk factor. There have been previous reports of increased PrCa risk in male BRCA1 mutation carriers in female breast cancer families, but there is a controversy as to whether this risk is substantiated. We sought to evaluate the role of germline BRCA1 mutations in PrCa predisposition by performing a candidate gene study in a large UK population sample set. Methods: We screened 913 cases aged 36–86 years for germline BRCA1 mutation, with the study enriched for cases with an early age of onset. We analysed the entire coding region of the BRCA1 gene using Sanger sequencing. Multiplex ligation-dependent probe amplification was also used to assess the frequency of large rearrangements in 460 cases. Results: We identified 4 deleterious mutations and 45 unclassified variants (UV). The frequency of deleterious BRCA1 mutation in this study is 0.45% three of the mutation carriers were affected at age ⩽65 years and one developed PrCa at 69 years. Using previously estimated population carrier frequencies, deleterious BRCA1 mutations confer a relative risk of PrCa of ∼3.75-fold, (95% confidence interval 1.02–9.6) translating to a 8.6% cumulative risk by age 65. Conclusion This study shows evidence for an increased risk of PrCa in men who harbour germline mutations in BRCA1. This could have a significant impact on possible screening strategies and targeted treatments. PMID:22516946

  11. A Functional Genomic Screen for Evolutionarily Conserved Genes Required for Lifespan and Immunity in Germline-Deficient C. elegans

    PubMed Central

    Sinha, Amit; Rae, Robbie

    2014-01-01

    The reproductive system regulates lifespan in insects, nematodes and vertebrates. In Caenorhabditis elegans removal of germline increases lifespan by 60% which is dependent upon insulin signaling, nuclear hormone signaling, autophagy and fat metabolism and their microRNA-regulators. Germline-deficient C. elegans are also more resistant to various bacterial pathogens but the underlying molecular mechanisms are largely unknown. Firstly, we demonstrate that previously identified genes that regulate the extended lifespan of germline-deficient C. elegans (daf-2, daf-16, daf-12, tcer-1, mir-7.1 and nhr-80) are also essential for resistance to the pathogenic bacterium Xenorhabdus nematophila. We then use a novel unbiased approach combining laser cell ablation, whole genome microarrays, RNAi screening and exposure to X. nematophila to generate a comprehensive genome-wide catalog of genes potentially required for increased lifespan and innate immunity in germline-deficient C. elegans. We find 3,440 genes to be upregulated in C. elegans germline-deficient animals in a gonad dependent manner, which are significantly enriched for genes involved in insulin signaling, fatty acid desaturation, translation elongation and proteasome complex function. Using RNAi against a subset of 150 candidate genes selected from the microarray results, we show that the upregulated genes such as transcription factor DAF-16/FOXO, the PTEN homolog lipid phosphatase DAF-18 and several components of the proteasome complex (rpn-6.1, rpn-7, rpn-9, rpn-10, rpt-6, pbs-3 and pbs-6) are essential for both lifespan and immunity of germline deficient animals. We also identify a novel role for genes including par-5 and T12G3.6 in both lifespan-extension and increased survival on X. nematophila. From an evolutionary perspective, most of the genes differentially expressed in germline deficient C. elegans also show a conserved expression pattern in germline deficient Pristionchus pacificus, a nematode species

  12. The APC I1307K allele conveys a significant increased risk for cancer.

    PubMed

    Leshno, Ari; Shapira, Shiran; Liberman, Eliezer; Kraus, Sarah; Sror, Miri; Harlap-Gat, Amira; Avivi, Doran; Galazan, Lior; David, Maayan; Maharshak, Nitsan; Moanis, Serhan; Arber, Nadir; Moshkowitz, Menachem

    2016-03-15

    This study is the first attempt to evaluate the association between the APC I1307K variant and overall cancer risk. It is unique in both its large sample size and in the reliability of data in the control group. The findings described in this article have major implications in terms of identifying asymptomatic individuals who are at increased risk to harbor cancer and therefore targeted to be enrolled in specific early detection and prevention programs. The prevalence of the APC I1307K missense mutation among Ashkenazi Jews is ∼ 6%. Carriers are at an increased risk for colorectal neoplasia. In this study, we examined the association of this variant with non-colorectal cancers. Consecutive 13,013 healthy subjects who underwent screening at the Integrated Cancer Prevention Center between 2006 and 2014 were enrolled. This population was supplemented with 1,611 cancer patients from the same institution. Demographics, medical history, and pathological data were recorded. Mortality data were obtained from the Ministry of Health's registry. The prevalence of APC I1307K in cancer patients and healthy subjects was compared. The APC I1307K variant was detected in 189 (11.8%) cancer patients compared to 614 (4.7%) healthy subjects, reflecting an adjusted age and sex odds ratio (OR) of 2.53 (p < 0.0001). History of two or more cancer types was associated with a positive carrier prevalence (OR = 4.38 p < 0.0001). Males had significantly increased carrier prevalence in lung, urologic, pancreatic, and skin cancers. The carrier prevalence among females was significantly higher only in breast and skin cancers. Female carriers developed cancer at a significantly older age compared to non-carriers (average 62.7 years vs. 57.8, respectively, p = 0.027), had better survival rates (HR = 0.58, p = 0.022) and overall increased longevity (average age of death 78.8 vs. 70.4 years, respectively, p = 0.003). In conclusion, the APC I1307K variant is a reliable marker for overall cancer risk

  13. Creation of an engineered APC system to explore and optimize the presentation of immunodominant peptides of major allergens.

    PubMed

    Rosskopf, Sandra; Jutz, Sabrina; Neunkirchner, Alina; Candia, Martín R; Jahn-Schmid, Beatrice; Bohle, Barbara; Pickl, Winfried F; Steinberger, Peter

    2016-01-01

    We have generated engineered APC to present immunodominant peptides derived from the major aero-allergens of birch and mugwort pollen, Bet v 1142-153 and Art v 125-36, respectively. Jurkat-based T cell reporter lines expressing the cognate allergen-specific T cell receptors were used to read out the presentation of allergenic peptides on the engineered APC. Different modalities of peptide loading and presentation on MHC class II molecules were compared. Upon exogenous loading with allergenic peptides, the engineered APC elicited a dose-dependent response in the reporter T cells and the presence of chemical loading enhancers strongly increased reporter activation. Invariant chain-based MHC class II targeting strategies of endogenously expressed peptides resulted in stronger activation of the reporters than exogenous loading. Moreover, we used Bet v 1 as model allergen to study the ability of K562 cells to present antigenic peptides derived from whole proteins either taken up or endogenously expressed as LAMP-1 fusion protein. In both cases the ability of these cells to process and present peptides derived from whole proteins critically depended on the expression of HLA-DM. We have identified strategies to achieve efficient presentation of allergenic peptides on engineered APC and demonstrate their use to stimulate T cells from allergic individuals. PMID:27539532

  14. Colon epithelial cell differentiation is inhibited by constitutive c-myb expression or mutant APC plus activated RAS.

    PubMed

    Ramsay, Robert G; Ciznadija, Daniel; Sicurella, Catherine; Reyes, Nancy; Mitchelhill, Ken; Darcy, Phillip K; D'Abaco, Giovanna; Mantamadiotis, Theo

    2005-01-01

    Blocked differentiation is a hallmark of cancer cells and the restoration of differentiation programs in vivo is an actively pursued clinical aim. Understanding the key regulators of cyto-differentiation may focus therapies on molecules that reactivate this process. c-myb expression declines rapidly when human colon cancer epithelial cells are induced to differentiate with the physiologically relevant short-chain fatty acid, sodium butyrate. These cells show increased expression of alkaline phosphatase and cytokeratin 8. Similarly, murine Immorto-epithelial cells derived from wild-type colon cells also show c-myb mRNA declines when induced to differentiate with sodium butyrate. Immorto-cells harboring a single APC mutation are indistinguishable from wild-type cells with regard to differentiation, while addition of activated RAS alone markedly enhances differentiation. In marked contrast, complete differentiation arrest occurs when both APC and RAS are mutated. Expression of MybER, a 4-hydroxytamoxifen-activatable form of c-Myb, blocks differentiation in wildtype and APC mutant Immorto-cell lines as well as LIM1215 human colon carcinoma cells. These data identify two pathways of oncogenic change that lead to retarded epithelial cell differentiation, one involving the presence of a single APC mutation in conjunction with activated RAS or alternatively constitutive c-myb expression. PMID:15684716

  15. Creation of an engineered APC system to explore and optimize the presentation of immunodominant peptides of major allergens

    PubMed Central

    Rosskopf, Sandra; Jutz, Sabrina; Neunkirchner, Alina; Candia, Martín R.; Jahn-Schmid, Beatrice; Bohle, Barbara; Pickl, Winfried F.; Steinberger, Peter

    2016-01-01

    We have generated engineered APC to present immunodominant peptides derived from the major aero-allergens of birch and mugwort pollen, Bet v 1142–153 and Art v 125–36, respectively. Jurkat-based T cell reporter lines expressing the cognate allergen-specific T cell receptors were used to read out the presentation of allergenic peptides on the engineered APC. Different modalities of peptide loading and presentation on MHC class II molecules were compared. Upon exogenous loading with allergenic peptides, the engineered APC elicited a dose-dependent response in the reporter T cells and the presence of chemical loading enhancers strongly increased reporter activation. Invariant chain-based MHC class II targeting strategies of endogenously expressed peptides resulted in stronger activation of the reporters than exogenous loading. Moreover, we used Bet v 1 as model allergen to study the ability of K562 cells to present antigenic peptides derived from whole proteins either taken up or endogenously expressed as LAMP-1 fusion protein. In both cases the ability of these cells to process and present peptides derived from whole proteins critically depended on the expression of HLA-DM. We have identified strategies to achieve efficient presentation of allergenic peptides on engineered APC and demonstrate their use to stimulate T cells from allergic individuals. PMID:27539532

  16. Loss of heterozygosity of the APC and MCC genes in squamous cell carcinoma of the head and neck

    SciTech Connect

    Nogueira, C.P.; Afridi, N.A.; Licameli, G.

    1994-09-01

    Squamous cell carcinoma of the head and neck offers a unique opportunity to study genetic mechanisms in human tumorigenesis. Two types of premalignant lesions can be identified clinically, suggesting a multistep process. The majority of the lesions are easy to identify and access. Despite these advantages, possible genetic models for these cancers remain relatively unexplored. Loss of heterozygosity of the tumor suppressor genes APC (adenomatous polyposis coli) and MCC (mutated in colon cancer) was investigated in 24 squamous cell carcinomas of the head and neck. Three sites were analyzed by assays based on the polymerase chain reaction: two RFLPs in the APC gene (Rsa I on exon 11; Ssp I on the 3{prime} untranslated region) and an insertion/deletion in exon 10 of the MCC gene. Sixty three percent of the individuals were informative for at least one marker. Loss of heterozygosity (LOH) was observed in 33% of the tumors, either at the APC or MCC locus. Two out of 11 informative tumors (18%) showed LOH on the APC gene. Three out of seven informative tumors (43%) showed LOH on the MCC gene. These results suggest that inactivation of the APC, MCC and/or a linked gene on chromosome 5q plays a important role in squamous cell carcinoma of the head and neck. The data is in agreement with another study, which detected 25% of loss of heterozygosity in the chromosomal region 5q by using microsatellite markers. These values are much lower than the ones observed in esophageal cancer, a closely related type of tumor, with the same etiology. This may be attributed to differences in the biology of these two cancers.

  17. Targeted apc;twist double-mutant mice: a new model of spontaneous osteosarcoma that mimics the human disease.

    PubMed

    Entz-Werlé, Natacha; Choquet, Philippe; Neuville, Agnès; Kuchler-Bopp, Sabine; Clauss, François; Danse, Jean-Marc; Simo-Noumbissie, Pauline; Guérin, Eric; Gaub, Marie-Pierre; Freund, Jean-Noel; Boehm, Nelly; Constantinesco, André; Lutz, Patrick; Guenot, Dominique; Perrin-Schmitt, Fabienne

    2010-01-01

    TWIST and adenomatosis polyposis coli (APC) are critical signaling factors in normal bone development. In previous studies examining a homogeneously treated cohort of pediatric osteosarcoma patients, we reported the frequent and concurrent loss of both TWIST and APC genes. On these bases, we created a related animal model to further explore the oncogenic cooperation between these two genes. We performed intercrosses between twist-null/+ and Apc1638N/+ mice and studied their progeny. The Apc1638N/+;twistnull/+ mice developed bone abnormalities observed by macroscopic skeletal analyses and in vivo imaging. Complementary histologic, cellular, and molecular analyses were used to characterize the identified bone tumors, including cell culture and immunofluorescence of bone differentiation markers. Spontaneous localized malignant bone tumors were frequently identified in Apc1638N/+;twist-null/+ mice by in vivo imaging evaluation and histologic analyses. These tumors possessed several features similar to those observed in human localized osteosarcomas. In particular, the murine tumors presented with fibroblastic, chondroblastic, and osteoblastic osteosarcoma histologies, as well as mixtures of these subtypes. In addition, cellular analyses and bone differentiation markers detected by immunofluorescence on tumor sections reproduced most murine and human osteosarcoma characteristics. For example, the early bone differentiation marker Runx2, interacting physically with hypophosphorylated pRb, was undetectable in these murine osteosarcomas, whereas phosphorylated retinoblastoma was abundant in the osteoblastic and chondroblastic tumor subtypes. These characteristics, similar to those observed in human osteosarcomas, indicated that our animal model may be a powerful tool to further understand the development of localized osteosarcoma. PMID:21151473

  18. Targeted Apc;Twist Double-Mutant Mice: A New Model of Spontaneous Osteosarcoma That Mimics the Human Disease123

    PubMed Central

    Entz-Werlé, Natacha; Choquet, Philippe; Neuville, Agnès; Kuchler-Bopp, Sabine; Clauss, François; Danse, Jean-Marc; Simo-Noumbissie, Pauline; Guérin, Eric; Gaub, Marie-Pierre; Freund, Jean-Noel; Boehm, Nelly; Constantinesco, André; Lutz, Patrick; Guenot, Dominique; Perrin-Schmitt, Fabienne

    2010-01-01

    TWIST and adenomatosis polyposis coli (APC) are critical signaling factors in normal bone development. In previous studies examining a homogeneously treated cohort of pediatric osteosarcoma patients, we reported the frequent and concurrent loss of both TWIST and APC genes. On these bases, we created a related animal model to further explore the oncogenic cooperation between these two genes. We performed intercrosses between twist-null/+ and Apc1638N/+ mice and studied their progeny. The Apc1638N/+;twistnull/+ mice developed bone abnormalities observed by macroscopic skeletal analyses and in vivo imaging. Complementary histologic, cellular, and molecular analyses were used to characterize the identified bone tumors, including cell culture and immunofluorescence of bone differentiation markers. Spontaneous localized malignant bone tumors were frequently identified in Apc1638N/+;twist-null/+ mice by in vivo imaging evaluation and histologic analyses. These tumors possessed several features similar to those observed in human localized osteosarcomas. In particular, the murine tumors presented with fibroblastic, chondroblastic, and osteoblastic osteosarcoma histologies, as well as mixtures of these subtypes. In addition, cellular analyses and bone differentiation markers detected by immunofluorescence on tumor sections reproduced most murine and human osteosarcoma characteristics. For example, the early bone differentiation marker Runx2, interacting physically with hypophosphorylated pRb, was undetectable in these murine osteosarcomas, whereas phosphorylated retinoblastoma was abundant in the osteoblastic and chondroblastic tumor subtypes. These characteristics, similar to those observed in human osteosarcomas, indicated that our animal model may be a powerful tool to further understand the development of localized osteosarcoma. PMID:21151473

  19. Germline TP53 Variants and Susceptibility to Osteosarcoma

    PubMed Central

    Yeager, Meredith; Mai, Phuong L.; Gastier-Foster, Julie M.; Gorlick, Richard; Khanna, Chand; Patiño-Garcia, Ana; Sierrasesúmaga, Luis; Lecanda, Fernando; Andrulis, Irene L.; Wunder, Jay S.; Gokgoz, Nalan; Barkauskas, Donald A.; Zhang, Xijun; Vogt, Aurelie; Jones, Kristine; Boland, Joseph F.; Chanock, Stephen J.; Savage, Sharon A.

    2015-01-01

    The etiologic contribution of germline genetic variation to sporadic osteosarcoma is not well understood. Osteosarcoma is a sentinel cancer of Li-Fraumeni syndrome (LFS), in which approximately 70% of families meeting the classic criteria have germline TP53 mutations. We sequenced TP53 exons in 765 osteosarcoma cases. Data were analyzed with χ2 tests, logistic regression, and Cox proportional hazards regression models. We observed a high frequency of young osteosarcoma cases (age <30 years) carrying a known LFS- or likely LFS-associated mutation (3.8%) or rare exonic variant (5.7%) with an overall frequency of 9.5%, compared with none in case patients age 30 years and older (P < .001). This high TP53 mutation prevalence in young osteosarcoma cases is statistically significantly greater than the previously reported prevalence of 3% (P = .0024). We identified a novel association between a TP53 rare variant and metastasis at diagnosis of osteosarcoma (rs1800372, odds ratio = 4.27, 95% confidence interval = 1.2 to 15.5, P = .026). Genetic susceptibility to young onset osteosarcoma is distinct from older adult onset osteosarcoma, with a high frequency of LFS-associated and rare exonic TP53 variants. PMID:25896519

  20. Germline TP53 variants and susceptibility to osteosarcoma.

    PubMed

    Mirabello, Lisa; Yeager, Meredith; Mai, Phuong L; Gastier-Foster, Julie M; Gorlick, Richard; Khanna, Chand; Patiño-Garcia, Ana; Sierrasesúmaga, Luis; Lecanda, Fernando; Andrulis, Irene L; Wunder, Jay S; Gokgoz, Nalan; Barkauskas, Donald A; Zhang, Xijun; Vogt, Aurelie; Jones, Kristine; Boland, Joseph F; Chanock, Stephen J; Savage, Sharon A

    2015-07-01

    The etiologic contribution of germline genetic variation to sporadic osteosarcoma is not well understood. Osteosarcoma is a sentinel cancer of Li-Fraumeni syndrome (LFS), in which approximately 70% of families meeting the classic criteria have germline TP53 mutations. We sequenced TP53 exons in 765 osteosarcoma cases. Data were analyzed with χ(2) tests, logistic regression, and Cox proportional hazards regression models. We observed a high frequency of young osteosarcoma cases (age <30 years) carrying a known LFS- or likely LFS-associated mutation (3.8%) or rare exonic variant (5.7%) with an overall frequency of 9.5%, compared with none in case patients age 30 years and older (P < .001). This high TP53 mutation prevalence in young osteosarcoma cases is statistically significantly greater than the previously reported prevalence of 3% (P = .0024). We identified a novel association between a TP53 rare variant and metastasis at diagnosis of osteosarcoma (rs1800372, odds ratio = 4.27, 95% confidence interval = 1.2 to 15.5, P = .026). Genetic susceptibility to young onset osteosarcoma is distinct from older adult onset osteosarcoma, with a high frequency of LFS-associated and rare exonic TP53 variants. PMID:25896519

  1. Tumour morphology predicts PALB2 germline mutation status

    PubMed Central

    Teo, Z L; Provenzano, E; Dite, G S; Park, D J; Apicella, C; Sawyer, S D; James, P A; Mitchell, G; Trainer, A H; Lindeman, G J; Shackleton, K; Cicciarelli, L; Buys, S S; Andrulis, I L; Mulligan, A M; Glendon, G; John, E M; Terry, M B; Daly, M; Odefrey, F A; Nguyen-Dumont, T; Giles, G G; Dowty, J G; Winship, I; Goldgar, D E; Hopper, J L; Southey, M C

    2013-01-01

    Background: Population-based studies of breast cancer have estimated that at least some PALB2 mutations are associated with high breast cancer risk. For women carrying PALB2 mutations, knowing their carrier status could be useful in directing them towards effective cancer risk management and therapeutic strategies. We sought to determine whether morphological features of breast tumours can predict PALB2 germline mutation status. Methods: Systematic pathology review was conducted on breast tumours from 28 female carriers of PALB2 mutations (non-carriers of other known high-risk mutations, recruited through various resources with varying ascertainment) and on breast tumours from a population-based sample of 828 Australian women diagnosed before the age of 60 years (which included 40 BRCA1 and 18 BRCA2 mutation carriers). Tumour morphological features of the 28 PALB2 mutation carriers were compared with those of 770 women without high-risk mutations. Results: Tumours arising in PALB2 mutation carriers were associated with minimal sclerosis (odds ratio (OR)=19.7; 95% confidence interval (CI)=6.0–64.6; P=5 × 10−7). Minimal sclerosis was also a feature that distinguished PALB2 mutation carriers from BRCA1 (P=0.05) and BRCA2 (P=0.04) mutation carriers. Conclusion: This study identified minimal sclerosis to be a predictor of germline PALB2 mutation status. Morphological review can therefore facilitate the identification of women most likely to carry mutations in PALB2. PMID:23787919

  2. The Mechanism of Germline Sex Determination in Vertebrates.

    PubMed

    Nishimura, Toshiya; Tanaka, Minoru

    2016-07-01

    Germ cells are the common cells of origin for the two different types of gametes, sperm and eggs. In vertebrates so far examined, the sex of germ cells is determined by gonadal somatic cells. However, influenced by the somatic cells, how germ cells adopt their sexual fates by intrinsic factors has long been unclear in vertebrates. We recently identified forkhead box L3 (FOXL3) as a germ cell-intrinsic factor involved in the sperm-egg fate decision in the teleost fish medaka (Oryzias latipes). On the basis of the results obtained by the analysis of foxl3/FOXL3 expression and loss-of-function mutants, we review when and how germ cell sex is regulated non-cell-autonomously and cell-autonomously. We then discuss the fact that the germline sex determination pathway is genetically distinct from other essential gametogenic pathways such as meiotic entry and the establishment of germline stem cells. Another extraordinary finding in the foxl3 mutant is that functional sperm can be produced in the ovary, which provides a new notion that gametogenesis can proceed regardless of the sex of the surrounding somatic cells once the sexual identity of germ cells is established in medaka. PMID:27009043

  3. Pituitary blastoma: a pathognomonic feature of germ-line DICER1 mutations

    PubMed Central

    de Kock, Leanne; Sabbaghian, Nelly; Plourde, François; Srivastava, Archana; Weber, Evan; Soglio, Dorothée Bouron-Dal; Hamel, Nancy; Choi, Joon Hyuk; Park, Sung-Hye; Deal, Cheri L.; Kelsey, Megan M.; Dishop, Megan K.; Esbenshade, Adam; Kuttesch, John F.; Jacques, Thomas S.; Perry, Arie; Leichter, Heinz; Maeder, Philippe; Brundler, Marie-Anne; Warner, Justin; Neal, James; Zacharin, Margaret; Korbonits, Márta; Cole, Trevor; Traunecker, Heidi; McLean, Thomas W.; Rotondo, Fabio; Lepage, Pierre; Albrecht, Steffen; Horvath, Eva; Kovacs, Kalman; Priest, John R.; Foulkes, William D.

    2014-01-01

    Individuals harboring germ-line DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 Syndrome or pleuropulmonary blastoma-familial tumor and dysplasia syndrome [online Mendelian inheritance in man (OMIM) #601200]. In addition, specific somatic mutations in the DICER1 RNase III catalytic domain have been identified in several DICER1-associated tumor types. Pituitary blastoma (PitB) was identified as a distinct entity in 2008, and is a very rare, potentially lethal early childhood tumor of the pituitary gland. Since the discovery by our team of an inherited mutation in DICER1 in a child with PitB in 2011, we have identified 12 additional PitB cases. We aimed to determine the contribution of germ-line and somatic DICER1 mutations to PitB. We hypothesized that PitB is a pathognomonic feature of a germ-line DICER1 mutation and that each PitB will harbor a second somatic mutation in DICER1. Lymphocyte or saliva DNA samples ascertained from ten infants with PitB were screened and nine were found to harbor a heterozygous germ-line DICER1 mutation. We identified additional DICER1 mutations in nine of ten tested PitB tumor samples, eight of which were confirmed to be somatic in origin. Seven of these mutations occurred within the RNase IIIb catalytic domain, a domain essential to the generation of 5p miRNAs from the 5′ arm of miRNA-precursors. Germ-line DICER1 mutations are a major contributor to PitB. Second somatic DICER1 “hits” occurring within the RNase IIIb domain also appear to be critical in PitB pathogenesis. PMID:24839956

  4. Mitochondrial DNA Polymerase POLG1 Disease Mutations and Germline Variants Promote Tumorigenic Properties

    PubMed Central

    Singh, Bhupendra; Owens, Kjerstin M.; Bajpai, Prachi; Desouki, Mohamed Mokhtar; Srinivasasainagendra, Vinodh; Tiwari, Hemant K.; Singh, Keshav K.

    2015-01-01

    Germline mutations in mitochondrial DNA polymerase gamma (POLG1) induce mitochondrial DNA (mtDNA) mutations, depletion, and decrease oxidative phosphorylation. Earlier, we identified somatic mutations in POLG1 and the contribution of these mutations in human cancer. However, a role for germline variations in POLG1 in human cancers is unknown. In this study, we examined a role for disease associated germline variants of POLG1, POLG1 gene expression, copy number variation and regulation in human cancers. We analyzed the mutations, expression and copy number variation in POLG1 in several cancer databases and validated the analyses in primary breast tumors and breast cancer cell lines. We discovered 5-aza-2'-deoxycytidine led epigenetic regulation of POLG1, mtDNA-encoded genes and increased mitochondrial respiration. We conducted comprehensive race based bioinformatics analyses of POLG1 gene in more than 33,000 European-Americans and 5,000 African-Americans. We identified a mitochondrial disease causing missense variation in polymerase domain of POLG1 protein at amino acid 1143 (E1143G) to be 25 times more prevalent in European-Americans (allele frequency 0.03777) when compared to African-American (allele frequency 0.00151) population. We identified T251I and P587L missense variations in exonuclease and linker region of POLG1 also to be more prevalent in European-Americans. Expression of these variants increased glucose consumption, decreased ATP production and increased matrigel invasion. Interestingly, conditional expression of these variants revealed that matrigel invasion properties conferred by these germline variants were reversible suggesting a role of epigenetic regulators. Indeed, we identified a set of miRNA whose expression was reversible after variant expression was turned off. Together, our studies demonstrate altered genetic and epigenetic regulation of POLG1 in human cancers and suggest a role for POLG1 germline variants in promoting tumorigenic

  5. APC mutations in colorectal tumors with mismatch repair deficiency.

    PubMed Central

    Huang, J; Papadopoulos, N; McKinley, A J; Farrington, S M; Curtis, L J; Wyllie, A H; Zheng, S; Willson, J K; Markowitz, S D; Morin, P; Kinzler, K W; Vogelstein, B; Dunlop, M G

    1996-01-01

    We have investigated the influence of genetic instability [replication error (RER) phenotype] on APC (adenomatous polyposis coli), a gene thought to initiate colorectal tumorigenesis. The prevalence of APC mutations was similar in RER and non-RER tumors, indicating that both tumor types share this step in neoplastic transformation. However, in a total of 101 sequenced mutations, we noted a substantial excess of APC frameshift mutations in the RER cases (70% in RER tumors versus 47% in non-RER tumors, P < 0.04). These frameshifts were characteristic of mutations arising in cells deficient in DNA mismatch repair, with a predilection for mononucleotide repeats in the RER tumors (P < 0.0002), particularly (A)n tracts (P < 0.00007). These findings suggest that the genetic instability that is reflected by the RER phenotype precedes, and is responsible for, APC mutation in RER large bowel tumors and have important implications for understanding the very earliest stages of neoplasia in patients with tumors deficient in mismatch repair. Images Fig. 2 PMID:8799152

  6. Belgian MSWI fly ashes and APC residues: a characterisation study.

    PubMed

    De Boom, Aurore; Degrez, Marc

    2012-06-01

    Municipal Solid Waste Incineration (MSWI) produces different sorts of residues, bottom ash, fly ashes and Air Pollution Control (APC) residues. Generally, fly ashes and APC residues are mixed at the MSWI plant and manage as a sole residue. In this study, fly ashes and APC residues have been sampled separately at different Belgian MSWI plant and analysed by X-ray fluorescence in order to highlight the composition differences that may appear between the solids. Ca and Cl are found to be the major elements in most of the samples. Lithophilic elements, such as Al and Si, are richer in furnace and boiler ashes, as can be expected. Leaching tests also show differences between the residues; leachates from furnace and boiler ashes are alkaline while those from bag filter residues present a pH value of 6, which impacts the leaching of heavy metals (Pb and Zn). The results suggest that it could be advantageous to manage fly ashes and APC residues separately by adjusting the treatment to their specificities. PMID:22244614

  7. Timing of APC/C substrate degradation is determined by fzy/fzr specificity of destruction boxes

    PubMed Central

    Zur, Amit; Brandeis, Michael

    2002-01-01

    The anaphase promoting complex/cyclosome (APC/C), activated by fzy and fzr, degrades cell cycle proteins that carry RXXL or KEN destruction boxes (d-boxes). APC/C substrates regulate sequential events and must be degraded in the correct order during mitosis and G1. We studied how d-boxes determine APC/Cfzy/APC/Cfzr specificity and degradation timing. Cyclin B1 has an RXXL box and is degraded by both APC/Cfzy and APC/Cfzr; fzy has a KEN box and is degraded by APC/Cfzr only. We characterized the degradation of substrates with swapped d-boxes. Cyclin B1 with KEN was degraded by APC/Cfzr only. Fzy with RXXL could be degraded by APC/Cfzy and APC/Cfzr. Interestingly, APC/Cfzy- but not APC/Cfzr-specific degradation is highly dependent on the location of RXXL. We studied degradation of tagged substrates in real time and observed that APC/Cfzr is activated in early G1. These observations demonstrate how d-box specificities of APC/Cfzy and APC/Cfzr, and the successive activation of APC/C by fzy and fzr, establish the temporal degradation pattern. Our observations can explain further why some endogenous RXXL substrates are degraded by APC/Cfzy, while others are restricted to APC/Cfzr. PMID:12198152

  8. Elevated germline mutation rate in teenage fathers.

    PubMed

    Forster, Peter; Hohoff, Carsten; Dunkelmann, Bettina; Schürenkamp, Marianne; Pfeiffer, Heidi; Neuhuber, Franz; Brinkmann, Bernd

    2015-03-22

    Men age and die, while cells in their germline are programmed to be immortal. To elucidate how germ cells maintain viable DNA despite increasing parental age, we analysed DNA from 24 097 parents and their children, from Europe, the Middle East and Africa. We chose repetitive microsatellite DNA that mutates (unlike point mutations) only as a result of cellular replication, providing us with a natural 'cell-cycle counter'. We observe, as expected, that the overall mutation rate for fathers is seven times higher than for mothers. Also as expected, mothers have a low and lifelong constant DNA mutation rate. Surprisingly, however, we discover that (i) teenage fathers already set out from a much higher mutation rate than teenage mothers (potentially equivalent to 77-196 male germline cell divisions by puberty); and (ii) ageing men maintain sperm DNA quality similar to that of teenagers, presumably by using fresh batches of stem cells known as 'A-dark spermatogonia'. PMID:25694621

  9. Comprehensive proteome analysis of an Apc mouse model uncovers proteins associated with intestinal tumorigenesis.

    PubMed

    Hung, Kenneth E; Faca, Vitor; Song, Kenneth; Sarracino, David A; Richard, Larissa Georgeon; Krastins, Bryan; Forrester, Sara; Porter, Andrew; Kunin, Alexandra; Mahmood, Umar; Haab, Brian B; Hanash, Samir M; Kucherlapati, Raju

    2009-03-01

    Tumor-derived proteins may occur in the circulation as a result of secretion, shedding from the cell surface, or cell turnover. We have applied an in-depth comprehensive proteomic strategy to plasma from intestinal tumor-bearing Apc mutant mice to identify proteins associated with tumor development. We used quantitative tandem mass spectrometry of fractionated mouse plasma to identify differentially expressed proteins in plasma from intestinal tumor-bearing Apc mutant mice relative to matched controls. Up-regulated proteins were assessed for the expression of corresponding genes in tumor tissue. A subset of proteins implicated in colorectal cancer were selected for further analysis at the tissue level using antibody microarrays, Western blotting, tumor immunohistochemistry, and novel fluorescent imaging. We identified 51 proteins that were elevated in plasma with concordant up-regulation at the RNA level in tumor tissue. The list included multiple proteins involved in colon cancer pathogenesis: cathepsin B and cathepsin D, cullin 1, Parkinson disease 7, muscle pyruvate kinase, and Ran. Of these, Parkinson disease 7, muscle pyruvate kinase, and Ran were also found to be up-regulated in human colon adenoma samples. We have identified proteins with direct relevance to colorectal carcinogenesis that are present both in plasma and in tumor tissue in intestinal tumor-bearing mice. Our results show that integrated analysis of the plasma proteome and tumor transcriptome of genetically engineered mouse models is a powerful approach for the identification of tumor-related plasma proteins. PMID:19240248

  10. Assessing the effects of a sequestered germline on interdomain lateral gene transfer in Metazoa.

    PubMed

    Jensen, Lindy; Grant, Jessica R; Laughinghouse, Haywood Dail; Katz, Laura A

    2016-06-01

    A sequestered germline in Metazoa has been argued to be an obstacle to lateral gene transfer (LGT), though few studies have specifically assessed this claim. Here, we test the hypothesis that the origin of a sequestered germline reduced LGT events in Bilateria (i.e., triploblast lineages) as compared to early-diverging Metazoa (i.e., Ctenophora, Cnidaria, Porifera, and Placozoa). We analyze single-gene phylogenies generated with over 900 species sampled from among Bacteria, Archaea, and Eukaryota to identify well-supported interdomain LGTs. We focus on ancient interdomain LGT (i.e., those between prokaryotes and multiple lineages of Metazoa) as systematic errors in single-gene tree reconstruction create uncertainties for interpreting eukaryote-to-eukaryote transfer. The breadth of the sampled Metazoa enables us to estimate the timing of LGTs, and to examine the pattern before versus after the evolution of a sequestered germline. We identified 58 LGTs found only in Metazoa and prokaryotes (i.e., bacteria and/or archaea), and seven genes transferred from prokaryotes into Metazoa plus one other eukaryotic clade. Our analyses indicate that more interdomain transfers occurred before the development of a sequestered germline, consistent with the hypothesis that this feature is an obstacle to LGT. PMID:27139503

  11. Redefining the subcellular location and transport of APC: new insights using a panel of antibodies

    PubMed Central

    Brocardo, Mariana; Näthke, Inke S; Henderson, Beric R

    2005-01-01

    Adenomatous polyposis coli (APC) is a tumour suppressor involved in colon cancer progression. We and others previously described nuclear–cytoplasmic shuttling of APC. However, there are conflicting reports concerning the localization of endogenous wild-type and tumour-associated, truncated APC. To resolve this issue, we compared APC localization using immunofluorescence (IF) microscopy and cell fractionation with nine different APC antibodies. We found that three commonly used APC antibodies showed nonspecific nuclear staining by IF and validated this conclusion in cells where APC was inactivated using small interfering RNA or Cre/Flox. Fractionation showed that wild-type and truncated APC from colon cancer cells were primarily cytoplasmic, but increased in the nucleus after leptomycin B treatment, consistent with CRM1-dependent nuclear export. In contrast to recent reports, our biochemical data indicate that APC nuclear localization is not regulated by changes in cell density, and that APC nuclear export is not prevented by truncating mutations in cancer. These results verify that the bulk of APC resides in the cytoplasm and indicate the need for caution when evaluating the nuclear accumulation of APC. PMID:15678162

  12. 42 CFR 419.31 - Ambulatory payment classification (APC) system and payment weights.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 3 2010-10-01 2010-10-01 false Ambulatory payment classification (APC) system and... Outpatient Services § 419.31 Ambulatory payment classification (APC) system and payment weights. (a) APC... OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM PROSPECTIVE PAYMENT SYSTEM FOR...

  13. MORC1 represses transposable elements in the mouse male germline

    PubMed Central

    Pastor, William A.; Stroud, Hume; Nee, Kevin; Liu, Wanlu; Pezic, Dubravka; Manakov, Sergei; Lee, Serena A.; Moissiard, Guillaume; Zamudio, Natasha; Bourc’his, Déborah; Aravin, Alexei A.; Clark, Amander T.; Jacobsen, Steven E.

    2014-01-01

    The Microrchidia (Morc) family of GHKL ATPases are present in a wide variety of prokaryotic and eukaryotic organisms but are of largely unknown function. Genetic screens in Arabidopsis thaliana have identified Morc genes as important repressors of transposons and other DNA-methylated and silent genes. MORC1-deficient mice were previously found to display male-specific germ cell loss and infertility. Here we show that MORC1 is responsible for transposon repression in the male germline in a pattern that is similar to that observed for germ cells deficient for the DNA methyltransferase homologue DNMT3L. Morc1 mutants show highly localized defects in the establishment of DNA methylation at specific classes of transposons, and this is associated with failed transposon silencing at these sites. Our results identify MORC1 as an important new regulator of the epigenetic landscape of male germ cells during the period of global de novo methylation. PMID:25503965

  14. Germline genome-editing research and its socioethical implications.

    PubMed

    Ishii, Tetsuya

    2015-08-01

    Genetically modifying eggs, sperm, and zygotes ('germline' modification) can impact on the entire body of the resulting individual and on subsequent generations. With the advent of genome-editing technology, human germline gene modification is no longer theoretical. Owing to increasing concerns about human germline gene modification, a voluntary moratorium on human genome-editing research and/or the clinical application of human germline genome editing has recently been called for. However, whether such research should be suspended or encouraged warrants careful consideration. The present article reviews recent research on mammalian germline genome editing, discusses the importance of public dialogue on the socioethical implications of human germline genome-editing research, and considers the relevant guidelines and legislation in different countries. PMID:26078206

  15. The E3 ligase APC/C(Cdh1) promotes ubiquitylation-mediated proteolysis of PAX3 to suppress melanocyte proliferation and melanoma growth.

    PubMed

    Cao, Juxiang; Dai, Xiangpeng; Wan, Lixin; Wang, Hongshen; Zhang, Jinfang; Goff, Philip S; Sviderskaya, Elena V; Xuan, Zhenyu; Xu, Zhixiang; Xu, Xiaowei; Hinds, Philip; Flaherty, Keith T; Faller, Douglas V; Goding, Colin R; Wang, Yongjun; Wei, Wenyi; Cui, Rutao

    2015-09-01

    The anaphase-promoting complex or cyclosome with the subunit Cdh1 (APC/C(Cdh1)) is an E3 ubiquitin ligase involved in the control of the cell cycle. Here, we identified sporadic mutations occurring in the genes encoding APC components, including Cdh1, in human melanoma samples and found that loss of APC/C(Cdh1) may promote melanoma development and progression, but not by affecting cell cycle regulatory targets of APC/C. Most of the mutations we found in CDH1 were those associated with ultraviolet light (UV)-induced melanomagenesis. Compared with normal human skin tissue and human or mouse melanocytes, the abundance of Cdh1 was decreased and that of the transcription factor PAX3 was increased in human melanoma tissue and human or mouse melanoma cell lines, respectively; Cdh1 abundance was further decreased with advanced stages of human melanoma. PAX3 was a substrate of APC/C(Cdh1) in melanocytes, and APC/C(Cdh1)-mediated ubiquitylation marked PAX3 for proteolytic degradation in a manner dependent on the D-box motif in PAX3. Either mutating the D-box in PAX3 or knocking down Cdh1 prevented the ubiquitylation and degradation of PAX3 and increased proliferation and melanin production in melanocytes. Knocking down Cdh1 in melanoma cells in culture or before implantation in mice promoted doxorubicin resistance, whereas reexpressing wild-type Cdh1, but not E3 ligase-deficient Cdh1 or a mutant that could not interact with PAX3, restored doxorubicin sensitivity in melanoma cells both in culture and in xenografts. Thus, our findings suggest a tumor suppressor role for APC/C(Cdh1) in melanocytes and that targeting PAX3 may be a strategy for treating melanoma. PMID:26329581

  16. Exome sequencing identifies potential novel candidate genes in patients with unexplained colorectal adenomatous polyposis.

    PubMed

    Spier, Isabel; Kerick, Martin; Drichel, Dmitriy; Horpaopan, Sukanya; Altmüller, Janine; Laner, Andreas; Holzapfel, Stefanie; Peters, Sophia; Adam, Ronja; Zhao, Bixiao; Becker, Tim; Lifton, Richard P; Holinski-Feder, Elke; Perner, Sven; Thiele, Holger; Nöthen, Markus M; Hoffmann, Per; Timmermann, Bernd; Schweiger, Michal R; Aretz, Stefan

    2016-04-01

    In up to 30% of patients with colorectal adenomatous polyposis, no germline mutation in the known genes APC, causing familial adenomatous polyposis, MUTYH, causing MUTYH-associated polyposis, and POLE or POLD1, causing Polymerase-Proofreading-associated polyposis can be identified, although a hereditary etiology is likely. To uncover new causative genes, exome sequencing was performed using DNA from leukocytes and a total of 12 colorectal adenomas from seven unrelated patients with unexplained sporadic adenomatous polyposis. For data analysis and variant filtering, an established bioinformatics pipeline including in-house tools was applied. Variants were filtered for rare truncating point mutations and copy-number variants assuming a dominant, recessive, or tumor suppressor model of inheritance. Subsequently, targeted sequence analysis of the most promising candidate genes was performed in a validation cohort of 191 unrelated patients. All relevant variants were validated by Sanger sequencing. The analysis of exome sequencing data resulted in the identification of rare loss-of-function germline mutations in three promising candidate genes (DSC2, PIEZO1, ZSWIM7). In the validation cohort, further variants predicted to be pathogenic were identified in DSC2 and PIEZO1. According to the somatic mutation spectra, the adenomas in this patient cohort follow the classical pathways of colorectal tumorigenesis. The present study identified three candidate genes which might represent rare causes for a predisposition to colorectal adenoma formation. Especially PIEZO1 (FAM38A) and ZSWIM7 (SWS1) warrant further exploration. To evaluate the clinical relevance of these genes, investigation of larger patient cohorts and functional studies are required. PMID:26780541

  17. Single-molecule spectroscopy and femtosecond transient absorption studies on the excitation energy transfer process in ApcE(1-240) dimers.

    PubMed

    Long, Saran; Zhou, Meng; Tang, Kun; Zeng, Xiao-Li; Niu, Yingli; Guo, Qianjin; Zhao, Kai-Hong; Xia, Andong

    2015-05-28

    ApcE(1-240) dimers with one intrinsic phycocyanobilin (PCB) chromophore in each monomer that is truncated from the core-membrane linker (ApcE) of phycobilisomes (PBS) in Nostoc sp. PCC 7120 show a sharp and significantly red-shifted absorption. Two explanations either conformation-dependent Förster resonance energy transfer (FRET) or the strong exciton coupling limit have been proposed for red-shifted absorption. This is a classic example of the special pair in the photosynthetic light harvesting proteins, but the mechanism of this interaction is still a matter of intense debate. We report the studies using single-molecule and transient absorption spectra on the interaction in the special pair of ApcE dimers. Our results demonstrate the presence of conformation-dependent FRET between the two PCB chromophores in ApcE dimers. The broad distributions of fluorescence intensities, lifetimes and polarization difference from single-molecule measurements reveal the heterogeneity of local protein-pigment environments in ApcE dimers, where the same molecular structures but different protein environments are the main reason for the two PCB chromophores with different spectral properties. The excitation energy transfer rate between the donor and the acceptor about (110 ps)(-1) is determined from transient absorption measurements. The red-shifted absorption in ApcE dimers could result from more extending conformation, which shows another type of absorption redshift that does not depend on strong exciton coupling. The results here stress the importance of conformation-controlled spectral properties of the chemically identical chromophores, which could be a general feature to control energy/electron transfer, widely existing in the light harvesting complexes. PMID:25925197

  18. Germ-line genetic enhancement and Rawlsian primary goods.

    PubMed

    Allhoff, Fritz

    2005-03-01

    Genetic interventions raise a host of moral issues and, of its various species, germ-line genetic enhancement is the most morally contentious. This paper surveys various arguments against germ-line enhancement and attempts to demonstrate their inadequacies. A positive argument is advanced in favor of certain forms of germ-line enhancements, which holds that they are morally permissible if and only if they augment Rawlsian primary goods, either directly or by facilitating their acquisition. PMID:15881795

  19. High-Throughput Cloning of Temperature-Sensitive Caenorhabditis elegans Mutants with Adult Syncytial Germline Membrane Architecture Defects

    PubMed Central

    Lowry, Josh; Yochem, John; Chuang, Chien-Hui; Sugioka, Kenji; Connolly, Amy A.; Bowerman, Bruce

    2015-01-01

    The adult Caenorhabditis elegans hermaphrodite gonad consists of two mirror-symmetric U-shaped arms, with germline nuclei located peripherally in the distal regions of each arm. The nuclei are housed within membrane cubicles that are open to the center, forming a syncytium with a shared cytoplasmic core called the rachis. As the distal germline nuclei progress through meiotic prophase, they move proximally and eventually cellularize as their compartments grow in size. The development and maintenance of this complex and dynamic germline membrane architecture are relatively unexplored, and we have used a forward genetic screen to identify 20 temperature-sensitive mutations in 19 essential genes that cause defects in the germline membrane architecture. Using a combined genome-wide SNP mapping and whole genome sequencing strategy, we have identified the causal mutations in 10 of these mutants. Four of the genes we have identified are conserved, with orthologs known to be involved in membrane biology, and are required for proper development or maintenance of the adult germline membrane architecture. This work provides a starting point for further investigation of the mechanisms that control the dynamics of syncytial membrane architecture during adult oogenesis. PMID:26311651

  20. The Birth of Animal Development: Multicellularity and the Germline.

    PubMed

    Woodland, Hugh R

    2016-01-01

    The evolution of multicellular animals has been attributed to many kinds of selective advantage; here I suggest that the evolution of somatic cells to feed and protect the germline was central to the appearance of animals. This would have been driven by selection for extreme anisogamy-the evolution of sperm and egg. Evidence is adduced from the germline stem cells of simple animals (defining germline as any cell that normally produces the next generation via the sexual process) and from the control circuitry ubiquitous in animal germlines. With the soma and its elaboration came animal development, as we understand it. PMID:26970004

  1. Female mice lack adult germ-line stem cells but sustain oogenesis using stable primordial follicles.

    PubMed

    Lei, Lei; Spradling, Allan C

    2013-05-21

    Whether or not mammalian females generate new oocytes during adulthood from germ-line stem cells to sustain the ovarian follicle pool has recently generated controversy. We used a sensitive lineage-labeling system to determine whether stem cells are needed in female adult mice to compensate for follicular losses and to directly identify active germ-line stem cells. Primordial follicles generated during fetal life are highly stable, with a half-life during adulthood of 10 mo, and thus are sufficient to sustain adult oogenesis without a source of renewal. Moreover, in normal mice or following germ-cell depletion with Busulfan, only stable, single oocytes are lineage-labeled, rather than cell clusters indicative of new oocyte formation. Even one germ-line stem cell division per 2 wk would have been detected by our method, based on the kinetics of fetal follicle formation. Thus, adult female mice neither require nor contain active germ-line stem cells or produce new oocytes in vivo. PMID:23630252

  2. Neurofibromatosis type 1 in two siblings due to maternal germline mosaicism.

    PubMed

    Trevisson, E; Forzan, M; Salviati, L; Clementi, M

    2014-04-01

    Neurofibromatosis type 1 (NF1) is caused by loss of function mutations of the NF1 gene, which are de novo in 50% of cases. Although this gene shows one of the highest mutation rates in the human genome, germline mosaicism is very rare in this condition. We describe the molecular analysis of a family in which neurofibromatosis type 1 occurred in two out of four siblings born to unaffected parents. Molecular analysis of the NF1 gene identified in both patients the same splicing mutation c.1392+1G>A, which was absent in parental lymphocytes. Microsatellite analysis showed that the two affected siblings shared the same maternal allele, however a specific PCR-RFLP assay excluded the presence of the NF1 splicing mutation in multiple maternal tissues. Our molecular and clinical findings are consistent with a germline mosaicism for the NF1 splicing mutation. This is the first case of maternal germline mosaicism for a NF1 mutation characterized so far at the molecular level. Our data confirm that germline mosaicism is rare in neurofibromatosis 1, but it has important implications for genetic counseling. PMID:23621909

  3. Caenorhabditis elegans atx-2 Promotes Germline Proliferation and the Oocyte Fate

    PubMed Central

    Maine, Eleanor M.; Hansen, Dave; Springer, Deborah; Vought, Valarie E.

    2004-01-01

    In the Caenorhabditis elegans germline, proliferation is induced by Notch-type signaling. Entry of germ cells into meiosis is triggered by activity of the GLD-1 and GLD-2 pathways, which function redundantly to promote meiosis and/or inhibit proliferation. Activation of the germline Notch-type receptor, GLP-1, ultimately inhibits the activities of the GLD-1 and GLD-2 pathways. We previously identified several ego (enhancer of glp-1) genes that promote germline proliferation and interact genetically with the GLP-1 signaling pathway. Here, we show that atx-2 is an ego gene. Our data suggest that ATX-2 is not a positive regulator of the GLP-1 signaling pathway and GLP-1 signaling is not the sole positive regulator of ATX-2 activity. Moreover, our data indicate that GLP-1 must have an additional function, which may be to repress activity of a third meiotic entry pathway that would work in parallel with the GLD-1 and GLD-2 pathways. In addition to its role in proliferation, ATX-2 acts downstream of FOG-2 to promote the female germline fate. PMID:15514056

  4. Caenorhabditis elegans atx-2 promotes germline proliferation and the oocyte fate.

    PubMed

    Maine, Eleanor M; Hansen, Dave; Springer, Deborah; Vought, Valarie E

    2004-10-01

    In the Caenorhabditis elegans germline, proliferation is induced by Notch-type signaling. Entry of germ cells into meiosis is triggered by activity of the GLD-1 and GLD-2 pathways, which function redundantly to promote meiosis and/or inhibit proliferation. Activation of the germline Notch-type receptor, GLP-1, ultimately inhibits the activities of the GLD-1 and GLD-2 pathways. We previously identified several ego (enhancer of glp-1) genes that promote germline proliferation and interact genetically with the GLP-1 signaling pathway. Here, we show that atx-2 is an ego gene. Our data suggest that ATX-2 is not a positive regulator of the GLP-1 signaling pathway and GLP-1 signaling is not the sole positive regulator of ATX-2 activity. Moreover, our data indicate that GLP-1 must have an additional function, which may be to repress activity of a third meiotic entry pathway that would work in parallel with the GLD-1 and GLD-2 pathways. In addition to its role in proliferation, ATX-2 acts downstream of FOG-2 to promote the female germline fate. PMID:15514056

  5. Paternal lifestyle as a potential source of germline mutations transmitted to offspring.

    PubMed

    Linschooten, Joost O; Verhofstad, Nicole; Gutzkow, Kristine; Olsen, Ann-Karin; Yauk, Carole; Oligschläger, Yvonne; Brunborg, Gunnar; van Schooten, Frederik J; Godschalk, Roger W L

    2013-07-01

    Paternal exposure to high levels of radioactivity causes heritable germline minisatellite mutations. However, the effect of more general paternal exposures, such as cigarette smoking, on germline mutations remains unexplored. We analyzed two of the most commonly used minisatellite loci (CEB1 and B6.7) to identify germline mutations in blood samples of complete mother-father-child triads from the Norwegian Mother and Child Cohort Study (MoBa). The presence of mutations was subsequently related to general lifestyle factors, including paternal smoking before the partner became pregnant. Paternally derived mutations at the B6.7 locus (mutation frequency 0.07) were not affected by lifestyle. In contrast, high gross yearly income as a general measure of a healthy lifestyle coincided with low-mutation frequencies at the CEB1 locus (P=0.047). Income was inversely related to smoking behavior, and paternally derived CEB1 mutations were dose dependently increased when the father smoked in the 6 mo before pregnancy, 0.21 vs. 0.05 in smoking and nonsmoking fathers, respectively (P=0.061). These results suggest that paternal lifestyle can affect the chance of heritable mutations in unstable repetitive DNA sequences. To our knowledge, this is the first study reporting an effect of lifestyle on germline minisatellite mutation frequencies in a human population with moderate paternal exposures. PMID:23538710

  6. A Cdh1-APC/FMRP Ubiquitin Signaling Link Drives mGluR-Dependent Synaptic Plasticity in the Mammalian Brain

    PubMed Central

    Huang, Ju; Ikeuchi, Yoshiho; Malumbres, Marcos; Bonni, Azad

    2015-01-01

    SUMMARY Deregulation of synaptic plasticity may contribute to the pathogenesis of developmental cognitive disorders. In particular, exaggerated mGluR-dependent LTD is featured in fragile X syndrome, but the mechanisms that regulate mGluR-LTD remain incompletely understood. We report that conditional knockout of Cdh1, the key regulatory subunit of the ubiquitin ligase Cdh1-anaphase promoting complex (Cdh1-APC), profoundly impairs mGluR-LTD in the hippocampus. Mechanistically, we find that Cdh1-APC operates in the cytoplasm to drive mGluR-LTD. We also identify the fragile X syndrome protein FMRP as a substrate of Cdh1-APC. Endogenous Cdh1-APC forms a complex with endogenous FMRP, and knockout of Cdh1 impairs mGluR-induced ubiquitination and degradation of FMRP in the hippocampus. Knockout of FMRP suppresses, and expression of an FMRP mutant protein that fails to interact with Cdh1 phenocopies, the Cdh1 knockout phenotype of impaired mGluR-LTD. These findings define Cdh1-APC and FMRP as components of a novel ubiquitin-signaling pathway that regulates mGluR-LTD in the brain. PMID:25913861

  7. CDH1 germline mutations and hereditary lobular breast cancer.

    PubMed

    Corso, Giovanni; Intra, Mattia; Trentin, Chiara; Veronesi, Paolo; Galimberti, Viviana

    2016-04-01

    Hereditary diffuse gastric cancer is an autosomal dominant inherited disease associated of CDH1 germline mutations (that encodes for the E-cadherin protein), and lobular breast cancer is the second most frequent type of neoplasia. Recently, novel E-cadherin constitutional alterations have been identified in pedigree clustering only for lobular breast carcinoma without evidence of diffuse gastric tumors and in absence of BRCA1/2 mutations. This first evidence opens novel questions about the inherited correlation between diffuse gastric and lobular breast cancers. In this brief review we revise the literature data about the CDH1 mutation frequency affecting exclusively lobular breast cancer, providing clinical recommendation for asymptomatic mutation carriers. PMID:26759166

  8. Cellular Factors Targeting APCs to Modulate Adaptive T Cell Immunity

    PubMed Central

    Do, Jeongsu; Min, Booki

    2014-01-01

    The fate of adaptive T cell immunity is determined by multiple cellular and molecular factors, among which the cytokine milieu plays the most important role in this process. Depending on the cytokines present during the initial T cell activation, T cells become effector cells that produce different effector molecules and execute adaptive immune functions. Studies thus far have primarily focused on defining how these factors control T cell differentiation by targeting T cells themselves. However, other non-T cells, particularly APCs, also express receptors for the factors and are capable of responding to them. In this review, we will discuss how APCs, by responding to those cytokines, influence T cell differentiation and adaptive immunity. PMID:25126585

  9. 75 FR 78246 - Medicare Program; Re-Chartering of the Advisory Panel on Ambulatory Payment Classification (APC...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-15

    ... Charter effective through November 21, 2012. FOR FURTHER INFORMATION CONTACT: Shirl Ackerman-Ross, (410... new technology APCs to clinical APCs). Evaluating APC group weights. Reviewing packaging the cost of... methodology for packaging and the impact of packaging on APC group structure and payment. Removing...

  10. Racial variations in frequency and phenotypes of APC and MUTYH mutations in 6,169 individuals undergoing genetic testing

    PubMed Central

    Inra, Jennifer A.; Steyerberg, Ewout W.; Grover, Shilpa; McFarland, Ashley; Syngal, Sapna; Kastrinos, Fay

    2016-01-01

    Purpose To assess whether differences in frequency and phenotype of APC and MUTYH mutations exist among racially/ethnically diverse populations. Methods 6169 individuals with personal and/or family history of colorectal cancer (CRC) and polyps were studied. APC testing involved full sequencing/large rearrangement analysis (FS/LRA); MUTYH involved “panel testing” (for Y165C, G382D mutations), or FS/LRA, performed by Myriad Genetics, a commercial laboratory. Subjects were identified as Caucasian, Asian, African American (AA), or Other. Statistical tests included Chi-Square, Fisher’s Exact, ANOVA and z-approximation. Results 17.5% had pathogenic APC mutations. 4.8% were biallelic MUTYH carriers. 18% were non-Caucasian with >100 adenomas and younger ages of adenoma or CRC diagnosis (p<0.0001) than Caucasians. The overall APC mutation rate was higher in Asians, AAs and Others compared to Caucasians (25.2%, 30.9%, 24%, 15.5%;p<0.0001) but similar in all groups when adjusted for polyp burden. More MUTYH biallelic carriers were Caucasian or Other than Asian or AA (5%, 7%, 2.7%, 0.3%;p<0.0001). Among Caucasians, 5% were biallelic carriers identified by panel testing versus 2% by sequencing/LRA (p=0.002). Among non-Caucasians, 3% undergoing panel testing were biallelic carriers versus 10% identified by sequencing/LRA(p<0.0002). Conclusion Non-Caucasians undergo genetic testing at more advanced stages of polyposis and/or younger ages of CRC/polyp diagnosis. Restricted MUTYH analysis may miss significant numbers of biallelic carriers, particularly in non-Caucasians. PMID:25590978

  11. Clerics urge ban on altering germline cells.

    PubMed

    Norman, C

    1983-06-24

    A resolution calling for a ban on genetic engineering of human reproductive cells has been signed by leaders of almost every major church group in the United States. Some of the religious leaders, while not certain that a total moratorium should be placed on altering germline cells, signed the statement in order to stimulate public debate on the issue. Legislation has recently been introduced in Congress to set up a committee to monitor genetic engineering and its human applications, but author Jeremy Rifkin, the impetus behind the church leaders' resolution, argues that such tampering threatens the gene pool and should be banned altogether. PMID:6574603

  12. APC/C is an essential regulator of centrosome clustering.

    PubMed

    Drosopoulos, Konstantinos; Tang, Chan; Chao, William C H; Linardopoulos, Spiros

    2014-01-01

    Centrosome amplification has been extensively associated with cancer. Cancer cells with extra centrosomes have the ability to cluster the extra centrosomes and divide in a bipolar fashion. Although a number of proteins have been shown to be involved in centrosome clustering, a mechanistic understanding of how this process is coordinated is not yet well defined. Here, to reveal regulators of centrosome clustering, we perform small interfering RNA (siRNA) screens with multiple assay readouts in a human isogenic cellular model. We find that APC/C activity is essential for centrosome clustering. We show that the motor kinesin Eg5 is a substrate of APC/C-CDH1, and that inhibition of APC/C results in stabilization of Eg5. Increased Eg5 protein levels disturb the balance of forces on the spindle and prevent centrosome clustering. This process is completely reversed after a short treatment with the Eg5 inhibitor, monastrol. These data advance our understanding of the regulation of centrosome clustering. PMID:24751481

  13. Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis.

    PubMed

    March, H Nikki; Rust, Alistair G; Wright, Nicholas A; ten Hoeve, Jelle; de Ridder, Jeroen; Eldridge, Matthew; van der Weyden, Louise; Berns, Anton; Gadiot, Jules; Uren, Anthony; Kemp, Richard; Arends, Mark J; Wessels, Lodewyk F A; Winton, Douglas J; Adams, David J

    2011-12-01

    The evolution of colorectal cancer suggests the involvement of many genes. To identify new drivers of intestinal cancer, we performed insertional mutagenesis using the Sleeping Beauty transposon system in mice carrying germline or somatic Apc mutations. By analyzing common insertion sites (CISs) isolated from 446 tumors, we identified many hundreds of candidate cancer drivers. Comparison to human data sets suggested that 234 CIS-targeted genes are also dysregulated in human colorectal cancers. In addition, we found 183 CIS-containing genes that are candidate Wnt targets and showed that 20 CISs-containing genes are newly discovered modifiers of canonical Wnt signaling. We also identified mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included those encoding members of the FGF signaling cascade. Some 70 genes had co-occurrence of CIS pairs, clustering into 38 sub-networks that may regulate tumor development. PMID:22057237

  14. EGO-1, a Putative RNA-Directed RNA Polymerase, Promotes Germline Proliferation in Parallel With GLP-1/Notch Signaling and Regulates the Spatial Organization of Nuclear Pore Complexes and Germline P Granules in Caenorhabditis elegans

    PubMed Central

    Vought, Valarie E.; Ohmachi, Mitsue; Lee, Min-Ho; Maine, Eleanor M.

    2005-01-01

    Caenorhabditis elegans EGO-1, a putative cellular RNA-directed RNA polymerase, promotes several aspects of germline development, including proliferation, meiosis, and gametogenesis, and ensures a robust response to RNA interference. In C. elegans, GLP-1/Notch signaling from the somatic gonad maintains a population of proliferating germ cells, while entry of germ cells into meiosis is triggered by the GLD-1 and GLD-2 pathways. GLP-1 signaling prevents germ cells from entering meiosis by inhibiting GLD-1 and GLD-2 activity. We originally identified the ego-1 gene on the basis of a genetic interaction with glp-1. Here, we investigate the role of ego-1 in germline proliferation. Our data indicate that EGO-1 does not positively regulate GLP-1 protein levels or GLP-1 signaling activity. Moreover, GLP-1 signaling does not positively regulate EGO-1 activity. EGO-1 does not inhibit expression of GLD-1 protein in the distal germline. Instead, EGO-1 acts in parallel with GLP-1 signaling to influence the proliferation vs. meiosis fate choice. Moreover, EGO-1 and GLD-1 act in parallel to ensure germline health. Finally, the size and distribution of nuclear pore complexes and perinuclear P granules are altered in the absence of EGO-1, effects that disrupt germ cell biology per se and probably limit germline growth. PMID:15911573

  15. EGO-1, a putative RNA-directed RNA polymerase, promotes germline proliferation in parallel with GLP-1/notch signaling and regulates the spatial organization of nuclear pore complexes and germline P granules in Caenorhabditis elegans.

    PubMed

    Vought, Valarie E; Ohmachi, Mitsue; Lee, Min-Ho; Maine, Eleanor M

    2005-07-01

    Caenorhabditis elegans EGO-1, a putative cellular RNA-directed RNA polymerase, promotes several aspects of germline development, including proliferation, meiosis, and gametogenesis, and ensures a robust response to RNA interference. In C. elegans, GLP-1/Notch signaling from the somatic gonad maintains a population of proliferating germ cells, while entry of germ cells into meiosis is triggered by the GLD-1 and GLD-2 pathways. GLP-1 signaling prevents germ cells from entering meiosis by inhibiting GLD-1 and GLD-2 activity. We originally identified the ego-1 gene on the basis of a genetic interaction with glp-1. Here, we investigate the role of ego-1 in germline proliferation. Our data indicate that EGO-1 does not positively regulate GLP-1 protein levels or GLP-1 signaling activity. Moreover, GLP-1 signaling does not positively regulate EGO-1 activity. EGO-1 does not inhibit expression of GLD-1 protein in the distal germline. Instead, EGO-1 acts in parallel with GLP-1 signaling to influence the proliferation vs. meiosis fate choice. Moreover, EGO-1 and GLD-1 act in parallel to ensure germline health. Finally, the size and distribution of nuclear pore complexes and perinuclear P granules are altered in the absence of EGO-1, effects that disrupt germ cell biology per se and probably limit germline growth. PMID:15911573

  16. The tissue effect of argon-plasma coagulation with prior submucosal injection (Hybrid-APC) versus standard APC: A randomized ex-vivo study

    PubMed Central

    Neugebauer, Alexander; Scharpf, Marcus; Braun, Kirsten; May, Andrea; Ell, Christian; Fend, Falko; Enderle, Markus D

    2014-01-01

    Background Thermal ablation for Barrett’s oesophagus has widely been established in gastrointestinal endoscopy during the last decade. The mainly used methods of radiofrequency ablation (RFA) and argon-plasma coagulation (APC) carry a relevant risk of stricture formation of up to 5–15%. Newer ablation techniques that are able to overcome this disadvantage would therefore be desirable. The aim of the present study was to compare the depth of tissue injury of the new method of Hybrid-APC versus standard APC within a randomized study in a porcine oesophagus model. Methods Using a total of eight explanted pig oesophagi, 48 oesophageal areas were ablated either by standard or Hybrid-APC (APC with prior submucosal fluid injection) using power settings of 50 and 70 W. The depth of tissue injury to the oesophageal wall was analysed macroscopically and histopathologically. Results Using 50 W, mean coagulation depth was 937 ± 469 µm during standard APC, and 477 ± 271 µm during Hybrid-APC (p = 0.064). Using 70 W, coagulation depth was 1096 ± 320 µm (standard APC) and 468 ± 136 µm (Hybrid-APC; p = 0.003). During all settings, damage to the muscularis mucosae was observed. Using standard APC, damage to the submucosal layer was observed in 4/6 (50 W) and 6/6 cases (70 W). During Hybrid-APC, coagulation of the submucosal layer occurred in 2/6 (50 W) and 1/6 cases (70 W). The proper muscle layer was only damaged during conventional APC (50 W: 1/6; 70 W: 3/6). Limitations Ex-vivo animal study with limited number of cases. Conclusions Hybrid-APC reduces coagulation depth by half in comparison with standard APC, with no thermal injury to the proper muscle layer. It may therefore lead to a lower rate of stricture formation during clinical application. PMID:25360316

  17. DNA Demethylation Dynamics in the Human Prenatal Germline.

    PubMed

    Gkountela, Sofia; Zhang, Kelvin X; Shafiq, Tiasha A; Liao, Wen-Wei; Hargan-Calvopiña, Joseph; Chen, Pao-Yang; Clark, Amander T

    2015-06-01

    Global DNA demethylation in humans is a fundamental process that occurs in pre-implantation embryos and reversion to naive ground state pluripotent stem cells (PSCs). However, the extent of DNA methylation reprogramming in human germline cells is unknown. Here, we performed whole-genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq) of human prenatal germline cells from 53 to 137 days of development. We discovered that the transcriptome and methylome of human germline is distinct from both human PSCs and the inner cell mass (ICM) of human blastocysts. Using this resource to monitor the outcome of global DNA demethylation with reversion of primed PSCs to the naive ground state, we uncovered hotspots of ultralow methylation at transposons that are protected from demethylation in the germline and ICM. Taken together, the human germline serves as a valuable in vivo tool for monitoring the epigenome of cells that have emerged from a global DNA demethylation event. PMID:26004067

  18. Nuclear-cytoplasmic shuttling of APC regulates beta-catenin subcellular localization and turnover.

    PubMed

    Henderson, B R

    2000-09-01

    Mutational inactivation of the APC gene is a key early event in the development of familial adenomatous polyposis and colon cancer. APC suppresses tumour progression by promoting degradation of the oncogenic transcriptional activator beta-catenin. APC gene mutations can lead to abnormally high levels of beta-catenin in the nucleus, and the consequent activation of transforming genes. Here, we show that APC is a nuclear-cytoplasmic shuttling protein, and that it can function as a beta-catenin chaperone. APC contains two active nuclear export sequences (NES) at the amino terminus, and mutagenesis of these conserved motifs blocks nuclear export dependent on the CRM1 export receptor. Treatment of cells with the CRM1-specific export inhibitor leptomycin B shifts APC from cytoplasm to nucleus. beta-catenin localization is also regulated by CRM1, but in an APC-dependent manner. Transient expression of wild-type APC in SW480 (APCmut/mut) colon cancer cells enhances nuclear export and degradation of beta-catenin, and these effects can be blocked by mutagenesis of the APC NES. These findings suggest that wild-type APC controls the nuclear accumulation of beta-catenin by a combination of nuclear export and cytoplasmic degradation. PMID:10980707

  19. Dietary Methyl Donor Depletion Protects Against Intestinal Tumorigenesis in ApcMin/+ Mice

    PubMed Central

    Kadaveru, Krishna; Protiva, Petr; Greenspan, Emily J; Kim, Young-In; Rosenberg, Daniel W

    2012-01-01

    Despite recent population data, the influence of dietary folate supplementation on colon cancer risk remains controversial. This study examines the effects of folate deficiency, in combination with choline, methionine and vitamin B12 depletion, on intestinal tumorigenesis in ApcMin/+ mice. Methyl donor sufficient (MDS) and deficient (MDD) diets were started at 5 or 10 weeks of age and tumors evaluated at 16 weeks. MDD suppressed intestinal tumor formation in ApcMin/+ mice (~80%) when started at 5 weeks of age. The protective effect was lost when MDD was initiated at 10 weeks of age, indicating an important time-dependency on cancer suppression. Concomitant with cancer protection, MDD restricted body weight gain. Therefore, a second study was conducted in which MDS was given ad libitum or pair-fed with MDD. While small intestinal tumors were reduced 54% in pair-fed MDS mice, MDD caused a further reduction (96%). In colon, although MDD did not affect tumor numbers, tumor size was reduced. Gene expression profiling of normal-appearing colonic mucosa after 11 weeks on MDD identified a total of 493 significantly down-regulated genes relative to the MDS group. Pathway analysis placed many of these genes within general categories of inflammatory signaling and cell cycle regulation, consistent with recently published human data obtained during folate depletion (1). Further studies are warranted to investigate the complex interplay of methyl donor status and cancer protection in high-risk populations. PMID:22677908

  20. Cdh1 regulates craniofacial development via APC-dependent ubiquitination and activation of Goosecoid.

    PubMed

    Shao, Rui; Liu, Jia; Yan, Guang; Zhang, Jinfang; Han, Yujiao; Guo, Jianfeng; Xu, Zhan; Yuan, Zhu; Liu, Jiankang; Malumbres, Marcos; Wan, Lixin; Wei, Wenyi; Zou, Weiguo

    2016-06-01

    Craniofacial anomalies (CFAs) characterized by birth defects of skull and facial bones are the most frequent congenital disease. Genomic analysis has identified multiple genes responsible for CFAs; however, the underlying genetic mechanisms for the majority of CFAs remain largely unclear. Our previous study revealed that the Wwp2 E3 ubiquitin ligase facilitates craniofacial development in part through inducing monoubiquitination and activation of the paired-like homeobox transcription factor, Goosecoid (Gsc). Here we report that Gsc is also ubiquitinated and activated by the APC(Cdh1) E3 ubiquitin ligase, leading to transcriptional activation of various Gsc target genes crucial for craniofacial development. Consistenly, neural crest-specific Cdh1-knockout mice display similar bone malformation as Wwp2-deficient mice in the craniofacial region, characterized by a domed skull, a short snout and a twisted nasal bone. Mechanistically, like Wwp2-deficient mice, mice with Cdh1 deficiency in neural crest cells exhibit reduced Gsc/Sox6 transcriptional activities. Simultaneous deletion of Cdh1 and Wwp2 results in a more severe craniofacial defect compared with single gene deletion, suggesting a synergistic augmentation of Gsc activity by these two E3 ubiquitin ligases. Hence, our study reveals a novel role for Cdh1 in craniofacial development through promoting APC-dependent non-proteolytic ubiquitination and activation of Gsc. PMID:27126000

  1. Germline EPHB2 Receptor Variants in Familial Colorectal Cancer

    PubMed Central

    Zogopoulos, George; Jorgensen, Claus; Bacani, Julinor; Montpetit, Alexandre; Lepage, Pierre; Ferretti, Vincent; Chad, Lauren; Selvarajah, Subani; Zanke, Brent; Hudson, Thomas J.; Pawson, Tony; Gallinger, Steven

    2008-01-01

    Familial clustering of colorectal cancer occurs in 15–20% of cases, however recognized cancer syndromes explain only a small fraction of this disease. Thus, the genetic basis for the majority of hereditary colorectal cancer remains unknown. EPHB2 has recently been implicated as a candidate tumor suppressor gene in colorectal cancer. The aim of this study was to evaluate the contribution of EPHB2 to hereditary colorectal cancer. We screened for germline EPHB2 sequence variants in 116 population-based familial colorectal cancer cases by DNA sequencing. We then estimated the population frequencies and characterized the biological activities of the EPHB2 variants identified. Three novel nonsynonymous missense alterations were detected. Two of these variants (A438T and G787R) result in significant residue changes, while the third leads to a conservative substitution in the carboxy-terminal SAM domain (V945I). The former two variants were found once in the 116 cases, while the V945I variant was present in 2 cases. Genotyping of additional patients with colorectal cancer and control subjects revealed that A438T and G787R represent rare EPHB2 alleles. In vitro functional studies show that the G787R substitution, located in the kinase domain, causes impaired receptor kinase activity and is therefore pathogenic, whereas the A438T variant retains its receptor function and likely represents a neutral polymorphism. Tumor tissue from the G787R variant case manifested loss of heterozygosity, with loss of the wild-type allele, supporting a tumor suppressor role for EPHB2 in rare colorectal cancer cases. Rare germline EPHB2 variants may contribute to a small fraction of hereditary colorectal cancer. PMID:18682749

  2. Src64 controls a novel actin network required for proper ring canal formation in the Drosophila male germline.

    PubMed

    Eikenes, Åsmund Husabø; Malerød, Lene; Lie-Jensen, Anette; Sem Wegner, Catherine; Brech, Andreas; Liestøl, Knut; Stenmark, Harald; Haglund, Kaisa

    2015-12-01

    In many organisms, germ cells develop as cysts in which cells are interconnected via ring canals (RCs) as a result of incomplete cytokinesis. However, the molecular mechanisms of incomplete cytokinesis remain poorly understood. Here, we address the role of tyrosine phosphorylation of RCs in the Drosophila male germline. We uncover a hierarchy of tyrosine phosphorylation within germline cysts that positively correlates with RC age. The kinase Src64 is responsible for mediating RC tyrosine phosphorylation, and loss of Src64 causes a reduction in RC diameter within germline cysts. Mechanistically, we show that Src64 controls an actin network around the RCs that depends on Abl and the Rac/SCAR/Arp2/3 pathway. The actin network around RCs is required for correct RC diameter in cysts of developing germ cells. We also identify that Src64 is required for proper germ cell differentiation in the Drosophila male germline independent of its role in RC regulation. In summary, we report that Src64 controls actin dynamics to mediate proper RC formation during incomplete cytokinesis during germline cyst development in vivo. PMID:26628094

  3. Heparan sulfate regulates the number and centrosome positioning of Drosophila male germline stem cells

    PubMed Central

    Levings, Daniel C.; Arashiro, Takeshi; Nakato, Hiroshi

    2016-01-01

    Stem cell division is tightly controlled via secreted signaling factors and cell adhesion molecules provided from local niche structures. Molecular mechanisms by which each niche component regulates stem cell behaviors remain to be elucidated. Here we show that heparan sulfate (HS), a class of glycosaminoglycan chains, regulates the number and asymmetric division of germline stem cells (GSCs) in the Drosophila testis. We found that GSC number is sensitive to the levels of 6-O sulfate groups on HS. Loss of 6-O sulfation also disrupted normal positioning of centrosomes, a process required for asymmetric division of GSCs. Blocking HS sulfation specifically in the niche, termed the hub, led to increased GSC numbers and mispositioning of centrosomes. The same treatment also perturbed the enrichment of Apc2, a component of the centrosome-anchoring machinery, at the hub–GSC interface. This perturbation of the centrosome-anchoring process ultimately led to an increase in the rate of spindle misorientation and symmetric GSC division. This study shows that specific HS modifications provide a novel regulatory mechanism for stem cell asymmetric division. The results also suggest that HS-mediated niche signaling acts upstream of GSC division orientation control. PMID:26792837

  4. The ins and outs of APC and β-catenin nuclear transport

    PubMed Central

    Henderson, Beric R.; Fagotto, Francois

    2002-01-01

    Adenomatous polyposis coli (APC) and β-catenin, two key interacting proteins implicated in development and cancer, were recently found to traffic into and out of the nucleus in response to internal and external signals. The two proteins can enter and exit the nucleus independently, a discovery that has prompted debate about the previously proposed role of APC as a β-catenin chaperone. Here, we review the regulation of APC and β-catenin subcellular localization, in particular in cancer cells. We speculate that, in non-stimulated cells, APC actively exports β-catenin from the nucleus to the cytoplasm where its levels are regulated by degradation; and, conversely, that, in cancer cells or those stimulated by Wnt signaling, β-catenin degradation is inhibited and the accruing protein is capable of moving between the nucleus and cytoplasm independently of APC. Models that link APC and β-catenin transport to function are discussed. PMID:12223464

  5. The ins and outs of APC and beta-catenin nuclear transport.

    PubMed

    Henderson, Beric R; Fagotto, Francois

    2002-09-01

    Adenomatous polyposis coli (APC) and beta-catenin, two key interacting proteins implicated in development and cancer, were recently found to traffic into and out of the nucleus in response to internal and external signals. The two proteins can enter and exit the nucleus independently, a discovery that has prompted debate about the previously proposed role of APC as a beta-catenin chaperone. Here, we review the regulation of APC and beta-catenin subcellular localization, in particular in cancer cells. We speculate that, in non-stimulated cells, APC actively exports beta-catenin from the nucleus to the cytoplasm where its levels are regulated by degradation; and, conversely, that, in cancer cells or those stimulated by Wnt signaling, beta-catenin degradation is inhibited and the accruing protein is capable of moving between the nucleus and cytoplasm independently of APC. Models that link APC and beta-catenin transport to function are discussed. PMID:12223464

  6. Colon and Endometrial Cancers with Mismatch Repair Deficiency can Arise from Somatic, Rather Than Germline, Mutations

    PubMed Central

    Haraldsdottir, Sigurdis; Hampel, Heather; Tomsic, Jerneja; Frankel, Wendy L.; Pearlman, Rachel; de la Chapelle, Albert; Pritchard, Colin C.

    2014-01-01

    Background & Aims Patients with Lynch syndrome carry germline mutations in single alleles of genes encoding the MMR proteins MLH1, MSH2, MSH6 and PMS2; when the second allele becomes mutated, cancer can develop. Increased screening for Lynch syndrome has identified patients with tumors that have deficiency in MMR, but no germline mutations in genes encoding MMR proteins. We investigated whether tumors with deficient MMR had acquired somatic mutations in patients without germline mutations in MMR genes using next-generation sequencing. Methods We analyzed blood and tumor samples from 32 patients with colorectal or endometrial cancer who participated in Lynch syndrome screening studies in Ohio and were found to have tumors with MMR deficiency (based on microsatellite instability and/or absence of MMR proteins in immunohistochemical analysis, without hypermethylation of MLH1), but no germline mutations in MMR genes. Tumor DNA was sequenced for MLH1, MSH2, MSH6, PMS2, EPCAM, POLE and POLD1 with ColoSeq and mutation frequencies were established. Results Twenty-two of 32 patients (69%) were found to have two somatic (tumor) mutations in MMR genes encoding proteins that were lost from tumor samples, based on immunohistochemistry. Of the 10 tumors without somatic mutations in MMR genes, 3 had somatic mutations with possible loss of heterozygosity that could lead to MMR deficiency, 6 were found to be false-positive results (19%), and 1 had no mutations known to be associated with MMR deficiency. All of the tumors found to have somatic MMR mutations were of the hypermutated phenotype (>12 mutations/Mb); 6 had mutation frequencies >200 per Mb, and 5 of these had somatic mutations in POLE, which encodes a DNA polymerase. Conclusions Some patients are found to have tumors with MMR deficiency during screening for Lynch syndrome, yet have no identifiable germline mutations in MMR genes. We found that almost 70% of these patients acquire somatic mutations in MMR genes, leading to

  7. The I1307K APC mutation in a high-risk clinic setting: a follow-up study.

    PubMed

    Regev, M; Barzilai, S-Eisenberg; Figer, A; Zidan, J; Fidder, H H; Friedman, E

    2005-04-01

    While the I1307K APC mutation clearly confers an increased lifetime risk for colorectal cancer, there is a paucity of data on the natural history of colonic neoplasia in symptomatic and asymptomatic mutation carriers. In this study, 51 Jewish I1307K APC mutation carriers were identified in a high-risk familial cancer clinic over a 4-year period, of whom 29 (56.8%) (four males and 25 females) were successfully telephone interviewed for 0.5-5 years (mean 2.4 +/- 1.4) after initial genetic testing. Of these 29 cases, one individual was diagnosed with colon cancer at the age of 45 years, five had adenomatous polyps (mean number of polyps = 1.8), 11 had breast cancer (mean age at diagnosis 49.5 +/- 10.5 years), and 12 were asymptomatic, at the time of the testing. During the follow-up period, new colonic polyps were diagnosed in three mutation carriers, two with previously diagnosed colon cancer and polyps and only one of the asymptomatic mutation carriers, and two additional previously affected patients had new cancer diagnoses: gastric cancer and melanoma. From this descriptive study, it seems that the short-term risk for colonic polyps in I1307K APC mutation is low, primarily affecting patients with previously diagnosed colon tumors. PMID:15733272

  8. Analytic Patch Configuration (APC) gateway version 1.0 user's guide

    NASA Technical Reports Server (NTRS)

    Bingel, Bradford D.

    1990-01-01

    The Analytic Patch Configuration (APC) is an interactive software tool which translates aircraft configuration geometry files from one format into another. This initial release of the APC Gateway accommodates six formats: the four accepted APC formats (89f, 89fd, 89u, and 89ud), the PATRAN 2.x phase 1 neutral file format, and the Integrated Aerodynamic Analysis System (IAAS) General Geometry (GG) format. Written in ANSI FORTRAN 77 and completely self-contained, the APC Gateway is very portable and was already installed on CDC/NOS, VAX/VMS, SUN, SGI/IRIS, CONVEX, and GRAY hosts.

  9. Apc bridges Wnt/{beta}-catenin and BMP signaling during osteoblast differentiation of KS483 cells

    SciTech Connect

    Miclea, Razvan L.; Horst, Geertje van der; Robanus-Maandag, Els C.; Loewik, Clemens W.G.M.; Oostdijk, Wilma; Wit, Jan M.; Karperien, Marcel

    2011-06-10

    The canonical Wnt signaling pathway influences the differentiation of mesenchymal cell lineages in a quantitative and qualitative fashion depending on the dose of {beta}-catenin signaling. Adenomatous polyposis coli (Apc) is the critical intracellular regulator of {beta}-catenin turnover. To better understand the molecular mechanisms underlying the role of Apc in regulating the differentiation capacity of skeletal progenitor cells, we have knocked down Apc in the murine mesenchymal stem cell-like KS483 cells by stable expression of Apc-specific small interfering RNA. In routine culture, KSFrt-Apc{sub si} cells displayed a mesenchymal-like spindle shape morphology, exhibited markedly decreased proliferation and increased apoptosis. Apc knockdown resulted in upregulation of the Wnt/{beta}-catenin and the BMP/Smad signaling pathways, but osteogenic differentiation was completely inhibited. This effect could be rescued by adding high concentrations of BMP-7 to the differentiation medium. Furthermore, KSFrt-Apc{sub si} cells showed no potential to differentiate into chondrocytes or adipocytes. These results demonstrate that Apc is essential for the proliferation, survival and differentiation of KS483 cells. Apc knockdown blocks the osteogenic differentiation of skeletal progenitor cells, a process that can be overruled by high BMP signaling.

  10. APC mutation and the crypt cycle in murine and human intestine.

    PubMed Central

    Bjerknes, M.; Cheng, H.; Hay, K.; Gallinger, S.

    1997-01-01

    Dysplastic colon adenomas are thought to arise from growth of clones of APC -/- colonic epithelial cells. Isolated clusters of dysplastic crypts are often observed in patients with familial adenomatous polyposis. These patients have genotype APC +/-, and the clusters of dysplastic crypts (called microadenoma or aberrant crypt foci) are thought to represent an early stage in the expansion of a mutant clone of APC -/- cells. It is thought that the growth of these clusters of mutant crypts results from crypt replication through a process similar to what occurs in the normal crypt cycle. We measured the relative replication rate of mutant crypts by analyzing the size of clusters of mutant crypts in APC +/- individuals and found that mutant APC -/- crypts replicate more rapidly than do normal APC +/- (i.e., nonneoplastic) crypts. In contrast, the replication rate of mutant crypts in Apc +/- mice is not significantly different from that of normal crypts, thus supporting previous findings that aberrant crypt foci do not contribute significantly to the colon adenoma population in adult Apc +/- mice. Intriguingly, we found an effect of Apc heterozygosity on the frequency of branching crypts in young mice. PMID:9060821

  11. APC: A New Code for Atmospheric Polarization Computations

    NASA Technical Reports Server (NTRS)

    Korkin, Sergey V.; Lyapustin, Alexei I.; Rozanov, Vladimir V.

    2014-01-01

    A new polarized radiative transfer code Atmospheric Polarization Computations (APC) is described. The code is based on separation of the diffuse light field into anisotropic and smooth (regular) parts. The anisotropic part is computed analytically. The smooth regular part is computed numerically using the discrete ordinates method. Vertical stratification of the atmosphere, common types of bidirectional surface reflection and scattering by spherical particles or spheroids are included. A particular consideration is given to computation of the bidirectional polarization distribution function (BPDF) of the waved ocean surface.

  12. A trellis-searched APC (adaptive predictive coding) speech coder

    SciTech Connect

    Malone, K.T. ); Fischer, T.R. . Dept. of Electrical and Computer Engineering)

    1990-01-01

    In this paper we formulate a speech coding system that incorporates trellis coded vector quantization (TCVQ) and adaptive predictive coding (APC). A method for optimizing'' the TCVQ codebooks is presented and experimental results concerning survivor path mergings are reported. Simulation results are given for encoding rates of 16 and 9.6 kbps for a variety of coder parameters. The quality of the encoded speech is deemed excellent at an encoding rate of 16 kbps and very good at 9.6 kbps. 13 refs., 2 figs., 4 tabs.

  13. Heterochromatin components in germline stem cell maintenance

    PubMed Central

    Xing, Yalan; Li, Willis X.

    2015-01-01

    Stem cell maintenance requires expression of genes essential for stemness and repression of differentiation genes. How this is achieved remains incompletely understood. Here we investigate the requirement for central components of heterochromatin, Heterochromatin Protein 1 (HP1) and the histone H3 lys9 methyltransferase Su(var)3-9, in the Drosophila male germline stem cell (GSC) self-renewal, a paradigm for studying adult stem cell behavior. We found that mutations or RNAi knock down of HP1 or Su(var)3-9 cause loss of GSCs, accompanied by defects in cell division or survival and premature expression of the differentiation gene bag of marbles (bam). Conversely, over-expressing HP1 increases GSC number in wildtype flies and, strikingly, restores fertility to the sterile hopscotch (hop) mutant flies that lack niche signals. These results suggest that the central components of heterochromatin play roles including repressing differentiation genes in Drosophila male GSC maintenance. PMID:26626305

  14. Human Germline CRISPR-Cas Modification: Toward a Regulatory Framework

    PubMed Central

    Evitt, Niklaus H.; Mascharak, Shamik; Altman, Russ B.

    2015-01-01

    CRISPR germline editing therapies (CGETs) hold unprecedented potential to eradicate hereditary disorders. However, the prospect of altering the human germline has sparked a debate over the safety, efficacy, and morality of CGETs, triggering a funding moratorium by the NIH. There is an urgent need for practical paths for the evaluation of these capabilities. We propose a model regulatory framework for CGET research, clinical development, and distribution. Our model takes advantage of existing legal and regulatory institutions but adds elevated scrutiny at each stage of CGET development to accommodate the unique technical and ethical challenges posed by germline editing. PMID:26632357

  15. Genetic disorders and the ethical status of germ-line gene therapy.

    PubMed

    Berger, E M; Gert, B M

    1991-12-01

    Recombinant DNA technology will soon allow physicians an opportunity to carry out both somatic cell- and germ-line gene therapy. While somatic cell gene therapy raises no new ethical problems, gene therapy of gametes, fertilized eggs or early embryos does raise several novel concerns. The first issue discussed here relates to making a distinction between negative and positive eugenics; the second issue deals with the evolutionary consequences of lost genetic diversity. In distinguishing between positive and negative eugenics, the concept of malady is applied as a definitional criterion for identifying genetic disorders that could qualify for germ-line therapy. Because gene replacement techniques are currently unavailable for humans, and because even if they were possible the number of people involved would be quite small, the loss of diversity concern seems moot. Finally, we discuss the issue of iatrogenic disorders associated with gene therapy and discuss several 'real world considerations.' PMID:1787394

  16. Germline melanoma susceptibility and prognostic genes: a review of the literature.

    PubMed

    Ward, Katherine A; Lazovich, DeAnn; Hordinsky, Maria K

    2012-11-01

    In recent years, there have been increasing efforts to identify germline genetic variants that may alter melanoma susceptibility and prognosis. The findings of these studies have indicated the presence of rare, high-penetrance alleles with large effects, such as CDKN2A and CDK4, more common, moderately penetrant genes like MC1R, and very common, low-penetrance polymorphisms with small effects that are related to pigmentation, nevus count, immune responses, DNA repair, metabolism, and the vitamin D receptor. The study of these low-penetrance single nucleotide polymorphisms is relatively new; thus many of them are termed 'candidate melanoma susceptibility or prognostic genes.' This review summarizes the research on germline polymorphisms that have been implicated in melanoma susceptibility and prognosis in order to provide a framework for additional studies to meet the ultimate goal of predicting a patient's risk of, and prognosis in, cutaneous malignant melanoma. PMID:22583682

  17. Rare De Novo Germline Copy-Number Variation in Testicular Cancer

    PubMed Central

    Stadler, Zsofia K.; Esposito, Diane; Shah, Sohela; Vijai, Joseph; Yamrom, Boris; Levy, Dan; Lee, Yoon-ha; Kendall, Jude; Leotta, Anthony; Ronemus, Michael; Hansen, Nichole; Sarrel, Kara; Rau-Murthy, Rohini; Schrader, Kasmintan; Kauff, Noah; Klein, Robert J.; Lipkin, Steven M.; Murali, Rajmohan; Robson, Mark; Sheinfeld, Joel; Feldman, Darren; Bosl, George; Norton, Larry; Wigler, Michael; Offit, Kenneth

    2012-01-01

    Although heritable factors are an important determinant of risk of early-onset cancer, the majority of these malignancies appear to occur sporadically without identifiable risk factors. Germline de novo copy-number variations (CNVs) have been observed in sporadic neurocognitive and cardiovascular disorders. We explored this mechanism in 382 genomes of 116 early-onset cancer case-parent trios and unaffected siblings. Unique de novo germline CNVs were not observed in 107 breast or colon cancer trios or controls but were indeed found in 7% of 43 testicular germ cell tumor trios; this percentage exceeds background CNV rates and suggests a rare de novo genetic paradigm for susceptibility to some human malignancies. PMID:22863192

  18. Germline Allele-Specific Expression of DAPK1 in Chronic Lymphocytic Leukemia

    PubMed Central

    Hielscher, Thomas; Mertens, Daniel; Raval, Aparna; Oakes, Christopher C.; Tanner, Stephan M.; de la Chapelle, Albert; Byrd, John C.; Stilgenbauer, Stephan; Plass, Christoph

    2013-01-01

    We previously reported a rare germline variant (c.1-6531) that resulted in allele–specific expression (ASE) of death-associated protein kinase 1 (DAPK1) and predisposition to chronic lymphocytic leukemia (CLL). We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE) with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2%) CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5′ upstream regulatory region, within distinct exons or in the 3′-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL. PMID:23383130

  19. Germlining of the HIV-1 broadly neutralizing antibody domain m36

    PubMed Central

    Chen, Weizao; Li, Wei; Ying, Tianlei; Wang, Yanping; Feng, Yang; Dimitrov, Dimiter S.

    2015-01-01

    Engineered antibody domains (eAds) have emerged as a novel class of HIV-1 inhibitors and are currently under preclinical testing as promising drug candidates for prevention and therapy of HIV-1 infection. Reverse mutation of antibodies to germline sequences (germlining) could not only identify less mutated variants with lower probability of immunogenicity and other improved properties but also help elucidate their mechanisms of action. In this study, we sequentially reverted the framework (FRs) and complementary determining regions (CDRs) of m36, a human antibody heavy chain variable domain-based eAd targeting the coreceptor binding site of the viral envelope glycoprotein gp120, back to germline sequences. Two types of amino acid mutations and one region in the antibody V segment were identified that are critical for HIV-1 neutralization. These include four mutations to acidic acid residues distributed in the CDR1 and CDR2, two mutations to hydrophobic residues in the FR3 and CDR3, and partial FR2 and FR3 sequences flanking the CDR2 that are derived from a different gene family. An m36 variant with all five mutations in the FRs reverted back to germline showed slightly increased neutralizing activity against two HIV-1 isolates tested. Another variant with seven of twelve mutations in the V segment reverted retained potency within three-fold of that of the mature antibody. These results, together with an analysis of m36-gp120-CD4 docking structures, could have implications for the further development of m36 as candidate therapeutics and elucidation of its mechanism of potent and broad HIV-1 neutralization. PMID:25676867

  20. Sipuleucel-T: APC 8015, APC-8015, prostate cancer vaccine--Dendreon.

    PubMed

    2006-01-01

    Sipuleucel-T [APC 8015, Provenge] is an autologous, dendritic cell-based vaccine under development with Dendreon Corporation for the treatment of androgen-independent and androgen-dependent prostate cancer. It was generated using the company's active immunotherapy platform to stimulate a patient's own immune system to specifically target and destroy cancer cells, while leaving healthy cells unharmed. This approach could provide patients with a meaningful survival benefit and an improved tolerability profile over existing anticancer therapies. Sipuleucel-T selectively targets the prostate-specific antigen (PSA) known as prostatic acid phosphatase (PAP) that is expressed in approximately 95% of prostate cancers. It is produced by ex vivo exposure of dendritic cell precursors to PA 2024, a recombinant fusion protein composed of the PAP target fused to granulocyte-macrophage colony-stimulating factor (GM-CSF) and incorporated into Dendreon's proprietary Antigen Delivery Cassette. Patients are typically administered three intravenous (IV)-infusions of the vaccine over a 1-month period as a complete course of therapy. It is undergoing late-stage clinical evaluation among patients with early and advanced prostate cancer. In November 2003, Kirin Brewery returned to Dendreon the full rights to Sipuleucel-T for Asia. In exchange, Dendreon licensed patent rights relating to the use of certain HLA-DR antibodies to Kirin for $US20 million. This amended agreement enables Dendreon to complete ongoing discussions for a worldwide marketing and sales partnership for Sipuleucel-T. Similarly, Kirin is able to develop its HLA-DR monoclonal antibodies free of potential infringement claims arising from Dendreon's patent rights to HLA-DR. The licensing agreement relates to patent rights owned by Dendreon relating to monoclonal antibodies against the HLA-DR antigen. In addition, Dendreon retains rights to develop and commercialise its two existing HLA-DR monoclonal antibodies, DN 1921 and

  1. Tumorigenic fragments of APC cause dominant defects in directional cell migration in multiple model systems.

    PubMed

    Nelson, Scott A; Li, Zhouyu; Newton, Ian P; Fraser, David; Milne, Rachel E; Martin, David M A; Schiffmann, David; Yang, Xuesong; Dormann, Dirk; Weijer, Cornelis J; Appleton, Paul L; Näthke, Inke S

    2012-11-01

    Nonsense mutations that result in the expression of truncated, N-terminal, fragments of the adenomatous polyposis coli (APC) tumour suppressor protein are found in most sporadic and some hereditary colorectal cancers. These mutations can cause tumorigenesis by eliminating β-catenin-binding sites from APC, which leads to upregulation of β-catenin and thereby results in the induction of oncogenes such as MYC. Here we show that, in three distinct experimental model systems, expression of an N-terminal fragment of APC (N-APC) results in loss of directionality, but not speed, of cell motility independently of changes in β-catenin regulation. We developed a system to culture and fluorescently label live pieces of gut tissue to record high-resolution three-dimensional time-lapse movies of cells in situ. This revealed an unexpected complexity of normal gut cell migration, a key process in gut epithelial maintenance, with cells moving with spatial and temporal discontinuity. Quantitative comparison of gut tissue from wild-type mice and APC heterozygotes (APC(Min/+); multiple intestinal neoplasia model) demonstrated that cells in precancerous epithelia lack directional preference when moving along the crypt-villus axis. This effect was reproduced in diverse experimental systems: in developing chicken embryos, mesoderm cells expressing N-APC failed to migrate normally; in amoeboid Dictyostelium, which lack endogenous APC, expressing an N-APC fragment maintained cell motility, but the cells failed to perform directional chemotaxis; and multicellular Dictyostelium slug aggregates similarly failed to perform phototaxis. We propose that N-terminal fragments of APC represent a gain-of-function mutation that causes cells within tissue to fail to migrate directionally in response to relevant guidance cues. Consistent with this idea, crypts in histologically normal tissues of APC(Min/+) intestines are overpopulated with cells, suggesting that a lack of migration might cause cell

  2. Identification of germline transcriptional regulatory elements in Aedes aegypti

    NASA Astrophysics Data System (ADS)

    Akbari, Omar S.; Papathanos, Philippos A.; Sandler, Jeremy E.; Kennedy, Katie; Hay, Bruce A.

    2014-02-01

    The mosquito Aedes aegypti is the principal vector for the yellow fever and dengue viruses, and is also responsible for recent outbreaks of the alphavirus chikungunya. Vector control strategies utilizing engineered gene drive systems are being developed as a means of replacing wild, pathogen transmitting mosquitoes with individuals refractory to disease transmission, or bringing about population suppression. Several of these systems, including Medea, UDMEL, and site-specific nucleases, which can be used to drive genes into populations or bring about population suppression, utilize transcriptional regulatory elements that drive germline-specific expression. Here we report the identification of multiple regulatory elements able to drive gene expression specifically in the female germline, or in the male and female germline, in the mosquito Aedes aegypti. These elements can also be used as tools with which to probe the roles of specific genes in germline function and in the early embryo, through overexpression or RNA interference.

  3. Germline and somatic FGFR1 abnormalities in dysembryoplastic neuroepithelial tumors.

    PubMed

    Rivera, Barbara; Gayden, Tenzin; Carrot-Zhang, Jian; Nadaf, Javad; Boshari, Talia; Faury, Damien; Zeinieh, Michele; Blanc, Romeo; Burk, David L; Fahiminiya, Somayyeh; Bareke, Eric; Schüller, Ulrich; Monoranu, Camelia M; Sträter, Ronald; Kerl, Kornelius; Niederstadt, Thomas; Kurlemann, Gerhard; Ellezam, Benjamin; Michalak, Zuzanna; Thom, Maria; Lockhart, Paul J; Leventer, Richard J; Ohm, Milou; MacGregor, Duncan; Jones, David; Karamchandani, Jason; Greenwood, Celia M T; Berghuis, Albert M; Bens, Susanne; Siebert, Reiner; Zakrzewska, Magdalena; Liberski, Pawel P; Zakrzewski, Krzysztof; Sisodiya, Sanjay M; Paulus, Werner; Albrecht, Steffen; Hasselblatt, Martin; Jabado, Nada; Foulkes, William D; Majewski, Jacek

    2016-06-01

    Dysembryoplastic neuroepithelial tumor (DNET) is a benign brain tumor associated with intractable drug-resistant epilepsy. In order to identify underlying genetic alterations and molecular mechanisms, we examined three family members affected by multinodular DNETs as well as 100 sporadic tumors from 96 patients, which had been referred to us as DNETs. We performed whole-exome sequencing on 46 tumors and targeted sequencing for hotspot FGFR1 mutations and BRAF p.V600E was used on the remaining samples. FISH, copy number variation assays and Sanger sequencing were used to validate the findings. By whole-exome sequencing of the familial cases, we identified a novel germline FGFR1 mutation, p.R661P. Somatic activating FGFR1 mutations (p.N546K or p.K656E) were observed in the tumor samples and further evidence for functional relevance was obtained by in silico modeling. The FGFR1 p.K656E mutation was confirmed to be in cis with the germline p.R661P variant. In 43 sporadic cases, in which the diagnosis of DNET could be confirmed on central blinded neuropathology review, FGFR1 alterations were also frequent and mainly comprised intragenic tyrosine kinase FGFR1 duplication and multiple mutants in cis (25/43; 58.1 %) while BRAF p.V600E alterations were absent (0/43). In contrast, in 53 cases, in which the diagnosis of DNET was not confirmed, FGFR1 alterations were less common (10/53; 19 %; p < 0.0001) and hotspot BRAF p.V600E (12/53; 22.6 %) (p < 0.001) prevailed. We observed overexpression of phospho-ERK in FGFR1 p.R661P and p.N546K mutant expressing HEK293 cells as well as FGFR1 mutated tumor samples, supporting enhanced MAP kinase pathway activation under these conditions. In conclusion, constitutional and somatic FGFR1 alterations and MAP kinase pathway activation are key events in the pathogenesis of DNET. These findings point the way towards existing targeted therapies. PMID:26920151

  4. Structural basis for germline antibody recognition of HIV-1 immunogens

    PubMed Central

    Scharf, Louise; West, Anthony P; Sievers, Stuart A; Chen, Courtney; Jiang, Siduo; Gao, Han; Gray, Matthew D; McGuire, Andrew T; Scheid, Johannes F; Nussenzweig, Michel C; Stamatatos, Leonidas; Bjorkman, Pamela J

    2016-01-01

    Efforts to elicit broadly neutralizing antibodies (bNAbs) against HIV-1 require understanding germline bNAb recognition of HIV-1 envelope glycoprotein (Env). The VRC01-class bNAb family derived from the VH1-2*02 germline allele arose in multiple HIV-1–infected donors, yet targets the CD4-binding site on Env with common interactions. Modified forms of the 426c Env that activate germline-reverted B cell receptors are candidate immunogens for eliciting VRC01-class bNAbs. We present structures of germline-reverted VRC01-class bNAbs alone and complexed with 426c-based gp120 immunogens. Germline bNAb–426c gp120 complexes showed preservation of VRC01-class signature residues and gp120 contacts, but detectably different binding modes compared to mature bNAb-gp120 complexes. Unlike typical antibody-antigen interactions, VRC01–class germline antibodies exhibited preformed antigen-binding conformations for recognizing immunogens. Affinity maturation introduced substitutions increasing induced-fit recognition and electropositivity, potentially to accommodate negatively-charged complex-type N-glycans on gp120. These results provide general principles relevant to the unusual evolution of VRC01–class bNAbs and guidelines for structure-based immunogen design. DOI: http://dx.doi.org/10.7554/eLife.13783.001 PMID:26997349

  5. BRCA somatic and germline mutation detection in paraffin embedded ovarian cancers by next-generation sequencing

    PubMed Central

    Mafficini, Andrea; Simbolo, Michele; Parisi, Alice; Rusev, Borislav; Luchini, Claudio; Cataldo, Ivana; Piazzola, Elena; Sperandio, Nicola; Turri, Giona; Franchi, Massimo; Tortora, Giampaolo; Bovo, Chiara; Lawlor, Rita T.; Scarpa, Aldo

    2016-01-01

    BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2. All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue. PMID:26745875

  6. POLD1 Germline Mutations in Patients Initially Diagnosed with Werner Syndrome

    PubMed Central

    Lessel, Davor; Hisama, Fuki M.; Szakszon, Katalin; Saha, Bidisha; Sanjuanelo, Alexander Barrios; Salbert, Bonnie A.; Steele, Pamela D.; Baldwin, Jennifer; Brown, W. Ted; Piussan, Charles; Plauchu, Henri; Szilvássy, Judit; Horkay, Edit; Hoögel, Josef; Martin, George M.; Herr, Alan J.; Oshima, Junko; Kubisch, Christian

    2015-01-01

    Segmental progeroid syndromes are rare, heterogeneous disorders characterized by signs of premature aging affecting more than one tissue or organ. A prototypic example is the Werner syndrome (WS), caused by biallelic germline mutations in the Werner helicase gene (WRN). While heterozygous lamin A/C (LMNA) mutations are found in a few nonclassical cases of WS, another 10%–15% of patients initially diagnosed with WS do not have mutations in WRN or LMNA. Germline POLD1 mutations were recently reported in five patients with another segmental progeroid disorder: mandibular hypoplasia, deafness, progeroid features syndrome. Here, we describe eight additional patients with heterozygous POLD1 mutations, thereby substantially expanding the characterization of this new example of segmental progeroid disorders. First, we identified POLD1 mutations in patients initially diagnosed with WS. Second, we describe POLD1 mutation carriers without clinically relevant hearing impairment or mandibular underdevelopment, both previously thought to represent obligate diagnostic features. These patients also exhibit a lower incidence of metabolic abnormalities and joint contractures. Third, we document postnatal short stature and premature greying/loss of hair in POLD1 mutation carriers. We conclude that POLD1 germline mutations can result in a variably expressed and probably underdiagnosed segmental progeroid syndrome. PMID:26172944

  7. POLD1 Germline Mutations in Patients Initially Diagnosed with Werner Syndrome.

    PubMed

    Lessel, Davor; Hisama, Fuki M; Szakszon, Katalin; Saha, Bidisha; Sanjuanelo, Alexander Barrios; Salbert, Bonnie A; Steele, Pamela D; Baldwin, Jennifer; Brown, W Ted; Piussan, Charles; Plauchu, Henri; Szilvássy, Judit; Horkay, Edit; Högel, Josef; Martin, George M; Herr, Alan J; Oshima, Junko; Kubisch, Christian

    2015-11-01

    Segmental progeroid syndromes are rare, heterogeneous disorders characterized by signs of premature aging affecting more than one tissue or organ. A prototypic example is the Werner syndrome (WS), caused by biallelic germline mutations in the Werner helicase gene (WRN). While heterozygous lamin A/C (LMNA) mutations are found in a few nonclassical cases of WS, another 10%-15% of patients initially diagnosed with WS do not have mutations in WRN or LMNA. Germline POLD1 mutations were recently reported in five patients with another segmental progeroid disorder: mandibular hypoplasia, deafness, progeroid features syndrome. Here, we describe eight additional patients with heterozygous POLD1 mutations, thereby substantially expanding the characterization of this new example of segmental progeroid disorders. First, we identified POLD1 mutations in patients initially diagnosed with WS. Second, we describe POLD1 mutation carriers without clinically relevant hearing impairment or mandibular underdevelopment, both previously thought to represent obligate diagnostic features. These patients also exhibit a lower incidence of metabolic abnormalities and joint contractures. Third, we document postnatal short stature and premature greying/loss of hair in POLD1 mutation carriers. We conclude that POLD1 germline mutations can result in a variably expressed and probably underdiagnosed segmental progeroid syndrome. PMID:26172944

  8. Patterns and functional implications of rare germline variants across 12 cancer types

    PubMed Central

    Lu, Charles; Xie, Mingchao; Wendl, Michael C.; Wang, Jiayin; McLellan, Michael D.; Leiserson, Mark D. M.; Huang, Kuan-lin; Wyczalkowski, Matthew A.; Jayasinghe, Reyka; Banerjee, Tapahsama; Ning, Jie; Tripathi, Piyush; Zhang, Qunyuan; Niu, Beifang; Ye, Kai; Schmidt, Heather K.; Fulton, Robert S.; McMichael, Joshua F.; Batra, Prag; Kandoth, Cyriac; Bharadwaj, Maheetha; Koboldt, Daniel C.; Miller, Christopher A.; Kanchi, Krishna L.; Eldred, James M.; Larson, David E.; Welch, John S.; You, Ming; Ozenberger, Bradley A.; Govindan, Ramaswamy; Walter, Matthew J.; Ellis, Matthew J.; Mardis, Elaine R.; Graubert, Timothy A.; Dipersio, John F.; Ley, Timothy J.; Wilson, Richard K.; Goodfellow, Paul J.; Raphael, Benjamin J.; Chen, Feng; Johnson, Kimberly J.; Parvin, Jeffrey D.; Ding, Li

    2015-01-01

    Large-scale cancer sequencing data enable discovery of rare germline cancer susceptibility variants. Here we systematically analyse 4,034 cases from The Cancer Genome Atlas cancer cases representing 12 cancer types. We find that the frequency of rare germline truncations in 114 cancer-susceptibility-associated genes varies widely, from 4% (acute myeloid leukaemia (AML)) to 19% (ovarian cancer), with a notably high frequency of 11% in stomach cancer. Burden testing identifies 13 cancer genes with significant enrichment of rare truncations, some associated with specific cancers (for example, RAD51C, PALB2 and MSH6 in AML, stomach and endometrial cancers, respectively). Significant, tumour-specific loss of heterozygosity occurs in nine genes (ATM, BAP1, BRCA1/2, BRIP1, FANCM, PALB2 and RAD51C/D). Moreover, our homology-directed repair assay of 68 BRCA1 rare missense variants supports the utility of allelic enrichment analysis for characterizing variants of unknown significance. The scale of this analysis and the somatic-germline integration enable the detection of rare variants that may affect individual susceptibility to tumour development, a critical step toward precision medicine. PMID:26689913

  9. CDKN2A and BAP1 germline mutations predispose to melanoma and mesothelioma.

    PubMed

    Betti, M; Aspesi, A; Biasi, A; Casalone, E; Ferrante, D; Ogliara, P; Gironi, L C; Giorgione, R; Farinelli, P; Grosso, F; Libener, R; Rosato, S; Turchetti, D; Maffè, A; Casadio, C; Ascoli, V; Dianzani, C; Colombo, E; Piccolini, E; Pavesi, M; Miccoli, S; Mirabelli, D; Bracco, C; Righi, L; Boldorini, R; Papotti, M; Matullo, G; Magnani, C; Pasini, B; Dianzani, I

    2016-08-10

    BAP1 germline mutations predispose to a cancer predisposition syndrome that includes mesothelioma, cutaneous melanoma, uveal melanoma and other cancers. This co-occurrence suggests that these tumors share a common carcinogenic pathway. To evaluate this hypothesis, we studied 40 Italian families with mesothelioma and/or melanoma. The probands were sequenced for BAP1 and for the most common melanoma predisposition genes (i.e. CDKN2A, CDK4, TERT, MITF and POT1) to investigate if these genes may also confer susceptibility to mesothelioma. In two out of six families with both mesothelioma and melanoma we identified either a germline nonsense mutation (c.1153C > T, p.Arg385*) in BAP1 or a recurrent pathogenic germline mutation (c.301G > T, p.Gly101Trp) in CDKN2A. Our study suggests that CDKN2A, in addition to BAP1, could be involved in the melanoma and mesothelioma susceptibility, leading to the rare familial cancer syndromes. It also suggests that these tumors share key steps that drive carcinogenesis and that other genes may be involved in inherited predisposition to malignant mesothelioma and melanoma. PMID:27181379

  10. Congenital B cell lymphocytosis explained by novel germline CARD11 mutations.

    PubMed

    Snow, Andrew L; Xiao, Wenming; Stinson, Jeffrey R; Lu, Wei; Chaigne-Delalande, Benjamin; Zheng, Lixin; Pittaluga, Stefania; Matthews, Helen F; Schmitz, Roland; Jhavar, Sameer; Kuchen, Stefan; Kardava, Lela; Wang, Wei; Lamborn, Ian T; Jing, Huie; Raffeld, Mark; Moir, Susan; Fleisher, Thomas A; Staudt, Louis M; Su, Helen C; Lenardo, Michael J

    2012-11-19

    Nuclear factor-κB (NF-κB) controls genes involved in normal lymphocyte functions, but constitutive NF-κB activation is often associated with B cell malignancy. Using high-throughput whole transcriptome sequencing, we investigated a unique family with hereditary polyclonal B cell lymphocytosis. We found a novel germline heterozygous missense mutation (E127G) in affected patients in the gene encoding CARD11, a scaffolding protein required for antigen receptor (AgR)-induced NF-κB activation in both B and T lymphocytes. We subsequently identified a second germline mutation (G116S) in an unrelated, phenotypically similar patient, confirming mutations in CARD11 drive disease. Like somatic, gain-of-function CARD11 mutations described in B cell lymphoma, these germline CARD11 mutants spontaneously aggregate and drive constitutive NF-κB activation. However, these CARD11 mutants rendered patient T cells less responsive to AgR-induced activation. By reexamining this rare genetic disorder first reported four decades ago, our findings provide new insight into why activating CARD11 mutations may induce B cell expansion and preferentially predispose to B cell malignancy without dramatically perturbing T cell homeostasis. PMID:23129749

  11. Congenital B cell lymphocytosis explained by novel germline CARD11 mutations

    PubMed Central

    Xiao, Wenming; Stinson, Jeffrey R.; Lu, Wei; Chaigne-Delalande, Benjamin; Zheng, Lixin; Pittaluga, Stefania; Matthews, Helen F.; Schmitz, Roland; Jhavar, Sameer; Kuchen, Stefan; Kardava, Lela; Wang, Wei; Lamborn, Ian T.; Jing, Huie; Raffeld, Mark; Moir, Susan; Fleisher, Thomas A.; Staudt, Louis M.; Su, Helen C.

    2012-01-01

    Nuclear factor-κB (NF-κB) controls genes involved in normal lymphocyte functions, but constitutive NF-κB activation is often associated with B cell malignancy. Using high-throughput whole transcriptome sequencing, we investigated a unique family with hereditary polyclonal B cell lymphocytosis. We found a novel germline heterozygous missense mutation (E127G) in affected patients in the gene encoding CARD11, a scaffolding protein required for antigen receptor (AgR)–induced NF-κB activation in both B and T lymphocytes. We subsequently identified a second germline mutation (G116S) in an unrelated, phenotypically similar patient, confirming mutations in CARD11 drive disease. Like somatic, gain-of-function CARD11 mutations described in B cell lymphoma, these germline CARD11 mutants spontaneously aggregate and drive constitutive NF-κB activation. However, these CARD11 mutants rendered patient T cells less responsive to AgR-induced activation. By reexamining this rare genetic disorder first reported four decades ago, our findings provide new insight into why activating CARD11 mutations may induce B cell expansion and preferentially predispose to B cell malignancy without dramatically perturbing T cell homeostasis. PMID:23129749

  12. Hermes (Rbpms) is a Critical Component of RNP Complexes that Sequester Germline RNAs during Oogenesis

    PubMed Central

    Aguero, Tristan; Zhou, Yi; Kloc, Malgorzata; Chang, Patrick; Houliston, Evelyn; King, Mary Lou

    2016-01-01

    The germ cell lineage in Xenopus is specified by the inheritance of germ plasm that assembles within the mitochondrial cloud or Balbiani body in stage I oocytes. Specific RNAs, such as nanos1, localize to the germ plasm. nanos1 has the essential germline function of blocking somatic gene expression and thus preventing Primordial Germ Cell (PGC) loss and sterility. Hermes/Rbpms protein and nanos RNA co-localize within germinal granules, diagnostic electron dense particles found within the germ plasm. Previous work indicates that nanos accumulates within the germ plasm through a diffusion/entrapment mechanism. Here we show that Hermes/Rbpms interacts with nanos through sequence specific RNA localization signals found in the nanos-3′UTR. Importantly, Hermes/Rbpms specifically binds nanos, but not Vg1 RNA in the nucleus of stage I oocytes. In vitro binding data show that Hermes/Rbpms requires additional factors that are present in stage I oocytes in order to bind nanos1. One such factor may be hnRNP I, identified in a yeast-2-hybrid screen as directly interacting with Hermes/Rbpms. We suggest that Hermes/Rbpms functions as part of a RNP complex in the nucleus that facilitates selection of germline RNAs for germ plasm localization. We propose that Hermes/Rbpms is required for nanos RNA to form within the germinal granules and in this way, participates in the germline specific translational repression and sequestration of nanos RNA. PMID:26998427

  13. Patterns and functional implications of rare germline variants across 12 cancer types.

    PubMed

    Lu, Charles; Xie, Mingchao; Wendl, Michael C; Wang, Jiayin; McLellan, Michael D; Leiserson, Mark D M; Huang, Kuan-Lin; Wyczalkowski, Matthew A; Jayasinghe, Reyka; Banerjee, Tapahsama; Ning, Jie; Tripathi, Piyush; Zhang, Qunyuan; Niu, Beifang; Ye, Kai; Schmidt, Heather K; Fulton, Robert S; McMichael, Joshua F; Batra, Prag; Kandoth, Cyriac; Bharadwaj, Maheetha; Koboldt, Daniel C; Miller, Christopher A; Kanchi, Krishna L; Eldred, James M; Larson, David E; Welch, John S; You, Ming; Ozenberger, Bradley A; Govindan, Ramaswamy; Walter, Matthew J; Ellis, Matthew J; Mardis, Elaine R; Graubert, Timothy A; Dipersio, John F; Ley, Timothy J; Wilson, Richard K; Goodfellow, Paul J; Raphael, Benjamin J; Chen, Feng; Johnson, Kimberly J; Parvin, Jeffrey D; Ding, Li

    2015-01-01

    Large-scale cancer sequencing data enable discovery of rare germline cancer susceptibility variants. Here we systematically analyse 4,034 cases from The Cancer Genome Atlas cancer cases representing 12 cancer types. We find that the frequency of rare germline truncations in 114 cancer-susceptibility-associated genes varies widely, from 4% (acute myeloid leukaemia (AML)) to 19% (ovarian cancer), with a notably high frequency of 11% in stomach cancer. Burden testing identifies 13 cancer genes with significant enrichment of rare truncations, some associated with specific cancers (for example, RAD51C, PALB2 and MSH6 in AML, stomach and endometrial cancers, respectively). Significant, tumour-specific loss of heterozygosity occurs in nine genes (ATM, BAP1, BRCA1/2, BRIP1, FANCM, PALB2 and RAD51C/D). Moreover, our homology-directed repair assay of 68 BRCA1 rare missense variants supports the utility of allelic enrichment analysis for characterizing variants of unknown significance. The scale of this analysis and the somatic-germline integration enable the detection of rare variants that may affect individual susceptibility to tumour development, a critical step toward precision medicine. PMID:26689913

  14. Co-repressor CBFA2T2 regulates pluripotency and germline development.

    PubMed

    Tu, Shengjiang; Narendra, Varun; Yamaji, Masashi; Vidal, Simon E; Rojas, Luis Alejandro; Wang, Xiaoshi; Kim, Sang Yong; Garcia, Benjamin A; Tuschl, Thomas; Stadtfeld, Matthias; Reinberg, Danny

    2016-06-16

    Developmental specification of germ cells lies at the heart of inheritance, as germ cells contain all of the genetic and epigenetic information transmitted between generations. The critical developmental event distinguishing germline from somatic lineages is the differentiation of primordial germ cells (PGCs), precursors of sex-specific gametes that produce an entire organism upon fertilization. Germ cells toggle between uni- and pluripotent states as they exhibit their own 'latent' form of pluripotency. For example, PGCs express a number of transcription factors in common with embryonic stem (ES) cells, including OCT4 (encoded by Pou5f1), SOX2, NANOG and PRDM14 (refs 2, 3, 4). A biochemical mechanism by which these transcription factors converge on chromatin to produce the dramatic rearrangements underlying ES-cell- and PGC-specific transcriptional programs remains poorly understood. Here we identify a novel co-repressor protein, CBFA2T2, that regulates pluripotency and germline specification in mice. Cbfa2t2(-/-) mice display severe defects in PGC maturation and epigenetic reprogramming. CBFA2T2 forms a biochemical complex with PRDM14, a germline-specific transcription factor. Mechanistically, CBFA2T2 oligomerizes to form a scaffold upon which PRDM14 and OCT4 are stabilized on chromatin. Thus, in contrast to the traditional 'passenger' role of a co-repressor, CBFA2T2 functions synergistically with transcription factors at the crossroads of the fundamental developmental plasticity between uni- and pluripotency. PMID:27281218

  15. A Germline Polymorphism of DNA Polymerase Beta Induces Genomic Instability and Cellular Transformation

    PubMed Central

    Keh, Agnes; Sweasy, Joann B.

    2012-01-01

    Several germline single nucleotide polymorphisms (SNPs) have been identified in the POLB gene, but little is known about their cellular and biochemical impact. DNA Polymerase β (Pol β), encoded by the POLB gene, is the main gap-filling polymerase involved in base excision repair (BER), a pathway that protects the genome from the consequences of oxidative DNA damage. In this study we tested the hypothesis that expression of the POLB germline coding SNP (rs3136797) in mammalian cells could induce a cancerous phenotype. Expression of this SNP in both human and mouse cells induced double-strand breaks, chromosomal aberrations, and cellular transformation. Following treatment with an alkylating agent, cells expressing this coding SNP accumulated BER intermediate substrates, including single-strand and double-strand breaks. The rs3136797 SNP encodes the P242R variant Pol β protein and biochemical analysis showed that P242R protein had a slower catalytic rate than WT, although P242R binds DNA similarly to WT. Our results suggest that people who carry the rs3136797 germline SNP may be at an increased risk for cancer susceptibility. PMID:23144635

  16. Protein Phosphatase 1ß Limits Ring Canal Constriction during Drosophila Germline Cyst Formation

    PubMed Central

    Yamamoto, Shinya; Bayat, Vafa; Bellen, Hugo J.; Tan, Change

    2013-01-01

    Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in Drosophila melanogaster germ cells and identified multiple alleles of flapwing (flw), a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early D. melanogaster oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the Drosophila myosin binding subunit of myosin phosphatase (DMYPT). This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in D. melanogaster and probably other species as well. PMID:23936219

  17. Drastic effect of germline TP53 missense mutations in Li-Fraumeni patients.

    PubMed

    Zerdoumi, Yasmine; Aury-Landas, Juliette; Bonaïti-Pellié, Catherine; Derambure, Céline; Sesboüé, Richard; Renaux-Petel, Mariette; Frebourg, Thierry; Bougeard, Gaëlle; Flaman, Jean-Michel

    2013-03-01

    In contrast to other tumor suppressor genes, the majority of TP53 alterations are missense mutations. We have previously reported that in the Li-Fraumeni syndrome (LFS), germline TP53 missense mutations are associated with an earlier age of tumor onset. In a larger series, we observed that mean age of tumor onset in patients harboring dominant negative missense mutations and clearly null mutations was 22.6 and 37.5 years, respectively. To assess the impact of heterozygous germline TP53 mutations in the genetic context of the patients, we developed a new functional assay of the p53 pathway on the basis of induction of DNA damage in Epstein-Barr-virus-immortalized lymphocytes, followed by comparative gene-expression profiling. In wild-type lymphocytes, we identified a core of 173 genes whose expression was induced more than twofold, of which 46 were known p53 target genes. In LFS lymphocytes with canonical missense mutations, the number of induced genes and the level of known p53 target genes induction were strongly reduced as compared with controls and LFS lymphocytes with null mutations. These results show that certain germline missense TP53 mutations, such as those with dominant negative effect, dramatically alter the response to DNA damage. This probably explains why TP53 alterations are predominantly missense mutations. PMID:23172776

  18. BRCA somatic and germline mutation detection in paraffin embedded ovarian cancers by next-generation sequencing.

    PubMed

    Mafficini, Andrea; Simbolo, Michele; Parisi, Alice; Rusev, Borislav; Luchini, Claudio; Cataldo, Ivana; Piazzola, Elena; Sperandio, Nicola; Turri, Giona; Franchi, Massimo; Tortora, Giampaolo; Bovo, Chiara; Lawlor, Rita T; Scarpa, Aldo

    2016-01-12

    BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2. All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue. PMID:26745875

  19. 42 CFR 419.31 - Ambulatory payment classification (APC) system and payment weights.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 3 2011-10-01 2011-10-01 false Ambulatory payment classification (APC) system and payment weights. 419.31 Section 419.31 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT... Outpatient Services § 419.31 Ambulatory payment classification (APC) system and payment weights. (a)...

  20. REMOVAL OF ARSENIC IN DRINKING WATER: ARS CFU-50 APC ELECTROFLOCCULATION AND FILTRATION WATER TREATMENT SYSTEM

    EPA Science Inventory

    ETV testing of the ARS CFU-50 APC Electroflocculation and Filtration Water Treatment System (ARS CFU-50 APC) for arsenic removal was conducted at the Town of Bernalillo Well #3 site from April 18 through May 2, 2006. The source water was chlorinated groundwater from two supply w...

  1. Modeling the Effect of APC Truncation on Destruction Complex Function in Colorectal Cancer Cells

    SciTech Connect

    Barua, Dipak; Hlavacek, William S.

    2013-09-26

    In colorectal cancer cells, APC, a tumor suppressor protein, is commonly expressed in truncated form. Truncation of APC is believed to disrupt degradation of β—catenin, which is regulated by a multiprotein complex called the destruction complex. The destruction complex comprises APC, Axin, β—catenin, serine/threonine kinases, and other proteins. The kinases CK1α and GSK–3β, which are recruited by Axin, mediate phosphorylation of β—catenin, which initiates its ubiquitination and proteosomal degradation. The mechanism of regulation of β—catenin degradation by the destruction complex and the role of truncation of APC in colorectal cancer are not entirely understood. Through formulation and analysis of a rule-based computational model, we investigated the regulation of β—catenin phosphorylation and degradation by APC and the effect of APC truncation on function of the destruction complex. The model integrates available mechanistic knowledge about site-specific interactions and phosphorylation of destruction complex components and is consistent with an array of published data. In this paper, we find that the phosphorylated truncated form of APC can outcompete Axin for binding to β—catenin, provided that Axin is limiting, and thereby sequester β—catenin away from Axin and the Axin-recruited kinases CK1α and GSK–3β. Full-length APC also competes with Axin for binding to β—catenin; however, full-length APC is able, through its SAMP repeats, which bind Axin and which are missing in truncated oncogenic forms of APC, to bring β—catenin into indirect association with Axin and Axin-recruited kinases. Because our model indicates that the positive effects of truncated APC on β—catenin levels depend on phosphorylation of APC, at the first 20-amino acid repeat, and because phosphorylation of this site is mediated by CK1ϵ, we suggest that CK1ϵ is a potential target for therapeutic intervention in colorectal cancer. Finally, specific inhibition

  2. Modeling the Effect of APC Truncation on Destruction Complex Function in Colorectal Cancer Cells

    DOE PAGESBeta

    Barua, Dipak; Hlavacek, William S.

    2013-09-26

    In colorectal cancer cells, APC, a tumor suppressor protein, is commonly expressed in truncated form. Truncation of APC is believed to disrupt degradation of β—catenin, which is regulated by a multiprotein complex called the destruction complex. The destruction complex comprises APC, Axin, β—catenin, serine/threonine kinases, and other proteins. The kinases CK1α and GSK–3β, which are recruited by Axin, mediate phosphorylation of β—catenin, which initiates its ubiquitination and proteosomal degradation. The mechanism of regulation of β—catenin degradation by the destruction complex and the role of truncation of APC in colorectal cancer are not entirely understood. Through formulation and analysis of amore » rule-based computational model, we investigated the regulation of β—catenin phosphorylation and degradation by APC and the effect of APC truncation on function of the destruction complex. The model integrates available mechanistic knowledge about site-specific interactions and phosphorylation of destruction complex components and is consistent with an array of published data. In this paper, we find that the phosphorylated truncated form of APC can outcompete Axin for binding to β—catenin, provided that Axin is limiting, and thereby sequester β—catenin away from Axin and the Axin-recruited kinases CK1α and GSK–3β. Full-length APC also competes with Axin for binding to β—catenin; however, full-length APC is able, through its SAMP repeats, which bind Axin and which are missing in truncated oncogenic forms of APC, to bring β—catenin into indirect association with Axin and Axin-recruited kinases. Because our model indicates that the positive effects of truncated APC on β—catenin levels depend on phosphorylation of APC, at the first 20-amino acid repeat, and because phosphorylation of this site is mediated by CK1ϵ, we suggest that CK1ϵ is a potential target for therapeutic intervention in colorectal cancer. Finally, specific

  3. Modeling the effect of APC truncation on destruction complex function in colorectal cancer cells.

    PubMed

    Barua, Dipak; Hlavacek, William S

    2013-01-01

    In colorectal cancer cells, APC, a tumor suppressor protein, is commonly expressed in truncated form. Truncation of APC is believed to disrupt degradation of β-catenin, which is regulated by a multiprotein complex called the destruction complex. The destruction complex comprises APC, Axin, β-catenin, serine/threonine kinases, and other proteins. The kinases CK1α and GSK -3β, which are recruited by Axin, mediate phosphorylation of β-catenin, which initiates its ubiquitination and proteosomal degradation. The mechanism of regulation of β-catenin degradation by the destruction complex and the role of truncation of APC in colorectal cancer are not entirely understood. Through formulation and analysis of a rule-based computational model, we investigated the regulation of β-catenin phosphorylation and degradation by APC and the effect of APC truncation on function of the destruction complex. The model integrates available mechanistic knowledge about site-specific interactions and phosphorylation of destruction complex components and is consistent with an array of published data. We find that the phosphorylated truncated form of APC can outcompete Axin for binding to β-catenin, provided that Axin is limiting, and thereby sequester β-catenin away from Axin and the Axin-recruited kinases CK1α and GSK -3β. Full-length APC also competes with Axin for binding to β-catenin; however, full-length APC is able, through its SAMP repeats, which bind Axin and which are missing in truncated oncogenic forms of APC, to bring β-catenin into indirect association with Axin and Axin-recruited kinases. Because our model indicates that the positive effects of truncated APC on β-catenin levels depend on phosphorylation of APC, at the first 20-amino acid repeat, and because phosphorylation of this site is mediated by CK1ε, we suggest that CK1ε is a potential target for therapeutic intervention in colorectal cancer. Specific inhibition of CK1ε is predicted to limit binding of

  4. Critical Current Properties in Longitudinal Magnetic Field of YBCO Superconductor with APC

    NASA Astrophysics Data System (ADS)

    Kido, R.; Kiuchi, M.; Otabe, E. S.; Matsushita, T.; Jha, A. K.; Matsumoto, K.

    The critical current density (Jc) properties of the Artificial Pinning Center (APC) introduced YBa2Cu3O7 (YBCO) films in the longitudinal magnetic field were measured. Y2O3 or Y2BaCuO5 (Y211) was introduced as APCs to YBCO, and YBCO films with APC were fabricated on SrTiO3 single crystal substrate. The sizes of Y2O3 and Y211 were 5-10 nm and 10-20 nm, respectively. As a result, Jc enhancement in the longitudinal magnetic field was observed in Y2O3 introduced YBCO films. However, it was not observed in Y211 introduced YBCO films. Therefore, it was considered that Jc properties in the longitudinal magnetic field were affected by introducing of small size APC, and it was necessary that APC does not disturb the current pathway in the superconductor.

  5. A knock-in mouse model reveals roles for nuclear Apc in cell proliferation, Wnt signal inhibition and tumor suppression.

    PubMed

    Zeineldin, M; Cunningham, J; McGuinness, W; Alltizer, P; Cowley, B; Blanchat, B; Xu, W; Pinson, D; Neufeld, K L

    2012-05-10

    Mutation of the tumor suppressor adenomatous polyposis coli (APC) is considered an initiating step in the genesis of the vast majority of colorectal cancers. APC inhibits the Wnt-signaling pathway by targeting the proto-oncogene β-catenin for destruction by cytoplasmic proteasomes. In the presence of a Wnt signal, or in the absence of functional APC, β-catenin can serve as a transcription cofactor for genes required for cell proliferation such as cyclin-D1 and c-Myc. In cultured cells, APC shuttles between the nucleus and the cytoplasm, with nuclear APC implicated in the inhibition of Wnt target gene expression. Adopting a genetic approach to evaluate the functions of nuclear APC in the context of a whole organism, we generated a mouse model with mutations that inactivate the nuclear localization signals (NLSs) of Apc (Apc(mNLS)). Apc(mNLS/mNLS) mice are viable and fractionation of mouse embryonic fibroblasts (MEFs) isolated from these mice revealed a significant reduction in nuclear Apc as compared with Apc(+/+) MEFs. The levels of Apc and β-catenin protein were not significantly altered in small intestinal epithelia from Apc(mNLS/mNLS) mice. Compared with Apc(+/+) mice, Apc(mNLS/mNLS) mice showed increased proliferation in epithelial cells from the jejunum, ileum and colon. These same tissues from Apc(mNLS/mNLS) mice showed more mRNA from three genes upregulated in response to canonical Wnt signal, c-Myc, axin-2 and cyclin-D1, and less mRNA from Hath-1, which is downregulated in response to Wnt. These observations suggest a role for nuclear Apc in the inhibition of canonical Wnt signaling and the control of epithelial proliferation in intestinal tissue. Furthermore, we found Apc(Min/+) mice, which harbor a mutation that truncates Apc, to have an increased polyp size and multiplicity if they also carry the Apc(mNLS) allele. Taken together, this analysis of the novel Apc(mNLS) mouse model supports a role for nuclear Apc in the control of Wnt target genes

  6. Loss of Rassf1a co-operates with ApcMin to accelerate intestinal tumourigenesis

    PubMed Central

    van der Weyden, L.; Arends, M.J.; Dovey, O.M.; Harrison, H. L.; Lefebvre, G.; Conte, N.; Gergely, F.V.; Bradley, A.; Adams, D.J.

    2013-01-01

    Promoter methylation of the RAS-association domain family 1, isoform A gene (RASSF1A) is one of the most frequent events found in human tumours. In this study we set out to test the hypothesis that loss of Rassf1a can co-operate with inactivation of the adenomatous polyposis coli (Apc) gene to accelerate intestinal tumourigenesis using the Apc-Min (ApcMin/+) mouse model, as mutational or deletional inactivation of APC is a frequent early event in the genesis of intestinal cancer. Further, loss of RASSF1A has also been reported to occur in premalignant adenomas of the bowel. RASSF1A has been implicated in an array of pivotal cellular processes, including regulation of the cell cycle, apoptosis, microtubule stability and most recently in the β-catenin signalling pathway. By interbreeding isoform specific Rassf1a knockout mice with Apc+/Min mice we showed that loss of Rassf1a results in a significant increase in adenomas of the small intestine and accelerated intestinal tumourigenesis leading to the earlier death of adenocarcinoma-bearing mice and decreased overall survival. Comparative genomic hybridization of adenomas from Rassf1a−/−; Apc+/Min mice revealed no evidence of aneuploidy or gross chromosomal instability (no difference to adenomas from Rassf1a+/+; Apc+/Min mice). Immunohistochemical analysis of adenomas revealed increased nuclear β-catenin accumulation in adenomas from Rassf1a−/−; Apc+/Min mice, compared to those from Rassf1a+/+; Apc+/Min mice, but no differences in proliferation marker (Ki67) staining patterns. Collectively these data demonstrate co-operation between inactivation of Rassf1a and Apc resulting in accelerated intestinal tumourigenesis, with adenomas showing increased nuclear accumulation of β-catenin, supporting a mechanistic link via loss of the known interaction of Rassf1 with β-TrCP that usually mediates degradation of β-catenin. PMID:18391979

  7. Establishing the germline in spiralian embyos.

    PubMed

    Rebscher, Nicole

    2014-01-01

    Elucidating the origin of germ cells in embryos and larvae is often obscured by the fact that the typical germ cell markers vasa, nanos and piwi are not exclusively expressed in primordial germ cells (PGCs), but are also commonly found in undifferentiated somatic tissues and stem cells as part of an evolutionary conserved 'germline multipotency program' (Juliano et al., 2010). Hidden in the crowd of undifferentiated cells, the PGCs have occasionally been overlooked and their formation during early embryogenesis was only revealed recently by new methodological approaches (e.g. Wu et al., 2011). Spiralians are excellent model organisms to deepen our understanding of PGC formation, given the highly stereotypical cleavage that occurs during embryogenesis. In these species, detailed cell lineage studies enable the tracing of single cells up to gastrulation stages. Here, I review our knowledge of the origin of PGCs in these invertebrates. Similarities in PGC formation among spiralian phyla as well as peculiarities of the highly derived clitellates are discussed with respect to developmental mode and evolution. Furthermore, the issue of gonad regeneration in platyhelminths and the asexually reproducing oligochaete Enchytraeus japonensis is addressed. An alternative strategy of compensating for caudal regeneration is presented for the polychaete Platynereis dumerilli. Finally, the molecular bases of PGC specification and the question of germplasm are discussed. PMID:25690958

  8. Source apportionment of gaseous atmospheric pollutants by means of an absolute principal component scores (APCS) receptor model.

    PubMed

    Bruno, P; Caselli, M; de Gennaro, G; Traini, A

    2001-12-01

    A multivariate statistical method has been applied to apportion the atmospheric pollutant concentrations measured by automatic gas analyzers placed on a mobile laboratory for air quality monitoring in Taranto (Italy). In particular, Principal Component Analysis (PCA) followed by Absolute Principal Component Scores (APCS) technique was performed to identify the number of emission sources and their contribution to measured concentrations of CO, NOx, benzene toluene m+p-Xylene (BTX). This procedure singled out two different sources that explain about 85% of collected data variance. PMID:11798109

  9. Viral receptor-binding site antibodies with diverse germline origins

    PubMed Central

    Schmidt, Aaron G.; Therkelsen, Matthew D.; Stewart, Shaun; Kepler, Thomas B.; Liao, Hua-Xin; Moody, M. Anthony; Haynes, Barton F.; Harrison, Stephen C.

    2015-01-01

    Vaccines for rapidly evolving pathogens will confer lasting immunity if they elicit antibodies recognizing conserved epitopes, such as a receptor-binding site (RBS). From characteristics of an influenza-virus RBS-directed antibody, we devised a signature motif to search for similar antibodies. We identified, from three vaccinees, over 100 candidates encoded by eleven different VH genes. Crystal structures show that antibodies in this class engage the hemagglutinin RBS and mimic binding of the receptor, sialic acid, by supplying a critical dipeptide on their projecting, heavy-chain third complementarity determining region. They share contacts with conserved, receptor-binding residues but contact different residues on the RBS periphery, limiting the likelihood of viral escape when several such antibodies are present. These data show that related modes of RBS recognition can arise from different germline origins and mature through diverse affinity maturation pathways. Immunogens focused on an RBS-directed response will thus have a broad range of B-cell targets. PMID:25959776

  10. Viral receptor-binding site antibodies with diverse germline origins.

    PubMed

    Schmidt, Aaron G; Therkelsen, Matthew D; Stewart, Shaun; Kepler, Thomas B; Liao, Hua-Xin; Moody, M Anthony; Haynes, Barton F; Harrison, Stephen C

    2015-05-21

    Vaccines for rapidly evolving pathogens will confer lasting immunity if they elicit antibodies recognizing conserved epitopes, such as a receptor-binding site (RBS). From characteristics of an influenza-virus RBS-directed antibody, we devised a signature motif to search for similar antibodies. We identified, from three vaccinees, over 100 candidates encoded by 11 different VH genes. Crystal structures show that antibodies in this class engage the hemagglutinin RBS and mimic binding of the receptor, sialic acid, by supplying a critical dipeptide on their projecting, heavy-chain third complementarity determining region. They share contacts with conserved, receptor-binding residues but contact different residues on the RBS periphery, limiting the likelihood of viral escape when several such antibodies are present. These data show that related modes of RBS recognition can arise from different germline origins and mature through diverse affinity maturation pathways. Immunogens focused on an RBS-directed response will thus have a broad range of B cell targets. PMID:25959776

  11. Mechanistic study of the anti-cancer effect of Gynostemma pentaphyllum saponins in the Apc(Min/+) mouse model.

    PubMed

    Tai, William Chi-Shing; Wong, Wing-Yan; Lee, Magnolia Muk-Lan; Chan, Brandon Dow; Lu, Cheng; Hsiao, Wen-Luan Wendy

    2016-05-01

    Gynostemma pentaphyllum saponins (GpS) have been shown to have anti-cancer activity. However, the underlying mechanisms remain unclear. In this study, we used the Apc(Min) (/+) colorectal cancer (CRC) mouse model to investigate the anti-cancer effect of GpS and we demonstrated that GpS treatment could significantly reduce the number and size of intestinal polyps in Apc(Min) (/+) mice. In order to identify the potential targets and mechanisms involved, a comparative proteomics analysis was performed and 40 differentially expressed proteins after GpS treatment were identified. Bioinformatics analyses suggested a majority of these proteins were involved in processes related to cellular redox homeostasis, and predicted Raf-1 as a potential target of GpS. The upregulation of two proteins known to be involved in redox homeostasis, peroxiredoxin-1 (Prdx1) and peroxiredoxin-2 (Prdx2), and the downregulation of Raf-1 were validated using Western blot analysis. After further investigation of the associated signaling networks, we postulated that the anti-cancer effect of GpS was mediated through the upregulation of Prdx1 and Prdx2, suppression of Ras, RAF/MEK/ERK/STAT, PI3K/AKT/mTOR signaling and modulation of JNK/p38 MAPK signaling. We also examined the potential combinatorial effect of GpS with the chemotherapeutic 5-fluorouracil (5-FU) and found that GpS could enhance the anti-cancer efficacy of 5-FU, further suppressing the number of polyps in Apc(Min/+) mice. Our findings highlight the potential of GpS as an anti-cancer agent, the potential mechanisms of its anti-cancer activities, and its effect as an adjuvant of 5-FU in the chemotherapy of CRC. PMID:26970558

  12. Use of Germline Polymorphisms in Predicting Concurrent Chemoradiotherapy Response in Esophageal Cancer

    SciTech Connect

    Chen, Pei-Chun; Chen, Yen-Ching; Lai, Liang-Chuan; Tsai, Mong-Hsun; Chen, Shin-Kuang; Yang, Pei-Wen; Lee, Yung-Chie; Hsiao, Chuhsing K.; Lee, Jang-Ming; Chuang, Eric Y.

    2012-04-01

    Purpose: To identify germline polymorphisms to predict concurrent chemoradiation therapy (CCRT) response in esophageal cancer patients. Materials and Methods: A total of 139 esophageal cancer patients treated with CCRT (cisplatin-based chemotherapy combined with 40 Gy of irradiation) and subsequent esophagectomy were recruited at the National Taiwan University Hospital between 1997 and 2008. After excluding confounding factors (i.e., females and patients aged {>=}70 years), 116 patients were enrolled to identify single nucleotide polymorphisms (SNPs) associated with specific CCRT responses. Genotyping arrays and mass spectrometry were used sequentially to determine germline polymorphisms from blood samples. These polymorphisms remain stable throughout disease progression, unlike somatic mutations from tumor tissues. Two-stage design and additive genetic models were adopted in this study. Results: From the 26 SNPs identified in the first stage, 2 SNPs were found to be significantly associated with CCRT response in the second stage. Single nucleotide polymorphism rs16863886, located between SGPP2 and FARSB on chromosome 2q36.1, was significantly associated with a 3.93-fold increase in pathologic complete response to CCRT (95% confidence interval 1.62-10.30) under additive models. Single nucleotide polymorphism rs4954256, located in ZRANB3 on chromosome 2q21.3, was associated with a 3.93-fold increase in pathologic complete response to CCRT (95% confidence interval 1.57-10.87). The predictive accuracy for CCRT response was 71.59% with these two SNPs combined. Conclusions: This is the first study to identify germline polymorphisms with a high accuracy for predicting CCRT response in the treatment of esophageal cancer.

  13. Methylation of the adenomatous polyposis coli (APC) gene in human placenta and hypermethylation in choriocarcinoma cells.

    PubMed

    Wong, N C; Novakovic, B; Weinrich, B; Dewi, C; Andronikos, R; Sibson, M; Macrae, F; Morley, R; Pertile, M D; Craig, J M; Saffery, R

    2008-09-01

    Methylation of the human APC gene promoter is associated with several different types of cancers and has also been documented in some pre-cancerous tissues. We have examined the methylation of APC gene promoters in human placenta and choriocarcinoma cells. This revealed a general hypomethylation of the APC-1b promoter and a pattern with monoallelic methylation of the APC-1a promoter in full term placental tissue. However, there was no evidence of a parent-of-origin effect, suggesting random post zygotic origin of methylation. Increased methylation of this promoter was observed in all choriocarcinoma-derived trophoblast cell lines, suggesting a trophoblastic origin of placental APC methylation and implicating APC hypermethylation in the development of this group of gestational tumours. Our demonstration of placental methylation of the APC-1a promoter represents the first observation of monoallelic methylation of this gene in early development, and provides further support for a role of canonical Wnt signalling in placental trophoblast invasiveness. This also implicates tumour suppressor gene silencing as an integral part of normal human placental development. PMID:18485586

  14. APC2 and Axin promote mitotic fidelity by facilitating centrosome separation and cytoskeletal regulation.

    PubMed

    Poulton, John S; Mu, Frank W; Roberts, David M; Peifer, Mark

    2013-10-01

    To ensure the accurate transmission of genetic material, chromosome segregation must occur with extremely high fidelity. Segregation errors lead to chromosomal instability (CIN), with deleterious consequences. Mutations in the tumor suppressor adenomatous polyposis coli (APC) initiate most colon cancers and have also been suggested to promote disease progression through increased CIN, but the mechanistic role of APC in preventing CIN remains controversial. Using fly embryos as a model, we investigated the role of APC proteins in CIN. Our findings suggest that APC2 loss leads to increased rates of chromosome segregation error. This occurs through a cascade of events beginning with incomplete centrosome separation leading to failure to inhibit formation of ectopic cleavage furrows, which result in mitotic defects and DNA damage. We test several hypotheses related to the mechanism of action of APC2, revealing that APC2 functions at the embryonic cortex with several protein partners, including Axin, to promote mitotic fidelity. Our in vivo data demonstrate that APC2 protects genome stability by modulating mitotic fidelity through regulation of the cytoskeleton. PMID:24026117

  15. Parafibromin and APC as screening markers for malignant potential in atypical parathyroid adenomas.

    PubMed

    Juhlin, C Christofer; Nilsson, Inga-Lena; Johansson, Kenth; Haglund, Felix; Villablanca, Andrea; Höög, Anders; Larsson, Catharina

    2010-09-01

    The identification of parathyroid carcinomas is based upon histopathological criteria in which an invasive growth pattern or distant metastasis is demonstrated. A dilemma arises when tumours present with atypical histopathological features but lack direct evidence of malignancy. Recently, reduced expression or loss of the tumour suppressor proteins parafibromin and adenomatous polyposis coli (APC) has been associated with parathyroid malignancy. We report results from APC and parafibromin expression analyses by immunohistochemistry and Western blot in five cases of atypical adenoma, a single case of carcinoma and 54 adenomas without atypical features. Complete loss of APC immunoreactivity and reduced expression of parafibromin was evident in two of the atypical adenomas and in the parathyroid carcinoma. By contrast, all adenomas displayed APC expression, including two cases with hyperparathyroidism 2 gene (HRPT2) mutations and loss of parafibromin expression. We conclude that loss of APC is a frequent molecular event in atypical adenomas and carcinomas, but not in adenomas. Following verification in an independent material, APC could become a valuable tool when assessing parathyroid tumours in the clinical setting. Furthermore, the molecular resemblance of atypical adenomas with carcinoma concerning parafibromin and APC expression indicates that atypical adenomas should be subjects to watchful follow-up. PMID:20473645

  16. Oscillation of APC/C activity during cell cycle arrest promotes centrosome amplification

    PubMed Central

    Prosser, Suzanna L.; Samant, Mugdha D.; Baxter, Joanne E.; Morrison, Ciaran G.; Fry, Andrew M.

    2014-01-01

    Centrosome duplication is licensed by the disengagement, or ‘uncoupling’, of centrioles during late mitosis. However, arrest of cells in G2 can trigger premature centriole disengagement. Here, we show that premature disengagement results from untimely activation of the APC/C leading to securin degradation and release of active separase. APC/C activation during G2 arrest is dependent on Plk1-mediated degradation of the APC/C inhibitor, Emi1, but Plk1 also has a second APC/C-independent role in promoting disengagement. Importantly, APC/C and Plk1 activity also stimulate centriole disengagement in response to hydroxyurea or DNA damage-induced cell cycle arrest and this leads to centrosome amplification. However, the re-duplication of disengaged centrioles is dependent on Cdk2 activity and Cdk2 activation coincides with a subsequent inactivation of the APC/C and re-accumulation of cyclin A. Release from these arrests leads to mitotic entry but, due to the presence of disengaged and/or amplified centrosomes, formation of abnormal mitotic spindles that lead to chromosome missegregation. Thus, oscillation of APC/C activity during cell cycle arrest promotes both centrosome amplification and genome instability. PMID:22956538

  17. The Expensive Germline and the Evolution of Ageing.

    PubMed

    Maklakov, Alexei A; Immler, Simone

    2016-07-11

    The trade-off between survival and reproduction is the bedrock of the evolutionary theory of ageing. The reproductive system regulates ageing of the soma, and removal of germ cells extends somatic lifespan and increases resistance to a broad variety of abiotic and biotic stresses. The general explanation for this somatic response is that reduced reproduction frees up resources for survival. Remarkably, however, the disruption of molecular signaling pathways that regulate ageing increases lifespan without the obligatory reduction in fecundity, thus challenging the key role of the survival-reproduction trade-off. Here, we review the diverse literature on the costs of lifespan extension and suggest that the current paradigm is overly centered on the trade-off between lifespan and fecundity, often neglecting key aspects of fitness, such as development time, defense against parasites and, in particular, the high costs of germline maintenance. Compromised germline maintenance increases germline mutation rate, which reduces offspring fitness and ultimately can terminate germline proliferation across generations. We propose that future work should incorporate the costs of germline maintenance in the study of ageing evolution, as well as in applied biomedical research, by assessing offspring fitness. PMID:27404253

  18. Prostate Cancer Induced by Loss of Apc Is Restrained by TGFβ Signaling

    PubMed Central

    Bjerke, Glen A.; Pietrzak, Karolina; Melhuish, Tiffany A.; Frierson Jr., Henry F.; Paschal, Bryce M.; Wotton, David

    2014-01-01

    Recent work with mouse models of prostate cancer (CaP) has shown that inactivation of TGFβ signaling in prostate epithelium can cooperate with deletion of the Pten tumor suppressor to drive locally aggressive cancer and metastatic disease. Here, we show that inactivating the TGFβ pathway by deleting the gene encoding the TGFβ type II receptor (Tgfbr2) in combination with a deletion of the Apc tumor suppressor gene specifically in mouse prostate epithelium, results in the rapid onset of invasive CaP. Micro-metastases were observed in the lymph nodes and lungs of a proportion of the double mutant mice, whereas no metastases were observed in Apc single mutant mice. Prostate-specific Apc;Tgfbr2 mutants had a lower frequency of metastasis and survived significantly longer than Pten;Tgfbr2 double mutants. However, all Apc;Tgfbr2 mutants developed invasive cancer by 30 weeks of age, whereas invasive cancer was rarely observed in Apc single mutant animals, even by one year of age. Further comparison of the Pten and Apc models of CaP revealed additional differences, including adenosquamous carcinoma in the Apc;Tgfbr2 mutants that was not seen in the Pten model, and a lack of robust induction of the TGFβ pathway in Apc null prostate. In addition to causing high-grade prostate intra-epithelial neoplasia (HGPIN), deletion of either Pten or Apc induced senescence in affected prostate ducts, and this restraint was overcome by loss of Tgfbr2. In summary, this work demonstrates that TGFβ signaling restrains the progression of CaP induced by different tumor suppressor mutations, suggesting that TGFβ signaling exerts a general tumor suppressive effect in prostate. PMID:24651496

  19. Tumorigenic fragments of APC cause dominant defects in directional cell migration in multiple model systems

    PubMed Central

    Nelson, Scott A.; Li, Zhouyu; Newton, Ian P.; Fraser, David; Milne, Rachel E.; Martin, David M. A.; Schiffmann, David; Yang, Xuesong; Dormann, Dirk; Weijer, Cornelis J.; Appleton, Paul L.; Näthke, Inke S.

    2012-01-01

    SUMMARY Nonsense mutations that result in the expression of truncated, N-terminal, fragments of the adenomatous polyposis coli (APC) tumour suppressor protein are found in most sporadic and some hereditary colorectal cancers. These mutations can cause tumorigenesis by eliminating β-catenin-binding sites from APC, which leads to upregulation of β-catenin and thereby results in the induction of oncogenes such as MYC. Here we show that, in three distinct experimental model systems, expression of an N-terminal fragment of APC (N-APC) results in loss of directionality, but not speed, of cell motility independently of changes in β-catenin regulation. We developed a system to culture and fluorescently label live pieces of gut tissue to record high-resolution three-dimensional time-lapse movies of cells in situ. This revealed an unexpected complexity of normal gut cell migration, a key process in gut epithelial maintenance, with cells moving with spatial and temporal discontinuity. Quantitative comparison of gut tissue from wild-type mice and APC heterozygotes (APCMin/+; multiple intestinal neoplasia model) demonstrated that cells in precancerous epithelia lack directional preference when moving along the crypt-villus axis. This effect was reproduced in diverse experimental systems: in developing chicken embryos, mesoderm cells expressing N-APC failed to migrate normally; in amoeboid Dictyostelium, which lack endogenous APC, expressing an N-APC fragment maintained cell motility, but the cells failed to perform directional chemotaxis; and multicellular Dictyostelium slug aggregates similarly failed to perform phototaxis. We propose that N-terminal fragments of APC represent a gain-of-function mutation that causes cells within tissue to fail to migrate directionally in response to relevant guidance cues. Consistent with this idea, crypts in histologically normal tissues of APCMin/+ intestines are overpopulated with cells, suggesting that a lack of migration might cause

  20. Inference on germline BAP1 mutations and asbestos exposure from the analysis of familial and sporadic mesothelioma in a high-risk area.

    PubMed

    Betti, Marta; Casalone, Elisabetta; Ferrante, Daniela; Romanelli, Antonio; Grosso, Federica; Guarrera, Simonetta; Righi, Luisella; Vatrano, Simona; Pelosi, Giuseppe; Libener, Roberta; Mirabelli, Dario; Boldorini, Renzo; Casadio, Caterina; Papotti, Mauro; Matullo, Giuseppe; Magnani, Corrado; Dianzani, Irma

    2015-01-01

    Inherited loss-of-function mutations in the BAP1 oncosuppressor gene are responsible for an inherited syndrome with predisposition to malignant mesothelioma (MM), uveal and keratinocytic melanoma, and other malignancies. Germline mutations that were inherited in an autosomal dominant fashion were identified in nine families with multiplex MM cases and 25 families with multiple melanoma, renal cell carcinoma, and other tumors. Germline mutations were also identified in sporadic MM cases, suggesting that germline mutations in BAP1 occur frequently. In this article, we report the analysis of BAP1 in five multiplex MM families and in 103 sporadic cases of MM. One family carried a new truncating germline mutation. Using immunohistochemistry, we show that BAP1 is not expressed in tumor tissue, which is in accordance with Knudson's two hits hypothesis. Interestingly, whereas the three individuals who were possibly exposed to asbestos developed MM, the individual who was not exposed developed a different tumor type, that is, mucoepidermoid carcinoma. This finding suggests that the type of carcinogen exposure may be important for the cancer type that is developed by mutation carriers. On the contrary, the other families or the 103 sporadic patients did not show germline mutations in BAP1. Our data show that BAP1 mutations are very rare in patients with sporadic MM, and we report a new BAP1 mutation, extend the cancer types associated with these mutations, and suggest the existence of other yet unknown genes in the pathogenesis of familial MM. PMID:25231345

  1. Characterization of an APC Promoter 1B deletion in a Patient Diagnosed with Familial Adenomatous Polyposis via Whole Genome Shotgun Sequencing

    PubMed Central

    Kalbfleisch, Ted; Brock, Pamela; Snow, Angela; Neklason, Deborah; Gowans, Gordon; Klein, Jon

    2015-01-01

    Recently, deletions have been identified and published as causal for Familial Adenomatous Polyposis in the 1B promoter region of the APC gene.  Those deletions were measured using multiplex ligation-dependent probe amplification.  Here, we present and characterize an ~11kb deletion identified by whole genome shotgun sequencing.  The deletion occurred in a patient diagnosed with Familial Adenomatous Polyposis, and was located on chr5, between bases 112,034,824 and 112,045,845, fully encompassing the 1B promoter region of the APC gene.   Results are presented here that include the sequence evidence supporting the presence of the deletion as well as base level characterization of the deletion site.  These results demonstrate the capacity of whole genome sequencing for the detection of large structural variants in single individuals. PMID:26213617

  2. Characterization of an APC Promoter 1B deletion in a Patient Diagnosed with Familial Adenomatous Polyposis via Whole Genome Shotgun Sequencing.

    PubMed

    Kalbfleisch, Ted; Brock, Pamela; Snow, Angela; Neklason, Deborah; Gowans, Gordon; Klein, Jon

    2015-01-01

    Recently, deletions have been identified and published as causal for Familial Adenomatous Polyposis in the 1B promoter region of the APC gene.  Those deletions were measured using multiplex ligation-dependent probe amplification.  Here, we present and characterize an ~11kb deletion identified by whole genome shotgun sequencing.  The deletion occurred in a patient diagnosed with Familial Adenomatous Polyposis, and was located on chr5, between bases 112,034,824 and 112,045,845, fully encompassing the 1B promoter region of the APC gene.   Results are presented here that include the sequence evidence supporting the presence of the deletion as well as base level characterization of the deletion site.  These results demonstrate the capacity of whole genome sequencing for the detection of large structural variants in single individuals. PMID:26213617

  3. Comprehensive analysis of BRCA1, BRCA2 and TP53 germline mutation and tumor characterization: a portrait of early-onset breast cancer in Brazil.

    PubMed

    Carraro, Dirce Maria; Koike Folgueira, Maria Aparecida Azevedo; Garcia Lisboa, Bianca Cristina; Ribeiro Olivieri, Eloisa Helena; Vitorino Krepischi, Ana Cristina; de Carvalho, Alex Fiorini; de Carvalho Mota, Louise Danielle; Puga, Renato David; do Socorro Maciel, Maria; Michelli, Rodrigo Augusto Depieri; de Lyra, Eduardo Carneiro; Grosso, Stana Helena Giorgi; Soares, Fernando Augusto; Achatz, Maria Isabel Alves de Souza Waddington; Brentani, Helena; Moreira-Filho, Carlos Alberto; Brentani, Maria Mitzi

    2013-01-01

    Germline mutations in BRCA1, BRCA2 and TP53 genes have been identified as one of the most important disease-causing issues in young breast cancer patients worldwide. The specific defective biological processes that trigger germline mutation-associated and -negative tumors remain unclear. To delineate an initial portrait of Brazilian early-onset breast cancer, we performed an investigation combining both germline and tumor analysis. Germline screening of the BRCA1, BRCA2, CHEK2 (c.1100delC) and TP53 genes was performed in 54 unrelated patients <35 y; their tumors were investigated with respect to transcriptional and genomic profiles as well as hormonal receptors and HER2 expression/amplification. Germline mutations were detected in 12 out of 54 patients (22%) [7 in BRCA1 (13%), 4 in BRCA2 (7%) and one in TP53 (2%) gene]. A cancer familial history was present in 31.4% of the unrelated patients, from them 43.7% were carriers for germline mutation (37.5% in BRCA1 and in 6.2% in the BRCA2 genes). Fifty percent of the unrelated patients with hormone receptor-negative tumors carried BRCA1 mutations, percentage increasing to 83% in cases with familial history of cancer. Over-representation of DNA damage-, cellular and cell cycle-related processes was detected in the up-regulated genes of BRCA1/2-associated tumors, whereas cell and embryo development-related processes were over-represented in the up-regulated genes of BRCA1/2-negative tumors, suggesting distinct mechanisms driving the tumorigenesis. An initial portrait of the early-onset breast cancer patients in Brazil was generated pointing out that hormone receptor-negative tumors and positive familial history are two major risk factors for detection of a BRCA1 germline mutation. Additionally, the data revealed molecular factors that potentially trigger the tumor development in young patients. PMID:23469205

  4. Somatic and occult germ-line mutations in SDHD, a mitochondrial complex II gene, in nonfamilial pheochromocytoma.

    PubMed

    Gimm, O; Armanios, M; Dziema, H; Neumann, H P; Eng, C

    2000-12-15

    Most pheochromocytomas are sporadic but about 10% are though to be hereditary. Although the etiology of most inherited pheochromocytoma is well known, little is known about the etiology of the more common sporadic tumor. Recently, germ-line mutations of SDHD, a mitochondria complex II gene, were found in patients with hereditary paraganglioma. We sought to determine whether SDHD plays a role in the development of sporadic pheochromocytomas and performed a mutation and deletion analysis of SDHD. Among 18 samples, we identified 4 heterozygous sequence variants (3 germ-line, 1 somatic). One germ-line SDHD mutation IVS1+2T>G (absent among 78 control alleles) is predicted to cause aberrant splicing. On reinvestigation, this patient was found to have a tumor of the carotid body, which was likely a paraganglioma. Another patient with malignant, extra-adrenal pheochromocytoma was found to have germ-line c.34G> A (G12S). However, this sequence variant was also found in 1 of 78 control alleles. The third, germ-line nonsense mutation R38X was found in a patient with extra-adrenal pheochromocytoma. The only somatic heterozygous mutation, c.242C>T (P81L), has been found in the germ line of two families with hereditary paraganglioma and is conserved among four eukaryotic multicellular organisms. Hence, this mutation is most likely of functional significance too. Overall, loss of heterozygosity in at least one of the two markers flanking SDHD was found in 13 tumors (72%). All of the tumors that already harbored intragenic SDHD mutations, whether germ-line or somatic, also had loss of heterozygosity. Our results indicate that SDHD plays a role in the pathogenesis of pheochromocytoma. Given the minimum estimated germline SDHD mutation frequency of 11% (maximum estimate up to 17%) in this set of apparently sporadic pheochromocytoma cases and if these data can be replicated in other populations, our observations might suggest that all such patients be considered for SDHD mutation

  5. Adenomatous polyposis coli (APC) regulates multiple signaling pathways by enhancing glycogen synthase kinase-3 (GSK-3) activity.

    PubMed

    Valvezan, Alexander J; Zhang, Fang; Diehl, J Alan; Klein, Peter S

    2012-02-01

    Glycogen synthase kinase-3 (GSK-3) is essential for many signaling pathways and cellular processes. As Adenomatous Polyposis Coli (APC) functions in many of the same processes, we investigated a role for APC in the regulation of GSK-3-dependent signaling. We find that APC directly enhances GSK-3 activity. Furthermore, knockdown of APC mimics inhibition of GSK-3 by reducing phosphorylation of glycogen synthase and by activating mTOR, revealing novel roles for APC in the regulation of these enzymes. Wnt signaling inhibits GSK-3 through an unknown mechanism, and this results in both stabilization of β-catenin and activation of mTOR. We therefore hypothesized that Wnts may regulate GSK-3 by disrupting the interaction between APC and the Axin-GSK-3 complex. We find that Wnts rapidly induce APC dissociation from Axin, correlating with β-catenin stabilization. Furthermore, Axin interaction with the Wnt co-receptor LRP6 causes APC dissociation from Axin. We propose that APC regulates multiple signaling pathways by enhancing GSK-3 activity, and that Wnts induce APC dissociation from Axin to reduce GSK-3 activity and activate downstream signaling. APC regulation of GSK-3 also provides a novel mechanism for Wnt regulation of multiple downstream effectors, including β-catenin and mTOR. PMID:22184111

  6. Sex Chromosome-wide Transcriptional Suppression and Compensatory Cis-Regulatory Evolution Mediate Gene Expression in the Drosophila Male Germline.

    PubMed

    Landeen, Emily L; Muirhead, Christina A; Wright, Lori; Meiklejohn, Colin D; Presgraves, Daven C

    2016-07-01

    The evolution of heteromorphic sex chromosomes has repeatedly resulted in the evolution of sex chromosome-specific forms of regulation, including sex chromosome dosage compensation in the soma and meiotic sex chromosome inactivation in the germline. In the male germline of Drosophila melanogaster, a novel but poorly understood form of sex chromosome-specific transcriptional regulation occurs that is distinct from canonical sex chromosome dosage compensation or meiotic inactivation. Previous work shows that expression of reporter genes driven by testis-specific promoters is considerably lower-approximately 3-fold or more-for transgenes inserted into X chromosome versus autosome locations. Here we characterize this transcriptional suppression of X-linked genes in the male germline and its evolutionary consequences. Using transgenes and transpositions, we show that most endogenous X-linked genes, not just testis-specific ones, are transcriptionally suppressed several-fold specifically in the Drosophila male germline. In wild-type testes, this sex chromosome-wide transcriptional suppression is generally undetectable, being effectively compensated by the gene-by-gene evolutionary recruitment of strong promoters on the X chromosome. We identify and experimentally validate a promoter element sequence motif that is enriched upstream of the transcription start sites of hundreds of testis-expressed genes; evolutionarily conserved across species; associated with strong gene expression levels in testes; and overrepresented on the X chromosome. These findings show that the expression of X-linked genes in the Drosophila testes reflects a balance between chromosome-wide epigenetic transcriptional suppression and long-term compensatory adaptation by sex-linked genes. Our results have broad implications for the evolution of gene expression in the Drosophila male germline and for genome evolution. PMID:27404402

  7. Sex Chromosome-wide Transcriptional Suppression and Compensatory Cis-Regulatory Evolution Mediate Gene Expression in the Drosophila Male Germline

    PubMed Central

    Landeen, Emily L.; Muirhead, Christina A.; Meiklejohn, Colin D.; Presgraves, Daven C.

    2016-01-01

    The evolution of heteromorphic sex chromosomes has repeatedly resulted in the evolution of sex chromosome-specific forms of regulation, including sex chromosome dosage compensation in the soma and meiotic sex chromosome inactivation in the germline. In the male germline of Drosophila melanogaster, a novel but poorly understood form of sex chromosome-specific transcriptional regulation occurs that is distinct from canonical sex chromosome dosage compensation or meiotic inactivation. Previous work shows that expression of reporter genes driven by testis-specific promoters is considerably lower—approximately 3-fold or more—for transgenes inserted into X chromosome versus autosome locations. Here we characterize this transcriptional suppression of X-linked genes in the male germline and its evolutionary consequences. Using transgenes and transpositions, we show that most endogenous X-linked genes, not just testis-specific ones, are transcriptionally suppressed several-fold specifically in the Drosophila male germline. In wild-type testes, this sex chromosome-wide transcriptional suppression is generally undetectable, being effectively compensated by the gene-by-gene evolutionary recruitment of strong promoters on the X chromosome. We identify and experimentally validate a promoter element sequence motif that is enriched upstream of the transcription start sites of hundreds of testis-expressed genes; evolutionarily conserved across species; associated with strong gene expression levels in testes; and overrepresented on the X chromosome. These findings show that the expression of X-linked genes in the Drosophila testes reflects a balance between chromosome-wide epigenetic transcriptional suppression and long-term compensatory adaptation by sex-linked genes. Our results have broad implications for the evolution of gene expression in the Drosophila male germline and for genome evolution. PMID:27404402

  8. Family history characteristics, tumor microsatellite instability and germline MSH2 and MLH1 mutations in hereditary colorectal cancer.

    PubMed

    Bapat, B V; Madlensky, L; Temple, L K; Hiruki, T; Redston, M; Baron, D L; Xia, L; Marcus, V A; Soravia, C; Mitri, A; Shen, W; Gryfe, R; Berk, T; Chodirker, B N; Cohen, Z; Gallinger, S

    1999-02-01

    Recent characterization of the molecular genetic basis of hereditary nonpolyposis colorectal cancer provides an important opportunity for identification of individuals and their families with germline mutations in mismatch repair genes. Cancer family history criteria that accurately define hereditary colorectal cancer are necessary for cost-effective testing for germline mutations in mismatch repair genes. The present report describes the results of analysis of 33 colorectal cancer cases/families that satisfy our modified family history criteria (Mount Sinai criteria) for colorectal cancer. Fourteen of these families met the more stringent Amsterdam criteria. Germline MSH2 and MLH1 mutations were identified by the reverse transcription-polymerase chain reaction and the protein truncation test, and confirmed by sequencing. Microsatellite instability analysis was performed on available tumors from affected patients. MSH2 or MLH1 mutations were detected in 8 of 14 Amsterdam criteria families and in 5 of the remaining 19 cases/families that only satisfied the Mount Sinai criteria. Three of the latter families had features of the Muir-Torre syndrome. A high level of microsatellite instability (MSI-H) was detected in almost all (16/18) colorectal cancers from individuals with MSH2 and MLH1 mutations, and infrequently (1/21) in colorectal cancer specimens from cases without detectable mutations. Families with germline MSH2 and MLH1 mutations tended to have individuals affected at younger ages and with multiple tumors. The Amsterdam criteria are useful, but not sufficient, for detecting hereditary colorectal cancer families with germline MSH2 and MLH1 mutations, since a proportion of cases and families with mutations in mismatch repair genes will be missed. Further development of cancer family history criteria are needed, using unbiased prospectively collected cases, to define more accurately those who will benefit from MSH2 and MLH1 mutation analysis. PMID:10190329

  9. β-Catenin destruction complex-independent regulation of Hippo–YAP signaling by APC in intestinal tumorigenesis

    PubMed Central

    Cai, Jing; Maitra, Anirban; Anders, Robert A.; Taketo, Makoto M.; Pan, Duojia

    2015-01-01

    Mutations in Adenomatous polyposis coli (APC) underlie familial adenomatous polyposis (FAP), an inherited cancer syndrome characterized by the widespread development of colorectal polyps. APC is best known as a scaffold protein in the β-catenin destruction complex, whose activity is antagonized by canonical Wnt signaling. Whether other effector pathways mediate APC's tumor suppressor function is less clear. Here we report that activation of YAP, the downstream effector of the Hippo signaling pathway, is a general hallmark of tubular adenomas from FAP patients. We show that APC functions as a scaffold protein that facilitates the Hippo kinase cascade by interacting with Sav1 and Lats1. Consistent with the molecular link between APC and the Hippo signaling pathway, genetic analysis reveals that YAP is absolutely required for the development of APC-deficient adenomas. These findings establish Hippo–YAP signaling as a critical effector pathway downstream from APC, independent from its involvement in the β-catenin destruction complex. PMID:26193883

  10. An adolescent case of familial hyperparathyroidism with a germline frameshift mutation of the CDC73 gene.

    PubMed

    Takeuchi, Takako; Yoto, Yuko; Tsugawa, Takeshi; Kamasaki, Hotaka; Kondo, Atsushi; Ogino, Jiro; Hasegawa, Tadashi; Yama, Naoya; Anan, Sawa; Uchino, Shinya; Ishikawa, Aki; Sakurai, Akihiro; Tsutsumi, Hiroyuki

    2015-10-01

    A 13-yr-old boy who complained of persistent nausea, vomiting and weight loss had hypercalcemia and an elevated intact PTH level. Computed tomography confirmed two tumors in the thyroid gland. The tumors were surgically removed and pathologically confirmed as parathyroid adenoma. Because his maternal aunt and grandmother both had histories of parathyroid tumors, genetic investigation was undertaken for him, and a germline frameshift mutation of the CDC73 gene was identified. CDC73 gene analysis should be done on individuals who are at risk of familial hyperparathyroidism, including those who are asymptomatic, and they should be followed for potential primary hyperparathyroidism and associated disorders including resultant parathyroid carcinoma. PMID:26568659

  11. Germline duplication of ATG2B and GSKIP predisposes to familial myeloid malignancies.

    PubMed

    Saliba, Joseph; Saint-Martin, Cécile; Di Stefano, Antonio; Lenglet, Gaëlle; Marty, Caroline; Keren, Boris; Pasquier, Florence; Valle, Véronique Della; Secardin, Lise; Leroy, Gwendoline; Mahfoudhi, Emna; Grosjean, Sarah; Droin, Nathalie; Diop, M'boyba; Dessen, Philippe; Charrier, Sabine; Palazzo, Alberta; Merlevede, Jane; Meniane, Jean-Côme; Delaunay-Darivon, Christine; Fuseau, Pascal; Isnard, Françoise; Casadevall, Nicole; Solary, Eric; Debili, Najet; Bernard, Olivier A; Raslova, Hana; Najman, Albert; Vainchenker, William; Bellanné-Chantelot, Christine; Plo, Isabelle

    2015-10-01

    No major predisposition gene for familial myeloproliferative neoplasms (MPN) has been identified. Here we demonstrate that the autosomal dominant transmission of a 700-kb duplication in four genetically related families predisposes to myeloid malignancies, including MPN, frequently progressing to leukemia. Using induced pluripotent stem cells and primary cells, we demonstrate that overexpression of ATG2B and GSKIP enhances hematopoietic progenitor differentiation, including of megakaryocytes, by increasing progenitor sensitivity to thrombopoietin (TPO). ATG2B and GSKIP cooperate with acquired JAK2, MPL and CALR mutations during MPN development. Thus, the germline duplication may change the fitness of cells harboring signaling pathway mutations and increases the probability of disease development. PMID:26280900

  12. A germline MTOR mutation in Aboriginal Australian siblings with intellectual disability, dysmorphism, macrocephaly, and small thoraces.

    PubMed

    Baynam, Gareth; Overkov, Angela; Davis, Mark; Mina, Kym; Schofield, Lyn; Allcock, Richard; Laing, Nigel; Cook, Matthew; Dawkins, Hugh; Goldblatt, Jack

    2015-07-01

    We report on three Aboriginal Australian siblings with a unique phenotype which overlaps with known megalencephaly syndromes and RASopathies, including Costello syndrome. A gain-of-function mutation in MTOR was identified and represents the first reported human condition due to a germline, familial MTOR mutation. We describe the findings in this family to highlight that (i) the path to determination of pathogenicity was confounded by the lack of genomic reference data for Australian Aboriginals and that (ii) the disease biology, functional analyses in this family, and studies on the tuberous sclerosis complex support consideration of an mTOR inhibitor as a therapeutic agent. PMID:25851998

  13. Contribution of BRCA1 and BRCA2 Germline Mutations to Early Algerian Breast Cancer

    PubMed Central

    Henouda, Sarra; Bensalem, Assia; Reggad, Rym; Serrar, Nedda; Rouabah, Leila; Pujol, Pascal

    2016-01-01

    Breast cancer is the most common female malignancy and the leading cancer mortality cause among Algerian women. Germline mutations in the BRCA1 and BRCA2 genes in patients with early-onset breast cancer have not been clearly identified within the Algerian population. It is necessary to study the BRCA1/2 genes involvement in the Algerian breast cancer occurrence. We performed this study to define germline mutations in BRCA1/2 and their implication in breast cancer among young women from eastern Algeria diagnosed or treated with primary invasive breast cancer at the age of 40 or less who were referred to Anti-Cancer Center of Setif, Algeria. Case series were unselected for family history. Eight distinct pathogenic mutations were identified in eight unrelated families. Three deleterious mutations and one large genomic rearrangement involving deletion of exon 2 were found in BRCA1 gene. In addition, four mutations within the BRCA2 gene and one large genomic rearrangement were identified. Novel mutation was found among Algerian population. Moreover, five variants of uncertain clinical significance and favor polymorphisms were identified. Our data suggest that BRCA1/2 mutations are responsible for a significant proportion of breast cancer in Algerian young women. PMID:26997744

  14. Contribution of BRCA1 and BRCA2 Germline Mutations to Early Algerian Breast Cancer.

    PubMed

    Henouda, Sarra; Bensalem, Assia; Reggad, Rym; Serrar, Nedda; Rouabah, Leila; Pujol, Pascal

    2016-01-01

    Breast cancer is the most common female malignancy and the leading cancer mortality cause among Algerian women. Germline mutations in the BRCA1 and BRCA2 genes in patients with early-onset breast cancer have not been clearly identified within the Algerian population. It is necessary to study the BRCA1/2 genes involvement in the Algerian breast cancer occurrence. We performed this study to define germline mutations in BRCA1/2 and their implication in breast cancer among young women from eastern Algeria diagnosed or treated with primary invasive breast cancer at the age of 40 or less who were referred to Anti-Cancer Center of Setif, Algeria. Case series were unselected for family history. Eight distinct pathogenic mutations were identified in eight unrelated families. Three deleterious mutations and one large genomic rearrangement involving deletion of exon 2 were found in BRCA1 gene. In addition, four mutations within the BRCA2 gene and one large genomic rearrangement were identified. Novel mutation was found among Algerian population. Moreover, five variants of uncertain clinical significance and favor polymorphisms were identified. Our data suggest that BRCA1/2 mutations are responsible for a significant proportion of breast cancer in Algerian young women. PMID:26997744

  15. Scratching the Niche That Controls C. elegans Germline Stem Cells

    PubMed Central

    Byrd, Dana T.; Kimble, Judith

    2010-01-01

    The C. elegans gonad provides a well-defined model for a stem cell niche and its control of self-renewal and differentiation. The distal tip cell (DTC) forms a mesenchymal niche that controls germline stem cells (GSCs), both to generate the germline tissue during development and to maintain it during adulthood. The DTC uses GLP-1/Notch signaling to regulate GSCs; germ cells respond to Notch signaling with a network of RNA regulators to control the decision between self-renewal and entry into the meiotic cell cycle. PMID:19765664

  16. Impact of Atomic Gap Size on Sensitivity and Backaction of APC Displacement Detectors

    NASA Astrophysics Data System (ADS)

    Flowers-Jacobs, N. E.; Lehnert, K. W.

    2008-03-01

    Recently our group created a mesoscopic displacement detector formed by coupling an atomic point contact (APC) to a nanomechanical beam and demonstrated a displacement imprecision limited by the fundamental shot-noise in the number of electrons that tunnel across the APC [1]. We continue this work by using a cryogenic apparatus that flexes the device substrate to mechanically adjust the size of the APC atomic gap in situ. The resulting changes in the APC displacement detector's intrinsic noise properties are measured by observing the 1 K random thermal motion of the nanomechanical beam at resonance frequencies up to 200 MHz. The goal of this work is to explore the effect of atomic gap size and shape on displacement sensitivity, understand the origin of the observed measurement backaction, and measure the recoil force of tunneling electrons. [1] N. E. Flowers-Jacobs, D. R. Schmidt, and K. W. Lehnert, Phys. Rev. Lett. 98, 096804 (2007)

  17. Quercetin Supplementation Attenuates the Progression of Cancer Cachexia in ApcMin/+ Mice123

    PubMed Central

    Velázquez, Kandy T.; Enos, Reilly T.; Narsale, Aditi A.; Puppa, Melissa J.; Davis, J. Mark; Murphy, E. Angela; Carson, James A.

    2014-01-01

    Although there are currently no approved treatments for cancer cachexia, there is an intensified interest in developing therapies because of the high mortality index associated with muscle wasting diseases. Successful treatment of the cachectic patient focuses on improving or maintaining body weight and musculoskeletal function. Nutraceutical compounds, including the natural phytochemical quercetin, are being examined as potential treatments because of their anti-inflammatory, antioxidant, and anticarcinogenic properties. The purpose of this study was to determine the effect of quercetin supplementation on the progression of cachexia in the adenomatous polyposis coli (Apc)Min/+ mouse model of colorectal cancer. At 15 wk of age, C57BL/6 and male ApcMin/+ mice were supplemented with 25 mg/kg of quercetin or vehicle solution mix of Tang juice and water (V) daily for 3 wk. Body weight, strength, neuromuscular performance, and fatigue were assessed before and after quercetin or V interventions. Indicators of metabolic dysfunction and inflammatory signaling were also assessed. During the treatment period, the relative decrease in body weight in the ApcMin/+ mice gavaged with V (ApcMin/+V; −14% ± 2.3) was higher than in control mice gavaged with V (+0.6% ± 1.0), control mice gavaged with quercetin (−2% ± 1.0), and ApcMin/+ mice gavaged with quercetin (ApcMin/+Q; −9% ± 1.3). At 18 wk of age, the loss of grip strength and muscle mass shown in ApcMin/+V mice was significantly attenuated (P < 0.05) in ApcMin/+Q mice. Furthermore, ApcMin/+V mice had an induction of plasma interleukin-6 and muscle signal transducer and activator of transcription 3 phosphorylation, which were significantly (P < 0.05) mitigated in ApcMin/+Q mice, despite having a similar tumor burden. Quercetin treatment did not improve treadmill run-time-to-fatigue, hyperglycemia, or hyperlipidemia in cachectic ApcMin/+ mice. Overall, quercetin supplementation positively affected several aspects of

  18. Evaluation and Analysis of Seasat a Scanning Multichannel Microwave Radiometer (SMMR) Antenna Pattern Correction (APC) Algorithm

    NASA Technical Reports Server (NTRS)

    Kitzis, S. N.; Kitzis, J. L.

    1979-01-01

    The accuracy of the SEASAT-A SMMR antenna pattern correction (APC) algorithm was assessed. Interim APC brightness temperature measurements for the SMMR 6.6 GHz channels are compared with surface truth derived sea surface temperatures. Plots and associated statistics are presented for SEASAT-A SMMR data acquired for the Gulf of Alaska experiment. The cross-track gradients observed in the 6.6 GHz brightness temperature data are discussed.

  19. Oral mucosal stigmata in hereditary-cancer syndromes: From germline mutations to distinctive clinical phenotypes and tailored therapies.

    PubMed

    Ponti, Giovanni; Tomasi, Aldo; Manfredini, Marco; Pellacani, Giovanni

    2016-05-10

    Numerous familial tumor syndromes are associated with distinctive oral mucosal findings, which may make possible an early diagnosis as an efficacious marker for the risk of developing visceral malignancies. In detail, Familial Adenomatous Polyposis (FAP), Gardner syndrome, Peutz-Jeghers syndrome, Cowden Syndrome, Gorlin Syndrome, Lynch/Muir-Torre Syndrome and Multiple Endocrine Neoplasia show specific lesions of the oral mucosa and other distinct clinical and molecular features. The common genetic background of the above mentioned syndromes involve germline mutations in tumor suppressor genes, such as APC, PTEN, PTCH1, STK11, RET, clearly implied in both ectodermal and mesodermal differentiation, being the oral mucosal and dental stigmata frequently associated in the specific clinical phenotypes. The oral and maxillofacial manifestations of these syndromes may become visible several years before the intestinal lesions, constituting a clinical marker that is predictive for the development of intestinal polyps and/or other visceral malignancies. A multidisciplinary approach is therefore necessary for both clinical diagnosis and management of the gene-carriers probands and their family members who have to be referred for genetic testing or have to be investigated for the presence of visceral cancers. PMID:26850131

  20. Germline Mutations in HOXB13 and Prostate-Cancer Risk

    PubMed Central

    Ewing, Charles M.; Ray, Anna M.; Lange, Ethan M.; Zuhlke, Kimberly A.; Robbins, Christiane M.; Tembe, Waibhav D.; Wiley, Kathleen E.; Isaacs, Sarah D.; Johng, Dorhyun; Wang, Yunfei; Bizon, Chris; Yan, Guifang; Gielzak, Marta; Partin, Alan W.; Shanmugam, Vijayalakshmi; Izatt, Tyler; Sinari, Shripad; Craig, David W.; Zheng, S. Lilly; Walsh, Patrick C.; Montie, James E.; Xu, Jianfeng; Carpten, John D.; Isaacs, William B.; Cooney, Kathleen A.

    2013-01-01

    BACKGROUND Family history is a significant risk factor for prostate cancer, although the molecular basis for this association is poorly understood. Linkage studies have implicated chromosome 17q21-22 as a possible location of a prostate-cancer susceptibility gene. METHODS We screened more than 200 genes in the 17q21-22 region by sequencing germline DNA from 94 unrelated patients with prostate cancer from families selected for linkage to the candidate region. We tested family members, additional case subjects, and control subjects to characterize the frequency of the identified mutations. RESULTS Probands from four families were discovered to have a rare but recurrent mutation (G84E) in HOXB13 (rs138213197), a homeobox transcription factor gene that is important in prostate development. All 18 men with prostate cancer and available DNA in these four families carried the mutation. The carrier rate of the G84E mutation was increased by a factor of approximately 20 in 5083 unrelated subjects of European descent who had prostate cancer, with the mutation found in 72 subjects (1.4%), as compared with 1 in 1401 control subjects (0.1%) (P = 8.5×10−7). The mutation was significantly more common in men with early-onset, familial prostate cancer (3.1%) than in those with late-onset, nonfamilial prostate cancer (0.6%) (P = 2.0×10−6). CONCLUSIONS The novel HOXB13 G84E variant is associated with a significantly increased risk of hereditary prostate cancer. Although the variant accounts for a small fraction of all prostate cancers, this finding has implications for prostate-cancer risk assessment and may provide new mechanistic insights into this common cancer. (Funded by the National Institutes of Health and others.) PMID:22236224

  1. Atomic structure of the APC/C and its mechanism of protein ubiquitination

    PubMed Central

    Yang, Jing; McLaughlin, Stephen H.; Barford, David

    2015-01-01

    The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex, and interphase inhibitor Emi1 ensures the correct order and timing of distinct cell cycle transitions. Here, we used cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module, and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of experiments to further investigate APC/C functions in vivo. PMID:26083744

  2. The APC/C Ubiquitin Ligase: From Cell Biology to Tumorigenesis

    PubMed Central

    Penas, Clara; Ramachandran, Vimal; Ayad, Nagi George

    2011-01-01

    The ubiquitin proteasome system (UPS) is required for normal cell proliferation, vertebrate development, and cancer cell transformation. The UPS consists of multiple proteins that work in concert to target a protein for degradation via the 26S proteasome. Chains of an 8.5-kDa protein called ubiquitin are attached to substrates, thus allowing recognition by the 26S proteasome. Enzymes called ubiquitin ligases or E3s mediate specific attachment to substrates. Although there are over 600 different ubiquitin ligases, the Skp1–Cullin–F-box (SCF) complexes and the anaphase promoting complex/cyclosome (APC/C) are the most studied. SCF involvement in cancer has been known for some time while APC/C’s cancer role has recently emerged. In this review we will discuss the importance of APC/C to normal cell proliferation and development, underscoring its possible contribution to transformation. We will also examine the hypothesis that modulating a specific interaction of the APC/C may be therapeutically attractive in specific cancer subtypes. Finally, given that the APC/C pathway is relatively new as a cancer target, therapeutic interventions affecting APC/C activity may be beneficial in cancers that are resistant to classical chemotherapy. PMID:22655255

  3. Atomic structure of the APC/C and its mechanism of protein ubiquitination.

    PubMed

    Chang, Leifu; Zhang, Ziguo; Yang, Jing; McLaughlin, Stephen H; Barford, David

    2015-06-25

    The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex and interphase early mitotic inhibitor 1 (Emi1) ensures the correct order and timing of distinct cell-cycle transitions. Here we use cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of future experiments to investigate APC/C functions in vivo. PMID:26083744

  4. Adenomatous Polyposis Coli (APC) Is Required for Normal Development of Skin and Thymus

    PubMed Central

    Kuraguchi, Mari; Wang, Xiu-Ping; Bronson, Roderick T; Rothenberg, Rebecca; Ohene-Baah, Nana Yaw; Lund, Jennifer J; Kucherlapati, Melanie; Maas, Richard L; Kucherlapati, Raju

    2006-01-01

    The tumor suppressor gene Apc (adenomatous polyposis coli) is a member of the Wnt signaling pathway that is involved in development and tumorigenesis. Heterozygous knockout mice for Apc have a tumor predisposition phenotype and homozygosity leads to embryonic lethality. To understand the role of Apc in development we generated a floxed allele. These mice were mated with a strain carrying Cre recombinase under the control of the human Keratin 14 (K14) promoter, which is active in basal cells of epidermis and other stratified epithelia. Mice homozygous for the floxed allele that also carry the K14-cre transgene were viable but had stunted growth and died before weaning. Histological and immunochemical examinations revealed that K14-cre–mediated Apc loss resulted in aberrant growth in many ectodermally derived squamous epithelia, including hair follicles, teeth, and oral and corneal epithelia. In addition, squamous metaplasia was observed in various epithelial-derived tissues, including the thymus. The aberrant growth of hair follicles and other appendages as well as the thymic abnormalities in K14-cre; ApcCKO/CKO mice suggest the Apc gene is crucial in embryonic cells to specify epithelial cell fates in organs that require epithelial–mesenchymal interactions for their development. PMID:17002498

  5. Fizzy is required for activation of the APC/cyclosome in Xenopus egg extracts.

    PubMed

    Lorca, T; Castro, A; Martinez, A M; Vigneron, S; Morin, N; Sigrist, S; Lehner, C; Dorée, M; Labbé, J C

    1998-07-01

    The Xenopus homologue of Drosophila Fizzy and budding yeast CDC20 has been characterized. The encoded protein (X-FZY) is a component of a high molecular weight complex distinct from the APC/cyclosome. Antibodies directed against FZY were produced and shown to prevent calmodulin-dependent protein kinase II (CaMKII) from inducing the metaphase to anaphase transition of spindles assembled in vitro in Xenopus egg extracts, and this was associated with suppression of the degradation of mitotic cyclins. The same antibodies suppressed M phase-promoting factor (MPF)-dependent activation of the APC/cyclosome in interphase egg extracts, although they did not appear to alter the pattern or extent of MPF-dependent phosphorylation of APC/cyclosome subunits. As these phosphorylations are thought to be essential for APC/cyclosome activation in eggs and early embryos, we conclude that at least two events are required for MPF to activate the APC/cyclosome, allowing both chromatid segregation and full degradation of mitotic cyclins. The first one, which does not require FZY function, is the phosphorylation of APC/cyclosome subunits. The second one, that requires FZY function (even in the absence of MAD2 protein and when the spindle assembly checkpoint is not activated) is not yet understood at its molecular level. PMID:9649427

  6. Copy number variants associated with 18p11.32, DCC and the promoter 1B region of APC in colorectal polyposis patients

    PubMed Central

    Masson, Amy L.; Talseth-Palmer, Bente A.; Evans, Tiffany-Jane; McElduff, Patrick; Spigelman, Allan D.; Hannan, Garry N.; Scott, Rodney J.

    2015-01-01

    Familial Adenomatous Polyposis (FAP) is the second most common inherited predisposition to colorectal cancer (CRC) associated with the development of hundreds to thousands of adenomas in the colon and rectum. Mutations in APC are found in ~ 80% polyposis patients with FAP. In the remaining 20% no genetic diagnosis can be provided suggesting other genes or mechanisms that render APC inactive may be responsible. Copy number variants (CNVs) remain to be investigated in FAP and may account for disease in a proportion of polyposis patients. A cohort of 56 polyposis patients and 40 controls were screened for CNVs using the 2.7M microarray (Affymetrix) with data analysed using ChAS (Affymetrix). A total of 142 CNVs were identified unique to the polyposis cohort suggesting their involvement in CRC risk. We specifically identified CNVs in four unrelated polyposis patients among CRC susceptibility genes APC, DCC, MLH1 and CTNNB1 which are likely to have contributed to disease development in these patients. A recurrent deletion was observed at position 18p11.32 in 9% of the patients screened that was of particular interest. Further investigation is necessary to fully understand the role of these variants in CRC risk given the high prevalence among the patients screened. PMID:26909336

  7. Copy number variants associated with 18p11.32, DCC and the promoter 1B region of APC in colorectal polyposis patients.

    PubMed

    Masson, Amy L; Talseth-Palmer, Bente A; Evans, Tiffany-Jane; McElduff, Patrick; Spigelman, Allan D; Hannan, Garry N; Scott, Rodney J

    2016-02-01

    Familial Adenomatous Polyposis (FAP) is the second most common inherited predisposition to colorectal cancer (CRC) associated with the development of hundreds to thousands of adenomas in the colon and rectum. Mutations in APC are found in ~ 80% polyposis patients with FAP. In the remaining 20% no genetic diagnosis can be provided suggesting other genes or mechanisms that render APC inactive may be responsible. Copy number variants (CNVs) remain to be investigated in FAP and may account for disease in a proportion of polyposis patients. A cohort of 56 polyposis patients and 40 controls were screened for CNVs using the 2.7M microarray (Affymetrix) with data analysed using ChAS (Affymetrix). A total of 142 CNVs were identified unique to the polyposis cohort suggesting their involvement in CRC risk. We specifically identified CNVs in four unrelated polyposis patients among CRC susceptibility genes APC, DCC, MLH1 and CTNNB1 which are likely to have contributed to disease development in these patients. A recurrent deletion was observed at position 18p11.32 in 9% of the patients screened that was of particular interest. Further investigation is necessary to fully understand the role of these variants in CRC risk given the high prevalence among the patients screened. PMID:26909336

  8. Germline oncopharmacogenetics, a promising field in cancer therapy.

    PubMed

    Pesenti, Chiara; Gusella, Milena; Sirchia, Silvia M; Miozzo, Monica

    2015-02-01

    Pharmacogenetics (PGx) is the study of the relationship between inter-individual genetic variation and drug responses. Germline variants of genes involved in drug metabolism, drug transport, and drug targets can affect individual response to medications. Cancer therapies are characterized by an intrinsically high toxicity; therefore, the application of pharmacogenetics to cancer patients is a particularly promising method for avoiding the use of inefficacious drugs and preventing the associated adverse effects. However, despite continuing efforts in this field, very few labels include information about germline genetic variants associated with drug responses. DPYD, TPMT, UGT1A1, G6PD, CYP2D6, and HLA are the sole loci for which the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) report specific information. This review highlights the germline PGx variants that have been approved to date for anticancer treatments, and also provides some insights about other germline variants with potential clinical applications. The continuous and rapid evolution of next-generation sequencing applications, together with the development of computational methods, should help to refine the implementation of personalized medicine. One day, clinicians may be able to prescribe the best treatment and the correct drug dosage based on each patient's genotype. This approach would improve treatment efficacy, reduce toxicity, and predict non-responders, thereby decreasing chemotherapy-associated morbidity and improving health benefits. PMID:25573079

  9. Timing, rates and spectra of human germline mutation

    PubMed Central

    Lindsay, Sarah J.; Hardwick, Robert J.; Alexandrov, Ludmil B.; Turki, Saeed Al; Dominiczak, Anna; Morris, Andrew; Porteous, David; Smith, Blair; Stratton, Michael R.; Hurles, Matthew E.

    2015-01-01

    Germline mutations are a driving force behind genome evolution and genetic disease. We investigated genome-wide mutation rates and spectra in multi-sibling families. Mutation rate increased with paternal age in all families, but the number of additional mutations per year differed more than two-fold between families. Meta-analysis of 6,570 mutations showed that germline methylation influences mutation rates. In contrast to somatic mutations, we found remarkable consistency of germline mutation spectra between the sexes and at different paternal ages. 3.8% of mutations were mosaic in the parental germline, resulting in 1.3% of mutations being shared between siblings. The number of these shared mutations varied significantly between families. Our data suggest that the mutation rate per cell division is higher during both early embryogenesis and differentiation of primordial germ cells, but is reduced substantially during post-pubertal spermatogenesis. These findings have important consequences for the recurrence risks of disorders caused by de novo mutations. PMID:26656846

  10. Germline replacement by blastula cell transplantation in the fish medaka.

    PubMed

    Li, Mingyou; Hong, Ni; Xu, Hongyan; Song, Jianxing; Hong, Yunhan

    2016-01-01

    Primordial germ cell (PGC) specification early in development establishes the germline for reproduction and reproductive technologies. Germline replacement (GR) is a powerful tool for conservation of valuable or endangered animals. GR is achievable by germ cell transplantation into the PGC migration pathway or gonads. Blastula cell transplantation (BCT) can also lead to the chimeric germline containing PGCs of both donor and host origins. It has remained largely unknown whether BCT is able to achieve GR at a high efficiency. Here we report efficient GR by BCT into blastula embryos in the fish medaka (Oryzias latipes). Specifically, dnd depletion completely ablated host PGCs and fertility, and dnd overexpression remarkably boosted PGCs in donor blastulae. BCT between normal donor and host produced a germline transmission rate of ~4%. This rate was enhanced up to ~30% upon PGC boosting in donors. Most importantly, BCT between PGC-boosted donors and PGC-ablated hosts led to more than 90% fertility restoration and 100% GR. Therefore, BCT features an extremely high efficiency of fertility recovery and GR in medaka. This finding makes medaka an ideal model to analyze genetic and physiological donor-host compatibilities for BCT-mediated surrogate production and propagation of endangered lower vertebrates and biodiversity. PMID:27406328

  11. Germline replacement by blastula cell transplantation in the fish medaka

    PubMed Central

    Li, Mingyou; Hong, Ni; Xu, Hongyan; Song, Jianxing; Hong, Yunhan

    2016-01-01

    Primordial germ cell (PGC) specification early in development establishes the germline for reproduction and reproductive technologies. Germline replacement (GR) is a powerful tool for conservation of valuable or endangered animals. GR is achievable by germ cell transplantation into the PGC migration pathway or gonads. Blastula cell transplantation (BCT) can also lead to the chimeric germline containing PGCs of both donor and host origins. It has remained largely unknown whether BCT is able to achieve GR at a high efficiency. Here we report efficient GR by BCT into blastula embryos in the fish medaka (Oryzias latipes). Specifically, dnd depletion completely ablated host PGCs and fertility, and dnd overexpression remarkably boosted PGCs in donor blastulae. BCT between normal donor and host produced a germline transmission rate of ~4%. This rate was enhanced up to ~30% upon PGC boosting in donors. Most importantly, BCT between PGC-boosted donors and PGC-ablated hosts led to more than 90% fertility restoration and 100% GR. Therefore, BCT features an extremely high efficiency of fertility recovery and GR in medaka. This finding makes medaka an ideal model to analyze genetic and physiological donor-host compatibilities for BCT-mediated surrogate production and propagation of endangered lower vertebrates and biodiversity. PMID:27406328

  12. GERM-LINE SPECIFIC FACTORS IN CHEMICAL MUTAGENESIS

    EPA Science Inventory

    Chemical mutagenesis test results ave not revealed evidence of germ-line specific mutagens. owever, conventional assays have indicated that there are male-female differences in mutagenic response, as well as quantitative/qualitative differences in induced mutations which depend u...

  13. Water quality assessment and apportionment of pollution sources using APCS-MLR and PMF receptor modeling techniques in three major rivers of South Florida.

    PubMed

    Haji Gholizadeh, Mohammad; Melesse, Assefa M; Reddi, Lakshmi

    2016-10-01

    In this study, principal component analysis (PCA), factor analysis (FA), and the absolute principal component score-multiple linear regression (APCS-MLR) receptor modeling technique were used to assess the water quality and identify and quantify the potential pollution sources affecting the water quality of three major rivers of South Florida. For this purpose, 15years (2000-2014) dataset of 12 water quality variables covering 16 monitoring stations, and approximately 35,000 observations was used. The PCA/FA method identified five and four potential pollution sources in wet and dry seasons, respectively, and the effective mechanisms, rules and causes were explained. The APCS-MLR apportioned their contributions to each water quality variable. Results showed that the point source pollution discharges from anthropogenic factors due to the discharge of agriculture waste and domestic and industrial wastewater were the major sources of river water contamination. Also, the studied variables were categorized into three groups of nutrients (total kjeldahl nitrogen, total phosphorus, total phosphate, and ammonia-N), water murkiness conducive parameters (total suspended solids, turbidity, and chlorophyll-a), and salt ions (magnesium, chloride, and sodium), and average contributions of different potential pollution sources to these categories were considered separately. The data matrix was also subjected to PMF receptor model using the EPA PMF-5.0 program and the two-way model described was performed for the PMF analyses. Comparison of the obtained results of PMF and APCS-MLR models showed that there were some significant differences in estimated contribution for each potential pollution source, especially in the wet season. Eventually, it was concluded that the APCS-MLR receptor modeling approach appears to be more physically plausible for the current study. It is believed that the results of apportionment could be very useful to the local authorities for the control and

  14. Prevalence of Germline TP53 Mutations in a Prospective Series of Unselected Patients with Adrenocortical Carcinoma

    PubMed Central

    Else, Tobias; Everett, Jessica N.; Long, Jessica M.; Gruber, Stephen B.; Hammer, Gary D.

    2013-01-01

    Purpose: Adrenocortical carcinoma (ACC) is a hallmark cancer in families with Li Fraumeni syndrome (LFS) caused by mutations in the TP53 gene. The prevalence of germline TP53 mutations in children diagnosed with ACC ranges from 50–97%. Although existing criteria advocate for TP53 testing in all patients with ACC regardless of age at diagnosis, the overall prevalence of germline mutations in patients diagnosed with ACC has not been well studied. Patients and Methods: A total of 114 patients with confirmed ACC evaluated in the University of Michigan Endocrine Oncology Clinic were prospectively offered genetic counseling and TP53 genetic testing, regardless of age at diagnosis or family history. Ninety-four of the 114 patients met with a genetic counselor (82.5%), with 53 of 94 (56.4%) completing TP53 testing; 9.6% (nine of 94) declined testing. The remainder (32 of 94; 34%) expressed interest in testing but did not pursue it for various reasons. Results: Four of 53 patients in this prospective, unselected series were found to have a TP53 mutation (7.5%). The prevalence of mutations in those diagnosed over age 18 was 5.8% (three of 52). There were insufficient data to estimate the prevalence in those diagnosed under age 18. None of these patients met clinical diagnostic criteria for classic LFS. Three of the families met criteria for Li Fraumeni-like syndrome; one patient met no existing clinical criteria for LFS or Li Fraumeni-like syndrome. Three of the four patients with mutations were diagnosed with ACC after age 45. Conclusions: Genetic counseling and germline testing for TP53 should be offered to all patients with ACC. Restriction on age at diagnosis or strength of the family history would fail to identify mutation carriers. PMID:23175693

  15. Expression patterns of prdm1 during chicken embryonic and germline development.

    PubMed

    Wan, Zhiyi; Rui, Lei; Li, Zandong

    2014-05-01

    PRDM1 (PR domain containing 1) is a transcriptional repressor that has been identified in various species and is crucial for cell growth, differentiation and development. However, the expression pattern and role of PRDM1 in development has not been sufficiently established in birds. We therefore investigate the spatio-temporal expression of PRDM1 in various tissues, especially in the germline, during chicken development, providing the basis for functional study. Our results show that prdm1 mRNA was expressed in blastodermal cells (BCs) at stage X and in various tissues including the liver, skin, lung, kidney, eye, bursa of fabricius, spleen, proventriculus, gizzard, intestine, testis, ovary, tongue, feathers and thymus but was not or was only sparcely present in the heart, brain and skeletal muscle. The level of prdm1 mRNA was highest in the BCs among all tissues tested and significantly changed during development in many tissues, such as the blastoderm, bursa of fabricius, spleen, feathers and germline. Furthermore, the expression of the PRDM1 protein generally paralleled the mRNA results, except for in the gizzard. Immunohistochemistry also revealed that PRDM1 was localized in the smooth muscle. In addition, during germline development, PRDM1 was found to be continuously expressed in the presumptive primordial germ cells (PGCs) at stage X, the circulating PGCs in blood and the germ cells in the gonads from embryonic day 6 to adult in both males and females. The expression pattern of PRDM1 in chicken thus suggests that this protein plays an important role during chicken development, such as in BC differentiation, feather formation and germ cell specification. PMID:24691770

  16. Germ-line deletion in DICER1 revealed by a novel MLPA assay using synthetic oligonucleotides.

    PubMed

    Sabbaghian, Nelly; Srivastava, Archana; Hamel, Nancy; Plourde, François; Gajtko-Metera, Malgorzata; Niedziela, Marek; Foulkes, William D

    2014-04-01

    DICER1 is an endoribonuclease responsible for the production of mature microRNAs which are small, single-stranded RNA molecules that regulate gene expression post-transcriptionally by binding to mRNA and repressing the expression of target genes. Germ-line mutations in DICER1 are responsible for a rare cancer syndrome, including tumors that can co-occur with multinodular goiter (MNG). Using Sanger sequencing, we screened all DICER1 exons and intron boundaries in 20 suspected mutation carriers: nine with ovarian sex cord-stromal tumors (including Sertoli-Leydig cell tumors (SLCTs)), five with pleuropulmonary blastoma, one with cystic nephroma, one with nasal chondromesenchymal hamartoma and four with more than one manifestation suggestive of a germ-line DICER1 mutation. All were negative for any apparently deleterious variants. We developed a Multiplex Ligation-based Probe Amplification assay for DICER1 to screen for large deletions or duplications. Synthetic oligonucleotides were designed to cover all exons in three probe-mixes. In a child with a SLCT and MNG, and in her mother and brother (both diagnosed with MNG), we identified a heterozygous germ-line deletion of approximately 3 kilobases that eliminates exon 21 of DICER1 and two-thirds of intron 21, accompanied by an insertion of a G nucleotide at the 3' end of the deletion (c.3270-6_4051-1280delinsG). This allele is expressed in the patient's cDNA, creating an out-of-frame deletion predicted to result in a truncated protein (r.3270_4050del; p.Tyr1091Ser*28). Our novel finding of a disease-causing large deletion in DICER1 emphasizes the need to include assays that can detect rearrangements, duplications and deletions in any DICER1 screening protocol. PMID:24065110

  17. Germ-line deletion in DICER1 revealed by a novel MLPA assay using synthetic oligonucleotides

    PubMed Central

    Sabbaghian, Nelly; Srivastava, Archana; Hamel, Nancy; Plourde, François; Gajtko-Metera, Malgorzata; Niedziela, Marek; Foulkes, William D

    2014-01-01

    DICER1 is an endoribonuclease responsible for the production of mature microRNAs which are small, single-stranded RNA molecules that regulate gene expression post-transcriptionally by binding to mRNA and repressing the expression of target genes. Germ-line mutations in DICER1 are responsible for a rare cancer syndrome, including tumors that can co-occur with multinodular goiter (MNG). Using Sanger sequencing, we screened all DICER1 exons and intron boundaries in 20 suspected mutation carriers: nine with ovarian sex cord-stromal tumors (including Sertoli–Leydig cell tumors (SLCTs)), five with pleuropulmonary blastoma, one with cystic nephroma, one with nasal chondromesenchymal hamartoma and four with more than one manifestation suggestive of a germ-line DICER1 mutation. All were negative for any apparently deleterious variants. We developed a Multiplex Ligation-based Probe Amplification assay for DICER1 to screen for large deletions or duplications. Synthetic oligonucleotides were designed to cover all exons in three probe-mixes. In a child with a SLCT and MNG, and in her mother and brother (both diagnosed with MNG), we identified a heterozygous germ-line deletion of approximately 3 kilobases that eliminates exon 21 of DICER1 and two-thirds of intron 21, accompanied by an insertion of a G nucleotide at the 3′ end of the deletion (c.3270-6_4051-1280delinsG). This allele is expressed in the patient's cDNA, creating an out-of-frame deletion predicted to result in a truncated protein (r.3270_4050del; p.Tyr1091Ser*28). Our novel finding of a disease-causing large deletion in DICER1 emphasizes the need to include assays that can detect rearrangements, duplications and deletions in any DICER1 screening protocol. PMID:24065110

  18. Human Immunodeficiencies Related to Defective APC/T Cell Interaction

    PubMed Central

    Kallikourdis, Marinos; Viola, Antonella; Benvenuti, Federica

    2015-01-01

    The primary event for initiating adaptive immune responses is the encounter between T lymphocytes and antigen presenting cells (APCs) in the T cell area of secondary lymphoid organs and the formation of highly organized intercellular junctions referred to as immune synapses (IS). In vivo live-cell imaging of APC–T cell interactions combined to functional studies unveiled that T cell fate is dictated, in large part, by the stability of the initial contact. Immune cell interaction is equally important during delivery of T cell help to B cells and for the killing of target cells by cytotoxic T cells and NK cells. The critical role of contact dynamics and synapse stability on the immune response is well illustrated by human immune deficiencies in which disease pathogenesis is linked to altered adhesion or defective cross-talk between the synaptic partners. The Wiskott–Aldrich syndrome (WAS) is a severe primary immunodeficiency caused by mutations in the Wiskott–Aldrich syndrome protein (WASp), a scaffold that promotes actin polymerization and links TCR stimulation to T cell activation. Absence or mutations in WASp affects intercellular APC–T cell communications by interfering with multiple mechanisms on both sides of the IS. The warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is caused by mutations in CXCR4, a chemokine receptor that in mutant form leads to impairment of APC–T cell interactions. Present evidences suggest that other recently characterized primary immune deficiencies caused by mutation in genes linked to actin cytoskeletal reorganization, such as WIP and DOCK8, may also depend on altered synapse stability. Here, we will discuss in details the mechanisms of disturbed APC–T cell interactions in WAS and WHIM. Moreover, we will summarize the evidence pointing to a compromised conjugate formation in WIP, DOCK8, and X-linked lymphoproliferative syndrome. PMID:26379669

  19. Splice site and Germline variations of the MGMT gene in Esophageal cancer from Kashmir Valley: India

    PubMed Central

    Shah, Mohd Amin; Shaffi, Sheikh M.; Lone, Ghulam Nabi; Jan, Syed Mudassar

    2013-01-01

    Objectives The aim of our investigation was to detect mutation or genetic polymorphisms in MGMT gene of esophageal cancer patients from Kashmir Valley (India) Methodology The genetic polymorphisms or mutations in the coding exons 2, 3, 4 and 5 of MGMT gene were searched for in DNA samples from the frozen tumor tissues of 30 esophageal cancer patients from Kashmir. The PCR products were sequenced with fluorescently labelled terminators and separated on automatic sequencer. We developed a new PCR based RFLP approach for genotyping c.459A>G (p.Gly153Gly) variation in 71 esophageal cancer patients and 60 healthy controls. Results Two somatic variations c.274 +4G>A and c.274 + 22G>A were identified in Exon3-intron 4 boundary. A novel germline variation c.459A>G (p.Gly153Gly) was found in the exon 5 of an esophageal cancer patient. This germline variation was not found in any of the studied esophageal cancer patients and healthy controls except the patient where it has been found by direct sequencing. Conclusion We identified novel sequence variants of the MGMT gene in esophageal cancer patients from Kashmir valley-India. PMID:24533020

  20. Identification of germline DICER1 mutations and loss of heterozygosity in familial Wilms tumour.

    PubMed

    Palculict, Timothy Blake; Ruteshouser, E Cristy; Fan, Yu; Wang, Wenyi; Strong, Louise; Huff, Vicki

    2016-06-01

    Wilms tumour (WT), a paediatric renal cancer, is the most common childhood kidney cancer. The aetiology of WT is heterogeneous with multiple genes known to result in WT tumorigenesis. However, these genes are rarely associated with familial Wilms tumour (FWT). To identify mutations predisposing to FWT, we performed whole-genome sequencing using genomic DNA from three affected/obligate carriers in a large WT family, followed by Sanger sequencing of candidate gene mutations in 47 additional WT families to determine their frequency in FWT. As a result, we identified two novel germline DICER1 mutations (G803R and R800Xfs5) co-segregating in two families, thus expanding the number of reported WT families with unique germline DICER1 mutations. The one large family was found to include individuals with multiple DICER1 syndrome phenotypes, including four WT cases. Interestingly, carriers of the DICER1 mutation displayed a greatly increased frequency of WT development compared with the penetrance observed in previously published pedigrees. Also uniquely, in one tumour this DICER1 mutant allele (G803R) was reduced to homozygosity in contrast to the somatic hotspot mutations typically observed in tumours in DICER1 families. PMID:26566882

  1. Differences in Strength and Timing of the mtDNA Bottleneck between Zebrafish Germline and Non-germline Cells.

    PubMed

    Otten, Auke B C; Theunissen, Tom E J; Derhaag, Josien G; Lambrichs, Ellen H; Boesten, Iris B W; Winandy, Marie; van Montfoort, Aafke P A; Tarbashevich, Katsiaryna; Raz, Erez; Gerards, Mike; Vanoevelen, Jo M; van den Bosch, Bianca J C; Muller, Marc; Smeets, Hubert J M

    2016-07-19

    We studied the mtDNA bottleneck in zebrafish to elucidate size, timing, and variation in germline and non-germline cells. Mature zebrafish oocytes contain, on average, 19.0 × 10(6) mtDNA molecules with high variation between oocytes. During embryogenesis, the mtDNA copy number decreases to ∼170 mtDNA molecules per primordial germ cell (PGC), a number similar to that in mammals, and to ∼50 per non-PGC. These occur at the same developmental stage, implying considerable variation in mtDNA copy number in (non-)PGCs of the same female, dictated by variation in the mature oocyte. The presence of oocytes with low mtDNA numbers, if similar in humans, could explain how (de novo) mutations can reach high mutation loads within a single generation. High mtDNA copy numbers in mature oocytes are established by mtDNA replication during oocyte development. Bottleneck differences between germline and non-germline cells, due to early differentiation of PGCs, may account for different distribution patterns of familial mutations. PMID:27373161

  2. Prevalence of SDHB, SDHC, and SDHD germline mutations in clinic patients with head and neck paragangliomas

    PubMed Central

    Baysal, B; Willett-Brozick, J; Lawrence, E; Drovdlic, C; Savul, S; McLeod, D; Yee, H; Brackmann, D; Slattery, W; Myers, E; Ferrell, R; Rubinstein, W

    2002-01-01

    Background: Paragangliomas are rare and highly heritable tumours of neuroectodermal origin that often develop in the head and neck region. Germline mutations in the mitochondrial complex II genes, SDHB, SDHC, and SDHD, cause hereditary paraganglioma (PGL). Methods: We assessed the frequency of SDHB, SDHC, and SDHD gene mutations by PCR amplification and sequencing in a set of head and neck paraganglioma patients who were previously managed in two otolaryngology clinics in the USA. Results: Fifty-five subjects were grouped into 10 families and 37 non-familial cases. Five of the non-familial cases had multiple tumours. Germline SDHD mutations were identified in five of 10 (50%) familial and two of 37 (∼5%) non-familial cases. R38X, P81L, H102L, Q109X, and L128fsX134 mutations were identified in the familial cases and P81L was identified in the non-familial cases. Both non-familial cases had multiple tumours. P81L and R38X mutations have previously been reported in other PGL families and P81L was suggested as a founder mutation. Allelic analyses of different chromosomes carrying these mutations did not show common disease haplotypes, strongly suggesting that R38X and P81L are potentially recurrent mutations. Germline SDHB mutations were identified in two of 10 (20%) familial and one of 33 (∼3%) non-familial cases. P131R and M71fsX80 were identified in the familial cases and Q59X was identified in the one non-familial case. The non-familial case had a solitary tumour. No mutations could be identified in the SDHC gene in the remaining four families and 20 sporadic cases. Conclusions: Mutations in SDHD are the leading cause of head and neck paragangliomas in this clinic patient series. SDHD and SDHB mutations account for 70% of familial cases and ∼8% of non-familial cases. These results also suggest that the commonness of the SDHD P81L mutation in North America is the result of both a founder effect and recurrent mutations. PMID:11897817

  3. APC promoter is frequently methylated in pancreatic juice of patients with pancreatic carcinomas or periampullary tumors

    PubMed Central

    Ginesta, Mireia M.; Diaz-Riascos, Zamira Vanessa; Busquets, Juli; Pelaez, Núria; Serrano, Teresa; Peinado, Miquel Àngel; Jorba, Rosa; García-Borobia, Francisco Javier; Capella, Gabriel; Fabregat, Joan

    2016-01-01

    Early detection of pancreatic and periampullary neoplasms is critical to improve their clinical outcome. The present authors previously demonstrated that DNA hypermethylation of adenomatous polyposis coli (APC), histamine receptor H2 (HRH2), cadherin 13 (CDH13), secreted protein acidic and cysteine rich (SPARC) and engrailed-1 (EN-1) promoters is frequently detected in pancreatic tumor cells. The aim of the present study was to assess their prevalence in pancreatic juice of carcinomas of the pancreas and periampullary area. A total of 135 pancreatic juices obtained from 85 pancreatic cancer (PC), 26 ampullary carcinoma (AC), 10 intraductal papillary mucinous neoplasm (IPMN) and 14 chronic pancreatitis (CP) patients were analyzed. The methylation status of the APC, HRH2, CDH13, SPARC and EN-1 promoters was analyzed using methylation specific-melting curve analysis (MS-MCA). Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were also tested with allele-specific quantitative polymerase chain reaction amplification. Out of the 5 promoters analyzed, APC (71%) and HRH2 (65%) were the most frequently methylated in PC juice. APC methylation was also detected at a high frequency in AC (76%) and IPMN (80%), but only occasionally observed in CP (7%). APC methylation had a high sensitivity (71–80%) for all types of cancer analyzed. The panel (where a sample scored as positive when ≥2 markers were methylated) did not outperform APC as a single marker. Finally, KRAS detection in pancreatic juice offered a lower sensitivity (50%) and specificity (71%) for detection of any cancer. APC hypermethylation in pancreatic juice, as assessed by MS-MCA, is a frequent event of potential clinical usefulness in the diagnosis of pancreatic and periampullary neoplasms. PMID:27602165

  4. Detection and Analysis of Cell Cycle-Associated APC/C-Mediated Cellular Ubiquitylation In Vitro and In Vivo.

    PubMed

    Cedeño, Cesyen; La Monaca, Esther; Esposito, Mara; Gutierrez, Gustavo J

    2016-01-01

    The anaphase-promoting complex or cyclosome (APC/C) is one of the major orchestrators of the cell division cycle in mammalian cells. The APC/C acts as a ubiquitin ligase that triggers sequential ubiquitylation of a significant number of substrates which will be eventually degraded by proteasomes during major transitions of the cell cycle. In this chapter, we present accessible methodologies to assess both in in vitro conditions and in cellular systems ubiquitylation reactions mediated by the APC/C. In addition, we also describe techniques to evidence the changes in protein stability provoked by modulation of the activity of the APC/C. Finally, specific methods to analyze interactors or posttranslational modifications of particular APC/C subunits are also discussed. Given the crucial role played by the APC/C in the regulation of the cell cycle, this review only focuses on its action and effects in actively proliferating cells. PMID:27613041

  5. Germline BAP1 Mutational Landscape of Asbestos-Exposed Malignant Mesothelioma Patients with Family History of Cancer.

    PubMed

    Ohar, Jill A; Cheung, Mitchell; Talarchek, Jacqueline; Howard, Suzanne E; Howard, Timothy D; Hesdorffer, Mary; Peng, Hongzhuang; Rauscher, Frank J; Testa, Joseph R

    2016-01-15

    Heritable mutations in the BAP1 tumor suppressor gene predispose individuals to mesothelioma and other cancers. However, a large-scale assessment of germline BAP1 mutation incidence and associated clinical features in mesothelioma patients with a family history of cancer has not been reported. Therefore, we examined the germline BAP1 mutation status of 150 mesothelioma patients with a family history of cancer, 50 asbestos-exposed control individuals with a family history of cancers other than mesothelioma, and 153 asbestos-exposed individuals without familial cancer. No BAP1 alterations were found in control cohorts, but were identified in nine of 150 mesothelioma cases (6%) with a family history of cancer. Alterations among these cases were characterized by both missense and frameshift mutations, and enzymatic activity of BAP1 missense mutants was decreased compared with wild-type BAP1. Furthermore, BAP1 mutation carriers developed mesothelioma at an earlier age that was more often peritoneal than pleural (five of nine) and exhibited improved long-term survival compared to mesothelioma patients without BAP1 mutations. Moreover, many tumors harboring BAP1 germline mutations were associated with BAP1 syndrome, including mesothelioma and ocular/cutaneous melanomas, as well as renal, breast, lung, gastric, and basal cell carcinomas. Collectively, these findings suggest that mesothelioma patients presenting with a family history of cancer should be considered for BAP1 genetic testing to identify those individuals who might benefit from further screening and routine monitoring for the purpose of early detection and intervention. PMID:26719535

  6. Scarce evidence of the causal role of germline mutations in UNC5C in hereditary colorectal cancer and polyposis

    PubMed Central

    Mur, Pilar; Elena, Sánchez-Cuartielles; Aussó, Susanna; Aiza, Gemma; Rafael, Valdés-Mas; Pineda, Marta; Navarro, Matilde; Brunet, Joan; Urioste, Miguel; Lázaro, Conxi; Moreno, Victor; Capellá, Gabriel; Puente, Xose S.; Valle, Laura

    2016-01-01

    Germline mutations in UNC5C have been suggested to increase colorectal cancer (CRC) risk, thus causing hereditary CRC. However, the evidence gathered thus far is insufficient to include the study of the UNC5C gene in the routine genetic testing of familial CRC. Here we aim at providing a more conclusive answer about the contribution of germline UNC5C mutations to genetically unexplained hereditary CRC and/or polyposis cases. To achieve this goal we sequenced the coding region and exon-intron boundaries of UNC5C in 544 familial CRC or polyposis patients (529 families), using a technique that combines pooled DNA amplification and massively parallel sequencing. A total of eight novel or rare variants, all missense, were identified in eight families. Co-segregation data in the families and association results in case-control series are not consistent with a causal effect for 7 of the 8 identified variants, including c.1882_1883delinsAA (p.A628K), previously described as a disease-causing mutation. One variant, c.2210G > A (p.S737N), remained unclassified. In conclusion, our results suggest that the contribution of germline mutations in UNC5C to hereditary colorectal cancer and to polyposis cases is negligible. PMID:26852919

  7. Germline-specific MATH-BTB substrate adaptor MAB1 regulates spindle length and nuclei identity in maize.

    PubMed

    Juranič, Martina; Srilunchang, Kanok-orn; Krohn, Nádia Graciele; Leljak-Levanic, Dunja; Sprunck, Stefanie; Dresselhaus, Thomas

    2012-12-01

    Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle-dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s). PMID:23250449

  8. Identification of Grandchildless Loci Whose Products Are Required for Normal Germ-Line Development in the Nematode Caenorhabditis Elegans

    PubMed Central

    Capowski, E. E.; Martin, P.; Garvin, C.; Strome, S.

    1991-01-01

    To identify genes that encode maternal components required for development of the germ line in the nematode Caenorhabditis elegans, we have screened for mutations that confer a maternal-effect sterile or ``grandchildless'' phenotype: homozygous mutant hermaphrodites produced by heterozygous mothers are themselves fertile, but produce sterile progeny. Our screens have identified six loci, defined by 21 mutations. This paper presents genetic and phenotypic characterization of four of the loci. The majority of mutations, those in mes-2, mes-3 and mes-4, affect postembryonic germ-line development; the progeny of mutant mothers undergo apparently normal embryogenesis but develop into agametic adults with 10-1000-fold reductions in number of germ cells. In contrast, mutations in mes-1 cause defects in cytoplasmic partitioning during embryogenesis, and the resulting larvae lack germ-line progenitor cells. Mutations in all of the mes loci primarily affect the germ line, and none disrupt the structural integrity of germ granules. This is in contrast to grandchildless mutations in Drosophila melanogaster, all of which disrupt germ granules and affect abdominal as well as germ-line development. PMID:1783292

  9. Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize[W

    PubMed Central

    Juranić, Martina; Srilunchang, Kanok-orn; Krohn, Nádia Graciele; Leljak-Levanić, Dunja; Sprunck, Stefanie; Dresselhaus, Thomas

    2012-01-01

    Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle–dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s). PMID:23250449

  10. Identification and evaluation of major histocompatibility complex antigens in chicken chimeras and their relationship to germline transmission.

    PubMed

    Bacon, L D; Zajchowski, L; Clark, M E; Etches, R J

    2002-10-01

    Chimeric chickens were evaluated as an intermediate for development of transgenic chickens. The transfer of Barred Plymouth Rock (BR) blastodermal cells into White Leghorn (WL) embryos results in BR-->WL chimeras, and some breeder males generate over 30% germline transmission of the BR genotype to offspring based on a feather-color trait. The objectives of the current study were to 1) identify the MHC (B haplotypes) in resident BR and WL lines, 2) establish that B antigens could be detected and quantified in red blood cells (RBC) of chimeras, 3) establish if there is a correlation in chimeras between percentage of RBC with donor B antigens and percentage germline transmission, and 4) evaluate if the MHC genotype influences chimera development. The RBC agglutination data indicated three B haplotypes were present in each line. The B*2-like, and B*19-like genes were unique to the WL line, and B*13-like and B-15-like genes were unique to the BR line, whereas a B*21-like gene was present in both lines. In adult BR-->WL chimeras, as well as 10- to 14 d-old WL-->WL chimeras, donor-type B antigens were detectable and quantifiable on RBC using flow cytometry. In BR-->WL chimeras, the percentage germline transmission was significantly correlated with the percentage of RBC with donor B antigen, as well as percentage of black feathers in the plumage. In a retrospective study using previously developed BR-->WL chimeras, the level of chimerism and germline transmission was higher in B*21/*21 type recipients, but this was not statistically significant in two prospective studies. It was concluded that MHC antigens on RBC can be used for identifying, quantifying, and selecting chicken chimeras developed by the transfer of blastodermal cells. PMID:12412906

  11. Discovery of germline-related genes in Cephalochordate amphioxus: A genome wide survey using genome annotation and transcriptome data.

    PubMed

    Yue, Jia-Xing; Li, Kun-Lung; Yu, Jr-Kai

    2015-12-01

    The generation of germline cells is a critical process in the reproduction of multicellular organisms. Studies in animal models have identified a common repertoire of genes that play essential roles in primordial germ cell (PGC) formation. However, comparative studies also indicate that the timing and regulation of this core genetic program vary considerably in different animals, raising the intriguing questions regarding the evolution of PGC developmental mechanisms in metazoans. Cephalochordates (commonly called amphioxus or lancelets) represent one of the invertebrate chordate groups and can provide important information about the evolution of developmental mechanisms in the chordate lineage. In this study, we used genome and transcriptome data to identify germline-related genes in two distantly related cephalochordate species, Branchiostoma floridae and Asymmetron lucayanum. Branchiostoma and Asymmetron diverged more than 120 MYA, and the most conspicuous difference between them is their gonadal morphology. We used important germline developmental genes in several model animals to search the amphioxus genome and transcriptome dataset for conserved homologs. We also annotated the assembled transcriptome data using Gene Ontology (GO) terms to facilitate the discovery of putative genes associated with germ cell development and reproductive functions in amphioxus. We further confirmed the expression of 14 genes in developing oocytes or mature eggs using whole mount in situ hybridization, suggesting their potential functions in amphioxus germ cell development. The results of this global survey provide a useful resource for testing potential functions of candidate germline-related genes in cephalochordates and for investigating differences in gonad developmental mechanisms between Branchiostoma and Asymmetron species. PMID:25847029

  12. Anillin is a substrate of anaphase-promoting complex/cyclosome (APC/C) that controls spatial contractility of myosin during late cytokinesis.

    PubMed

    Zhao, Wei-Meng; Fang, Guowei

    2005-09-30

    Anillin, an actin-binding protein localized at the cleavage furrow, is required for cytokinesis. Through an in vitro expression screen, we identified anillin as a substrate of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. We found that the levels of anillin fluctuate in the cell cycle, peaking in mitosis and dropping drastically during mitotic exit. Ubiquitination of anillin required a destruction-box and was mediated by Cdh1, an activator of APC/C. Overexpression of Cdh1 reduced the levels of anillin, whereas inactivation of APC/C(Cdh1) increased the half-life of anillin. Functionally, anillin was required for the completion of cytokinesis. In anillin knockdown cells, the cleavage furrow ingressed but failed to complete the ingression. At late cytokinesis, the cytosol and DNA in knockdown cells underwent rapid myosin-based oscillatory movement across the furrow. During this movement, RhoA and active myosin were absent from the cleavage furrow, and myosin was redistributed to cortical patches, which powers the random oscillatory movement. We concluded that anillin functions to maintain the localization of active myosin, thereby ensuring the spatial control of concerted contraction during cytokinesis. PMID:16040610

  13. Point Mutations in Exon 1B of APC Reveal Gastric Adenocarcinoma and Proximal Polyposis of the Stomach as a Familial Adenomatous Polyposis Variant.

    PubMed

    Li, Jun; Woods, Susan L; Healey, Sue; Beesley, Jonathan; Chen, Xiaoqing; Lee, Jason S; Sivakumaran, Haran; Wayte, Nicci; Nones, Katia; Waterfall, Joshua J; Pearson, John; Patch, Anne-Marie; Senz, Janine; Ferreira, Manuel A; Kaurah, Pardeep; Mackenzie, Robertson; Heravi-Moussavi, Alireza; Hansford, Samantha; Lannagan, Tamsin R M; Spurdle, Amanda B; Simpson, Peter T; da Silva, Leonard; Lakhani, Sunil R; Clouston, Andrew D; Bettington, Mark; Grimpen, Florian; Busuttil, Rita A; Di Costanzo, Natasha; Boussioutas, Alex; Jeanjean, Marie; Chong, George; Fabre, Aurélie; Olschwang, Sylviane; Faulkner, Geoffrey J; Bellos, Evangelos; Coin, Lachlan; Rioux, Kevin; Bathe, Oliver F; Wen, Xiaogang; Martin, Hilary C; Neklason, Deborah W; Davis, Sean R; Walker, Robert L; Calzone, Kathleen A; Avital, Itzhak; Heller, Theo; Koh, Christopher; Pineda, Marbin; Rudloff, Udo; Quezado, Martha; Pichurin, Pavel N; Hulick, Peter J; Weissman, Scott M; Newlin, Anna; Rubinstein, Wendy S; Sampson, Jone E; Hamman, Kelly; Goldgar, David; Poplawski, Nicola; Phillips, Kerry; Schofield, Lyn; Armstrong, Jacqueline; Kiraly-Borri, Cathy; Suthers, Graeme K; Huntsman, David G; Foulkes, William D; Carneiro, Fatima; Lindor, Noralane M; Edwards, Stacey L; French, Juliet D; Waddell, Nicola; Meltzer, Paul S; Worthley, Daniel L; Schrader, Kasmintan A; Chenevix-Trench, Georgia

    2016-05-01

    Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present. PMID:27087319

  14. LATS1 and LATS2 phosphorylate CDC26 to modulate assembly of the tetratricopeptide repeat subcomplex of APC/C.

    PubMed

    Masuda, Kenta; Chiyoda, Tatsuyuki; Sugiyama, Naoyuki; Segura-Cabrera, Aldo; Kabe, Yasuaki; Ueki, Arisa; Banno, Kouji; Banno, Koji; Suematsu, Makoto; Aoki, Daisuke; Ishihama, Yasushi; Saya, Hideyuki; Kuninaka, Shinji

    2015-01-01

    In budding yeast, the Mitotic Exit Network (MEN) regulates anaphase promoting complex/cyclosome (APC/C) via the Dbf2-Cdc14 signaling cascade. Dbf2 kinase phosphorylates and activates Cdc14 phosphatase, which removes the inhibitory phosphorylation of the APC/C cofactor Cdh1. Although each component of the MEN was highly conserved during evolution, there is presently no evidence supporting direct phosphorylation of CDC14 by large tumor suppressor kinase 1 (LATS1), the human counterpart of Dbf2; hence, it is unclear how LATS1 regulates APC/C. Here, we demonstrate that LATS1 phosphorylates the Thr7 (T7) residue of the APC/C component CDC26 directly. Nocodazole-induced phosphorylation of T7 was reduced by knockdown of LATS1 and LATS2 in HeLa cells, indicating that both of these kinases contribute to the phosphorylation of CDC26 in vivo. The T7 residue of CDC26 is critical for its interaction with APC6, a tetratricopeptide repeat-containing subunit of APC/C, and mutation of this residue to Asp (T7D) reduced the interaction of CDC26 with APC6. Replacement of endogenous CDC26 in HeLa cells with exogenous phosphor-mimic T7D-mutated CDC26 increased the elution size of APC/C subunits in a gel filtration assay, implying a change in the APC/C assembly upon phosphorylation of CDC26. Furthermore, T7D-mutated CDC26 promoted the ubiquitination of polo-like kinase 1, a well-known substrate of APC/C. Overall, these results suggest that LATS1/2 are novel kinases involved in APC/C phosphorylation and indicate a direct regulatory link between LATS1/2 and APC/C. PMID:25723520

  15. LATS1 and LATS2 Phosphorylate CDC26 to Modulate Assembly of the Tetratricopeptide Repeat Subcomplex of APC/C

    PubMed Central

    Masuda, Kenta; Chiyoda, Tatsuyuki; Sugiyama, Naoyuki; Segura-Cabrera, Aldo; Kabe, Yasuaki; Ueki, Arisa; Banno, Koji; Suematsu, Makoto; Aoki, Daisuke; Ishihama, Yasushi; Saya, Hideyuki; Kuninaka, Shinji

    2015-01-01

    In budding yeast, the Mitotic Exit Network (MEN) regulates anaphase promoting complex/cyclosome (APC/C) via the Dbf2-Cdc14 signaling cascade. Dbf2 kinase phosphorylates and activates Cdc14 phosphatase, which removes the inhibitory phosphorylation of the APC/C cofactor Cdh1. Although each component of the MEN was highly conserved during evolution, there is presently no evidence supporting direct phosphorylation of CDC14 by large tumor suppressor kinase 1 (LATS1), the human counterpart of Dbf2; hence, it is unclear how LATS1 regulates APC/C. Here, we demonstrate that LATS1 phosphorylates the Thr7 (T7) residue of the APC/C component CDC26 directly. Nocodazole-induced phosphorylation of T7 was reduced by knockdown of LATS1 and LATS2 in HeLa cells, indicating that both of these kinases contribute to the phosphorylation of CDC26 in vivo. The T7 residue of CDC26 is critical for its interaction with APC6, a tetratricopeptide repeat-containing subunit of APC/C, and mutation of this residue to Asp (T7D) reduced the interaction of CDC26 with APC6. Replacement of endogenous CDC26 in HeLa cells with exogenous phosphor-mimic T7D-mutated CDC26 increased the elution size of APC/C subunits in a gel filtration assay, implying a change in the APC/C assembly upon phosphorylation of CDC26. Furthermore, T7D-mutated CDC26 promoted the ubiquitination of polo-like kinase 1, a well-known substrate of APC/C. Overall, these results suggest that LATS1/2 are novel kinases involved in APC/C phosphorylation and indicate a direct regulatory link between LATS1/2 and APC/C. PMID:25723520

  16. The Apc(min) mouse has altered hematopoietic stem cell function and provides a model for MPD/MDS.

    PubMed

    Lane, Steven W; Sykes, Stephen M; Al-Shahrour, Fatima; Shterental, Sebastian; Paktinat, Mahnaz; Lo Celso, Cristina; Jesneck, Jonathan L; Ebert, Benjamin L; Williams, David A; Gilliland, D Gary

    2010-04-29

    Apc, a negative regulator of the canonical Wnt signaling pathway, is a bona-fide tumor suppressor whose loss of function results in intestinal polyposis. APC is located in a commonly deleted region on human chromosome 5q, associated with myelodysplastic syndrome (MDS), suggesting that haploinsufficiency of APC contributes to the MDS phenotype. Analysis of the hematopoietic system of mice with the Apc(min) allele that results in a premature stop codon and loss of function showed no abnormality in steady state hematopoiesis. Bone marrow derived from Apc(min) mice showed enhanced repopulation potential, indicating a cell intrinsic gain of function in the long-term hematopoietic stem cell (HSC) population. However, Apc(min) bone marrow was unable to repopulate secondary recipients because of loss of the quiescent HSC population. Apc(min) mice developed a MDS/myeloproliferative phenotype. Our data indicate that Wnt activation through haploinsufficiency of Apc causes insidious loss of HSC function that is only evident in serial transplantation strategies. These data provide a cautionary note for HSC-expansion strategies through Wnt pathway activation, provide evidence that cell extrinsic factors can contribute to the development of myeloid disease, and indicate that loss of function of APC may contribute to the phenotype observed in patients with MDS and del(5q). PMID:20197553

  17. The Spindle Assembly Checkpoint Is Not Essential for Viability of Human Cells with Genetically Lowered APC/C Activity

    PubMed Central

    Wild, Thomas; Larsen, Marie Sofie Yoo; Narita, Takeo; Schou, Julie; Nilsson, Jakob; Choudhary, Chunaram

    2016-01-01

    Summary The anaphase-promoting complex/cyclosome (APC/C) and the spindle assembly checkpoint (SAC), which inhibits the APC/C, are essential determinants of mitotic timing and faithful division of genetic material. Activation of the APC/C is known to depend on two APC/C-interacting E2 ubiquitin-conjugating enzymes—UBE2C and UBE2S. We show that APC/C activity in human cells is tuned by the combinatorial use of three E2s, namely UBE2C, UBE2S, and UBE2D. Genetic deletion of UBE2C and UBE2S, individually or in combination, leads to discriminative reduction in APC/C function and sensitizes cells to UBE2D depletion. Reduction of APC/C activity results in loss of switch-like metaphase-to-anaphase transition and, strikingly, renders cells insensitive to chemical inhibition of MPS1 and genetic ablation of MAD2, both of which are essential for the SAC. These results provide insights into the regulation of APC/C activity and demonstrate that the essentiality of the SAC is imposed by the strength of the APC/C. PMID:26904940

  18. AGILE integration into APC for high mix logic fab

    NASA Astrophysics Data System (ADS)

    Gatefait, M.; Lam, A.; Le Gratiet, B.; Mikolajczak, M.; Morin, V.; Chojnowski, N.; Kocsis, Z.; Smith, I.; Decaunes, J.; Ostrovsky, A.; Monget, C.

    2015-09-01

    mix logic Fab) in term of product and technology portfolio AGILE corrects for up to 120nm of product topography error on process layer with less than 50nm depth of focus Based on tool functionalities delivered by ASML and on high volume manufacturing requirement, AGILE integration is a real challenge. Regarding ST requirements "Automatic AGILE" functionality developed by ASML was not a turnkey solution and a dedicated functionality was needed. A "ST homemade AGILE integration" has been fully developed and implemented within ASML and ST constraints. This paper describes this integration in our Advanced Process Control platform (APC).

  19. Polarizing T and B Cell Responses by APC-Targeted Subunit Vaccines

    PubMed Central

    Grødeland, Gunnveig; Fossum, Even; Bogen, Bjarne

    2015-01-01

    Current influenza vaccines mostly aim at the induction of specific neutralizing antibodies. While antibodies are important for protection against a particular virus strain, T cells can recognize epitopes that will offer broader protection against influenza. We have previously developed a DNA vaccine format by which protein antigens can be targeted specifically to receptors on antigen presenting cells (APCs). The DNA-encoded vaccine proteins are homodimers, each chain consisting of a targeting unit, a dimerization unit, and an antigen. The strategy of targeting antigen to APCs greatly enhances immune responses as compared to non-targeted controls. Furthermore, targeting of antigen to different receptors on APCs can polarize the immune response to different arms of immunity. Here, we discuss how targeting of hemagglutinin to MHC class II molecules increases Th2 and IgG1 antibody responses, whereas targeting to chemokine receptors XCR1 or CCR1/3/5 increases Th1 and IgG2a responses, in addition to CD8+ T cell responses. We also discuss these results in relation to work published by others on APC-targeting. Differential targeting of APC surface molecules may allow the induction of tailor-made phenotypes of adaptive immune responses that are optimal for protection against various infectious agents, including influenza virus. PMID:26257735

  20. Polyethylene glycol inhibits intestinal neoplasia and induces epithelial apoptosis in Apc(min) mice.

    PubMed

    Roy, Hemant K; Gulizia, James; DiBaise, John K; Karolski, William J; Ansari, Sajid; Madugula, Madhavi; Hart, John; Bissonnette, Marc; Wali, Ramesh K

    2004-11-01

    Efficacy of a safe and clinically utilized polyethylene glycol formulation (PEG-3350) to suppress intestinal tumors was investigated in the Apc(min) mouse-model of experimental carcinogenesis. Furthermore, based on our previous finding on the induction of apoptosis in HT-29 cells by PEG, we evaluated its ability to stimulate epithelial cell apoptosis in both Apc(min) mouse as well as AOM-treated rat as a potential molecular mechanism of chemoprevention. Twenty-two Apc(min) mice were randomized equally to PEG or vehicle (control) supplementation. Tumors were scored and uninvolved intestinal mucosal apoptosis was assayed using a modified terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) assay and by immunohistochemical detection of cleaved caspase-3. Supplementation of Apc(min) mice with 10% PEG 3350 (in drinking water) resulted in a 48% (P<0.05) reduction in intestinal tumor burden and induced 2-3 fold increase in mucosal apoptosis. Dietary supplementation of polyethylene glycol (5%) also stimulated colonic mucosal apoptosis 4-5 fold in AOM-treated rats, the regimen that we previously reported to reduce tumor burden by 76% (P<0.05). In summary, we demonstrate, for the first time, that PEG does protect against Apc(min) mouse tumorigenesis. The correlation between pro-apoptotic actions and chemopreventive efficacy of PEG in these models strongly implicates induction of apoptosis as one of the impending mechanisms of chemoprevention. PMID:15374630

  1. Production of geopolymers using glass produced from DC plasma treatment of air pollution control (APC) residues.

    PubMed

    Kourti, Ioanna; Rani, D Amutha; Deegan, D; Boccaccini, A R; Cheeseman, C R

    2010-04-15

    Air pollution control (APC) residues are the hazardous waste produced from cleaning gaseous emissions at energy-from-waste (EfW) facilities processing municipal solid waste (MSW). APC residues have been blended with glass-forming additives and treated using DC plasma technology to produce a high calcium alumino-silicate glass. This research has investigated the optimisation and properties of geopolymers prepared from this glass. Work has shown that high strength geopolymers can be formed and that the NaOH concentration of the activating solution significantly affects the properties. The broad particle size distribution of the APC residue glass used in these experiments results in a microstructure that contains unreacted glass particles included within a geopolymer binder phase. The high calcium content of APC residues may cause the formation of some amorphous calcium silicate hydrate (C-S-H) gel. A mix prepared with S/L=3.4, Si/Al=2.6 and [NaOH]=6M in the activating solution, produced high strength geopolymers with compressive strengths of approximately 130 MPa. This material had high density (2070 kg/m(3)) and low porosity. The research demonstrates for the first time that glass derived from DC plasma treatment of APC residues can be used to form high strength geopolymer-glass composites that have potential for use in a range of applications. PMID:20022170

  2. Modulation of APC Function and Anti-Tumor Immunity by Anti-Cancer Drugs

    PubMed Central

    Martin, Kea; Schreiner, Jens; Zippelius, Alfred

    2015-01-01

    Professional antigen-presenting cells (APCs), such as dendritic cells (DCs), are central to the initiation and regulation of anti-cancer immunity. However, in the immunosuppressive environment within a tumor APCs may antagonize anti-tumor immunity by inducing regulatory T cells (Tregs) or anergy of effector T cells due to lack of efficient costimulation. Hence, in an optimal setting, anti-cancer drugs have the power to reduce tumor size and thereby may induce the release of tumor antigens and, at the same time, modulate APC function toward efficient priming of antigen-specific effector T cells. Selected cytotoxic agents may revert APC dysfunction either by directly maturing DCs or through induction of immunogenic tumor cell death. Furthermore, specific cytotoxic agents may support adaptive immunity by selectively depleting regulatory subsets, such as Tregs or myeloid-derived suppressor cells. Perspectively, this will allow developing effective combination strategies with novel immunotherapies to exert complementary pressure on tumors via direct toxicity as well as immune activation. We, here, review our current knowledge on the capacity of anti-cancer drugs to modulate APC functions to promote durable anti-cancer immune responses. PMID:26483791

  3. Spliced DNA Sequences in the Paramecium Germline: Their Properties and Evolutionary Potential

    PubMed Central

    Catania, Francesco; McGrath, Casey L.; Doak, Thomas G.; Lynch, Michael

    2013-01-01

    Despite playing a crucial role in germline-soma differentiation, the evolutionary significance of developmentally regulated genome rearrangements (DRGRs) has received scant attention. An example of DRGR is DNA splicing, a process that removes segments of DNA interrupting genic and/or intergenic sequences. Perhaps, best known for shaping immune-system genes in vertebrates, DNA splicing plays a central role in the life of ciliated protozoa, where thousands of germline DNA segments are eliminated after sexual reproduction to regenerate a functional somatic genome. Here, we identify and chronicle the properties of 5,286 sequences that putatively undergo DNA splicing (i.e., internal eliminated sequences [IESs]) across the genomes of three closely related species of the ciliate Paramecium (P. tetraurelia, P. biaurelia, and P. sexaurelia). The study reveals that these putative IESs share several physical characteristics. Although our results are consistent with excision events being largely conserved between species, episodes of differential IES retention/excision occur, may have a recent origin, and frequently involve coding regions. Our findings indicate interconversion between somatic—often coding—DNA sequences and noncoding IESs, and provide insights into the role of DNA splicing in creating potentially functional genetic innovation. PMID:23737328

  4. Germline variants in the SEMA4A gene predispose to familial colorectal cancer type X

    PubMed Central

    Schulz, Eduard; Klampfl, Petra; Holzapfel, Stefanie; Janecke, Andreas R.; Ulz, Peter; Renner, Wilfried; Kashofer, Karl; Nojima, Satoshi; Leitner, Anita; Zebisch, Armin; Wölfler, Albert; Hofer, Sybille; Gerger, Armin; Lax, Sigurd; Beham-Schmid, Christine; Steinke, Verena; Heitzer, Ellen; Geigl, Jochen B.; Windpassinger, Christian; Hoefler, Gerald; Speicher, Michael R.; Richard Boland, C.; Kumanogoh, Atsushi; Sill, Heinz

    2014-01-01

    Familial colorectal cancer type X (FCCTX) is characterized by clinical features of hereditary non-polyposis colorectal cancer with a yet undefined genetic background. Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy. Compared with wild-type protein, SEMA4AV78M demonstrates significantly increased MAPK/Erk and PI3K/Akt signalling as well as cell cycle progression of SEMA4A-deficient HCT-116 colorectal cancer cells. In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser. This SNP is highly associated with the FCCTX phenotype exhibiting increased risk for colorectal cancer (OR 6.79, 95% CI 2.63 to 17.52). Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families. PMID:25307848

  5. The Wnt pathway limits BMP signaling outside of the germline stem cell niche in Drosophila ovaries.

    PubMed

    Mottier-Pavie, Violaine I; Palacios, Victor; Eliazer, Susan; Scoggin, Shane; Buszczak, Michael

    2016-09-01

    The mechanisms that modulate and limit the signaling output of adult stem cell niches remain poorly understood. To gain further insights into how these microenvironments are regulated in vivo, we performed a candidate gene screen designed to identify factors that restrict BMP signal production to the cap cells that comprise the germline stem cell (GSC) niche of Drosophila ovaries. Through these efforts, we found that disruption of Wnt4 and components of the canonical Wnt pathway results in a complex germ cell phenotype marked by an expansion of GSC-like cells, pre-cystoblasts and cystoblasts in young females. This phenotype correlates with an increase of decapentaplegic (dpp) mRNA levels within escort cells and varying levels of BMP responsiveness in the germline. Further genetic experiments show that Wnt4, which exhibits graded expression in somatic cells of germaria, activates the Wnt pathway in posteriorly positioned escort cells. The activation of the Wnt pathway appears to be limited by the BMP pathway itself, as loss of Mad in escort cells results in the expansion of Wnt pathway activation. Wnt pathway activity changes within germaria during the course of aging, coincident with changes in dpp production. These data suggest that mutual antagonism between the BMP and Wnt pathways in somatic cells helps to regulate germ cell differentiation. PMID:27364467

  6. Hereditary leiomyomatosis and renal cell cancer in families referred for fumarate hydratase germline mutation analysis.

    PubMed

    Smit, D L; Mensenkamp, A R; Badeloe, S; Breuning, M H; Simon, M E H; van Spaendonck, K Y; Aalfs, C M; Post, J G; Shanley, S; Krapels, I P C; Hoefsloot, L H; van Moorselaar, R J A; Starink, T M; Bayley, J-P; Frank, J; van Steensel, M A M; Menko, F H

    2011-01-01

    Heterozygous fumarate hydratase (FH) germline mutations cause hereditary leiomyomatosis and renal cell cancer (HLRCC), an autosomal dominant syndrome characterized by multiple cutaneous piloleiomyomas, uterine leiomyomas and papillary type 2 renal cancer. The main objective of our study was to evaluate clinical and genetic data from families suspected of HLRCC on a nationwide level. All families referred for FH mutation analysis in the Netherlands were assessed. We performed FH sequence analysis and multiplex ligation-dependent probe amplification. Families with similar FH mutations were examined for haplotype sharing. In 14 out of 33 families, we identified 11 different pathogenic FH germline mutations, including 4 novel mutations and 1 whole-gene deletion. Clinical data were available for 35 FH mutation carriers. Cutaneous leiomyomas were present in all FH mutation carriers older than 40 years of age. Eleven out of 21 female FH mutation carriers underwent surgical treatment for symptomatic uterine leiomyomas at an average of 35 years. Two FH mutation carriers had papillary type 2 renal cancer and Wilms' tumour, respectively. We evaluated the relevance of our findings for clinical practice and have proposed clinical diagnostic criteria, indications for FH mutation analysis and recommendations for management. PMID:20618355

  7. Identification of Two Novel HOXB13 Germline Mutations in Portuguese Prostate Cancer Patients

    PubMed Central

    Maia, Sofia; Cardoso, Marta; Pinto, Pedro; Pinheiro, Manuela; Santos, Catarina; Peixoto, Ana; Bento, Maria José; Oliveira, Jorge; Henrique, Rui; Jerónimo, Carmen; Teixeira, Manuel R.

    2015-01-01

    The HOXB13 germline variant G84E (rs138213197) was recently described in men of European descent, with the highest prevalence in Northern Europe. The G84E mutation has not been found in patients of African or Asian ancestry, which may carry other HOXB13 variants, indicating allelic heterogeneity depending on the population. In order to gain insight into the full scope of coding HOXB13 mutations in Portuguese prostate cancer patients, we decided to sequence the entire coding region of the HOXB13 gene in 462 early-onset or familial/hereditary cases. Additionally, we searched for somatic HOXB13 mutations in 178 prostate carcinomas to evaluate their prevalence in prostate carcinogenesis. Three different patients were found to carry in their germline DNA two novel missense variants, which were not identified in 132 control subjects. Both variants are predicted to be deleterious by different in silico tools. No somatic mutations were found. These findings further support the hypothesis that different rare HOXB13 mutations may be found in different ethnic groups. Detection of mutations predisposing to prostate cancer may require re-sequencing rather than genotyping, as appropriate to the population under investigation. PMID:26176944

  8. Diversity and Functional Consequences of Germline and Somatic PTPN11 Mutations in Human Disease

    PubMed Central

    Tartaglia, Marco; Martinelli, Simone; Stella, Lorenzo; Bocchinfuso, Gianfranco; Flex, Elisabetta; Cordeddu, Viviana; Zampino, Giuseppe; Burgt, Ineke van der; Palleschi, Antonio; Petrucci, Tamara C.; Sorcini, Mariella; Schoch, Claudia; Foà, Robin; Emanuel, Peter D.; Gelb, Bruce D.

    2006-01-01

    Germline mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome (NS) and the clinically related LEOPARD syndrome (LS), whereas somatic mutations in the same gene contribute to leukemogenesis. On the basis of our previously gathered genetic and biochemical data, we proposed a model that splits NS- and leukemia-associated PTPN11 mutations into two major classes of activating lesions with differential perturbing effects on development and hematopoiesis. To test this model, we investigated further the diversity of germline and somatic PTPN11 mutations, delineated the association of those mutations with disease, characterized biochemically a panel of mutant SHP-2 proteins recurring in NS, LS, and leukemia, and performed molecular dynamics simulations to determine the structural effects of selected mutations. Our results document a strict correlation between the identity of the lesion and disease and demonstrate that NS-causative mutations have less potency for promoting SHP-2 gain of function than do leukemia-associated ones. Furthermore, we show that the recurrent LS-causing Y279C and T468M amino acid substitutions engender loss of SHP-2 catalytic activity, identifying a previously unrecognized behavior for this class of missense PTPN11 mutations. PMID:16358218

  9. Prevalence of deleterious ATM germline mutations in gastric cancer patients

    PubMed Central

    He, Xu-Jun; Long, Ming; Yu, Sheng; Xia, Ying-Jie; Wei, Zhang; Xiong, Zikai; Jones, Sian; He, Yiping; Yan, Hai; Wang, Xiaoyue

    2015-01-01

    Besides CDH1, few hereditary gastric cancer predisposition genes have been previously reported. In this study, we discovered two germline ATM mutations (p.Y1203fs and p.N1223S) in a Chinese family with a history of gastric cancer by screening 83 cancer susceptibility genes. Using a published exome sequencing dataset, we found deleterious germline mutations of ATM in 2.7% of 335 gastric cancer patients of different ethnic origins. The frequency of deleterious ATM mutations in gastric cancer patients is significantly higher than that in general population (p=0.0000435), suggesting an association of ATM mutations with gastric cancer predisposition. We also observed biallelic inactivation of ATM in tumors of two gastric cancer patients. Further evaluation of ATM mutations in hereditary gastric cancer will facilitate genetic testing and risk assessment. PMID:26506520

  10. Nonrandom Germline Transmission of Mouse Spermatogonial Stem Cells.

    PubMed

    Kanatsu-Shinohara, Mito; Naoki, Honda; Shinohara, Takashi

    2016-08-01

    Genes are thought to be transmitted to offspring by random fertilization of a small number of oocytes with numerous spermatozoa. Here we analyzed the dynamics of male germline transmission by genetic marking and transplantation of spermatogonial stem cells (SSCs). We found that offspring deriving from a small number of specific SSCs appear within a limited time. Interestingly, the same SSC clones reappear later with an average functional lifespan of ∼124.4 days. Cyclic offspring production from SSCs was not caused by changes in SSC self-renewal activity because lineage-tracing analyses suggested that all SSCs actively proliferated. Selection appears to occur during the differentiating spermatogonia stage, when extensive apoptosis was observed. The pattern of germline transmission could be predicted using a mathematical model in which SSCs repeat cycles of transient spermatogenic burst and refractory periods. Thus, spermatogenesis is a regulated process whereby specific SSC clones are repeatedly recruited for fertilization with long-term cycles. PMID:27505415

  11. Prevalence of deleterious ATM germline mutations in gastric cancer patients.

    PubMed

    Huang, Dong-Sheng; Tao, Hou-Quan; He, Xu-Jun; Long, Ming; Yu, Sheng; Xia, Ying-Jie; Wei, Zhang; Xiong, Zikai; Jones, Sian; He, Yiping; Yan, Hai; Wang, Xiaoyue

    2015-12-01

    Besides CDH1, few hereditary gastric cancer predisposition genes have been previously reported. In this study, we discovered two germline ATM mutations (p.Y1203fs and p.N1223S) in a Chinese family with a history of gastric cancer by screening 83 cancer susceptibility genes. Using a published exome sequencing dataset, we found deleterious germline mutations of ATM in 2.7% of 335 gastric cancer patients of different ethnic origins. The frequency of deleterious ATM mutations in gastric cancer patients is significantly higher than that in general population (p=0.0000435), suggesting an association of ATM mutations with gastric cancer predisposition. We also observed biallelic inactivation of ATM in tumors of two gastric cancer patients. Further evaluation of ATM mutations in hereditary gastric cancer will facilitate genetic testing and risk assessment. PMID:26506520

  12. Stand Still, a Drosophila Gene Involved in the Female Germline for Proper Survival, Sex Determination and Differentiation

    PubMed Central

    Pennetta, G.; Pauli, D.

    1997-01-01

    We identified a new gene, stand still (stil), required in the female germline for proper survival, sex determination and differentiation. Three strong loss-of-function alleles were isolated. The strongest phenotype exhibited by ovaries dissected from adult females is the complete absence of germ cells. In other ovaries, the few surviving germ cells frequently show a morphology typical of primary spermatocytes. still is not required either for fly viability or for male germline development. The gene was cloned and found to encode a novel protein. still is strongly expressed in the female germ cells. Using P[stil(+)] transgenes, we show that stil and a closely localized gene are involved in the modification of the ovarian phenotypes of the dominant alleles of ovo caused by heterozygosity of region 49 A-D. The similarity of the mutant phenotypes of stil to that of otu and ovo suggests that the three genes function in a common or in parallel pathways necessary in the female germline for its survival, sex determination and differentiation. PMID:9093851

  13. Improved Detection of Germline Mutations in Korean VHL Patients by Multiple Ligation-dependent Probe Amplification Analysis

    PubMed Central

    Cho, Hyun-Jung; Ki, Chang-Seok

    2009-01-01

    von Hippel-Lindau (VHL) disease is an autosomal dominant inherited tumor syndrome characterized by the development of tumors in the eye, brain, spinal cord, inner ear, adrenal gland, pancreas, kidney, and epididymis, associated with germline mutations in the VHL gene. We used sequentially sequencing method and multiple ligation-dependent probe amplification (MLPA) analysis and detected germline mutations in the VHL in 15/15 (100%) of VHL patients fulfilling the clinical criteria. Of the 15 distinct mutations detected, large deletions were detected in 5/15 (33.3%) patients, including 4/15 (26.7%) partial deletions and 1/15 (6.6%) deletion of the entire VHL gene by MLPA and the remainder were point mutations detected by sequencing method, of which five mutations were novel. Using MLPA analysis, we detected large deletions including both partial deletions and complete gene deletion, which has not been reported in Korean VHL patients. In conclusion, sequential application of sequencing method and MLPA analysis might make possible to identify germline mutations in most patients with VHL. PMID:19270817

  14. Germline Heterozygous Variants in SEC23B Are Associated with Cowden Syndrome and Enriched in Apparently Sporadic Thyroid Cancer

    PubMed Central

    Yehia, Lamis; Niazi, Farshad; Ni, Ying; Ngeow, Joanne; Sankunny, Madhav; Liu, Zhigang; Wei, Wei; Mester, Jessica L.; Keri, Ruth A.; Zhang, Bin; Eng, Charis

    2015-01-01

    Cancer-predisposing genes associated with inherited cancer syndromes help explain mechanisms of sporadic carcinogenesis and often inform normal development. Cowden syndrome (CS) is an autosomal-dominant disorder characterized by high lifetime risks of epithelial cancers, such that ∼50% of affected individuals are wild-type for known cancer-predisposing genes. Using whole-exome and Sanger sequencing of a multi-generation CS family affected by thyroid and other cancers, we identified a pathogenic missense heterozygous SEC23B variant (c.1781T>G [p.Val594Gly]) that segregates with the phenotype. We also found germline heterozygous SEC23B variants in 3/96 (3%) unrelated mutation-negative CS probands with thyroid cancer and in The Cancer Genome Atlas (TCGA), representing apparently sporadic cancers. We note that the TCGA thyroid cancer dataset is enriched with unique germline deleterious SEC23B variants associated with a significantly younger age of onset. SEC23B encodes Sec23 homolog B (S. cerevisiae), a component of coat protein complex II (COPII), which transports proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Interestingly, germline homozygous or compound-heterozygous SEC23B mutations cause an unrelated disorder, congenital dyserythropoietic anemia type II, and SEC23B-deficient mice suffer from secretory organ degeneration due to ER-stress-associated apoptosis. By characterizing the p.Val594Gly variant in a normal thyroid cell line, we show that it is a functional alteration that results in ER-stress-mediated cell-colony formation and survival, growth, and invasion, which reflect aspects of a cancer phenotype. Our findings suggest a different role for SEC23B, whereby germline heterozygous variants associate with cancer predisposition potentially mediated by ER stress “addiction.” PMID:26522472

  15. Ability to develop broadly neutralizing HIV-1 antibodies is not restricted by the germline Ig gene repertoire.

    PubMed

    Scheepers, Cathrine; Shrestha, Ram K; Lambson, Bronwen E; Jackson, Katherine J L; Wright, Imogen A; Naicker, Dshanta; Goosen, Mark; Berrie, Leigh; Ismail, Arshad; Garrett, Nigel; Abdool Karim, Quarraisha; Abdool Karim, Salim S; Moore, Penny L; Travers, Simon A; Morris, Lynn

    2015-05-01

    The human Ig repertoire is vast, producing billions of unique Abs from a limited number of germline Ig genes. The IgH V region (IGHV) is central to Ag binding and consists of 48 functional genes. In this study, we analyzed whether HIV-1-infected individuals who develop broadly neutralizing Abs show a distinctive germline IGHV profile. Using both 454 and Illumina technologies, we sequenced the IGHV repertoire of 28 HIV-infected South African women from the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 002 and 004 cohorts, 13 of whom developed broadly neutralizing Abs. Of the 259 IGHV alleles identified in this study, approximately half were not found in the International Immunogenetics Database (IMGT). This included 85 entirely novel alleles and 38 alleles that matched rearranged sequences in non-IMGT databases. Analysis of the rearranged H chain V region genes of mAbs isolated from seven of these women, as well as previously isolated broadly neutralizing Abs from other donors, provided evidence that at least eight novel or non-IMGT alleles contributed to functional Abs. Importantly, we found that, despite a wide range in the number of IGHV alleles in each individual, including alleles used by known broadly neutralizing Abs, there were no significant differences in germline IGHV repertoires between individuals who do and do not develop broadly neutralizing Abs. This study reports novel IGHV repertoires and highlights the importance of a fully comprehensive Ig database for germline gene usage prediction. Furthermore, these data suggest a lack of genetic bias in broadly neutralizing Ab development in HIV-1 infection, with positive implications for HIV vaccine design. PMID:25825450

  16. Ability to develop broadly neutralizing HIV-1 antibodies is not restricted by the germline immunoglobulin gene repertoire1

    PubMed Central

    Scheepers, Cathrine; Shrestha, Ram K.; Lambson, Bronwen E.; Jackson, Katherine J. L.; Wright, Imogen A.; Naicker, Dshanta; Goosen, Mark; Berrie, Leigh; Ismail, Arshad; Garrett, Nigel; Karim, Quarraisha Abdool; Karim, Salim S. Abdool; Moore, Penny L.; Travers, Simon A.; Morris, Lynn

    2015-01-01

    The human immunoglobulin repertoire is vast, producing billions of unique antibodies from a limited number of germline immunoglobulin genes. The immunoglobulin heavy chain variable region (IGHV) is central to antigen binding and is comprised of 48 functional genes. Here we analyzed whether HIV-1 infected individuals who develop broadly neutralizing antibodies show a distinctive germline IGHV profile. Using both 454 and Illumina technologies we sequenced the IGHV repertoire of 28 HIV-infected South African women from the Center for the AIDS Programme of Research in South African (CAPRISA) 002 and 004 cohorts, 13 of whom developed broadly neutralizing antibodies. Of the 259 IGHV alleles identified in this study, approximately half were not found in the International Immunogenetics Database (IMGT). This included 85 entirely novel alleles and 38 alleles that matched rearranged sequences in non-IMGT databases. Analysis of the rearranged H chain V region genes of monoclonal antibodies isolated from 7 of the CAPRISA women and previously isolated broadly neutralizing antibodies from other donors provided evidence that at least 8 novel or non-IMGT alleles contributed to functional antibodies. Importantly, we found that despite a wide range in the number of IGHV alleles in each individual, including alleles used by known broadly neutralizing antibodies, there were no significant differences in germline IGHV repertoires between individuals who do and do not develop broadly neutralizing antibodies. This study reports novel IGHV repertoires and highlights the importance of a fully comprehensive immunoglobulin database for germline gene usage prediction. Furthermore, these data suggest a lack of genetic bias in broadly neutralizing antibody development in HIV-1 infection, with implications for HIV vaccine design. PMID:25825450

  17. Production of Interspecific Germline Chimeras via Embryo Replacement.

    PubMed

    Choi, Hee Jung; Lee, Hyung Chul; Kang, Kyung Soo; Lee, Hyo Gun; Ono, Tamao; Nagai, Hiroki; Sheng, Guojun; Han, Jae Yong

    2015-08-01

    In avian species, primordial germ cells (PGCs) use the vascular system to reach their destination, the genital ridge. Because of this unique migratory route of avian germ cells, germline chimera production can be achieved via germ cell transfer into a blood vessel. This study was performed to establish an alternative germ cell-transfer system for producing germline chimeras by replacing an original host embryo with a donor embryo, while retaining the host extraembryonic tissue and yolk, before circulation. First, to test the migratory capacity of PGCs after embryo replacement, Korean Oge (KO) chick embryos were used to replace GFP transgenic chick embryos. Four days after replacement, GFP-positive cells were detected in the replaced KO embryonic gonads, and genomic DNA PCR analysis with the embryonic gonads demonstrated the presence of the GFP transgene. To produce an interspecific germline chimera, the original chick embryo proper was replaced with a quail embryo onto the chick yolk. To detect the gonadal PGCs in the 5.5-day-old embryonic gonads, immunohistochemistry was performed with monoclonal antibodies specific to either quail or chick PGCs, i.e., QCR1 and anti-stage-specific embryonic antigen-1 (SSEA-1), respectively. Both the QCR1-positive and SSEA-1-positive cells were detected in the gonads of replaced quail embryos. Forty percent of the PGC population in the quail embryos was occupied by chick extraembryonically derived PGCs. In conclusion, replacement of an embryo onto the host yolk before circulation can be applied to produce interspecies germline chimeras, and this germ cell-transfer technology is potentially applicable for reproduction of wild or endangered bird species. PMID:26063873

  18. Germline Variants and Advanced Colorectal Adenomas: Adenoma Prevention with Celecoxib Trial Genomewide Association Study

    PubMed Central

    Wang, Jiping; Carvajal-Carmona, Luis G.; Chu, Jen-Hwa; Zauber, Ann G.; Kubo, Michikai; Matsuda, Koichi; Dunlop, Malcolm; Houlston, Richard S.; Sieber, Oliver; Lipton, Lara; Gibbs, Peter; Martin, Nicholas G.; Montgomery, Grant W.; Young, Joanne; Baird, Paul N.; Ratain, Mark J.; Nakamura, Yusuke; Weiss, Scott T.; Tomlinson, Ian; Bertagnolli, Monica M.

    2014-01-01

    Purpose Identification of single nucleotide polymorphisms (SNPs) associated with development of advanced colorectal adenomas. Experimental Design Discovery Phase: 1,406 Caucasian patients (139 advanced adenoma cases and 1,267 controls) from the Adenoma Prevention with Celecoxib (APC) trial were included in a genome-wide association study (GWAS) to identify variants associated with post-polypectomy disease recurrence. Genome-wide significance was defined as false discovery rate < 0.05, unadjusted p=7.4×10−7. Validation Phase: Results were further evaluated using 4,175 familial colorectal adenoma or CRC cases and 5,036 controls from patients of European ancestry (COloRectal Gene Identification consortium, Scotland, Australia and VQ58). Results Our study identified eight SNPs associated with advanced adenoma risk in the APC trial (rs2837156, rs7278863, rs2837237, rs2837241, rs2837254, rs741864 at 21q22.2, and rs1381392 and rs17651822 at 3p24.1, at p<10–7 level with odds ratio – OR>2). Five variants in strong pairwise linkage disequilbrium (rs7278863, rs2837237, rs741864, rs741864 and rs2837241, r2=0.8–1) are in or near the coding region for the tight junction adhesion protein, IGSF5. An additional variant associated with advanced adenomas, rs1535989 (minor allele frequency 0.11; OR 2.09; 95% confidence interval 1.50–2.91), also predicted CRC development in a validation analysis (p=0.019) using a series of adenoma cases or CRC (CORGI study) and 3 sets of CRC cases and controls (Scotland, VQ58 and Australia, N=9,211). Conclusions Our results suggest that common polymorphisms contribute to the risk of developing advanced adenomas and might also contribute to the risk of developing CRC. The variant at rs1535989 may identify patients whose risk for neoplasia warrants increased colonoscopic surveillance. PMID:24084763

  19. The CDK-APC/C Oscillator Predominantly Entrains Periodic Cell-Cycle Transcription.

    PubMed

    Rahi, Sahand Jamal; Pecani, Kresti; Ondracka, Andrej; Oikonomou, Catherine; Cross, Frederick R

    2016-04-01

    Throughout cell-cycle progression, the expression of multiple transcripts oscillate, and whether these are under the centralized control of the CDK-APC/C proteins or can be driven by a de-centralized transcription factor (TF) cascade is a fundamental question for understanding cell-cycle regulation. In budding yeast, we find that the transcription of nearly all genes, as assessed by RNA-seq or fluorescence microscopy in single cells, is dictated by CDK-APC/C. Three exceptional genes are transcribed in a pulsatile pattern in a variety of CDK-APC/C arrests. Pursuing one of these transcripts, the SIC1 inhibitor of B-type cyclins, we use a combination of mathematical modeling and experimentation to provide evidence that, counter-intuitively, Sic1 provides a failsafe mechanism promoting nuclear division when levels of mitotic cyclins are low. PMID:27058667

  20. Germline ETV6 Mutations Confer Susceptibility to Acute Lymphoblastic Leukemia and Thrombocytopenia

    PubMed Central

    Jacobs, Lauren; Maria, Ann; Villano, Danylo; Gaddam, Pragna; Wu, Gang; McGee, Rose B.; Quinn, Emily; Inaba, Hiroto; Hartford, Christine; Pui, Ching-hon; Pappo, Alberto; Edmonson, Michael; Zhang, Michael Y.; Stepensky, Polina; Steinherz, Peter; Schrader, Kasmintan; Lincoln, Anne; Bussel, James; Lipkin, Steve M.; Goldgur, Yehuda; Harit, Mira; Stadler, Zsofia K.; Mullighan, Charles; Weintraub, Michael; Shimamura, Akiko; Zhang, Jinghui; Downing, James R.; Nichols, Kim E.; Offit, Kenneth

    2015-01-01

    Somatic mutations affecting ETV6 often occur in acute lymphoblastic leukemia (ALL), the most common childhood malignancy. The genetic factors that predispose to ALL remain poorly understood. Here we identify a novel germline ETV6 p. L349P mutation in a kindred affected by thrombocytopenia and ALL. A second ETV6 p. N385fs mutation was identified in an unrelated kindred characterized by thrombocytopenia, ALL and secondary myelodysplasia/acute myeloid leukemia. Leukemic cells from the proband in the second kindred showed deletion of wild type ETV6 with retention of the ETV6 p. N385fs. Enforced expression of the ETV6 mutants revealed normal transcript and protein levels, but impaired nuclear localization. Accordingly, these mutants exhibited significantly reduced ability to regulate the transcription of ETV6 target genes. Our findings highlight a novel role for ETV6 in leukemia predisposition. PMID:26102509

  1. Mutation rates and the evolution of germline structure

    PubMed Central

    2016-01-01

    Genome sequencing studies of de novo mutations in humans have revealed surprising incongruities in our understanding of human germline mutation. In particular, the mutation rate observed in modern humans is substantially lower than that estimated from calibration against the fossil record, and the paternal age effect in mutations transmitted to offspring is much weaker than expected from our long-standing model of spermatogenesis. I consider possible explanations for these discrepancies, including evolutionary changes in life-history parameters such as generation time and the age of puberty, a possible contribution from undetected post-zygotic mutations early in embryo development, and changes in cellular mutation processes at different stages of the germline. I suggest a revised model of stem-cell state transitions during spermatogenesis, in which ‘dark’ gonial stem cells play a more active role than hitherto envisaged, with a long cycle time undetected in experimental observations. More generally, I argue that the mutation rate and its evolution depend intimately on the structure of the germline in humans and other primates. This article is part of the themed issue ‘Dating species divergences using rocks and clocks'. PMID:27325834

  2. Mutation rates and the evolution of germline structure.

    PubMed

    Scally, Aylwyn

    2016-07-19

    Genome sequencing studies of de novo mutations in humans have revealed surprising incongruities in our understanding of human germline mutation. In particular, the mutation rate observed in modern humans is substantially lower than that estimated from calibration against the fossil record, and the paternal age effect in mutations transmitted to offspring is much weaker than expected from our long-standing model of spermatogenesis. I consider possible explanations for these discrepancies, including evolutionary changes in life-history parameters such as generation time and the age of puberty, a possible contribution from undetected post-zygotic mutations early in embryo development, and changes in cellular mutation processes at different stages of the germline. I suggest a revised model of stem-cell state transitions during spermatogenesis, in which 'dark' gonial stem cells play a more active role than hitherto envisaged, with a long cycle time undetected in experimental observations. More generally, I argue that the mutation rate and its evolution depend intimately on the structure of the germline in humans and other primates.This article is part of the themed issue 'Dating species divergences using rocks and clocks'. PMID:27325834

  3. Timing, rates and spectra of human germline mutation.

    PubMed

    Rahbari, Raheleh; Wuster, Arthur; Lindsay, Sarah J; Hardwick, Robert J; Alexandrov, Ludmil B; Al Turki, Saeed; Dominiczak, Anna; Morris, Andrew; Porteous, David; Smith, Blair; Stratton, Michael R; Hurles, Matthew E

    2016-02-01

    Germline mutations are a driving force behind genome evolution and genetic disease. We investigated genome-wide mutation rates and spectra in multi-sibling families. The mutation rate increased with paternal age in all families, but the number of additional mutations per year differed by more than twofold between families. Meta-analysis of 6,570 mutations showed that germline methylation influences mutation rates. In contrast to somatic mutations, we found remarkable consistency in germline mutation spectra between the sexes and at different paternal ages. In parental germ line, 3.8% of mutations were mosaic, resulting in 1.3% of mutations being shared by siblings. The number of these shared mutations varied significantly between families. Our data suggest that the mutation rate per cell division is higher during both early embryogenesis and differentiation of primordial germ cells but is reduced substantially during post-pubertal spermatogenesis. These findings have important consequences for the recurrence risks of disorders caused by de novo mutations. PMID:26656846

  4. Germline BAP1 mutations misreported as somatic based on tumor-only testing.

    PubMed

    Abdel-Rahman, Mohamed H; Rai, Karan; Pilarski, Robert; Davidorf, Frederick H; Cebulla, Colleen M

    2016-04-01

    We present three unrelated patients with germline mutations in BAP1 misreported as somatic mutations. All had strong family histories of cancer. One of these patients presented with an invasive breast cancer with the tumor tissue showing partial loss of the mutant rather than the wild type allele, suggesting that the germline BAP1 mutation didn't contribute to breast cancer development in this patient. This data highlights the importance of sequencing matching germline and tumor DNA for proper assessment of somatic versus germline mutation status. In patients with somatic mutations reported from laboratories carrying out tumor-only genomic testing, the possibility that a variant may be a germline mutation should be considered, especially if the personal and/or family history suggests hereditary cancer predisposition. Since tumor-only testing can reveal germline mutations, ethical issues for patients being tested should be considered including proper consent and genetic counseling. PMID:26748926

  5. Characterization of elastic-plastic properties of AS4/APC-2 thermoplastic composite

    NASA Technical Reports Server (NTRS)

    Sun, C. T.; Yoon, K. J.

    1988-01-01

    Elastic and inelastic properties of AS4/APC-2 composites were characterized with respect to temperature variation by using a one-parameter orthotropic plasticity model and a one parameter failure criterion. Simple uniaxial off-axis tension tests were performed on coupon specimens of unidirectional AS4/APC-2 thermoplastic composite at various temperatures. To avoid the complication caused by the extension-shear coupling effect in off-axis testing, new tabs were designed and used on the test specimens. The experimental results showed that the nonlinear behavior of constitutive relations and the failure strengths can be characterized quite well using the one parameter plasticity model and the failure criterion, respectively.

  6. Orthotropic elasto-plastic behavior of AS4/APC-2 thermoplastic composite in compression

    NASA Technical Reports Server (NTRS)

    Sun, C. T.; Rui, Y.

    1989-01-01

    Uniaxial compression tests were performed on off-axis coupon specimens of unidirectional AS4/APC-2 thermoplastic composite at various temperatures. The elasto-plastic and strength properties of AS4/APC-2 composite were characterized with respect to temperature variation by using a one-parameter orthotropic plasticity model and a one-parameter failure criterion. Experimental results show that the orthotropic plastic behavior can be characterized quite well using the plasticity model, and the matrix-dominant compressive strengths can be predicted very accurately by the one-parameter failure criterion.

  7. Germline and somatic mutations in meningiomas.

    PubMed

    Smith, Miriam J

    2015-04-01

    Meningiomas arise from the arachnoid layer of the meninges that surround the brain and spine. They account for over one third of all primary central nervous system tumors in adults and confer a significant risk of location-dependent morbidity due to compression or displacement. A significant increase in risk of meningiomas is associated with neurofibromatosis type 2 (NF2) disease through mutation of the NF2 gene. In addition, approximately 5% of individuals with schwannomatosis disease develop meningiomas, through mutation of the SWI/SNF chromatin remodeling complex subunit, SMARCB1. Recently, a second SWI/SNF complex subunit, SMARCE1, was identified as a cause of clear cell meningiomas, indicating a wider role for this complex in meningioma disease. The sonic hedgehog (SHH)-GLI1 signaling pathway gene, SUFU, has also been identified as the cause of hereditary multiple meningiomas in a large Finnish family. The recent identification of somatic mutations in components of the SHH-GLI1 and AKT1-MTOR signaling pathways indicates the potential for cross talk of these pathways in the development of meningiomas. This review describes the known meningioma predisposition genes and their links to the recently identified somatic mutations. PMID:25857641

  8. An APC/C-Cdh1 Biosensor Reveals the Dynamics of Cdh1 Inactivation at the G1/S Transition

    PubMed Central

    Ondracka, Andrej; Robbins, Jonathan A.; Cross, Frederick R.

    2016-01-01

    B-type cyclin-dependent kinase activity must be turned off for mitotic exit and G1 stabilization. B-type cyclin degradation is mediated by the anaphase-promoting complex/cyclosome (APC/C); during and after mitotic exit, APC/C is dependent on Cdh1. Cdh1 is in turn phosphorylated and inactivated by cyclin-CDK at the Start transition of the new cell cycle. We developed a biosensor to assess the cell cycle dynamics of APC/C-Cdh1. Nuclear exit of the G1 transcriptional repressor Whi5 is a known marker of Start; APC/C-Cdh1 is inactivated 12 min after Whi5 nuclear exit with little measurable cell-to-cell timing variability. Multiple phosphorylation sites on Cdh1 act in a redundant manner to repress its activity. Reducing the number of phosphorylation sites on Cdh1 can to some extent be tolerated for cell viability, but it increases variability in timing of APC/C-Cdh1 inactivation. Mutants with minimal subsets of phosphorylation sites required for viability exhibit striking stochasticity in multiple responses including budding, nuclear division, and APC/C-Cdh1 activity itself. Multiple cyclin-CDK complexes, as well as the stoichiometric inhibitor Acm1, contribute to APC/C-Cdh1 inactivation; this redundant control is likely to promote rapid and reliable APC/C-Cdh1 inactivation immediately following the Start transition. PMID:27410035

  9. The Controversy, Challenges, and Potential Benefits of Putative Female Germline Stem Cells Research in Mammals

    PubMed Central

    Pan, Zezheng; Sun, Mengli; Liang, Xia; Li, Jia; Zhou, Fangyue; Zhong, Zhisheng; Zheng, Yuehui

    2016-01-01

    The conventional view is that female mammals lose their ability to generate new germ cells after birth. However, in recent years, researchers have successfully isolated and cultured a type of germ cell from postnatal ovaries in a variety of mammalian species that have the abilities of self-proliferation and differentiation into oocytes, and this finding indicates that putative germline stem cells maybe exist in the postnatal mammalian ovaries. Herein, we review the research history and discovery of putative female germline stem cells, the concept that putative germline stem cells exist in the postnatal mammalian ovary, and the research progress, challenge, and application of putative germline stem cells in recent years. PMID:26788065

  10. The Controversy, Challenges, and Potential Benefits of Putative Female Germline Stem Cells Research in Mammals.

    PubMed

    Pan, Zezheng; Sun, Mengli; Liang, Xia; Li, Jia; Zhou, Fangyue; Zhong, Zhisheng; Zheng, Yuehui

    2016-01-01

    The conventional view is that female mammals lose their ability to generate new germ cells after birth. However, in recent years, researchers have successfully isolated and cultured a type of germ cell from postnatal ovaries in a variety of mammalian species that have the abilities of self-proliferation and differentiation into oocytes, and this finding indicates that putative germline stem cells maybe exist in the postnatal mammalian ovaries. Herein, we review the research history and discovery of putative female germline stem cells, the concept that putative germline stem cells exist in the postnatal mammalian ovary, and the research progress, challenge, and application of putative germline stem cells in recent years. PMID:26788065

  11. Germline PARP4 mutations in patients with primary thyroid and breast cancers.

    PubMed

    Ikeda, Yuji; Kiyotani, Kazuma; Yew, Poh Yin; Kato, Taigo; Tamura, Kenji; Yap, Kai Lee; Nielsen, Sarah M; Mester, Jessica L; Eng, Charis; Nakamura, Yusuke; Grogan, Raymon H

    2016-03-01

    Germline mutations in the PTEN gene, which cause Cowden syndrome, are known to be one of the genetic factors for primary thyroid and breast cancers; however, PTEN mutations are found in only a small subset of research participants with non-syndrome breast and thyroid cancers. In this study, we aimed to identify germline variants that may be related to genetic risk of primary thyroid and breast cancers. Genomic DNAs extracted from peripheral blood of 14 PTEN WT female research participants with primary thyroid and breast cancers were analyzed by whole-exome sequencing. Gene-based case-control association analysis using the information of 406 Europeans obtained from the 1000 Genomes Project database identified 34 genes possibly associated with the phenotype with P < 1.0 × 10(-3). Among them, rare variants in the PARP4 gene were detected at significant high frequency (odds ratio = 5.2; P = 1.0 × 10(-5)). The variants, G496V and T1170I, were found in six of the 14 study participants (43%) while their frequencies were only 0.5% in controls. Functional analysis using HCC1143 cell line showed that knockdown of PARP4 with siRNA significantly enhanced the cell proliferation, compared with the cells transfected with siControl (P = 0.02). Kaplan-Meier analysis using Gene Expression Omnibus (GEO), European Genome-phenome Archive (EGA) and The Cancer Genome Atlas (TCGA) datasets showed poor relapse-free survival (P < 0.001, Hazard ratio 1.27) and overall survival (P = 0.006, Hazard ratio 1.41) in a PARP4 low-expression group, suggesting that PARP4 may function as a tumor suppressor. In conclusion, we identified PARP4 as a possible susceptibility gene of primary thyroid and breast cancer. PMID:26699384

  12. An AT-rich region in the APC gene may cause misinterpretation of familial adenomatous polyposis molecular screening.

    PubMed

    Palmirotta, Raffaele; De Marchis, Maria Laura; Ludovici, Giorgia; Leone, Barbara; Valente, Maria Giovanna; Alessandroni, Jhessica; Spila, Antonella; Della-Morte, David; Guadagni, Fiorella

    2012-05-01

    Familial adenomatous polyposis (FAP) is an autosomal-dominant condition mainly due to a mutation of the adenomatous polyposis coli (APC) gene. The present study reports evidence of a technical issue occurring during the mutational analysis of APC exon 4. Genetic conventional direct sequence analysis of a repetitive AT-rich region in the splice acceptor site of APC intron 3 could be misinterpreted as a pathogenetic frameshift result. However, this potential bias may be bypassed adopting a method for random mutagenesis of DNA based on the use of a triphosphate nucleoside analogues mixture. Using this method as a second-level analysis, we also demonstrated the nonpathogenic nature of the variant in the poly A trait in APC exon 4 region (c.423-4delA) that do not result in aberrant splicing of APC exons 3-4; conversely, we did not find a previously reported T deletion/insertion polymorphism. PMID:22447671

  13. Metals accumulations during thermal processing of sewage sludge - characterization of bottom ash and air pollution control (APC) residues

    NASA Astrophysics Data System (ADS)

    Kasina, Monika; Kowalski, Piotr R.; Michalik, Marek

    2016-04-01

    Due to increasing mass of sewage sludge, problems in its management have appeared. Over years sewage sludge was landfilled, however due to EU directives concerning environmental issues this option is no longer possible. This type of material is considered hazardous due to highly concentrated metals and harmful elements, toxic organic substances and biological components (e.g. parasites, microbes). Currently in Europe, incineration is considered to be the most reasonable method for sewage sludge treatment. As a result of sludge incineration significant amount of energy is recovered due to high calorific value of sewage sludge but bottom ash and APC residues are being produced. In this study we show the preliminary results of chemical and mineral analyses of both bottom ash and APC residues produced in fluidized bed boiler in sewage sludge incineration plant in Poland, with a special emphasis on metals which, as a part of incombustible fraction can accumulate in the residual materials after thermal processing. The bottom ash was a SiO2-P2O5-Fe2O3-CaO-Al2O3 dominated material. Main mineral phases identified in X-ray diffraction patterns were: quartz, feldspar, hematite, and phosphates (apatite and scholzite). The bottom ash was characterized by high content of Zn - 4472 mg kg‑1, Cu - 665.5 mg kg‑1, Pb - 138 mg kg‑1, Ni - 119.5 mg kg‑1, and interestingly high content of Au - 0.858 mg kg‑1 The APC residues composition was dominated by soluble phases which represent more than 90% of the material. The XRD patterns indicated thenardite, halite, anhydrite, calcite and apatite as main mineral phases. The removal of soluble phases by dissolution in deionised water caused a significant mass reduction (ca. 3% of material remained on the filters). Calcite, apatite and quartz were main identified phases. The content of metals in insoluble material is relatively high: Zn - 6326 mg kg‑1, Pb - 514.3 mg kg‑1, Cu - 476.6 mg kg‑1, Ni - 43.3 mg kg‑1. The content of Cd

  14. APC/C-Cdh1 coordinates neurogenesis and cortical size during development

    NASA Astrophysics Data System (ADS)

    Delgado-Esteban, Maria; García-Higuera, Irene; Maestre, Carolina; Moreno, Sergio; Almeida, Angeles

    2013-12-01

    The morphology of the adult brain is the result of a delicate balance between neural progenitor proliferation and the initiation of neurogenesis in the embryonic period. Here we assessed whether the anaphase-promoting complex/cyclosome (APC/C) cofactor, Cdh1—which regulates mitosis exit and G1-phase length in dividing cells—regulates neurogenesis in vivo. We use an embryo-restricted Cdh1 knockout mouse model and show that functional APC/C-Cdh1 ubiquitin ligase activity is required for both terminal differentiation of cortical neurons in vitro and neurogenesis in vivo. Further, genetic ablation of Cdh1 impairs the ability of APC/C to promote neurogenesis by delaying the exit of the progenitor cells from the cell cycle. This causes replicative stress and p53-mediated apoptotic death resulting in decreased number of cortical neurons and cortex size. These results demonstrate that APC/C-Cdh1 coordinates cortical neurogenesis and size, thus posing Cdh1 in the molecular pathogenesis of congenital neurodevelopmental disorders, such as microcephaly.

  15. The future of APCs: a look at Medicare's 2005 changes and industry trends.

    PubMed

    Leary, Renee S; Farley, Dean

    2005-03-01

    As the healthcare industry continues to struggle with an ever-evolving APC-based hospital outpatient PPS, it's important to be aware of some of the latest changes affecting providers and to recognize areas that may present particular challenges for private payers. PMID:17233243

  16. Control of APC/C-dependent ubiquitin chain elongation by reversible phosphorylation

    PubMed Central

    Craney, Allison; Kelly, Aileen; Jia, Luying; Fedrigo, Indro; Yu, Hongtao; Rape, Michael

    2016-01-01

    Most metazoan E3 ligases contain a signature RING domain that promotes the transfer of ubiquitin from the active site of E2 conjugating enzymes to lysine residues in substrates. Although these RING-E3s depend on E2 enzymes for catalysis, how they turn on their E2s at the right time and place remains poorly understood. Here we report a phosphorylation-dependent mechanism that ensures timely activation of the E2 Ube2S by its RING-E3, the anaphase-promoting complex (APC/C); while phosphorylation of a specific serine residue in the APC/C coactivator Cdc20 prevents delivery of Ube2S to the APC/C, removal of this mark by PP2AB56 allows Ube2S to bind the APC/C and catalyze ubiquitin chain elongation. PP2AB56 also stabilizes kinetochore–microtubule attachments to shut off the spindle checkpoint, suggesting that cells regulate the E2–E3 interplay to coordinate ubiquitination with critical events during cell division. PMID:26811472

  17. Control of APC/C-dependent ubiquitin chain elongation by reversible phosphorylation.

    PubMed

    Craney, Allison; Kelly, Aileen; Jia, Luying; Fedrigo, Indro; Yu, Hongtao; Rape, Michael

    2016-02-01

    Most metazoan E3 ligases contain a signature RING domain that promotes the transfer of ubiquitin from the active site of E2 conjugating enzymes to lysine residues in substrates. Although these RING-E3s depend on E2 enzymes for catalysis, how they turn on their E2s at the right time and place remains poorly understood. Here we report a phosphorylation-dependent mechanism that ensures timely activation of the E2 Ube2S by its RING-E3, the anaphase-promoting complex (APC/C); while phosphorylation of a specific serine residue in the APC/C coactivator Cdc20 prevents delivery of Ube2S to the APC/C, removal of this mark by PP2A(B56) allows Ube2S to bind the APC/C and catalyze ubiquitin chain elongation. PP2A(B56) also stabilizes kinetochore-microtubule attachments to shut off the spindle checkpoint, suggesting that cells regulate the E2-E3 interplay to coordinate ubiquitination with critical events during cell division. PMID:26811472

  18. Application of accelerated carbonation on MSW combustion APC residues for metal immobilization and CO2 sequestration.

    PubMed

    Cappai, G; Cara, S; Muntoni, A; Piredda, M

    2012-03-15

    The present study focuses on the application of an aqueous phase accelerated carbonation treatment on air pollution control (APC) residues from municipal solid waste combustion, aimed at assessing its influence on the environmental behaviour of the residue under concern, as well as the potential of the process in terms of sequestration of the CO2. APC residues are considered hazardous waste and must be treated before final disposal in order to achieve the immobilization/mobilization of critical contaminants such as heavy metals as well as mobilization of soluble salts. The treatment applied proved to be effective in reducing the mobility of Pb, Zn, Cr, Cu and Mo, the optimum final pH for the carbonated APC residues being in a range of 10-10.5, whilst a mobilization effect was noticed for Sb and no effect was assessed for chlorides. The effect of carbonation treatment on the contaminant release was further evaluated by means of a sequential extraction procedure, indicating that the distribution of contaminants on water soluble, exchangeable and carbonate fraction was modified after treatment. The CO2 sequestration potential assessed for the APC residues showed that the carbonation technology could be a technically viable option in order to reduce emissions from WtE plants. PMID:21601357

  19. PIK3CA and APC Mutations are Synergistic in the Development of Intestinal Cancers

    PubMed Central

    Deming, Dustin A.; Leystra, Alyssa A.; Nettekoven, Laura; Sievers, Chelsea; Miller, Devon; Middlebrooks, Malisa; Clipson, Linda; Albrecht, Dawn; Bacher, Jeff; Washington, Mary Kay; Weichert, Jamey; Halberg, Richard B.

    2013-01-01

    Human colorectal cancers are known to possess multiple mutations, though how these mutations interact in tumor development and progression has not been fully investigated. We have previously described the FCPIK3ca* murine colon cancer model which expresses a constitutively activated phosphoinositide-3 kinase (PI3K) in the intestinal epithelium. The expression of this dominantly active form of PI3K results in hyperplasia and invasive mucinous adenocarcinomas. These cancers form via a non-canonical mechanism of tumor initiation that is mediated through activation of PI3K and not through aberrations in WNT signaling. Since the Adenomatous Polyposis Coli (APC) gene is mutated in the vast majority of human colon cancers and often occurs simultaneously with PIK3CA mutations, we sought to better understand the interaction between APC and PIK3CA mutations in the mammalian intestine. In this study, we have generated mice in which the expression of a constitutively active PI3K and the loss of APC occur simultaneously in the distal small intestine and colon. Here we demonstrate that expression of a dominant active PI3K synergizes with loss of APC activity resulting in a dramatic changes in tumor multiplicity, size, morphology, and invasiveness. Activation of the PI3K pathway is not able to directly activate WNT signaling through the nuclear localization of CTNNB1 (β-catenin) in the absence of aberrant WNT signaling. Alterations at the transcriptional level, including increased CCND1, may be the etiology of synergy between these activated pathways. PMID:23708654

  20. Essential elements for translation: the germline factor Vasa functions broadly in somatic cells

    PubMed Central

    Yajima, Mamiko; Wessel, Gary M.

    2015-01-01

    ABSTRACT Vasa is a conserved RNA-helicase found in the germ lines of all metazoans tested. Whereas Vasa presence is often indicated as a metric for germline determination in animals, it is also expressed in stem cells of diverse origin. Recent research suggests, however, that Vasa has a much broader function, including a significant role in cell cycle regulation. Results herein indicate that Vasa is utilized widely, and often induced transiently, during development in diverse somatic cells and adult precursor tissues. We identified that Vasa in the sea urchin is essential for: (1) general mRNA translation during embryogenesis, (2) developmental re-programming upon manipulations to the embryo and (3) larval wound healing. We also learned that Vasa interacted with mRNAs in the perinuclear area and at the spindle in an Importin-dependent manner during cell cycle progression. These results suggest that, when present, Vasa functions are essential to contributing to developmental regulation. PMID:25977366

  1. Multifunctionality of PIWI proteins in control of germline stem cell fate.

    PubMed

    Yakushev, E Y; Sokolova, O A; Gvozdev, V A; Klenov, M S

    2013-06-01

    PIWI proteins interacting with specific type of small RNAs (piRNAs) repress transposable elements in animals. Besides, they have been shown to participate in various cellular processes: in the regulation of heterochromatin formation including telomere structures, in the control of translation and the cell cycle, and in DNA rearrangements. PIWI proteins were first identified by their roles in the self-renewal of germline stem cells. PIWI protein functions are not limited to gonadogenesis, but the role in determining the fate of stem cells is their specific feature conserved throughout the evolution of animals. Molecular mechanisms underlying these processes are far from being understood. This review focuses on the role of PIWI proteins in the control of maintenance and proliferation of germinal stem cells and its relation to the known function of PIWI in transposon repression. PMID:23980885

  2. Tumor promotion and inhibition by phenobarbital in livers of conditional Apc-deficient mice.

    PubMed

    Braeuning, Albert; Gavrilov, Alina; Geissler, Miriam; Wenz, Christine; Colnot, Sabine; Templin, Markus F; Metzger, Ute; Römer, Michael; Zell, Andreas; Schwarz, Michael

    2016-06-01

    Activation of Wnt/β-catenin signaling is important for human and rodent hepatocarcinogenesis. In mice, the tumor promoter phenobarbital (PB) selects for hepatocellular tumors with activating β-catenin mutations via constitutive androstane receptor activation. PB-dependent tumor promotion was studied in mice with genetic inactivation of Apc, a negative regulator of β-catenin, to circumvent the problem of randomly induced mutations by chemical initiators and to allow monitoring of PB- and Wnt/β-catenin-dependent tumorigenesis in the absence of unknown genomic alterations. Moreover, the study was designed to investigate PB-induced proliferation of liver cells with activated β-catenin. PB treatment provided Apc-deficient hepatocytes with only a minor proliferative advantage, and additional connexin 32 deficiency did not affect the proliferative response. PB significantly promoted the outgrowth of Apc-deficient hepatocellular adenoma (HCA), but simultaneously inhibited the formation of Apc-deficient hepatocellular carcinoma (HCC). The probability of tumor promotion by PB was calculated to be much lower for hepatocytes with loss of Apc, as compared to mutational β-catenin activation. Comprehensive transcriptomic and phosphoproteomic characterization of HCA and HCC revealed molecular details of the two tumor types. HCC were characterized by a loss of differentiated hepatocellular gene expression, enhanced proliferative signaling, and massive over-activation of Wnt/β-catenin signaling. In conclusion, PB exerts a dual role in liver tumor formation by promoting the growth of HCA but inhibiting the growth of HCC. Data demonstrate that one and the same compound can produce opposite effects on hepatocarcinogenesis, depending on context, highlighting the necessity to develop a more differentiated view on the tumorigenicity of this model compound. PMID:26838046

  3. A novel tankyrase small-molecule inhibitor suppresses APC mutation-driven colorectal tumor growth.

    PubMed

    Lau, Ted; Chan, Emily; Callow, Marinella; Waaler, Jo; Boggs, Jason; Blake, Robert A; Magnuson, Steven; Sambrone, Amy; Schutten, Melissa; Firestein, Ron; Machon, Ondrej; Korinek, Vladimir; Choo, Edna; Diaz, Dolores; Merchant, Mark; Polakis, Paul; Holsworth, Daniel D; Krauss, Stefan; Costa, Mike

    2013-05-15

    Most colorectal cancers (CRC) are initiated by mutations of APC, leading to increased β-catenin-mediated signaling. However, continued requirement of Wnt/β-catenin signaling for tumor progression in the context of acquired KRAS and other mutations is less well-established. To attenuate Wnt/β-catenin signaling in tumors, we have developed potent and specific small-molecule tankyrase inhibitors, G007-LK and G244-LM, that reduce Wnt/β-catenin signaling by preventing poly(ADP-ribosyl)ation-dependent AXIN degradation, thereby promoting β-catenin destabilization. We show that novel tankyrase inhibitors completely block ligand-driven Wnt/β-catenin signaling in cell culture and display approximately 50% inhibition of APC mutation-driven signaling in most CRC cell lines. It was previously unknown whether the level of AXIN protein stabilization by tankyrase inhibition is sufficient to impact tumor growth in the absence of normal APC activity. Compound G007-LK displays favorable pharmacokinetic properties and inhibits in vivo tumor growth in a subset of APC-mutant CRC xenograft models. In the xenograft model most sensitive to tankyrase inhibitor, COLO-320DM, G007-LK inhibits cell-cycle progression, reduces colony formation, and induces differentiation, suggesting that β-catenin-dependent maintenance of an undifferentiated state may be blocked by tankyrase inhibition. The full potential of the antitumor activity of G007-LK may be limited by intestinal toxicity associated with inhibition of Wnt/β-catenin signaling and cell proliferation in intestinal crypts. These results establish proof-of-concept antitumor efficacy for tankyrase inhibitors in APC-mutant CRC models and uncover potential diagnostic and safety concerns to be overcome as tankyrase inhibitors are advanced into the clinic. PMID:23539443

  4. Germline transmission in transgenic Huntington’s disease monkeys

    PubMed Central

    Moran, Sean; Chi, Tim; Prucha, Melinda S.; Ahn, Kwang Sung; Connor-Stroud, Fawn; Jean, Sherrie; Gould, Kenneth; Chan, Anthony W. S.

    2015-01-01

    Transgenic nonhuman primate models are increasingly popular model for neurological and neurodegenerative disease because their brain functions and neural anatomies closely resemble those of humans [1–6]. Transgenic Huntington’s disease monkeys (HD monkeys) developed clinical features similar to those seen in HD patients, making the monkeys suitable for preclinical study of HD [6–12]. However, until HD monkey colonies can be readily expanded, their use in preclinical studies will be limited [1, 13, 14]. In the present study, we confirmed germline transmission of the mutant huntingtin (mHTT) transgene in both embryonic stem cells (ESCs) generated from three male HD monkey founders (F0), as well as in second-generation offspring (F1) produced via artificial insemination by using intrauterine insemination (IUI) technique. A total of five offspring were produced from fifteen females that were inseminated by IUI using semen collected from the three HD founders (5/15; 33%). Thus far, sperm collected from HD founder (rHD8) has led to two F1 transgenic HD moenkys with germline transmission rate at 100% (2/2). mHTT expression was confirmed by quantitative real-time PCR (qPCR) using skin fibroblasts from the F1 HD monkeys, as well as induced pluripotent stem cells (iPSCs) established from one of the F1 HD monkeys (rHD8-2). Here we report the stable germline transmission and expression of the mHTT transgene in HD monkeys, which suggest possible expansion of HD monkey colonies for preclinical and biomedical researches. PMID:25917881

  5. Source apportionment of ambient non-methane hydrocarbons in Hong Kong: application of a principal component analysis/absolute principal component scores (PCA/APCS) receptor model.

    PubMed

    Guo, H; Wang, T; Louie, P K K

    2004-06-01

    Receptor-oriented source apportionment models are often used to identify sources of ambient air pollutants and to estimate source contributions to air pollutant concentrations. In this study, a PCA/APCS model was applied to the data on non-methane hydrocarbons (NMHCs) measured from January to December 2001 at two sampling sites: Tsuen Wan (TW) and Central & Western (CW) Toxic Air Pollutants Monitoring Stations in Hong Kong. This multivariate method enables the identification of major air pollution sources along with the quantitative apportionment of each source to pollutant species. The PCA analysis identified four major pollution sources at TW site and five major sources at CW site. The extracted pollution sources included vehicular internal engine combustion with unburned fuel emissions, use of solvent particularly paints, liquefied petroleum gas (LPG) or natural gas leakage, and industrial, commercial and domestic sources such as solvents, decoration, fuel combustion, chemical factories and power plants. The results of APCS receptor model indicated that 39% and 48% of the total NMHCs mass concentrations measured at CW and TW were originated from vehicle emissions, respectively. 32% and 36.4% of the total NMHCs were emitted from the use of solvent and 11% and 19.4% were apportioned to the LPG or natural gas leakage, respectively. 5.2% and 9% of the total NMHCs mass concentrations were attributed to other industrial, commercial and domestic sources, respectively. It was also found that vehicle emissions and LPG or natural gas leakage were the main sources of C(3)-C(5) alkanes and C(3)-C(5) alkenes while aromatics were predominantly released from paints. Comparison of source contributions to ambient NMHCs at the two sites indicated that the contribution of LPG or natural gas at CW site was almost twice that at TW site. High correlation coefficients (R(2) > 0.8) between the measured and predicted values suggested that the PCA/APCS model was applicable for estimation

  6. Germ-line gene therapy and the medical imperative.

    PubMed

    Munson, Ronald; Davis, Lawrence H

    1992-06-01

    Somatic cell gene therapy has yielded promising results. If germ cell gene therapy can be developed, the promise is even greater: hundreds of genetic diseases might be virtually eliminated. But some claim the procedure is morally unacceptable. We thoroughly and sympathetically examine several possible reasons for this claim but find them inadequate. There is no moral reason, then, not to develop and employ germ-line gene therapy. Taking the offensive, we argue next that medicine has a prima facie moral obligation to do so. PMID:11645742

  7. Repression of somatic cell fate in the germline.

    PubMed

    Robert, Valérie J; Garvis, Steve; Palladino, Francesca

    2015-10-01

    Germ cells must transmit genetic information across generations, and produce gametes while also maintaining the potential to form all cell types after fertilization. Preventing the activation of somatic programs is, therefore, crucial to the maintenance of germ cell identity. Studies in Caenorhabditis elegans, Drosophila melanogaster, and mouse have revealed both similarities and differences in how somatic gene expression is repressed in germ cells, thereby preventing their conversion into somatic tissues. This review will focus on recent developments in our understanding of how global or gene-specific transcriptional repression, chromatin regulation, and translational repression operate in the germline to maintain germ cell identity and repress somatic differentiation programs. PMID:26043973

  8. Germline HABP2 Mutation Causing Familial Nonmedullary Thyroid Cancer

    PubMed Central

    Gara, Sudheer Kumar; Jia, Li; Merino, Maria J.; Agarwal, Sunita K.; Zhang, Lisa; Cam, Maggie; Patel, Dhaval; Kebebew, Electron

    2015-01-01

    SUMMARY Familial nonmedullary thyroid cancer accounts for 3 to 9% of all cases of thyroid cancer, but the susceptibility genes are not known. Here, we report a germline variant of HABP2 in seven affected members of a kindred with familial nonmedullary thyroid cancer and in 4.7% of 423 patients with thyroid cancer. This variant was associated with increased HABP2 protein expression in tumor samples from affected family members, as compared with normal adjacent thyroid tissue and samples from sporadic cancers. Functional studies showed that HABP2 has a tumor-suppressive effect, whereas the G534E variant results in loss of function. PMID:26222560

  9. E proteins are required to activate germline transcription of the TCR Vbeta8.2 gene.

    PubMed

    Jia, Jingquan; Dai, Meifang; Zhuang, Yuan

    2008-10-01

    Each TCR Vbeta gene is regulated by an individual Vbeta promoter, which becomes active prior to V(D) J recombination and drives germline transcription. It has been shown that Vbeta gene locus activation and recombination are dependent on the Vbeta promoter. However, transcription factors that regulate Vbeta germline transcription remain largely undefined. A major challenge in studying Vbeta gene germline transcription is the quantitative assessment of relatively low-level transcripts in T-cell progenitors. Here we used the established Vbeta8.2(CD2) knock-in mouse model to assess functions of E-protein transcription factors in Vbeta8.2 germline transcription. We show that E proteins are required for the activation but not the maintenance of the Vbeta8.2 germline transcription during thymocyte development. The activation of Vbeta8.2 germline transcription depends more on the E proteins encoded by the E2A gene than by the HEB gene. We further show that IL-7 receptor (IL-7R)-mediated signals are essential for Vbeta8.2 germline transcription. We provide evidence that IL-7R expression is only partially controlled by E2A, suggesting a role for E2A in driving Vbeta8.2 germline transcription independent of IL-7R activation. PMID:18958875

  10. Prevalence of low-penetrant germline TP53 D49H mutation in Japanese cancer patients.

    PubMed

    Yamaguchi, Ken; Urakami, Kenichi; Nagashima, Takeshi; Shimoda, Yuji; Ohnami, Shumpei; Ohnami, Sumiko; Ohshima, Keiichi; Mochizuki, Tohru; Hatakeyama, Keiichi; Serizawa, Masakuni; Akiyama, Yasuto; Maruyama, Kouji; Katagiri, Hirohisa; Ishida, Yuji; Takahashi, Kaoru; Nishimura, Seiichiro; Terashima, Masanori; Kawamura, Taiichi; Kinugasa, Yusuke; Yamakawa, Yushi; Onitsuka, Tetsuro; Ohde, Yasuhisa; Sugino, Takashi; Ito, Ichiro; Matsubayashi, Hiroyuki; Horiuchi, Yasue; Mizuguchi, Maki; Yamazaki, Mutsumi; Inoue, Kengo; Wakamatsu, Kimiko; Sugiyama, Misato; Uesaka, Katsuhiko; Kusuhara, Masatoshi

    2016-01-01

    Using whole exome sequencing data obtained from 1,685 Japanese cancer patients, we examined genetic variations of germline TP53 and found 10 types of non-synonymous single nucleotide variants. In the present study, we focused on 6 patients with germline D49H mutation located in the transactivation domain 2 of p53 protein, since the mutation seemed to be prevalent in cancer patients and to be pathogenic. According to the initial survey for family history of the proband with the germline TP53 D49H mutation, one osteosarcoma patient and his pedigree fulfill the criteria for Li-Fraumeni-like syndrome and the 2009 Chompret criteria for germline TP53 mutation screening. Since this patient possesses double germline mutations of TP53 D49H and A159D, further studies are required to evaluate contribution of the D49H mutation in this morbidity. The remaining 5 patients had family histories of cancer, but none fulfills the criteria either for the Li-Fraumeni/Li-Fraumeni-like syndromes or the 2009 Chompret criteria for germline TP53 mutation screening. It is possible to postulate that the germline TP53 D49H mutation is likely to be low-penetrant in some pedigrees. The present study also indicates that the survey for the germline TP53 mutation plays an important role in clinical practice as it will prevent mistaking cancer patients with unusual heredities for sporadic cases. PMID:27545002

  11. Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein.

    PubMed Central

    Munemitsu, S; Albert, I; Souza, B; Rubinfeld, B; Polakis, P

    1995-01-01

    The APC tumor-suppressor protein associates with beta-catenin, a cell adhesion protein that is upregulated by the WNT1 oncogene. We examined the effects of exogenous APC expression on the distribution and amount of beta-catenin in a colorectal cancer cell containing only mutant APC. Expression of wild-type APC caused a pronounced reduction in total beta-catenin levels by eliminating an excessive supply of cytoplasmic beta-catenin indigenous to the SW480 colorectal cancer cell line. This reduction was due to an enhanced rate of beta-catenin protein degradation. Truncated mutant APC proteins, characteristic of those associated with cancer, lacked this activity. Mutational analysis revealed that the central region of the APC protein, which is typically deleted or severely truncated in tumors, was responsible for the down-regulation of beta-catenin. These results suggest that the tumor-suppressor activity of mutant APC may be compromised due to a defect in its ability to regulate beta-catenin. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7708772

  12. Cryo-EM of Mitotic Checkpoint Complex-Bound APC/C Reveals Reciprocal and Conformational Regulation of Ubiquitin Ligation.

    PubMed

    Yamaguchi, Masaya; VanderLinden, Ryan; Weissmann, Florian; Qiao, Renping; Dube, Prakash; Brown, Nicholas G; Haselbach, David; Zhang, Wei; Sidhu, Sachdev S; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A

    2016-08-18

    The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). APC/C(CDC20) and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/C(CDC20)-UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here electron microscopy reveals conformational variation of APC/C(CDC20)-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly and conformational variability of APC/C modulate UBE2C-catalyzed ubiquitination of MCC's CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/C(CDC20)-MCC modulate E2 activation and determine distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation. PMID:27522463

  13. Germline ESR2 mutation predisposes to medullary thyroid carcinoma and causes up-regulation of RET expression.

    PubMed

    Smith, Joel; Read, Martin L; Hoffman, Jon; Brown, Rachel; Bradshaw, Beth; Campbell, Christopher; Cole, Trevor; Navas, Johanna Dieguez; Eatock, Fiona; Gundara, Justin S; Lian, Eric; Mcmullan, Dom; Morgan, Neil V; Mulligan, Lois; Morrison, Patrick J; Robledo, Mercedes; Simpson, Michael A; Smith, Vicki E; Stewart, Sue; Trembath, Richard C; Sidhu, Stan; Togneri, Fiona S; Wake, Naomi C; Wallis, Yvonne; Watkinson, John C; Maher, Eamonn R; McCabe, Christopher J; Woodward, Emma R

    2016-05-01

    Familial medullary thyroid cancer (MTC) and its precursor, C cell hyperplasia (CCH), is associated with germline RET mutations causing multiple endocrine neoplasia type 2. However, some rare families with apparent MTC/CCH predisposition do not have a detectable RET mutation. To identify novel MTC/CCH predisposition genes we undertook exome resequencing studies in a family with apparent predisposition to MTC/CCH and no identifiable RET mutation. We identified a novel ESR2 frameshift mutation, c.948delT, which segregated with histological diagnosis following thyroid surgery in family members and demonstrated loss of ESR2-encoded ERβ expression in the MTC tumour. ERα and ERβ form heterodimers binding DNA at specific oestrogen-responsive elements (EREs) to regulate gene transcription. ERβ represses ERα-mediated activation of the ERE and the RET promoter contains three EREs. In vitro, we showed that ESR2 c.948delT results in unopposed ERα mediated increased cellular proliferation, activation of the ERE and increased RET expression. In vivo, immunostaining of CCH and MTC using an anti-RET antibody demonstrated increased RET expression. Together these findings identify germline ESR2 mutation as a novel cause of familial MTC/CCH and provide important insights into a novel mechanism causing increased RET expression in tumourigenesis. PMID:26945007

  14. APC Activation Restores Functional CD4+CD25+ Regulatory T Cells in NOD Mice that Can Prevent Diabetes Development

    PubMed Central

    Manirarora, Jean N.; Kosiewicz, Michele M.; Parnell, Sarah A.; Alard, Pascale

    2008-01-01

    Background Defects in APC and regulatory cells are associated with diabetes development in NOD mice. We have shown previously that NOD APC are not effective at stimulating CD4+CD25+ regulatory cell function in vitro. We hypothesize that failure of NOD APC to properly activate CD4+CD25+ regulatory cells in vivo could compromise their ability to control pathogenic cells, and activation of NOD APC could restore this defect, thereby preventing disease. Methodology/Principal Findings To test these hypotheses, we used the well-documented ability of complete Freund's adjuvant (CFA), an APC activator, to prevent disease in NOD mice. Phenotype and function of CD4+CD25+ regulatory cells from untreated and CFA-treated NOD mice were determined by FACS, and in vitro and in vivo assays. APC from these mice were also evaluated for their ability to activate regulatory cells in vitro. We have found that sick NOD CD4+CD25+ cells expressed Foxp3 at the same percentages, but decreased levels per cell, compared to young NOD or non-NOD controls. Treatment with CFA increased Foxp3 expression in NOD cells, and also increased the percentages of CD4+CD25+Foxp3+ cells infiltrating the pancreas compared to untreated NOD mice. Moreover, CD4+CD25+ cells from pancreatic LN of CFA-treated, but not untreated, NOD mice transferred protection from diabetes. Finally, APC isolated from CFA-treated mice increased Foxp3 and granzyme B expression as well as regulatory function by NOD CD4+CD25+ cells in vitro compared to APC from untreated NOD mice. Conclusions/Significance These data suggest that regulatory T cell function and ability to control pathogenic cells can be enhanced in NOD mice by activating NOD APC. PMID:19011680

  15. Sugar-carrying Polystyrenes Facilitate Harvesting of APCs from MLRs: Possible Application of Sugar-carrying Polystyrenes to Immunotherapy.

    PubMed

    Imaizumi, Akira; Onishi, Hideya; Yamasaki, Akio; Kawamoto, Makoto; Morisaki, Takashi; Goto, Mitsuaki; Iwama, Masamichi; Akaike, Toshihiro; Hasumi, Kenichiro

    2016-02-01

    Antigen-presenting cells (APCs) play a pivotal role in cancer immunotherapy. APCs in conventionally used flasks are harvested by enzymatic digestion or cell scraping for application to cancer immunotherapy. However, these methods may impair functional molecules expressed on the APC surface and reduce their effects in cancer immunotherapy. Recently, we found that APCs could be harvested by shaking at 4°C in flasks coated with poly[N-p-vinylbenzyl-O-2-acetoamide-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetoamide-2-deoxy-β-D-gluconamide] (PVGlcNAc) or a copolymer consisting of sulfonylurea (SU) linked to poly[N-p-vinyl-benzyl-4-O-β-D-galactopyranosyl-D-gluconamide] [P(VLA-co-SU)]. In the present study, we compared the functions of cytotoxic T-lymphocytes (CTLs) induced by APCs generated in PVGlcNAc- or P(VLA-co-SU)-coated flasks and conventional flasks. APCs from PVGlcNAc- or P(VLA-co-SU)-coated flasks showed higher expression of cluster of differentiation (CD)80/86, CD11c, and major histocompatibility complex class II alloantigen I-A(d), and higher cytotoxicity than APCs from conventional flasks. These results suggest that the use of PVGlcNAc- or P(VLA-co-SU)-coated flasks is optimal for harvesting APCs. The generated APCs also have a higher antigen-presenting ability compared to those generated in conventional flasks. Our results may contribute to the development of effective cancer immunotherapies. PMID:26851023

  16. Selective accumulation of germ-line associated gene products in early development of the sea star and distinct differences from germ-line development in the sea urchin

    PubMed Central

    Fresques, Tara; Zazueta-Novoa, Vanesa; Reich, Adrian; Wessel, Gary M.

    2014-01-01

    Background Echinodermata is a diverse Phylum, a sister group to chordates, and contains diverse organisms that may be useful to understand varied mechanisms of germ-line specification. Results We tested 23 genes in development of the sea star Patiria miniata that fall into five categories: 1) Conserved germ-line factors; 2) Genes involved in the inductive mechanism of germ-line specification; 3) Germ-line associated genes; 4) Molecules involved in left-right asymmetry; and 5) Genes involved in regulation and maintenance of the genome during early embryogenesis. Overall, our results support the contention that the posterior enterocoel is a source of the germ line in the sea star P. miniata. Conclusion The germ line in this organism appears to be specified late in embryogenesis, and in a pattern more consistent with inductive interactions amongst cells. This is distinct from the mechanism seen in sea urchins, a close relative of the sea star clad. We propose that P. miniata may serve as a valuable model to study inductive mechanisms of germ-cell specification and when compared to germ-line formation in the sea urchin S. purpuratus may reveal developmental transitions that occur in the evolution of inherited and inductive mechanisms of germ-line specification. PMID:24038550

  17. Immunohistological techniques for studying the Drosophila male germline stem cell.

    PubMed

    Singh, Shree Ram; Hou, Steven X

    2008-01-01

    Stem cells are undifferentiated cells that have a remarkable ability to self-renew and produce differentiated cells that support normal development and tissue homeostasis. This unique capacity makes stem cells a powerful tool for future regenerative medicine and gene therapy. Accumulative evidence suggests that stem cell self-renewal or differentiation is controlled by both intrinsic and extrinsic factors, and that deregulation of stem cell behavior results in cancer formation, tissue degeneration, and premature aging. The Drosophila testis provides an excellent in vivo model for studying and understanding the fundamental cellular and molecular mechanisms controlling stem cell behavior and the relationship between niches and stem cells. At the tip of the Drosophila testes, germline stem cells (GSCs) and somatic stem cells (SSCs) contact each other and share common niches (known as a hub) to maintain spermatogenesis. Signaling pathways, such as the Janus kinase (JAK)/signal transducer and activator of transcription (STAT), bone morphogenetic protein (BMP), ras-associated protein-guanine nucleotide exchange factor for small GTPase (Rap-GEF), and epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK), are known to regulate self-renewal or differentiation of Drosophila male germline stem cells. We describe the detailed in vivo immunohistological protocols that mark GSCs, SSCs, and their progeny in Drosophila testes. PMID:18370050

  18. Epigenetic inheritance systems contribute to the evolution of a germline

    PubMed Central

    Libby, Eric

    2016-01-01

    Differentiation within multicellular organisms is controlled by epigenetic markers transmitted across cell division. The process of differentiation will modify these epigenetic markers so that information that one cell type possesses can be lost in the transition to another. Many of the systems that encode these markers also exist in unicellular organisms but do not control differentiation. Thus, during the evolution of multicellularity, epigenetic inheritance systems were probably exapted for their current use in differentiation. We show that the simultaneous use of an information carrier for differentiation and transmission across generations can lead to the evolution of cell types that do not directly contribute to the progeny of the organism and ergo a germ–soma distinction. This shows that an intrinsic instability during a transition from unicellularity to multicellularity may contribute to widespread evolution of a germline and its maintenance, a phenomenon also relevant to the evolution of eusociality. The difference in epigenetic information contents between different cell lines in a multicellular organism is also relevant for the full-success cloning of higher animals, as well as for the maintenance of single germlines over evolutionary timescales. This article is part of the themed issue ‘The major synthetic evolutionary transitions’. PMID:27431523

  19. 8-oxoguanine causes spontaneous de novo germline mutations in mice

    NASA Astrophysics Data System (ADS)

    Ohno, Mizuki; Sakumi, Kunihiko; Fukumura, Ryutaro; Furuichi, Masato; Iwasaki, Yuki; Hokama, Masaaki; Ikemura, Toshimichi; Tsuzuki, Teruhisa; Gondo, Yoichi; Nakabeppu, Yusaku

    2014-04-01

    Spontaneous germline mutations generate genetic diversity in populations of sexually reproductive organisms, and are thus regarded as a driving force of evolution. However, the cause and mechanism remain unclear. 8-oxoguanine (8-oxoG) is a candidate molecule that causes germline mutations, because it makes DNA more prone to mutation and is constantly generated by reactive oxygen species in vivo. We show here that endogenous 8-oxoG caused de novo spontaneous and heritable G to T mutations in mice, which occurred at different stages in the germ cell lineage and were distributed throughout the chromosomes. Using exome analyses covering 40.9 Mb of mouse transcribed regions, we found increased frequencies of G to T mutations at a rate of 2 × 10-7 mutations/base/generation in offspring of Mth1/Ogg1/Mutyh triple knockout (TOY-KO) mice, which accumulate 8-oxoG in the nuclear DNA of gonadal cells. The roles of MTH1, OGG1, and MUTYH are specific for the prevention of 8-oxoG-induced mutation, and 99% of the mutations observed in TOY-KO mice were G to T transversions caused by 8-oxoG; therefore, we concluded that 8-oxoG is a causative molecule for spontaneous and inheritable mutations of the germ lineage cells.

  20. Epigenetic inheritance systems contribute to the evolution of a germline.

    PubMed

    Lachmann, Michael; Libby, Eric

    2016-08-19

    Differentiation within multicellular organisms is controlled by epigenetic markers transmitted across cell division. The process of differentiation will modify these epigenetic markers so that information that one cell type possesses can be lost in the transition to another. Many of the systems that encode these markers also exist in unicellular organisms but do not control differentiation. Thus, during the evolution of multicellularity, epigenetic inheritance systems were probably exapted for their current use in differentiation. We show that the simultaneous use of an information carrier for differentiation and transmission across generations can lead to the evolution of cell types that do not directly contribute to the progeny of the organism and ergo a germ-soma distinction. This shows that an intrinsic instability during a transition from unicellularity to multicellularity may contribute to widespread evolution of a germline and its maintenance, a phenomenon also relevant to the evolution of eusociality. The difference in epigenetic information contents between different cell lines in a multicellular organism is also relevant for the full-success cloning of higher animals, as well as for the maintenance of single germlines over evolutionary timescales.This article is part of the themed issue 'The major synthetic evolutionary transitions'. PMID:27431523

  1. In Vitro Cytotoxicity of Nanoparticles in Mammalian Germline Stem Cells

    PubMed Central

    Braydich-Stolle, Laura; Hussain, Saber; Schlager, John J.; Hofmann, Marie-Claude

    2010-01-01

    Gametogenesis is a complex biological process that is particularly sensitive to environmental insults such as chemicals. Many chemicals have a negative impact on the germline, either by directly affecting the germ cells, or indirectly through their action on the somatic nursing cells. Ultimately, these effects can inhibit fertility, and they may have negative consequences for the development of the offspring. Recently, nanomaterials such as nanotubes, nanowires, fullerene derivatives (buckyballs), and quantum dots have received enormous national attention in the creation of new types of analytical tools for biotechnology and the life sciences. Despite the wide application of nanomaterials, there is a serious lack of information concerning their impact on human health and the environment. Thus, there are limited studies available on toxicity of nanoparticles for risk assessment of nanomaterials. The purpose of this study was to assess the suitability of a mouse spermatogonial stem cell line as a model to assess nanotoxicity in the male germline in vitro. The effects of different types of nanoparticles on these cells were evaluated by light microscopy, and by cell proliferation and standard cytotoxicity assays. Our results demonstrate a concentration-dependent toxicity for all types of particles tested, whereas the corresponding soluble salts had no significant effect. Silver nanoparticles were the most toxic while molybdenum trioxide (MoO3) nanoparticles were the least toxic. Our results suggest that this cell line provides a valuable model with which to assess the cytotoxicity of nanoparticles in the germ line in vitro. PMID:16014736

  2. Hidden genetic variation in the germline genome of Tetrahymena thermophila.

    PubMed

    Dimond, K L; Zufall, R A

    2016-06-01

    Genome architecture varies greatly among eukaryotes. This diversity may profoundly affect the origin and maintenance of genetic variation within a population. Ciliates are microbial eukaryotes with unusual genome features, such as the separation of germline and somatic genomes within a single cell and amitotic division. These features have previously been proposed to increase the rate of molecular evolution in these species. Here, we assessed the fitness effects of genetic variation in the two genomes of natural isolates of the ciliate Tetrahymena thermophila. We find more extensive genetic variation in fitness in the transcriptionally silent germline genome than in the expressed somatic genome. Surprisingly, this variation is not primarily deleterious, but has both beneficial and deleterious effects. We conclude that Tetrahymena genome architecture allows for the maintenance of genetic variation that would otherwise be eliminated by selection. We consider the effect of selection on the two genomes and the impacts of reproductive strategies and the mechanism of sex determination on the structure of this variation. PMID:26998689

  3. APC/β-catenin-rich complexes at membrane protrusions regulate mammary tumor cell migration and mesenchymal morphology

    PubMed Central

    2013-01-01

    Background The APC tumor suppressor is mutated or downregulated in many tumor types, and is prominently localized to punctate clusters at protrusion tips in migratory cells, such as in astrocytes where it has been implicated in directed cell motility. Although APC loss is considered an initiating event in colorectal cancer, for example, it is less clear what role APC plays in tumor cell motility and whether loss of APC might be an important promoter of tumor progression in addition to initiation. Methods The localization of APC and β-catenin was analyzed in multiple cell lines, including non-transformed epithelial lines treated with a proteasome inhibitor or TGFβ to induce an epithelial-to-mesenchymal transition (EMT), as well as several breast cancer lines, by immunofluorescence. APC expression was knocked down in 4T07 mammary tumor cells using lentiviral-mediated delivery of APC-specific short-hairpin (sh) RNAs, and assessed using quantitative (q) reverse-transcriptase (RT)-PCR and western blotting. Tumor cell motility was analyzed by performing wound-filling assays, and morphology via immunofluorescence (IF) and phase-contrast microscopy. Additionally, proliferation was measured using BrdU incorporation, and TCF reporter assays were performed to determine β-catenin/TCF-mediated transcriptional activity. Results APC/β-catenin-rich complexes were observed at protrusion ends of migratory epithelial cells treated with a proteasome inhibitor or when EMT has been induced and in tumor cells with a mesenchymal, spindle-like morphology. 4T07 tumor cells with reduced APC levels were significantly less motile and had a more rounded morphology; yet, they did not differ significantly in proliferation or β-catenin/TCF transcriptional activity. Furthermore, we found that APC/β-catenin-rich complexes at protrusion ends were dependent upon an intact microtubule cytoskeleton. Conclusions These findings indicate that membrane protrusions with APC/β-catenin-containing puncta

  4. Paternal B Vitamin Intake Is a Determinant of Growth, Hepatic Lipid Metabolism and Intestinal Tumor Volume in Female Apc1638N Mouse Offspring

    PubMed Central

    Sabet, Julia A.; Park, Lara K.; Iyer, Lakshmanan K.; Tai, Albert K.; Koh, Gar Yee; Pfalzer, Anna C.; Parnell, Laurence D.; Mason, Joel B.; Liu, Zhenhua; Byun, Alexander J.; Crott, Jimmy W.

    2016-01-01

    Background The importance of maternal nutrition to offspring health and risk of disease is well established. Emerging evidence suggests paternal diet may affect offspring health as well. Objective In the current study we sought to determine whether modulating pre-conception paternal B vitamin intake alters intestinal tumor formation in offspring. Additionally, we sought to identify potential mechanisms for the observed weight differential among offspring by profiling hepatic gene expression and lipid content. Methods Male Apc1638N mice (prone to intestinal tumor formation) were fed diets containing replete (control, CTRL), mildly deficient (DEF), or supplemental (SUPP) quantities of vitamins B2, B6, B12, and folate for 8 weeks before mating with control-fed wild type females. Wild type offspring were euthanized at weaning and hepatic gene expression profiled. Apc1638N offspring were fed a replete diet and euthanized at 28 weeks of age to assess tumor burden. Results No differences in intestinal tumor incidence or burden were found between male Apc1638N offspring of different paternal diet groups. Although in female Apc1638N offspring there were no differences in tumor incidence or multiplicity, a stepwise increase in tumor volume with increasing paternal B vitamin intake was observed. Interestingly, female offspring of SUPP and DEF fathers had a significantly lower body weight than those of CTRL fed fathers. Moreover, hepatic trigylcerides and cholesterol were elevated 3-fold in adult female offspring of SUPP fathers. Weanling offspring of the same fathers displayed altered expression of several key lipid-metabolism genes. Hundreds of differentially methylated regions were identified in the paternal sperm in response to DEF and SUPP diets. Aside from a few genes including Igf2, there was a striking lack of overlap between these genes differentially methylated in sperm and differentially expressed in offspring. Conclusions In this animal model, modulation of

  5. High prevalence of germline STK11 mutations in Hungarian Peutz-Jeghers Syndrome patients

    PubMed Central

    2010-01-01

    Background Peutz-Jeghers syndrome (PJS) is a rare autosomal dominantly inherited disease characterized by gastrointestinal hamartomatous polyposis and mucocutaneous pigmentation. The genetic predisposition for PJS has been shown to be associated with germline mutations in the STK11/LKB1 tumor suppressor gene. The aim of the present study was to characterize Hungarian PJS patients with respect to germline mutation in STK11/LKB1 and their association to disease phenotype. Methods Mutation screening of 21 patients from 13 PJS families were performed using direct DNA sequencing and multiplex ligation-dependent probe amplification (MLPA). Comparative semi-quantitative sequencing was applied to investigate the mRNA-level effects of nonsense and splice-affecting mutations. Results Thirteen different pathogenic mutations in STK11, including a high frequency of large genomic deletions (38%, 5/13), were identified in the 13 unrelated families studied. One of these deletions also affects two neighboring genes (SBNO2 and GPX4), located upstream of STK11, with a possible modifier effect. The majority of the point mutations (88%, 7/8) can be considered novel. Quantification of the STK11 transcript at the mRNA-level revealed that the expression of alleles carrying a nonsense or frameshift mutation was reduced to 30-70% of that of the wild type allele. Mutations affecting splice-sites around exon 2 displayed an mRNA processing pattern indicative of co-regulated splicing of exons 2 and 3. Conclusions A combination of sensitive techniques may assure a high (100%) STK11 mutation detection frequency in PJS families. Characterization of mutations at mRNA level may give a deeper insight into the molecular consequences of the pathogenic mutations than predictions made solely at the genomic level. PMID:21118512

  6. Germline genetics of cancer of unknown primary (CUP) and its specific subtypes

    PubMed Central

    Hemminki, Kari; Chen, Bowang; Kumar, Abhishek; Melander, Olle; Manjer, Jonas; Hallmans, Göran; Pettersson-Kymmer, Ulrika; Ohlsson, Claes; Folprecht, Gunnar; Löffler, Harald; Krämer, Alwin; Försti, Asta

    2016-01-01

    Cancer of unknown primary site (CUP) is a fatal cancer diagnosed through metastases at various organs. Little is known about germline genetics of CUP which appears worth of a search in view of reported familial associations in CUP. In the present study, samples from CUP patients were identified from 2 Swedish biobanks and a German clinical trial, totaling 578 CUP patients and 7628 regionally matched controls. Diagnostic data specified the organ where metastases were diagnosed. We carried out a genome-wide association study on CUP cases and controls. In the whole sample set, 6 loci reached an allelic p-value in the range of 10−7 and were supported by data from the three centers. Three associations were located next to non-coding RNA genes. rs2660852 flanked 5′UTR of LTA4H (leukotriene A4 hydrolase), rs477145 was intronic to TIAM1 (T-cell lymphoma invasion and metastases) and rs2835931 was intronic to KCNJ6 (potassium channel, inwardly rectifying subfamily J, member 6). In analysis of subgroups of CUP patients (smokers, non-smokers and CUP with liver metastases) genome-wide significant associations were noted. For patients with liver metastases associations on chromosome 6 and 11, the latter including a cluster of genes DHCR7 and NADSYN1, encoding key enzymes in cholesterol and NAD synthesis, and KRTAP5-7, encoding a keratin associated protein. This first GWAS on CUP provide preliminary evidence that germline genes relating to inflammation (LTA4H), metastatic promotion (TIAM1) in association with lipid metabolic disturbance (chromosome 11 cluster) may contribute to the risk of CUP. PMID:26959888

  7. Quantification of the paternal allele bias for new germline mutations in the retinoblastoma gene

    SciTech Connect

    Morrow, J.F.; Rapaport, J.M.; Dryia, T.P.

    1994-09-01

    New germline mutations in the human retinoblastoma gene preferentially arise on a paternally derived allele. In nonhereditary retinoblastoma, the initial somatic mutation seems to have no such bias. The few previous reports of these phenomena included relatively few cases (less than a dozen new germline or initial somatic mutations), so that the magnitude of the paternal allele bias for new germline mutations is not known. Knowledge of the magnitude of the bias is valuable for genetic counseling, since, for example, patients with new germline mutations who reproduce transmit risk for retinoblastoma according to the risk that the transmitted allele has a germline mutation. We sought to quantitate the paternal allele bias and to determine whether paternal age is a factor possibly accounting for it. We studied 311 families with retinoblastoma (261 simplex, 50 multiplex) that underwent clinical genetic testing and 5 informative families recruited from earlier research. Using RFLPs and polymorphic microsatellites in the retinoblastoma gene, we could determine the parental origin of 45 new germline mutations and 44 probable initial somatic mutations. Thirty-seven of the 45 new germline mutations, or 82%, arose on a paternal allele while only 24 of the 44 initial somatic mutations (55%) did so. Increased paternal age does not appear to account for the excess of new paternal germline mutations, since the average age of fathers of children with new germline mutations (29.4 years, n=26, incomplete records on 11) was not significantly different from the average age of fathers of children with maternal germline mutations or somatic initial mutations (29.8 years, n=35, incomplete records on 17).

  8. APC alterations are frequently involved in the pathogenesis of acinar cell carcinoma of the pancreas, mainly through gene loss and promoter hypermethylation.

    PubMed

    Furlan, Daniela; Sahnane, Nora; Bernasconi, Barbara; Frattini, Milo; Tibiletti, Maria Grazia; Molinari, Francesca; Marando, Alessandro; Zhang, Lizhi; Vanoli, Alessandro; Casnedi, Selenia; Adsay, Volkan; Notohara, Kenji; Albarello, Luca; Asioli, Sofia; Sessa, Fausto; Capella, Carlo; La Rosa, Stefano

    2014-05-01

    Genetic and epigenetic alterations involved in the pathogenesis of pancreatic acinar cell carcinomas (ACCs) are poorly characterized, including the frequency and role of gene-specific hypermethylation, chromosome aberrations, and copy number alterations (CNAs). A subset of ACCs is known to show alterations in the APC/β-catenin pathway which includes mutations of APC gene. However, it is not known whether, in addition to mutation, loss of APC gene function can occur through alternative genetic and epigenetic mechanisms such as gene loss or promoter methylation. We investigated the global methylation profile of 34 tumor suppressor genes, CNAs of 52 chromosomal regions, and APC gene alterations (mutation, methylation, and loss) together with APC mRNA level in 45 ACCs and related peritumoral pancreatic tissues using methylation-specific multiplex ligation probe amplification (MS-MLPA), fluorescence in situ hybridization (FISH), mutation analysis, and reverse transcription-droplet digital PCR. ACCs did not show an extensive global gene hypermethylation profile. RASSF1 and APC were the only two genes frequently methylated. APC mutations were found in only 7 % of cases, while APC loss and methylation were more frequently observed (48 and 56 % of ACCs, respectively). APC mRNA low levels were found in 58 % of cases and correlated with CNAs. In conclusion, ACCs do not show extensive global gene hypermethylation. APC alterations are frequently involved in the pathogenesis of ACCs mainly through gene loss and promoter hypermethylation, along with reduction of APC mRNA levels. PMID:24590585

  9. Reversible modification of adenomatous polyposis coli (APC) with K63-linked polyubiquitin regulates the assembly and activity of the β-catenin destruction complex.

    PubMed

    Tran, Hoanh; Polakis, Paul

    2012-08-17

    The adenomatous polyposis coli (APC) tumor suppressor forms a complex with Axin and GSK3β to promote the phosphorylation and degradation of β-catenin, a key co-activator of Wnt-induced transcription. Here, we establish that APC is modified predominantly with K63-linked ubiquitin chains when it is bound to Axin in unstimulated HEK293 cells. Wnt3a stimulation induced a time-dependent loss of K63-polyubiquitin adducts from APC, an effect synchronous with the dissociation of Axin from APC and the stabilization of cytosolic β-catenin. RNAi-mediated depletion of Axin or β-catenin, which negated the association between APC and Axin, resulted in the absence of K63-adducts on APC. Overexpression of wild-type and phosphodegron-mutant β-catenin, combined with analysis of thirteen human cancer cell lines that harbor oncogenic mutations in APC, Axin, or β-catenin, support the hypothesis that a fully assembled APC-Axin-GSK3β-phospho-β-catenin complex is necessary for the K63-polyubiquitylation of APC. Intriguingly, the degree of this modification on APC appears to correlate inversely with the levels of β-catenin in cells. Together, our results indicate that K63-linked polyubiquitin adducts on APC regulate the assembly and/or efficiency of the β-catenin destruction complex. PMID:22761442

  10. Germline loss-of-function mutations in LZTR1 predispose to an inherited disorder of multiple schwannomas.

    PubMed

    Piotrowski, Arkadiusz; Xie, Jing; Liu, Ying F; Poplawski, Andrzej B; Gomes, Alicia R; Madanecki, Piotr; Fu, Chuanhua; Crowley, Michael R; Crossman, David K; Armstrong, Linlea; Babovic-Vuksanovic, Dusica; Bergner, Amanda; Blakeley, Jaishri O; Blumenthal, Andrea L; Daniels, Molly S; Feit, Howard; Gardner, Kathy; Hurst, Stephanie; Kobelka, Christine; Lee, Chung; Nagy, Rebecca; Rauen, Katherine A; Slopis, John M; Suwannarat, Pim; Westman, Judith A; Zanko, Andrea; Korf, Bruce R; Messiaen, Ludwine M

    2014-02-01

    Constitutional SMARCB1 mutations at 22q11.23 have been found in ∼50% of familial and <10% of sporadic schwannomatosis cases. We sequenced highly conserved regions along 22q from eight individuals with schwannomatosis whose schwannomas involved somatic loss of one copy of 22q, encompassing SMARCB1 and NF2, with a different somatic mutation of the other NF2 allele in every schwannoma but no mutation of the remaining SMARCB1 allele in blood and tumor samples. LZTR1 germline mutations were identified in seven of the eight cases. LZTR1 sequencing in 12 further cases with the same molecular signature identified 9 additional germline mutations. Loss of heterozygosity with retention of an LZTR1 mutation was present in all 25 schwannomas studied. Mutations segregated with disease in all available affected first-degree relatives, although four asymptomatic parents also carried an LZTR1 mutation. Our findings identify LZTR1 as a gene predisposing to an autosomal dominant inherited disorder of multiple schwannomas in ∼80% of 22q-related schwannomatosis cases lacking mutation in SMARCB1. PMID:24362817

  11. Evidence for a founder effect of the germline fumarate hydratase gene mutation R58P causing hereditary leiomyomatosis and renal cell cancer (HLRCC).

    PubMed

    Heinritz, W; Paasch, U; Sticherling, M; Wittekind, C; Simon, J C; Froster, U G; Renner, R

    2008-01-01

    We report on the results of clinical investigation, pedigree analysis, mutation screening and haplotyping in a family with the syndrome of multiple cutaneous and uterine leiomyomas (MCUL1) and a germline missense mutation (R58P) in the fumarate hydratase gene (FH). We provide evidence for a founder effect for the identified mutation and distant relationship of our family to another familial case of MCUL1 associated with renal cell cancer, which was recently published with the same mutation. PMID:17908262

  12. Whole exome sequencing reveals a C-terminal germline variant in CEBPA-associated acute myeloid leukemia: 45-year follow up of a large family

    PubMed Central

    Pathak, Anand; Seipel, Katja; Pemov, Alexander; Dewan, Ramita; Brown, Christina; Ravichandran, Sarangan; Luke, Brian T.; Malasky, Michael; Suman, Shalabh; Yeager, Meredith; Gatti, Richard A.; Caporaso, Neil E.; Mulvihill, John J.; Goldin, Lynn R.; Pabst, Thomas; McMaster, Mary L.; Stewart, Douglas R.

    2016-01-01

    Familial acute myeloid leukemia is rare and linked to germline mutations in RUNX1, GATA2 or CCAAT/enhancer binding protein-α (CEBPA). We re-evaluated a large family with acute myeloid leukemia originally seen at NIH in 1969. We used whole exome sequencing to study this family, and conducted in silico bioinformatics analysis, protein structural modeling and laboratory experiments to assess the impact of the identified CEBPA Q311P mutation. Unlike most previously identified germline mutations in CEBPA, which were N-terminal frameshift mutations, we identified a novel Q311P variant that was located in the C-terminal bZip domain of C/EBPα. Protein structural modeling suggested that the Q311P mutation alters the ability of the CEBPA dimer to bind DNA. Electrophoretic mobility shift assays showed that the Q311P mu-tant had attenuated binding to DNA, as predicted by the protein modeling. Consistent with these findings, we found that the Q311P mutation has reduced transactivation, consistent with a loss-of-function mutation. From 45 years of follow up, we observed incomplete penetrance (46%) of CEBPA Q311P. This study of a large multi-generational pedigree reveals that a germline mutation in the C-terminal bZip domain can alter the ability of C/EBP-α to bind DNA and reduces transactivation, leading to acute myeloid leukemia. PMID:26721895

  13. Whole exome sequencing reveals a C-terminal germline variant in CEBPA-associated acute myeloid leukemia: 45-year follow up of a large family.

    PubMed

    Pathak, Anand; Seipel, Katja; Pemov, Alexander; Dewan, Ramita; Brown, Christina; Ravichandran, Sarangan; Luke, Brian T; Malasky, Michael; Suman, Shalabh; Yeager, Meredith; Gatti, Richard A; Caporaso, Neil E; Mulvihill, John J; Goldin, Lynn R; Pabst, Thomas; McMaster, Mary L; Stewart, Douglas R

    2016-07-01

    Familial acute myeloid leukemia is rare and linked to germline mutations in RUNX1, GATA2 or CCAAT/enhancer binding protein-α (CEBPA). We re-evaluated a large family with acute myeloid leukemia originally seen at NIH in 1969. We used whole exome sequencing to study this family, and conducted in silico bioinformatics analysis, protein structural modeling and laboratory experiments to assess the impact of the identified CEBPA Q311P mutation. Unlike most previously identified germline mutations in CEBPA, which were N-terminal frameshift mutations, we identified a novel Q311P variant that was located in the C-terminal bZip domain of C/EBPα. Protein structural modeling suggested that the Q311P mutation alters the ability of the CEBPA dimer to bind DNA. Electrophoretic mobility shift assays showed that the Q311P mu-tant had attenuated binding to DNA, as predicted by the protein modeling. Consistent with these findings, we found that the Q311P mutation has reduced transactivation, consistent with a loss-of-function mutation. From 45 years of follow up, we observed incomplete penetrance (46%) of CEBPA Q311P. This study of a large multi-generational pedigree reveals that a germline mutation in the C-terminal bZip domain can alter the ability of C/EBP-α to bind DNA and reduces transactivation, leading to acute myeloid leukemia. PMID:26721895

  14. Geopolymers prepared from DC plasma treated air pollution control (APC) residues glass: properties and characterisation of the binder phase.

    PubMed

    Kourti, Ioanna; Devaraj, Amutha Rani; Bustos, Ana Guerrero; Deegan, David; Boccaccini, Aldo R; Cheeseman, Christopher R

    2011-11-30

    Air pollution control (APC) residues have been blended with glass-forming additives and treated using DC plasma technology to produce a high calcium aluminosilicate glass (APC glass). This has been used to form geopolymer-glass composites that exhibit high strength and density, low porosity, low water absorption, low leaching and high acid resistance. The composites have a microstructure consisting of un-reacted residual APC glass particles imbedded in a complex geopolymer and C-S-H gel binder phase, and behave as particle reinforced composites. The work demonstrates that materials prepared from DC plasma treated APC residues have potential to be used to form high quality pre-cast products. PMID:21963174

  15. Specific immunotherapy of experimental myasthenia by genetically engineered APCs: the "guided missile" strategy.

    PubMed

    Drachman, D B; Wu, J-M; Miagkov, A; Williams, M A; Adams, R N; Wu, B

    2003-09-01

    Although treatment of MG with general immunosuppressive agents is often effective, it has important drawbacks, including suppression of the immune system as a whole, with the risks of infection and neoplasia, and numerous other adverse side effects. Ideally, treatment of MG should eliminate the specific pathogenic autoimmune response to AChR, without otherwise suppressing the immune system or producing other adverse side effects. Although antibodies to AChR are directly responsible for the loss of AChRs at neuromuscular junctions in MG, the AChR antibody response is T cell-dependent, and immunotherapy directed at T cells can abrogate the autoantibody response, with resulting benefit. As in other autoimmune diseases, the T cell response in MG is highly heterogeneous. The design of specific immunotherapy must take this heterogeneity into account and target the entire repertoire of AChR-specific T cells. We describe our investigation of a novel strategy for specific immunotherapy of MG, involving gene transfer to convert antigen-presenting cells (APCs) to "guided missiles" that target AChR-specific T cells, and that induce apoptosis and elimination of those T cells. This strategy uses the ability of APCs from a given individual to present the entire spectrum of AChR epitopes unique for that individual, and thereby to target the entire repertoire of antigen-specific T cells of the same individual. Using viral vectors, we have genetically engineered the APCs to process and present the most important domain of the AChR molecule, and to express a "warhead" of Fas ligand (FasL) to eliminate the activated AChR-specific T cells with which they interact. Our results show that the APCs express the appropriate gene products, and effectively and specifically eliminate AChR-specific T cells by the Fas/FasL pathway, while sparing T cells of other specificities. PMID:14592923

  16. Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C.

    PubMed

    Brown, Nicholas G; VanderLinden, Ryan; Watson, Edmond R; Weissmann, Florian; Ordureau, Alban; Wu, Kuen-Phon; Zhang, Wei; Yu, Shanshan; Mercredi, Peter Y; Harrison, Joseph S; Davidson, Iain F; Qiao, Renping; Lu, Ying; Dube, Prakash; Brunner, Michael R; Grace, Christy R R; Miller, Darcie J; Haselbach, David; Jarvis, Marc A; Yamaguchi, Masaya; Yanishevski, David; Petzold, Georg; Sidhu, Sachdev S; Kuhlman, Brian; Kirschner, Marc W; Harper, J Wade; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A

    2016-06-01

    Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination. PMID:27259151

  17. The Specificity of Targeted Vaccines for APC Surface Molecules Influences the Immune Response Phenotype

    PubMed Central

    Grødeland, Gunnveig; Mjaaland, Siri; Tunheim, Gro; Fredriksen, Agnete B.; Bogen, Bjarne

    2013-01-01

    Different diseases require different immune responses for efficient protection. Thus, prophylactic vaccines should prime the immune system for the particular type of response needed for protection against a given infectious agent. We have here tested fusion DNA vaccines which encode proteins that bivalently target influenza hemagglutinins (HA) to different surface molecules on antigen presenting cells (APC). We demonstrate that targeting to MHC class II molecules predominantly induced an antibody/Th2 response, whereas targeting to CCR1/3/5 predominantly induced a CD8+/Th1 T cell response. With respect to antibodies, the polarizing effect was even more pronounced upon intramuscular (i.m) delivery as compared to intradermal (i.d.) vaccination. Despite these differences in induced immune responses, both vaccines protected against a viral challenge with influenza H1N1. Substitution of HA with ovalbumin (OVA) demonstrated that polarization of immune responses, as a consequence of APC targeting specificity, could be extended to other antigens. Taken together, the results demonstrate that vaccination can be tailor-made to induce a particular phenotype of adaptive immune responses by specifically targeting different surface molecules on APCs. PMID:24244595

  18. The utility of Apc-mutant rats in modeling human colon cancer

    PubMed Central

    Irving, Amy A.; Yoshimi, Kazuto; Hart, Marcia L.; Parker, Taybor; Clipson, Linda; Ford, Madeline R.; Kuramoto, Takashi; Dove, William F.; Amos-Landgraf, James M.

    2014-01-01

    Prior to the advent of genetic engineering in the mouse, the rat was the model of choice for investigating the etiology of cancer. Now, recent advances in the manipulation of the rat genome, combined with a growing recognition of the physiological differences between mice and rats, have reignited interest in the rat as a model of human cancer. Two recently developed rat models, the polyposis in the rat colon (Pirc) and Kyoto Apc Delta (KAD) strains, each carry mutations in the intestinal-cancer-associated adenomatous polyposis coli (Apc) gene. In contrast to mouse models carrying Apc mutations, in which cancers develop mainly in the small intestine rather than in the colon and there is no gender bias, these rat models exhibit colonic predisposition and gender-specific susceptibility, as seen in human colon cancer. The rat also provides other experimental resources as a model organism that are not provided by the mouse: the structure of its chromosomes facilitates the analysis of genomic events, the size of its colon permits longitudinal analysis of tumor growth, and the size of biological samples from the animal facilitates multiplexed molecular analyses of the tumor and its host. Thus, the underlying biology and experimental resources of these rat models provide important avenues for investigation. We anticipate that advances in disease modeling in the rat will synergize with resources that are being developed in the mouse to provide a deeper understanding of human colon cancer. PMID:25288683

  19. Cdh1-APC/C, cyclin B-Cdc2, and Alzheimer's disease pathology

    SciTech Connect

    Aulia, Selina; Tang, Bor Luen . E-mail: bchtbl@nus.edu.sg

    2006-01-06

    The anaphase-promoting complex/cyclosome (APC/C) is a key E3 ubiquitin ligase complex that functions in regulating cell cycle transitions in proliferating cells and has, as revealed recently, novel roles in postmitotic neurons. Regulated by its activator Cdh1 (or Hct1), whose level is high in postmitotic neurons, APC/C seems to have multiple functions at different cellular locations, modulating diverse processes such as synaptic development and axonal growth. These processes do not, however, appear to be directly connected to cell cycle regulation. It is now shown that Cdh1-APC/C activity may also have a basic role in suppressing cyclin B levels, thus preventing terminally differentiated neurons from aberrantly re-entering the cell cycle. The result of an aberrant cyclin B-induced S-phase entry, at least for some of these neurons, would be death via apoptosis. Cdh1 thus play an active role in maintaining the terminally differentiated, non-cycling state of postmitotic neurons-a function that could become impaired in Alzheimer's and other neurodegenerative diseases.

  20. Ecdysone response gene E78 controls ovarian germline stem cell niche formation and follicle survival in Drosophila

    PubMed Central

    Ables, Elizabeth T.; Bois, Kelly E.; Garcia, Caroline A.; Drummond-Barbosa, Daniela

    2015-01-01

    Nuclear hormone receptors have emerged as important regulators of mammalian and Drosophila adult physiology, affecting such seemingly diverse processes as adipogenesis, carbohydrate metabolism, circadian rhythm, stem cell function, and gamete production. Although nuclear hormone receptors Ecdysone Receptor (EcR) and Ultraspiracle (Usp) have multiple known roles in Drosophila development and regulate key processes during oogenesis, the adult function of the majority of nuclear hormone receptors remains largely undescribed. Ecdysone-induced protein 78C (E78), a nuclear hormone receptor closely related to Drosophila E75 and to mammalian Rev-Erb and Peroxisome Proliferator Activated Receptors, was originally identified as an early ecdysone target; however, it has remained unclear whether E78 significantly contributes to adult physiology or reproductive function. To further explore the biological function of E78 in oogenesis, we used available E78 reporters and created a new E78 loss-of-function allele. We found that E78 is expressed throughout the germline during oogenesis, and is important for proper egg production and for the maternal control of early embryogenesis. We showed that E78 is required during development to establish the somatic germline stem cell (GSC) niche, and that E78 function in the germline promotes the survival of developing follicles. Consistent with its initial discovery as an ecdysone-induced target, we also found significant genetic interactions between E78 and components of the ecdysone signaling pathway. Taken together with the previously described roles of EcR, Usp, and E75, our results suggest that nuclear hormone receptors are critical for the broad transcriptional control of a wide variety of cellular processes during oogenesis. PMID:25624267

  1. Transcriptome Analysis of the Arabidopsis Megaspore Mother Cell Uncovers the Importance of RNA Helicases for Plant Germline Development

    PubMed Central

    Schmidt, Anja; Wuest, Samuel E.; Vijverberg, Kitty; Baroux, Célia; Kleen, Daniela; Grossniklaus, Ueli

    2011-01-01

    Germ line specification is a crucial step in the life cycle of all organisms. For sexual plant reproduction, the megaspore mother cell (MMC) is of crucial importance: it marks the first cell of the plant “germline” lineage that gets committed to undergo meiosis. One of the meiotic products, the functional megaspore, subsequently gives rise to the haploid, multicellular female gametophyte that harbours the female gametes. The MMC is formed by selection and differentiation of a single somatic, sub-epidermal cell in the ovule. The transcriptional network underlying MMC specification and differentiation is largely unknown. We provide the first transcriptome analysis of an MMC using the model plant Arabidopsis thaliana with a combination of laser-assisted microdissection and microarray hybridizations. Statistical analyses identified an over-representation of translational regulation control pathways and a significant enrichment of DEAD/DEAH-box helicases in the MMC transcriptome, paralleling important features of the animal germline. Analysis of two independent T-DNA insertion lines suggests an important role of an enriched helicase, MNEME (MEM), in MMC differentiation and the restriction of the germline fate to only one cell per ovule primordium. In heterozygous mem mutants, additional enlarged MMC-like cells, which sometimes initiate female gametophyte development, were observed at higher frequencies than in the wild type. This closely resembles the phenotype of mutants affected in the small RNA and DNA-methylation pathways important for epigenetic regulation. Importantly, the mem phenotype shows features of apospory, as female gametophytes initiate from two non-sister cells in these mutants. Moreover, in mem gametophytic nuclei, both higher order chromatin structure and the distribution of LIKE HETEROCHROMATIN PROTEIN1 were affected, indicating epigenetic perturbations. In summary, the MMC transcriptome sets the stage for future functional characterization as

  2. Increased IκBα expression is essential for the tolerogenic property of TGF-β-exposed APCs

    PubMed Central

    Ghafoori, Paiman; Yoshimura, Takeru; Turpie, Bruce; Masli, Sharmila

    2009-01-01

    IκBα is an inhibitor of the transcriptional factor NF-κB, and it is an essential component of the signaling pathways that lead to expression of inflammatory molecules. These include cytokines and costimulatory molecules associated with antigen presentation in an inflammatory immune response. In this study, we report that antigen-presenting cells exposed to TGF-β induce peripheral tolerance by increasing IκBα expression. Exposure of antigen presenting cells (APCs) to TGF-β is known to impair their ability to secrete IL-12, and such impairment correlated with reduced NF-κB activity as indicated by significantly reduced nuclear levels of p50, an essential subunit of NF-κB for IL-12 transcription. Blockade of increased nuclear IκBα in APCs by expression of small interfering RNA molecules (siRNAs) targeting IκBα transcripts prevented IL-12 impairment and the decline in nuclear p50 levels. Furthermore, such IκBα blockade also interfered with the tolerogenic property of TGF-β- exposed APCs. However, increased expression of IκBα in APCs, independent of TGF-β exposure, reduced nuclear p50 levels and permitted tolerance induction by APCs. Thus, our findings attribute a direct and significant role to IκBα in the tolerogenic potential of APCs. Increased IκBα expression in APCs may therefore offer a therapeutic approach to achieve antigen-specific immunomodulation.—Ghafoori, P., Yoshimura, T., Turpie, B., Masli, S. Increased IκBα expression is essential for the tolerogenic property of TGF-β-exposed APCs. PMID:19237504

  3. Germline mutations in the VHL tumor suppresssor gene are similar to somatic VHL aberrations in sporadic renal cell carcinoma

    SciTech Connect

    Whaley, J.M.; Naglich, J.; Gelbert, L.

    1994-09-01

    A candidate gene for von Hippel Lindau disease was recently identified that led to the isolation of a partial cDNA clone with extended open reading frame without significant homology to known genes or obvious functional motifs, except for an acidic pentamer repeat domain. To further characterize the functional domains of the VHL gene and assess its involvement in hereditary and non-hereditary tumors, we performed mutation analyses and studied its expresssion in normal and tumor tissue. We identified germline mutations in 39% of VHL disease families. Moreover, 33% of sporadic RCCs, and all (6/6) sporadic RCC cell lines analyzed, showed mutations within the VHL gene. Both germline and somatic mutations included deletions, insertions, splice site mutations, missense and nonsense mutations, all of which clustered at the 3{prime} end of the corresponding partial VHL cDNA open reading frame including an alternatively-spliced exon of 123 nucleotides in length, suggesting functionally important domains encoded by the VHL gene in this region. Over 180 sporadic tumors of other types have shown no detectable base changes within the presumed coding sequence of the VHL gene to date. We conclude that the gene causing VHL has an important and specific role in the etiology of sporadic renal cell carcinomas, acts as a recessive tumor suppressor gene, and appears to encode important functional domains within the 3{prime} end of the known open reading frame.

  4. Somatic mutations and germline sequence variants in the expressed tyrosine kinase genes of patients with de novo acute myeloid leukemia

    PubMed Central

    Xiang, Zhifu; Walgren, Richard; Zhao, Yu; Kasai, Yumi; Miner, Tracie; Ries, Rhonda E.; Lubman, Olga; Fremont, Daved H.; McLellan, Michael D.; Payton, Jacqueline E.; Westervelt, Peter; DiPersio, John F.; Link, Daniel C.; Walter, Matthew J.; Graubert, Timothy A.; Watson, Mark; Baty, Jack; Heath, Sharon; Shannon, William D.; Nagarajan, Rakesh; Bloomfield, Clara D.; Mardis, Elaine R.; Wilson, Richard K.; Ley, Timothy J.

    2008-01-01

    Activating mutations in tyrosine kinase (TK) genes (eg, FLT3 and KIT) are found in more than 30% of patients with de novo acute myeloid leukemia (AML); many groups have speculated that mutations in other TK genes may be present in the remaining 70%. We performed high-throughput resequencing of the kinase domains of 26 TK genes (11 receptor TK; 15 cytoplasmic TK) expressed in most AML patients using genomic DNA from the bone marrow (tumor) and matched skin biopsy samples (“germline”) from 94 patients with de novo AML; sequence variants were validated in an additional 94 AML tumor samples (14.3 million base pairs of sequence were obtained and analyzed). We identified known somatic mutations in FLT3, KIT, and JAK2 TK genes at the expected frequencies and found 4 novel somatic mutations, JAK1V623A, JAK1T478S, DDR1A803V, and NTRK1S677N, once each in 4 respective patients of 188 tested. We also identified novel germline sequence changes encoding amino acid substitutions (ie, nonsynonymous changes) in 14 TK genes, including TYK2, which had the largest number of nonsynonymous sequence variants (11 total detected). Additional studies will be required to define the roles that these somatic and germline TK gene variants play in AML pathogenesis. PMID:18270328

  5. A Dual Model for Prioritizing Cancer Mutations in the Non-coding Genome Based on Germline and Somatic Events

    PubMed Central

    Li, Jia; Poursat, Marie-Anne; Drubay, Damien; Motz, Arnaud; Saci, Zohra; Morillon, Antonin; Michiels, Stefan; Gautheret, Daniel

    2015-01-01

    We address here the issue of prioritizing non-coding mutations in the tumoral genome. To this aim, we created two independent computational models. The first (germline) model estimates purifying selection based on population SNP data. The second (somatic) model estimates tumor mutation density based on whole genome tumor sequencing. We show that each model reflects a different set of constraints acting either on the normal or tumor genome, and we identify the specific genome features that most contribute to these constraints. Importantly, we show that the somatic mutation model carries independent functional information that can be used to narrow down the non-coding regions that may be relevant to cancer progression. On this basis, we identify positions in non-coding RNAs and the non-coding parts of mRNAs that are both under purifying selection in the germline and protected from mutation in tumors, thus introducing a new strategy for future detection of cancer driver elements in the expressed non-coding genome. PMID:26588488

  6. A novel AXIN2 germline variant associated with attenuated FAP without signs of oligondontia or ectodermal dysplasia.

    PubMed

    Rivera, B; Perea, J; Sánchez, E; Villapún, M; Sánchez-Tomé, E; Mercadillo, F; Robledo, M; Benítez, J; Urioste, M

    2014-03-01

    Truncating mutations in the AXIN2 gene, a key regulator of β-catenin degradation in the Wnt pathway, have been reported in three families with gastrointestinal adenomatous polyposis and features of ectodermal dysplasia. However, the role of AXIN2 in familial adenomatous polyposis (FAP) syndrome is not completely understood. We performed an in-depth study of APC and MUTYH, and ruled out their implication in 23 FAP families. We then investigated the role of other genes involved in the Wnt pathway, including AXIN2, and identified a novel missense variant in AXIN2 in one family with attenuated FAP. Carriers of the variant exhibited a variable number of polyps but none showed any sign of ectodermal dysplasia. We have demonstrated the pathogenicity of this novel variant by establishing its low frequency in controls as well as by LOH analysis, a segregation study, and immunofluorescent staining of AXIN2 and β-catenin proteins. This report expands the phenotype known to be related to AXIN2 alterations and raises the question of whether to screen AXIN2 in FAP cases negative for alterations in APC and MUTYH. PMID:23838596

  7. Association between aberrant APC promoter methylation and breast cancer pathogenesis: a meta-analysis of 35 observational studies.

    PubMed

    Zhou, Dan; Tang, Weiwei; Wang, Wenyi; Pan, Xiaoyan; An, Han-Xiang; Zhang, Yun

    2016-01-01

    Background. Adenomatous polyposis coli (APC) is widely known as an antagonist of the Wnt signaling pathway via the inactivation of β-catenin. An increasing number of studies have reported that APC methylation contributes to the predisposition to breast cancer (BC). However, recent studies have yielded conflicting results. Methods. Herein, we systematically carried out a meta-analysis to assess the correlation between APC methylation and BC risk. Based on searches of the Cochrane Library, PubMed, Web of Science and Embase databases, the odds ratio (OR) with 95% confidence interval (CI) values were pooled and summarized. Results. A total of 31 articles involving 35 observational studies with 2,483 cases and 1,218 controls met the inclusion criteria. The results demonstrated that the frequency of APC methylation was significantly higher in BC cases than controls under a random effect model (OR = 8.92, 95% CI [5.12-15.52]). Subgroup analysis further confirmed the reliable results, regardless of the sample types detected, methylation detection methods applied and different regions included. Interestingly, our results also showed that the frequency of APC methylation was significantly lower in early-stage BC patients than late-stage ones (OR = 0.62, 95% CI [0.42-0.93]). Conclusion. APC methylation might play an indispensable role in the pathogenesis of BC and could be regarded as a potential biomarker for the diagnosis of BC. PMID:27478702

  8. Interphase APC/C–Cdc20 inhibition by cyclin A2–Cdk2 ensures efficient mitotic entry

    PubMed Central

    Hein, Jamin B.; Nilsson, Jakob

    2016-01-01

    Proper cell-cycle progression requires tight temporal control of the Anaphase Promoting Complex/Cyclosome (APC/C), a large ubiquitin ligase that is activated by one of two co-activators, Cdh1 or Cdc20. APC/C and Cdc20 are already present during interphase but APC/C–Cdc20 regulation during this window of the cell cycle, if any, is unknown. Here we show that cyclin A2–Cdk2 binds and phosphorylates Cdc20 in interphase and this inhibits APC/C–Cdc20 activity. Preventing Cdc20 phosphorylation results in pre-mature activation of the APC/C–Cdc20 and several substrates, including cyclin B1 and A2, are destabilized which lengthens G2 and slows mitotic entry. Expressing non-degradable cyclin A2 but not cyclin B1 restores mitotic entry in these cells. We have thus uncovered a novel positive feedback loop centred on cyclin A2–Cdk2 inhibition of interphase APC/C–Cdc20 to allow further cyclin A2 accumulation and mitotic entry. PMID:26960431

  9. Regulation of APC and AXIN2 expression by intestinal tumor suppressor CDX2 in colon cancer cells.

    PubMed

    Olsen, Anders Krüger; Coskun, Mehmet; Bzorek, Michael; Kristensen, Michael Holmsgaard; Danielsen, Erik Thomas; Jørgensen, Steffen; Olsen, Jørgen; Engel, Ulla; Holck, Susanne; Troelsen, Jesper Thorvald

    2013-06-01

    Wnt signaling is often constitutively active in colorectal cancer cells. The expression of the intestinal specific transcription factor CDX2 is found to be transiently decreased in invasive cells at the tumor/stroma interface. A recent ChIP-Seq study has indicated that several Wnt signaling-related genes are regulated by CDX2. The aim was to investigate the role of decreased CDX2 level on the expression of APC, AXIN2 and GSK3β in migrating colon cancer cells at the invasive front. CDX2-bound promoter and enhancer regions from APC, AXIN2 and GSK3β were analyzed for gene regulatory activity and the expression pattern of APC and GSK3β at the invasive front was evaluated by immunohistochemical procedures. Transfection of intestinal and non-intestinal cell lines demonstrated that CDX2 activated APC and AXIN2 promoter activities via intestinal cell-specific enhancer elements. Suppressed CDX2 expression was associated with endogenous downregulation of APC and AXIN2 expression in Caco-2 cells but did not affect GSK3β expression. Furthermore, elevated levels of nuclear β-catenin and reduced levels of cytoplasmic APC were correlated to a low CDX2 expression in migrating colon cancer cells in vivo. These results suggest that a low CDX2 level has influence on the Wnt signaling in invasive colon cancer cells possibly promoting cellular migration. PMID:23393221

  10. The APC tumor suppressor binds to C-terminal binding protein to divert nuclear beta-catenin from TCF.

    PubMed

    Hamada, Fumihiko; Bienz, Mariann

    2004-11-01

    Adenomatous polyposis coli (APC) is an important tumor suppressor in the colon. APC antagonizes the transcriptional activity of the Wnt effector beta-catenin by promoting its nuclear export and its proteasomal destruction in the cytoplasm. Here, we show that a third function of APC in antagonizing beta-catenin involves C-terminal binding protein (CtBP). APC is associated with CtBP in vivo and binds to CtBP in vitro through its conserved 15 amino acid repeats. Failure of this association results in elevated levels of beta-catenin/TCF complexes and of TCF-mediated transcription. Notably, CtBP is neither associated with TCF in vivo nor does mutation of the CtBP binding motifs in TCF-4 alter its transcriptional activity. This questions the idea that CtBP is a direct corepressor of TCF. Our evidence indicates that APC is an adaptor between beta-catenin and CtBP and that CtBP lowers the availability of free nuclear beta-catenin for binding to TCF by sequestering APC/beta-catenin complexes. PMID:15525529

  11. Association between aberrant APC promoter methylation and breast cancer pathogenesis: a meta-analysis of 35 observational studies

    PubMed Central

    Zhou, Dan; Tang, Weiwei; Wang, Wenyi; Pan, Xiaoyan

    2016-01-01

    Background. Adenomatous polyposis coli (APC) is widely known as an antagonist of the Wnt signaling pathway via the inactivation of β-catenin. An increasing number of studies have reported that APC methylation contributes to the predisposition to breast cancer (BC). However, recent studies have yielded conflicting results. Methods. Herein, we systematically carried out a meta-analysis to assess the correlation between APC methylation and BC risk. Based on searches of the Cochrane Library, PubMed, Web of Science and Embase databases, the odds ratio (OR) with 95% confidence interval (CI) values were pooled and summarized. Results. A total of 31 articles involving 35 observational studies with 2,483 cases and 1,218 controls met the inclusion criteria. The results demonstrated that the frequency of APC methylation was significantly higher in BC cases than controls under a random effect model (OR = 8.92, 95% CI [5.12–15.52]). Subgroup analysis further confirmed the reliable results, regardless of the sample types detected, methylation detection methods applied and different regions included. Interestingly, our results also showed that the frequency of APC methylation was significantly lower in early-stage BC patients than late-stage ones (OR = 0.62, 95% CI [0.42–0.93]). Conclusion. APC methylation might play an indispensable role in the pathogenesis of BC and could be regarded as a potential biomarker for the diagnosis of BC. PMID:27478702

  12. Germline mutations in BAP1 predispose to melanocytic tumors

    PubMed Central

    Wiesner, Thomas; Obenauf, Anna C.; Murali, Rajmohan; Fried, Isabella; Griewank, Klaus G.; Ulz, Peter; Windpassinger, Christian; Wackernagel, Werner; Loy, Shea; Wolf, Ingrid; Viale, Agnes; Lash, Alex E.; Pirun, Mono; Socci, Nicholas D.; Rütten, Arno; Palmedo, Gabriele; Abramson, David; Offit, Kenneth; Ott, Arthur; Becker, Jürgen C.; Cerroni, Lorenzo; Kutzner, Heinz; Bastian, Boris C.; Speicher, Michael R.

    2012-01-01

    Common acquired melanocytic nevi are benign neoplasms that are composed of small uniform melanocytes and typically present as flat or slightly elevated, pigmented lesions on the skin. We describe two families with a new autosomal dominant syndrome characterized by multiple skin-colored, elevated melanocytic tumors. In contrast to common acquired nevi, the melanocytic neoplasms in affected family members ranged histopathologically from epithelioid nevi to atypical melanocytic proliferations that showed overlapping features with melanoma. Some affected patients developed uveal or cutaneous melanomas. Segregating with this phenotype, we found inactivating germline mutations of the BAP1 gene. The majority of melanocytic neoplasms lost the remaining wild-type allele of BAP1 by various somatic alterations. In addition, we found BAP1 mutations in a subset of sporadic melanocytic neoplasms showing histologic similarities to the familial tumors. These findings suggest that loss of BAP1 is associated with a clinically and morphologically distinct type of melanocytic neoplasm. PMID:21874003

  13. Biased DNA segregation in Drosophila male germline stem cells.

    PubMed

    Yamashita, Yukiko M

    2013-01-01

    The immortal strand hypothesis, which emerged four decades ago, proposes that certain cells retain a template copy of chromosomal DNA to protect against replication-induced mutations. As the interest in stem cells rose in recent years, researchers speculated that stem cells, which must maintain proliferative capacity throughout the life of the organism, may be the population that most needs the strong protection afforded by immortal strand segregation. Alternative hypotheses have also been proposed to explain observed non-random sister chromatid segregation. We recently found that Drosophila male germline stem cells segregate sister chromatids non-randomly, but such bias was limited to the sex chromosomes. Interestingly, the biased segregation does not lead to immortal strand segregation. We will discuss the implications of this observation and molecular mechanisms, which might be applicable to non-random sister chromatid segregation in other systems as well. PMID:23707893

  14. The Development of Germline Stem Cells in Drosophila

    PubMed Central

    Dansereau, David A.; Lasko, Paul

    2009-01-01

    Summary Germline stem cells (GSCs) in Drosophila are a valuable model to explore of how adult stem cells are regulated in vivo. Genetic dissection of this system has shown that stem cell fate is determined and maintained by the stem cell’s somatic microenvironment or niche. In Drosophila gonads, the stem cell niche—the cap cell cluster in females and the hub in males—acts as a signaling center to recruit GSCs from among a small population of undifferentiated primordial germ cells (PGCs). Short-range signals from the niche specify and regulate stem cell fate by maintaining the undifferentiated state of the PGCs next to the niche. Germline cells that do not receive the niche signals because of their location assume the default fate and differentiate. Once GSCs are specified, adherens junctions maintain close association between the stem cells and their niche and help to orient stem cell division so that one daughter is displaced from the niche and differentiates. In females, stem cell fate depends on bone morphogenetic protein (BMP) signals from the cap cells; in males, hub cells express the cytokine-like ligand Unpaired, which activates the Janus kinase-signal transducers and activators of transcription (Jak-Stat) pathway in stem cells. Although the signaling pathways operating between the niche and stem cells are different, there are common general features in both males and females, including the arrangement of cell types, many of the genes used, and the logic of the system that maintains stem cell fate. PMID:18370048

  15. Closing the circle of germline and stem cells: the Primordial Stem Cell hypothesis

    PubMed Central

    2013-01-01

    Background Germline determination is believed to occur by either preformation or epigenesis. Animals that undergo germ cell specification by preformation have a continuous germline. However, animals with germline determination by epigenesis have a discontinuous germline, with somatic cells intercalated. This vision is contrary to August Weismann’s Germ Plasm Theory and has led to several controversies. Recent data from metazoans as diverse as planarians, annelids and sea urchins reveal the presence of pluripotent stem cell populations that express germ plasm components, despite being considered to be somatic. These data also show that germ plasm is continuous in some of these animals, despite their discontinuous germline. Presentation of the hypothesis Here, based on recent molecular data on germ plasm components, I revise the germline concept. I introduce the concept of primordial stem cells, which are evolutionarily conserved stem cells that carry germ plasm components from the zygote to the germ cells. These cells, delineated by the classic concept of the Weismann barrier, can contribute to different extents to somatic tissues or be present in a rudimentary state. The primordial stem cells are a part of the germline that can drive asexual reproduction. Testing the hypothesis Molecular information on the expression of germ plasm components is needed during early development of non-classic model organisms, with special attention to those capable of undergoing asexual reproduction and regeneration. The cell lineage of germ plasm component-containing cells will also shed light on their position with respect to the Weismann barrier. This information will help in understanding the germline and its associated stem cells across metazoan phylogeny. Implications of the hypothesis This revision of the germline concept explains the extensive similarities observed among stem cells and germline cells in a wide variety of animals, and predicts the expression of germ plasm

  16. Protein synthesis and degradation are essential to regulate germline stem cell homeostasis in Drosophila testes.

    PubMed

    Yu, Jun; Lan, Xiang; Chen, Xia; Yu, Chao; Xu, Yiwen; Liu, Yujuan; Xu, Lingna; Fan, Heng-Yu; Tong, Chao

    2016-08-15

    The homeostasis of self-renewal and differentiation in stem cells is controlled by intrinsic signals and their niche. We conducted a large-scale RNA interference (RNAi) screen in Drosophila testes and identified 221 genes required for germline stem cell (GSC) maintenance or differentiation. Knockdown of these genes in transit-amplifying spermatogonia and cyst cells further revealed various phenotypes. Complex analysis uncovered that many of the identified genes are involved in key steps of protein synthesis and degradation. A group of genes that are required for mRNA splicing and protein translation contributes to both GSC self-renewal and early germ cell differentiation. Loss of genes in the protein degradation pathway in cyst cells leads to testis tumors consisting of overproliferated germ cells. Importantly, in the Cullin 4-RING E3 ubiquitin ligase (CRL4) complex, we identified multiple proteins that are crucial to GSC self-renewal: pic/DDB1, a CRL4 linker protein, is not only required for GSC self-renewal in flies but also for maintenance of spermatogonial stem cells (SSCs) in mice. PMID:27471256

  17. Drosophila Homologues of Adenomatous Polyposis Coli (APC) and the Formin Diaphanous Collaborate by a Conserved Mechanism to Stimulate Actin Filament Assembly*

    PubMed Central

    Jaiswal, Richa; Stepanik, Vince; Rankova, Aneliya; Molinar, Olivia; Goode, Bruce L.; McCartney, Brooke M.

    2013-01-01

    Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad range of animal phyla. PMID:23558679

  18. Identification of regions interacting with ovo{sup D} mutations: Potential new genes involved in germline sex determination or differentiation in Drosophila melanogaster

    SciTech Connect

    Pauli, D.; Oliver, B.; Mahowald, A.P.

    1995-02-01

    Only a few Drosophila melanogaster germline sex determination genes are known, and there have been no systematic screens to identify new genes involved in this important biological process. The ovarian phenotypes produced by females mutant for dominant alleles of the ovo gene are modified in flies with altered doses of other loci involved in germline sex determination in Drosophila (Sex-lethal{sup +}, snas fille{sup +} and ovarian tumor{sup +}). This observation constitutes the basis for a screen to identify additional genes required for proper establishment of germline sexual identity. We tested 300 deletions, which together cover {approximately}58% of the euchromatic portion of the genome, for genetic interactions with ovo{sup D}. Hemizygosity for more than a dozen small regions show interactions that either partially suppress or enhance the ovarian phenotypes of females mutant for one or more of the three dominant ovo mutations. These regions probably contain genes whose products act in developmental heirarchies that include ovo{sup +} protein. 40 refs, 7 figs., 5 tabs.

  19. A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice.

    PubMed

    Waaler, Jo; Machon, Ondrej; Tumova, Lucie; Dinh, Huyen; Korinek, Vladimir; Wilson, Steven Ray; Paulsen, Jan Erik; Pedersen, Nina Marie; Eide, Tor J; Machonova, Olga; Gradl, Dietmar; Voronkov, Andrey; von Kries, Jens Peter; Krauss, Stefan

    2012-06-01

    Increased nuclear accumulation of β-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/β-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identified a novel small molecule inhibitor of the β-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the β-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the β-catenin destruction complex, followed by increased degradation of β-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of β-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our findings provide a novel chemotype for targeting canonical Wnt/β-catenin signaling through inhibiting the PARP domain of TNKS1/2. PMID:22440753

  20. Germline mutations in PMS2 and MLH1 in individuals with solitary loss of PMS2 expression in colorectal carcinomas from the Colon Cancer Family Registry Cohort

    PubMed Central

    Rosty, Christophe; Clendenning, Mark; Walsh, Michael D; Eriksen, Stine V; Southey, Melissa C; Winship, Ingrid M; Macrae, Finlay A; Boussioutas, Alex; Parry, Susan; Arnold, Julie; Young, Joanne P; Casey, Graham; Haile, Robert W; Gallinger, Steven; Le Marchand, Loïc; Newcomb, Polly A; Potter, John D; DeRycke, Melissa; Lindor, Noralane M; Thibodeau, Stephen N; Baron, John A; Win, Aung Ko; Hopper, John L; Jenkins, Mark A; Buchanan, Daniel D

    2016-01-01

    Objectives Immunohistochemistry for DNA mismatch repair proteins is used to screen for Lynch syndrome in individuals with colorectal carcinoma (CRC). Although solitary loss of PMS2 expression is indicative of carrying a germline mutation in PMS2, previous studies reported MLH1 mutation in some cases. We determined the prevalence of MLH1 germline mutations in a large cohort of individuals with a CRC demonstrating solitary loss of PMS2 expression. Design This cohort study included 88 individuals affected with a PMS2-deficient CRC from the Colon Cancer Family Registry Cohort. Germline PMS2 mutation analysis (long-range PCR and multiplex ligation-dependent probe amplification) was followed by MLH1 mutation testing (Sanger sequencing and multiplex ligation-dependent probe amplification). Results Of the 66 individuals with complete mutation screening, we identified a pathogenic PMS2 mutation in 49 (74%), a pathogenic MLH1 mutation in 8 (12%) and a MLH1 variant of uncertain clinical significance predicted to be damaging by in silico analysis in 3 (4%); 6 (9%) carried variants likely to have no clinical significance. Missense point mutations accounted for most alterations (83%; 9/11) in MLH1. The MLH1 c.113A> G p.Asn38Ser mutation was found in 2 related individuals. One individual who carried the MLH1 intronic mutation c.677+3A>G p.Gln197Argfs*8 leading to the skipping of exon 8, developed 2 tumours, both of which retained MLH1 expression. Conclusions A substantial proportion of CRCs with solitary loss of PMS2 expression are associated with a deleterious MLH1 germline mutation supporting the screening for MLH1 in individuals with tumours of this immunophenotype, when no PMS2 mutation has been identified. PMID:26895986

  1. Tumor Mismatch Repair Immunohistochemistry and DNA MLH1 Methylation Testing of Patients With Endometrial Cancer Diagnosed at Age Younger Than 60 Years Optimizes Triage for Population-Level Germline Mismatch Repair Gene Mutation Testing

    PubMed Central

    Buchanan, Daniel D.; Tan, Yen Y.; Walsh, Michael D.; Clendenning, Mark; Metcalf, Alexander M.; Ferguson, Kaltin; Arnold, Sven T.; Thompson, Bryony A.; Lose, Felicity A.; Parsons, Michael T.; Walters, Rhiannon J.; Pearson, Sally-Ann; Cummings, Margaret; Oehler, Martin K.; Blomfield, Penelope B.; Quinn, Michael A.; Kirk, Judy A.; Stewart, Colin J.; Obermair, Andreas; Young, Joanne P.; Webb, Penelope M.; Spurdle, Amanda B.

    2014-01-01

    Purpose Clinicopathologic data from a population-based endometrial cancer cohort, unselected for age or family history, were analyzed to determine the optimal scheme for identification of patients with germline mismatch repair (MMR) gene mutations. Patients and Methods Endometrial cancers from 702 patients recruited into the Australian National Endometrial Cancer Study (ANECS) were tested for MMR protein expression using immunohistochemistry (IHC) and for MLH1 gene promoter methylation in MLH1-deficient cases. MMR mutation testing was performed on germline DNA of patients with MMR-protein deficient tumors. Prediction of germline mutation status was compared for combinations of tumor characteristics, age at diagnosis, and various clinical criteria (Amsterdam, Bethesda, Society of Gynecologic Oncology, ANECS). Results Tumor MMR-protein deficiency was detected in 170 (24%) of 702 cases. Germline testing of 158 MMR-deficient cases identified 22 truncating mutations (3% of all cases) and four unclassified variants. Tumor MLH1 methylation was detected in 99 (89%) of 111 cases demonstrating MLH1/PMS2 IHC loss; all were germline MLH1 mutation negative. A combination of MMR IHC plus MLH1 methylation testing in women younger than 60 years of age at diagnosis provided the highest positive predictive value for the identification of mutation carriers at 46% versus ≤ 41% for any other criteria considered. Conclusion Population-level identification of patients with MMR mutation-positive endometrial cancer is optimized by stepwise testing for tumor MMR IHC loss in patients younger than 60 years, tumor MLH1 methylation in individuals with MLH1 IHC loss, and germline mutations in patients exhibiting loss of MSH6, MSH2, or PMS2 or loss of MLH1/PMS2 with absence of MLH1 methylation. PMID:24323032

  2. A Small RNA-Catalytic Argonaute Pathway Tunes Germline Transcript Levels to Ensure Embryonic Divisions.

    PubMed

    Gerson-Gurwitz, Adina; Wang, Shaohe; Sathe, Shashank; Green, Rebecca; Yeo, Gene W; Oegema, Karen; Desai, Arshad

    2016-04-01

    Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here, we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by the density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to the emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition. PMID:27020753

  3. Fast Functional Germline and Epigenetic Assays in the Nematode Caenorhabditis elegans.

    PubMed

    Lundby, Zachary; Camacho, Jessica; Allard, Patrick

    2016-01-01

    Germ cells are unique in their ability to transfer traits and genetic information from one generation to the next. The proper development and integrity of their genome are therefore of utmost importance for the health of organisms and survival of species. Many features of mammalian germ cells, including their long development span and difficulty of access, present challenges for their study in the context of toxicity assays. In light of these barriers, the model system Caenorhabditis elegans shows great potential given its ease of manipulation and genetic tractability which can be easily adapted for high-throughput analysis. In this chapter, we discuss the advantages of examining germ cell processes in C. elegans, and describe three functional germline assays for the examination of chemical impact on germline maintenance and function including assays probing germ cell differentiation, germline apoptosis, and germline epigenetic regulation. PMID:27518628

  4. A C. elegans Screening Platform for the Rapid Assessment of Chemical Disruption of Germline Function

    PubMed Central

    Allard, Patrick; Kleinstreuer, Nicole C.; Knudsen, Thomas B.

    2013-01-01

    Background: Despite the developmental impact of chromosome segregation errors, we lack the tools to assess environmental effects on the integrity of the germline in animals. Objectives: We developed an assay in Caenorhabditis elegans that fluorescently marks aneuploid embryos after chemical exposure. Methods: We qualified the predictive value of the assay against chemotherapeutic agents as well as environmental compounds from the ToxCast Phase I library by comparing results from the C. elegans assay with the comprehensive mammalian in vivo end point data from the ToxRef database. Results: The assay was highly predictive of mammalian reproductive toxicities, with a 69% maximum balanced accuracy. We confirmed the effect of select compounds on germline integrity by monitoring germline apoptosis and meiotic progression. Conclusions: This C. elegans assay provides a comprehensive strategy for assessing environmental effects on germline function. PMID:23603051

  5. Transposon-Based Reporter Marking Provides Functional Evidence for Intercellular Bridges in the Male Germline of Rabbits.

    PubMed

    Hoffmann, Orsolya I; Kerekes, Andrea; Lipták, Nandor; Hiripi, Laszlo; Bodo, Szilard; Szaloki, Gabor; Klein, Sabine; Ivics, Zoltan; Kues, Wilfried A; Bosze, Zsuzsanna

    2016-01-01

    The Sleeping Beauty transposon system was established as a robust and efficient method for germline transgenesis in different mammalian species. The generation of transgenic mice, rats, rabbits and swine carrying an identical Venus reporter construct delivered by transposon-mediated gene transfer enables comparative studies of gene expression in these lines of mammalian models. Whereas comparable expression patterns of the Venus reporter were found in somatic tissues, preliminary studies suggested that a striking difference in reporter expression may exist in mature spermatozoa of these species. Here we clearly show the differential expression of Venus reporter protein during spermatogenesis of the two compared species, the laboratory rabbit and mice. We provide evidence for the functionality of intercellular bridges in the male germline and genotype-independent transgenic phenotype of rabbit spermatids. Our data suggest that the reporter rabbit line may be a suitable tool to identify molecular mechanisms in testicular development, and may contribute to develop better animal models for male infertility in men. PMID:27148973

  6. Maternal Germline-Specific Genes in the Asian Malaria Mosquito Anopheles stephensi: Characterization and Application for Disease Control

    PubMed Central

    Biedler, James K.; Qi, Yumin; Pledger, David; James, Anthony A.; Tu, Zhijian

    2014-01-01

    Anopheles stephensi is a principal vector of urban malaria on the Indian subcontinent and an emerging model for molecular and genetic studies of mosquito biology. To enhance our understanding of female mosquito reproduction, and to develop new tools for basic research and for genetic strategies to control mosquito-borne infectious diseases, we identified 79 genes that displayed previtellogenic germline-specific expression based on RNA-Seq data generated from 11 life stage–specific and sex-specific samples. Analysis of this gene set provided insights into the biology and evolution of female reproduction. Promoters from two of these candidates, vitellogenin receptor and nanos, were used in independent transgenic cassettes for the expression of artificial microRNAs against suspected mosquito maternal-effect genes, discontinuous actin hexagon and myd88. We show these promoters have early germline-specific expression and demonstrate 73% and 42% knockdown of myd88 and discontinuous actin hexagon mRNA in ovaries 48 hr after blood meal, respectively. Additionally, we demonstrate maternal-specific delivery of mRNA and protein to progeny embryos. We discuss the application of this system of maternal delivery of mRNA/miRNA/protein in research on mosquito reproduction and embryonic development, and for the development of a gene drive system based on maternal-effect dominant embryonic arrest. PMID:25480960

  7. Transposon-Based Reporter Marking Provides Functional Evidence for Intercellular Bridges in the Male Germline of Rabbits

    PubMed Central

    Hoffmann, Orsolya I.; Kerekes, Andrea; Lipták, Nandor; Hiripi, Laszlo; Bodo, Szilard; Szaloki, Gabor; Klein, Sabine; Ivics, Zoltan; Kues, Wilfried A.; Bosze, Zsuzsanna

    2016-01-01

    The Sleeping Beauty transposon system was established as a robust and efficient method for germline transgenesis in different mammalian species. The generation of transgenic mice, rats, rabbits and swine carrying an identical Venus reporter construct delivered by transposon-mediated gene transfer enables comparative studies of gene expression in these lines of mammalian models. Whereas comparable expression patterns of the Venus reporter were found in somatic tissues, preliminary studies suggested that a striking difference in reporter expression may exist in mature spermatozoa of these species. Here we clearly show the differential expression of Venus reporter protein during spermatogenesis of the two compared species, the laboratory rabbit and mice. We provide evidence for the functionality of intercellular bridges in the male germline and genotype-independent transgenic phenotype of rabbit spermatids. Our data suggest that the reporter rabbit line may be a suitable tool to identify molecular mechanisms in testicular development, and may contribute to develop better animal models for male infertility in men. PMID:27148973

  8. An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline.

    PubMed

    Frøkjær-Jensen, Christian; Jain, Nimit; Hansen, Loren; Davis, M Wayne; Li, Yongbin; Zhao, Di; Rebora, Karine; Millet, Jonathan R M; Liu, Xiao; Kim, Stuart K; Dupuy, Denis; Jorgensen, Erik M; Fire, Andrew Z

    2016-07-14

    Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here, we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-base pair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure. PMID:27374334

  9. Germline mutations in the proof-reading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas

    PubMed Central

    Palles, Claire; Cazier, Jean-Baptiste; Howarth, Kimberley M; Domingo, Enric; Jones, Angela M.; Broderick, Peter; Kemp, Zoe; Spain, Sarah L; Almeida, Estrella Guarino; Salguero, Israel; Sherborne, Amy; Chubb, Daniel; Carvajal-Carmona, Luis G; Ma, Yusanne; Kaur, Kulvinder; Dobbins, Sara; Barclay, Ella; Gorman, Maggie; Martin, Lynn; Kovac, Michal B; Humphray, Sean; Lucassen, Anneke; Holmes, Christopher; Bentley, David; Donnelly, Peter; Taylor, Jenny; Petridis, Christos; Roylance, Rebecca; Sawyer, Elinor J; Kerr, David J.; Clark, Susan; Grimes, Jonathan; Kearsey, Stephen E; Thomas, Huw JW; McVean, Gilean; Houlston, Richard S; Tomlinson, Ian

    2013-01-01

    Many individuals with multiple or large colorectal adenomas, or early-onset colorectal cancer (CRC), have no detectable germline mutations in the known cancer predisposition genes. Using whole-genome sequencing, supplemented by linkage and association analysis, we identified specific heterozygous POLE or POLD1 germline variants in several multiple adenoma and/or CRC cases, but in no controls. The susceptibility variants appear to have high penetrance. POLD1 is also associated with endometrial cancer predisposition. The mutations map to equivalent sites in the proof-reading (exonuclease) domain of DNA polymerases ε and δ, and are predicted to impair correction of mispaired bases inserted during DNA replication. In agreement with this prediction, mutation carriers’ tumours were microsatellite-stable, but tended to acquire base substitution mutations, as confirmed by yeast functional assays. Further analysis of published data showed that the recently-described group of hypermutant, microsatellite-stable CRCs is likely to be caused by somatic POLE exonuclease domain mutations. PMID:23263490

  10. Hoyeraal-Hreidarsson syndrome caused by a germline mutation in the TEL patch of the telomere protein TPP1.

    PubMed

    Kocak, Hande; Ballew, Bari J; Bisht, Kamlesh; Eggebeen, Rebecca; Hicks, Belynda D; Suman, Shalabh; O'Neil, Adri; Giri, Neelam; Maillard, Ivan; Alter, Blanche P; Keegan, Catherine E; Nandakumar, Jayakrishnan; Savage, Sharon A

    2014-10-01

    Germline mutations in telomere biology genes cause dyskeratosis congenita (DC), an inherited bone marrow failure and cancer predisposition syndrome. DC is a clinically heterogeneous disorder diagnosed by the triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia; Hoyeraal-Hreidarsson syndrome (HH), a clinically severe variant of DC, also includes cerebellar hypoplasia, immunodeficiency, and intrauterine growth retardation. Approximately 70% of DC cases are associated with a germline mutation in one of nine genes, the products of which are all involved in telomere biology. Using exome sequencing, we identified mutations in Adrenocortical Dysplasia Homolog (ACD) (encoding TPP1), a component of the telomeric shelterin complex, in one family affected by HH. The proband inherited a deletion from his father and a missense mutation from his mother, resulting in extremely short telomeres and a severe clinical phenotype. Characterization of the mutations revealed that the single-amino-acid deletion affecting the TEL patch surface of the TPP1 protein significantly compromises both telomerase recruitment and processivity, while the missense mutation in the TIN2-binding region of TPP1 is not as clearly deleterious to TPP1 function. Our results emphasize the critical roles of the TEL patch in proper stem cell function and demonstrate that TPP1 is the second shelterin component (in addition to TIN2) to be implicated in DC. PMID:25233904

  11. Eukaryotic translation initiation factor 5B activity regulates larval growth rate and germline development in Caenorhabditis elegans.

    PubMed

    Yu, Xiang; Vought, Valarie E; Conradt, Barbara; Maine, Eleanor M

    2006-09-01

    In C. elegans, a population of proliferating germ cells is maintained via GLP-1/Notch signaling; in the absence of GLP-1 signaling, germ cells prematurely enter meiosis and differentiate. We previously identified ego (enhancer of glp-1) genes that promote germline proliferation and interact genetically with the GLP-1 signaling pathway. Here, we report that iffb-1 (initiation factor five B) is an ego gene. iffb-1 encodes the sole C. elegans isoform of eukaryotic translation initiation factor 5B, a protein essential for translation. We have used RNA interference and a deletion mutation to determine the developmental consequences of reduced iffb-1 activity. Our data indicate that maternal iffb-1 gene expression is sufficient for embryogenesis, and zygotic iffb-1 expression is required for development beyond late L1/early L2 stage. Partial reduction in iffb-1 expression delays larval development and can severely disrupt proliferation and differentiation of germ cells. We hypothesize that germline development is particularly sensitive to iffb-1 expression level. PMID:16937415

  12. Germline viral “fossils” guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus

    PubMed Central

    Smith, Richard H.; Hallwirth, Claus V.; Westerman, Michael; Hetherington, Nicola A.; Tseng, Yu-Shan; Cecchini, Sylvain; Virag, Tamas; Ziegler, Mona-Larissa; Rogozin, Igor B.; Koonin, Eugene V.; Agbandje-McKenna, Mavis; Kotin, Robert M.; Alexander, Ian E.

    2016-01-01

    Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV “fossils” provide novel capsid sequences for use in translational research and clinical applications. PMID:27377618

  13. Struma Ovarii With Malignant Transformation and Germline KIT Mutation: A Case Report With Review of the Literature.

    PubMed

    Ma, Deqin; Guseva, Natalya V; Dahmoush, Laila; Robinson, Robert A

    2016-09-01

    Struma ovarii accounts for 5% of ovarian teratomas. Malignant transformation occurs in <0.3%, however, the underlying molecular mechanism is unknown. We report a patient with follicular variant and tall cell variant of papillary thyroid carcinoma (PTC) arising from struma ovarii and coexisting incidental PTC in the thyroid. Mutation analysis by next-generation sequencing identified a novel germline mutation, KIT p.V530I mutation in the tumors and normal ovarian and thyroid tissue. Immunohistochemical staining showed loss of KIT expression in the PTCs. Activating mutations in KIT play an important role in diagnosis and prognosis of multiple malignancies including mastocytosis, gastrointestinal stromal tumors, and a subset of melanoma and acute myeloid leukemia. The p.V530I mutation has only been reported in 3 previous cases: acute myeloid leukemia, aggressive fibromatosis, and adenocarcinoma of the colon. In the case of aggressive fibromatosis, the patient responded well to imatinib treatment. KIT mutations have never been reported in thyroid carcinomas. This is the first case of PTC-harboring KIT mutation. Although more work needs to be done to elucidate the significance of this germline mutation, the response of the fibromatosis patient to imatinib may shed light on targeted therapy in PTC harboring this mutation. PMID:27258816

  14. Germline viral "fossils" guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus.

    PubMed

    Smith, Richard H; Hallwirth, Claus V; Westerman, Michael; Hetherington, Nicola A; Tseng, Yu-Shan; Cecchini, Sylvain; Virag, Tamas; Ziegler, Mona-Larissa; Rogozin, Igor B; Koonin, Eugene V; Agbandje-McKenna, Mavis; Kotin, Robert M; Alexander, Ian E

    2016-01-01

    Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV "fossils" provide novel capsid sequences for use in translational research and clinical applications. PMID:27377618

  15. Copy number variants and rasopathies: germline KRAS duplication in a patient with syndrome including pigmentation abnormalities.

    PubMed

    Gilbert-Dussardier, Brigitte; Briand-Suleau, Audrey; Laurendeau, Ingrid; Bilan, Frédéric; Cavé, Hélène; Verloes, Alain; Vidaud, Michel; Vidaud, Dominique; Pasmant, Eric

    2016-01-01

    RAS/MAPK pathway germline mutations were described in Rasopathies, a class of rare genetic syndromes combining facial abnormalities, heart defects, short stature, skin and genital abnormalities, and mental retardation. The majority of the mutations identified in the Rasopathies are point mutations which increase RAS/MAPK pathway signaling. Duplications encompassing RAS/MAPK pathway genes (PTPN11, RAF1, MEK2, or SHOC2) were more rarely described. Here we report, a syndromic familial case of a 12p duplication encompassing the dosage sensitive gene KRAS, whose phenotype overlapped with rasopathies. The patient was referred because of a history of mild learning disabilities, small size, facial dysmorphy, and pigmentation abnormalities (café-au-lait and achromic spots, and axillar lentigines). This phenotype was reminiscent of rasopathies. No mutation was identified in the most common genes associated with Noonan, cardio-facio-cutaneous, Legius, and Costello syndromes, as well as neurofibromatosis type 1. The patient constitutional DNA exhibited a ~10.5 Mb duplication at 12p, including the KRAS gene. The index case's mother carried the same chromosome abnormality and also showed development delay with short stature, and numerous café-au-lait spots. Duplication of the KRAS gene may participate in the propositus phenotype, in particular of the specific pigmentation abnormalities. Array-CGH or some other assessment of gene/exon CNVs of RAS/MAPK pathway genes should be considered in the evaluation of individuals with rasopathies. PMID:27450488

  16. Human germline and pan-cancer variomes and their distinct functional profiles

    PubMed Central

    Pan, Yang; Karagiannis, Konstantinos; Zhang, Haichen; Dingerdissen, Hayley; Shamsaddini, Amirhossein; Wan, Quan; Simonyan, Vahan; Mazumder, Raja

    2014-01-01

    Identification of non-synonymous single nucleotide variations (nsSNVs) has exponentially increased due to advances in Next-Generation Sequencing technologies. The functional impacts of these variations have been difficult to ascertain because the corresponding knowledge about sequence functional sites is quite fragmented. It is clear that mapping of variations to sequence functional features can help us better understand the pathophysiological role of variations. In this study, we investigated the effect of nsSNVs on more than 17 common types of post-translational modification (PTM) sites, active sites and binding sites. Out of 1 705 285 distinct nsSNVs on 259 216 functional sites we identified 38 549 variations that significantly affect 10 major functional sites. Furthermore, we found distinct patterns of site disruptions due to germline and somatic nsSNVs. Pan-cancer analysis across 12 different cancer types led to the identification of 51 genes with 106 nsSNV affected functional sites found in 3 or more cancer types. 13 of the 51 genes overlap with previously identified Significantly Mutated Genes (Nature. 2013 Oct 17;502(7471)). 62 mutations in these 13 genes affecting functional sites such as DNA, ATP binding and various PTM sites occur across several cancers and can be prioritized for additional validation and investigations. PMID:25232094

  17. Multiple Pathways Suppress Telomere Addition to DNA Breaks in the Drosophila Germline

    PubMed Central

    Beaucher, Michelle; Zheng, Xiao-Feng; Amariei, Flavia; Rong, Yikang S.

    2012-01-01

    Telomeres protect chromosome ends from being repaired as double-strand breaks (DSBs). Just as DSB repair is suppressed at telomeres, de novo telomere addition is suppressed at the site of DSBs. To identify factors responsible for this suppression, we developed an assay to monitor de novo telomere formation in Drosophila, an organism in which telomeres can be established on chromosome ends with essentially any sequence. Germline expression of the I-SceI endonuclease resulted in precise telomere formation at its cut site with high efficiency. Using this assay, we quantified the frequency of telomere formation in different genetic backgrounds with known or possible defects in DNA damage repair. We showed that disruption of DSB repair factors (Rad51 or DNA ligase IV) or DSB sensing factors (ATRIP or MDC1) resulted in more efficient telomere formation. Interestingly, partial disruption of factors that normally regulate telomere protection (ATM or NBS) also led to higher frequencies of telomere formation, suggesting that these proteins have opposing roles in telomere maintenance vs. establishment. In the ku70 mutant background, telomere establishment was preceded by excessive degradation of DSB ends, which were stabilized upon telomere formation. Most strikingly, the removal of ATRIP caused a dramatic increase in telomeric retrotransposon attachment to broken ends. Our study identifies several pathways thatsuppress telomere addition at DSBs, paving the way for future mechanistic studies. PMID:22446318

  18. Germline De Novo Mutations in GNB1 Cause Severe Neurodevelopmental Disability, Hypotonia, and Seizures.

    PubMed

    Petrovski, Slavé; Küry, Sébastien; Myers, Candace T; Anyane-Yeboa, Kwame; Cogné, Benjamin; Bialer, Martin; Xia, Fan; Hemati, Parisa; Riviello, James; Mehaffey, Michele; Besnard, Thomas; Becraft, Emily; Wadley, Alexandrea; Politi, Anya Revah; Colombo, Sophie; Zhu, Xiaolin; Ren, Zhong; Andrews, Ian; Dudding-Byth, Tracy; Schneider, Amy L; Wallace, Geoffrey; Rosen, Aaron B I; Schelley, Susan; Enns, Gregory M; Corre, Pierre; Dalton, Joline; Mercier, Sandra; Latypova, Xénia; Schmitt, Sébastien; Guzman, Edwin; Moore, Christine; Bier, Louise; Heinzen, Erin L; Karachunski, Peter; Shur, Natasha; Grebe, Theresa; Basinger, Alice; Nguyen, Joanne M; Bézieau, Stéphane; Wierenga, Klaas; Bernstein, Jonathan A; Scheffer, Ingrid E; Rosenfeld, Jill A; Mefford, Heather C; Isidor, Bertrand; Goldstein, David B

    2016-05-01

    Whole-exome sequencing of 13 individuals with developmental delay commonly accompanied by abnormal muscle tone and seizures identified de novo missense mutations enriched within a sub-region of GNB1, a gene encoding the guanine nucleotide-binding protein subunit beta-1, Gβ. These 13 individuals were identified among a base of 5,855 individuals recruited for various undiagnosed genetic disorders. The probability of observing 13 or more de novo mutations by chance among 5,855 individuals is very low (p = 7.1 × 10(-21)), implicating GNB1 as a genome-wide-significant disease-associated gene. The majority of these 13 mutations affect known Gβ binding sites, which suggests that a likely disease mechanism is through the disruption of the protein interface required for Gα-Gβγ interaction (resulting in a constitutively active Gβγ) or through the disruption of residues relevant for interaction between Gβγ and certain downstream effectors (resulting in reduced interaction with the effectors). Strikingly, 8 of the 13 individuals recruited here for a neurodevelopmental disorder have a germline de novo GNB1 mutation that overlaps a set of five recurrent somatic tumor mutations for which recent functional studies demonstrated a gain-of-function effect due to constitutive activation of G protein downstream signaling cascades for some of the affected residues. PMID:27108799

  19. Somatic and germ-line mosaicism in Rubinstein-Taybi syndrome.

    PubMed

    Chiang, Pei-Wen; Lee, Ni-Chung; Chien, Nancy; Hwu, Wuh-Liang; Spector, Elaine; Tsai, Anne Chun-Hui

    2009-07-01

    Rubinstein-Taybi syndrome (RSTS) is a rare autosomal dominant genetic disease and is characterized by mental retardation, distinctive facial features, broad and often angulated thumbs and great toes, short stature, and growth retardation. CREBBP and EP300 are the only genes currently known to be associated with RSTS. Mutations in CREBBP and EP300 were identified in approximately 50% and 3% of RSTS patients, respectively. To date, most of CREBBP mutations were de novo mutations and the recurrence rate in a family was low. Families with more than one affected child are extremely rare. In this study, we have shown a family with two affected siblings; the same mutation was found in both siblings. However, the mutation was not found in the blood or saliva DNA samples from the parents, suggesting the mechanism of germ-line mosaicism. In addition, we identified low-level mosaicism of a CREBBP mutation in the father from a second family with one affected child. Among the three analyzed tissue samples from the father, low-level mosaicism is present only significantly in the blood sample. We hypothesize mutations in CREBBP in these two families occur in the postzygotic stage in one of the parents (one generation ahead) of the affected individual. Additional family studies are required to determine how common somatic and/or gonadal mosaicism is present in RSTS patients. PMID:19533794

  20. Alterations in K-ras, APC and p53-multiple genetic pathway in colorectal cancer among Indians.

    PubMed

    Malhotra, Pooja; Anwar, Mumtaz; Nanda, Neha; Kochhar, Rakesh; Wig, Jai Dev; Vaiphei, Kim; Mahmood, Safrun

    2013-06-01

    The incidence of colorectal cancer (CRC) is increasing rapidly in Asian countries during the past few decades, but no comprehensive analysis has been done to find out the exact cause of this disease. In this study, we investigated the frequencies of mutations and expression pattern of K-ras, APC (adenomatosis polyposis coli) and p53 in tumor, adjoining and distant normal mucosa and to correlate these alterations with patients clinicopathological parameters as well as with the survival. Polymerase chain reaction (PCR)-restriction digestion was used to detect mutations in K-ras and PCR-SSCP (Single Strand Conformation Polymorphism) followed by DNA sequencing was used to detect mutations in APC and p53 genes. Immunohistochemistry was used to detect the expression pattern of K-ras, APC and p53 proteins. The frequencies of mutations of K-ras, APC and p53 in 30 tumor tissues samples were 26.7 %, 46.7 % and 20 %, respectively. Only 3.3 % of tumors contained mutations in all the three genes. The most common combination of mutation was APC and p53 whereas mutation in both p53 and K-ras were extremely rare. There was no association between the mutations and expression pattern of K-ras, APC and p53 (p>0.05). In Indians, the frequency of alterations of K-ras and APC is similar as in Westerns, whereas the frequency of p53 mutation is slightly lower. The lack of multiple mutations in tumor specimens suggests that these genetic alterations might have independent influences on CRC development and there could be multiple alternative genetic pathways to CRC in our present study cohort. PMID:23526092

  1. Promoter methylation of APC and RAR-β genes as prognostic markers in non-small cell lung cancer (NSCLC).

    PubMed

    Feng, Hongxiang; Zhang, Zhenrong; Qing, Xin; Wang, Xiaowei; Liang, Chaoyang; Liu, Deruo

    2016-02-01

    Aberrant promoter hypermethylations of tumor suppressor genes are promising markers for lung cancer diagnosis and prognosis. The purpose of this study was to determine methylation status at APC and RAR-β promoters in primary NSCLC, and whether they have any relationship with survival. APC and RAR-β promoter methylation status were determined in 41 NSCLC patients using methylation specific PCR. APC promoter methylation was detectable in 9 (22.0%) tumor samples and 6 (14.6%) corresponding non-tumor samples (P=0.391). RAR-β promoter methylation was detectable in 13 (31.7%) tumor samples and 4 (9.8%) corresponding non-tumor samples (P=0.049) in the NSCLC patients. APC promoter methylation was found to be associated with T stage (P=0.046) and nodal status (P=0.019) in non-tumor samples, and with smoking (P=0.004) in tumor samples. RAR-β promoter methylation was found associated with age (P=0.031) in non-tumor samples and with primary tumor site in tumor samples. Patients with APC promoter methylation in tumor samples showed significantly longer survival than patients without it (Log-rank P=0.014). In a multivariate analysis of prognostic factors, APC methylation in tumor samples was an independent prognostic factor (P=0.012), as were N1 positive lymph node number (P=0.025) and N2 positive lymph node number (P=0.06). Our study shows that RAR-β methylation detected in lung tissue may be used as a predictive marker for NSCLC diagnosis and that APC methylation in tumor sample may be a useful marker for superior survival in NSCLC patients. PMID:26681652

  2. Study of Cyclin Proteolysis in Anaphase-Promoting Complex (APC) Mutant Cells Reveals the Requirement for APC Function in the Final Steps of the Fission Yeast Septation Initiation Network

    PubMed Central

    Chang, Louise; Morrell, Jennifer L.; Feoktistova, Anna; Gould, Kathleen L.

    2001-01-01

    Cytokinesis in eukaryotic cells requires the inactivation of mitotic cyclin-dependent kinase complexes. An apparent exception to this relationship is found in Schizosaccharomyces pombe mutants with mutations of the anaphase-promoting complex (APC). These conditional lethal mutants arrest with unsegregated chromosomes because they cannot degrade the securin, Cut2p. Although failing at nuclear division, these mutants septate and divide. Since septation requires Cdc2p inactivation in wild-type S. pombe, it has been suggested that Cdc2p inactivation occurs in these mutants by a mechanism independent of cyclin degradation. In contrast to this prediction, we show that Cdc2p kinase activity fluctuates in APC cut mutants due to Cdc13/cyclin B destruction. In APC-null mutants, however, septation and cutting do not occur and Cdc13p is stable. We conclude that APC cut mutants are hypomorphic with respect to Cdc13p degradation. Indeed, overproduction of nondestructible Cdc13p prevents septation in APC cut mutants and the normal reorganization of septation initiation network components during anaphase. PMID:11533255

  3. Refurbishing the germline epigenome: Out with the old, in with the new.

    PubMed

    Hogg, Kirsten; Western, Patrick S

    2015-09-01

    Mammalian germline reprogramming involves the erasure and re-establishment of epigenetic information critical for germ cell function and inheritance in offspring. The bi-faceted nature of such reprogramming ensures germline repression of somatic programmes and the establishment of a carefully constructed epigenome essential for fertilisation and embryonic development in the next generation. While the majority of the germline epigenome is erased in preparation for embryonic development, certain genomic sequences remain resistant to this and may represent routes for transmission of epigenetic changes through the germline. Epigenetic reprogramming is regulated by highly conserved epigenetic modifiers, which function to establish, maintain and remove DNA methylation and chromatin modifications. In this review, we discuss recent findings from a considerable body of work illustrating the critical requirement of epigenetic modifiers that influence the epigenetic signature present in mature gametes, and have the potential to affect developmental outcomes in the offspring. We also briefly discuss the similarities of these mechanisms in the human germline and consider the potential for inheritance of epigenetically induced germline genetic errors that could impact on offspring phenotypes. PMID:26597001

  4. The Four Canonical TPR Subunits of Human APC/C Form Related Homo-Dimeric Structures and Stack in Parallel to Form a TPR Suprahelix☆

    PubMed Central

    Zhang, Ziguo; Chang, Leifu; Yang, Jing; Conin, Nora; Kulkarni, Kiran; Barford, David

    2013-01-01

    The anaphase-promoting complex or cyclosome (APC/C) is a large E3 RING-cullin ubiquitin ligase composed of between 14 and 15 individual proteins. A striking feature of the APC/C is that only four proteins are involved in directly recognizing target proteins and catalyzing the assembly of a polyubiquitin chain. All other subunits, which account for > 80% of the mass of the APC/C, provide scaffolding functions. A major proportion of these scaffolding subunits are structurally related. In metazoans, there are four canonical tetratricopeptide repeat (TPR) proteins that form homo-dimers (Apc3/Cdc27, Apc6/Cdc16, Apc7 and Apc8/Cdc23). Here, we describe the crystal structure of the N-terminal homo-dimerization domain of Schizosaccharomyces pombe Cdc23 (Cdc23Nterm). Cdc23Nterm is composed of seven contiguous TPR motifs that self-associate through a related mechanism to those of Cdc16 and Cdc27. Using the Cdc23Nterm structure, we generated a model of full-length Cdc23. The resultant “V”-shaped molecule docks into the Cdc23-assigned density of the human APC/C structure determined using negative stain electron microscopy (EM). Based on sequence conservation, we propose that Apc7 forms a homo-dimeric structure equivalent to those of Cdc16, Cdc23 and Cdc27. The model is consistent with the Apc7-assigned density of the human APC/C EM structure. The four canonical homo-dimeric TPR proteins of human APC/C stack in parallel on one side of the complex. Remarkably, the uniform relative packing of neighboring TPR proteins generates a novel left-handed suprahelical TPR assembly. This finding has implications for understanding the assembly of other TPR-containing multimeric complexes. PMID:23583778

  5. Germline MLH1 and MSH2 mutational spectrum including frequent large genomic aberrations in Hungarian hereditary non-polyposis colorectal cancer families: Implications for genetic testing

    PubMed Central

    Papp, Janos; Kovacs, Marietta E; Olah, Edith

    2007-01-01

    AIM: To analyze the prevalence of germline MLH1 and MSH2 gene mutations and evaluate the clinical characteristics of Hungarian hereditary non-polyposis colorectal cancer (HNPCC) families. METHODS: Thirty-six kindreds were tested for mutations using conformation sensitive gel electrophoreses, direct sequencing and also screening for genomic rearrangements applying multiplex ligation-dependent probe amplification (MLPA). RESULTS: Eighteen germline mutations (50%) were identified, 9 in MLH1 and 9 in MSH2. Sixteen of these sequence alterations were considered pathogenic, the remaining two were non-conservative missense alterations occurring at highly conserved functional motifs. The majority of the definite pathogenic mutations (81%, 13/16) were found in families fulfilling the stringent Amsterdam I/II criteria, including three rearrangements revealed by MLPA (two in MSH2 and one in MLH1). However, in three out of sixteen HNPCC-suspected families (19%), a disease-causing alteration could be revealed. Furthermore, nine mutations described here are novel, and none of the sequence changes were found in more than one family. CONCLUSION: Our study describes for the first time the prevalence and spectrum of germline mismatch repair gene mutations in Hungarian HNPCC and suspected-HNPCC families. The results presented here suggest that clinical selection criteria should be relaxed and detection of genomic rearrangements should be included in genetic screening in this population. PMID:17569143

  6. Characterization of multigene families in the micronuclear genome of Paramecium tetraurelia reveals a germline specific sequence in an intron of a centrin gene.

    PubMed Central

    Vayssié, L; Sperling, L; Madeddu, L

    1997-01-01

    In Paramecium, as in other ciliates, the transcriptionally active macronucleus is derived from the germline micronucleus by programmed DNA rearrangements, which include the precise excision of thousands of germline-specific sequences (internal eliminated sequences, IESs). We report the characterization of micronuclear versions of genes encoding Paramecium secretory granule proteins (trichocyst matrix proteins, TMPs) and Paramecium centrins. TMP and centrin multigene families, previously studied in the macronuclear genome, consist of genes that are co-expressed to provide mixtures of related polypeptides that co-assemble to form respectively the crystalline trichocyst matrix and the infraciliary lattice, a contractile cytoskeletal network. We present evidence that TMP and centrin genes identified in the macronucleus are also present in the micronucleus, ruling out the possibility that these novel multigene families are generated by somatic rearrangements during macronuclear development. No IESs were found in TMP genes, however, four IESs in or near germline centrin genes were characterized. The only intragenic IES is 75 bp in size, interrupts a 29 bp intron and is absent from at least one other closely related centrin gene. This is the first report of an IES in an intron in Paramecium. PMID:9023115

  7. Expanding the spectrum of phenotypes associated with germline PIGA mutations: a child with developmental delay, accelerated linear growth, facial dysmorphisms, elevated alkaline phosphatase, and progressive CNS abnormalities.

    PubMed

    van der Crabben, Saskia N; Harakalova, Magdalena; Brilstra, Eva H; van Berkestijn, Frédérique M C; Hofstede, Floris C; van Vught, Adrianus J; Cuppen, Edwin; Kloosterman, Wigard; Ploos van Amstel, Hans Kristian; van Haaften, Gijs; van Haelst, Mieke M

    2014-01-01

    Phosphatidyl inositol glycan (PIG) enzyme subclasses are involved in distinct steps of glycosyl phosphatidyl inositol anchor protein biosynthesis. Glycolsyl phosphatidyl inositol-anchored proteins have heterogeneous functions; they can function as enzymes, adhesion molecules, complement regulators and co-receptors in signal transduction pathways. Germline mutations in genes encoding different members of the PIG family result in diverse conditions with (severe) developmental delay, (neonatal) seizures, hypotonia, CNS abnormalities, growth abnormalities, and congenital abnormalities as hallmark features. The variability of clinical features resembles the typical diversity of other glycosylation pathway deficiencies such as the congenital disorders of glycosylation. Here, we report the first germline missense mutation in the PIGA gene associated with accelerated linear growth, obesity, central hypotonia, severe refractory epilepsy, cardiac anomalies, mild facial dysmorphic features, mildly elevated alkaline phosphatase levels, and CNS anomalies consisting of progressive cerebral atrophy, insufficient myelinization, and cortical MRI signal abnormalities. X-exome sequencing in the proband identified a c.278C>T (p.Pro93Leu) mutation in the PIGA gene. The mother and maternal grandmother were unaffected carriers and the mother showed 100% skewing of the X-chromosome harboring the mutation. These results together with the clinical similarity of the patient reported here and the previously reported patients with a germline nonsense mutation in PIGA support the determination that this mutation caused the phenotype in this family. PMID:24259184

  8. Nasal chondromesenchymal hamartomas arise secondary to germline and somatic mutations of DICER1 in the pleuropulmonary blastoma tumor-predisposition disorder

    PubMed Central

    Stewart, Douglas R.; Messinger, Yoav; Williams, Gretchen M.; Yang, Jiandong; Field, Amanda; Schultz, Kris Ann P.; Harney, Laura A.; Doros, Leslie A.; Dehner, Louis P.; Hill, D. Ashley

    2014-01-01

    Background Nasal chondromesenchymal hamartoma (NCMH) is a rare nasal tumor that typically presents in young children. We previously reported on NCMH occurrence in children with pleuropulmonary blastoma (PPB), a rare pulmonary dysembryonic sarcoma that is the hallmark neoplasm in the PPB-associated DICER1 tumor predisposition disorder. Methods Original pathologic materials from individuals with a PPB, PPB-associated tumor and/or a DICER1 mutation were centrally reviewed by the International PPB Registry. Paraffin-embedded NCMH tumor tissue was available in three cases. Laser-capture microdissection was used to isolate mesenchymal spindle cells and cartilage in one case for Sanger sequencing of DICER1. Results Nine patients (5F/4M) had PPB and NCMH. NCMH was diagnosed at a median age of 10 years (range 6-21years). NCMH developed 4.5 - 13 years after PPB. Presenting NCMH symptoms included chronic sinusitis and nasal congestion. Five patients had bilateral tumors. Local NCMH recurrences required several surgical resections in two patients, but all nine patients were alive at 0 – 16 years of follow-up. Pathogenic germline DICER1 mutations were found in 6/8 NCMH patients tested. In 2 of the patients with germline DICER1 mutations, somatic DICER1 missense mutations were also identified in their NCMH (E1813D; n=2). Three additional PPB patients developed other nasal lesions seen in the general population (a Schneiderian papilloma, chronic sinusitis with cysts, and allergic nasal polyps with eosinophils). Two of these patients had germline DICER1 mutations. Conclusion Pathogenic germline and somatic mutations of DICER1 in NCMH establishes that the genetic etiology of NCMH is similar to PPB, despite the disparate biological potential of these neoplasms. PMID:25118636

  9. Kinesin-2 and Apc function at dendrite branch points to resolve microtubule collisions.

    PubMed

    Weiner, Alexis T; Lanz, Michael C; Goetschius, Daniel J; Hancock, William O; Rolls, Melissa M

    2016-01-01

    In Drosophila neurons, kinesin-2, EB1 and Apc are required to maintain minus-end-out dendrite microtubule polarity, and we previously proposed they steer microtubules at branch points. Motor-mediated steering of microtubule plus ends could be accomplished in two ways: 1) by linking a growing microtubule tip to the side of an adjacent microtubule as it navigates the branch point (bundling), or 2) by directing a growing microtubule after a collision with a stable microtubule (collision resolution). Using live imaging to distinguish between these two mechanisms, we found that reduction of kinesin-2 did not alter the number of microtubules that grew along the edge of the branch points where stable microtubules are found. However, reduction of kinesin-2 or Apc did affect the number of microtubules that slowed down or depolymerized as they encountered the side of the branch opposite to the entry point. These results are consistent with kinesin-2 functioning with Apc to resolve collisions. However, they do not pinpoint stable microtubules as the collision partner as stable microtubules are typically very close to the membrane. To determine whether growing microtubules were steered along stable ones after a collision, we analyzed the behavior of growing microtubules at dendrite crossroads where stable microtubules run through the middle of the branch point. In control neurons, microtubules turned in the middle of the crossroads. However, when kinesin-2 was reduced some microtubules grew straight through the branch point and failed to turn. We propose that kinesin-2 functions to steer growing microtubules along stable ones following collisions. PMID:26785384

  10. Dietary acrylamide intake and the risk of colorectal cancer with specific mutations in KRAS and APC.

    PubMed

    Hogervorst, Janneke G F; de Bruijn-Geraets, Daisy; Schouten, Leo J; van Engeland, Manon; de Kok, Theo M C M; Goldbohm, R Alexandra; van den Brandt, Piet A; Weijenberg, Matty P

    2014-05-01

    Acrylamide, a probable human carcinogen, is present in heat-treated carbohydrate-rich foods. Epidemiological studies have not shown a clear association between acrylamide intake and colorectal cancer (CRC) risk. This may be due to the molecular heterogeneity in colorectal tumors, which was not taken into consideration before. Since the acrylamide metabolite glycidamide induces specific DNA mutations in rodents, we investigated whether acrylamide is associated with CRC risk characterized by mutations in Kirsten-ras (KRAS) and adenomatous polyposis coli (APC); key genes in colorectal carcinogenesis. This case-cohort analysis, within the Netherlands Cohort Study on diet and cancer, was based on 7.3 years of follow-up. Acrylamide intake was assessed with a food frequency questionnaire. Mutation analysis of codons 1286-1520 in exon 15 in APC and codons 12 and 13 in exon 1 in KRAS was performed on tumor tissue of 733 cases. Hazard ratios (HR) were calculated using Cox proportional hazards analysis. Among men, acrylamide intake was statistically significantly associated with an increased risk of particularly tumors with an activating KRAS mutation {HR fourth versus first quartile: 2.12 [95% confidence interval (CI): 1.16-3.87], P trend: 0.01}. Among women, acrylamide intake was statistically significantly associated with a decreased risk of particularly tumors with a truncating APC mutation (fourth versus first quartile: 0.47 (95% CI: 0.23-0.94), P trend: 0.02), but only in the highest quartile of intake. This is the first study to show that acrylamide might be associated with CRC with specific somatic mutations, differentially in men and women. More research is needed to corroborate or refute these findings. PMID:24398672

  11. PLK1 regulates spindle formation kinetics and APC/C activation in mouse zygote.

    PubMed

    Baran, Vladimir; Brzakova, Adela; Rehak, Pavol; Kovarikova, Veronika; Solc, Petr

    2016-06-01

    Polo-like kinase 1 (PLK1) is involved in essential events of cell cycle including mitosis in which it participates in centrosomal microtubule nucleation, spindle bipolarity establishment and cytokinesis. Although PLK1 function has been studied in cycling cancer cells, only limited data are known about its role in the first mitosis of mammalian zygotes. During the 1-cell stage of mouse embryo development, the acentriolar spindle is formed and the shift from acentriolar to centrosomal spindle formation progresses gradually throughout the preimplantation stage, thus providing a unique possibility to study acentriolar spindle formation. We have shown previously that PLK1 activity is not essential for entry into first mitosis, but is required for correct spindle formation and anaphase onset in 1-cell mouse embryos. In the present study, we extend this knowledge by employing quantitative confocal live cell imaging to determine spindle formation kinetics in the absence of PLK1 activity and answer the question whether metaphase arrest at PLK1-inhibited embryos is associated with low anaphase-promoting complex/cyclosome (APC/C) activity and consequently high securin level. We have shown that inhibition of PLK1 activity induces a delay in onset of acentriolar spindle formation during first mitosis. Although these PLK1-inhibited 1-cell embryos were finally able to form a bipolar spindle, not all chromosomes were aligned at the metaphase equator. PLK1-inhibited embryos were arrested in metaphase without any sign of APC/C activation with high securin levels. Our results document that PLK1 controls the onset of spindle assembly and spindle formation, and is essential for APC/C activation before anaphase onset in mouse zygotes. PMID:26174739

  12. STS-34 crewmembers sit in M1-13 APC during emergency egress training at KSC

    NASA Technical Reports Server (NTRS)

    1989-01-01

    STS-34 crewmembers sit in M1-13 Armored Personnel Carrier (APC) during emergency egress training at KSC's shuttle landing facility (SLF) prior to terminal countdown demonstration test (TCDT) activities. Wearing launch and entry suits (LESs), are (from left) Mission Specialist (MS) Ellen S. Baker, MS Shannon W. Lucid, Commander Donald E. Williams (right side, in back), MS Franklin R. Chang-Diaz, and Pilot Michael J. McCulley (holding headset). View provided by KSC with alternate number KSC-89PC-871.

  13. The ubiquitin ligase APC/CCdh1 puts the brakes on DNA-end resection

    PubMed Central

    Lafranchi, Lorenzo; Sartori, Alessandro A

    2015-01-01

    DNA double-strand breaks (DSBs) are highly deleterious lesions and their misrepair can promote genomic instability, a hallmark of cancer. DNA-end resection is a cell cycle-regulated mechanism that is required for the faithful repair of DSBs. We recently discovered that the anaphase-promoting complex/cyclosome-Cdh1 (APC/CCdh1) ubiquitin ligase is responsible for the timely degradation of CtBP-interacting protein (CtIP), a key DNA-end resection factor, providing a new layer of regulation of DSB repair in human cells. PMID:27308488

  14. Intestinal APCs of the endogenous nanomineral pathway fail to express PD-L1 in Crohn’s disease

    PubMed Central

    Robertson, Jack; Haas, Carolin T.; Pele, Laetitia C.; Monie, Tom P.; Charalambos, Charles; Parkes, Miles; Hewitt, Rachel E.; Powell, Jonathan J.

    2016-01-01

    Crohn’s disease is a chronic inflammatory condition most commonly affecting the ileum and colon. The aetiology of Crohn’s disease is complex and may include defects in peptidoglycan recognition, and/or failures in the establishment of intestinal tolerance. We have recently described a novel constitutive endogenous delivery system for the translocation of nanomineral-antigen-peptidoglycan (NAP) conjugates to antigen presenting cells (APCs) in intestinal lymphoid patches. In mice NAP conjugate delivery to APCs results in high surface expression of the immuno-modulatory molecule programmed death receptor ligand 1 (PD-L1). Here we report that NAP conjugate positive APCs in human ileal tissues from individuals with ulcerative colitis and intestinal carcinomas, also have high expression of PD-L1. However, NAP-conjugate positive APCs in intestinal tissue from patients with Crohn’s disease show selective failure in PD-L1 expression. Therefore, in Crohn’s disease intestinal antigen taken up by lymphoid patch APCs will be presented without PD-L1 induced tolerogenic signalling, perhaps initiating disease. PMID:27226337

  15. Aggregate material formulated with MSWI bottom ash and APC fly ash for use as secondary building material

    SciTech Connect

    Valle-Zermeño, R. del; Formosa, J.; Chimenos, J.M.; Martínez, M.; Fernández, A.I.

    2013-03-15

    Highlights: ► A concrete formulation was optimized using Bottom Ash and APC ash. ► 10% of APC ash achieves good compromise between economic and performance aspects. ► The crushed concrete was evaluated as secondary building granular material. ► The environmental behavior allows its use as secondary material. ► The abrasion resistance is not good enough for its use as a road sub-base material. - Abstract: The main goal of this paper is to obtain a granular material formulated with Municipal Solid Waste Incineration (MSWI) bottom ash (BA) and air pollution control (APC) fly ash to be used as secondary building material. Previously, an optimum concrete mixture using both MSWI residues as aggregates was formulated. A compromise between the environmental behavior whilst maximizing the reuse of APC fly ash was considered and assessed. Unconfined compressive strength and abrasion resistance values were measured in order to evaluate the mechanical properties. From these results, the granular mixture was not suited for certain applications owing to the high BA/APC fly ash content and low cement percentages used to reduce the costs of the final product. Nevertheless, the leaching test performed showed that the concentrations of all heavy metals were below the limits established by the current Catalan legislation for their reutilization. Therefore, the material studied might be mainly used in embankments, where high mechanical properties are not needed and environmental safety is assured.

  16. Irreversible APC(Cdh1) Inactivation Underlies the Point of No Return for Cell-Cycle Entry.

    PubMed

    Cappell, Steven D; Chung, Mingyu; Jaimovich, Ariel; Spencer, Sabrina L; Meyer, Tobias

    2016-06-30

    Proliferating cells must cross a point of no return before they replicate their DNA and divide. This commitment decision plays a fundamental role in cancer and degenerative diseases and has been proposed to be mediated by phosphorylation of retinoblastoma (Rb) protein. Here, we show that inactivation of the anaphase-promoting complex/cyclosome (APC(Cdh1)) has the necessary characteristics to be the point of no return for cell-cycle entry. Our study shows that APC(Cdh1) inactivation is a rapid, bistable switch initiated shortly before the start of DNA replication by cyclin E/Cdk2 and made irreversible by Emi1. Exposure to stress between Rb phosphorylation and APC(Cdh1) inactivation, but not after APC(Cdh1) inactivation, reverted cells to a mitogen-sensitive quiescent state, from which they can later re-enter the cell cycle. Thus, APC(Cdh1) inactivation is the commitment point when cells lose the ability to return to quiescence and decide to progress through the cell cycle. PMID:27368103

  17. New origin firing is inhibited by APC/CCdh1 activation in S-phase after severe replication stress

    PubMed Central

    Ercilla, Amaia; Llopis, Alba; Feu, Sonia; Aranda, Sergi; Ernfors, Patrik; Freire, Raimundo; Agell, Neus

    2016-01-01

    Defects in DNA replication and repair are known to promote genomic instability, a hallmark of cancer cells. Thus, eukaryotic cells have developed complex mechanisms to ensure accurate duplication of their genomes. While DNA damage response has been extensively studied in tumour cells, the pathways implicated in the response to replication stress are less well understood especially in non-transformed cells. Here we show that in non-transformed cells, APC/CCdh1 is activated upon severe replication stress. Activation of APC/CCdh1 prevents new origin firing and induces permanent arrest in S-phase. Moreover, Rad51-mediated homologous recombination is also impaired under these conditions. APC/CCdh1 activation in S-phase occurs after replication forks have been processed into double strand breaks. Remarkably, this activation, which correlates with decreased Emi1 levels, is not prevented by ATR/ATM inhibition, but it is abrogated in cells depleted of p53 or p21. Importantly, we found that the lack of APC/CCdh1 activity correlated with an increase in genomic instability. Taken together, our results define a new APC/CCdh1 function that prevents cell cycle resumption after prolonged replication stress by inhibiting origin firing, which may act as an additional mechanism in safeguarding genome integrity. PMID:26939887

  18. The ubiquitin ligase APC(Cdh1) is required to maintain genome integrity in primary human cells.

    PubMed

    Engelbert, D; Schnerch, D; Baumgarten, A; Wäsch, R

    2008-02-01

    Ensuring precise DNA replication and chromosome segregation is essential during cell division in order to provide genomic stability and avoid malignant growth. Proteolytic control of cell cycle regulators by the anaphase-promoting complex, activated by Cdh1 (APC(Cdh1)), is responsible for a stable G1 phase after mitotic exit allowing accurate preparation for DNA replication in the following S phase. APC(Cdh1) target proteins are frequently upregulated in tumor cells and the inactivation of human Cdh1 might interfere with genome integrity by target stabilization. Here we show that APC(Cdh1) is required for maintaining genomic integrity in primary human cells. Lentiviral-delivered strong and stable suppression of Cdh1 by RNA interference (RNAi) causes aberrant accumulation of several APC(Cdh1) target proteins, such as cyclin A, B, Aurora A or Plk1, which control accurate and equal distribution of the genetic information to daughter cells. This induces a premature and prolonged S phase, mitotic-entry delay and defects in chromosome separation and cytokinesis. Cell cycle deregulation by stable knockdown of Cdh1 leads to activation of p53/p21 and genomic instability, which is further increased by codepletion of p53. Thus, stabilization of APC(Cdh1) targets may initiate aberrant DNA replication and chromosome separation, and trigger a p53 response by deregulating G1 in primary human cells. PMID:17700535

  19. The Cdk1-APC/C cell cycle oscillator circuit functions as a time-delayed, ultrasensitive switch

    PubMed Central

    Yang, Qiong; Ferrell, James E.

    2013-01-01

    Despite the complexity and variety of biological oscillators, their core design invariably includes an essential negative feedback loop. In the Xenopus laevis embryonic cell cycle oscillator, this loop consists of the kinase cyclin B-Cdk1 and the ubiquitin ligase APC/CCdc20; active Cdk1 activates APC/CCdc20, which then brings about cyclin B degradation and inactivates Cdk1. Here we ask how this negative feedback loop functions quantitatively, with the aim of understanding what mechanisms keep the Cdk1-APC/CCdc20 system from settling into a stable steady state with intermediate levels of Cdk1 and APC/CCdc20 activity. We found that the system operates as a time-delayed, digital switch, with a time lag of ~15 min between Cdk1 and APC/CCdc20 activation and a tremendously high degree of ultrasensitivity (nH ≈ 17). Computational modeling shows how these attributes contribute to the generation of robust, clock-like oscillations. Principles uncovered here may also apply to other activator-repressor oscillators and help in designing robust synthetic clocks. PMID:23624406

  20. New origin firing is inhibited by APC/CCdh1 activation in S-phase after severe replication stress.

    PubMed

    Ercilla, Amaia; Llopis, Alba; Feu, Sonia; Aranda, Sergi; Ernfors, Patrik; Freire, Raimundo; Agell, Neus

    2016-06-01

    Defects in DNA replication and repair are known to promote genomic instability, a hallmark of cancer cells. Thus, eukaryotic cells have developed complex mechanisms to ensure accurate duplication of their genomes. While DNA damage response has been extensively studied in tumour cells, the pathways implicated in the response to replication stress are less well understood especially in non-transformed cells. Here we show that in non-transformed cells, APC/C(Cdh1) is activated upon severe replication stress. Activation of APC/C(Cdh1) prevents new origin firing and induces permanent arrest in S-phase. Moreover, Rad51-mediated homologous recombination is also impaired under these conditions. APC/C(Cdh1) activation in S-phase occurs after replication forks have been processed into double strand breaks. Remarkably, this activation, which correlates with decreased Emi1 levels, is not prevented by ATR/ATM inhibition, but it is abrogated in cells depleted of p53 or p21. Importantly, we found that the lack of APC/C(Cdh1) activity correlated with an increase in genomic instability. Taken together, our results define a new APC/C(Cdh1) function that prevents cell cycle resumption after prolonged replication stress by inhibiting origin firing, which may act as an additional mechanism in safeguarding genome integrity. PMID:26939887

  1. Intestinal APCs of the endogenous nanomineral pathway fail to express PD-L1 in Crohn's disease.

    PubMed

    Robertson, Jack; Haas, Carolin T; Pele, Laetitia C; Monie, Tom P; Charalambos, Charles; Parkes, Miles; Hewitt, Rachel E; Powell, Jonathan J

    2016-01-01

    Crohn's disease is a chronic inflammatory condition most commonly affecting the ileum and colon. The aetiology of Crohn's disease is complex and may include defects in peptidoglycan recognition, and/or failures in the establishment of intestinal tolerance. We have recently described a novel constitutive endogenous delivery system for the translocation of nanomineral-antigen-peptidoglycan (NAP) conjugates to antigen presenting cells (APCs) in intestinal lymphoid patches. In mice NAP conjugate delivery to APCs results in high surface expression of the immuno-modulatory molecule programmed death receptor ligand 1 (PD-L1). Here we report that NAP conjugate positive APCs in human ileal tissues from individuals with ulcerative colitis and intestinal carcinomas, also have high expression of PD-L1. However, NAP-conjugate positive APCs in intestinal tissue from patients with Crohn's disease show selective failure in PD-L1 expression. Therefore, in Crohn's disease intestinal antigen taken up by lymphoid patch APCs will be presented without PD-L1 induced tolerogenic signalling, perhaps initiating disease. PMID:27226337

  2. Autism-like behaviours and germline transmission in transgenic monkeys overexpressing MeCP2.

    PubMed

    Liu, Zhen; Li, Xiao; Zhang, Jun-Tao; Cai, Yi-Jun; Cheng, Tian-Lin; Cheng, Cheng; Wang, Yan; Zhang, Chen-Chen; Nie, Yan-Hong; Chen, Zhi-Fang; Bian, Wen-Jie; Zhang, Ling; Xiao, Jianqiu; Lu, Bin; Zhang, Yue-Fang; Zhang, Xiao-Di; Sang, Xiao; Wu, Jia-Jia; Xu, Xiu; Xiong, Zhi-Qi; Zhang, Feng; Yu, Xiang; Gong, Neng; Zhou, Wen-Hao; Sun, Qiang; Qiu, Zilong

    2016-02-01

    Methyl-CpG binding protein 2 (MeCP2) has crucial roles in transcriptional regulation and microRNA processing. Mutations in the MECP2 gene are found in 90% of patients with Rett syndrome, a severe developmental disorder with autistic phenotypes. Duplications of MECP2-containing genomic segments cause the MECP2 duplication syndrome, which shares core symptoms with autism spectrum disorders. Although Mecp2-null mice recapitulate most developmental and behavioural defects seen in patients with Rett syndrome, it has been difficult to identify autism-like behaviours in the mouse model of MeCP2 overexpression. Here we report that lentivirus-based transgenic cynomolgus monkeys (Macaca fascicularis) expressing human MeCP2 in the brain exhibit autism-like behaviours and show germline transmission of the transgene. Expression of the MECP2 transgene was confirmed by western blotting and immunostaining of brain tissues of transgenic monkeys. Genomic integration sites of the transgenes were characterized by a deep-sequencing-based method. As compared to wild-type monkeys, MECP2 transgenic monkeys exhibited a higher frequency of repetitive circular locomotion and increased stress responses, as measured by the threat-related anxiety and defensive test. The transgenic monkeys showed less interaction with wild-type monkeys within the same group, and also a reduced interaction time when paired with other transgenic monkeys in social interaction tests. The cognitive functions of the transgenic monkeys were largely normal in the Wisconsin general test apparatus, although some showed signs of stereotypic cognitive behaviours. Notably, we succeeded in generating five F1 offspring of MECP2 transgenic monkeys by intracytoplasmic sperm injection with sperm from one F0 transgenic monkey, showing germline transmission and Mendelian segregation of several MECP2 transgenes in the F1 progeny. Moreover, F1 transgenic monkeys also showed reduced social interactions when tested in pairs, as

  3. Phenotypic expression in von Hippel-Lindau disease: correlations with germline VHL gene mutations.

    PubMed Central

    Maher, E R; Webster, A R; Richards, F M; Green, J S; Crossey, P A; Payne, S J; Moore, A T

    1996-01-01

    Von Hippel-Lindau disease is an autosomal dominantly inherited familial cancer syndrome predisposing to retinal and central nervous system haemangioblastomas, renal cell carcinoma, and phaeochromocytoma. VHL disease shows variable expression and interfamilial differences in predisposition to phaeochromocytoma. In a previous study of 65 VHL kindreds with defined VHL mutations we detected significant differences between VHL families with and without phaeochromocytoma such that missense mutations were more common and large deletions or protein truncating mutations less frequent in phaeochromocytoma positive families. To investigate the significance and cause of this association further, we studied 138 VHL kindreds for germline mutations and calculated the age related tumour risks for different classes of VHL gene mutations. Using SSCP, heteroduplex and Southern analysis we identified a germline VHL gene mutation in 101 families (73%). Direct sequencing of the VHL coding region further increased the mutation detection rate to 81%. In addition to precise presymptomatic diagnosis, identification of a VHL gene mutation can provide an indication of the likely phenotype. We found that large deletions and mutations predicted to cause a truncated protein were associated with a lower risk of phaeochromocytoma (6% and 9% at 30 and 50 years, respectively) than missense mutations (40% and 59%, respectively) and that missense mutations at codon 167 were associated with a high risk of phaeochromocytoma (53% and 82% at ages 30 and 50 years). Cumulative probabilities of renal cell carcinoma did not differ between the two groups (deletion/ truncation mutations: 8% and 60%, and missense mutations: 10% and 64% at ages 30 and 50 years, respectively). Age related risks for haemangioblastoma were similar in the two mutation groups, with the age related risks of cerebellar haemangioblastoma slightly less (35% and 64% v 38% and 75% at ages 30 and 50 years) and retinal haemangioblastoma

  4. Germline-specific H1 variants: the "sexy" linker histones.

    PubMed

    Pérez-Montero, Salvador; Carbonell, Albert; Azorín, Fernando

    2016-03-01

    The eukaryotic genome is packed into chromatin, a nucleoprotein complex mainly formed by the interaction of DNA with the abundant basic histone proteins. The fundamental structural and functional subunit of chromatin is the nucleosome core particle, which is composed by 146 bp of DNA wrapped around an octameric protein complex formed by two copies of each core histone H2A, H2B, H3, and H4. In addition, although not an intrinsic component of the nucleosome core particle, linker histone H1 directly interacts with it in a monomeric form. Histone H1 binds nucleosomes near the exit/entry sites of linker DNA, determines nucleosome repeat length and stabilizes higher-order organization of nucleosomes into the ∼30 nm chromatin fiber. In comparison to core histones, histone H1 is less well conserved through evolution. Furthermore, histone H1 composition in metazoans is generally complex with most species containing multiple variants that play redundant as well as specific functions. In this regard, a characteristic feature is the presence of specific H1 variants that replace somatic H1s in the germline and during early embryogenesis. In this review, we summarize our current knowledge about their structural and functional properties. PMID:25921218

  5. Germ-line engineering, freedom, and future generations.

    PubMed

    Cooke, Elizabeth F

    2003-02-01

    New technologies in germ-line engineering have raised many questions about obligations to future generations. In this article, I focus on the importance of increasing freedom and the equality of freedom for present and future generations, because these two ideals are necessary for a just society and because they are most threatened by the wide-scale privatisation of GLE technologies. However, there are ambiguities in applying these ideals to the issue of genetic technologies. I argue that Amartya Sen's capability theory can be used as a framework to ensure freedom and equality in the use of GLE technology. Capability theory articulates the goal of equalising real freedom by bringing all people up to a threshold of basic human capabilities. Sen's capability theory can clarify the proper moral goal of GLE insofar as this technology could be used to bring people up to certain basic human capabilities, thereby increasing their real freedom. And by increasing the freedom of those who lack basic human capabilities, GLE can aid in decreasing the inequalities of freedom among classes of people. PMID:12718332

  6. Germline Competent Pluripotent Mouse Stem Cells Generated by Plasmid Vectors.

    PubMed

    Chen, Chien-Hong; Su, Yu-Hsiu; Lee, Kun-Hsiung; Chuang, Chin-Kai

    2016-07-01

    We developed nonintegrated methods to reprogram mouse embryonic fibroblast (MEF) cells into induced pluripotent stem cells (iPSCs) using pig pOct4, pSox2, and pc-Myc as well as human hKLF4, hAID, and hTDG that were carried by plasmid vectors. The 4F method employed pOct4, pSox2, pc-Myc, and hKLF4 to derive iPSC clones with naive embryonic stem cell (ESC)-like morphology. These 4F clones expressed endogenous mouse Nanog protein and could generate chimeras. In addition to the four conventional reprogramming factors used in the 4F method, hAID and hTDG were utilized in a 6F method to increase the conversion efficiency of reprogramming by approximately five-fold. One of the 6F plasmid derived iPSC (piPSC) clones was shown to be germline transmission competent. PMID:26980563

  7. Interspecific Germline Transmission of Cultured Primordial Germ Cells

    PubMed Central

    van de Lavoir, Marie-Cecile; Collarini, Ellen J.; Leighton, Philip A.; Fesler, Jeffrey; Lu, Daniel R.; Harriman, William D.; Thiyagasundaram, T. S.; Etches, Robert J.

    2012-01-01

    In birds, the primordial germ cell (PGC) lineage separates from the soma within 24 h following fertilization. Here we show that the endogenous population of about 200 PGCs from a single chicken embryo can be expanded one million fold in culture. When cultured PGCs are injected into a xenogeneic embryo at an equivalent stage of development, they colonize the testis. At sexual maturity, these donor PGCs undergo spermatogenesis in the xenogeneic host and become functional sperm. Insemination of semen from the xenogeneic host into females from the donor species produces normal offspring from the donor species. In our model system, the donor species is chicken (Gallus domesticus) and the recipient species is guinea fowl (Numida meleagris), a member of a different avian family, suggesting that the mechanisms controlling proliferation of the germline are highly conserved within birds. From a pragmatic perspective, these data are the basis of a novel strategy to produce endangered species of birds using domesticated hosts that are both tractable and fecund. PMID:22629301

  8. Multiplex screening for RB1 germline mutations in 106 patients with hereditary retinoblastoma

    SciTech Connect

    Lohmann, D.R.; Brandt, B.; Passarge, E.

    1994-09-01

    The identification of germline mutations in the retinoblastoma susceptibility gene (RB1) is important for genetic counseling in hereditary retinoblastoma. Due to the complex genomic organization of this gene and the heterogeneity of mutations, efficient screening procedures are important for rapid mutation detection. We have developed methods based on simultaneous analysis of multiple regions of this gene in an ABI automated DNA fragment analyzer to examine 106 patients with hereditary retinoblastoma in which no alteration was identified by Southern blot hybridization. Primers for the amplification of all 27 exons of the RB1 gene as well as the promoter and poly(A) signal sequences were labelled with distinct fluorescent dyes (FAM, HEX, TAMRA) to enable simultaneous electrophoretic analysis of PCR products with similar mobility. PCR fragments distinguishable by size or color were co-amplified by multiplex PCR and analyzed for length by GENESCAN analysis. Using this approach, small deletions ranging from 1 bp to 22 bp were identified in 24 patients (23%). Short sequence repeats or polypyrimidine runs were present in the vicinity of most of these deletions. In 4 patients (4%), insertions from 1 bp to 4 bp were found. The majority of length mutations resulted in a truncated gene product due to frameshift and premature termination. No mutation was identified in exons 25 to 27 possibly indicating that the encoded protein domains have minor functional importance. In order to screen for base substitutions that are not detectable by fragment length analysis, we adapted heteroduplex analysis for the use in the DNA fragment analyzer. During the optimization of this method we detected 10 single base substitutions most of which generated stop codons. Intriguingly, two identical missense mutations were identified in two unrelated families with a low-penetrance phenotype.

  9. Silver nanoparticles disrupt germline stem cell maintenance in the Drosophila testis

    NASA Astrophysics Data System (ADS)

    Ong, Cynthia; Lee, Qian Ying; Cai, Yu; Liu, Xiaoli; Ding, Jun; Yung, Lin-Yue Lanry; Bay, Boon-Huat; Baeg, Gyeong-Hun

    2016-02-01

    Silver nanoparticles (AgNPs), one of the most popular nanomaterials, are commonly used in consumer products and biomedical devices, despite their potential toxicity. Recently, AgNP exposure was reported to be associated with male reproductive toxicity in mammalian models. However, there is still a limited understanding of the effects of AgNPs on spermatogenesis. The fruit fly Drosophila testis is an excellent in vivo model to elucidate the mechanisms underlying AgNP-induced defects in spermatogenesis, as germ lineages can be easily identified and imaged. In this study, we evaluated AgNP-mediated toxicity on spermatogenesis by feeding Drosophila with AgNPs at various concentrations. We first observed a dose-dependent uptake of AgNPs in vivo. Concomitantly, AgNP exposure caused a significant decrease in the viability and delay in the development of Drosophila in a dose-dependent manner. Furthermore, AgNP-treated male flies showed a reduction in fecundity, and the resulting testes contained a decreased number of germline stem cells (GSCs) compared to controls. Interestingly, testes exposed to AgNPs exhibited a dramatic increase in reactive oxygen species levels and showed precocious GSC differentiation. Taken together, our study suggests that AgNP exposure may increase ROS levels in the Drosophila testis, leading to a reduction of GSC number by promoting premature GSC differentiation.

  10. Silver nanoparticles disrupt germline stem cell maintenance in the Drosophila testis

    PubMed Central

    Ong, Cynthia; Lee, Qian Ying; Cai, Yu; Liu, Xiaoli; Ding, Jun; Yung, Lin-Yue Lanry; Bay, Boon-Huat; Baeg, Gyeong-Hun

    2016-01-01

    Silver nanoparticles (AgNPs), one of the most popular nanomaterials, are commonly used in consumer products and biomedical devices, despite their potential toxicity. Recently, AgNP exposure was reported to be associated with male reproductive toxicity in mammalian models. However, there is still a limited understanding of the effects of AgNPs on spermatogenesis. The fruit fly Drosophila testis is an excellent in vivo model to elucidate the mechanisms underlying AgNP-induced defects in spermatogenesis, as germ lineages can be easily identified and imaged. In this study, we evaluated AgNP-mediated toxicity on spermatogenesis by feeding Drosophila with AgNPs at various concentrations. We first observed a dose-dependent uptake of AgNPs in vivo. Concomitantly, AgNP exposure caused a significant decrease in the viability and delay in the development of Drosophila in a dose-dependent manner. Furthermore, AgNP-treated male flies showed a reduction in fecundity, and the resulting testes contained a decreased number of germline stem cells (GSCs) compared to controls. Interestingly, testes exposed to AgNPs exhibited a dramatic increase in reactive oxygen species levels and showed precocious GSC differentiation. Taken together, our study suggests that AgNP exposure may increase ROS levels in the Drosophila testis, leading to a reduction of GSC number by promoting premature GSC differentiation. PMID:26847594

  11. Bacterial Folates Provide an Exogenous Signal for C. elegans Germline Stem Cell Proliferation.

    PubMed

    Chaudhari, Snehal N; Mukherjee, Madhumati; Vagasi, Alexandra S; Bi, Gaofeng; Rahman, Mohammad M; Nguyen, Christine Q; Paul, Ligi; Selhub, Jacob; Kipreos, Edward T

    2016-07-11

    Here we describe an in vitro primary culture system for Caenorhabditis elegans germline stem cells. This culture system was used to identify a bacterial folate as a positive regulator of germ cell proliferation. Folates are a family of B-complex vitamins that function in one-carbon metabolism to allow the de novo synthesis of amino acids and nucleosides. We show that germ cell proliferation is stimulated by the folate 10-formyl-tetrahydrofolate-Glun both in vitro and in animals. Other folates that can act as vitamins to rescue folate deficiency lack this germ cell stimulatory activity. The bacterial folate precursor dihydropteroate also promotes germ cell proliferation in vitro and in vivo, despite its inability to promote one-carbon metabolism. The folate receptor homolog FOLR-1 is required for the stimulation of germ cells by 10-formyl-tetrahydrofolate-Glun and dihydropteroate. This work defines a folate and folate-related compound as exogenous signals to modulate germ cell proliferation. PMID:27404357

  12. Germline BAP1 mutations predispose also to multiple basal cell carcinomas.

    PubMed

    de la Fouchardière, A; Cabaret, O; Savin, L; Combemale, P; Schvartz, H; Penet, C; Bonadona, V; Soufir, N; Bressac-de Paillerets, B

    2015-09-01

    The BRCA1-associated protein 1 (BAP1) gene encodes a nuclear deubiquitin enzyme which acts as a tumour suppressor. Loss of function germline mutations of BAP1 have been associated with an enhanced risk of uveal and cutaneous melanomas, mesothelioma, clear cell renal cancer and atypical cutaneous melanocytic proliferations. In two independent BAP1 families, we noticed an unusual frequency of basal cell carcinomas (BCCs). Indeed, 19 BCCs were diagnosed in four patients, either of superficial (13/19) or nodular (6/19) subtype; they were all located in chronic sun-exposed areas (limbs, head or neck). Immunohistochemistry (IHC) identified in the 19 tumours, complete or partial loss of BAP1 protein nuclear expression, restricted to the BCC nests. A control study was conducted in 22 sporadic BCCs in 22 subjects under 65 without known associated BAP1 tumours: no loss of BAP1 expression was found. Overall, our observations suggest that BCCs are part of the BAP1 cancer syndrome, perhaps in relation with chronic sun exposure and melanocortin 1 receptor (MC1R) variants. In conclusion, cutaneous follow-up of BAP1 carriers should not only aim to detect melanocytic neoplasms but also BCCs. PMID:25080371

  13. Novel Germline Mutation in the Transmembrane Domain of HER2 in Familial Lung Adenocarcinomas

    PubMed Central

    2014-01-01

    We encountered a family of Japanese descent in which multiple members developed lung cancer. Using whole-exome sequencing, we identified a novel germline mutation in the transmembrane domain of the human epidermal growth factor receptor 2 (HER2) gene (G660D). A novel somatic mutation (V659E) was also detected in the transmembrane domain of HER2 in one of 253 sporadic lung adenocarcinomas. Because the transmembrane domain of HER2 is considered to be responsible for the dimerization and subsequent activation of the HER family and downstream signaling pathways, we performed functional analyses of these HER2 mutants. Mutant HER2 G660D and V659E proteins were more stable than wild-type protein. Both the G660D and V659E mutants activated Akt. In addition, they activated p38, which is thought to promote cell proliferation in lung adenocarcinoma. Our findings strongly suggest that mutations in the transmembrane domain of HER2 may be oncogenic, causing hereditary and sporadic lung adenocarcinomas. PMID:24317180

  14. Novel germline mutation in the transmembrane domain of HER2 in familial lung adenocarcinomas.

    PubMed

    Yamamoto, Hiromasa; Higasa, Koichiro; Sakaguchi, Masakiyo; Shien, Kazuhiko; Soh, Junichi; Ichimura, Koichi; Furukawa, Masashi; Hashida, Shinsuke; Tsukuda, Kazunori; Takigawa, Nagio; Matsuo, Keitaro; Kiura, Katsuyuki; Miyoshi, Shinichiro; Matsuda, Fumihiko; Toyooka, Shinichi

    2014-01-01

    We encountered a family of Japanese descent in which multiple members developed lung cancer. Using whole-exome sequencing, we identified a novel germline mutation in the transmembrane domain of the human epidermal growth factor receptor 2 (HER2) gene (G660D). A novel somatic mutation (V659E) was also detected in the transmembrane domain of HER2 in one of 253 sporadic lung adenocarcinomas. Because the transmembrane domain of HER2 is considered to be responsible for the dimerization and subsequent activation of the HER family and downstream signaling pathways, we performed functional analyses of these HER2 mutants. Mutant HER2 G660D and V659E proteins were more stable than wild-type protein. Both the G660D and V659E mutants activated Akt. In addition, they activated p38, which is thought to promote cell proliferation in lung adenocarcinoma. Our findings strongly suggest that mutations in the transmembrane domain of HER2 may be oncogenic, causing hereditary and sporadic lung adenocarcinomas. PMID:24317180

  15. Berberine Inhibits Intestinal Polyps Growth in Apc (min/+) Mice via Regulation of Macrophage Polarization.

    PubMed

    Piao, Meiyu; Cao, Hailong; He, NaNa; Yang, Boli; Dong, Wenxiao; Xu, Mengque; Yan, Fang; Zhou, Bing; Wang, Bangmao

    2016-01-01

    Antitumor effect of berberine has been reported in a wide spectrum of cancer, however, the mechanisms of which are not fully understood. The aim of this study was to investigate the hypothesis that berberine suppresses tumorigenesis in the familial adenomatous polyposis (FAP) by regulating the macrophage polarization in Apc (min/+) mouse model. Berberine was given to Apc (min/+) mice for 12 weeks. Primary macrophages were isolated; after berberine treatment, the change in signaling cascade was determined. The total number and size of polyps were reduced remarkably in berberine group, compared with control group. A significant decrease in protein levels of F4/80, mannose receptor (MR), and COX-2 in stroma of intestinal polyps and an increase in the level of iNOS were observed after berberine treatment. The mRNA level of MR and Arg-1 in berberine group was significantly lower than those in IL-10 or IL-4 group, while no significant difference in mRNA levels of iNOS and CXCL10 was observed. The migration and invasiveness assays in vitro showed that berberine could reduce the capability of migration and invasiveness. These findings suggest that berberine attenuates intestinal tumorigenesis by inhibiting the migration and invasion of colorectal tumor cells via regulation of macrophage polarization. PMID:27493671

  16. Increased variability in ApcMin/+ intestinal tissue can be measured with microultrasound

    NASA Astrophysics Data System (ADS)

    Fatehullah, A.; Sharma, S.; Newton, I. P.; Langlands, A. J.; Lay, H.; Nelson, S. A.; McMahon, R. K.; McIlvenny, N.; Appleton, P. L.; Cochran, S.; Näthke, I. S.

    2016-07-01

    Altered tissue structure is a feature of many disease states and is usually measured by microscopic methods, limiting analysis to small areas. Means to rapidly and quantitatively measure the structure and organisation of large tissue areas would represent a major advance not just for research but also in the clinic. Here, changes in tissue organisation that result from heterozygosity in Apc, a precancerous situation, are comprehensively measured using microultrasound and three-dimensional high-resolution microscopy. Despite its normal appearance in conventionally examined cross-sections, both approaches revealed a significant increase in the variability of tissue organisation in Apc heterozygous tissue. These changes preceded the formation of aberrant crypt foci or adenoma. Measuring these premalignant changes using microultrasound provides a potential means to detect microscopically abnormal regions in large tissue samples, independent of visual examination or biopsies. Not only does this provide a powerful tool for studying tissue structure in experimental settings, the ability to detect and monitor tissue changes by microultrasound could be developed into a powerful adjunct to screening endoscopy in the clinic.

  17. Increased variability in ApcMin/+ intestinal tissue can be measured with microultrasound

    PubMed Central

    Fatehullah, A.; Sharma, S.; Newton, I. P.; Langlands, A. J.; Lay, H.; Nelson, S. A.; McMahon, R. K.; McIlvenny, N.; Appleton, P. L.; Cochran, S.; Näthke, I. S.

    2016-01-01

    Altered tissue structure is a feature of many disease states and is usually measured by microscopic methods, limiting analysis to small areas. Means to rapidly and quantitatively measure the structure and organisation of large tissue areas would represent a major advance not just for research but also in the clinic. Here, changes in tissue organisation that result from heterozygosity in Apc, a precancerous situation, are comprehensively measured using microultrasound and three-dimensional high-resolution microscopy. Despite its normal appearance in conventionally examined cross-sections, both approaches revealed a significant increase in the variability of tissue organisation in Apc heterozygous tissue. These changes preceded the formation of aberrant crypt foci or adenoma. Measuring these premalignant changes using microultrasound provides a potential means to detect microscopically abnormal regions in large tissue samples, independent of visual examination or biopsies. Not only does this provide a powerful tool for studying tissue structure in experimental settings, the ability to detect and monitor tissue changes by microultrasound could be developed into a powerful adjunct to screening endoscopy in the clinic. PMID:27406832

  18. Berberine Inhibits Intestinal Polyps Growth in Apc (min/+) Mice via Regulation of Macrophage Polarization

    PubMed Central

    Piao, Meiyu; Cao, Hailong; He, NaNa; Yang, Boli; Dong, Wenxiao; Xu, Mengque; Yan, Fang; Zhou, Bing

    2016-01-01

    Antitumor effect of berberine has been reported in a wide spectrum of cancer, however, the mechanisms of which are not fully understood. The aim of this study was to investigate the hypothesis that berberine suppresses tumorigenesis in the familial adenomatous polyposis (FAP) by regulating the macrophage polarization in Apc (min/+) mouse model. Berberine was given to Apc (min/+) mice for 12 weeks. Primary macrophages were isolated; after berberine treatment, the change in signaling cascade was determined. The total number and size of polyps were reduced remarkably in berberine group, compared with control group. A significant decrease in protein levels of F4/80, mannose receptor (MR), and COX-2 in stroma of intestinal polyps and an increase in the level of iNOS were observed after berberine treatment. The mRNA level of MR and Arg-1 in berberine group was significantly lower than those in IL-10 or IL-4 group, while no significant difference in mRNA levels of iNOS and CXCL10 was observed. The migration and invasiveness assays in vitro showed that berberine could reduce the capability of migration and invasiveness. These findings suggest that berberine attenuates intestinal tumorigenesis by inhibiting the migration and invasion of colorectal tumor cells via regulation of macrophage polarization. PMID:27493671

  19. Lovastatin, but not orlistat, reduces intestinal polyp volume in an ApcMin/+ mouse model.

    PubMed

    Notarnicola, Maria; Barone, Michele; Francavilla, Antonio; Tutino, Valeria; Bianco, Giusy; Tafaro, Angela; Minoia, Mario; Polimeno, Lorenzo; Napoli, Anna; Scavo, Maria Principia; Caruso, Maria Gabriella

    2016-08-01

    The statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and orlistat, an inhibitor of fatty acid synthase (FAS), inhibit tumor cell growth by restricting cholesterol and fatty acid synthesis, respectively. We previously demonstrated that an omega (ω)-3 polyunsaturated fatty acid (PUFA)- or olive oil-enriched diet reduced the polyp number and volume in ApcMin/+ mice. This phenomenon was associated with a significant inhibition of FAS and HMGCoAR, as well as an increase in the estrogen receptor (ER)β/α ratio. Herein, we evaluated the effect of lovastatin and orlistat on polyp development and ER expression in ApcMin/+ mice, in order to confirm previous data obtained with ω‑3-PUFAs and olive oil. As expected, the use of lovastatin and orlistat significantly reduced HMGCoAR and FAS enzymatic activities and gene expression in colonic tissues, but did not affect the number of intestinal polyps, while there was a statistically significant reduction in polyp volume only in the mouse group treated with lovastatin. In the mice receiving orlistat, we observed a significant increase in cell proliferation in the polyp tissue, as well as enhanced expression of ERα. Moreover, the overexpression of ERα was associated with a statistically significant increase in PES1, Shh and Gli1 protein levels, considered ERα-related molecular targets. PMID:27277576