1,733 research outputs found
Loss of APC induces polyploidy as a result of a combination of defects in mitosis and apoptosis
No full textMutations in the adenomatous polyposis coli (APC) tumor suppressor gene initiate a majority of colorectal cancers. Acquisition of chromosomal instability is an early event in these tumors. We provide evidence that the loss of APC leads to a partial loss of inter-kinetochore tension at metaphase and alters mitotic progression. Furthermore, we show that inhibition of APC in U2OS cells compromises the mitotic spindle checkpoint. This is accompanied by a decrease in the association of the checkpoint proteins Bub1 and BubR1 with kinetochores. Additionally, APC depletion reduced apoptosis. As expected from this combination of defects, tetraploidy and polyploidy are consequences of APC inhibition in vitro and in vivo. The removal of APC produced the same defects in HCT1 16 cells that have constitutively active P-catenin. These data show that the loss of APC immediately induces chromosomal instability as a result of a combination of mitotic and apoptotic defects. We suggest that these defects amplify each other to increase the incidence of tetra- and polyploidy in early stages of tumorigenesis
Precocious activation of APC/C-Cdh1 at pre-anaphase causes genome instability
No full textFaithful chromosome segregation and thereby accurate gene transmission are crucial for all organisms. Until proper attachment of the mitotic spindle to the kinetochore is established, the ubiquitin ligase (E3) Cdc20-activated APC/C (anaphase promoting complex/cyclosome) is repressed by the spindle assembly checkpoint (SAC) and sister chromatin cohesion is protected. Mutants defective in SAC fail to arrest at metaphase even in the presence of damaged microtubules. Interestingly, a similar phenomenon occurs in yeast cells defective in Bub2, a negative factor of the mitotic exit network (MEN), which is required for telophase onset, although its precise molecular mechanism is unknown. Here, we show that chromosome missegregation occurs frequently in bub2∆ cells in the presence of damaged microtubules. The loss of Bub2 caused precocious activation of APC/C-Cdh1/Hct1 at pre-anaphase, leading to securin degradation and then separase-mediated cohesin cleavage. Overexpression of CDH1 and CDC14, encoding Cdc14 phosphatase, at pre-anaphase similarly caused chromosome missegregation. Thus, sequential activation of APC/C-Cdc20 and then APC/C-Cdh1 is critical for precise chromosome segregation and precocious activation of APC/C-Cdh1 at pre-anaphase causes genomic instability. Since degradation of human securin is also mediated by APC/C-Cdc20 and APC/C-Cdh1, this study predicts that precocious activation APC/C-Cdh1 in human cells similarly causes genomic instability, and thereby cell death or tumorigenesis
Càncer colorectal hereditari: Aplicacions diagnòstiques de l'estudi de la dosi dels gens APC, MLH1 i MSH2
No full text[cat] Les síndromes de càncer colorectal (CCR) hereditari representen entre un 3% i un 5% de tots els casos de CCR i inclouen tots aquells individus amb un elevat grau d'agregació familiar. La més freqüent és la síndrome de Lynch, causada per la presència de mutacions en els gens reparadors del DNA, majoritàriament MLH1 i MSH2. La poliposi adenomatosa familiar (FAP) és la segona en incidència, es caracteritza per l'aparició de pòlips precursors a la neoplàsia colorectal i la seva causa principal és la presència de mutacions en el gen supressor tumoral APC. En els últims anys s'ha descobert que els grans reordenaments d'aquests gens són responsables de la malaltia en una part de les famílies que pateixen aquestes síndromes. A més, estudis d'expressió d'aquests gens en línia germinal han demostrat l'existència de desequilibris al·lèlics tant en famílies portadores de mutacions com en famílies on no es detecten mutacions en el DNA. L'objectiu d'aquesta tesi és l'estudi de la dosi en la dels gens MLH1, MSH2 i APC, implicats en la síndrome de Lynch i la FAP. D'aquesta forma, hem analitzat la dosi tant a nivell de DNA (grans reordenaments) com a nivell d'RNA (expressió específica d'al·lel), sempre treballant amb línia germinal de pacients. Els resultats aquí recollits permeten millorar l'estratègia de diagnòstic molecular de les famílies amb síndrome de Lynch i FAP que són ateses al nostre centre, al mateix temps que la comprensió del procés tumorogènic.[eng] Hereditary colorectal cancer (CRC) syndromes represent about 3% to 5% of all cases of CRC and include all those individuals with high familiar aggregation. The most frequent syndrome is Lynch syndrome, caused by the presence of mutations in the mismatch repair (MMR) genes, mostly MLH1 and MSH2. Familiar adenomatous polyposis (FAP) is the second in incidence, is characterized by precursor polyps and its mainly caused by mutations in the tumoral suppressor gene APC. Recently, it has been discovered that gross rearrangements of these genes are responsible of these two syndromes. Also, expression analyses of these genes in the germline have demonstrated the existence of allelic imbalances in both families carrying pathogenic mutations and families without detected mutations. Our aim was to study de dose of MLH1, MSH2 and APC genes in the germline of Lynch syndrome and polyposis families, respectively. To that end, we analyzed the dose at DNA level (gross rearrangements) and at RNA level (allele-specific expression) of these patients. The results summarized in this thesis permit improving the molecular diagnostic strategy in Lynch syndrome and FAP families, and also improve the knowledge of the tumorogenic process
The <i>Adenomatous Polyposis Coli</i> protein contributes to normal compaction of mitotic chromatin
No full textThe tumour suppressor <i>Adenomatous Polyposis Coli</i> (APC) is required for proper mitosis; however, the exact role of APC in mitosis is not understood. Using demembranated sperm chromatin exposed to meiotic <i>Xenopus</i> egg extract and HeLa cells expressing fluorescently labelled histones, we established that APC contributes to chromatin compaction. Sperm chromatin in APC-depleted <i>Xenopus</i> egg extract frequently formed tight round or elongated structures. Such abnormally compacted chromatin predominantly formed spindles with low microtubule content. Furthermore, in mitotic HeLa cells expressing GFP- and mCherry-labelled H2B histones, depletion of APC caused a decrease in the donor fluorescence lifetime of neighbouring fluorophores, indicative of excessive chromatin compaction. Profiling the chromatin-associated proteome of sperm chromatin incubated with <i>Xenopus</i> egg extracts revealed temporal APC-dependent changes in the abundance of histones, closely mirrored by chromatin-associated Topoisomerase IIa, condensin I complex and Kif4. In the absence of APC these factors initially accumulated on chromatin, but then decreased faster than in controls. We also found and validated significant APC-dependent changes in chromatin modifiers Set-a and Rbbp7. Both were decreased on chromatin in APC-depleted extract; in addition, the kinetics of association of Set-a with chromatin was altered in the absence of APC
Gene expression analyses on skin lesions from patients with familial adenomatous polyposis
No full textFamilial adenomatous polyposis coli (FAP) is an autosomal dominant colorectal cancer predisposition syndrome caused by germline mutations in the APC gene. It is characterized by an increased risk for the development of both several internal cancers and benign skin tumors such as fibromas, lipomas, and epidermal cysts occurring with different frequencies early in life. The molecular mechanisms underlying these skin lesions are still poorly understood.
In this study we aimed to clarify the underlying molecular mechanisms in the development of FAP-associated skin lesions. Such mechanisms were hypothesized to either follow the APC second hit model or to include other genes, possibly such independent of Wnt signaling.
To this end we analyzed 9 fibromas, 3 lipomas, and 3 epidermal cysts from 14 FAP patients of 7 families with pathogenic APC germline mutations for somatic alterations by direct sequencing of the mutation cluster region (MCR), exon-overlapping cDNA analysis, and locus-specific marker analysis. Somatic changes were found in two skin lesions, one lipoma and one epidermal cyst. Both lesions displayed loss of heterozygosity (LOH) at APC marker locus D5S346. The epidermal cyst in addition carried a somatic mutation (c.4778delA) in the MCR of APC. These results suggest that somatic APC alterations may influence the development of FAP-associated lipomas and epidermal cysts.
For the investigation of APC-independent processes we analyzed in total 5 fibromas, 6 lipomas and 3 epidermal cysts compared to healthy skin of 13 FAP patients by whole genome expression analysis and confirmed targets of highest expression changes by qPCR. We show that genes mostly changed in fibromas and lipomas of FAP patients mainly function in cell proliferation processes. Therefore we suggest that FAP-associated cutaneous neoplasia might develop by the influence of activated proto-oncogenes and deactivated tumor suppressors similar to other tumors. We suppose that an invasive growth is prevented by increased expression of tumor suppressors in those benign neoplasms. In comparison to the general population expression results of FAP lipomas have also been compared to similar lesions of non-FAP individuals. Non-FAP lipomas tend to be mainly influenced by genes involved in lipid metabolism.
In conclusion, we assume that FAP-associated skin lesions are mostly not caused by APC second hits. In contrast, we rather suppose Wnt independent mechanisms. In addition, we suggest that lipomas develop differentially in FAP patients and in the general population
lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2
No full textBackground: Ubiquitin-dependent protein degradation is a critical step in key cell cycle events, such as metaphase-anaphase transition and mitotic exit. The anaphase promoting complex/cyclosome (APC/C) plays a pivotal role in these transitions by recognizing and marking regulatory proteins for proteasomal degradation. Its overall structure and function has been elucidated mostly in yeasts and mammalian cell lines. The APC/C is, however, a multisubunit assembly with at least 13 subunits and their function and interaction within the complex is still relatively uncharacterized, particularly in metazoan systems. Here, lemming (lmg) mutants were used to study the APC/C subunit, Apc11, and its interaction partners in Drosophila melanogaster. Results: The lmg gene was initially identified through a pharate adult lethal P element insertion mutation expressing developmental abnormalities and widespread apoptosis in larval imaginal discs and pupal abdominal histoblasts. Larval neuroblasts were observed to arrest mitosis in a metaphase-like state with highly condensed, scattered chromosomes and frequent polyploidy. These neuroblasts contain high levels of both cyclin A and cyclin B. The lmg gene was cloned by virtue of the lmg(03424) P element insertion which is located in the 5' untranslated region. The lemming locus is transcribed to give a 2.0 kb mRNA that contains two ORFs, lmgA and lmgB. The lmgA ORF codes for a putative protein with more than 80% sequence homology to the APC11 subunit of the human APC/C. The 85 amino acid protein also contains a RING-finger motif characteristic of known APC11 subunits. The lmgA ORF alone was sufficient to rescue the lethal and mitotic phenotypes of the lmg(138) null allele and to complement the temperature sensitive lethal phenotype of the APC11-myc9 budding yeast mutant. The LmgA protein interacts with Mr/Apc2, and they together form a binding site for Vihar, the E2-C type ubiquitin conjugating enzyme. Despite being conserved among Drosophila species, the LmgB protein is not required for viability or fertility. Conclusions: Our work provides insight into the subunit structure of the Drosophila APC/C with implications for its function. Based on the presented data, we suggest that the Lmg/Apc11 subunit recruits the E2-C type ubiquitin conjugating enzyme, Vihar, to the APC/C together with Mr/Apc2 by forming a ternary complex
The Initiation and Progression of Neoplasia in Inherited and Sporadic Colorectal Cancer.
No full textPhDThis dissertation describes investigations into aspects of neoplastic initiation and
progression in the gut, in the context of inherited and sporadic gastrointestinal
cancer.
In familial adenomatous polyposis, there is marked locoregional variation in
polyp density. This work investigated the relationship between gut location and
somatic inactivation of APC. It demonstrated that the frequency of APC loss
of heterozygosity (LOR) varied along the colon, suggesting a colonic gradient
in "just right" signalling. It investigated adenomas of the ileoanal pouch, where
small bowel functions as rectum. A longitudinal study revealed the largely indolent
natural history of these polyps, while mutational analysis demonstrated
that they differed from polyps arising in colon, by usually retaining 2-3 remaining
20-amino-acid repeats. A comparison of pouch and rectal polyps showed
differences in their histogenesis.
Neoplastic initiation was investigated arising in the context of a f!t' new phenotype
of young-onset colorectal cancer, with multiple adenomas and neurological
deficit. A novel homozygous mutation involving PMS2 and FSCJV was found.
Although neoplastic lesions showed nuclear beta-catenin expression, no mutated
wnt pathway genes were identified. No evidence was found that PMS2 or FSCN
mutation was causative in other patients with related multiple adenoma or neurological
phenotypes.
Key stepwise events in the adenoma-carcinoma sequence were investigated in
sporadically occurring adenomas with contiguous carcinoma. Laser microdissection
allowed crypt-by-crypt analysis of APC, TP53, KRAS2, and LOR at
17p and 18q, in order to establish the presence of cryptal heterogeneity and
the inferred order of genetic alterations. Using similar contiguous paired lesions,
a further study investigated neoplastic aneuploidy using image cytometry~
genome-wide LOR analysis and individual SNP analysis for LOR 5q, 17p, and
18q. Aneuploidy did not cluster as tetraploidy. The degree of aneuploidy correlated
well with the degree of LOR ascertained by genome-wide analysis, with
LOR at 17p and 18q (but not 5q), and with nuclear beta-catenin accumulation
Identification of novel germline mutations in hereditary colorectal cancer patients and characterization of somatic alterations in their tumors
No full textColorectal cancer has been reported as the third leading cause of cancer related death in the world. About 5-10% of colorectal cancers are due to an inherited predisposition. This thesis focuses on investigating the prevalence of large genomic rearrangements and other types of germline mutations in novel cancer susceptibility genes in two major hereditary colorectal cancer syndromes, hereditary nonpolyposis colorectal cancer (HNPCC) and familial adenomatous polyposis (FAP). Furthermore, the second somatic mutations were characterized in the tumors from DNA mismatch repair (HNPC) and APC gene mutation carriers (FAP) to address the mechanism(s) of tumorigenesis in these two syndromes. All these investigations aim to understand tumor initiation and progression in hereditary colorectal cancer syndromes in order to enable early and reliable presymptomatic diagnosis of a person at increased risk and offer optimal medical management to prevent cancer. HNPCC is an autosomal dominantly inherited cancer predisposition syndrome caused by germline mutations in DNA mismatch repair (MMR) genes. Prescreening methods are routinely applied to detect MMR gene sequence alterations, but inevitably miss large genomic rearrangements. Here, two novel PCR-based methods to study gene dosage were introduced in 35 MLH/MSH2 HNPCC patients in whom no mutation could be identified by conventional screening methods. These methods are QMPA (quantitative multiplex PCR amplification) and MLPA (multiplex ligation dependent probe amplification). Three patients were found to carry large deletions by QMPA and MLPA. In 1 patient, however, QMPA yielded a false positive result. Both methods, QMPA and MLPA appear to be of comparable sensitivity albeit with different specificity. Since the QMPA technique is difficult to set up and to standardize the PCR conditions, the MLPA assay is better suited to routinely search for large genomic rearrangements. The investigations subsequently continued to detect the frequency and nature of LOH as second, somatic event in tumors from MLH/MSH2 germline deletion carriers. MLPA technique was applied to analyze 18 cancer specimens from two independent sets of Swiss and Finnish MLH1/MSH2 deletion carriers. Results revealed that somatic deletions identical to the ones in the germline occur frequently (55%) in CRCs and that this type of loss of the wild type allele is also present in extracolonic HNPCC associated tumors. Chromosome specific marker analysis implies that loss of the wild type allele predominantly occurs through locus restricted recombination events, i.e. gene conversion, rather than mitotic recombination or deletion of the respective gene
locus. The same investigation was carried on a 31 years old colorectal cancer patient who carries de novo mutation (c.666dupA) in the MLH1 gene. The tumor analysis of this patient showed a similar somatic mutation mechanism to the large genomic deletion carriers. Prior to our analysis of the somatic hits in the attenuated form of familial adenomatous polyposis (AFAP), earlier investigations had shown that in classical FAP the "two hits" in the APC (Adenomatosis polyposis coli) gene are not occurring randomly but are in fact interdependent. AFAP is clinically characterized by fewer than 100 adenomatous polyps in the colorectum and presents with a milder phenotype compared to classical FAP. APC mutations in AFAP patients are typically located in the very 5’ and 3’ gene regions as well as in the alternatively spliced region of exon 9. In a collaborative effort we investigated the somatic alterations in 235 tumors of 35 AFAP patients. Adenomas of AFAP patients were often found to actually exhibit ‘three hits’ at the APC gene that mostly result in loss of the allele carrying the germline APC mutation. We assume that this actually leads to an optimization of the beta-catenin level, hence positively regulating the Wnt signal. Recently, bi-allelic germline mutations in the base excision repair gene MutY homologue (MYH) have been associated with an autosomal recessively inherited predisposition to multiple colorectal adenomas. They are also referred to as MYH-associated polyposis (MAP). Here, we assessed the prevalence of MYH germline alteration in 79 unrelated polyposis patients in whom no APC mutation could be detected. The aims of the study were i) to assess the MYH mutation carrier frequency among Swiss APC mutation negative patients and (ii) to identify phenotypic differences between MYH mutation carriers and APC / MYH mutation negative polyposis patients. dHPLC and direct genomic DNA sequencing were applied to screen for mutation. Overall, 7 biallelic and 9 monoallelic MYH germline mutation carriers were identified. 1 out of 10 classical polyposis and 6 out of 35 attenuated polyposis patients carried biallelic MYH alterations, 2 of which represent novel gene variants (p.R 171q and p.R 231H). On the basis of our finding and earlier reports, MYH mutation screening should be considered if all of the following criteria are fulfilled: (1) presence of classical or attenuated polyposis coli, 2) absence of a pathogenic APC mutation, and 3) a family history compatible with an autosomal recessive mode of inheritance
Hanseniaspora meyeri (APC 12.1) genome annotation
No full textAnnotation of the Hanseniaspora meyeri (APC 12.1) genome that is deposited at NCBI under the bioproject PRJNA907227
Pichia kluyveri (APC 11.10 B) genome annotation
No full textAnnotation of the Pichia kluyveri (APC 11.10 B) genome that is deposited at NCBI under the bioproject XXXX
- …
