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The acetylene-vinylidene system serves as a benchmark for investigations of ultrafast dynamical processes where the coupling of the electronic and nuclear degrees of freedom provides a fertile playground to explore the femto- and sub-femto-second physics with coherent extreme-ultraviolet (EUV) photon sources both on the table-top as well as free-electron lasers. We focus on detailed investigations of this molecular system in the photon energy range 19-40 eV where EUV pulses can probe the dynamics effectively. We employ photoelectron-photoion coincidence (PEPICO) spectroscopy to uncover hitherto unrevealed aspects of this system. In this work, the role of excited states of the C2H2+ cation, the primary photoion, is specifically addressed. From photoelectron energy spectra and angular distributions, the nature of the dissociation and isomerization channels is discerned. Exploiting the 4π-collection geometry of the velocity map imaging spectrometer, we not only probe pathways where the efficiency of photoionization is inherently high but also perform PEPICO spectroscopy on relatively weak channels.
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Embedded atoms or molecules in a photoexcited He nanodroplet are well-known to be ionized through inter-atomic relaxation in a Penning process. In this work, we investigate the Penning ionization of acetylene oligomers occurring from the photoexcitation bands of He nanodroplets. In close analogy to conventional Penning electron spectroscopy by thermal atomic collisions, the n = 2 photoexcitation band plays the role of the metastable atomic 1s2s 3,1S He*. This facilitates electron spectroscopy of acetylene aggregates in the sub-Kelvin He environment, providing the following insight into their structure: the molecules in the dopant cluster are loosely bound van der Waals complexes rather than forming covalent compounds. In addition, this work reveals a Penning process stemming from the n = 4 band where charge-transfer from autoionized He in the droplets is known to be the dominant relaxation channel. This allows for excited states of the remnant dopant oligomer Penning-ions to be studied. Hence, we demonstrate Penning ionization electron spectroscopy of doped droplets as an effective technique for investigating dopant oligomers which are easily formed by attachment to the host cluster.
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BACKGROUND: Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer. Preclinical studies show that anti-FGFR3 treatment slows down tumor growth, suggesting that this tyrosine kinase receptor is a candidate for personalized bladder cancer treatment, particularly in patients with mutated FGFR3. We addressed tumor heterogeneity in a large multicenter, multi-laboratory study, as this may have significant impact on therapeutic response. PATIENTS AND METHODS: We evaluated possible FGFR3 heterogeneity by the PCR-SNaPshot method in the superficial and deep compartments of tumors obtained by transurethral resection (TUR, n = 61) and in radical cystectomy (RC, n = 614) specimens and corresponding cancer-positive lymph nodes (LN+, n = 201). RESULTS: We found FGFR3 mutations in 13/34 (38%) T1 and 8/27 (30%) ≥T2-TUR samples, with 100% concordance between superficial and deeper parts in T1-TUR samples. Of eight FGFR3 mutant ≥T2-TUR samples, only 4 (50%) displayed the mutation in the deeper part. We found 67/614 (11%) FGFR3 mutations in RC specimens. FGFR3 mutation was associated with pN0 (P < 0.001) at RC. In 10/201 (5%) LN+, an FGFR3 mutation was found, all concordant with the corresponding RC specimen. In the remaining 191 cases, RC and LN+ were both wild type. CONCLUSIONS: FGFR3 mutation status seems promising to guide decision-making on adjuvant anti-FGFR3 therapy as it appeared homogeneous in RC and LN+. Based on the results of TUR, the deep part of the tumor needs to be assessed if neoadjuvant anti-FGFR3 treatment is considered. We conclude that studies on the heterogeneity of actionable molecular targets should precede clinical trials with these drugs in the perioperative setting.
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Biomarcadores Tumorais/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Tomada de Decisão Clínica , Cistectomia , Feminino , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Humanos , Linfonodos/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Período Perioperatório , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgiaRESUMO
We report a comparative study of the features in dissociative double ionization by high energy electron impact of N2 and CO molecules. The ratio of cross-section of charge symmetric dissociative ionization to non-dissociative ionization (CSD-to-ND ratio) and the kinetic energy release (KER) spectra of dissociation are experimentally measured and carefully corrected for various ion transmission losses and detector inefficiencies. Given that the double ionization cross sections of these iso-electronic diatomics are very similar, the large difference in the CSD-to-ND ratios must be attributable to the differences in the evolution dynamics of the dications. To understand these differences, potential energy curves (PECs) of dications have been computed using multi-reference configuration interaction method. The Franck-Condon factors and tunneling life times of vibrational levels of dications have also been computed. While the KER spectrum of N2 (++) can be readily explained by considering dissociation via repulsive states and tunneling of meta-stable states, indirect dissociation processes such as predissociation and autoionization have to be taken into account to understand the major features of the KER spectrum of CO(++). Direct and indirect processes identified on the basis of the PECs and experimental KER spectra also provide insights into the differences in the CSD-to-ND ratios.
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Fragmentation kinematics of CS2 following various S(2p) Auger transitions is studied. Employing a combination of electron energy analysis and recoil ion momentum spectroscopy, changes in the dissociation channel yields, as well as the differences in the kinematical parameters for various bands of Auger hole states are presented. The fragmentation mechanism for dissociative channels leading to complete atomization of CS2(2+) molecular ion is studied in detail. We find that CS2(2+) does not retain linear geometry and is bent before undergoing concerted break-up. It is also observed that different geometric configurations of the CS2(2+) precursor result in different kinetic energy release values.
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Alterations of the human MSH2 gene, a homologue of the bacterial MutS mismatch repair gene, co-segregate with the majority of hereditary non-polyposis colon cancer (HNPCC) cases. We have generated homozygous MSH2-/- mice. Surprisingly, these mice were found to be viable, produced offspring in a mendelian ratio and bred through at least two generations. Starting at two months of age homozygous-/- mice began, with high frequency, to develop lymphoid tumours that contained microsatellite instabilities. These data establish a direct link between MSH2 deficiency and the pathogenesis of cancer. These mutant mice should be good models to study the progression of tumours and also to screen carcinogenic and anti-cancer agents.
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Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA/genética , DNA de Neoplasias/análise , DNA Satélite/análise , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas , Marcação de Genes , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animais , Sequência de Bases , Transformação Celular Neoplásica/genética , DNA de Neoplasias/genética , Feminino , Genótipo , Humanos , Tecido Linfoide/patologia , Masculino , Meiose , Camundongos , Camundongos Knockout , Camundongos Mutantes , Dados de Sequência Molecular , Proteína 2 Homóloga a MutS , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Especificidade da EspécieRESUMO
Isolated human microphthalmia/anophthalmia, a cause of congenital blindness, is a clinically and genetically heterogeneous developmental disorder characterized by a small eye and other ocular abnormalities. Three microphthalmia/anophthalmia loci have been identified, and two others have been inferred by the co-segregation of translocations with the phenotype. We previously found that mice with ocular retardation (the or-J allele), a microphthalmia phenotype, have a null mutation in the retinal homeobox gene Chx10 (refs 7,8). We report here the mapping of a human microphthalmia locus on chromosome 14q24.3, the cloning of CHX10 at this locus and the identification of recessive CHX10 mutations in two families with non-syndromic microphthalmia (MIM 251600), cataracts and severe abnormalities of the iris. In affected individuals, a highly conserved arginine residue in the DNA-recognition helix of the homeodomain is replaced by glutamine or proline (R200Q and R200P, respectively). Identification of the CHX10 consensus DNA-binding sequence (TAATTAGC) allowed us to demonstrate that both mutations severely disrupt CHX10 function. Human CHX10 is expressed in progenitor cells of the developing neuroretina and in the inner nuclear layer of the mature retina. The strong conservation in vertebrates of the CHX10 sequence, pattern of expression and loss-of-function phenotypes demonstrates the evolutionary importance of the genetic network through which this gene regulates eye development.
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Proteínas de Homeodomínio/genética , Microftalmia/genética , Fatores de Transcrição/genética , Adulto , Mapeamento Cromossômico , Cromossomos Humanos Par 14/genética , Análise Mutacional de DNA , Éxons , Saúde da Família , Evolução Fatal , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes/genética , Genes Homeobox/genética , Humanos , Lactente , Íntrons , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Retina/crescimento & desenvolvimento , Retina/metabolismoRESUMO
BACKGROUND: In colorectal cancer (CRC), tumour microsatellite instability (MSI) status and CpG island methylator phenotype (CIMP) status are indicators of patient outcome, but the molecular events that give rise to these outcomes remain largely unknown. Wnt5a is a critical regulator of non-canonical Wnt activity and promoter hypermethylation of this gene has emerging prognostic roles in CRC; however the frequency and prognostic significance of this epigenetic event have not been explored in the context of colorectal tumour subtype. Consequently, we investigated the frequency and prognostic significance of Wnt5a methylation in a large cohort of MSI-stratified CRCs. METHODS: Methylation was quantified in a large cohort of 1232 colorectal carcinomas from two clinically distinct populations from Canada. Associations were examined between methylation status and clinicopathlogical features, including tumour MSI status, BRAF V600E mutation, and patient survival. RESULTS: In Ontario, Wnt5a methylation was strongly associated with MSI tumours after adjustment for age, sex, and tumour location (odds ratio (OR)=4.2, 95% confidence interval (CI)=2.4-7.4, P<10(-6)) and with BRAF V600E mutation, a marker of CIMP (OR=12.3, 95% CI=6.9-21.7, P<10(-17)), but was not associated with patient survival. Concordant results were obtained in Newfoundland. CONCLUSION: Methylation of Wnt5a is associated with distinct tumour subtypes, strengthening the evidence of an epigenetic-mediated Wnt bias in CRC.
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Neoplasias Colorretais/genética , Metilação de DNA , Instabilidade de Microssatélites , Mutação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Wnt/genética , Adulto , Idoso , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Wnt-5aRESUMO
BACKGROUND AND AIMS: Colorectal cancer (CRC) is the second most frequent cancer in developed countries. Newfoundland has the highest incidence of CRC in Canada and the highest rate of familial CRC yet reported in the world. To determine the impact of mutations in known CRC susceptibility genes and the contribution of the known pathways to the development of hereditary CRC, an incident cohort of 750 patients with CRC (708 different families) from the Newfoundland population was studied. METHODS: Microsatellite instability (MSI) testing was performed on tumours, together with immunohistochemistry analysis for mismatch repair (MMR) genes. Where indicated, DNA sequencing and multiplex ligation-dependent probe amplifications of MMR genes and APC was undertaken. DNA from all patients was screened for MUTYH mutations. The presence of the BRAF variant, p.V600E, and of MLH1 promoter methylation was also tested in tumours. RESULTS: 4.6% of patients fulfilled the Amsterdam criteria (AC), and an additional 44.6% fulfilled the revised Bethesda criteria. MSI-high (MSI-H) was observed in 10.7% (n=78) of 732 tumours. In 3.6% (n=27) of patients, CRC was attributed to 12 different inherited mutations in six known CRC-related genes associated with chromosomal instability or MSI pathways. Seven patients (0.9%) carried a mutation in APC or biallelic mutations in MUTYH. Of 20 patients (2.7%) with mutations in MMR genes, 14 (70%) had one of two MSH2 founder mutations. 17 of 28 (61%) AC families did not have a genetic cause identified, of which 15 kindreds fulfilled the criteria for familial CRC type X (FCCTX). CONCLUSIONS: Founder mutations accounted for only 2.1% of cases and this was insufficient to explain the high rate of familial CRC. Many of the families classified as FCCTX may have highly penetrant mutations segregating in a Mendelian-like manner. These families will be important for identifying additional CRC susceptibility loci.
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Neoplasias Colorretais/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Distribuição por Idade , Idoso , Neoplasias Colorretais/epidemiologia , Metilação de DNA , Reparo de Erro de Pareamento de DNA/genética , DNA de Neoplasias/genética , Feminino , Efeito Fundador , Predisposição Genética para Doença , Humanos , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Mutação , Proteínas de Neoplasias/genética , Terra Nova e Labrador/epidemiologia , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas B-raf/genética , Sistema de RegistrosRESUMO
ApoE single nucleotide polymorphisms (SNPs) Cys112Arg (Epsilon-4), and Arg158Cys (Epsilon-2) have been implicated in cardiovascular and Alzheimer's disease, but their role in colorectal cancer (CRC) has not been extensively studied. We investigated whether ApoE polymorphisms alone or in combination with dietary factors selectively contribute to mismatch-repair (MMR) proficient (microsatellite stable/low or MSS/L) vs deficient (microsatellite unstable or MSI-H) CRCs. We carried out a case-control study with 906 CRC cases and 911 unaffected controls to examine the associations between ApoE polymorphisms and dietary factors and assessed their contribution to MSS/L and MSI-H CRCs. We used unconditional logistic regression to evaluate the associations between ApoE SNPs, tumour MSI status, and dietary factors after adjusting for age and sex. All statistical tests were two-sided. No significant differences in ApoE genotype frequencies were observed between CRC cases and unaffected controls. We observed that increased dietary intake of total fat, saturated fat, cholesterol, and red meat was significantly associated with CRC. Among non-ApoE4 carriers, 2-4 and >4 red meat servings/week were associated with developing MSS/L CRC (OR=1.51, 95% CI 1.10-2.07 and OR=1.80, 95% CI 1.30-2.48, respectively), whereas among ApoE4 allele carriers, four or more red meat servings/week were associated with MSI-H CRC (OR=4.62, 95% CI 1.20-17.77) when compared with the controls. ApoE isoforms modulate the risk of MSI-H and MSS/L CRCs among high red meat consumers.
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Apolipoproteínas E/genética , Neoplasias Colorretais/genética , Reparo do DNA/genética , Dieta , Polimorfismo de Nucleotídeo Único/genética , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Masculino , Carne , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Fatores de Risco , Adulto JovemRESUMO
The synovial fluid or group II secretory phospholipase A2 (sPLA2) has been implicated in various inflammatory processes and has been shown to release arachidonic acid for prostaglandin biosynthesis. In human colorectal cancer, both arachidonic acid and eicosanoid levels are elevated. Recently, sPLA2 has been identified as a candidate gene that modifies the Apc gene in the Min mouse, a murine model for familial adenomatous polyposis (FAP). Loss of sPLA2 gene function results in susceptibility to the Min phenotype and the formation of multiple intestinal polyps, whereas mice expressing an active sPLA2 gene are resistant to polyp formation. Therefore, there are two potentially contrasting roles for sPLA2 in colon cancer; one is protection against polyp formation, and the other, the release of arachidonic acid for prostaglandin production and subsequent tumor promotion. To investigate these contrasting dual roles of sPLA2, we have examined the expression and sequence of the sPLA2 mRNA in normal mucosa and duodenal and colorectal polyps from FAP patients. In 11 of 14 patients, there was a significant increase in sPLA2 mRNA levels in the adenoma over the normal tissue. In some cases, there was over 100-fold increase in mRNA levels in the adenoma compared with normal tissue. Analysis of multiple adenomatous polyps from individual patients revealed that not all polyps contained elevated levels of sPLA2 mRNA. Immunoblot analysis also showed that sPLA2 protein expression was elevated in adenoma over normal tissue in five of six FAP patients analyzed. Furthermore, sequence analysis of sPLA2 mRNA present in these samples did not reveal mutations in the coding region. The implications of the up-regulation of sPLA2 in FAP is not clear, but unlike the Min mouse model, it does not seem to have a significant effect on polyp formation. In contrast, the high level of sPLA2 expression is more likely contributing to the elevated levels of arachidonic acid found in colorectal cancer and, in conjunction with the elevated expression of cyclooxygenase-2, could be another factor in tumor formation.
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Adenoma/enzimologia , Polipose Adenomatosa do Colo/enzimologia , Neoplasias Colorretais/enzimologia , Regulação Neoplásica da Expressão Gênica , Isoenzimas/biossíntese , Proteínas de Neoplasias/biossíntese , Fosfolipases A/biossíntese , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Adenoma/genética , Polipose Adenomatosa do Colo/genética , Animais , Ácido Araquidônico/metabolismo , Neoplasias Colorretais/genética , Ciclo-Oxigenase 2 , Análise Mutacional de DNA , Modelos Animais de Doenças , Neoplasias Duodenais/enzimologia , Indução Enzimática , Fosfolipases A2 do Grupo II , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana , Camundongos , Proteínas de Neoplasias/genética , Fosfolipases A/genética , Fosfolipases A2 , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/genética , RNA Neoplásico/genéticaRESUMO
Aberrant crypt foci (ACF) are microscopic lesions which have been postulated to precede the development of adenomatous polyps, the precursors to colorectal cancer. APC and ras gene mutations have been shown to be important early molecular events in the development of colorectal neoplasms. The objective of this study was to establish the nature and frequency of these two genetic alterations in ACF harvested from human colorectal resection specimens. One hundred and fifty-four ACF comprised of between 1 and 56 crypts were harvested from the grossly normal mucosa of colorectal resection specimens of 28 patients with varying pathological diagnoses. One hundred and twenty-five ACF from 20 colons were screened for the presence of K-ras codon 12 mutations with a polymerase chain reaction/restriction enzyme-based method. The APC gene mutation cluster region was screened in 65 ACF from 20 colons using a polymerase chain reaction/single strand conformation polymorphism technique. Putative mutations were confirmed by direct sequencing. K-ras codon 12 mutations were identified in 13% (16 of 125) of ACF. We also identified APC mutations in 4.6% (3 of 65) of ACF. The results of this study demonstrate that both APC and K-ras mutations occur in ACF. These observations support the role of the ACF as a colorectal cancer precursor and provide further insight into the early genetic changes which occur during colorectal tumorigenesis.
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Pólipos Adenomatosos/genética , Códon/genética , Pólipos do Colo/genética , Genes APC/genética , Genes ras/genética , Lesões Pré-Cancerosas/genética , Sequência de Bases , Primers do DNA/genética , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodosRESUMO
Germline mutations of the hMSH2 gene are responsible for many cases of hereditary nonpolyposis colorectal cancer. While screening for hMSH2 gene mutations in hereditary nonpolyposis colorectal cancer kindreds, we observed that a previously reported germline mutation is in fact a common, alternatively spliced variant in the population. Using RT-PCR and the protein truncation test, the hMSH2 exon 13 deletion variant was found in more than 90% of individuals. The exon 13 deletion transcript was only present in lymphocyte RNA, no abnormalities were detected in genomic DNA flanking exon 13, and the deletion transcript is apparently not translated. These findings highlight further that caution should be exercised in providing genetic risk assessment on the basis of currently used germline mutation detection strategies.
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Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais/prevenção & controle , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas , Aconselhamento Genético , Testes Genéticos , Variação Genética , Deleção de Sequência , Neoplasias Colorretais/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Análise Mutacional de DNA , Proteínas de Ligação a DNA/química , Éxons/genética , Reações Falso-Positivas , Regulação Neoplásica da Expressão Gênica , Humanos , Linfócitos/química , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase , Splicing de RNA , RNA Mensageiro/genética , RNA Neoplásico/genética , Medição de RiscoRESUMO
Some colorectal tumors with wild-type adenomatous polyposis coli gene have activating mutations in beta-catenin (encoded by CTNNB1) that result in decreased phosphorylation by GSK-3beta and increased signaling through the Tcf/Lef transcription factors. To investigate the relationship between CTNNB1 mutations and underlying pathways of genomic instability, we examined 80 colorectal cancers stratified by the presence or absence of microsatellite instability (MSI). CTNNB1 mutations were identified in 13 (25%) of 53 cancers with high frequency MSI (MSI-H), including 12 point mutations at exon 3 phosphorylation sites (codons 41 and 45) and one deletion of the entire exon 3 degradation box. No CTNNB1 mutations were identified in 27 microsatellite stable or low frequency MSI (MSI-L) colorectal cancers (P < 0.01). In contrast, CTNNB1 mutations were identified in 3 of 9 (33%) MSI-H and 10 of 20 (50%) MSS/MSI-L endometrial carcinomas, suggesting a more generalized involvement in these tumors. Only six (46%) of the endometrial carcinoma CTNNB1 mutations occurred at residues directly phosphorylated by GSK-3beta, and only one of these was at either codon 41 or 45. All point mutations in MSI-H cancers were transitions, whereas 64% of those in MSS/MSI-L cancers were transversions (P < 0.01). The differences in the mutation profiles suggest that there may be molecular fingerprints of CTNNB1 mutations, determined by biological factors related to both tumor type and underlying pathways of genomic instability.
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Adenocarcinoma/genética , Substituição de Aminoácidos , Transformação Celular Neoplásica/genética , Neoplasias do Colo/genética , Proteínas do Citoesqueleto/genética , Neoplasias do Endométrio/genética , Repetições de Microssatélites , Mutação Puntual , Transativadores , Adenoma/genética , Polipose Adenomatosa do Colo/genética , Diferenciação Celular , Códon/genética , Análise Mutacional de DNA , DNA de Neoplasias/genética , Éxons/genética , Feminino , Genes APC , Humanos , Masculino , Especificidade de Órgãos , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de Sinais/genética , beta CateninaRESUMO
Hereditary nonpolyposis colorectal cancer is associated with defects in DNA mismatch repair. Here, we characterize tumor susceptibility of the recently described Msh2-deficient mouse model. Within the first year of observation, all homozygous mice succumbed to disease, with lymphomas observed in at least 80% of the cases. The majority (70%) of animals 6 months or older developed intestinal neoplasms associated with APC inactivation. Microsatellite instability was more common in carcinomas than in adenomas, but uncommon in normal tissues. Some animals (7%) developed a variety of skin neoplasms analogous to the Muir-Torre syndrome. Msh2-/- mice implicate a direct role for mismatch repair in several neoplasms with striking phenotypic similarities to humans.
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Reparo do DNA , Proteínas de Ligação a DNA/análise , Proteínas Fúngicas , Neoplasias Intestinais/etiologia , Neoplasias Cutâneas/etiologia , Proteína da Polipose Adenomatosa do Colo , Animais , Sequência de Bases , Proteínas do Citoesqueleto/análise , Proteínas de Ligação a DNA/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Repetições de Microssatélites , Dados de Sequência Molecular , Proteína 2 Homóloga a MutSRESUMO
Accelerated intestinal tumorigenesis is probable in hereditary nonpolyposis colorectal cancer, a condition associated with germ line DNA mismatch repair (MMR) gene defects, and is believed to be caused by rapid accumulation of replication errors in critical genes, such as the APC (adenomatous polyposis coli) tumor suppressor gene. To study the potential contribution of MMR genes to accelerated intestinal tumorigenesis, we crossed the Min mouse, heterozygous for a germ line mutation of Apc, with an MMR gene (Msh2)-deficient mouse. MSH2 deficiency resulted in the development of many colonic aberrant crypt foci, as well as reduced survival of the mice, secondary to both a greater number and more rapidly developing adenomas. The mechanism of inactivation of the wild-type Apc allele depended on MSH2 status. In the presence of functional MSH2, all tumors demonstrated loss of heterozygosity. In contrast, whereas all adenomas were APC negative by immunostaining, only 5 of 34 adenomas from Apc+/-/Msh2-/- mice demonstrated loss of heterozygosity of the wild-type Apc allele, suggesting that somatic Apc mutations are responsible for the additional tumors. These findings provide evidence for the important role of MMR genes in accelerated intestinal tumorigenesis, thus supporting more aggressive surveillance strategies to prevent colorectal cancer in hereditary nonpolyposis colorectal cancer.
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Reparo do DNA/genética , Proteínas de Ligação a DNA , Genes APC , Neoplasias Intestinais/genética , Proteínas Proto-Oncogênicas/deficiência , Adenoma/genética , Animais , DNA de Neoplasias/genética , DNA Satélite/genética , Feminino , Deleção de Genes , Mutação em Linhagem Germinativa , Heterozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 2 Homóloga a MutS , Proteínas Proto-Oncogênicas/genéticaRESUMO
Aberrant activation of the Wnt pathway is observed in numerous cancers, and is particularly important in colon cancer. We demonstrate that Rac1 GTPase can significantly increase the signaling activity of beta-catenin in cells with inherent dysregulation of the canonical Wnt signaling pathway. Expression of dominant-negative (N17)Rac1 mutant in colon cancer cells caused a marked inhibition of Wnt signaling, as determined by the TCF/LEF-responsive (TOPFLASH) transcription assay. Expression of a constitutively active (V12)Rac1 mutant caused up to 40-fold induction from the TOPFLASH promoter, and this was dependent on the presence of stabilized beta-catenin. This induction was completely blocked by the expression of dominant-negative TCF-4, suggesting that beta-catenin and TCF-4 complex formation is required for Rac1-mediated transcription. Furthermore, we show that Cyclin D1, an important biological Wnt target gene, is regulated by Rac1 in a beta-catenin/TCF-dependent manner. We observed that Rac1 co-immunoprecipitates with beta-catenin and TCF-4 only in its active GTP-bound form. Both cell fractionation studies and fluorescence microscopy indicate that overexpression of V12Rac1 results in increased cytosolic and nuclear expression of beta-catenin. Interestingly, mutation of the polybasic region of Rac1, which prevents its nuclear localization, also caused an appreciable decrease in nuclear localization of beta-catenin, and effectively abolished its beta-catenin-dependent transcription co-activator function. Taken together, our data demonstrate a novel mechanism of Wnt pathway regulation whereby activation of Rac1 amplifies the signaling activity of stabilized/mutated beta-catenin by promoting its accumulation in the nucleus, and synergizing with beta-catenin to augment TCF/LEF-dependent gene transcription.
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Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/fisiologia , Ativação Transcricional/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Genes Reporter , Humanos , Imuno-Histoquímica , Fatores de Transcrição TCF , Proteína 2 Semelhante ao Fator 7 de Transcrição , Proteínas Wnt , beta CateninaRESUMO
Periampullary adenomas in the duodenum of Familial Adenomatous Polyposis (FAP) patients are among the most frequent and clinically important extracolonic neoplasms in FAP. The purpose of this study was to characterize the frequency and nature of somatic adenomatous polyposis coli (APC) gene and K-ras codon 12 mutations in periampullary adenomas and carcinomas in FAP. These molecular changes have been shown to be important during the early stages of colorectal carcinogenesis. DNA was prepared from endoscopic periampullary biopsies and paraffin blocks from 49 FAP patients. Of 143 samples, 77 were histologically normal, 29 were biopsies from small periampullary adenomas, 29 biopsies were from 19 large adenomas and eight samples were from periampullary cancers. APC mutations in the mutation cluster region and K-ras codon 12 mutations were detected by polymerase chain reaction based techniques. Somatic APC mutations consisting of deletions at codons 1464 and 1465 were detected in one small and two large periampullary adenomas. Loss of heterozygosity was seen in one periampullary carcinoma. K-ras codon 12 mutations were detected in seven of 19 large periampullary adenomas and in one of eight periampullary carcinomas. These data suggest that K-ras codon 12 mutations may be important during periampullary tumorigenesis in FAP but somatic APC mutations in the mutation cluster region are infrequent. Local environmental factors in the duodenum may contribute to differences in the molecular changes which occur during the adenoma-to-carcinoma sequence in periampullary compared to colonic tumorigenesis.
Assuntos
Adenoma/genética , Polipose Adenomatosa do Colo/genética , Carcinoma/genética , Neoplasias do Colo/genética , Genes ras , Sequência de Bases , Primers do DNA/química , Genes APC , Humanos , Dados de Sequência Molecular , Mutação , Polimorfismo Conformacional de Fita SimplesRESUMO
Aggressive fibromatosis (also called desmoid tumor) occurs as a sporadic lesion or as part of Familial Adenomatous Polyposis, which is caused by germ line mutations in the Adenomatous polyposis Coli (APC) gene. APC is involved in the regulation of the cellular level of beta-catenin, which is a mediator in Wnt signaling. Mutational analysis of the beta-catenin and APC genes was performed in 42 sporadic aggressive fibromatoses. Nine tumors had mutations in APC, and 22 had a point mutation in beta-catenin at either codon 45 or codon 41 (producing a stabilized beta-catenin protein product). Immunohistochemistry showed an elevated beta-catenin protein level in all tumors, regardless of mutational status. Beta-catenin localized to the nucleus, and was not tyrosine phosphorylated in the six tumors in which this was tested. The demonstration of mutations in two mediators in the Wnt-APC-beta-catenin pathway implicates beta-catenin stabilization as the key factor in the pathogenesis of aggressive fibromatosis. This is the first demonstration of somatic beta-catenin mutations in a locally invasive, but non metastatic lesion composed of spindle cells, illustrating the importance of beta-catenin stabilization in a variety of cell types and neoplastic processes. Moreover, this tumor has one of the highest reported frequencies of beta-catenin mutations of any tumor type.