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1.
Genetics ; 212(3): 655-665, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31126976

RESUMO

Microsatellite sequences have an enhanced susceptibility to mutation, and can act as sentinels indicating elevated mutation rates and increased risk of cancer. The probability of mutant fixation within the intestinal epithelium is dictated by a combination of stem cell dynamics and mutation rate. Here, we exploit this relationship to infer microsatellite mutation rates. First a sensitive, multiplexed, and quantitative method for detecting somatic changes in microsatellite length was developed that allowed the parallel detection of mutant [CA]n sequences from hundreds of low-input tissue samples at up to 14 loci. The method was applied to colonic crypts in Mus musculus, and enabled detection of mutant subclones down to 20% of the cellularity of the crypt (∼50 of 250 cells). By quantifying age-related increases in clone frequencies for multiple loci, microsatellite mutation rates in wild-type and Msh2-deficient epithelium were established. An average 388-fold increase in mutation per mitosis rate was observed in Msh2-deficient epithelium (2.4 × 10-2) compared to wild-type epithelium (6.2 × 10-5).


Assuntos
Células-Tronco Adultas/metabolismo , Mucosa Intestinal/citologia , Repetições de Microssatélites , Proteína 2 Homóloga a MutS/genética , Taxa de Mutação , Células-Tronco Adultas/citologia , Animais , Feminino , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitose , Proteína 2 Homóloga a MutS/deficiência
2.
Methods ; 156: 79-84, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30578845

RESUMO

A major concern of CRISPR and related genome engineering technologies is off-target mutagenesis from prolonged exposure to Cas9 and related editing enzymes. To help mitigate this concern we added a loxP site to the 3'-LTR of an HIV-based lentiviral vector capable of expressing Cas9/gRNA complexes in a wide variety of mammalian cell types. Transduction of susceptible target cells yields an integrated provirus that expresses the desired Cas9/gRNA complex. The reverse transcription process also results in duplication of the 3'-LTR such that the integrated provirus becomes flanked by loxP sites (floxed). Subsequent expression of Cre recombinase results in loxP-to-loxP site-specific recombination that deletes the Cas9/gRNA payload and effectively prevents additional Cas9-mediated mutations. This construct also expresses a gRNA with a single transcription termination sequence, which results in higher expression levels and more efficient genome engineering as evidenced by disruption of the SAMHD1 gene. This hit-and-run CRISPR approach was validated by recreating a natural APOBEC3B deletion and by disrupting the mismatch repair gene MSH2. This hit-and-run strategy may have broad utility in many areas and especially those where cell types are difficult to engineer by transient delivery of ribonucleoprotein complexes.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Integrases/genética , Lentivirus/genética , RNA Guia/genética , Recombinação Genética , Pareamento de Bases , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Citidina Desaminase/deficiência , Citidina Desaminase/genética , Éxons , Deleção de Genes , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Integrases/metabolismo , Íntrons , Lentivirus/metabolismo , Células MCF-7 , Antígenos de Histocompatibilidade Menor/genética , Proteína 2 Homóloga a MutS/deficiência , Proteína 2 Homóloga a MutS/genética , RNA Guia/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/deficiência , Proteína 1 com Domínio SAM e Domínio HD/genética
3.
Anticancer Res ; 38(11): 6399-6404, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30396964

RESUMO

BACKGROUND/AIM: The benefit of IFL (irinotecan, fluorouracil and leucovorin) regimen for metastatic colorectal cancer patients (mCRCs) with high levels of microsatellite instability (MSI-H) or loss of mismatch repair (dMMR) protein expression, is uncertain. This study investigated the association of tumour MMR-status and VEGF-1 expression with response to first-line IFL regimen in mCRCs. PATIENTS AND METHODS: This prospective study analyzed patients diagnosed with mCRC between August 1st, 1998, and August 30th, 2003, at the Turku University Hospital, Finland. All patients received postoperative IFL regimen. Tumour expression of the MMR proteins, hMLH1 and hMSH2, and VEGF-1 expression were assessed by immunohistochemistry (IHC). Tumours with dMMR were those demonstrating loss of MMR protein expression, and tumours with high VEGF-1 expression were those showing moderate or strong cytoplasmic staining. The primary endpoint was the association between tumour hMLH1 or/and hMSH2-deficient and VEGF-1 expression; the relation between tumour MMR-status and IFL response rate was the secondary endpoint. RESULTS: Of the 67 mCRCs patients, 29 (43%) were hMLH1 or/and hMSH2-deficient and 15 (22%) were pMMR mCRCs. At diagnosis, patients with hMLH1 or/and hMSH2-deficient tumours expressed lower levels of VEGF-1 compared to pMMR tumour patients (p=0.01). More than half (n=17, 59%) of those with dMMR were chemosensitive to first-line IFL regimen, while just one-fifth (n=3, 20%) of those with pMMR were chemosensitive to the IFL regimen (p=0.045). CONCLUSION: Association between MMR-status and VEGF-1 expression predicts clinical outcome in mCRC patients.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Camptotecina/análogos & derivados , Neoplasias Colorretais/tratamento farmacológico , Proteína 1 Homóloga a MutL/deficiência , Proteína 2 Homóloga a MutS/deficiência , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Camptotecina/administração & dosagem , Camptotecina/uso terapêutico , Neoplasias Colorretais/metabolismo , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/uso terapêutico , Humanos , Leucovorina/administração & dosagem , Leucovorina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estudos Prospectivos , Análise de Sobrevida , Resultado do Tratamento
4.
DNA Repair (Amst) ; 70: 25-36, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30103093

RESUMO

We investigated the homology dependency of recombination in thymidine kinase (tk)-deficient mouse fibroblasts. Cells were transfected with DNA constructs harboring a herpes tk gene (the "recipient") rendered non-functional by an oligonucleotide containing the recognition site for endonuclease I-SceI. Constructs also contained a "donor" tk sequence that could restore function to the recipient gene through spontaneous gene conversion or via repair of a double-strand break (DSB) at the I-SceI site. Recombination events were recoverable by selection for tk-positive clones. Three different donors were used containing 16, 25, or 33 mismatches relative to the recipient. The mismatches were clustered, forming an interval of "homeology" relative to the recipient sequences. We show that when homeologous sequences were surrounded by high homology, mismatches were frequently included in gene conversion events. Notably, conversion tracts from spontaneous recombination included either all or none of the mismatches, suggesting that recombination must begin and end in high homology. This requirement was relaxed for events that occurred near an induced DSB, as a significant number of these latter conversion tracts had one end positioned within homeology. Knock-down of mismatch repair showed that incorporation of mismatches into gene conversion tracts can involve repair of mismatched heteroduplex intermediates, indicating that mismatch repair does not necessarily impede homeologous genetic exchange. Our results illustrate (1) genetic exchange between homeologous sequences in a mammalian genome is enabled by nearby homology, (2) proximity to a DSB impacts the homology requirements for where genetic exchange may begin and end, and (3) mismatch correction and previously documented anti-recombination activity are separable functions of the mismatch repair machinery in mammalian cells.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Fibroblastos/metabolismo , Recombinação Homóloga , Animais , Sequência de Bases , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Camundongos , Proteína 2 Homóloga a MutS/deficiência , Timidina Quinase/deficiência
5.
J Gastrointest Cancer ; 49(3): 379-384, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29974347

RESUMO

BACKGROUND: The frequency of colorectal cancer (CRC) with defective mismatch repair (dMMR) is estimated between 5 and 15%. In our population, the frequency of dMMR is unknown. Our objective was to show the frequency of dMMR. METHODS: Determination of dMMR with immunohistochemistry was performed prospectively for 202 patients who presented consecutively with CRC for the first time at our institution. RESULTS: The median age was 59 years (IQR 47 to 68), 119 (58.9%) were women, and 43 (21.3%) cases showed dMMR. The only clinicopathological characteristics associated with dMMR were the location in the right colon and the presence of a family history of cancer. In the multivariate analysis, only the presence of the tumor in the right colon was associated with dMMR (OR = 5.823, 95%-C.I. = 2.653-12.784, p < .001). CONCLUSION: The 21.3% of the cases demonstrated a dMMR and the only clinical-pathological characteristic associated with dMMR was location in the right colon.


Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Instabilidade de Microssatélites , Proteína 1 Homóloga a MutL/provisão & distribução , Proteína 2 Homóloga a MutS/deficiência , Idoso , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Proteína 1 Homóloga a MutL/análise , Proteína 2 Homóloga a MutS/análise , Estadiamento de Neoplasias , Fatores de Risco
6.
Skinmed ; 15(4): 259-264, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28859734

RESUMO

Muir-Torre syndrome is a rare genodermatosis inherited most frequently in an autosomal dominant fashion. Current criteria for its diagnosis include at least one sebaceous tumor and an underlying visceral malignancy. Muir-Torre syndrome is strongly associated with a germline mutation in DNA mismatch repair genes. We report two patients with a history of colorectal carcinoma who presented with sebaceous neoplasms on the face and trunk. Immunohistochemical staining of the sebaceous neoplasms demonstrated absence of mismatch repair proteins MSH2 and MSH6. Genetic studies confirmed deletions in the MSH2 gene, and a diagnosis of Lynch syndrome was made. Immunohistochemical staining for mismatch repair genes MLH1, MSH2, MSH6 and PMS2 may aid in the diagnosis of Muir-Torre syndrome in cases where there is high suspicion. Genetic testing is an important final step in the confirmation of Muir-Torre syndrome.


Assuntos
Proteínas de Ligação a DNA/deficiência , Síndrome de Muir-Torre/genética , Síndrome de Muir-Torre/metabolismo , Proteína 2 Homóloga a MutS/deficiência , Neoplasias Nasais/genética , Neoplasias Nasais/metabolismo , Couro Cabeludo , Proteínas de Ligação a DNA/genética , Feminino , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome de Muir-Torre/diagnóstico , Síndrome de Muir-Torre/patologia , Proteína 2 Homóloga a MutS/genética , Neoplasias Nasais/diagnóstico , Neoplasias Nasais/patologia , Deleção de Sequência
7.
PLoS One ; 12(8): e0182175, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28767666

RESUMO

Whereas transformation events in hematopoietic malignancies may occur at different developmental stages, the initial mutation originates in hematopoietic stem cells (HSCs), creating a preleukemic stem cell (PLSC). Subsequent mutations at either stem cell or progenitor cell levels transform the PLSC into lymphoma/leukemia initiating cells (LIC). Thymic lymphomas have been thought to develop from developing thymocytes. T cell progenitors are generated from HSCs in the bone marrow (BM), but maturation and proliferation of T cells as well as T-lymphomagenesis depends on both regulatory mechanisms and microenvironment within the thymus. We studied PLSC linked to thymic lymphomas. In this study, we use MSH2-/- mice as a model to investigate the existence of PLSC and the evolution of PLSC to LIC. Following BM transplantation, we found that MSH2-/- BM cells from young mice are able to fully reconstitute multiple hematopoietic lineages of lethally irradiated wild-type recipients. However, all recipients developed thymic lymphomas within three and four months post transplantation. Transplantation of different fractions of BM cells or thymocytes from young health MSH2-/- mice showed that an HSC enriched fraction always reconstituted hematopoiesis followed by lymphoma development. In addition, lymphomas did not occur in thymectomized recipients of MSH2-/- BM. These results suggest that HSCs with DNA repair defects such as MSH2-/- are PLSCs because they retain hematopoietic function, but also carry an obligate lymphomagenic potential within their T-cell progeny that is dependent on the thymic microenvironment.


Assuntos
Células-Tronco Hematopoéticas/citologia , Linfoma/patologia , Proteína 2 Homóloga a MutS/deficiência , Neoplasias do Timo/patologia , Animais , Transplante de Medula Óssea , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/metabolismo , Linfoma/genética , Linfoma/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Linfócitos T/citologia , Linfócitos T/patologia , Neoplasias do Timo/genética , Neoplasias do Timo/metabolismo , Microambiente Tumoral
8.
Clin Cancer Res ; 23(11): e32-e37, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28572265

RESUMO

Replication proofreading is crucial to avoid mutation accumulation in dividing cells. In humans, proofreading and replication repair is maintained by the exonuclease domains of DNA polymerases and the mismatch repair system. Individuals harboring germline mutations in genes involved in this process are at increased risk of early cancers from multiple organs. Biallelic mutations in any of the four mismatch repair genes MSH2, MSH6, MLH1, and PMS2 result in one of the most aggressive childhood cancer predisposition syndromes, termed constitutional mismatch repair deficiency or constitutional mismatch repair deficiency syndrome (CMMRD). Data gathered in the last decade allow us to better define the clinical manifestations, tumor spectrum, and diagnostic algorithms for CMMRD. In this article, we summarize this information and present a comprehensive consensus surveillance protocol for these individuals. Ongoing research will allow for further definition of replication repair-deficient cancer syndromes, assessing the cost-effectiveness of such surveillance protocols and potential therapeutic interventions for these children and families. Clin Cancer Res; 23(11); e32-e37. ©2017 AACRSee all articles in the online-only CCR Pediatric Oncology Series.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Colorretais/genética , Mutação em Linhagem Germinativa/genética , Instabilidade de Microssatélites , Síndromes Neoplásicas Hereditárias/genética , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/epidemiologia , Neoplasias Encefálicas/patologia , Criança , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Detecção Precoce de Câncer , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento/deficiência , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Proteína 1 Homóloga a MutL/deficiência , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/deficiência , Proteína 2 Homóloga a MutS/genética , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/epidemiologia , Síndromes Neoplásicas Hereditárias/patologia
9.
Int J Cancer ; 141(7): 1365-1380, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28577310

RESUMO

In a proportion of patients presenting mismatch repair (MMR)-deficient tumors, no germline MMR mutations are identified, the so-called Lynch-like syndrome (LLS). Recently, MMR-deficient tumors have been associated with germline mutations in POLE and MUTYH or double somatic MMR events. Our aim was to elucidate the molecular basis of MSH2-deficient LS-suspected cases using a comprehensive analysis of colorectal cancer (CRC)-associated genes at germline and somatic level. Fifty-eight probands harboring MSH2-deficient tumors were included. Germline mutational analysis of MSH2 (including EPCAM deletions) and MSH6 was performed. Pathogenicity of MSH2 variants was assessed by RNA analysis and multifactorial likelihood calculations. MSH2 cDNA and methylation of MSH2 and MSH6 promoters were studied. Matched blood and tumor DNA were analyzed using a customized next generation sequencing panel. Thirty-five individuals were carriers of pathogenic or probably pathogenic variants in MSH2 and EPCAM. Five patients harbored 4 different MSH2 variants of unknown significance (VUS) and one had 2 novel MSH6 promoter VUS. Pathogenicity assessment allowed the reclassification of the 4 MSH2 VUS and 6 probably pathogenic variants as pathogenic mutations, enabling a total of 40 LS diagnostics. Predicted pathogenic germline variants in BUB1, SETD2, FAN1 and MUTYH were identified in 5 cases. Three patients had double somatic hits in MSH2 or MSH6, and another 2 had somatic alterations in other MMR genes and/or proofreading polymerases. In conclusion, our comprehensive strategy combining germline and somatic mutational status of CRC-associated genes by means of a subexome panel allows the elucidation of up to 86% of MSH2-deficient suspected LS tumors.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Proteína 2 Homóloga a MutS/deficiência , Proteína 2 Homóloga a MutS/genética , DNA Glicosilases/genética , Metilação de DNA , Análise Mutacional de DNA , Proteínas de Ligação a DNA/deficiência , Molécula de Adesão da Célula Epitelial/genética , Exodesoxirribonucleases/genética , Sequenciamento de Nucleotídeos em Larga Escala , Histona-Lisina N-Metiltransferase/genética , Humanos , Perda de Heterozigosidade , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética
10.
Int J Biol Markers ; 32(3): e352-e356, 2017 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-28525661

RESUMO

BACKGROUND: DNA mismatch repair deficiency is an important molecular mechanism of genetic instability in gastric cancer, and a high instability at microsatellites is associated with favorable prognosis. We compared mononucleotide and dinucleotide microsatellite instability (MSI) marker panels in 56 paired gastric tumor and normal samples. METHODS: The mononucleotide marker panel (mono panel) consisted of 8 markers: BAT25, BAT26, BAT40, BAT-RII, NR21, NR22, NR24 and NR27. The dinucleotide marker panel (di panel) contained D2S123, D5S346, D17S250, D17S261, D17S520, D18S34 and D18S58. The NCI panel was used as reference panel. RESULTS: Among 13 gastric tumors showing no hMLH1 or hMSH2 expression, 8 MSI-H (high) and 5 MSI-L (low) were identified. The analytical sensitivities of the NCI, mono and di panels to detect unstable MSI were 61.5% (8/13), 76.9% (10/13) and 84.6% (11/13), respectively. The size change of allele shift was statistically greater in the mono panel than in the di panel (p = 0.02 by Mann-Whitney U-test). The BAT40 (69.2%, 9/13) and D18S34 (76.9%, 10/13) markers showed high sensitivity for determination of MSI status. CONCLUSIONS: To improve the detection rate of MSI in gastric cancer with loss of hMLH1 or hMSH2 expression, the kind of MSI marker may need to be considered more, instead of the repetitive type of marker. Thus, an MSI panel designed with a combination of both BAT40 and D18S34 is suggested for providing more accurate and sensitive MSI analysis in gastric cancer.


Assuntos
Reparo de Erro de Pareamento de DNA , Proteína 1 Homóloga a MutL/deficiência , Proteína 2 Homóloga a MutS/deficiência , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Humanos , Instabilidade de Microssatélites , Repetições de Microssatélites , Proteína 1 Homóloga a MutL/biossíntese , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/biossíntese , Proteína 2 Homóloga a MutS/genética , Neoplasias Gástricas/patologia
11.
Sci Rep ; 6: 30757, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27476972

RESUMO

A feature in patients with constitutional DNA-mismatch repair deficiency is agenesis of the corpus callosum, the cause of which has not been established. Here we report a previously unrecognized consequence of deficiency in MSH2, a protein known primarily for its function in correcting nucleotide mismatches or insertions and deletions in duplex DNA caused by errors in DNA replication or recombination. We documented that Msh2 deficiency causes dysmyelination of the axonal projections in the corpus callosum. Evoked action potentials in the myelinated corpus callosum projections of Msh2-null mice were smaller than wild-type mice, whereas unmyelinated axons showed no difference. Msh2-null mice were also impaired in locomotive activity and had an abnormal response to heat. These findings reveal a novel pathogenic consequence of MSH2 deficiency, providing a new mechanistic hint to previously recognized neurological disorders in patients with inherited DNA-mismatch repair deficiency.


Assuntos
Corpo Caloso , Reparo de Erro de Pareamento de DNA , Doenças Desmielinizantes , Potenciais Evocados , Locomoção , Proteína 2 Homóloga a MutS/deficiência , Animais , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Corpo Caloso/fisiopatologia , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/fisiopatologia , Camundongos , Camundongos Knockout , Proteína 2 Homóloga a MutS/metabolismo
12.
DNA Repair (Amst) ; 42: 26-32, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27131875

RESUMO

Trinucleotide repeat (TNR) expansion beyond a certain threshold results in some 20 incurable neurodegenerative disorders where disease anticipation positively correlates with repeat length. Long TNRs typically display a bias toward further expansion during germinal transmission from parents to offspring, and then are highly unstable in somatic tissues of affected individuals. Understanding mechanisms of TNR instability will provide insights into disease pathogenesis. Previously, we showed that enhanced convergent transcription at long CAG repeat tracks induces TNR instability and cell death via ATR activation. Components of TC-NER (transcription-coupled nucleotide excision repair) and RNaseH enzymes that resolve RNA/DNA hybrids oppose cell death, whereas the MSH2 component of MMR (mismatch repair) enhances cell death. The exact role of the MMR pathway during convergent transcription-induced cell death at CAG repeats is not well understood. In this study, we show that siRNA knockdowns of MMR components-MSH2, MSH3, MLHI, PMS2, and PCNA-reduce DNA toxicity. Furthermore, knockdown of MSH2, MLH1, and PMS2 significantly reduces the frequency of ATR foci formation. These observations suggest that MMR proteins activate DNA toxicity by modulating ATR foci formation during convergent transcription.


Assuntos
Reparo de Erro de Pareamento de DNA , Transcrição Genética/genética , Repetições de Trinucleotídeos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sequência de Bases , Morte Celular/genética , Linhagem Celular , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Ativação Enzimática/genética , Técnicas de Silenciamento de Genes , Humanos , Proteína 1 Homóloga a MutL/deficiência , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/deficiência , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS , RNA Interferente Pequeno/genética
14.
Hum Mutat ; 36(4): 482-7, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25726753

RESUMO

The fragile X-related disorders (FXDs) are members of the group of diseases known as the repeat expansion diseases. The FXDs result from expansion of an unstable CGG/CCG repeat tract in the 5' UTR of the FMR1 gene. Contractions are also seen, albeit at lower frequency. We have previously shown that ERCC6/CSB plays an auxiliary role in promoting germ line and somatic expansions in a mouse model of the FXDs. However, work in model systems of other repeat expansion diseases has suggested that CSB may protect against expansions by promoting contractions. Since FXD mice normally have such a high expansion frequency, it is possible that such a protective effect would have been masked. We thus examined the effect of the loss of CSB in an Msh2(+/-) background where the germ line expansion frequency is reduced and in an Msh2(-/-) background where expansions do not occur, but contractions do. Our data show that in addition to promoting repeat expansion, CSB does in fact protect the genome from germ line expansions in the FXD mouse model. However, it likely does so not by promoting contractions but by promoting an error-free process that preserves the parental allele.


Assuntos
Enzimas Reparadoras do DNA/genética , Síndrome do Cromossomo X Frágil/genética , Transcrição Genética , Expansão das Repetições de Trinucleotídeos , Animais , Enzimas Reparadoras do DNA/deficiência , Modelos Animais de Doenças , Feminino , Deleção de Genes , Instabilidade Genômica , Genótipo , Masculino , Camundongos , Camundongos Knockout , Proteína 2 Homóloga a MutS/deficiência , Proteínas de Ligação a Poli-ADP-Ribose
15.
Cancer Med ; 4(6): 897-902, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25773971

RESUMO

Inherited as well as acquired deficiencies in specific DNA mismatch repair (MMR) components are associated with the development of a wide range of benign and malignant neoplasms. Loss of key members such as MSH2 and MLH1 severely cripples the ability of the cell to recognize and correct such lesions as base:base mismatches and replicative DNA polymerase errors such as slippages at repetitive sequences. Genomic instability resulting from MMR deficiency not only predisposes cells to malignant transformation but may also promote tumor progression. To test the latter, we interbred Msh2(-/-) mice with the K-ras(LA1/+) transgenic line that spontaneously develops a range of premalignant and malignant lung lesions. Compared to K-ras(LA1/+) mice, K-ras(LA1/+); Msh2(-/-) mice developed lung adenomas and adenocarcinomas at an increased frequency and also demonstrated evidence of accelerated adenocarcinoma growth. Since MMR defects have been identified in some human lung cancers, the mutant mice may not only be of preclinical utility but they will also be useful in identifying gene alterations able to act in concert with Kras mutants to promote tumor progression.


Assuntos
Adenocarcinoma/genética , Reparo de Erro de Pareamento de DNA/genética , Genes ras/genética , Neoplasias Pulmonares/genética , Proteína 2 Homóloga a MutS/deficiência , Proteínas Proto-Oncogênicas p21(ras)/deficiência , Animais , Transformação Celular Neoplásica/genética , Progressão da Doença , Camundongos Transgênicos , Mutação/genética
16.
J Gynecol Oncol ; 26(1): 40-5, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25310854

RESUMO

OBJECTIVE: Recent investigations have revealed DNA mismatch repair (MMR) gene mutations are closely related with carcinogenesis of endometrial cancer; however the impact of MMR protein expression on prognosis is not determined. Correlations between MMR-related protein expression and clinicopathological factors of endometrial cancers are analyzed in the present study. METHODS: A total of 191 endometrial cancer tissues treated between 1990 and 2007 in our hospital were enrolled. Immunoreactions for MSH2, MLH1, MSH6, and PMS2 on tissue microarray specimens and clinicopathological features were analyzed retrospectively. RESULTS: Seventy-six cases (40%) had at least one immunohistochemical alteration in MMR proteins (MMR-deficient group). There were statistically significant differences of histology, International Federation of Gynecology and Obstetrics (FIGO) stage, and histological grade between MMR-deficient group and the other cases (MMR-retained group). Response rate of first-line chemotherapy in evaluable cases was slightly higher in MMR-deficient cases (67% vs. 44%, p=0.34). MMR-deficient cases had significantly better progression-free and overall survival (OS) compared with MMR-retained cases. Multivariate analysis revealed MMR status was an independent prognostic factor for OS in endometrial cancers. CONCLUSION: MMR-related proteins expression was identified as an independent prognostic factor for OS, suggesting that MMR was a key biomarker for further investigations of endometrial cancers.


Assuntos
Biomarcadores Tumorais/metabolismo , Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias do Endométrio/diagnóstico , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimioterapia Adjuvante , Enzimas Reparadoras do DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/deficiência , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Prognóstico , Estudos Retrospectivos
17.
Am J Surg Pathol ; 38(11): 1501-9, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25229768

RESUMO

Lynch syndrome (LS) is an autosomal dominant inherited disorder caused by germline mutations in DNA mismatch repair (MMR) genes. Mutation carriers are at substantially increased risk of developing cancers of the colorectum and endometrium, among others. Given recent recommendations for universal, cost-effective screening of all patients with newly diagnosed colorectal cancer using MMR protein immunohistochemistry, we evaluated MMR protein expression in a series of endometrial cancers in the general population. A total of 605 consecutive cases of primary endometrial cancer at a single institution (1997 to 2013) were evaluated regardless of age, family history, or histologic features. Evaluation methods consisted of immunohistochemistry for the MMR proteins MLH1, MSH2, MSH6, and PMS2, followed by DNA methylation analysis for cases with MLH1/PMS2 deficiency. Germline mutation testing was performed on a subset of cases. Forty MMR-deficient, nonmethylated endometrial cancers were identified: 3 MLH1/PMS2 and 37 MSH6/MSH2 protein deficiencies. Only 25% occurred in women below 50 years of age (range, 39 to 88 y), 1 of which was in a risk-reducing hysterectomy specimen. Only 15% of patients had a prior history of carcinoma, including only 2 patients with prior colorectal carcinoma. Most (80%) of the endometrial cancers were purely endometrioid; there were 2 mixed endometrioid/mucinous, 1 mucinous, 1 serous, 2 clear cell, and 2 carcinosarcoma cases. When grading was applicable, 40% of the endometrial malignancies were FIGO grade 1, 34% grade 2, and 26% grade 3. Thirteen percent arose in the lower uterine segment, and 23% had tumor-infiltrating lymphocytes. Of the tumors with known germline testing, 41% with a LS-associated germline mutation were not associated with any of the traditional indicators that have been recommended for LS screening (ie, age 50 y or younger, personal/family cancer pedigree that meets Bethesda guideline criteria, presence of MMR-associated tumor morphology, or location in the lower uterine segment). These data suggest that a significant number of LS-associated endometrial carcinomas are missed using clinical, histologic, and locational screening parameters and provide support for universal screening of all newly diagnosed endometrial cancers.


Assuntos
Biomarcadores Tumorais/deficiência , Neoplasias Colorretais Hereditárias sem Polipose/química , Reparo de Erro de Pareamento de DNA , Neoplasias do Endométrio/química , Programas de Rastreamento , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Adenosina Trifosfatases/deficiência , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biópsia , California , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo de Erro de Pareamento de DNA/genética , Análise Mutacional de DNA , Enzimas Reparadoras do DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Humanos , Imuno-Histoquímica , Programas de Rastreamento/métodos , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/deficiência , Gradação de Tumores , Proteínas Nucleares/deficiência , Valor Preditivo dos Testes , Medição de Risco , Fatores de Risco
18.
Gastroenterology ; 147(5): 1064-72.e5, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25088490

RESUMO

BACKGROUND & AIMS: Lynch syndrome, a nonpolyposis form of hereditary colorectal cancer, is caused by inherited defects in DNA mismatch repair (MMR) genes. Most patients carry a germline mutation in 1 allele of the MMR genes MSH2 or MLH1. With spontaneous loss of the wild-type allele, cells with defects in MMR exist among MMR-proficient cells, as observed in healthy intestinal tissues from patients with Lynch syndrome. We aimed to create a mouse model of this situation to aid in identification of environmental factors that affect MMR-defective cells and their propensity for oncogenic transformation. METHODS: We created mice in which the MMR gene Msh2 can be inactivated in a defined fraction of crypt base columnar stem cells to generate MSH2-deficient intestinal crypts among an excess of wild-type crypts (Lgr5-CreERT2;Msh2(flox/-) mice). Intestinal tissues were collected; immunohistochemical analyses were performed for MSH2, along with allele-specific PCR assays. We traced the fate of MSH2-deficient crypts under the influence of different external factors. RESULTS: Lgr5-CreERT2;Msh2(flox/-) mice developed more adenomas and adenocarcinomas than control mice; all tumors were MSH2 deficient. Exposure of Lgr5-CreERT2;Msh2(flox/-) mice to the methylating agent temozolomide caused MSH2-deficient intestinal stem cells to proliferate more rapidly than wild-type stem cells. The MSH2-deficient intestinal stem cells were able to colonize the intestinal epithelium and many underwent oncogenic transformation, forming intestinal neoplasias. CONCLUSIONS: We developed a mouse model of Lynch syndrome (Lgr5-CreERT2;Msh2(flox/-) mice) and found that environmental factors can modify the number and mutability of the MMR-deficient stem cells. These findings provide evidence that environmental factors can promote development of neoplasias and tumors in patients with Lynch syndrome.


Assuntos
Adenocarcinoma/induzido quimicamente , Adenocarcinoma/genética , Adenoma/induzido quimicamente , Adenoma/genética , Neoplasias Colorretais Hereditárias sem Polipose/induzido quimicamente , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Dacarbazina/análogos & derivados , Intestinos/efeitos dos fármacos , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenoma/metabolismo , Adenoma/patologia , Animais , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Dacarbazina/toxicidade , Modelos Animais de Doenças , Feminino , Mucosa Intestinal/metabolismo , Intestinos/patologia , Masculino , Camundongos Knockout , Proteína 2 Homóloga a MutS/deficiência , Proteína 2 Homóloga a MutS/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fatores de Risco , Temozolomida , Fatores de Tempo
19.
Am J Surg Pathol ; 38(11): 1494-500, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24921635

RESUMO

Lynch syndrome (LS) is an autosomal dominant inherited disease that is associated with an increased risk for colorectal and endometrial cancer due to germline mutations in mismatch-repair (MMR) genes. Whereas primary tumors in this syndrome are widely recognized, the relative risk(s) of secondary malignancies, particularly breast cancer, in LS patients are still poorly characterized. To provide an improved assessment of these risks, MMR status was evaluated in secondary tumors from a series of patients with index tumors of known MMR status (both proficient and deficient). A total of 1252 tumors (index tumors) and all secondary malignancies were tested for MMR by immunohistochemistry (MSH2, MSH6, MLH1, PMS2) between 1992 and 2013. Tumors with MLH1/PMS2 deficiency were tested for hypermethylation or BRAF mutation, when appropriate. Of the 1252 index tumors, 162 were MMR deficient (dMMR), and, of that subset, 32 secondary tumors were identified (19.7%). In contrast, 80 secondary tumors were identified in the proficient (intact) group (7.3%). Although secondary malignancies were more common in the dMMR group (P=0.0001), there was no trend in tumor type. Specifically, breast cancer was not overly represented in the dMMR group. When secondary tumors had dMMR, they were more likely to have deficiency in MSH2/MSH6 than in MLH1/PMS2 (P=0.01). Of the patients with tumors exhibiting dMMR, women were more likely to have a dMMR secondary tumor in this series (P=0.0001); however, breast cancer was not overly represented, and our study provides no evidence that it is more frequent in LS. MSH2/MSH6 deficiency is more commonly associated with a secondary tumor compared with MLH1/PMS2 deficiency, when methylation/BRAF status is taken into account.


Assuntos
Biomarcadores Tumorais/deficiência , Neoplasias da Mama/química , Neoplasias Colorretais Hereditárias sem Polipose/química , Reparo de Erro de Pareamento de DNA , Segunda Neoplasia Primária , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Adenosina Trifosfatases/deficiência , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Metilação de DNA , Reparo de Erro de Pareamento de DNA/genética , Análise Mutacional de DNA , Enzimas Reparadoras do DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/deficiência , Mutação , Proteínas Nucleares/deficiência , Estudos Prospectivos , Proteínas Proto-Oncogênicas B-raf/genética , Estudos Retrospectivos , Fatores de Risco , Adulto Jovem
20.
J Nutrigenet Nutrigenomics ; 7(4-6): 299-313, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-26022687

RESUMO

Red meat may increase promutagenic lesions in the colon. Resistant starch (RS) can reduce these lesions and chemically induced colon tumours in rodents. Msh2 is a mismatch repair (MMR) protein, recognising unrepaired promutagenic adducts for removal. We determined if red meat and/or RS modulated DNA adducts or oncogenesis in Msh2-deficient mice. A total of 100 Msh2-/- and 60 wild-type mice consumed 1 of 4 diets for 6 months: control, RS, red meat and red meat+RS. Survival time, aberrant crypt foci (ACF), colon and small intestinal tumours, lymphoma, colonic O6-methyl-2-deoxyguanosine (O6MeG) adducts, methylguanine methyltransferase (MGMT) and cell proliferation were examined. In Msh2-/- mice, red meat enhanced survival compared to control (p<0.01) and lowered total tumour burden compared to RS (p<0.167). Msh2-/- mice had more ACF than wild-type mice (p<0.014), but no colon tumours developed. Msh2-/- increased cell proliferation (p<0.001), lowered DNA O6MeG adducts (p<0.143) and enhanced MGMT protein levels (p<0.001) compared to wild-type mice, with RS supplementation also protecting against DNA adducts (p<0.01). No link between red meat-induced promutagenic adducts and risk for colorectal cancer was observed after 6 months' feeding. Colonic epithelial changes after red meat and RS consumption with MMR deficiency will differ from normal epithelial cells.


Assuntos
Adutos de DNA/biossíntese , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Neoplasias Intestinais/etiologia , Linfoma/etiologia , Proteína 2 Homóloga a MutS/deficiência , Carne Vermelha , Amido/administração & dosagem , Neoplasias do Timo/etiologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Anticarcinógenos/administração & dosagem , Reparo de Erro de Pareamento de DNA , Feminino , Neoplasias Intestinais/patologia , Neoplasias Intestinais/prevenção & controle , Linfoma/patologia , Linfoma/prevenção & controle , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Homóloga a MutS/genética , Mutagênicos/metabolismo , Nutrigenômica , Carne Vermelha/efeitos adversos , Fatores de Risco , Neoplasias do Timo/patologia , Neoplasias do Timo/prevenção & controle
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