RESUMEN
DNA methylation of CpG sites in promoter regions of several cancer related genes, such as O6-MGMT, hMLH1, p14ARF, p16INK4a, RASSF1A and APC1A, has been actively explored recently. Much of the data has been obtained using a variation of an allele-specific PCR assay known as methylation specific PCR. This technique is objectionable for a number of methodological limitations and drawbacks. We wanted to study the promoter regions of the above mentioned genes using bisulfite-treated genomic DNA amplified by PCR, using primers designed to bind only to CpG-free sequences. The methylated fraction (%) of each CpG site was measured by Pyrosequencing technology. For three of the genes, O6-MGMT, hMLH1 and p14ARF, two amplicons were designed to cover all relevant CpG sites. Several of the amplicons were analyzed by two different Pyrosequencing assays. In all, we designed nine optimized PCR protocols and 13 Pyrosequencing assays covering the promoters of all six studied genes. In all cases, standard PCR generated sufficient quantities of pure amplicons to be further analyzed by Pyrosequencing technology. Thus, a total of 119 CpG sites in six genes could be quantified. We conclude that standard PCR followed by Pyrosequencing is a workable, more specific and quantitative alternative to 'methylation specific' PCR. This approach provides a more comprehensive picture of the distribution of DNA methylation throughout the promoter regions of the studied set of six genes, which will be of benefit in oncological research.
Asunto(s)
Islas de CpG/genética , Metilación de ADN/genética , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Bases , Metilasas de Modificación del ADN/genética , Cartilla de ADN , Enzimas Reparadoras del ADN/genética , Genes p16 , Humanos , Datos de Secuencia Molecular , Homólogo 1 de la Proteína MutL , Proteínas Nucleares/genética , Proteína p14ARF Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
The mechanism of how organ shape emerges and specifies cell fate is not understood. Pancreatic duct and endocrine lineages arise in a spatially distinct domain from the acinar lineage. Whether these lineages are pre-determined or settle once these niches have been established remains unknown. Here, we reconcile these two apparently opposing models, demonstrating that pancreatic progenitors re-localize to establish the niche that will determine their ultimate fate. We identify a p120ctn-regulated mechanism for coordination of organ architecture and cellular fate mediated by differential E-cadherin based cell sorting. Reduced p120ctn expression is necessary and sufficient to re-localize a subset of progenitors to the peripheral tip domain, where they acquire an acinar fate. The same mechanism is used re-iteratively during endocrine specification, where it balances the choice between the alpha and beta cell fates. In conclusion, organ patterning is regulated by p120ctn-mediated cellular positioning, which precedes and determines pancreatic progenitor fate.
Asunto(s)
Tipificación del Cuerpo/genética , Cateninas/genética , Páncreas/crecimiento & desarrollo , Conductos Pancreáticos/crecimiento & desarrollo , Animales , Cadherinas/genética , Diferenciación Celular/genética , Linaje de la Célula/genética , Movimiento Celular/genética , Desarrollo Embrionario/genética , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Humanos , Islotes Pancreáticos/crecimiento & desarrollo , Islotes Pancreáticos/metabolismo , Ratones , Páncreas/metabolismo , Receptores Notch/genética , Transducción de Señal/genética , Células Madre/metabolismo , Catenina deltaRESUMEN
This corrects the article DOI: 10.1038/ncb3628.
RESUMEN
Apicobasal polarity is known to affect epithelial morphogenesis and cell differentiation, but it remains unknown how these processes are mechanistically orchestrated. We find that ligand-specific EGFR signalling via PI(3)K and Rac1 autonomously modulates apicobasal polarity to enforce the sequential control of morphogenesis and cell differentiation. Initially, EGF controls pancreatic tubulogenesis by negatively regulating apical polarity induction. Subsequently, betacellulin, working via inhibition of atypical protein kinase C (aPKC), causes apical domain constriction within neurogenin3+ endocrine progenitors, which results in reduced Notch signalling, increased neurogenin3 expression, and ß-cell differentiation. Notably, the ligand-specific EGFR output is not driven at the ligand level, but seems to have evolved in response to stage-specific epithelial influences. The EGFR-mediated control of ß-cell differentiation via apical polarity is also conserved in human neurogenin3+ cells. We provide insight into how ligand-specific EGFR signalling coordinates epithelial morphogenesis and cell differentiation via apical polarity dynamics.
Asunto(s)
Polaridad Celular/fisiología , Receptores ErbB/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiología , Organogénesis/fisiología , Transducción de Señal/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Células Epiteliales/metabolismo , Ratones , Ratones Noqueados , Morfogénesis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuropéptidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
We quantitated the methylated fraction of CpG sites in the promoter regions of O6-MGMT, p14ARF, p16INK4a, RASSF1A and APC1A in tumor tissue from patients with colorectal cancer (CRC) in order to determine if promoter hypermethylation of any of these genes predicts survival. DNA was isolated from 111 primary CRC and 46 matched normal colorectal mucosa samples from the same patients, obtained at primary surgery and DNA methylation was examined by Pyrosequencing®. Follow-up time was up to 20 years. Patients showed partial promoter methylation in the following frequencies: O6-MGMT, 34%; p14ARF, 29%; p16INK4a, 28%; RASSF1A, 14%; and APC1A, 27%. Normal mucosa was always unmethylated. CRC patients with methylated p14ARF gene promoter had significantly worse prognosis (p=0.036), whereas those with methylated O6-MGMT had significantly better prognosis through the first 60 months post-treatment (RR 0.36; p=0.023). Methylation of one or more of the genes from the set p14ARF, RASSF1A and APC1A, was significantly (p=0.021) associated with worse prognosis even adjusting for tumor stage and differentiation (RR 2.2, p=0.037). Thus, DNA methylation of the p14ARF, RASSF1A and APC1A genes, diagnosed by Pyrosequencing, defines a poor prognosis subset of CRC patients independently of both tumor stage and differentiation. O6-MGMT methylation may play a protective role.
Asunto(s)
Adenocarcinoma/genética , Proteína de la Poliposis Adenomatosa del Colon/genética , Neoplasias Colorrectales/genética , Metilación de ADN , Proteína p14ARF Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Colon/metabolismo , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Humanos , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Pronóstico , Regiones Promotoras Genéticas/genética , Recto/metabolismo , Tasa de SupervivenciaRESUMEN
DNA methylation status in the CpG sites of promoter regions in cancer-related genes, such as PTCH, has traditionally been investigated using either dye-terminator sequencing or methylation-specific PCR. We aimed to study the PTCH gene promoter methylation in gynecological cancers, with a method that gives a quantitative measure of the methylation status of the promoter region of the studied gene, and for this purpose, we designed novel Pyrosequencing-based assays. Bisulfite-treated genomic DNA (bsDNA) was amplified by standard PCR and applied to novel Pyrosequencing® assays, in order to measure the methylated fraction (%) at each CpG site of the PTCH gene promoter. We analyzed 22 squamous cell cervical cancer tissue specimens (11 with good and 11 with poor outcomes after radiotherapy) and 5 ovarian cancer tissue specimens matched with 5 normal ovarian tissue specimens. Six optimized PCR protocols which generated 8 Pyrosequencing assays covering 63 CpG sites in the promoter regions 1 and 2 as well as the previously unanalyzed promoter region 3 in the PTCH gene were developed. The 27 tumor tissue specimens and 5 normal tissues did not show any methylation within any of the 63 CpG sites. Our data suggest that methylation of the PTCH promoter is not a high-prevalence feature of squamous cell cervical cancer or ovarian cancer, but Pyrosequencing assays are a good method for studying promoter methylation.
Asunto(s)
Metilación de ADN , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Regiones Promotoras Genéticas , Receptores de Superficie Celular/genética , Análisis de Secuencia de ADN/métodos , Neoplasias del Cuello Uterino/genética , Secuencia de Bases , Islas de CpG , Metilación de ADN/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica , Frecuencia de los Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Análisis por Apareamiento , Datos de Secuencia Molecular , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patología , Receptores Patched , Receptor Patched-1 , Regiones Promotoras Genéticas/genética , Receptores de Superficie Celular/fisiología , Neoplasias del Cuello Uterino/patologíaRESUMEN
Hypermethylation of the O6-MGMT, p14ARF, p16INK4a, RASSF1A and APC1A genes are unfavourable prognostic markers in colorectal cancer (CRC). We hypothesized that they could be related to prognosis also in cervical cancer. Methylation was studied in DNA extracts from surgical specimens of cancer tissue by novel pyrosequencing methods. In 109 patients (90 squamous cell carcinomas, 19 adenocarcinomas), we found that hypermethylation of the APC1A gene promoter occurred in 8.3% of patients, and of p16INK4a in 1.8%. APC1A hypermethylation was significantly related to more advanced FIGO stage of the tumor (P=0.013), larger tumor diameter (P=0.049) and distant recurrence-free survival (P=0.0007), but not with locoregional recurrence rate, age, HPV status, DNA ploidy, tumor grade or malignancy grading score. We conclude that methylation of the APC1A promoter in cervical cancer, as diagnosed by pyrosequencing, is significantly related to major biological characteristics of the tumor, and may be a new predictor of poor prognosis in cervical cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN , Neoplasias del Cuello Uterino/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Supervivencia sin Enfermedad , Epigenómica , Femenino , Humanos , Pronóstico , Regiones Promotoras Genéticas , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
Telomeres generally shorten with age. An accelerated shortening of the telomeres has been linked to several age-related disorders. We hypothesized that the relative length of telomeres could discriminate between patients with late-onset Alzheimer's disease (AD) and vascular dementia (VaD). A quantitative real-time PCR method was used to calculate the relative telomere length in 76 age-matched and sex-matched, newly diagnosed late-onset AD or VaD patients recruited from our Memory Unit. No significant difference was found in the relative telomere length between AD and VaD cases neither in men (P=0.315) nor women (P=0.12). Thus, we could not confirm that the length of telomeres would predict which form of dementia, late-onset AD or VaD that develops.