RESUMEN
T-lymphocyte maturation associated protein, MAL, has been described as a tumour-suppressor gene with diagnostic value in colorectal and oesophageal cancers, and can be inactivated by promoter hypermethylation. The aim of this study was to analyse the prevalence of MAL promoter hypermethylation and the association with mRNA expression in gastric cancers and to correlate methylation status to clinicopathological data. Bisulphite-treated DNA isolated from formalin-fixed and paraffin-embedded samples of 202 gastric adenocarcinomas and 22 normal gastric mucosae was subjected to real-time methylation-specific PCR (Q-MSP). Two regions within the MAL promoter (M1 and M2) were analysed. In addition, 17 frozen gastric carcinomas and two gastric cancer cell lines were analysed both by Q-MSP and real-time RT-PCR. Methylation of M1 and M2 occurred in 71 and 80% of the gastric cancers, respectively, but not in normal gastric mucosa tissue. Hypermethylation of M2, but not M1, correlated with significantly better disease-free survival in a univariate (P=0.03) and multivariate analysis (P=0.03) and with downregulation of expression (P=0.01). These results indicate that MAL has a putative tumour-suppressor gene function in gastric cancer, and detection of promoter hypermethylation may be useful as a prognostic marker.
Asunto(s)
Biomarcadores de Tumor/genética , Metilación de ADN , Genes Supresores de Tumor , Proteínas de Transporte de Membrana/genética , Proteínas de la Mielina/genética , Regiones Promotoras Genéticas , Proteolípidos/genética , Neoplasias Gástricas/genética , Anciano , Anciano de 80 o más Años , Femenino , Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Proteínas de Transporte de Membrana/biosíntesis , Persona de Mediana Edad , Proteínas de la Mielina/biosíntesis , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito , Pronóstico , Proteolípidos/biosíntesis , ARN Mensajero/análisis , Curva ROC , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/mortalidadRESUMEN
We previously showed that silencing of TSLC1, recently renamed CADM1, is functionally involved in high-risk HPV-mediated cervical carcinogenesis. CADM1 silencing often results from promoter methylation. Here, we determined the extent of CADM1 promoter methylation in cervical (pre)malignant lesions and its relation to anchorage-independent growth and gene silencing to select a CADM1-based methylation marker for identification of women at risk of cervical cancer. Methylation-specific PCRs targeting three regions within the CADM1 promoter were performed on high-risk HPV-containing cell lines, PBMCs, normal cervical smears, and (pre)malignant lesions. CADM1 protein expression in cervical tissues was analysed by immunohistochemistry. All statistical tests were two-sided. Density of methylation was associated with the degree of anchorage-independent growth and CADM1 gene silencing in vitro. In cervical squamous lesions, methylation frequency and density increased with severity of disease. Dense methylation (defined as >or= 2 methylated regions) increased from 5% in normal cervical samples to 30% in CIN3 lesions and 83% in squamous cell carcinomas (SCCs) and was significantly associated with decreased CADM1 protein expression (p < 0.00005). The frequency of dense methylation was significantly higher in >or= CIN3 compared with
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/genética , Inmunoglobulinas/genética , Proteínas de la Membrana/genética , Regiones Promotoras Genéticas , Proteínas Supresoras de Tumor/genética , Displasia del Cuello del Útero/genética , Neoplasias del Cuello Uterino/genética , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Estudios de Casos y Controles , Molécula 1 de Adhesión Celular , Moléculas de Adhesión Celular , Línea Celular Transformada , Metilación de ADN , Femenino , Silenciador del Gen , Humanos , Inmunoglobulinas/análisis , Inmunohistoquímica , Modelos Logísticos , Proteínas de la Membrana/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/análisis , Neoplasias del Cuello Uterino/metabolismo , Displasia del Cuello del Útero/metabolismoRESUMEN
We aimed to link DNA methylation events occurring in cervical carcinomas to distinct stages of HPV-induced transformation. Methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) analysis of cervical carcinomas revealed promoter methylation of 12 out of 29 tumour suppressor genes analysed, with MGMT being most frequently methylated (92%). Subsequently, consecutive stages of HPV16/18-transfected keratinocytes (n=11), ranging from pre-immortal to anchorage-independent phenotypes, were analysed by MS-MLPA. Whereas no methylation was evident in pre-immortal cells, progression to anchorage independence was associated with an accumulation of frequent methylation events involving five genes, all of which were also methylated in cervical carcinomas. TP73 and ESR1 methylation became manifest in early immortal cells followed by RARbeta and DAPK1 methylation in late immortal passages. Complementary methylation of MGMT was related to anchorage independence. Analysis of nine cervical cancer cell lines, representing the tumorigenic phenotype, revealed in addition to these five genes frequent methylation of CADM1, CDH13 and CHFR. In conclusion, eight recurrent methylation events in cervical carcinomas could be assigned to different stages of HPV-induced transformation. Hence, our in vitro model system provides a valuable tool to further functionally address the epigenetic alterations that are common in cervical carcinomas.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/virología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virología , Infecciones por Papillomavirus/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Adenocarcinoma/patología , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Metilación de ADN , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Femenino , Perfilación de la Expresión Génica , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , Infecciones por Papillomavirus/complicaciones , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas/genética , Sensibilidad y Especificidad , Neoplasias del Cuello Uterino/patologíaRESUMEN
BACKGROUND: Supervision and feedback are essential factors that contribute to the learning environment in the context of workplace learning and their frequency and quality can be improved. Assessment is a powerful tool with which to influence students' learning and supervisors' teaching and thus the learning environment. OBJECTIVE: To investigate an in-training assessment (ITA) programme in action and to explore its effects on supervision and feedback. DESIGN: A qualitative study using individual, semistructured interviews. SUBJECTS AND SETTING: Eight students and 17 assessors (9 members of staff and 8 residents) in the internal medicine undergraduate clerkship at Vrije Universiteit Medical Centre, Amsterdam, the Netherlands. RESULTS: The ITA programme in action differed from the intended programme. Assessors provided hardly any follow-up on supervision and feedback given during assessments. Although students wanted more supervision and feedback, they rarely asked for it. Students and assessors failed to integrate the whole range of competencies included in the ITA programme into their respective learning and supervision and feedback. When giving feedback, assessors rarely gave borderline or fail judgements. DISCUSSION AND CONCLUSION: If an ITA programme in action is to be congruent with the intended programme, the implementation of the programme must be monitored. It is also necessary to provide full information about the programme and to ensure this information is given repeatedly. Introducing an ITA programme that includes the assessment of several competencies does not automatically lead to more attention being paid to these competencies in terms of supervision and feedback. Measures that facilitate change in the learning environment seem to be a prerequisite for enabling the assessment programme to steer the learning environment.
Asunto(s)
Competencia Clínica/normas , Medicina Clínica/normas , Educación de Postgrado en Medicina/métodos , Educación de Pregrado en Medicina/métodos , Retroalimentación , Países Bajos , Administración de PersonalRESUMEN
CPT-11 (irinotecan or 7-ethyl-10[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an anticancer agent in use for the treatment of colon cancer. In order to be fully active, CPT-11 needs to be converted into SN-38 (7-ethyl-10-hydroxycamptothecin) by the enzyme carboxylesterase (CE). In humans, only a minority of CPT-11 is converted to SN-38. To increase the antitumour effect of CPT-11 by gene-directed enzyme prodrug therapy, we constructed a replication-deficient adenoviral vector Ad.C28-sCE2 containing a fusion gene encoding a secreted form of human liver CE2 targeted to the surface antigen epithelial cell adhesion molecule (EpCAM) that is highly expressed on most colon carcinoma cells. By targeting CE2 to EpCAM, the enzyme should accumulate specifically in tumours and leakage into the circulation should be minimised. Ad.C28-sCE2-transduced colon carcinoma cells expressed and secreted active CE that bound specifically to EpCAM-expressing cells. In sections of three-dimensional colon carcinoma spheroids transduced with Ad.C28-sCE2, it was shown that C28-sCE2 was capable of binding untransduced cells. Most importantly, treatment of these spheroids with nontoxic concentrations of CPT-11 resulted in growth inhibition comparable to treatment with SN-38. Therefore, Ad.C28-sCE2 holds promise in gene therapy approaches for the treatment of colon carcinoma.