RESUMEN
BACKGROUND: Necdin (NDN) expression is downregulated in telomerase-immortalised normal human urothelial cells. Telomerase-immortalised normal human urothelial cells have no detected genetic alterations. Accordingly, many of the genes whose expression is altered following immortalisation are those for which epigenetic silencing is reported. METHODS: NDN expression was examined in normal tissues and tumour cell lines by quantitative real-time PCR and immunoblotting. Immunohistochemistry was performed on urothelial carcinoma (UC). Urothelial carcinoma and UC cell lines were subject to HumanMethylation27 BeadChip Array-based methylation analyses. Mutation screening was performed. The functional significance of NDN expression was investigated using retroviral-mediated downregulation or overexpression. RESULTS: NDN protein was widely expressed in normal tissues. Loss of expression was observed in 38 out of 44 (86%) of UC cell lines and 19 out of 25 (76%) of non-UC cell lines. Loss of NDN protein was found in the majority of primary UC. Oncomine analysis demonstrated downregulation of expression in multiple tumour types. In UC, tumour-specific hypermethylation of NDN and key CpG sites where hypermethylation correlated with reduced expression were identified. Six novel mutations, including some of predicted functional significance, were identified in colorectal and ovarian cancer cell lines. Functional studies showed that NDN could suppress colony formation at low cell density and affect anchorage-independent growth and anoikis in vitro. CONCLUSION: NDN is a novel tumour suppressor candidate that is downregulated and hypermethylated or mutated in cancer.
Asunto(s)
Biomarcadores de Tumor/genética , Metilación de ADN , Genes Supresores de Tumor , Mutación/genética , Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Proliferación Celular , Células Cultivadas , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Immunoblotting , Técnicas para Inmunoenzimas , Neoplasias/patología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Urotelio/metabolismoRESUMEN
The CDKN2A locus is frequently inactivated in urothelial cell carcinoma (UCC), yet how this alteration contributes to bladder tumorigenesis is not known. Although most UCC express telomerase, inactivation of the p16/Rb pathway is generally required for in vitro immortalisation. This and the involvement of p16 in senescence of normal human urothelial cells (NHUC) suggest that CDKN2A deletion may aid bypass of senescence and allow immortalisation. CDKN2A encodes p16 and p14ARF and therefore inactivation of this locus can disrupt both the Rb and p53 tumour suppressor pathways. Retrovirus-mediated transduction was used to specifically modulate the p16/Rb and/or p53 tumour suppressor pathways in NHUC and to express human telomerase reverse transcriptase (hTERT). Expression of hTERT bypassed Rb and p53 pathway-dependent barriers to proliferation and immortalised NHUC. TERT-NHUC had normal karyotypes, were non-tumorigenic and unexpectedly retained CDKN2A. Thus, the phenotypic significance of inactivation of CDKN2A in UCC may not be solely related to bypass of senescence. Phenotypic assays in human urothelial cells have relied on cell strains derived from invasive tumours or NHUC immortalised by expression of SV40-large T. The production of genetically normal but immortal NHUC lines now provides a valuable platform for experiments to examine the timing and combination of events necessary for UCC tumorigenesis.
Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/genética , Genes de Retinoblastoma , Genes p16 , Telomerasa/genética , Urotelio/patología , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Humanos , Urotelio/citología , Urotelio/metabolismoRESUMEN
Claims for clinical negligence against nursing and medical staff can be a daunting prospect for those involved. This article provides practical advice on situations where nurses may become involved and actions that should be taken when a serious, potentially litigative incident occurs. It also recommends how nurses can avoid claims by handling patients' complaints satisfactorily.