RESUMEN
Physical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations (R24P, G101W and N71S) and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant (IVS1, +37 G>C) of uncertain significance was detected in a non-carrier melanoma case. The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families. Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Quinasas Ciclina-Dependientes/genética , Mutación de Línea Germinal , Melanoma/genética , Proteínas Proto-Oncogénicas , Neoplasias Cutáneas/genética , Adulto , Anciano , Ciclo Celular , División Celular , Quinasa 4 Dependiente de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Análisis Mutacional de ADN , ADN de Neoplasias/análisis , Susceptibilidad a Enfermedades , Exones , Femenino , Fase G1 , Ligamiento Genético , Pruebas Genéticas , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Transcripción GenéticaRESUMEN
The p16INK4a cyclin-dependent kinase inhibitor is implicated in replicative senescence, the state of permanent growth arrest provoked by cumulative cell divisions or as a response to constitutive Ras-Raf-MEK signalling in somatic cells. Some contribution to senescence presumably underlies the importance of p16INK4a as a tumour suppressor but the mechanisms regulating its expression in these different contexts remain unknown. Here we demonstrate a role for the Ets1 and Ets2 transcription factors based on their ability to activate the p16INK4a promoter through an ETS-binding site and their patterns of expression during the lifespan of human diploid fibroblasts. The induction of p16INK4a by Ets2, which is abundant in young human diploid fibroblasts, is potentiated by signalling through the Ras-Raf-MEK kinase cascade and inhibited by a direct interaction with the helix-loop-helix protein Id1 (ref. 11). In senescent cells, where the Ets2 levels and MEK signalling decline, the marked increase in p16INK4a expression is consistent with the reciprocal reduction of Id1 and accumulation of Ets1.