RESUMEN
A hallmark of cancer is genomic instability that is considered to provide the adaptive capacity of cancers to thrive under conditions in which the normal precursors would not survive. Recent genomic analysis has revealed a very high degree of genomic instability in melanomas, although the mechanism by which this instability arises is not known. Here we report that a high proportion (68%) of melanoma cell lines are either partially (40%) or severely (28%) compromised for the G2 phase decatenation checkpoint that normally functions to ensure that the sister chromatids are able to separate correctly during mitosis. The consequence of this loss of checkpoint function is a severely reduced ability to partition the replicated genome in mitosis and thereby increase genomic instability. We also demonstrate that decatenation is dependent on both TopoIIα and ß isoforms. The high incidence of decatenation checkpoint defect is likely to be a major contributor to the high level of genomic instability found in melanomas.
Asunto(s)
Genes cdc/genética , Inestabilidad Genómica/genética , Melanoma/genética , Melanoma/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , ADN-Topoisomerasas de Tipo II/genética , Fase G2/genética , Humanos , Mitosis/genética , ARN Interferente Pequeño/genética , Intercambio de Cromátides Hermanas/genéticaRESUMEN
UVR is a major environmental risk factor for the development of melanoma. Here we describe a coupled DNA-damage tolerance (DDT) mechanism and G2-phase cell cycle checkpoint induced in response to suberythemal doses of UVR that is commonly defective in melanomas. This coupled response is triggered by a small number of UVR-induced DNA lesions incurred during G1 phase that are not repaired by nucleotide excision repair (NER). These lesions are detected during S phase, but rather than stalling replication, they trigger the DDT-dependent formation of single-stranded DNA (ssDNA) gaps. The ssDNA attracts replication protein A (RPA), which initiates ATR-Chk1 (ataxia telangiectasia and Rad3-related/checkpoint kinase 1) G2-phase checkpoint signaling, and colocalizes with components of the RAD18 and RAD51 postreplication repair pathways. We demonstrate that depletion of RAD18 delays both the resolution of RPA foci and exit from the G2-phase arrest, indicating the involvement of RAD18-dependent postreplication repair in ssDNA gap repair during G2 phase. Moreover, the presence of RAD51 and BRCA1 suggests that an error-free mechanism may also contribute to repair. Loss of the UVR-induced G2-phase checkpoint results in increased UVR signature mutations after exposure to suberythemal UVR. We propose that defects in the UVR-induced G2-phase checkpoint and repair mechanism are likely to contribute to melanoma development.
Asunto(s)
Replicación del ADN/genética , ADN de Cadena Simple/genética , Fase G2/efectos de la radiación , Melanoma/patología , Neoplasias Cutáneas/patología , Proteína Quinasa CDC2/metabolismo , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Ciclina B1/metabolismo , Replicación del ADN/efectos de la radiación , Proteínas de Unión al ADN/fisiología , Células Epidérmicas , Epidermis/efectos de la radiación , Fase G1/genética , Fase G1/efectos de la radiación , Fase G2/genética , Humanos , Melanoma/genética , Mutación/genética , Mutación/efectos de la radiación , Fase S/genética , Fase S/efectos de la radiación , Neoplasias Cutáneas/genética , Rayos Ultravioleta/efectos adversosRESUMEN
Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis.