RESUMEN
The spindle assembly checkpoint (SAC) is a cellular surveillance mechanism that ensures the fidelity of chromosomes segregation. Reduced expression of some of its components weakens the SAC and induces chromosome instability and aneuploidy, which are both well-known hallmarks of cancer cells. Centromere protein-E (CENP-E) is a crucial component of the SAC and its function is to facilitate kinetochore microtubule attachment required to achieve and maintain chromosome alignment. The present study investigates the possible role of p14ARF as a controller of aneuploid cells proliferation. We used RNA interference to induce aneuploidy by partial depletion of CENP-E in human primary fibroblasts (IMR90) and in near diploid tumor cells (HCT116). In contrast to IMR90 aneuploid cell number, which was drastically reduced and leaned towards the WT condition, HCT116 aneuploid cell numbers were slightly decreased at later time points. This euploidy restoration was accompanied by increased p14ARF expression in IMR90 cells and followed ectopic p14ARF re-expression in p14ARF-null HCT116 cells. Collectively, our results suggest that hampering proliferation of aneuploid cells could be an additional role of the p14ARF tumor suppressor.
Asunto(s)
Aneuploidia , Proteínas Cromosómicas no Histona/genética , Fibroblastos/citología , Proteínas Oncogénicas/genética , Línea Celular , Proliferación Celular , Supervivencia Celular , Proteínas Cromosómicas no Histona/metabolismo , Genes Supresores de Tumor , Células HCT116 , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Proteínas Oncogénicas/metabolismo , ARN Interferente PequeñoRESUMEN
Weakening the Spindle Assembly Checkpoint by reduced expression of its components induces chromosome instability and aneuploidy that are hallmarks of cancer cells. The tumor suppressor p14(ARF) is overexpressed in response to oncogenic stimuli to stabilize p53 halting cell progression. Previously, we found that lack or reduced expression of p14(ARF) is involved in the maintenance of aneuploid cells in primary human cells, suggesting that it could be part of a pathway controlling their proliferation. To investigate this aspect further, p14(ARF) was ectopically expressed in HCT116 cells after depletion of the Spindle Assembly Checkpoint MAD2 protein that was used as a trigger for aneuploidy. p14(ARF) Re-expression reduced the number of aneuploid cells in MAD2 post-transcriptionally silenced cells. Also aberrant mitoses, frequently displayed in MAD2-depleted cells, were decreased when p14(ARF) was expressed at the same time. In addition, p14(ARF) ectopic expression in MAD2-depleted cells induced apoptosis associated with increased p53 protein levels. Conversely, p14(ARF) ectopic expression did not induce apoptosis in HCT116 p53KO cells. Collectively, our results suggest that the tumor suppressor p14(ARF) may have an important role in counteracting proliferation of aneuploid cells by activating p53-dependent apoptosis.
Asunto(s)
Aneuploidia , Apoptosis/fisiología , Proteína p14ARF Supresora de Tumor/fisiología , Proteína p53 Supresora de Tumor/fisiología , Apoptosis/genética , Proliferación Celular/genética , Proliferación Celular/fisiología , Técnicas de Inactivación de Genes , Células HCT116 , Humanos , Puntos de Control de la Fase M del Ciclo Celular/genética , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Proteínas Mad2/genética , Proteínas Mad2/fisiología , Mitosis/genética , Mitosis/fisiología , Interferencia de ARN , Proteína p14ARF Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Most human tumors are characterized by aneuploidy that is believed to be the consequence of chromosomal instability (CIN). The mechanism(s) leading to aneuploidy and the pathways that allow its tolerance are not completely understood. The Spindle Assembly Checkpoint (SAC) is a cellular surveillance mechanism working during mitosis, and alterations of genes that encode components of the SAC weakening the mitotic checkpoint, induce aneuploidy by chromosome mis-segregation. We induced aneuploidy in near-diploid tumor cells by simultaneous depletion of the SAC proteins MAD2 and BUBR1 by RNA interference in the attempt to gain further insight on the cellular responses to aneuploidy. Individual reduction of MAD2 and BUBR1 protein levels caused defective mitosis and aneuploidy, while co-depletion of MAD2 and BUBR1 caused cell cycle arrest and cell death in addition to aneuploidy. The simultaneous reduction of the two SAC proteins induced high percentage of hyperdiploid cells and p53 stabilization suggesting that hyperdiploidy could activate a p53 controlled pathway. The results indicate that p53 is required to induce cell cycle arrest and cell death when the mitotic checkpoint is strongly perturbed, thereby preventing aneuploid cell propagation.