Viviana Barra

MAD2 depletion triggers premature cellular senescence in human primary fibroblasts by activating a P53 pathway preventing aneuploid cells propagation

The spindle assembly checkpoint (SAC) is a cellular surveillance mechanism that ensures faithful chromosome segregation during mitosis and its failure can result in aneuploidy. Previously, it was suggested that reduction of the MAD2 gene, encoding a major component of the SAC, induced aneuploidy in human tumor cells. However, tumor cell lines contain multiple mutations that might affect or exacerbate the cellular response to Mad2 depletion. Thus, the scenario resulting by Mad2 depletion in primary human cells could be different and more complex that the one depicted so far. We used primary human fibroblasts (IMR90) and epithelial breast cells (MCF10A) to gain further insight on the effects of genomic instability caused by transient Mad2 depletion. To this aim we depleted Mad2 by RNAi to a level shown by Mad2 haplo‐insufficient cells and found that induced aneuploidy caused premature cellular senescence in IMR90 cells. IMR90 cells showed typical features of senescent cells, like senescence‐associated (SA)

Bypass of cell cycle arrest induced by transient DNMT1 post-transcriptional silencing triggers aneuploidy in human cells

{\rm {\tilde {\beta }}}

DNA demethylation caused by 5-Aza-2’-deoxycytidine induces mitotic alterations and aneuploidy

galactosidase expression, including up‐regulation of p53 and p14ARF proteins and of p21waf1 as well, but not of p16(INK4A) cyclin‐dependent kinase (Cdk) inhibitor. In contrast, after MAD2 post‐transcriptional silencing MCF10A cells in which the INK4A/ARF locus is deleted, showed both aneuploidy and a small increase of p53 and p21waf1 proteins, but not premature cellular senescence. Finally, our results provides an explanation of how a p53 controlled pathway, involving initially p21waf1 and then p14ARF, could minimize the occurrence of genomic alterations derived from chromosome instability induced by low amounts of MAD2 protein. J. Cell. Physiol. 227: 3324–3332, 2012.

p14(ARF) Prevents Proliferation of Aneuploid Cells by Inducing p53-Dependent Apoptosis

BackgroundAneuploidy has been acknowledged as a major source of genomic instability in cancer, and it is often considered the result of chromosome segregation errors including those caused by defects in genes controlling the mitotic spindle assembly, centrosome duplication and cell-cycle checkpoints. Aneuploidy and chromosomal instability has been also correlated with epigenetic alteration, however the molecular basis of this correlation is poorly understood.ResultsTo address the functional connection existing between epigenetic changes and aneuploidy, we used RNA-interference to silence the DNMT1 gene, encoding for a highly conserved member of the DNA methyl-transferases. DNMT1 depletion slowed down proliferation of near-diploid human tumor cells (HCT116) and triggered G1 arrest in primary human fibroblasts (IMR90), by inducing p53 stabilization and, in turn, p21waf1 transactivation. Remarkably, p53 increase was not caused by DNA damage and was not observed after p14-ARF post-transcriptional silencing. Interestingly, DNMT1 silenced cells with p53 or p14-ARF depleted did not arrest in G1 but, instead, underwent DNA hypomethylation and became aneuploid.ConclusionOur results suggest that DNMT1 depletion triggers a p14ARF/p53 dependent cell cycle arrest to counteract the aneuploidy induced by changes in DNA methylation.

Proliferation of aneuploid cells induced by CENP-E depletion is counteracted by the p14ARF tumor suppressor

Aneuploidy, the unbalanced number of chromosomes in a cell, is considered a prevalent form of genetic instability and is largely acknowledged as a condition implicated in tumorigenesis. Epigenetic alterations like DNA hypomethylation have been correlated with cancer initiation/progression. Furthermore, a growing body of evidence suggests the involvement of epigenome-wide disruption as a cause of global DNA hypomethylation in aneuploidy generation. Here, we report that the DNA hypomethylating drug 5-aza-2′-deoxycytidine (DAC), affects the correct ploidy of nearly diploid HCT-116 human cells by altering the methylation pattern of the chromosomes. Specifically, we show that a DAC-induced reduction of 5-Methyl Cytosine at the pericentromeric region of chromosomes correlates with aneuploidy and mitotic defects. Our results suggest that DNA hypomethylation leads to aneuploidy by altering the DNA methylation landscape at the centromere that is necessary to ensure proper chromosomes segregation by recruiting the proteins necessary to build up a functional kinetochore.

“DNA Methyl transferase 1 post-trascriptional silencing indues aneuploidy and cell cycle arrest in human cells”,

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 p14ARF is overexpressed in response to oncogenic stimuli to stabilize p53 halting cell progression. Previously, we found that lack or reduced expression of p14ARF 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, p14ARF was ectopically expressed in HCT116 cells after depletion of the Spindle Assembly Checkpoint MAD2 protein that was used as a trigger for aneuploidy. p14ARF 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 p14ARF was expressed at the same time. In addition, p14ARF ectopic expression in MAD2‐depleted cells induced apoptosis associated with increased p53 protein levels. Conversely, p14ARF ectopic expression did not induce apoptosis in HCT116 p53KO cells. Collectively, our results suggest that the tumor suppressor p14ARF may have an important role in counteracting proliferation of aneuploid cells by activating p53‐dependent apoptosis. J. Cell. Physiol. 231: 336–344, 2016.

Aneuploidy induced by MAD2 haploinsufficiency triggers premature senescence in human primary fibroblast

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.