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) beta galactosidase expression, including up-regulation of p53 and p14ARF proteins and of p21(waf1) 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 p21(waf1) proteins, but not premature cellular senescence. Finally, our results provides an explanation of how a p53 controlled pathway, involving initially p21(waf1) and then p14ARF, could minimize the occurrence of genomic alterations derived from chromosome instability induced by low amounts of MAD2 protein.

Lentini L ，Barra V ，Schillaci T ，Di Leonardo A ... -  《-》

p31comet Induces cellular senescence through p21 accumulation and Mad2 disruption.

Yun M ，Han YH ，Yoon SH ，Kim HY ，Kim BY ，Ju YJ ，Kang CM ，Jang SH ，Chung HY ，Lee SJ ，Cho MH ，Yoon G ，Park GH ，Kim SH ，Lee KH ... -  《MOLECULAR CANCER RESEARCH》

Single-cell analysis of p16(INK4a) and p21(WAF1) expression suggests distinct mechanisms of senescence in normal human and Li-Fraumeni Syndrome fibroblasts.

Herein we used single-cell observation methods to gain insight into the roles of p16(INK4A) and p21(WAF1) (hereafter p16 and p21) in replicative senescence and ionizing radiation-induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li-Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence-associated beta-galactosidase. In addition, proliferating early-passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53-proficient (normal and AT) fibroblasts. In p53-deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation-triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild-type p53 activity. The possibility that one of the tumor-suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results.

Mirzayans R ，Andrais B ，Scott A ，Paterson MC ，Murray D ... -  《-》

MAD2DeltaC induces aneuploidy and promotes anchorage-independent growth in human prostate epithelial cells.

To-Ho KW ，Cheung HW ，Ling MT ，Wong YC ，Wang X ... -  《-》

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

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.

Veneziano L ，Barra V ，Lentini L ，Spatafora S ，Di Leonardo A ... -  《-》