Centro Of Oncobiologia Sperimentale

Palermo, Italy

Centro Of Oncobiologia Sperimentale

Palermo, Italy
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Ferretti C.,CNR Institute of Neuroscience | Totta P.,CNR Institute of Neuroscience | Fiore M.,CNR Institute of Neuroscience | Mattiuzzo M.,CNR Institute of Neuroscience | And 6 more authors.
Cell Cycle | Year: 2010

Highly Expressed in Cancer protein 1 (Hec1) is a subunit of the Ndc80 complex, a constituent of the mitotic kinetochore. HEC1 has been shown to be overexpressed in many cancers, suggesting that HEC1 upregulation is involved in the generation and/or maintenance of the tumour phenotype. However, the regulation of Hec1 expression in normal and tumour cells and the molecular alterations promoting accumulation of this protein in cancer cells are still unknown. Here we show that elevated Hec1 protein levels are characteristic of transformed cell lines of different origins and that kinetochore recruitment of this protein is also increased in cancer cell lines in comparison with normal human cells. Using different cell synchronization strategies, Hec1 expression was found to be tightly regulated during the cell cycle in both normal and cancer cells. A limited proteasome-dependent degradation of Hec1 cellular content was observed at mitotic exit, with no evident differences between normal and cancer cells. Interestingly, increased expression of HEC1 mRNA and Hec1 protein was observed after transient silencing of the retinoblastoma gene by siRNA or following microRNA-mediated permanent depletion of the retinoblastoma protein in HCT116 cells. Our data provide evidence for a functional link between Hec1 expression and the pRb pathway. These observations suggest that disruption of pRb function may lead to chromosome segregation errors and mitotic defects through Hec1 overexpression. This may importantly contribute to aneuploidy and chromosomal instability in RB-defective cancer cells. © 2010 Landes Bioscience.

Barra V.,University of Palermo | Schillaci T.,University of Palermo | Lentini L.,University of Palermo | Costa G.,University of Palermo | And 2 more authors.
Cell Division | Year: 2012

Background: Aneuploidy 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.Results: To 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, p21 waf1transactivation. 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.Conclusion: Our results suggest that DNMT1 depletion triggers a p14ARF/p53 dependent cell cycle arrest to counteract the aneuploidy induced by changes in DNA methylation. © 2012 Barra et al; licensee BioMed Central Ltd.

Lentini L.,University of Palermo | Barra V.,University of Palermo | Schillaci T.,University of Palermo | Di Leonardo A.,University of Palermo | Di Leonardo A.,Centro Of Oncobiologia Sperimentale
Journal of Cellular Physiology | Year: 2012

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) ${\rm {\tilde {\beta }}}$ 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. © 2011 Wiley Periodicals, Inc.

Sancataldo G.,University of Palermo | Vetri V.,University of Palermo | Vetri V.,National Research Council Italy | Fodera V.,Copenhagen University | And 6 more authors.
PLoS ONE | Year: 2014

Oxidative damages are linked to several aging-related diseases and are among the chemical pathways determining protein degradation. Specifically, interplay of oxidative stress and protein aggregation is recognized to have a link to the loss of cellular function in pathologies like Alzheimer's and Parkinson's diseases. Interaction between protein and reactive oxygen species may indeed induce small changes in protein structure and lead to the inhibition/modification of protein aggregation process, potentially determining the formation of species with different inherent toxicity. Understanding the temperate relationship between these events can be of utmost importance in unraveling the molecular basis of neurodegeneration. In this work, we investigated the effect of hydrogen peroxide oxidation on Human Serum Albumin (HSA) structure, thermal stability and aggregation properties. In the selected conditions, HSA forms fibrillar aggregates, while the oxidized protein undergoes aggregation via new routes involving, in different extents, specific domains of the molecule. Minute variations due to oxidation of single residues affect HSA tertiary structure leading to protein compaction, increased thermal stability, and reduced association propensity. © 2014 Sancataldo et al.

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