BackgroundAneuploidy is a hallmark of most human cancers that arises as a consequence of chromosomal instability and it is frequently associated with centrosome amplification. Functional inactivation of the Retinoblastoma protein (pRb) has been indicated as a cause promoting chromosomal instability as well centrosome amplification. However, the underlying molecular mechanism still remains to be clarified.ResultsHere we show that pRb depletion both in wild type and p53 knockout HCT116 cells was associated with the presence of multipolar spindles, anaphase bridges, lagging chromosomes and micronuclei harbouring whole chromosomes. In addition aneuploidy caused by pRb acute loss was not affected by p53 loss.Quantitative real-time RT-PCR showed that pRB depletion altered expression of genes involved in centrosome duplication, kinetochore assembly and in the Spindle Assembly Checkpoint (SAC). However, despite MAD2 up-regulation pRb-depleted cells seemed to have a functional SAC since they arrested in mitosis after treatments with mitotic poisons. Moreover pRb-depleted HCT116 cells showed BRCA1 overexpression that seemed responsible for MAD2 up-regulation.Post-transcriptional silencing of CENPA by RNA interference, resulting in CENP-A protein levels similar to those present in control cells greatly reduced aneuploid cell numbers in pRb-depleted cells.ConclusionAltogether our findings indicate a novel aspect of pRb acute loss that promotes aneuploidy mainly by inducing CENPA overexpression that in turn might induce micronuclei by affecting the correct attachment of spindle microtubules to kinetochores.

The presence in the mRNA of premature stop codons (PTCs) results in protein truncation responsible for several inherited (genetic) diseases. A well-known example of these diseases is cystic fibrosis (CF), where approximately 10% (worldwide) of patients have nonsense mutations in the CF transmembrane regulator (CFTR) gene. PTC124 (3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)-benzoic acid), also known as Ataluren, is a small molecule that has been suggested to allow PTC readthrough even though its target has yet to be identified. In the lack of a general consensus about its mechanism of action, we experimentally tested the ability of PTC124 to promote the readthrough of premature termination codons by using a new reporter. The reporter vector was based on a plasmid harboring the H2B histone coding sequence fused in frame with the green fluorescent protein (GFP) cDNA, and a TGA stop codon was introduced in the H2B-GFP gene by site-directed mutagenesis. Additionally, an unprecedented computational study on the putative supramolecular interaction between PTC124 and an 11-codon (33-nucleotides) sequence corresponding to a CFTR mRNA fragment containing a central UGA nonsense mutation showed a specific interaction between PTC124 and the UGA codon. Altogether, the H2B-GFP-opal based assay and the molecular dynamics (MD) simulation support the hypothesis that PTC124 is able to promote the specific readthrough of internal TGA premature stop codons.

Human experience of physical space and places is a complex phenomenon that includes geographical and sensorial, as well as more social and interpersonal dimensions. We investigate theoretical insights from computing research and environmental psychology on space and place to determine the different dimensions of the experience of physical space. Empirical results from a case study on creative activities for environment exploration are also presented. We indicate five dimensions that encompass the different ways of apprehending our environment, as well as the emotional relationships we develop toward it through personal and interpersonal experiences-in-place. To us, technology should be examined in terms of its potentiality for supporting rich experiences of and in physical space. We assume that the identified dimensions can serve as basis for the development of technological tools to be used in that perspective.

Background: Changes in chromosome number or structure as well as supernumerary centrosomes and multipolar mitoses are commonly observed in human tumors. Thus, centrosome amplification and mitotic checkpoint dysfunctions are believed possible causes of chromosomal instability. The Retinoblastoma tumor suppressor (RB) participates in the regulation of synchrony between DNA synthesis and centrosome duplication and it is involved in transcription regulation of some mitotic genes. Primary human fibroblasts were transfected transiently with short interfering RNA (siRNA) specific for human pRb to investigate the effects of pRb acute loss on chromosomal stability.

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.