Site-Specific RNA Editing of Stop Mutations in the CFTR mRNA of Human Bronchial Cultured Cells.

It is reported that about 10% of cystic fibrosis (CF) patients worldwide have nonsense (stop) mutations in the CFTR gene, which cause the premature termination of CFTR protein synthesis, leading to a truncated and non-functional protein. To address this issue, we investigated the possibility of rescuing the CFTR nonsense mutation (UGA) by sequence-specific RNA editing in CFTR mutant CFF-16HBEge, W1282X, and G542X human bronchial cells. We used two different base editor tools that take advantage of ADAR enzymes (adenosine deaminase acting on RNA) to edit adenosine to inosine (A-to-I) within the mRNA: the REPAIRv2 (RNA Editing for Programmable A to I Replacement, version 2) and the minixABE (A to I Base Editor). Immunofluorescence experiments show that both approaches were able to recover the CFTR protein in the CFTR mutant cells. In addition, RT-qPCR confirmed the rescue of the CFTR full transcript. These findings suggest that site-specific RNA editing may efficiently correct the UGA premature stop codon in the CFTR transcript in CFF-16HBEge, W1282X, and G542X cells. Thus, this approach, which is safer than acting directly on the mutated DNA, opens up new therapeutic possibilities for CF patients with nonsense mutations.

Investigating the Inhibition of FTSJ1, a Tryptophan tRNA-Specific 2'-O-Methyltransferase by NV TRIDs, as a Mechanism of Readthrough in Nonsense Mutated CFTR.

Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene, coding for the CFTR chloride channel. About 10% of the CFTR gene mutations are "stop" mutations that generate a premature termination codon (PTC), thus synthesizing a truncated CFTR protein. A way to bypass PTC relies on ribosome readthrough, which is the ribosome's capacity to skip a PTC, thus generating a full-length protein. "TRIDs" are molecules exerting ribosome readthrough; for some, the mechanism of action is still under debate. We investigate a possible mechanism of action (MOA) by which our recently synthesized TRIDs, namely NV848, NV914, and NV930, could exert their readthrough activity by in silico analysis and in vitro studies. Our results suggest a likely inhibition of FTSJ1, a tryptophan tRNA-specific 2'-O-methyltransferase.

Aneuploid IMR90 cells induced by depletion of pRB, DNMT1 and MAD2 show a common gene expression signature.

Chromosome segregation defects lead to aneuploidy which is a major feature of solid tumors. How diploid cells face chromosome mis-segregation and how aneuploidy is tolerated in tumor cells are not completely defined yet. Thus, an important goal of cancer genetics is to identify gene networks that underlie aneuploidy and are involved in its tolerance. To this aim, we induced aneuploidy in IMR90 human primary cells by depleting pRB, DNMT1 and MAD2 and analyzed their gene expression profiles by microarray analysis. Bioinformatic analysis revealed a common gene expression profile of IMR90 cells that became aneuploid. Gene Set Enrichment Analysis (GSEA) also revealed gene-sets/pathways that are shared by aneuploid IMR90 cells that may be exploited for novel therapeutic approaches in cancer. Furthermore, Protein-Protein Interaction (PPI) network analysis identified TOP2A and KIF4A as hub genes that may be important for aneuploidy establishment.

Targeting Nonsense: Optimization of 1,2,4-Oxadiazole TRIDs to Rescue CFTR Expression and Functionality in Cystic Fibrosis Cell Model Systems.

Cystic fibrosis (CF) patients develop a severe form of the disease when the cystic fibrosis transmembrane conductance regulator (CFTR) gene is affected by nonsense mutations. Nonsense mutations are responsible for the presence of a premature termination codon (PTC) in the mRNA, creating a lack of functional protein. In this context, translational readthrough-inducing drugs (TRIDs) represent a promising approach to correct the basic defect caused by PTCs. By using computational optimization and biological screening, we identified three new small molecules showing high readthrough activity. The activity of these compounds has been verified by evaluating CFTR expression and functionality after treatment with the selected molecules in cells expressing nonsense-CFTR-mRNA. Additionally, the channel functionality was measured by the halide sensitive yellow fluorescent protein (YFP) quenching assay. All three of the new TRIDs displayed high readthrough activity and low toxicity and can be considered for further evaluation as a therapeutic approach toward the second major cause of CF.

Investigating REPAIRv2 as a Tool to Edit CFTR mRNA with Premature Stop Codons.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the transmembrane conductance regulator (CFTR) protein. Some CF patients are compound heterozygous or homozygous for nonsense mutations in the CFTR gene. This implies the presence in the transcript of premature termination codons (PTCs) responsible for a truncated CFTR protein and a more severe form of the disease. Aminoglycoside and PTC124 derivatives have been used for the read-through of PTCs to restore the full-length CFTR protein. However, in a precision medicine framework, the CRISPR/dCas13b-based molecular tool "REPAIRv2" (RNA Editing for Programmable A to I Replacement, version 2) could be a good alternative to restore the full-length CFTR protein. This RNA editing approach is based on the targeting of the deaminase domain of the hADAR2 enzyme fused to the dCas13b protein to a specific adenosine to be edited to inosine in the mutant mRNA. Targeting specificity is allowed by a guide RNA (gRNA) complementarily to the target region and recognized by the dCas13b protein. Here, we used the REPAIRv2 platform to edit the UGA PTC to UGG in different cell types, namely IB3-1 cells, HeLa, and FRT cells engineered to express H2BGFPopal and CFTRW1282X, respectively.

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

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.

Strategies against Nonsense: Oxadiazoles as Translational Readthrough-Inducing Drugs (TRIDs).

This review focuses on the use of oxadiazoles as translational readthrough-inducing drugs (TRIDs) to rescue the functional full-length protein expression in mendelian genetic diseases caused by nonsense mutations. These mutations in specific genes generate premature termination codons (PTCs) responsible for the translation of truncated proteins. After a brief introduction on nonsense mutations and their pathological effects, the features of various classes of TRIDs will be described discussing differences or similarities in their mechanisms of action. Strategies to correct the PTCs will be presented, particularly focusing on a new class of Ataluren-like oxadiazole derivatives in comparison to aminoglycosides. Additionally, recent results on the efficiency of new candidate TRIDs in restoring the production of the cystic fibrosis transmembrane regulator (CFTR) protein will be presented. Finally, a prospectus on complementary strategies to enhance the effect of TRIDs will be illustrated together with a conclusive paragraph about perspectives, opportunities, and caveats in developing small molecules as TRIDs.

NOTCH3 expression is linked to breast cancer seeding and distant metastasis.

BACKGROUND: Development of distant metastases involves a complex multistep biological process termed the invasion-metastasis cascade, which includes dissemination of cancer cells from the primary tumor to secondary organs. NOTCH developmental signaling plays a critical role in promoting epithelial-to-mesenchymal transition, tumor stemness, and metastasis. Although all four NOTCH receptors show oncogenic properties, the unique role of each of these receptors in the sequential stepwise events that typify the invasion-metastasis cascade remains elusive. METHODS: We have established metastatic xenografts expressing high endogenous levels of NOTCH3 using estrogen receptor alpha-positive (ERα+) MCF-7 breast cancer cells with constitutive active Raf-1/mitogen-associated protein kinase (MAPK) signaling (vMCF-7Raf-1) and MDA-MB-231 triple-negative breast cancer (TNBC) cells. The critical role of NOTCH3 in inducing an invasive phenotype and poor outcome was corroborated in unique TNBC cells resulting from a patient-derived brain metastasis (TNBC-M25) and in publicly available claudin-low breast tumor specimens collected from participants in the Molecular Taxonomy of Breast Cancer International Consortium database. RESULTS: In this study, we identified an association between NOTCH3 expression and development of metastases in ERα+ and TNBC models. ERα+ breast tumor xenografts with a constitutive active Raf-1/MAPK signaling developed spontaneous lung metastases through the clonal expansion of cancer cells expressing a NOTCH3 reprogramming network. Abrogation of NOTCH3 expression significantly reduced the self-renewal and invasive capacity of ex vivo breast cancer cells, restoring a luminal CD44low/CD24high/ERαhigh phenotype. Forced expression of the mitotic Aurora kinase A (AURKA), which promotes breast cancer metastases, failed to restore the invasive capacity of NOTCH3-null cells, demonstrating that NOTCH3 expression is required for an invasive phenotype. Likewise, pharmacologic inhibition of NOTCH signaling also impaired TNBC cell seeding and metastatic growth. Significantly, the role of aberrant NOTCH3 expression in promoting tumor self-renewal, invasiveness, and poor outcome was corroborated in unique TNBC cells from a patient-derived brain metastasis and in publicly available claudin-low breast tumor specimens. CONCLUSIONS: These findings demonstrate the key role of NOTCH3 oncogenic signaling in the genesis of breast cancer metastasis and provide a compelling preclinical rationale for the design of novel therapeutic strategies that will selectively target NOTCH3 to halt metastatic seeding and to improve the clinical outcomes of patients with breast cancer.

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.

Toward a rationale for the PTC124 (Ataluren) promoted readthrough of premature stop codons: a computational approach and GFP-reporter cell-based assay.

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.

Simultaneous reduction of MAD2 and BUBR1 expression induces mitotic spindle alterations associated with p53 dependent cell cycle arrest and death.

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.

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.

Specific Irreversible Cell-Cycle Arrest and Depletion of Cancer Cells Obtained by Combining Curcumin and the Flavonoids Quercetin and Fisetin.

Background: Induced senescence could be exploited to selectively counteract the proliferation of cancer cells and target them for senolysis. We examined the cellular senescence induced by curcumin and whether it could be targeted by fisetin and quercetin, flavonoids with senolytic activity. Methods: Cell-cycle profiles, chromosome number and structure, and heterochromatin markers were evaluated via flow cytometry, metaphase spreads, and immunofluorescence, respectively. The activation of p21waf1/cip1 was assessed via RT-qPCR and immunoblotting. Senescent cells were detected via SA-ß-Galactosidase staining. Results: We report that curcumin treatment specifically triggers senescence in cancer cells by inducing mitotic slippage and DNA damage. We show that curcumin-induced senescence is p21waf1/cip1-dependent and characterized by heterochromatin loss. Finally, we found that flavonoids clear curcumin-induced senescent cancer cells. Conclusions: Our findings expand the characterization of curcumin-induced cellular senescence in cancer cells and lay the foundation for the combination of curcumin and flavonoids as a possible anti-cancer therapy.

Transcriptomic Changes Following Partial Depletion of CENP-E in Normal Human Fibroblasts.

The centromere is a fundamental chromosome structure in which the macro-molecular kinetochore assembles and is bound by spindle microtubules, allowing the segregation of sister chromatids during mitosis. Any alterations in kinetochore assembly or functioning or kinetochore-microtubule attachments jeopardize chromosome stability, leading to aneuploidy, a common feature of cancer cells. The spindle assembly checkpoint (SAC) supervises this process, ensuring a faithful segregation of chromosomes. CENP-E is both a protein of the kinetochore and a crucial component of the SAC required for kinetochore-microtubule capture and stable attachment, as well as congression of chromosomes to the metaphase plate. As the function of CENP-E is restricted to mitosis, its haploinsufficiency has been used to study the induced cell aneuploidy; however, the gene expression profile triggered by CENP-E reduction in normal cells has never been explored. To fill this gap, here we investigated whether a gene network exists that is associated with an siRNA-induced 50% reduction in CENP-E and consequent aneuploidy. Gene expression microarray analyses were performed at early and late timepoints after transfection. Initially, cell cycle regulation and stress response pathways were downregulated, while afterwards pathways involved in epithelial-mesenchymal transition, hypoxia and xenobiotic metabolism were altered. Collectively, our results suggest that CENP-E reduction triggers a gene expression program that recapitulates some features of tumor cells.

A new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19.

Inflammation is a physiological process whose deregulation causes some diseases including cancer. Nuclear Factor kB (NF-kB) is a family of ubiquitous and inducible transcription factors, in which the p65/p50 heterodimer is the most abundant complex, that play critical roles mainly in inflammation. Glucocorticoid Receptor (GR) is a ligand-activated transcription factor and acts as an anti-inflammatory agent and immunosuppressant. Thus, NF-kB and GR are physiological antagonists in the inflammation process. Here we show that in mice and humans there is a spliced variant of p65, named p65 iso5, which binds the corticosteroid hormone dexamethasone amplifying the effect of the glucocorticoid receptor and is expressed in the liver of patients with hepatic cirrhosis and hepatocellular carcinoma (HCC). Furthermore, we have quantified the gene expression level of p65 and p65 iso5 in the PBMC of patients affected by SARS-CoV-2 disease. The results showed that in these patients the p65 and p65 iso5 mRNA levels are higher than in healthy subjects. The ability of p65 iso5 to bind dexamethasone and the regulation of the glucocorticoid (GC) response in the opposite way of the wild type improves our knowledge and understanding of the anti-inflammatory response and identifies it as a new therapeutic target to control inflammation and related diseases.

Pyrazole[3,4-d]pyrimidine derivatives loaded into halloysite as potential CDK inhibitors.

Uncontrolled cell proliferation is a hallmark of cancer as a result of rapid and deregulated progression through the cell cycle. The inhibition of cyclin-dependent kinases (CDKs) activities is a promising therapeutic strategy to block cell cycle of tumor cells. In this work we reported a new example of nanocomposites based on halloysite nanotubes (HNTs)/pyrazolo[3,4-d]pyrimidine derivatives (Si306 and Si113) as anticancer agents and CDK inhibitors. HNTs/Si306 and HNTs/Si113 nanocomposites were synthesized and characterized. The release kinetics were also investigated. Antitumoral activity was evaluated on three cancer cell lines (HeLa, MDA-MB-231 and HCT116) and the effects on cell cycle arrest in HCT116 cells were evaluated. Finally, molecular dynamics simulations were performed of the complexes between Si113 or Si306 and the active site of both CDK 1 and 2.

P14ARF: The Absence that Makes the Difference.

P14ARF is a tumor suppressor encoded by the CDKN2a locus that is frequently inactivated in human tumors. P14ARF protein quenches oncogene stimuli by inhibiting cell cycle progression and inducing apoptosis. P14ARF functions can be played through interactions with several proteins. However, the majority of its activities are notoriously mediated by the p53 protein. Interestingly, recent studies suggest a new role of p14ARF in the maintenance of chromosome stability. Here, we deepened this new facet of p14ARF which we believe is relevant to its tumor suppressive role in the cell. To this aim, we generated a monoclonal HCT116 cell line expressing the p14ARF cDNA cloned in the piggyback vector and then induced aneuploidy by treating HCT116 cells with the CENP-E inhibitor GSK923295. P14ARF ectopic re-expression restored the near-diploid phenotype of HCT116 cells, confirming that p14ARF counteracts aneuploid cell generation/proliferation.

Inhalable nano into micro dry powders for ivacaftor delivery: The role of mannitol and cysteamine as mucus-active agents.

In this paper the innovative approach of nano into micro dry powders (NiM) was applied to incorporate into mannitol or mannitol/cysteamine micromatrices ivacaftor-loaded nanoparticles for pulmonary delivery in CF. Nanoparticles composed by a mixture of two polyhydrohydroxyethtylaspartamide copolymers containing loaded with ivacaftor at 15.5% w/w were produced. The nanoparticles were incorporated into microparticles to obtain NiM that were fully characterized in terms of size, morphology, interactions with artificial Cf mucus (CF-AM) as well as for aerodynamic behaviour. Finally the activity of ivacaftor-containing NiM was evaluated by in vitro preliminary experiments. NiM at matrix composed by a mixture of mannitol:cysteamine showed greater ability to reduce CF-AM viscosity whereas that based on just mannitol showed better aerodynamic properties with a FPF of about 25%. All produced NiM showed very good cytocompatibility and the released ivacaftor was able to restore the chroride transport in vitro.

RNAi mediated acute depletion of retinoblastoma protein (pRb) promotes aneuploidy in human primary cells via micronuclei formation.

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. RESULTS: Acutely pRb-depleted fibroblasts showed altered expression of genes necessary for cell cycle progression, centrosome homeostasis, kinetochore and mitotic checkpoint proteins. Despite altered expression of genes involved in the Spindle Assembly Checkpoint (SAC) the checkpoint seemed to function properly in pRb-depleted fibroblasts. In particular AURORA-A and PLK1 overexpression suggested that these two genes might have a role in the observed genomic instability. However, when they were post-transcriptionally silenced in pRb-depleted fibroblasts we did not observe reduction in the number of aneuploid cells. This finding suggests that overexpression of these two genes did not contribute to genomic instability triggered by RB acute loss although it affected cell proliferation. Acutely pRb-depleted human fibroblasts showed the presence of micronuclei containing whole chromosomes besides the presence of supernumerary centrosomes and aneuploidy. CONCLUSION: Here we show for the first time that RB acute loss triggers centrosome amplification and aneuploidy in human primary fibroblasts. Altogether, our results suggest that pRb-depleted primary human fibroblasts possess an intact spindle checkpoint and that micronuclei, likely caused by mis-attached kinetochores that in turn trigger chromosome segregation errors, are responsible for aneuploidy in primary human fibroblasts where pRb is acutely depleted.

CENPA overexpression promotes genome instability in pRb-depleted human cells.

BACKGROUND: Aneuploidy 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. RESULTS: Here 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. CONCLUSION: Altogether 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.