The tumor suppressor p53 is a cell cycle checkpoint protein that contributes to the preservation of genetic stability by mediating either a G1 arrest or apoptosis in response to DNA damage. Recent reports suggest that p53 causes growth arrest through transcriptional activation of the cyclin-dependent kinase (Cdk)-inhibitor Cip1. Here, we characterize the p53-dependent G1 arrest in several normal human diploid fibroblast (NDF) strains and p53-deficient cell lines treated with 0.1-6 Gy gamma radiation. DNA damage and cell cycle progression analyses showed that NDF entered a prolonged arrest state resembling senescence, even at low doses of radiation. This contrasts with the view that p53 ensures genetic stability by inducing a transient arrest to enable repair of DNA damage, as reported for some myeloid leukemia lines. Gamma radiation administered in early to mid-, but not late, G1 induced the arrest, suggesting that the p53 checkpoint is only active in G1 until cells commit to enter S phase at the G1 restriction point. A log-linear plot of the fraction of irradiated G0 cells able to enter S phase as a function of dose is consistent with single-hit kinetics. Cytogenetic analyses combined with radiation dosage data indicate that only one or a small number of unrepaired DNA breaks may be sufficient to cause arrest. The arrest also correlated with long-term elevations of p53 protein, Cip1 mRNA, and Cip1 protein. We propose that p53 helps maintain genetic stability in NDF by mediating a permanent cell cycle arrest through long-term induction of Cip1 when low amounts of unrepaired DNA damage are present in G1 before the restriction point.

Cells with a functional p53 pathway undergo a G0/G1 arrest or apoptosis when treated with gamma radiation or many chemotherapeutic drugs. It has been proposed that DNA damage is the exclusive signal that triggers the arrest response. However, we found that certain ribonucleotide biosynthesis inhibitors caused a p53-dependent G0 or early G1 arrest in the absence of replicative DNA synthesis or detectable DNA damage in normal human fibroblasts. CTP, GTP, or UTP depletion alone was sufficient to induce arrest. In contrast to the p53-dependent response to DNA damage, characterized by long-term arrest and irregular cellular morphologies, the antimetabolite-induced arrest was highly reversible and cellular morphologies remained relatively normal. Both arrest responses correlated with prolonged induction of p53 and the Cdk inhibitor P21(WAF1/CIP1/SDI1) and with dephosphorylation of pRb. Thus, we propose that p53 can serve as a metabolite sensor activated by depletion of ribonucleotides or products or processes dependent on ribonucleotides. Accordingly, p53 may play a role in inducing a quiescence-like arrest state in response to nutrient challenge and a senescence-like arrest state in response to DNA damage. These results have important implications for the mechanisms by which p53 prevents the emergence of genetic variants and for developing more effective approaches to chemotherapy based on genotype.

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.

In this study, we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic kinase that ultimately drives the transition from an epithelial to a highly invasive mesenchymal phenotype in estrogen receptor α-positive (ERα⁺) breast cancer cells. The transition from an epithelial- to a mesenchymal-like phenotype was characterized by reduced expression of ERα, HER-2/Neu overexpression and loss of CD24 surface receptor (CD24^−/low). Importantly, expression of key epithelial-to-mesenchymal transition (EMT) markers and upregulation of the stemness gene SOX2 was linked to acquisition of stem cell-like properties such as the ability to form mammospheres in vitro and tumor self-renewal in vivo. Moreover, aberrant Aurora-A kinase activity induced phosphorylation and nuclear translocation of SMAD5, indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in the development of EMT, stemness and ultimately tumor progression. Importantly, pharmacological and molecular inhibition of Aurora-A kinase activity restored a CD24⁺ epithelial phenotype that was coupled to ERα expression, downregulation of HER-2/Neu, inhibition of EMT and impaired self-renewal ability, resulting in the suppression of distant metastases. Taken together, our findings show for the first time the causal role of Aurora-A kinase in the activation of EMT pathway responsible for the development of distant metastases in ERα⁺ breast cancer cells. Moreover, this study has important translational implications because it highlights the mitotic kinase Aurora-A as a novel promising therapeutic target to selectively eliminate highly invasive cancer cells and improve the disease-free and overall survival of ERα⁺ breast cancer patients resistant to conventional endocrine therapy.

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.

Incorrect segregation of whole chromosomes or parts of chromosome leads to aneuploidy commonly observed in cancer. The correct centrosome duplication, assuring assembly of a bipolar mitotic spindle, is essential for chromosome segregation fidelity and preventing aneuploidy. Alteration of p53 and pRb functions by expression of HPV16-E6 and E7 oncoproteins has been associated with centrosome amplification. However, these last findings could be the result of targeting cellular proteins in addition to pRb by HPV16-E7 oncoprotein. To get a more detailed picture on the role of pRb in chromosomal instability and centrosome amplification, we analyzed the effects of the acute loss of retinoblastoma gene function in primary conditional Rb deficient mouse embryonic fibroblasts (MEFs). Moreover, since pRb is a transcriptional repressor, microarray analysis was done on pRb-competent and pRb-deficient MEFs to evaluate changes in expression of genes for centrosome homeostasis and for correct mitosis. Acute loss of pRb induces centrosome amplification and aneuploidy in the vast majority of cells analyzed. A time course analysis shows a decrease of cells with amplified centrosomes after 40 days from the adenoviral infection. At this time only 12% of cells still show amplified centrosomes. Interestingly, cells with pRb constitutive loss show a similar percentage of cells with amplified centrosomes. DNA-Chip analyses in MEFs wt (mock infected) and pRb depleted (Ad-Cre infected) cells reveal differential expression of genes controlling both centrosome duplication and mitotic progression. Our findings suggest a direct link between pRb status, centrosome amplification and chromosomal instability, and define specific mitotic genes as targets whose gene expression has to be altered to achieve or maintain aneuploidy.