Age-related accumulation of ploidy changes is associated with decreased expression of genes controlling chromosome segregation and cohesin functions. To determine the consequences of whole chromosome instability (W-CIN) we down-regulated the spindle assembly checkpoint component BUB1 and the mitotic cohesin SMC1A, and used four-color-interphase-FISH coupled with BrdU incorporation and analyses of senescence features to reveal the fate of W-CIN cells. We observed significant correlations between levels of not-diploid cells and senescence-associated features (SAFs). W-CIN induced DNA double strand breaks and elevated oxidative stress, but caused low apoptosis. SAFs of W-CIN cells were remarkably similar to those induced by replicative senescence but occurred in only 13 days versus 4 months. Cultures enriched with not-diploid cells acquired a senescence-associated secretory phenotype (SASP) characterized by IL1B, CXCL8, CCL2, TNF, CCL27 and other pro-inflammatory factors including a novel SASP component CLEC11A. These findings suggest that W-CIN triggers premature senescence, presumably to prevent the propagation of cells with an abnormal DNA content. Cells deviating from diploidy have the ability to communicate with their microenvironment by secretion of an array of signaling factors. Our results suggest that aneuploid cells that accumulate during aging in some mammalian tissues potentially contribute to age-related pathologies and inflammation through SASP secretion.

Early genetic instability induced in dividing V79-Cl3 Chinese hamster cells by inorganic arsenic, as demonstrated in our previous investigation, was evidenced by aneuploidy and nuclear abnormalities, but not by chromosomal rearrangements. Here we report the results of cytogenetic and morphological analyses performed on the progeny of cells dividing at the end of sodium arsenite treatment after they had been expanded through 120 generations (ASO cells) and then cloned. The acquired genetic instability persisted and was increased by highly unstable chromosomal rearrangements, namely dicentric chromosomes and telomeric associations, which were not seen following acute exposure. A peculiar finding was the preferential involvement of a particular chromosome in dicentric rearrangements observed in some isolated ASO clones. Interestingly, by immunostaining with anti-5-methylcytosine antibodies the genome-wide DNA hypomethylation, induced by arsenic immediately after the acute treatment, was found to affect those ASO clones characterized by aneuploidy and chromosomal rearrangements. These findings demonstrate that short-term exposure to arsenic has long-term effects and suggest that genome-wide DNA hypomethylation enhances genetic instability.

Fanconi Anemia (FA) is a rare disease characterized by congenital defects, progressive bone marrow failure and heightened cancer susceptibility. The FA proteins, BRCA1, and FANCD1/BRCA2, function cooperatively in the FA-BRCA pathway, to repair damaged DNA. Activation of the FA-BRCA pathway occurs via the monoubiquitination of the FANCD2 and FANCI proteins, targeting these proteins to discrete nuclear foci where they function in DNA repair. The cellular regulation of FANCD2/I monoubiquitination, however, remains poorly understood. In this study, we have examined the roles of the p53 tumor suppressor protein, as well as its downstream target the p21Cip1/Waf1 cyclin-dependent kinase inhibitor, in the regulation of the activation of the FA-BRCA pathway. We demonstrate that, in contrast to p53, p21 plays a major role in the regulation of the activation of the FA-BRCA pathway: p21 promotes S-phase and DNA damage-inducible FANCD2/I monoubiquitination and nuclear foci formation. Several lines of evidence establish that this effect is not a consequence of a defective G1-S checkpoint or altered cell cycle progression in the absence of p21. Instead, we demonstrate that p21 is required for the transcriptional repression of the USP1 deubiquitinating enzyme upon exposure to DNA damaging agents. In the absence of p21, persistent USP1 expression precludes the DNA damage-inducible accumulation of monoubiquitinated FANCD2 and FANCI. Consequently, p21−/− cells exhibit increased levels of mitomycin C-inducible complex chromosomal aberrations and elevated γ-H2AX nuclear foci formation. Our results demonstrate that p21 plays a critical role in the regulation of the activation of the FA-BRCA pathway and suggest a broader role for p21 in the orchestration of DNA repair processes following exposure to DNA crosslinking agents.

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Sirolimus (rapamycin) is an immunosuppressive drug used in transplantation. One of its major side effects is the increased risk of diabetes mellitus; however, the exact mechanisms underlying such association have not been elucidated. Here we show that sirolimus impairs glucose-stimulated insulin secretion both in human and murine pancreatic islets and in clonal β cells in a dose- and time-dependent manner. Importantly, we demonstrate that sirolimus markedly depletes calcium (Ca2+) content in the endoplasmic reticulum and significantly decreases glucose-stimulated mitochondrial Ca2+ uptake. Crucially, the reduced mitochondrial Ca2+ uptake is mirrored by a significant impairment in mitochondrial respiration. Taken together, our findings indicate that sirolimus causes depletion of intracellular Ca2+ stores and alters mitochondrial fitness, eventually leading to decreased insulin release. Our results provide a novel molecular mechanism underlying the increased incidence of diabetes mellitus in patients treated with this drug.

p21 is a well-established regulator of cell cycle progression. The role of p21 in DNA repair, however, remains poorly characterized. Here, we describe a critical role of p21 in a replication-coupled DNA double-strand break (DSB) repair that is mechanistically distinct from its cell cycle checkpoint function. We demonstrate that p21-deficient cells exhibit elevated chromatid-type aberrations, including gaps and breaks, dicentrics and radial formations, following exposure to several DSB-inducing agents. p21−/− cells also exhibit an increased DNA damage-inducible DNA-PKCS S2056 phosphorylation, indicative of elevated non-homologous DNA end joining. Concomitantly, p21−/− cells are defective in replication-coupled homologous recombination (HR), exhibiting decreased sister chromatid exchanges and HR-dependent repair as determined using a crosslinked GFP reporter assay. Importantly, we establish that the DSB hypersensitivity of p21−/− cells is associated with increased cyclin-dependent kinase (CDK)-dependent BRCA2 S3291 phosphorylation and MRE11 nuclear foci formation and can be rescued by inhibition of CDK or MRE11 nuclease activity. Collectively, our results uncover a novel mechanism by which p21 regulates the fidelity of replication-coupled DSB repair and the maintenance of chromosome stability distinct from its role in the G1-S phase checkpoint.

The Fanconi anemia (FA)-BRCA pathway is critical for the repair of DNA interstrand crosslinks (ICLs) and the maintenance of chromosome stability. A key step in FA-BRCA pathway activation is the covalent attachment of monoubiquitin to FANCD2 and FANCI. Monoubiquitinated FANCD2 and FANCI localize in chromatinassociated nuclear foci where they interact with several well-characterized DNA repair proteins. Importantly, very little is known about the structure, function, and regulation of FANCD2. Herein, we describe the identification and characterization of a CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) ubiquitin-binding domain (UBD) in FANCD2, and demonstrate that the CUE domain mediates noncovalent binding to ubiquitin in vitro. We show that although mutation of the CUE domain destabilizes FANCD2, the protein remains competent for DNA damage-inducible monoubiquitination and phosphorylation. Importantly, we demonstrate that the CUE domain is required for interaction with FANCI, retention of monoubiquitinated FANCD2, and FANCI in chromatin, and for efficient ICL repair. Our results suggest a model by which heterodimerization of monoubiquitinated FANCD2 and FANCI in chromatin is mediated in part through a noncovalent interaction between the FANCD2 CUE domain and monoubiquitin covalently attached to FANCI, and that this interaction shields monoubiquitinated FANCD2 from polyubiquitination and proteasomal degradation. (Blood. 2012;120(10):2109-2117) IntroductionFanconi anemia (FA) is a rare, recessive disease characterized by congenital abnormalities, bone marrow failure, hematologic malignancies, and elevated cancer risk. 1 FA is caused by biallelic mutation in any 1 of the following 15 genes: FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BRIP1, FANCL, FANCM, FANCN/PALB2, FANCO/ RAD51C, and FANCP/SLX4. The FA proteins together with BRCA1 function cooperatively in the FA-BRCA pathway to repair damaged DNA and prevent cellular transformation. 2 Disruption of the FA-BRCA pathway leads to cellular hypersensitivity to the cytotoxic and clastogenic effects of DNA interstrand crosslinking agents. 3 The FA-BRCA pathway is activated after exposure to DNA damaging agents and during S-phase of the cell cycle. 4,5 Activation of the pathway occurs when the core FA complex composed of FANCA, B, C, E, F, G, L, and M, and other proteins, assembles in the nucleus and monoubiquitinates the paralogous proteins FANCD2 and FANCI. 4,6,7 FANCL, a RING domain-containing protein is the catalytic E3 ubiquitin ligase subunit of the FA core complex, whereas UBE2T is the E2 conjugating enzyme. [8][9][10] Monoubiquitination of FANCD2 and FANCI targets these proteins to discrete chromatin-associated nuclear foci, where they interact with several key DNA repair proteins, including BRCA1, FANCD1/BRCA2, and RAD51. 4,5,11 After repair, FANCD2 and FANCI are deubiquitinated by the USP1/UAF1 complex facilitating the release of these proteins from chromatin. 12,13 Recent studies indicate that ...

Here, we report the effects of exposure of mammalian cells to α-pinene, a bicyclic monoterpene used in insecticides, solvents and perfumes. Morphological analysis, performed in V79-Cl3 cells exposed for 1 h to increasing concentrations (25 up to 50 μM) of α-pinene, indicated a statistically significant increase in micronucleated and multinucleated cell frequencies; apoptotic cells were seen at 40 and 50 μM. This monoterpene caused genomic instability by interfering with mitotic process; in fact, 50% of cells (versus 19% of control cells) showed irregular mitosis with multipolar or incorrectly localised spindles. Cytogenetic analysis demonstrated high-frequency hypodiploid metaphases as well as endoreduplicated cells and chromosome breaks. Clastogenic damage was prevalent over aneuploidogenic damage as demonstrated by the higher proportion of kinetochore-negative micronuclei. Alkaline comet confirmed that monoterpene exposure caused DNA lesions in a concentration-dependent manner. This damage probably arose by increased reactive oxygen species (ROS) production. In order to assess the generation of ROS, the cells were incubated with CM-H(2)DCFDA and then analysed by flow cytometry. Results demonstrated an increase in fluorescence intensity after α-pinene treatment indicating increased oxidative stress. On the whole, these findings strongly suggest that α-pinene is able to compromise genome stability preferentially through mitotic alterations and to damage DNA through ROS production.