DNA Damage Checkpoint Activation Research Articles

Oxidative Stress and Replication-Independent DNA Breakage Induced by Arsenic in Saccharomyces cerevisiae

Arsenic is a well-established human carcinogen of poorly understood mechanism of genotoxicity. It is generally accepted that arsenic acts indirectly by generating oxidative DNA damage that can be converted to replication-dependent DNA double-strand breaks (DSBs), as well as by interfering with DNA repair pathways and DNA methylation. Here we show that in budding yeast arsenic also causes replication and transcription-independent DSBs in all phases of the cell cycle, suggesting a direct genotoxic mode of arsenic action. This is accompanied by DNA damage checkpoint activation resulting in cell cycle delays in S and G2/M phases in wild type cells. In G1 phase, arsenic activates DNA damage response only in the absence of the Yku70–Yku80 complex which normally binds to DNA ends and inhibits resection of DSBs. This strongly indicates that DSBs are produced by arsenic in G1 but DNA ends are protected by Yku70–Yku80 and thus invisible for the checkpoint response. Arsenic-induced DSBs are processed by homologous recombination (HR), as shown by Rfa1 and Rad52 nuclear foci formation and requirement of HR proteins for cell survival during arsenic exposure. We show further that arsenic greatly sensitizes yeast to phleomycin as simultaneous treatment results in profound accumulation of DSBs. Importantly, we observed a similar response in fission yeast Schizosaccharomyces pombe, suggesting that the mechanisms of As(III) genotoxicity may be conserved in other organisms.

DNA resection proteins Sgs1 and Exo1 are required for G1 checkpoint activation in budding yeast

Double-strand breaks (DSBs) in budding yeast trigger activation of DNA damage checkpoints, allowing repair to occur. Although resection is necessary for initiating damage-induced cell cycle arrest in G2, no role has been assigned to it in the activation of G1 checkpoint. Here we demonstrate for the first time that the resection proteins Sgs1 and Exo1 are required for efficient G1 checkpoint activation. We find in G1 arrested cells that histone H2A phosphorylation in response to ionizing radiation is independent of Sgs1 and Exo1. In contrast, these proteins are required for damage-induced recruitment of Rfa1 to the DSB sites, phosphorylation of the Rad53 effector kinase, cell cycle arrest and RNR3 expression. Checkpoint activation in G1 requires the catalytic activity of Sgs1, suggesting that it is DNA resection mediated by Sgs1 that stimulates the damage response pathway rather than protein–protein interactions with other DDR proteins. Together, these results implicate DNA resection, which is thought to be minimal in G1, as necessary for activation of the G1 checkpoint.

Suppression of Chromosome Healing and Anticheckpoint Pathways in Yeast Postsenescence Survivors

Telomere repeat-like sequences at DNA double-strand breaks (DSBs) inhibit DNA damage signaling and serve as seeds to convert DSBs to new telomeres in mutagenic chromosome healing pathways. We find here that the response to seed-containing DSBs differs fundamentally between budding yeast (Saccharomyces cerevisiae) cells that maintain their telomeres via telomerase and so-called postsenescence survivors that use recombination-based alternative lengthening of telomere (ALT) mechanisms. Whereas telomere seeds are efficiently elongated by telomerase, they remain remarkably stable without de novo telomerization or extensive end resection in telomerase-deficient (est2Δ, tlc1Δ) postsenescence survivors. This telomere seed hyper-stability in ALT cells is associated with, but not caused by, prolonged DNA damage checkpoint activity (RAD9, RAD53) compared to telomerase-positive cells or presenescent telomerase-negative cells. The results indicate that both chromosome healing and anticheckpoint activity of telomere seeds are suppressed in yeast models of ALT pathways.

Sen1p Contributes to Genomic Integrity by Regulating Expression of Ribonucleotide Reductase 1 (RNR1) in Saccharomyces cerevisiae

Gene expression is a multi-step process which requires recruitment of several factors to promoters. One of the factors, Sen1p is an RNA/DNA helicase implicated in transcriptional termination and RNA processing in yeast. In the present study, we have identified a novel function of Sen1p that regulates the expression of ribonucleotide reductase RNR1 gene, which is essential for maintaining genomic integrity. Cells with mutation in the helicase domain or lacking N-terminal domain of Sen1p displayed a drastic decrease in the basal level transcription of RNR1 gene and showed enhanced sensitivity to various DNA damaging agents. Moreover, SEN1 mutants [Sen1-1 (G1747D), Sen1-2 (Δ1-975)] exhibited defects in DNA damage checkpoint activation. Surprisingly, CRT1 deletion in Sen1p mutants (Sen1-1, Sen1-2) was partly able to rescue the slow growth phenotype upon genotoxic stress. Altogether, our observations suggest that Sen1p is required for cell protection against DNA damage by regulating the expression of DNA repair gene RNR1. Thus, the misregulation of Sen1p regulated genes can cause genomic instability that may lead to neurological disorders and premature aging.

Abstract 3420: The novel Chk1 inhibitor MK8776 sensitizes AML cells to HDAC inhibitors by targeting the intra-S checkpoint and DNA replication and repair.

Abstract While histone deacetylase inhibitors (HDACIs, e.g., vorinostat) are approved for CTCL, they have limited single-agent activity in AML. Consequently, new mechanisms and targeted agents capable of improving HDACI anti-leukemic efficacy are urgently needed. Recently, new insights into HDACI mechanisms of actions have implicated interruption of DNA replication and DNA damage checkpoints, raising the possibility that agents targeting Chk1, a key modulator that monitors the DNA replication process and mediates activation of DNA damage checkpoints, may improve HDACI activity. To test this hypothesis, interactions between the novel Chk1 inhibitor MK8776 and the HDAC inhibitor (HDACI) vorinostat were examined in human AML cells harboring wild-type (wt) or deficient p53 and/or FLT3-ITD. MK-8776 synergistically potentiated vorinostat-mediated apoptosis in various AML cell lines, although lethality was significantly more pronounced in p53-deficient cells. Notably, AML cells carrying FLT3-ITD were particularly sensitive to the MK8776/vorinostat regimen, regardless of p53 status. Synergistic interactions were associated with inhibition of Chk1 activity (e.g., diminished Ser296 phosphorylation), interference with the intra-S phase checkpoint (cell cycle analysis), disruption of DNA replication (EdU incorporation), and down-regulation of proteins involved in DNA replication (e.g., CDT1) and repair (e.g., CtIP and BRCA1), resulting in sharp increases in DNA damage, reflected by enhanced γH2A.X formation and increased Chk2 Thr68 phosphorylation, and apoptosis. Moreover, AML cells expressing kinase-dead Chk1 (D130A) or Chk1 shRNA were significantly more sensitive to HDACIs compared to their wt-counterparts, and displayed down-regulation of CtIP and BRCA1 phosphorylation following HDACI exposure. Interestingly, MK8776 induced Ser15, but not Ser20, phosphorylation and accumulation of p53 in p53-wt cells, events that were largely blocked by HDACIs. Moreover, p53 knock down by shRNA significantly sensitized these cells to MK8776 or HDACIs, particularly in combination. Finally, the MK8776/vorinostat regimen was active in primary AML blasts, particularly against CD34+/CD38-/CD123+ populations enriched for leukemia-initiating cells. In contrast, identical regimens were relatively sparing toward normal cord blood CD34+ cells. Together, these findings indicate that the novel Chk1 inhibitor MK8776 markedly potentiates HDACI lethality in AML cells displaying various genetic backgrounds through mechanisms involving disruption of DNA replication, the intra-S checkpoint, and DNA repair. They also argue that leukemic cells, and particularly those bearing oncogenic mutations associated with poor prognosis e.g., p53 deletion/mutation or FLT3-ITD, may be more susceptible than their wt-counterparts to this strategy. Citation Format: Shuang Chen, Maciej Kmieciak, Liang Zhou, Hui Lin, Xin-Yan Pei, Yun Dai, Steven Grant. The novel Chk1 inhibitor MK8776 sensitizes AML cells to HDAC inhibitors by targeting the intra-S checkpoint and DNA replication and repair. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3420. doi:10.1158/1538-7445.AM2013-3420

Abstract 1770: Frequent BRCA1 promoter methylation and low 53BP1 status in sporadic triple-negative breast cancer.

Abstract Purpose: BRCA1 plays a key role in DNA repair and cellular stress response. Inhibitors of PARP show promising clinical activity in metastatic, triple-negative BRCA-mutated breast cancer. P53 binding protein 1 (53BP1) is a protein involved in DNA damage checkpoint activation and DNA repair that is involved in both non homologous end-joining and homology-mediated repair of double-strand DNA breaks. When both BRCA1 and 53BP1 are lost, homology-mediated repair appears to be restored and the sensitivity to DNA damaging agents is reduced. Loss of 53BP1 leads to resistance to cis-platinum and PARP inhibitors in BRCA1-deficient cells, suggesting that 53BP1 is a critical modulator of repair choice in BRCA1 mutant, and possibly epigenetically inactivated cells. Reduced 53BP1 expression has been reported in sporadic basal-like and triple-negative and BRCA-associated breast cancers. It thus appears important to simultaneously evaluate 53BP1 status altogether with BRCA1 mutation/methylation in order to estimate the homology-mediated functionality in breast cancer tumors. Methods: We investigated cytosolic 53BP1 levels and BRCA1 gene promoter hypermethylation profile in 151 surgical tumor samples from patients treated in our institution between January 2006 and November 2009 and evaluated their statistical association with classical breast cancer predictive and prognostic factors. DNA methylation patterns in the CpG islands of BRCA1 gene promoter were determined by a methylation specific PCR distinguishing unmethylated from methylated alleles in a given gene on the basis of sequence changes produced following bisulfite treatment of DNA. Cytosolic 53BP1 level was evaluated using a commercially available assay kit. Results: Median age was 54 years (range 29-75 years). Median 53BP1 protein level (pg/mg of cytosolic protein) was 9.5. Using this cut-off, low 53BP1 levels were significantly associated with negative ER and PR status, triple-negative profile, low cytosolic PARP activity, BRCA1 promoter hypermethylation and absence of nodal metastases. The hypermethylation status was significantly associated with UPA and PAI-1 levels, SBR grade, SBR Mitotic count score, ER, PR and HER-2 negative status, and then conversely triple negative (ER, PR and HER-2 negative) profile. Twenty nine percent of the triple negative tumors presented a hypermethylated status, comparing to 5 and 2% for HR+/HER-2- and HER-2+ tumors respectively. In the triple negative population (N=48), only 2 of the 13 tumors with BRCA1 promoter hypermethylation, presented high (>9.5 pg/mg) 53BP1 protein levels Conclusion: Triple negative breast cancers harboring simultaneously a high level of 53BP1 and BRCA1 promoter hypermethylation are a putative target population for development of drugs targeting DNA repair. However, this population appears to be restricted to a small subgroup of triple negative breast cancers. Citation Format: William Jacot, Simon Thezenas, Anne-Claire Laberenne, Romain Senal, Cathy Viglianti, Frédéric Bibeau, Jean-Pierre Bleuse, Gilles Romieu, Pierre-Jean Lamy. Frequent BRCA1 promoter methylation and low 53BP1 status in sporadic triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1770. doi:10.1158/1538-7445.AM2013-1770

Checkpoint kinase 1 in DNA damage response and cell cycle regulation

Originally identified as a mediator of DNA damage response (DDR), checkpoint kinase 1 (Chk1) has a broader role in checkpoint activation in DDR and normal cell cycle regulation. Chk1 activation involves phosphorylation at conserved sites. However, recent work has identified a splice variant of Chk1, which may regulate Chk1 in both DDR and normal cell cycle via molecular interaction. Upon activation, Chk1 phosphorylates a variety of substrate proteins, resulting in the activation of DNA damage checkpoints, cell cycle arrest, DNA repair, and/or cell death. Chk1 and its related signaling may be an effective therapeutic target in diseases such as cancer.

Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM

A major hurdle to evolutionary engineering approaches for multigenic phenotypes is the ability to simultaneously modify multiple genes rapidly and selectively. Here, we describe a method for in vivo-targeted mutagenesis in yeast, targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM). By fusing the yeast 3-methyladenine DNA glycosylase MAG1 to a tetR DNA-binding domain, we are able to elevate mutation rates >800 fold in a specific ∼20-kb region of the genome or on a plasmid that contains an array of tetO sites. A wide spectrum of transitions, transversions and single base deletions are observed. We provide evidence that TaGTEAM generated point mutations occur through error-prone homologous recombination (HR) and depend on resectioning and the error-prone polymerase Pol ζ. We show that HR is error-prone in this context because of DNA damage checkpoint activation and base pair lesions and use this knowledge to shift the primary mutagenic outcome of targeted endonuclease breaks from HR-independent rearrangements to HR-dependent point mutations. The ability to switch repair in this way opens up the possibility of using targeted endonucleases in diverse organisms for in vivo-targeted mutagenesis.

DNA Damage and Repair in Cancer Therapy

Most cancer therapies have their main mechanism of action DNA damage. Tumor cells have several ways to respond to this DNA damage: removal of the damaged area and reconstruction of the strand of DNA, activation of DNA damage checkpoint, changes in transcription profile of benefit to the cell and apoptosis in the case in which the DNA is seriously damaged. We summarize in this article the most important cellular responses to DNA damage caused by chemotherapy. Bcl-2 family, other than in the follicular B-cell lymphoma, is involved in a number of cancers, including melanoma and breast carcinoma, prostate and lung. Its expression was associated with good prognosis in non-small-cell lung cancer. Cytotoxicity of many chemotherapeutic agents is induced through the Bcl-2 apoptotic pathway; overexpression of BCL2 is associated with increased resistance to these agents.

Saccharomyces cerevisiae as a Model to Study Replicative Senescence Triggered by Telomere Shortening

In many somatic human tissues, telomeres shorten progressively because of the DNA-end replication problem. Consequently, cells cease to proliferate and are maintained in a metabolically viable state called replicative senescence. These cells are characterized by an activation of DNA damage checkpoints stemming from eroded telomeres, which are bypassed in many cancer cells. Hence, replicative senescence has been considered one of the most potent tumor suppressor pathways. However, the mechanism through which short telomeres trigger this cellular response is far from being understood. When telomerase is removed experimentally in Saccharomyces cerevisiae, telomere shortening also results in a gradual arrest of population growth, suggesting that replicative senescence also occurs in this unicellular eukaryote. In this review, we present the key steps that have contributed to the understanding of the mechanisms underlying the establishment of replicative senescence in budding yeast. As in mammals, signals stemming from short telomeres activate the DNA damage checkpoints, suggesting that the early cellular response to the shortest telomere(s) is conserved in evolution. Yet closer analysis reveals a complex picture in which the apparent single checkpoint response may result from a variety of telomeric alterations expressed in the absence of telomerase. Accordingly, the DNA replication of eroding telomeres appears as a critical challenge for senescing budding yeast cells and the easy manipulation of S. cerevisiae is providing insights into the way short telomeres are integrated into their chromatin and nuclear environments. Finally, the loss of telomerase in budding yeast triggers a more general metabolic alteration that remains largely unexplored. Thus, telomerase-deficient S. cerevisiae cells may have more common points than anticipated with somatic cells, in which telomerase depletion is naturally programed, thus potentially inspiring investigations in mammalian cells.

The MRN protein complex genes: MRE11 and RAD50 and susceptibility to head and neck cancers

BackgroundThe members of MRE11/RAD50/NBN (MRN) protein complex participates in DNA double-strand break repair and DNA-damage checkpoint activation. We have previously shown that the p.I171V NBN gene mutation may contribute to the development of laryngeal cancer. This study tested the hypothesis that variants of the MRE11 and RAD50 genes, previously described as cancer risk factors, predispose to increased susceptibility to head and neck cancer.FindingsIn this study we analyzed the RAD50 and MRE11 genes in 358 patients: 175 with a single laryngeal cancer (LC), 115 with multiple primary tumors but one malignancy (primary or second) localized in the larynx (MPT-LC), 68 patients with multiple primary tumors localized in the head or neck (MPT) and 506 controls. No carriers of previously reported mutation in the MRE11 or RAD50 gene (particularly the pathogenic c.687delT) were detected in the present study. We identified the p.V127I variant (2/175 LC, 2/506 controls; OR=2.91; 95% CI 0.41-20.85) and p.V315L variant (2/115 MPT-LC, 1/506 controls; OR=8.96; 95% CI 0.81-99.68) of the RAD50 gene.ConclusionsOur data indicated that previously described common genetic variations in the MRE11 and RAD50 genes do not contribute to an increased risk of laryngeal cancer and second primary tumors localized in the head and neck. Prospective studies with larger groups of patients may reveal the possible impact of these genes in tumor occurrence.

Multiple phosphorylation of Rad9 by CDK is required for DNA damage checkpoint activation

The DNA damage checkpoint controls cell cycle arrest in response to DNA damage, and activation of this checkpoint is in turn cell cycle-regulated. Rad9, the ortholog of mammalian 53BP1, is essential for this checkpoint response and is phosphorylated by the cyclin-dependent kinase (CDK) in the yeast Saccharomyces cerevisiae. Previous studies suggested that the CDK consensus sites of Rad9 are important for its checkpoint activity. However, the precise CDK sites of Rad9 involved have not been determined. Here we show that CDK consensus sites of Rad9 function in parallel to its BRCT domain toward checkpoint activation, analogous to its fission yeast ortholog Crb2. Unlike Crb2, however, mutation of multiple rather than any individual CDK site of Rad9 is required to completely eliminate its checkpoint activity in vivo. Although Dpb11 interacts with CDK-phosphorylated Rad9, we provide evidence showing that elimination of this interaction does not affect DNA damage checkpoint activation in vivo, suggesting that additional pathway(s) exist. Taken together, these findings suggest that the regulation of Rad9 by CDK and the role of Dpb11 in DNA damage checkpoint activation are more complex than previously suggested. We propose that multiple phosphorylation of Rad9 by CDK may provide a more robust system to allow Rad9 to control cell cycle-dependent DNA damage checkpoint activation.

A Role for Homologous Recombination and Abnormal Cell-Cycle Progression in Radioresistance of Glioma-Initiating Cells

Glioblastoma multiforme (GBM) is the most common form of brain tumor with a poor prognosis and resistance to radiotherapy. Recent evidence suggests that glioma-initiating cells play a central role in radioresistance through DNA damage checkpoint activation and enhanced DNA repair. To investigate this in more detail, we compared the DNA damage response in nontumor forming neural progenitor cells (NPC) and glioma-initiating cells isolated from GBM patient specimens. As observed for GBM tumors, initial characterization showed that glioma-initiating cells have long-term self-renewal capacity. They express markers identical to NPCs and have the ability to form tumors in an animal model. In addition, these cells are radioresistant to varying degrees, which could not be explained by enhanced nonhomologous end joining (NHEJ). Indeed, NHEJ in glioma-initiating cells was equivalent, or in some cases reduced, as compared with NPCs. However, there was evidence for more efficient homologous recombination repair in glioma-initiating cells. We did not observe a prolonged cell cycle nor enhanced basal activation of checkpoint proteins as reported previously. Rather, cell-cycle defects in the G(1)-S and S-phase checkpoints were observed by determining entry into S-phase and radioresistant DNA synthesis following irradiation. These data suggest that homologous recombination and cell-cycle checkpoint abnormalities may contribute to the radioresistance of glioma-initiating cells and that both processes may be suitable targets for therapy.

Voltage-gated potassium channel EAG2 controls mitotic entry and tumor growth in medulloblastoma via regulating cell volume dynamics

Medulloblastoma (MB) is the most common pediatric CNS malignancy. We identify EAG2 as an overexpressed potassium channel in MBs across different molecular and histological subgroups. EAG2 knockdown not only impairs MB cell growth in vitro, but also reduces tumor burden in vivo and enhances survival in xenograft studies. Mechanistically, we demonstrate that EAG2 protein is confined intracellularly during interphase but is enriched in the plasma membrane during late G2 phase and mitosis. Disruption of EAG2 expression results in G2 arrest and mitotic catastrophe associated with failure of premitotic cytoplasmic condensation. While the tumor suppression function of EAG2 knockdown is independent of p53 activation, DNA damage checkpoint activation, or changes in the AKT pathway, this defective cell volume control is specifically associated with hyperactivation of the p38 MAPK pathway. Inhibition of the p38 pathway significantly rescues the growth defect and G2 arrest. Strikingly, ectopic membrane expression of EAG2 in cells at interphase results in cell volume reduction and mitotic-like morphology. Our study establishes the functional significance of EAG2 in promoting MB tumor progression via regulating cell volume dynamics, the perturbation of which activates the tumor suppressor p38 MAPK pathway, and provides clinical relevance for targeting this ion channel in human MBs.

The Rad4TopBP1 ATR-Activation Domain Functions in G1/S Phase in a Chromatin-Dependent Manner

DNA damage checkpoint activation can be subdivided in two steps: initial activation and signal amplification. The events distinguishing these two phases and their genetic determinants remain obscure. TopBP1, a mediator protein containing multiple BRCT domains, binds to and activates the ATR/ATRIP complex through its ATR-Activation Domain (AAD). We show that Schizosaccharomyces pombe Rad4TopBP1 AAD–defective strains are DNA damage sensitive during G1/S-phase, but not during G2. Using lacO-LacI tethering, we developed a DNA damage–independent assay for checkpoint activation that is Rad4TopBP1 AAD–dependent. In this assay, checkpoint activation requires histone H2A phosphorylation, the interaction between TopBP1 and the 9-1-1 complex, and is mediated by the phospho-binding activity of Crb253BP1. Consistent with a model where Rad4TopBP1 AAD–dependent checkpoint activation is ssDNA/RPA–independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4TopBP1 AAD is important for Chk1 phosphorylation when resection is limited in G2 by ablation of the resecting nuclease, Exo1. We also show that the Rad4TopBP1 AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD–dependent Rad3ATR checkpoint amplification is particularly important when DNA resection is limiting. In S. pombe, this manifests in G1/S phase and relies on protein–chromatin interactions.

Mechanism and Regulation of the Helicase‐driven Path of DNA End Resection in Saccharomyces cerevisiae

The ends of DNA double‐strand breaks (DSBs) are resected in a cell‐cycle dependent fashion, to yield 3′ overhanging single‐stranded DNA (ssDNA) for the recruitment of factors critical for DNA damage checkpoint activation and repair by homologous recombination. As defined in studies in the budding yeast Saccharomyces cerevisiae, the DNA end resection process involves multiple, redundant pathways, one of which entails the Sgs1 helicase and its partners Rmi1‐Top3, Dna2 helicase/nuclease, Mre11‐Rad50‐Xrs2 (MRX) complex, and the heterotrimeric ssDNA‐binding protein RPA. We have reconstituted the DNA end resection reaction using the aforementioned factors. To gain further insight into how Cdk1 kinase activity regulates the motor‐driven resection pathway, we have purified the Cdk1‐Clb2 complex and examined its effects both in our reconstituted system and in conjunction with genetic analysis. Our studies indicate that one means of Cdk1 regulation is via direct phosphorylation of Dna2 at three consensus sites located within its amino‐terminus, which promotes efficient recruitment of Dna2 to DSBs. However, phospho‐mimic mutations of Dna2 cannot fully bypass the requirement of Cdk1 activity in vivo, raising the possibility that Cdk1 also targets other factor(s), an idea that is being examined using our reconstituted system. The biochemical and genetic systems that we have established should serve as a useful model for delineating the mechanistic intricacy of the DNA break resection process in eukaryotes.

Down-regulated expression of apoptosis-associated genes APIP and UACA in non-small cell lung carcinoma

The Apaf-1 interacting protein (APIP) and the uveal autoantigen with coiled coil domains and ankyrin repeats (UACA) belong to endogenous regulators of the apoptosome apparatus, but their role in tumourigenesis and progression of non-small cell lung carcinoma (NSCLC) is not known. Previous studies demonstrated that APIP inhibits the apoptosome-mediated procaspase-9 activation while UACA induces translocation of Apaf-1 from the cytoplasm into the nucleus. Here, we report for the first time that the expression of APIP and UACA genes is down-regulated on the level of both mRNA and protein in NSCLC cells and tumours. In particular, the expression of APIP protein was strikingly decreased and the expression of UACA mRNA and protein was frequently down-regulated in NSCLC tumours of different histopathological types. Moreover, stage IA NSCLC tumours showed significantly lower expression of UACA mRNA compared to higher stage tumours. The weak increase of both APIP and UACA mRNA levels in the 5-aza-2'-deoxycytidine-treated NSCLC cells indicates that mechanisms other than DNA methylation are involved in the regulation of APIP and UACA gene expression in these cancer cells. Taken together, the down-regulation of APIP and UACA expression suggests that the threshold to activate the apoptosome apparatus may be decreased in NSCLC cells due to the lack of APIP-mediated suppression and UACA-assisted Apaf-1 nuclear entry. Moreover, the loss of UACA-assisted Apaf-1 nuclear translocation may underlie the failure of DNA damage checkpoint activation in NSCLC cells leading to their genomic instability.

Nondisjunction of a Single Chromosome Leads to Breakage and Activation of DNA Damage Checkpoint in G2

The resolution of chromosomes during anaphase is a key step in mitosis. Failure to disjoin chromatids compromises the fidelity of chromosome inheritance and generates aneuploidy and chromosome rearrangements, conditions linked to cancer development. Inactivation of topoisomerase II, condensin, or separase leads to gross chromosome nondisjunction. However, the fate of cells when one or a few chromosomes fail to separate has not been determined. Here, we describe a genetic system to induce mitotic progression in the presence of nondisjunction in yeast chromosome XII right arm (cXIIr), which allows the characterisation of the cellular fate of the progeny. Surprisingly, we find that the execution of karyokinesis and cytokinesis is timely and produces severing of cXIIr on or near the repetitive ribosomal gene array. Consequently, one end of the broken chromatid finishes up in each of the new daughter cells, generating a novel type of one-ended double-strand break. Importantly, both daughter cells enter a new cycle and the damage is not detected until the next G2, when cells arrest in a Rad9-dependent manner. Cytologically, we observed the accumulation of damage foci containing RPA/Rad52 proteins but failed to detect Mre11, indicating that cells attempt to repair both chromosome arms through a MRX-independent recombinational pathway. Finally, we analysed several surviving colonies arising after just one cell cycle with cXIIr nondisjunction. We found that aberrant forms of the chromosome were recovered, especially when RAD52 was deleted. Our results demonstrate that, in yeast cells, the Rad9-DNA damage checkpoint plays an important role responding to compromised genome integrity caused by mitotic nondisjunction.

Distinct pattern of oxidative DNA damage and DNA repair in follicular thyroid tumours

Increased oxidative stress has been linked to thyroid carcinogenesis. In this paper, we investigate whether oxidative DNA damage and DNA repair differ in follicular adenoma (FA) and follicular thyroid carcinoma (FTC). 7,8-Dihydro-8-oxoguanine (8-OxoG) formation was analysed by immunohistochemistry in 46 FAs, 52 FTCs and 18 normal thyroid tissues (NTs). mRNA expression of DNA repair genes OGG1, Mut Y homologue (MUTYH) and endonuclease III (NTHL1) was analysed by real-time PCR in 19 FAs, 25 FTCs and 19 NTs. Induction and repair of oxidative DNA damage were studied in rat FRTL-5 cells after u.v. irradiation. Moreover, activation of DNA damage checkpoints (ataxia telangiectasia mutated (ATM) and H2A histone family, member X (H2AFX (H2AFX))) and proliferation index (MIB-1) were quantified in 28 non-oxyphilic and 24 oxyphilic FTCs. Increased nuclear and cytosolic 8-OxoG formation was detected in FTC compared with follicular adenoma, whereby cytosolic 8-OxoG formation was found to reflect RNA oxidation. Significant downregulation of DNA repair enzymes was detected in FTC compared with FA. In vitro experiments mirrored the findings in FTC with oxidative stress-induced DNA checkpoint activation and downregulation of OGG1, MUTYH and NTHL1 in FRTL-5 cells, an effect that, however, was reversible after 24 h. Further analysis of FTC variants showed decreased oxidative DNA damage, sustained checkpoint activation and decreased proliferation in oxyphilic vs non-oxyphilic FTC. Our data suggest a pathophysiological scenario of accumulating unrepaired DNA/RNA damage in FTC vs counterbalanced DNA/RNA damage and repair in FA. Furthermore, this study provides the first evidence for differences in oxidative stress defence in FTC variants with possible implications for therapeutic response and prognostic outcome.

Histone H3 Lysine 14 Acetylation Is Required for Activation of a DNA Damage Checkpoint in Fission Yeast

Histone lysine acetylation has emerged as a key regulator of genome organization. However, with a few exceptions, the contribution of each acetylated lysine to cellular functions is not well understood because of the limited specificity of most histone acetyltransferases and histone deacetylases. Here we show that the Mst2 complex in Schizosaccharomyces pombe is a highly specific H3 lysine 14 (H3K14) acetyltransferase that functions together with Gcn5 to regulate global levels of H3K14 acetylation (H3K14ac). By analyzing the effect of H3K14ac loss through both enzymatic inactivation and histone mutations, we found that H3K14ac is critical for DNA damage checkpoint activation by directly regulating the compaction of chromatin and by recruiting chromatin remodeling protein complex RSC.