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Replication of the genome must be coordinated with gene transcription and cellular metabolism, especially following replication stress in the presence of limiting deoxyribonucleotides. The Saccharomyces cerevisiae Rad53 (CHEK2 in mammals) checkpoint kinase plays a major role in cellular responses to DNA replication stress. Cell cycle regulated, genome-wide binding of Rad53 to chromatin was examined. Under replication stress, the kinase bound to sites of active DNA replication initiation and fork progression, but unexpectedly to the promoters of about 20% of genes encoding proteins involved in multiple cellular functions. Rad53 promoter binding correlated with changes in expression of a subset of genes. Rad53 promoter binding to certain genes was influenced by sequence-specific transcription factors and less by checkpoint signaling. However, in checkpoint mutants, untimely activation of late-replicating origins reduces the transcription of nearby genes, with concomitant localization of Rad53 to their gene bodies. We suggest that the Rad53 checkpoint kinase coordinates genome-wide replication and transcription under replication stress conditions.

Upon replication fork arrest, the replication checkpoint kinase Cds1 is stimulated to preserve genome integrity. Robust activation of Cds1 in response to hydroxyurea prevents the endonuclease Mus81 from cleaving the stalled replication fork inappropriately. However, we find that the response is different in temperature-sensitive mcm4 mutants, affecting a subunit of the MCM replicative helicase. We show that Cds1 inhibition of Mus81 promotes genomic instability and allows mcm4-dg cells to evade cell cycle arrest. Cds1 regulation of Mus81 activity also contributes to the formation of the replication stress-induced DNA damage markers replication protein A (RPA) and Ku. These results identify a surprising role for Cds1 in driving DNA damage and disrupted chromosomal segregation under certain conditions of replication stress. [ABSTRACT FROM AUTHOR]

Target of rapamycin (TOR) is an evolutionarily conserved protein kinase that plays an important role in the regulation of cell growth and the sensing of nutrient and energy status in eukaryotes. In yeasts and mammals, the roles of TOR have been very well described and various functions of TOR signaling in plant lineages have also been revealed over the past 20 years. In the case of microalgae, the functions of TOR have been primarily studied in the model green alga Chlamydomonas reinhardtii and were summarized in an earlier single review article. However, the recent development of tools for the functional analysis of TOR has helped to reveal the involvement of TOR in various functions, including autophagy, transcription, translation, accumulation of energy storage molecules, etc., in microalgae. In the present review, we discuss recent novel findings relating to TOR signaling and its roles in microalgae along with relevant information on land plants and also provide details of topics that must be addressed in future studies to reveal how TOR regulates various physiological functions in microalgae. [ABSTRACT FROM AUTHOR]

Background: As a component of the EIF3 complex, EIF3C is essential for several steps in protein synthesis initiation. Recently, it has been addressed that EIF3C is overexpressed in several human cancers and plays a pivotal role in cell proliferation and tumorigenesis. Materials and methods: Immunohistochemistry, quantitative real-time PCR (qPCR), and Western blotting assays were employed to determine the expression of EIF3C in osteosarcoma (OsC) tissues obtained from 60 patients. The levels of EIF3C mRNA and protein were assessed by qPCR and Western blotting, respectively. The effect of EIF3C knockdown on OsC cell proliferation was detected by MTT and colony formation assays, respectively. Cell apoptosis induced by EIF3C silencing was analyzed by flow cytometric analysis. PathScan stress and apoptosis signaling antibody array kit was used to analyze the potential effects of EIF3C knockdown on OsC cells. Results: The levels of EIF3C were high in OsC tissues and cell lines. In addition, EIF3C knockdown by lentivirus-mediated shRNA targeting EIF3C significantly suppressed cell proliferation and colony formation and induced apoptosis in U-2OS cells. Moreover, EIF3C knockdown led to the upregulated expression of CASP3/7, Chk1/2, and SAPK/JNK, indicating that the downregulated expression of EIF3C might be associated with pro-apoptosis of U-2OS cells. Conclusion: EIF3C may be a promising target for gene therapy of human OsC. However, the precise mechanisms behind the effect of EIF3C on OsC tumorigenesis require further analysis. [ABSTRACT FROM AUTHOR]

Targeting of oncogene-driven replicative stress as therapeutic option for high-risk medullobastoma was assessed using a panel of medulloblastoma cells differing in their c-Myc expression [i.e. group SHH (c-Myc low) vs. group 3 (c-Myc high)]. High c-Myc levels were associated with hypersensitivity to pharmacological Chk1 and ATR inhibition but not to CDK inhibition nor to conventional (genotoxic) anticancer therapeutics. The enhanced sensitivity of group 3 medulloblastoma cells to Chk1 inhibitors likely results from enhanced damage to intracellular organelles, elevated replicative stress and DNA damage and activation of apoptosis/necrosis. Furthermore, Chk1 inhibition differentially affected c-Myc expression and functions. In c-Myc high cells, Chk1 blockage decreased c-Myc and p-GSK3α protein and increased p21 and GADD45A mRNA expression. By contrast, c-Myc low cells revealed increased p-GSK3β protein and CHOP and DUSP1 mRNA levels. Inhibition of Chk1 sensitized medulloblastoma cells to additional replication stress evoked by cisplatin independent of c-Myc. Importantly, Chk1 inhibition only caused minor toxicity in primary rat neurons in vitro. Collectively, targeting of ATR/Chk1 effectively triggers death in high-risk medulloblastoma, potentiates the anticancer efficacy of cisplatin and is well tolerated in non-cancerous neuronal cells. [ABSTRACT FROM AUTHOR]

AZD1775 is a small molecule WEE1 inhibitor used in combination with DNA-damaging agents to cause premature mitosis and cell death in p53-mutated cancer cells. Here we sought to determine the mechanism of action of AZD1775 in combination with chemotherapeutic agents in light of recent findings that AZD1775 can cause double-stranded DNA (DS-DNA) breaks. AZD1775 significantly improved the cytotoxicity of 5-FU in a p53-mutated colorectal cancer cell line (HT29 cells), decreasing the IC50from 9.3 μM to 3.5 μM. Flow cytometry showed a significant increase in the mitotic marker pHH3 (3.4% vs. 56.2%) and DS-DNA break marker γH2AX (5.1% vs. 50.7%) for combination therapy compared with 5-FU alone. Combination therapy also increased the amount of caspase-3 dependent apoptosis compared with 5-FU alone (4% vs. 13%). The addition of exogenous nucleosides to combination therapy significantly rescued the increased DS-DNA breaks and caspase-3 dependent apoptosis almost to the levels of 5-FU monotherapy. In conclusion, AZD1775 enhances 5-FU cytotoxicity through increased DS-DNA breaks, not premature mitosis, in p53-mutated colorectal cancer cells. This finding is important for designers of future clinical trials when considering the optimal timing and duration of AZD1775 treatment. [ABSTRACT FROM PUBLISHER]

Background: More effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide. We therefore explored the efficacy of the combination of gemcitabine and AZD7762, a checkpoint kinase 1/2 (CHK1/2) inhibitor, for bladder cancer. Methods: Viability, clonogenicity, cell cycle distribution and apoptosis were assessed in urothelial cancer cell lines and various non-malignant urothelial cells treated with gemcitabine and AZD7762. DNA damage was assessed by γH2A.X and 53-BP1 staining and checkpoint activation was followed by Western blotting. Pharmacological inhibition of CHK1 and CHK2 was compared to downregulation of either CHK1 or CHK2 using siRNAs. Results: Combined use of gemcitabine and AZD7762 synergistically reduced urothelial carcinoma cell viability and colony formation relative to either single treatment. Non-malignant urothelial cells were substantially less sensitive to this drug combination. Gemcitabine plus AZD7762 inhibited cell cycle progression causing cell accumulation in S-phase. Moreover, the combination induced pronounced levels of apoptosis as indicated by an increase in the fraction of sub-G1 cells, in the levels of cleaved PARP, and in caspase 3/7 activity. Mechanistic investigations showed that AZD7762 treatment inhibited the repair of gemcitabine-induced double strand breaks by interference with CHK1, since siRNA-mediated depletion of CHK1 but not of CHK2 mimicked the effects of AZD7762. Conclusions: AZD7762 enhanced sensitivity of urothelial carcinoma cells to gemcitabine by inhibiting DNA repair and disturbing checkpoints. Combining gemcitabine with CHK1 inhibition holds promise for urothelial cancer therapy. [ABSTRACT FROM AUTHOR]

The cell cycle checkpoint kinase Mec1 ATR and its integral partner Ddc2 ATRIP are vital for the DNA damage and replication stress response. Mec1-Ddc2 "senses" single-stranded DNA (ssDNA) by being recruited to the ssDNA binding Replication Protein A (RPA) via Ddc2. In this study, we show that a DNA damage-induced phosphorylation circuit modulates checkpoint recruitment and function. We demonstrate that Ddc2-RPA interactions modulate the association between RPA and ssDNA and that Rfa1-phosphorylation aids in the further recruitment of Mec1-Ddc2. We also uncover an underappreciated role for Ddc2 phosphorylation that enhances its recruitment to RPA-ssDNA that is important for the DNA damage checkpoint in yeast. The crystal structure of a phosphorylated Ddc2 peptide in complex with its RPA interaction domain provides molecular details of how checkpoint recruitment is enhanced, which involves Zn 2+ . Using electron microscopy and structural modeling approaches, we propose that Mec1-Ddc2 complexes can form higher order assemblies with RPA when Ddc2 is phosphorylated. Together, our results provide insight into Mec1 recruitment and suggest that formation of supramolecular complexes of RPA and Mec1-Ddc2, modulated by phosphorylation, would allow for rapid clustering of damage foci to promote checkpoint signaling.

Background: Although progress in children, in adults, ALL still carries a dismal outcome. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), a potent, selective ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2). Methods: The effectiveness of PF-00477736 as single agent in B-/T-ALL was evaluated in vitro and in vivo studies as a single agent. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B-/T-ALL cell lines. Finally, the action of PF-00477736 was assessed in vivo using leukemic mouse generated by a single administration of the tumorigenic agent N-ethyl-N-nitrosourea. Results: Chk1 and Chk2 are overexpressed concomitant with the presence of genetic damage as suggested by the nuclear labeling for γ-H2A.X (Ser139) in 68 % of ALL patients. In human B- and T-ALL cell lines, inhibition of Chk1/2 as a single treatment strategy efficiently triggered the Chk1-Cdc25-Cdc2 pathway resulting in a dose- and time-dependent cytotoxicity, induction of apoptosis, and increased DNA damage. Moreover, treatment with PF-00477736 showed efficacy ex vivo in primary leukemic blasts separated from 14 adult ALL patients and in vivo in mice transplanted with T-ALL, arguing in favor of its future clinical evaluation in leukemia. Conclusions: In vitro, ex vivo, and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia, and they provide a strong rationale for its future clinical investigation. [ABSTRACT FROM AUTHOR]

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress. [ABSTRACT FROM AUTHOR]

Replication of the genome must be coordinated with gene transcription and cellular metabolism, especially following replication stress in the presence of limiting deoxyribonucleotides. The Saccharomyces cerevisiae Rad53 (CHEK2 in mammals) checkpoint kinase plays a major role in cellular responses to DNA replication stress. Cell cycle regulated, genome-wide binding of Rad53 to chromatin was examined. Under replication stress, the kinase bound to sites of active DNA replication initiation and fork progression, but unexpectedly to the promoters of about 20% of genes encoding proteins involved in multiple cellular functions. Rad53 promoter binding correlated with changes in expression of a subset of genes. Rad53 promoter binding to certain genes was influenced by sequence-specific transcription factors and less by checkpoint signaling. However, in checkpoint mutants, untimely activation of late-replicating origins reduces the transcription of nearby genes, with concomitant localization of Rad53 to their gene bodies. We suggest that the Rad53 checkpoint kinase coordinates genome-wide replication and transcription under replication stress conditions., Competing Interests: YS, RK, JG No competing interests declared, BS Reviewing editor, eLife, (© 2022, Sheu et al.)

Checkpoint kinase (Chk) inhibitors are thought to increase the cytotoxic effects of DNA-damaging agents and are undergoing clinical trials. The present study was aimed to assess the potential to use the Chk1 and Chk2 inhibitor, AZD7762, with other anticancer agents in chemotherapy to treat ovarian clear cell carcinoma.Four ovarian clear cell carcinoma cell lines were used in this study. We treated the cells with AZD7762 and anticancer agents, then assessed cell viability, cell cycle distribution, apoptosis, and the expression of protein in apoptotic pathways and molecules downstream of the Chk signaling pathways. We also investigated the effects of these drug combinations on tumor growth in a nude mouse xenograft model.Synergistic effects from the combination of AZD7762 and cisplatin were observed in all 4 cell lines. However, we observed additive effects when AZD7762 was combined with paclitaxel on all cell lines tested. AZD7762 effectively suppressed the Chk signaling pathways activated by cisplatin, dramatically enhanced expression of phosphorylated H2A.X, cleaved caspase 9 and PARP, decreased the proportion of cells in the gap 0/ gap 1 phase and the synthesis-phase fraction, and increased apoptotic cells. Combinations of small interfering RNA against Chk 1 and small interfering RNA against Chk2 enhanced the cytotoxic effect of cisplatin in both RMG-I and KK cells. Finally, treating mice-bearing RMG-I with AZD7762 and cisplatin significantly suppressed growth of tumors in a xenograft model.The present study indicates that chemotherapy with AZD7762 and cisplatin should be explored as a treatment modality for women with ovarian clear cell carcinoma. [ABSTRACT FROM AUTHOR]

Cullin4, Ddb1 and Cdt2 are core subunits of the ubiquitin ligase complex CRL4Cdt2, which controls genome stability by targeting Spd1 for degradation during DNA replication and repair in fission yeast. Spd1 has an inhibitory effect on ribonucleotide reductase (RNR), the activity of which is required for deoxynucleotide (dNTP) synthesis. The failure to degrade Spd1 in mutants where CRL4Cdt2 is defective leads to DNA integrity checkpoint activation and dependency. This correlates with a lower dNTP pool. Pools are restored in a spd1- deleted background and this also suppresses checkpoint activation and dependency. We hypothesized that fission yeast with RNR hyperactivity would display a mutator phenotype on their own, but also possibly repress aspects of the phenotype associated with the inability to target Spd1 for degradation. Here, we report that a mutation in the R1 subunit of ribonucleotide reductase cdc22 (cdc22- D57N), which alleviated allosteric feedback inhibition, caused a highly elevated dNTP pool that was further increased by deleting spd1. The Dspd1 cdc22-D57N double mutant had elevated mutation rates and was sensitive to damaging agents that cause DNA strand breaks, demonstrating that Spd1 can protect the genome when dNTP pools are high. In ddb1-deleted cells, cdc22-D57N also potently elevated RNR activity, but failed to allow cell growth independently of the intact checkpoint. Our results provide evidence that excess Spd1 interferes with other functions in addition to its inhibitory effect on ribonucleotide reduction to generate replication stress and genome instability. [ABSTRACT FROM AUTHOR]

Background There are 600 million betel quid chewers around the world. betel quid chewing is a major risk factor of oral cancer. Why betel quid components induce oral cancer is not clear. Methods Cytotoxicity of areca nut extract and arecoline (an areca nut alkaloid) to SAS oral epithelial cell line was evaluated by trypan blue dye exclusion and MTT assays. Cell cycle distribution and apoptosis was analyzed by propidium iodide staining flow cytometry. Chk1 and chk2 activation was analyzed by Pathscan phospho-enzyme-linked immunosorbent assay. Metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinase (TIMPs) production was measured by enzyme-linked immunosorbent assay. Results Areca nut extract (800 μg/mL) and arecoline (>0.4 mmol/L) caused cell death, apoptosis, and cell cycle arrest of SAS cells. Areca nut extract and arecoline stimulated Chk1 and Chk2 phosphorylation in SAS cells. Areca nut extract stimulated cellular MMP-9 but suppressed TIMP-1 and TIMP-2 production. Conclusions Areca nut components activate Chk1/Chk2, alter cell cycle regulation/apoptosis, MMP-9, and TIMPs production, contributing to the pathogenesis of oral carcinogenesis. © 2012 Wiley Periodicals, Inc. Head Neck, 2013 [ABSTRACT FROM AUTHOR]

Abstract: Arsenic trioxide (ATO) is widely used in tumor treatment, but excessive arsenic exposure can have adverse effects. We recently found that, in primary osteoblasts, ATO produces oxidative stress and causes DNA tailing, but does not induce apoptosis. We further examined the signaling pathway by which osteoblasts survive ATO treatment, and found that they were arrested at G2/M phase of the cell cycle at 30h and overrode the G2/M boundary at 48h. After treatment for 30h, there was increased Cdc2 phosphorylation and expression of Wee1, a Cdc2 kinase, and expression of the cell cycle inhibitor, p21waf1/cip1, which interacts with Cdc2. Furthermore, levels of the phosphatase Cdc25C, which activates Cdc2, were decreased, while the ratio of its phosphorylated/inactivated form to the total amount was increased. Moreover, phosphorylation/activation of the checkpoint kinases Chk1, Chk2 and p53 levels were increased, as were levels of activated ATM and γ-H2AX. The cell viability was decreased as an ATM inhibitor was added. Additionally, these effects of ATO on γ-H2AX, Chk1, Chk2, p53, and p21waf1/cip1 were reduced by an ATM inhibitor. These findings suggest that G2/M phase arrest of osteoblasts is mediated by Chk1/Chk2 activation via an ATM-dependent pathway by which osteoblasts survive. [Copyright &y& Elsevier]

Abstract: Pyridyl aminothiazoles comprise a novel class of ATP-competitive Chk1 inhibitors with excellent inhibitory potential. Modification of the core with ethylenediamine amides provides compounds with low picomolar potency and very high residence times. Investigation of binding parameters of such compounds using X-ray crystallography and molecular dynamics simulations revealed multiple hydrogen bonds to the enzyme backbone as well as stabilization of the conserved water molecules network in the hydrophobic binding region. [Copyright &y& Elsevier]

Abstract: The effects of DNA damage on NO production have not been completely elucidated. Using ultraviolet (UV) irradiation as a DNA-damaging agent, we studied its effect on NO production in bovine aortic endothelial cells (BAEC). UV irradiation acutely increased NO production, the phosphorylation of endothelial NO synthase (eNOS) at serine 1179, and eNOS activity. No alterations in eNOS expression nor phosphorylation at eNOS Thr497 or eNOS Ser116 were found. SB218078, a checkpoint kinase 1 (Chk1) inhibitor, inhibited UV-irradiation-stimulated eNOS-Ser1179 phosphorylation and NO production. Similarly, ectopic expression of small interference RNA for Chk1 or a dominant-negative Chk1 repressed the UV-irradiation stimulatory effect, whereas wild-type Chk1 increased basal eNOS-Ser1179 phosphorylation. Purified Chk1 directly phosphorylated eNOS Ser1179 in vitro. Confocal microscopy and coimmunoprecipitation studies revealed a colocalization of eNOS and Chk1. In basal BAEC, heat shock protein 90 (Hsp90) predominantly interacted with Chk1. This interaction, which decreased significantly in response to UV irradiation, was accompanied by increased interaction of Hsp90 with eNOS. The Hsp90 inhibitor geldanamycin attenuated UV-irradiation-stimulated eNOS-Ser1179 phosphorylation by dissociating Hsp90 from eNOS. UV irradiation and geldanamycin did not alter the interaction between eNOS and Chk1. Overall, this is the first study demonstrating that Chk1 directly phosphorylates eNOS Ser1179 in response to UV irradiation, which is dependent on Hsp90 interaction. [Copyright &y& Elsevier]

Abstract: Pixantrone is a promising anti-cancer aza-anthracenedione that has prompted the development of new anthracenediones incorporating symmetrical side-chains of increasing length varying from two to five methylene units in each pair of drug side-chains. A striking relationship has emerged in which anthracenedione-induced growth inhibition and apoptosis was inversely associated with side-chain length, a relationship that was attributable to a differential ability to stabilise the topoisomerase II (TOP2) cleavage complex. Processing of the complex to a DNA double strand break (DSB) flanked by γH2AX in nuclear foci is likely to occur, as the generation of the primary lesion was antecedent to γH2AX induction. M2, bearing the shortest pair of side-chains, induced TOP2-mediated DSBs efficiently and activated cell cycle checkpoints via Chk1 and Chk2 phosphorylation, implicating the involvement of ATM and ATR, and induced a protracted S phase and subsequent G2/M arrest. The inactive analogue M5, containing the longest pair of side-chains, only weakly stimulated any of these responses, suggesting that efficient stabilisation of the TOP2 cleavage complex was crucial for eliciting a strong DNA damage response (DDR). An M2 induced DDR in p53-defective MDA-MB-231 cells was abrogated by UCN-01, a ubiquitous inhibitor of kinases including Chk1, in a response associated with substantial mitotic catastrophe and strong synergy. The rational selection of checkpoint kinase inhibitors may significantly enhance the therapeutic benefit of anthracenediones that efficiently stabilise the TOP2 cleavage complex. [Copyright &y& Elsevier]

Abstract: Chronic ethanol abuse results in hepatocyte injury and impairs hepatocyte replication. We have previously shown that ethanol metabolism results in cell cycle arrest at the G2/M transition, which is partially mediated by inhibitory phosphorylation of the cyclin-dependent kinase, Cdc2. To further delineate the mechanisms by which ethanol metabolism mediates this G2/M arrest, we investigated the involvement of upstream regulators of Cdc2 activity. Cdc2 is activated by the phosphatase Cdc25C. The activity of Cdc25C can, in turn, be regulated by the checkpoint kinase, Chk2, which is regulated by the kinase ataxia telangiectasia mutated (ATM). To investigate the involvement of the regulators of Cdc2 activity, VA-13 cells, which are Hep G2 cells modified to efficiently express alcohol dehydrogenase, were cultured in the presence or absence of 25 mM ethanol. Immunoblots were performed to determine the effects of ethanol metabolism on the activation of Cdc25C, Chk2, and ATM. Ethanol metabolism increased the active forms of ATM and Chk2, as well as the phosphorylated form of Cdc25C. Additionally, inhibition of ATM resulted in approximately 50% of the cells being rescued from the G2/M cell cycle arrest and ameliorated the inhibitory phosphorylation of Cdc2. Our findings demonstrated that ethanol metabolism activates ATM. ATM can activate the checkpoint kinase Chk2, resulting in phosphorylation of Cdc25C and ultimately in the accumulation of inactive Cdc2. This may, in part, explain the ethanol metabolism–mediated impairment in hepatocyte replication, which may be important in the initiation and progression of alcoholic liver injury. [Copyright &y& Elsevier]

Abstract: Fertilization relieves the oocyte from a cell cycle arrest, inducing progression towards mitotic cycles. While the signalling pathways involved in oocyte to embryo transition have been widely investigated, how they specifically trigger DNA replication is still unclear. We used sea urchin eggs whose oocytes are arrested in G1 to investigate in vivo the molecular mechanisms regulating initiation of replication after fertilization. Unexpectedly, we found that CDC6, Cdt1 and MCM3, components of the pre-replication complexes (pre-RC) which license origins for replication, were already loaded on female chromatin before fertilization. This is the first demonstration of a cell cycle arrest in metazoan in which chromatin is already licensed for replication. In contrast pre-RC assemble on chromatin post-fertilization as in other organisms. These differences in the timing of pre-RC assembly are accompanied by differences in Cdk2 requirement for DNA replication initiation between female and male chromatin post-fertilization. Finally, we demonstrated that a concomitant inhibition of MAP kinase and ATM/ATR pathways releases the block to DNA synthesis. Our findings provide new insight into the mechanisms contributing to the release of G1 arrest and the control of S-phase entry at fertilization. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: The fruits and seeds of Semecarpus anacardium are used widely for the treatment of human cancers and other diseases in the Ayurvedic and Sidda systems of medicine in India. Aim of the study: The principal aim of this investigation was to isolate and characterize the anticancer compound from the kernel of Semecarpus anacardium nut. Materials and methods: The bioactivity-tailored isolation and detailed chemical characterization were used to identify the active compound. Cytotoxicity, apoptosis, cell cycle arrest as well as synergism between the identified anticancer compound and doxorubicin in human tumor cell lines were analyzed. Results: GC/MS, IR, proton NMR, carbon NMR and collisionally induced dissociation (CID) spectra analysis showed that the isolated active compound is 3-(8′(Z),11′(Z)-pentadecadienyl) catechol (SA-3C). SA-3C is cytotoxic to tumor cell lines with IC50 values lower than doxorubicin and even multidrug resistant tumor cell lines were equally sensitive to SA-3C. SA-3C induced apoptosis in human leukemia cell lines in a dose-dependent manner and showed synergistic cytotoxicity with doxorubicin. The cell cycle arrest induced by SA-3C at S- and G2/M-phases correlated with inhibition of checkpoint kinases. Conclusion: SA-3C isolated from the kernel of Semecarpus anacardium can be developed as an important anticancer agent for single agent and/or multiagent cancer therapy. [Copyright &y& Elsevier]

Gene-environment interactions are implicated in congenital human disorders. Accordingly, there is a pressing need to develop animal models of human disease, which are the product of defined gene-environment interactions. Previously, our laboratory demonstrated that gestational salt stress of bradykinin B2 receptor (B2R)-null mice induces renal dysgenesis and early death of the offspring (El-Dahr SS, Harrison Bernard LM, Dipp S, Yosipiv IV, and Meleg-Smith S. Physiol Genomics 3: 121-131, 2000). In contrast, salt-stressed B2R +/+ or +/- littermates have normal development. The present study investigates the mechanisms underlying the susceptibility of B2R-null mice to renal dysgenesis. Proteomic and conventional Western blot screens identified E-cadherin among the differentially repressed proteins in B2R-/- kidneys, whereas the checkpoint kinase Chk1 and its substrate P-Ser20 p53 were induced. We tested the hypothesis that p53 mediates repression of E-cadherin gene expression and is causally linked to the renal dysgenesis. Genetic crosses between B2R -/- and p53+/- mice revealed that germline reduction of p53 gene dosage rescues B2R-/- mice from renal dysgenesis and restores kidney E-cadherin gene expression. Furthermore, γ-irradiation induces repression of E-cadherin gene expression in p53+/+ but not -/- cells. In transient transfection assays, p53 repressed human E-cadherin promoter-driven reporter activity, whereas a mutant p53, which cannot bind DNA, did not. Functional promoter analysis indicated the presence of a p53-responsive element in exon 1, which partially mediates p53-induced repression. Chromatin immunoprecipitation assays revealed that p53 inhibits histone acetylation of the E-cadherin promoter. Treatment with a histone deacetylase inhibitor reversed both p53-mediated promoter repression and deacetylation. In conclusion, this study demonstrates that gene-environment interactions cooperate to induce congenital defects through p53 activation. [ABSTRACT FROM AUTHOR]

Naturally occurring neuronal death (NOND) has been described in the postnatal cerebellum of several species, mainly affecting the cerebellar granule cells (CGCs) by an apoptotic mechanism. However, little is known about the cellular pathway(s) of CGC apoptosis in vivo. By immunocytochemistry, in situ detection of fragmented DNA, electron microscopy, and Western blotting, we demonstrate here the existence of two different molecular mechanisms of apoptosis in the rabbit postnatal cerebellum. These two mechanisms affect CGCs at different stages of their maturation and migration. In the external granular layer, premigratory CGCs undergo apoptosis upon phosphorylation of checkpoint kinase 1 (Chk1), and hyperphosphorylation of retinoblastoma protein. In postmigratory CGCs within the internal granular layer, caspase 3 and to a lesser extent 7 and 9 are activated, eventually leading to poly-ADP-ribose polymerase-1 (PARP-1) cleavage and programmed cell death. We conclude that NOND of premigratory CGCs is linked to activation of DNA checkpoint and alteration of normal cell cycle, whereas in postmigratory CGCs apoptosis is, more classically, dependent upon caspase 3 activation. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 437-452, 2004 [ABSTRACT FROM AUTHOR]

Chk2 is a key player of the DNA damage signalling pathway. To identify new regulators of this kinase, we performed a yeast two-hybrid screen and found that Chk2 associated with the B′ regulatory subunit of protein phosphatase PP2A. In vitro GST-Chk2 pulldowns demonstrated that B′γ isoforms bound to Chk2 with the strongest apparent affinity. This was confirmed in cellulo by co-immunoprecipitation after overexpression of the respective partners in HEK293 cells. The A and C subunits of PP2A were present in the complexes, suggesting that Chk2 was associated with a functionnal PP2A. In vitro kinase assays showed that B′γ3 was a potent Chk2 substrate. This phosphorylation increased the catalytic phosphatase activity of PP2A measured on MAP kinase-phosphorylated myelin basic protein as well as on autophosphorylated Chk2. Finally, we demonstrated that overexpressing B′γ3 in HEK293 suppressed the phosphorylation of Chk2 induced by a genotoxic treatment, suggesting that PP2A may counteract the action of the checkpoint kinase in living cells. [Copyright &y& Elsevier]

Regulation of the vertebrate checkpoint kinase Chk1 involves several protein complexes including the recently identified protein Claspin. Claspin associates with Chk1 upon replication stress and DNA damage and is required for Chk1 activation in both Xenopus and human systems. More importantly, Claspin is involved in regulation of cell cycle checkpoints. Here, we discuss the emerging roles of Claspin in the Chk1 pathway and its functions in checkpoint control. [Copyright &y& Elsevier]

Replication protein A (RPA) is a conserved single-stranded DNA (ssDNA) binding protein with well-characterized roles in DNA metabolism. RPA is phosphorylated in response to genotoxic stress and is required for efficient checkpoint function, although these aspects of RPA function are not well understood. We have investigated the association between RPA and the checkpoint kinase Mec1 in yeast. RPA and Mec1 were found to be physically associated during unperturbed cell growth and in response to DNA damage. Using a Mec1 immunoprecipitate (IP)-kinase assay, we show that the two large subunits, RPA1 and RPA2, are good substrates for Mec1 kinase. The major phosphorylation site of RPA1 was further investigated as it was found to be localized to its amino terminus (RPA1N), which is a non-ssDNA binding domain implicated in regulatory function. This phosphorylation site mapped to serine 178 and phosphorylation-defective mutant protein, expressed from rfa1-S178A, showed reduced physical interaction with Mec1. Phenotypic analysis in vivo revealed that the rfa1-S178A mutation affected the kinetics of RPA1 and Rad53 phosphorylation but did not otherwise affect the checkpoint response. We suggest that phosphorylation of RPA1N by Mec1 may function together with other checkpoint events to regulate the checkpoint response. [Copyright &y& Elsevier]

Background: Malignant glioma (MG) is an aggressive malignant brain tumor. Despite advances in multidisciplinary treatment, overall survival rates remain low. A trifluoroethyl amide derivative of 2-cyano-3-,12-dioxoolean-1,9-dien-28-oic acid (CDDO), CDDO–trifluoroethyl amide (CDDO–TFEA) is a nuclear erythroid 2-related factor 2/antioxidant response element pathway activator. RTA404 is used to inhibit proliferation and induce apoptosis in cancer cells. However, its effect on tumorigenesis in glioma is unclear. Methods: This in vitro study evaluated the effects of RTA404 on MG cells. We treated U87MG cell lines with RTA404 and performed assessments of apoptosis and cell cycle distributions. DNA content and apoptosis induction were subjected to flow cytometry analysis. The mitotic index was assessed based on MPM-2 expression. Protein expression was analyzed through Western blotting. Results: RTA404 significantly inhibited the cell viability and induced cell apoptosis on the U87MG cell line. The Annexin-FITC/PI assay revealed significant changes in the percentage of apoptotic cells. Treatment with RTA404 led to a significant reduction in the U87MG cells' mitochondrial membrane potential. A significant rise in the percentage of caspase-3 activity was detected in the treated cells. In addition, these results suggest that cells pass the G2 checkpoint without cell cycle arrest by RTA404 treatment in the MPM-2 staining. An analysis of CHK1, CHK2, and p-CHK2 expression suggested that the DNA damage checkpoint system seems also to be activated by RTA404 treatment in established U87MG cells. Therefore, RTA404 may not only activate the DNA damage checkpoint system, it may also exert apoptosis in established U87MG cells. Conclusions: RTA404 inhibits the cell viability of gliomas and induces cancer cell apoptosis through intrinsic apoptotic pathway in Malignant glioma. In addition, the DNA damage checkpoint system seems also to be activated by RTA404. Taken together, RTA404 activated the DNA damage checkpoint system and induced apoptosis through intrinsic apoptotic pathways in established U87MG cells. [ABSTRACT FROM AUTHOR]

Summary Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2’s recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage. [ABSTRACT FROM AUTHOR]

DNA damage repair is essential for cellular homeostasis and cell survival. Cytostatic drugs and ionizing radiation cause an enormous amount of DNA damage and efficient repair is crucial for sustained cell growth. Hence manipulating DNA damage repair offers a new therapeutic principle, which together with conventional treatment strategies can increase treatment efficiency. In this issue we would like to give a brief overview of the current status of DNA damage repair factors as clinical markers and therapeutic targets. Ongoing and future clinical trials will determine the therapeutic potential of this new target for cancer treatment. [ABSTRACT FROM AUTHOR]

DNA damage response plays a critical role in tumor growth, but little is known about its potential role in bone metabolism. We employed selective inhibitors of Chk1 and examined their effects on the proliferation and migration of mammary tumor cells as well as the development of osteoblasts and osteoclasts. Further, using a mouse model of bone metastasis we evaluated the effects of Chk1 inhibitors on bone quality. Chk1 inhibitors blocked the proliferation, survival, and migration of tumor cells in vitro and suppressed the development of bone-resorbing osteoclasts by downregulating NFATc1. In the mouse model, Chk1 inhibitor reduced osteolytic lesions and prevented mechanical weakening of the femur and tibia. Analysis of RNA-seq expression data indicated that the observed effects were mediated through the regulation of eukaryotic translation initiation factor 2 alpha, stress to the endoplasmic reticulum, S100 proteins, and bone remodeling-linked genes. Our findings suggest that targeting Chk1 signaling without adding DNA damaging agents may protect bone from degradation while suppressing tumor growth and migration., Competing Interests: CONFLICTS OF INTEREST The authors report no conflicts of interest.

Background: More effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide. We therefore explored the efficacy of the combination of gemcitabine and AZD7762, a checkpoint kinase 1/2 (CHK1/2) inhibitor, for bladder cancer., Methods: Viability, clonogenicity, cell cycle distribution and apoptosis were assessed in urothelial cancer cell lines and various non-malignant urothelial cells treated with gemcitabine and AZD7762. DNA damage was assessed by γH2A.X and 53-BP1 staining and checkpoint activation was followed by Western blotting. Pharmacological inhibition of CHK1 and CHK2 was compared to downregulation of either CHK1 or CHK2 using siRNAs., Results: Combined use of gemcitabine and AZD7762 synergistically reduced urothelial carcinoma cell viability and colony formation relative to either single treatment. Non-malignant urothelial cells were substantially less sensitive to this drug combination. Gemcitabine plus AZD7762 inhibited cell cycle progression causing cell accumulation in S-phase. Moreover, the combination induced pronounced levels of apoptosis as indicated by an increase in the fraction of sub-G1 cells, in the levels of cleaved PARP, and in caspase 3/7 activity. Mechanistic investigations showed that AZD7762 treatment inhibited the repair of gemcitabine-induced double strand breaks by interference with CHK1, since siRNA-mediated depletion of CHK1 but not of CHK2 mimicked the effects of AZD7762., Conclusions: AZD7762 enhanced sensitivity of urothelial carcinoma cells to gemcitabine by inhibiting DNA repair and disturbing checkpoints. Combining gemcitabine with CHK1 inhibition holds promise for urothelial cancer therapy.

PNAS-4, a novel pro-apoptotic gene, was activated during the early response to DNA damage. Our previous study has shown that PNAS-4 induces S phase arrest and apoptosis when overexpressed in A549 lung cancer cells. However, the underlying action mechanism remains far from clear. In this work, we found that PNAS-4 expression in lung tumor tissues is significantly lower than that in adjacent lung tissues; its expression is significantly increased in A549 cells after exposure to cisplatin, methyl methane sulfonate, and mitomycin; and its overexpression induces S phase arrest and apoptosis in A549 (p53 WT), NCI-H460 (p53 WT), H526 (p53 mutation), and Calu-1 (p53(-/-)) lung cancer cells, leading to proliferation inhibition irrespective of their p53 status. The S phase arrest is associated with up-regulation of p21(Waf1/Cip1) and inhibition of the Cdc25A-CDK2-cyclin E/A pathway. Up-regulation of p21(Waf1/Cip1) is p53-independent and correlates with activation of ERK. We further showed that the intra-S phase checkpoint, which occurs via DNA-dependent protein kinase-mediated activation of Chk1 and Chk2, is involved in the S phase arrest and apoptosis. Gene silencing of Chk1/2 rescues, whereas that of ATM or ATR does not affect, S phase arrest and apoptosis. Furthermore, human PNAS-4 induces DNA breaks in comet assays and γ-H2AX staining. Intriguingly, caspase-dependent cleavage of Chk1 has an additional role in enhancing apoptosis. Taken together, our findings suggest a novel mechanism by which elevated PNAS-4 first causes DNA-dependent protein kinase-mediated Chk1/2 activation and then results in inhibition of the Cdc25A-CDK2-cyclin E/A pathway, ultimately causing S phase arrest and apoptosis in lung cancer cells., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

CDC25B is one of the three human phosphatases that activate the CDK-cyclin complexes, thereby triggering cell-cycle progression and division. Commitment to early mitotic events depends on the activation of a centrosomal pool of CDK1--cyclin-B1, and CDC25B is thought to be involved in initiating this centrosomal CDK1--cyclin-B1 activity. Centrosome-associated checkpoint kinase 1 (CHK1) has been proposed to contribute to the proper timing of a normal cell division cycle by inhibiting the activation of the centrosomal pool of CDK1. Here, we show that CDC25B is phosphorylated by CHK1 in vitro on multiple residues, including S230 and S563. We demonstrate these phosphorylations occur in vivo and that they are dependent on CHK1 activity. S230 CHK1-mediated phosphorylation is detected in cell extracts during S phase and G2 phase in the absence of DNA damage. We show that the S230-phosphorylated form of CDC25B is located at the centrosome from early S phase until mitosis. Furthermore, mutation of S230 to alanine increases the mitotic-inducing activity of CDC25B. Our results support a model in which, under normal cell cycle conditions and in the absence of DNA damage, CHK1 constitutively phosphorylates CDC25B during interphase and thus prevents the premature initiation of mitosis by negatively regulating the activity of CDC25B at the centrosome. [ABSTRACT FROM AUTHOR]

The nucleus/cytoplasm (N/C) ratio controls S phase dynamics in many biological systems, most notably the abrupt remodeling of the cell cycle that occurs at the midblastula transition in early Xenopus laevis embryos. After an initial series of rapid cleavage cycles consisting only of S and M phases, a critical N/C ratio is reached, which causes a sharp increase in the length of S phase as the cell cycle is reconfigured to resemble somatic cell cycles. How the N/C ratio determines the length of S phase has been a longstanding problem in developmental biology. Using Xenopus egg extracts, we show that DNA replication at high N/C ratio is restricted by one or more limiting substances. We report here that the protein phosphatase PP2A, in conjunction with its B55α regulatory subunit, becomes limiting for replication origin firing at high N/C ratio, and this in turn leads to reduced origin activation and an increase in the time required to complete S phase. Increasing the levels of PP2A catalytic subunit or B55α experimentally restores rapid DNA synthesis at high N/C ratio. Inversely, reduction of PP2A or B55α levels sharply extends S phase even in low N/C extracts. These results identify PP2A-B55α as a link between DNA replication and N/C ratio in egg extracts and suggest a mechanism that may influence the onset of the midblastula transition in vivo.