Neocarzinostatin Treatment Research Articles

Studies on melatonin intervention in reactive oxygen species-linked autophagy of neocarzinostatin-induced varicocele infertility in male Swiss Albino mice

BackgroundVaricocele is known to be a condition involving the impairment of spermatogenesis, thereby leading to low sperm production and decreased sperm quality and ultimately one of the major cause of male infertility. Melatonin has shown to directly neutralize a number of free radicals responsible for toxicity in the cell. PurposeTherefore, this, study is designed to evaluate the induction of varicocele following neocarzinostatin treatment and also to investigate possible ameliorative effect of melatonin against varicocele induction. MethodIn this study, male albino mice (aged 6 weeks and weighing 25 gs) were divided into eight groups (n = 10). Group I (Control), Group II -Varicocele (Positive Control), Group III -NCS (5 mg/kg.bwt), Group IV -NCS (10 mg/kg.bwt), Group V - NCS (20 mg/kg.bwt), Group VI- Varicocele + Melatonin (10 mg/kg.bwt), Group VII -NCS (10 mg/kg.bwt) + Melatonin (10 mg/kg.bwt), Group VIII -NCS (20 mg/kg.bwt) + Melatonin (10 mg/kg.bwt). Intraperitoneal injection of a mixture of ketamine and xylazine was administered into each mouse in group II and served as the positive control. Group III, IV and V received 5, 10 and 20 mg/kg.bwt of Neocarzinostatin daily respectively. Five mice from each group was sacrificed 24 hour (h) after the twenty-eight days treatment for further experiments while the remaining animals were used for fertility study. ResultsAt the end of the experiment, NCS treatment for 28 days reduced the body weight significantly decreased across the groups in a dose dependent manner (group II, III, IV, VI, VII and VIII). Swollen of the testis and enlarge blood vein were observed confirming the induction of varicocele by NCS-treated groups. Tubular Differential index (%) and Johnsen score (%) significantly reduced (p < 0.001) (p < 0.05). The levels of reactive oxygen species and malondialdehyde significantly increased (p<0.001) (p<0.05). The activities of glutathione, superoxide dismutase and catalase significantly reduced (p < 0.001) (p < 0.05). The level of Luteinizing Hormone, FSH and testosterone significantly reduced (p < 0.001) (p < 0.05). ConclusionIn conclusion, the results of this present study clearly showed that neocarzinostatin treatment leads to induction of varicocele infertility. Also, this study gives an insight that co-treatment of melatonin is able to reduce the hazardous effect of neocarzinostatin-induced varicocele and cytotoxicity in the testis of mice.

Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System.

DNA repair pathways trigger robust downstream responses, making it challenging to select suitable reference genes for comparative studies. In this study, our goal was to identify the most suitable housekeeping genes to perform comparable molecular analyses for DNA damage-related studies. Choosing the most applicable reference genes is important in any kind of target gene expression-related quantitative study, since using the housekeeping genes improperly may result in false data interpretation and inaccurate conclusions. We evaluated the expressional changes of eight well-known housekeeping genes (i.e., 18S rRNA, B2M, eEF1α1, GAPDH, GUSB, HPRT1, PPIA, and TBP) following treatment with the DNA-damaging agents that are most frequently used: ultraviolet B (UVB) non-ionizing irradiation, neocarzinostatin (NCS), and actinomycin D (ActD). To reveal the significant changes in the expression of each gene and to determine which appear to be the most acceptable ones for normalization of real-time quantitative polymerase chain reaction (RT-qPCR) data, comparative and statistical algorithms (such as absolute quantification, Wilcoxon Rank Sum Test, and independent samples T-test) were conducted. Our findings clearly demonstrate that the genes commonly employed as reference candidates exhibit substantial expression variability, and therefore, careful consideration must be taken when designing the experimental setup for an accurate and reproducible normalization of RT-qPCR data. We used the U2OS cell line since it is generally accepted and used in the field of DNA repair to study DNA damage-induced cellular responses. Based on our current data in U2OS cells, we suggest using 18S rRNA, eEF1α1, GAPDH, GUSB, and HPRT1 genes for UVB-induced DNA damage-related studies. B2M, HPRT1, and TBP genes are recommended for NCS treatment, while 18S rRNA, B2M, and PPIA genes can be used as suitable internal controls in RT-qPCR experiments for ActD treatment. In summary, this is the first systematic study using a U2OS cell culture system that offers convincing evidence for housekeeping gene selection following treatment with various DNA-damaging agents. Here, we unravel an indispensable issue for performing and assessing trustworthy DNA damage-related differential gene expressional analyses, and we create a "zero set" of potential reference gene candidates.

Nuclear Ceramide Is Associated with Ataxia Telangiectasia Mutated Activation in the Neocarzinostatin-Induced Apoptosis of Lymphoblastoid Cells.

Ceramide is a bioactive sphingolipid that mediates ionizing radiation- and chemotherapy-induced apoptosis. Neocarzinostatin (NCS) is a genotoxic anti-cancer drug that induces apoptosis in response to DNA double-strand breaks (DSBs) through ataxia telangiectasia mutated (ATM) activation. However, the involvement of ceramide in NCS-evoked nuclear events such as DSB-activated ATM has not been clarified. Here, we found that nuclear ceramide increased by NCS-mediated apoptosis through the enhanced assembly of ATM and the meiotic recombination 11/double-strand break repair/Nijmengen breakage syndrome 1 (MRN) complex proteins in human lymphoblastoid L-39 cells. NCS induced an increase of ceramide production through activation of neutral sphingomyelinase (nSMase) and suppression of sphingomyelin synthase (SMS) upstream of DSB-mediated ATM activation. In ATM-deficient lymphoblastoid AT-59 cells compared with L-39 cells, NCS treatment showed a decrease of apoptosis even though ceramide increase and DSBs were observed. Expression of wild-type ATM, but not the kinase-dead mutant ATM, in AT-59 cells increased NCS-induced apoptosis despite similar ceramide accumulation. Interestingly, NCS increased ceramide content in the nucleus through nSMase activation and SMS suppression and promoted colocalization of ceramide with phosphorylated ATM and foci of MRN complex. Inhibition of ceramide generation by the overexpression of SMS suppressed NCS-induced apoptosis through the inhibition of ATM activation and assembly of the MRN complex. In addition, inhibition of ceramide increased by the nSMase inhibitor GW4869 prevented NCS-mediated activation of the ATM. Therefore, our findings suggest the involvement of the nuclear ceramide with ATM activation in NCS-mediated apoptosis. SIGNIFICANCE STATEMENT: This study demonstrates that regulation of ceramide with neutral sphingomyelinase and sphingomyelin synthase in the nucleus in double-strand break-mimetic agent neocarzinostatin (NCS)-induced apoptosis. This study also showed that ceramide increase in the nucleus plays a role in NCS-induced apoptosis through activation of the ataxia telangiectasia mutated/meiotic recombination 11/double-strand break repair/Nijmengen breakage syndrome 1 complex in human lymphoblastoid cells.

Chemotherapy of HER2- and MDM2-Enriched Breast Cancer Subtypes Induces Homologous Recombination DNA Repair and Chemoresistance.

Simple SummaryMDM2 is a protein responsible for negative regulation of the p53 tumor suppressor. In addition, MDM2 exhibits chaperone-like properties similar to the HSP90 molecular chaperone. Multiple studies revealed that MDM2 is deeply involved in cancer development and progression. Some recently published results indicate that the role of MDM2 in DNA repair inhibition is more complex than previously thought. We show that MDM2 is directly involved in the homologous recombination DNA repair, and its chaperone-like activity is crucial for this function. The DNA repair inhibition is a result of inefficient MDM2 dissociation from the NBN protein complex. When cancer cells are treated with chemotherapy, MDM2 can be easily released from the interaction and degraded, resulting in effective homologous recombination DNA repair, which translates into the acquisition of a chemoresistant phenotype by the tumor. This knowledge may allow for identification of the patients that are at particular risk of tumor chemoresistance. Analyzing the TCGA breast cancer database, we discovered that patients with the HER2 cancer subtype and overexpression of MDM2 exhibited decreased post-treatment survival. Inhibition of MDM2 expression in the SKBR3 cell line (HER2 subtype) diminished the survival of cancer cells treated with doxorubicin, etoposide, and camptothecin. Moreover, we demonstrated that inhibition of MDM2 expression diminished DNA repair by homologous recombination (HR) and sensitized SKBR3 cells to a PARP inhibitor, olaparib. In H1299 (TP53−/−) cells treated with neocarzinostatin (NCS), overexpression of MDM2 WT or E3-dead MDM2 C478S variant stimulated the NCS-dependent phosphorylation of ATM, NBN, and BRCA1, proteins involved in HR DNA repair. However, overexpression of chaperone-dead MDM2 K454A variant diminished phosphorylation of these proteins as well as the HR DNA repair. Moreover, we demonstrated that, upon NCS treatment, MDM2 K454A interacted with NBN more efficiently than MDM2 WT and that MDM2 WT was degraded more efficiently than MDM2 K454A. Using a proliferation assay, we showed that overexpression of MDM2 WT, but not MDM2 K454A, led to acquisition of resistance to NCS. The presented results indicate that, following chemotherapy, MDM2 WT was released from MDM2-NBN complex and efficiently degraded, hence allowing extensive HR DNA repair leading to the acquisition of chemoresistance by cancer cells.

TDP1 suppresses mis-joining of radiomimetic DNA double-strand breaks and cooperates with Artemis to promote optimal nonhomologous end joining.

The Artemis nuclease and tyrosyl-DNA phosphodiesterase (TDP1) are each capable of resolving protruding 3′-phosphoglycolate (PG) termini of DNA double-strand breaks (DSBs). Consequently, both a knockout of Artemis and a knockout/knockdown of TDP1 rendered cells sensitive to the radiomimetic agent neocarzinostatin (NCS), which induces 3′-PG-terminated DSBs. Unexpectedly, however, a knockdown or knockout of TDP1 in Artemis-null cells did not confer any greater sensitivity than either deficiency alone, indicating a strict epistasis between TDP1 and Artemis. Moreover, a deficiency in Artemis, but not TDP1, resulted in a fraction of unrepaired DSBs, which were assessed as 53BP1 foci. Conversely, a deficiency in TDP1, but not Artemis, resulted in a dramatic increase in dicentric chromosomes following NCS treatment. An inhibitor of DNA-dependent protein kinase, a key regulator of the classical nonhomologous end joining (C-NHEJ) pathway sensitized cells to NCS, but eliminated the sensitizing effects of both TDP1 and Artemis deficiencies. These results suggest that TDP1 and Artemis perform different functions in the repair of terminally blocked DSBs by the C-NHEJ pathway, and that whereas an Artemis deficiency prevents end joining of some DSBs, a TDP1 deficiency tends to promote DSB mis-joining.

Persistent 3′-phosphate termini and increased cytotoxicity of radiomimetic DNA double-strand breaks in cells lacking polynucleotide kinase/phosphatase despite presence of an alternative 3′-phosphatase

Polynucleotide kinase/phosphatase (PNKP) has been implicated in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). To assess the consequences of PNKP deficiency for NHEJ of 3′-phosphate-ended DSBs, PNKP-deficient derivatives of HCT116 and of HeLa cells were generated using CRISPR/CAS9. For both cell lines, PNKP deficiency conferred sensitivity to ionizing radiation as well as to neocarzinostatin (NCS), which specifically induces DSBs bearing protruding 3′-phosphate termini. Moreover, NCS-induced DSBs, detected as 53BP1 foci, were more persistent in PNKP −/− HCT116 cells compared to their wild-type (WT) counterparts. Surprisingly, PNKP-deficient whole-cell and nuclear extracts were biochemically competent in removing both protruding and recessed 3′-phosphates from synthetic DSB substrates, albeit much less efficiently than WT extracts, suggesting an alternative 3′-phosphatase. Measurements by ligation-mediated PCR showed that PNKP-deficient HeLa cells contained significantly more 3′-phosphate-terminated and fewer 3′-hydroxyl-terminated DSBs than parental cells 5–15 min after NCS treatment, but this difference disappeared by 1 h. These results suggest that, despite presence of an alternative 3′-phosphatase, loss of PNKP significantly sensitizes cells to 3′-phosphate-terminated DSBs, due to a 3′-dephosphorylation defect.

DNA damage induces a SAMHD1-mediated block to the infection of macrophages by HIV-1

Monocyte-derived macrophages (MDMs) are an important target for HIV-1 despite SAMHD1, a myeloid restriction factor for which HIV-1 lacks a counteracting accessory protein. The antiviral activity of SAMHD1 is modulated by phosphorylation of T592 by cyclin-dependent kinases (CDK). We show that treatment of MDMs with neocarzinostatin, a compound that introduces double strand breaks (DBS) in genomic DNA, results in the decrease of phosphorylated SAMHD1, activating its antiviral activity and blocking HIV-1 infection. The effect was specific for DSB as DNA damage induced by UV light irradiation did not affect SAMHD1 phosphorylation and did not block infection. The block to infection was at reverse transcription and was counteracted by Vpx, demonstrating that it was caused by SAMHD1. Neocarzinostatin treatment also activated an innate immune response that induced interferon-stimulated genes but this was not involved in the block to HIV-1 infection, as it was not relieved by an interferon-blocking antibody. In response to Neocarzinostatin-induced DNA damage, the level of the CDK inhibitor p21cip1 increased which could account for the decrease of phosphorylated SAMHD1. The results show that the susceptibility of MDMs to HIV-1 infection can be affected by stimuli that alter the phosphorylation state of SAMHD1, one of which is the DNA damage response.

MP76-10 MSH5 DEFICIENCIES IN A SUBSET OF NOA MEN

You have accessJournal of UrologyInfertility: Basic Research, Physiology, Pathophysiology1 Apr 2015MP76-10 MSH5 DEFICIENCIES IN A SUBSET OF NOA MEN Koji Chiba, Alex Ridgeway, Larry Lipshultz, Masato Fujisawa, and Dolores Lamb Koji ChibaKoji Chiba More articles by this author , Alex RidgewayAlex Ridgeway More articles by this author , Larry LipshultzLarry Lipshultz More articles by this author , Masato FujisawaMasato Fujisawa More articles by this author , and Dolores LambDolores Lamb More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2795AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Spermatogenesis requires prolific cellular division and high fidelity DNA replication, as well as homologous recombination during meiosis. DNA repair systems are needed to correct cellular insults or to commit the cell to apoptosis if repair is not possible. MSH5, which is mainly expressed in testis, is a key component of the DNA repair pathways required for meiosis. MSH5 plays an important role in the repair of DNA double-strand breaks (DSB). When DSB occur, MSH5 interacts with c-abl. The activation of C-ABL then induces p73 accumulation. Because MSH5 and p73 are required in mouse models for spermatogenesis, we sought to define their expression in infertile men. METHODS Fibroblasts were cultured using testicular biopsy samples obtained from non-obstructive azoospermia (NOA) men (n=31). For fertile controls (n=14), men who underwent vasectomy reversal provided the fibroblasts. A global DNA methylation profile using the Infinium HumanMethylation450 BeadChip was obtained on these patients. MSH5 gene expression was quantified by qPCR and Western blot. Fibroblasts were then treated with Neocarzinostatin (NCS), a potent drug that induces DSB. Cell growth after NCS treatment was evaluated using WST assay, and comet assay was performed to evaluate DSB formation. MSH5, p73 gene expression was quantified by qPCR before and after treatment with NCS. RESULTS DNA methylation was significantly increased at a CpG island that spans the promoter region to exon 2 of MSH5. Specifically, five terminal CpGs in exon 2 of MSH5 were hypermethylated in a subset of NOA men tested (6 out of 31). This cohort also displayed decreased MSH5 gene expression and reduced MSH5 protein. The cell growth assay showed that NCS severely inhibited fibroblast growth after treatment in NOA- hypermethylated MSH5 group, whereas only mild cell growth inhibition was evident in fertile group. A comet assay showed a significant comet halo after NCS treatment in both groups, showing that DSB was induced by NCS treatment. Fertile control cells rapidly repaired the DSB whereas cells with hypermethylated MSH5 did not. NCS administration to the fertile group increased MSH5 and p73 gene expression. In contrast, expression of these genes remained low after NCS treatment of the NOA group. CONCLUSIONS NOA patients frequently show increased DNA methylation of MSH5 with down-regulation of its gene expression. In these men, MSH5 and p73 gene expression failed to respond to DSB formation caused by NCS. Down-regulation of the MSH5 and p73 pathways may be involved in spermatogenic failure in NOA patients and underlie other additional health concerns for the patient. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e987-e988 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Koji Chiba More articles by this author Alex Ridgeway More articles by this author Larry Lipshultz More articles by this author Masato Fujisawa More articles by this author Dolores Lamb More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Ceramide Regulates MRN Complex‐ATM‐Dependent Apoptotic Pathway in Human Lymphoblastoids

Ceramide is a key lipid second messenger for apoptotic stresses. However, it remains unknown how ceramide is involved in genotoxic apoptotic pathway such as DNA double-strand breaks (DSBs). Ataxia telangiectasia mutated (ATM) plays crucial roles in cellular responses including DNA repair, cell cycle control and apoptosis following DNA DSBs. We herein demonstrated the requirement of ceramide for ATM signaling in genotoxic drug neocarzinostatin (NCS)-induced apoptotic pathway in lymphoblastoids. NCS treatment produces ceramide by synergistic effect of neutral sphingomyelinase (SMase) activation and sphingomyelin synthase (SMS) inactivation. The ceramide production activates ATM and leads to caspase-3-dependent apoptosis. Nuclear study indicates that ceramide regulates ATM activity through MRN (Mre11/ Rad50/NBS1) complex that is a primary factor to detect DSBs (figure). Thus, we demonstrate that ceramide acts as an early trigger for the activation of MRN complex-ATM pathway in nuclear upon DNA damage stress, and finally exert caspase-3-dependent apoptosis.

The nuclear oncogene SET controls DNA repair by KAP1 and HP1 retention to chromatin.

Cells experience damage from exogenous and endogenous sources that endanger genome stability. Several cellular pathways have evolved to detect DNA damage and mediate its repair. Although many proteins have been implicated in these processes, only recent studies have revealed how they operate in the context of high-ordered chromatin structure. Here, we identify the nuclear oncogene SET (I2PP2A) as a modulator of DNA damage response (DDR) and repair in chromatin surrounding double-strand breaks (DSBs). We demonstrate that depletion of SET increases DDR and survival in the presence of radiomimetic drugs, while overexpression of SET impairs DDR and homologous recombination (HR)-mediated DNA repair. SET interacts with the Kruppel-associated box (KRAB)-associated co-repressor KAP1, and its overexpression results inthe sustained retention of KAP1 and Heterochromatin protein 1 (HP1) on chromatin. Our results are consistent with a model in which SET-mediated chromatin compaction triggers an inhibition of DNA end resection and HR.

Abstract 1774: Emerging role of Smad7 in DNA damage response pathway as a positive regulator.

Abstract The cellular response to DNA damage is essential in maintaining genomic stability. As DNA damage accumulates, the occurrence rate of cancer is significantly enhanced. Smad7 is known as an inhibitory Smad which negatively regulates TGF-β-induced signaling pathway. Smad7 is predominantly localized in the nucleus even without TGF-β stimulation. However, TGF-β-independent Smad7 function in the nucleus is still not clear. Here, we report Smad7 regulates DNA damage signaling in response to double-strand break induced by radiomimetic drug, neocarzinostatin (NCS). Smad7 overexpression accelerates DNA repair by enhancing phosphorylation of ATM and γH2AX, major DNA damage response signaling, after DNA damage. In Smad7−/− MEF cell, loss of Smad7 leads to failure of ATM phosphorylation and nuclear foci formation at the site of DNA damage upon NCS treatment. We show that Smad7 interacts with activated ATM/MRN complex and this interaction is significantly increased after DNA damage. Additionally, Smad7 co-localizes with pATM, MRN complex and γH2AX at the sites of DNA damage. Taken together, our data suggests that Smad7 plays a critical role in DNA damage response pathway through interaction with activated ATM. In this study, we reveal the de novo function of Smad7 in the nucleus. Citation Format: Sujin Park, Jin Muk Kang, Staci Jakyong Kim, Hyojung Kim, Seong-Jin Kim. Emerging role of Smad7 in DNA damage response pathway as a positive regulator. [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 1774. doi:10.1158/1538-7445.AM2013-1774

Inhibitor of growth 1 (ING1) acts at early steps of multiple DNA repair pathways

ING proteins are tumor suppressors involved in the regulation of gene transcription, cell cycle arrest, apoptosis, and senescence. Here, we show that ING1b expression is upregulated by several DNA-damaging agents, in a p53-independent manner. ING1b stimulates DNA repair of a variety of DNA lesions requiring activation of multiple DNA repair pathways. Moreover, Ing1(-/-) cells showed impaired genomic DNA repair after H2O2 and neocarzinostatin treatment and this defect was reverted by overexpression of ING1b. Two tumor-derived ING1 mutants failed to promote DNA repair highlighting the physiological importance of the integrity of the PHD domain for ING1b DNA repair activity and suggesting a role in the prevention of tumor progression. Ing(-/-) cells showed higher basal levels of γ-H2AX and, upon DNA damage, γ-H2AX increase was greater and with faster kinetics compared to wild-type cells. Chromatin relaxation by Trichostatin A led to an exacerbated damage signal in both types of cells, but this effect was dependent on Ing1 status, and more pronounced in wild-type cells. Our results suggest that ING1 acts at early stages of the DNA damage response activating a variety of repair mechanisms and that this function of ING1 is targeted in tumors.

DNA Double Strand Breaks but Not Interstrand Crosslinks Prevent Progress through Meiosis in Fully Grown Mouse Oocytes

There is some interest in how mammalian oocytes respond to different types of DNA damage because of the increasing expectation of fertility preservation in women undergoing chemotherapy. Double strand breaks (DSBs) induced by ionizing radiation and agents such as neocarzinostatin (NCS), and interstrand crosslinks (ICLs) induced by alkylating agents such as mitomycin C (MMC), are toxic DNA lesions that need to be repaired for cell survival. Here we examined the effects of NCS and MMC treatment on oocytes collected from antral follicles in mice, because potentially such oocytes are readily collected from ovaries and do not need to be in vitro grown to achieve meiotic competency. We found that oocytes were sensitive to NCS, such that this ionizing radiation mimetic blocked meiosis I and caused fragmented DNA. In contrast, MMC had no impact on the completion of either meiosis I or II, even at extremely high doses. However, oocytes treated with MMC did show γ-H2AX foci and following their in vitro maturation and parthenogenetic activation the development of the subsequent embryos was severely compromised. Addition of MMC to 1-cell embryos caused a similarly poor level of development, demonstrating oocytes have eventual sensitivity to this ICL-inducing agent but this does not occur during their meiotic division. In oocytes, the association of Fanconi Anemia protein, FANCD2, with sites of ICL lesions was not apparent until entry into the embryonic cell cycle. In conclusion, meiotic maturation of oocytes is sensitive to DSBs but not ICLs. The ability of oocytes to tolerate severe ICL damage and yet complete meiosis, means that this type of DNA lesion goes unrepaired in oocytes but impacts on subsequent embryo quality.

USP4 inhibits p53 through deubiquitinating and stabilizing ARF-BP1

Tumour suppressor p53 levels in the cell are tightly regulated by controlled degradation through ubiquitin ligases including Mdm2, COP1, Pirh2, and ARF-BP1. The ubiquitination process is reversible via deubiquitinating enzymes, such as ubiquitin-specific peptidases (USPs). In this study, we identified ubiquitin-specific peptidase 4 (USP4) as an important regulator of p53. USP4 interacts directly with and deubiquitinates ARF-BP1, leading to the stabilization of ARF-BP1 and subsequent reduction of p53 levels. Usp4 knockout mice are viable and developmentally normal, but showed enhanced apoptosis in thymus and spleen in response to ionizing radiation. Compared with wild-type mouse embryonic fibroblasts (MEFs), Usp4-/- MEFs exhibited retarded growth, premature cellular senescence, resistance to oncogenic transformation, and hyperactive DNA damage checkpoints, consistent with upregulated levels and activity of p53 in the absence of USP4. Finally, we showed that USP4 is overexpressed in several types of human cancer, suggesting that USP4 is a potential oncogene.

EDD Inhibits ATM-mediated Phosphorylation of p53

The EDD (E3 identified by differential display) gene, first identified as a progestin-induced gene in T-47D breast cancer cells, encodes an E3 ubiquitin ligase with a HECT domain. It was reported that EDD is involved in the G(2)/M progression through ubiquitination of phospho-katanin p60. Previous study has also shown that EDD can act as a transcription cofactor independently of its E3 ligase activity. In this study, we uncover a new role for EDD during cell cycle progression in an E3 ligase-independent manner. We demonstrate that EDD can physically interact with p53 and that this interaction blocks the phosphorylation of p53 by ataxia telangiectasia mutated (ATM). Silencing of EDD induces phosphorylation of p53 at Ser(15) and activates p53 target genes in fibroblasts and some transformed cells without activation of DNA damage response. The G(1)/S arrest induced by EDD depletion depends on p53. On the other hand, overexpression of EDD inhibits p53-Ser(15) phosphorylation and suppresses the induction of p53 target genes during DNA damage, and this effect does not require its E3 ligase activity. Thus, through binding to p53, EDD actively inhibits p53 phosphorylation by ATM and plays a role in ensuring smooth G(1)/S progression.

Mismatch-repair protein MSH6 is associated with Ku70 and regulates DNA double-strand break repair

MSH6, a key component of the MSH2–MSH6 complex, plays a fundamental role in the repair of mismatched DNA bases. Herein, we report that MSH6 is a novel Ku70-interacting protein identified by yeast two-hybrid screening. Ku70 and Ku86 are two key regulatory subunits of the DNA-dependent protein kinase, which plays an essential role in repair of DNA double-strand breaks (DSBs) through the non-homologous end-joining (NEHJ) pathway. We found that association of Ku70 with MSH6 is enhanced in response to treatment with the radiomimetic drug neocarzinostatin (NCS) or ionizing radiation (IR), a potent inducer of DSBs. Furthermore, MSH6 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci after NCS or IR treatment. MSH6 colocalizes with γ-H2AX at sites of DNA damage after NCS or IR treatment. Cells depleted of MSH6 accumulate high levels of persistent DSBs, as detected by formation of γ-H2AX foci and by the comet assay. Moreover, MSH6-deficient cells were also shown to exhibit impaired NHEJ, which could be rescued by MSH6 overexpression. MSH6-deficient cells were hypersensitive to NCS- or IR-induced cell death, as revealed by a clonogenic cell-survival assay. These results suggest a potential role for MSH6 in DSB repair through upregulation of NHEJ by association with Ku70.

Protein Phosphatase 5 Regulates the Function of 53BP1 after Neocarzinostatin-induced DNA Damage

53BP1 (p53-binding protein 1) is a conserved nuclear protein that is phosphorylated in response to DNA damage and rapidly recruited to the site of DNA double strand breaks, demonstrating its role in the early events to DNA damage and repair of damaged DNA. In this study, we used the yeast two-hybrid system to identify proteins that interact with 53BP1. Identification and characterization of 53BP1 protein interactions may help to further elucidate the function and regulation of 53BP1. We identified protein phosphatase 5 (PP5), a serine/threonine phosphatase that has been implicated in multiple cellular function, as a 53BP1-binding protein. This interaction further confirmed that 53BP1 interacts with PP5 in PP5-overexpressing U2OS cells, after radiomimetic agent neocarzinostatin (NCS) treatment. 53BP1 dephosphorylation at Ser-25 and Ser-1778 was accelerated in PP5-overexpressing U2OS cells following NCS treatment, and its dephosphorylation was correlated with reduced phospho-53BP1 foci formation. In contrast, the overexpression of PP5 had no effect on NCS-activated BRCA1-Ser-1524 phosphorylation. Additionally, PP5 down-regulation inhibited the dephosphorylation of 53BP1 on Ser-1778 and the disappearance of phospho-53BP1 foci following NCS treatment. Moreover, non-homologous end-joining activity was reduced in PP5-overexpressing U2OS cells. These findings indicate that PP5 plays an important role in the regulation of 53BP1 phosphorylation and activity in vivo.

ATM Signaling Facilitates Repair of DNA Double-Strand Breaks Associated with Heterochromatin

Ataxia Telangiectasia Mutated (ATM) signaling is essential for the repair of a subset of DNA double-strand breaks (DSBs); however, its precise role is unclear. Here, we show that < or =25% of DSBs require ATM signaling for repair, and this percentage correlates with increased chromatin but not damage complexity. Importantly, we demonstrate that heterochromatic DSBs are generally repaired more slowly than euchromatic DSBs, and ATM signaling is specifically required for DSB repair within heterochromatin. Significantly, knockdown of the transcriptional repressor KAP-1, an ATM substrate, or the heterochromatin-building factors HP1 or HDAC1/2 alleviates the requirement for ATM in DSB repair. We propose that ATM signaling temporarily perturbs heterochromatin via KAP-1, which is critical for DSB repair/processing within otherwise compacted/inflexible chromatin. In support of this, ATM signaling alters KAP-1 affinity for chromatin enriched for heterochromatic factors. These data suggest that the importance of ATM signaling for DSB repair increases as the heterochromatic component of a genome expands.

Poly(ADP-ribose) polymerase-1 inhibits ATM kinase activity in DNA damage response

DNA double-strand breaks (DSB) mobilize DNA-repair machinery and cell cycle checkpoint by activating the ataxia-telangiectasia (A-T) mutated (ATM). Here we show that ATM kinase activity is inhibited by poly(ADP-ribose) polymerase-1 (PARP-1) in vitro. It was shown by biochemical fractionation procedure that PARP-1 as well as ATM increases at chromatin level after induction of DSB with neocarzinostatin (NCS). Phosphorylation of histone H2AX on serine 139 and p53 on serine 15 in Parp-1 knockout (Parp-1−/−) mouse embryonic fibroblasts (MEF) was significantly induced by NCS treatment compared with MEF derived from wild-type (Parp-1+/+) mouse. NCS-induced phosphorylation of histone H2AX on serine 139 in Parp-1−/− embryonic stem cell (ES) clones was also higher than that in Parp-1+/+ ES clone. Furthermore, in vitro, PARP-1 inhibited phosphorylation of p53 on serine 15 and 32P-incorporation into p53 by ATM in a DNA-dependent manner. These results suggest that PARP-1 negatively regulates ATM kinase activity in response to DSB.

Poly(ADP-ribose) polymerase-1 inhibits ATM kinase activity in DNA damage response

DNA double-strand breaks (DSB) mobilize DNA-repair machinery and cell cycle checkpoint by activating the ataxia-telangiectasia (A-T) mutated (ATM). Here we show that ATM kinase activity is inhibited by poly(ADP-ribose) polymerase-1 (PARP-1) in vitro. It was shown by biochemical fractionation procedure that PARP-1 as well as ATM increases at chromatin level after induction of DSB with neocarzinostatin (NCS). Phosphorylation of histone H2AX on serine 139 and p53 on serine 15 in Parp-1 knockout (Parp-1−/−) mouse embryonic fibroblasts (MEF) was significantly induced by NCS treatment compared with MEF derived from wild-type (Parp-1+/+) mouse. NCS-induced phosphorylation of histone H2AX on serine 139 in Parp-1−/− embryonic stem cell (ES) clones was also higher than that in Parp-1+/+ ES clone. Furthermore, in vitro, PARP-1 inhibited phosphorylation of p53 on serine 15 and 32P-incorporation into p53 by ATM in a DNA-dependent manner. These results suggest that PARP-1 negatively regulates ATM kinase activity in response to DSB.