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Artemis is a multifunctional phospho-protein with roles in V(D)J recombination, repair of double-strand breaks by nonhomologous end-joining and regulation of cell-cycle checkpoints after DNA damage. Here, we describe a new function of Artemis as a negative regulator of p53 in response to oxidative stress in both primary cells and cancer cell lines. We show that depletion of Artemis under typical culture conditions (21% oxygen) leads to a spontaneous phosphorylation and stabilization of p53, and resulting cellular G1 arrest and apoptosis. These effects are suppressed by co-depletion of DNA-PKcs, but not ATM, indicating that Artemis is an inhibitor of DNA–PKcs-mediated stabilization of p53. Culturing of cellsat 3% oxygen or treatment with an antioxidant abrogated p53 stabilization, indicating that oxidative stress is the responsible cellular stimulus. Treatment with ionizing radiation or hydrogen peroxide did not cause activation of this signaling pathway, whereas inhibitors of mitochondrial electron transport were effective in reducing its activation. In addition, we show that p53-inducible genes involved in reducing reactive oxygen species are upregulated by Artemis depletion. These findings indicate that Artemis and DNA-PKcs participate in a new, signaling pathway to modulate p53 function in response to oxidative stress produced by mitochondrial respiration.Oncogene (2009) 28, 2196–2204; doi:10.1038/onc.2009.100; published online 27 April 2009 [ABSTRACT FROM AUTHOR]

The human tumor suppressor gene ataxia telangiectasia mutated (ATM) encodes a 3056 amino-acid protein kinase that regulates cell cycle checkpoints. ATM is defective in the neurodegenerative and cancer predisposition syndrome ataxia-telangiectasia. ATM protein kinase is activated by DNA damage and responds by phosphorylating downstream effectors involved in cell cycle arrest and DNA repair, such as p53, MDM2, CHEK2, BRCA1 and H2AX. ATM is probably a component of, or in close proximity to, the double-stranded DNA break-sensing machinery. We have observed purified human ATM protein, ATM-DNA and ATM-DNA-avidin bound complexes by single-particle electron microscopy and obtained three-dimensional reconstructions which show that ATM is composed of two main domains comprising a head and an arm. DNA binding to ATM induces a large conformational movement of the arm-like domain. Taken together, these three structures suggest that ATM is capable of interacting with DNA, using its arm to clamp around the double helix.Oncogene (2003) 22, 3867-3874. doi:10.1038/sj.onc.1206649 [ABSTRACT FROM AUTHOR]

Certain growth regulatory kinases contain a common domain related to the phospho-inositol 3 (PI-3) kinase catalytic site. These include the ATM gene product, DNA-PKcs, and the target of rapamycin (TOR in yeast; and FRAP in mammalian cells). Rapamycin inhibits growth factor signalling and induces G1 arrest in many cell types. Some growth regulatory PI-3 kinases appear functionally linked to p53 and we have explored potential links between cellular effects induced by rapamycin and p53. In p53 null cells rapamycin inhibited cell cycling but did not induce G1 arrest. In cells which showed selective G1 arrest in response to rapamycin, rapamycin had no effect on basal levels of p53 protein. Similarly p21(WAF1) protein was not induced by rapamycin. The kinetics of the cellular p53/p21(WAF1) response to ionising radiation was unaffected by rapamycin; and the ability of growth factor to protect against p53-mediated apoptosis in response to DNA damage was also unaffected by rapamycin. The ATM gene is mutated in the cancer susceptibility syndrome ataxia telangiectasia (AT) but such mutant cells showed a similar sensitivity to rapamycin compared to their normal counterparts. RKO cell lines of common genetic background, but with different levels of functional p53 protein, also responded similarly to rapamycin. Thus, although rapamycin and p53 are each able to induce G1 arrest, they appear to act through independent growth regulatory pathways.

Several newly identified tumor suppressor genes including ATM, NBS1, BRCA1 and BRCA2 are involved in DNA double-strand break repair (DSBR) and DNA damage-induced checkpoint activation. Many of the gene products involved in checkpoint control and DSBR have been studied in great detail in yeast. In addition to evolutionarily conserved proteins such as Chk1 and Chk2, studies in mammalian cells have identified novel proteins such as p53 in executing checkpoint control. DSBR proteins including Mre11, Rad50, Rad51, Rad54, and Ku are present in yeast and in mammals. Many of the tumor suppressor gene products interact with these repair proteins as well as checkpoint regulators, thus providing a biochemical explanation for the pleiotropic phenotypes of mutant cells. This review focuses on the proteins mediating G1/S, S, and G2/M checkpoint control in mammalian cells. In addition, mammalian DSBR proteins and their activities are discussed. An intricate network among DNA damage signal transducers, cell cycle regulators and the DSBR pathways is illustrated. Mouse knockout models for genes involved in these processes have provided valuable insights into their function, establishing genomic instability as a major contributing factor in tumorigenesis. [ABSTRACT FROM AUTHOR]

We investigated the intracellular mechanisms of retinoic acid (9-cis-RA, 13-cis-RA or all-trans-RA) and a cyclic AMP analog 8-Cl-cAMP on growth-inhibition and apoptosis in human ovarian cancer NIH: OVCAR-3 and OVCAR-8 cells. The cyclic AMP analog, 8-Cl-cAMP, acted synergistically with RA in inducing and activating retinoic acid receptor β (RARβ) which correlated with the growth inhibition, cell cycle arrest, and apoptosis in both cell types. In addition, combined treatment of cells with RA plus 8-Cl-cAMP resulted in the release of cytochrome c, loss in mitochondrial membrane potential and activation of caspase-3 followed by cleavage of anti-poly(ADP-ribose)polymerase and DNA-dependent protein kinase (catalytic subunit). Interestingly, inhibition of caspase-3 activation blocked RA plus 8-Cl-cAMP induced apoptosis. Furthermore, mutations in a CRE-related motif within the RARβ promoter resulted in loss of both transcriptional activation of RARβ and synergy between RA and 8-Cl-cAMP. Thus, RARβ can mediate RA and/or cyclic AMP action in ovarian cancer cells by promoting apoptosis. Loss of RARβ expression, therefore, may contribute to the tumorigenicity of human ovarian cancer cells. These findings suggest that RA and 8-Cl-cAMP act in a synergistic fashion in inducing apoptosis via caspase-3 activation, and may have potential for combination biotherapy for the treatment of malignant disease such as ovarian cancer. [ABSTRACT FROM AUTHOR]

The recently cloned gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is involved in DNA damage response at different cell cycle checkpoints and also appears to have a wider role in signal transduction. Antibodies prepared against peptides from the predicted protein sequence detected a ∼ 350 kDa protein corresponding to the open reading frame, which was absent in 13/23 A-T homozygotes. Subcellular fractionation, immunoelectronmicroscopy and immunofluorescence showed that the ATM protein is present in the nucleus and cytoplasmic vesicles. This distribution did not change after irradiation. We also provide evidence that ATM protein binds to p53 and this association is defective in A-T cells compatible with the defective p53 response in these cells. These results provide further support for a role for the ATM protein as a sensor of DNA damage and in a more general role in cell signalling, compatible with the broader phenotype of the syndrome. [ABSTRACT FROM AUTHOR]

In the presence of sustained DNA damage occurring in S-phase or G2, normal cells arrest before mitosis and eventually become senescent. The checkpoint kinases Chk1/Chk2 and the CDK inhibitor p21 are known to have important complementary roles in this process, in G2 arrest and cell cycle exit, respectively. However, additional checkpoint roles have been reported for these regulators and it is not clear to what extent their functions are redundant. Here we compared the respective roles of Chk1, Chk2 and p21 in DNA damage-induced G2 arrest in normal human fibroblasts, normal epithelial cells and frequently used p53 proficient cancer cells. We show that in normal cells, Chk1, but not Chk2, is involved in G2 arrest whereas neither are essential. In contrast, p21 is required. However, Chk1, but not Chk2, becomes necessary for arrest in U2OS osteosarcoma cells. We find that their ATM/p53/p21 response in G2 phase is defective, like in other cancer cells with wild-type p53, and conclude that cross-talk between the Chk1 and p21 pathways allows them to switch dependency for G2 arrest onto Chk1. Using the specific ATM inhibitor KU-55933 we confirm the essential role of ATM in the induction of p21 for G2 arrest of normal cells. Efficient p21 induction is required for nuclear sequestration of inactive cyclin B1-Cdk1 complexes preceding irreversible cell cycle exit in G2. Our results demonstrate that p21 is able to fulfill the Chk1 functions in G2 arrest under continuous genotoxic stress, which has important implications for cancer chemotherapy. [ABSTRACT FROM AUTHOR]

Cells experiencing DNA damage undergo a complex response entailing cell-cycle arrest, DNA repair and apoptosis, the relative importance of the three being modulated by the extent of the lesion. The observation that Abl interacts in the nucleus with several proteins involved in different aspects of DNA repair has led to the hypothesis that this kinase is part of the damage-sensing mechanism. However, the mechanistic details underlying the role of Abl in DNA repair remain unclear. Here, I will review the evidence supporting our current understanding of Abl activation following DNA insults, while focusing on the relevance of these mechanisms in protecting DNA-injured germ cells. Early studies have shown that Abl transcripts are highly expressed in the germ line. Abl-deficient mice exhibit multiple abnormalities, increased perinatal mortality and reduced fertility. Recent findings have implicated Abl in a cisplatin-induced signaling pathway eliciting death of immature oocytes. A p53-related protein, TAp63, is an important immediate downstream effector of this pathway. Of note, pharmacological inhibition of Abl protects the ovarian reserve from the toxic effects of cisplatin. This suggests that the extent of Abl catalytic outputs may shift the balance between survival (likely through DNA repair) and activation of a death response. Taken together, these observations are consistent with the evolutionary conserved relationship between DNA damage and activation of the p53 family of transcription factors, while shedding light on the key role of Abl in dictating the fate of germ cells upon genotoxic insults. [ABSTRACT FROM AUTHOR]

V(D)J recombination is essential for the maturation of lymphocytes. Because of the involvement of cutting and joining DNA double strands, this recombination activity is strictly contained within the noncycling phases of the cell cycle. Such containment is crucial for the maintenance of genomic integrity. The ataxia telangiectasia mutated (ATM) gene is known to have a central role in sensing general DNA damage and mediating cell-cycle checkpoint. In this study, we investigated the role of ATM and its downstream targets in the cell-cycle control of V(D)J recombination in vivo. Our results revealed the persistence of double-strand breaks (DSBs) throughout the cell cycle in ATM−/− and p53−/− thymocytes, but the cell-cycle regulation of a V(D)J recombinase, Rag-2, was normal. The histone variant H2AX, which is phosphorylated during normal V(D)J recombination, was dispensable for containing DSBs. H2AX was still phosphorylated at V(D)J loci in the absence of ATM. Therefore, V(D)J recombination, a physiological DNA rearrangement process, activates the ATM/p53 pathway to contain DNA breaks within the noncycling cells and surprisingly this pathway is not important for containing Rag-2 activity. This study shows the dynamic multiple functions of ATM in maintaining genomic stability and preventing tumorigenesis in developing lymphocytes. [ABSTRACT FROM AUTHOR]

Ku70 forms a heterodimer with Ku80, called Ku that is well known for repairing DNA double-strand breaks through non-homologous end joining. As a result, deletion of either causes a very similar phenotype in mice that includes hypersensitivity to clastogens and early aging. In addition, deletion of Ku80 along with the cell cycle checkpoint protein, p53, dramatically increases the incidence of pro-B-cell lymphoma. Even though Ku70- p53-mutant mice have not been analysed, a logical assumption is they would exhibit the same cancer phenotype. Here, we test this assumption by comparing p53-mutant littermates deleted for either Ku70 or Ku80 or both. We find this assumption to be incorrect as p53-mutants live significantly longer when deleted for Ku70 rather than Ku80 or Ku70+Ku80. We also find the former cohort displays much lower levels of pro-B-cell lymphoma than the latter two cohorts. As pro-B-cell lymphoma is caused by a translocation between chromosomes 12 and 15, we tested fibroblasts for DNA repair capacity, and found that p53-mutant fibroblasts are more sensitive to streptonigrin and paraquat when deleted for Ku80 as compared with Ku70. Thus, Ku80 may function outside the Ku heterodimer to influence DNA damage repair presenting the possibility that Ku80 influenced the open coding ends in a manner that suppressed a cancer-causing translocation.Oncogene (2009) 28, 1875–1878; doi:10.1038/onc.2009.57; published online 30 March 2009 [ABSTRACT FROM AUTHOR]

P53 wild-type and p53-null or mutant cells undergo a G2-phase cell-cycle arrest in response to ionizing radiation (IR). In this study we examined the effect of heat-shock protein 90 (HSP90) inhibitor, geldanamycin (GA), on IR-induced G2 arrest in human colon adenocarcinoma cells with different p53 status. We show that GA treatment abrogates IR-induced G2-phase arrest in cells null or mutant for p53. Specifically, GA treatment pushed irradiated p53 signaling-defective cells into a premature mitosis characterized by aberrant mitotic figures, increased γH2AX expression and formation of micronucleated cells. Cells expressing wild-type p53 were resistant to GA-induced G2 checkpoint abrogation. Notably, GA treatment decreased levels of G2 regulatory proteins Wee1 and Chk1, and inhibitory phosphorylation of Cdc2, independent of p53 status. Further investigation identified p21 as the potential downstream effector of p53 that mediates resistance to G2 checkpoint abrogation. Clonogenic survival studies demonstrated higher sensitivity to GA alone or combination IR plus GA treatment in p53 and p21-null cells. Collectively, these data demonstrate potential mechanisms through which HSP90 inhibition can enhance the effects of ionizing radiation in p53-compromised cancer cells. Combination IR plus HSP90 inhibitor therapies may be particularly useful in treating cancers that lack wild-type p53.Oncogene (2008) 27, 5567–5577; doi:10.1038/onc.2008.172; published online 26 May 2008 [ABSTRACT FROM AUTHOR]

We used intrachromosomal substrates to directly monitor the effect of the cell cycle on the efficiency and the accuracy of nonhomologous end joining (NHEJ) in mammalian cells. We show that both KU and KU-independent (KU-alt) pathways are efficient when maintaining cells in G1/S, in G2/M or during dynamic progression through S phase. In addition, the accuracy of NHEJ is barely altered when the cells are blocked in G1/S or in G2/M. However, progression through S phase increases the frequency of deletions, which is a hallmark of the KU-alt pathway. Moreover, we show that the intermediates that are generated by the KU-dependent end joining of non-fully complementary ends, and which contain mismatches, nicks or gap intermediates, are less accurately processed when the cells progress through S phase. In conclusion, both KU and KU-alt processes are active throughout the cell cycle, but the repair is more error prone during S phase, both by increasing the mutagenic KU-alt pathway and decreasing the accuracy of the repair of the intermediates generated by the KU-dependent pathway.Oncogene (2008) 27, 1726–1736; doi:10.1038/sj.onc.1210807; published online 24 September 2007 [ABSTRACT FROM AUTHOR]

DNA viruses promote cell cycle progression, stimulate unscheduled DNA synthesis, and present the cell with an extraordinary amount of exogenous DNA. These insults elicit vigorous responses mediated by cellular factors that govern cellular homeostasis. To ensure productive infection, adenovirus has developed means to inactivate these intracellular antiviral responses. Among the challenges to the host cell is the viral DNA genome, which is viewed as DNA damage and elicits a cellular response to inhibit replication. Adenovirus therefore encodes proteins that dismantle the cellular DNA damage machinery. Studying virus–host interactions has yielded insights into the molecular functioning of fundamental cellular mechanisms. In addition, it has suggested ways that viral cytotoxicity can be exploited to offer a selective means of restricted growth in tumor cells as a therapy against cancer. In this review, we discuss aspects of the intracellular response that are unique to adenovirus infection and how adenoviral proteins produced from the early region E4 act to neutralize antiviral defenses, with a particular focus on DNA damage signaling.Oncogene (2005) 24, 7686–7696. doi:10.1038/sj.onc.1209063 [ABSTRACT FROM AUTHOR]

The activation of caspases is a critical event for the execution phase of programmed cell death. Caspases are highly specific in their ability to activate or inhibit many crucial proteins in the cell via cleavage. In this study, we report the identification of several caspase-3-like cleavage sites in the cell-cycle checkpoint protein Rad9. We demonstrate that human Rad9 can be specifically cleaved in cells induced to enter apoptosis by both DNA damage and staurosporine treatment. Indeed, we show that human Rad9 can be effectively cleaved both in vitro and in vivo, which can be inhibited by either a pan-caspase inhibitor or a caspase-3-specific inhibitor. Additionally, no cleavage of Rad9 can be seen in the caspase-3-deficient cell line MCF-7. Site-directed mutagenesis of three of the most conserved cleavage sites dramatically abrogates cleavage of Rad9 by caspase-3 in vitro, and in intact cells after DNA damage. Expression of the cleavage-resistant mutant Rad9 DDD/AAA appears to protect the cell from DNA damage-induced apoptosis. Immunofluorescence studies of Rad9 localization before and after induction of apoptosis show a translocation of Rad9 from the nucleus to the cytosol, concomitant to the appearance of apoptotic morphology. Furthermore, analysis of a truncated Rad9 mutant that corresponds to a putative N-terminal cleavage fragment shows that the N-terminal portion of Rad9 localizes in the cytosol, binds to Bcl-XL, and induces apoptosis. These results support a dual role for cleavage of Rad9: (1) the liberation and translocation of the BH3 domain-containing N-terminus of Rad9 to the cytosol, as a means of promoting apoptosis via antagonism of Bcl-XL, and (2) the disruption of the Rad9-Rad1-Hus1 DNA damage checkpoint complex.Oncogene (2003) 22, 6340-6346. doi:10.1038/sj.onc.1206729 [ABSTRACT FROM AUTHOR]

In the last few years, the ubiquitin(Ub)/proteasome system has become increasingly recognized as a controller of numerous physiological processes, including signal transduction, DNA repair, chromosome maintenance, transcriptional activation, cell cycle progression, cell survival, and certain immune cell functions. This is in addition to its more established roles in the removal of misfolded, damaged, and effete proteins. This review examines the role of the Ub/proteasome system in processes underlying the classical effects of irradiation on cells, such as radiation-induced gene expression, DNA repair and chromosome instability, oxidative damage, cell cycle arrest, and cell death. Furthermore, recent evidence suggests that the proteasome is a redox-sensitive target for ionizing radiation and other oxidative stress signals. In other words, the Ub/proteasome system may not simply be a passive player in radiation-induced responses, but may modulate them. The extent of the modulation will be influenced by the functional and structural diversity that is expressed by the system. Cell types vary in the Ub/proteasome structures they possess and the level at which they function, and this changes as they go from the normal to the cancerous condition. Cancer-related functional changes within the Ub/proteasome system may therefore present unique targets for cancer therapy, especially when targeting agents are used in combination with radio- or chemotherapy. The peptide boronic acid compound PS-341, which was designed to inhibit proteasome chymotryptic activity, is in clinical trials for the treatment of solid and hematogenous tumors. It has shown some efficacy on its own and in combination with chemotherapy. Preclinical studies have shown that PS-341 will also potentiate the cytotoxic effects of radiation therapy. In addition, other drugs in common clinical use have been shown to affect proteasome function, and their activities may be valuably reconsidered from this perspective.Oncogene (2003) 22, 5755-5773. doi:10.1038/sj.onc.1206676 [ABSTRACT FROM AUTHOR]

Ku antigen is necessary for DNA double-strand break (DSB) repair through its ability to bind DNA ends with high affinity and to recruit the catalytic subunit of DNA-PK to the DSBs. Ku-deficient cells are hypersensitive to agents causing DSBs in DNA but also to the DNA topoisomerase II (topo II) inhibitor ICRF-193, which does not induce DSBs. This suggests a new role of Ku antigen, that is independent of DSB repair by DNA-PK. Here we characterize the basis for the hypersensitivity of Ku-deficient cells to ICRF-193. Chromosome condensation and segregation, which are dependent on topo II, but also the catalytic activity of topo II in late S-G2 were inhibited to a comparable extent when ICRF-193 was applied to Ku-deficient cells or wild-type cells. However, mutant cells arrested in G2 by ICRF-193 treatment were unable to progress into M phase upon drug removal, although drug-trapped topo II complexes were removed from DNA and the two isoforms of topo II recovered their catalytic activity as in wild-type cells. The reversibility of G2 arrest was recovered by complementation of mutant cells with a human Ku86 cDNA. Notably, chromosome condensation was abnormal in Ku-deficient cells after suppression of the G2 arrest by caffeine, even in the absence of ICRF-193. These results reflect the involvement of Ku-antigen in the cellular response to topo II inhibition, more particularly in relieving G2 arrest caused by topo II inhibition in late S/G2 and the subsequent recovery of chromosome condensation. Oncogene (2001) 20, 1990–1999. [ABSTRACT FROM AUTHOR]

Ku antigen is a complex of Ku70 and Ku80 subunits and plays an important role in not only DNA double-strand breaks (DSB) repair and V(D)J recombination, but also in growth regulation. Ku is generally believed to always form and function as heterodimers on the basis of in vitro observations. Here we demonstrate that the localization of Ku80 does not completely coincide with that of Ku70. Ku70 and Ku80 were colocalized in the nucleus in the interphase but not in the late telophase/early G1 phase of the cell cycle. Since the in vivo function of Ku might be partially regulated by the control of its transport, we attempted to investigate the molecular mechanisms underlying the nuclear translocation of Ku. The nuclear translocation of Ku80 started during the late telophase/early G1 phase after the nuclear envelope was formed and this was preceded by the nuclear translocation of Ku70. Furthermore, we found that the Ku80 protein was transported to the nucleus without heterodimerization with Ku70. To understand in detail the mechanism of transport of Ku80, we attempted to identify the nuclear localization signal (NLS) of Ku80 and defined to a region spanning nine amino acid residues (positions 561 – 569). The Ku80 NLS was demonstrated to be mediated to the nuclear rim by two components of PTAC58 and PTAC97. All these findings support the idea that Ku80 can translocate to the nucleus using its own NLS independent of the translocation of Ku70. [ABSTRACT FROM AUTHOR]

There is now evidence to suggest that BRCA1 and BRCA2 are involved in the response of cells to DNA damage and cell cycle checkpoint control. This report examines the death pathways of human cells with various BRCA1 and BRCA2 genotypes after exposure to gamma-rays. A lack of functional BRCA1 and BRCA2 led to defective repair of DNA double-strand breaks in irradiated cells. This impairment resulted in a relaxation of cell cycle checkpoints, production of micronuclei, and a loss of proliferative capacity. Heterozygous BRCA1 and BRCA2 mutations also led to enhanced radiosensitivity, with an impaired proliferative capacity after irradiation. The existence of a phenotype related to radiosensitivity in BRCA1+/- and BRCA2+/- cells raises the question of the response of heterozygous women to radiation. [ABSTRACT FROM AUTHOR]

ATM is a member of the large phosphatidylinositol-3 kinase family and plays an important role in cellular response to DNA damage. To further define the physiological roles of ATM at the cellular level, we created an isogenic set of stable cell lines differing only in their ATM status from the chicken B cell line DT40 by targeted integration. These stable DT40 cell lines, as most of transformed chicken cell lines, do not express p53. However, ATM-/- DT40 cells displayed retarded cellular proliferation, defective G2/M checkpoint control and radio-resistant DNA synthesis. Furthermore, ATM-/- DT40 cells were sensitive to ionizing radiation and showed highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In addition, a slight but significant reduction in targeted integration frequency was observed in ATM-/- DT40 cells. These results suggest that ATM has multiple p53-independent functions in cell cycle checkpoint control and in maintenance of chromosomal DNA. These ATM deficient DT40 clones therefore provide a useful model system for analysing p53-independent ATM functions. [ABSTRACT FROM AUTHOR]