Induces DNA Damage Signaling Research Articles

Poly (ADP-ribose) polymerase inhibitor sensitized DNA damage caused by an alkylating pyrrole-imidazole polyamide targeting MYCN in neuroblastoma cells

MYCN amplification (MYCN-amp) is a significant prognostic factor and early genetic marker of high-risk neuroblastoma (NB). MYCN induces the DNA damage response (DDR) and modulates the insensitivity of NB cells to Poly (ADP-ribose) polymerase (PARP) inhibitors. We previously reported that CCC-002, a DNA-alkylating agent conjugated with pyrrole-imidazole polyamide targeting MYCN, inhibits NB cell proliferation and induces DNA damage signaling. In this study, we investigated the synergistic effects of CCC-002 and PARP inhibitors on MYCN-amp NB cells. Combination treatment with PARP inhibitors significantly enhanced the sensitivity of MYCN-amp NB cells to CCC-002. DNA damage signals, such as phosphorylation of H2AX and RPA32 elicited after CCC-002 treatment, were further enhanced by PARP inhibitors, as detected through western blotting and immunofluorescence analyses. The potent cytotoxicity of this combination treatment was confirmed by the significant increase in the subG0-G1 phase. Although MYCN knockdown showed no synergistic effect with PARP inhibitors, fluorescence in situ hybridization and quantitative PCR analyses indicated that PARP inhibitors enhanced the effect of CCC-002 to reduce MYCN copy number and suppress its expression. Overall, our study provides novel insights into a therapeutic approach that combines CCC-002 and PARP inhibition to effectively induce DNA damage and apoptosis in MYCN-amp NB cells.

Leishmania major telomerase RNA knockout: From altered cell proliferation to decreased parasite infectivity

This study focuses on the biological impacts of deleting the telomerase RNA from Leishmania major (LeishTER), a parasite responsible for causing leishmaniases, for which no effective treatment or prevention is available. TER is a critical player in the telomerase ribonucleoprotein complex, containing the template sequence copied by the reverse transcriptase component during telomere elongation. The success of knocking out both LeishTER alleles was confirmed, and no off-targets were detected. LmTER−/− cells share similar characteristics with other TER-depleted eukaryotes, such as altered growth patterns and partial G0/G1 cell cycle arrest in early passages, telomere shortening, and elevated TERRA expression. They also exhibit increased γH2A phosphorylation, suggesting that the loss of LeishTER induces DNA damage signaling. Moreover, pro-survival autophagic signals and mitochondrion alterations were shown without any detectable plasma membrane modifications. LmTER−/− retained the ability to transform into metacyclics, but their infectivity capacity was compromised. Furthermore, the overexpression of LeishTER was also deleterious, inducing a dominant negative effect that led to telomere shortening and growth impairments. These findings highlight TER's vital role in parasite homeostasis, opening discussions about its potential as a drug target candidate against Leishmania.

O-GlcNAc transferase couples MRE11 to transcriptionally active chromatin to suppress DNA damage

BackgroundTranscription, metabolism and DNA damage response are tightly regulated to preserve the genomic integrity, and O-GlcNAc transferase (OGT) is positioned to connect the three. Prostate cancer is the most common cancer in men, and androgen-ablation therapy halts disease progression. However, a significant number of prostate cancer patients develop resistance against anti-androgens, and this incurable disease is termed castration-resistant prostate cancer (CRPC). We have shown that combined inhibition of OGT and the transcription elongation kinase CDK9 induce CRPC-selective anti-proliferative effects. Here, we explain the functional basis for these combinatorial effects.MethodsWe used comprehensive mass spectrometry profiling of short-term CDK9 inhibitor effects on O-GlcNAcylated proteins in an isogenic cell line system that models transition from PC to CRPC. In addition, we used both ChIP-seq and RNA-seq profiling, and pulldown experiments in multiple CRPC models. Finally, we validated our findings in prostate cancer patient samples.ResultsInhibition of CDK9 results in an OGT-dependent remodeling of the proteome in prostate cancer cells. More specifically, the activity of the DNA damage repair protein MRE11 is regulated in response to CDK9 inhibition in an OGT-dependent manner. MRE11 is enriched at the O-GlcNAc-marked loci. CDK9 inhibition does not decrease the expression of mRNAs whose genes are bound by both O-GlcNAc and MRE11. Combined inhibition of CDK9 and OGT or MRE11 further decreases RNA polymerase II activity, induces DNA damage signaling, and blocks the survival of prostate cancer cells. These effects are seen in CRPC cells but not in normal prostate cells. Mechanistically, OGT activity is required for MRE11 chromatin-loading in cells treated with CDK9 inhibitor. Finally, we show that MRE11 and O-GlcNAc are enriched at the prostate cancer-specific small nucleotide polymorphic sites, and the loss of MRE11 activity results in a hyper-mutator phenotype in patient tumors.ConclusionsBoth OGT and MRE11 are essential for the repair of CDK9 inhibitor-induced DNA damage. Our study raises the possibility of targeting CDK9 to elicit DNA damage in CRPC setting as an adjuvant to other treatments.Graphical

Venetoclax and dexamethasone synergize with inotuzumab ozogamicin–induced DNA damage signaling in B-lineage ALL

AbstractAdult patients with relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) have a dismal prognosis. To improve pharmacotherapy, we analyzed induction of apoptosis by venetoclax and inotuzumab ozogamicin in terms of cytotoxicity and mode of action. Flow cytometry–based analyses of mitochondrial outer membrane permeabilization (MOMP) and ataxia telangiectasia mutated activation demonstrate rapid induction of MOMP by venetoclax and DNA damage signaling by inotuzumab ozogamicin, respectively. In primary ALL samples and patient-derived xenograft (PDX) models, venetoclax and inotuzumab ozogamicin cooperated and synergized in combination with dexamethasone in vitro in all tested samples of ALL. In murine PDX models, inotuzumab ozogamicin, but not venetoclax, induced complete remission in a dose-dependent manner but constantly failed to achieve relapse-free survival. In contrast, combination therapy with venetoclax, dexamethasone, and inotuzumab ozogamicin induced long-term leukemia-free survival and treatment-free survival in all 3 ALL-PDX models tested. These data demonstrate synergistic and highly efficient pharmacotherapy in preclinical models that qualify for evaluation in clinical trials.

Inter and intra-tumor somatostatin receptor 2 heterogeneity influences peptide receptor radionuclide therapy response.

Patients with neuroendocrine tumors (NETs) can be treated with peptide receptor radionuclide therapy (PRRT). Here, the somatostatin analogue octreotate radiolabeled with lutetium-177 is targeted to NET cells by binding to the somatostatin receptor subtype 2 (SST2). During radioactive decay, DNA damage is induced, leading to NET cell death. Although the therapy proves to be effective, mortality rates remain high. To appropriately select more optimal treatment strategies, it is essential to first better understand the radiobiological responses of tumor cells to PRRT.Methods: We analyzed PRRT induced radiobiological responses in SST2 expressing cells and xenografted mice using SPECT/MRI scanning and histological and molecular analyses. We measured [177Lu]Lu-DOTA-TATE uptake and performed analyses to visualize induction of DNA damage, cell death and other cellular characteristics.Results: The highest accumulation of radioactivity was measured in the tumor and kidneys. PRRT induced DNA damage signaling and repair in a time-dependent manner. We observed intra-tumor heterogeneity of DNA damage and apoptosis, which was not attributed to proliferation or bioavailability. We found a strong correlation between high DNA damage levels and high SST2 expression. PRRT elicited a different therapeutic response between models with different SST2 expression levels. Heterogeneous SST2 expression levels were also confirmed in patient NETs.Conclusion: Heterogeneous SST2 expression levels within NETs cause differentially induced DNA damage levels, influence recurrent tumor phenotypes and impact the therapeutic response in different models and potentially in patients. Our results contribute to a better understanding of PRRT effects, which might impact future therapeutic outcome of NET patients.

Hydroxyl metabolite of PCB 180 induces DNA damage signaling and enhances the DNA damaging effect of benzo[a]pyrene

Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) and their hydroxyl metabolites (OH-PCBs) are ubiquitous environmental contaminants in human tissues and blood. The toxicological impact of these metabolites is poorly understood. In this study rats were exposed to ultrapure PCB180 (10–1000mg/kgbw) for 28days and induction of genotoxic stress in liver was investigated. DNA damage signaling proteins (pChk1Ser317 and γH2AXSer319) were increased dose dependently in female rats. This increase was paralleled by increasing levels of the metabolite 3′-OH-PCB180. pChk1 was the most sensitive marker. In in vitro studies HepG2 cells were exposed to 1μM of PCB180 and 3′-OH-PCB180 or the positive control benzo[a]pyrene (BaP, 5μM). 3′-OH-PCB180, but not PCB180, induced CYP1A1 mRNA and γH2AX. CYP1A1 mRNA induction was seen at 1h, and γH2AX at 3h. The anti-oxidant N-Acetyl-l-Cysteine (NAC) completely prevented, and 17β-estradiol amplified the γH2AX induction by 3′-OH-PCB180. As 3′-OH-PCB180 induced CYP1A1, a major BaP-metabolizing and activating enzyme, interactions between 3′-OH-PCB180 and BaP was also studied. The metabolite amplified the DNA damage signaling response to BaP. In conclusion, metabolism of PCB180 to its hydroxyl metabolite and the subsequent induction of CYP1A1 seem important for DNA damage induced by PCB180 in vivo. Amplification of the response with estradiol may explain why DNA damage was only seen in female rats.

DNA damage signaling, impairment of cell cycle progression, and apoptosis triggered by 5‐ethynyl‐2′‐deoxyuridine incorporated into DNA

The "click chemistry" approach utilizing 5-ethynyl-2'-deoxyuridine (EdU) as a DNA precursor was recently introduced to assess DNA replication and adapted to flow- and imaging-cytometry. In the present study, we observed that EdU, once incorporated into DNA, induces DNA damage signaling (DDS) such as phosphorylation of ATM on Ser1981, of histone H2AX on Ser139, of p53 on Ser15, and of Chk2 on Thr68. It also perturbs progression of cells through the cell cycle and subsequently induces apoptosis. These effects were observed in non-small cell lung adenocarcinoma A549 as well as in B-cell human lymphoblastoid TK6 and WTK1 cells, differing in the status of p53 (wt versus mutated). After 1 h EdU pulse-labeling, the most affected was cells progression through the S phase subsequent to that at which they had incorporated EdU. This indicates that DNA replication using the template containing incorporated EdU is protracted and triggers DDS. Furthermore, progression of cells having DNA pulse-labeled with EdU led to accumulation of cells in G2 , likely by activating G2 checkpoint. Consistent with the latter was activation of p53 and Chk2. Although a correlation was observed in A549 cells between the degree of EdU incorporation and the extent of γH2AX induction, such correlation was weak in TK6 and WTK1 cells. The degree of perturbation of the cell cycle kinetics by the incorporated EdU was different in the wt p53 TK6 cells as compared to their sister WTK1 cell line having mutated p53. The data are thus consistent with the role of p53 in modulating activation of cell cycle checkpoints in response to impaired DNA replication. The confocal microscopy analysis of the 3D images of cells exposed to EdU for 1 h pulse and then grown for 24 or 48 h revealed an increased number of colocalized γH2AX and p53BP1 foci considered to be markers of DNA double-strand breaks and enlarged nuclei.

Abstract 4041: Wild-type p53-induced phosphatase 1 (Wip1) prevents cellular senescence at physiological oxygen levels by regulating DNA damage signaling during DNA replication.

Abstract Wip1 (PPM1D) is a nuclear serine/threonine protein phosphatase that is induced by p53 following the activation of DNA damage response (DDR) signaling. Upon repair of damage, Wip1 phosphatase activity dampens stress signaling and facilitates the return to homeostasis, thus, regulating recovery from DDR activation. Wip1 is amplified in several types of human primary cancer and has been shown to promote tumorigenesis by inhibiting several tumor suppressors, including p53, therefore its oncogenic potential is now recognized. Interestingly, while Wip1 knockout (Ppm1d–/–) mice are resistant to tumorigenesis, they exhibit reproductive and immune system defects, increased stress sensitivity and a moderately reduced lifespan. Furthermore, Ppm1d–/– Mouse Embryonic Fibroblasts (MEFs) exhibit premature senescence under conventional culture conditions. Little is known, however, about the mechanism by which the deletion of Ppm1d induces premature senescence. Therefore, we have investigated the role of Wip1 in the regulation of cellular senescence in MEFs. We found that at physiological oxygen tension (3% oxygen), early passage Ppm1d–/– MEFs underwent increased cellular senescence as compared with wild–type (Wt) MEFs, as shown by reduced proliferation rate, increased number of flattened enlarged cells, increased SA–β–Gal positive cells, and reduced BrdU incorporation. Moreover, under 3% oxygen, early passage Ppm1d–/– MEFs exhibited more γ–H2AX positive foci, increased activation of p53 and increased levels of p21 than Wt MEFs. Interestingly, we found that the increased levels of γ–H2AX in Ppm1d–/– MEFs were primarily associated with S–phase cells. Given that S–phase cells are more susceptible to oxidative damage, the increased levels of γ–H2AX may result from increased production of reactive oxygen species (ROS) in Ppm1d–/– MEFs. However, primary passage Wt and Ppm1d–/– MEFs cultured under 3% oxygen exhibited no significant difference in intracellular–ROS levels. Ppm1d–/– MEFs did show sustained activation of DDR signaling (γ–H2AX, p–p53Ser15, p21) after H2O2 treatment compared with Wt MEFs. Furthermore, Ppm1d–/– MEFs exhibited increased activation of Chk1, indicating that there is increased replicative stress signaling. These findings suggest that Wip1 prevents the induction of cellular senescence by regulating both oxidative stress– and replicative stress–induced DNA damage signaling during DNA replication. Citation Format: Hiroyasu Sakai, Christophe E. Redon, William M. Bonner, Ettore Appella, Sharlyn Mazur. Wild-type p53-induced phosphatase 1 (Wip1) prevents cellular senescence at physiological oxygen levels by regulating DNA damage signaling during DNA replication. [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 4041. doi:10.1158/1538-7445.AM2013-4041

Targeting of Nrf2 induces DNA damage signaling and protects colonic epithelial cells from ionizing radiation

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a key transcriptional regulator for antioxidant and anti-inflammation enzymes that binds to its endogenous inhibitor protein, Kelch-like ECH (erythroid cell-derived protein with CNC homology)-associated protein 1, in the cytoplasm under normal conditions. Various endogenous or environmental oxidative stresses, such as ionizing radiation (IR), can disrupt the Nrf2-Kelch-like ECH-associated protein 1 complex. This allows Nrf2 to translocate from the cytoplasm into the nucleus to induce transcription of heme oxygenase-1 and other cytoprotective enzymes through binding to antioxidant responsive elements. However, how Nrf2 protects cells from IR-induced damage remains unclear. Here, we report that Nrf2 activation by the synthetic triterpenoids, bardoxolone methyl (BARD) and 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid-ethyl amide, protects colonic epithelial cells against IR-induced damage, in part, by enhancing signaling of the DNA damage response. Pretreatment with BARD reduced the frequency of both G1 and S/G2 chromosome aberrations and enhanced the disappearance of repairosomes (C-terminal binding protein interacting protein, Rad51, and p53 binding protein-1 foci) after IR. BARD protected cells from IR toxicity in a Nrf2-dependent manner. The p53 binding protein-1 promoter contains three antioxidant responsive elements in which Nrf2 directly binds following BARD treatment. In addition, 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid-ethyl amide provided before exposure to a lethal dose of whole-body irradiation protected WT mice from DNA damage and acute gastrointestinal toxicity, which resulted in improved overall survival. These results demonstrate that Nrf2 activation by synthetic triterpenoids is a promising candidate target to protect the gastrointestinal tract against acute IR in vitro and in vivo.

Abstract SY23-02: A telomere-dependent DNA damage checkpoint induced by prolonged mitotic arrest

Abstract Telomere shortening and disruption of telomeric components are pathways that induce telomere deprotection. Here we describe another pathway, where prolonged mitotic arrest induces damage signals at telomeres. Exposure to microtubule drugs, kinesin inhibitors, proteasome inhibitors or the disruption of proper chromosome alignment resulted in the formation of damage-foci at telomeres. Induction of mitotic telomere deprotection coincided with dissociation of TRF2 from telomeres, telomeric 3′-overhang degradation and ATM activation, and could be suppressed by TRF2 overexpression or inhibition of Aurora B kinase. Normal cells that escape from prolonged mitotic arrest halted in the following G1 phase, whereas cells lacking p53 continued to cycle and became aneuploid. We propose a telomere dependent mitotic duration checkpoint that ensures elimination of cells, which fail to progress through mitosis. To avoid unwanted checkpoint activation by natural chromosome ends, cells have evolved telomeres. Human telomeres are composed of double stranded TTAGGG repeats and a single stranded G rich 3′ overhang, which are covered and protected by shelterin. Among the six shelterin components TRF2 and POT1 have predominantly been implicated in chromosome end protection by preventing ATM- and ATR-dependent checkpoint activation. Upon disruption of TRF2 or POT1 telomeres are recognized as sites of DNA damage, resulting in phosphorylation of histone H2AX (γ-H2AX) within the telomeric and sub-telomeric chromatin and association of 53BP1 with the chromosome ends. The co-localization of DNA-damage response factors and chromosome ends can be visualized as telomere dysfunction-induced foci (TIF). TIF have also been intimately linked to replicative senescence and shown to occur spontaneously in cancer cell lines. Cells arrested in mitosis are known to either die during mitotic arrest, or skip cytokinesis and “slip” into the subsequent G1 phase of the cell cycle. Mitotic slippage occurs through the degradation of Cyclin B1 in the presence of the active spindle assembly checkpoint (SAC). Cells that exit from prolonged mitotic arrest or progress through mitotic slippage exhibit various fates, including apoptosis or p53-dependent cell cycle arrest. In both normal and cancer cells, cell death during mitotic arrest, or apoptosis or senescence after escape from prolonged mitotic arrest are crucial for preventing chromosome instability. A failure to remove cells from the cycling population following prolonged mitotic arrest may allow cells to continue propagating with an abnormal number of chromosomes. However, despite intense research, the molecular mechanisms that trigger growth arrest or death in mitotically arrested cultures have not yet been identified. In the course of experimentation to explore putative telomeric functions for cohesin, we found that mitotic arrest per se induces telomere deprotection in primary and transformed human cells. Telomere deprotection during mitotic arrest associated with loss of the telomeric 3′-overhangs, led to ATM activation and was ATM dependent. TRF2 was dissociated from telomeres during prolonged mitotic arrest, providing the molecular basis for overhang loss and ATM activation, which was emphasized by the finding that TRF2 overexpression protected telomeres from the damage machinery during mitotic arrest. Inhibition of Aurora B kinase suppressed the telomere deprotection phenotype, suggesting the involvment of the SAC. Cells suffering from mitotic telomere deprotection underwent p53 dependent cell cycle arrest in the following G1 phase after mitotic release, while cells lacking p53 function continued to cycle and became aneuploid. Our findings provide a molecular mechanism explaining the induction of DNA damage signaling, cell cycle arrest or apoptosis following prolonged mitotic arrest, and explain the mechanism of action of therapeutic drugs, such as Taxol, Vinblastine and Velcade, which all inhibit mitotic progression. We propose that telomeric destabilization during mitotic arrest induces DNA damage signaling and serves as a mitotic duration checkpoint, responsible for eliminating cells that fail to progress through mitosis properly. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY23-02. doi:1538-7445.AM2012-SY23-02

Induction of DNA Damage Signaling upon Rift Valley Fever Virus Infection Results in Cell Cycle Arrest and Increased Viral Replication

Rift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus infecting a wide range of vertebrate hosts. Of particular interest is the nonstructural NSs protein, which forms large filamentous fibril bundles in the nucleus. Past studies have shown NSs to be a multifaceted protein important for virulence through modulation of the interferon response as well acting as a general inhibitor of transcription. Here we investigated the regulation of the DNA damage signaling cascades by RVFV infection and found virally inducted phosphorylation of the classical DNA damage signaling proteins, ataxia-telangiectasia mutated (ATM) (Ser-1981), Chk.2 (Thr-68), H2A.X (Ser-139), and p53 (Ser-15). In contrast, ataxia-telangiectasia mutated and Rad3-related kinase (ATR) (Ser-428) phosphorylation was decreased following RVFV infection. Importantly, both the attenuated vaccine strain MP12 and the fully virulent strain ZH548 showed strong parallels in their up-regulation of the ATM arm of the DNA damage response and in the down-regulation of the ATR pathway. The increase in DNA damage signaling proteins did not result from gross DNA damage as no increase in DNA damage was observed following infection. Rather the DNA damage signaling was found to be dependent on the viral protein NSs, as an NSs mutant virus was not found to induce the equivalent signaling pathways. RVFV MP12-infected cells also displayed an S phase arrest that was found to be dependent on NSs expression. Use of ATM and Chk.2 inhibitors resulted in a marked decrease in S phase arrest as well as viral production. These results indicate that RVFV NSs induces DNA damage signaling pathways that are beneficial for viral replication.

Transcription-dependent Activation of Ataxia Telangiectasia Mutated Prevents DNA-dependent Protein Kinase-mediated Cell Death in Response to Topoisomerase I Poison

Camptothecin (CPT) is a topoisomerase I inhibitor, derivatives of which are being used for cancer chemotherapy. CPT-induced DNA double-strand breaks (DSBs) are considered a major cause of its tumoricidal activity, and it has been shown that CPT induces DNA damage signaling through the phosphatidylinositol 3-kinase-related kinases, including ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase). In addition, CPT causes DNA strand breaks mediated by transcription, although the downstream signaling events are less well characterized. In this study, we show that CPT-induced activation of ATM requires transcription. Mechanistically, transcription inhibition suppressed CPT-dependent activation of ATM and blocked recruitment of the DNA damage mediator p53-binding protein 1 (53BP1) to DNA damage sites, whereas ATM inhibition abrogated CPT-induced G(1)/S and S phase checkpoints. Functional inactivation of ATM resulted in DNA replication-dependent hyperactivation of DNA-PK in CPT-treated cells and dramatic CPT hypersensitivity. On the other hand, simultaneous inhibition of ATM and DNA-PK partially restored CPT resistance, suggesting that activation of DNA-PK is proapoptotic in the absence of ATM. Correspondingly, comet assay and cell cycle synchronization experiments suggested that transcription collapse occurring as the result of CPT treatment are converted to frank double-strand breaks when ATM-deficient cells bypass the G(1)/S checkpoint. Thus, ATM suppresses DNA-PK-dependent cell death in response to topoisomerase poisons, a finding with potential clinical implications.

Abstract 4385: Testosterone induces DNA damage signaling in response to oxidative stress in prostate cancer cells

Abstract Introduction: In prostatic tissues, DNA damage response is specially activated in prostatic intraepithelial neoplasias (PINs) that are generally regarded as precursors of prostate cancer. The process of oncogenic transformation leads to enhanced DNA damage and activates the checkpoint network as an inducible barrier against cancer progression. In this study we analyzed the effects of testosterone under oxidative stress in DNA damage response to understand the biological function and the network of this signaling. Methods: We tested the expression level of PSA and the activation of DNA damage response proteins including ATM (ataxia-telangiectasia-mutated kinase), H2AX (histone H2AX variant) and Chk2 (checkpoint kinase2) in prostate cancer cell lines (LNCaP and PC-3) with various concentration of H2O2. Apoptosis is quantified by flow cytometry and PARP cleavage. Results: H2O2 induces apoptosis and the phosphorylation of ATM, H2AX and Chk2 in LNCaP cells. ATM inhibitor Ku55933 can block the phospholyration of H2AX and Chk2, but induced the activation of alternative pathways, p38 and ERK. Activation of DNA damage response and PARP cleavage was increased by testosterone. H2O2 did not induce phosphorylation of ATM, CHK2 and H2AX in PC-3 cells because of losing their expression. ATM inhibitor Ku55933 can reduce the apoptotic cells in LNCaP cells but PC-3 cells by the treatment of H2O2. Conclusion: Oxidative stress has been associated with tumor formation. On the other hand, DNA damage pathway plays important roles in the maintenance of the cell homeostasis in response to oxidative stress. Our results indicate that androgen signaling have tumor suppressive effects in prostate carcinogenesis through DNA damage response pathways under oxidative stress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4385.

Regulator of G protein Signaling 6 (RGS6) Modulates Doxorubicin‐induced DNA Damage Signaling by Regulating ATM Activity

RGS proteins are well known negative regulators of heterotrimeric G protein signaling. We have shown previously that RGS6 sensitizes breast cancer cells to doxorubicin (Dox)‐induced DNA damage signaling (ATM and p53 phosphorylation), suggesting a possible role of RGS6 in tumor suppression. Unexpectedly, these actions of RGS6 were not dependent on its interaction with G proteins. Here we further reveal the mechanism by which RGS6 modulates DNA damage signaling. Expression of a dominant negative form of ATM completely blocked the ability of RGS6 to promote p53 phosphorylation in response to a submaximal dose of Dox in MCF7 cells. Thus, RGS6‐induced activation of p53 is dependent upon ATM activity. Moreover, ATM co‐immunoprecipitates with RGS6 under basal conditions, demonstrating that these two proteins form a complex without induction of DNA damage. RGS6 does not enhance phosphorylation of CHK2 in response to Dox suggesting that RGS6 interactions with ATM may modulate its activity on specific substrates. RGS6 knockdown in MCF7 cells by RNAi significantly reduces p53 activation in response to Dox, demonstrating that RGS6 is required, at least in part, for Dox‐induced DNA damage signaling. Our data indicate that RGS6 plays an important role in modulating chemosensitivity in cancer cells and identify RGS6 as a novel chemotherapeutic target. (NIH GM075033)

Cell cycle-dependent DNA damage signaling induced by ICRF-193 involves ATM, ATR, CHK2, and BRCA1

Topoisomerase II is essential for cell proliferation and survival and has been a target of various anticancer drugs. ICRF-193 has long been used as a catalytic inhibitor to study the function of topoisomerase II. Here, we show that ICRF-193 treatment induces DNA damage signaling. Treatment with ICRF-193 induced G2 arrest and DNA damage signaling involving γ-H2AX foci formation and CHK2 phosphorylation. DNA damage by ICRF-193 was further demonstrated by formation of the nuclear foci of 53BP1, NBS1, BRCA1, MDC1, and FANCD2 and increased comet tail moment. The DNA damage signaling induced by ICRF-193 was mediated by ATM and ATR and was restricted to cells in specific cell cycle stages such as S, G2, and mitosis including late and early G1 phases. Downstream signaling of ATM and ATR involved the phosphorylation of CHK2 and BRCA1. Altogether, our results demonstrate that ICRF-193 induces DNA damage signaling in a cell cycle-dependent manner and suggest that topoisomerase II might be essential for the progression of the cell cycle at several stages including DNA decondensation.

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