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Project description:For many decades genomic instability is considered one of the hallmarks of cancer. Role of the tumor suppressor WWOX (WW domain-containing oxidoreductase) in DNA damage response upon double strand breaks has been recently revealed. Here we demonstrate unforeseen functions for WWOX upon DNA single strand breaks (SSBs) checkpoint activation. We found that WWOX levels are induced following SSBs and accumulate in the nucleus. WWOX deficiency is associated with reduced activation of ataxia telangiectasia and Rad3-related protein (ATR) checkpoint proteins and increased chromosomal breaks. At the molecular level, we show that upon SSBs WWOX is modified at lysine 274 by ubiquitination mediated by the ubiquitin E3 ligase ITCH and interacts with ataxia telangiectasia-mutated (ATM). Interestingly, ATM inhibition was associated with reduced activation of ATR checkpoint proteins suggesting that WWOX manipulation of ATR checkpoint proteins is ATM-dependent. Taken together, the present findings indicate that WWOX plays a key role in ATR checkpoint activation, while its absence might facilitate genomic instability.
| S-EPMC4826209 | biostudies-literature
Project description:The translesion DNA synthesis (TLS) polymerase REV1 is implicated in the bypass of the irreparable DNA damage such as interstrand crosslinks (ICLs). However, the potential role of REV1 in DNA damage response (DDR) pathway has not been determined. In this research communication, we provide evidence to demonstrate that REV1 plays a previously unidentified but important role in the ATR-Chk1 checkpoint activation in response to mitomycin C (MMC)-induced ICLs in Xenopus egg extracts. We further pinpointed that REV1 plays a downstream role of a checkpoint protein complex assembly including ATR, ATRIP, TopBP1 and the Rad9-Rad1-Hus1 complex to MMC-induced ICLs on chromatin in the DDR pathway. Notably, domain dissection analysis demonstrates that a C-terminal domain, but not the individual ubiquitin binding motifs, of REV1 is important for the binding of REV1 to MMC-damaged chromatin and the MMC-induced Chk1 phosphorylation. Yet, the ATR-Chk1 DDR pathway appears to be dispensable for the preferential association of REV1 to MMC-damaged chromatin. Taken together, REV1 is important for the DDR pathway in Xenopus egg extracts.
| S-EPMC4426025 | biostudies-literature
Project description:The mismatch repair pathway (MMR) is essential for removing DNA polymerase errors, thereby maintaining genomic stability. Loss of MMR function increases mutation frequency and is associated with tumorigenesis. However, how MMR is executed at active DNA replication forks is unclear. This has important implications for understanding how MMR repairs O6-methylguanine/thymidine (MeG/T) mismatches created upon exposure to DNA alkylating agents. If MeG/T lesion recognition by MMR initiates mismatch excision, the reinsertion of a mismatched thymidine during resynthesis could initiate futile repair cycles. One consequence of futile repair cycles might be a disruption of overall DNA replication in the affected cell. Herein, we show that in MMR-proficient HeLa cancer cells, treatment with a DNA alkylating agent slows S phase progression, yet cells still progress into the next cell cycle. In the first S phase following treatment, they activate ataxia telangiectasia and Rad3-related (ATR)-Checkpoint Kinase 1 (Chk1) signaling, which limits DNA damage, while inhibition of ATR kinase activity accelerates DNA damage accumulation and sensitivity to the DNA alkylating agent. We also observed that exposure of human embryonic stem cells to alkylation damage severely compromised DNA replication in a MMR-dependent manner. These cells fail to activate the ATR-Chk1 signaling axis, which may limit their ability to handle replication stress. Accordingly, they accumulate double-strand breaks and undergo immediate apoptosis. Our findings implicate the MMR-directed response to alkylation damage as a replication stress inducer, suggesting that repeated MMR processing of mismatches may occur that can disrupt S phase progression.
| S-EPMC5816205 | biostudies-literature
Project description:Cell division cycle 6 (CDC6), an androgen receptor (AR) target gene, is implicated in regulating DNA replication and checkpoint mechanisms. CDC6 expression is increased during prostate cancer (PCa) progression and positively correlates with AR in PCa tissues. AR or CDC6 knockdown, together with AZD7762, a Chk1/2 inhibitor, results in decreased TopBP1-ATR-Chk1 signaling and markedly increased ataxia-telangiectasia-mutated (ATM) phosphorylation, a biomarker of DNA damage, and synergistically increases treatment efficacy. Combination treatment with the AR signaling inhibitor enzalutamide (ENZ) and the Chk1/2 inhibitor AZD7762 demonstrates synergy with regard to inhibition of AR-CDC6-ATR-Chk1 signaling, ATM phosphorylation induction, and apoptosis in VCaP (mutant p53) and LNCaP-C4-2b (wild-type p53) cells. CDC6 overexpression significantly reduced ENZ- and AZD7762-induced apoptosis. Additive or synergistic therapeutic activities are demonstrated in AR-positive animal xenograft models. These findings have important clinical implications, since they introduce a therapeutic strategy for AR-positive, metastatic, castration-resistant PCa, regardless of p53 status, through targeting AR-CDC6-ATR-Chk1 signaling.
| S-EPMC5349188 | biostudies-literature
Project description:RNF8 plays a critical role in DNA damage response (DDR) to initiate ubiquitination-dependent signaling. To better characterize the role of RNF8 in UV-induced DDR, we searched for novel substrates of RNF8 and identified NONO as one intriguing substrate. We found that: (i) RNF8 ubiquitinates NONO and (ii) UV radiation triggers NONO ubiquitination and its subsequent degradation. Depletion of RNF8 inhibited UV-induced degradation of NONO, suggesting that RNF8 targets NONO for degradation in response to UV damage. In addition, we found that 3 NONO lysine residues (positions 279, 290 and 295) are important for conferring its instability in UV-DDR. Depletion of RNF8 or expression of NONO with lysine to arginine substitutions at positions 279, 290 and 295 prolonged CHK1 phosphorylation over an extended period of time. Furthermore, expression of the stable mutant, but not wild-type NONO, induced a prolonged S phase following UV exposure. Stable cell lines expressing the stable NONO mutant showed increased UV sensitivity in a clonogenic survival assay. Since RNF8 recruitment to the UV-damaged sites is dependent on ATR, we propose that RNF8-mediated NONO degradation and subsequent inhibition of NONO-dependent chromatin loading of TOPBP1, a key activator of ATR, function as a negative feedback loop critical for turning off ATR-CHK1 checkpoint signaling in UV-DDR.
| S-EPMC6344893 | biostudies-literature
Project description:Oncolytic adenoviruses replicate selectively within and lyse malignant cells. As such, they are being developed as anticancer therapeutics. However, the sensitivity of ovarian cancers to adenovirus cytotoxicity varies greatly, even in cells of similar infectivity. Using both the adenovirus E1A-CR2 deletion mutant dl922-947 and WT adenovirus serotype 5 in a panel of human ovarian cancer cell lines that cover a 3-log range of sensitivity, we observed profound overreplication of genomic DNA only in highly sensitive cell lines. This was associated with the presence of extensive genomic DNA damage. Inhibition of ataxia telangiectasia and Rad3-related checkpoint kinase 1 (ATR-Chk1), but not ataxia telangiectasia mutated (ATM), promoted genomic DNA damage and overreplication in resistant and partially sensitive cells. This was accompanied by increased adenovirus cytotoxicity both in vitro and in vivo in tumor-bearing mice. We also demonstrated that Cdc25A was upregulated in highly sensitive ovarian cancer cell lines after adenovirus infection and was stabilized after loss of Chk1 activity. Knockdown of Cdc25A inhibited virus-induced DNA damage in highly sensitive cells and blocked the effects of Chk1 inhibition in resistant cells. Finally, inhibition of Chk1 decreased homologous recombination repair of virus-induced genomic DNA double-strand breaks. Thus, virus-induced host cell DNA damage signaling and repair are key determinants of oncolytic adenoviral activity, and promoting unscheduled DNA synthesis and/or impeding homologous recombination repair could potentiate the effects of oncolytic adenoviruses in the treatment of ovarian cancer.
| S-EPMC3069766 | biostudies-literature
Project description:DNA damage can induce autophagy; however, the underlying mechanism remains largely unknown. Here we report that DNA damage leads to autophagy through ATR/Chk1/RhoB-mediated lysosomal recruitment of TSC complex and subsequent mTORC1 inhibition. DNA damage caused by ultraviolet light (UV) or alkylating agent methyl methanesulphonate (MMS) results in phosphorylation of small GTPase RhoB by Chk1. Phosphorylation of RhoB enhances its interaction with the TSC2, and promotes its sumoylation by PIAS1, which is required for RhoB/TSC complex to translocate to lysosomes. As a result, mTORC1 is inhibited, and autophagy is activated. Knockout of RhoB severely attenuates lysosomal translocation of TSC complex and the DNA damage-induced autophagy. Reintroducing wild-type but not sumoylation-resistant RhoB into RhoB-/- cells restores the onset of autophagy. Hence, our study identifies a molecular mechanism for translocation of TSC complex to lysosomes in response to DNA damage, which depends on ATR/Chk1-mediated RhoB phosphorylation and sumoylation.
| S-EPMC6175864 | biostudies-other
Project description:Chk1 kinase inhibitors are currently under clinical investigation as potentiators of cytotoxic chemotherapy and demonstrate potent activity in combination with anti-metabolite drugs that increase replication stress through the inhibition of nucleotide or deoxyribonucleotide biosynthesis. Inhibiting other metabolic pathways critical for the supply of building blocks necessary to support DNA replication may lead to increased DNA damage and synergy with an inhibitor of Chk1. A screen of small molecule metabolism modulators identified combinatorial activity between a Chk1 inhibitor and chloroquine or the LDHA/LDHB inhibitor GSK 2837808A. Compounds, such as 2-deoxyglucose or 6-aminonicotinamide, that reduced the fraction of cells undergoing active replication rendered tumour cells more resistant to Chk1 inhibitor-induced DNA damage. Withdrawal of glucose or glutamine induced G1 and G2/M arrest without increasing DNA damage and reduced Chk1 expression and activation through autophosphorylation. This suggests the expression and activation of Chk1 kinase is associated with cells undergoing active DNA replication. Glutamine starvation rendered tumour cells more resistant to Chk1 inhibitor-induced DNA damage and reversal of the glutamine starvation restored the sensitivity of tumour cells to Chk1 inhibitor-induced DNA damage. Chk1 inhibitors may be a potentially useful therapeutic treatment for patients whose tumours contain a high fraction of replicating cells.
| S-EPMC5247758 | biostudies-literature
Project description:Lung cancer has the highest mortality rate worldwide and is often diagnosed at late stages, requiring genotoxic chemotherapy with significant side effects. Cancer prevention has become a major focus, including the use of dietary and supplemental antioxidants. Thus, we investigated the ability of an antioxidant formulation (AOX1) to reduce DNA damage in human bronchial epithelial cells (BEAS-2B) with and without the combination of apple peel flavonoid fraction (AF4), or its major constituent quercetin (Q), or Q-3-O-d-glucoside (Q3G) in vitro. To model smoke-related genotoxicity, we used cigarette-smoke hydrocarbon 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) as well as methotrexate (MTX) to induce DNA damage in BEAS-2B cells. DNA fragmentation, γ-H2AX immunofluorescence, and comet assays were used as indicators of DNA damage. Pre-exposure to AOX1 alone or in combination with AF4, Q, or Q3G before challenging with NNKOAc and MTX significantly reduced intracellular reactive oxygen species (ROS) levels and DNA damage in BEAS-2B cells. Although NNKOAc-induced DNA damage activated ATM-Rad3-related (ATR) and Chk1 kinase in BEAS-2B cells, pre-exposure of the cells with tested antioxidants prior to carcinogen challenge significantly reduced their activation and levels of γ-H2AX (p ≤ 0.05). Therefore, AOX1 alone or combined with flavonoids holds promise as a chemoprotectant by reducing ROS and DNA damage to attenuate activation of ATR kinase following carcinogen exposure.
| S-EPMC8615515 | biostudies-literature
Project description:The genetic syndrome Fanconi anemia (FA) is characterized by aplastic anemia, cancer predisposition and hypersensitivity to DNA interstrand crosslinks (ICLs). FA proteins (FANCs) are thought to work in pathway(s) essential for dealing with crosslinked DNA. FANCs interact with other proteins involved in both DNA repair and S-phase checkpoint such as BRCA1, ATM and the RAD50/MRE11/NBS1 (RMN) complex. We deciphered the previously undefined pathway(s) leading to the ICLs-induced S-phase checkpoint and the role of FANCs in this process. We found that ICLs activate a branched pathway downstream of the ATR kinase: one branch depending on CHK1 activity and the other on the FANCs-RMN complex. The transient slow-down of DNA synthesis was abolished in cells lacking ATR, whereas CHK1-siRNA-treated cells, NBS1 or FA cells showed partial S-phase arrest. CHK1 RNAi in NBS1 or FA cells abolished the S-phase checkpoint, suggesting that CHK1 and FANCs/NBS1 proteins work on parallel pathways. Furthermore, we found that ICLs trigger ATR-dependent FANCD2 phosphorylation and FANCD2/ATR colocalization. This study demonstrates a novel relationship between the FA pathway(s) and the ATR kinase.
| S-EPMC380971 | biostudies-literature