Long non-coding RNA ENST00000414355 as a biomarker of cadmium exposure regulates DNA damage and apoptosis.

Human exposure to cadmium (Cd) may induce severe effects in different organs. Recent studies suggest that long non-coding RNAs (lncRNAs) are closely involved in the pathophysiological mechanisms of Cd-related diseases. This study evaluated the use of lncRNA (ENST00000414355) as an expression signature of Cd exposure and assessed its ability to modulate DNA damage and apoptosis by measuring the expression of ATM serine/threonine kinase (ATM) and mitochondrial membrane potential (ΔΨm) in Cd-exposed workers. A total of 139 (74 non-smokers and 65 smokers) participants from a Cd battery manufacturer were included in the study. Venous blood samples were collected to determine the blood Cd level and detect blood ENST00000414355 and its target gene (ATM) using real-time reverse transcription-polymerase chain reaction (qRT-PCR). Mitochondrial membrane potential was used to assess the Cd effect on mitochondrial permeability. Our results indicated a significant positive correlation between blood Cd level and lncRNA-ENST00000414355 and ATM expression and a significant negative correlation between blood Cd level and ΔΨm (p < 0.0001). Moreover, significant correlations were observed between the expression of lncRNA-ENST00000414355 and ATM expression and ΔΨm (p < 0.0001). Statistical significance was found in the blood Cd level, lncRNA-ENST00000414355 expression, ATM expression, and ΔΨm (p < 0.0001) between smokers and non-smokers. This study confirmed the upregulation of the lncRNA-ENST00000414355 expression, DNA damage-checkpoint-related gene (ATM), and decreased ΔΨm in Cd-exposed workers. Thus, lncRNA-ENST00000414355 may serve as a valuable biomarker for the exposure and toxicity of Cd.

DNA Double-Strand Breaks Induced in Human Cells by Twelve Metallic Species: Quantitative Inter-Comparisons and Influence of the ATM Protein.

Despite a considerable amount of data, the molecular and cellular bases of the toxicity due to metal exposure remain unknown. Recent mechanistic models from radiobiology have emerged, pointing out that the radiation-induced nucleo-shuttling of the ATM protein (RIANS) initiates the recognition and the repair of DNA double-strand breaks (DSB) and the final response to genotoxic stress. In order to document the role of ATM-dependent DSB repair and signalling after metal exposure, we applied twelve different metal species representing nine elements (Al, Cu, Zn Ni, Pd, Cd, Pb, Cr, and Fe) to human skin, mammary, and brain cells. Our findings suggest that metals may directly or indirectly induce DSB at a rate that depends on the metal properties and concentration, and tissue type. At specific metal concentration ranges, the nucleo-shuttling of ATM can be delayed which impairs DSB recognition and repair and contributes to toxicity and carcinogenicity. Interestingly, as observed after low doses of ionizing radiation, some phenomena equivalent to the biological response observed at high metal concentrations may occur at lower concentrations. A general mechanistic model of the biological response to metal exposure based on the nucleo-shuttling of ATM is proposed to describe the metal-induced stress response and to define quantitative endpoints for toxicity and carcinogenicity.

Usnic Acid Inhibits Proliferation and Migration through ATM Mediated DNA Damage Response in RKO Colorectal Cancer Cell.

BACKGROUND: Usnic Acid (UA), also known as lichenol, has been reported to have inhibitory effects on a variety of cancer cells, but its specific mechanism remained to be elucidated. Tumor chemotherapy drugs, especially DNA damage chemotherapeutic drugs, target Chromosomal DNA, but their spontaneous and acquired drug resistance are also an urgent problem to be solved. Therefore, drug combination research has become the focus of researchers. METHODS: Here, we evaluated the tumor-suppressing molecular mechanism of UA in colorectal cancer cells RKO from the perspective of the ATM-mediated DNA damage signaling pathway through H2O2 simulating DNA damage chemotherapeutic drugs. CCK8 cell proliferation assay was used to determine the inhibition of RKO cells by hydrogen peroxide and UA alone or in combination, and wound healing assay was applied to determine the effect of the drug on cell migration. RESULTS: Transfected cells with miRNA18a-5p mimics and inhibitors, MDC and DCFH-DA staining for the measurement of autophagy and ROS, cell cycle and apoptosis were detected by flow cytometry, expressions of microRNA and mRNA were determined by fluorescence quantitative PCR, and protein by Western blot. DISCUSSION: We found that UA can upregulate ATM via miR-18a to activate the DNA damage signaling pathway and inhibit the proliferation and migration of RKO cells in a concentration-dependent manner. CONCLUSION: At the same time, DNA damage responses, including cell cycle, autophagy, apoptosis and ROS levels, are also regulated by UA. Therefore, UA combined with DNA damage chemotherapeutic drugs may be an effective treatment for cancer.

Involvement of Host ATR-CHK1 Pathway in Hepatitis B Virus Covalently Closed Circular DNA Formation.

The covalently closed circular (CCC) DNA of hepatitis B virus (HBV) functions as the only viral transcriptional template capable of producing all viral RNA species and is essential to initiate and sustain viral replication. CCC DNA is converted from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. As RC DNA mimics damaged cellular DNA, the host cell DNA damage repair (DDR) system is thought to be responsible for HBV CCC DNA formation. The potential role of two major cellular DDR pathways, the ataxia telangiectasia mutated (ATM) pathway and the ATM and Rad3-related (ATR) pathway, in HBV CCC DNA formation was thus investigated. Inhibition, or expression knockdown, of ATR and its downstream signaling factor CHK1, but not of ATM, decreased CCC DNA formation during de novo HBV infection, as well as intracellular CCC DNA amplification, when RC DNA from extracellular virions and intracellular nucleocapsids, respectively, is converted to CCC DNA. Furthermore, a novel RC DNA processing product with 5' truncated minus strands was detected when the ATR-CHK1 pathway was inhibited, further indicating that this pathway controls RC DNA processing during its conversion to CCC DNA. These results provide new insights into how host cells recognize and process HBV RC DNA in order to produce CCC DNA and have implications for potential means to block CCC DNA production.IMPORTANCE Hepatitis B virus (HBV) chronically infects hundreds of millions of people and remains a major cause of viral hepatitis, cirrhosis, and liver cancer. HBV persistence is sustained by a viral nuclear episome that directs all viral gene expression needed to support viral replication. The episome is converted from an incomplete DNA precursor in viral particles in an ill-understood process. We report here that the incomplete DNA precursor is recognized by the host cell in a way similar to the sensing of damaged cellular DNA for subsequent repair to form the nuclear episome. Intense efforts are ongoing to develop novel antiviral strategies to eliminate CCC DNA so as to cure chronic HBV infection. Our results here provide novel insights into, and suggest novel ways of perturbing, the process of episome formation. Furthermore, our results inform mechanisms of cellular DNA damage recognition and repair, processes essential for normal cell growth.

Mathematical Model of ATM Activation and Chromatin Relaxation by Ionizing Radiation.

We propose a comprehensive mathematical model to study the dynamics of ionizing radiation induced Ataxia-telangiectasia mutated (ATM) activation that consists of ATM activation through dual mechanisms: the initiative activation pathway triggered by the DNA damage-induced local chromatin relaxation and the primary activation pathway consisting of a self-activation loop by interplay with chromatin relaxation. The model is expressed as a series of biochemical reactions, governed by a system of differential equations and analyzed by dynamical systems techniques. Radiation induced double strand breaks (DSBs) cause rapid local chromatin relaxation, which is independent of ATM but initiates ATM activation at damage sites. Key to the model description is how chromatin relaxation follows when active ATM phosphorylates KAP-1, which subsequently spreads throughout the chromatin and induces global chromatin relaxation. Additionally, the model describes how oxidative stress activation of ATM triggers a self-activation loop in which PP2A and ATF2 are released so that ATM can undergo autophosphorylation and acetylation for full activation in relaxed chromatin. In contrast, oxidative stress alone can partially activate ATM because phosphorylated ATM remains as a dimer. The model leads to predictions on ATM mediated responses to DSBs, oxidative stress, or both that can be tested by experiments.

ATM and ATR Expression Potentiates HBV Replication and Contributes to Reactivation of HBV Infection upon DNA Damage.

Chronic hepatitis B virus infection (CHB) caused by the hepatitis B virus (HBV) is one of the most common viral infections in the world. Reactivation of HBV infection is a life-threatening condition observed in patients with CHB receiving chemotherapy or other medications. Although HBV reactivation is commonly attributed to immune suppression, other factors have long been suspected to play a role, including intracellular signaling activated in response to DNA damage. We investigated the effects of DNA-damaging factors (doxorubicin and hydrogen peroxide) on HBV reactivation/replication and the consequent DNA-damage response. Dose-dependent activation of HBV replication was observed in response to doxorubicin and hydrogen peroxide which was associated with a marked elevation in the mRNA levels of ataxia-telangiectasia mutated (ATM) and ATM- and RAD3-related (ATR) kinases. Downregulation of ATM or ATR expression by shRNAs substantially reduced the levels of HBV RNAs and DNA. In contrast, transcriptional activation of ATM or ATR using CRISPRa significantly increased HBV replication. We conclude that ATM and ATR are essential for HBV replication. Furthermore, DNA damage leading to the activation of ATM and ATR transcription, results in the reactivation of HBV replication.

Liposomal prodigiosin and plasmid encoding serial GCA nucleotides reduce inflammation in microglial and astrocyte cells by ATM/ATR signaling.

The aim of this study was to use liposomal structure consisting prodigiosin and plasmid encoding serial GCA nucleotides (LP/pSGCAN) to reduce inflammation in microglial cells (MGCs) and astrocyte cells (ACCs) by ATM/ATR signaling. Here, it was shown that LP/pSGCAN decreased cell viability and total RNA level. Importantly, LP/pSGCAN had more effect on ACCs than MGCs (P < 0.05). Moreover, increase of apoptosis was seen with increase of concentration. The expression of IL-1 and IL-6 were decreased and the expression of ATM and ATR were increased in treated MGCs and ACCs, which showed LP/pSGCAN could inhibit inflammation by activation of ATM/ATR pathway.

[Topoisomerase inhibitor upregulates MICA/B expression in breast cancer cells through ATM/ATR and NF-κB pathway].

OBJECTIVE: To investigate the effects of chemotherapeutic agents widely used in clinical practice on major histocompatibility complex class I-related chain A and B (MICA/B) expression in breast cancer cells, and to explore the molecular mechanisms involved. METHODS: We examined MICA/B mRNA and surface protein expressions in breast cancer cells treated with chemotherapeutic agents by real-time RT-PCR and flow cytometry respectively. The blocking effects of ataxia telangiectasia mutated and Rad3-related kinase (ATM/ATR) inhibitor caffeine and nuclear factor κB (NF-κB) inhibitor pynolidine dithiocarbamate (PDTC) on etoposide-upregulated MICA/B mRNA and surface protein expressions were investigated. Electrophoretic mobility shift assay (EMSA) was taken to investigate whether etoposide enhanced the binding of NF-κB to MICA/B gene promoter. RESULTS: Three topoisomerase inhibitors etoposide, camptothecin and doxorubicine upregulated MICA and MICB mRNA expressions in breast cancer cell MCF-7. Comparing to no-drug-treated cells, MICA mRNA levels increased to (1.68±0.17), (2.54±0.25) and (3.42±0.15) fold, and levels of MICB mRNA increased to (1.82±0.24), (1.56±0.05) and (5.84±0.57) fold respectively in cancer cells treated by etoposide at the concentrations of 5, 20 and 100 µmol/L (P<0.05). MICA and MICB mRNA levels also increased significantly when MCF-7 cells were incubated with camptothecin or doxorubicine at the specific concentrations (P<0.05). MICB mRNA expression also increased slightly in another breast cancer cell SK-BR-3 treated by topoisomerase II inhibitors etoposide and camptothecin (P<0.05). Furthermore, etoposide and camptothecin upregulated MICA/B surface protein expression in MCF-7 cells (P<0.05), and the upregulation was found in both living and apoptotic cells. Our study showed that etoposide induced-MICA/B expression in MCF-7 was inhibited by caffeine at different concentrations. When cancer cells were treated by caffeine with 1, 5 and 10 mmol/L, MICA mRNA levels decreased from (3.75±0.25) to (0.89±0.05), (0.81±0.02) and (0.48±0.04) fold respectively (P<0.001), and MICB mRNA levels decreased from (6.85±0.35) to (1.36±0.13), (0.76±0.06) and (0.56±0.03) fold (P<0.05), while MICA/B protein levels decreased from (3.42±0.05) to (1.32±0.03), (1.21±0.06) and (1.14±0.03) fold (P<0.001), indicating that etoposide-induced MICA/B expression was inhibited by ATM/ATR inhibitor. Similarly, NF-κB inhibitor PDTC also inhibited MICA/B mRNA and protein expressions induced by etoposide significantly when MCF-7 cells were incubated with PDTC at the concentrations of 10, 50 and 100 µmol/L (P<0.05), indicating that NF-κB was also involved in this process. EMSA showed that the binding of NF-κB to MICA/B promoter enhanced in MCF-7 cells after etoposide treatment. CONCLUSION: Topoisomerase inhibitor increased MICA/B mRNA and protein expressions in breast cancer cells, indicating that chemotherapeutic agents might increase the recognizing and killing ability of immunocytes to breast cancer cells. ATM/ATR and NF-κB pathways might be involved in it.

Acute Megakaryoblastic Leukemia with Myelodysplasia-related Changes Associated with ATM Gene Deletion.

Ataxia telangiectasia mutated (ATM) is a tumor suppressor gene, and its somatic inactivation plays a role in the pathogenesis of lymphoid malignancies. However, the role of ATM in patients with myeloid malignancies is still unknown. We herein report a case of acute megakaryoblastic leukemia (AMKL) with ATM gene deletion. An 84-year-old Japanese woman presenting with a pale face and pancytopenia was admitted to our institution and diagnosed to have AMKL with ATM gene deletion. She was treated with intravenous azacitidine. The azacitidine treatment was effective for approximately 1 year. Somatic inactivation of the ATM gene may therefore be involved in the pathogenesis of AMKL.

The isopeptidase inhibitor 2cPE triggers proteotoxic stress and ATM activation in chronic lymphocytic leukemia cells.

Relapse after treatment is a common and unresolved problem for patients suffering of the B-cell chronic lymphocytic leukemia (B-CLL). Here we investigated the ability of the isopeptidase inhibitor 2cPE to trigger apoptosis in leukemia cells in comparison with bortezomib, another inhibitor of the ubiquitin-proteasome system (UPS). Both inhibitors trigger apoptosis in CLL B cells and gene expression profiles studies denoted how a substantial part of genes up-regulated by these compounds are elements of adaptive responses, aimed to sustain cell survival. 2cPE treatment elicits the up-regulation of chaperones, proteasomal subunits and elements of the anti-oxidant response. Selective inhibition of these responses augments apoptosis in response to 2cPE treatment. We have also observed that the product of the ataxia telangiectasia mutated gene (ATM) is activated in 2cPE treated cells. Stimulation of ATM signaling is possibly dependent on the alteration of the redox homeostasis. Importantly ATM inhibition, mutations or down-modulation increase cell death in response to 2cPE. Overall this work suggests that 2cPE could offer new opportunities for the treatment of B-CLL.

Modulation of DNA damage response and induction of apoptosis mediates synergism between doxorubicin and a new imidazopyridine derivative in breast and lung cancer cells.

DNA damage response machinery (DDR) is an attractive target of cancer therapy. Modulation of DDR network may alter the response of cancer cells to DNA damaging anticancer drugs such as doxorubicin. The aim of the present study is to investigate the effects of a newly developed imidazopyridine (IAZP) derivative on the DDR after induction of DNA damage in cancer cells by doxorubicin. Cytotoxicity sulphrhodamine-B assay showed a weak anti-proliferative effect of IAZP alone on six cancer cell lines (MCF7, A549, A549DOX11, HepG2, HeLa and M8) and a normal fibroblast strain. Combination of IAZP with doxorubicin resulted in synergism in lung (A549) and breast (MCF7) cancer cells but neither in the other cancer cell lines nor in normal fibroblasts. Molecular studies revealed that synergism is mediated by modulation of DNA damage response and induction of apoptosis. Using constant-field gel electrophoresis and immunofluorescence detection of γ-H2AX foci, IAZP was shown to inhibit the repair of doxorubicin-induced DNA damage in A549 and MCF7 cells. Immunoblot analysis showed that IAZP suppresses the phosphorylation of the ataxia lelangiectasia and Rad3 related (ATR) protein, which is an important player in the response of cancer cells to chemotherapy-induced DNA damage. Moreover, IAZP augmented the doxorubicin-induced degradation of p21, activation of p53, CDK2, caspase 3/7 and phosphorylation of Rb protein. These effects enhanced doxorubicin-induced apoptosis in both cell lines. Our results indicate that IAZP is a promising agent that may enhance the cytotoxic effects of doxorubicin on some cancer cells through targeting the DDR. It is a preliminary step toward the clinical application of IAZP in combination with anticancer drugs and opens the avenue for the development of compounds targeting the DDR pathway that might improve the therapeutic index of anticancer drugs and enhance their cure rate.

The natural anticancer compound rocaglamide selectively inhibits the G1-S-phase transition in cancer cells through the ATM/ATR-mediated Chk1/2 cell cycle checkpoints.

Targeting the cancer cell cycle machinery is an important strategy for cancer treatment. Cdc25A is an essential regulator of cycle progression and checkpoint response. Over-expression of Cdc25A occurs often in human cancers. In this study, we show that Rocaglamide-A (Roc-A), a natural anticancer compound isolated from the medicinal plant Aglaia, induces a rapid phosphorylation of Cdc25A and its subsequent degradation and, thereby, blocks cell cycle progression of tumor cells at the G1-S phase. Roc-A has previously been shown to inhibit tumor proliferation by blocking protein synthesis. In this study, we demonstrate that besides the translation inhibition Roc-A can induce a rapid degradation of Cdc25A by activation of the ATM/ATR-Chk1/Chk2 checkpoint pathway. However, Roc-A has no influence on cell cycle progression in proliferating normal T lymphocytes. Investigation of the molecular basis of tumor selectivity of Roc-A by a time-resolved microarray analysis of leukemic vs. proliferating normal T lymphocytes revealed that Roc-A activates different sets of genes in tumor cells compared with normal cells. In particular, Roc-A selectively stimulates a set of genes responsive to DNA replication stress in leukemic but not in normal T lymphocytes. These findings further support the development of Rocaglamide for antitumor therapy.

SB202190 affects cell response to hydroxyurea-induced genotoxic stress in root meristems of Vicia faba.

Genotoxic stress caused by a variety of chemical and physical agents may lead to DNA breaks and genome instability. Response to DNA damage depends on ATM/ATR sensor kinases and their downstream proteins, which arrange cell cycle checkpoints. Activation of ATM (ataxia-telangiectasia-mutated)/ATR (ATM and Rad 3-related) signaling pathway triggers cell cycle arrest (by keeping cyclin-Cdk complexes inactive), combined with gamma-phosphorylation of histone H2A.X and induction of DNA repair processes. However, genotoxic stress activates also mitogen-activated protein kinases (MAPKs) which may control the functions of checkpoint proteins both directly, by post-translational modifications, or indirectly, by regulation of their expression. Our results indicate that in root meristem cells of Vicia faba, MAP kinase signaling pathway takes part in response to hydroxyurea-induced genotoxic stress. It is shown that SB202190, an inhibitor of p38 MAP kinase, triggers PCC (premature chromosome condensation) more rapidly, but only if cell cycle checkpoints are alleviated by caffeine. Since SB202190 and, independently, caffeine reduces HU-mediated histone H4 Lys5 acetylation, it may be that there is a cooperation of MAP kinase signaling pathways and ATM/ATR-dependent checkpoints during response to genotoxic stress.