Ataxia Telangiectasia Mutated Promoter Research Articles

A Novel ATM Antisense Transcript ATM-AS Positively Regulates ATM Expression in Normal and Breast Cancer Cells

ObjectiveThe ataxia telangiectasia mutated (ATM) gene is a master regulator in cellular DNA damage response. The dysregulation of ATM expression is frequent in breast cancer, and is known to be involved in the carcinogenesis and prognosis of cancer. However, the underlying mechanism remains unclear. The bioinformatic analysis predicted a potential antisense transcript ATM-antisense (AS) from the opposite strand of the ATM gene. The purpose of this study was to identify ATM-AS and investigate the possible effect of ATM-AS on the ATM gene regulation.MethodsSingle strand-specific RT-PCR was performed to verify the predicted antisense transcript ATM-AS within the ATM gene locus. qRT-PCR and Western blotting were used to detect the expression levels of ATM-AS and ATM in normal and breast cancer cell lines as well as in tissue samples. Luciferase reporter gene assays, biological mass spectrometry, ChIP-qPCR and RIP were used to explore the function of ATM-AS in regulating the ATM expression. Immunofluorescence and host-cell reactivation (HCR) assay were performed to evaluate the biological significance of ATM-AS in ATM-mediated DNA damage repair. Breast cancer tissue samples were used for evaluating the correlation of the ATM-AS level with the ATM expression as well as prognosis of the patients.ResultsThe ATM-AS significantly upregulated the ATM gene activity by recruiting KAT5 histone acetyltransferase to the gene promoter. The reduced ATM-AS level led to the abnormal downregulation of ATM expression, and impaired the ATM-mediated DNA damage repair in normal breast cells in vitro. The ATM-AS level was positively correlated with the ATM expression in the examined breast cancer tissue samples, and the patient prognosis.ConclusionThe present study demonstrated that ATM-AS, an antisense transcript located within the ATM gene body, is an essential positive regulator of ATM expression, and functions by mediating the binding of KAT5 to the ATM promoter. These findings uncover the novel mechanism underlying the dysregulation of the ATM gene in breast cancer, and enrich our understanding of how an antisense transcript regulates its host gene.

Acquired temozolomide resistance in MGMTlow gliomas is associated with regulation of homologous recombination repair by ROCK2

It was reported that MGMTlow gliomas may still be resistant to TMZ, while the mechanisms remain poorly understood. In this study, we demonstrated that rho-associated kinase 2 (ROCK2), a cytoskeleton regulator, was highly expressed in MGMTlow recurrent gliomas, and its expression strongly correlated with poor overall survival (OS) time in a subset of MGMTlow recurrent gliomas patients with TMZ therapy. And we also found that overactive ROCK2 enhanced homologous recombination repair (HR) in TMZ-resistant (TMZ-R) glioma cell lines with low MGMT expression. Silencing ROCK2 impaired HR repair, and induced double-strand break (DSB) and eradicated TMZ-R glioma cells in culture. Notably, in MGMTlow TMZ-R models, as a key factor of HR, ataxia telangiectasia-mutated (ATM) expression was upregulated directly by hyper-activation of ROCK2 to improve HR efficiency. ROCK2 enhanced the binding of transcription factor zinc finger E-box binding homeobox 1 (ZEB1) to ATM promoter for increasing ATM expression. Moreover, ROCK2 transformed ZEB1 into a gene activator via Yes-associated protein 1 (YAP1). These results provide evidence for the use of ROCK inhibitors in the clinical therapy for MGMTlow TMZ-resistant glioma. Our study also offered novel insights for improving therapeutic management of MGMTlow gliomas.

SCGB3A1 gene DNA methylation status is associated with breast cancer in Egyptian female patients

BackgroundIn recent years, hypermethylation of gene promoters has emerged as one of the fundamental mechanisms for the inactivation of tumor suppressor genes and has a potential role in the early detection of breast cancer. The present study is a case-control study aimed to quantify the methylation levels in the promoters of secretoglobin 3A1 (SCGB3A1), and ataxia-telangiectasia mutated (ATM) genes and evaluate their relation to clinicopathological features of the tumor in a cohort of Egyptian female patients with breast cancer.MethodsGenomic deoxyribonucleic acid (DNA) was extracted from 100 tissue samples, 50 breast cancer tissues and 50 adjacent non-cancerous breast tissues, then, it was subjected to bisulfite conversion. The converted DNA was amplified by real-time PCR; then, pyrosequencing was performed to quantify DNA methylation levels in four CpG sites in ATM and SCGB3A1 gene promoters. The methylation data were presented as the percentage of average methylation of all the observed CpG sites and were calculated for each sample and each gene.ResultsThe percentage of DNA methylation of the SCGB3A1 promoter was significantly higher in the tumor group than in the normal group (P= 0.001). However, a non-statistical significance difference was found in the DNA methylation percentage of the ATM promoter in the tumor group compared to the normal group (P = 0.315). The SCGB3A1 promoter methylation frequency was significantly associated with estrogen receptors (ER) and progesterone receptors (PR) positive tumors, lymph node metastasis, and lymphovascular invasion. However, no association was found between ATM methylation status and the different clinicopathological features of the tumor.ConclusionsThe findings of this work showed that the SCGB3A1 promoter methylation was significantly higher in the tumor group and was significantly associated with different clinicopathologic features in breast cancer. It may be considered as a suitable biomarker for diagnosis and prognosis. However, the promoter methylation levels of the ATM gene in breast cancer cases were unable to distinguish between breast cancer tissues and adjacent normal tissues, and there is no evidence that epigenetic silencing by ATM methylation has a role in breast cancer pathogenesis.

Methylation of the ataxia telangiectasia mutated gene (ATM) promoter as a radiotherapy outcome biomarker in patients with hepatocellular carcinoma.

The goal of this study was to evaluate the contribution of ataxia telangiectasia mutated (ATM) gene promoter methylation to hepatocellular carcinoma (HCC) and the predictive value of radiotherapy outcome. ATM promoter methylation status was detected using methylation-specific PCR in 118 HCC, 50 adjacent liver, and 20 normal liver samples. PCR products were verified by bisulfite sequencing PCR. ATM expression was detected by quantitative PCR (qPCR) and immunohistochemistry (IHC) in 50 paired HCC and adjacent normal tissues and 68 locally advanced HCC biopsy tissues. Furthermore, radiotherapy outcomes in 68 locally advanced HCC patients were determined using European Association for the Study of Liver criteria and survival analysis. The results revealed that the methylation frequency of the ATM promoter was significantly higher in HCC tissues than in normal liver tissues (χ2 = 16.830, P < .001). Quantitative PCR (qPCR) and IHC results showed a significant association between ATM promoter methylation and ATM expression in HCC (χ2 = 10.510, P < .001), and methylated ATM was correlated with lower ATM expression compared with unmethylated ATM (r = 0.356, P < .001). Furthermore, methylation of the ATM promoter was significantly associated with superior outcomes in patients with locally advanced HCC who initially received radiotherapy. Together, these results indicate that ATM promoter methylation might increase the risk of HCC by regulating ATM expression, and thus may function as a potential biomarker for predicting radiotherapy outcomes in HCC patients.

Cellular cytokine receptor signaling and ATM pathway intersections affect hepatic DNA repair

Pathways involving ataxia telangiectasia mutated (ATM) gene and its downstream partners and effectors are critical for the DNA damage response. Cell survival, proliferation and tissue homeostasis are dependent upon preservation of DNA integrity but additional intracellular mechanisms contribute in these processes. As receptor-mediated signaling with beneficial intersections in ATM pathways could have therapeutic significance, we interrogated such intersections with assays using HuH-7 cells (hepatocytes). These cells were subjected to acetaminophen toxicity, which is a leading cause of hepatic injury and acute liver failure in people. The ATM pathway was examined in HuH-7-ATM-Prom-tdT cells containing fluorescent td-Tomato transgene reporter for ATM promoter activity. Titrated doses of specific growth factors were used as ligands for receptor-mediated signaling. The contribution of JAK/STAT3 signaling was defined by the loss-of-function approach with the JAK antagonist, ruxolitinib. In these assays, impairment in ATM-related DNA damage response following acetaminophen toxicity was ameliorated by selected growth factors, including fibroblast growth factors, granulocyte colony stimulating factor and vascular endothelial growth factor. The JAK/STAT3 signaling was exclusive to granulocyte colony stimulating factor but concerned additional pathways in cases of other growth factors. Antagonism of JAK/STAT3 by ruxolitinib abrogated benefits in ATM pathway-mediated DNA repair; and identification of the ruxolitinib-sensitive component of cytoprotection allowed separations of these pathway intersections. Therefore, this subtractive approach for ATM and other regulators in pathways will be informative for DNA damage response. These mechanisms will benefit therapeutic development for ATM-related tissue and organ injuries.

Loss of the tumor suppressor BIN1 enables ATM Ser/Thr kinase activation by the nuclear protein E2F1 and renders cancer cells resistant to cisplatin

The tumor suppressor bridging integrator 1 (BIN1) is a corepressor of the transcription factor E2F1 and inhibits cell-cycle progression. BIN1 also curbs cellular poly(ADP-ribosyl)ation (PARylation) and increases sensitivity of cancer cells to DNA-damaging therapeutic agents such as cisplatin. However, how BIN1 deficiency, a hallmark of advanced cancer cells, increases cisplatin resistance remains elusive. Here, we report that BIN1 inactivates ataxia telangiectasia-mutated (ATM) serine/threonine kinase, particularly when BIN1 binds E2F1. BIN1 + 12A (a cancer-associated BIN1 splicing variant) also inhibited cellular PARylation, but only BIN1 increased cisplatin sensitivity. BIN1 prevented E2F1 from transcriptionally activating the human ATM promoter, whereas BIN1 + 12A did not physically interact with E2F1. Conversely, BIN1 loss significantly increased E2F1-dependent formation of MRE11A/RAD50/NBS1 DNA end-binding protein complex and efficiently promoted ATM autophosphorylation. Even in the absence of dsDNA breaks (DSBs), BIN1 loss promoted ATM-dependent phosphorylation of histone H2A family member X (forming γH2AX, a DSB biomarker) and mediator of DNA damage checkpoint 1 (MDC1, a γH2AX-binding adaptor protein for DSB repair). Of note, even in the presence of transcriptionally active (i.e. proapoptotic) TP53 tumor suppressor, BIN1 loss generally increased cisplatin resistance, which was conversely alleviated by ATM inactivation or E2F1 reduction. However, E2F2 or E2F3 depletion did not recapitulate the cisplatin sensitivity elicited by E2F1 elimination. Our study unveils an E2F1-specific signaling circuit that constitutively activates ATM and provokes cisplatin resistance in BIN1-deficient cancer cells and further reveals that γH2AX emergence may not always reflect DSBs if BIN1 is absent.

ATM expression is attenuated by promoter hypermethylation in human ovarian endometriotic stromal cells.

A number of genes involved in the pathogenesis of endometriosis are silenced by the hypermethylation of their promoter regions. We assessed the effect and mechanism of the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) (10μM) on the cell cycle in human endometriotic cyst stromal cells (ECSCs) and normal endometrial stromal cells (NESCs) by flow cytometry. The DNA methylation status of G2/M checkpoint regulators were investigated by methylation-specific PCR. The expression of ataxia telangiectasia mutated (ATM) and the effect of 5-aza-dC on its expression were also evaluated by quantitative RT-PCR and western blotting analysis. 5-aza-dC treatment resulted in the cell cycle arrest of ECSCs at the G2/M phase. In contrast, 5-aza-dC did not affect the cell cycle of NESCs. The promoter region of the ATM gene was hypermethylated in ECSCs, but not in NESCs. ATM mRNA expression was attenuated in ECSCs compared to that in NESCs. Further, 5-aza-dC was found to restore ATM expression in ECSCs by its promoter demethylation. Our findings indicate that ATM promoter hypermethylation occurs in endometriosis, and that ATM silencing is involved in tumorigenesis during this disease; moreover, selective DNA demethylating agents and molecular target drugs against ATM silencing are promising for the treatment of endometriosis.

Arecoline-regulated ataxia telangiectasia mutated expression level in oral cancer progression.

Ataxia telangiectasia mutated (ATM) regulates DNA repair and cell cycle. The present study analyzed arecoline-induced ATM expression during oral cancer progression. In vitro studies were performed using oral squamous cell carcinoma (OSCC) cell lines treated with arecoline to analyze cell response and ATM regulation. in vivo studies were performed using immunohistochemistry to detect ATM expression in normal, oral potentially malignant disorder (OPMD), and OSCC tissues. Low-dose arecoline induced cell proliferation, ATM promoter activity, and DNA repair. High-dose arecoline induced cell cycle arrest, apoptosis, and DNA damage. ATM was overexpressed in OPMD tissues but was downregulated in OSCC tissues. ATM expression level was associated with the risk of developing dysplasia, buccal-OSCC, and with OSCC survival rate. High ATM expression helps DNA repair mechanisms to maintain the cells in the OPMD stage, but low ATM expression causes DNA damage accumulation to increase cell malignancy.

Polymorphism rs189037C\xa0>\xa0T in the promoter region of the ATM gene may associate with reduced risk of T2DM in older adults in China: a case control study

BackgroundRecent evidence indicates that ataxia telangiectasia mutated (ATM) is a cytoplasmic protein that involves in insulin signaling pathways. When ATM gene is mutated, this event appears to contribute to the development of insulin resistance and type 2 diabetes mellitus (T2DM). Up to date, little information about the relationship between ATM gene polymorphism and T2DM is available. This study aimed to explore potential association between a genetic variant [single nucleotide polymorphism (SNP), i.e. rs189037C > T] in the ATM promoter region and T2DM in older adults in China.MethodsWe conducted a 1:1 age- and sex-matched case-control study. It enrolled 160 patients including 80 type 2 diabetic and 80 nondiabetic patients who were aged 60 years and above. Genotyping of the polymorphism rs189037 in the promoter of the ATM gene was performed using polymerase chain reaction-restriction fragment length polymorphism. Chi-square test or Fisher’s exact test (when an expected cell count was <5) and unpaired Student’s t test were used for categorical and continuous variables, respectively. Logistic regression was used to estimate odds ratio (OR) and 95% confidence interval (CI) with adjustment for factors associated with T2DM.ResultsSignificant association was found between the genotypes of the ATM rs189037 polymorphism and T2DM (P = 0.037). The frequency of CT genotype is much higher in patients without T2DM than in diabetics (60.0% versus 40.0%, P = 0.012). After adjustment of the major confounding factors, such difference remained significant (OR for non-T2DM is 2.62, 95%CI = 1.05–6.53, P = 0.038). Similar effect of CT genotype on T2DM was observed in male population (adjusted: OR = 0.27, 95%CI = 0.09–0.84, P = 0.024). In addition, the percentage of TT genotype in diabetics with coronary artery disease (CAD) was considerably lower than in those without CAD (17.9% versus 61.5%, P = 0.004).ConclusionsOur study suggests that the ATM rs189037 polymorphism is associated with reduced risk of T2DM in older adult population in China. Specifically, CT heterozygote seems to be associated with a lower risk of T2DM than CC or TT genotype, especially in male older adults. Moreover, TT genotype may reduce the risk of CAD in diabetic patients.

Promoter Hypermethylation of the ATM Gene as a Novel Biomarker for Breast Cancer

Background:Breast cancer may be induced by activation of protooncogenes to oncogenes and in many cases inactivation of tumor suppressor genes. Ataxia telangiectasia mutated (ATM) is an important tumor suppressor gene which plays central roles in the maintenance of genomic integrity by activating cell cycle checkpoints and promoting repair of double-strand breaks of DNA. In breast cancer, decrease ATM expression correlates with a poor outcome; however, the molecular mechanisms underlying downregulation are still unclear. Promoter hypermethylation may contribute in downregulation. Hence the present investigation was designed to evaluate promoter methylation and expression of the ATM gene in breast cancer cases, and to determine links with clinical and demographic manifestations, in a South Indian population.Methods:Tumor biopsy samples were collected from 50 pathologically confirmed sporadic breast cancer cases. DNA was isolated from tumor and adjacent non-tumorous regions, and sodium bisulfite conversion and methylation-specific PCR were performed using MS-PCR primers for the ATM promoter region. In addition, ATM mRNA expression was also analyzed for all samples using real-time PCR.Results:Fifty eight percent (58%) of cancer tissue samples showed promoter hypermethylation for the ATM gene, in contrast to only 4.44% of normal tissues (p= 0.0001). Furthermore, ATM promoter methylation was positively associated with age (p = 0.01), tumor size (p=0.045) and advanced stage of disease i.e. stages III and IV (p =0.019). An association between promoter hypermethylation and lower expression of ATM mRNA was also found (p=0.035).Conclusion:We report for the first time that promoter hypermethylation of ATM gene may be useful as a potential new biomarker for breast cancer, especially in the relatively young patients.

Linking ATM Promoter Methylation to Cell Cycle Protein Expression in Brain Tumor Patients: Cellular Molecular Triangle Correlation in ATM Territory

Ataxia telangiectasia mutated (ATM) is a key gene in DNA double-strand break (DSB), and therefore, most of its disabling genetic alterations play an important initiative role in many types of cancer. However, the exact role of ATM gene and its epigenetic alterations, especially promoter methylation in different grades of brain tumors, remains elusive. The current study was conducted to query possible correlations among methylation statue of ATM gene, ATM/ retinoblastoma (RB) protein expression, D1853N ATM polymorphism, telomere length (TL), and clinicopathological characteristics of various types of brain tumors. Isolated DNA from 30 fresh tissues was extracted from different types of brain tumors and two brain tissues from deceased normal healthy individuals. DNAs were treated with bisulfate sodium using DNA modification kit (Qiagen). Methylation-specific polymerase chain reaction (MSP-PCR) was implicated to determine the methylation status of treated DNA templates confirmed by promoter sequencing. Besides, the ATM and RB protein levels were determined by immunofluorescence (IF) assay using monoclonal mouse antihuman against ATM, P53, and RB proteins. To achieve an interactive correlation, the methylation data were statistically analyzed by considering TL and D1853N ATM polymorphism. More than 73% of the brain tumors were methylated in ATM gene promoter. There was strong correlation between ATM promoter methylation and its protein expression (p < 0.001). As a triangle, meaningful correlation was also found between methylated ATM promoter and ATM protein expression with D1853N ATM polymorphism (p = 0.01). ATM protein expression was not in line with RB protein expression while it was found to be significantly correlated with ATM promoter methylation (p = 0.01). There was significant correlation between TL neither with ATM promoter methylation nor with ATM protein expression nor with D1853N polymorphism. However, TL has shown strong correlation with patient's age and tumor grade (p = 0.01). Given the important role of cell cycle checkpoint proteins as well as RB and ATM in TL and cancer evolution, further assessment is warranted to shed more light on the pathway linking the telomere instability to tumor progression. High ATM methylation rate in brain tumor patients could open a new avenue toward early screening and cancer therapy.

Evaluation of cytotoxicity and DNA damage response with analysis of intracellular ATM signaling pathways.

Maintenance of genome integrity by preventing and overcoming DNA damage is critical for cell survival. Deficiency or aberrancy in the DNA damage response, for example, through ataxia telangiectasia mutated (ATM) signaling, lead to pathophysiological perturbations in organs throughout the body. Therefore, control of DNA damage is of major interest for development of therapeutic agents. Such efforts will greatly benefit from convenient and simple diagnostic and/or drug development tools to demonstrate whether ATM and related genes have been activated and to then determine whether these have been returned to normal levels of activity because pathway members sense and also repair DNA damage. To overcome difficulties in analyzing differences in multitudinous ATM pathway members following DNA damage, we measured ATM promoter activity with a fluorescent td-Tomato reporter gene to interrogate the global effects of ATM signaling pathways. In cultured HuH-7 cell line derived from human hepatocellular carcinoma, cis-platinum, acetaminophen, or hydrogen peroxide caused DNA strand breaks and ATM pathway activation as shown by γH2AX expression, which in turn, led to rapid and sustained increases in ATM promoter activity. This assay of ATM promoter activity identified biological agents capable of controlling cellular DNA damage in toxin-treated HuH-7 cells and in mice after onset of drug-induced acute liver failure. Therefore, the proposed assay of ATM promoter activity in HuH-7 cells was appropriately informative for treating DNA damage. High-throughput screens using ATM promoter activation will be helpful for therapeutic development in DNA damage-associated abnormal ATM signaling in various cell types and organs.

Abstract 1312: ATM transcriptional regulation mediated by BRCA1/E2F1 axis controls DNA damage response in prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death affecting men worldwide. Chromosomal instability is a key feature in PCa progression. Using genome-wide screen for factor binding in combination with expression profiles, we previously reported BRCA1 binding to several promoters involved in cell cycle regulation and DNA damage response. We also generated a BRCA1 depleted xenograft model in order to study the role of BRCA1 role in DNA damage response in vivo. We demonstrated that BRCA1 expression status plays a central role in doxorubicin resistance in PCa. One of the BRCA1 targets that emerged from these analyses was ATM (ataxia telangiectasia mutated).Although it is well accepted that the kinase protein ATM is a pivotal mediator in genotoxic stress, it is unknown if its transcriptional regulation plays a role in the DNA damage response. Our goal was to investigate ATM transcription regulation in PCa under different genotoxic insults. Here we exposed PC3 cells to different genotoxic agents and the ATM promoter activity was determined by a luciferase reporter assay. We found that the topoisomerase II inhibitors doxorubicin and mitoxantrone repressed ATM transcription; however etoposide and methotrexate did not show significant changes. Using BRCA1-overexpressing PC3 cell lines, we found that BRCA1 increases ATM mRNA and promoter activity. Accordingly, BRCA1 depletion by shRNA abolished ATM transcription induction. Furthermore, BRCT domain loss (BRCA1ΔBRCT) impaired the ability of BRCA1 to regulate ATM promoter activity, strongly suggesting that BRCT domain is essential for ATM regulation. Xenograft tumors generated by BRCA1 depleted PC3 cells injected in nu/nu mice demonstrated that BRCA1 knock-down abolished ATM transcriptional induction. Considering ATM phosphorylates BRCA1, we investigated BRCA's ability to activate ATM promoter after inhibition of the ATM kinase activity by KU55933. BRCA1 overexpressing PC3 cells exposed to KU55933 showed significant decreased ATM promoter activity compared to control cells suggesting ATM regulation by BRCA1 is not mediated by ATM kinase activity. In addition, we performed BRCA1-ChIP-qPCR using primers spanning every 500bp along ATM promoter; we found that BRCA1 binds at 500bp upstream of the ATM transcription start site which was disrupted by doxorubicin. We identified one E2F1 putative DNA binding site at this region suggesting that BRCA1 association to ATM promoter could be E2F1 mediated. Finally, E2F1 transfection in PC3 cells significant decreased ATM transcription which was impaired by E2F1 dominant negative (E1-363). In summary, BRCA1/E2F1 complex binds and induces ATM transcription. After genotoxic stress BRCA1 protein is displaced from the ATM promoter and E2F1 downregulates ATM transcription. Thus, BRCA1/E2F1 axis controls DNA damage response in PCa through ATM transcriptional regulation. 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 1312. doi:1538-7445.AM2012-1312

Dynamic Coregulatory Complex Containing BRCA1, E2F1 and CtIP Controls ATM Transcription

Chromosomal instability is a key feature in cancer progression. Recently we have reported that BRCA1 regulates the transcription of several genes in prostate cancer, including ATM (ataxia telangiectasia mutated). Although it is well accepted that ATM is a pivotal mediator in genotoxic stress, it is unknown whether ATM transcription is regulated during the molecular response to DNA damage. Here we investigate ATM transcription regulation in human prostate tumor PC3 cell line. We have found that doxorubicin and mitoxantrone repress ATM transcription in PC3 cells but etoposide and methotrexate do not affect ATM expression. We have demonstrated that BRCA1 binds to ATM promoter and after doxorubicin exposure, it is released. BRCA1 overexpression increases ATM transcription and this enhancement is abolished by BRCA1 depletion. Moreover, BRCA1-BRCT domain loss impairs the ability of BRCA1 to regulate ATM promoter activity, strongly suggesting that BRCT domain is essential for ATM regulation by BRCA1. BRCA1-overexpressing PC3 cells exposed to KU55933 ATM kinase inhibitor showed significant decreased ATM promoter activity compared to untreated cells, suggesting that ATM transcriptional regulation by BRCA1 is partially mediated by the ATM kinase activity. In addition, we have demonstrated E2F1 binding to ATM promoter before and after doxorubicin exposure. E2F1 overexpression diminishes ATM transcription after doxorubicin exposure which is impaired by E2F1 dominant negative mutants. Finally, the co-regulator of transcription CtIP increases ATM transcription. CtIP increases ATM transcription. Altogether, BRCA1/E2F1/CtIP binding to ATM promoter activates ATM transcription. Doxorubicin exposure releases BRCA1 and CtIP from ATM promoter still keeping E2F1 recruited and, in turn, represses ATM expression.

Modulation of DNA Repair Capacity by Ataxia Telangiectasia Mutated Gene Polymorphisms among Polycyclic Aromatic Hydrocarbons–Exposed Workers

The purpose of this study was to address the association between the ataxia telangiectasia mutated (ATM) gene polymorphisms and susceptibility to DNA repair capacity (DRC) among polycyclic aromatic hydrocarbons (PAHs)-exposed workers. Polymorphisms of ATM were genotyped. DRC was determined by comet assay. Chromosomal damage was detected by cytokinesis-block micronucleus (CBMN) assay. Flow cytometry was used to detect the distributions of cell cycle. Expressions of ATM and rH2AX were determined by immunoblotting analysis. Luciferase assays were performed to determine the functional difference of ATM promoter region allele. Subjects carrying T allele of rs228589 had significantly lower DRC compared with those with AA genotype. Subjects carrying G allele of rs652311 had significantly lower DRC than those with zero copy number of haplotype CGGT. SH ataxia telangiectasia mutated (SHATM) cells had significantly lower DRC than SH green fluorescent protein (SHGFP) cells induced by bleomycin and higher CBMN frequencies treated by benzo(a)pyrene [B(a)P] than SHGFP cells. After B(a)P treatment, a decrease in the percentage of G1 phase cells was observed in SHATM cells compared with SHGFP cells, rH2AX expressions were increased in SHATM cells and SHGFP cells, but ATM expressions had no change in 16HBE-SHGFP cells and HEK-SHGFP cells. Luciferase expression was not different between rs228589T and rs228589A plasmid constructs. In conclusions, it is suggested that ATM polymorphisms are associated with DRC among PAHs-exposed workers and ATM plays key roles in repair of chromosomal damage and cell cycle control with the treatment of B(a)P.

HMGA proteins promote ATM expression and enhance cancer cell resistance to genotoxic agents

DNA-damaging therapies represent a keystone in cancer treatment. Unfortunately, many tumors often relapse because of a group of cancer cells, which are resistant to conventional therapies. High-mobility group A (HMGA) proteins has a key role in cell transformation, and their overexpression is a common feature of human malignant neoplasias, representing a poor prognostic index often correlated to anti-cancer drug resistance. Our previous results demonstrated that HMGA1 is a substrate of ataxia-telangiectasia mutated (ATM), the main cellular sensor of genotoxic stress. Here we also report thatHMGA2, the other member of the HMGA family, is a novel substrate of ATM. Interestingly, we found that HMGA proteins positively regulate ATM gene expression. Moreover, induction of ATM kinase activity by DNA-damaging agents enhances HMGA-dependent transcriptional activation of ATM promoter, suggesting that ATM expression is modulated by a DNA-damage- and HMGA-dependent positive feedback loop. Finally, inhibition of HMGA expression in mouse embryonic fibroblasts and in cancer cells strongly reduces ATM protein levels, impairing the cellular DNA-damage response and enhancing the sensitivity to DNA-damaging agents. These findings indicate this novel HMGA-ATM pathway as a new potential target to improve the effectiveness of conventional anti-neoplastic treatments on the genotoxic-drug resistant cancer cells.

Phospho-ΔNp63α/Rpn13-dependent regulation of LKB1 degradation modulates autophagy in cancer cells.

Oxidative stress was shown to promote the translocation of Ataxia-telangiectasia mutated (ATM) to cytoplasm and trigger the LKB1-AMPK-tuberin pathway leading to a down-regulation of mTOR and subsequently inducing the programmed cell death II (autophagy). Cisplatin was previously found to induce the ATM-dependent phosphorylation of ΔNp63α in squamous cell carcinoma (SCC) cells. In this study, phosphorylated (p)-ΔNp63α was shown to bind the ATM promoter, to increase the ATM promoter activity and to enhance the ATM cytoplasmic accumulation. P-ΔNp63α protein was further shown to interact with the Rpn13 protein leading to a proteasome-dependent degradation of p-ΔNp63α and thereby protecting LKB1 from the degradation. In SCC cells (with an altered ability to support the ATM-dependent ΔNp63α phosphorylation), the non-phosphorylated ΔNp63α protein failed to form protein complexes with the Rpn13 protein and thereby allowing the latter to bind and target LKB1 into a proteasome-dependent degradation pathway thereby modulating a cisplatin-induced autophagy. We thus suggest that SCC cells sensitive to cisplatin-induced cell death are likely to display a greater ratio of p-ΔNp63α/non-phosphorylated ΔNp63α than cells with the innate resistant/impaired response to a cisplatin-induced cell death. Our data also suggest that the choice made by Rpn13 between p-ΔNp63α or LKB1 to be targeted for degradation is critical for cell death decision made by cancer cells in response to chemotherapy.

Down‐regulation of ATM protein in HRS cells of nodular sclerosis Hodgkin's lymphoma in children occurs in the absence of ATM gene inactivation

The tumour component of classical Hodgkin's lymphoma (cHL), Hodgkin Reed-Sternberg (HRS) cells, are believed to be derived from germinal centre (GC) B cells but intriguingly display a characteristic loss of B cell receptor (BCR) expression. The precise mechanisms by which BCR-negative HRS cell progenitors survive negative selection during the GC reaction remain obscure. Individuals with ataxia telangiectasia, caused by biallelic inactivation of the DNA damage response gene, ataxia telangiectasia mutated (ATM), have a higher risk of cHL development. Here we show that, in contrast to normal GC B cells that expressed low but detectable ATM protein, ATM protein was not detected in HRS cells of 17/18 cases of paediatric cHL, all but one with nodular sclerosis (NS) subtype. A comprehensive analysis of the ATM gene in microdissected HRS cells of nine representative tumours showed no evidence of either loss of heterozygosity or consistent pathogenic mutations. Furthermore, bisulphite sequencing of the ATM promoter from HRS cells of five tumours also revealed the absence of hypermethylation. Since our microarray data suggested significantly reduced ATM transcription in HRS cells compared to GC B cells, we conclude that loss of ATM expression could be the result of alterations in upstream regulators of ATM transcription. Importantly, ATM loss in paediatric cHLs has clinical implications and could be potentially exploited to guide future, less toxic, tumour-specific treatments.

Down-regulation of ATM Protein Sensitizes Human Prostate Cancer Cells to Radiation-induced Apoptosis

Treatment with the protein kinase C activator 12-O-tetradecanoylphorbol 12-acetate (TPA) enables radiation-resistant LNCaP human prostate cancer cells to undergo radiation-induced apoptosis, mediated via activation of the enzyme ceramide synthase (CS) and de novo synthesis of the sphingolipid ceramide (Garzotto, M., Haimovitz-Friedman, A., Liao, W. C., White-Jones, M., Huryk, R., Heston, D. W. W., Cardon-Cardo, C., Kolesnick, R., and Fuks, Z. (1999) Cancer Res. 59, 5194-5201). Here, we show that TPA functions to decrease the cellular level of the ATM (ataxia telangiectasia mutated) protein, known to repress CS activation (Liao, W.-C., Haimovitz-Friedman, A., Persaud, R., McLoughlin, M., Ehleiter, D., Zhang, N., Gatei, M., Lavin, M., Kolesnick, R., and Fuks, Z. (1999) J. Biol. Chem. 274, 17908-17917). Gel shift analysis in LNCaP and CWR22-Rv1 cells demonstrated a significant reduction in DNA binding of the Sp1 transcription factor to the ATM promoter, and quantitative reverse transcription-PCR showed a 50% reduction of ATM mRNA between 8 and 16 h of TPA treatment, indicating that TPA inhibits ATM transcription. Furthermore, treatment of LNCaP, CWR22-Rv1, PC-3, and DU-145 human prostate cells with antisense-ATM oligonucleotides, which markedly reduced cellular ATM levels, significantly enhanced radiation-induced CS activation and apoptosis, leading to apoptosis at doses as a low as 1 gray. These data suggest that the CS pathway initiates a generic mode of radiation-induced apoptosis in human prostate cancer cells, regulated by a suppressive function of ATM, and that ATM might represent a potential target for pharmacologic inactivation with potential clinical applications in human prostate cancer.