Depletion of ATR selectively sensitizes ATM-deficient human mammary epithelial cells to ionizing radiation and DNA-damaging agents.

DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G2/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G2/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G2/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G2/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.

Genome-wide small RNA sequencing and gene expression analysis reveals a microRNA profile of cancer susceptibility in ATM-deficient human mammary epithelial cells.

Deficiencies in the ATM gene are the underlying cause for ataxia telangiectasia, a syndrome characterized by neurological, motor and immunological defects, and a predisposition to cancer. MicroRNAs (miRNAs) are useful tools for cancer profiling and prediction of therapeutic responses to clinical regimens. We investigated the consequences of ATM deficiency on miRNA expression and associated gene expression in normal human mammary epithelial cells (HME-CCs). We identified 81 significantly differentially expressed miRNAs in ATM-deficient HME-CCs using small RNA sequencing. Many of these have been implicated in tumorigenesis and proliferation and include down-regulated tumor suppressor miRNAs, such as hsa-miR-29c and hsa-miR-16, as well as over-expressed pro-oncogenic miRNAs, such as hsa-miR-93 and hsa-miR-221. MicroRNA changes were integrated with genome wide gene expression profiles to investigate possible miRNA targets. Predicted mRNA targets of the miRNAs significantly regulated after ATM depletion included many genes associated with cancer formation and progression, such as SOCS1 and the proto-oncogene MAF. While a number of miRNAs have been reported as altered in cancerous cells, there is little understanding as to how these small RNAs might be driving cancer formation or how they might be used as biomarkers for cancer susceptibility. This study provides preliminary data for defining miRNA profiles that may be used as prognostic or predictive biomarkers for breast cancer. Our integrated analysis of miRNA and mRNA expression allows us to gain a better understanding of the signaling involved in breast cancer predisposition and suggests a mechanism for the breast cancer-prone phenotype seen in ATM-deficient patients.

Dissecting cellular responses to irradiation via targeted disruptions of the ATM-CHK1-PP2A circuit.

Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints and promoting DNA damage repair. The phosphoinositide 3-kinase-related kinases (PIKKs) ataxia telangiectasia-mutated (ATM), ATM and Rad 3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are crucial for sensing lesions and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anticancer regimens. This study employed cell lines stably depleted for CHK1, ATM or both for dissecting cross-talk and compensatory effects on G(2)/M checkpoint in response to ionizing radiation (IR). We show that a 90% depletion of CHK1 renders cells radiosensitive without abrogating their IR-mediated G(2)/M checkpoint arrest. ATM phosphorylation is enhanced in CHK1-deficient cells compared with their wild-type counterparts. This correlates with lower nuclear abundance of the PP2A catalytic subunit in CHK1-depleted cells. Stable depletion of CHK1 in an ATM-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels and resulted in an additive attenuation of the G(2)/M checkpoint response compared with the individual knockdowns. ATM inhibition and 90% CHK1 depletion abrogated the early G(2)/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time points. Our data indicates that dual targeting of ATM and CHK1 functionalities disrupts the compensatory response to DNA damage and could be exploited for developing efficient anti-neoplastic treatments.

DNA damage activates a complex transcriptional response in murine lymphocytes that includes both physiological and cancer-predisposition programs.

BACKGROUND: Double strand (ds) DNA breaks are a form of DNA damage that can be generated from both genotoxic exposures and physiologic processes, can disrupt cellular functions and can be lethal if not repaired properly. Physiologic dsDNA breaks are generated in a variety of normal cellular functions, including the RAG endonuclease-mediated rearrangement of antigen receptor genes during the normal development of lymphocytes. We previously showed that physiologic breaks initiate lymphocyte development-specific transcriptional programs. Here we compare transcriptional responses to physiological DNA breaks with responses to genotoxic DNA damage induced by ionizing radiation. RESULTS: We identified a central lymphocyte-specific transcriptional response common to both physiologic and genotoxic breaks, which includes many lymphocyte developmental processes. Genotoxic damage causes robust alterations to pathways associated with B cell activation and increased proliferation, suggesting that genotoxic damage initiates not only the normal B cell maturation processes but also mimics activated B cell response to antigenic agents. Notably, changes including elevated levels of expression of Kras and mmu-miR-155 and the repression of Socs1 were observed following genotoxic damage, reflecting induction of a cancer-prone phenotype. CONCLUSIONS: Comparing these transcriptional responses provides a greater understanding of the mechanisms cells use in the differentiation between types of DNA damage and the potential consequences of different sources of damage. These results suggest genotoxic damage may induce a unique cancer-prone phenotype and processes mimicking activated B cell response to antigenic agents, as well as the normal B cell maturation processes.

Rapid and transient recruitment of DNMT1 to DNA double-strand breaks is mediated by its interaction with multiple components of the DNA damage response machinery.

DNA methylation is an epigenetic mark critical for regulating transcription, chromatin structure and genome stability. Although many studies have shed light on how methylation impacts transcription and interfaces with the histone code, far less is known about how it regulates genome stability. We and others have shown that DNA methyltransferase 1 (DNMT1), the maintenance methyltransferase, contributes to the cellular response to DNA damage, yet DNMT1's exact role in this process remains unclear. DNA damage, particularly in the form of double-strand breaks (DSBs), poses a major threat to genome integrity. Cells therefore possess a potent system to respond to and repair DSBs, or to initiate cell death. In the current study, we used a near-infrared laser microirradiation system to directly study the link between DNMT1 and DSBs. Our results demonstrate that DNMT1 is rapidly but transiently recruited to DSBs. DNMT1 recruitment is dependent on its ability to interact with both PCNA and the ATR effector kinase CHK1, but is independent of its catalytic activity. In addition, we show for the first time that DNMT1 interacts with the 9-1-1 PCNA-like sliding clamp and that this interaction also contributes to DNMT1 localization to DNA DSBs. Finally, we demonstrate that DNMT1 modulates the rate of DSB repair and is essential for suppressing abnormal activation of the DNA damage response in the absence of exogenous damage. Taken together, our studies provide compelling additional evidence for DNMT1 acting as a regulator of genome integrity and as an early responder to DNA DSBs.

Triuret as a potential hypokalemic agent: Structure characterization of triuret and triuret-alkali metal adducts by mass spectrometric techniques.

Triuret (also known as carbonyldiurea, dicarbamylurea, or 2,4-diimidotricarbonic diamide) is a byproduct of purine degradation in living organisms. An abundant triuret precursor is uric acid, whose level is altered in multiple metabolic pathologies. Triuret can be generated via urate oxidation by peroxynitrite, the latter being produced by the reaction of nitric oxide radical with superoxide radical anion. From this standpoint, an excess production of superoxide radical anions could indirectly favor triuret formation; however very little is known about the potential in vivo roles of this metabolite. Triuret's structure is suggestive of its ability to adopt various conformations and act as a flexible ligand for metal ions. In the current study, HPLC-MS/MS, energy-resolved mass spectrometry, selected ion monitoring, collision-induced dissociation, IRMPD spectroscopy, Fourier transform-ion cyclotron resonance mass spectrometry and computational methods were employed to characterize the structure of triuret and its metal complexes, to determine the triuret-alkali metal binding motif, and to evaluate triuret affinity toward alkali metal ions, as well as its affinity for Na(+) and K(+) relative to other organic ligands. The most favored binding motif was determined to be a bidentate chelation of triuret with the alkali metal cation involving two carbonyl oxygens. Using the complexation selectivity method, it was observed that in solution triuret has an increased affinity for potassium ions, compared to sodium and other alkali metal ions. We propose that triuret may act as a potential hypokalemic agent under pathophysiological conditions conducive to its excessive formation and thus contribute to electrolyte disorders. The collision- or photo-induced fragmentation channels of deprotonated and protonated triuret, as well as its alkali metal adducts, are likely to mimic the triuret degradation pathways in vivo.

Targeting retinal and choroid neovascularization using the small molecule inhibitor carboxyamidotriazole.

Neovascular ocular diseases as exemplified by proliferative diabetic retinopathy (PDR), exudative age-related macular degeneration (AMD), and retinopathy of prematurity (ROP) are severe diseases affecting all age groups in the US. We asked whether a small molecule, carboxyamidotriazole (CAI) known for its anti-angiogenic and anti-tumor effects and its ability to be administered orally in humans, could have anti-angiogenic effects in ocular in vitro and in vivo angiogenesis models. The anti-proliferative effects of CAI were examined by BrdU incorporation using human retinal and dermal endothelial cells and human pigment epithelial cells. The effect of CAI was determined using the Matrigel tube formation assay. The mouse model of choroidal neovascularization (CNV) initiated by laser rupture of Bruch's membrane was used to quantify in vivo effects of aqueous beta-hydroxypropyl cyclodextrin (bHPCD) formulations of CAI on neovascularization. The pharmacokinetics (PK) of CAI after intravitreal administration of bHPCD-CAI was studied in rabbit. The intravitreal toxicology of bHPCD-CAI was also examined in rat ocular tissue. We observed that CAI treatment of human endothelial cells decreased cell proliferation in a dose-dependent manner. In the in vivo tests bHPCD-CAI treatment reduced choroidal neovascular lesion volume, also in a dose-dependent manner. The intravitreal PK of bHPCD-CAI demonstrated that highly efficacious concentrations of CAI are reached in the vitreous compartment. No ocular toxicology was observed with intravitreous injection of CAI. These studies support the potential of developing intravitreal CAI in an bHPCD ocular formulation for treatment of proliferative retinopathies in humans.

Despite increased ATF4 binding at the C/EBP-ATF composite site following activation of the unfolded protein response, system A transporter 2 (SNAT2) transcription activity is repressed in HepG2 cells.

The activated amino acid response (AAR) and unfolded protein response (UPR) stress signaling pathways converge at the phosphorylation of translation initiation factor eIF2alpha. This eIF2alpha modification suppresses global protein synthesis but enhances translation of selected mRNAs such as that for activating transcription factor 4 (ATF4). An ATF4 target gene, SNAT2 (system A sodium-dependent neutral amino acid transporter 2), contains a C/EBP-ATF site that binds ATF4 and triggers increased transcription during the AAR. However, the present studies show that despite increased ATF4 binding to the SNAT2 gene during UPR activation in HepG2 human hepatoma cells, transcription activity was not enhanced. Hyperacetylation of histone H3 and recruitment of the general transcription factors at the HepG2 SNAT2 promoter occurred in response to the AAR but not the UPR. In contrast, the UPR did enhance transcription from a plasmid-based reporter gene driven by a SNAT2 genomic fragment containing the C/EBP-ATF site. Simultaneous activation of the AAR and the UPR pathways revealed that the UPR actually suppressed the increased SNAT2 transcription by the AAR pathway, demonstrating that the UPR pathway generates a repressive signal that acts downstream of ATF4 binding.

Nonpeptide somatostatin receptor agonists specifically target ocular neovascularization via the somatostatin type 2 receptor.

PURPOSE: To define the molecular pharmacology underlying the antiangiogenic effects of nonpeptide imidazolidine-2,4-dione somatostatin receptor agonists (NISAs) and evaluate the efficacy of NISA in ocular versus systemic delivery routes in ocular disease models. METHODS: Functional inhibitory effects of the NISAs and the somatostatin peptide analogue octreotide were evaluated in vitro by chemotaxis, proliferation, and tube-formation assays. The oxygen-induced retinopathy (OIR) model and the laser model of choroidal neovascularization (CNV) were used to test the in vivo efficacy of NISAs. Transscleral permeability of a candidate NISA was also measured. RESULTS: NISAs inhibited growth factor-induced HREC proliferation, migration and tube formation with submicromolar potencies (IC(50), 0.1-1.0 microM) comparable to octreotide. In the OIR model, systemic administration of the NISAs RFE-007 and RFE-011 inhibited retinal neovascularization in a dose-dependent manner, comparable to octreotide. In the CNV model, intravitreal RFE-011 resulted in a 56% reduction (P < 0.01) in CNV lesion area, whereas systemic administration resulted in a 35% reduction (P < 0.05) in lesion area. RFE-011 demonstrated transscleral penetration. CONCLUSIONS: Micromolar concentrations of octreotide and NISAs are necessary for antiangiogenic effects, whereas nanomolar concentrations are effective for endocrine inhibition. This suggests that the antiangiogenic activity of NISAs and octreotide is mediated by an overall much less efficient downstream coupling mechanism than is growth hormone release. As a result, the intravitreal or transscleral route of administration should be seriously considered for future clinical studies of SSTR2 agonists used for treatment of ocular neovascularization to ensure efficacious concentrations in the target retinal and choroidal tissue.

DNA methylation inhibitor 5-Aza-2'-deoxycytidine induces reversible genome-wide DNA damage that is distinctly influenced by DNA methyltransferases 1 and 3B.

Genome-wide DNA methylation patterns are frequently deregulated in cancer. There is considerable interest in targeting the methylation machinery in tumor cells using nucleoside analogs of cytosine, such as 5-aza-2'-deoxycytidine (5-azadC). 5-azadC exerts its antitumor effects by reactivation of aberrantly hypermethylated growth regulatory genes and cytoxicity resulting from DNA damage. We sought to better characterize the DNA damage response of tumor cells to 5-azadC and the role of DNA methyltransferases 1 and 3B (DNMT1 and DNMT3B, respectively) in modulating this process. We demonstrate that 5-azadC treatment results in growth inhibition and G(2) arrest-hallmarks of a DNA damage response. 5-azadC treatment led to formation of DNA double-strand breaks, as monitored by formation of gamma-H2AX foci and comet assay, in an ATM (ataxia-telangiectasia mutated)-dependent manner, and this damage was repaired following drug removal. Further analysis revealed activation of key strand break repair proteins including ATM, ATR (ATM-Rad3-related), checkpoint kinase 1 (CHK1), BRCA1, NBS1, and RAD51 by Western blotting and immunofluorescence. Significantly, DNMT1-deficient cells demonstrated profound defects in these responses, including complete lack of gamma-H2AX induction and blunted p53 and CHK1 activation, while DNMT3B-deficient cells generally showed mild defects. We identified a novel interaction between DNMT1 and checkpoint kinase CHK1 and showed that the defective damage response in DNMT1-deficient cells is at least in part due to altered CHK1 subcellular localization. This study therefore greatly enhances our understanding of the mechanisms underlying 5-azadC cytotoxicity and reveals novel functions for DNMT1 as a component of the cellular response to DNA damage, which may help optimize patient responses to this agent in the future.

Epigenetic control of tumor suppression.

Epigenetic silencing of tumor suppressor genes is a major contributor to neoplastic transformation and is an area of intense research. Identification of genes that undergo cancer-specific CpG island hypermethylation in combination with repressive histone tail modifications (deacetylation and methylation) and correlation of these data with tumor stage, progression, and long-term prognosis are becoming increasingly common. The efforts directed toward elucidating the mechanisms of neoplastic tumor suppression catalyzed the convergence of epigenetics, chromatin remodeling, and pharmacology of epigenome-altering drugs. This review discusses the key findings and current concepts concerning the epigenetic control of tumor suppression and analyzes the role of DNA hypermethylation in conjunction with histone deacetylation and methylation profiles of tumor suppressor genes as it relates to epigenetic loss of function in malignancy. Examples arguing for hierarchic control and interdependent regulation within the cellular tumor suppression networks are also presented. Finally, the necessity of a human epigenome database integrating the continually produced experimental information for use by both researchers and clinicians for prospective translational multidisciplinary studies of tumor suppressor networks is rationalized.

Medical treatment of diabetic retinopathy with somatostatin analogues.

Traditional management strategies for retinal neovascularisation accompanying proliferative diabetic retinopathy include photocoagulation laser therapy. The development of preventative pharmacological treatments aimed at replacing or delaying this acute intervention has been an active research area and somatostatin analogues have shown promise in reducing the progression of retinal vascular pathologies. This review summarises the present knowledge on the molecular and cellular mechanisms of neovascularisation, and the rationale for the therapeutic use of somatostatin analogues as well as the results of two key recent clinical trials using octreotide. The potential use of octreotide and other somatostatin analogues in reducing the risk of severe visual impairment in proliferative diabetic retinopathy is discussed and pharmacological treatment regimens are proposed as an additional strategy or a less invasive alternative to laser therapy.

Characterization of the amino acid response element within the human sodium-coupled neutral amino acid transporter 2 (SNAT2) System A transporter gene.

The neutral amino acid transport activity, System A, is enhanced by amino acid limitation of mammalian cells. Of the three gene products that encode System A activity, the one that exhibits this regulation is SNAT2 (sodium-coupled neutral amino acid transporter 2). Fibroblasts that are deficient in the amino acid response pathway exhibited little or no induction of SNAT2 mRNA. Synthesis of SNAT2 mRNA increased within 1-2 h after amino acid removal from HepG2 human hepatoma cells. The amino acid responsive SNAT2 genomic element that mediates the regulation has been localized to the first intron. Increased binding of selected members of the ATF (activating transcription factor) and C/EBP (CCAAT/enhancer-binding protein) families to the intronic enhancer was established both in vitro and in vivo. In contrast, there was no significant association of these factors with the SNAT2 promoter. Expression of exogenous individual ATF and C/EBP proteins documented that specific family members are associated with either activation or repression of SNAT2 transcription. Chromatin immunoprecipitation analysis established in vivo that amino acid deprivation led to increased RNA polymerase II recruitment to the SNAT2 promoter.

Transcriptional control of the human sodium-coupled neutral amino acid transporter system A gene by amino acid availability is mediated by an intronic element.

System A amino acid transporter (SNAT2) gene expression is up-regulated at the transcriptional level in response to amino acid deprivation. Functional analysis of genomic fragments 5' upstream of the transcription start site, for both human and mouse SNAT2 genes showed that these regions exhibit promoter activity, but were amino acid unresponsive. However, when the human and mouse constructs were extended to include intron 1, it was observed that the rate of transcription was increased following amino acid deprivation. Deletion analysis of the human gene identified an intron 1 sequence spanning 54 nucleotides that was sufficient for conferring amino acid-dependent regulation to a minimal SNAT2 promoter. Alignment of the corresponding region from the human, mouse, and rat genomes revealed three highly conserved sequences. From site-directed mutagenesis, it was concluded that one of these sites functions as an amino acid response element (AARE) to regulate transcription. The core sequence of this site is identical to the AARE in the human CHOP gene. The SNAT2 AARE, along with a nearby conserved CAAT box, has enhancer activity in that it functions in an orientation and position independent manner, and it confers regulated transcription to a heterologous promoter.

The mechanism for transcriptional activation of the human ATA2 transporter gene by amino acid deprivation is different than that for asparagine synthetase.

After amino acid deprivation, the mRNA content for both asparagine synthetase (AS) and the system A transporter ATA2 is increased. The purpose of the reported experiments was to characterize the molecular mechanism for the ATA2 gene and to contrast the ATA2 regulatory characteristics with those of AS. Amino acid limitation was initiated by incubation of HepG2 human hepatoma cells in either amino acid-free Krebs-Ringer bicarbonate buffer or culture medium lacking the single amino acid histidine. For ATA2, like AS, the elevated mRNA content was due to increased transcription. However, there were fundamental differences between the mechanisms for nutrient regulation of the AS and ATA2 genes. When cells were deprived of amino acids, there was a lag period of approximately 4 h before an increase in AS mRNA occurred, whereas the elevation of ATA2 mRNA was readily detectable at 2-4 h. Consistent with these observations, de novo protein synthesis was absolutely required for the activation of the AS gene, but the increase in ATA2 mRNA was largely independent of protein synthesis. Furthermore, in contrast to AS, transcription from the ATA2 gene was not increased by glucose deprivation. Given this lack of ATA2 transcriptional activation by glucose starvation and that the induction of the AS gene by glucose or amino acid starvation is mediated by common genomic elements, it is likely that the ATA2 gene does not contain the same genomic amino acid-responsive cis-elements as the AS gene.