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PubMed2000

Analysis of pressure sensitive adhesive tape: I. Evaluation of infrared ATR accessory advances

Attenuated total reflection (ATR), also known as internal reflection spectroscopy (IRS), is a forensically accepted method for infrared (IR) analysis of pressure sensitive adhesive tapes. Advancements of ATR accessories in the last decade have provided the forensic examiner several ATR methods to choose from. These accessories offer a variety of ATR crystal choices with a variety of prices and capabilities. Four different types of pressure sensitive adhesive tapes including duct tape, electrical tape, packaging tape and office tape have been used to compare six different ATR methods. Each of the methods tested offers both benefits and limitaltions which must be considered for the type of sample to be analyze. The intent of this paper is to assist the reader in better understanding ATR techniques, the many differences among currently available ATR accessories and how the method is applied to the analysis of pressure sensitive adhesive tape.

Journal Article↗
PubMed2026

ATR-dependent phosphorylation of the histone acetyltransferase HBO1 suppresses chromatin binding and promotes replication stress responses.

Mounting evidence has shown that histone acetyltransferase binding to ORC1 (HBO1) serves as an oncoprotein, warranting the use of the small molecule inhibitor WM-3835 for cancer therapy. However, HBO1 is ubiquitously expressed in both tumor and normal tissues, with potential to increase the risk of systemic toxicity. This unmet need highlights the importance of identifying suitable biomarkers to predict the sensitivity to HBO1 inhibitor. Here, we show that ATR, a key regulator of DNA replication stress, is a novel interacting partner of HBO1. In addition, we reveal a regulatory function of HBO1 in DNA replication stress responses, in an ATR-dependent manner. Mechanistically, ATR mediated HBO1 Ser50/53 phosphorylation interferes with the genomic binding of HBO1 and regulates gene expression. Notably, overexpression of HBO1 mutated at the ATR phosphorylation site (S50/53A) dampens the expression of DNA repair related genes and suppresses tumor colony formation, consistent with the observations of WM-3835 treatment. Inhibition of ATR significantly antagonized the sensitivity to WM-3835 treatment. Collectively, our findings uncovered a previously unidentified role of HBO1 in the regulation of replication stress and discovered ATR as a potential biomarker for WM-3835 treatment.

ATR↗
PubMed2000

Distribution of Atr protein in primary spermatocytes of a mouse chromosomal mutant: a comparison of preparation techniques.

In this study, we examined the suitability of a three dimensional preparation technique for studying chromosome behaviour in the first meiotic prophase in the mouse chromosomal mutant T(1;13)H/T(1;13)Wa. To preserve cellular shape, primary spermatocytes were encapsulated in a fibrin clot. Conventionally sedimented prophase nuclei served as controls. Axial elements and lateral synaptonemal complex components were subsequently stained by immunofluorescence and the presence of axial elements at the pachytene stage was highlighted with indirect immunofluorescence against the Atr protein. We compared the distribution of Atr signal in the fibrin-embedded spermatocytes with surface-spread preparations and immunohistochemically stained histological sections of seminiferous tubules. Furthermore, fluorescence in situ hybridisation of the mouse minor satellite DNA was done on fibrin-embedded spermatocytes. The Atr signal is most conspicuous in fibrin-embedded nuclei on unpaired axial elements during pachytene, both for sex chromosomal and for autosomal segments, and expanding from these elements into the surrounding chromatin. Both spread and encapsulated zygotene nuclei with extended axial element formation proved to be positive for Atr. Mid- to late zygotene nuclei were devoid of 3,3'-diaminodibenzene deposition in the histological sections. Highlighting the unpaired axial elements in the small heteromorphic 1(13)H;1(13)Wa bivalent with an Atr signal enabled meiotic analysis of this bivalent to be carried out in a three-dimensional context. Thus, proximity of this bivalent with the sex chromosomes is found more often in three-dimensional preparations than in spread preparations. Furthermore, the development of the Atr signal over the sex chromosomes as pachytene proceeds helps in substaging of this long and heterogeneous meiotic phase, in sedimented but especially in fibrin-encapsulated nuclei.

Animals↗
PubMed2006

Applications of ATR-FTIR spectroscopic imaging to biomedical samples.

FTIR spectroscopic imaging in ATR (Attenuated Total Reflection) mode is a powerful tool for studying biomedical samples. This paper summarises recent advances in the applications of ATR-FTIR imaging to dissolution of pharmaceutical formulations and drug release. The use of two different ATR accessories to obtain chemical images of formulations in contact with water as a function of time is demonstrated. The innovative use of the diamond ATR accessory allowed in situ imaging of tablet compaction and dissolution. ATR-FTIR imaging was also applied to obtain images of the surface of skin and the spatial distribution of protein and lipid rich domains was obtained. Chemical images of cross-section of rabbit aorta were obtained using a diamond ATR accessory and the possibility of in situ imaging of arterial samples in contact with aqueous solution was demonstrated for the first time. This experiment opens an opportunity to image arterial samples in contact with solutions containing drug molecules. This approach may help in understanding the mechanisms of treatment of atherosclerosis.

Animals↗
PubMed2005

Inhibition of Atm and/or Atr disrupts gene silencing on the inactive X chromosome.

ATM and ATR are well documented for their roles in maintaining the integrity of genomic DNA by responding to DNA damage and preparing the cell for repair. Since ATM and ATR have been reported to exist in complexes with histone deacetylases, we asked whether Atm and Atr might also uphold gene silencing by heterochromatin. We show that the Atm/Atr inhibitor 2-aminopurine causes the inactive X chromosome to accumulate abnormal chromatin and undergo unwanted gene reactivation. We provide evidence that this gene expression from the inactive X chromosome is not a byproduct of the accumulation of DNA breaks. Individually inhibiting Atm and Atr by either small interfering RNA or the expression of dominant-negative ATM and ATR constructs also compromised X-inactivation. Atm and Atr, therefore, not only function in responding to DNA damage but perhaps also are involved in gene silencing via the maintenance of heterochromatin.

2-Aminopurine↗
PubMed2005

Alterations of DNA damage-response genes ATM and ATR in pyothorax-associated lymphoma.

Pyothorax-associated lymphoma (PAL) is non-Hodgkin's lymphoma that develops from chronic inflammation. Free radicals and oxidative stress generated in the inflammatory lesions could cause DNA damage and thus provide a basis for lymphomagenesis. Ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) genes are responsive genes for DNA damage, therefore potential involvement of these genes in PAL lymphomagenesis was examined in eight PAL cell lines and clinical samples from five cases. ATM mutations were detected in five of eight PAL lines. All but one of these mutations affected the phosphatidylinositol 3-kinase domain, indicating the loss-of-function mutation of ATM gene. Heterozygous mutations of ATR were found in two of eight lines; one a missense and the other a truncation mutation. ATR mutations were also detected in two of five cases in clinical samples from PAL. PAL cells with ATR mutation showed a delay or abrogation in repair for ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) or ultraviolet (UV)-induced DNA single-strand breaks (SSBs), and exhibited a defect in p53 accumulation and failure in cell cycle checkpoint at G1-S phase. These findings showed that mutations of ATR gene result in failure for DNA DSB and SSB repair, suggesting the role of ATM and ATR gene mutations in PAL lymphomagenesis.

Aged↗
PubMed2005

Regulation of NF-kappaB and p53 through activation of ATR and Chk1 by the ARF tumour suppressor.

The ARF tumour suppressor is a central component of the cellular defence against oncogene activation. In addition to activating p53 through binding Mdm2, ARF possesses other functions, including an ability to repress the transcriptional activity of the antiapoptotic RelA(p65) NF-kappaB subunit. Here we demonstrate that ARF induces the ATR- and Chk1-dependent phosphorylation of the RelA transactivation domain at threonine 505, a site required for ARF-dependent repression of RelA transcriptional activity. Consistent with this effect, ATR and Chk1 are required for ARF-induced sensitivity to tumour necrosis factor alpha-induced cell death. Significantly, ATR activity is also required for ARF-induced p53 activity and inhibition of proliferation. ARF achieves these effects by activating ATR and Chk1. Furthermore, ATR and its scaffold protein BRCA1, but not Chk1, relocalise to specific nucleolar sites. These results reveal novel functions for ARF, ATR and Chk1 together with a new pathway regulating RelA NF-kappaB function. Moreover, this pathway provides a mechanism through which ARF can remodel the cellular response to an oncogenic challenge and execute its function as a tumour suppressor.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2002

Preferential binding of ATR protein to UV-damaged DNA.

The ATR protein is a member of the phosphoinositide 3-kinase-related kinase family and plays an important role in UV-induced DNA damage checkpoint response. Its role as a signal transducer in cell cycle checkpoint is well established, but it is currently unclear whether ATR functions as a damage sensor as well. Here we have purified the ATR protein and investigated its interaction with DNA by using biochemical analysis and electron microscopy. We find that ATR is a DNA-binding protein with higher affinity to UV-damaged than undamaged DNA. In addition, damaged DNA stimulates the kinase activity of ATR to a significantly higher level than undamaged DNA. Our data suggest that ATR may function as an initial sensor in the DNA damage checkpoint response.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2001

S phase and G2 arrests induced by topoisomerase I poisons are dependent on ATR kinase function.

ATR, a human phosphatidylinositol 3-kinase-related kinase, is an important component of the cellular response to DNA damage. In the present study, we evaluated the role of ATR in modulating the response of cells to S phase-associated DNA double-stranded breaks induced by topoisomerase poisons. Prolonged exposure to low doses of the topoisomerase I poison topotecan (TPT) resulted in S phase slowing because of diminished DNA synthesis at late-firing replicons. In contrast, brief TPT exposure, as well as prolonged exposure to the topoisomerase II poison etoposide, resulted in subsequent G(2) arrest. These responses were associated with phosphorylation of the checkpoint kinase Chk1. The cell cycle responses and phosphorylation of Chk1 were markedly diminished by forced overexpression of a dominant negative, kinase-inactive allele of ATR. In contrast, deficiency of the related kinase ATM had no effect on these events. The loss of ATR-dependent checkpoint function sensitized GM847 human fibroblasts to the cytotoxic effects of the topoisomerase I poisons TPT and 7-ethyl-10-hydroxycamptothecin, as assessed by inhibition of colony formation, increased trypan blue uptake, and development of apoptotic morphological changes. Expression of kdATR also sensitized GM847 cells to the cytotoxic effects of prolonged low dose etoposide and doxorubicin, albeit to a smaller extent. Collectively, these results not only suggest that ATR is important in responding to the replication-associated DNA damage from topoisomerase poisons, but also support the view that ATM and ATR have unique roles in activating the downstream kinases that participate in cell cycle checkpoints.

Animals↗
PubMed2007

Phosphatase type 2A-dependent and -independent pathways for ATR phosphorylation of Chk1.

ATM and Rad3-related (ATR) is a regulatory kinase that, when activated by hydroxyurea, UV, or human immunodeficiency virus-1 Vpr, causes cell cycle arrest through Chk1-Ser(345) phosphorylation. We demonstrate here that of these three agents only Vpr requires protein phosphatase type 2A (PP2A) to activate ATR for Chk1-Ser(345) phosphorylation. A requirement for PP2A by Vpr was first shown with the PP2A-specific inhibitor okadaic acid, which reduced Vpr-induced G(2) arrest and Cdk1-Tyr(15) phosphorylation. Using small interference RNA to down-regulate specific subunits of PP2A indicated that the catalytic beta-isoform PP2A(Cbeta) and the A regulatory alpha-isoform PP2A(Aalpha) are involved in the G(2) induction, and these downregulations decreased the Vpr-induced, ATR-dependent phosphorylations of Cdk1-Tyr(15) and Chk1-Ser(345). In contrast, the same down-regulations had no effect on hydroxyurea- or UV-activated ATR-dependent Chk1-Ser(345) phosphorylation. Vpr and hydroxyurea/UV all induce ATR-mediated gammaH2AX-Ser(139) phosphorylation and foci formation, but down-regulation of PP2A(Aalpha) or PP2A(Cbeta) did not decrease gammaH2AX-Ser(139) phosphorylation by any of these agents or foci formation by Vpr. Conversely, H2AX down-regulation had little effect on PP2A(Aalpha/Cbeta)-mediated G(2) arrest and Chk1-Ser(345) phosphorylation by Vpr. The expression of vpr increases the amount and phosphorylation of Claspin, an activator of Chk1 phosphorylation. Down-regulation of either PP2A(Cbeta) or PP2A(Aalpha) had little effect on Claspin phosphorylation, but the amount of Claspin was reduced. Claspin may then be one of the phosphoproteins through which PP2A(Aalpha/Cbeta) affects Chk1 phosphorylation when ATR is activated by human immunodeficiency virus-1 Vpr.

Adaptor Proteins, Signal Transducing↗
PubMed2006

ATM regulates ATR chromatin loading in response to DNA double-strand breaks.

DNA double-strand breaks (DSBs) are among the most deleterious lesions that can challenge genomic integrity. Concomitant to the repair of the breaks, a rapid signaling cascade must be coordinated at the lesion site that leads to the activation of cell cycle checkpoints and/or apoptosis. In this context, ataxia telangiectasia mutated (ATM) and ATM and Rad-3-related (ATR) protein kinases are the earliest signaling molecules that are known to initiate the transduction cascade at damage sites. The current model places ATM and ATR in separate molecular routes that orchestrate distinct pathways of the checkpoint responses. Whereas ATM signals DSBs arising from ionizing radiation (IR) through a Chk2-dependent pathway, ATR is activated in a variety of replication-linked DSBs and leads to activation of the checkpoints in a Chk1 kinase-dependent manner. However, activation of the G2/M checkpoint in response to IR escapes this accepted paradigm because it is dependent on both ATM and ATR but independent of Chk2. Our data provides an explanation for this observation and places ATM activity upstream of ATR recruitment to IR-damaged chromatin. These data provide experimental evidence of an active cross talk between ATM and ATR signaling pathways in response to DNA damage.

Ataxia Telangiectasia↗
PubMed2006

Phosphorylation of Xenopus Rad1 and Hus1 defines a readout for ATR activation that is independent of Claspin and the Rad9 carboxy terminus.

The DNA damage checkpoint pathways sense and respond to DNA damage to ensure genomic stability. The ATR kinase is a central regulator of one such pathway and phosphorylates a number of proteins that have roles in cell cycle progression and DNA repair. Using the Xenopus egg extract system, we have investigated regulation of the Rad1/Hus1/Rad9 complex. We show here that phosphorylation of Rad1 and Hus1 occurs in an ATR- and TopBP1-dependent manner on T5 of Rad1 and S219 and T223 of Hus1. Mutation of these sites has no effect on the phosphorylation of Chk1 by ATR. Interestingly, phosphorylation of Rad1 is independent of Claspin and the Rad9 carboxy terminus, both of which are required for Chk1 phosphorylation. These data suggest that an active ATR signaling complex exists in the absence of the carboxy terminus of Rad9 and that this carboxy-terminal domain may be a specific requirement for Chk1 phosphorylation and not necessary for all ATR-mediated signaling events. Thus, Rad1 phosphorylation provides an alternate and early readout for the study of ATR activation.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Rad50 depletion impacts upon ATR-dependent DNA damage responses.

The Mre11/Rad50/NBS1 (MRN) complex is mutated in inherited genomic instability syndromes featuring cancer predisposition, mental retardation and immunodeficiency. It functions both in DNA double-strand break repair and in controlling the ataxia telangiectasia mutated (ATM) kinase during the response to these lesions. Patients inheriting homozygosity for an NBS1 hypomorphic allele display reduced phosphorylation of signaling factors such as Chk1, but not of chromatin-associated factor H2AX, after stresses that activate the ATM-related kinase, ATR. Therefore, we tested whether MRN has a global controlling role over the ATR kinase through the study of MRN deficiencies generated via RNA interference. We show for the first time that MRN is required for ATR-dependent phosphorylation of structural maintenance of chromosomes 1 (Smc1), which acts within chromatin to ensure sister chromatid cohesion and to effect several DNA damage responses. We have uncovered novel phenotypes caused by MRN deficiency that support a functional link between this complex, ATR and Smc1, including hypersensitivity to UV exposure, a defective UV responsive intra-S phase checkpoint and a specific pattern of genomic instability. In addition, certain ATR-dependent responses do not require MRN. These studies demonstrate that there is indeed a controlling role for MRN over the ATR kinase and have established that the downstream events under this control are broad, including both chromatin-associated and diffuse signaling factors, but may not be universal. These studies contribute to our understanding of the central role that MRN plays in damage detection and signaling, which serve to maintain genomic stability and resist neoplastic transformation.

Acid Anhydride Hydrolases↗
PubMed2005

ATM and ATR make distinct contributions to chromosome end protection and the maintenance of telomeric DNA in Arabidopsis.

Here we examine the function of ATM and ATR at telomeres in Arabidopsis. Although plants lacking ATM or ATR display wild-type telomere length homeostasis, chromosome end protection is compromised in atm atr mutants. Moreover, atm tert Arabidopsis experience an abrupt, early onset of genome instability, arguing that ATM is required for protection of short telomeres. ATR, by contrast, is required for maintenance of telomeric DNA as telomere shortening is dramatically accelerated in atr tert mutants relative to tert plants. Thus, ATM and ATR make essential and distinct contributions to chromosome end protection and telomere maintenance in higher eukaryotes.

Arabidopsis↗
PubMed2004

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Animals↗
PubMed2006

ATR-dependent phosphorylation of DNA-dependent protein kinase catalytic subunit in response to UV-induced replication stress.

Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) upon ionizing radiation (IR) is essential for cellular radioresistance and nonhomologous-end-joining-mediated DNA double-strand break repair. In addition to IR induction, we have previously shown that DNA-PKcs phosphorylation is increased upon camptothecin treatment, which induces replication stress and replication-associated double-strand breaks. To clarify the involvement of DNA-PKcs in this process, we analyzed DNA-PKcs phosphorylation in response to UV irradiation, which causes replication stress and activates ATR (ATM-Rad3-related)/ATM (ataxia-telangiectasia mutated) kinases in a replication-dependent manner. Upon UV irradiation, we observed a rapid DNA-PKcs phosphorylation at T2609 and T2647, but not at S2056, distinct from that induced by IR. UV-induced DNA-PKcs phosphorylation occurs specifically only in replicating cells and is dependent on ATR kinase. Inhibition of ATR activity via caffeine, a dominant-negative kinase-dead mutant, or RNA interference led to the attenuation of UV-induced DNA-PKcs phosphorylation. Furthermore, DNA-PKcs associates with ATR in vivo and is phosphorylated by ATR in vitro, suggesting that DNA-PKcs could be the direct downstream target of ATR. Taken together, these results strongly suggest that DNA-PKcs is required for the cellular response to replication stress and might play an important role in the repair of stalled replication forks.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2006

The Achilles tendon Total Rupture Score (ATRS): development and validation.

BACKGROUND: There is a need for a patient-relevant instrument to evaluate outcome after treatment in patients with a total Achilles tendon rupture. PURPOSE: To develop and validate a new patient-reported instrument for measuring outcome after treatment for total Achilles tendon rupture. STUDY DESIGN: Cohort study (diagnosis); Level of evidence, 1. METHODS: Development of this instrument consisted of item generation and test construction, item reduction, validation, evaluation of structure and internal consistency, test-retest, and test for responsiveness. The final version, the Achilles tendon Total Rupture Score (ATRS), was tested for validity, structure, and internal consistency (Cronbach's alpha) on 82 patients and 52 healthy persons. A correlation analysis was performed of the ATRS with the 2 validated foot/ankle/Achilles tendon scores, the Foot and Ankle Outcome Score (FAOS) and the Swedish version of the Victorian Institute of Sports Assessment-Achilles questionnaire (VISA-A-S). Structure was evaluated with factor analysis. Test-retest reliability was evaluated on 43 patients. The ATRS responsiveness was tested on 43 patients by calculating the effect size. RESULTS: The total score for the patients ranged from 17 to 100 with a mean (median) of 77 (85) and a standard deviation (interquartile range) of 21.4 (23). A significantly (P < .0001) higher total score was found for the healthy subjects, ranging from 94 to 100 with a mean (median) of 99.8 (100) and a standard deviation (interquartile range) of 1.1 (0). The ATRS correlated significantly (P < .01) with all subscales of the FAOS (r = 0.60-0.84) and the VISA-A-S (r = 0.78). The factor analysis gave 1 factor of importance. The internal consistency was 0.96 as measured with Cronbach's alpha. The test-retest produced an intraclass correlation coefficient of 0.98. The tests for responsiveness showed an effect size between 0.87 and 2.21. CONCLUSION: The ATRS is a patient-reported instrument with high reliability, validity, and sensitivity for measuring outcome after treatment in patients with a total Achilles tendon rupture. CLINICAL RELEVANCE: The ATRS is a self-administered instrument with high clinical utility, and we suggest the score for measuring the outcome, related to symptoms and physical activity, after treatment in patients with a total Achilles tendon rupture.

Achilles Tendon↗
PubMed2005

Mutation analysis of the ATR gene in breast and ovarian cancer families.

INTRODUCTION: Mutations in BRCA1, BRCA2, ATM, TP53, CHK2 and PTEN account for only 20-30% of the familial aggregation of breast cancer, which suggests the involvement of additional susceptibility genes. The ATR (ataxia-telangiectasia- and Rad3-related) kinase is essential for the maintenance of genomic integrity. It functions both in parallel and cooperatively with ATM, but whereas ATM is primarily activated by DNA double-strand breaks induced by ionizing radiation, ATR has been shown to respond to a much broader range of DNA damage. Upon activation, ATR phosphorylates several important tumor suppressors, including p53, BRCA1 and CHK1. Based on its central function in the DNA damage response, ATR is a plausible candidate gene for susceptibility to cancer. METHODS: We screened the entire coding region of the ATR gene for mutations in affected index cases from 126 Finnish families with breast and/or ovarian cancer, 75 of which were classified as high-risk and 51 as moderate-risk families, by using conformation sensitive gel electrophoresis and direct sequencing. RESULTS: A large number of novel sequence variants were identified, four of which -- Glu254Gly, Ser1142Gly, IVS24-48G>A and IVS26+15C>T -- were absent from the tested control individuals (n = 300). However, the segregation of these mutations with the cancer phenotype could not be confirmed, partly because of the lack of suitable DNA samples. CONCLUSION: The present study does not support a major role for ATR mutations in hereditary susceptibility to breast and ovarian cancer.

Adult↗