A novel approach using ATR-FTIR spectroscopy and chemometric analysis to distinguish male and female human hair samples.

This article presents an attempt to discriminate between human male and female hair samples using a single strand of scalp hair. The methodology involves the non-destructive application of ATR-FTIR spectroscopy coupled with chemometric analysis. A total of 96 hair samples, evenly distributed between 48 male and 48 female volunteers from India, were collected. Spectral analysis revealed subtle differences between the two groups, and reliance on visual interpretation might introduce biasness. To avoid subjective biases, chemometric techniques such as principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) were employed for enhanced data visualization and separation. PCA results revealed that the first 10 principal components accounted for 93% of the total variance, with three significant PCs. The PLS-DA model demonstrated a remarkable sensitivity and specificity in sex discrimination from hair samples, establishing its efficacy as a robust classification tool. Furthermore, the proposed model exhibited 100% accuracy in predicting unknown samples, underscoring its potential applicability in real-world scenarios. These outcomes affirm the viability of our approach for non-invasive classification of human male and female hair based on single-strand scalp hair analysis.

Estimation of time since deposition of semen stain on different fabric types using ATR-FTIR spectroscopy and chemometrics.

Various body fluids such as blood, semen, vaginal secretions, and saliva are frequently encountered at crime scene. In cases of sexual assault, semen stains are one of the most reliable evidence of biological origin. In this study, our objective was to develop a method for estimating the time since deposition of semen stains on five different fabric types using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy, with a focus on a time frame of up to 8 weeks. Semen samples from six different volunteers were dripped onto five distinct fabric materials, and ATR-FTIR measurements were obtained at 17 different time points. Principal component analysis (PCA) and partial least squares (PLS) methods were employed to differentiate semen stains on various fabric samples and estimate the age of semen stains. Models constructed using PCA and PLSR achieved high R2 values and low root-mean-square error (RMSE). While the performance varies depending on fabric types, it was observed that age estimation of semen stains can be made within following intervals: 0.39-0.76 days for 0-7 day range, 2.59-3.38 days for the 1-8 week range, and 3.98-8.1 days for the 0-56 day range. This study demonstrates the effectiveness of using ATR-FTIR spectroscopy in combination with chemometrics to estimate the age of human semen stains on various fabric types based on time-dependent spectral changes.

Studying the effect of cocaine on the structure of cosmetically treated human hair using micro-Attenuated Total Reflection spectroscopy (ATR).

It has been demonstrated that hair can accumulate and trap drugs over time with exposure and use, thus being able to provide a useful indication of long-term exposure. Studying changes in the structural components of hair, which may occur as a result of drug binding processes, can be a useful tool in understanding the issues involving false positive results from hair drug testing. Furthermore, the effect of cosmetic treatments, such as bleaching and straightening on hair structural changes, may help to understand the effect of drugs on human hair structure with pre-treatment history. In this investigation, changes resulting from exposure of bleached, unbleached, and straightened, neat blonde Caucasian hair to external cocaine were examined. Cocaine was chosen as the test drug as it is among the most widely abused substances. The results from infrared spectra and their second derivatives with aid of multivariate statistical method, PCA analysis demonstrated that changes in the hair structure occurred as a result of these treatments.

Assessment of Measurement of Salivary Urea by ATR-FTIR Spectroscopy to Screen for CKD.

Stages of CKD are currently defined by eGFR and require measurement of serum creatinine concentrations. Previous studies have shown a good correlation between salivary and serum urea levels and the stage of CKD. However, quantitative salivary urea assays in current clinical use require costly and labor-intensive commercial kits, which restricts the advantage of using saliva and limits wider applicability as a quick and easy means of assessing renal function. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy has been shown to provide a potentially straightforward, reagent-free method for the identification of a range of disease-related biomarkers and is in current clinical use for analyses of the chemical composition of kidney stones. We assessed the feasibility of ATR-FTIR spectroscopy as an alternative method to measure salivary urea in patients with different stages of CKD. The ATR-FTIR spectra of dried saliva samples from six healthy controls and 20 patients with CKD (stages 1-5) were analyzed to provide their urea concentrations. The lower limit of detection of salivary urea by the ATR-FTIR spectroscopy method was 1-2 mM, at the lower end of the clinically relevant range. Statistically significant differences in salivary urea concentrations were demonstrated between healthy subjects (4.1±0.5 mM) and patients with CKD stages 3-5 (CKD stage 3, 6.8±0.7 mM; CKD stage 4, 9.1±1 mM; CKD stage 5, 14.8±1.6 mM). These salivary urea concentrations correlated well with serum urea levels in the same patients measured by an automated analyzer (Spearman rank correlation coefficient of 0.71; P<0.001). The ability of the method to detect and stage CKD was assessed from the sensitivity and specificity parameters of a receiver operating characteristics (ROC) curve analysis. This proof-of-concept study demonstrates that quantitation of salivary urea by ATR-FTIR spectroscopy could provide a viable tool for rapid and cost-effective diagnosis of stages 3-5 CKD.

Abnormal expression of p-ATM/CHK2 in nasal extranodal NK/T cell lymphoma, nasal type, is correlated with poor prognosis.

AIMS: The aim of this study is to investigate the expression profiles of cell cycle related proteins in nasal extranodal NK/T cell lymphoma, nasal type (ENKTCL). METHODS: The expression profiles of cell cycle related proteins were assessed with a cell cycle antibody array and validated by immunohistochemistry. Correlations between the expression levels of proteins and clinical outcomes of patients with nasal ENKTCL were evaluated. RESULTS: The expression of full length ataxia telangiectasia mutated (ATM) in nasal ENKTCL significantly decreased compared with that in nasal benign lymphoid proliferative disease (NBLPD), but the expression levels of p-ATM, CHK2 and RAD51 significantly increased in nasal ENKTCL compared with that in NBLPD. Kaplan-Meier analysis showed that the expression levels of p-ATM and CHK2 in nasal ENKTCL were inversely related to overall survival (p=0.011 and p=0.025, respectively). CONCLUSION: Abnormalities in the ATM pathway may play a crucial role in the oncogenesis and chemoradiotherapy resistance of nasal ENKTCL.

Quantitative analysis of ATM phosphorylation in lymphocytes.

Since many anticancer therapies target DNA and DNA damage response pathways, biomarkers of DNA damage endpoints may prove valuable in basic and clinical cancer research. Ataxia telangiectasia-mutated (ATM) kinase is the principal regulator of cellular responses to DNA double-strand breaks (DSBs). In humans, ATM autophosphorylation at serine 1981 (p-S1981) is an immediate molecular response to nascent DSBs and ionizing radiation (IR). Here we describe the analytical characteristics and fit-for-purpose validation of a quantitative dual-labeled immunoblot that simultaneously measures p-S1981-ATM and pan-ATM in human peripheral blood mononuclear cells (PBMCs) following ex vivo exposure to 2 Gy IR, facilitating the calculation of %p-ATM. To validate our assay, we isolated PBMCs from 41 volunteers. We report that the median basal level of p-S1981-ATM and pan-ATM was 2.4 and 49.5 ng/107 PBMCs, respectively, resulting in %p-ATM of 4%. Following exposure of PBMCs to 2 Gy IR, p-S1981-ATM levels increased 12-fold to 29.8 ng/107 PBMCs resulting in %p-ATM of 63%. Interestingly, we show that PBMCs from women have a 2.6-fold greater median p-S1981-ATM level following IR exposure than men (44.4 versus 16.9 ng/107 cells; p < 0.01). This results in a significantly greater %p-ATM for women (68% versus 49%; p <  0.01). Our rigorous description of the analytical characteristics and reproducibility of phosphoprotein immunoblotting, along with our finding that the ATM DNA damage response is greater in women, has far reaching implications for biomedical researchers.

ATM-Mediated Phosphorylation of Cortactin Involved in Actin Polymerization Promotes Breast Cancer Cells Migration and Invasion.

BACKGROUND/AIMS: The ataxia-telangiectasia mutated (ATM) protein kinase is critical for the maintenance of genomic stability and acts as tumor suppressor. Although evidence shows that a DNA damage-independent ATM (oxidized ATM) may be involved in cancer progression, the underlying mechanism is still unclear. METHODS: Immunohistochemistry, immunofluorescence and western blotting were applied to detect the levels of oxidized ATM. Transwell assay was used to detect the cell migration and invasion abilities in different treatments. Quantitative phosphoproteome analysis was performed using hypoxic BT549 cells, in the presence or absence of Ku60019, a specific inhibitor of ATM kinase. The phosphorylated cortactin, the target protein of oxidized ATM, was confirmed by immunoprecipitation-western blots and in vitro kinase assay. The functions of phosphorylated cortactin were studied by specific short hairpin RNA, site-directed mutation, transwell assay, and actin polymerization assay. RESULTS: Enhanced oxidized ATM proteins were present not only in the advanced and invasive breast tumor tissues but also malignant hypoxic breast cancer cells, in the absence of DNA damage. Loss of ATM expression or inhibiting oxidized ATM kinase activity reduced breast cancer cell migration and invasion. Using quantitative phosphoproteomics approach, 333 oxidized ATM target proteins were identified, some of these proteins govern key signaling associated with gap junction, focal adhesion, actin cytoskeleton rearrangement. Cortactin, one of the biggest changed phospho-protein, is a novel oxidized ATM-dependent target in response to hypoxia. Mechanically, we reveal that hypoxia-activated ATM can enhance the binding affinity of cortactin with Arp2/3 complex by phosphorylating cortactin at serine 113, and as a result, in favor of breast cancer cell migration and invasion. CONCLUSION: Oxidized ATM can phosphorylate cortactin at serine 113, playing a critical role in promoting breast tumor cell mobility and invasion via actin polymerization.

[Clinicopathologic characteristics of Burkitt-like lymphoma with chromosome 11q aberration].

Objective: To analyze clinical, pathological, molecular and genetic characteristics of Burkitt-like lymphoma with chromosome 11q aberration. Methods: A case of Burkitt-like lymphoma with 11q aberration was presented at Beijing Friendship Hospital in November 2016 with detailed clinicopathological features, immunophenotypes, Epstein-Barr virus(EBV) status and molecular genetic characteristics. Results: The patient was a 38-year-old man presenting with the cervical lymphadenopathy. In morphology, the tumor had the similar characteristics of Burkitt lymphoma, including diffuse infiltration of medium to large lymphoid cells, and presence of"starry sky"phenomenon. Immunophenotypically, the tumor cells were positive for CD20, CD10, bcl-6, but negative for bcl-2. MUM-1 showed weak and patchy positivity. Ki-67 index was more than 95%. C-MYC expression was seen in about 50% of tumor cells. EBV in situ hybridization was negative. IgH and IgK genes were clonally rearranged.Fluorescence in situ hybrization detection using MYC break probe was negative but ATM gene amplification on chromosome 11q was detected. The patient did not receive any chemotherapy or radiotherapy and had not recurrence during the 10 months follow-up. Conclusion: Burkitt-like lymphoma with chromosome 11q aberration has similar clinical, morphological and immunological characteristics to classic Burkitt's lymphoma.

Low-Dose Hypersensitive Response for Residual pATM and γH2AX Foci in Normal Fibroblasts of Cancer Patients.

PURPOSE: To define the dose-response relationship for initial and residual pATM and γH2AX foci and temporal response of pATM foci in fibroblasts of 4 hyper-radiosensitivity (HRS)-positive cancer patients and 8 HRS-negative cancer patients and answer the question regarding the role of DNA double-strand break (DSB) recognition and repair in the mechanism of HRS. METHODS AND MATERIALS: The cells were irradiated with single doses (0.1-4 Gy) of 6-MV X rays. The number of initial and residual pATM and γH2AX foci was assessed 1 hour and 24 hours after irradiation, respectively. Kinetics of DSB recognition and repair was estimated by pATM foci assay after irradiation with 0.2 and 2 Gy. RESULTS: Hyper-radiosensitivity response (confirmed by the induced-repair model) was clearly evident for residual pATM and γH2AX foci in fibroblasts of HRS-positive patients but not in fibroblasts of HRS-negative patients. Significantly less DSB was recognized by pATM early (10-30 minutes) after irradiation with 0.2 Gy in HRS-positive compared with HRS-negative fibroblasts. CONCLUSIONS: The present results provide evidence for the role of DSB recognition by pATM and repair in the mechanism of HRS and seem to support the idea of nucleo-shuttling of the pATM protein to be involved in HRS response.

Quantitative Mass Spectrometry by Isotope Dilution and Multiple Reaction Monitoring (MRM).

Selected reaction monitoring (SRM) is used in molecular profiling to detect and quantify specific known proteins in complex mixtures. Using isotope dilution (Barnidge et al., Anal Chem 75(3):445-451, 2003) methodologies, peptides can be quantified without the need for an antibody-based method. Selected reaction monitoring assays employ electrospray ionization mass spectrometry (ESI-MS) followed by two stages of mass selection: a first stage where the mass of the peptide ion is selected and, after fragmentation by collision-induced dissociation (CID), a second stage (tandem MS) where either a single (e.g., SRM) or multiple (multiple reaction monitoring, MRM) specific peptide fragment ions are transmitted for detection. The MRM experiment is accomplished by specifying the parent masses of the selected endogenous and isotope-labeled peptides for MS/MS fragmentation and then monitoring fragment ions of interest, using their intensities/abundances and relative ratios to quantify the parent protein of interest. In this example protocol, we will utilize isotope dilution MRM-MS to quantify in absolute terms the total levels of the protein of interest, ataxia telangiectasia mutated (ATM) serine/threonine protein kinase. Ataxia telangiectasia mutated (ATM) phosphorylates several key proteins that initiate activation of the DNA damage checkpoint leading to cell cycle arrest.

Activation of ATR-Chk1 pathway facilitates EBV-mediated transformation of primary tonsillar B-cells.

Primary infection of the immunocompromised host with the oncovirus Epstein-Barr virus (EBV) that targets mainly B-cells is associated with an increased risk for EBV-associated tumors. The early events subsequent to primary infection with potential for B-cell transformation are poorly studied. Here, we modeled in vitro the primary infection by using B-cells isolated from tonsils, the portal of entry of EBV, since species specificity of EBV hampers modeling in experimental animals. Increasing evidence indicates that the host DNA damage response (DDR) can influence and be influenced by EBV infection. Thus, we inoculated tonsillar B-cells (TBCs) with EBV-B95.8 and investigated cell proliferation and the DDR during the first 96 hours thereafter. We identified for the first time that EBV infection of TBCs induces a period of hyperproliferation 48-96 hours post infection characterized by the activation of ataxia telangiectasia and Rad3-releated (ATR) and checkpoint kinase-1 (Chk1). Whereas inhibition of Chk1 did not affect B-cell transformation, the specific inhibition of ATR robustly decreased the transformation efficiency of EBV. Our results suggest that activation of ATR is key for EBV-induced B-cell transformation. Thus, targeting the interaction between ATR/Chk1 and EBV could offer new options for the treatment of EBV-associated malignancies.

Low Ki67/high ATM protein expression in malignant tumors predicts favorable prognosis in a retrospective study of early stage hormone receptor positive breast cancer.

INTRODUCTION: This study was designed to investigate the combined influence of ATM and Ki67 on clinical outcome in early stage hormone receptor positive breast cancer (ES-HPBC), particularly in patients with smaller tumors (< 4 cm) and fewer than four positive lymph nodes. METHODS: 532 formalin-fixed paraffin-embedded specimens of resected primary breast tumors were used to construct a tissue microarray. Samples from 297 patients were suitable for final statistical analysis. We detected ATM and Ki67 proteins using fluorescence and brightfield immunohistochemistry respectively, and quantified their expression with digital image analysis. Data on expression levels were subsequently correlated with clinical outcome. RESULTS: Remarkably, ATM expression was useful to stratify the low Ki67 group into subgroups with better or poorer prognosis. Specifically, in the low Ki67 subgroup defined as having smaller tumors and no positive nodes, patients with high ATM expression showed better outcome than those with low ATM, with estimated survival rates of 96% and 89% respectively at 15 years follow up (p = 0.04). Similarly, low-Ki67 patients with smaller tumors, 1-3 positive nodes and high ATM also had significantly better outcomes than their low ATM counterparts, with estimated survival rates of 88% and 46% respectively (p = 0.03) at 15 years follow up. Multivariable analysis indicated that the combination of high ATM and low Ki67 is prognostic of improved survival, independent of tumor size, grade, and lymph node status (p = 0.02). CONCLUSIONS: These data suggest that the prognostic value of Ki67 can be improved by analyzing ATM expression in ES-HPBC.

Identification of Group B Streptococci Using 16S rRNA, cfb, scpB, and atr Genes in Pregnant Women by PCR.

Streptococcus agalactiae is acommensalorganism, but it may cause infection in susceptible hosts. The aim of this study was to evaluate PCR assay compared with conventional culture method for direct detection of Streptococcus agalactiae. Total of 203 paired low vaginal swabs were collected from women at 35-37 weeks of pregnancy from June 2013 through February 2014 for detection of Streptococcus agalactiae using PCR assay targeting 16S rRNA, cfb, scpB, and atr genes and culture method following broth enrichment. The results were recorded and evaluated for determining of sensitivity, specificity, positive and negative predictive values of PCR assaycompared with culture method. Prevalence of Streptococcus agalactiae was determined as 7.39% (n=15) using culture method; 19.70% (n=40) by PCR targeting 16S rRNA gene; 18.23% (n=37) by targeting atr gene; 17.24% (n=35) by cfb gene; and 8.87% (n=18) by scpB gene. Generally, a total of 49 specimens were considered true positive (27 samples by PCR assay using the four genes in sum, 4 samples only by atr gene PCR, 3 samples only by cfb gene PCR, 2 samples only by culture method, and 13 samples by PCR assay and culture method in common) and prevalence of Streptococcus agalactiae determined 24.14% in Hamadan. The current data demonstrated that performing only culture method for detecting GBS from pregnant women leads to missed false negative carrier individuals. Thus, it is recommended that both the PCR assay and conventional culture method to be performed in order to detect Streptococcus agalactiae.

CDKN2A (p14(ARF)/p16(INK4a)) and ATM promoter methylation in patients with impalpable breast lesions.

Early detection of breast cancer increases the chances of cure, but the reliable identification of impalpable lesions is still a challenge. In spite of the advances in breast cancer detection, the molecular basis of impalpable lesions and the corresponding circulating biomarkers are not well understood. Impalpable lesions, classified by radiologists according to the Breast Imaging Reporting and Data System in the categories 3 and 4, can be either benign or malignant (slow growing or aggressive). In this article, we report the DNA methylation pattern in CDKN2A (p14(ARF)/p16(INK4a)) and in ATM gene promoters from 62 impalpable lesions, 39 peripheral blood samples, and 39 saliva samples, assessed by methylation-specific polymerase chain reaction method. ATM showed the greatest percentage of methylation in DNA from lesions (benign and malignant), blood (even with p16(INK4a)), and saliva, followed by p16(INK4a) and p14(ARF). Among the malignant cases, ATM promoter was the most hypermethylated in lesion DNA and in blood and saliva DNAs, and p14(ARF), the least. The highest percentage of p16(INK4a) methylation was found in the blood. Finally, our data are relevant because they were obtained using impalpable breast lesions from patients who were carefully recruited in 2 public hospitals of Rio de Janeiro.

Assessment of the Radiation Effects of Cardiac CT Angiography Using Protein and Genetic Biomarkers.

OBJECTIVES: The purpose of this study was to evaluate whether radiation exposure from cardiac computed tomographic angiography (CTA) is associated with deoxyribonucleic acid (DNA) damage and whether damage leads to programmed cell death and activation of genes involved in apoptosis and DNA repair. BACKGROUND: Exposure to radiation from medical imaging has become a public health concern, but whether it causes significant cell damage remains unclear. METHODS: We conducted a prospective cohort study in 67 patients undergoing cardiac CTA between January 2012 and December 2013 in 2 U.S. medical centers. Median blood radiation exposure was estimated using phantom dosimetry. Biomarkers of DNA damage and apoptosis were measured by flow cytometry, whole genome sequencing, and single cell polymerase chain reaction. RESULTS: The median dose length product was 1,535.3 mGy·cm (969.7 to 2,674.0 mGy·cm). The median radiation dose to the blood was 29.8 mSv (18.8 to 48.8 mSv). Median DNA damage increased 3.39% (1.29% to 8.04%, p < 0.0001) and median apoptosis increased 3.1-fold (interquartile range [IQR]: 1.4- to 5.1-fold, p < 0.0001) post-radiation. Whole genome sequencing revealed changes in the expression of 39 transcription factors involved in the regulation of apoptosis, cell cycle, and DNA repair. Genes involved in mediating apoptosis and DNA repair were significantly changed post-radiation, including DDB2 (1.9-fold [IQR: 1.5- to 3.0-fold], p < 0.001), XRCC4 (3.0-fold [IQR: 1.1- to 5.4-fold], p = 0.005), and BAX (1.6-fold [IQR: 0.9- to 2.6-fold], p < 0.001). Exposure to radiation was associated with DNA damage (odds ratio [OR]: 1.8 [1.2 to 2.6], p = 0.003). DNA damage was associated with apoptosis (OR: 1.9 [1.2 to 5.1], p < 0.0001) and gene activation (OR: 2.8 [1.2 to 6.2], p = 0.002). CONCLUSIONS: Patients exposed to >7.5 mSv of radiation from cardiac CTA had evidence of DNA damage, which was associated with programmed cell death and activation of genes involved in apoptosis and DNA repair.

DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1ß, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

Aberrant TCRδ rearrangement underlies the T-cell lymphocytopenia and t(12;14) translocation associated with ATM deficiency.

Ataxia telangiectasia mutated (ATM) is a protein kinase and a master regulator of DNA-damage responses. Germline ATM inactivation causes ataxia-telangiectasia (A-T) syndrome with severe lymphocytopenia and greatly increased risk for T-cell lymphomas/leukemia. Both A-T and T-cell prolymphoblastic leukemia patients with somatic mutations of ATM frequently carry inv(14;14) between the T-cell receptor α/δ (TCRα/δ) and immunoglobulin H loci, but the molecular origin of this translocation remains elusive. ATM(-/-) mice recapitulate lymphocytopenia of A-T patients and routinely succumb to thymic lymphomas with t(12;14) translocation, syntenic to inv(14;14) in humans. Here we report that deletion of the TCRδ enhancer (Eδ), which initiates TCRδ rearrangement, significantly improves αß T cell output and effectively prevents t(12;14) translocations in ATM(-/-) mice. These findings identify the genomic instability associated with V(D)J recombination at the TCRδ locus as the molecular origin of both lymphocytopenia and the signature t(12;14) translocations associated with ATM deficiency.

Ataxia-telangiectasia-mutated protein kinase levels stratify patients with pancreatic adenocarcinoma into prognostic subgroups with loss being a strong indicator of poor survival.

OBJECTIVES: Recently, aberrations in the gene encoding for ataxia-telangiectasia-mutated (ATM) protein kinase have been reported for pancreatic ductal adenocarcinomas (PDAC). These findings argue that ATM deficiency may play a role during carcinogenesis. Therefore, in this study, we investigated the clinical relevance of ATM expression and ATM activation in PDAC. METHODS: Both ATM expression and nuclear phosphoSer1981-ATM levels were assessed by immunohistochemistry in a cohort of 133 PDAC and correlated with clinicopathological parameters. RESULTS: We found stratification in prognostic subgroups. Complete loss of Ser1981-ATM was indicative of the worst prognosis (median survival, 10.8 vs 14.3 months [low expression] vs 31.1 months [high expression], P < 0.001). Similarly, analysis of ATM expression demonstrated absent expression levels of ATM to be associated with dismal prognosis (median survival, 9.6 months), whereas expression of ATM in general was associated with increased survival (17.7 months, P = 0.001). CONCLUSIONS: Our analysis shows that both ATM expression and activated ATM are prognostic markers in PDAC with respect to standard clinicopathological parameters. These results suggest that ATM should be further explored as prognostic as well as predictive factor with respect to conventional chemotherapies and for putative synthetic lethal approaches.

Polymerase η suppresses telomere defects induced by DNA damaging agents.

Telomeres at chromosome ends are normally masked from proteins that signal and repair DNA double strand breaks (DSBs). Bulky DNA lesions can cause DSBs if they block DNA replication, unless they are bypassed by translesion (TLS) DNA polymerases. Here, we investigated roles for TLS polymerase η, (polη) in preserving telomeres following acute physical UVC exposure and chronic chemical Cr(VI) exposure, which both induce blocking lesions. We report that polη protects against cytotoxicity and replication stress caused by Cr(VI), similar to results with ultraviolet C light (UVC). Both exposures induce ataxia telangiectasia and Rad3-related (ATR) kinase and polη accumulation into nuclear foci and localization to individual telomeres, consistent with replication fork stalling at DNA lesions. Polη-deficient cells exhibited greater numbers of telomeres that co-localized with DSB response proteins after exposures. Furthermore, the genotoxic exposures induced telomere aberrations associated with failures in telomere replication that were suppressed by polη. We propose that polη's ability to bypass bulky DNA lesions at telomeres is critical for proper telomere replication following genotoxic exposures.

Interplay between the DNA damage proteins MDC1 and ATM in the regulation of the spindle assembly checkpoint.

To avoid genomic instability, cells have developed surveillance mechanisms such as the spindle assembly checkpoint (SAC) and the DNA damage response. ATM and MDC1 are central players of the cellular response to DNA double-strand breaks. Here, we identify a new role for these proteins in the regulation of mitotic progression and in SAC activation. MDC1 localizes at mitotic kinetochores following SAC activation in an ATM-dependent manner. ATM phosphorylates histone H2AX at mitotic kinetochores, and this phosphorylation is required for MDC1 localization at kinetochores. ATM and MDC1 are needed for kinetochore localization of the inhibitory mitotic checkpoint complex components, Mad2 and Cdc20, and for the maintenance of the mitotic checkpoint complex integrity. This probably relies on the interaction of MDC1 with the MCC. In this work, we have established that ATM and MDC1 maintain genomic stability not only by controlling the DNA damage response, but also by regulating SAC activation, providing an important link between these two essential biological processes.