An essential function for the ATR-Activation-Domain (AAD) of TopBP1 in mouse development and cellular senescence

ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins – Dpb11TopBP1, Ddc1Rad9 and Dna2 - all interact with and activate Mec1ATR. Each contains an ATR activation domain (ADD) that interacts directly with the Mec1ATR:Ddc2ATRIP complex. Any of the Dpb11TopBP1, Ddc1Rad9 or Dna2 ADDs is sufficient to activate Mec1ATR in vitro. All three can also independently activate Mec1ATR in vivo: the checkpoint is lost only when all three AADs are absent. In metazoans, only TopBP1 has been identified as a direct ATR activator. Depletion-replacement approaches suggest the TopBP1-AAD is both sufficient and necessary for ATR activation. The physiological function of the TopBP1 AAD is, however, unknown. We created a knock-in point mutation (W1147R) that ablates mouse TopBP1-AAD function. TopBP1-W1147R is early embryonic lethal. To analyse TopBP1-W1147R cellular function in vivo, we silenced the wild type TopBP1 allele in heterozygous MEFs. AAD inactivation impaired cell proliferation, promoted premature senescence and compromised Chk1 signalling following UV irradiation. We also show enforced TopBP1 dimerization promotes ATR-dependent Chk1 phosphorylation. Our data suggest that, unlike the yeast models, the TopBP1-AAD is the major activator of ATR, sustaining cell proliferation and embryonic development

Identification of an autoantibody panel to separate lung cancer from smokers and nonsmokers

<p>Abstract</p> <p>Background</p> <p>Sera from lung cancer patients contain autoantibodies that react with tumor associated antigens (TAAs) that reflect genetic over-expression, mutation, or other anomalies of cell cycle, growth, signaling, and metabolism pathways.</p> <p>Methods</p> <p>We performed immunoassays to detect autoantibodies to ten tumor associated antigens (TAAs) selected on the basis of previous studies showing that they had preferential specificity for certain cancers. Sera examined were from lung cancer patients (22); smokers with ground-glass opacities (GGOs) (46), benign solid nodules (55), or normal CTs (35); and normal non-smokers (36). Logistic regression models based on the antibody biomarker levels among the high risk and lung cancer groups were developed to identify the combinations of biomarkers that predict lung cancer in these cohorts.</p> <p>Results</p> <p>Statistically significant differences in the distributions of each of the biomarkers were identified among all five groups. Using Receiver Operating Characteristic (ROC) curves based on age, c-myc, Cyclin A, Cyclin B1, Cyclin D1, CDK2, and survivin, we obtained a sensitivity = 81% and specificity = 97% for the classification of cancer vs smokers(no nodules, solid nodules, or GGO) and correctly predicted 31/36 healthy controls as noncancer.</p> <p>Conclusion</p> <p>A pattern of autoantibody reactivity to TAAs may distinguish patients with lung cancer versus smokers with normal CTs, stable solid nodules, ground glass opacities, or normal healthy never smokers.</p

Studying the Underlying Event in Drell-Yan and High Transverse Momentum Jet Production at the Tevatron

We study the underlying event in proton-antiproton collisions by examining the behavior of charged particles (transverse momentum pT > 0.5 GeV/c, pseudorapidity |\eta| < 1) produced in association with large transverse momentum jets (~2.2 fb-1) or with Drell-Yan lepton-pairs (~2.7 fb-1) in the Z-boson mass region (70 < M(pair) < 110 GeV/c2) as measured by CDF at 1.96 TeV center-of-mass energy. We use the direction of the lepton-pair (in Drell-Yan production) or the leading jet (in high-pT jet production) in each event to define three regions of \eta-\phi space; toward, away, and transverse, where \phi is the azimuthal scattering angle. For Drell-Yan production (excluding the leptons) both the toward and transverse regions are very sensitive to the underlying event. In high-pT jet production the transverse region is very sensitive to the underlying event and is separated into a MAX and MIN transverse region, which helps separate the hard component (initial and final-state radiation) from the beam-beam remnant and multiple parton interaction components of the scattering. The data are corrected to the particle level to remove detector effects and are then compared with several QCD Monte-Carlo models. The goal of this analysis is to provide data that can be used to test and improve the QCD Monte-Carlo models of the underlying event that are used to simulate hadron-hadron collisions.Comment: Submitted to Phys.Rev.

Measurement of the W+W−W^+W^- Production Cross Section and Search for Anomalous WWγWW\gamma and WWZWWZ Couplings in ppˉp \bar p Collisions at s=1.96\sqrt{s} = 1.96 TeV

This Letter describes the current most precise measurement of the $W$ boson pair production cross section and most sensitive test of anomalous $WW\gamma$ and $WWZ$ couplings in $p \bar p$ collisions at a center-of-mass energy of 1.96 TeV. The $WW$ candidates are reconstructed from decays containing two charged leptons and two neutrinos, where the charged leptons are either electrons or muons. Using data collected by the CDF II detector from 3.6 fb$^{-1}$ of integrated luminosity, a total of 654 candidate events are observed with an expected background contribution of $320 \pm 47$ events. The measured total cross section is $\sigma (p \bar p \to W^+ W^- + X) = 12.1 \pm 0.9 \textrm{(stat)} ^{+1.6}_{-1.4} \textrm{(syst)}$ pb, which is in good agreement with the standard model prediction. The same data sample is used to place constraints on anomalous $WW\gamma$ and $WWZ$ couplings.Comment: submitted to Phys. Rev. Let

α-Adducin Gly460Trp Gene Mutation and Essential Hypertension in a Chinese Population: A Meta-Analysis including 10960 Subjects

BACKGROUND: The α-adducin Gly460Trp (G460W) gene polymorphism may be associated with susceptibility to essential hypertension (EH), but this relationship remains controversial. In an attempt to resolve this issue, we conducted a meta-analysis. METHODS: Twenty-three separated studies involving 5939 EH patients and 5021 controls were retrieved and analyzed. Four ethnicities were included: Han, Kazakh, Mongolian, and She. Eighteen studies with 5087 EH patients and 4183 controls were included in the Han subgroup. Three studies with 636 EH patients and 462 controls were included in the Kazakh subgroup. The Mongolian subgroup was represented by only one study with 100 EH patients and 50 controls; similarly, only one study with 116 EH patients and 326 controls was available for the She subgroup. The pooled and ethnic group odds ratios (ORs) along with the corresponding 95% confidence intervals (95% CI) were assessed using a random effects model. RESULTS: There was a significant association between the α-adducin G460W gene polymorphism and EH in the pooled Chinese population under both an allelic genetic model (OR: 1.12, 95% CI: 1.04-1.20, P = 0.002) and a recessive genetic model (OR: 1.40, 95% CI: 1.16-1.70, P = 0.0005). In contrast, no significant association between the α-adducin G460W gene polymorphism and EH was observed in the dominant genetic model (OR: 0.88, 95% CI: 0.72-1.09, P = 0.24). In stratified analysis by ethnicity, significantly increased risk was detected in the Han subgroup under an allelic genetic model (OR: 1.13, 95% CI: 1.04-1.23, P = 0.003) and a recessive genetic model (OR: 1.43, 95% CI: 1.17-1.75, P = 0.0006). CONCLUSIONS: In a Chinese population of mixed ethnicity, the α-adducin G460W gene polymorphism was linked to EH susceptibility, most strongly in Han Chinese

TGF-β1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-β1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models.</p> <p>Methods</p> <p>The mRNA expression levels of TGF-β isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-β1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays.</p> <p>Results</p> <p>In general, TGF-β2, TβRI and TβRII are over-expressed in more aggressive cells, except for TβRI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-β1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-β1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-β1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-β1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-β1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors.</p> <p>Conclusion</p> <p>Altogether, our results support that TGF-β1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-β1 still remains a promising target for breast cancer treatment.</p

Activation of Host Translational Control Pathways by a Viral Developmental Switch

In response to numerous signals, latent herpesvirus genomes abruptly switch their developmental program, aborting stable host–cell colonization in favor of productive viral replication that ultimately destroys the cell. To achieve a rapid gene expression transition, newly minted capped, polyadenylated viral mRNAs must engage and reprogram the cellular translational apparatus. While transcriptional responses of viral genomes undergoing lytic reactivation have been amply documented, roles for cellular translational control pathways in enabling the latent-lytic switch have not been described. Using PEL-derived B-cells naturally infected with KSHV as a model, we define efficient reactivation conditions and demonstrate that reactivation substantially changes the protein synthesis profile. New polypeptide synthesis correlates with 4E-BP1 translational repressor inactivation, nuclear PABP accumulation, eIF4F assembly, and phosphorylation of the cap-binding protein eIF4E by Mnk1. Significantly, inhibiting Mnk1 reduces accumulation of the critical viral transactivator RTA through a post-transcriptional mechanism, limiting downstream lytic protein production, and impairs reactivation efficiency. Thus, herpesvirus reactivation from latency activates the host cap-dependent translation machinery, illustrating the importance of translational regulation in implementing new developmental instructions that drastically alter cell fate

Nucleophosmin Phosphorylation by v-Cyclin-CDK6 Controls KSHV Latency

Nucleophosmin (NPM) is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199) in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA

Diverse Roles and Interactions of the SWI/SNF Chromatin Remodeling Complex Revealed Using Global Approaches

A systems understanding of nuclear organization and events is critical for determining how cells divide, differentiate, and respond to stimuli and for identifying the causes of diseases. Chromatin remodeling complexes such as SWI/SNF have been implicated in a wide variety of cellular processes including gene expression, nuclear organization, centromere function, and chromosomal stability, and mutations in SWI/SNF components have been linked to several types of cancer. To better understand the biological processes in which chromatin remodeling proteins participate, we globally mapped binding regions for several components of the SWI/SNF complex throughout the human genome using ChIP-Seq. SWI/SNF components were found to lie near regulatory elements integral to transcription (e.g. 5′ ends, RNA Polymerases II and III, and enhancers) as well as regions critical for chromosome organization (e.g. CTCF, lamins, and DNA replication origins). Interestingly we also find that certain configurations of SWI/SNF subunits are associated with transcripts that have higher levels of expression, whereas other configurations of SWI/SNF factors are associated with transcripts that have lower levels of expression. To further elucidate the association of SWI/SNF subunits with each other as well as with other nuclear proteins, we also analyzed SWI/SNF immunoprecipitated complexes by mass spectrometry. Individual SWI/SNF factors are associated with their own family members, as well as with cellular constituents such as nuclear matrix proteins, key transcription factors, and centromere components, implying a ubiquitous role in gene regulation and nuclear function. We find an overrepresentation of both SWI/SNF-associated regions and proteins in cell cycle and chromosome organization. Taken together the results from our ChIP and immunoprecipitation experiments suggest that SWI/SNF facilitates gene regulation and genome function more broadly and through a greater diversity of interactions than previously appreciated