Regulation of PCNA polyubiquitination in human cells

<p>Abstract</p> <p>Background</p> <p>The ubiquitin-based molecular switch dictating error free versus error prone repair has been conserved throughout eukaryotic evolution. A central component of this switch is the homotrimeric clamp PCNA, which is ubiquitinated in response to genotoxic stress allowing recovery of replication forks blocked at sites of DNA damage. The particulars of PCNA ubiquitination have been elucidated in yeast and to a further extent recently in human cells. However, gaps in the detailed mechanism and regulation of PCNA polyubiquitination still persist in human cells.</p> <p>Findings</p> <p>We expand upon several studies and show that PCNA is polyubiquitnated in normal skin fibroblasts, and that this ubiquitination is dependant on RAD18. Furthermore we define the types of DNA damage that induce ubiquitination on PCNA. Cisplatin, methylmethane sulphonate and benzo(a)pyrene-diol-epoxide induce the polyubiquitination of PCNA to the same extent as UV while polyubiquitination is not detected after X-ray treatment. Moreover, we show that ubiquitination of PCNA is not regulated by cell cycle checkpoint kinases ATM-Chk2 or ATR-Chk1. Significantly, we report that PCNA polyubiquitination is negatively regulated by USP1.</p> <p>Conclusions</p> <p>Our results demonstrate the importance of PCNA polyubiquitination in human cells and define the key regulator of this ubiquitination.</p

Lysine 63-Polyubiquitination Guards against Translesion Synthesis–Induced Mutations

Eukaryotic cells possess several mechanisms to protect the integrity of their DNA against damage. These include cell-cycle checkpoints, DNA-repair pathways, and also a distinct DNA damage–tolerance system that allows recovery of replication forks blocked at sites of DNA damage. In both humans and yeast, lesion bypass and restart of DNA synthesis can occur through an error-prone pathway activated following mono-ubiquitination of proliferating cell nuclear antigen (PCNA), a protein found at sites of replication, and recruitment of specialized translesion synthesis polymerases. In yeast, there is evidence for a second, error-free, pathway that requires modification of PCNA with non-proteolytic lysine 63-linked polyubiquitin (K63-polyUb) chains. Here we demonstrate that formation of K63-polyUb chains protects human cells against translesion synthesis–induced mutations by promoting recovery of blocked replication forks through an alternative error-free mechanism. Furthermore, we show that polyubiquitination of PCNA occurs in UV-irradiated human cells. Our findings indicate that K63-polyubiquitination guards against environmental carcinogenesis and contributes to genomic stability

Hedgehog Pathway as a Potential Intervention Target in Esophageal Cancer

Esophageal cancer (EC) is an aggressive disease with a poor prognosis. Treatment resistance is a major challenge in successful anti-cancer therapy. Pathological complete response after neoadjuvant chemoradiation (nCRT) is low, thus requiring therapy optimization. The Hedgehog (HH) pathway has been implicated in therapy resistance, as well as in cancer stemness. This article focusses on the HH pathway as a putative target in the treatment of EC. Immunohistochemistry on HH members was applied to EC patient material followed by modulation of 3D-EC cell cultures, fluorescence-activated cell sorting (FACS), and gene expression analysis after HH pathway modulation. Sonic Hedgehog (SHH) and its receptor Patched1 (PTCH1) were significantly enriched in EC resection material of patients with microresidual disease (mRD) after receiving nCRT, compared to the control group. Stimulation with SHH resulted in an up-regulation of cancer stemness in EC sphere cultures, as indicated by increased sphere formation after sorting for CD44+/CD24- EC cancer stem-like cell (CSC) population. On the contrary, inhibiting this pathway with vismodegib led to a decrease in cancer stemness and both radiation and carboplatin resistance. Our results strengthen the role of the HH pathway in chemoradiotherapy resistance. These findings suggest that targeting the HH pathway could be an attractive approach to control CSCs.</p

The In Vitro Response of Tissue Stem Cells to Irradiation With Different Linear Energy Transfers

PURPOSE: A reduction in the dose, irradiated volume, and sensitivity of, in particular, normal tissue stem cells is needed to advance radiation therapy. This could be obtained with the use of particles for radiation therapy. However, the radiation response of normal tissue stem cells is still an enigma. Therefore, in the present study, we developed a model to investigate the in vitro response of stem cells to particle irradiation. METHODS AND MATERIALS: We used the immortalized human salivary gland (HSG) cell line resembling salivary gland (SG) cells to translate the radiation response in 2-dimensional (2D) to 3-dimensional (3D) conditions. This response was subsequently translated to the response of SG stem cells (SGSCs). Dispersed single cells were irradiated with photons or carbon ions at different linear energy transfers (LETs; 48.76 ± 2.16, 149.9 ± 10.8, and 189 ± 15 keV/μm). Subsequently, 2D or 3D clonogenicity was determined by counting the colonies or secondary stem cell-derived spheres in Matrigel. γH2AX immunostaining was used to assess DNA double strand break repair. RESULTS: The 2D response of HSG cells showed a similar increase in dose response to increasing higher LET irradiation as other cell lines. The 3D response of HSG cells to increasing LET irradiation was reduced compared with the 2D response. Finally, the response of mouse SGSCs to photons was similar to the 3D response of HSG cells. The response to higher LET irradiation was reduced in the stem cells. CONCLUSIONS: Mouse SGSC radiosensitivity seems reduced at higher LET radiation compared with transformed HSG cells. The developed model to assess the radiation response of SGSCs offers novel possibilities to study the radiation response of normal tissue in vitro

Clinical relevance of the radiation dose bath in lower grade glioma, a cross-sectional pilot study on neurocognitive and radiological outcome

AIM: To investigate the clinical relevance of the radiotherapy (RT) dose bath in patients treated for lower grade glioma (LGG). METHODS: Patients (n = 17) treated with RT for LGG were assessed with neurocognitive function (NCF) tests and structural Magnetic Resonance Imaging (MRI) and categorized in subgroups based on tumour lateralisation. RT dose, volumetric results and cerebral microbleed (CMB) number were extracted for contralateral cerebrum, contralateral hippocampus, and cerebellum. The RT clinical target volume (CTV) was included in the analysis as a surrogate for focal tumour and other treatment effects. The relationships between RT dose, CTV, NCF and radiological outcome were analysed per subgroup. RESULTS: The subgroup with left-sided tumours (n = 10) performed significantly lower on verbal tests. The RT dose to the right cerebrum, as well as CTV, were related to poorer performance on tests for processing speed, attention, and visuospatial abilities, and more CMB. In the subgroup with right-sided tumours (n = 7), RT dose in the left cerebrum was related to lower verbal memory performance, (immediate and delayed recall, r = −0.821, p = 0.023 and r = −0.937, p = 0.002, respectively), and RT dose to the left hippocampus was related to hippocampal volume (r = −0.857, p = 0.014), without correlation between CTV and NCF. CONCLUSION: By using a novel approach, we were able to investigate the clinical relevance of the RT dose bath in patients with LGG more specifically. We used combined MRI-derived and NCF outcome measures to assess radiation-induced brain damage, and observed potential RT effects on the left-sided brain resulting in lower verbal memory performance and hippocampus volume

hMMS2 serves a redundant role in human PCNA polyubiquitination

<p>Abstract</p> <p>Background</p> <p>In yeast, DNA damage leads to the mono and polyubiquitination of the sliding clamp PCNA. Monoubiquitination of PCNA is controlled by RAD18 (E3 ligase) and RAD6 (E2 conjugating enzyme), while the extension of the monoubiquitinated PCNA into a polyubiquitinated substrate is governed by RAD5, and the heterodimer of UBC13/MMS2. Each modification directs a different branch of the DNA damage tolerance pathway (DDT). While PCNA monoubiquitination leads to error-prone bypass via TLS, biochemical studies have identified MMS2 along with its heteromeric partner UBC13 to govern the error-free repair of DNA lesions by catalyzing the formation of lysine 63-linked polyubiquitin chains (K63-polyUb). Recently, it was shown that PCNA polyubiquitination is conserved in human cells and that this modification is dependent on RAD18, UBC13 and SHPRH. However, the role of hMMS2 in this process was not specifically addressed.</p> <p>Results</p> <p>In this report we show that mammalian cells in which MMS2 was reduced by siRNA-mediated knockdown maintains PCNA polyubiquitination while a knockdown of RAD18 or UBC13 abrogates PCNA ubiquitination. Moreover, the additional knockdown of a UEV1A (MMS2 homolog) does not deplete PCNA polyubiquitination. Finally, mouse embryonic stem cells null for MMS2 with or without the additional depletion of mUEV1A continue to polyubiquitinated PCNA with normal kinetics.</p> <p>Conclusion</p> <p>Our results point to a high level of redundancy in the DDT pathway and suggest the existence of another hMMS2 variant (hMMSv) or complex that can compensate for its loss.</p

Centrality Dependence of the High p_T Charged Hadron Suppression in Au+Au collisions at sqrt(s_NN) = 130 GeV

PHENIX has measured the centrality dependence of charged hadron p_T spectra from central Au+Au collisions at sqrt(s_NN)=130 GeV. The truncated mean p_T decreases with centrality for p_T > 2 GeV/c, indicating an apparent reduction of the contribution from hard scattering to high p_T hadron production. For central collisions the yield at high p_T is shown to be suppressed compared to binary nucleon-nucleon collision scaling of p+p data. This suppression is monotonically increasing with centrality, but most of the change occurs below 30% centrality, i.e. for collisions with less than about 140 participating nucleons. The observed p_T and centrality dependence is consistent with the particle production predicted by models including hard scattering and subsequent energy loss of the scattered partons in the dense matter created in the collisions.Comment: 7 pages text, LaTeX, 6 figures, 2 tables, 307 authors, resubmitted to Phys. Lett. B. Revised to address referee concerns. Plain text data tables for the points plotted in figures for this and previous PHENIX publications are publicly available at http://www.phenix.bnl.gov/phenix/WWW/run/phenix/papers.htm

Measurement of the cross-section and charge asymmetry of WW bosons produced in proton-proton collisions at s=8\sqrt{s}=8 TeV with the ATLAS detector

This paper presents measurements of the $W^+ \rightarrow \mu^+\nu$ and $W^- \rightarrow \mu^-\nu$ cross-sections and the associated charge asymmetry as a function of the absolute pseudorapidity of the decay muon. The data were collected in proton--proton collisions at a centre-of-mass energy of 8 TeV with the ATLAS experiment at the LHC and correspond to a total integrated luminosity of 20.2~\mbox{fb^{-1}}. The precision of the cross-section measurements varies between 0.8% to 1.5% as a function of the pseudorapidity, excluding the 1.9% uncertainty on the integrated luminosity. The charge asymmetry is measured with an uncertainty between 0.002 and 0.003. The results are compared with predictions based on next-to-next-to-leading-order calculations with various parton distribution functions and have the sensitivity to discriminate between them.Comment: 38 pages in total, author list starting page 22, 5 figures, 4 tables, submitted to EPJC. All figures including auxiliary figures are available at https://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/STDM-2017-13

Search for chargino-neutralino production with mass splittings near the electroweak scale in three-lepton final states in √s=13 TeV pp collisions with the ATLAS detector

A search for supersymmetry through the pair production of electroweakinos with mass splittings near the electroweak scale and decaying via on-shell W and Z bosons is presented for a three-lepton final state. The analyzed proton-proton collision data taken at a center-of-mass energy of √s=13  TeV were collected between 2015 and 2018 by the ATLAS experiment at the Large Hadron Collider, corresponding to an integrated luminosity of 139  fb−1. A search, emulating the recursive jigsaw reconstruction technique with easily reproducible laboratory-frame variables, is performed. The two excesses observed in the 2015–2016 data recursive jigsaw analysis in the low-mass three-lepton phase space are reproduced. Results with the full data set are in agreement with the Standard Model expectations. They are interpreted to set exclusion limits at the 95% confidence level on simplified models of chargino-neutralino pair production for masses up to 345 GeV

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente