Misregulation of DNA damage repair pathways in HPV-positive head and neck squamous cell carcinoma contributes to cellular radiosensitivity

Patients with human papillomavirus type 16 (HPV)-associated oropharyngealsquamous cell carcinomas (OPSCC) display increased sensitivity to radiotherapy andimproved survival rates in comparison to HPV-negative forms of the disease. Howeverthe cellular mechanisms responsible for this characteristic difference are unclear.Here, we have investigated the contribution of DNA damage repair pathways to thein vitro radiosensitivity of OPSCC cell lines. We demonstrate that two HPV-positiveOPSCC cells are indeed more radiosensitive than two HPV-negative OPSCC cells, whichcorrelates with reduced efficiency for the repair of ionising radiation (IR)-inducedDNA double strand breaks (DSB). Interestingly, we show that HPV-positive OPSCCcells consequently have upregulated levels of the proteins XRCC1, DNA polymerase β,PNKP and PARP-1 which are involved in base excision repair (BER) and single strandbreak (SSB) repair. This translates to an increased capacity and efficiency for therepair of DNA base damage and SSBs in these cells. In addition, we demonstratethat HPV-positive but interestingly more so HPV-negative OPSCC display increasedradiosensitivity in combination with the PARP inhibitor olaparib. This suggests thatPARP inhibition in combination with radiotherapy may be an effective treatmentfor both forms of OPSCC, particularly for HPV-negative OPSCC which is relativelyradioresistant

Bim Links ER Stress and Apoptosis in Cells Expressing Mutant SOD1 Associated with Amyotrophic Lateral Sclerosis

Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1

Mammographic density and breast cancer risk: current understanding and future prospects

Variations in percent mammographic density (PMD) reflect variations in the amounts of collagen and number of epithelial and non-epithelial cells in the breast. Extensive PMD is associated with a markedly increased risk of invasive breast cancer. The PMD phenotype is important in the context of breast cancer prevention because extensive PMD is common in the population, is strongly associated with risk of the disease, and, unlike most breast cancer risk factors, can be changed. Work now in progress makes it likely that measurement of PMD will be improved in the near future and that understanding of the genetics and biological basis of the association of PMD with breast cancer risk will also improve. Future prospects for the application of PMD include mammographic screening, risk prediction in individuals, breast cancer prevention research, and clinical decision making

Cotranslational folding of spectrin domains via partially structured states.

How do the key features of protein folding, elucidated from studies on native, isolated proteins, manifest in cotranslational folding on the ribosome? Using a well-characterized family of homologous α-helical proteins with a range of biophysical properties, we show that spectrin domains can fold vectorially on the ribosome and may do so via a pathway different from that of the isolated domain. We use cryo-EM to reveal a folded or partially folded structure, formed in the vestibule of the ribosome. Our results reveal that it is not possible to predict which domains will fold within the ribosome on the basis of the folding behavior of isolated domains; instead, we propose that a complex balance of the rate of folding, the rate of translation and the lifetime of folded or partly folded states will determine whether folding occurs cotranslationally on actively translating ribosomes.Supported by grants from the Swedish Cancer Foundation, the Swedish Research Council and the Knut and Alice Wallenberg Foundation (to G.v.H.); the Wellcome Trust (WT095195 to J.C.) and the European Research Council (ERC-2008-AdG 232648, to R.B.). J.C. is a Wellcome Trust Senior Research Fellow

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response

Endonuclease VIII-like protein 1 (NEIL1) is a DNA glycosylase involved in initiating the base excision repair pathway, the major cellular mechanism for repairing DNA base damage. Here, we have purified the major E3 ubiquitin ligases from human cells responsible for regulation of NEIL1 by ubiquitylation. Interestingly, we have identified two enzymes that catalyse NEIL1 polyubiquitylation, Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26). We demonstrate that these enzymes are capable of polyubiquitylating NEIL1 in vitro, and that both catalyse ubiquitylation of NEIL1 within the same C-terminal lysine residues. An siRNA-mediated knockdown of Mule or TRIM26 leads to stabilisation of NEIL1, demonstrating that these enzymes are important in regulating cellular NEIL1 steady state protein levels. Similarly, a mutant NEIL1 protein lacking residues for ubiquitylation is more stable than the wild type protein in vivo. We also demonstrate that cellular NEIL1 protein is induced in response to ionising radiation (IR), although this occurs specifically in a Mule-dependent manner. Finally we show that stabilisation of NEIL1, particularly following TRIM26 siRNA, contributes to cellular resistance to IR. This highlights the importance of Mule and TRIM26 in maintaining steady state levels of NEIL1, but also those required for the cellular DNA damage response

Complex DNA damage induced by high-LET a-particles and protons triggers a specific cellular DNA damage response

Purpose Complex DNA damage (CDD), where two or more DNA lesions are induced in close proximity, is a signature of ionising radiation (IR) that contributes significantly to cell killing due to the difficult nature of its repair. However the precise mechanism of recognition and processing of CDD in cellular DNA, where the DNA is wrapped around histone proteins to form chromatin, is currently unknown. Methods and Materials HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A and UMSCC6) cells were irradiated with high linear energy transfer (LET) -particles or protons, versus low-LET protons and x-rays. At various time points post-irradiation, site-specific histone post-translational modifications (PTMs) were analysed by quantitative Western blotting, DNA damage and repair was measured by different versions of the comet assay, and cell survival was determined using clonogenic assays. Results Site-specific histone PTMs following low and high LET radiation, particularly proton irradiation, were screened aiming to identify those responsive to CDD. We demonstrate that histone H2B ubiquitylated on lysine 120 (H2Bub) is specifically induced several hours post-irradiation in response to high-LET α-particles and protons, but not by low-LET protons or x-rays. This is associated with increased levels of CDD and which contributes to decreased cell survival. We further discovered that modulation of H2Bub is under the control of two E3 ubiquitin ligases, MSL2 and RNF20/RNF40 complex, whose depletion leads to defective processing and further persistence of CDD, and to additional decreased cell survival post-irradiation. Conclusion This study demonstrates that the signalling and repair of CDD, particularly induced by high-LET IR is co-ordinated through the specific induction of H2Bub catalysed by MSL2 and RNF20/40, a mechanism which contributes significantly to cell survival post-irradiation.</p