Helmy Institute for Medical science
Helmy Institute for Medical science
Walker C.,University of Sheffield |
Herranz-Martin S.,University of Sheffield |
Karyka E.,University of Sheffield |
Liao C.,University of Sheffield |
And 21 more authors.
Nature Neuroscience | Year: 2017
Hexanucleotide repeat expansions represent the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, though the mechanisms by which such expansions cause neurodegeneration are poorly understood. We report elevated levels of DNA-RNA hybrids (R-loops) and double strand breaks in rat neurons, human cells and C9orf72 ALS patient spinal cord tissues. Accumulation of endogenous DNA damage is concomitant with defective ATM-mediated DNA repair signaling and accumulation of protein-linked DNA breaks. We reveal that defective ATM-mediated DNA repair is a consequence of P62 accumulation, which impairs H2A ubiquitylation and perturbs ATM signaling. Virus-mediated expression of C9orf72-related RNA and dipeptide repeats in the mouse central nervous system increases double strand breaks and ATM defects and triggers neurodegeneration. These findings identify R-loops, double strand breaks and defective ATM-mediated repair as pathological consequences of C9orf72 expansions and suggest that C9orf72-linked neurodegeneration is driven at least partly by genomic instability.
Alagoz M.,University of Sussex |
Chiang S.-C.,University of Sussex |
Sharma A.,University of Sussex |
El-Khamisy S.F.,University of Sussex |
And 2 more authors.
PLoS ONE | Year: 2013
Accumulation of peptide-linked DNA breaks contributes to neurodegeration in humans. This is typified by defects in tyrosyl DNA phosphodiesterase 1 (TDP1) and human hereditary ataxia. TDP1 primarily operates at single-strand breaks (SSBs) created by oxidative stress or by collision of transcription machinery with topoisomerase I intermediates (Top1-CCs). Cellular and cell-free studies have shown that Top1 at stalled Top1-CCs is first degraded to a small peptide resulting in Top1-SSBs, which are the primary substrates for TDP1. Here we established an assay to directly compare Top1-SSBs and Top1-CCs. We subsequently employed this assay to reveal an increased steady state level of Top1-CCs in neural cells lacking Atm; the protein mutated in ataxia telangiectasia. Our data suggest that the accumulation of endogenous Top1-CCs in Atm-/- neural cells is primarily due to elevated levels of reactive oxygen species. Biochemical purification of Top1-CCs from neural cell extract and the use of Top1 poisons further confirmed a role for Atm during the formation/resolution of Top1-CCs. Finally, we report that global transcription is reduced in Atm-/- neural cells and fails to recover to normal levels following Top1-mediated DNA damage. Together, these data identify a distinct role for ATM during the formation/resolution of neural Top1-CCs and suggest that their accumulation contributes to the neuropathology of ataxia telangiectasia. © 2013 Alagoz et al.
Meisenberg C.,University of Sussex |
Ward S.E.,University of Sussex |
Schmid P.,University of Sussex |
Schmid P.,Queen Mary, University of London |
And 3 more authors.
Journal of Cancer Science and Therapy | Year: 2014
Background and objective: Small cell lung cancer (SCLC) is one of the most challenging tumors to treat due to high proliferation rate, early metastatic dissemination and rapid development of chemotherapy resistance. The current treatment protocols involve the use of topoisomerase 1 (TOP1) poisons such as irinotecan and topotecan in combination with platinum-based compounds. TOP1 poisons kill cancer cells by trapping TOP1 on DNA, generating lethal DNA double-strand breaks. A potential mechanism employed by cancer cells to resist killing by TOP1 poisons is to overexpress enzymes involved in the repair of TOP1-DNA breaks. Tyrosyl DNA phosphodiesterase 1 (TDP1) is a key player in this process and despite its importance, no data is currently available to correlate TDP1 protein and mRNA levels with catalytic activity in SCLC. In addition, it is not known if TDP1 and TOP1 protein levels correlate with the cellular response of SCLC to TOP1 based therapies. Methods and results: We report a remarkable variation in TDP1 and TOP1 protein levels in a panel of SCLC cell lines. TDP1 protein level correlates well with TDP1 mRNA and TDP1 catalytic activity, as measured by two newly developed independent activity assays, suggesting the potential utility of immunohistochemistry in assessing TDP1 levels in SCLC tissues. We further demonstrate that whilst TDP1 protein level alone does not correlate with topotecan sensitivity, TDP1/TOP1 ratio correlates well with sensitivity in 8 out of 10 cell lines examined. Conclusion: This study provides the first cellular analyses of TDP1 and TOP1 in SCLC and suggests the potential utility of TDP1/TOP1 ratio to assess the response of SCLC to topotecan. The establishment and validation of an easy-to-use TDP1 enzymatic assay in cell extracts could be exploited as a diagnostic tool in the clinic. These findings may help in stratifying patients that are likely to benefit from TOP1 poisons and TDP1 inhibitors currently under development.
Carroll J.,University of Sussex |
Page T.K.,University of Sussex |
Chiang S.-C.,University of Sheffield |
Kalmar B.,University College London |
And 8 more authors.
Human Molecular Genetics | Year: 2015
Aprataxin (APTX) deficiency causes progressive cerebellar degeneration, ataxia and oculomotor apraxia in man. Cell free assays and crystal structure studies demonstrate a role for APTX in resolving 5--adenylated nucleic acid breaks, however, APTX function in vertebrates remains unclear due to the lack of an appropriate model system. Here, we generated a murine model in which a pathogenic mutant of superoxide dismutase 1 (SOD1G93A) is expressed in an Aptx-/- mouse strain. We report a delayed population doubling and accelerated senescence in Aptx-/- primary mouse fibroblasts, which is not due to detectable telomere instability or cell cycle deregulation but is associated with a reduction in transcription recovery following oxidative stress. Expression of SOD1G93A uncovers a survival defect ex vivo in cultured cells and in vivo in tissues lacking Aptx. The surviving neurons feature numerous and deep nuclear envelope invaginations, a hallmark of cellular stress. Furthermore, they possess an elevated number of high-density nuclear regions and a concomitant increase in histone H3 K9 trimethylation, hallmarks of silenced chromatin. Finally, the accelerated cellular senescence was also observed at the organismal level as shown by down-regulation of insulin-like growth factor 1 (IGF-1), a hallmark of premature ageing. Together, this study demonstrates a protective role of Aptx in vivo and suggests that its loss results in progressive accumulation of DNA breaks in the nervous system, triggering hallmarks of premature ageing, systemically. © The Author 2014. Published by Oxford University Press.
Zuniga-Hertz J.P.,University of Sao Paulo |
Zuniga-Hertz J.P.,University of California at San Diego |
Rebelato E.,University of Sao Paulo |
Kassan A.,University of California at San Diego |
And 5 more authors.
Journal of Endocrinology | Year: 2015
Results from previous investigations have indicated that glucose-stimulated insulin secretion (GSIS) is affected by changes in cholesterol and its intermediates, but the precise link between secretion and cholesterol has not been thoroughly investigated. In this study, we show the contribution of both protein isoprenylation and cholesterol-dependent plasma membrane structural integrity to insulin secretion in INS-1E cells andmouse islets. Acute (2 h) inhibition of hydroxyl-methylglutaryl-CoA reductase by simvastatin (SIM) resulted in inhibition of GSIS without reduction in total cellular cholesterol content. This effect was prevented by cell loading with the isoprenyl molecule geranylgeranyl pyrophosphate. Chronic (24 h) inhibition of cholesterol biosynthesis resulted in inhibition of GSIS with a significant reduction in total cellular cholesterol content, which was also observed after the inhibition of cholesterol biosynthesis downstream of isoprenoid formation. Electron paramagnetic resonance analyses of INS-1E cells showed that the SIM-induced reduction in cholesterol increased plasma membrane fluidity. Thus, the blockade of cholesterol biosynthesis resulted in the reduction of availability of isoprenoids, followed by a reduction in the total cholesterol content associated with an increase in plasma membrane fluidity. Herein, we show the different contributions of cholesterol biosynthesis to GSIS, and propose that isoprenoid molecules and cholesterol-dependent signaling are dual regulators of proper β-cell function. © 2015 Society for Endocrinology.
PubMed | University of Sheffield, Helmy Institute for Medical science and University of Sussex
Type: | Journal: Nucleic acids research | Year: 2016
The topoisomerase I (TOP1) inhibitor irinotecan triggers cell death by trapping TOP1 on DNA, generating cytotoxic protein-linked DNA breaks (PDBs). Despite its wide application in a variety of solid tumors, the mechanisms of cancer cell resistance to irinotecan remains poorly understood. Here, we generated colorectal cancer (CRC) cell models for irinotecan resistance and report that resistance is neither due to downregulation of the main cellular target of irinotecan TOP1 nor upregulation of the key TOP1 PDB repair factor TDP1. Instead, the faster repair of PDBs underlies resistance, which is associated with perturbed histone H4K16 acetylation. Subsequent treatment of irinotecan-resistant, but not parental, CRC cells with histone deacetylase (HDAC) inhibitors can effectively overcome resistance. Immunohistochemical analyses of CRC tissues further corroborate the importance of histone H4K16 acetylation in CRC. Finally, the resistant clones exhibit cross-resistance with oxaliplatin but not with ionising radiation or 5-fluoruracil, suggesting that the latter two could be employed following loss of irinotecan response. These findings identify perturbed chromatin acetylation in irinotecan resistance and establish HDAC inhibitors as potential therapeutic means to overcome resistance.
PubMed | Helmy Institute for Medical science, Alexandria University and Biomedical Technology
Type: Journal Article | Journal: BMC complementary and alternative medicine | Year: 2016
Virus-induced dendritic cells (DCs) functional deficiency leads to sub-optimal initiation of adaptive immune responses and consequently chronic infection establishment. The present study reports an advanced hepatitis C virus (HCV) therapeutic vaccine model based on In vivo enrichment of DCs with barberry ethanolic crude extract (BCE) then pulsing them with HCV core protein.DCs were enriched by BCE intravenous injection in BALB/c mice. Vaccine efficiency was assessed by flow cytometric analysis of splenocytes of immunized mice, cytokine profiling, cytotoxic T lymphocyte assay, and humoral immune response assessment.There was no significant difference in surface phenotypic characterization of splenocytes from mice immunized with non-BCE-enriched-core-pulsed DCs (iDcs-core) compared to those from mice injected with RPMI-1640 medium. However, splenocytes from mice immunized with BCE-enriched-core-pulsed DCs showed 197% increase in CD16+ population, 33% increase in MHCII(+) population, and 43% decrease in CD3(+) population. In iDCs-core group, 57.9% greater anti-core cytotoxic T lymphocyte activity, up-regulation in interferon gamma and interleukin (IL) -12 expression, and down-regulation in IL-4 and IL-10 were recorded. Moreover, sustained specific anti-core antibodies were detected only in sera of the same group.results indicate that BCE-enriched-core-transduced DCs may serve as a new model for immunotherapy of HCV chronic infection.
PubMed | University of Bonn, Suez Canal University, Taibah University, Genetic Engineering & Biotechnology Research Institute and 2 more.
Type: | Journal: Evidence-based complementary and alternative medicine : eCAM | Year: 2015
Berberine is a plant alkaloid that has several pharmacological effects such as antioxidant, antilipidemic, and anti-inflammatory effects. Nonalcoholic steatohepatitis (NASH) triggers different aspects of disorders such as impaired endogenous lipid metabolism, hypercholesterolemia, oxidative stress, and neurotoxicity. In this study, we examined the mechanism by which NASH induces neurotoxicity and the protective effect of berberine against both NASH and its associated neurotoxicity. NASH induced rats showed significant impairments in lipid metabolism with increased serum triglycerides, cholesterol, and low-density lipoprotein (LDL). The NASH induced group also demonstrated a significant oxidative stress which is characterized by increased TBARs level and decreased antioxidant capacity such as GSH and SOD levels. Moreover, the NASH induction was associated with inflammation which was demonstrated by increased TNF and nitric oxide levels. Hyperglycemia and hyperinsulinemia were observed in the NASH induced group. Also, our results showed a significant increase in the expression of the acetylcholine esterase (AChE) and amyloid beta precursor protein (APP). These changes were significantly correlated with decreased insulin degrading enzyme (IDE) and beta-amyloid40 (A 40) and increased beta-amyloid42 (A 42) in the hippocampal region. Daily administration of berberine (50mg/kg) for three weeks ameliorated oxidative stress, inflammation, hyperlipidemia, hyperglycemia, hyperinsulinemia, and the observed neurotoxicity.
Eskander E.F.,National Research Center of Egypt |
Abd-Rabou A.A.,National Research Center of Egypt |
Abd-Rabou A.A.,Helmy Institute for Medical science |
Ahmed H.H.,National Research Center of Egypt
Indian Journal of Clinical Biochemistry | Year: 2013
Hormones play an important role in the digestive system. The main hormones that control digestion are gastrin, secretin, and cholecystokinin. Herein, the current study is concerned with assessing the effect of spasmo canulase and librax drugs on the human hormones profile. Blood samples were withdrawn from adult patients to measure serum FSH, E2, LH, prolactin, progesterone, DHEAS, testosterone, TSH, T3, T4, fasting insulin, and cortisol. All hormone concentrations were determined quantitatively using ELISA procedure. Intriguingly, the present study showed putative changes including thyroid and sex hormonal profiles. Eventually, we concluded that the prospective study could be important in drug dose optimization and providing new medical guidelines to avoid side effects that could harm patients. © 2013 Association of Clinical Biochemists of India.
PubMed | University of Sheffield, Helmy Institute for Medical science and Cairo University
Type: | Journal: Mechanisms of ageing and development | Year: 2016
Despite being an invaluable chemotherapeutic agent for several types of cancer, the clinical utility of doxorubicin is hampered by its age-related and dose-dependent cardiotoxicity. Co-administration of dexrazoxane as a cardioprotective agent has been proposed, however recent studies suggest that it attenuates doxorubicin-induced antitumor activity. Since compounds of natural origin present a rich territory for drug discovery, we set out to identify putative natural compounds with the view to mitigate or minimize doxorubicin cardiotoxicity. We identify the DYRK1A kinase inhibitor harmine, which phosphorylates Tau that is deregulated in Alzheimers disease, as a potentiator of cell death induced by non-toxic doses of doxorubicin. These observations suggest that harmine or other compounds that target the DYRK1A kinase my offer a new therapeutic opportunity to suppress doxorubicin age-related and dose-dependent cardiotoxicity.