151 research outputs found

    ATM inhibition blocks glucose metabolism and amplifies the sensitivity of resistant lung cancer cell lines to oncogene driver inhibitors

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    Background: ATM is a multifunctional serine/threonine kinase that in addition to its well-established role in DNA repair mechanisms is involved in a number of signaling pathways including regulation of oxidative stress response and metabolic diversion of glucose through the pentose phosphate pathway. Oncogene-driven tumorigenesis often implies the metabolic switch from oxidative phosphorylation to glycolysis which provides metabolic intermediates to sustain cell proliferation. The aim of our study is to elucidate the role of ATM in the regulation of glucose metabolism in oncogene-driven cancer cells and to test whether ATM may be a suitable target for anticancer therapy. Methods: Two oncogene-driven NSCLC cell lines, namely H1975 and H1993 cells, were treated with ATM inhibitor, KU55933, alone or in combination with oncogene driver inhibitors, WZ4002 or crizotinib. Key glycolytic enzymes, mitochondrial complex subunits (OXPHOS), cyclin D1, and apoptotic markers were analyzed by Western blotting. Drug-induced toxicity was assessed by MTS assay using stand-alone or combined treatment with KU55933 and driver inhibitors. Glucose consumption, pyruvate, citrate, and succinate levels were also analyzed in response to KU55933 treatment. Both cell lines were transfected with ATM-targeted siRNA or non-targeting siRNA and then exposed to treatment with driver inhibitors. Results: ATM inhibition deregulates and inhibits glucose metabolism by reducing HKII, p-PKM2Tyr105, p-PKM2Ser37, E1α subunit of pyruvate dehydrogenase complex, and all subunits of mitochondrial complexes except ATP synthase. Accordingly, glucose uptake and pyruvate concentrations were reduced in response to ATM inhibition, whereas citrate and succinate levels were increased in both cell lines indicating the supply of alternative metabolic substrates. Silencing of ATM resulted in similar changes in glycolytic cascade and OXPHOS levels. Furthermore, the driver inhibitors amplified the effects of ATM downregulation on glucose metabolism, and the combined treatment with ATM inhibitors enhanced the cytotoxic effect of driver inhibitors alone by increasing the apoptotic response. Conclusions: Inhibition of ATM reduced both glycolytic enzymes and OXPHOS levels in oncogene-driven cancer cells and enhanced apoptosis induced by driver inhibitors thus highlighting the possibility to use ATM and the driver inhibitors in combined regimens of anticancer therapy in vivo

    Inflammation and DNA damage: cause, effect or both

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    : Inflammation is a biological response involving immune cells, blood vessels and mediators induced by endogenous and exogenous stimuli, such as pathogens, damaged cells or chemicals. Unresolved (chronic) inflammation is characterized by the secretion of cytokines that maintain inflammation and redox stress. Mitochondrial or nuclear redox imbalance induces DNA damage, which triggers the DNA damage response (DDR) that is orchestrated by ATM and ATR kinases, which modify gene expression and metabolism and, eventually, establish the senescent phenotype. DDR-mediated senescence is induced by the signalling proteins p53, p16 and p21, which arrest the cell cycle in G1 or G2 and promote cytokine secretion, producing the senescence-associated secretory phenotype. Senescence and inflammation phenotypes are intimately associated, but highly heterogeneous because they vary according to the cell type that is involved. The vicious cycle of inflammation, DNA damage and DDR-mediated senescence, along with the constitutive activation of the immune system, is the core of an evolutionarily conserved circuitry, which arrests the cell cycle to reduce the accumulation of mutations generated by DNA replication during redox stress caused by infection or inflammation. Evidence suggests that specific organ dysfunctions in apparently unrelated diseases of autoimmune, rheumatic, degenerative and vascular origins are caused by inflammation resulting from DNA damage-induced senescence

    Loss of p53 activates thyroid hormone via type 2 deiodinase and enhances DNA damage

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    : The Thyroid Hormone (TH) activating enzyme, type 2 Deiodinase (D2), is functionally required to elevate the TH concentration during cancer progression to advanced stages. However, the mechanisms regulating D2 expression in cancer still remain poorly understood. Here, we show that the cell stress sensor and tumor suppressor p53 silences D2 expression, thereby lowering the intracellular THs availability. Conversely, even partial loss of p53 elevates D2/TH resulting in stimulation and increased fitness of tumor cells by boosting a significant transcriptional program leading to modulation of genes involved in DNA damage and repair and redox signaling. In vivo genetic deletion of D2 significantly reduces cancer progression and suggests that targeting THs may represent a general tool reducing invasiveness in p53-mutated neoplasms

    5-Hydroxytryptamine Modulates Maturation and Mitochondria Function of Human Oligodendrocyte Progenitor M03-13 Cells

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    Inside the adult CNS, oligodendrocyte progenitor cells (OPCS) are able to proliferate, migrate and differentiate into mature oligodendrocytes (OLs) which are responsible for the production of myelin sheet and energy supply for neurons. Moreover, in demyelinating diseases, OPCs are recruited to the lesion areas where they undergo differentiation and myelin synthesis. Serotonin (5-hydroxytryptamine, 5-HT) is involved in OLs’ development and myelination, but so far the molecular mechanisms involved or the effects of 5-HT on mitochondria function have not yet been well documented. Our data show that 5-HT inhibits migration and proliferation committing cells toward differentiation in an immortalized human oligodendrocyte precursor cell line, M03-13. Migration blockage is mediated by reactive oxygen species (ROS) generation since antioxidants, such as Vit C and Cu-Zn superoxide dismutase, prevent the inhibitory effects of 5-HT on cell migration. 5-HT inhibits OPC migration and proliferation and increases OL phenotypic markers myelin basic protein (MBP) and Olig-2 via protein kinase C (PKC) activation since the inhibitor of PKC, bis-indolyl-maleimide (BIM), counteracts 5-HT effects. NOX inhibitors as well, reverse the effects of 5-HT, indicating that 5-HT influences the maturation process of OPCs by NOX-dependent ROS production. Finally, 5-HT increases mitochondria function and antioxidant activity. The identification of the molecular mechanisms underlying the effects of 5-HT on maturation and energy metabolism of OPCs could pave the way for the development of new treatments for autoimmune demyelinating diseases such as Multiple Sclerosis where oligodendrocytes are the primary target of immune attack

    Tracing and tracking epiallele families in complex DNA populations

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    DNA methylation is a stable epigenetic modification, extremely polymorphic and driven by stochastic and deterministic events. Most of the current techniques used to analyse methylated sequences identify methylated cytosines (mCpGs) at a single-nucleotide level and compute the average methylation of CpGs in the population of molecules. Stable epialleles, i.e. CpG strings with the same DNA sequence containing a discrete linear succession of phased methylated/non-methylated CpGs in the same DNA molecule, cannot be identified due to the heterogeneity of the 5′–3′ ends of the molecules. Moreover, these are diluted by random unstable methylated CpGs and escape detection. We present here MethCoresProfiler, an R-based tool that provides a simple method to extract and identify combinations of methylated phased CpGs shared by all components of epiallele families in complex DNA populations. The methylated cores are stable over time, evolve by acquiring or losing new methyl sites and, ultimately, display high information content and low stochasticity. We have validated this method by identifying and tracing rare epialleles and their families in synthetic or in vivo complex cell populations derived from mouse brain areas and cells during postnatal differentiation

    Targeted DNA oxidation by LSD1–SMAD2/3 primes TGF-β1/ EMT genes for activation or repression

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    Abstract The epithelial-to-mesenchymal transition (EMT) is a complex transcriptional program induced by transforming growth factor β1 (TGF-β1). Histone lysine-specific demethylase 1 (LSD1) has been recognized as a key mediator of EMT in cancer cells, but the precise mechanism that underlies the activation and repression of EMT genes still remains elusive. Here, we characterized the early events induced by TGF-β1 during EMT initiation and establishment. TGF-β1 triggered, 30–90 min post-treatment, a nuclear oxidative wave throughout the genome, documented by confocal microscopy and mass spectrometry, mediated by LSD1. LSD1 was recruited with phosphorylated SMAD2/3 to the promoters of prototypic genes activated and repressed by TGF-β1. After 90 min, phospho-SMAD2/3 downregulation reduced the complex and LSD1 was then recruited with the newly synthesized SNAI1 and repressors, NCoR1 and HDAC3, to the promoters of TGF-β1-repressed genes such as the Wnt soluble inhibitor factor 1 gene (WIF1), a change that induced a late oxidative burst. However, TGF-β1 early (90 min) repression of transcription also required synchronous signaling by reactive oxygen species and the stress-activated kinase c-Jun N-terminal kinase. These data elucidate the early events elicited by TGF-β1 and the priming role of DNA oxidation that marks TGF-β1-induced and -repressed genes involved in the EMT

    DNA damage signatures in peripheral blood cells as biomarkers in prodromal huntington disease

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    Easily accessible biomarkers in Huntington disease (HD) are actively searched. We investigated telomere length (TL) and DNA double-strand breaks (histone variant pγ-H2AX) as predictive disease biomarkers in peripheral blood mononuclear cells (PBMC) from 25 pre-manifest, 58 HD patients, with similar CAG expansion in the huntingtin gene (HTT) gene and 44 healthy controls (HC). PBMC from PRE-HD and HD groups showed shorter telomeres (p<0.0001) and a significant increase of pγ-H2AX compared to the controls (p<0.0001). The levels of pγ-H2AX correlated robustly with the presence of the mutated gene in PRE-HD and HD. The availability of a potentially reversible biomarker (pγ-H2AX) in the pre-manifest stage of HD, negligible in HC, provides a novel tool to monitor pre-manifest subjects and find patient-specific drugs

    Selective demethylation of two CpG sites causes postnatal activation of the Dao gene and consequent removal of D-serine within the mouse cerebellum

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    Programmed epigenetic modifications occurring at early postnatal brain developmental stages may have a long-lasting impact on brain function and complex behavior throughout life. Notably, it is now emerging that several genes that undergo perinatal changes in DNA methylation are associated with neuropsychiatric disorders. In this context, we envisaged that epigenetic modifications during the perinatal period may potentially drive essential changes in the genes regulating brain levels of critical neuromodulators such as D-serine and D-aspartate. Dysfunction of this fine regulation may contribute to the genesis of schizophrenia or other mental disorders, in which altered levels of D-amino acids are found. We recently demonstrated that Ddo, the D-aspartate degradation gene, is actively demethylated to ultimately reduce D-aspartate levels. However, the role of epigenetics as a mechanism driving the regulation of appropriate D-ser levels during brain development has been poorly investigated to date
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