Increased hepatic telomerase activity in a rat model of iron overload: A role for altered thiol.....Materials and methodsAnimalsHepatic iron concentrationDetermination of telomere restriction fragment lengthTelomerase activityQuantitation of rat TERT expression by real-time PCRImmunoprecipitation of rat TERTHPLC analysis of thiol poolsGlutamate cysteine ligase activityWestern blotsStatistical analysisResultsEffects of iron loadingEffects of iron loading on telomere lengthEffects of iron loading on telomerase activityEffects of iron loading on hepatic glutathione metabolismDiscussionAcknowledgmentsReferencesOriginal ContributionIncreased hepatic telomerase activity in a rat model of iron overload:A role for altered thiol redox state?Kyle E. Browna,b,c,⁎, M. Meleah Mathahsa, Kimberly A. Broadhursta, Mitchell C. Colemanc,Lisa A. Ridnourd, Warren N. Schmidta,b, Douglas R. SpitzcaIowa City Veterans Administration Medical Center, Iowa City, IA, USAbDivision of Gastroenterology–Hepatology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242, USAcProgram in Free Radical and Radiation Biology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242, USAdRadiation Biology Branch, National Cancer Institute, Bethesda, MD 20892, USAReceived 28 June 2006; revised 3 October 2006; accepted 10 October 2006Available online 17 October 2006AbstractTelomeres are repeated sequences at chromosome ends that are incompletely replicated during mitosis. Telomere shortening caused byproliferation or oxidative damage culminates in replicative arrest and senescence, which may impair regeneration during chronic liver injury.Whereas the effects of experimental liver injury on telomeres have received little attention, prior studies suggest that telomerase, the enzymecomplex that catalyzes the addition of telomeric repeats, is protective in some rodent liver injury models. Thus, the aim of this study was todetermine the effects of iron overload on telomere length and telomerase activity in rat liver. Mean telomere lengths were similar in iron−loadedand control livers. However, telomerase activity was increased 3-fold by iron loading, with no change in levels of TERT mRNA or protein.Because thiol redox state has been shown to modulate telomerase activity in vitro, hepatic thiols were assessed. Significant increases in GSH (1.5-fold), cysteine (15-fold), and glutamate cysteine ligase activity (1.5-fold) were observed in iron-loaded livers, whereas telomerase activity wasinhibited by treatment with N-ethylmaleimide. This is the first demonstration of increased telomerase activity associated with thiol alterations invivo. Enhanced telomerase activity may be an important factor contributing to the resistance of rodent liver to iron-induced damage.© 2006 Elsevier Inc. All rights reserved.Keywords: Glutamate cysteine ligase; Glutathione; Hemochromatosis; Oxidative stress; Telomeres; Free radicalsTelomeres are repeated sequences (TTAGGGn) at the ends ofchromosomes that are incompletely copied when DNA isreplicated during mitosis. In cells lacking a mechanism to restoretelomeric sequences, telomeres therefore shorten progressivelywith each round of cell division. When telomeres reach athreshold length, cells withdraw from the cell cycle and acquire asenescent phenotype. Thus, the inexorable shortening oftelomeres with each round of cell division is regarded as a“mitotic clock” that records the number of antecedent celldivisions and signals the onset of phenotypic alterationsassociated with aging [1,2].A substantial body of data indicates that telomere attrition ismodulated by ox idant –antioxidant balance. For example,human cells cultured under hyperoxic conditions demonstrateaccelerated loss of telomeric sequences and diminishedreplicative capacity compared to cells grown under morephysiologic oxygen concentrations, an effect that is attenuatedin cell lines with more robust antioxidant capacities [3,4].Telomere shortening under these circumstances is accompaniedby accumulation of single-strand breaks along the telomeres [5],which may reflect the enhanced susceptibility of telomericsequences to oxidan t-mediated cleavage. Data from in vitrostudies indi cate that the guanine-rich telomeric repeats arepreferential sites for sequestration of transition metals that mayparticipate in the generation of the oxidizing species responsiblefor strand breaks [6]. These observations suggest that, along withcell division, oxidative damag e is a major factor contributing totelomere shortening.Free Radical Biology & Medicine 42 (2007) 228 – 235www.elsevier.com/locate/freeradbiomed⁎Corresponding author. Division of Gastroenterology-Hepatology, 4553 JCP,200 Hawkins Drive, Iowa City, IA 52242, USA. Fax: +1 319 356 7918.E-mail address: [email protected] (K.E. Brown).0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2006.10.039Conversely, oxidant–antioxidant balance may impact ontelomere length through modulation of telomerase activity.Telomerase is a ribonucleoprotein enzyme co mplex thatcatalyzes the addition of telomeric repeats to chromosomeends, thereby counteracting the effects of telomere shortening.Although most differentiated human somatic cells lackdetectable telomerase activity, germ cells, some stem cells,and most cancers possess telomerase activity. Furthermore,unlike humans, adult rodents retain telomerase activity in manydifferentiated cell types. In the murine 3T3 fibroblast cell lineand in some human and rat cancer cell lines, telomerase activityhas been shown to be sensitive to thiol redox state, with activityincreasing after addition of glutathione or dith iothreitol anddecreasing after inhibition of glutathione synthesis or treatmentwith N-ethylma leimide [7,8]. Taken together wi th the observa-tions regarding accelerated telomere shortening under condi-tions of oxidative stress, these data provide evidence thattelomere biology may be profoundly affected by oxidant–antioxidant balance. However, as all of these studies have beenperformed on cultured cells, it is unknown whether suchalterations have a similar effect in the whole animal.Several lines of evidence indicate that telomere shorteningmay play a role in the evolution of chronic liver disease. Thisconcept i s su pported b y stud ies d emonstrating that thetelomeres of chronically diseased human livers are shorterthan those
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