细胞死亡及周期阻滞基本信号通路

CELLDEATHANDCELL-CYCLECHECKPOINTPATHWAYSDURINGDNADAMAGEJIAHong-Ti（贾弘禔）DEPTBIOCHEMMOLBIOLHSC,[e-mail]损伤时细胞死亡和细胞周期监控点（调控的基本）信号途径1.有哪些因素可引起DNA损伤？DNA损伤的结局如何？2.什么分子可作为DNA双链断裂/损伤的标志？用什么方法测定？3.简述MicroRNA在DDR中的作用。（听完本讲座，结合自己研究课题和文献检索）试举一例（可能）与自己工作领域相关的MicroRNA功能及其生物学或临床意义。4.写出G1、G2/Mcheckpoint基本信号通路主要分子成分。5.程序性细胞死亡（PCD）都有哪些形式？I型（凋亡）、II型（自噬）（自噬定义）PCD主要形态学特征如何？如何检测或鉴定？其基本信号通路所涉及的主要激活分子是什么？各有何意义？本讲基本要求——思考题诱导DNA损伤与细胞死亡原因*——1.各种射线照射/光动力学治疗（光敏反应）2.化疗药物/细胞毒、基因毒试剂3.缺血/缺氧/氧化应激4.血清/营养因子/氨基酸撤出5.营养过剩6.过度兴奋/刺激7.中毒/钙超载8.感染/炎症9.其它*常伴有或直接、间接引起DNA损伤该讲的普遍意义——多种原因相关的DNA损伤引起细胞死亡和细胞周期阻滞1.THEPLETHORAOFDAMAGESINDNA(DNA损伤的多样性/复杂性）2.THECONSEQUENCESOFDNAINJURY(DNA损伤的结局)3.CELL-CYCLECHECKPOINTPATHWAYS(细胞周期监控点基本信号途径)4.PROGRAMMED(REGULATED)CELLDEATH(程序性细胞死亡,PCD)本讲提纲1.APLETHORAOFDAMAGESINDNAAPERPLEXINGDIVERSITYOFDNALESIONSARISESFROMTHREEMAINCAUSESEnvironmentalagents(环境因素)Productsofnormalcellularmetabolisminmitochondrion（线粒体代谢产物——ROS）SomechemicalbondsinDNAspontaneouslydisintegrateunderphysiologicalconditions（DNA自发裂解）(A)EnvironmentalAgents:ultraviolet(UV),ionizingradiation…(环境因素)genotoxicchemicalsvirusinfectionAlterationsinDNAstructure:AdjacentthyminedimerDNAsinglestrandedbreaks(DNASSBs)DNAdoublestrandedbreaks(DNADSBs)Cross-linking(intra-andinter-cross-linking)…DNA损伤的多样性及主要诱因(B)ProductsofNormalCellularMetabolisminMitochondrion:(线粒体代谢产物——ROS）Reactiveoxygenspecies(ROS)derivedfromoxidativerespirationandproductsoflipidperoxidation.OxidativemodificationsinDNA.e.g.,8-OxoGAntioxidantdefencesystemcomposedofenzymatic(superoxidedismutase/SOD,catalase,glutathioneperoxidase/GPxandperoxyredoxins/GR)andlowmolecular-massscavengers(suchasglutathione/GSH).Fig-ProductionofROSandantioxidantdefencesystemsinhippocampalneuronsunderkainate-inducedoxidativestress.(LiSYetal.FreeRadBiolMed,48:597-608,2010)ROS产生——海人酸引起的神经元过度兴奋(举例)Fig-GenotoxicchemicalexposureinducesDNADSBs.(A)IdentificationofDNADSBsbyPFGE.(B)Immunocytochemicalstaininggamma-H2AXfoci.(C)Testinggamma-H2AX(H2AX-Ser139)byWesternblotting.(YangSY,etal.BiochemPharmacol,77:433-443,2009)DNA损伤——双链断裂的(3种)鉴定（举例）Fig-Theeffectsofkanicacid-inducedROSproductionontheintegrityofthegenomeinculturedneurons.ThegenomicDNAdamagewastestedusingfluoresceinisothiocyanate(FITC)-conjugatedavidin,whichbindsto8-oxodeoxyguanosine(8-oxodG)withastructuralsimilaritytobiotin(RadiskyDCetal.Nature,2005.436:123-127)抗生物素蛋白检测DNA氧化损伤（8-O-鸟苷）鉴定（举例）彗星实验检测DSBs（举例）Fig-Cometassay.ToscorethepercentageofDNAinthetail,theimageanalysissystemwasused.Thepercentageofcomettailarea(DNAtailarea/totalDNAarea)andthecomettaillength(fromthecenteroftheDNAheadtotheendoftheDNAtail)wereanalyzedin50cellsforoneslide.(AndersonDetal.MutatRes1994;307:261-271)Con0.2μM2μM10μM(C)SomeChemicalBondsinDNASpontaneouslyDisintegrateunderPhysiologicalConditions（DNA自发裂解）:Hydrolysisofnucleotideresiduesleavesnon-instructiveabasicsites.Deaminationofcytosine,adenine,guanineor5-methylcytosineconvertsthesebasestothemiscodinguracil,hypoxanthine,xanthineandthymine,respectively.Wholechromosomalinstability(W-CIN);segmentalchromosomalinstability(S-CIN);genomicinstability(GIN)/基因组不稳定性（Geigl,J.B.etal.(2008)Defining‘chromosomalinstability’.TrendsGenet.24,64–69RickeRMetal.(2008)Wholechromosomeinstabilityandcancer:acomplexrelationship.TrendsGenet24:475-466）诺贝尔化学奖评审委员会2015年10月7日在瑞典皇家科学院宣布，2015年度诺贝尔化学奖由英国弗朗西斯·克里克研究所名誉教授/瑞典人托马斯·林达尔、美国科学家/杜克大学医学院教授保罗·莫德里和拥有美国和土耳其双重国籍的科学家/美国北卡罗来纳大学医学院教授阿齐兹·桑贾尔分享，以表彰他们在DNA损伤修复机制——BER、NER和MMR方面的研究成果；他们的发现揭示了细胞是如何维持基因组DNA稳定性的，在以DNA损伤为基础的肿瘤治疗以及在衰老中的作用。此前与DNA损伤及结局相关研究而获奖NobelPrize的有DNADamage&Mutation-HermannMuller(NobelPrize,1946)showedthattherateofmutationwasproportionaltothedoseofirradiation.GeorgeBeadle(NobelPrize,1958)showedthattheeffectofX-irradiationonmetabolismwasduetomutationsofgenes.EdwardTatum(NobelPrize,1958)furthershowedthatamutationofasinglegeneresultedonlyinasinglechemicalreaction,whichgaveevidencetotheconceptofonegene,oneenzyme.CellCycleCheckpointSignaling-LalandHartwell,TimothyHuntandPaulNursewereawardedNobelPrizein2001fortheircontributionsinelucidatingthecellcycleregulation.Apoptosis-SydneyBrenner,RobertHorvitzandJohnESulstonwereawardedNobelPrizein2002fortheircontributionsinapoptosismechanisms.2015年度诺贝尔化学奖与DNA损伤修复TomasLindahl发现E.coliUracilDNAglycosidase及碱基切除修复(BEP)机制托马斯•林达尔(LindahlT.AnN-glycosidasefromEscherichiacolithatreleasesfreeuracilfromDNAcontainingdeaminatedcytosineresidues.ProcNatlAcadSciUSA1974;71(9):3649-3653BarnersDE,LindahlT.RepairandgeneticconsequencesofendogenousDNAbasedamageinmammaliancells.AnnRevGenet2004;38:445-476)2015年度诺贝尔化学奖——Lindahl与BEPAzizSancar利用纯化的UvrA、UvrB、UvrC重建了核苷酸切除修复(NER)系统，揭示了NER的分子机制。该机制可帮助细胞修复UV引起的DNA损伤。(SancarA.RuppWD.Anovelrepairenzyme:UVRABCexcisionnucleaseofEscherichiacolicutsaDNAstrandonbothsidesofthedamagedregion.Cell1983;33(1):249-260SancarA,RupertCS.CloningofthephrgeneandamplificationofphotolyaseinEscherichiacoli.Gene1978;4(4):295-308SancarA.Regulationofthemammaliancircadianclockbycryptochrome.JBiolChem2004;279(33):34079-34082)阿齐兹•桑加尔2015年度诺贝尔化学奖——Sancar与NEPPaulModrich利用重建错配修复(MMR)体外系统，探索从大肠杆菌到哺乳动物细