肿瘤分子生物学3细胞永生化与肿瘤发生教材

细胞永生化与肿瘤发生

Xi’anJiaotongUniversity

2016.2.Page

2肿瘤概念

Cancerisageneticdisease,arisingfromanaccumulationofmutationsthatpromoteclonalselectionofcellswithincreasinglyaggressivebehavior.Thevastmajorityofmutationsincanceraresomaticandarefoundonlyinanindividual'scancercells.However,about1%ofallcancersariseinindividualswithanunmistakablehereditarycancersyndrome.Theseindividualscarryaparticulargermlinemutationineverycelloftheirbody.ScienceVol.278.no.5340,pp.1043–1050,7November1997

肿瘤是机体在各种致瘤因素的作用下，局部组织的细胞在基因水平上失去对其生长的正常调控，导致克隆性异常增生而形成的新生物（病理学－卫生部统编教材第五版2001）。Page

3癌细胞的基本特征1、细胞生长与增殖失去控制2、具有浸润性和扩散性3、细胞间相互作用改变4、蛋白表达谱系或蛋白活性改变5、mRNA转录谱系改变6、体外培养的恶性转化细胞的特征二、癌基因与抑癌基因癌基因－控制细胞生长和分裂的正常基因的一种突变形式抑癌基因－正常细胞增殖过程中的负调控因子三、肿瘤的发生是基因突变逐渐积累的结果Page

4Normalcellpopulationsregisterthenumberofcellgenerations

C.elegance-Thelineageofall959somaticcellsintheadultbody(couldbe)hasbeentracedtotheirfounderandcanbedepictedasapedigreePage

5发育生物学：关注的是不同细胞谱系中的细胞个体如何从其周围获取信息，使其进入特定的分化程序，而不关注与肿瘤发生最相关的问题，有无特定的

控制系统决定一个生物体特定的细胞谱系一生中能够传多少代？一个细胞系谱的分支是否能够无限制生长（grow),或者每一个细胞谱系分裂次数是否为预先设定，有限的？

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61960LeonardHayflik’sworkbycountingthenumberoftimesthatpopulationofcellshaddoubled.Whenthecellsenterintosenescence,theycouldremainviablebutnonproliferatingforaslongasayearPage

7LossofproliferativecapacitywithagePage

8Senescentcells--

Whenthecellsenterintosenescence,theyceaseproliferatingbutremainviable,“Friedeggappearance”isthemorphologicalfeaturebecauseoftheenlargedcytoplasm.Metabolically,senescentcellscharacteristicallyexpressthesenescence-associated,acidicβ-galactosidaseenzyme,whichcanbedetectedbysupplyingthemwithsubstratethatturnsblueuponcleavagebythisenzymePage

9CancercellsneedtobecomeimmortalinordertoformcancerGenerationsofcellsformingatumor(a)1cm3=109cell,life-threateningtumor103cm3≈1012cell,103≈210,hence1012≈240cyclesCellPD601018cell≈109cm3≈106kgPage

10Generationsofcellsformingatumor(b)

CellpopulationsthatareevolvingtowardtheneoplasiticstateandthosethatarealreadyneoplasticexperiencesubstantialattritionduringeachcellgenerationHowcannormalcellsthroughoutthebodypossiblyremembertheirreplicativehistory?howcancancercellserasethememoryofthishistoryandacquiretheabilitytoproliferateindefinitely?Page

11CellphysiologicstressesimposealimitationonreplicationInfluenceofcultureconditionsontheonsetofsenescenceOxygenconcentrationPlasticorfeedercellinthecultureinfluencetheexpressionoftumorsuppressorgeneexpression,sotothesenescenceInvitromechanism-senescence-Page

12Increasedexpressionofp16andp21progressivelyduringextendedcultureinvitroEctopicexpressionofp16incellscausedthemtodevelopmanyoftheattributesofreplicativesenescenceNormalcellsForcedexpressionofP16SenescentcellsActinstressfibers(orange)Focalcontactswiththesubstrate(yellow)Page

13RoleoflargeTantigenincircumventingsenescenceInactivationofbothpRBandp53isneededtoensurethatthesecellsdonotsenesceinculture.ThiscanbearchivedthroughtheexpressionoftheSV40largeTantigeninthetargetcellsPage

14EvidenceofsenescentcellsinlivingtissuesAdefinitiveproofthatsenescenceisaninvivophenomenoniscriticaltoourunderstandingofcancerdevelopment.Brca1mutantwhichisinvolvedinmaintaininggenomicintegrityThepresenceofsenescenthumanmelanocytswithindysplasticneviTreatmentoftumorswithchemotherapeuticdrugscarboplatinandtaxolpriortosurgicalexcisionofthetumorPage

15Senescence

representsahaltincellproliferationwithretentionofcellviabilityoverextendedperiodsoftime,while

Crisis

involvesdeathbyapoptosis.Senescentcellsseemtohaveareasonably(butnottotally)stablekaryotype,whilecellsincrisisshowwidespreadkaryotypicinstability危象的时相、形态变化提示其触发机制是独立于衰老的，启动危象的分子装置确实是细胞谱系从胚胎早期（进入growth-and-divisioncycle)以后记录细胞连续传代的功能性计数装置TheproliferationofculturedcellsislimitedbythetelomeresoftheirchromosomesPage

16BarbaraMcClintock1941年报道了玉米染色人体端粒和转座子，获得1983年诺奖TelomeresdetectedbyFISH(left),(right)karyotypeseeninleftcomparedwiththatofcellshavebeendeprivedofTRF2实验室DNA转染技术显现出哺乳动物染色体线状结构的致命缺陷。真核细胞转染DNA需要线状化-使其不稳定TA-质粒Page

17Shortening

oftelomericDNA(5′-TTAGGG-3′tandemlyrepeatedhexanucleotidesequence)inconcertwithcellproliferationPage

18Dicentricchromosomes,anaphasebridges,andinternuclearbridgesPage

19Mechanismsofbreakage-fusion-bridgecyclesPage

20端粒与细胞危象进入危象的细胞准确的呈现染色体缺失端粒时的核型紊乱模式。这些细胞内融合的染色体（breakage-

fusion-bridge）端粒很短，甚至端粒全无。提示触发细胞危象分子机制存在于端粒内。Page

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测量端粒DNA的长度不能准确预测细胞的增殖潜能即便已知一个细胞端粒DNA的长度，该细胞距离危象的增殖代数并不能精确预测。一个细胞内的端粒缩短速率不同，细胞内最短的端粒可能决定细胞的增殖潜能，因为这个端粒将决定最早的断裂-融合-桥接出现时间，从而发生危象。此外，我们不知道端粒短到何种程度才失去其保护功能。有时数Kb长的端粒已失去保护染色体端-端融合的能力。Page

22IncipientCancerCellsCanEscapeCrisisByExpressingTelomeraseTheTelomericRepeatAmplificationProtocolassayTRAPassayPage

23ActivationoftelomeraseactivityfollowingescapefromcrisisPage

24Preventionofcrisisbyexpressionoftelomerase体外培养细胞的危象及重生是体内癌前细胞克隆的重现.癌前细胞克隆进入永生的途径—重建端粒端粒酶是端粒重建的关键

85%-90%人体肿瘤可检测到明显的端粒酶活性

AcquisitionoftelomeraseactivityisaCAUSEofescapefromcrisisratherthanaCORRELATEPage

251、端粒酶全酶是由多种、不同的（multiple,distinct）亚单位构成，在正常、危象前的人体细胞缺失的唯一亚单位是催化亚单位。端粒酶全酶其他亚单位（包括hTR-端粒酶相关RNA分子）在危象前的细胞均适量储存。2、端粒酶的表达（而不是其它酶）允许细胞规避危象，因为端粒酶特异地作用于端粒DNA，所以缩短的端粒

确实是危象的关键原因。3、实验显示，端粒酶活性的获得（从大量进入危象的细胞群中自主产生的变异细胞）足以使细胞逃逸危象，其产生的子代细胞能够无限制生长（永生化）。Page

26TelomeraseplaysakeyroleintheproliferationofhumancancercellsPage

27TelomeraseactivityandtheprognosisofpediatrictumorsPage

28Earlypassagecells40populationdoublingslaterhTERTisadistantrelativeofthereversetranscriptaseofHIVCopychoice

MismatchRepairmechanismmaybeinvolved,becausetheALTstateisreadilyactivatedwhensomeofthegenesweredeprived.OneofthefunctionsofmismatchrepairistosuppressrecombinationbetweenimperfectlyhomologousDNAsequencesSomeimmortalizedcellscanmaintaintelomereswithouttelomerasePage

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TheALTisassociatedpreferentiallywithaspecificsubsetoftumors,suchassofttissuesarcoma,osteosarcoma,glioblastoma…CellsInALTstate:TelomericDNA＞30kbTelomerasepositivecells5-10kbdnTERTfailstoinducecrisisinALT+tumorcellline●ALTpositivecellsdonotdependontelomerasefortheirgrowth●dnhTERTenzymeisnotintrinsicallycytotoxic

hTERTisadistantrelativeofthereversetranscriptaseofHIVPage

30TelomeresplaydifferentrolesincellsofLabmiceandinhumancellsErosionoftelomeresovermultipleorganismicgenerationsinpopulationsofmTR-/-miceTelomericDNAhuman/mice6-8kb/30-40kbMitoseshuman/micelifetime1016/1011,0.1%ofhumancelland1%ofhumanlifespanHumancellimmortalization,theintroductionofboththeSV40largeToncogene(toavoidsenescence)andthehTERTgene(toavoidcrisis).Page

31Telomereandbonemarrowcollapseofdyskeratosiscongenita

X-linkedformofthedisease,aproteinknownasdyskerinisabsen