《癌癥分子生物學(xué)》(研究生)全冊(cè)配套完整課件

《癌癥分子生物學(xué)》(研究生)全冊(cè)配套完整課件Class1TheNatureofCancerTumor,

likenormaltissue,arecomposeofmassofcells,butareunabletoassembleandcreatetissueofnormalformandfunctionCancer,isadiseaseofmalfunctioningcellsWhatisatumororcancer?TheBiologyofCancer2007腫瘤產(chǎn)生于正常組織normalmucosa(正常粘膜)dysplasticmucosa(異常粘膜)adinocarcinoma

(腺癌)小腸(回腸)內(nèi)壁切片顯示了正常組織與癌組織之間的延續(xù)性TheBiologyofCancer2007Stroma(mixtureoffibroblasts,adipocytesandcollagen)Stroma(基質(zhì))MilkductBreastadenoma與正常組織一樣，腫瘤也是有細(xì)胞組成正常乳腺切片

乳腺癌組織切片TheBiologyofCancer2007StromaStroma(基質(zhì))MilkductBreastcarcinoma腫瘤能侵入鄰近正常組織正常乳腺切片

乳腺癌組織切片TheBiologyofCancer2007B16鼠黑色素瘤細(xì)胞尾靜脈注射2周后在WT和

g-/-

小鼠肺的黑色素瘤轉(zhuǎn)移灶Metastases-newsettlementsbeyondoriginaltumorsitesWTg

-/-腫瘤能轉(zhuǎn)移到其他正常組織形成轉(zhuǎn)移灶大腸癌的肝轉(zhuǎn)移(白色部分)乳腺癌的腦轉(zhuǎn)移

Primarytumor(原發(fā)瘤): tumoratoriginaltumorsitesMetastasis(轉(zhuǎn)移瘤,轉(zhuǎn)移灶): newsettlementsbeyondoriginaltumorsitesBenigntumor(良性瘤): withoutinvadingadjacenttissuesMalignant

tumor(惡性瘤): invadednearbytissuesandspawnedmetastasesAdenoma(腺瘤): premalignantepitheliagrowthCarcinoma(癌): malignantepitheliagrowthInfact,thegreatmajorityoftumorinhumanarebenignandharmless,exceptfor:1.localizedmesstopressonvitalorgan 2.thyroidadenomas

hyperthyroidism(甲狀腺功能亢進(jìn)癥) 3.pituitaryadenomas acromegaly(肢端肥大癥）Metastasisareresponsibleforsome90%ofdeathsfromcancer常見與腫瘤相關(guān)術(shù)語或名詞I.Epithelialcells: carcinoma (癌）squamouscellcarcinoma adenocarcinomaII.Nonepithelialcells:1.Connectivetissues:sarcoma(肉瘤,frommesenchymalcells)2.Hematopoietictissue：

leukemia，lymphoma3.Nervoussystem:neuroectodermaltumors(神經(jīng)上皮瘤)III.Sometypesoftumorsnotfittingthemajorclassification:Smallcelllungcarcinomas(SCLCs)腫瘤由體內(nèi)多種特殊化細(xì)胞產(chǎn)生Adenomasorcarciomas:Readilybedetectedwithnakedeyes，dysplasticundermicroscopeNotpenetratedbasemembraneNeoplasia(瘤生):

bothbenignandmalignanttumorMetastases:formnewclonesinremotesitesMultiplestepsinCancerdevelopmentMetaplasia:正常細(xì)胞被原來不在同一區(qū)域的另一種細(xì)胞取代，anearlyindicationofpremalignantchange(組織變形)Hyperplasia:細(xì)胞數(shù)量增加，但與正常組織相比細(xì)胞學(xué)上只有些微差異(增生)Dysplasia:細(xì)胞學(xué)異常、細(xì)胞形態(tài)異常

variabilityofnuclearsize&shapeincreasedratioofnuclearversuscytoplasmicsizeIncreasemitoticactivitylackofcytoplasmicfeaturesofdifferentiatedcellscytologicalchanges:(異常)MetastasesHyperplasia(增生)Dysplasia

(異常)Neoplasia(瘤生)Normal(轉(zhuǎn)移)Normalmicro-structureofepitheliumacollectingtubeofthekidneyThecolumnarepitheliumofthegallbladderthebronchioleofthelungTheendometriumoftheuterusTheBiologyofCancer2007TheBiologyofCancer2007雞角膜表皮掃描電鏡顯微照片Normalultrastructureofepithelium小鼠氣管表皮切片透射電鏡顯微照片HyperplasiaMammaryepithelialcellshavebeguntopileupandtoprotrudeintolumina.Moreadvancedhyperplasticmammaryductshowepithelialcellsalmostcompletelyfillthelumen,buttheyhavenotpenetratedthebasementmembraneyet.TheBiologyofCancer2007StromaMilkductMetaplasiaTheBiologyofCancer2007Thesquamouscellsinesophagusisreplacedbysecretorycellsmigratedfromstomach.Toincrease38foldsformalignancy.Figure2.14TheBiologyofCancer(?GarlandScience2007)DysplasiaTheBiologyofCancer2007DysplasiaisatransitionalstatebetweencompletebenigngrowthandpremalignantTheBiologyofCancer2007Adenoma,orpolypspapilomasorcarcinoma,(orwartinskin)BreastcarcinomasColonicadenomasTheBiologyofCancer2007Epithelialcells: squamouscells

squamous

cellcarcinoma(鱗狀細(xì)胞癌) secretorycells

adenocarcinoma

(腺癌)

responsiblefor>80%ofthecancer-relateddeathinwesternworldTheBiologyofCancer2007Squamouscells(鱗狀細(xì)胞)/Squamouscellscarcinomas(鱗狀細(xì)胞癌)宮頸上皮皮膚上皮正在侵入基質(zhì)的食管癌胃壁上形成小囊，排列整齊的粘液分泌細(xì)胞小腸壁上單個(gè)粘液分泌細(xì)胞Secretoryepithelia/adenocarcinomasAdenocarcinomasinstomach（胃腺癌）Adenocarcinomasincolon（大腸腺癌）Epithelialcells: carcinoma squamouscellcarcinoma adenocarcinomaNonepithelialcells:1.Connectivetissues: sarcoma(frommesenchymalcells); ~1% 2.Hematopoietictissues:

3.Nervoussystem:>80%腫瘤由體內(nèi)多種特殊化細(xì)胞產(chǎn)生Leiomyosarcoma(平滑肌瘤)sarcomasderivedfrommesenchymalcellsinconnectivetissuesOsteosarcoma(骨瘤)Liposarcoma(脂肪瘤)TheBiologyofCancer2007TumorsarisefrommanyspecializedcelltypesthroughthebodyEpithelialcells: carcinoma squamouscellcarcinoma adenocarcinomaNonepithelialcells:1.Connectivetissues: sarcoma(frommesenchymalcells); ~1%

2.Hematopoietictissues:leukemia

lymphoma:TandBlymphocytesaggregatedsolidmessinlymphnodeerythocytesantibody-secretingcellsTandBlymphocytes3.Nervoussystem:>80%CommonhematopoieticmalignancyALLAMLCLLCMLMMALLAMLCMLMMerythroleukemiaTheBiologyofCancer2007TumorsarisefrommanyspecializedcelltypesthroughthebodyEpithelialcells: carcinoma squamouscellcarcinoma adenocarcinomaNonepithelialcells:1.Connectivetissues: sarcoma(frommesenchymalcells); ~1% 2.Hematopoietictissues:leukemia

lymphoma:TandBlymphocytesaggregatedsolidmessinlymphnodeerythocytesantibody-secretingcellsTandBlymphocytes3.Nervoussystem:neuroecodermaltumors>80%~2.5%TheBiologyofCancer2007GangliacellsGranularlayer(GL)正常神經(jīng)組織各種神經(jīng)瘤Sometypesoftumorsdonotfitintotheclassification 1.melanomas 2.small-celllungcarcinomas 3.epithelian-mesenchymaltransition(EMT) 4.anaplastic(退行發(fā)育)ordedifferenciation(去分化)MelanocytesandmelanomasMelanocytesarepigmentedcellsoftheskinandretina,Developedfromneurocrest,butnodirectedconnectionwithnervoussystemTheBiologyofCancer2007MelanocytesinskinpigmentgranulesinamelanocyteviewedbyEMMelanomametastasesSmallcelllungcarcinomas(SCLCs)Containcellshavingmanyattributions

ofneurosecretorycellsResponsetoneuronalsignalingSecretebiologicallyactivepeptidesFrequentlyseenintobaccousersNotclearabouttheoriginoftheSCLCs

Epithelial-mesenchymaltransitionEpitheliacancercellsattheborderofmanycarcinomasoftenchangeshapeandgeneexpressionprogramsandtakeonattributesofnearbystromalcellsofmesenchymalorigin.Anaplastic(退行發(fā)育)tumorsTheBiologyofCancer2007Intheabovethreesituation,cancercellsalmostalwaysretainsomeofthedistinctiveattributesascriticalcluesforpathologiststodefinetheoriginofcancercells.BUT…MultiplestepsinCancerdevelopmentMetastasesHyperplasia(增生)Dysplasia

(異常)Neoplasia(瘤生)Normal(轉(zhuǎn)移)TheBiologyofCancer2007monoclonalityversuspolyclonalityofTumorsTheBiologyofCancer2007TumorsaremonoclonalgrowthIHforG6PDofintestinesectionsTheBiologyofCancer2007腫瘤的單克隆起源TheBiologyofCancer2007腫瘤單克隆起源的其他證據(jù)－－骨髓瘤細(xì)胞只產(chǎn)生一種抗體TheBiologyofCancer2007腫瘤單克隆起源的其他證據(jù)不同人種的腫瘤發(fā)生率是sameordifferent？？每個(gè)成年人個(gè)體平均細(xì)胞數(shù)： 1013每個(gè)成年人一世平均細(xì)胞數(shù)： 1016每個(gè)人每秒鐘死亡或被替換的細(xì)胞數(shù)： 107Somearesame,samedifferent

dependingontumortypes.Somecausedbyrandom,unavoidableaccidentsofnatureoccuratsimilarfrequenciesinvarioushumanpopulations,egforcertainpediatrictumors.TheBiologyofCancer2007TumorincidenceisdifferentindifferenthumanpopulationTheBiologyofCancer2007EnvironmentsaffecttumorincidenceLifestyleaffectstumorincidenceTheBiologyofCancer2007TheBiologyofCancer20071915年，Dr.KatsusaburoYamagiwa用煤焦油首次在兔耳成功誘導(dǎo)腫瘤TheBiologyofCancer2007TheBiologyofCancer2007StructureofcarcinogenichydrocarbonsFigure2.23aTheBiologyofCancer(?GarlandScience2007)Figure2.23bTheBiologyofCancer(?GarlandScience2007)Figure2.24TheBiologyofCancer(?GarlandScience2007)Figure2.25TheBiologyofCancer(?GarlandScience2007)Table2.8TheBiologyofCancer(?GarlandScience2007)leiomyosarcomasarcomasderivedfrommesenchymalcellsinconnectivetissuesAdipocytes:liposarcomaOsteoblasts:osteosarcomaMyocytes:leiomyosarcomaFibroblasts:fibrosarcoma…osteosarcomaLiposarcomaFigure2.16aTheBiologyofCancer(?GarlandScience2007)Figure2.16bTheBiologyofCancer(?GarlandScience2007)InvasivecarcinomaTumorareconsideredmalignantonlyaftertheyhavepenetratedbasemembraneandinvadedintonearbysurrondingstroma.BreastductalcarcinomTheBiologyofCancerClass2TumorViruses腫瘤病毒在人類癌癥研究中的地位病毒導(dǎo)致人類許多人類疾病(如狂犬病，

天花，感冒等）絕大部分病毒在細(xì)胞內(nèi)瘋狂復(fù)制增殖,殺死細(xì)胞而引起疾病某些病毒的增殖不殺死細(xì)胞，還導(dǎo)致細(xì)胞不受控制地瘋狂生長

腫瘤病毒－誘發(fā)腫瘤的病毒。1970’s腫瘤病毒研究證實(shí)：人類腫瘤一小部分是腫瘤病毒誘發(fā)的。腫瘤病毒研究提供了揭開隱藏?cái)?shù)百年的人類腫瘤秘密的鑰匙癌癥是一種基因病。可用分子生物學(xué)和遺傳學(xué)進(jìn)行分析研究腫瘤病毒革命了人類癌癥病理學(xué)的研究PeytonRous－

腫瘤病毒學(xué)的奠基人1910年，發(fā)現(xiàn)勞氏肉瘤病毒(Roussarcomavirus,RSV)引發(fā)雞腫瘤50多年后(1966)獲得NobelPrizeinMedicineandPhysiologyTheBiologyofCancer,20071876年，一位俄國科學(xué)家發(fā)現(xiàn)通過組織移植腫瘤在不同的狗之間轉(zhuǎn)移，

提出腫瘤也是一種傳染病的假說1908年，兩位哥本哈根科學(xué)家從雞白血病細(xì)胞制備出一種可過濾的雞白血病致病原Roussarcomavirus(RSV)

induceschickentumorsTheBiologyofCancer,2007提供了一種隨意誘導(dǎo)腫瘤產(chǎn)生的研究腫瘤的工具，腫瘤研究不必依賴不可預(yù)期的動(dòng)物或人類自發(fā)腫瘤的出現(xiàn)。A

setbackinresearchontumorviruses1913年丹麥人JohannesGribFibiger報(bào)道了大鼠胃中的螺旋菌導(dǎo)致胃瘤

1926年JohannesGribFibiger獲得了NobelPrize所稱胃瘤實(shí)際上是一些組織變形的胃表皮(meteplasticstomachepithelia).

組織變形的原因是嚴(yán)重缺乏維生素丟掉了Fibiger的實(shí)驗(yàn)動(dòng)物丟棄了感染性致病因子會(huì)引發(fā)腫瘤的學(xué)說相差光鏡照片掃描電鏡照片正常CEFRSV轉(zhuǎn)化的CEFRSV轉(zhuǎn)化的CEF細(xì)胞失去了接觸抑制特性

在長滿細(xì)胞的培養(yǎng)皿中形成fociTheBiologyofCancer,20071950代后期加州理工學(xué)院的博后HurryRubin和博士生HowardTermin發(fā)現(xiàn)：

1.RSV能感染培養(yǎng)皿中的雞胚成纖微(CEF)細(xì)胞,并復(fù)制病毒

2.RSV感染的CEF不裂解，能無限生長,即RSV能轉(zhuǎn)化CEF

—RSV能在培養(yǎng)皿中轉(zhuǎn)化(transform)CEF細(xì)胞RSV能使正常細(xì)胞轉(zhuǎn)化成腫瘤細(xì)胞。TheBiologyofCancer,2007RenatoDulbeccoHowardTemin1975的醫(yī)學(xué)和生理學(xué)NobelPrizeAnRSV-inducedfocus腫瘤病毒研究的再生及意義意義：腫瘤的發(fā)生可以從個(gè)體細(xì)胞水平進(jìn)行研究。他們的發(fā)現(xiàn)再次改變了20世紀(jì)的癌癥研究進(jìn)程。Questions:是RSV病毒感染、轉(zhuǎn)化了theprogenitorcelloffocus,并且RSV病毒繼續(xù)存在與子代細(xì)胞中維持轉(zhuǎn)化狀態(tài)？或:RSV病毒以

“hitandrun”的方式最初作用于progenitorcell,造成轉(zhuǎn)化細(xì)胞的一系列形態(tài)和生理變化，然后RSV病毒從犯罪現(xiàn)場消失了？維持細(xì)胞的轉(zhuǎn)化狀態(tài)依賴于RSV存在和活性TheBiologyofCancer,20071970年，UniversityofCaliforniaBerkeley分校發(fā)現(xiàn)了一株溫度敏感突變型的RSV病毒AnchorageindependentgrowthRSV轉(zhuǎn)化CEF細(xì)胞在軟瓊脂中形成克隆andtumorigenecityTheBiologyofCancer,2007在athymicnudemice形成腫瘤轉(zhuǎn)化細(xì)胞的特征細(xì)胞形態(tài)的改變(圓形,在相差顯微鏡下有折射性)；丟失接觸抑制(contactinhibition)特征(abilitytogrowoveronanother)，能高密度生長；

無需附著固體生長的能力(anchorageindependencegrowth)；無限生長的能力

(immotalization)；減少對(duì)生長因子需求或依賴性；增加了葡萄糖運(yùn)輸能力；具有成瘤性。1910 Roussarcomavirus(RSV)50s-60s

Murinemammarytumorvirus(MMTV)

HumanT-cellleukemiavirus(HTLV-1)

Avianleukosisvirus(ALV)

…1930sPapillomavirus(to

inducepapilloma

(乳頭(狀)瘤)

inrats）50s-60sHepatitisBvirus(HBV，肝炎病毒)

SV40

(能在猴細(xì)胞中復(fù)制增殖,但只能轉(zhuǎn)化鼠細(xì)胞）

polyomavirus

(多瘤病毒，polyoma

inmice）

Humanpapilloma16(HPV)Humanadenovirus5

(人上呼吸道感染，誘導(dǎo)倉鼠腫瘤）

…DNAviruses:RNAviruses:RNA和DNA病毒都能誘導(dǎo)腫瘤HaraldzurHausenTheNobelPrizein2008（發(fā)現(xiàn)人乳頭瘤病毒誘發(fā)宮頸癌）Papovavirus的DNA基因組的電鏡照片一個(gè)RSV相關(guān)病毒的RNA基因組電鏡照片(A)和示意圖(B)RNA病毒和DNA病毒含有不同的遺傳物質(zhì)RSV病毒顆粒含有兩份RNA線性基因組大部分DNA病毒顆粒含有一份DNA環(huán)狀基因組DNAviruses:RNAviruses:宿主DNA聚合酶;RNA聚合酶;

核糖體自身編碼以RNA為模板的

RNA聚合酶;宿主核糖體自身編碼以RNA為模板的

DNA聚合酶;宿主核糖體RNA病毒顆粒和DNA病毒顆粒結(jié)構(gòu)不同TheBiologyofCancer,2007RSV病毒Shopepapillomavirus電鏡照片

Shopepapillomavirus冰凍蝕刻電鏡照片根據(jù)X光衍射推出的SV40病毒結(jié)構(gòu)DNA病毒EnterhostcellsMultiplyQuicklykilltheirhosts,ReleaseprogenyvirusparticlesfromdyingcellsRe-infectothersusceptiblecellsinthevicinity.SimplelifecycleformostofDNAvirusesTheBiologyofCancer,2007RSV和其他RNA病毒以出芽的方式從細(xì)胞釋放TheBiologyofCancer,2007Murinelekemiavirus病毒感染細(xì)胞的掃描電鏡照片

TheBiologyofCancer,2007DNA病毒誘導(dǎo)的細(xì)胞轉(zhuǎn)化也依賴于DNA病毒的全部或部分基因組在細(xì)胞表達(dá)LargeT是SV40DNA病毒的一個(gè)腫瘤相關(guān)抗原SV40DNA病毒誘導(dǎo)的轉(zhuǎn)化細(xì)胞的細(xì)胞核呈現(xiàn)LargeT抗原陽性TheBiologyofCancerTransmittedviavirusinfection?NewSV40virusesareonlymadeduringlyticcycleofSV40TransformedcellsarenonpermissiveforproducingnewvirusesTransmittedviacelldivision?viralDNAsreplicateasautonomous,extrachromosomalmol.Nonpermissivenesspreventstherepl.ofSV40virualDNALimitednumberofSV40DNAmoleculesinthetransformedcells.Question:

HowdoesSV40DNAvirusgenomeinatransformedcelltransmitovermanycellgeneration?IntegrationofSV40DNAintochromosomalDNADNAmoleculesfromSV40-transformedcellsandSV40DNAmoleculesfromfreeSV40viruswereco-centrifugedinalkaline-sucrosegradient.ThefractionationscollectedwerethenanalyzedbyUV-spectrophotometerandDNAhybridization.TheBiologyofCancer99.7%人宮頸癌細(xì)胞攜帶人HPV病毒基因組含致癌基因的DNA片斷，而不是整個(gè)HPV病毒基因組。。Humancervicalcarcinoma:

Figure3.6TheBiologyofCancer(?GarlandScience2007)PuzzleaboutthestabletransmissionofRSVgenome重復(fù)感染假說某些RSV病毒突變株能感染和轉(zhuǎn)化細(xì)胞，

但不能在被轉(zhuǎn)化的細(xì)胞中復(fù)制2

.某些細(xì)胞能被轉(zhuǎn)化，但不能被再感染。HowardTemin3

.

抑制DNA復(fù)制或DNA-dependentRNA

合成可抑制RSV病毒的復(fù)制。AnyQuestion??TheBiologyofCancer,2007HowardTeminProvirusTheory(ThelifecycleofRetrovirus)1970年，HowardTermin和DavidBaltimore發(fā)現(xiàn)反轉(zhuǎn)錄酶1975的醫(yī)學(xué)和生理學(xué)NobelPrizeTheBiologyofCancer,2007ImportantdistinctionbetweenintegrationmechanismsusedbyretrovirusandDNAvirusRetrovirus:normal&essentialpartofreplicationcyclewholegenomeintegratedatmosttimeDNAvirus:rareaccident(<<1/1000infection)onlypartofgenomeintegratedatmosttimeTheBiologyofCancer,2007RSVviruscarriesanextratransforminggene,srcRSV病毒就有兩種突變株:一種突變株能在感染細(xì)胞后產(chǎn)生新病毒，但不能轉(zhuǎn)化細(xì)胞，

另一種突變株感染細(xì)胞后能轉(zhuǎn)化細(xì)胞但不會(huì)產(chǎn)生新病毒。Question:

為什么有些病毒是致瘤，

而有些不致瘤？

Question:

這些病毒攜帶的oncogene的起源？

TheBiologyofCancer,2007Preparationofsrc-specificDNAprobe1975,MichaelBishopHaroldE.VarmusTheBiologyofCancer,2007Srcgenepresentsinthegenomeofuninfectedchickencellsandallothervertebratessrc

基因的進(jìn)化樹Creatingarevolutioninthinkingabouttheoriginofcancer鴨火雞雞鵪鶉鴯鹋RSV轉(zhuǎn)化的大鼠細(xì)胞TheBiologyofCancer,2007Puzzle:c-srcvsv-src

c-src:ensconcedincellulargenome;actsasanormalcellulargene,compatiblewithnormalcellularbehaviorandorganismdevelopmentv-src:bornebyRSVgenome;actsasapotentoncogene,capableoftransforminganormalcellintoatumorcellc-srcandv-srcaretwocloselyrelatedgenesTheBiologyofCancer,2007Captureofproto-oncogene,c-src,fromcellulargenomebyavianleukosisvirusc-src原癌基因的發(fā)現(xiàn)再次革新了癌癥起源的認(rèn)識(shí)既然反轉(zhuǎn)錄病毒能夠激活原癌基因，那么不改變?cè)┗蛟谌旧w上的位置，其他基因突變機(jī)制也可能將原癌基因激活成為一個(gè)癌基因。可能誘導(dǎo)癌癥的信息造就存在于正常細(xì)胞基因組中，正等待著被發(fā)現(xiàn)。單個(gè)致癌基因能導(dǎo)致細(xì)胞在形態(tài)，代謝以及生長行為等一系列變化。少量幾個(gè)基因足以將正常細(xì)胞轉(zhuǎn)化成腫瘤細(xì)胞。其他具轉(zhuǎn)化能力的病毒可能獲取了與src無關(guān)的細(xì)胞基因。細(xì)胞基因組藏有許多其他細(xì)胞原癌基因？J.MichaelBishopHaroldE.VarmusTheNobelPrize1989fortheirdiscoveryofthecellularoriginofretroviraloncogenes急性轉(zhuǎn)化反轉(zhuǎn)錄病毒及其攜帶的致癌基因Puzzle:Howslowlytransformingretrovirusesw/ooncogeneinduceamalignancy??快轉(zhuǎn)化和慢轉(zhuǎn)化反轉(zhuǎn)錄病毒差異rapidly

transformingretrovirus,e.g.RSV,MC29slowlytransformingretrovirus，e.g.MLV,ALVTheBiologyofCancer,2007>80%雞白血病ALV原病毒都整合在c-myc座位ALVprovirusesintegratedat5’ofc-mycin>80%ofchickenleukemiaMostintegratedinthesametranscriptionalorientationasthatofc-mycIn1981TheBiologyofCancer,2007慢轉(zhuǎn)化反轉(zhuǎn)錄病毒通過插入誘變激活原癌基因TheBiologyofCancer,2007Examplesofcellulargenesfoundtobeactivatedbyinsertionalmutagenesis(Wnt-1)(FGF)(notchreceptor)insertionalmutagenesiscanbeusedasastrategytoscreenfornewproto-oncogenesSomeretrovirusesnaturallycarryoncogenesHTLV-1,belongtothethirdclassofretroviruses,naturallycarryingoncogenes;infects~1%oftheinhabitantsofasouthislandofJapan,andsomeislandsoftheCaribbean,3-4%riskofdevelopingadultT-cellleukemia;provirusesareintegratedrandomly;carryataxgene,encodingaproteinrequiredfortranscriptionofprovirusgenome;alsoactivatetranscriptionoftwocellulargenes,IL-2&GM-CSFIL-2&GM-CSFstimulateproliferationofsomehematopoieticcells,suchinducedproliferatedcellsdoesnotdirectlycreateleukemia,butthesecellsmayprogressatlowfrequencytoneoplastia.基因治療引起的悲劇1.MLV-derivedretrovirusvectorwasusedforgenetherapyforX-linkedsevercombinedimmunodeficiency.2.2.5yearslater(2003orlater),threeofninedevelopedleukemia3.Why???基因治療引起的悲劇1.MLV-derivedretrovirusvectorwasusedforgenetherapyforX-linkedsevercombinedimmunodeficiency.2.2.5yearslater(2003orlater),threeofninedevelopedleukemia3.ProvirusintegratedLMO2genelocus,LMO2wasknowntobeactivatedinhumanleukemiaTheBiologyofCancerClass3CellularOncogenes通過DNA/RNA病毒尋找人類癌癥基因的失敗上世紀(jì)70年代證明腫瘤病毒能轉(zhuǎn)化鼠細(xì)胞和雞細(xì)胞,許多病毒能在人群中傳播;

假設(shè)：腫瘤病毒可能也可以轉(zhuǎn)化人細(xì)胞。But,

絕大多數(shù)的人的癌癥不傳染大多數(shù)人腫瘤中不能分離得到病毒

100多種臨床常見腫瘤中只有兩種(宮頸癌和肝癌)被證明語病度感染相關(guān)。假說：

1.Endougenousprovirusesisresponsibleforhumancancers

2.CellularoncogenesareresponsibleforhumancancersA.1915年，Dr.Yamagiwa

用煤焦油在兔耳朵成功地誘發(fā)皮膚瘤B.1920’s，巴黎的一篇博士論文報(bào)道了100多位曾經(jīng)從事X-射線管工作的工作人員得癌癥的病例1.物理或化學(xué)因子能致癌Dr.Yamagiwa和他誘發(fā)的兔耳皮膚瘤樣本2.某些小鼠結(jié)締組織細(xì)胞培養(yǎng)時(shí)可用BrdU誘發(fā)生產(chǎn)病毒內(nèi)源性原病毒誘發(fā)腫瘤學(xué)說的實(shí)驗(yàn)基礎(chǔ)TheBiologyofCancer,2007內(nèi)源原病毒的起源和誘發(fā)腫瘤的方式Theevidencesofpresenceofendogenousretrovirus(ERV)inmiceandhumanVariousmousegenomicDNAprobedwithXenotropicmurineretrovirous(Tomonaga,K.,Violo.73,1999)VarioushumangenomicDNAprobedwithhumanERVfragment(Hughes,JF.,PNAS101,2004)Canhumancancersbetriggedbyactivationofendogenousretrovirus?1.有關(guān)感染性反轉(zhuǎn)錄病毒的報(bào)告在人腫瘤樣品中不能重復(fù)。2.人腫瘤(癌細(xì)胞)無法分離獲得攜帶反轉(zhuǎn)錄酶的病毒。8%的人基因組DNA來源于內(nèi)源性反轉(zhuǎn)錄病毒(ERV)DNA，它們是在5百萬年前被整合到人基因組中的。但只有四萬分子幾的ERV片段是完整的，是可能有功能的。But,noneofthemwasfoundtoproduceinfectiousvirusorhavevirusinhumantumor.A.1915年，Dr.Yamagiwa

用煤焦油在兔耳朵成功地誘發(fā)皮膚瘤B.1920’s，巴黎的一篇博士論文報(bào)道了100多位曾經(jīng)從事X-射線管工作的工作人員得癌癥的病例1.物理或化學(xué)因子能致癌Dr.Yamagiwa和他誘發(fā)的兔耳皮膚瘤樣本2.人基因組有原癌基因存在，可以被誘變成的致癌基因細(xì)胞原癌基因誘發(fā)腫瘤學(xué)說的實(shí)驗(yàn)基礎(chǔ)Adauntingchallenge:尋找化學(xué)誘變腫瘤中的致癌基因

lookingforatinyneedleinverylargehaystacksTheBiologyofCancer細(xì)胞原癌基因誘發(fā)腫瘤學(xué)說的實(shí)驗(yàn)證據(jù)Transfection,apowerfulmethodtodetecttheoncogenefrom

tumorDNA實(shí)驗(yàn)成功關(guān)鍵：引入DNA的方法：合適的受體細(xì)胞：合適的供體腫瘤細(xì)胞：磷酸鈣轉(zhuǎn)法染(1972)NIH3T3細(xì)胞小鼠C3H10T2

/1成纖微細(xì)胞株TheBiologyofCancer化學(xué)誘變過的小鼠C3H10T2

/1細(xì)胞的DNA能轉(zhuǎn)化NIH3T3細(xì)胞而未處理C3H10T2

/1細(xì)胞的DNA不能細(xì)胞原癌基因誘發(fā)腫瘤學(xué)說的實(shí)驗(yàn)證據(jù)意義：

細(xì)胞原癌基因可以通過非病毒途徑被誘變成致癌基因2.人腫瘤細(xì)胞也有能轉(zhuǎn)化小鼠細(xì)胞的致癌基因。TheBiologyofCancer人腫瘤細(xì)胞的DNA也能轉(zhuǎn)化NIH3T3細(xì)胞T24人膀胱癌細(xì)胞株DNA轉(zhuǎn)化的NIH3T3形成的Foci圖A

foci中轉(zhuǎn)化細(xì)胞的高倍鏡照片正常NIH3T3細(xì)胞的高倍鏡照片人腫瘤細(xì)胞的致癌基因與

腫瘤病毒的致癌基因的關(guān)系??腫瘤病毒攜帶的致癌基因是從截取，并進(jìn)一步突變而來的

人腫瘤細(xì)胞的致癌基因是細(xì)胞基因的突變結(jié)果。這兩種來源不同的致癌基因是同一類細(xì)胞基因？還是屬于根本不同的兩類基因？如何鑒別？？TheBiologyofCancerSouthern

orNorthernBlotTheBiologyofCancer人腫瘤細(xì)胞的(轉(zhuǎn)化)致癌基因與反轉(zhuǎn)錄病毒致癌基因具有同源性不同的轉(zhuǎn)化NIH3T3細(xì)胞株正常NIH3T3人致癌基因轉(zhuǎn)化細(xì)胞基因組DNA與Harvey病毒H-Ras致癌基因的SouthernBolt雜交結(jié)果探針：Harvey病毒的H-RasDNA活化原癌基因的機(jī)制?腫瘤病毒攜帶的癌基因不受控制地?zé)o限表達(dá)

正常細(xì)胞中原癌基因的表達(dá)受到嚴(yán)格調(diào)控normallyexpressedatverylowlevel,gohighwhencellneedit。e.g.Mycexpressionistightlycontroledbyextracellulargrowthsignals人細(xì)胞原癌基因如何被誘變成致癌基因？？

Then,to

cloneRasoncogene

Question：HowtocloneRasoncogene？？TheBiologyofCancerThestrategyforcloninghumanH-RasRasoncogene1x106Alu/cellT24/EJhumanbladdertumorcellcellDNA1Alu/cell1x103Alu/cell建立轉(zhuǎn)化細(xì)胞的基因文庫，以人Alu

DNA為探針篩選獲得一個(gè)能轉(zhuǎn)化小鼠細(xì)胞的6.6kb人基因組DNA片斷CloninghumanH-RasRasoncogene今天，如果知道T24人膀胱癌細(xì)胞株有一段能轉(zhuǎn)化NIH3T3細(xì)胞、并與v-Src同源的oncogene，你如何更簡單，快速的克隆該oncogene？？TheBiologyofCancer人H-Rasoncogene的精細(xì)定位TheBiologyofCancerhumanH-Rasoncogenecontainsapointmutation(GlyVal)這個(gè)H-Ras的發(fā)現(xiàn)代表著癌癥研究的又一個(gè)里程碑。首次發(fā)現(xiàn)一個(gè)基因的單個(gè)突變可誘發(fā)人類腫瘤的生長。這是一個(gè)體細(xì)胞突變產(chǎn)生的遺傳變異。許多人腫瘤攜帶Ras

點(diǎn)突變H-Ras(sameasthatinHarveysarcomarvirus)K-Ras(sameasthatinKirstensarcomarvirus)N-RasMutatedatresidues12,61or(lessfrequently)13TheBiologyofCancerTheBiologyofCancer各種人腫瘤攜帶的致癌基因與腫瘤病毒致癌基因的相關(guān)性原癌基因點(diǎn)突變?cè)《静迦爰せ?/p>

???活化原癌基因的機(jī)制TheBiologyofCancer原癌基因點(diǎn)突變?cè)《静迦爰せ?/p>

基因擴(kuò)增

染色體易位基因的部分缺失突變活化原癌基因的機(jī)制TheBiologyofCancerTheBiologyofCancer人乳腺癌細(xì)胞基因組erbB2/neu/HER2致癌基因的擴(kuò)增TheBiologyofCancer人乳腺癌erbB2(neu,HER2)原癌基因擴(kuò)增和過表達(dá)1.基因擴(kuò)增2.基因轉(zhuǎn)錄控制失調(diào)(增強(qiáng))3.蛋白生產(chǎn)增強(qiáng)降解減緩TheBiologyofCancer人染色體17長臂上的部分基因與Her2同時(shí)過表達(dá)360個(gè)原發(fā)乳腺瘤360個(gè)基因TheBiologyofCancererbB2/neu/HER2致癌基因擴(kuò)增與乳腺癌

患者生存率反相關(guān)TheBiologyofCancerMyc基因家族的染色體內(nèi)、外的擴(kuò)增N-mycamplifiedin~30％ofchildhoodneuroblastomac-mycamplifiedin~10％humanleukemeaand~50%carcinomaL-mycamplifiedin~10％ofhuamnlungcarcinomaHomogeneousstainingregions(HSRs)N-Myc的染色體內(nèi)擴(kuò)增doubleminutes(DMs)N-Myc的的染色體外擴(kuò)增FISH:FluorescenceinsituhybridizationTheBiologyofCancerN-myc擴(kuò)增與神經(jīng)母細(xì)胞瘤的預(yù)后N-myc高擴(kuò)增(>10拷貝)病人在癌癥確診和治療后的復(fù)發(fā)和癌癥相關(guān)癥狀再現(xiàn)率的比例遠(yuǎn)高于N-myc低擴(kuò)增病人，

時(shí)間也提早很多。經(jīng)常被擴(kuò)增的染色體區(qū)域和其攜帶的已知基因TheBiologyofCancer

并非所有擴(kuò)增基因都高表達(dá)表達(dá)未增加的擴(kuò)增基因可能受復(fù)雜的負(fù)反饋機(jī)制調(diào)控?cái)U(kuò)增基因過表達(dá)常伴隨有轉(zhuǎn)錄因子失調(diào)改變轉(zhuǎn)錄啟動(dòng)子腫瘤細(xì)胞基因組染色體擴(kuò)增區(qū)基因，40－60％擴(kuò)增基因高表達(dá)。系統(tǒng)性分析表明：原癌基因點(diǎn)突變?cè)《静迦爰せ罨驍U(kuò)增染色體易位：

1.導(dǎo)致原癌基因過表達(dá)活化原癌基因的機(jī)制TheBiologyofCancerChromosomaltranslocationsinBurkitt’slymphoma14:8heavychain75％22:8lchain(light)16％2:8

chain

(light)9％TheBiologyofCancerB-細(xì)胞染色體易位導(dǎo)致正常c-myc基因在重鏈基因啟動(dòng)子控制下組成型高表達(dá)cataloged310distinctrecurringtranslocationClonedmorethan100ofthenovelhybridgenescreatedbytranslocation~adozendistincttranslocationsderegulatingknownproto-oncogenes染色體易位與人類癌癥FromG.M.Cooper,Oncogenes,1995TheBiologyofCancer1.人類腫瘤的染色體易位導(dǎo)致原癌基因表達(dá)失控(過表達(dá))變成癌基因原癌基因點(diǎn)突變?cè)《静迦爰せ罨驍U(kuò)增染色體易位：

1.導(dǎo)致原癌基因過表達(dá)活化原癌基因的機(jī)制2.產(chǎn)生新結(jié)構(gòu)新功能的致癌蛋白

TheBiologyofCancer(9:22)染色體易位形成bcr-abl致癌基因，導(dǎo)致慢性骨髓性白血病(chronicmyelogenousleukemia)染色體易位產(chǎn)生新結(jié)構(gòu)新功能的致癌蛋白

abl:

最初在Abelsonmurineleukemiavirus發(fā)現(xiàn)的致癌基因bcr:

breakpointclusterregionabl:

最初在Abelsonmurineleukemiavirus發(fā)現(xiàn)的致癌基因bcr:

breakpointclusterregionTheBiologyofCancerbcr-abl融合產(chǎn)生導(dǎo)致三種不同白血病的三種新致癌蛋白AcutelymphocyticleukeamiaChronicmyelogenousleukeamiaChronicneutrophilicleukeamiaTheBiologyofCancer染色體易位與人類癌癥2.人類腫瘤的染色體易位導(dǎo)致具有新的結(jié)構(gòu)和功能的致癌融合蛋白FromG.M.Cooper,Oncogenes,1995原癌基因點(diǎn)突變基因擴(kuò)增原病毒插入激活

染色體易位：

1.導(dǎo)致原癌基因過表達(dá)基因的部分缺失突變活化原癌基因的機(jī)制2.產(chǎn)生新結(jié)構(gòu)新功能的致癌蛋白

TheBiologyofCancer基因的部分缺失突變導(dǎo)致生長因子受體永久性激活1/3glioblastomas,EGF受體缺少extracellulardomain原癌基因點(diǎn)突變基因擴(kuò)增原病毒插入激活

染色體易位導(dǎo)致原癌基因過表達(dá)染色體易位產(chǎn)生新結(jié)構(gòu)新功能的致癌蛋白基因的部分缺失突變

活化原癌基因的機(jī)制TheBiologyofCancer與人類癌癥相關(guān)的病毒1/5deathfromcancerworldwideareassociatedwithinonewayoranotherwithinfectiousagents9%cancermortalityfromstomachcancer helicobacterpylori6%cancermortalityfromlivercancer hepatitisB&Cinfection5%cancermortalitycausedbycervicalcarcinoma HPVinfectionTheBiologyofCancerBurkitt’slymphomaincidenceinAfiricaBurkitt’slymphoma與EpisteinBar病毒和瘧原蟲感染的流行病因子的相關(guān)性TheBiologyofCancerBurkitt’slymphomaincidenceinAfirica慢性瘧疾降低兒童的免疫抵抗能力，使得他們?nèi)菀资蹺BV病毒EBV病毒刺激被感染的B淋巴細(xì)胞的不斷增殖，導(dǎo)致體內(nèi)B淋巴細(xì)胞大量累積B淋巴細(xì)胞抗體基因重組酶出錯(cuò)，導(dǎo)致染色體易位，myc過表達(dá)，最后導(dǎo)致淋巴癌。Burkitt’slymphoma與EpisteinBar病毒和瘧原蟲感染的流行病因子的相關(guān)性可能：TheBiologyofCancerClass4GrowthFactors,Receptors,andCancerTheBiologyofCancer正常組織內(nèi)細(xì)胞共同決定有關(guān)細(xì)胞的增殖、維持組織結(jié)構(gòu)小腸絨毛吸收細(xì)胞粘液分泌細(xì)胞間質(zhì)細(xì)胞體隱窩間質(zhì)細(xì)胞巨噬細(xì)胞TheBiologyofCancer

granulescontainingPDGFandothersurvivalfactors血小板的電鏡照片血小板中存貯大量PDGF

和其他存活生長因子TheBiologyofCancerPDGF-R

+/+PDGF-R

-/--PDGFEffectsofgrowthfactors(PDGF)oncells+PDGFTheBiologyofCancerCEFRSVw/osrcinfectedCEFwtRSVinfectedCEF最初的Src蛋白免疫沉淀實(shí)驗(yàn)

1，4，7：Normalrabbitserum2，5，8：Tumor-bearingrabbitserum3，6，9：Tumor-bearingrabbitserumpreincubatedwithlysateofRSVparticle.TheBiologyofCancerNormalrabbitserumSerumfromarabbitbearinganRSV-inducedtumorCEFALVinfectedCEFwtRSVinfectedCEFtdRSVinfectedCEFlgGphosphorylatedbypp60V-SrcPhosphorylationofaprecipitatingantibodymoleculebySrcMethod:

SrcproteinswerefirstIP-edfromvariouscelllysatesbyanti-SrcAbs.

Thenimmunoprecipitateswereincubatedwith32P-

-ATPandsubjectedPAGEandautoradiographTheBiologyofCancerNormalrabbitserumSerumfromarabbitbearinganRSV-inducedtumorCEFALVinfectedCEFwtRSVinfectedCEFtdRSVinfectedCEFlgGphosphorylatedbypp60V-SrcPhosphorylationofaprecipitatingantibodymoleculebySrc

IgG磷酸化的證據(jù):

1.分子量53000

2.變性PAGE中同位素標(biāo)記的蛋白與

考馬斯藍(lán)染色的IgGHC的電泳位置相同。在無巰基乙醇的PAGE中同位素標(biāo)記的蛋白與高分子量的IgG共電泳。磷酸氨基酸分析顯示：磷酸化IgG只含有磷酸絲氨酸Kinase:anenzymethatremovesahigh-energyphosphategroupfromATPandtransferittosuitablesubstrate.SrcoperatesasaproteinkinaseTheBiologyofCancerSrcoperatesasaproteinkinaseanditselfisaphosphoproteinSrcdoesnotnormallyphosphorylateantibodymoleculesSrccanphosphorylatemultiple,distinctsubstrateproteins(>50)withincell.Srcisaphosphoprotein(pp60src),indicatingSrccanbephosphorylatedanotherkinase

orphosphorylateitself(autophosphorylation).TheBiologyofCancerPleiotropicactionsofaproteinkinaseEachphosphorylatedsubstratemaybefunctionallyalteredandproceed,inturn,toalterthefunctionofitsowndownstreamtargets.TheBiologyofCancerSrc是一個(gè)酪氨酸蛋白激酶99%磷酸氨基酸:

phosphoserineandphosphothreonine0.05-0.15％磷酸氨基酸

:phosphotyrosin在src-轉(zhuǎn)化的NIH3T3cells>1%磷酸氨基酸是phosphotyrosine細(xì)胞有兩種蛋白激酶:Ser/Thr激酶和Tyr激酶TheBiologyofCancer不同的信號(hào)傳遞通道使用兩種不同的蛋白激酶Tyrosineproteinkinases:

largelyusedbymitogensignalingpathwaysinmammals

Ser/Thrproteinkinases:

almostexclusivelyusedbyothersignalingpathwaysincellsIdentificationofEGFandEGF-REGF(epithelialgrowthfactor,表皮細(xì)胞生長因