癌基因与抑癌基因课件.pptx

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1、,癌基因与抑癌基因Oncogene&Tumor Suppressor Gene,癌生物学课程,提 纲,一、癌基因二、抑癌基因三、癌基因/抑癌基因与多步骤癌变四、癌基因/抑癌基因的鉴定,肿瘤发生机理,物理因子,化学因子,生物因子，如病毒,遗传物质改变,正常细胞,癌细胞,a genetic disease characterized by uncontrolled cell growth,一、癌基因 Oncogene,1.癌基因的发现,Non-Transforming(Avian Leukemia Viruses,ALV),Ellerman and Bang,1908,1,Induce leuke

2、mia after long latency periods2,Do not“transform”tissue culture cells,Virus and Cancer,Transforming(Sarcoma Viruses,RSV),Peyton Rous,1911-cell free lysates could induce sarcomas in other chickens,1，Acute;2，Transform cultured cells,病毒致癌-Nobel Prize!1966,Isolation of Retroviral Transforming Genes,RSV(

3、gag,pol,env,src),R.T.(1975 Nobel),gag,pol env,src,cDNA,RSV(td:gag,pol,env),gag,pol,env,Denature and Hybridize1975,1977,unhybridizedsequences,hybridizedsequences,gag,pol,env,src,genomic RNA,probe,RSV-InfectedCEF(+control),“Normal”chick DNA,Mouse,Drosophila,Human,+,+,+,+,+,1989 Nobel Prize for Bishop

4、and Varmus,Thus:a proto-oncogene is the NORMAL progenitor gene of a viral oncogene,Cont.,1980,1st oncogene identified,Src（v-onc,viral oncogene）,Human Bladder Tumor cell line DNA,Isolate high MW DNA,Isolate DNA fragments,Restriction endonuclease,Transfection,NIH 3T3fibroblasts,Transformation,Isolate

5、DNA(99%mouse+8-10 human genes),Transformation,Identify Human DNA,Activated Proto-oncogenes from DNA transfection,RESULT:RAS“Activation”is due to a SINGLE point mutation(gly val)at codon 12,Use Alu probe,Isolate Human DNA,RESULT:A SINGLE human gene is responsible for transforming capability,Sequencin

6、g,RESULT:The gene is the HUMAN c-H-ras gene,Compare sequenceto NORMAL gene,1st human oncogene mutationRas G12V was identified!,1982,Robert Weinberg,etc three groups,Cont.,2.癌基因定义 Definition,Viral Oncogene(v-onc):a gene carried by a tumor virus(RNA or DNA),the expression of which is necessary and suf

7、ficient to induce transformation in tissue culture cells and tumors in the appropriate animal.V-onc is encoded by cellular sequences that have become inserted into the viral genome.,Oncogene:an altered gene whose product can act in a dominant fashion to help make a cell cancerous.Typically,an oncoge

8、ne is a mutant form of a normal gene(proto-oncogene)involved in the control of cell growth or division.,Proto-oncogene(c-onc):a normal cellular form of a gene that controls cell proliferation and can be converted into a cancer-promoting gene by mutation,whose continued activation leads to continued

9、signal transduction,and whose aberrant expression or activity may contribute to tumorigenesis.,原癌基因特点,1Controls cell proliferation and survival;2Can be converted into oncogene,and induce transformation,3Conserved across organisms4Tissue-specific,Physiological function:cell signaling pathways,tightly

10、 controlled,“Hyper”-functional,3.原癌基因激活机制,Transduction via retroviruses,Viral RNA,PackagingOf retrovirus,gag pol env,Proto-oncogene is“captured”or“usurped”from host cell genome,Retrovirus without oncogene,LTR,LTR,gag pol env Src,Retrovirus with oncogene,Retroviral promoter/enhancer insertion,Chronic

11、 Myelogenous Leukemia(CML)：chromosome 9q34(c-abl)chromosom22q11.2(bcr)，proto-oncogene c-abl is activated,Chromosomal translocation,Gain of multiple copies of defined chromosomal regions(1)Homogeneously staining region(HSR)(2)Double minute chromosomes(DM),Amplification,Cellular Proto-oncogenes amplif

12、ied in human tumors,HSR,DM,Ras：P21 transforming protein c-H-ras-Harvey rat sarcoma virus c-K-ras-Kirsten rat sarcoma virus N-ras-Neuroblastoma,Point mutation,Hot mutation points:12,13,61 12 Gly-Val,wt-Ras,GTP,wt-Ras,GDP,Active form,Inactive form,mt-Ras,GTP,mt-Ras,GDP,Constitutively active,According

13、to location and function of proto-oncogene products,4.癌基因分类,cAMP DG IP3 Ca2+,Overview:Classes of oncogenes A.Secreted Growth Factors(e.g.SIS,TGF-,etc)induce cell growth by mobilisation of energy stores,differentiation and entry into the cell cycle.B.Receptors(cell surface)different cells have differ

14、ent receptors,thereby a signal can produce a response in some cell type but not others Cell surface receptor with protein tyrosine kinase(PTK)activity(erbB,neu/erbB-2,ros,fms)C.Intracellular Transducers act as second messengers which alter transcription,either by allowing new genes to be expressed o

15、r by modifying levels of expression of already active genes a-Protein Tyrosine Kinase(src,yes,fps,abl,met)b-Protein-Serine/Threonine kinases(akt,mos,raf)c-Ras proteins(Ha-ras,Ki-ras,N-ras)d-Adaptors(crk)D.Nuclear Transcription Factors specific binding proteins that recognise short sequence motifs wi

16、thin the promoters and enhancers.These factors then accelerate or retard the rate of initiation of transcripts by RNA polymerase II a-jun,fos b-myc,N-myc,myb,ski,rel,Basic Cellular Signaling Machinery:for example Kinase signaling and Cancer,Receptor Protein Tyrosine Kinase Signaling,Non-receptor Pro

17、tein Tyrosine Kinase Signaling,Intracellular Serine/Threonine Kinase Signaling,5.癌基因功能,GTPase proteins,Nuclear transcription factor,Growth Factor Receptors,trans-membrane(glyco)proteinspossess intrinsic protein tyrosine kinase(PTK)activitydimerize(homo and heterodimers)auto-,trans-phosphorylationrec

18、ruit signaling molecules at specified phosphorylated sitesAberrantly expressed in many tumors,P,P,P,P,P,P,P,P,P,Receptor Protein Tyrosine Kinase Signaling,Collection of Receptor protein Tyrosine Kinases(RPTK/RTK),RECEPTORTYROSINEKINASE,GRB-2,SOS,RAS,RAF,MAPKK,MEMBRANE,MAPK,Dimerization;autophosphory

19、lation,Interaction ofsignaling molecules,Activation of“downstream”kinases,JAK,STAT,NUCLEUS,MAPK,Phosphorylation of transcription factors,STAT,GENE EXPRESSION,i.e.,fos,jun,myc,CYTOPLASM,RPTK SIGNALING CASCADES,25,Receptor Protein Tyrosine Kinases and Cancer,Non-receptor Protein Tyrosine Kinase Signal

20、ing,Non-receptor Protein Tyrosine Kinases and Cancer,Src Structure,Basal Activty(discerned from crystal structure),Activated Kinase,Src domains and Src activity,PI-3-kinase,Src Family,ras,myc,mitosis,mitotic functions,stress pathways,extracellular matrix,cytoskeletal reorganization,antigens,oxidativ

21、e stress,cytokines,G protein coupled receptors,RPTKs,angiogenesis,The World According to Src,RAS superfamly,GTPase proteins,1.H-ras,chr11;K-ras,chr12;N-ras,chr12.5 exons,188-189 aa,MW 21 KD3.GTPase,RAS signaling,RAS,PI3K,PIP3,Akt/PKB,Rho-GEF,Raf(MAPKKK),MEK(MAPKK),Erk1/2(MAPK),Mnk1,RSK,Ets,Elk-1,SAP

22、-1,Ral-GEF,Ral-A,Ral-B,Cdc42,Rac,The World According to RAS,RAS mutation and Cancer,Catalytic,reg,PH,T308*,S473*,Lipid binding,Ser/thr kinase,*Full activation of kinase requires phosphorylation of both T308 and S473,Akt structure,Intracellular Serine/Threonine Kinase Signaling,PI3Kactivation,PI(3,4,

23、5,)P3,PDK1,P,P,PDK2,PDK1,ILK,Catalytic,reg,PH,T308,S473,P,P,PI(3,4,5,)P3,NUCLEUS,Growth factor receptors,PI(4,5,)P2,Akt activation,Catalytic,reg,PH,T308,S473,GSK3,PFK-2,PDE-3B,mTOR,IkB,Bad,p21,Forkhead,Glycogensynthesis,Proteinsynth,glycolysis,cAMP,translation,Expression of Fas ligand,ExpressionOf a

24、ntiapoptotic genes,Bcl-XLBcl-2,Cell cycle,Akt/PKB-mediated signals,PI3K and MEK pathway in cancer,1.c-myc,8q24,439aa;N-myc,2p23-24,456aa;L-myc,1p32,364aa2.Nuclear transcription factor,Nuclear transcription factor,Myc,MYC regulation,二、抑癌基因 Tumor Suppressor Gene,Evidences for Tumor Suppressor Genes,So

25、matic Cell Genetic Studies,1969,Ephrussi and Harris,Ras oncogene-NIH3T3-transformationRas oncogene-CHO-NO transformation,DNA transfection,1.抑癌基因的发现,PEDIGREE of a family with familial retinoblastoma was published by Thaddeus P.Dryja and his collaborators.Affected members are indicated by solid circle

26、s(females)or squares(males).Five children in the second generation developed the tumor.One son who was unaffected had nonetheless inherited a mutated chromosome 13:two of his daughters were affected.13q14,Cytogenetic Analysis of familial cancers,Cloning and Identification of RB gene,1986 Cloning of

27、Rb genePut Rb gene back into Rb cells-lose transforming ability such as tumorigenicity,soft agar colony forming ability,1st tumor suppressor gene identified!,Terminology:Tumor suppressor genes,Anti-oncogenes,Recessive oncogenes,Tumor Suppressor Gene：a gene whose product can negatively control cell g

28、rowth and suppress cell transformation.,1.Negatively control cell growth and division2.Suppress cell transformation3.Mutated and inactivated in cancer4.tissue-specific,Criteria:,A.Expresses in normal tissuesB.Mutated in cancer：DNA,mRNA,proteinC.Re-introduction make cancer cell lose transforming abil

29、ity,Features:,2.抑癌基因定义,Knudsons Two-hit theory,Definition:inactivation of one allele by mutations or small deletions and loss of the second allele,usually by chromosome loss(loss of heterozygosity).-chromosome loss-mutations-small intragenic deletions-promoter methylation(epigenetics),Haploinsuffici

30、ecy Inactivation of one allele is enough to lead to tumor growth,3.抑癌基因失活机制,Epigentic deregulation The methylation states of promoter and histone affect the expression level of gene,1st hit,2nd hit,Tumor Suppressor Genes Associated With a Cancer Syndrome,Receptor：PTCH DCCIntermediate regulators：NF1

31、PTEN APC NF2Transcription factors/activators：p53 WT1 RB1 VHLCell cycle inhibitors：p16 p21 p15DNA repair：MSH2 MLH1 PMS2 ATM BRCA1 BRCA2,According to localization and molecular function,4.抑癌基因分类,Gatekeepers vs.Caretakers,Gatekeepers Genes directly regulating tumor growth by inhibiting their growth or

32、promoting their death.Ex.PTEN,p53,RBCaretakers Genes that do not directly suppress tumor growth,i.e.,inactivation of these genes leads to genetic instability that indirectly promotes tumor growth.Ex.BRCA1,BRCA2,5.抑癌基因功能,1.17p13.12.11 exons,393 aa,MW 53 KD3.Transactivator(TA),Transcriptional Activati

33、on domain,DNA binding domain,Tetramerization domain,Gene and protein structure,TP53,Evidences:,Mutations in cancers:Loss of Heterozygosity Colorectal cancers,Inhibition of transformation,A p53 null mouse,viable loss of one or both p53 alleles gives a tumor phenotype loss of p53 cooperates with other

34、 alterations to increase tumor incidence,p53:Li-Fraumeni Syndrome,-an inherited predisposition to cancer,-various kinds of cancers occur:osteosarcomas,soft tissue sarcomas,etc,-multiple tumors in the same individual,A tumor suppressor gene,Classification of p53 mutations,1.Loss-of-function,2.Dominan

35、t-negative,Mutant:loss of functionWide type:normal,3.Gain-of-function,Mutant:loss of functionWide type:loss of function,p53,p53,p53,p53,Transcriptional activation,Mutantp53,p53,p53,p53,Binds and regulates transcriptionof other genes MDR-1,c-myc,p53 functions,Transcriptional Activator:binds to cis-el

36、ement Consensus sequence:P：G/A Py：C/T AT PPPC GPyPyPy TA,Cell cycle regulatory genes p21/WAF1/Cip1,GADD45,cyclin GApoptosis genes Bax,Puma,Noxa,etcRegulators of itself Mdm2,ARF,Inactivation of p53,Ubiquitination and degradation by Mdm2(E3 ligase),p53,mdm2,p53,E2,E1,p53,UB,UB,mdm2,Interaction with vi

37、rus proteins:SV40 large T Ag,HPV16E6,E1B,T Ag,DNA binding,Dominant-negativeMutant p53,Activation of p53:increase of stability,Inhibition of Mdm2,p53,Mdm2,p19Arf,Phosphorylation of p53,Ultraviolet light Ionizing radiation Drugs(cisplatin,adriamycin),ATMATRChk1Chk2DNA-PK,p53,mdm2,out,Acetylation of p5

38、3,Acetyltransferase:p300/CBP,PCAF,etc,Sumoylation of p53(Small ubiquitin related molecules),p53,The p53 signaling network,PTEN(phosphatase and tensin homolog deleted on chromosome 10,PTEN);MMAC1(mutated in multiple advanced cancers 1);TEP1(TGF-regulated and epithelial cell-enriched phosphatase 1),Ge

39、ne structure,PTEN/MMAC1,discovered in 1997,Regulation of PTEN,A.Constitutive Phosphorylation(by casein kinase 2),C.Ubiquitination(proteasome degradation),B.Lipid interaction(PIP2)(Membrane localization),D.Protein interactions(stability,etc),PI 3.4.5 P3 PI-4.5 P2,PTEN,PI3K,PTEN activity,Canonical PTE

40、NPI3KAKTmTOR pathway,Biological functions of PTEN in tumorigenesis,PTEN,Akt,Forkhead,p27 Down-regulation,GSK-3,cyclin D1 degradation,2.Cell cycle arrest in G1 phase,Rac,Rho,MMP-2,3.Inhibits migration and invasion,migration,invasion,HIF-1,VEGF,angiogenesis,4.Inhibits angiogenesis,mTOR,elF4E disfuncti

41、on,1.protein synthesisinhibition,PTEN in cancer,Cont.,Schematic of RB in cell cycle control.,Alterations of the CdKRBE2F pathway in human cancer,三、癌基因/抑癌基因与多步骤癌变Synergy of Oncogenes&Tumor Suppressor Genes in Multi-step Tumorigenesis,1.Multi-hit requirements for carcinogenesis,NIH3T3 transfected assa

42、y is an one-hit mechanism(Ras transformation),Epidemiology supports multi-hit requirements for most tumors,Only 20%of human tumor DNA produce foci in NIH3T3 transfection assay,Problems:NIH3T3 is a immortal cell line.NIH3T3 cell is not exactly normal!How about foci-forming assays using embryo fibrobl

43、asts?,Transfection of Rat Diploid Embryo Fibroblasts,REF,NO FOCI!,Ras Gene,REF,NO FOCI!,v-myc Gene,+Ras,FOCI,CONCLUSION:At least two genes are required for transformation of“normal”cells,Complementation groups of Proto-oncogenes,ESTABLISHMENT(IMMORTALIZATION)GENES,c-myc,N-mycE1A(adenovirus)Large T(p

44、olyoma),NUCLEAR,TRANSFORMING GENES,c-K-ras,C-H-rasN-rasc-neu,CYTOPLASMIC,Tumor formation can be mimicked in the lab.By delivery of the following genes:Catalytic hTERT subunit of telomerase:Maintains telomere length2.SV40 large T antigen:inactivates both the p53 and Rb tumor suppressor geneActivated

45、oncogenic ras oncogene:induces transformation to a cancerous state allowing cells to grow indefinitely in the absence of growth factors,Disruption of the intracelluar pathways regulated by the large-T,oncogenic ras and telomerase suffice to create a human tumor cell.,Creation of human tumor cells wi

46、th defined genetic elements,Hahn,W.C.,Counter,C.M.,Lundberg,A.S.,Beijersbergen,R.L.,Brooks,M.W.,and Weinberg,R.A.,Nature 400,464-468,1999,1.Whitehead Institute for Biomedical Research2.Department of Biology,Massachusetts Institute of Technology,Cambridge,Massachusetts 02142,Cancer Cell,Vol.6,171-183

47、,August 2004,Annapoorni Rangarajan,1 Sue J.Hong,2 Annie Gifford,1 and Robert A.Weinberg1,2,*,Species-and cell type-specific requirements for cellular transformation,The Long Road to Cancer,Initiation,Promotion,Progression,Invasion and Metastasis,2.Synergy of Multi-hits in Multi-step Human Carcinogen

48、esis,Model for Colon Cancer Development and Progression,After Vogelsteinand colleagues,APC,3.Beyond Oncogenes and Tumor Suppressor Genes,Cell,Vol.100,5770,January 7,2000,The Hallmarks of Cancer,Douglas Hanahan*and Robert A.Weinberg,Cell,Vol.144,646674,January 7,2011,Douglas Hanahan*and Robert A.Wein

49、berg,四、癌基因/抑癌基因的鉴定Identification of Oncogene/Tumor Suppressor Genes,Cancer:a disease from genome on,HCC is the most common primary liver malignancyWorldwide incidence 600,000 cases per yearMore common in men than women(4:1)For resection,rate of recurrence can be as high as 50%at 2 yearsOnly 12%are e

50、ligible for resection or for transplant80%-90%of HCC cases occur in cirrhotic livers,International Agency for Cancer Research.Globocan 2002.Available at:http:/www-dep.iarc.fr.Accessed February 19,2008;Parkin DM et al.Int J Cancer.2001;94;153-156;American Cancer Society.Cancer Facts 127:S27-S34.,HCC