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1、赵莹珺复旦大学生物医学研究院 复旦大学附属肿瘤医院/肿瘤研究所癌基因与抑癌基因癌基因与抑癌基因 Oncogene 2，Transform cultured cells病毒致癌- Nobel Prize! 1966BIsolation of Retroviral Transforming GenesIsolation of Retroviral Transforming GenesRSV (gag, pol, env, src)R.T.(1975 Nobel)gag, pol envsrccDNARSV (td: gag, pol, env)gag, pol, envDenature and H
2、ybridize 1975,1977unhybridized sequenceshybridized sequencesgag, pol, envsrcgenomic RNACprobeRSV-Infected CEF (+ control)“Normal” chick DNAMouseDrosophilaHuman+1989 Nobel Prize for Bishop and VarmusThus: a proto-oncogene is the NORMAL progenitor gene of a viral oncogeneCont.Cont.1980, 1st oncogene i
3、dentifiedSrc （v-onc, viral oncogene）Human Bladder Tumorcell line DNAIsolate high MW DNAIsolate DNAfragmentsRestriction endonucleaseTransfectionNIH 3T3 fibroblastsTransformationIsolate DNA (99% mouse + 8-10 human genes)TransformationIdentify Human DNAActivated Proto-oncogenes from DNA transfectionDRE
4、SULT: RAS “Activation” is due to a SINGLE point mutation (gly val) at codon 12Use Alu probeIsolate Human DNARESULT: A SINGLE human gene is responsible for transformingcapabilitySequencingRESULT: The gene is the HUMANc-H-ras geneCompare sequence to NORMAL gene1st human oncogene mutation Ras G12V was
5、identified !1982, Robert Weinberg, etc three groupsCont.Cont.2.癌基因定义 DefinitionViral Viral OncogeneOncogene (v-(v-onconc): ): a gene carried by a tumor virus (RNA or DNA), the expression of which is necessary and sufficient to induce transformation in tissue culture cells and tumors in the appropria
6、te animal. V-onc is encoded by cellular sequences that have become inserted into the viral genome. OncogeneOncogene: : an altered gene whose product can act in a dominant fashion to help make a cell cancerous. Typically, an oncogene is a mutant form of a normal gene (proto-oncogene) involved in the
7、control of cell growth or division. Proto-oncogene Proto-oncogene (c-(c-onconc): ): 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 signal transduction, and whose aberrant e
8、xpression or activity may contribute to tumorigenesis . 原癌基因特点1Controls cell proliferation and survival;2Can be converted into oncogene, and induce transformation3Conserved across organisms4Tissue-specificPhysiological function: cell signaling pathways tightly controlled“Hyper”-functional3.原癌基因激活机制T
9、ransduction via retrovirusesLTRLTRViral RNAPackaging Of retrovirusgag pol envProto-oncogene is “captured” or “usurped” from host cell genomeRetrovirus without oncogeneLTRLTRgag pol env Src Retrovirus with oncogeneARetroviral promoter/enhancer insertionBChronic Myelogenous Leukemia (CML)：chromosome 9
10、q34 (c-abl) chromosom22q11.2 (bcr)，proto-oncogene c-abl is activatedChromosomal translocationq349535322q11.2ablder 9Phabl9q34 22q11.222q11.2 9q34Der (22)DNA RNA Proteinbcr bcrbcr/abl Fusion proteinCGain of multiple copies of defined chromosomal regions(1) Homogeneously staining region (HSR)(2) Doubl
11、e minute chromosomes (DM) Tumor OncogeneAmplificationSmall cell lung cancer c-myc Up to 8OXN-myc Up to 50XL-myc Up to 20XNeuroblastomas N-myc Up to 250XGlioblastomas c-erbB1 (EGFR) Up to 50XMammary carcinoma c-erbB2 (HER2) Up to 30Xc-myc Up to 50XCyclin D1 Up to 30XAmplificationCellular Proto-oncoge
12、nes amplified in human tumorsDHSRDMRas：P21 transforming proteinc-H-ras-Harvey rat sarcoma virusc-K-ras-Kirsten rat sarcoma virusN-ras-NeuroblastomaPoint mutationHot mutation points: 12, 13, 6112 Gly-Valwt-RasGTP wt-RasGDPActive formInactive formmt-RasGTP mt-RasGDPConstitutively activeEgrowth factors
13、 (sis)GTPase proteins (ras)guanine nucleotide Exchange proteins (rho)Cytoplasmic Membrane associated Tyrosine kinases (src)growth factor receptors (erb-b)nuclear transcription factors (myc)cytoplasmic serine Threonine kinases (akt)According to location and function of proto-oncogene products4.癌基因分类c
14、AMP 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 different receptors, thereby a signal can produce a respo
15、nse in some cell type but not others Cell surface receptor with protein tyrosine kinase (PTK) activity (erbB, neu/erbB-2, ros, fms)C. Intracellular Transducersact as second messengers which alter transcription, either by allowing new genes to be expressed or by modifying levels of expression of alre
16、ady 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 Factorsspecific binding proteins that recognise short sequence motifs within the promoters and enhancers. These factors then accelerate or retard t
