医学精品课件：2011-12-06 张令强研究生课 细胞周期调控与癌症 分子生物学课.ppt

1、Lingqiang Zhang,Ph.DProfessor,Principal InvestigatorDept.Proteomics&Genomics(P&G)Beijing Inst.Radiation Medicine(BIRM)Tel:66931216Email: Cancer 10 million people are diagnosed with and 6 millions die of cancer each year worldwide.2 million people are diagnosed with and 1.5 millions die of cancer in

2、China.1.2 million people are diagnosed with and 550,000 die of cancer in the US each year.Cancer is the#1 killer for people under 85 and cost(health care and lose of productivity)$100 billion/year in US.1.1.从世界范围看，恶性肿瘤的发病率和死亡率呈上升从世界范围看，恶性肿瘤的发病率和死亡率呈上升趋势。除宫颈癌和食管癌外，几乎所有的恶性肿瘤趋势。除宫颈癌和食管癌外，几乎所有的恶性肿瘤均呈上升趋

3、势。均呈上升趋势。2.2.肺癌成为主要的恶性肿瘤，年发病人数达肺癌成为主要的恶性肿瘤，年发病人数达120120万，死万，死亡亡110110万。万。3.3.全球发病顺位：肺癌、乳腺癌、结直肠癌、胃癌；全球发病顺位：肺癌、乳腺癌、结直肠癌、胃癌；死亡顺位：肺癌、胃癌、肝癌、结直肠癌。死亡顺位：肺癌、胃癌、肝癌、结直肠癌。恶性肿瘤的发病率与死亡率呈上升趋势世界恶性肿瘤新病例（万）世界恶性肿瘤新病例（万）0 02 20 00 04 40 00 06 60 00 08 80 00 01 10 00 00 01 12 20 00 02 20 00 00 0年年2 20 01 10 0年年2 20 02 2

4、0 0年年2 20 05 50 0年年亚亚洲洲欧欧洲洲北北美美南南美美非非洲洲大大洋洋洲洲世界恶性肿瘤死亡病例（万）世界恶性肿瘤死亡病例（万）-1 10 00 01 10 00 03 30 00 05 50 00 07 70 00 09 90 00 01 11 10 00 02 20 00 00 0年年2 20 01 10 0年年2 20 02 20 0年年2 20 05 50 0年年亚亚洲洲欧欧洲洲北北美美南南美美非非洲洲大大洋洋洲洲亚洲国家的形势最为严峻20062006年城市因病死亡人口构成年城市因病死亡人口构成恶性肿瘤占恶性肿瘤占25.0%25.0%，第一位，第一位20062006年农村

5、因病死亡人口构成年农村因病死亡人口构成恶性肿瘤占恶性肿瘤占21.0%21.0%，第二位，第二位其他疾病其他疾病泌尿生殖泌尿生殖系病系病其余其余内分泌，营内分泌，营养代谢疾病养代谢疾病消化系病消化系病损伤和中毒损伤和中毒外部原因外部原因呼吸系病呼吸系病恶性肿瘤恶性肿瘤心脏病心脏病脑血管病脑血管病内分泌.营内分泌.营养代谢疾养代谢疾病病消化系病消化系病损伤和中毒损伤和中毒外部原因外部原因泌尿生殖泌尿生殖系统疾病系统疾病其他疾病其他疾病其余其余心脏病心脏病呼吸系病呼吸系病恶性肿瘤恶性肿瘤脑血管病脑血管病1 1，肺癌，肺癌2 2，胃癌，胃癌3 3，肝癌，肝癌1 1，胃癌，胃癌2 2，肝癌，肝癌3 3，

6、食管癌，食管癌恶性肿瘤是我国城乡居民因病死亡的首要原因1.1.累积死亡率（累积死亡率（0 06464岁）显著高于全球平均水平：全球平均累积死亡率岁）显著高于全球平均水平：全球平均累积死亡率为为10%10%；中国大城市累积死亡率为；中国大城市累积死亡率为20%20%，高发区及农村，高发区及农村25%25%50%50%2.2.肺癌已代替肝癌成为我国首位恶性肿瘤死亡原因，占全部恶性肿瘤死亡肺癌已代替肝癌成为我国首位恶性肿瘤死亡原因，占全部恶性肿瘤死亡的的22.7%22.7%（2008.11)2008.11)3.3.过去过去3030年肺癌和乳腺癌上升幅度最大（年肺癌和乳腺癌上升幅度最大（465%465

7、%和和96%96%），食管癌、胃癌、），食管癌、胃癌、宫颈癌、鼻咽癌死亡率呈明显下降趋势，其中宫颈癌下降幅度最大。宫颈癌、鼻咽癌死亡率呈明显下降趋势，其中宫颈癌下降幅度最大。4.4.每年用于癌症病人医疗费约每年用于癌症病人医疗费约800800亿元，占卫生总费用亿元，占卫生总费用20%20%，远高于其他慢，远高于其他慢性病的医疗药费。性病的医疗药费。我国恶性肿瘤的特征、严重危害与经济损失原癌基因：促进癌症发生、发展原癌基因：促进癌症发生、发展抑癌基因：抑制癌症发生、发展抑癌基因：抑制癌症发生、发展恶性肿瘤是医学领域一大科学奥秘和一大科学难题DNA synthesisChromosome dupl

8、icationCell with duplicated chromosomeChromosome separationMitosisCell with chromosome in the nucleusSG1MG2OncogenesActivity:Stimulating cell cycleMutation in cancer:Gain-of-functionExample:Ras,Myc,cyclin D1,MDM2Tumor suppressorsActivity:Inhibiting cell cycleMutation in cancer:Loss-of-functionExampl

9、e:Rb,p53,p16,ARF,PTENCell Cycle and Cancer?The Rb pathwayThe p53 pathwayCell cycle and cancer Cdk and cyclinThe spindle checkpoint2001年Nobel生理学和医学奖LELAND H.HARTWELL,R.TIMOTHY HUNT and PAUL M.NURSE for their discoveries of key regulators of the cell cycle.2001年生理学和医学Nobel奖Hartwell L.H贡献：鉴定了贡献：鉴定了100多

10、种参与细胞多种参与细胞周期调控的基因周期调控的基因细胞分裂周期基因细胞分裂周期基因(cdc)lCDC28启动基因启动基因(p34cdc2)lcheckpoint：有监视、调控周期时相精确运行Hunt R.T贡献贡献lCyclins，10种，Cyclin的转录、合成、降解随细胞周期不同时相 而变化；lCyclin选择性地同不同CDK（催化亚基）结合，只有结合才有激酶活性。CDK在一个细胞中的表达量恒定，其活性随Cyclin的调节功能而变化。Hunt R.T 周期蛋白是一件幸运的发现。1982年7月22日，那是一个非常 宁静的日子，我纯属处于好奇，作了一个简单的实验。我将35S 标记的蛋氨酸加到受

11、精的和单性发育激活了的海胆卵子，并在 一定的时间间隔取样，置于SDS-PAGE胶上电泳，以比较受精卵 和单性发育激活卵中蛋白合成的类型和速率。结果发现，当绝大多数蛋白带随着发育越来越强时，一个早期表现很强的蛋白带在1小时后消失了。这一结果是完全没有料到的，具有很强的诱惑力。反复的进一步实验表明，这一蛋白还可再出现和消失，其出现和消失呈现出周期性，因而被命名为周期蛋白。它消失于细胞分裂前10分钟。当用秋水仙素或用抑制DNA合成来抑制细胞分裂时，周期蛋白的降解则被延缓或不降解。这些结果在极为欣快的心境中写就成文Cell,1983,33,389-396.Nurse P.Ml鉴定了调节cell cyc

12、le的许多 基因，例如染色体分离时调控进入有丝 分裂通路的基因cdc2、cdc25lCDK克隆，并在人体细胞鉴定了启动基因CDK-1/cdc2lCDK活性受cyclin调节；磷酸化与去磷酸化调节；CKIS调节。The Cell CycleCell cycle:细胞前一次分裂结束开始生长，到下一次分裂终了细胞前一次分裂结束开始生长，到下一次分裂终了所经历的过程。所经历的过程。G1期：期：gap1 phase，从有丝分裂完成到，从有丝分裂完成到DNA复制之前的间隙期复制之前的间隙期S期：期：synthesis phase，DNA复制期复制期G2期：期：gap2 phase，从，从DNA复制完成到有

13、丝分裂开始第二次间复制完成到有丝分裂开始第二次间隙期隙期M期：期：mitosis phase，从有丝分裂开始到结束，即有丝分裂期，从有丝分裂开始到结束，即有丝分裂期就一个有机体或一个组织来说，其细胞群体大多由可进行连续分就一个有机体或一个组织来说，其细胞群体大多由可进行连续分裂的周期细胞、处于静止状态的裂的周期细胞、处于静止状态的G0细胞和不再进行分裂而注定细胞和不再进行分裂而注定要死亡的终端分化细胞的混合群体所组成。要死亡的终端分化细胞的混合群体所组成。The Cell CycleThe cycle must be regulated so events occur with the rig

14、ht timing,in the right order and only once per cycle.FACS is Used Widely to Determine Cell Cycle Characteristics and To Obtain Populations of Cells at Particular Cell Cycle StagesThe stages of mitosis and cytokinesisRoad to the discovery of CDKDifferent experimental systems offer unique advantagesYe

15、ast geneticsSea urchin embryo development biologyFrog oocyte biochemistryLay down the concept,logically and thoughtfullyDo the experiment,solidly and patientlyMPF=CDC2+cyclin B p45 cyclin B1 p34 CDC2CDC2CDC2Cyclin Binavtive cyclin activation cyclin degradationactiveMPF(mitosis-promoting factor;matur

16、ation promoting factor):p34cdc2-cyclinB 19711990The Cell CycleDNA synthesisChromosome duplicationCell with duplicated chromosomeChromosome separationMitosisCell with chromosome in the nucleusSG1MG2CDKcyclin?The First Layer of Cell Cycle Control Cyclin-Dependent Protein Kinases G1-Cdk G1/S-Cdk S-Cdk

17、M-CdkCore of the Cell Cycle EngineCdk(cyclin-dependent kinase)complexes Heterodimeric(two different subunits)protein kinases that regulate cell cycle Cyclin:regulatory subunit Cdk(cyclin-dependent kinase):catalytic subunit Phosphorylate proteins involved in cell cycle Different Cdk complexes for dif

18、ferent cell-cycle phases(G1,S,M)Cyclins Regulatory subunits of Cdk(cyclin-dependent kinase)complexes(heterodimeric protein kinases)Turn on kinase activity(phosphorylation)Levels vary cyclically during cell cycle Degraded by proteolysis at specific points in the cell cycleYeastCyclin levels vary cycl

19、icallyMammalCyclin levels vary cyclicallyRestriction point(START)Point at which cell is irreversibly committed to traversing the cell cycle Mammals:restriction point Yeast:START Cell cycle proceeds without influence from environment(only stopped by damage)Late in G1YeastMammalRestriction point/START

20、 in late G1Cdks(cyclin-dependent kinases)Catalytic subunits of Cdk(cyclin-dependent kinase)complexes(heterodimeric protein kinases)Kinase activity(phosphorylation)is turned on by association with cyclins Activities cyclically vary during cell cycle Phosphorylate proteins involved in cell cycle and c

21、ell-cycle regulationYeastMammalCyclin and Cdk types Yeast:9 cyclins and 1 Cdk(cdc28)Mammals:4 cyclin families and 4 CdksCdks and Cyclins Pair Combinatorially in Vertebrates and More Simply in Yeast Yeast:9 cyclins and 1 Cdk(cdc28)Mammals:4 cyclin families and 4 CdksFull Cdk Activation Requires Two S

22、teps:Cyclin Binding and Phosphorylation by a CAKAdditional Layers of Cdk Control-Reversible Phosphorylation and Dephosphorylation of CdkThe Wee-1 kinase and Cdc25 phosphatase are major regulators of M-Cdk.Additional Layers of Cdk Control-Cdk Inhibitors(CKIs)Inactivate CdksCKIs are involved primarily

23、 in control of G1 and S.The p21 Family of CDK inhibitors(p21CIP1/WAF1,p27KIP1,p57KIP2)CDKCyclinactivep21+inactiveCDKCyclinp21p27Kip1Cyclin ACDK2Russo et al.(1996)Nature 382:325Cyclin ACDK2Jeffrey et al.(1995)Nature 376:313Cip-kip(Cyclin inhibition protein-1 or kinase inhibition protein-1)The INK4 Fa

24、mily of CDK inhibitors(p16INK4a,p15INK4b,p18INK4c,p19INK4d)INK4+CDK4/6Cyclin DactiveCDK4/6Cyclin DINK4+inactiveRusso et al.(1998)Nature 395:237Brotherton et al.(1998)Nature 395:244Regulation of CDKinactiveCDKCDKCyclinactive cyclin activationSubstratePPPSubstrateCDKCyclinCKIinactive CKI inhibition CK

25、I degradation cyclin degradationHow G1 cyclins stimulate S entry?How CDK inhibitors arrest cell cycle in G1 The retinoblastoma 视网膜母细胞瘤视网膜母细胞瘤RB1 Gene is mutated in human cancers Reference:Lohmann,D.R.(1999)Human Mutation 14:283-288 Putative consequence Type of mutations All Single base substitution

26、Small deletion Small insertions Complex mutations All 227 Premature termination Splicing Missense ofIn-frame Regulatory 158 41 24 4 96 38 7 368 78 35 7 278 14 4-3 -58-28-4Cell cycle alterationsfaster doublingreduced serum response and contact inhibitionimmortalcomprised G1 arrest after DNA damagesus

27、ceptible to oncogene transformationDevelopment defectshematopoiesis(RBC)central nervous system(spinal cord,ventricles)peripheral nervous system(dorsal root ganglia)skeletal system(delayed ossification)myogenesis(skeletal,cardiac)Tumor formationpituitary tumor(100%)retinoblastoma neuroendocrine organ

28、 tumors(thyroid,parathyroid,adrenal,islet cell)Broad function of Rb gene family:Rb,p107,p130XE2FHDACG1 controlp15p16 p18 p19p21 p27p57Mitosis-G1RbG1 to SCDK4-DsCDK2-EsHDACPRbPPPPS phaseE2FDP?The Rb pathwayCKIG1 CDKsRbG1Scell proliferationE2FsThe Rb tumor suppression pathwayINK4(p16INK4a,p15INK4b,p18

29、INK4c,p19INK4d)CDK4,CDK6Rb(Rb,p107,p130)Cyclin D(Cyclin D1,cyclin D2,cyclin D3)G1SXretinoblastomalung cancerosteosarcomamelanomaleukemiabreast cancerhead&neck cancerparathyroid tumoresophagus cancerXmany type tumorscell proliferationp16 is frequently mutated in human tumorsTumor type Lines(n)Deletio

30、ns(n)Deletions(%)Astrocytoma 17 14 82 Bladder 15 5 33 Breast 10 6 60 Colon 20 0 0 Glioma 35 25 71 Leukemia 4 1 25 Lung 59 15 25 Melanoma 99 57 58 Neuroblastoma 10 0 0 Osteosarcoma 5 3 60 Ovary 7 2 29 Renal 9 5 56 Total 290 133 46Table 1.Deletions in tumor cells and primary tumors.See Kamb et al.(199

31、4)Science 264:436;Nobori et al.(1994)Nature 368:753 for detail9p21INK4:Inhibitor of CDK4ARF:Alternative Reading Frame of INK4aThe ARF-p16 locusExon2 GTCATGATGATGGGCAGCGCC p16:V M M M G S A ARF:H D D G Q R PExon 1 Exon 1 Exon 2Exon 3p14ARF*ATGATGp16INK4a*Mice deficient for p16,retaining Arf,are prone

32、 to tumor developmentExon 1 Exon 1 Exon 2Exon 3p14ARF*ATGATGp16INK4a*XTime(weeks)Tumor-free survival(%)Sharpless et al(2001)Nature 413:86;Krimpenfort et al.(2001),Nature 413:83Mice deficient for ARF,retaining p16,develop spontanous tumors at early ageK5 M 18 none Fibrosarcoma K11 M 8 none Metastatic

33、 salivary gland carcinoma K17 F 21 none Thymoma K75 F 11 none Maligant fibrous histiocytoma K116 M 18 none Lymphoma K199 M 16 none Fibrosarcoma K86 F 12 DMBA Epidemal papilloma K88 F 20 DMBA Epidemal papilloma K90 F 14 DMBA Lymphoma,epidemal papilloma K98 F 13 DMBA Metastatic epidermoid carcinoma K1

34、06 M 9 DMBA Invasive epidermoidcarcinoma K149 M 12 DMBA Fibrosarcoma,maligant adenexal tumor K150 M 14 DMBA Epidemal papilloma K151 F 13 DMBA Fibrosarcoma,epidemal papilloma K163 M 13 DMBA Epidemal papilloma K173 M 12 Irrdiation Fibrosarcoma K175 M 12 Irrdiation Lymphoma K178 F 13 Irrdiation Fibrosa

35、rcoma K185 M 19 Irrdiation LymphomaMouseTreatmentSpontanous and induced tumors in p19ARF-deficient miceSex Age(weeks)HistologySee Kamijo et al.(1997)Cell 91:649-659 for more details.Exon 1 Exon 1 Exon 2Exon 3p14ARF*ATGATGp16INK4a*XARF binds MDM2Key+His-Hisp16+MDM2p16+CDK4ARF+VectorARF+MDM2Zhang et a

36、l.(1998)Cell 92:725SV40 large T protein binds to p53T53 K612345IPNRSanti-TLane,D.and Crawford,L.(1979)Nature 278:261Linzer DI,and Levine,AJ.(1979)Cell 17:43p53 is frequently mutated in human cancerLung(897)56%Adrenal (31)23%Colon(960)50%Breast (1536)22%Esophagus(279)45%Endometrium(224)22%Ovary(386)4

37、4%Mesothelioma(23)22%Pancreas(170)44%Renal(102)19%Skin (220)44%Thyroid(299)13%Gastric (314)41%Hematological (1916)12%Head&neck(524)37%Carcinoid (13)11%Bladder (308)34%Melanoma (70)9%Sarcoma (339)31%Parathyroid(13)8%Prostate (87)30%Cevix(350)7%Hepatocellular (716)29%Neuroblastoma(212)1%Brain (456)25%

38、others(155)0%Tumor type(n)p53 mutationT umor type(n)p53 mutationAll tumors:37%Greenblatt et al.(1995)Cancer Res.54:4855p53 and Arf-p16 are two most frequently mutated genes in human tumorsLocusChromosome locationtype of alterationsestimated frequency of alterationsp53ARF-p1617p139p21nucleotide subst

39、itutionhomozygous deletion nucleotide substitution small deletion/insertion promoter methylation 50%40%1979年，被克隆发现年，被克隆发现 1993年，被年，被Science（科学）杂志评为年度明星分子（科学）杂志评为年度明星分子(Molecule of the Year 1993)人体一半以上的肿瘤有人体一半以上的肿瘤有p53基因的突变基因的突变/变异，另外一半的肿瘤有变异，另外一半的肿瘤有p53相关通路分子的异常相关通路分子的异常 是迄今为止已发现的与人类肿瘤发生相关性最高的抑癌基因是迄

40、今为止已发现的与人类肿瘤发生相关性最高的抑癌基因 被称为被称为“基因组守护神基因组守护神”、“分子警察分子警察”全世界有近全世界有近5万篇万篇p53相关的研究论文发表，平均相关的研究论文发表，平均3篇篇/天天 科学家、制药公司、临床医生关注的热点分子科学家、制药公司、临床医生关注的热点分子p53：与癌症发生、发展最为密切的抑癌基因：与癌症发生、发展最为密切的抑癌基因发展中国家发展中国家发达国家发达国家肺癌肺癌直肠癌直肠癌 乳腺癌乳腺癌胃癌胃癌 肝癌肝癌 前列腺癌前列腺癌 宫颈癌宫颈癌 头颈癌头颈癌 食管癌食管癌 白血病白血病 淋巴瘤淋巴瘤 卵巢癌卵巢癌 膀胱癌膀胱癌 肺癌肺癌胃癌胃癌 肝癌肝癌

41、恶性肿瘤中恶性肿瘤中p53基因发生突变、失去基因发生突变、失去“警察警察”功能功能 p53的结构域组成的结构域组成p53 调控多种重要的生理学与病理学过程调控多种重要的生理学与病理学过程Karen H.Vousden and Carol Prives.Cell(2009)137,413p53?How is p53 regulated?Normal cellTumor cellp53激活的模式激活的模式Jan-Philipp Kruse and Wei Gu.Cell(2009)137,609Jan-Philipp Kruse and Wei Gu.Cell(2009)137,609p53翻译后

42、修饰与翻译后修饰与p53调控调控p53的磷酸化修饰的磷酸化修饰Lys363SIRT1p53相互作用蛋白与相互作用蛋白与p53调控调控Karen H.Vousden and Carol Prives.Cell(2009)137,413Identification of MDM2 oncogeneMDM2:mouse double minutesN/Mdm2 12/12 R/Mdm2 4/4 N/Mdm1 1/8 R/Mdm1 0/4 N/Mdm3 0/8 R/Mdm3 0/2 N/pCV001 0/8 R/pCV001 2/8Cell lineaTumorigenicitybTumorigeni

43、city testing of transfectantsTumorigenicityCell linea:1-5 x106 cells(expressing different mdm genes)were injected into each nude mice and monitored over a period of 12-15 weeks.b:Tumorigenicity is defined as number of tumors/number of injections.Fakharzadeh et al.(1991)EMBO J.10:1565MDM2 binds to p5

44、312345IP-T-p53p90/MDM2p53Momand et al.(1992)Cell 69:1237MDM2 binds to p53Kussue et al.(1996)Science 274:948MDM2 inhibits p53p53-dependent CAT activity50-2:wild type p53-responsive CAT reporter CV001:a cosmid vectorMomand et al.(1992)Cell 69:1237p53 is the major target of MDM2 Wild-typeMdm2-/-Mdm2-/-

45、p53-/-Jones Et al.(1995)Nature 378:206Luna et al.(1995)Nature 378:203Vogelstein B et al.Nature,2000,408:307MDM2 degrades p53Kubbutat et al.(1997)Nature 387:299(mutated for MDM2 binding)-MDM2-MDM2+MDM2+MDM2MDM2 ubiquitinates p53MDM2-1+2+3-4+5+6poly ubiquitinated p53UbcH5+-+-Honda et al.(1997)FEBS Let

46、ter 420:25 p53MDM2How is MDM2 regulated?Normal cellTumor cell?ARF inhibits MDM2Relative Amount of p53(%)Chase(hour)0 1 2 3 4 5 6 710090807060504030201001234vectorp53p53+MDM2p53+MDM2+ARFp53Zhang et al.(1998)Cell 92:725Who regulates ARF?p53MDM2?ARFNormal cellTumor cellHigh level of Myc oncogene induce

47、s ARF expressionZindy et al.(1998)Genes&Dev.12:2424p53 can be activated by multiple stressesp53MDM2Oncogenic insultsARFDNA damageKinases(ATM,Chk1/2)Normal cellTumor cellDNA damage-kinases-Mdm2-p53 pathwayp2114-3-3s sBaxPIGAIP1DNA damage(Ionizing radiation)Chk2ATMMDM2p53Cell cycle arrestapoptosisPp53

48、MDM2ATM and Chk2 genes are mutated in human cancersATMExonNumber of mutationsMutations in the ATM gene detected in Ataxia telangiectasia patientsSource:www.vmresearch.org/atm.htmChk2Bartek J.and Lukas,J.(2003)Cancer Cell 3:421 Control of p53nucleusOncogenic insultsp53MDM2ARFE2Ubcell cycle arrestor a

49、poptosisp53p53MDM2Ubp53Ub Ub Ub Ub UbDNA damage MDM2Pp53Pp53Pp53p53p53PPPAAAAUbp53p53PAp53UbUbUbUbp53p53 target genesHAUSPApoptosisMDM2PhosphorylationATM,Chk1,Chk2,etcPID/HDAC1Other factors:COP1,YY1,Pirh2,gankyrin26SSome corepressor:CTBP2,IFI61,mSin3aOncogeneARFSir 2AcetylationCBP/P300,etcBcl2/Bcl X

50、LUbp5326SMDM2ApoptosisCell cycle arrestDNA repairSenescenceE4Cellular stressp53的调控网络的调控网络Rb and p53 tumor suppression pathwaysRbG0/G1SNormal cellCDKp53MDM2oncogenesARFmitogencyclinDNA damageKinases(ATM,Chk1/2)diferentiationsenescenceCKIXexcessive cell proliferationXTumor cellXXXSmart tumor virusesVi