（优选）肿瘤分子生物学细胞永生化（优选）肿瘤分子生物学细胞永生化与肿瘤发生与肿瘤发生Page 2癌细胞的基本特征癌细胞的基本特征1、细胞生长与增殖失去控制细胞生长与增殖失去控制2、具有浸润性和扩散性、具有浸润性和扩散性3、细胞间相互作用改变、细胞间相互作用改变4、蛋白表达谱系或蛋白活性改变、蛋白表达谱系或蛋白活性改变5、mRNA转录谱系改变转录谱系改变6、体外培养的恶性转化细胞的特征、体外培养的恶性转化细胞的特征二、癌基因与抑癌基因二、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三、肿瘤的发生是基因突变逐渐积累的结果Page 3Normal cell populations register the number of cell generations C. elegance-The lineage of all 959 somatic cells in the adult body( could be) has been traced to their founder and can be depicted as a pedigreePage 4发育生物学：发育生物学：关注的是不同细胞谱系中的细胞个体如何从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，其周围获取信息，使其进入特定的分化程序， 而不关注与而不关注与肿瘤发生最相关的问题，肿瘤发生最相关的问题， 有无特定的有无特定的 控制系统控制系统 决定一个生物体特定的细胞谱系一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生中能够传多少代？一个细胞系谱的分支 是否能够无限制是否能够无限制生长（生长（grow),或者每一个细胞谱系分裂次数是否为预先设或者每一个细胞谱系分裂次数是否为预先设定，有限的？定，有限的？ Page 51960 Leonard Hayfliks work by counting the number of times that population of cells had doubled . When the cells enter into senescence , they could remain viable but nonproliferating for as long as a yearPage 6Loss of proliferative capacity with agePage 7Senescent cells- When the cells enter into senescence, they cease proliferating but remain viable, “Fried egg appearance” is the morphological feature because of the enlarged cytoplasm. Metabolically, senescent cells characteristically express the senescence-associated, acidic -galactosidase enzyme, which can be detected by supplying them with substrate that turns blue upon cleavage by this enzyme Page 8 Cancer cells need to become immortal in order to form cancerGenerations of cells forming a tumor(a)1cm3=109 cell, life-threatening tumor 103cm3 1012 cell,103 210 , hence 1012 240 cyclesCell PD 60 1018cell 109cm3 106kgPage 9Generations of cells forming a tumor(b) Cell populations that are evolving toward the neoplasitic state and those that are already neoplastic experience substantial attrition during each cell generationHow can normal cells throughout the body possibly remember their replicative history ? how can cancer cells erase the memory of this history and acquire the ability to proliferate indefinitely?Page 10Cell physiologic stresses impose a limitation on replicationInfluence of culture conditions on the onset of senescenceOxygen concentrationPlastic or feeder cell in the culture influence the expression of tumor suppressor gene expression, so to the senescenceIn vitro mechanism - senescence -Page 11Increased expression of p16 and p21 progressively during extended culture in vitroEctopic expression of p16 in cells caused them to develop many of the attributes of replicative senescence Normal cellsForced expression of P16Senescent cellsActin stress fibers (orange) Focal contacts with thesubstrate (yellow)Page 12Role of large T antigen in circumventing senescence Inactivation of both pRB and p53 is needed to ensure that these cells do not senesce in culture. This can be archived through the expression of the SV40 large T antigen in the target cellsPage 13Evidence of senescent cells in living tissuesA definitive proof that senescence is an in vivo phenomenon is critical to our understanding of cancer development. Brca1 mutant which is involved in maintaining genomic integrityThe presence of senescent human melanocyts within dysplastic nevi Treatment of tumors with chemotherapeutic drugs carboplatin and taxol prior to surgical excision of the tumorPage 14Senescence represents a halt in cell proliferation with retention of cell viability over extended periods of time, while Crisis involves death by apoptosis . Senescent cells seem to have a reasonably(but not totally) stable karyotype, while cells in crisis show widespread karyotypic instability危象的时相、形态变化提示其触发机制是独立于衰老的，危象的时相、形态变化提示其触发机制是独立于衰老的， 启动危象的分启动危象的分子装置确实是子装置确实是 细胞谱系从胚胎早期（进入细胞谱系从胚胎早期（进入 growth-and-division cycle)以后记录细胞连续传代的功能性计数装置以后记录细胞连续传代的功能性计数装置The proliferation of cultured cells is limited by the telomeres of their chromosomesPage 15Barbara McClintock 1941 年报道了玉米染色年报道了玉米染色人体端粒和转座子，获人体端粒和转座子，获得得1983年诺奖年诺奖 Telomeres detected by FISH(left),( right)karyotype seen in left compared with that of cells have been deprived of TRF2实验室实验室DNA 转染技术显现出哺乳动物染色体线状转染技术显现出哺乳动物染色体线状结构的致命缺陷。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 16Shortening of telomeric DNA (5-TTAGGG-3 tandemly repeated hexanucleotide sequence) in concert with cell proliferationPage 17Dicentric chromosomes, anaphase bridges, and internuclear bridgesPage 18Mechanisms of breakage- fusion-bridge cyclesPage 19端粒与细胞危象端粒与细胞危象 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型紊乱模式 。 这些细胞内融合的染色体（这些细胞内融合的染色体（breakage- fusion-bridge）端粒很短，）端粒很短， 甚至端粒全无。甚至端粒全无。 提示触发细胞危象分子机制提示触发细胞危象分子机制 存在于端粒内。存在于端粒内。Page 20 测量端粒测量端粒DNA的长度不能准确预测细胞的增殖潜能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即便已知一个细胞端粒DNA的长度，该细胞距离危象的增的长度，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，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，细胞内最短的端粒可能决定细胞的增殖潜能， 因为这个端粒因为这个端粒将决定最早的断裂将决定最早的断裂-融合融合-桥接出现时间，从而发生危象。此外，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长的端粒已失去保护染色体端-端融合的能力。端融合的能力。Page 21Incipient Cancer Cells Can Escape Crisis By Expressing TelomeraseThe Telomeric Repeat Amplification Protocol assay TRAP assayPage 22Activation of telomerase activity following escape from crisisPage 23Prevention of crisis by expression of telomerase 体外培养细胞的危象及体外培养细胞的危象及重生是体内癌前细胞克隆的重生是体内癌前细胞克隆的重现重现. 癌前细胞克隆进入永生癌前细胞克隆进入永生的途径的途径重建端粒重建端粒端粒酶是端粒重建的关键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 Acquisition of telomerase activity is a CAUSE of escape from crisis rather than a CORRELATEPage 241、端粒酶全酶是由多种、不同的（、端粒酶全酶是由多种、不同的（multiple, distinct） 亚亚单位构成，在正常单位构成，在正常、 危象前的人体细胞缺失的唯一亚单位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（包括是催化亚单位。端粒酶全酶其他亚单位（包括hTR-端粒酶相端粒酶相关关RNA分子分子）在危象前的细胞均适量储存。）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、端粒酶的表达（而不是其它酶）允许细胞规避危象，因为端为端粒粒酶特异酶特异地地作用于端粒作用于端粒DNA， 所以缩短的端粒所以缩短的端粒 确实是确实是危象的关键原因。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、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）。的子代细胞能够无限制生长（永生化）。Page 25Telomerase plays a key role in the proliferation of human cancer cellsPage 26Telomerase activity and the prognosis of pediatric tumorsPage 27Early passage cells40 population doublings laterhTERT is a distant relative of the reverse transcriptase of HIVCopy choice Mismatch Repair mechanism may be involved, because the ALT state is readily activated when some of the genes were deprived. One of the functions of mismatch repair is to suppress recombination between imperfectly homologous DNA sequences Some immortalized cells can maintain telomeres without telomerasePage 28 The ALT is associated preferentially with a specific subset of tumors, such as soft tissue sarcoma, osteosarcoma, glioblastoma Cells In ALT state: Telomeric DNA 30kb Telomerase positive cells 5-10kb dn TERT fails to induce crisis in ALT+ tumor cell line ALT positive cells do not depend on telomerase for their growth dn hTERT enzyme is not intrinsically cytotoxic hTERT is a distant relative of the reverse transcriptase of HIVPage 29Telomeres play different roles in cells of Lab mice and in human cellsErosion of telomeres over multiple organismic generations in populations of mTR-/- miceTelomeric DNA human/ mice 6-8kb /30-40kbMitoses human/mice lifetime 1016/1011, 0.1% of human cell and 1% of human lifespanHuman cell immortalization , the introduction of both the SV40 large T oncogene (to avoid senescence) and the hTERT gene (to avoid crisis).