肿瘤分子生物学3细胞永生化与肿瘤发生.ppt

Page 1,肿瘤概念,Cancer is a genetic disease, arising from an accumulation of mutations that promote clonal selection of cells with increasingly aggressive behavior. The vast majority of mutations in cancer are somatic and are found only in an individuals cancer cells. However, about 1% of all cancers arise in individuals with an unmistakable hereditary cancer syndrome. These individuals carry a particular germline mutation in every cell of their body. Science Vol. 278. no. 5340, pp. 1043 1050, 7 November 1997,肿瘤是机体在各种致瘤因素的作用下，局部组织的细胞在基因水平上失去对其生长的正常调控，导致克隆性异常增生而形成的新生物（病理学卫生部统编教材第五版2001） 。,Page 2,癌细胞的基本特征 1、细胞生长与增殖失去控制 2、具有浸润性和扩散性 3、细胞间相互作用改变 4、蛋白表达谱系或蛋白活性改变 5、mRNA转录谱系改变 6、体外培养的恶性转化细胞的特征 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 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,Page 3,Normal 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 pedigree,Page 4,发育生物学：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， 而不关注与肿瘤发生最相关的问题， 有无特定的 控制系统 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 是否能够无限制生长（grow),或者每一个细胞谱系分裂次数是否为预先设定，有限的？,Page 5,1960 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 year,Page 6,Loss of proliferative capacity with age,Page 7,Senescent 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 cancer,Generations of cells forming a tumor(a) 1cm3=109 cell, life-threatening tumor 103cm3 1012 cell, 103 210 , hence 1012 240 cycles Cell PD 60 1018cell 109cm3 106kg,Page 9,Generations 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 generation,How 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 10,Cell physiologic stresses impose a limitation on replication,Influence of culture conditions on the onset of senescence Oxygen concentration Plastic or feeder cell in the culture influence the expression of tumor suppressor gene expression, so to the senescence In vitro mechanism - senescence -,Page 11,Increased expression of p16 and p21 progressively during extended culture in vitro,Ectopic expression of p16 in cells caused them to develop many of the attributes of replicative senescence,Normal cells,Forced expression of P16,Senescent cells,Actin stress fibers (orange) Focal contacts with the substrate (yellow),Page 12,Role 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 cells,Page 13,Evidence of senescent cells in living tissues A 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 integrity,The presence of senescent human melanocyts within dysplastic nevi,Treatment of tumors with chemotherapeutic drugs carboplatin and taxol prior to surgical excision of the tumor,Page 14,Senescence 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， 启动危象的分子装置确实是 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,The proliferation of cultured cells is limited by the telomeres of their chromosomes,Page 15,Barbara McClintock 1941 年报道了玉米染色人体端粒和转座子，获得1983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DNA 转染技术显现出哺乳动物染色体线状结构的致命缺陷。 真核细胞转染DNA 需要线状 化-使其不稳定 TA-质粒,Page 16,Shortening of telomeric DNA (5-TTAGGG-3 tandemly repeated hexanucleotide sequence) in concert with cell proliferation,Page 17,Dicentric chromosomes, anaphase bridges, and internuclear bridges,Page 18,Mechanisms of breakage- fusion-bridge cycles,Page 19,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型紊乱模式 。 这些细胞内融合的染色体（breakage- fusion-bridge）端粒很短， 甚至端粒全无。 提示触发细胞危象分子机制 存在于端粒内。,Page 20,测量端粒DNA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DNA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， 细胞内最短的端粒可能决定细胞的增殖潜能， 因为这个端粒将决定最早的断裂-融合-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Kb长的端粒已失去保护染色体端-端融合的能力。,Page 21,Incipient Cancer Cells Can Escape Crisis By Expressing Telomerase,The Telomeric Repeat Amplification Protocol assay TRAP assay,Page 22,Activation of telomerase activity following escape from crisis,Page 23,Prevention of crisis by expression of telomerase,体外培养细胞的危象及重生是体内癌前细胞克隆的重现. 癌前细胞克隆进入永生的途径重建端粒 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 CORRELATE,Page 24,1、端粒酶全酶是由多种、不同的（multiple, distinct） 亚单位构成，在正常、 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hTR-端粒酶相关RNA分子）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，因为端粒酶特异地作用于端粒DNA， 所以缩短的端粒 确实是危象的关键原因。 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）。,Page 25,Telomerase plays a key role in the proliferation of human cancer cells,Page 26,Telomerase activity and the prognosis of pediatric tumors,Page 27,Early passage cells,40 population doublings later hTERT is a distant relative of the reverse transcriptase of HIV,Copy 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 telomerase,Page 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 HIV,Page 29,Telomeres play different roles in cells of Lab mice and in human cells,Erosion of telomeres over multiple organismic generations in populations of mTR-/- mice,Telomeric DNA human/ mic