腫瘤分子生物學(xué)3細(xì)胞永生化與腫瘤發(fā)生

1、Page 1,腫瘤概念,,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 a

2、re found only in an individual's 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 ce

3、ll of their body. Science Vol. 278. no. 5340, pp. 1043 – 1050, 7 November 1997,腫瘤是機體在各種致瘤因素的作用下，局部組織的細(xì)胞在基因水平上失去對其生長的正常調(diào)控，導(dǎo)致克隆性異常增生而形成的新生物（病理學(xué)－衛(wèi)生部統(tǒng)編教材第五版2001） 。,Page 2,癌細(xì)胞的基本特征1、細(xì)胞生長與增殖失去控制2、具有浸潤性和擴散性3、細(xì)胞間相互作用改變4、蛋白表達(dá)

4、譜系或蛋白活性改變5、mRNA轉(zhuǎn)錄譜系改變6、體外培養(yǎng)的惡性轉(zhuǎn)化細(xì)胞的特征二、癌基因與抑癌基因癌基因－控制細(xì)胞生長和分裂的正常基因的一種突變形式抑癌基因－正常細(xì)胞增殖過程中的負(fù)調(diào)控因子三、腫瘤的發(fā)生是基因突變逐漸積累的結(jié)果,Page 3,Normal cell populations register the number of cell generations,C. elegance-The lineage of al

5、l 959 somatic cells in the adult body( could be) has been traced to their founder and can be depicted as a pedigree,Page 4,發(fā)育生物學(xué)：關(guān)注的是不同細(xì)胞譜系中的細(xì)胞個體如何從其周圍獲取信息，使其進入特定的分化程序， 而不關(guān)注與腫瘤發(fā)生最相關(guān)的問題， 有無特定的 控制系統(tǒng) 決定一個生物體特定的細(xì)胞譜系一生中能夠

6、傳多少代？一個細(xì)胞系譜的分支 是否能夠無限制生長（grow),或者每一個細(xì)胞譜系分裂次數(shù)是否為預(yù)先設(shè)定，有限的？,Page 5,1960 Leonard Hayflik’s work by counting the number of times that population of cells had doubled . When the cells enter into senescence , they could remain

7、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 a

8、ppearance” is the morphological feature because of the enlarged cytoplasm. Metabolically, senescent cells characteristically express the senescence-associated, acidic β-galactosidase enzyme, which can be detected by sup

9、plying 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 1

10、03cm3 ≈ 1012 cell,103 ≈210 , hence 1012 ≈240 cyclesCell 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 a

11、re 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 h

12、istory 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 senescenceOxygen concentrationPlastic or

13、feeder cell in the culture influence the expression of tumor suppressor gene expression, so to the senescenceIn vitro mechanism - senescence -,Page 11,Increased expression of p16 and p21 progressively during extended

14、 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 cont

15、acts with thesubstrate (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 throu

16、gh the expression of the SV40 large T antigen in the target cells,Page 13,Evidence of senescent cells in living tissuesA definitive proof that senescence is an in vivo phenomenon is critical to our understanding of c

17、ancer 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 p

18、rior 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 cell

19、s seem to have a reasonably(but not totally) stable karyotype, while cells in crisis show widespread karyotypic instability,危象的時相、形態(tài)變化提示其觸發(fā)機制是獨立于衰老的， 啟動危象的分子裝置確實是 細(xì)胞譜系從胚胎早期（進入 growth-and-division cycle)以后記錄細(xì)胞連續(xù)傳代的功能性計數(shù)裝置

20、,The proliferation of cultured cells is limited by the telomeres of their chromosomes,Page 15,Barbara McClintock 1941 年報道了玉米染色人體端粒和轉(zhuǎn)座子，獲得1983年諾獎,Telomeres detected by FISH(left),( right)karyotype seen in left compared wi

21、th that of cells have been deprived of TRF2實驗室DNA 轉(zhuǎn)染技術(shù)顯現(xiàn)出哺乳動物染色體線狀結(jié)構(gòu)的致命缺陷。 真核細(xì)胞轉(zhuǎn)染DNA 需要線狀 化-使其不穩(wěn)定 TA-質(zhì)粒,Page 16,Shortening of telomeric DNA (5′-TTAGGG-3′ tandemly repeated hexanucleotide sequence) in co

22、ncert with cell proliferation,Page 17,Dicentric chromosomes, anaphase bridges, and internuclear bridges,Page 18,Mechanisms of breakage- fusion-bridge cycles,Page 19,端粒與細(xì)胞危象 進入危象的細(xì)胞準(zhǔn)確的呈現(xiàn)染色體缺失端粒時的核型紊亂模式 。 這些細(xì)胞內(nèi)融合的染色

23、體（breakage- fusion-bridge）端粒很短， 甚至端粒全無。 提示觸發(fā)細(xì)胞危象分子機制 存在于端粒內(nèi)。,Page 20,測量端粒DNA的長度不能準(zhǔn)確預(yù)測細(xì)胞的增殖潛能 即便已知一個細(xì)胞端粒DNA的長度，該細(xì)胞距離危象的增殖代數(shù)并不能精確預(yù)測。一個細(xì)胞內(nèi)的端?？s短速率不同， 細(xì)胞內(nèi)最短的端粒可能決定細(xì)胞的增殖潛能， 因為這個端粒將決定最早的斷裂-融合-橋接出現(xiàn)時間，從而發(fā)生危象。此外，我們不知道端粒

24、短到何種程度才失去其保護功能。有時數(shù)Kb長的端粒已失去保護染色體端-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 f

25、ollowing escape from crisis,Page 23,Prevention of crisis by expression of telomerase,體外培養(yǎng)細(xì)胞的危象及重生是體內(nèi)癌前細(xì)胞克隆的重現(xiàn). 癌前細(xì)胞克隆進入永生的途徑—重建端粒端粒酶是端粒重建的關(guān)鍵 85%-90% 人體腫瘤可檢測到明顯的端粒酶活性 Acquisition of telomerase activity i

26、s a CAUSE of escape from crisis rather than a CORRELATE,Page 24,1、端粒酶全酶是由多種、不同的（multiple, distinct） 亞單位構(gòu)成，在正常、 危象前的人體細(xì)胞缺失的唯一亞單位是催化亞單位。端粒酶全酶其他亞單位（包括hTR-端粒酶相關(guān)RNA分子）在危象前的細(xì)胞均適量儲存。 2、端粒酶的表達(dá)（而不是其它酶）允許細(xì)胞規(guī)避危象，因為端粒酶特異地作用于端粒DNA，

27、 所以縮短的端粒 確實是危象的關(guān)鍵原因。3、實驗顯示，端粒酶活性的獲得（從大量進入危象的細(xì)胞群中自主產(chǎn)生的變異細(xì)胞）足以使細(xì)胞逃逸危象，其產(chǎn)生的子代細(xì)胞能夠無限制生長（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 p

28、ediatric tumors,Page 27,Early passage cells,40 population doublings laterhTERT is a distant relative of the reverse transcriptase of HIV,Copy choice Mismatch Repair mechanism may be involved, because the ALT stat

29、e 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 telomere

30、s 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 p

31、ositive 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

32、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

33、in populations of mTR-/- mice,Telomeric 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

34、 large T oncogene (to avoid senescence) and the hTERT gene (to avoid crisis).,Page 30,Telomere and bone marrow collapse of dyskeratosis congenita X-linked form of the disease, a protein known as dyskerin is absent or

35、 malfunctioning. Dyskerin associates with the telomerase-associated RNA subunit (hTR) and helps hTR to assemble in the telomerase holoenzyme. An autosomal dominant version of the same disease is caused by the inheritanc