1、CHAPTER 14細胞衰老、死亡與癌變 Cell aging, Death and Cancer 1OUTLINE14.1 細胞衰老 (Cell Aging)14.1.1 衰老的概念14.1.2 細胞的壽限14.1.3 細胞衰老的特征14.1.4 細胞衰老的理論14.2 細胞死亡 (Cell Death)14.2.1 程序性細胞死亡的基本特性14.2.2 程序性細胞死亡的機理14.2.3 程序性細胞死亡的生物學意義14.3 癌細胞 （Cancer）14.3.1 癌生物學14.3.2 癌的起因：物理和化學致癌物14.3.3 腫瘤發生的遺傳學2314.1 細胞衰老14.1.1 衰老的概念衰老(s

2、enescing，aging)是機體在退化時期生理功能下降和紊亂的綜合表現,是不可逆的生命過程。細胞的衰老與死亡是新陳代謝的自然現象。機體的衰老與細胞的衰老相關聯。 現代人類面臨著3種衰老：生理性衰老病理性衰老心理性衰老 414.1.2 細胞的壽限：1961年，Hayflick 首次報道了體外培養的人成纖維細胞具有增殖分裂的極限,且分裂能力與個體的年齡有關。來源于胚胎 分裂傳代50次后開始死亡來源于成年組織 培養1530代就開始死亡動物體細胞在體外可傳代的次數，與物種的壽命有關。5體外培養的年輕和老的人成纖維細胞的顯微形態6物種的壽命與體外培養時細胞傳代次數的關系長壽物種的細胞體外培養的代數比

3、短壽物種的細胞代數多7 Hayflick界限 (Hayflick life span): 細胞至少是體外培養細胞的增殖能力不是無限的，而是有一定界限，細胞的衰老控制著細胞的分裂次數, 進而控制著細胞的數量。細胞的壽限：各類細胞本身的壽命差別顯著, 一般說來, 能夠保持持續分裂能力的細胞是不容易衰老的。分化程度高又不分裂的細胞壽命是有限的。814.1.3 細胞衰老的特征細胞內水分減少色素生成和色素顆粒沉積衰老過程中細胞質膜的變化：流動性降低；興奮性降低；配體受體復合物形成下降衰老過程中線粒體的變化數量減少，體積變大，膜破壞，DNA突變細胞核的變化核膜內折；染色質固縮；端粒縮短細胞骨架的變化蛋白質

4、合成的變化合成速度降低，蛋白定位改變2022/9/299MorphologySenescent cells become flattened, enlarged and have increased -galactosidase ( -半乳糖苷酶) activityIncreased size of nucleus and nucleoliIncreased number of multinucleated cellsIncreased number of lysosomes, Golgi and cytoplasmic microfilaments2022/9/2910Senescent c

5、ells undergo three phenotypic changes1114.1.4 細胞衰老的理論細胞衰老的線粒體損傷論 (Mitochondria ageing)自由基理論：氧自由基 (Reactive oxygen species (ROS)細胞衰老的端粒假說 (Replicative senescence and telomere shortening)細胞衰老的表觀調控: (Epigenetic regulation of senescence) 衰老的進化論衰老的突變積聚、互逆多效理論12自由基攻擊細胞的證據2022/9/2913Replicative senescence

6、is the progressive shortening of telomeres at chromosome endsCritically short telomeres trigger activation of cell cycle checkpointsPermanent cell cycle growth arrest due to activated cell cycle checkpoints, similar to those activated upon double strand breakageCells metabolically active but cannot

7、continue to divide, unlike quiesence 靜止Replicative senescence2022/9/2914In the early embryonic period, cells have a determined length of telomere endings. As organism develops by cell differentiation, cells keep proliferating and during each division, telomeres get shorter because of replication mec

8、hanism specificity. So nature determined that as organism get older, telomeres get shorter and cell goes to death. 2022/9/2915Telomere dysfunction contributes to cancer X-rays UV OthersOxidative stressOxidative stressTelomeredysfunctionDNA damageCheckpoint activationSenescenceor apoptosisAccumulatio

9、n During lifeInactivatingmutationsp19ARFp53Stem CellCancerSenescence2022/9/2916Epigenetic Regulation of Senescence Epigenetics entails the study of the switching on and off of genes during development, cell proliferation, senescence and also by environmental insults. Genome modifications resulting f

10、rom epigenetic changes appear to play a critical role in the cellular senescene. Scatter experimental evidence suggests that epigenetic changes could also be critical determinants of cellular senescence and organisms senescence. 2022/9/2917 Histone deacetylases (HDACs) participate in senescence Elev

11、ated HDAC activity appears causally related to cellular senescence, as overexpression of a p300 mutant protein, or treatment with a specific chemical inhibitor of p300, results in irreversible growth arrest and senescence of normal human cells. DNA methylationSequential loss of DNA methylation could

12、 act as an alternative counting mechanism. A progressive loss of 5-methylcytosine in genomic DNA occurs during serial passage of normal cells in culture. The extent of CpG methylation also decreases during aging of organisms. On the other hand, immortal cell lines maintain constant levels of DNA met

13、hylation.2022/9/2918Chromatin remodeling and senescenceGenes in the p53, Rb, and ING (inhibitor of growth) pathways affect cell senescence and are capable of regulating gene expression through chromatin remodeling.p16INK4a is required for hSNF5 chromatin-remodeler induced cellular senescence in mali

14、gnant tumor cells PASG, an SNF2 family member, is essential for properly maintaining normal DNA methylation and gene expression patterns. Disruption of PASG leads to accumulation of senescence-associated tumor suppressor genes, and increased senescence- associated galactosidase as well as age-relate

15、d phenotypes.2022/9/2919Modification of different amino acid residues in histone H3 leads either to activation or repression of transcription.2022/9/2920RNA Degradation and Aging Model of age-related changes in AU-rich elements (ARE)-directed mRNA decay. As cells age, HuR levels decline, shifting th

16、e balance to mRNA degradation. Many ARE-mRNAs encoding proteins that contribute to proliferation, thus, decline which contributes to the phenotype of senescence. 2022/9/2921The role of genetics in determining life-span is complex and paradoxical. Although the heritability of life-span is relatively

17、minor, some genetic variants significantly modify senescence of mammals and invertebrates, with both positive and negative impacts on age-related disorders and life-spans. The Role of Genetics in Senescence It appears certain that DNA mutations and chromosomal abnormalities increase with age in mice

18、 and humans 2022/9/2922A mutant model mouse is useful for studies of aging. The klotho phenotype (premature aging) is caused by a disruption of the single gene, klotho. 2022/9/2923Cellular senescence involved in genetic errorsThere is an invariant relationship between life span and the number of ran

19、dom mutations. A number of studies at a number of gene loci have shown that somatic mutations of a variety of types accumulate with age. Deficient in DNA Repair and Transcription induce Premature Aging in Mice.TTD and XPD, genes for DNA repair and replication. SCIENCE VOL 296 17 MAY 2002Photograph o

20、f a 3-week-old XPA/TTD double-mutant (left), TTD (middle), and XPA (right) mouse.2022/9/2924Evolutionary Theory of SenescenceAging is a by-product of natural selection due to lack of selective pressure for the post-reproductive individual. Any individual has a probability to reproduce. It is zero at

21、 birth and reaches a peak in young adults. Then, it decreases due to the increased probability of death linked to various external (predators, illnesses, accidents) and internal causes (aging).25早老癥兒童26細胞衰老的分子途徑p19ARF/p53/p21Cip1 : p16INK4a/Rb端粒-p53-PGCCDK inhibitorsSenescence signal27Role of p53 in

22、 G1 arrest induced by DNA damage Induction of p21 via p53 activation p21: Cdk inhibitor. inhibit DNA synthesis by interacting with PCNA (a subunit of DNA polymerase ) 28Rb蛋白對細胞周期的調節p16INK4a2022/9/2929 Polycomb group protein BMI1 has been linked to proliferation, senescence and apoptosis.30Telomere-p

23、53-PGC PGC (perioxisome proliferator-activated receptor gamma coactivator)細胞代謝及線粒體功能的主要調控因子3114.2 細胞死亡細胞死亡概念：細胞死亡的一般定義是細胞生命現象不可逆的停止。細胞死亡有兩種形式：一種為壞死性死亡，另一種為程序性死亡。3214.2.1 程序性細胞死亡及其特性程序性細胞死亡(programmed cell death, PCD), 又稱細胞凋亡(apoptosis)是指為維持內環境穩定，由基因控制的細胞自主的有序性的死亡，它涉及一系列基因的激活、表達以及調控等的作用，因而是具有生理性和選擇性的

24、。 Apoptosis：希臘語，是指樹葉或花的自然凋落；33程序性細胞死亡34程序性死亡細胞的形態結構變化細胞變圓，染色質聚集、分塊，胞質皺縮35程序性死亡細胞的DNA降解PCD生化特征：染色質DNA的有控裂解：核DNA在核小體連接處斷裂成核小體片段，200bp的倍數DNA Ladder36細胞壞死與程序性細胞死亡凋亡小體 (apoptotic body)37比較內容程序性細胞死亡細胞壞死質膜不破裂發生破裂細胞核固縮，DNA片段化彌漫性降解細胞質由質膜包圍溢出形成凋亡小體細胞破裂成碎片溶酶體的酶增多溶酶體解體蛋白質合成有無基因活動由基因調控無基因調控自吞噬常見缺少線粒體自身吞噬腫脹誘發因素生理

25、性信號強烈刺激信號對個體影響生長、發育、引起炎癥生存所必需細胞壞死與程序性細胞死亡比較3814.2.2 程序性細胞死亡的機理2002年的諾貝爾生理學和醫學獎:英國的Brenner、Sulston和美國的Horvitz，用C. elegans 研究了調控器官發育程序性細胞死亡的關鍵基因及其功能，并進一步在高等哺乳動物中發現了相關功能基因。Caenorhabditis elegans (C. elegans) 雌雄同體 39程序性細胞死亡的過程死亡激活期 (activation phase)：接收death signal死亡執行期 (execution phase)：執行一套死亡程序40 apop

26、tosis related genes in C elegans :決定死亡的兩個基因，即ces-1(ces表示CE細胞存活的調控基因)和ces-2基因執行死亡的4個基因：ced-3、ced-4、ced-9和egl-1基因：“死亡機器”(death machinery)7個與死亡細胞被吞噬細胞所吞噬的基因，即ced-1、ced-2、ced-5、ced-6、ced-7、ced-10和ced-11。ced-9可抵消ced-3和ced-4 的作用，防止細胞被殺死，因此是存活因子; 死亡細胞在吞噬體中被降解的基因細胞凋亡的機理：基因調控作用的結果41Apoptotic genes in C. eleg

27、ans42 Caspase自殺性蛋白水解酶是天冬氨酸特異性半胱氨酸蛋白酶(cysteine-containing aspartate specific protease)，簡稱caspase;caspase-3、6、7和8 在FAS/TNF介導的程序性細胞死亡途徑中起作用；caspase-9和3 一起參與線粒體中Apaf-I、細胞色素c介導的程序性細胞死亡;在人類，已經鑒定了10種不同的caspase。Apoptotic genes in mammalian cells43Apoptotic protease cascade in mammalian cells自殺性蛋白酶家族自我切割蛋白降解

28、級聯44執行者caspase在程序性細胞死亡中的作用45能夠被caspase切割的靶蛋白蛋白激酶核纖層蛋白細胞結構蛋白與DNA修復相關的酶類caspase激活的DNase抑制蛋白2022/9/2946Death receptors: CD95 (or Fas) TNFR1 (TNF receptor-1) DR4 and DR5. 細胞外信號（The extrinsic death pathway） 對程序性細胞死亡的激發47腫瘤壞死因子 （tumor necrosis factor, TNF）48細胞內信號（The intrinsic death pathway ）對程序性細胞死亡的激發內源

29、信號DNA損傷細胞質中Ca2+ 濃度過高極度氧脅迫（產生大量的自由基）正控制信號 促進細胞死亡,如細胞色素C，凋亡蛋白酶激活因子（apoptotic protease-activating factor，Apaf）負控制信號抑制細胞死亡，如哺乳動物中的BCL-2和BCL-x蛋白。49細胞內源信號激發細胞程序性死亡50 Apoptosis Regulators and effectors2022/9/2951Apoptotic proteins p53Bax線粒體外膜通透性2022/9/2952BidApoptotic proteins Bcl-2 FamilyBid2022/9/2953Apo

30、ptotic proteins Caspase family2022/9/2954Apoptotic proteins IAP family （inhibitor of apoptosis protein)livin2022/9/2955Apoptosis and senescence both are a failsafe (錯誤消除) mechanisms in cellCells respond to a number of potentially oncogenic stimuli by adopting a senescent or apoptosis, suggesting tha

31、t both are fail-safe mechanisms that protects cells from tumorigenic transformation. potentially oncogenic stimuliNormal SenescenceNormal ApoptosisNormal Cancer2022/9/2956Through accumulated genetic mutations, cell can be transformed, leading to tumors. Nature stops this tumorigenesis process throug

32、h apoptosis or senescence. However, as organisms age, the accumulation of senescent cells can create a pro-tumorigenic tissue environment. 2022/9/2957At the cellular level, activated p53 induced checkpoints in the cell-division cycle, permanent cell-division arrest (senescence) and cell death. At th

33、e whole-organism level, p53 activation results in a lower cancer incidence. But Tyner et al.3 show that p53 can also promote ageing. p53- common in apoptosis and senescence 2022/9/2958Apoptosis：a two-edged swordIn the reproductive years：providing critical tumor surveillancein a post reproductive per

34、iod：contributing to agingApoptosis and lifespan:Apoptosis vs Senescence lifespanApoptosis Senecence ( ageing )5914.2.3 程序性細胞死亡的意義動物機體靠對細胞增殖和細胞周期的正負控制以及對程序性細胞死亡的正負控制來維持細胞總數的平衡和機體的生命活力。程序性細胞死亡在形態建成中起重要作用。60動物細胞數量控制的途徑61程序性細胞死亡在小鼠腳趾形成中的作用62蝌蚪向蛙發育的變態反應中程序性細胞死亡的作用63程序性細胞死亡對發育中神經細胞數量的調節競爭上崗64癌細胞所謂癌細胞實際上是一

35、種突變的體細胞，這種突變體脫離了細胞社會關于增殖和存活的控制，因此可以無限制的增殖產生腫瘤(Tumor)。分良性及惡性腫瘤。6514.1 癌生物學惡性腫瘤“癌”的類群癌(carcinoma)：上皮和內皮，內外胚層瘤(sarcoma)：結締組織、肌肉，中胚層淋巴瘤(lymphoma)和白血病(leukemia)：是由淋巴和血液產生的癌，白血病主要是指癌細胞已經大量進入血液中。中胚層來源畸胎瘤（teratoma）早期胚胎細胞轉化，有良惡之分癌細胞離體培養時接觸抑制缺失癌細胞的生物學特性：無限增殖Colony formation/ transformation assay癌細胞的生物學特性：侵襲及轉

36、移原位癌 vs 繼發癌68癌細胞轉移69PET全稱為正電子發射計算機斷層顯像，是反映病變的基因、分子、代謝及功能狀態的顯像設備。它是利用正電子核素標記葡萄糖等人體代謝物作為顯像劑，通過病灶對顯像劑的攝取來反映其代謝變化，從而為臨床提供疾病的生物代謝信息。Benign tumorMalignant tumor癌細胞惡性程度越高，分化程度越低快速增殖、不播散快速增殖、播散轉移71 “癌”細胞形態外形：變圓細胞骨架結構紊亂核異常細胞質膜結構改變：細胞間連接、細胞表面受體癌細胞的染色體異常，如多倍體Pancreas cancer73“癌”細胞生理功能異常無限分裂、無接觸抑制細胞黏著性、貼壁性減弱：Ce

37、ll-cell 、Cell-ECMeg：Fibronectin、cadherin、GAG易于凝集：凝集素74“癌”細胞生化異常細胞質膜成份改變：糖脂糖蛋白減少高爾基體成分變化：糖基轉移酶缺乏纖連蛋白分泌減少新的膜抗原生成：MHA丟失生長因子需求降低分泌多種蛋白水解酶：MMPs75癌細胞的自分泌生長刺激7614.2 癌的起因： 物理和化學致癌物化學致癌物輻射對癌的誘發病毒77化學致癌物7814. 3 腫瘤發生遺傳學癌發生的進程Normal cell Benign tumor Malignant tumor腫瘤抑制基因、癌基因與原癌基因79結腸癌的多步發展過程80癌基因與原癌基因癌基因(oncog

38、ene)：癌基因是細胞加速器，它們編碼的蛋白使細胞生長不受控制，并促進細胞癌變細胞癌基因 (c-onc)：由細胞原癌基因突變而來；病毒癌基因 (v-onc)：大約已經鑒定了100多種不同的癌基因，它們中的大多數屬于RNA腫瘤病毒基因 組中的基因。1976年發現，正常雞細胞核DNA中，具有與V-onc的同源序列，稱正常細胞中的同源序列為原癌基因（protooncogene）,或C-oncogene.v-onc和c-onc的關系：c-onc來自v-onc. 理由如下:1、v-onc對病毒復制和生存都是不必要的；而c-onc對細胞重要功能和活動不可缺少；提示c-onc是進化中保存下來的細胞重要結構元

39、件。2、發現的30種c-onc是依靠病毒的v-onc探針找到的；并不是所有c-onc都有對應的同源v-onc。3、v-src缺失3/4不能致癌的RSV,注入雞體內，發現缺失的v-src與c-src發生重組，v-src回復并致癌。82原癌基因（proto-oncogene）:原本是細胞的正常基因，它們編碼的蛋白質在正常細胞中通常參與細胞的生長與增殖的調節。但突變后成為促癌的癌基因(cancer-promoting oncogene)，導致細胞癌變。原癌基因突變成癌基因，稱為原癌基因的激活。83原癌基因激活成癌基因84RNA腫瘤病毒癌基因起源的假說模型Proto-oncogenes Proto-oncogens: Gain-of-function mutationsRas oncogene: Retain bind GTPBcl-2 oncogene: Prevent apoptosis87癌基因轉化腫瘤抑制基因(tumor suppressor gene)，抑癌基因（tumor suppressor gene）兩個拷貝，只有當兩個拷貝都丟失了或兩個拷貝都失活了才會使細胞失去增殖的控制， 如：RB, P53Harris(1968):癌細胞系與同組織正常細胞融合雜交細胞無惡性表型，也不致癌； 隨著染色體丟失則可能恢復致癌（Rb1