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发表于 2012-3-23 18:54 |只看该作者 |倒序浏览 |打印

过去10 年间，全球癌症的发病及死亡增长了约22%，世界卫生组织预测，2020年全球癌症新发病例将达到2000万，1200万人死于癌症[1,2]。癌症的发病率和死亡率多年来踞高不下，一个重要原因是当前癌变机制不甚明了，癌症治疗存在相当大的盲目性。我们通过分析当前研究人员对癌变的不同认识，努力在他们的认识基础上分析癌变的具体机制。

一．肿瘤和癌变的特征
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在分析癌变机制之前，有必要首先分析肿瘤和癌变(carcinogenesis)的特征。一个世纪前病理学家Hansemann[3]用去分化一词来概括肿瘤的本质特征：“某种因素将细胞推向某些完全新的方向——该方向在所有的肿瘤甚至在同一种肿瘤中都不一样，……细胞通过一些未明确的因素不断破坏有丝分裂，同时产生未分化的细胞，这种不分化或逆分化意味着肿瘤细胞是一种新的生物而不是一种单纯的胚胎细胞，它与个体发生的任何时期的正常细胞都不一样，但是……这个新生物并没有完全丢失其起源细胞的特征……它已经变化的行为只是夸大或减弱了其起源细胞的某些方面的特征。” 下面我们列出肿瘤和癌变的具体特征。
肿瘤的特征：1）不受控制的（自主性）异常增生,恶性肿瘤还具有侵袭和转移的能力[3.4]；2）大部分肿瘤可以自发演进(progression)，表现为不可逆性[5]，但是在适当的条件下肿瘤的表型可以被逆转[6-8]；3）肿瘤组织细胞表型存在很大的异质性(heterogeneity)，在形态、代谢、分化程度、增殖能力、抗原性、侵袭和转移能力等方面几乎不存在两个完全一样的肿瘤细胞[3]；4）肿瘤细胞与正常细胞融合产生的细胞失去癌的表型特征，提示肿瘤表型为隐性(recessive)遗传方式[9]；5）肿瘤细胞体外培养可以无限传代（永生化,immortality）[3]；6）肿瘤细胞可以在不同种属的动物体内移植(transplantation) [3]；7）几乎所有癌细胞基因组不稳定(genetic instability)[10]；8）实体瘤的核型几乎都是非整倍体(aneuploidy)核型，慢性粒细胞白血病急变期伴随核型非整倍体化[3,5,10]；9）所有的肿瘤细胞都表现为分化异常甚至间变(anaplasia)[3,7]。5 g* z# i1 X9 P# t* `2 B! d  g: A
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癌变的特征：1）除了少数由急性转化病毒引起的细胞转化过程外，癌变是一个漫长的过程，几个月甚至几十年，一般可以分为几个阶段：过度增生(hyperplasia)、不典型增生(atypical hyperplasia)、良性肿瘤和恶性肿瘤[3-5]；2）大部分肿瘤单克隆起源（moloclonality）[11]；3）肿瘤发病率与年龄成指数关系[12]；4）癌变是一个小概率事件[3,4,12]。
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虽然肿瘤和癌变的特征存在于多个方面，但是肿瘤的本质是细胞异常增殖，这一点主要体现在肿瘤概念的演变过程。20世纪40年代，Ewing提出肿瘤是一种自主性过度生长的新生组织[13]。1967年Willis将肿瘤定义为一种肿块，其生长和周围正常组织不一致，而且在缺乏诱发刺激时仍能存留和生长[14]。我国医学本科生病理学教材[15]将肿瘤定义为：机体在各种致癌因素作用下，局部组织的细胞异常增生形成的新生物。至今，虽然研究人员对肿瘤的定义多少有些差别，但是都强调肿瘤是一种细胞异常自主性增生的疾病。0 Z9 D" p4 e+ {/ G
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二．目前对癌变机理的研究进展和存在问题

至今虽然人类在肿瘤研究领域取得很大的进展，但是关于癌变的具体机理还存在争议和分歧[6,16,17]。主要存在体细胞突变（somatic mutation）假说和表型遗传修饰（epigenesis）改变假说[6,16,17]，其中体细胞突变假说主要包括基因突变(gene mutation)假说[18,19]和非整倍体(aneuploidy)假说[20,21]。假说的重要特征是可以解释已经观察到的现象和预测未知的实验结果。下面我们通过分析上述假说对肿瘤特征的解释和预测，分析这些假说的科学性和局限性，在此基础上我们提出新的癌变机理模式。
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（一）体细胞突变假说! O- u' k+ ]# U: W
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大约一个世纪前，Hansemann和Boveri提出肿瘤是体细胞遗传物质发生“突变”的结果，其中Boveri比较完整地提出癌变的非整倍体假说[3,20]。后来由于Morgan在基因研究方面的贡献和Muller发现X射线可以导致基因突变，促使研究人员将注意力集中在基因方面，因此在随后几十年内非整倍体假说没有得到继续发展[3]。上世纪70－80年代，研究人员在逆转录病毒和癌变关系的研究过程中发现了原癌基因（protooncogene）[23]。Weinberg等[24]的癌基因转化实验和Ames提出“致癌剂就是致突变剂”促进“癌基因”(oncogeng)假说[25]的成熟。随着Knudson提出癌变的“二次打击学说”[26,27]和DNA病毒致癌机理的研究以及细胞融合实验的事实，研究人员很快克隆出Rb抑癌基因[28]。癌基因和抑癌基因(anti-oncogene)的发现以及对这些基因功能的研究，结合细胞转化实验和化学诱癌经验，研究人员从分子水平提出癌变是一个多步骤多阶段的过程[11,29]。90年代初Vogelstein等[11]提出结肠癌癌变模式加强了人们对癌变的多步骤多阶段假说的认识。但是随着新的癌基因和抑癌基因不断被发现，引起了一些研究人员对基因突变假说的质疑[3,30]。虽然信号转导(signal transduction)理论逐渐理顺了癌基因和抑癌基因在细胞中的作用以及它们与细胞周期(cell cycle)的关系[31-34]，但是基因突变假说变得越来越复杂已经远远超越了70－80年代研究人员的推测。90年代末Weinberg等[35]首次宣称应用3个不同的基因可以转化(transform)人类细胞，从一定意义上证明了基因突变假说，但是其转化用了6个月的时间不能排除其它的因素也参与了转化过程。到目前为止人类还没有找到癌变所需的共有的基因突变，研究人员不得不将原来基因突变假说推测的至少3－7个基因的突变改为至少3个信号转导通路的改变[18,19]。从历史的角度看，体细胞突变假说后来基本演变为基因突变假说，一些研究人员甚至认为Boveri提出的促进细胞增殖的染色体其实就是癌基因，抑制细胞增殖的染色体就是抑癌基因[36]，而且近年来教科书中已经不再提及癌变的非整倍体假说。虽然基因突变假说被很多研究人员接受了，但是其本身存在的缺陷和越来越复杂的理论促使Duesberg等[37]在90年代末又重新提出非整倍体假说，虽然该假说目前仍然存在很大争议，但是它至少引起了很多研究人员对非整倍体的重新重视[38]。下面我们分析这两种假说的科学性和局限性。) x3 P# z! l, s
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1．基因突变假说

基因突变主要指DNA碱基序列的改变[19]。经典的基因突变假说认为3－7个基因突变可以导致细胞癌变，癌基因突变激活和抑癌基因突变失活导致细胞周期失控是癌细胞异常增殖的主要原因[4,39,59]。
基因突变假说的主要依据有：1）病毒癌基因的DNA序列与细胞原癌基因的DNA序列同源性很高[23]；2）化学诱癌产生的突变基因能够转化细胞[40-43]；3）体外细胞转化实验表明3－4个基因可以导致人类细胞发生转化[44,45]；4）转基因和基因剔除动物癌变率升高[46]；5）阻断突变的癌基因信号可以使肿瘤细胞失去肿瘤表型或凋亡[47,48]；6）重要基因突变的遗传病人癌症发病率升高[49]；7）很多致癌剂是致突变剂[51,52]；8）大部分肿瘤单克隆起源[11]。

基因突变假说对肿瘤和癌变现象的解释：1）肿瘤是在自然选择的作用下基因突变不断积累的结果[4,52]；2）肿瘤异常增殖的原因是癌基因激活和抑癌基因失活导致细胞周期失控的结果[39,53,54]；3）癌变所需的多个基因突变积累需要很长的时间[12,52]；4）基因突变积累导致肿瘤的不断演进[12,55]；5）肿瘤细胞的异质性表型、非整倍体核型以及基因组不稳定是基因突变的结果[12,38,56,57]；6）癌变需要多个基因独立突变来完成，该过程决定了癌变是一个小概率事件[4,12]。  Z3 l9 r7 v% d- m
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基因突变假说存在如下问题：1）从相关性来看，至今没有发现共有的基因突变[4,20,58]；2）基因突变率和癌变率之间存在难以调和的关系：如果癌变需要3－7个基因突变，从统计概率计算的癌变率远远小于实际癌变发生率[3,60]，为了解释该问题，vogelstein等提出癌变细胞首先发生基因组不稳定，之后基因组不稳定加速基因突变[61]；Loeb等提出肿瘤是突变表型(mutator phenotype)的概念，即肿瘤细胞中存在成千上万个基因突变[62,63]，目前这个问题仍在争论之中[64]；3）基因突变假说认为癌基因的激活和抑癌基因的失活导致细胞周期异常从而导致肿瘤发生，根据该假说得出推论：癌变的根本原因是肿瘤的细胞周期快于正常细胞的细胞周期。但是许多研究表明肿瘤细胞的细胞周期并不缩短甚至延长，肿瘤并不是简单的细胞周期问题[65]；4）基因突变假说认为癌变过程是选择扩增获得突变优势细胞的过程[52]，事实上，肿瘤细胞存在很大的异质性，对肿瘤增殖起决定作用的只是少数肿瘤干细胞，并不是所有的肿瘤细胞，也就是说基因突变并不能够赋予所有的肿瘤细胞相同的遗传表型和生存优势[66-68]；5）基因突变的性质是不可逆和积累性的，因此肿瘤细胞表型应当是不可逆和积累性的，但是事实上肿瘤的表型总是处于变化之中，各种表型独立演进甚至还可以逆转[3,6-8,69]；6）癌细胞与正常细胞融合的细胞总失去癌的表型，该结果支持存在抑癌基因而不是癌基因[9,70]；7）基因变异假说认为致癌因素都是通过基因突变导致肿瘤发生，但是现在发现许多化学物质不能引起基因突变却可以诱导肿瘤发生[3,71]；9）突变的发生是瞬间的事件，在致突变因素作用下细胞基因的变异会很快发生，因此细胞转化应当与致癌基因的突变几乎同时发生，但是在致癌因素作用后几年或数十年细胞才发生恶性转化[3]；10）根据基因变异假说肿瘤细胞应当是二倍体(diploidy)细胞，与正常细胞的差别只是因为其基因组的某些的癌基因被激活和/或另一些抑癌基因被灭活，但事实上除了很少的由逆转录病毒转化的二倍体细胞存在外，几乎所有的实体肿瘤细胞都是非整倍体核型[3,20,72,73]；12）随着年龄的增加肿瘤发生的几率增加1000倍[20,12]，基因变异假说认为这是由于细胞中变异基因积累的结果，由于基因变异可以遗传，因此年轻人应当具有较高肿瘤发生率才对，可事实上老年人的肿瘤率发生最高；13）体外转化实验所用的启动子都是病毒的启动子，它们调控的基因表达量远远高于细胞本身基因的表达水平，因此基因表达量的差别在转化实验中的作用无法排除[45,74]；14)不存在真正的癌基因，一些经典的癌基因在不同的组织细胞中的作用不同，在一些细胞中促进细胞增殖[29]，但是在另一些细胞中促进细胞分化衰老凋亡[75,76]。/ x( S) I6 H) M- ?& R0 u# v

2．非整倍体假说
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非整倍体指染色体结构和数目的改变[3,20]。Boveri首先提出非整倍体假说，主要观点是非整倍体通过保留具有促进细胞增殖的染色体而丢失具有抑制细胞增殖的染色体，产生特定染色体组合（核型）是癌变的重要原因[20,77]。最近Duesberg等重新提出的非整倍体假说，虽然他们也认为特定的核型是癌变的根本原因，但是他们更强调非整倍体导致细胞内正常基因的表达比例失衡而不是个别少数突变基因是癌变的主要原因[3,20-22]。

该假说的主要依据是：1）从相关性分析，几乎所有的实体肿瘤都是非整倍体核型[3,20,37]；2）虽然部分肿瘤细胞非整倍体的产生与基因突变有关[38]，但是很多肿瘤的非整倍体产生与有丝分裂错误、端粒缩短引起基因组不稳定、致癌因素直接作用等等有关，因此非整倍体的产生与基因突变不存在确定的因果关系，非整倍体可以是癌变的原始启动因素[3,78-80]；3）癌变早期和癌前病变细胞就已经出现非整倍体核型[81-84]；4）非整倍体的程度基本与肿瘤的恶性程度成正比[3,20,55,81]；5）生化代谢与细胞表型关系的数学模型验证了非整倍假说对肿瘤发生与年龄关系的解释，较好的说明了癌的发生和演进的动力学过程, [85-88]；6）首次被转化的人类细胞是非整倍体核型[21,89]。
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非整倍体假说对肿瘤和癌变过程的解释：1）非整倍体导致肿瘤细胞转变为一种新的“寄生”物种，因此其增殖不再受机体调节而转变为自由的增殖状态[3,20]；2）非整倍体自身催化进化成为肿瘤细胞核型需要漫长的细胞增殖过程[20]；3）肿瘤的各种异常表型和核型是非整倍体产生的结果[20]；4）非整倍体催化染色体重新组合的是肿瘤细胞多药耐药的根源[90,91]；5）非整倍体可以直接导致细胞基因组不稳定[10,20]；6）肿瘤细胞与正常细胞融合的细胞失去了肿瘤特定的染色体组合特征，因此失去癌细胞表型，但是当部分染色体丢失而重新获得肿瘤特定的染色体组合时细胞就又获得肿瘤的表型特征[3,20]；7）非整倍体假说较合理的解释了肿瘤演进的Foulds的规则[3]；8)非整倍体自身催化进化产生肿瘤细胞特定的核型即特定的染色体组合是一个小概率事件[3,20]。

非整倍体假说存在如下问题：1）真正二倍体的肿瘤是否存在还存在争议[92-98]；2）关于非整倍体的产生问题存在争论[20,38,78-80]；3)根据非整倍体假说可能得出肿瘤异质性是随机性的，但事实表明肿瘤的异质性具有分化级别关系[66-68]；4）非整倍体假说难以解释在特定的条件下不改变细胞的核型，但可逆转肿瘤细胞表型的现象[6-8,30]。2 F3 H* ~* D4 N& a5 M5 b) a
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（二）表型遗传修饰改变假说
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早期研究人员发现化学诱癌实验产生的肿瘤，如果停止使用致癌剂和/或促癌剂，相当一部分肿瘤会通过重新分化而自发消失[30]。上世纪40年代年Furth发现卵巢细胞移植到脾脏可以自发癌变[6]。60年代King和McKinnell等将青蛙的肾癌细胞核转移到正常卵细胞中，发现这种卵细胞依然可以发育成正常的蝌蚪[99,100]。70年代，Mintz和Illmensee等[101,102]发现将畸胎瘤细胞移植至正常同系动物的胚泡内，结果产生不长肿瘤的嵌合型小鼠。Howell等[103]发现肿瘤细胞和正常细胞的融合产生的细胞失去恶性表型。基于上述发现，1979年Holliday首先提出表型遗传修饰改变假说[104]。80年代Sachs等[105]将白血病细胞向同系动物的早期胚胎移植，发现白血病细胞可参与正常动物血液系统的发育，动物发育成熟后各系血液细胞均可发现白血病细胞的基因标记。90年代McCullough等[106]将肝癌细胞向同系成年动物肝组织移植，发现癌细胞可参与正常肝脏细胞的更新；最近研究发现致癌剂作用于间质细胞却可以产生实质细胞肿瘤[107]。这些实验结果均支持表型遗传修饰改变假说。1994年Prehn提出肿瘤产生突变而不是突变产生肿瘤比较合理的解释了突变在癌变过程中的作用[30]，在理论方面有力的支持了表型遗传修饰改变假说。现在该假说基本演绎为癌变的组织结构场理论（tissue organization field theory ,TOFT）[108,109]。 " Y3 k% @7 q: d  M( U4 h
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表型遗传修饰改变假说认为肿瘤是细胞表型遗传修饰改变即基因表达异常的结果[6,30,104]。主要依据有：1）化学诱癌早期，一部分肿瘤可以自发逆转[30,110,111]；2）适当的环境可以逆转或降低肿瘤的恶性表型[6-8,108]；3）表型遗传修饰的改变可以导致基因突变和非整倍体[112,113]；4）一部分致癌剂并不是致突变剂[3,114]；5）相同的遗传信息（DNA序列）可以产生不同的细胞表型，肿瘤细胞的表型并没有完全超越正常细胞的表型，只是增加或减少了某方面的特征[30]；6）致癌剂作用于间质细胞，可以产生实质细胞的肿瘤[107]；7）胚胎细胞错位移植可以导致肿瘤发生[115]；8）蛋白酶抑制剂可以抑制细胞转化[116]。: C5 q- N) u/ o# l& ^; Q, A- R

表型遗传修饰改变假说对癌变的解释：1）癌变过程是致癌因素导致细胞表型遗传改变的结果[6,30]；2）肿瘤细胞表型异质性是基因表达异常的结果[6,30,104,108]；3）基因突变是癌变过程中细胞对那些经常不转录的基因位点修复很慢或者不进行必要的修复的结果，肿瘤细胞不断演进的原因是在选择压力的作用下，肿瘤细胞中不转录的基因位点不断发生基因突变，导致细胞的表型遗传无法逆转的结果，从这个意义上说，突变的作用是通过维持一定的表型遗传方式维持肿瘤的表型状态，这与基因突变假说认为肿瘤的表型直接依赖基因突变的观点完全相反[30]；4）适当条件下肿瘤表型可以被逆转是通过改变肿瘤细胞的表型遗传方式导致肿瘤细胞分化的结果[30,108]；5）非整倍体和基因突变只是癌变的结果或表象[6,108,111]；6）细胞融合实验结果可以解释为表型遗传修饰改变的结果[6,117]。9 u: x9 I& c* C1 L, r' Z
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表型遗传修饰改变假说存在下列问题：1）难以合理解释大部分肿瘤单克隆起源[11]；2）肿瘤表型逆转只是少数现象，需要苛刻的条件[30,108]；3）难以解释某些肿瘤细胞存在非随机的染色体改变以及大部分肿瘤是非整倍体核型的现象[3,20]；4）难以解释肿瘤细胞表型异质性和基因型不稳定现象[3,20]；5）癌变的动力学过程不清楚。 8 f6 V% P. x% {+ o4 E

（三）肿瘤干细胞假说

约150年前，病理学家Virchow等认为肿瘤来源于发育过程中某些潜伏的胚胎残余组织，这种推测基于对胚胎发育和某些肿瘤例如畸胎瘤的相似性的观察[118]。1961年Bruce等[119]发现只有1－4%的鼠类淋巴瘤细胞可以在被移植动物的脾脏形成克隆(clones)，1973年McCulloch等[120]发现只有不到1%的髓白血病细胞可以在体外形成克隆。1977年Hamburger等[121]发现1000－5000个实体瘤细胞中仅有1个细胞可以在软琼脂中形成细胞克隆。随着这些研究结果不断出现，上世纪70年代Potter和Pierce等[122,123]分别提出肿瘤是干细胞分化成熟被抑制的结果。直到1997年Dick实验室分离到急性髓白血病干细胞[124]，肿瘤干细胞(tumor stem cells)的研究才逐渐升温，至今研究人员已经分别从慢性髓白血病、胶质瘤和乳腺癌中分离到具有特定免疫表型的肿瘤干细胞[66-68,125,126]。

肿瘤干细胞假说的主要观点是：肿瘤干细胞是肿瘤异常增殖、侵袭、转移、耐药以及复发等的根源[66,127]。

肿瘤干细胞假说主要依据：1）细胞琼脂克隆实验表明血液肿瘤和实体瘤的肿瘤细胞中只有少数细胞具有形成克隆的能力[66]；2）利用特异分子标志和细胞分离技术，发现不论血液肿瘤还是实体瘤中只有少数细胞具有在动物体内重新产生肿瘤的能力，这些细胞再次产生的肿瘤与原发肿瘤具有类似的特征[67,68,128,129]；3）目前发现所有的肿瘤都是一个具有分化差别的异质性细胞群体，这种分化差别类似干细胞产生不同分化程度的细胞群体[127]。
肿瘤干细胞假说对癌变的解释：1）肿瘤干细胞是肿瘤不断异常增殖的根源[127]；2）肿瘤细胞异质性是肿瘤干细胞产生不同分化程度的肿瘤细胞的结果[66,127]；3）肿瘤复发和耐药是抗癌药不能杀死肿瘤干细胞的结果[124-129]；4）肿瘤的侵袭和转移是肿瘤干细胞扩散的结果[127]。% N. Z: \+ K: [5 A* O+ O/ U
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肿瘤干细胞研究是当前肿瘤研究的一个热点，由于肿瘤干细胞的研究主要得益于对正常干细胞的研究成果，而且这些研究刚刚起步，因此存在很多问题。当然这些问题可能并不是该假说本身存在的问题，而是从该假说出发研究肿瘤所面临的一些实际问题。这些问题主要有：1）肿瘤干细胞的细胞起源问题[127]；2）肿瘤干细胞产生机制问题[66]？3）肿瘤干细胞与肿瘤的演进是什么关系[66]？4）针对肿瘤干细胞治疗肿瘤问题[126,130]；5）正常干细胞与肿瘤干细胞有什么相似性和差异性等等[66]？
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（四）对称分裂抑制机制紊乱假说

许多研究人员围绕细胞周期进行研究，认为这些问题的解决可以帮助解决肿瘤问题。病理学家Prehn在1992年指出[65]:虽然提高细胞增殖率可以加速肿瘤的生长和演进，但是细胞分裂本身似乎不能区分肿瘤和正常组织，器官和肿瘤的生长依赖细胞产生和死亡的比例（生长率,growth ratio）的失衡，显然生长率与细胞的增殖速率没有绝对的关系，肿瘤并不是简单的细胞周期紊乱的疾病，肿瘤的根源在于干细胞的分裂方式问题。他提醒肿瘤研究人员应当认识到存在两种不同的细胞分裂方式，一种是普通的有丝分裂，这种分裂方式只是简单的细胞增殖，只涉及细胞分化但不涉及细胞的自我更新，例如普通细胞虽然具有一定的分裂能力，但是它们在分裂有限次数后全部分化而失去增殖能力；另一种有丝分裂是干细胞的增殖方式，这种增殖方式与细胞的自我更新和分化关系密切。在体内，干细胞大多数以不对称分裂方式增殖，结果是一半子代细胞成为干细胞而另一半子代细胞趋向分化死亡，显然只有干细胞的增殖方式改变才可能改变生长率，而细胞周期的改变与生长率没有必然的关系。他进一步提出这两种增殖方式所受的调节方式不同，细胞周期的控制只由有丝分裂抑制因子(mitosis preventers)控制，这些抑制因子主要由分化细胞和干细胞产生。干细胞的增殖方式主要由对称分裂抑制因子（symmetric preventers）控制，这种因子主要由干细胞产生。因此正常组织的生长率能够保持在1/1是由干细胞产生的对称分裂抑制因子和由分化细胞和干细胞产生的有丝分裂抑制因子共同作用的结果。癌的产生主要是癌细胞不能产生或产生无效的对称分裂抑制因子的结果。# L# @) Z! M& {3 i- N( U& O" V# m
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1．对称分裂抑制机制紊乱假说的主要依据：
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(1) 细胞的增殖和分化

从进化的观点来看，生命的本质就是自我复制，细胞作为生命的最重要的形式，其天然的特性就是在适当的环境条件下可以不断复制增殖，例如单细胞生物，只要环境适合，就能够以指数方式无限增殖[65]。对于多细胞生物来说，其生命的维持离不开其体内干细胞的不断增殖，如果干细胞失去增殖能力，生命的个体也就进入衰老死亡阶段[131,132]。但是对于多细胞生物来说，其必须有能力控制其体内细胞的增殖，因为细胞的指数增殖会导致多细胞生物体积持续增长，这不可避免对宿主带来自然选择压力，因此保持适当的细胞数目和维持一定的体积对于多细胞生物来说至关重要[133,134]。4 w: L+ ?/ p" C6 h, @" Y; @$ s& N
多细胞生物控制体积的机制与细胞对整体体积的感应有关，许多研究表明细胞的有丝分裂与生物的体积具有密切关系，当体积小于应有的体积时，细胞分裂速度加快，当达到适当的体积后细胞的增殖就被抑制。这表明存在一种细胞有丝分裂抑制因子，其浓度和/或效能与生物的整体细胞数量成正比[65,133]。! z6 Q* i, B$ f
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随着细胞的分化和不同组织器官的产生，需要更加特异的机制来调节这些组织器官的体积，特别是那些经常处于细胞更新状态的组织器官。细胞分化即细胞分裂过程中一半细胞趋向分化死亡可能是一种有效的机制。对于处于不断更新的组织（例如皮肤）来说，细胞是否趋向分化主要由干细胞来决定，干细胞可能通过产生对称分裂抑制因子来保证干细胞的子代细胞中一半趋向分化另一半保留了干细胞的特征。细胞分化不可能由已经分化的细胞决定，因为如果细胞分化主要由已经分化的细胞来决定，那么去除分化细胞后，干细胞就只能产生干细胞而不能产生趋向分化的细胞了[65,132]。& e2 _% l% N0 v
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(2) 有丝分裂和组织生长

高等动物的大多数组织细胞都处于不断更新状态[132]，干细胞不断产生新的细胞用来补充那些分化衰老脱落的细胞，当干细胞分裂时其一半子代细胞保留了干细胞的特征而另一半子代细胞趋向分化衰老死亡（生长率等于1/1），这时组织保持在一定水平不发生变化；只有当子代细胞中多于一半的细胞成为干细胞时（生长率大于1/1），组织才可能生长[65]。因此肿瘤最可能是生长率的失衡而不是细胞有丝分裂速度的问题。当然一旦生长率大于1/1，增加细胞有丝分裂率会加速组织或肿瘤的生长。不过需要注意的是生长率和有丝分裂率之间似乎没有确定的关系，因为一些肿瘤细胞的有丝分裂率接近于甚至小于正常细胞有丝分裂率，但是一些体积和细胞数目并不增加的组织（例如皮肤和小肠上皮）的细胞有丝分裂率却很快。从上面分析来看，只增加细胞有丝分裂率但不增加生长率的所谓生长因子不能称为严格意义上的生长因子，只有那些能够增加生长率的生长因子才是真正的生长因子[65]。& y$ d2 J8 t. L

综上所述，生长率主要由对称分裂抑制因子控制，有丝分裂抑制因子与生长率没有绝对的关系，当然在生长率改变后，改变有丝分裂率会增加或减弱组织的生长速度。因此肿瘤主要是对称分裂抑制因子缺陷的结果[65]。
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三．癌变机理新假说――干细胞不对称分裂机制紊乱假说
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我们通过分析上述不同的癌变机理假说，发现每种假说都只是强调某种因素的重要性，而忽略或者否定其它因素的作用。例如基因突变假说强调基因序列改变是癌变的根源，而非整倍体和表型遗传修饰改变只是结果或者表象[4]；非整倍体假说认为非整倍体是癌变的唯一根源，基因突变和表型遗传修饰改变只是结果或者表象[20]；同样表型遗传修饰改变假说认为表型遗传修饰改变是癌变的真正根源，基因突变和非整倍体只是结果或者表象[6]。我们认为肿瘤是一种遗传因素改变的疾病，基因、染色体和表型遗传修饰都属于遗传信息,它们在所有的肿瘤细胞中都发生不同程度改变，因此都可能参与癌变过程。不同研究人员强调某一方面重要性的原因可能与研究人员的视角和他们选用的实验模型不同。基因突变假说支持者总在寻找特定的基因突变与癌变的关系，以及其是否有能力转化细胞；非整倍体假说的支持者则总在证明非整倍体与癌变存在很大的相关性，以及其确实可以引起细胞各种表型的改变；表型遗传修饰假说的支持者一再强调癌变可以逆转，因此任何不可逆的因素都不可能是癌变的真正原因。上述不同假说的对立在一定程度上反映了它们自身的局限性。肿瘤的本质是细胞自主性异常增殖，Prehn深刻地揭示了这种自主性异常增殖的根源在于干细胞分裂方式（不对称分裂机制）改变[65]，因此如果认为那种因素是癌变的根源，那么这种因素一定是通过改变干细胞不对称分裂机制达到癌变的目的。下面我们分别分析基因突变、非整倍体和表型遗传修饰改变对干细胞不对称分裂机制的影响，从而判断癌变的真实根源。

（一）干细胞不对称分裂的概念

成体干细胞的有丝分裂通常是不对称分裂（asymmetric division），分裂结果产生两个具有不同生物学功能的子代细胞，其中一个子代细胞具有干细胞功能，另一个细胞发生分化[131,132]。研究表明干细胞不对称分裂与有丝分裂过程中细胞内的蛋白质和mRNA不均等分配以及细胞的微环境差别有关[135-138]，最近研究表明成体干细胞的不对称分裂与染色体非随机分配具有密切关系[138-142]，因此可以推测成体干细胞的不对称分裂可能是一个涉及DNA、mRNA和蛋白质的非随机分配的协同过程。与干细胞不对称分裂对应，总是获得含有DNA永生化链的染色体的子代细胞成为干细胞，而获得含有新合成链染色体的子代细胞发生分化死亡脱落(图1A)[138-143]。在多细胞生物发育成熟前和组织损伤时，干细胞的增殖方式趋向对称分裂方式，这时含有DNA永生化链的染色体和含有新合成链的染色体发生对称分配（随机分配），导致干细胞发生扩增(图1[139,143,144]。我们推测：含有DNA永生化链的染色体可! {: ~8 @' V, _9 _0 q$ O% p9 E" o

图1 成体干细胞与肿瘤干细胞的增殖方式

能与细胞自我更新能力有关，而含有新合成链的染色体可能与细胞分化有关，这种差别可能是DNA半保留复制过程中，细胞对DNA永生化链和新合成链不同表型遗传修饰的结果，不同的表型遗传和不对称分配结合可能正是干细胞不对称分裂的一个重要遗传基础（图1A）。

(二) 基因突变与干细胞不对称紊乱

许多研究表明基因突变在癌变过程中具有重要作用，研究人员一般认为基因突变通过改变细胞周期、细胞凋亡和分化等机制参与癌变过程[4,5,11,19]。从干细胞不对称分裂机制紊乱方面分析，基因突变可能引起干细胞不对称分裂紊乱从而导致细胞癌变。事实上一些经典的癌基因和抑癌基因如p53、APC、PTEN和notch等确实参与干细胞不对称分裂过程[140,142,144-149]。但是基因突变可能不是癌变的唯一原因。一个重要原因就是干细胞的不对称分裂过程可能受很多方面机制调节。在发育成熟前，多细胞生物体内胚胎干细胞数目是以指数方式扩增，维持生物个体体积的持续增长；成熟后，其体内的成体干细胞以不对称分裂方式增殖，子代细胞中只有一个成为干细胞，另一个最终分化衰老死亡，干细胞数目不再扩增同时生物个体体积基本不再发生变化。同样现象在体外也类似，研究人员很容易在体外培养扩增胚胎干细胞但是很难扩增成体干细胞[146]。胚胎干细胞相对于成体干细胞来说的分化程度更低，因此分化差别可能是胚胎干细胞和成体干细胞扩增能力差别的重要原因。如果维持这种分化差别的基因失活，成体干细胞则可能恢复胚胎干细胞的功能而发生扩增，有可能转变为肿瘤干细胞。大部分癌基因的激活可以抑制细胞的分化，而抑癌基因可以促进细胞分化[117,150,151]，因此特定癌基因的激活和抑癌基因的失活可能导致干细胞分化功能异常而转变为肿瘤干细胞。但是事实上，这种只有基因突变的肿瘤好像还不存在或者发生机会非常小[3,20]；另外，如果肿瘤只是基因突变的结果，那么肿瘤的所有或大部分细胞都应当是肿瘤干细胞，可事实上几乎不存在这种肿瘤。因此我们认为基因突变可能参与癌变，但是不可能是癌变的唯一原因。9 F" j% R* F# k! s$ z
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（三）非整倍体与干细胞不对称分裂紊乱
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细胞遗传学资料表明几乎所有的实体瘤细胞都是非整倍体核型[3,10,20,72,73]，虽然Boveri认为非整倍体通过保留具有促进细胞增殖的染色体而丢失具有抑制细胞增殖的染色体，从而产生特定染色体组合（核型）是癌变的重要原因[77]。但是至今关于非整倍体在癌变过程中的作用仍然存在很大的争议。表型遗传改变假说和经典的基因突变假说认为非整倍体只是癌变的一种结果或者表象[4,6]，但是Duesberg等认为非整倍体是癌变的唯一原因，另一些研究人员认为非整倍体通过加速基因突变从而促进肿瘤发生[152]，还有人认为非整倍体引起的基因表达量的改变和基因突变协同导致癌变发生[153]。我们认为非整倍体可能导致干细胞不对称分裂机制紊乱从而促进癌变。0 t8 s: Q8 B* O0 v1 P& F
约30年前Cairns提出DNA永生化链（模板链）的假说[143]：成体干细胞（adult stem cells）在分裂过程中，含有相对比较古老的DNA链（永生化链）的染色体总是被分配给将要成为干细胞的子代细胞，含有相对新合成DNA链的染色体被分配给趋向分化并最终衰老死亡的另一个子代细胞（图1A）。最近研究人员从不同的角度证实了Cairns的假说[139,142]。根据这些事实，Cairns提出了干细胞与癌变关系的新假说[154]，该假说从基因突变假说出发，推测癌变的发生频率与干细胞的死亡频率成正相关，因为只有部分干细胞的死亡才可能导致其它干细胞的扩增，以补充损失的干细胞。干细胞扩增时需要建立新的DNA永生化DNA链，该过程可能导致错误碱基嵌入DNA永生化链，造成基因突变在干细胞内的积累，这种情况持续发生最终可能导致癌变发生。该论点与认为增加细胞分裂频率可增加癌变几率的论点一致[155-159]。但是，增加干细胞的扩增频率只是通过增加基因突变的频率来导致肿瘤发生？我们认为DNA永生化链的作用至少有3方面的生物学意义：1）防止基因突变在干细胞内积累[143,154]；2）决定细胞是否分化，DNA永生化链可能具有促进细胞永生化（自我更新）的能力，而含有新合成链的染色体可能具有赋予细胞分化的能力，这种功能上的差别可能是细胞对DNA永生化链和新合成链不同表型遗传修饰的结果[160,161]；3）保护干细胞染色体端粒，
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图2 DNA复制与端粒长度变化& H. R. P' m# L5 K+ ]! G
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从而避免因为染色体端粒缩短引起的基因组不稳定和非整倍体发生（图2，3）[139]。由于永生化DNA链将基因突变、非整倍体和表型遗传修饰差别联系起来，因此它本身的紊乱可能正是癌变的根源。
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干细胞的扩增过程表明干细胞的两个子代细胞分别获得一半含有DNA永生化链的染色体，每个子代细胞的另一半DNA永生化链只能由新合成链重新建立（图1.B）。由于细胞分裂过程中DNA半保留复制方式和细胞内的端粒酶活性不足以有效地合成损失的端粒部分，导致含有新合成链的染色体的端粒部分丢失（图2，3）[162-164]。因此，如果干细胞不断扩增，最终会因为含有新合成链的染色体的端粒耗尽，导致细胞内不同染色体之间的末端融合，形成双着丝粒的染色体，在下次细胞分裂时可能导致染色体不分离或双着丝粒的染色体异常断裂，这样循环往复最终导致干细胞成为非整倍体核型[78-80]。由于DNA永生化链可赋予干细胞自我更新的能力，因此我们认为其可能恰好被非整倍体利用从而成为肿瘤发生的有效工具。因为非整倍体本身可促进干细胞中DNA永生化链的扩增和有丝分裂紊乱从而可能导致干细胞不断扩增，即肿瘤干细胞产生（如图1.C,D）。相反，如果干细胞总不发生扩增而一直以不对称方式分裂，总获得含有永生化DNA链的染色体，那么其端粒永远不会缩短和发生结构变化，因此不会因为端粒问题而发生非整倍体化。
总之，即使按照Cairns的假说，干细胞持续扩增的结果也必然导致非整倍体的产生，肿瘤的发生仍然难以摆脱非整倍体，不但如此，还提示非整倍体可能导致DNA永生化链的扩增和重建，从而可能促进干细胞向肿瘤干细胞方向转变（如图1.C,D）。8 G. q! R$ b9 a7 v+ |  I2 `
图3 干细胞不对称分裂与端粒变化关系

（四）表型遗传改变与干细胞不对称分裂紊乱7 o* {( y9 F6 T/ R
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表型遗传修饰改变与癌变关系的研究是当前肿瘤研究领域的一个热点[165]。许多研究报道肿瘤组织整体甲基化水平降低，部分癌基因甲基化降低，抑癌基因的甲基化水平升高，一些等位基因发生杂合性甲基化，类似抑癌基因的杂合性丢失[166,167]。虽然这些发现与基因突变假说基本一致，但是表型遗传修饰改变是不是启动因素还没有确定的证据[166,167]。我们推测发生这些变化可能与DNA永生化链趋向随机分配有关。DNA永生化链非随机分配导致子代细胞具有不同的生物功能，提示它们的子代细胞获得的DNA的表型遗传修饰不同，一般情况下，癌基因抑制细胞分化而抑癌基因促进细胞分化，因此在正常的发育过程中，细胞可能通过表型遗传改变方式抑制癌基因而激活抑癌基因完成细胞的分化过程，但是一旦这种表型遗传修饰不同的染色体分配发生紊乱，就可能导致细胞的分化异常以及癌基因和抑癌基因甲基化修饰紊乱，因此我们认为肿瘤细胞中的表型遗传修饰改变可能是DNA永生化链分配紊乱的结果。当然，由于癌变是一个漫长的过程，因此并不能排除作为基因突变或者非整倍体结果的表型遗传改变在后来癌变过程中具促进癌变的作用。2 B" \; A& {* c% w8 W( J

（五）细胞永生化与干细胞不对称分裂机制紊乱

肿瘤的一个重要问题是细胞永生化，这里的“永生化”一般指细胞在体外可以无限传代能力。需要说明的是这种“永生化”其实只是细胞能否传代问题，与“永生化”本来的意义没有必然关系。从广义的角度来讲，在生物领域“永生化”只能指物种的延续，对于单细胞生物来讲其本身就是永生化细胞，对于多细胞生物来说，永生化主要指生殖细胞的延续。从严格意义上来说，只有这些细胞是天然永生化的细胞。干细胞虽然不是严格意义上的永生化细胞，但是其具有自我更新（永生化）功能，因此可以被看作是一种永生化细胞。
上世纪20年代，Carrel等人认为只要环境条件适合多细胞生物的组织细胞就可以像单细胞生物一样在体外无限传代培养[164]。60年代Hayflick通过实验证明了Carrel的错误，宣布原代培养的细胞只能传代有限次数,后来大多数研究人员接受了Hayflick限制这个概念[164]。人类组织细胞在原代培养过程中，一般经历2个阶段，分别称为M1和M2期，原代细胞经过一段旺盛的增殖阶段后进入M1期，这时细胞保持代谢活性但是不再增殖，如果p53等基因发生突变，这些细胞又可以继续增殖一段时间，进入M2期，这时大部分细胞因为端粒缩短到极限而凋亡，只有少数细胞（1/107）能够存活下来，成为永生化细胞[168]。由于组织细胞中只有干细胞具有无限传代能力，因此细胞能否无限传代首先与这些细胞中的干细胞的能否无限扩增有关[169]。很多研究表明成体干细胞在体外很难发生扩增，根源是干细胞的不对称分裂问题[146]。由于不对称分裂导致干细胞只能增殖但不能扩增，结果是干细胞数目不变但增殖能力有限的细胞不断增加，这样随着细胞不断传代培养不断稀释干细胞的数目，最终必然导致干细胞丢失而不能无限传代[169]。因此细胞培养所说的永生化首先涉及干细胞能否扩增的问题。我们认为M1期可能是干细胞数量减少而分化细胞逐渐增多的必然结果，这种推测与克隆丢失学说一致[170,171]。事实上p53等抑癌基因确实参与干细胞的不对称分裂过程，它们失活可能导致干细胞具有更多的机会扩增，从而跨过M1期[146,148,169]。另外，永生化的细胞大部分表现为非整倍体核型，因此我们认为非整倍体在永生化过程中的作用可能是通过促进干细胞不对称分裂机制紊乱而促进干细胞扩增，从而促进细胞“永生化”（在体外无限传代）。

（六）干细胞不对称分裂紊乱假说对癌变的解释和预测
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1）肿瘤干细胞来源于正常干细胞，其永生化功能是继承了干细胞自我更新功能的结果，其持续不断的扩增是肿瘤持续不断生长的根本原因；2）癌基因的激活和抑癌基因的失活可能具有促进肿瘤干细胞不断扩增的作用，但不可能是肿瘤干细胞不断扩增唯一原因；3）非整倍体可能引起肿瘤干细胞内DNA永生化链不断重建、扩增和分配紊乱，从而促进肿瘤干细胞扩增，因此可以容易理解几乎所有的肿瘤细胞都是非整倍体核型和慢性粒细胞白血病急变期总伴随核型非整倍体化的现象；4）肿瘤演进可能是基因突变积累和非整倍体程度增加导致永生化DNA链重建、扩增和分配紊乱机会增加的过程，该过程也是肿瘤干细胞扩增机会不断增加的过程；5）非整倍体细胞是癌前细胞，同样癌基因和/或抑癌基因突变的细胞也是癌前细胞，因此染色体异常和重要基因突变的遗传病患者肿瘤的发生率增加；6）化学诱癌过程中部分肿瘤自发消退是因为遗传改变不足于引起干细胞的不对称分裂方式完全转变为自发的对称分裂方式，或者在致癌剂的作用下，干细胞的不对称分裂方式被转变为对称分裂方式，但是致癌因素去除后，干细胞又恢复原来的不对称分裂方式，从而使这些病变通过分化渐渐逆转；7）胚胎环境、正常间质细胞等因素可以逆转肿瘤表型是因为这些因素可能激活其它调控干细胞不对称分裂的机制，导致这些肿瘤细胞由对称分裂方式恢复为不对称分裂方式的结果；8）细胞错位移植产生肿瘤和致癌剂作用于间质细胞产生实质细胞肿瘤是实质干细胞不对称分裂紊乱的结果。- ~( g* l% t: Z# n2 o
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四．结语
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我们通过分析当前关于癌变机理的主要研究进展，分析了研究人员对癌变过程的不同认识，提出了癌变机理新假说：基因突变和非整倍体协同作用，引起干细胞的永生化DNA链不断重建、扩增和分配紊乱，导致干细胞数目开始不断扩增，促使干细胞向肿瘤干细胞方向转化，最终导致癌变发生。

作者：张丰李青，第四军医大学病理学教研室/西京医院病理科7 N; ?6 B9 R$ `$ q4 |

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（选自樊代明主编的《癌症研究的前沿》第4卷，1－38页。2006年第1次印刷# b2 m) I5 D: D
# `9 m! J( ]$ }) g
选自张丰的博士毕业论文《癌变机理新假说及其初步验证》文献回顾部分。 . C, |7 o$ A+ n  I% e5 t# k

癌变机理分析(肿瘤学）.doc (198.5k) ' J; u1 |1 C+ e4 C

癌变机理分析(肿瘤学）.doc (198.5k), t/ L7 M9 O( j; b  i
人类进化的过程就是基因不断重组和突变的过程;亲代将遗传信息传递给子代的同时基因重组,伴随着少量的信息传递错误(突变),但在我们眼里重组与突变不太好区别.实际上重组的后果有时候比"突变"更象"突变".2 }6 G( q+ E* m/ u; J
肿瘤细胞所谓基因"突变"有无可能就是的基因重组?
肿瘤细胞的增殖周期与凋亡率并不高于正常细胞,那么有无足够的时间产生足够的子代来积累足够的基因突变致癌 ?1 k) _6 M2 q2 g* {* n
如果说癌的产生是基因重组的结果,启动在哪?0 ]# p% N+ G; o9 g6 X& _; T: ~
唯一欣慰的是这种想法与肿瘤单克隆起源不矛盾．. I" d3 _5 \7 A; D! e* _& y, b
癌症本质/ O* X; p( G0 m
Acta Oncol. 1995;34(1):3-21.
The nature of cancer: morphogenesis and progressive (self)-disorganization in neoplastic development and progression.Clark WH Jr.6 ?: f% |$ p& k! I
Department of Pathology, Harvard Medical School, Beth Israel Hospital, Boston, MA 02215.

The aberrant forms of life, neoplasia and cancer, are discussed under the events at the beginning of neoplasia and under five classes of neoplastic lesions. The lesional classes are: 1) The precursor state; 2) Intermediate lesions; 3) Primary cancer; 4) Metastasis; and 5) Metastasis from metastasis. The events at the beginning are a diverse group of agents and mechanisms that induce the lesions of the precursor state, not cancer. The lesions and events produced by induction are similar regardless of the agent. Thus, there must be similar biological principles and mechanisms operative in different neoplastic systems. The classes of neoplastic lesions and cancer are described and a theory derived therefrom. The theory is: Any perturbation that alters a cell or group of cells and their stroma so that they no longer respond appropriately to the forces of tissue, organ, and organismal maintenance, may induce a neoplastic system. The sequential progression of lesions of the induced neoplastic system is the result of a successive series of flaws in the continuum of reciprocal interactions between a group of cells and their stroma. The flaws, appearing seriatim, produce progressive (self)-disorganization of the lesions and progressive loss of response to the forces of tissue and organ maintenance.$ e) O5 {7 Q4 a/ V
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PMID: 7865232 [PubMed - indexed for MEDLINE]

1: Br J Cancer. 1991 Oct;64(4):631-44. Links ) Q( r( L+ ~: T
Tumour progression and the nature of cancer.Clark WH.
Pigmented Lesion Study Group, University of Pennsylvania, School of Medicine, Philadelphia., [. O4 I/ M2 b6 l+ k5 H2 ^; T
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The nature of neoplasia and its sometime end result, cancer, has been studied by exposition and explanation of the sequential lesions of tumour progression. Neoplastic lesions were divided into four classes on the basis of growth characteristics and whether lesional growth is confined to one or more tissue compartments. Class IA, the initial lesion, an orderly, probably clonal growth, usually differentiates and disappears. Class IB: Failure to differentiate accompanied by disorderly growth. Class IC: Randomly dispersed atypical cells, constituting a precursor state. Class II, intermediate lesions, apparently arising from the atypical cells, show temporally unrestricted growth within the tissue compartment of origin. Class III lesions, primary invasive cancers, show temporally unrestricted growth in two or more tissue compartments and metastasise along different paths, a property associated with extracellular matrix interaction. The metastatic pathways may result from different subsets of cells in the primary cancer. Class IV lesions are the metastases. It was concluded that, all neoplasms develop in the same way, have the same general behavioural characteristics, and, when malignant, all interact with the extracellular matrix of the primary and the secondary sites. The origins and development of cancer are considered to be pluralistic and not due to a discrete change in a cell, whose progeny, as a result of that discrete change, carries all of the information required to explain the almost limitless events of a neoplastic system.

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美国癌症协会对肿瘤干细胞2006年开会综述：
Cancer Stem Cells—Perspectives on Current Status and Future
Directions: AACR Workshop on Cancer Stem Cells
Michael F. Clarke,1 John E. Dick,2 Peter B. Dirks,3 Connie J. Eaves,4 Catriona H.M. Jamieson,55 [! ^9 o5 d) Q7 t' ]; U/ v/ r
D. Leanne Jones,6 Jane Visvader,7 Irving L. Weissman,8 and Geoffrey M. Wahl6$ R, `+ ^; o& @
1Stanford University School of Medicine, Stanford, California; 2University Health Network; 3University of Toronto Hospital for Sick Children,
Toronto, Ontario, Canada; 4Terry Fox Laboratory BC Cancer Research Center, Vancouver, British Columbia, Canada; 5Moores
University of California San Diego Cancer Center; 6The Salk Institute for Biological Studies, La Jolla, California; 7Walter and
Eliza Hall Institute, Parkville, Victoria, Australia; and 8Stanford University Medical Center, Palo Alto, California& ]. t) u0 R6 N1 R1 w5 a: [; M7 K
篇号：2

5.pdf (203.6k)
篇号：38 @  D7 v3 ?5 W5 `
杂志全名及年卷期： Annu Rev Med. 2006 Sep 26; [Epub ahead of print] Links .
文题：Cancer Stem Cells: Models and Concepts.
作者：Dalerba P, Cho RW, Clarke MF. * S. k% K4 W3 q' F7 O, _- o0 r; W5 p/ r
PMID: 17002552
全文链接：　>
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4.rar (225.68k)3 A, v9 j! `& c( ]
楼主可以给出<<癌变机理的研究进展>>的全文链接吗？或者把文章中的图贴上来？
gaoy98 wrote:3 R: s- k3 e* {) |9 D4 E3 B' D1 J
楼主可以给出<<癌变机理的研究进展>>的全文链接吗？或者把文章中的图贴上来？' S7 f& |/ L; f- |* ~! ~

上面有图呀,见文章的附件部分0 d2 R( a. S9 P. s$ Z& L" t

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我打开看了几份，有些觉得不是和标题和相称的感觉。/ B; s2 [; Y( F0 x. J. M" F2 G6 l
Proc Natl Acad Sci U S A. 2006 Oct 31;103(44):16466-71. ( H9 ]+ C) g2 h7 \9 j

Angiogenesis-independent tumor growth mediated by stem-like cancer cells.; _$ U& y3 o* c" [

Sakariassen PO, Prestegarden L, Wang J, Skaftnesmo KO, Mahesparan R, Molthoff C, Sminia P, Sundlisaeter E, Misra A, Tysnes BB, Chekenya M, Peters H, Lende G, Kalland KH, Oyan AM, Petersen K, Jonassen I, van der Kogel A, Feuerstein BG, Terzis AJ, Bjerkvig R, Enger PO. & E' Z, Q7 f6 z3 U; L3 w# ?& {( Y
NorLux NeuroOncology, Department of Biomedicine, University of Bergen, N-5020 Bergen, Norway.# x7 G9 |$ X$ @. F% k% `. P
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In this work, highly infiltrative brain tumors with a stem-like phenotype were established by xenotransplantation of human brain tumors in immunodeficient nude rats. These tumors coopted the host vasculature and presented as an aggressive disease without signs of angiogenesis. The malignant cells expressed neural stem cell markers, showed a migratory behavior similar to normal human neural stem cells, and gave rise to tumors in vivo after regrafting. Serial passages in animals gradually transformed the tumors into an angiogenesis-dependent phenotype. This process was characterized by a reduction in stem cells markers. Gene expression profiling combined with high throughput immunoblotting analyses of the angiogenic and nonangiogenic tumors identified distinct signaling networks in the two phenotypes. Furthermore, proinvasive genes were up-regulated and angiogenesis signaling genes were down-regulated in the stem-like tumors. In contrast, proinvasive genes were down-regulated in the angiogenesis-dependent tumors derived from the stem-like tumors. The described angiogenesis-independent tumor growth and the uncoupling of invasion and angiogenesis, represented by the stem-like cancer cells and the cells derived from them, respectively, point at two completely independent mechanisms that drive tumor progression. This article underlines the need for developing therapies that specifically target the stem-like cell pools in tumors.4 ?( Z1 _! E$ n) E6 V
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（缩略图，点击图片链接看原图）
Identification of a Retroviral Transforming Gene* I' a" I2 ?& N4 ~; `0 R- q/ Y4 [+ f

Steven Martin Our current understanding of the molecular origins of cancer derives from many sources, such as the recognition that many chemical carcinogens act as mutagens, the discovery of tumor suppressor genes inactivated in familial cancers, and the identification of tumor virus genes that can cause the alteration to malignancy ("transformation") (see GENES 2000: 29.4 Transforming viruses carry oncogenes). Here I will focus on one of these threads, the identification of the transforming (cancer-causing) gene of Rous sarcoma virus (RSV). I first review the work on RSV that suggested that its genome might include a gene responsible for transformation. I then describe the experiments that led to the identification of the src gene, the RSV gene responsible for the induction and maintenance of transformation. Finally I briefly describe how work on src has enriched our understanding of signaling in normal cells, the mechanism of malignant transformation, and the role of genetic change in human cancer.

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In 1908 Ellerman and Bang reported that an avian leukosis could be transmitted by a filterable agent, that is, by a virus (Ellerman and Bang, 1908). Although this report now stands as the first description of a tumor virus, at that time leukemia was not regarded as a form of cancer, and their paper did not arouse great interest or opposition. Three years later Peyton Rous described the discovery of the virus that is now called Rous sarcoma virus (Rous, 1911). Rous’s report that a virus could induce "authentic" cancers met with considerable skepticism, because cancers were believed to be of local origin, and not dependent on infection: one oncologist told him that "this can’t be cancer, because you know its cause". Many argued that virus production was a consequence of tumor growth, and not its cause. Thus in 1928 Boycott (Boycott, 1928) wrote that "all the evidence seems to concur in indicating that the Rous virus arises de novo in each tumor." Although RSV does indeed induce tumors upon injection into susceptible birds, it does not spread by infection in natural populations, and there was a germ of truth in Boycott’s statement. Thanks to the work of Bishop and Varmus, described in the following essay, we now know that transforming viruses do indeed arise "de novo " by recombination between viral and cellular genomes. In the case of RSV, this recombination event presumably occurred in the tumor from which Peyton Rous first isolated the virus. In any event Boycott’s comment predated an understanding of the origin of retroviral transforming genes by some fifty years.+ V% s, @5 q3 J% C: B9 l) t

And so for several decades these avian viruses were regarded as curiosities with no relevance to mammalian cancer. But by the 1950s it had become apparent that a variety of viruses could cause tumors in susceptible animal hosts. Renato Dulbecco and his colleagues at Caltech realized that animal tumor viruses could provide an entry to an understanding of cancer at the molecular level. The first challenge was the development of an in vitro system in which the mechanism of malignant change could be studied outside the animal host. There were already several reports that chicken embryo fibroblasts (CEF) could be morphologically altered by RSV (Halberstaedter, Doljanski, and Tenenbaum, 1941; Lo, Gey, and Shapras, 1955; Manaker and Groupé, 1956). This morphological alteration was used by Temin and Rubin (Temin and Rubin, 1958) as the basis for the focus assay, which formed the foundation for all subsequent studies on transformation by RSV. In this assay, infection of a single cell by a single infectious particle results in the formation - by cell division and successive rounds of infection - of a cluster or "focus" of morphologically distinct cells. Cells from these foci produce tumors in vivo and are said to have undergone malignant transformation, or simply, "transformation". In cell culture they display altered growth properties, such as the ability to grow independently of anchorage when suspended in a semi-solid agar medium.3 q: p/ Y, N: L; I

A key issue in those early days was whether the tumor viruses were perpetuated with the transformed cells, and if so, how the viral genome was maintained. A model was provided by the phenomenon of bacterial lysogeny, characterized by André Lwoff and his colleagues in the 1940s and 1950s. As early as 1955, Harry Rubin showed that each cell in a Rous sarcoma virus-induced tumor could release infectious virus (Rubin, 1955). Moreover, in contrast to the situation observed with temperate (lysogenic) bacteriophages, the virus-producing tumor cells survived. He therefore suggested that "The virus plays a direct and continuing role in perpetuating the cell in its malignant state." In the mid-1960s Temin made the controversial proposal that the viral genome is perpetuated as an integrated DNA (a provirus) (Temin, 1964); however the physical demonstration of integrated viral genomes in transformed cells, and an understanding of how these genomes are generated and integrated, came only much later, and is a separate story.( A. [9 I  O/ E* E. t+ v; y& q

The finding that tumor viruses were permanently associated with transformed cells raised the possibility that transformation might result from the expression of viral gene products. In the case of Rous sarcoma virus, the first key observation was made by Howard Temin in 1960. He showed that a mutant of Rous sarcoma virus could cause the production of morphologically distinct, fusiform or spindle-shaped cells, readily distinguishable from the rounded cells that resulted from infection by wild-type virus (Temin, 1960). Temin therefore concluded that the morphology of the transformed cell is controlled by a genetic property of the virus.) p2 b6 {6 m$ }7 f& C
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Replication and Transformation Properties of RSV are Separable 4 w+ r% Z" [. |% H' F8 _
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During the decade that followed the biology of Rous sarcoma virus was intensively investigated. Two important findings emerged from work in the laboratories of Harry Rubin, Howard Temin, Peter Vogt and Saburo and Teruko Hanafusa, and are illustrated in Figure 1. First, Rubin and Vogt isolated replication-competent viruses (the RIFs, or Rous-interfering factors, and RAVs, or Rous-associated viruses) that were clearly related to RSV, but did not transform the infected cells (Rubin and Vogt, 1962). This finding again raised the possibility that the ability to transform was a specific genetic property of Rous sarcoma virus that distinguished it from its relatives, and was independent of the replication cycle of the virus. Second, one strain of Rous sarcoma virus, the Bryan strain, proved to be replication-defective (Hanafusa and T. Hanafusa, 1963). Cells infected with the Bryan virus became transformed but, as a result of the viral replication defect, yielded only non-infectious viral particles. Secondary "superinfection" of these transformed cells with a non-transforming "helper virus" (such as one of the Rous-associated viruses) could, however, rescue virus production in these cells. Thus the production of infectious virus was not necessary for transformation. Taken together, these two findings suggested that virus replication and malignant transformation might be separable genetic properties of Rous sarcoma virus.
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Clearly the definitive way to test the idea that transforming ability was a distinct genetic function of the virus would be to isolate transformation-defective mutants of RSV. Two strains of RSV, the Schmidt-Ruppin and Prague strains, had been shown to be replication-competent (that is, non-defective). These virus strains were evidently good substrates for genetic analysis, because clonal stocks of mutant viruses could be readily isolated and propagated. One approach would be to isolate mutants non-conditionally defective for transformation, which would biologically resemble the RIFs and RAVs, and such mutants were identified by Alice Goldé (Golde, 1970) and by Toyoshima, Friis and Vogt (Toyoshima, Friis, and Vogt, 1970). A second approach would be to look for temperature-sensitive (ts ) mutants. One advantage of such mutants is that they allow the mutant function to be switched on and off by temperature-shifts. The systematic use of ts mutants had been pioneered by Edgar in studies on the replication cycle of bacteriophage T4 (Epstein, 1963). Moreover Mike Fried had isolated a temperature-sensitive mutant of polyoma (a DNA tumor virus) that was unable to initiate transformation at the non-permissive temperature (Fried, 1965). I was familiar with the utility of ts mutants from my graduate work in Sydney Brenner’s lab. So when I moved to Harry Rubin’s lab in 1968, the isolation of ts mutants seemed like a plausible strategy to identify a transforming function of RSV. Peter Vogt’s laboratory was also looking for ts mutants, and in 1969 reported the isolation of two temperature-sensitive mutants of the avian sarcoma virus B77 (Toyoshima and Vogt, 1969). These mutants however were defective in virus replication, and thus did not define an independent transforming function.

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To isolate ts transformation-defective mutants of RSV, I subjected a stock of Schmidt-Ruppin RSV to mutagenesis with the mutagen N-methyl-N’-nitro-N-nitrosoguanidine ("nitrosoguanidine"). The survival of infectious progeny was about 10-3. (I chose to use this "withering dose of chemical mutagen" (Bishop, 1985) because I was aware that much of the decrease in infectivity would be due, not to mutations, but to interaction of the mutagen with protein components of the virus particle or to modifications of the genome that directly block replication; in any event I never determined whether this drastic mutagenesis procedure was necessary for the isolation of mutants). A simple screen sufficed to examine surviving virus for temperature-sensitive mutants. The mutagenized virus was used to infect susceptible CEF, which were then plated in agar suspension at 36°C. Clonal stocks generated by picking the transformed colonies onto monolayer cultures were then tested for their ability to form foci at 36°C and 41°C. Six of the two hundred and sixty clones tested were unable to produce foci at 41°C. Because the frequency of ts mutants amongst the survivors was only 2%, I decided not to worry whether the mutants contained multiple mutations, and went on to characterize one of them, which I imaginatively named T1.

Was the mutant defective only in its ability to transform, or did the mutation also affect the ability to replicate? To test the ability of the mutant virus to replicate at the non-permissive temperature, I infected CEF with wild-type or mutant virus and then held the infected cultures at 36°C or 41°C. As shown in Figure 2, cells infected with wild-type virus became morphologically transformed at both temperatures, whereas the cells infected with mutant virus became transformed at 36°C and not at 41°C. However, as shown in Figure 3, the mutant virus replicated at the same rate as wild-type virus, both at 36°C and 41°C. Moreover the morphologically normal cells infected by the mutant at 41°C became resistant to superinfection by wild-type virus. Resistance to superinfection by RIF- or RAV-infected cells was known to result from blockade of virus receptors by the envelope protein of the virus. The high degree of superinfection resistance exhibited by the mutant-infected cells indicated that almost all of the cells in the culture were infected. Thus the mutant virus could replicate at the non-permissive temperature without inducing morphological transformation. 9 }/ L$ D3 Z( c8 q5 U. M4 D( g! A
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At this point two possible roles could be imagined for the transforming function of the virus. They are distinguished in Figure 4. One possibility was that the function was required only to initiate transformation: that is the virus might transform by a "hit-and-run" mechanism, so that once the cell was transformed the function would be dispensable. If that were case, mutant-infected cells would be expected to remain transformed if an infection was first established at 36°C and the cells were then shifted to the non-permissive temperature. Alternatively the transforming function might be required continuously to maintain the transformed state. In the latter case, mutant-infected cells would be expected to revert to the normal phenotype after a shift to the non-permissive temperature. Temperature-shift experiments of this type indicated that the mutant-infected cells did in fact revert to the normal morphology following a shift from 36°C to 41°C, and would then re-transform when shifted back to 36°C. Similarly, the mutant-infected cells could not grow into colonies in agar suspension at 41°C even if first grown at 37°C for a few days, whereas transformed colonies did appear in cultures held at 41°C and then shifted to 37°C ( Figure 4). Thus the viral function was required continuously to maintain both morphological transformation in monolayer culture and anchorage-independent growth in suspension cultures.

The legacy

The isolation of ts transformation defective mutants of RSV raised a series of questions: can the transforming gene be identified physically? what is its protein product? and how does that protein product induce transformation? In 1970—the same year that the temperature-sensitive mutants were first described—Peter Duesberg and Peter Vogt demonstrated that the RNA genomes of wild-type replication-competent RSV strains were larger than those of non-conditional transformation-defective mutants (or non-transforming RAVs) (Duesberg and Vogt, 1970). They concluded that the transformation defect of these mutants resulted from the deletion of a gene required for transformation. Genetic crosses subsequently demonstrated that the ts mutations all fell within the region deleted in the non-conditional transformation-defective mutants, thus identifying the same transforming gene (Bernstein, 1976). Peter Duesberg’s laboratory went on to define the region deleted in the non-conditional transformation-defective mutants—the src gene—by oligonucleotide fingerprinting (Lai et al., 1973). It was not until the end of the decade, when the moratorium on cloning was over, that the RSV genome and the v-src gene were sequenced. Meanwhile, the product of the src gene was identified by Brugge and Erikson as a 60 kDa phosphoprotein that could be immunoprecipitated from RSV-transformed cells (Brugge and Erikson, 1977). One year later the Bishop and Erikson labs showed that this protein had protein kinase activity (Collet and Erikson, 1978; Levinson et al., 1978), and in 1980 Hunter and Sefton demonstrated that the kinase specifically phosphorylated protein substrates at tyrosine residues (Hunter and Sefton, 1980). Growth factor receptors also proved to have tyrosine kinase activity, providing the first biochemical link between malignant transformation and growth control in normal cells., [" f0 r: w( h8 [; P

The isolation of temperature-sensitive mutants also made possible a detailed examination of the biochemical events that occur during transformation. Temperature-shift experiments showed that a number of membrane-associated events occurred early in the transformation process, and could occur in the absence of protein synthesis. In a classic experiment, Beug and Graf showed that cells infected with ts mutant virus could undergo morphological transformation even when enucleated, although later events were blocked by enucleation (Beug et al., 1978). The picture that emerged from these early studies was that the transformation was initiated at the plasma membrane - where the Src protein was found to reside - and that signaling pathways then conveyed signals to the nucleus.
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The discovery of tyrosine kinase activity of Src made it possible to examine the nature of the signaling pathways responsible for transformation. We and others identified many Src substrates. But the fundamental raison d’être of tyrosine phosphorylation did not become apparent until the end of the 1980s, when Tony Pawson noticed a region of homology in the non-catalytic domain of Src and a related non-receptor tyrosine kinase, Fps (Sadowski, Stone, and Pawson, 1986). This region of homology, the Src homology 2 (SH2) domain, was subsequently identified in many other signaling proteins, and was shown by Pawson’s and Hanafusa’s groups to specifically recognize phosphotyrosine (Matsuda et al., 1990; Beug et al., 1978). The interaction between SH2 domains and phosphotyrosine residues is now understood to be the key step in the assembly of signaling complexes and in signal transduction at the plasma membrane (see GENES 2000: 27.9 Receptor kinases activate signal transduction pathways).
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But the most significant questions raised by the definition of the v-src gene concerned its origin: why did the virus carry a gene that was not required for replication? and where did it come from? Consideration of these questions led Bishop and Varmus to look for a cellular homolog of v-src. The identification of this gene, and how this discovery led to an understanding of the molecular basis of cancer, are described in the following essay.1 h* X: f9 y; G4 L

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The Discovery of Oncogenes in Human Tumors

Robert Weinberg By the mid-1970s, the notion that cancer was a genetically templated disease was deeply imprinted in the minds of many cancer researchers. A series of tumor viruses had been shown able to transform normal cells into tumor cells using the oncogenes carried in their relatively small genomes. Some speculated that, by extension, mutant cancer-causing genes must lie within all types of tumor cells, even those that lacked evidence of tumor virus infections. This gave rise to the idea that cells contain "proto-oncogenes"—genes that are concerned with normal aspects of cell behavior. The proto-oncogenes might can be mutated to give "oncogenes"—genes that have the ability to convert normal cells to a cancerous state (see GENES 2000: 29 Oncogenes and cancer). This essay describes how oncogenes originating in cells were first discovered and characterized. . {( g$ f4 a' m& K+ j; i

Background
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The notion that cancer cells carried mutant cellular genes had been articulated time and again throughout the middle decades of the 20th century. The greatest impact on my own thinking was made by the work of Bruce Ames published in 1975 (McCann et al., 1975). He developed a bacterial test for measuring the mutagenic potency of various compounds and used this test to analyze a number of compounds known to cause cancer in laboratory rodents. This test allowed him to report a correlation over 6 orders of magnitude between the mutagenic and carcinogenic potencies of these compounds.
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The logic flowing from Ames’s work was simple and straightforward. Carcinogens appeared to act through their ability to damage the genes of target cells. Hence, cancer cells must carry mutant genes. Moreover, these genes, in mutated form, must confer a growth advantage. Many naysayers soon pointed out that some carcinogens were not mutagenic at all, rendering this logic foolish, but I was not troubled. I thought that a good, simple idea should not be undermined by complicated facts.

A second input came from the work of Dominique Stehelin in the laboratory of Harold Varmus and Mike Bishop. The oncogene of Rous sarcoma virus had previously been identified and named as the src gene after the virus. To indicate its presence in the viral genome, it was more fully described as v-src. The discovery of a counterpart in uninfected cells (Stehelin et al., 1976), which was called c-src to indicate its presence in the cellular genome, provided specificity to the scheme that I, and others, had in mind. One reading of this work was that the genomes of normal cells harbored proto-oncogenes that, when perturbed in one way or another, assumed growth-promoting powers—precisely the notion implied by the somatic mutation theory of cancer. The Varmus/Bishop work indicated that at least one such proto-oncogene (c-src ) could be activated to give an oncogene (v-src ) by a retrovirus (see GENES 2000: 29.7 Retroviral oncogenes have cellular counterparts). A simple extension of this was that somatic mutations of normal cellular genes could also yield the same end result./ n1 ?" w  I  g# J
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Transfection as a way of finding cellular oncogenes

Gene transfer was ultimately the way that my lab was able to address whether such somatically mutated genes actually existed. This procedure, later called transfection, was first described for mammalian cells by Graham and van der Eb in 1972 (Graham and van der Eb, 1973). Some in my group had started using it and by 1978 had become quite adept at it. Our only innovation of substance was our discovery that NIH-3T3 cells, a line of immortalized mouse fibroblasts, were particularly adept at taking up and expressing DNA transfected by this method.
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The idea here was to use the gene transfer procedure to test whether the DNAs of cancer cells harbored genes that programmed their transformed growth. In principal, transfer of the DNA and thus the hypothetical transforming genes from the cancer cells should convert untransformed recipient cells to a transformed phenotype. Success in this effort would prove that transforming genetic information resides in the genomes of the cancer cells, and could be transferred via naked DNA from this cell into hitherto normal recipients. Furthermore, this would indicate that the transformed growth properties of cancer cells derived from genetic mechanisms. This would diminishing the candidacy of an alternative theory that was popular at the time—that cancerous growth is only a reflection of some epigenetic changes in the cells, which rearrange their gene expression programs to mimic normal processes that occur during differentiation., [  Y' S; K( |7 _% W

We agonized for a while on what type of cancer cells to use for the preparation of the transforming DNA. Ultimately, I chose the most conservative course, using the DNA from mouse cells that had originally been transformed in culture through exposure to chemical carcinogens. These seemed to be ideally suited to our purposes: like the NIH-3T3 cells, they were cells of murine origin and they had been transformed by the actions of a carcinogen that ostensibly had inflicted damage on their genomes. , {1 [- T' |1 |

By late 1978, we had the first glimmers of success transfecting the DNA of these chemically transformed mouse cells into NIH-3T3 cells. Our strategy here was further inspired by observations with a variety of tumor viruses. When they infected normal cells, such cells no longer exhibited the trait of contact inhibition. As a consequence, the descendants of a virus-transformed cell would pile up in a discrete area of a Petri dish, forming a clump of cells many layers thick. Such a clump, termed a focus, contrasted to the behavior of the surrounding normal cells, whose behavior continued to be governed by contact inhibition; as a consequence, these normal cells stopped growing after they had formed a single-cell-thick sheet termed a monolayer. A keen eye could easily pick out and count the foci at the bottom of a Petri dish.) K4 M' `  x/ }5 `% B) B1 ]3 R
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Figure 1 shows the approach taken by my student, Chiaho Shih. He exposed NIH-3T3 cells to DNAs prepared from several methyl-cholanthrene-transformed mouse cell lines. He then cultured the recipient cells for several weeks, looking for foci of transformed cells in the resulting cultures. Occasionally foci did appear, but more often than not they were spurious, reflections of the tendency of the NIH-3T3 cells to spontaneously undergo some type of mysterious morphologic transformation. Sometimes the apparently bona fide foci were distinguishable from these spontaneous transformants, but sometimes they were not. We clearly were working with an experimental system that had strong noise and weak signal.
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The convincing evidence only came when Chiaho carried out blind experiments in which he was oblivious to the identity of the donor DNAs (including as controls DNAs prepared from normal, untransformed cells). In the early months of 1979, decoding of such an blind experiment revealed clear-cut evidence that the DNA prepared from methylcholanthrene-transformed mouse cells induced far more foci of transformed recipient cells than did control normal mouse DNA (Shih et al., 1979). We had the proof in hand that the DNA of these transformed cells was different from the DNA of normal cells! Transformed cells contained a gene or genes that induced cell transformation. This provided the first information that cancer cells induced by chemicals indeed carried mutant transforming genetic information.+ T5 B- q( P; L% X. Y# G2 ?2 O$ T

Characterization of cellular oncogenes * s* L! x+ f, I& n  l" f: J# w
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We needed now to identify the actual oncogene. Soon my lab and that of Geoffrey Cooper were able to use the same procedure to show that cancer cell DNA prepared from human tumor cells also carried transforming information (Shih et al., 1981; Krontiris and Cooper, 1981). This indicated that the chemically transformed mouse cells with which we had begun our work were indeed good models of the cancer cells that arise in human beings. The latter, by implication, must also carry genes that are mutated by processes similar to those occurring in the cultured mouse cells exposed to carcinogens. Moreover, it was clear that at least one human oncogene worked perfectly well in mouse cells to induce cell transformation. Hence, there were no inter-species incompatibilities in the action of these transforming genes.

Still, this work did not shed light on one very important puzzle: Did the transforming information transferred from donor to recipient cells reside in a single gene or in a cohort of genes that were simultaneously passed during the transfection process? Among the other outcomes of Michael Wigler’s work was the conclusion that the efficiency of transfer of even a single gene was extraordinarily low (Wigler, 1978); hence, the concomitant transfer of two unlinked genes from donor to recipient cell was astronomically unlikely.
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The resulting conclusion that only a single transforming gene—an oncogene —was being transfected left us with another puzzle. We had been preparing DNAs from four distinct lines of chemically transformed mouse fibroblasts, each transformed on a separate occasion by exposure to methylcholanthrene. Had this carcinogen struck the same normal gene on these four different occasions? Or had four different genes been mutated, each yielding a distinct mutant oncogene?
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Ben-Zion Shilo working in my lab provided one answer to this puzzle. Figure 2 and Figure 3 show that when the transforming DNAs were treated with a series of restriction enzymes prior to transfection, all four genes were susceptible to inactivation by the same set of enzymes and resistant to inactivation by another set of enzymes (Shilo and Weinberg, 1981 ). This implied that they shared the same physical structure and that they all derived originally from the same antecedent normal proto-oncogene gene., V/ u* \$ y; S# v; G. Q8 G
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Cloning of a cellular oncogene

The similarities in behavior between the oncogenes transfected from a variety of tumor cells and those carried by retroviruses such as Rous sarcoma virus were striking. In both cases, an agent—either a virus or a chemical carcinogen—had apparently succeeded in converting a normal cellular gene into a transforming oncogene. This provoked a not-too-subtle speculation: that a common proto-oncogene could be activated into a potent oncogene either by a retrovirus or by a mutagenic chemical. So we used the then-recently invented Southern blotting technique to check whether the transfected oncogenes had sequences related to the known retrovirus-activated oncogenes.8 |% W3 y! k  I4 A
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Our initial tests came out negative and so Chiaho Shih pursued an elaborate strategy for cloning a human bladder carcinoma oncogene. The logic of his cloning strategy is shown in Figure 4, and depended on the fact that human cells were known to carry hundreds of thousands of Alu repeats scattered randomly throughout their genome. We imagined, therefore, that virtually every human gene, including the transfected human oncogenes, were closely linked to one or more of these Alu sequences. Hence, when transfected into recipient mouse cells, these human oncogenes would carry Alu sequences along for the ride. While mouse cells also carried highly repeated sequences scattered through their genome, these were sufficiently different in sequence from the human Alu sequences that the two could be easily distinguished using a DNA probe specific for the human repeat sequence.

So Chiaho looked for human Alu sequences that co-segregated with a human bladder carcinoma oncogene through successive cycles of transfection. In 1982, he had cloned out an Alu-containing DNA fragment from a transfected NIH-3T3 cell that also possessed potent transforming activity (Shih and Weinberg, 1982). This DNA segment had the properties that we had previously associated with a cellular oncogene—a single, contiguous stretch of DNA with strong transforming powers. Moreover, it seemed as if all the transforming power initially associated with the genome of the human bladder carcinoma cells could be ascribed to this single, discrete stretch of DNA. Michael Wigler’s group used an elegant technique that derived from his studies of "co-transfection" to clone out the same gene at the same time (Goldfarb et al., 1982). & k3 _! F' t' @3 t" L
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When used as a probe in a Southern blot, shown in Figure 5, this bladder carcinoma oncogene revealed that a gene of virtually identical structure resided within normal human DNA (Shih and Weinberg, 1982). Hence, the bladder carcinoma oncogene was indeed a mutant version of a pre-existing normal human gene! Provocatively, this proto-oncogene had a structure that was indistinguishable in its restriction enzyme map from the active oncogene. The difference between the two forms of the gene (one potent in transforming cells, the other inactive in transformation) was clearly very subtle.
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Mechanism of activation of a cellular oncogene

Within several months of this gene cloning, we confronted another surprise. Both Geoffrey Cooper’s lab and my own discovered that the human bladder carcinoma oncogene was in fact closely related to the ras oncogene (Parada et al., 1982; Der, 1982). Edward Scolnick’s group had previously shown that a ras oncogene was carried in the genome of Harvey sarcoma virus, a retrovirus of mixed rat-mouse origin whose v-ras oncogene arose much like the v-src oncogene of Rous sarcoma virus. Like v-src, v- ras had been acquired via retrovirus transduction and activation of a preexisting normal cellular proto-oncogene (c-ras ; Ellis et al., 1980).
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This discovery was both exciting and painful. The excitement came from the resulting conclusion that the same normal cellular gene could be activated into a potent oncogene either by a retrovirus or by a somatic mutation of the sort inflicted by mutagenic carcinogens. The pain came from the realization that the discovery of this homology could easily have been made two years earlier, thereby sparing us the complex and challenging gene cloning procedure that Chiaho had used to isolate the bladder carcinoma oncogene.$ k- y  O$ a$ G) h  R, A9 ~
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While the human bladder carcinoma oncogene was clearly derived via mutation from a normal cellular proto-oncogene, the precise nature of the mutation remained elusive. We only knew that the difference between the two versions of the gene was very subtle. A three-way race soon began between my own lab and those of Michael Wigler and Mariano Barbacid. The three groups crossed the finishing line at about the same time with a startling discovery: the essential difference between the two alleles was a single base substitution in the twelfth codon of the ras gene which caused the replacement of a glycine by a valine (Tabin et al., 1982; Reddy et al., 1982; Taparowsky et al., 1982). Within several years, it became clear that about one quarter of all human tumors, derived from a variety of organs, carry point mutations in either the 12th or 61st codon of a ras gene.5 A- W1 C3 N7 E8 q- W( U8 F8 @0 |
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These discoveries provoked great interest from those studying signal transduction biochemistry. They soon launched a multi-pronged attack on the signaling cascade in which the Ras protein, normal or oncogenic, plays a central role. Soon Scolnick demonstrated that the Ras protein, termed p21, behaved much like the alpha subunit of heterotrimeric G proteins; like these others, p21ras switched back and forth from an active, signal-emitting configuration that bound GTP to a silent state that bound GDP. By hydrolyzing its bound GTP, p21 ensured that its excited state persisted for only a brief period of time. It was then demonstrated that the oncogenic form of p21ras encoded by mutant ras oncogenes lacked substantial GTPase activity, thereby trapping p21 in an excited, signal-emitting configuration for extended periods of time (Sweet, 1984).
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The mutations that led to p21 activation and the resulting biochemical perturbation were peculiar to these genes and their encoded proteins. Within months of the discovery of the point mutation that activated the ras bladder carcinoma oncogene, other cellular genes such as myc were also found in mutant form in human tumor DNAs. We now know that cancer can be provoked by a wide variety of somatic mutations.; y/ Y: L1 D& g% ^' l

Oncogene collaboration ( A, y* F. H: H- ^* V& a8 U
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The discovery of the point-mutated ras oncogene in a human bladder carcinoma genome suggested a deceptively simple mechanism by which tumors arose: A mutagenic carcinogen entered a target cell and damaged a critical nucleotide in the normal ras gene; the resulting mutated cell then began to proliferate, spawning the large flock of descendant cells that formed a macroscopic tumor.

Tumorigenesis seemed to be a much more complex phenomenon however. It seemed to involve a gestation period of many years, and histopathological analyses of tissues strongly suggested that the process of forming a tumor involved multiple steps. This suggested in turn that real human tumors carried multiple mutated genes, and that a single mutated gene, on its own, was insufficient to create a malignant cell.

We soon tested this notion by studying the behavior of rat embryo fibroblasts rather than the NIH-3T3 cells that we had used for many years. Because the NIH-3T3 cells derived from an immortalized cell line, we imagined that they had already undergone some of the initial changes that normal cells underwent in the human body during their multi-step evolution toward a malignant growth state. The rat embryo fibroblasts (REFs), in contrast, had been recently explanted from a rat embryo, and as such, were presumed to be fully normal.( l5 ~& |; V& |4 m+ ^& H/ F. s8 W
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In 1983, Hartmut Land transfected the cloned ras oncogene into these REFs and found that they did not yield any foci of transformants in response. A cloned myc oncogene was similarly unable to evoke transformation of REFs. However, when the ras and myc oncogenes were co-transfected into REFs, foci of transformants arose, as shown in Figure 6. Moreover, these foci were shown to contain tumorigenic cells, as evidenced by the large tumors formed in hosts when cells from such foci were implanted in young, syngeneic rats or nude mice (Land, Parada, and Weinberg, 1983).# L3 O) H3 y3 G8 J
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This oncogene collaboration indicated that cellular oncogenes did not constitute a single, analogously functioning group of genes. Instead, these two oncogenes—ras and myc—seemed to work in distinct, complementary ways on cell phenotype. We speculated that by extension, the genomes of human tumor cells also carried two or more mutated genes that collaborate to drive the cell into a malignant growth state.2 p  C+ Q3 p6 q* I3 L4 [7 \

Only later did it become apparent that the human genes that participate in mutant form in cancer pathogenesis encompass a wider spectrum, including tumor suppressor genes and those involved the maintenance of genomic integrity. In the best-studied of human cancers—colon carcinoma—mutation of a ras gene represents only one of four or five distinct genetic alterations that contribute to the phenotype of the malignant tumor cells. So the discovery of the cellular ras oncogene was a start, but no more than that. 2 c) {! T- I# _  P8 [
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The author

Robert A. Weinberg is a founding Member of the Whitehead Institute for Biomedical Research and a Professor in the MIT Department of Biology. He received both his undergraduate and graduate degrees at MIT and returned to MIT in 1972 after post-doctoral training at the Weizmann Institute in Rehovoth, Israel and the Salk Institute in San Diego, California. His research in the early 1970s focused on the replication strategies of DNA and RNA tumor viruses. At the end of the decade, his work took a new direction. Thereafter, his laboratory concentrated its efforts on studying cellular oncogenes and tumor suppressor genes. His laboratory has isolated the human H-ras oncogene from a bladder carcinoma, the neu oncogene (later termed HER2 ) from a rat neuroectodermal tumor, the human Rb (retinoblastoma) tumor suppressor gene, and recently the hTERT gene which specifies the catalytic subunit of the human telomerase holoenzyme and plays a critical role in human tumorigenesis.

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