第十二章    肿 瘤

 

   一、教学目的与要求

通过本章学习,应该了解肿瘤发生、发展的内外源性因素;掌握癌基因、抑癌基因概念及在肿瘤发生、发展中的作用;熟悉化学致癌物活化相关的代谢酶基因在肿瘤发生、发展中的作用;熟悉凋亡基因和抗凋亡基因在肿瘤的发生、发展中的作用;熟悉细胞癌变是一个多基因、多阶段的过程;掌握细胞黏附分子、基质分解酶、细胞运动因子、血管生成因子在肿瘤转移中的作用;了解与肿瘤转移有关的基因(转移基因、抑制转移基因);熟悉肿瘤转移的多步骤较漫长过程;了解肿瘤对机体的影响。

二、重点与难点

肿瘤的发病机制(癌基因、抑癌基因、DNA修复基因、化学致癌物活化相关的代谢酶基因、凋亡基因和抗凋亡基因在肿瘤发生、发展中的作用);细胞癌变是一个多基因、多阶段的过程;肿瘤转移的相关因素(细胞黏附分子、基质分解酶、细胞运动因子、血管生成因子、转移基因、抑制转移基因)在肿瘤转移中的作用;肿瘤转移的多步骤较漫长过程(7个过程)。

肿瘤从本质上讲是一种基因病,细胞癌变是一个受多因素作用(包括外源性因素:化学、物理、生物、营养等与内源性因素:遗传、免疫、激素、精神等)及多阶段的基因(癌基因、抑癌基因、DNA修复基因和化学致癌物活化相关的代谢酶基因、凋亡基因及抗凋亡基因、肿瘤转移基因、抑制转移基因等)改变的积累过程,因此使恶性肿瘤具有细胞增殖失控、凋亡受阻、分化障碍、侵袭与转移的特性。癌细胞缺乏正常细胞的形态和功能,如癌细胞缺乏接触抑制和密度依赖抑制,对血清依赖性降低,具有无限生长能力成为永生化细胞,无锚泊依赖性生长;癌细胞的微丝排列松散使癌细胞出现不规则的形态,细胞易变形和易活动;癌细胞的微绒毛多、密、细长,对氨基酸等摄取增加;癌细胞纤维连接蛋白减少、层黏蛋白大大增加、Ⅳ型胶原增加;癌细胞表面ATP酶活性与磷酸二酯酶活性增高,腺苷酸环化酶活性下降,癌细胞内cAMP水平降低;细胞内溶酶体酶活性增高,癌基因编码癌蛋白增加等。

 

  三、主要相关知识 

一.   关于引起肿瘤的外源性、内源性因素

(一)外源性因素

1.化学致癌物

化学致癌物(chemical carcinogen)是指能引起人或动物形成肿瘤的化学物质。目前已发现2000余种化学物质有致癌作用,而且它们常与人们饮食、生活方式有着密切联系,它已成为最主要的肿瘤病因。估计约80%人类癌症的病因与化学因素有关,如在高加索人中男性肺癌96%由吸烟引起。随着分子生物学理论及技术的发展,为研究化学致癌物致癌机制及肿瘤的预防提供实验和理论的依据。

1)有机化合物

常见有机化合物类的致癌物有以下几种:

①多环芳烃类(polycyclic  aromatic  hydrocarbons , PAH) 是迄今已知的致癌物中数量最多、分布最广、与人关系最密切、对人的健康威胁最大的一类化学致癌物。如34苯并芘、3-甲基胆蒽等,它们可以与细胞核、线粒体中大分子物质及目标DNA结合形成加合物(致癌物将烷基、芳香基等转移到生物大分子上形成的共价化合物),诱导产生肿瘤。多环芳烃类存在于矿物燃料中,是污染大气的主要成分之一。它们常与肺癌、皮肤癌发生有关。

②亚硝基化合物(N-nitroso compounds, NOC) 分为亚硝胺和亚硝酰胺两类,能溶于水和脂肪中,具有分布广、致癌性强、能够引起胃、食道、肝、尿路、膀胱、肺等多种脏器肿瘤。亚硝胺化学性质稳定,在紫外线照射下可发生光解作用,在体内经酶作用转化成具有致癌作用的代谢产物。亚硝酰胺化学性质活泼,可直接致癌。常与多种脏器肿瘤发生有关。

③烷化剂类(alkylating agents) 是一类具有烷化作用、化学性质活泼、极不稳定而且易于分解的有机物。可引起基因突变、染色体畸变,直接引起细胞癌变。如芥子气、抗癌药物氮芥、环磷酰胺等。常与多种肿瘤发生有关。

④芳香胺类(aromatic amines) 包括芳香胺、芳香酰胺和杂环胺。芳香胺存在于各种着色剂及人工合成染料中,可引起转移性膀胱癌;芳香酰胺为一种杀虫剂,可诱发大鼠多种器官的肿瘤。杂环胺是肉、鱼中氨基酸、肌氨酸在高温时分解、浓缩而成,有时它分布在烟雾中。常与膀胱癌有关。

⑤偶氮染料(azodyes) 广泛用作纺织品、食品、饮料等工业生产中的着色剂,如奶油黄、偶氮萘、酸性猩红等。主要引起肝脏、膀胱肿瘤。

⑥烟草  吸烟引起肿瘤主要是烟草中尼古丁可抑制细胞凋亡,使肺、子宫颈、喉内加合物形成,造成姊妹染色体交换率增加,微核体形成,泛黄嘌呤核糖核酸转移酶的突变和P53CT易位等。我国80%肺癌是由吸烟引起的。

2)无机元素及其化合物(inorganic elements and their compounds

铬、镉、砷、镍、铍等及其化合物均为人类确定致癌物。这些物质可通过职业性接触、周边环境污染或食物摄入途径而致癌,主要诱发皮肤肿瘤和呼吸系统肿瘤。

总之,化学致癌物可引起癌基因、抑癌基因等发生点突变、扩增、易位、重排、缺失等使癌基因活化,抑癌基因失活,导致细胞的增殖、分化、凋亡异常引起肿瘤。

3)化学致癌的特点

①致癌性与化学致癌物分子结构有关  不同种类的化学致癌物的分子结构不同,其致癌活性也不同。如多环芳烃类以45个环的致癌性最强,小于3个环的致癌性较弱。

②致癌性与化学致癌物剂量有关  机体接受致癌物的剂量越大,肿瘤出现就越早。

③化学致癌物之间致癌性具有相加(summation)、协同 (synergistion) 和拮抗(antagonistion)作用  相加作用:两种或两种以上致癌物同时或相继作用于机体,其复合效应等于单独作用之和;协同作用:两种或两种以上致癌物同时或相继作用于机体,其复合效应超过单独作用之和,如二乙基亚硝胺与4-二甲基氨基偶氮苯合用,只需二者单用量的66%,二者显示协同作用;拮抗作用:两种或两种以上致癌物同时或相继作用于机体,其复合效应低于单独作用之和 ,也即一种致癌物能抑制另一种致癌物的致癌效应,如N-丁基亚硝胺诱发大鼠膀胱癌的过程中,若加用N-亚硝基哌啶可明显减少膀胱癌的发生,二者显示拮抗作用。

④致癌性可垂直传播  化学致癌剂所造成的细胞损伤在细胞分裂过程中可遗传到子代细胞。如果这种损伤发生在生殖细胞,还能遗传给所形成的胚胎,引发流产、先天畸形或儿童恶性肿瘤。

总之,化学致癌物可引起癌基因、抑癌基因等发生点突变、扩增、易位、重排、缺失等使癌基因活化,抑癌基因失活,导致细胞的增殖、分化、凋亡异常引起肿瘤。

 

2.病毒感染致癌 

全世界1/7肿瘤与病毒感染有关,病毒分为RNA病毒和DNA病毒。RNA病毒中只有逆转录病毒具有致癌性,称为RNA肿瘤病毒。DNA病毒的6大家族中5个具有细胞转化潜能,称为DNA肿瘤病毒。

1RNA肿瘤病毒  它首先吸附到宿主细胞膜上,以其鞘膜外侧的糖蛋白与宿主细胞表面特异性的膜相关蛋白受体结合并产生细胞膜融合,向细胞内释放出病毒核心颗粒,后者进一步解聚释放出病毒RNA基因组,病毒编码的逆转录酶将单链RNA基因组转化为双链DNA拷贝,然后以共价键形式整合到宿主细胞染色体上,在逆转录过程中异常的链转移是造成基因重组和重排(缺失、重复、倒位)的原因,逆转录病毒转导的病毒癌基因(V-onc)是目前最强的致癌物质。如人T细胞白血病病毒-12HTLV-12)、艾滋病病毒(HIV)、小鼠乳腺肿瘤病毒(MMTV)分别引起成人T-淋巴细胞白血病、卡波氏肉瘤、人类乳腺癌。

RNA肿瘤病毒如猿猴肉瘤病毒的病毒癌基因v-sis编码产物P28sisPDGF样作用,禽类红细胞生成病毒的病毒癌基因v-erbB编码产物P68erbBEGFR样作用,Rous病毒的病毒癌基因v-scr编码产物P68src为酪氨酸蛋白激酶样作用,Harvey大鼠肉瘤病毒的病毒癌基因Ha-ras编码产物P21H-ras为结合GTP/GDP样作用,禽类髓细胞瘤病毒的病毒癌基因v-myc编码产物P110gag-mycDNA结合蛋白样作用等。这些病毒癌基因编码的产物可使细胞增殖、转化,导致肿瘤的发生。

2DNA肿瘤病毒  它能将基因直接整合到宿主细胞染色体DNA中,复制后以溶细胞的方式释放到胞外再感染其他细胞,由于它们引起细胞死亡,限制了它们在宿主细胞中长期存在。因此DNA肿瘤病毒往往使不能复制病毒的细胞转化,不同DNA肿瘤病毒致癌潜伏期和致癌机制并不相同。如人类乳头状瘤病毒(HPV)引起宫颈癌、EB病毒(EBV)引起鼻咽癌、嗜肝病毒(乙型肝炎病毒HBV)引起肝癌等。DNA肿瘤病毒致癌机制尚未完全清楚。以HBV诱发肝癌为例叙述可能的机制。当HBV感染后,HBV基因组中X基因编码X蛋白(HBX抗原),属核蛋白,也可出现胞质中,可作为核转录活化因子而发挥作用,X蛋白与一种或多种细胞因子结合后,再作用于靶基因,使肝细胞发生转化;X蛋白与v-abl蛋白同源,也具有酪氨酸蛋白激酶活性,它可促进胞浆信号转导途径(Ras-Raf-MAPK途径);可结合抑癌基因产物P53并使之失活;肝癌中有X蛋白表达,转入X基因成功诱发肝癌,因此X基因为致癌基因。HBV感染还能诱导肝细胞坏死,继而产生炎症反应、 再生和纤维化,并释放细胞因子激活机体免疫系统。已有人发现HBVDNA可插入至细胞周期素A(cyclin A)基因的一个内含子中,使肝细胞的cyclin A mRNA转录增加,cyclin AS期与CDK2cyclin dependent kinase 2)结合参与调控DNA复制,与cdc2(cell division cycle 2)结合参与G2/M过渡,使细胞增殖。这可能与人原发性肝癌的发生有一定关系。总之,整合人肝细胞中HBVDNA使细胞的遗传性状发生改变,细胞的代谢和调节功能发生紊乱,使肝细胞的分裂、增殖失去控制而癌变。

肿瘤病毒致癌机制是RNA肿瘤病毒和DNA肿瘤病毒都可以将其自身的病毒基因序列整合到宿主细胞基因组中,通过改变细胞基因的转录水平、结构完整性等影响细胞的增殖、分化和凋亡,使之获得恶性转化表型。

 

(二)内源性因素

肿瘤逃逸免疫攻击的机制:

   1)肿瘤抗原性表达减弱: 人类肿瘤细胞表面都存在B7等共刺激分子表达缺乏或低下,是造成机体免疫系统对肿瘤细胞异常生长无免疫应答的原因之一。B7分子是T淋巴细胞活化所必须的共刺激因子,在T细胞活化增殖过程中起着重要的第二信号的作用,仅有单一抗原刺激(第一信号)并不能激活T淋巴细胞,有时反而会导致免疫耐受。有人将B7基因导入B7分子表达缺乏或低下肿瘤细胞内,提高肿瘤免疫原性,加强机体对肿瘤细胞免疫反应,从而达到抗肿瘤效应。

   2) 机体主动免疫被抑制:  肿瘤细胞可表达FasL,与淋巴细胞表面的Fas受体结合,诱导淋巴细胞凋亡;肿瘤细胞还可释放可溶性黏附分子如LCAM-1与免疫细胞表面的配体结合,使免疫细胞不能与肿瘤细胞结合而发挥免疫效应。

    肿瘤细胞具有抗原性并能引起机体免疫应答反应,这为肿瘤免疫诊断、免疫治疗、免疫预防的可能性提供了理论基础。生物反应调节物如单克隆抗体及其偶联物、细胞因子(IL-2IFN-γ等)、过继免疫活性细胞治疗(CTLLAKTIL细胞)、肿瘤疫苗治疗等,1985年定为肿瘤治疗(继手术、化疗、放疗之后)第四种抗癌疗法(生物疗法)。

 

.关于癌基因

(一)常见的癌基因

1c-sis 基因  位于染色体22q,其表达产物P28sis,是编码血小板源生长因

-β链(platelet derived growth factor, PDGF-β)的基因。是细胞有丝分裂原,正常表达对细胞生长、分裂、分化的正常调控具有重要作用。当 P28sis高表达时,能刺激多种细胞增殖,主要在神经胶质细胞瘤、骨肉瘤、纤维肉瘤、横纹肌肉瘤中表达。

2c-erbB基因  位于染色体7q12-14,其表达产物gP170c-erbB,是编码生长因

子受体的基因,它的产物与表皮生长因子(epidermal growth factor, EGF)受体高度同源,可与配体EGF结合或没有配体结合时都能传入生长信号。

3c-src 基因  位于染色体20q13.3,其表达产物P60 c-src,是编码酪氨酸蛋白激酶(protein tyrosine kinase, TPK)的基因。它多定位在细胞膜上,可使底物中酪氨酸残基磷酸化,将信息从活化的受体传至细胞内信号转导系统,促进细胞增殖、移动等。TPKc-src 家族的显著特征之一,该酶活性与细胞恶性有密切关系。主要在造血系统恶性肿瘤中高表达。

4c-ras 基因家族  该家族基因包括c-Ha-rasc-Ki-rasN-ras,分别位于染色体11p15.212p12.11p22,所编码的蛋白质分子量均为P21c-ras,它能与鸟嘌呤核苷酸(GTPGDP)有高度亲和力,并且具有GTP酶活性而水解GTPGDPP21ras参与细胞内信号转导。P21c-ras热点突变是N端第12135961位氨基酸,使Ras蛋白的GTP酶活性下降,而且对GTP酶激活蛋白(GAP)作用产生耐受,因此Ras- GTP长期处于激活状态,连续产生细胞生长信号,引起细胞永生化。ras 基因家族的表达有相对的组织特异性:rasH主要在泌尿道肿瘤中表达,rasK主要在肺癌和结肠癌中表达,rasN主要在造血系统恶性肿瘤及神经母细胞瘤中表达。

5c-raf 基因  位于染色体3p25,其表达产物P74 c-raf,是编码丝/苏氨酸蛋白

激酶的基因。定位细胞质内,可作为丝裂原活化蛋白激酶的激酶的激酶(MAPKKK),逐级将丝/苏氨酸残基磷酸化形成MAPK,引起细胞增殖反应。

6c-myc 基因  定位于8q24,其表达产物P64 c-myc,定位于细胞核内,属DNA

结合蛋白。当P64 c-myc高表达时,与DNA上的位点结合,激活与生长增殖有关基因的转录,抑制与分化有关的基因表达,使细胞获得无限增殖、永生化的功能。myc基因家族在泌尿生殖系统、造血系统、消化系统、神经系统、呼吸系统等多种肿瘤均有高表达,而且以扩增形式进行,出现均匀染色区和双微核。

(二)癌基因产物的作用

    大多数癌基因表达产物都是细胞信号转导系统的组成成分,作为细胞生长

因子和细胞生长因子受体;具有酪氨酸蛋白激酶活性及丝/苏氨酸蛋白激酶活

性;具有GTP结合蛋白活性;作为核转录因子或转录调节蛋白,促进细胞有丝

分裂及参与细胞周期的调控等。它们可以从多个环节改变和扰乱细胞正常代谢、

生长、分化等基本过程,使这些细胞具备了恶性转化的基础,并在某些促进因

素的作用下,加深了转化的进程,逐步演变为恶性肿瘤。

1.某些癌基因能编码表达生长因子样物质:sis癌基因编码产物P28PDGFβ链同源;int-2癌基因编码产物与FGF同源等。当癌基因激活时它编码产物增加,它们与细胞膜上生长因子受体结合,不断刺激细胞生长繁殖。

2.某些癌基因能编码生长因子受体:如erb-B癌基因编码产物P60EGF

受体同源;kit癌基因编码产物与PDGF受体同源等。当癌基因激活时生长因子受体表达增加,生长因子与其结合后导致细胞生长的信号转录处于持续激活状态;有些受体不需与生长因子结合,持续发出细胞生长的信号。

3.某些癌基因能编码具有酪氨酸、丝氨酸、苏氨酸蛋白激酶活性的蛋白:如srcablyes等可编码PTKrafmilmos等可编码丝氨酸、苏氨酸蛋白激酶。当癌基因激活时它们高表达可对下游信号转导分子磷酸化,促进细胞增殖。

4.某些癌基因能编码信号转导蛋白(G蛋白):ras癌基因编码产物P21Ras蛋白)与Gα亚基同源,当ras癌基因121361位突变,使Ras-GTP长期处于结合状态,Ras-MAPK途径激活,连续产生细胞增殖信号。

5.某些癌基因编码核转录因子或转录调节蛋白:mycfosjun癌基因表达产物位于核内,能与DNA结合,具有直接调节转录活性的转录因子(AP-1)样作用,激活基因转录,DNA合成增加,促进肿瘤发生;rel癌基因编码NF-kB家族蛋白癌基因产物的作用。

肿瘤细胞信号转导的改变是多成分、多环节的,表现在肿瘤细胞可以自分泌生长因子,并可有多种受体(生长因子、细胞因子、激素、死亡受体等)以及受

体后信号转导成分异常等,引起肿瘤细胞表型改变。

 

三.关于抑癌基因

作为抑癌基因有3个条件:在该肿瘤起源的相应正常组织中,这种基因必须表达正常;在该肿瘤组织中,这种基因必须有所缺陷;将这种基因(野生型)导入恶性肿瘤细胞,可部分或全部改变其恶性表现。

(一)常见抑癌基因:

1Rb基因  视网膜母细胞瘤易感基因(retinoblastoma, Rb基因)定位于染色体13q14.127个外显子,DNA长度约为200kbRb基因编码产物P105 Rb是由928个氨基酸组成的核磷蛋白。正常情况下c-fosc-mycTGF-β等基因上有视网膜母细胞瘤控制元件(RCE),未磷酸化的Rb蛋白可与它结合,阻止了腺病毒2启动活化因子(E2F,它是一类基因转录活化因子)与RCE结合而起活化转录的作用,细胞阻滞于G1期;当Rb蛋白磷酸化时,不能阻止E2FRCE结合而起活化转录作用,启动DNA合成,细胞进入S期,完成整个细胞周期,因此Rb蛋白作为一种重要的细胞周期调节因子,未磷酸化的Rb蛋白可抑制细胞增殖,而磷酸化的Rb蛋白则无此作用。Rb基因异常存在于视网膜母细胞瘤、骨肉瘤、软 组织肉瘤、小细胞肺癌、乳腺癌、食管癌及前列腺癌等多种肿瘤。

2p53基因  p53基因定位于染色体17p13.1,有11个外显子和10个内含子,DNA长度为20kbp53基因编码产物 P53蛋白(野生型)是由393个氨基酸组成的核蛋白,生物学功能是细胞DNA损伤的“检查点(checkpoint)”,具有控制细胞周期运行速度和促使细胞凋亡的生理作用。若细胞DNA受损, P53表达水平很快升高,使细胞阻滞在G1期,以便细胞进行DNA损伤后的修复;当DNA损伤过于严重不能修复时,就触发细胞凋亡,参与细胞生长的负调控。P53是一种能与DNA结合的蛋白,可与c-mycc-fosc-junDNA序列结合,干扰DNA聚合酶与DNA复制物的作用因而阻止DNA复制。P53还是一种转录激活因子,能激活p53-DNA结合部位邻近基因的表达,活化细胞生长的其他负调控基因。P53除抑制细胞生长外,还能诱导细胞分化,正常p53基因表达可诱导K562红白血病细胞分化。实验显示将典型的致癌物作用于无p53的小鼠,可以导致良性乳头瘤,而这些瘤极易癌变,说明p53失活在癌前病变中的作用。p53基因突变或缺失,即可引起细胞周期运行失控和细胞凋亡减弱导致肿瘤发生。有资料表明50%以上的人类肿瘤的p53基因突变发生在进化上高度保守区的第58个外显子,包括肺癌、软组织肉瘤、肝癌、胃癌、结直肠癌、膀胱癌、乳腺癌、头颈部磷状细胞癌、前列腺癌、胶质细胞瘤、淋巴造血系统肿瘤等;30%肿瘤患者血清中存在p53抗体阳性,故认为p53抗体是一种肿瘤发生的危险因子。

  P53促进凋亡的生化机制可能如下:①许多凋亡因子基因fasbax等启动子

区都存在有P53反应元件(PRE),结合P53后被激活转录表达,促进凋亡;而P53却能抑制抗凋亡基因(bcl-2基因)的表达,降低其抗凋亡作用。②生长阻止和DNA损伤诱导蛋白质45GADD45)基因启动子也有PRE,故P53也可促进GADD45表达,后者结合增殖细胞核抗原(proliferationg cell nuclear antigen, PCNA)而减弱DNA的修复。③P53可促进细胞内质网颗粒释放出TNF-α和Fas ,加速凋亡。④P53本身还可结合DNA复制蛋白ARPA)而阻止DNA的复制。因此P53是促进细胞凋亡的强诱导因子。当p53基因突变或缺失,它的促进细胞凋亡功能受到抑制,该死的细胞不死,逐渐发展成为肿瘤。

3.神经纤维瘤病基因 (nervoufibromatoma type 1gene, NF1基因 )  NF1基因

位于染色体17q11.2,编码由2458个氨基酸组成的蛋白质-NF1蛋白。NF1蛋白具有GTP酶活化蛋白作用,可与P21ras结合,激活其GTP酶活性,使P21rasGTP变为P21rasGDP,下调其介导细胞信号转导的活性。当NF1蛋白表达低下时,P21rasGTP增多,细胞信号转导的活性加强,与周围神经系统的神经纤维瘤发生有关。

 NF2基因位于人染色体22q12,编码由587个氨基酸组成的磷酸化蛋白,NF2蛋白是细胞膜的整合蛋白和细胞骨架蛋白的连接物。与前庭神经鞘瘤、乳腺癌、黑色素瘤发生有关。

4.肾母细胞瘤易感基因 (Wilms tumor gene, WT基因 )  WT1基因位于染色

11p13WT1基因编码的锌指蛋白为转录抑制因子,可识别DNA上一段特异序列并与之结合,抑制转录。它能抑制胰岛素样生长因子-Ⅱ(insulin-like growth factor-, IGF-Ⅱ)受体基因的表达,从而抑制细胞生长。WT1基因的缺失或突变,变异的WT1蛋白不能与DNA结合,因此失去抑制转录的作用,常见于肾母细胞瘤。

5.结肠癌相关抑癌基因

1)结肠腺瘤样息肉基因(adenomatous polyplsis of colon, APC基因)  它位于染色体5q21,有15个外显子,它编码分子量为300kD蛋白,APC蛋白可能通过与β-catenin (β-连环蛋白)E-钙粘素细胞连接蛋白结合并调节其水平,参与细胞内的信号传导和细胞间粘附,从而发挥抑癌作用。APC基因的突变常与结直肠癌有关。生殖细胞APC基因突变使家族性腺瘤样多发性息肉病几乎100%地发展成为结直肠癌;6880%的散发性结直肠癌中有APC基因体细胞突变。

2)结直肠癌缺失基因(deleted in colonrectal cancer, DCC基因 )  它位于染色体18q21.3,编码产物为免疫球蛋白超家族成员,与神经细胞粘附分子有同源性,参与细胞粘附功能,从而发挥抑癌作用。DCC基因在70%以上的结直肠癌中缺失或突变。

3MSH2MLH1基因  MSH2基因位于染色体2pMLH1基因位于染色体3p。它们有识别和修复DNA碱基错配的功能,能维持基因组和染色体的稳定,降低细胞自发性突变。当它们基因组中重复序列发生改变,增加了基因的不稳定性,因而不能有效地修复DNA,常与遗传性非息肉型多发性肠癌(HNPCC)、肾癌、肺癌发生有关。

6.乳腺癌相关抑癌基因-BRCA基因(breast  cancer基因)

1BRCA1基因  位于染色体17q12DNA长度为100kb,含22个外显子,编码由1863个氨基酸组成的蛋白质,似为转录因子,是乳腺组织特异性的抑癌基因。当其突变时,增加了乳腺细胞基因的不稳定性,常与遗传性乳腺癌、卵巢癌发生有关。

2BRCA2基因  位于染色体13 q12,该基因突变常可引起较年轻妇女的乳腺癌。

7.细胞周期相关的抑癌基因

1CIP1/WAF1基因(CDK-interacted protein 1 gene,CIP1/wild-type p53 activated fragment 1 gene,WAF1) 此基因定位于染色体6 p21.2DNA长度2.1 kb,其编码蛋白为P21(又称p 21基因),含495个氨基酸。P21可与细胞周期素竞争与CDK结合,细胞周期素不能与CDK结合,则使PRB保持未磷酸化状态,使细胞停滞于G1期而不进入S期,抑制细胞增殖。P21还可直接抑制增殖细胞核抗原(PCNA)而抑制细胞增殖。CIP1/WAF1基因异常可见于结肠癌、脑瘤、肺癌等。

2)多瘤抑制1基因(multiple tumor suppressor 1 gene,MTS1基因) 它定位于染色体9p21,由两个内含子和三个外显子组成,DNA长度为8.5kb,该基因编码产物为P16,故又称为p16基因。P16蛋白生物学功能与细胞周期的调控有关,它是一种CDK4的特异性抑制因子,CDK4可与周期素D1(推进细胞由G1期进入S期蛋白)结合,使细胞进入S期。P16蛋白能与CDK4直接结合,阻止CDK4与周期素D1结合而抑制细胞生长。p16基因突变使P16发生改变,不能与CDK4结合,CDK4与周期素D1结合而刺激细胞增殖。p16基因异常见于恶性黑色素瘤、肺癌、胰腺癌、膀胱癌、脑瘤、头颈部肿瘤及白血病等。

多瘤抑制2基因(MTS2基因)定位于染色体9p21,与p16基因相邻,它编码产物为P15,故又称为p15基因。P15P1690%同源性,P15能与CDK4结合,抑制其与相应的周期素结合,从而抑制细胞增殖。

8.胰腺癌缺失基因(deleted in pancreatic cancer Locus 4, DPC4基因) 它

位于人染色体18q21.1,参与TGF-β的信号转导。90%的胰腺癌染色体18q存在LOH(杂合性丢失),其中50%证实有DPC4基因的缺失或突变,也见于部分直肠癌、胆管癌。

(二)抑癌基因产物的作用:

1.编码转录因子或作为细胞周期调节因子参与细胞增殖、分化的调控,如Rbp53WTBRCA1BRCA2MTS1CIP/WAF1等。

2.参与DNA损伤后的修复、复制,保证DNA遗传的稳定性,如p53MSH2MLH1等。

3.与细胞内骨架蛋白相连,如APCNF2等。

4.基因产物为细胞粘附分子,如DCC基因。

5.编码GTP酶活化蛋白或磷酸酶,通过阻断癌基因产物如Ras蛋白或蛋白激酶的活性而发挥抑癌效应,如NF1等。

6.参与细胞内TGF-β的信号转导。如DPC4等。

 

四.关于凋亡基因、抗凋亡基因

细胞凋亡是由基因控制的细胞自主的有序死亡,基因控制包括凋亡基因和抗凋亡基因,凋亡基因常见p53fasbaxrelICE、腺病毒EIA等;抗凋亡基因常见bcl-2rasfosjunIAP、突变型p53等。研究表明肿瘤不仅是细胞增殖异常,也是细胞凋亡异常过程。

1fas/apo-1 基因  fas基因对细胞凋亡有促进作用,它编码Fas蛋白是一种跨膜蛋白,分子量为36000的细胞表面受体,受体的胞质部分有一段与TNF受体高度同源;apo-1是存在细胞表面分子量为52000的蛋白,它与Fas蛋白具有相似的作用。,当FasLFas结合,导致受体三聚化,再与受体连接蛋白结合形成死亡诱导信号复合体,其中的死亡效应区激活caspases级联反应,引起细胞凋亡。

2bcl-2基因  它是细胞凋亡研究中最受重视的癌基因之一,目前发现该家族具有抗凋亡作用的基因有bcl-2bcl-xbcl-w等,具有促进凋亡的基因有baxbcl-xshinbid等。它表达的蛋白质B淋巴细胞瘤/白血病-2B cell lymphoma/Leukemia-2Bcl-2)是由229个氨基酸组成的膜蛋白,主要存在于线粒体膜、内质网膜、核膜等处。Bcl-2抗凋亡作用机制:①主要维护线粒体膜的跨膜压和阻止线粒体膜通透性转换孔开放,阻抑线粒体内释放出凋亡因子(细胞色素CAIFSmacpro-caspase 2379),从而阻止凋亡小体的形成,防止细胞凋亡;②Bcl-2还能结合线粒体内释放的凋亡因子如pro-caspases,并维持其非活性状态,阻止了caspases级联反应,抑制线粒体介导的细胞凋亡信号转导途径;③直接或间接与P53结合而调控凋亡;④抗氧化作用或抑制自由基的产生;⑤可抑制正在发生凋亡的细胞内质网释放钙。实验发现抗凋亡基因bcl-2在前列腺癌、结肠癌、神经母细胞瘤、白血病时都有过高表达。在上述疾病过程中,如肿瘤组织免疫组化显示bcl-2阳性细胞超过20%,提示预后不良。

当凋亡基因失活、抗凋亡基因激活,细胞凋亡减弱,该“死”的细胞不死,让它继续存活,一方面使细胞数量增加,另一方面该细胞染色体不稳定即脆性增大,基因容易突变、染色体也容易易位、对致癌剂的敏感性增大,增加了癌变机会。同时已形成的肿瘤细胞有高表达FasL,借以来凋亡淋巴细胞;同时又低表达Fas,降低被凋亡的危险。这样可使肿瘤细胞形成能逃避免疫攻击和凋亡耐受的特性,肿瘤细胞表现为凋亡过低、增殖增加,肿瘤得以不断的发展。

病毒感染宿主细胞后,可分泌病毒因子,这些病毒因子有些与细胞生长因子、生长因子受体或信号转导分子同源,促进宿主细胞增殖与转化;有些是阻止宿主细胞的凋亡。因此某些病毒长期感染有严重的致癌风险。如人乳头状病毒(HPV)分泌的病毒因子B6蛋白,它可促进P53分解,引起细胞周期失控和阻止细胞凋亡导致细胞癌变,常与宫颈癌、生殖道肿瘤发生有关;乙型肝炎病毒(HBV)分泌的病毒因子HBX,它可结合P53,从而抑制凋亡,常与肝癌发生有关。其他一些病毒如杆状病毒分泌的病毒因子P35,其分子结构与Bcl-2部分同源,也具有抑制凋亡作用;牛痘病毒分泌的病毒因子为细胞因子反应修饰因子A,它具有抑制caspase3活性而起到抗凋亡作用;腺病毒与肉瘤疱疹病毒分泌的病毒因子可结合P53而抑制细胞凋亡。因此它们也与肿瘤的发生、发展有关。

综上可见,肿瘤的发生、发展与细胞凋亡有关,因此治疗肿瘤的新措施是诱导肿瘤细胞凋亡,临床上对癌症病人的化疗、放疗、热疗、生物性治疗等的重要机制是诱导肿瘤细胞凋亡。

 

五.关于肿瘤细胞增殖

肿瘤细胞呈失控性增生及不协调性生长。增生的肿瘤细胞在形态、功能、代谢等多方面与相应的正常细胞有着质的区别。肿瘤细胞的细胞周期时间(TC)与同型的正常细胞大致接近,如人的正常肠上皮细胞TC13d,结肠癌细胞TC2.5d;人的正常白细胞TC1d,急性白血病细胞TC24d。但正常细胞增殖到一定程度就会停止,其增加的细胞数仅仅补充死亡的细胞数,而肿瘤细胞则不同,其增加的细胞数远远超过死亡的细胞数,只要条件合适,增殖永不停止,瘤体不断长大,肿瘤细胞增殖失控是肿瘤细胞重要特性之一。肿瘤细胞增殖失控的可能机制如下:

(一)      癌基因与细胞增殖

有些癌基因能编码生长因子和生长因子受体,生长因子与其受体结合后,受

体蛋白内的酪氨酸蛋白激酶磷酸化,使含有SH2结构域的生长因子受体蛋白与核苷酸交换蛋白磷酸化,与RasGTP结合,RasGTP使Raf蛋白磷酸化,又可激活丝裂原活化蛋白激酶的激酶(mitogen activated kinase kinase,MAPKK),MAPKK再激活丝裂原活化蛋白激酶(mitogen activated kinase ,MAPK),MAPK通过磷酸化激活核糖S6蛋白(RSK)和Fos蛋白,磷酸化RSK又可磷酸化Jun蛋白,激活的Fos蛋白与Jun蛋白成为活性转录因子,与近旁myc癌基因的特异序列结合而激活myc癌基因,编码Myc蛋白,激活其下游周期素D1CYC D1)基因,CYC D1基因是生长因子感受器,协助细胞做出有丝分裂的“决定” ,使细胞由G1期进入S期,细胞增殖。此外,生长因子与其受体结合后,可激活磷脂酰肌醇信号转导途径,激活PKC,使立早基因c-fos c-jun反式作用子磷酸化,加快转录,产生第三信使,第三信使跨越核膜进入核内经磷酸化修饰后,活化晚期反应基因并转录,导致细胞增殖。癌基因编码产物大都属于细胞信号转导系统的组成成分,它们能从多个途径介入或干扰细胞内与生长有关的信号转导过程,使细胞增殖失控,而细胞增殖失控正是肿瘤细胞最明显的特征之一。

(二)抑癌基因与细胞增殖

有些抑癌基因失活,对细胞的增殖抑制作用减弱,也可引起细胞增殖。如视网膜母细胞瘤基因(Rb),它编码RB蛋白,当RB蛋白去磷酸化,可阻止转录因子E2F作用,DNA合成减弱,细胞被阻滞在G1期,还可延缓周期素AEDmRNA表达,从而抑制细胞增殖;当RB蛋白磷酸化,促进E2F作用,DNA合成增加,细胞进入S期,促进细胞增殖。在异常情况下,Rb基因突变、缺失或与肿瘤病毒的癌蛋白结合时,Rb基因失去活性(失去Rb蛋白去磷酸化活性),对细胞周期的控制作用丧失而导致细胞不断增殖。又如p53基因,它编码P53蛋白,P53蛋白能使细胞阻滞在G1期并参与DNA修复,如DNA损伤不能有效修复时,P53蛋白就诱导细胞凋亡,因此P53蛋白具有抑制细胞无限增殖的功能。当p53基因突变或缺失,即可引起细胞周期运行失控和细胞凋亡减弱导致肿瘤细胞增殖。此外,抑癌基因CIP/WAF1p21基因)、MTS1(p16基因)失活都可使细胞周期失控,细胞无限增殖。

(三)细胞周期调控与细胞增殖

 细胞生长是通过细胞周期来实现的,是由于不同基因按照时间顺序活化和

表达,使周期内出现的一系列有序的生化反应和结构的变化。肿瘤细胞增殖是由于细胞周期调控障碍,即驱使细胞周期的力量(cyclinCDK)增加、检查机制失控与抑制细胞周期的力量(CDI)不足。

1.细胞周期素(cyclin)过量表达   主要是cyclinDE的过量表达,cyclin作为

调节亚基与不同的催化亚基CDK结合形成cyclin/CDK复合物而参与细胞周期的调控,实验证实cyclinD是生长因子感受器,因此当生长因子与受体结合后,通过细胞内信号转导级联反应,使cyclinD基因激活,产生大量cyclinD,促使CDK活化,驱使细胞周期的力量增加。在乳腺癌、胃肠癌、食道癌、B细胞淋巴瘤等中发现cyclinD过量表达,乳腺癌中cyclinE也有高表达。

2CDK的过量表达   主要是CDK4CDK6的过量表达,TGF-β抑制增

殖的靶蛋白可能就是CDK4 ,实验证实用TGF-β处理可引起细胞内CDK4的减少。在诱导肿瘤细胞分化过程中,常有CDK4表达下降;而CDK4高表达时,与细胞周期素形成复合物,常常抑制细胞分化,促进细胞增殖,驱使细胞周期的力量增加。

    3.检查机制失控   细胞周期各时相存在多个检查点(分别位于G1/SG2/M交界处),决定细胞是否继续增殖或进入静止状态。当各种因素造成DNA损伤时,就会停止细胞周期进程,在检查点准确调控下,确保细胞准确有序地进行。P53作为重要的DNA损伤检查点分子,当检查到细胞存在严重DNA损伤时会诱导细胞阻滞在G1期,进行DNA的修复;如果不能修复DNA损伤则触发凋亡。P53可能直接激活Bax等凋亡基因,或抑制Bcl-2等抗凋亡基因,避免癌前病变细胞进入S期。当P53丢失或突变时,损伤的DNA不能修复进入有丝分裂期,增加了癌变的机会。另外DNA双链断裂还可在G2/M期激活DNA损伤检查点,细胞阻滞在G2期,完成DNA断裂修复,如果失去G2/M期检查点的阻滞,则可引起染色体重排和基因扩增,增加了癌变的机会。

4.细胞周期蛋白依赖性蛋白激酶抑制因子(cyclin dependent kinase inhibitor,CDI)表达不足   CDI通过与CDK非共价结合而抑制CDK的活性,是与cyclin-CDK作用相对抗的重要机制之一,参与细胞周期检查机制。CDI包括:①MTS1p16)基因,②MTS2(p15)基因,③Cip/1Waf1p21)基因,(①②③在抑癌基因中已介绍)④p27基因  编码蛋白为P27,可抑制所有CDK的活性,但呈化学剂量依赖性。⑤GADD45(growth arrest and DNA damage inducible 45)基因,它可使细胞阻滞于G1期,有利于维持基因组DNA的稳定性。当CDI表达不足时对CDK抑制作用减少,cyclin-CDK复合物活性增高,促进细胞增殖。

(四)端粒、端粒酶与细胞增殖

1988年发现人类和其他脊椎动物染色体端粒DNA序列是5ˊ-TTAGGG-3ˊ,

串联重复2501000次,长度为515Kb。细胞分裂时,由于DNA复制难题,

5ˊ末端有一个缺口,端粒序列不断丢失(50200 bp/次),导致端粒长度缩短,当缩短到一定长度时,细胞停止分裂,启动凋亡。因此端粒功能:维持染色体的稳定性、保证基因编码区的正常复制、作为“有丝分裂钟(生物钟)”,调控细胞寿命。1989年发现人Hela细胞中存在端粒酶。它由端粒酶RNAhTR)、端粒酶催化亚单位(hTERT)和端粒酶相关蛋白1Htep1)组成。端粒酶RNA基因定位于3q26.3,由445bp组成,其中11bp5ˊ-CUAACCCUAAC-3ˊ)是编码端粒重复序列的模板,hTR行使逆转录酶功能,直接合成串联重复序列,转移到染色体3ˊ末端,维持端粒长度,解决末端复制的难题。hTERT分子量127KD,由1132个氨基酸多肽链组成,与端粒酶活性有关,hTERT基因定位5p15.3Htep12627个氨基酸组成,与端粒酶RNA结合,且可与hTERT免疫共沉淀,与端粒酶活性有关,是端粒酶翻译后修饰的调控元件。生物胚胎期端粒酶活性很高,出生后即受到严格抑制,仅在睾丸、卵巢保持活性,增殖旺盛细胞(造血干细胞、激活淋巴细胞、上皮基底细胞)有弱活性。当组织癌变时,肿瘤细胞端粒酶再次被激活,端粒酶能在端粒显著缩短后,以稳定已降解的末端,使细胞维持不死,无限增殖。Herley等认为端粒酶的激活是细胞永生化的关键和必需,而细胞永生化也是肿瘤细胞特征之一。从此揭开了端粒、端粒酶与肿瘤细胞增殖癌变关系的研究序幕。实验证明绝大多数恶性肿瘤细胞端粒酶活性增高,PKC激动剂佛波酯可以使端粒酶活性增高,而PKC抑制剂H7能抑制端粒酶活性,因此PKC可能在端粒酶的激活过程中发挥重要作用。

除此之外,凋亡基因失活、抗凋亡基因激活,“该”死的细胞不死,使细胞持续生长,细胞数目增多;胞内Ca2+-CaM增加,使胞质微管形态解聚,也能促进细胞增殖;PKC激活Na+/H+交换,使细胞内碱化(pH↑),有利于细胞增殖;

黏附分子如整合素与配体结合后,可激活多条信号转导途径,如Ras-Raf-MAPKJNK通路,MAPKJNK可协同调节细胞周期蛋白D1的促进子,从而促使细胞通过G1期,加快细胞周期进程。导致细胞的增殖。

 

六.关于肿瘤细胞分化障碍

正常细胞呈有控分化,以功能专一化作为特征,其主要标志是合成具有特殊功能的蛋白质、停止或失去细胞分裂功能、出现该细胞特有的表型结构。肿瘤细胞分化障碍(未分化或去分化),其主要标志是肿瘤细胞异形性:细胞大小不等、形态各异、核增大不规则、核/质比例增大、核仁多而大、染色体呈多倍体或非整倍体、细胞分裂相增加、细胞器结构趋于简单化;肿瘤细胞极性消失:细胞排列紊乱、细胞核长轴方向与细胞生长轴方向不一致;肿瘤细胞幼稚性:具有胚胎细胞的特征、结构上常有表面糖蛋白脂肪蛋白质的异常、细胞表面不规则突起多等。从发育生长学观点可认为恶性肿瘤是一类分化障碍性疾病,细胞分化障碍是肿瘤细胞又一重要的特性。

有关肿瘤细胞异型性解释,作者认为肿瘤组织中除了肿瘤细胞外,还有肿瘤血管内皮细胞、成纤维细胞,同时肿瘤细胞通过旁分泌作用,影响周围正常细胞的生物学行为,使正常细胞发生转化,也具有肿瘤细胞一些特性。这部分细胞由于不是肿瘤细胞增殖分裂产生,而是肿瘤细胞对正常细胞作用的结果,因此二者形态结构可以不同,产生肿瘤细胞异型性。作者用肿瘤细胞培养上清液诱导正常血管内皮细胞、成纤维细胞80代后,发现这些诱导细胞生物学行为与亲代细胞不同,反而具有肿瘤细胞的特征,如端粒酶活性增加,细胞群体倍增时间缩短、分裂指数增高,细胞内蛋白质合成增加,而且bcl-2MMP-9PCNA TGF-β1TN表达增强,细胞游走、侵袭及血管形成能力增加,用此来证实体内肿瘤细胞对周围正常细胞的转化作用。

肿瘤细胞分化障碍的机制如下。

(一)细胞增殖和分化脱偶联: 

在干细胞成熟初期阶段,细胞一方面定向分化,另一方面以较快的速度分裂增殖。其子代细胞大多数是分化不全的细胞,经过一定时间后,随着细胞分化程度的增强,其增殖速度也逐渐减慢,以至于不再分裂,但尚可继续分化,直至终末分化为完全成熟的细胞。但在部分组织和器官还保留少量的未分化的干细胞,这些干细胞在必要时能扩增并分化成一定类型的细胞,用以补充损伤的或死亡的细胞。实验证明在致癌因素作用下,这种分化不全的干细胞大量增殖即可成为肿瘤细胞(因细胞分化不全对致癌物分解解毒能力差)(肿瘤起源于未分化的干细胞);致癌物作用于接近分化的终末细胞,结果形成良性肿瘤;各种类型白血病发生可能是细胞分化过程中不同分化阶段被阻断的结果。如肿瘤起源于成熟细胞或功能细胞,肿瘤的发生是生物学上逆分化或去分化。分化信号可使细胞从细胞周期撤离,限制细胞分裂,使细胞出现终末分化;增殖信号可使细胞进入细胞周期,细胞继续分裂,但不出现细胞分化。现在认为细胞恶变是细胞增殖失控和分化受阻,使两者偶联解除而平衡失调的结果,在肿瘤的发生中首先是细胞不断增殖而永生化,在此基础上细胞分化受阻,是增殖与分化不全并存。实验证明:将分化缺陷或增殖突变的细胞分别注入裸鼠,各只有10%-20%发生恶性肿瘤,而注入两者都有缺陷的突变细胞,有66%发生恶性肿瘤。现在发现了介导增殖与分化的信号转导途径及它们与转录因子之间的交叉连系,人们可藉此研究抑制细胞增殖,促进细胞分化的方法和途径。

(二)基因表达失调

1.细胞癌基因的激活  细胞癌基因在细胞生长和分化中起着十分重要的调控作用,各基因产物之间的协调作用形成一个有序的细胞调节网络。细胞分化障碍可能是由于致癌因素直接或间接作用于细胞基因组,使与增殖有关的细胞癌基因启动,其产物为生长因子、生长因子受体、具有G蛋白与PTK的活性、核转录因子,这些产物可抑制细胞分化。如erb-A产物为甲状腺素受体拮抗物,可阻止细胞对甲状腺素的反应,抑制靶细胞的分化;M1白血病细胞中有C-myb表达↑,用细胞分化诱导剂促进肿瘤细胞分化时常伴C-myb表达↓。

2.抑癌基因的缺失或失活  抑癌基因中不少为促分化基因,如视网膜母细胞瘤易感基因(Rb基因,它在13q14),其编码产物促进视网膜母细胞分化,如果Rb基因有遗传性缺失或后天性失活,可产生视网膜母细胞瘤;肾母细胞癌易感基因(WT1),其编码产物促进肾母细胞发育分化,WT1基因丢失或失活,可引起肾母细胞瘤;神经纤维瘤基因(NF1)的编码产物促进神经细胞发育分化,其丢失或失活,可导致神经纤维瘤。

3.胚胎性和∕或体细胞基因表达失调  细胞分化是选择性基因激活、转录和翻译的结果。致癌因子使胚胎性基因重新表达时,它们的编码产物往往在某些肿瘤中高表达,如肝癌患者血中出现高浓度甲胎蛋白,肝癌细胞表达胎儿型醛缩酶A(成人为B型)。成熟体细胞特异性基因表达受阻,使肿瘤细胞缺乏正常细胞的功能,细胞分化不全,如胰岛细胞瘤不能合成胰岛素,结肠肿瘤不能合成粘蛋白。

根据肿瘤的形成上由于基因表达失调的理论,人们提出用分化诱导剂来诱导肿瘤细胞分化,将其恶性状态重新恢复为正常状态。肿瘤的诱导分化治疗为肿瘤治疗开辟了新的途径。

 

七.关于肿瘤细胞侵袭、转移 

肿瘤细胞在体内增殖生长到一定程度,部分癌细胞从原发瘤部位向邻近组织侵犯和占领,并在该处继续增殖生长,从而破坏邻近组织,这个过程称为侵袭。肿瘤细胞从原位瘤上脱离下来,通过静脉、淋巴等体内管道,在身体其它部位继续增殖生长(形成继发瘤),这个过程称为转移。肿瘤细胞侵袭转移是很复杂的过程,肿瘤细胞脱离原发瘤,穿出瘤体周围基底膜,穿过血管或淋巴管基底膜进入血管或淋巴管,经循环运行在适当部位穿出管壁基底膜,继续增殖生长形成转移灶。这过程还需肿瘤细胞的多次黏附、运动、基质降解酶产生等,同时也还需要肿瘤组织内基质细胞(血管内皮细胞、成纤维细胞等)共同参与来完成。肿瘤细胞侵袭转移也是肿瘤细胞重要的特征。

1.肿瘤的新生血管

一个肿瘤血管内皮细胞可支持50100个肿瘤细胞生长,因此肿瘤细胞的生长和转移都依赖于新生血管形成。肿瘤新生血管与正常新生血管不同,它血管壁薄弱,只有一层内皮细胞缺乏平滑肌、神经末梢及相应的淋巴管道,肿瘤血管内皮细胞的线粒体、核糖体、粗面内质网等细胞器增多,显示高度扩增状态,其扩增率高出正常的50100倍;并出现异常表达,如上调整合素、血管生长因子受体、CD44等,下调ICAM-1等,导致其渗透性、凝血控制、白细胞黏附的特性改变;还表达一些扩增相关抗原,如前列腺特异膜抗原、EndosialinEndoglin等;Croix等用SAGE方法发现肿瘤血管内皮细胞有79个异常转录子,有36个上调至少10倍,其中6个编码新生血管标志物,7个参与胞外基质形成与重塑。肿瘤血管内皮细胞分泌胶原酶和纤溶酶原激活因子使肿瘤细胞从原发瘤部位脱落;由于肿瘤新生血管内皮细胞的基底膜不完整,使肿瘤细胞更易穿透,并比正常成熟毛细血管更易接受肿瘤细胞,而且其本身也具有侵袭性,这样为肿瘤细胞的转移提供途径。随肿瘤新生血管密度增加,肿瘤细胞容易进入血液循环,从而转移也明显增加,因此肿瘤新生血管密度可作为判断预后一项指标。如光镜检查每个200倍视野内血管数为3467根,转移率45%;血管数为68100根,转移率71%;血管数大于100根,转移率100%。

肿瘤组织中的新生血管是由邻近组织中小血管壁芽生并向肿瘤组织内生长、成熟形成的。肿瘤组织上调血管内皮细胞生长因子(VEGF),VEGF特异地作用于血管内皮细胞,发挥促分裂、趋化作用;VEGF增加血管通透性,使管内纤维蛋白原等血浆蛋白外渗,形成纤维蛋白凝胶体,为内皮细胞与成纤维细胞的迁移、新生毛细血管网的建立提供必要的基质;VEGF诱导血管内皮细胞产生的间质胶原酶、纤溶酶原激活物,不仅满足血管新生对基质降解的要求,而且也有助于肿瘤细胞从肿瘤组织中脱离。新生血管的基底膜不完整也为肿瘤的侵袭转移提供便利条件。其他血管生成诱导因子如生长因子(TGF-α、PDGFHGFEGF等)、细胞因子(IL-8等)、黏附分子(E-选择素、αβ整合素等)、蛋白水解酶等通过直接或间接途径刺激肿瘤血管内皮细胞增殖、游走、管腔样形成。肿瘤新生血管参与了肿瘤发生、发展、转移的分子生物学改变。

体内也存在血管新生抑制因子如血管新生抑制素、内皮细胞抑制素、金属蛋白酶组织抑制物(TIMPs)、软骨源性抑制剂、凝血酶敏感蛋白-1、血小板因子-4、干扰素诱导蛋白-10、迁移抑制因子等,它们大都是通过对肿瘤血管内皮细胞的间接作用或对内皮细胞胞外成分作用而实现对血管新生的抑制。在肿瘤发生时它们被抑制,血管新生正负调节因子之间失去平衡,血管新生增加。

针对肿瘤的血管新生异常,近年来提出了抗血管新生治疗,为治疗肿瘤提供了新途径。

2.肿瘤组织内的成纤维细胞

80年代开始研究肿瘤组织中的成纤维细胞与肿瘤细胞相互作用,肿瘤细胞促进成纤维细胞形态与生理功能发生变化,表达多种细胞因子(如IGF-1TGF-α、bFGFVEGFHGF等)、蛋白酶(如PAMMP-129等)、黏附分子(如FNTN等);而成纤维细胞表达这些物质也能增加肿瘤细胞的恶性表型,对肿瘤发生、生长、血管形成、侵袭与转移有重要作用。

1)成纤维细胞促进肿瘤的发生:合并成纤维细胞皮下注射低于致瘤量(102)的鼠源、人源肿瘤细胞裸鼠移植瘤实验,其肿瘤发生率和生长率均比单独进行裸鼠移植瘤实验显著增加,而且潜伏期缩短。

2)成纤维细胞促进肿瘤生长:在非小细胞性肺癌(NSCLC)中IGF-1高出1.47倍,而NSCLC株只表达IGF-1R,体外发现成人肺成纤维细胞分泌IGF-1样蛋白质,并且使IGF-1R活性增加3倍,因此成纤维细胞通过分泌IGF-1及活化IGF-1R而刺激体内NSCLC生长;卵巢癌成纤维细胞分泌TGF-α刺激自身与卵巢癌上皮细胞生长。

3)成纤维细胞促进肿瘤血管形成:有学者用免疫电镜与原位杂交研究肝细胞性肝癌(HCC)血管形成机制,发现HCC基质成纤维细胞有VEGF表达,其水平随血管化程度增加而增高;用三维培养研究前列腺癌血管化时发现,胶质基质中包被成纤维细胞时血管芽形成明显。

4)成纤维细胞促进肿瘤细胞侵袭、转移:①成纤维细胞表达促进肿瘤细胞侵袭转移的细胞因子   成纤维细胞分泌HGF/SF,能促进肝癌细胞分裂增生及刺激细胞游走;硬胃癌中成纤维细胞分泌大量的HGFTGF-β显著刺激硬胃癌细胞的侵袭力;胆囊成纤维细胞分泌大量HGF促进胆囊癌侵袭与转移;肺与瘤周成纤维细胞产生移动刺激因子(FMSFs),特异刺激肺转移肉瘤细胞SYN-1移动。②成纤维细胞表达促进肿瘤细胞侵袭转移的蛋白酶   定位研究发现肿瘤组织内成纤维细胞有uPA表达,uPA参与组织降解。正常喉粘膜向肿瘤转化时伴有uPA水平及活性的增加(P<0.01,并且与肿瘤转移能力正相关(P<0.05)。有报道结肠癌组织中成纤维细胞可表达基质降解酶与明胶酶A。在乳腺癌组织中,成纤维细胞表达MMP-1MMP-9。③免疫组化与原位杂交发现肿瘤组织成纤维细胞上蛋白酶活化受体(PAR-1/PAR-2)明显上调,而正常、良性瘤组织成纤维细胞未见表达。基质细胞因子-1SDF-1/CXCR4受体配体系统促进肿瘤细胞游走与血管形成,在胰腺癌发展过程中有重要作用,定位研究发现胰腺癌组织成纤维细胞表达SDF-1,胰腺癌细胞与瘤周血管内皮细胞表达CXCR4。④成纤维细胞表达促进肿瘤细胞侵袭转移的黏附分子,黑色素瘤细胞表达的TGFβ-1可刺激成纤维细胞表达纤维连接素(FN),FN能明显增强高、降低转移黑色素瘤的游走能力。许多实体瘤成纤维细胞过表达细胞黏合素(TN),TN参与细胞许多功能:黏附、游走、胚胎发育、伤口愈合与致瘤转移,TN对胶质瘤细胞低剂量黏附,高剂量促游走。卵巢癌基质TN表达与强度明显高于卵巢瘤基质,卵巢癌腹水成纤维细胞TN高出肿瘤细胞100多倍。

成纤维细胞活化蛋白(fibroblast activation protein, FAP)在90%的肺癌、结肠癌、乳腺癌等上皮性肿瘤活化基质成纤维细胞中选择性表达,无个体差异;成纤维细胞基因稳定,不易耐药;FAP成纤维细胞构成了5095%肿瘤基质成分,靶位丰富;FAP基质细胞在血管内皮细胞周围,药物易达到。因此FAP成为体内诊治很有前景的靶分子,鼠源FAPmAbF19已用于临床诊断与实验性治疗(可消除肿瘤的结构与功能支持系统,以便有效地消灭肿瘤),也为肿瘤治疗提供新途径。

 

四、英语学习材料

NEOPLASIA

by Robert C. Mellors, M.D., Ph.D.

Etiology of Cancer: Carcinogenesis

Introduction

Animal tumors and the neoplastic transformation of cultured cells can be induced experimentally by well-characterized biological, chemical, and physical agents (carcinogens). Some of the known carcinogenic agents are also natural causes of cancers in man and animals. Oncogenic RNA retroviruses are etiological agents of spontaneously arising cancers in several species of animals, including primates, other mammals, birds, and reptiles. One must be aware also that the bacterial pathogen Helicobacter pylorus, a common cause of gastric ulcer, is considered a major factor in the development of human gastric cancer. Chemical carcinogenesis by tobacco smoke products is a major cause of common lung cancers. Physical carcinogenesis by ionizing radiations poses a potential world-wide threat in this nuclear age. Skin carcinogenesis by solar ultraviolet radiation is expected to increase even above its present high incidence as the ozone layer of the atmosphere undergoes depletion. Nevertheless, the specific causes of most common human cancers - of breast, colon, rectum, lymph nodes, uterus, bladder, pancreas, bone marrow, stomach, and so on - remain unknown.

As with many other diseases, both genetic and environmental factors are implicated in the etiology of human cancers. Recent advances in the molecular biology of cancer, stemming from the study of oncogenic viruses and transforming DNA, are providing new ways of investigating transforming genes ("oncogenes") and cellular pathways which are involved in the mechanisms of viral, chemical, and physical carcinogenesis. New findings in the molecular genetics of cancer hold promise of leading to a better understanding of the underlying mechanisms of common human cancers of unknown etiology.

Oncogenic Viruses


Oncogenic viruses can produce tumors in animals (mammals, birds, and other vertebrates) or transform cultured cells to a neoplastic state and comprise DNA viruses and RNA viruses.

Oncogenic DNA Viruses

These include some members of five of six major families of DNA viruses, namely: papova-, hepatitis B-, adeno-, herpes-, and poxviruses. In general, the infection of cells with an oncogenic DNA virus may result either in productive lytic infection with cell death and release of newly formed virus or in cell transformation to the neoplastic state with little or no virus production but with integration of viral genetic information into the cell DNA.

The papovavirus family consists of: the papilloma viruses of cattle, cottontail rabbit, and humans; the polyomavirus of the mouse; and the simian virus 40, originally termed simian vacuolating agent. The genomes of polyomavirus and SV40 are double-stranded circular DNA molecules with sizes of about 5 kilobase pairs (kb) and comprise two chief groups of genes which are associated with early and late events in the replication cycle. The "early" genes are transcribed soon after infection of a cell and are involved in producing "functional" proteins which participate in viral DNA synthesis but are not found in the virions themselves. The "late" genes encode structural proteins of the viral coat and capsid. In productive lytic infection by these viruses, in permissive hosts, early proteins are formed but then disappear and the structural proteins are assembled into viral particles. In non-permissive cells, derived from an animal species which is not a natural host of the virus, neoplastic transformation can occur. When stable transformation does take place, viral DNA is inserted or "integrated" into the cellular chromosomal DNA; some of the early proteins are persistently synthesized; and viral particles are not produced.

The early proteins found in tumors induced by polyomavirus and SV40 are termed "T" (for tumor) antigens. Polyomavirus produces three (large, middle, small) T antigens, of which middle T antigen is necessary for transformation. This early protein is bound to the plasma membrane of transformed cells. SV40 produces only two (large, small) T antigens. SV40 large T antigen maintains the transformed state.

Adenoviruses are found in many species of animals, including humans. The genomes of adenoviruses are double-stranded linear DNA molecules with sizes of about 35-40 kb. In cells transformed by oncogenic adenoviruses, a region of the genome encoding early proteins is always present, and a variety of early proteins can be detected. Among them, the major E1a proteins function in the initiation and maintenance of transformation.

The genomes of herpesviruses are double-stranded linear DNA molecules with sizes in the range of 140-170 kb. The initiation of transformation by oncogenic herpesviruses appears to be related to the presence of certain DNA sequences although no single T antigen is found.

In summary, the mechanisms of DNA virus oncogenesis indicate that oncogenesis is an attribute of the viral DNA, that the viral DNA is integrated into the host cell DNA, and that the protein products of viral genes maintain transformation to the neoplastic state.

DNA viruses, along with other environmental and hereditary factors, are associated with the etiology of several types of human cancer. The Epstein-Barr virus (EBV), a type of herpesvirus and the cause of infectious mononucleosis, may be involved in the causation of Burkitt's lymphoma in Africa and sporadic cases elsewhere, as well as nasopharyngeal carcinoma, and viral DNA and various EBV-determined antigens are detectable in the tumor cells. Hepatitis B virus (HBV) is considered to have a causal role in primary hepatocellular carcinoma, one of the most common forms of cancer in Asia and worldwide, and viral DNA is integrated into the tumor cells in some cases. Hepatitis C virus (HCV) is similarly involved in hepatic carcinogenesis. A newly recognized human herpes virus, first described in 1994 and now designated HHV type 8 or KSHV (Kaposi sarcoma-associated herpes virus), is implicated as a candidate etiologic agent in AIDS-associated KS, the most common malignant tumor seen in patients with acquired immunodeficiency syndrome, as well as in classic (sporadic) KSV unrelated to AIDS and in AIDS-associated B-cell lymphoma of body cavities (primary effusion lymphoma). Some types of sexually transmitted human papilloma viruses (HPV) are associated with precursor lesions of squamous carcinoma of the uterine cervix. HPV viral DNA is extrachromosomal in the precursor lesions, and infectious virus is produced. HPV types 16 and 18 are associated both with precursor lesions and with invasive cervical carcinoma. Viral DNA is integrated into the cancer cells in many studied cases, but additional agents or factors may be involved at different stages of the progression to invasive carcinoma.

Oncogenic RNA Viruses

Of the many families of RNA viruses, only members of the retrovirus family are capable of inducing animal tumors and transforming cultured cells. The oncogenic RNA viruses (RNA tumor viruses) are a subfamily of the retroviruses and are a cause of naturally occurring tumors and leukemias in a wide range of vertebrate animals, including mammalian, avian, and reptilian species. The RNA tumor viruses are classified according to their natural host, such as avian, murine, feline, and primate leukemia/ sarcoma virus species. Two unique types of human retroviruses, human T-cell leukemia viruses (HTLV) types 1 and 2 are etiologically associated with human leukemias.Human immunodeficiency virus (HIV), a member of the lentivirus subfamily of retroviruses and the causative agent of AIDS, predisposes to opportunistic infections, including those caused by tumor-related viruses such as KSHV and HPVs.

The genomes of retroviruses are diploid and are single-stranded RNA molecules with a size range of 3-9 kb. All retroviruses contain a reverse transcriptase (RNA-directed DNA-polymerase), and their replication requires the synthesis of a double-stranded DNA intermediate of the RNA genome. Some of the virally determined DNA becomes inserted, or integrated, into the host cell DNA as provirus DNA. Typically, there are three retroviral genes which encode proteins necessary for viral replication: gag (group-specific antigen) gene which encodes internal structural proteins of the virus; pol gene which encode reverse transcriptase;and env gene which encodes envelope proteins that enclose the virus particles and largely determine the host range.

Most oncogenic retroviruses also have another gene known as a transforming gene or oncogene and termed v-onc (and usually identified by a 3-letter code, such as src in the prototype Rous sarcoma virus). Under the influence of the viral promoter sequence, the v-onc gene is transcribed along with other viral genes and is responsible for neoplastic transformation of the cell. All rapidly transforming retroviruses possess one, or rarely two, unique oncogenes of which more than 20 have been isolated and characterized. A short list of retroviral oncogenes is given in the table.

Table: Retroviral Oncogenes (partial list)

Oncogene (v-onc)

Prototype Retrovirus

Species of Origin


src

Rous sarcoma virus

Chicken

myc

Avian myelocytomatosis virus

Chicken

erb A, erb B

Avian erythroblastosis virus

Chicken

myb

Avian myeloblastosis virus

Chicken

H-ras

Harvey rat sarcoma virus

Rat

K-ras

Kirsten murine sarcoma virus

Mouse

abl

Abelson murine leukemia virus

Mouse

fes

Feline sarcoma virus

Cat

sis

Simian sarcoma virus

Monkey


Oncogenes and Proto-oncogenes


It was early postulated (oncogene hypothesis) that retroviral oncogenes or their precursors (protovirus hypothesis) were naturally present in the genomes of virtually all normal vertebrate cells.

Molecular evidence suggesting the normal cellular origin of retroviral oncogenes was first obtained by showing that radiolabelled DNA from the avian retroviral oncogene src hybridized specifically to normal uninfected avian cellular DNA as well as to normal mammalian DNA and even normal human cellular DNA. All retroviral oncogenes are now known to have hybridizing homologs, that is, close relatives, in the genomes of virtually all normal vertebrate cells. The normal host cell homologs of v-onc genes are called c-onc genes or proto-oncogenes.

The retroviral homologs found in normal cellular DNA have the usual structure of cellular genes, possessing both exons and introns, whereas the oncogenes in retroviruses do not have introns. It has been concluded that the oncogene analogs found in normal cellular DNA represent native cellular DNA and are not of viral origin. It appears, then, that retroviral oncogenes are copies (allowing for subtle differences in gene sequences) of normal cellular genes which were picked up and transduced into the retroviral genome by pre-existing retroviruses. Nevertheless, retroviral oncogenes usually show structural mutations or changes in expression relative to the corresponding proto-oncogenes. Since proto-oncogenes are highly conserved in vertebrates and are demonstrable in the cellular genomes of virtually all metazoans, their roles in cellular functions are apparently of fundamental importance. The gene products of proto-oncogenes have been identified as proteins (growth factors, growth-factor receptors, signal transducer proteins) with known functions in normal cells.

Direct evidence for the activation of proto-oncogenes to transforming genes in-vivo, independently of any retroviral gene participation, has been obtained by isolating proto-oncogenes, attaching them to promotor or enhancer sequences, and introducing this DNA into one-cell mouse embryos (fertilized eggs) and thus ultimately the germ cells of so-called transgenic mice. The promotor causes a high rate of transcription ("activation") of the introduced proto-oncogene resulting in a high incidence of malignant tumors in some of the progeny mice. In other experiments, "weak" retroviruses which lack a separate viral oncogene have been shown to produce tumors only when, by chance, the proviral DNA is inserted into the cellular genome next to a cellular proto-oncogene, which is then activated through the effect of the inserted viral promoter.

In summary, the oncogene theory postulates that the oncogenes of transforming retroviruses are derived from normal cellular genes (proto-oncogenes); and that an increased expression (activation) of proto-oncogenes or an inappropriate expression of mutated forms of proto-oncogenes, occurring spontaneously or induced by cancer-causing agents, contributes to neoplastic transformation and the development of cancer, including human cancer.

It is now recognized, as you shall learn, that sequential mutations or inappropriate expressions of several different classes of cellular genes [oncogenes, tumor suppressor genes, DNA nucleotide mismatch repair genes, and genes that mediate programmed cell death (apoptosis)] are probably involved in the usual multiple-step process that leads to human cancer.

Oncogenes and Human Cancers


Most of the known retroviral oncogenes have hybridizing homologs in normal human cellular DNA (proto-oncogenes). Oncogenes are activated in somatic cells in many forms of human cancer, including carcinoma, sarcoma, leukemia, and lymphoma. The chief mechanisms of oncogene activation are chromosomal translocation, point mutation, and gene amplification (Table).

Table: Activation of Cellular Proto-oncogenes in Human Cancers

Proto-oncogene

Activation by

Chromosomal Change

Associated Cancer


c-myc

Genetic rearrangement

Translocation: 8-14, 8-2, or 8-22

Burkitt's lymphoma

c-abl

Genetic rearrangement

Translocation: 9-22

Chronic myeloid leukemia

c-H-ras

Point mutation

 

Bladder carcinoma

c-K-ras

Point mutation

 

Lung and colon carcinoma

N-myc

Gene amplification

 

Neuroblastoma


Burkitt's Lymphoma

In the cells of this human B cell lymphoma there is often a translocation between chromosomes 8 and 14, or between 8 and 2 or 22. This change transposes the cellular myc gene, which is normally on chromosome 8, into proximity with a predictably active promotor (in B lymphocytes) involved in immunoglobulin (Ig) synthesis. (Ig heavy chain loci are on chromosome 14, Ig light chain loci are on chromosomes 2 and 22). The translocation interrupts the normal transcriptional control of the myc gene and leads to amplification of the amount of the myc-encoded protein, thereby thrusting the myc proto-oncogene into the role of an oncogene.

Chronic Myelogenous Leukemia (CML)

The leukemia cells of virtually all (90-95%) patients with CML have a characteristic cytogenitic abnormality called Philadelphia chromosome, Ph', (Nowell, P.C., and Humperford, D.A., Science 132:1497, 1960) resulting from a reciprocal translocation between the long arms of chromosomes 9 and 22 (Rowley, J.D., Nature 243:290, 1973).

In this translocation, the proto-oncogene c-abl is translocated from its normal location on chromosome 9 to the break point region in chromosome 22, resulting in the generation of a fusion gene and its protein product (BCR-ABL), a constitutively activated mutant tyrosine kinase with proven oncogenic activity. Recent clinical trials with a selective inhibitor (Imatinib, formerly STI 571) of BCR-ABL tyrosine kinase in CML provide cytogenetic and hematologic evidence of improvement in patients with the chronic phase of CML (Drucker, B.J., et al., N Eng J Med 344: 1031-37, 2001; Kantarjian, H., et al., ibid. 346:645-52, 2002).

Human Carcinomas

The cellular DNA of tumor cells can be extracted and, following co-precipitation with calcium phosphate, introduced into the nuclei of selected cells (usually the mouse fibroblast cell line NIH 3T3) in vitro, a process termed transfection. Those cells which take up intact segments of DNA that induced the neoplastic state become transformed themselves. One can then isolate and sequence the transforming genes from these cells. In one of the earliest reported studies, such a transforming gene was identified in cells of the human bladder carcinoma cell line T24. When sequenced, this human oncogene in T24 cells was found to be a homolog of the v-H-ras gene (known to induce sarcomas and leukemias in susceptible murine animals). Furthermore, the human oncogene differed from the corresponding normal human proto-oncogene by only a single base substitution (G for T), a point mutation leading to the substitution of a single aminoacid (valine for glycine) at position 12 of the 21,000 m.wt. encoded protein.

Other transfection studies have shown that the transforming genes of a number of other human cancers are also homologs of members of the ras family: for example, the transforming genes of cell lines and primary carcinomas of human lung and colon are homologs of the v-K-ras gene. (as noted in the previous table)

An important finding is that when primary cultures of mouse embryo fibroblasts, rather than established cell lines such as NIH 3T3, are used as the DNA recipients in transfection assays, at least two co-operating oncogenes (often myc and ras) are usually required for complete neoplastic or "tumorigenic" conversion. This observation is consistent with the prevailing view that carcinogenesis is a multiple-step process. At this writing, more than 60 different oncogenes have been characterized in avian and mammalian, including human, species.

Role of Individual Cellular Oncogenes in Neoplastic Transformation

One of the simplest classifications of oncogenes, on the basis of the localization of their encoded proteins either in the cytoplasm or the nucleus, is useful in grouping those with similar roles. The accompanying table indicates the cellular localization of proteins encoded by some cellular proto-oncogenes and, for comparison, transforming proteins encoded by oncogenic DNA viruses.

Table: Proteins Encoded by Proto-oncogenes and DNA Tumor Viruses

Localization

Proteins Encoded by

 

Cellular Proto-oncogene

DNA Tumor Virus


Nuclear

myc

SV40 large T

 

N-myc

Polyoma large T

 

myb

Adenovirus E1a

Cytoplasmic

ras

Polyoma middle T

 

abl

 

 

src

 

 

erb B

 

 

sis

 


Most of the nuclear cellular oncogenes, that is, those with nuclear localization of the encoded proteins, appear to have similar, although not identical, roles. The most predictably observed trait which, as oncogenes, they confer on the affected cells is "immortalization", or the ability to be passaged in cell culture continuously without limit. Related traits which appear to be conferred are an incresed plating efficiency and the ability of cells to divide in the presence of low concentrations (0.5%) of serum. Nuclear cellular oncogenes show little ability to confer anchorage independence (growth in the absence of an added support structure) on cells in which they are activated and likewise only minimally induce morphologic changes, such as multilayering, of cultured cells.

Cellular oncogenes with cytoplasmic protein products are relatively "weak" in inducing immortalization of cells in culture, but, in contrast to nuclear oncogenes, are strong in conferring the morphologic changes of transformation as well as in conferring anchorage independence.

Neither nuclear nor cytoplasmic cellular oncogenes, acting alone, appear able to induce full transformation of cells. Their actions in transformation appear to be complementary. Many types of cells have been transformed, and rendered "tumorigenic" in animals, by transfection with collaborating pairs (one nuclear, one cytoplasmic) of cellular oncogenes in various combinations.

Conversion of Cellular Proto-oncogenes to Oncogenes

The known mechanisms of proto-oncogene activation and well-studied examples include:

·                              translocation: c-myc, c-abl;

·                              promotor insertion: c-myc, c-erb B;

·                              point mutation: c-ras;

·                              deletional mutation: c-erb B;

·                              amplification: c-myc.

Nuclear oncogenes of cellular origin are converted from proto-oncogenes by processes (translocation, promotor insertion, amplification) which lead to an increased level (or amount) of their encoded proteins. The disordered regulation of c-myc expression related to the 8-14 chromosomal translocation in Burkitt's lymphoma is the best documented example. In this genetic rearrangement, the normal promotor-enhancer regulators for c-myc are removed and replaced by those from predictably active immunoglobulin genes, resulting in amplification of the amount of the myc-encoded protein.

Most of the cytoplasmic oncogenes of cellular origin are derived from proto-oncogenes by mutations which alter the structure of the encoded proteins. The c-H-ras gene whose activation is associated with several human carcinomas is an important example. Point mutations of the proto-oncogene involving aminoacids at residues 12, 13, or 61 induce transformation even when the 21,000 m.w. protein, p21ras, is present at low levels. Further studies of cytoplasmic cellular oncogenes, such as src, erb B, and neu (related to erb B), show that structurally altered protein products cause transformation, while over-expression of the normal protein brings about little change.

An important cytoplasmic proto-oncogene is the abl gene which is present at the site of the 9-22 translocation resulting in the formation of the Philadelphia (Ph') chromosome typically seen in chronic myelogenous leukemia (CML). The abl protein is present in increased amounts in the tumor cells, but, apparently more important, the protein is altered (the translocation removes its amino terminus). Over-expression of the normal intact abl protein seems to cause no effect.

In summary, many cytoplasmic proto-oncogenes appear to be converted to oncogenes by mutations which alter the structure of their encoded proteins. In these circumstances, the amount of the normal protein appears to be of little importance in cellular transformation.

While the genetic changes in Burkitt's lymphoma and chronic myelogenous leukemia occur in cells of hematologic origin, no single genetic abnormality has thus far been identified as the transforming event in common human cancers of epithelial origin.

Cellular Functions of Oncogene-Encoded Proteins

The functions of proteins encoded by selected protooncogenes (and oncogenes) are given in the accompanying table.

Table: Cellular Functions of Proto-oncogene Encoded Proteins

Function

Protein

Protoonco-
gene

Associated Human Cancers


Growth factor

PDGF

sis

Osteosarcoma

Growth-factor receptor

EGF receptor

erb B

Breast, lung, ovarian cancer

Post-receptor signal transduction

GTP-binding protein

ras

Lung, colon, pancreatic cancer

Nuclear transcription regulator

myc protein

myc

Breast, colon, lung cancer, Burkitt's lymphoma


Most, if not all, of the many proteins encoded by cellular oncogenes are involved in the "growth factor-receptor-response" pathways of transmission of growth stimulatory signals from the cell surface to the nucleus, culminating in the transcription of certain genes and in DNA synthesis.

Aberrations in the normal process of transmembrane signaling involving growth factors (GFs), GF receptors, post-receptor "transducer" proteins, and nuclear controls may be caused by activated oncogenes and result in the abnormal growth characteristics of neoplastic cells.

·             Growth factors (GFs) are ubiquitous polypeptides that are produced and secreted by cells locally and that stimulate cell proliferation by binding to specific cell-surface receptors on the same cells (autocrine or autostimulation) or on neighboring cells (paracrine stimulation).

·             Conceptually, uncontrolled autostimulation from the persistent over-production of GFs may convert a cell to the neoplastic state. Human platelet-derived growth factor (PDGF), so named because it is released from platelets during blood clotting, is a major growth factor recoverable from serum. PDGF is apparently encoded by c-sis, the normal analog of v-sis which is the transforming gene of simian sarcoma virus (SSV). Cultured cells infected with SSV produce a PDGF-like mitogen which binds to PDGF receptors and stimulates the cells to proliferate in an uncontrolled manner, resulting in transformation.

·             Structural changes or amplification of GF receptors may promote neoplastic transformation. If GF receptors are changed in a way that continually presents cells with growth stimulatory signals, even in the absence of GFs, cells may respond as though high levels of GFs were present. One of several examples, all in the group of cytoplasmic oncogenes, can be mentioned. A portion of the human epidermal, growth factor receptor protein (now called Her-2/neu or Her-2) is similar to the proto-oncogene encoded erb B protein. Her-2 protein is over expressed in tissue and serum of ~25-30% of patients with breast cancer. Trastuzumab (Herceptin) is a recombinant humanized monoclonal anitbody that selectively binds to and mediates antibody-dependent cytolytic destruction of Her-2 protein and is approved for use in patients with metastatic breast cancer whose tumors over express Her-2.

·             Autonomy to GFs may be produced by changes in post-receptor "transducer" proteins that enable them to transmit growth stimulatory signals without prompting by a GF receptor. Ras-encoded proteins are guanosine triphosphate (GTP)-binding proteins that function as post-receptor signal transducers (cyclic on-off switches) for growth stimulatory signals.. Ras proteins are located in the plasma membrane and mediate the passage of growth signals from outside to inside the cell and to the cell nucleus, thus initiatiating the cell cycle and DNA synthesis. Mutations in ras proteins, potentially associated with a continuous growth signal that cannot be deactivated, are found in about 30% of human cancers (Wittinghofer, F., Nature 394: 317, 1998).

·             The cell division cycle is normally monitored at critical check points along the mitogenic signaling pathway. Deregulation of the cell cycle at the critical transition phase from G1 (resting) to S (synthesis of DNA) occurs in many types of human cancer. Transcriptional amplification of myc and related nuclear proteins is also associated with unregulated cell proliferation and transformation. Additionally, the microtubule network is necessary for interphase and mitotic cellular events, and paclitaxel (Taxol) which disrupts microtuble assembly is approved for use in advanced cancers of breast, ovary and lung.

In summary, the activation of proto-oncogenes to cancer-associated oncogenes represents a "gain of gene function" (dominant allele) mutation involving pathways of transmission and transduction of mitogenic signals from cell to cell and from cell surface to nucleus and the activation of certain nuclear genes, culminating in DNA synthesis, unregulated cell division, and neoplastic transformation.

Tumor Suppressor Genes


Most studies of genes and growth factors involved in carcinogenesis have focused on functions that have a positive stimulatory effect on cell proliferation and, hence, neoplastic transformation. While the activation of some genes may result in cancer, it is reasonable to suppose that the activation of other genes may suppress the development of cancer. The existence of tumor suppressor genes, or so-called "anti-oncogenes", in normal cells is suggested by the results of somatic cell hybridization experiments in which fusions of malignant tumor cells, including those with activated oncogenes, with normal diploid fibroblasts were usually found to yield non-malignant hybrid cells. It is possible, therefore, that the loss or inactivation of putative tumor suppressing genes may remove a block to cell proliferation and the development of the neoplastic state. Supporting evidence linking the loss of genes to the development of human cancer comes from studies of heritable cancers, among them retinoblastoma of childhood.

Retinoblastoma


Retinoblastoma, the most common primary malignant eye tumor of children, occurs in both a heritable (autosomally dominant) form characterized by early onset and bilateral eye involvement and a sporadic form with later onset and unilateral involvement.

Sequential deletions or mutational losses of function of both allelic genes ("two-hit" model) at the Rb locus on chromosome 13q14 are required for the development of retinoblastoma of either form. In this genetic model for the two forms of retinoblastoma, first proposed by Knudson and now a paradigm for some other forms of hereditary cancer, the first mutation (first "hit") affecting retinal cells may be one that is inherited at birth through the germ line (heritable form) or that is acquired later by somatic mutation (sporadic form). The second mutation (second "hit") at the same locus is somatic and random in either form, and both allelic (homozygous) mutations are necessary for neoplastic transformation of a retinal cell to retinoblastoma. The sequential mutations necessary for transformation of a retinal cell are much (>1000-fold) more likely to occur in a cell that already has one inherited mutant allele than in a cell that has none, consistent with the clinical characteristics of this and some other forms of hereditary cancer: early onset, multiple primaries, bilateral involvement of paired organs, and Mendelian pattern of inheritance.

Germ line inheritance of a mutated Rb allele also predisposes to the development of a second form of childhood cancer, notably osteosarcoma in adolescence. Somatic mutations (deletions, frameshifts, etc.) of Rb are also found in various forms of adult cancer of lung, breast, prostate, and other sites.

The normal (wild-type) Rb gene on human chromosome 13q14 is a transcription regulator, and its protein product pRb regulates a critical check point in the cell cycle, namely progression from a G1 (resting) phase to the S (DNA-synthetic) phase. Loss of function mutation of both Rb alleles removes this regulatory control.

There are many regulator controls in the cell cycle. Very briefly, cyclin proteins regulate timing mechanisms through CDKs (cyclin-dependent kinases) that enzymatically phosphorylate and activate proteins critical to the cell cycle, particularly at the transition phase from G1 (rest) to S (synthesis of DNA). pRb normally binds to and inactivates at least two protein enzymes that are essential for the synthesis of DNA precursors and the promotion of DNA replication. The hyper-phosphorylation of pRb by CDKs releases and reactivates these enzymes and thus abolishes the inhibitory control of pRb in the cell cycle.

The majority of human cancers appear to have alterations in at least one of the main regulatory controls of the G1-S transition point in the cell cycle (Russo, A.A., et al., Nature 395:237-43, 1998).

Colorectal Cancer


Carcinoma of colon and rectum ranks second to lung cancer as a cause of cancer mortality in males and third (after breast and lung cancer) in females and accounts for about 60,000 deaths annually in the U.S. The majority of colorectal carcinomas arise from adenomatous polyps (adenomas) that develop in the bowel mucosa.

Familial adenomatous polyposis (FAP), an autosomal dominant trait with a prevalence of about 1 in 10,000, predisposes to the development of multiple intestinal polyps and colorectal carcinoma with early age of onset (age 30 or so). Recent molecular genetic studies of FAP and its progression into cancer indicate that normal colonic epithelial cells evolve into polyps and subsequently into cancer through a sequence of mutations and interactions of two kinds of genes: tumor suppressor genes and oncogenes.

The first genetic change in the development of FAP is the mutational inactivation of a tumor suppressor gene called APC (adenomatous polyposis coli) on chromosome 5(q21) of normal colonic epithelial cells (Kinzler, K.W., et al. Identification of FAP locus genes from chromosome 5q21. Science 253:661-664, 1991). The subsequent progression of polyp into colonic cancer involves stepwise changes, beginning with mutational activation of the ras gene on chromosome 12 and followed by deletions of tumor suppressor genes on chromosomes 18 and17, and perhaps others. The observed sequence of mutations involving APC, ras, DCC ("Deleted in Colon Cancer"), and p53 is also found during tumor development in some, but not all, patients with non-familial (sporadic) colorectal cancer, indicating that this is not the only genetic pathway to this common cancer.

p53 Tumor Suppressor Gene


A normal (wild-type) nuclear protein of 53 kilodaltons called p53 protein is the product of the p53 tumor suppressor gene on human chromosome 17(p13). As previously noted with the Rb gene, mutated or deleted tumor suppressor genes such as mutant p53, while heritable as a dominant allele, are recessive to the normal (wild-type) allele in somatic cells, and mutations or deletions in both p53 alleles ("two hits") are required for loss of function. Patients with germline mutations at the p53 locus are at very high risk for cancer development, as seen in the Li-Fraumeni hereditary cancer syndrome characterized by early onset of breast carcinoma, childhood sarcomas, and other tumors.

Somatic mutations at the p53 locus, usually point mutations substituting one amino acid for another and inactivating suppressor activity, are the most common genetic change in human cancers and occur in about 50% of them, including carcinoma of breast, colon, stomach, bladder, and testis, melanoma, and soft-part sarcoma.

Normal p53 protein is a transcription factor (increases gene expression) and mediates several cellular functions: regulation of the cell division cycle, DNA repair, and programmed cell death. In response to various forms of genomic DNA damage (caused by oncogene activation, radiation, cytotoxic drugs, hypoxia, certain viruses), the p53 protein can arrest the cell cycle at the G1 to S transition point, thus affording time for DNA repair and preventing duplication of a mutant cell or, alternatively, failing DNA repair, p53 protein can implement programmed cell death (apoptosis). Accordingly, p53 has been dubbed the "guardian of the genome." The cellular process of DNA replication is initiated by the formation of complexes between proteins called cyclins and enzymes termed cyclin-dependent kinases (CDKs). The formation of cyclin-CDK complexes is inhibited by p53 protein and other cell-growth inhibitors.

As previuosly noted, mutated or deleted tumor suppressor genes, such as mutant p53 or mutant Rb, while heritable as a dominant allele are recessive to the normal (wild-type) allele in somatic cells, and mutations or deletions in both alleles are required for loss of gene function.

DNA Mismatch-Repair Genes


A new class of cancer susceptibility genes was recently identified in hereditary non-polyposis colorectal cancer (HNPCC):a defective hMSH2 gene located on human chromosome 2p and a defective hMLH1 gene located on chromosome 3p. These defective genes are associated with widespread instability of microsatellite DNA and other short repeat sequences in HNPCC cells and with the accumulation of mutations, both germ line and somatic, throughout the genome of some HNPCC patients (Peltomaki,P.,et al., Science 260: 810-812, 1993; Ionov,Y.,et al.,Nature 363:558-561, 1993).Other studies indicate that natural (wild-type) hMSH2 and hMLH1 are human homologs, respectively, of a bacterial gene and a yeast gene that encode a base-binding enzyme involved in DNA mismatch repair.Genetic defects in DNA repair genes not only contribute to the development of HNPCC but may also be involved in other hereditary cancer syndromes as well as in the genetic instability (heterogeneity) commonly shown by cancer cells.

It is estimated that about 0.5% of the general population (~ 1 million Americans) may carry this or other mutator genes, along with a greatly increased risk of colorectal carcinoma or other cancers, such as, ovarian, uterine, and renal.

Apoptosis


Apoptosis (Gr. apo, away; ptosis, falling) is a normal process of non-random cell death that occurs in many biological conditions, among them: embryological development; perinatal thymus selection and deletion of self-responsive T-cells; normal cell turnover throughout life; and in response to abnormal stimuli, such as genomic DNA damage, but without concurrent pathological necrosis and inflammation.

Apoptosis (programmed cell death) is an active, energy-dependent process characterized by the rapid occurrence of distinctive morphological and biochemical changes in the cell. These changes include: the formation of cytoplasmic blebs; chromatin condensation at the nuclear membrane; and cleavage of chromatin by an endonuclease that is exclusively activated in apoptosis and that yields a distinctive 'chromatin ladder' pattern of DNA fragments, as shown by gel electrophoresis. Specialized proteases called caspases (cysteine aspartases), associated with mitochondria and the cytochrome C respiratory pathway, accelerate the cell-death response (Earnshaw, W. C., Nature 397: 387-389, 1999).

As noted elsewhere (see: p53 Tumor suppressor gene), p53 protein expression in response to genomic DNA damage can arrest the cell cycle (at G1) for DNA repair, thus preventing duplication of a mutant cell or, alternatively, failing DNA repair, implement cell suicide through programmed cell death.

If oncogenes are likened to an "accelerator" of cell proliferation or transformation and tumor suppressor genes to a "brake", then apoptosis is a final "suicidal crash". Thus, at least two signals ( and obviously multiple genetic controls) are required for cell proliferation or transformation: one that drives cell proliferation; and one that blocks cell death. Surprisingly, the protein product of the myc proto-oncogene has one domain that mediates cell proliferation and another one that, in the absence of required growth factors, nutrients, or other gene products, induces apoptosis. On the other hand, the bcl-2 proto-oncogene (known to be activated by chromosomal translocation in a variety of B-cell lymphomas) encodes an antiapoptosis protein; the bcl-2 protein product functions as an inhibitor of apoptotic cell death.

Human Cancer Cells Created with Defined Genetic Elements


Sequential mutations or inappropriate expressions of different classes of cellular genes (oncogenes, tumor suppressor genes, mismatch repair genes, and genes that mediate apoptosis) are involved in the usually multiple-step process that leads to human cancer.

This concept was put to critical test in a recent landmark study: normal human epithelial cells (and fibroblasts) in culture were transformed into cancer cells by the insertion of defined genetic elements (Hahn, W.C., Counter, C. M., et al., Nature 400: 464-468, 1999). The three necessary genetic elements were serially inserted and included: a subunit of the telomerase gene, to immortalize the cells by telomere maintenance, i.e., preventing telomere shortening at each cell division (see: Neoplasia VIII. Chromosomal Abnormalities: Telomeres)); an activated ras oncogene, known to be mutated in many kinds of human cancers; and a viral oncoprotein gene (SV 40 large T), known to inhibit the tumor suppressor proteins p53 and pRb.

When injected into immunodeficient mice, the transformed cells formed malignant tumors having similar identifying cellular and molecular genetic characteristics as the injected cells, thus suggesting that the tumorigenic growth was not the consequence of some other rare random event occurring in vivo after inoculation of these cells. Telomere maintenance appeared to be essential for the formation of human cancer cells.

Cancer Susceptibility Genes - Summary


The following table gives only a short list of the ever increasing number of cellular genes that, through inherited or somatic mutations and a gain (dominant allele) or loss (recessive allele) of function, are associated with the development of certain forms of human cancer. Prototype examples of affected genes and associated cancers were previously discussed.

Activating (gain of function) somatic mutations of proto-oncogenes (such as ras, myc, abl, etc.,) are present in a variety of sporadic human cancers (Table). Surprisingly, inherited mutations of proto-oncogenes are not regularly found in hereditary cancers with the following notable exception: inherited germ line mutation in the ret (RET) proto-oncogene confers a genetic predisposition to multiple endocrine neoplasia.

Germ line and subsequently somatic (loss of function) mutations associated with human cancer susceptibility and expression (see Table) mainly involve tumor suppressor genes (established examples include: APC, Rb,& p53 which is mutated in many forms of cancer; BRCA1, BRCA2, NF1, WT1, and DNA repair genes (such as hMSH2).In familial breast cancer, an inherited germ line mutation of the BRCA1 gene located on chromosome 17(q21) confers a predisposition to early-onset (~ premenopausal) breast cancer and ovarian cancer; and an inherited mutation of the BRCA2 gene located on chromosome 13(q12-13 is also linked to early-onset breast cancer. Mutations in BRCA1 and BRCA2 are each estimated to account for less than 5% of the total of all female breast cancer cases (~180,000) occurring annually in the U.S.

Table: Cellular Genes Associated with Human Cancer Susceptibility and Expression

Human Genes Associated with Cancer Susceptibility and Expressions

Affected Gene

Chromosome

Associated Cancer


Oncogenes:

 

 

abl

9(q24)

Chronic myeloid leukemia

c-myc

8(q24)

Burkitt's lymphoma

ras

12(p)

Variety of cancers: colon, lung, pancreas, leukemia

N-myc

2(p)

Neuroblastoma, small cell cancer of lunh

RET

10(q11)

Medullary thyroid carcinoma, multiple endocrine neoplasias

PML/RAR-alpha

t(15;17)

Acute promyelocytic leukemia

Tumor Suppressor Genes:

 

 

APC

5(q21)

Colon carcinoma

BRCA 1

17(q21)

Breast and ovarian carcinoma

BRCA 2

13(q12-13)

Breast carcinoma

p53

17(p13)

Variety of cancers, Li-Fraumeni syndrome

NF1

17(q11)

Neurofibromatosis type 1

RB

13(q14)

Retinoblastoma, osteosarcoma

WT1

11(p13)

Wilms' tumor

Mismatch Repair Genes:

 

 

hMSH2

2(p16)

Colon carcinoma


Chemical and Physical Carcinogenesis


Introduction

The discovery of chemical carcinogenesis was made by Sir Percival Pott (1713-1788), English surgeon, who related the cause of scrotal skin cancer in a number of his patients to a common history of occupational exposure to large amounts of coal soot as chimney sweepers when they were boys. Industrial development, which began in the 18th century and continues to this day, has exposed many workers to the hazards of carcinogenic agents. The accompanying table lists many of the physical and chemical agents that have been established over time as causes of occupational cancers.

Table: Occupational Cancers

Agent

Occupation

Cancer Site


Ionizing radiations

 

 

radon

certain underground miners (uranium, fluorspar,etc.)

bronchus

X-rays, radium

radiologists, radiographers

skin

radium

luminous dial painters

bone to 1491

Ultraviolet radiation

farmers, sailors, etc.

skin

Polycyclic hydrocarbons in soot, tar, oil

chimney sweepers, manufacturers of coal gas, many other groups of exposed industrial workers

scrotum, skin, bronchus

2-Naphthylamine; 1-naph-thylamine

chemical workers, rubber workers, manufacturers of coal gas

bladder

Benzidine; 4-aminobiphenyl

chemical workers

bladder

Asbestos

asbestos workers, shipyard and insulation workers

bronchus, pleura, and peritoneum

Arsenic

sheep dip manufacturers, gold miners, some vineyard workers and ore smelters

skin and bronchus

Bis(chloromethyl) ether

makers of ion-exchange resins

bronchus

Benzene

workers with glues, varnishes, etc.

marrow (leukemia)

Mustard gas

poison gas makers

bronchus, larynx, nasal sinuses

Vinyl chloride

PVC manufacturers

liver (angio-sarcoma)


1491 Osteogenic sarcoma of femur

An occupational cancer in a radium dial painter. Neoplastic osteocytes embedded in malignant osteoid matrix (the homogeneous substance between tumor cells).

In addition to occupational exposure to carcinogens, medical treatment with agents such as ionizing radiations and natural exposure to solar ultraviolet radiation were early recognized as causes of human cancers. Occupational cancers comprise a small but preventable part of the worldwide incidence of human cancers. It is paradoxical that, more than two centuries after the discovery of the carcinogenic hazards of coal soot, a smoke product of another source, associated with life-style rather than occupation, is etiologically related to one of the most prevalent human cancers today, namely, bronchogenic carcinoma of tobacco smokers.

Classification of Chemical Carcinogens

Chemical carcinogens are of synthetic ("man made") or natural origin, are extremely diverse in structure without any common feature, and are classified into two categories:

·                              direct-acting (DNA-reactive, activation independent, genotoxic) carcinogens that bind covalently to cellular genomic DNA and are mutagens;

·                              procarcinogens (activation dependent) that require metabolic conversion to metabolites ("ultimate carcinogens") capable of transforming cells and inducing tumors.

Procarcinogens are among the most potent chemical carcinogens.

Table: Major Chemical Carcinogens


Pro-carcinogens (require metabolic activation to "ultimate carcinogens")

Polycyclic aromatic hydrocarbons

·                                                      benzanthracene (first pure carcinogen)

·                                                      3,4-benzpyrene (isolated from coal tar)

·                                                      3-methylcholanthrene (prepared from a steroid, deoxycholic acid)

·                                                      7,12-dimethylbenzanthracene (most potent carcinogen)

Aromatic Amines and Azo Dyes

·                                                      2-naphtylamine (produces bladder carcinoma)

·                                                      benzidine (produces bladder carcinoma)

·                                                      2-acetylaminofluorene

·                                                      4-dimethylaminoazobenzene (produces liver tumors)

Natural Products

·                                                      aflatoxin B1 (potent hepatocarcinogen produced by mold contamination of food)

·                                                      mitomycin C

Other

·                                                      nitrosamine (can be formed by action of nitrites on foods)

·                                                      some insecticides (chlordane and others)

·                                                      some metals (chromium and nickel)

·                                                      carbon tetrachloride

Direct-Acting Carcinogens (DNA-reactive)

Alkylating agents

·                                                      anticancer chemotherapeutic drugs (cyclophosphamide, busulfan, chlorambucil)

·                                                      beta-propiolactone

·                                                      bis(chloromethyl)ether

Acetylating agents

·                                                      1-acetylimidazole


Biological Aspects of Chemical Carcinogenesis

Carcinogenesis is a multiple step process. One of the characteristics of chemical or physical carcinogenesis is the usually extended period of time (latent period) between contact with the carcinogen and the appearance of a tumor. The latent periods of occupational cancers may extend from one to several years and commonly to several decades, as noted in the accompanying table.

Table: Latent Periods of Representative Occupational Cancers

 

 

 

LatentPeriod (years)

Site of Cancer

Type of Cancer

Agent

Average

Range

 

Skin

Epidermoid and basal cell carcinomas

Arsenic
Coal tar & pitch
Ionizing radiation
Solar radiation

25
20-24
7
20-30

4-46
1-50
1-12
15-40

Lung

Bronchogenic carcinoma

Asbestos
Ionizing radiation

18
25-35

15-48
7-50

Bone marrow

Leukemia

Benzene
Ionizing radiation

 

3-19
3-15

Bladder

Squamous cell carcinoma

Aromatic amines

11-15

2-40

Bone

Osteogenic sarcoma

Ionizing radiation

 

10-25

 

The latency between exposure to a carcinogen and cancer formation is explained in part by the results of animal experiments which show that skin carcinogenesis in the rabbit and mouse is divisible into two stages, tumor initiation and promotion. Following single exposure to a subcarcinogenic dose of a carcinogen ("initiation"), the latent period can be shortened and the tumor yield increased by treatment with certain "promoting agents" (croton oil, phorbol esters, others) which are not carcinogenic in themselves, or very weakly so, but cause vigorous cell proliferation in target tissue, a process that may be necessary for the "fixation" or expression of tumor initiation. Wound healing also has a tumor promoting effect: wounding of an area of skin treated with a carcinogen brings out tumors along the edge of the wound.

A scheme of tumor initiation-promotion phases is given in the following diagram.

Table: Initiation-Promotion Phases of Experimental Carcinogenesis of Mouse Skin

 

      I________________________________________> No Tumors
              
      I____________P_P_P_P_P_P_________________> Tumors
 
      I______________________P_P_P_P_P_P_______> Tumors
 
      P_P_P_P_P_P_P____________________________> No Tumors
 
      P_P_P_P_P_P_P_I__________________________> No Tumors
 
                       Time_____> (15-70 weeks)

I, initiation by single application of subcarcinogenic dose of polycyclic hydrocarbon.

P, promotion by spaced applications of promoting agent, such as phorbol ester.

Tumors are skin papillomas and carcinomas.

 

Initiation and promotion are two stages in the development of tumors. Initiation is caused by chemical, physical, or biological agents which irreversibly and heritably alter the cell genome.

The mechanism of promotion is not well understood. There are many kinds of promoting agents with diverse molecular structures: phorbol esters, estrogen, prolactin, other endogenous hormones, drugs, and others. Some of the promotors exhibit specific interaction with cell receptors. For example, phorbol esters bind with a surface receptor identified as protein kinase C which also mediates the effect of platelet derived growth factor (PDGF), a mitogen encoded by the proto-oncogene c-sis. The protein kinase becomes activated and in turn causes a change in Pi metabolism, an increase in intracellular Ca++, and a rise in intracellular pH. These changes trigger cell proliferation, an apparently necessary process in the "fixation" or expression of tumor initiation.

Progression

Tumor promotion as studied in the animal model of skin carcinogenesis results mainly in the formation of papillomas and occasionally in the progression of papillomas to carcinomas. Progression, the third definable stage of neoplastic development, is separable from promotion. To illustrate, phorbol esters are strong promotors but weak progressing agents. Furthermore, following the initiation-promotion stages of induction of skin carcinogenesis, a high incidence of carcinomas can be produced by subsequent applications of a different initiating agent, suggesting a second event ("second hit") in the induction of carcinomas. As previously noted, molecular genetic mechanisms are implicated in tumor progression, among them, chromosomal rearrangements or mutations that activate proto-oncogenes.

Thus, it appears that of the three stages of carcinogenesis - initiation, promotion, and progression - initiation, most certainly, and progression, most likely, involve molecular genetic changes.

Biochemical Aspects of Chemical Carcinogenesis

Most chemical carcinogens are , or are metabolically converted into, electrophilic reactants (electron-attracting chemicals) that cause their biological effects by covalent binding with cellular proteins and nucleic acids, particularly chromosomal DNA. The most frequent reaction sites in DNA are with guanine.

The majority of chemical carcinogens are procarcinogens and require metabolic conversion into chemically reactive forms (ultimate carcinogens). Many chemical pathways (oxidation, reduction, hydroxylation, hydrolysis, conjugation, etc.) lead to metabolic conversion of procarcinogens to intermediate metabolites (proximate carcinogens) and finally to ultimate carcinogens which react with cellular DNA to cause neoplastic transformation.

Most procarcinogens are activated by microsomal enzymes in the endoplasmic reticulum. The conversion of polycyclic aromatic hydrocarbons to ultimate carcinogens is initiated by aryl hydrocarbon hydroxylase (AHH). Cytochrome P-450, a terminal component of an electron transport system present in liver microsomes, is also involved in the metabolic activation of procarcinogens.

Tests for mutagenicity indicate that virtually all ultimate carcinogens are mutagenic. Conversely, most mutagens show carcinogenic activity. In the Ames screening test for mutagenicity, a putative carcinogen is incubated with liver microsomes and an indicator microorganism. An increase in the frequency of specific mutants above control levels is scored as a positive result .

Modifying Factors in Carcinogenesis

Host factors (genetics, gender, hormones, aging) and environmental factors may have a modifying role in increasing, or decreasing, the susceptibility to carcinogens. With procarcinogens, activating enzyme systems must be present or inducible in target cells. This genetically determined activity explains the organ and species specificity of some procarcinogens.

Microsomal enzymes in the liver degrade (detoxify) a large part of a procarcinogen to non-carcinogenic products. Enzymes can be induced which accelerate detoxification. A variety of naturally occurring compounds, such as indole, flavones, and related compounds that occur in vegetables (brussels sprouts, cabbage, broccoli, cauliflower) have a protective action in animals exposed to carcinogenic polycyclic hydrocarbons.

Endogenous (and exogenous) sex hormones are important factors apparently in the promotion stage of human carcinomas of breast, endometrium, and prostate. Additionally, a history of exposure in-utero to the synthetic estrogen diethylstilbestrol (DES) is strongly associated with the development of carcinoma of the vagina and cervix in some young women.

Other exogenous factors in human carcinogenesis include dietary excesses and deficiencies and, most notably, tobacco smoking which is a major factor associated with lung carcinoma.

Non-genotoxic Carcinogens

The actions previously described are those of agents which react with cellular DNA and cause genomic alterations. As more and more chemicals are tested for carcinogenicity, a number are now being recognized as "non-genotoxic". These chemicals do not form stable covalent bonds with cellular DNA or other macromolecules. Solid state materials (asbestos) are an example.

DNA Damage and Repair

Ultimate carcinogens are mutagens and cause point mutations (base-pair substitutions) and frame-shift mutations. The activation of ras proto-oncogenes by point mutations is associated with chemical carcinogenesis in experimental animals and with many types of human cancers. Ultraviolet and ionizing radiations are also mutagenic and produce several types of lesions (strand breaks, cross-links, base alterations) in cellular DNA. Ultraviolet radiation also produces dimers between adjacent thymidines.

Excision repair of damaged DNA occurs to a greater or lesser extent and involves the excision of the damaged strand, synthesis of a patch, and rejoining of the strand by a DNA ligase.

Several autosomal recessive disorders associated with an increased incidence of cancer exhibit defects in the repair and maintenance of DNA. Notably, the repair of damage caused by ultraviolet radiation is defective in patients with xeroderma pigmentosa (autosomal recessive) who have a high incidence of skin cancer in areas exposed to sunlight. Bloom's syndrome, a condition characterized by multiple chromosomal breaks and a high incidence of leukemia or intestinal cancer, is associated with a defect in a DNA ligase.

                           

 

五、练习题

(一)选择题

A型题

1.黄曲霉素常可引起下列哪种肿瘤  B

A.     肺癌  B.肝癌  C.膀胱癌  D.卵巢癌  E.胃癌

2.下列哪种细胞不参与细胞免疫介导的抗肿瘤效应  D

ACTL细胞  BNK细胞  CK细胞  D.上皮细胞  E.巨噬细胞

3.肿瘤逃避免疫攻击的机制下列哪项是错误的  C

A.肿瘤抗原性弱  B.肿瘤抗原调变  C.抗原提呈细胞功能亢进

D.免疫抑制细胞(TS)活化  E.免疫抑制因子释放

4.鼻咽癌发生与下列哪种病毒有关  C

A.乳头状瘤病毒  B.腺病毒  CEB病毒 

D.单纯疱疹病毒  E.猿猴病毒

5.霉变食物中含有下列哪种致癌物  B

   A.芳香胺类  B.黄曲霉素  C.多环芳烃类  D.亚硝胺类  E.烷化剂类

6.癌基因激活方式中下列哪项是错误的  E

   A.基因点突变  B.基因插入诱变  C.基因易位

 D.基因扩增  E.基因缺失

7.癌基因产物的作用下面哪项是错误的  B

   A.作为生长因子及生长因子受体  B.作为第二信使

B.     具有GTP结合蛋白活性  D.具有酪氨酸蛋白激酶活性 

E.作为核转录因子

8.抑癌基因失活方式下列哪项是错误的  C

   A.点突变  B.等位基因缺失  C.基因扩增 

D.抑癌基因产物失活  EDNA高甲基化

9.肿瘤发生机制与下列哪项无关  B

   A.癌基因激活  B.凋亡基因激活  C.抑癌基因失活 

DDNA修复基因失活  E.代谢酶基因多态性

10.下列哪项不属于黏附分子  A

   A.免疫复合物  B.整合素  C.钙黏素  DCD44  E.选择素

11.与肿瘤转移过程有关的因素下列哪项是产物的  E

   A.细胞黏附分子  B.基质分解酶  C.血管生成因子

C.     细胞运动因子  E.致癌物的性质

12.肿瘤时机体临床表现下列哪项是错误的  D

A.     发热  B.疼痛  C.贫血  D.一般无感染发生  E.恶病质

13.致癌过程下列哪项是错误的  B

A.多基因参与  B.立即发生  C.内外因素共同起作用

D.     多阶段性  E.停止接触致癌物后仍可致癌

14.肝癌发生与下列哪种病毒感染有关  C

A.人类乳头状瘤病毒  BEB病毒  C.乙型肝炎病毒

D.腺病毒  E.单纯疱疹病毒

二、多项选择题

15.细胞癌基因激活的机制是由于  ABDE

A.基因点突变  B.基因易位  C.基因缺失

D.基因插入诱变  E.基因扩增

16.致癌过程一般是  ACDE

    A.多基因参与  B.立即发生  C.内外因素共同起作用

D.停止接触致癌物后仍可致癌  E.多阶段过程

17.肿瘤病人最常见的合并症是  ABCE

A.感染  B.贫血  C.疼痛  D.低血钙  E.发热

18.肿瘤遗传易感性的遗传本质是  ABC

A.遗传缺陷  B.代谢酶活性异常  C.染色体异常

D.激素失衡  E.营养不良

19.体外能直接杀伤肿瘤细胞的细胞有  AC

ACTL细胞  BB淋巴细胞  CNK细胞 

E.     上皮细胞  E.肥大细胞

20.肿瘤细胞逃避机体免疫反应的攻击可能的机制是  ABCDE

A.肿瘤细胞表面的抗原发生调变  B.肿瘤细胞产生免疫抑制因子

C.肿瘤细胞表面的抗原性减弱  D.免疫抑制细胞(Ts)活化

E.抗原提呈细胞功能降低

二、    名词解释

1.肿瘤遗传易感性

2DNA修复基因

3.代谢酶基因多态性

4.原癌基因

5.肿瘤转移

三、    思考题

1.试述癌基因与抑癌基因的区别?

2.试述癌基因产物在肿瘤发生、发展中的作用?

3.举例说明正常细胞恶变过程是受多基因影响的?

4.肿瘤转移与哪些因素有关?

5.试述肿瘤转移的多步骤过程?

                                                           

 

六、主要参考文献

                        

1.   陈主初.病理生理学(供7年制临床医学等专业用). 北京. 人民卫生出版社. 2001.

 P226260

2.   金惠铭. 卢 建. 殷莲华. 细胞分子病理生理学. 河南. 郑州大学出版社. 2002.

P304324P110118P266267

3.   宋恕平. 临床肿瘤转移学.济南. 山东科学出版社. 2001. P17

4.   徐从高. 茂宏. 杨兴季等.癌-肿瘤学原理和实践(第5版). Vincen T. De Vita, Jr.Samud Hellman, Steven A. Rosenberg . Cancer principles &Practice of Oncology 5 th Edition. 济南. 山东科学出版社. 2001. P197231

5.   钱垂文.正常细胞如何人工诱变成癌细胞. 国外医学肿瘤学分册. 2000. 274):211212

6.   朱红梅.汤为学.成纤维细胞对肿瘤发生发展的作用研究.医学综述.200286):229-231.

7.   Takakura M, Kyo S, Tanaka M, et al. Expression of human telomerase subunits and correlation with telomerase activity in cervical cancer. [J]. Cancer Res. 1998, 58:1558-1561.

8.   Esteller M,Tortolas S, Toyota M, et al. Hypermethylation-associated inactivation of p14 (ARF) is independent of p16 (INK4a) methylation and p53 mutational status. [J]. Cancer Res. 2000, 60:129-133.

9.   Silva JM, Dominguez G, Gonzalez R, et al. Presence of  tumor DNA in plasma of breast cancer patients: clinico-pathological correlation. [J]. Cancer Res.1999, 59:3252-3256.

10.Croix BS, Rago C, Velculescu V, et al. Genes expression in human tumor endothelium. Science. 2000,289:1197-1202.

11.Loizidou MC,Carpenter R, Laurie H, et al. Growth enhancement of implanted human colorectal cancer cells by the addition of fibroblasts in vivo. Br J Surg. 1996, 83 (1):24-28.

12.Himelstein BP, Lee EJ, Sato H,et al. Tumor cell contact mediated transcriptional activation of the fibroblast matrix metalloproteinase-9 gene: involvement of multiple transcription factors Ets and an alternating purine-pyrimidine repeat. Clin Exp Metastasis. 1998, 16 (2):169-177.

 

(汤为学)