正常细胞癌变的原因和HPV病毒诱导的宫颈癌的分子机制.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,本文档所提供的信息仅供参考之用，不能作为科学依据，请勿模仿；如有不当之处，请联系网站或本人删除。,一个正常的细胞如何变为一个癌细胞呢?,癌症的本质？,癌症源于体细胞突变。,基因发生突变的体细胞，在与正常体细胞生存竞争的过程中，不断进化。最终变成永生不死的癌细胞。癌细胞无限制的增殖，进而导致肿瘤的发生。,癌变的原因,原癌基因突变,抑癌基因突变,癌细胞,物理致癌因子,化学致癌因子,病毒致癌因子,正常细胞,作用,肿瘤的发生是基因突变逐渐累积的结果,eg:,已知的癌症危险因素,吸烟,肺癌、膀胱癌、食道癌、胰腺癌、肝癌、口腔癌、鼻腔癌还有很多其他癌症都与烟草的使用有关，据估计90%的男性肺癌死亡的原因可以归为吸烟。烟草所产生的烟雾是复杂的化学物质混合体。当这些物质被吸入肺部，它们可以在局部或远程引发DNA损伤并改变细胞生长和增殖状态。,已知的癌症危险因素,饮食,仅次于烟草的关键致癌危险因素，这些因素包括摄入的饮食类化学成分以及总体上能量的消耗。14%20%的癌症死亡率与超重和肥胖率有关；红肉已经证实与肠癌有关，可能由于防腐处理以及热处理的肉里含有亚硝胺(nitosamine)和杂环胺(heterocyclic amine)等致癌物。,职业,许多致癌物被鉴定是以人类触及因工业化而导致的癌症为代价的,致癌物的类型,物理致癌物,生化致癌物,化学致癌物必须通过代谢激活为亲电性的中间物，与蛋白质、RNA或DNA形成共价加合物（化学致癌性的亲电性理论）,其与DNA结合而改变其完整性，导致突变。,物理致癌物,紫外线,（,主要是波长在100400nm）有足够的能量引起光化学损伤，导致皮肤癌的形成。,离子辐射,离子辐射的能量很高，足以从与其碰撞的原子或者分子上移除一个电子。,在UV辐射中，DNA是主要的靶标，DNA经过UV辐射后会形成嘧啶二聚体。当这些损伤没修复时，会产生DNA突变。标志性的损害时C T或者CC GG。,阳光辐射照成的基因突变有,a.p53(如鳞状细胞癌，squamous cell carcinama,SCC；基底细胞癌，basal cell carcinoma,BCC),b.p16(如黑色素瘤),c.PTCH(如BCC，也可能导致SCC),经过UV辐射，皮肤角质形成细胞中很多信号通路会改变，如生长停滞和DNA损伤应答基因(如p53，GADD45，错配修复基因)、凋亡信号分子(如bcl-2)和促细胞分裂信号(如Ras),生物致癌物,传染因子是仅次于烟草的潜在致癌物，15%30%的癌症与它相关。,传染因子引起癌症的机制有三大类：,a.通过感染源引起持久的感染伴随慢性炎症，导致巨噬细胞在感染的位点形成活性的氧化和氮化物，这些活性分子能够损伤DNA、蛋白质和膜，导致肿瘤的形成。,b.感染因子通过激活细胞的癌基因通道或者使一个抑癌基因失活，直接参与细胞的癌变。,c.与人类免疫缺陷病毒（human immunodeficiency virus,HIV）相关,感染可能导致免疫抑制，宿主免疫系统识别感染或癌变细胞的能力降低。,生物致癌物,化学致癌物,有机致癌物,苯（Benzene）,多环芳烃（polycyclic aromatic hydrocarbon,PAH）:PAH可以被转变为苯环“湾区”的二醇环氧物（diol epoxide）,这些环氧物可以与DNA形成共价的化合物。,黄曲霉素B1（aflatoxinB1,AFB1）:最强的肝癌致癌物。,无机致癌物,镉：能在体内积累，可能通过表观遗传的机制激活原癌基因，破坏细胞的正常过程。,砷：接触会产生活性自由基，导致DNA及蛋白质的损伤。,激素,己烯雌酚（diethyl-stilbestrol,DES）,小鼠皮肤致癌的多步骤模型,原癌基因和抑癌基因及相关的癌症,HPV诱发宫颈癌（cervical cancer）的过程,HPV感染建立在鳞状上皮细胞损伤，表皮防护缺失的基础上，而同时HPV感染又阻碍了鳞状上皮细胞的修复，引起恶性循环,高危型HPV持续感染可引起宫颈上皮内瘤变CIN（cervical intraepithelial neoplasia）和宫颈鳞癌。,HPV感染经过漫长的过程发展为宫颈癌,宫颈不典型增生原位癌早期浸润癌宫颈癌,),HPV诱发宫颈癌的分子机制,HPV（Human Papillomavirus,HPV）人类乳头瘤病毒，是一种属于乳多空病毒科的乳头瘤空泡病毒A属，是球形DNA病毒，能引起人体皮肤黏膜的鳞状上皮增殖。目前已分离出130多种，该病毒只侵犯人类，对其它动物无致病性。,高危的HPV-16病毒,HPV16基因组可分为三个不同区域：,a.早期区域，编码参与病毒DNA复制、转录调节和细胞转化的蛋白（分别编码为E1、E2、E3、E4、E5、E6、E7、E8等8个早期蛋白）,b.晚期区域，编码病毒大（L1）和小（L2）衣壳蛋白,c.长控制区域，又称上游调节区域（upstream regulatory region,URR）,不包括任何OPF，有顺式调节元件，包括起始子和重要的转录增强子,HPV16基因及其蛋白作用,基因,碱基数,碱基序列,基因表达产物的作用,E1,954 bp,8592 813,控制病毒DNA的复制,E2,1127 bp,2 7253 852,负调节E6、E7，负责细胞周期和凋亡的调控,E4,287 bp,3 3323 619,捆绑于细胞角蛋白，破坏并结合细胞角蛋白网,E5,236 bp,3 8634 099,活化生长因子受体,E6,494 bp,65559,捆绑于p53蛋白，抑制p53而阻断细胞凋亡,E7,314 bp,544858,捆绑于Rb蛋白，抑制pRB而使细胞周期失控,L1,1628 bp,5 5267 154,构成较大病毒壳体,L2,1523 bp,4 1335 656,构成较小病毒壳体,URR,813 bp,7 1557 904/064,内含启动子，构成反馈调节环,HPV诱发宫颈癌的分子机制,E6和E7的主要细胞靶点分别是肿瘤抑制蛋白p53和pRB。高危型HPV游离基因通过非同源重组整合至宿主DNA后，主要的改变是原癌基因E6和E7的高水平稳定表达分别导致抑癌基因p53和Rb失活,小DNA肿瘤病毒（如多瘤病毒，腺病毒，癌症相关的HPV病毒）有一个普遍的机制：编码的致癌蛋白能够和关键的细胞调节蛋白相互作用。,病毒癌蛋白的主要癌变活性，各自与pRB形成复合物，并将相应的“口袋蛋白”失活，激活转录因子E2F家族控制的基因，导致细胞增殖。,HPV诱发宫颈癌的分子机制,E6与p53的相互作用是非直接的，受一个细胞蛋白E6-相关蛋白（E6-associated protein,E6AP）的调节，E6AP是一种泛素蛋白连接酶，当存在E6时，其直接参与p53泛素化，多泛素化的p53被识别后被26S蛋白酶体降解，破坏了p53转录激活和抑制特性，干扰野生型p53在DNA损伤应答中调节细胞周期阻滞的能力。,HPV诱发宫颈癌的分子机制,分化的上皮细胞早已退出细胞周期,E7与pRB结合，释放转录因子E2F家族，刺激细胞增殖并驱使细胞进入S期，重新激活细胞周期的DNA复制。,E7与细胞周期蛋白依赖性激酶（CDK）的抑制因子相互作用,E7蛋白在正常的人类细胞中引起基因组的不稳定性，诱导G1/S和有丝分裂细胞周期检验点缺陷，导致错误分离和非整倍体。,HPV诱发宫颈癌的分子机制,分化的角质细胞是HPV感染的正常宿主,分化的角质层细胞早已退出细胞周期,G1期是唯一可以接受外界传入的细胞增殖和抑制增殖信号的周期,HPV诱发宫颈癌的分子机制,HPV16病毒E6和E7基因片段是HPV16转化正常细胞的关键早期基因。,E2基因存在于HPV病毒基因上，其表达的蛋白E2蛋白是一种特殊的DNA结合蛋白，它抑制病毒感染细胞从而抑制病毒周期，进一步抑制了病毒DNA的复制，促进了基因维护，使病毒转录降低。正常情况下，E2蛋白起到抑制HPV16的启动子的作用，减少了病毒基因E6或E7的转录，从而下调E6或E7的表达，促进了正常细胞的衰老死亡。,病毒基因整合的一个重要作用就是解除病毒E6、E7的表达控制。当E2基因断裂时，病毒蛋白E2表达下降，丧失了对E6，E7基因的抑制，病毒基因E6、E7过表达，E6，E7蛋白产生过多，刺激细胞，使其复制，随着复制的逐渐增多，宫颈病变持续加重，最终恶变。因此常将E2开放阅读框架的断裂作为HPV病毒整合到宫颈癌宿主细胞的重要标志。,共表达E6和E7就足以使原代人类细胞永生，最显著的是使原代人类角质化细胞永生，其为HPV病毒正常的宿主。与HPV-16和HPV-18中E6/E7蛋白具有使细胞永生的特性相反，低风险的病毒里E6/E7蛋白没有活性或活性很低。,Nucleotide Deficiency Promotes Genomic Instability in Early Stages of Cancer Development,SUMMARY,Chromosomal instability in early cancer stages is caused by stress on DNA replication.We studied the replication dynamics in cells in which a regulator of S phase entry and cell proliferation,the Rb-E2F pathway,is aberrantly activated.Aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes significantly decreased the cellular nucleotide levels in the newly transformed cells.Exogenously supplied nucleosides rescued the replication stress and DNA damage and dramatically decreased oncogene induced transformation.Increased transcription of nucleotide biosynthesis genes,mediated by expressing the transcription factor c-myc,increased the nucleotide pool and also rescued the replication-induced DNA damage.Our results suggest a model for early oncogenesis in which uncoordinated activation of factors regulating cell proliferation leads to insufficient nucleotides that fail to support normal replication and genome stability。,Nucleotide Deficiency Promotes Genomic Instability in Early Stages of Cancer Development,DSBs chromosomal instability,With an insufficent pool of nucleotide,aberrantly,HPV-16 E6/E7,Cyclin E,active,Cell proliferation,enforce,Rb-E2F pathway,cause,a.Retinoblastoma(Rb)E2F pathway(Rb-E2F)is an important regulator of cell proliferation.Rb is the key player in cell-cycle regulation,restricting cell proliferation by direct inhibition of the E2F family of transcription factor.,b.HPV16 E7 binds and degrades Rb.,c.Cyclin E regulates S phase entry by Rb phosphorylation and inactivation,facilitating E2F release.additional mutations abrogating the DNA damage response are required to overcome the apoptosis/senescence barrier,Nucleotide Deficiency Promotes Genomic Instability in Early Stages of Cancer Development,primary keratinocytes derived from adult skin biopsies,which have a very poor proliferation capacity ex vivo.The cells were infected with a LXSN-based retroviral vector,which did not affect cell proliferation or DNA replication.Keratinocytes from the same donor were infected with the LXSN vector containing the HPV-16 E6 and E7 viral genes.,the E6/E7-expressing cells continued to proliferate at least 100 days,indicating their successful immortalization.These results show that E6/E7 genes were expressed and enforced continuous proliferation of the infected keratinocytes.,In order to investigate early events induced by E6/E7 expression,the experiments were performed in newly transformed cells 26 weeks following E6/E7 infection and before anaphase bridges and micronuclei are visible.,HPV-16 E6/E7 Expression Generates Stresson the Cellular DNA Replication,Use DNA combing approach to investigate the effect of HPV on the cellular DNA replication.,The replication dynamics were studied,in normal primary keratinocytes,obtained from adult skin samples of two individuals and,in keratinocytes,from the same donors,expressing E6/E7proteins for 26 weeks,.,Altogether,the replication dynamic results show that,in newly transformed E6/E7-expressing cells,the host cell DNA replication is dramatically perturbed,.,A Low-Nucleotide Pool in Cells Expressing HPV-16 E6/E7 Leads to Replication Stress and Genome Instability,we studied the effect of E6/E7 on the nucleotide pool using the high-performance liquid chromatography(HPLC)method.,Our results show a 2-to 5-fold decrease in the level of the four dNTPs following E6/E7 expression for 24 weeks,These results suggest that the replication perturbation and genomic instability found in the E6/E7-expressing cells result from an insufficient nucleotide pool required to support extensive proliferation.,A Low-Nucleotide Pool in Cells Expressing HPV-16 E6/E7 Leads to Replication Stress and Genome Instability,For this,primary keratinocytes and keratinocytes expressing E6/E7 for 24 weeks were grown for 48 hr in a medium supplemented with the four nucleosides(A,U,C,and G).,A model for the events leading to genomic instability in early stages of cancer development,Oncogene expression forces cell proliferation by aberrant activation of cell-cycle regulators(Rb-E2F).Insufficient activation of the nucleotide biosynthesis pathways results in a low-nucleotide pool that fails to support normal DNA replication.This leads to replication stress and promotes genomic instability during early stages of cancer development.Additional factors contribute to genomic instability in different stages of tumorigenesis,such as reactive oxygen species(ROS),telomere loss,hypoxia,abrogated mitotic checkpoints,and loss of the DNA damage response(DDR).,Thank you for your attention,