正常細(xì)胞癌變的原因及hpv病毒誘導(dǎo)的宮頸癌的分子機(jī)制_ppt課件

正常細(xì)胞癌變的原因 和HPV病毒誘導(dǎo)宮頸癌的分子機(jī)制,一個(gè)正常的細(xì)胞如何變?yōu)橐粋€(gè)癌細(xì)胞呢?,癌癥的本質(zhì)？ 癌癥源于體細(xì)胞突變。基因發(fā)生突變的體細(xì)胞，在與正常體細(xì)胞生存競爭的過程中，不斷進(jìn)化。最終變成永生不死的癌細(xì)胞。癌細(xì)胞無限制的增殖，進(jìn)而導(dǎo)致腫瘤的發(fā)生。,癌變的原因,腫瘤的發(fā)生是基因突變逐漸累積的結(jié)果,eg:,已知的癌癥危險(xiǎn)因素,吸煙肺癌、膀胱癌、食道癌、胰腺癌、肝癌、口腔癌、鼻腔癌還有很多其他癌癥都與煙草的使用有關(guān)，據(jù)估計(jì)90%的男性肺癌死亡的原因可以歸為吸煙。煙草所產(chǎn)生的煙霧是復(fù)雜的化學(xué)物質(zhì)混合體。當(dāng)這些物質(zhì)被吸入肺部，它們可以在局部或遠(yuǎn)程引發(fā)DNA損傷并改變細(xì)胞生長和增殖狀態(tài)。,物理致癌物,紫外線（主要是波長在100400nm）有足夠的能量引起光化學(xué)損傷，導(dǎo)致皮膚癌的形成。 離子輻射離子輻射的能量很高，足以從與其碰撞的原子或者分子上移除一個(gè)電子。 在UV輻射中，DNA是主要的靶標(biāo)，DNA經(jīng)過UV輻射后會(huì)形成嘧啶二聚體。當(dāng)這些損傷沒修復(fù)時(shí)，會(huì)產(chǎn)生DNA突變。標(biāo)志性的損害時(shí)C T或者CC GG。 陽光輻射照成的基因突變有 a. p53(如鱗狀細(xì)胞癌，squamous cell carcinama,SCC；基底細(xì)胞癌，basal cell carcinoma,BCC) b. p16(如黑色素瘤) c. PTCH(如BCC，也可能導(dǎo)致SCC) 經(jīng)過UV輻射，皮膚角質(zhì)形成細(xì)胞中很多信號(hào)通路會(huì)改變，如生長停滯和DNA損傷應(yīng)答基因(如p53，GADD45，錯(cuò)配修復(fù)基因)、凋亡信號(hào)分子(如bcl-2)和促細(xì)胞分裂信號(hào)(如Ras),生物致癌物,傳染因子是僅次于煙草的潛在致癌物，15%30%的癌癥與它相關(guān)。 傳染因子引起癌癥的機(jī)制有三大類： a.通過感染源引起持久的感染伴隨慢性炎癥，導(dǎo)致巨噬細(xì)胞在感染的位點(diǎn)形成活性的氧化和氮化物，這些活性分子能夠損傷DNA、蛋白質(zhì)和膜，導(dǎo)致腫瘤的形成。 b.感染因子通過激活細(xì)胞的癌基因通道或者使一個(gè)抑癌基因失活，直接參與細(xì)胞的癌變。 c.與人類免疫缺陷病毒（human immunodeficiency virus,HIV）相關(guān),感染可能導(dǎo)致免疫抑制，宿主免疫系統(tǒng)識(shí)別感染或癌變細(xì)胞的能力降低。,生物致癌物,化學(xué)致癌物,有機(jī)致癌物 苯（Benzene） 多環(huán)芳烴（polycyclic aromatic hydrocarbon,PAH）:PAH可以被轉(zhuǎn)變?yōu)楸江h(huán)“灣區(qū)”的二醇環(huán)氧物（diol epoxide）,這些環(huán)氧物可以與DNA形成共價(jià)的化合物。 黃曲霉素B1（aflatoxinB1,AFB1）:最強(qiáng)的肝癌致癌物。,無機(jī)致癌物 鎘：能在體內(nèi)積累，可能通過表觀遺傳的機(jī)制激活原癌基因，破壞細(xì)胞的正常過程。 砷：接觸會(huì)產(chǎn)生活性自由基，導(dǎo)致DNA及蛋白質(zhì)的損傷。,激素 己烯雌酚（diethyl-stilbestrol,DES）,小鼠皮膚致癌的多步驟模型,原癌基因和抑癌基因及相關(guān)的癌癥,HPV誘發(fā)宮頸癌（cervical cancer）的過程,HPV感染建立在鱗狀上皮細(xì)胞損傷，表皮防護(hù)缺失的基礎(chǔ)上，而同時(shí)HPV感染又阻礙了鱗狀上皮細(xì)胞的修復(fù)，引起惡性循環(huán) 高危型HPV持續(xù)感染可引起宮頸上皮內(nèi)瘤變CIN（cervical intraepithelial neoplasia）和宮頸鱗癌。 HPV感染經(jīng)過漫長的過程發(fā)展為宮頸癌 宮頸不典型增生原位癌早期浸潤癌宮頸癌),HPV誘發(fā)宮頸癌的分子機(jī)制,HPV（Human Papillomavirus,HPV ）人類乳頭瘤病毒，是一種屬于乳多空病毒科的乳頭瘤空泡病毒A屬，是球形DNA病毒，能引起人體皮膚黏膜的鱗狀上皮增殖。目前已分離出130多種，該病毒只侵犯人類，對(duì)其它動(dòng)物無致病性。,高危的HPV-16病毒 HPV16基因組可分為三個(gè)不同區(qū)域： a.早期區(qū)域，編碼參與病毒DNA復(fù)制、轉(zhuǎn)錄調(diào)節(jié)和細(xì)胞轉(zhuǎn)化的蛋白（分別編碼為E1、E2、E3、E4、E5、E6、E7、E8等8個(gè)早期蛋白 ） b.晚期區(qū)域，編碼病毒大（L1）和?。↙2）衣殼蛋白 c.長控制區(qū)域，又稱上游調(diào)節(jié)區(qū)域（upstream regulatory region,URR）,不包括任何OPF，有順式調(diào)節(jié)元件，包括起始子和重要的轉(zhuǎn)錄增強(qiáng)子,HPV16基因及其蛋白作用,HPV誘發(fā)宮頸癌的分子機(jī)制,E6和E7的主要細(xì)胞靶點(diǎn)分別是腫瘤抑制蛋白p53和pRB。高危型HPV游離基因通過非同源重組整合至宿主DNA后，主要的改變是原癌基因E6和E7的高水平穩(wěn)定表達(dá)分別導(dǎo)致抑癌基因p53和Rb失活 小DNA腫瘤病毒（如多瘤病毒，腺病毒，癌癥相關(guān)的HPV病毒）有一個(gè)普遍的機(jī)制：編碼的致癌蛋白能夠和關(guān)鍵的細(xì)胞調(diào)節(jié)蛋白相互作用。 病毒癌蛋白的主要癌變活性，各自與pRB形成復(fù)合物，并將相應(yīng)的“口袋蛋白”失活，激活轉(zhuǎn)錄因子E2F家族控制的基因，導(dǎo)致細(xì)胞增殖。,HPV誘發(fā)宮頸癌的分子機(jī)制,E6與p53的相互作用是非直接的，受一個(gè)細(xì)胞蛋白E6-相關(guān)蛋白（E6-associated protein,E6AP）的調(diào)節(jié)，E6AP是一種泛素蛋白連接酶，當(dāng)存在E6時(shí)，其直接參與p53泛素化，多泛素化的p53被識(shí)別后被26S蛋白酶體降解，破壞了p53轉(zhuǎn)錄激活和抑制特性，干擾野生型p53在DNA損傷應(yīng)答中調(diào)節(jié)細(xì)胞周期阻滯的能力。,HPV誘發(fā)宮頸癌的分子機(jī)制,分化的上皮細(xì)胞早已退出細(xì)胞周期 E7與pRB結(jié)合，釋放轉(zhuǎn)錄因子E2F家族，刺激細(xì)胞增殖并驅(qū)使細(xì)胞進(jìn)入S期，重新激活細(xì)胞周期的DNA復(fù)制。 E7與細(xì)胞周期蛋白依賴性激酶（CDK）的抑制因子相互作用 E7蛋白在正常的人類細(xì)胞中引起基因組的不穩(wěn)定性，誘導(dǎo)G1/S和有絲分裂細(xì)胞周期檢驗(yàn)點(diǎn)缺陷，導(dǎo)致錯(cuò)誤分離和非整倍體。,HPV誘發(fā)宮頸癌的分子機(jī)制,分化的角質(zhì)細(xì)胞是HPV感染的正常宿主 分化的角質(zhì)層細(xì)胞早已退出細(xì)胞周期 G1期是唯一可以接受外界傳入的細(xì)胞增殖和抑制增殖信號(hào)的周期,HPV誘發(fā)宮頸癌的分子機(jī)制,HPV16病毒E6和E7基因片段是HPV16轉(zhuǎn)化正常細(xì)胞的關(guān)鍵早期基因。 E2基因存在于HPV病毒基因上，其表達(dá)的蛋白E2蛋白是一種特殊的DNA結(jié)合蛋白，它抑制病毒感染細(xì)胞從而抑制病毒周期，進(jìn)一步抑制了病毒DNA的復(fù)制，促進(jìn)了基因維護(hù)，使病毒轉(zhuǎn)錄降低。正常情況下，E2蛋白起到抑制HPV16的啟動(dòng)子的作用，減少了病毒基因E6或E7的轉(zhuǎn)錄，從而下調(diào)E6或E7的表達(dá)，促進(jìn)了正常細(xì)胞的衰老死亡。 病毒基因整合的一個(gè)重要作用就是解除病毒E6、E7的表達(dá)控制。當(dāng)E2基因斷裂時(shí)，病毒蛋白E2表達(dá)下降，喪失了對(duì)E6，E7基因的抑制，病毒基因E6、E7過表達(dá)，E6，E7蛋白產(chǎn)生過多，刺激細(xì)胞，使其復(fù)制，隨著復(fù)制的逐漸增多，宮頸病變持續(xù)加重，最終惡變。因此常將E2開放閱讀框架的斷裂作為HPV病毒整合到宮頸癌宿主細(xì)胞的重要標(biāo)志。 共表達(dá)E6和E7就足以使原代人類細(xì)胞永生，最顯著的是使原代人類角質(zhì)化細(xì)胞永生，其為HPV病毒正常的宿主。與HPV-16和HPV-18中E6/E7蛋白具有使細(xì)胞永生的特性相反，低風(fēng)險(xiǎn)的病毒里E6/E7蛋白沒有活性或活性很低。,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,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 Stress on 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,