Human papillomaviruses (HPVs) are small DNA tumor viruses that cause benign and malignant tumors of squamous epithelia.(Fig1) Among all the subtypes of HPVs, HPV-16 and HPV-18 are identified as high risk HPVs which are mainly responsible for HPV correlated human cancer. HPVs contain a double stranded DNA genome of approximately 8,000 base pairs and up to 10 open reading frames (ORFs).(Fig2) ORFs within the early region code for proteins like E6 and E7 which are involved in the regulation of viral replication and the viral life cycle, HPV-16 E6 and E7 oncoproteins can abrogate negative growth regulatory signaling pathways of the host cell through interaction with p53 and pRB tumor suppressor proteins. As a result, high-risk HPVs infected cells proliferation become de-regulated, and then, transformation develops.

E6 oncoprotein

The E6 protein is one of the key cancer-causing proteins expressed by the Human papillomavirus (HPV). Among the strains of HPV known to cause physical changes associated with cancer and pre-cancerous lesions, three oncoproteins are recognized: E5, E6 and E7. Although low-risk HPV strains also produce these proteins, the four major high-risk strains—HPV-16, HPV-18, HPV-31, and HPV-45—all exhibit E6 and E7 proteins. E6's activity in the high-risk strains can be oncogenic, or cancer-promoting. Therefore, it is the strains which exhibit these proteins which are associated with cervical cancer and pre-cancerous lesion development in women. E6 is a 151 amino-acid peptide that incorporates a type 1 motif with a consensus sequence –(T/S)-(X)-(V/I)-COOH. It also has two zinc finger motifs.
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E6 is of particular interest because it appears to have multiple roles in the cell and to interact with many other proteins. E6 primarily causes cancer by associating with and thereby inactivating P53 or Rb proteins, which act as tumor suppressors. When tumor suppressor proteins are inactivated tumor growth proceeds unchecked. E6's interaction with p53 and Rb marks these proteins for degradation by ubiquitylation and ubiquitin ligase. E6 is proven to act on other cellular proteins, and to positively affect telomerase activity, thus inactivating one of the ways by which cells are normally prevented from dividing unchecked. Additionally, E6 can act as a transcriptional cofactor—specifically, a transcription activator—when interacting with the cellular transcription factor, E2F1/DP1. E6 can also bind to PDZ-domains, short sequences which are often found in signalling proteins. E6's structural motif allows for interaction with PDZ domains on DLG (discs large) and hDLG (Drosophila large) tumor suppressor genes. Binding at these locations causes transformation of the DLG protein and disruption of its suppressor function. E6 proteins also interact with the MAGUK (membrane-associated guanylate kinase family) proteins. These proteins, including MAGI-1, MAGI-2, and MAGI-3 are usually structural proteins, and can help with signaling. More significantly, they are believed to be involved with DLG's suppression activity. When E6 complexes with the PDZ domains on the MAGI proteins, it distorts their shape and thereby impedes their function. Overall, the E6 protein serves to impede normal protein activity in such a way as to allow a cell to grow and multiply at the increased rate characteristic of cancer. The full length HPV E6 genes encode a 160 amino acids protein, which contains two domains including zinc binding Cys-X-X-Cys motifs. High risk HPV E6 proteins both have anti-apoptotic activities and can interfere with the antiproliferative functions of p53, a tumor suppressor in cell proliferation. To do this, E6 first forms a complex with a cellular ubiquitin- protein ligase E6AP, the E6/E6AP complex then acts as a p53-specific ubiquitin-protein ligase to accelerate degradation of p53. Also, E6 can significantly reduce the half life of p53 in vivo from several hours to 20 minutes. Moreover, E6 oncoprotein interferes with the stabilization and activation of p53 in response to genotoxic and cytotoxic stresses. As a result, the negative growth regulatory function of p53 is effectively abrogated by E6-stimulated degradation in high-risk HPV-infected cells.

E7 oncoprotein

E7, a protein encoded by Human papillomavirus which inhibits pRb. Smaller than high risk HPV E6 protein, E7 proteins are about 100 amino acids in size and also contain 2 copies of Cys-X-X-Cys domains as well as E6. As an oncoprotein, E7 functions through forming complex with and promote proteolysis of hypophosphorylated pRB, the active form of retinoblastoma tumor suppressor gene product. Formation of E7/pRB complex interfere the complex formation of pRB with E2F. So E2F is released from inactivated combination with pRB and continues its work as a transcription activator to promote DNA synthesis and cell proliferation.(Fig. 3)
                                                     
Co-expression of high risk HPV E6/E7

In high risk HPV infected cells, E6 and E7 are usually co-expressed. The high-risk HPV E7 oncoproteins bind and degrade the pRB and the related pRB family members p107 and p130.17, as well as inactivate the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1.1820 As a result, there comes aberrantly increased expression of cyclin E, cyclin A and aberrant CDK2 activity mediated by E2F. E2F can also activate p14ARF to stabilze p53. As cooperation with E7 to promote cell proliferation, the high-risk HPV E6 oncoprotein induces the rapid proteasomal degradation of p53 by interacting with E6-AP, a host cell protein. By respective dysfunction of p53 and pRB tumor suppressor pathways, high-risk HPV E6 and E7 proteins immortalize and transform human cells effectively. (Fig. 4)

E6 / E7在cell line上的應用

<以下列文獻為例>

高危HPV所致宫颈癌的预防与治疗
(文章来源:丁香园)


子宫颈癌的发生与人乳头瘤病毒(HPV)感染密切相关。人体内确定的HPV有85种以上。其中与宫颈癌发生有关的有20多种,可分为高危型、中危型、低危型三种,其中高危型组的HPV16、HPV18等与宫颈癌有密切联系。现将高危HPV所致宫颈癌的预防与治疗总结如下:

1.制备基因疫苗

HPV基因功能区的L区包括L1和L2,L1晚期基因能刺激机体产生保护性抗体,因此该基因的克隆在制备基因工程疫苗方面成为可行性。Donnelly用编码乳头瘤病毒L壳蛋白的DNA疫苗,在兔体内的抗感染效果,说明了基因疫苗用于治疗也是一个有效途径。Conson J在防御16型乳头瘤病毒感染的疫苗中提到:子宫颈分泌物中存在的抗HPV16核壳蛋白的中和抗体可能具有保护作用,故而可制备HPV16疫苗以预防原发 HPV16感染。现在对这种疫苗的研究已取得相当进展,据澳大利亚布里斯班消息:美国PDA已批准在美国进行宫颈癌的人乳头瘤病毒Ⅱ预防疫苗的Ⅰ期临床试验。

2.构建HPV16型E6、E7基因反义质粒

E6、E7两个开放读码框架及其表达产物在宫颈癌细胞中常被检出,且这两个框架具有转化能力,E6、E7基因的表达与否是宫颈癌发生及维持的必要条件,故而研究E6、E7基因的反义质粒干扰E6、E7基因的表达成为一种治疗宫颈癌患者的手段。曾萍、司静懿等在人乳头瘤病毒16型E6、E7反义质粒对人宫颈癌细胞恶性的逆转作用中,采用构建可为地塞米松诱导表达的人乳头瘤病毒16型E6、E7反义质粒利用磷酸钙沉淀法将其分别转染到HPV16阳性的宫颈癌细胞Cask:和HPV阴性的宫颈癌细胞C-33A中,地塞米松诱导反义质粒表达后,Cask:细胞失去其恶性表型,而C-33A细胞生长特性及恶性行为未发生变化,证明了E6、E7基因反义质粒可成为一种治疗宫颈癌的方法。

3.重组病毒载体肿瘤疫苗的应用

由于HPV宿主细胞通过下调MHC1表达位点突变,下调抗原呈递,降低E6、E7抗原性,使肿瘤细胞免疫原性降低,逃避免疫系统的杀伤,这样使得肿瘤生物治疗效果很低,因此有人提出将肿瘤抗原肽的编码基因导入病毒载体,可能诱导出有效的杀肿瘤作用,故而有人把HPV16的E7基因重组于牛痘病毒表达载体,作为重组病毒载体治疗疫苗与E7蛋白分别免疫小鼠,均获得特异性CTL,据此 Boassneu MEG用表达HPV16E6、E7(经处理失去致癌性,保留抗原性)的重组病毒载体治疗免疫8例晚期宫颈癌病人,所有病人均产生抗牛痘LgG抗体,3人出现HPV特异性抗体,HPV特异性CTL在免疫疫苗后前两周可以检测到,有2例分别在15和21个月后仍保持临床良好。说明疫苗诱导的ADCC和CTL有一定效应,但仍存在E6、E7表达量较低,免疫原性不高的问题。但随着对重组病毒载体肿瘤疫苗研究的逐步深入使之应用于临床治疗将成为可能。


4.P53的应用

已经证实在人体内有抑癌基因P53,恶性肿瘤的发生均是致癌基因与抑癌基因相互作用的结果,HPV所致宫颈癌也不例外,关于P53与HPV的相互作用已在致癌机制中作了详细叙述。由于P53有很强的抑制细胞凋亡的能力,目前已被用于临床上作为基因治疗的一种方法。除此之外,P53蛋白的表达受DNA损伤的影响很大,而多数抗癌药物均属DNA损伤剂,因此P53的功能与肿瘤对某种特定抗癌药物的反应有很密切关系,P53在肿瘤中的突变会影响抗癌药物杀伤肿瘤的能力,有结果表明,含有突变型无活性的P53的肿瘤预后往往很差,由于P53与预后治疗上的重要关系,可以采用激活宫颈癌内已丧失活性的P53或重新引入有活性的P53,从而达到治疗宫颈癌的目的。

5.其它

目前较流行的治疗方式是局部有破坏的治疗,如刀切、激光灼烧、液氮冷冻、电热凝、三氯乙酸或者普达非伦脂腐蚀等。但这些方法只是针对可见的或病变的局部进行治疗,而HPV还可在临近接受治疗的组织中潜伏。因此,治疗后HPV感染的再发生率高。 5-Fu是限制HPV的DNA和RNA合成的细胞毒性药物,局部注射收效显著,但它可造成化学性阴道炎,长期使用还可导致阴道粘膜的损伤,甚至阴道狭窄。也有人利用干扰素的抗病毒、抗增殖和免疫调节等作用,通过肌肉和病变局部联合用药的方法治疗HPV收效比较显著,但关于HPV所致宫颈癌的预防与诊疗至今还没有一个十分完美和实际可行的方法,对HPV感染的预防以及如何迅速准确的诊断,安全有效的治疗有待进一步解决。


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