Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation

Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 that is limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells.     

The human papillomavirus type 16 E7 oncoprotein targets Myc-interacting zinc-finger protein-1

We demonstrate that HPV-16 E7 forms a complex with Miz-1. UV-induced expression of the CDK-inhibitor p21^Cip1 and subsequent cell cycle arrest depends upon endogenous Miz-1 in HPV-negative C33A cervical cancer cells containing mutated p53. Transient expression of E7 in C33A inhibits UV-induced expression of p21^Cip1 and overcomes Miz-1-induced G1-phase arrest. The C-terminal E7Δ79LEDLL83-mutant with reduced Miz-1-binding capacity was impaired in its capability to repress p21^Cip1 expression; whereas the pRB-binding-deficient E7C24G-mutant inhibited p21^Cip1 expression similar to wild-type E7. Using ChIP, we demonstrate that endogenous E7 is bound to the endogenous p21^Cip1 core-promoter in CaSki cells and RNAi-mediated knock down of Miz-1 abrogates E7-binding to the p21^Cip1 promoter. Co-expression of E7 with Miz-1 inhibited Miz-1-induced p21^Cip1 expression from the minimal-promoter via Miz-1 DNA-binding sites. Co-expression of E7Δ79LEDLL83 did not inhibit Miz-1-induced p21^Cip1 expression. E7C24G retained E7-wild-type capability to inhibit Miz-1-dependent transactivation. These findings suggest that HPV-16 E7 can repress Miz-1-induced p21^Cip1 gene expression.     

Phage display for site-specific immunization and characterization of high-risk human papillomavirus specific E7 monoclonal antibodies

The paper describes a method to use filamentous phage to display specific regions of proteins for immunization in order to direct the immune response towards a pre-defined region of the protein. The method called site-specific immunization (SSI) was evaluated using the E7 protein of oncogenic (high-risk) human papillomavirus (HPV) type 16 as a model system. This protein consists of sequence blocks also present in other viral and cellular proteins and in the corresponding protein of low-risk HPVs. A fragment of the HPV16 E7 oncoprotein specific for a group of high-risk viruses was identified by sequence comparison and displayed on filamentous phages in fusion with the major phage coat protein VIII. The recombinant phages triggered an immune response in mice against the full-length HPV16 E7 protein. Fusion of B-lymphocytes from the immunized animals with myeloma cells resulted in three hybridomas producing monoclonal antibodies (MAbs) with reactivity against the endogenous E7 protein. The specificity of the MAbs for the HPV16 E7 protein in cancer cell lines was confirmed by Western blot analyses and immunocytochemistry. The epitope of each MAb was roughly mapped by determining the reactivity against overlapping E7 fragments displayed on phage particles. The mimotopes of the MAbs were further determined by biopanning against a randomized peptide library displayed on phage and found to be unique for a sub-set of high-risk HPV E7 proteins. The combination of different phage display techniques for immunization and epitope mapping was efficient for generation and characterization of highly specific MAbs.     

RNA干扰法建立人NADH-细胞色素b5还原酶缺陷的细胞表型

目的 探索经载体介导的RNA干扰法建立人NADH-细胞色素b5还原酶(EC1.6.2.2,NADH-cytochrome b5 reductase,b5R)缺陷细胞系的可行性.方法 设计并构建b5R的两种小干扰RNA(small inter-ference RNA,siRNA)表达载体,脂质体转染法瞬时转染BEL-7402细胞,半定量逆转录-PCR分析重组载体的干扰效应;G418筛选两种表达载体稳定转染的BEL-7402细胞,并通过稳定转染的细胞克隆在b5R mRNA水平,酶活性,酶含量等方面干扰效果的鉴定,获得b5R缺陷的细胞克隆;噻唑蓝法测定b5R缺陷细胞的生长曲线.结果 获得两个靶向b5R基因的siRNA重组表达载体pSib5R-1和pSib5R-2,其中pSib5R-2瞬时转染对BEL-7402细胞b5R mRNA的抑制率达68.3%.获得18个稳定转染的细胞克隆,pSib5R-2转染的克隆中有两个克隆MR mRNA抑制率达48.2%和56.2%,酶活性抑制率为54.6%和63.5%,酶含量也有明显降低.b5R酶缺陷没有改变细胞的生长速度.结论 b5R基因的表达可被载体介导的RNA干扰有效抑制,成功建立了b5R缺陷的细胞表型,为进一步研究b5R基因的功能以及探讨Ⅱ型隐性先天性高缺血红蛋白症的发生机制提供了实验依据和材料.     

Oncogene lineages of human papillomavirus type 16 E6, E7 and E5 in preinvasive and invasive cervical squamous cell carcinoma.

Human papillomavirus (HPV)16 accounts for about 60% of the HPV infections in invasive cervical cancer (ICC). There are many sequence variations within HPV16, some of which have been associated with different biological properties, although no definite correlations have yet been established. However, the definition 'variant' has been a source of confusion in research and diagnosis, since it is based on all sequence deviations from a randomly selected prototype. This study has sequenced the HPV16 oncogenes E6, E7 and E5 from 61 Swedish cases with cervical intraepithelial neoplasia grade III (CIN III) or ICC. Clustering the sequence variations at the three common sites of variation (nucleotide 350 in E6, which has previously been associated with the progression from CIN III to ICC, and nucleotides 3979 and 4042 in E5) resulted in the distinction of three major oncogene lineages encompassing more than 95% of the cases, and two minor oncogene lineages. Simple comparison of the distribution of the individual variations or oncogene lineages between CIN III and ICC showed no significant difference, but the number of variations in addition to the three common ones was significantly higher in ICC. This novel classification scheme, based on the variations in the E6, E7 and E5 region, is considered to be a major improvement over the classical 'prototype-variant' classification, and can help to clarify the interpretation of HPV sequence data in relation to the progression of cervical cancer.     

小干扰RNA对人乳头状瘤病毒6bE7基因表达的沉默作用

目的研究靶向人乳头状瘤病毒HPV-6b型E7基因的二聚体小干扰RNA(siRNA- HPV-6bE7)对靶基因表达的沉默作用。方法建立并筛选稳定表达HPV-6bE7基因的B16和293T转染细胞株,用脂质体转染法将体外合成的siRNA-HPV-6bE7转染上述细胞株,采用实时荧光定量PCR分析靶基因HPV-6bE7的mRNA表达情况。结果用不同浓度的siRNA-HPV-6bE7转染细胞48 h,50 nmol/L浓度对B16细胞中靶基因表达的抑制作用最强(抑制率87.05%),1nmol/L抑制作用较低(9.14%);而在293T细胞,10nmol/L的siRNA对靶基因表达的抑制效应最大(78.87%),1nmol/L仍有一定抑制作用(46.92%)。50 nmol/L siRNA-HPV-6bE7转染B16细胞后,靶基因的mRNA表达在24 h内开始受抑制(32.47%),48h作用最强(74.72%),96h作用很低(8.91%);25nmol/L和10nmol/L的siRNA转染293T细胞后,均在24h内起效(26.66%、20.31%),抑制作用至少能维持72h(65.93%、35.23%)。结论siRNA-HPV-6bE7对B16和293T细胞外源性靶基因表达均有较强的特异性沉默作用,在不同细胞株达到最大抑制效应的siRNA浓度不同,但时效曲线的变化趋势基本一致,siRNA均在24h内起效,48～72h达到高峰,抑制作用至少能维持72h。本研究结果为下一步在动物或临床进行siRNA干扰试验提供了实验依据。     

Silencing of African horse sickness virus VP7 protein expression in cultured cells by RNA interference

RNA interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of homologous mRNA. Short interfering RNAs (siRNAs) are the mediators of RNAi and represent powerful tools to silence gene expression in mammalian cells including genes of viral origin. In this study, we applied siRNAs targeting the VP7 gene of African horse sickness virus (AHSV) that encodes a structural protein required for stable capsid assembly. Using a VP7 expression reporter plasmid and an in vitro model of infection, we show that synthetic siRNA molecules corresponding to the AHSV VP7 gene silenced effectively VP7 protein and mRNA expression, and decreased production of infectious virus particles as evidenced by a reduction in the progeny virion titres when compared to control cells. This work establishes RNAi as a genetic tool for the study of AHSV and offers new possibilities for the analysis of viral genes important for AHSV physiology.     

Microarray analysis identifies differentiation-associated genes regulated by human papillomavirus type 16 E6

In this study, we used oligonucleotide microarray analysis to determine which cellular genes are regulated by the human papillomavirus type 16 (HPV-16) E6 oncoprotein. We found that E6 causes the downregulation of a large number of cellular genes involved in keratinocyte differentiation, including genes such as small proline-rich proteins, transglutaminase, involucrin, elafin, and cytokeratins, which are normally involved in the production of the cornified cell envelope. In contrast, E6 upregulates several genes, such as vimentin, that are usually expressed in mesenchymal lineages. E6 also modulates levels of genes involved in inflammation, including Cox-1 and Nag-1. By using E6 mutants that differentially target p53 for degradation, we determined that E6 regulates cellular genes by both p53-dependent and independent mechanisms. The microarray data also indicate that HPV-16 E6 modulates certain effects of HPV-16 E7 on cellular gene expression. The identification of E6-regulated genes in this analysis provides a basis for further studies on their role in HPV infection and cellular transformation.     

The epidemiology of human papillomavirus infection and cervical cancer

Cervical cancer has been recognized as a rare outcome of a common Sexually Transmitted Infection (STI). The etiologic association is restricted to a limited number of viral types of the family of the Human Papillomaviruses (HPVs). The association is causal in nature and under optimal testing systems, HPV DNA can be identified in all specimens of invasive cervical cancer. As a consequence, it has been claimed that HPV infection is a necessary cause of cervical cancer. The evidence is consistent worldwide and implies both the Squamous Cell Carcinomas (SCC), the adenocarcinomas and the vast majority (i.e. > 95%) of the immediate precursors, namely High Grade Squamous Intraepithelial Lesions (HSIL)/Cervical Intraepithelial Neoplasia 3 (CIN3)/Carcinoma in situ. Co-factors that modify the risk among HPV DNA positive women include the use of oral contraceptives (OC) for five or more years, smoking, high parity (five or more full term pregnancies) and previous exposure to other sexually transmitted diseases such as Chlamydia Trachomatis (CT) and Herpes Simplex Virus type 2 (HSV-2). Women exposed to the Human Immunodeficiency Virus (HIV) are at high risk for HPV infection, HPV DNA persistency and progression of HPV lesions to cervical cancer.     

RNA quality and gene expression analysis of ovarian tumor tissue undergoing repeated thaw-freezing

Gene expression profiles evaluated by microarray-based quantization of RNA are used in studies of differential diagnosis and prognosis in cancer. RNA of good quality is mandatory for this evaluation. The RNA most often comes from tumor banks with limited amount of tissue, and the tissue often undergoes repeated thawing and freezing. We evaluated the influence of repeated division of tumor samples at room temperature, on RNA quality and quantity, in addition to the gene expression profile. Sixteen ovarian tumor samples were divided in three aliquots each, undergoing respectively one, two, and three thaw-freeze cycles. RNA from each aliquot was extracted on the day of division, and quantity and quality were evaluated. RNA from all three aliquots of four tumor samples underwent microarray analysis on Affymetrix Human Genome U133A 2.0 arrays. Microarray data were evaluated using both unsupervised, and supervised multivariate statistical methods, reliability analysis, as well as verification using published gene lists in ovarian cancer studies. RNA quality and quantity did not change during the division procedure and microarray data showed insignificant difference in gene expression. Tumor samples from tumor banks can be frozen and thawed at least three times without compromising the RNA integrity and genetic expression profile.     

人乳头瘤病毒16型E7核酸疫苗的构建及鉴定

人乳头瘤病毒16 型在引起宫颈上皮转化过程中, 持续表达E7 蛋白, 有可能成为肿瘤特异性移植抗原。本文将E7蛋白的编码基因定向克隆于真核表达载体构建了HPV16E7 HB核酸疫苗, 将该疫苗直接注射BALB/c 小鼠和Wistar 大鼠股四头肌, 动态观察, 28d 后仍能测出E7 DNA 的扩增。提取肌组织RNA, RT PCR检测老鼠特异性E7 的转录情况, 3/3 大鼠,1/10 小鼠为阳性; 用ELISA法在免疫小鼠血清中检出了特异性抗E7 抗体。证明, HPV16E7 HB 核酸疫苗构建正确且能够在哺乳动物体内和体外有效表达     

人乳头瘤病毒16型E6E7反义RNA抑制宫颈癌细胞恶性表型的作用

目的研究癌基因的特异性反义ＲＮＡ对癌细胞生长繁殖和恶性程度的影响。方法用逆转录病毒载体将人乳头瘤病毒（ＨＰＶ）－１６Ｅ６Ｅ７反义ＲＮＡ导入ＨＰＶ－１６ＤＮＡ阳性的宫颈癌细胞株ＣａＳｋｉ中，观察该细胞在导入反义ＲＮＡ后其表型特征和在裸鼠体内致癌能力的变化。结果ＨＰＶ－１６Ｅ６Ｅ７反义ＲＮＡ能降低宫颈癌细胞ＣａＳｋｉ的生长速率，抑制其在软琼脂上的集落形成能力，并能明显地抑制其在裸鼠体内的致癌能力。Ｗｅｓｔｅｒｎｂｌｏｔ分析发现ＨＰＶ－１６Ｅ６Ｅ７反义ＲＮＡ能使宫颈癌细胞中病毒ＨＰＶ－１６Ｅ６基因的表达水平降低。结论ＨＰＶ－１６Ｅ６Ｅ７反义ＲＮＡ能使宫颈癌细胞ＣａＳｋｉ恶性表型逆转；由其引起的癌细胞中ＨＰＶ－１６癌基因表达水平的降低可能是癌细胞表型逆转的原因之所在；ＨＰＶ－１６癌基因的表达水平对维持癌细胞的恶性表型起着重要作用。     

RNA干扰技术在胰腺癌基因治疗中的研究进展

RNA干扰（RNAi）是生物体内由具有同源性的双链RNA（dsRNA）引发的序列特异的转录后基因沉默。该技术能够快速、高效、特异地抑制靶基因的表达。从1998年发现RNA干扰现象至今,该技术得到了广泛的应用。胰腺癌是一种恶性度高的肿瘤,但是由于各种原因,至今尚无有效的治疗方法。RNA干扰技术是一种十分有前途的基因治疗方法,已广泛应用到胰腺癌的基因治疗研究之中,包括采用RNA干扰技术抑制癌基因、抗凋亡因子;利用RNA干扰技术抑制胰腺癌侵袭、转移蛋白来探讨治疗胰腺癌;利用RNA干扰技术提高胰腺癌对化疗的敏感性等。     

携带甲胎蛋白基因小干扰RNA慢病毒载体的构建及其对靶基因的沉默效率

目的构建携带甲胎蛋白（AFP）基因小干扰RNA（siRNA）的慢病毒载体并转染肝癌细胞,评价其对AFP基因的沉默效率。方法设计和构建针对AFP基因的阳性siRNA及不针对任何已知基因的阴性siRNA,将其分别插入携带绿色荧光蛋白（GFP）基因的重组慢病毒载体,然后转染到表达AFP基因的人肝癌细胞HepG2并筛选阳性表达细胞株,分别为慢病毒转染阳性siRNA组和慢病毒转染阴性siRNA组。同时设立脂质体转染阳性siRNA组、脂质体转染阴性siRNA组以及加AFPsiRNA而未用转染试剂的siRNA组、空白对照组。荧光显微镜观察评估转染效率,荧光定量PCR和免疫印迹法检测各组HepG2细胞APFmRNA及蛋白的相对表达量,比较不同转染方法对AFP基因表达的抑制率。结果慢病毒能够有效地转染siRNA到HepG2细胞内。荧光定量PCR显示慢病毒转染siRNA对AFPmRNA表达的抑制率显著高于脂质体转染siRNA（92．1％比74．3％,P〈0．05）。免疫印迹亦显示慢病毒转染siRNA对AFP蛋白表达的抑制率显著高于脂质体转染siRNA（88．2％比63．7％,P〈0．05）。结论慢病毒转染AFPsiRNA能够更有效地抑制HepG2细胞内AFP基因表达。     

Prevalence of human papillomavirus types in women screened by cytology in Germany

Incidence and mortality rates of cervical cancer are higher in Germany than in other Western European countries. Type-specific human papillomavirus (HPV) distribution was investigated for the first time in Germany in an epidemiological study including 8,101 women. Women above the age of 30 years, self-referring for cervical cancer screening, were enrolled in two study centers in Hannover (Northern Germany) and Tübingen (Southern Germany). Participants were screened by the Pap smear and the hybrid capture 2 (HC2) test using the high-risk probe. All samples that were positive by the HC2 test were genotyped using the prototype PGMY09/11 PCR line blot assay. Most women in the study population had a negative Pap smear (96.7%). Prevalence of high-risk type HPV detected by HC2 was 6.4% and prevalence of carcinogenic types detected by PGMY09/11 was 4.3%. Of the PGMY09/11 PCR-positive women, 70.2% had a single infection, 28.1% had multiple infections and 1.7% remained uncharacterized. 32 different HPV types were detected using PGMY09/11 PCR. HPV 16, 31, 52, 51, 18, and 45 were the most common carcinogenic types in the study population. Among women with histologically confirmed high-grade lesions HPV 16, 45, 58, 18, 31, 33, and 52 were the predominant types. These results provide valuable information for the management of HPV infections in Germany, both in terms of future strategies of screening and vaccination.     

RNAi技术抑制HCMV-IE1表达的在体实验研究

目的:研究RNA干扰(RNA interference,RNAi) 对体内人类巨细胞病毒-立即早期基因1(human cytomegalovirus immediate-early gene 1,HCMV-IE1)表达的沉默作用,为靶向HCMV-IE1的基因治疗提供科学的实验依据.方法:选取60只雄性昆明种小鼠,并分为3组,分别为实验对照组(注射生理盐水)、病毒感染组(注射HCMVAD169株病毒液)和RNA干扰组(染毒后第2天注射转染了表达RNAi载体的人胚肺成纤维细胞液);采用PCR法检测小鼠体内HCMV基因的表达水平;采用ELISA检测动物血清中HCMV-IgM的含量;采用免疫组织化学和Western免疫印迹技术分别测定小鼠体内HCMV-IE1的蛋白水平.结果:PCR结果表明干扰组比病毒感染组的HCMV的基因表达下调20%;ELISA结果表明RNAi能使动物血清中HCMV的含量降低为19%;免疫组织化学和Western免疫印迹结果证实,干扰组和病毒感染组的HCMV-IE1蛋白水平分别降低为17.6%和18.5%.结论:RNAi技术能有效地抑制目的基因在体内的表达,有可能用于靶向HCMV-IE的基因治疗.     

The human papillomavirus (HPV) vaccine,HPV related diseases and cervical cancer in the post-reproductive years

Prophylactic HPV vaccines have demonstrated high efficacy against a range of HPV related diseases. They have been widely adopted as population health interventions in many jurisdictions and their routine use has been endorsed by the WHO. The development of these vaccines comes after an increased understanding of the natural history and epidemiology of HPV infection and disease in both males and females. Persistent HPV infection with oncogenic types induces malignant transformation in a range of epithelia including the cervix, anogenital regions, the penis and a number of head and neck sites. In relation to HPV disease prevention in the post-reproductive years, most infections occur soon after commencement of sexual activity but new infections do occur throughout the age spectrum. This reduces the likely impact of prophylactic vaccines in this population. The major impact on HPV related disease in this age group will come from advances in screening and early detection of HPV and neoplastic precursors. The most appropriate prevention for any individual man or women in this age group will be an individualised combination of vaccination, screening and early detection depending on the individual's own circumstances.