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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. 相似文献
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应用核酸原位杂交和免疫组织化学技术,检测人子宫颈癌中人乳头瘤病毒(HPV)16型E6ORF与抑癌基因产物P53,RB和增殖细胞核抗原(PCNA)。在44例宫颈癌石蜡切片中,原位杂交检测出HPV16E6ORF阳性27例(61.36%),其中免疫组化检测出P53、RB、PCNA阳性分别为8例(29.63%),14例(52.85%)、20例(74.07%),而在17例HPV16E6阴性标本中P53、RB、PCNA阳性分别为7例(41.17%),9例(52.94%)、12例(70.58%)。而在5例正常宫颈组织中未测出HPV16E6ORF,PCNA只在宫颈组织上皮基底层细胞中表达。统计学分析表明,HPV16E6与宫颈癌密切相关(P<0.05),PCNA在宫颈癌与正常宫颈组织中有显著性差异(P<0.05)。未能发现宫颈癌组织中HPV16E6ORF与P53蛋白相关性(P>0.05)。 相似文献
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乳头瘤病毒16型E6,P53,RB,PCNA在宫颈癌中的表达… 总被引:1,自引:0,他引:1
应用核酸原位杂交和免疫组织化学技术,检测人子宫颈癌中人乳头瘤病毒(HPV)16型E6RF与抑癌基因产物P53,RB和增殖细胞核抗原(PCNA)。在44例宫颈癌石蜡切片中,原位杂交检测出HPV16E6ORF阳性27例(61.36%),其中免疫组化检测出P53、RB、PCNA一分别为8例(29.63#),14例(52.85%)、20例(74.07%),而在17例HPV16E6阴性 本中P53、RB、P 相似文献
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Sequence analysis of human papillomavirus type 16 E6E7 gene from cervical carcinoma biopsies of Chinese patients in Shandong province 总被引:1,自引:0,他引:1
XU Xue mei SONG Guo xing SI Jing yiDepartment of Biophysics Institute of Basic Medical Science Chinese Academy of Medical Sciences School of Basic Medicine Peking Union Medical College Beijing China 《中国生物医学工程学报(英文版)》2000,(2)
INTRODUCTION Humanpapillomavirustype16(HPV16)hasastrongassociationwithcervicalcarcinoma,Itrepresentsabout50%ofcervicalcancer-associatedHPVinfectionsworldwide.TheexpressionofitsearlyproteinsE6andE7contributestothetrans-formationprocessinvitro.Continuned… 相似文献
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人乳头瘤病毒16型E6和E7基因特征分析 总被引:1,自引:0,他引:1
目的 对北京15例宫颈癌病变组织中的人乳头瘤病毒(HPV)16型E6和E7基因进行扩增及序列测定,分析E6和E7基因突变特征,并探讨宫颈癌病变中HPV16的感染情况.方法 自行设计HPVl6型E6和E7基因扩增引物,采用PCR法扩增15例宫颈癌组织中HPV16 E6和E7片段,将PCR产物克隆到TA载体,进行序列测定.通过Sequencer、Bioedit、Mega等生物学软件对E6和E7基因进行核苷酸和氨基酸序列分析.结果 15例宫颈癌中8例鳞癌检出E6和E7基因,检出率为8/15.2例腺癌、1例腺鳞癌和其他4例鳞癌中均未检出HPV16 E6E7 DNA.8例鳞癌中的4例检出的HPVl6为亚洲类似株,在E6基因178位(T→G,D25E)和E7基因647位(A→G,N29S)发生突变;另外4例为欧洲类似株,其中1例(BJ16)的HPV16在E6基因335位点发生突变(C→T,H78Y).结论 HPV16是致宫颈癌的重要因素;宫颈鳞状细胞癌HPV16感染的发生频率较腺癌和腺鳞癌高;E6基因178位点可能是区分亚洲株和欧洲株的重要位点;E6基因178位点和E7基因647位点是突变频率较高的位点,可能导致HPV16致癌能力发生改变. 相似文献
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宫颈癌组织中人乳头瘤病毒16型E7蛋白致癌机理初探 总被引:3,自引:0,他引:3
目的 研究宫颈癌组织中人乳头瘤病毒(HPV)16-E7蛋白对视网膜母细胞瘤基因(Retinoblastoma)Rb蛋白及E2F-1的作用的机制,探讨HPV16-E7蛋白与宫颈癌发生的关系。方法 采用聚合酶链反应检测宫颈癌及正常宫颈组织中HPV16感染等,用蛋白印迹技术对HPV16 DNA阳性的宫颈癌组织中是否存在HPV16-E7蛋白和R6蛋白-E2F-1形成的复合物进行检测。正常宫颈组织作为对照, 相似文献
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宫颈癌组织中HPV16,18E6蛋白表达的观察 总被引:12,自引:0,他引:12
目的观察HPV16、18早期蛋白E6在人宫颈鳞状细胞癌中的表达并评价E6单抗的应用价值。方法采用SP免疫组化染色法,检测40例宫颈鳞状细胞癌,30例慢性宫颈炎及30例正常宫颈组织中HPV16、18E6蛋白的表达。结果癌组中E6的阳性率为67.5%(27/40),慢性宫颈炎中为3.3%(1/30),正常宫颈组织中均为阴性,良、恶组HPV16、18E6的阳性率差异有显著意义(P<0.01)。结论HPV16、18感染与本地区宫颈癌病因学密切相关,E6单抗可作为预测HPV16、18感染及宫颈癌早期诊断的标记之一。 相似文献
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Expression of human papillomavirus type 16 E7 gene induces DNA synthesis of rat 3Y1 cells 总被引:7,自引:0,他引:7
Human papillomavirus type 16 (HPV 16) open reading frames (ORF) E6, E7, and E6E7, placed under the control of dexamethasone-inducible mouse mammary tumor virus long terminal repeat, were introduced into rat 3Y1 cells, an immortalized fibroblast line, with the aid of neomycin-selection. The cell clones containing inducible HPV 16 ORFs were selected and examined for DNA synthesis. Following induction of HPV mRNA synthesis by the hormone, DNA synthesis was stimulated in the cells containing E7 or E6E7 ORFs. The data indicate that expression of the HPV 16 E7 gene is mitogenic for rat 3Y1 cells. 相似文献
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Simon N. Stacey Anna Ghosh Jennifer S. Bartholomew Robert W. Tindle Peter L. Stern Michael Mackett John R. Arrand 《Journal of medical virology》1993,40(1):14-21
Although the presence of serum antibodies against the human papillomavirus type 16 (HPV-16) E7 protein has been linked with cervical cancer, currently available assays detect antibodies in only ca. 40% of carcinoma patients. The dependence of these serological assays on synthetic target antigens which present only linear epitopes may be a limiting factor. In order to produce a more realistic target antigen for use in serological assays, we have expressed the HPV-16 E7 protein in insect cells using a recombinant baculovirus vector. Two major E7 forms of ca. 18kDa and 16kDa were produced and characterised. The 16kDa component was shown to be truncated at the N-terminus. A radioimmunoprecipitation assay was developed for the detection of anti-E7 antibodies in human sera. This assay showed a marked increase in detection rate compared with a western blotting method based on bacterially derived E7 fusion proteins. © 1993 Wiley-Liss, Inc. 相似文献
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修饰后中国山东地方株HPV16 E6E7基因的转化活性检测及免疫原性研究 总被引:2,自引:0,他引:2
目的 利用突变修饰后消除转化活性并保留抗原性的中国山东地方株人乳头瘤病毒16型(HPV16)E6E7基因,研制HPV16 DNA疫苗。方法 定点突变E6的终止密码,并保证E7读码框架不定;定点突变E7蛋白的Rb结合区中对其转化活性维持起关键作用的第24位氨基酸。突变修饰后的基因命名为fmE6E7。PCR扩增fmE6E7,重组人pLNCX载体,脂质体法转染3T3细胞,免疫荧光组织化学及Western blot检测转染细胞蛋白的表达。经软琼脂集落培养法和BALB/c裸鼠皮下接种法检测fmE6E7的转化活性。然后PCR扩增fmE6E7,构建pVR1012-fmE6E7真核表达质粒,于C57BL/6小鼠肌肉内直接进行裸DNA免疫,^51Cr释放法体外分析免疫鼠的细胞毒性T淋巴细胞活性,间接ELISA法检测免疫鼠血清中E7特异性抗体。结果 测序证实获得了预期的突变结果。pLNCX-fmE6E7转染细胞体外软琼脂培养3周未见集落形成;裸鼠皮下接种2月后未见移植瘤形成(0/3)。免疫鼠获得了较好的E7特异性的抗体E和抗原特异性的CTL。结论 修饰后E6E7基因可融合表达,转化活性消除的同时还可诱发特异的细胞免疫和体液免疫,表明中国山东地方株的E6E7基因可作为HPV16治疗性DNA疫苗的靶基因。 相似文献
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Marc F. D. Baay Paul Herbrink Ernst Stolz Jitze M. Duk Matth P. M. Burger Henk W. A. De Bruijn 《Journal of medical virology》1995,45(3):342-347
A synthetic peptide comprising amino acids 6-35 of HPV-16 E7 was used in an ELISA to screen sera taken from 31 cervical carcinoma patients. Sera obtained before and during treatment, and in follow-up, were tested for the presence of antibodies to this peptide. Sixteen patients with negative pretreatment serum determination remained negative during treatment and follow-up. Of the 15 patients with positive pretreatment sera, 12 showed a decrease in anti-E7 6–35 antibody level during treatment. During follow-up an increase in anti-E76–35 antibody level was observed in 6 out of 7 patients with progressive or recurrent disease, whereas all patients who remained in complete remission showed stable or further decreasing antibody levels. During the course of disease of the 15 seropositive patients, serum anti-E76–35 antibody levels were compared with serum squamous cell carcinoma antigen (SCC-Ag) profiles, a clinically useful tumor marker in the management of cervical cancer patients. Similar patterns were observed in 10 out of 15 patients. The results of this study suggest that in a subset of cervical cancer patients, anti-E76–35 antibody response against HPV-16 E7 at least partially depends on the presence of viable tumor lesions, and that to some extent the anti-E7 profile reflects the course of disease. © 1995 Wiley-Liss, inc. 相似文献
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Antibodies to human papillomavirus type 16 E7 related to clinicopathological data in patients with cervical carcinoma. 总被引:1,自引:0,他引:1
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M F Baay J M Duk M P Burger J Walboomers J ter Schegget K H Groenier H W de Bruijn E Stolz P Herbrink 《Journal of clinical pathology》1995,48(5):410-414
AIMS--To investigate the correlation between antibodies to the transforming protein E7 of human papillomavirus (HPV) type 16 and clinicopathological indices in women with cervical squamous carcinoma. METHODS--A synthetic peptide of the HPV type 16 E7 protein (amino acids 6 to 35) was used to screen sera from 29 children, 130 women with cervical intraepithelial neoplasia, 443 women with cervical cancer, and 222 controls, for antibodies against this viral antigen. Bivariate and multivariate analyses were used to investigate the correlation between the serological status in the pretreatment sera and clinicopathological indices (size of the lesions, histological grade, stomal infiltration, vascular invasion, and nodal spread). Survival analysis was done using the Cox regression model for all FIGO stages and stages IB and ILA. RESULTS--Cervical carcinoma patients had a significantly higher prevalence of antibodies to synthetic peptide E7/6-35 than women with cervical intraepithelial neoplasia (17.7% v 7%, p < 0.005) or controls (17.7% v 11%, p < 0.05). Bivariate analysis of the data on the presence of anti-E7/6-35 antibodies in the pretreatment sera from these patients and clinicopathological indices showed a significant correlation between the presence of anti-E7/6-35 antibodies and the size of the lesion (p = 0.0009), histological grade (p = 0.0031), and lymph node metastasis (p = 0.01). 0.011). In addition, the Cox regression model, analysing four risk factors which can be determined before treatment, showed a significant correlation between the presence of anti-E7/6-35 antibodies and a worse prognosis (p = 0.003). Survival analysis revealed that both for all FIGO stages (p = 0.0005) and for stages IB and IIA alone (p = 0.0021), anti-E7/6-35 positive patients before treatment had a significantly shorter life expectancy. CONCLUSIONS--The presence of antibodies against E7/6-35 in pretreatment sera from patients with cervical carcinoma correlates with the size of the lesions, lymph node involvement, and a worse prognosis. 相似文献
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目的研究人乳头瘤病毒(HPV)相关肿瘤患者的HPV16E6抗体水平及其流行病学意义;探讨用杆状病毒-昆虫细胞表达载体系统表达的HPV16E6蛋白的抗原性。方法用PCR从HPV16基因组中扩增出HPV16E6完整基因,克隆至转移载体pVL1393中,重组质粒DNA与线性杆状病毒DNA共转染昆虫细胞Sf-9,经噬斑筛选获得带有编码E6基因的重组杆状病毒株,并在昆虫细胞中表达。结果Westernblot和高效液相色谱法检测HPV16E6表达蛋白,其分子量约为18000。免疫印迹检测显示其能与兔抗HPV16E6多抗特异性结合。酶联免疫吸附试验表明此重组蛋白能被人HPV16阳性血清所识别。结论昆虫细胞表达的HPV16E6蛋白,具有良好的抗原性,可用于检测HPV16E6特异性免疫球蛋白IgG和IgM抗体 相似文献
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Oncogene lineages of human papillomavirus type 16 E6, E7 and E5 in preinvasive and invasive cervical squamous cell carcinoma. 总被引:6,自引:0,他引:6
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. 相似文献
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利用突变修饰后消除转化活性并保留抗原性的中国山东地方株人乳头瘤病毒16型(human papillomavirus type 16,HPVl6)E6E7融合基因(fmE6E7),研制治疗HPVl6相关疾病的DNA疫苗。用PCR扩增fmE6E7基因后,插人真核表达质粒获得pVRl012-fmE6E7,瞬时转染Cos-7细胞,免疫荧光法检测证实其表达后,在C57BL/6小鼠后腿肌肉进行裸DNA免疫,5lCr释放法体外分析免疫鼠的细胞毒性T淋巴细胞活性Cytotoxic T lymphocyte,CTL),间接ELISA法检测免疫鼠血清中E7特异性抗体。研究表明修饰后的中国地方株E6E7融合基因可诱导机体产生特异的抗体反应和CTL反应,与单独野生型E7基因免疫相比,E6E7融和基因可更好的活化CTT反应。表明修饰后消除转化活性的中国地方株E6E7融合基因可作为HPVl6治疗性DNA疫苗的靶基因。 相似文献