HPV16/18和HPV16E6/E7DNA在宫颈癌组织中的表达

目的检测HPV16/18和HPV16E6/E7 DNA在宫颈癌组织中的表达,探讨其在宫颈癌发病中的作用.方法应用PCR和琼脂糖凝胶电泳方法检测46例宫颈癌组织中HPV16/18和HPV16E6/E7DNA.结果 46例宫颈癌中56.5%(26/46)扩增HPV16/18 DNA,其中宫颈鳞癌25例,宫颈腺癌1例.正常对照组20例HPV16/18DNA均为阴性,与宫颈癌组相比差异有显著性(P<0.01).HPV16/18 DNA阳性拷贝对数值为4.32±2.45.HPV16E6,E7DNA分别有53.8%(14/26)、46.2%(12/26)扩增.结论 HPV16/18和HPV16E6/E7 DNA与宫颈癌的发生密切相关,是宫颈癌恶性转化的关键之一,预示着宫颈癌有较强的增殖能力和转移能力.     

Analysis of human papillomavirus type-16 variants in Italian women with cervical intraepithelial neoplasia and cervical cancer

Human papillomavirus type 16 (HPV-16) classes (E, AA, As, Af1, Af2) and their variants have different geographic distribution and different degrees of association with cervical lesions. This study was designed to examine HPV-16 variants among Italian women and their prevalence in case patients (affected by invasive cervical carcinoma or cervical intraepithelial neoplasia grade 2-3 and cervical intraepithelial neoplasia grade 1), versus control subjects with normal cervical epithelium (controls). A total of 90 HPV-16 positive cervical samples from women of Italian Caucasian descent have been tested, including 36 invasive cervical carcinomas, 21 with cervical intraepithelial neoplasias grade 2-3, 17 with cervical intraepithelial neoplasia grade 1 and 16 controls. HPV-16 was detected with an E6/E7 gene-specific polymerase chain reaction, and variant HPV-16 classes and subclasses were identified by direct nucleotide sequencing of the region coding for the E6 and the E7 oncoproteins, the MY09/11-amplified highly conserved L1 region, and the long control region (LCR). Among the 90 HPV-16 samples, nine viral variants have been identified belonging to the European (Ep-T350 and E-G350) and non-European (AA and Af-1) branches. The E-G350 is the prevalent variant in all analyzed different disease stages being present in 55.5% of ICC, 52.4% of cervical intraepithelial neoplasias 2-3, 47.1% of cervical intraepithelial neoplasia grade 1, and 50.0% of control samples. The non-European variants AA and Af1, rarely detected in control samples, represent 33.3% of all HPV-16 infections in invasive cervical carcinoma (with a peak of 19.4% and 13.9%, respectively), showing a statistically significant increase in frequency in more advanced lesions (chi(2) trend = 7.2; P < 0.05). The prevalence of HPV-16 Ep-T350, however, is higher in controls (43.7%) and in of cervical intraepithelial neoplasia grade 1 (41.2%) than in cervical intraepithelial neoplasia grade 2-3 (28.6%) and in invasive cervical carcinoma (11.1%) cases strongly suggesting lack of progression for pre-neoplastic lesions associated with such variant. The increased frequency of non-European variants in invasive lesions suggests that they are more oncogenic than European variants. This could have implications for future diagnostic and therapeutic strategies.     

Genetic variability and functional implication of HPV16 from cervical intraepithelial neoplasia in Shanghai women

Human papillomavirus (HPV)16 gene mutation is usually associated with persistent HPV infection and cervical intraepithelial neoplasia (CIN). However, the functional implications of HPV16 mutations remain poorly understood.145 LCR/E6/E7 of the HPV16 isolates were amplified and sequenced, and HPV16 integration status was detected. In total, 89 SNPs (68 in the LCR, 13 in E6, 8 in E7) were discovered, 11 of which were nonsynonymous mutations (8 in E6, 3 in E7). The H85Y and E120D variants in E6 were significantly reduced in the high-grade squamous intraepithelial lesion (HSIL) group compared to the <HSIL group (P = .046 and .005), conversely the N29S in E7(P = .01). Amino acid substitutions (D32N/E, E36Q, H85Y, and E120D in E6 and N29H/S and R77C in E7) were predicted to have an effect on conserved structural and functional residues, and five amino acid substitutions (H85Y, E36Q, I34L, and D32E in E6; R77C in E7) would potentially change the secondary structure. “6329G>T,” a potential binding site for TATA-binding protein, is the most common in LCR variants. A4 (Asian) was associated with an increased risk of HSIL compared to A1–3(P = .009). The H85/E120 in E6 and N29 in HPV16 E7 might play a critical role in carcinogenesis by disrupting p53 and Rb degradation due to affecting their interaction, respectively. In a word, the findings in this study provide preventative and therapeutic interventions of HPV16 -related cervical lesions/cancer.     

HPV16 E6E7抗原表达模型的建立

目的 用基因重组技术构建pcDNA-E6E7真核表达载体.方法 经限制性内切酶和序列分析,用脂质体转染技术将其转入B16细胞,G418稳定筛选后IFA法检测其表达,RT-PCK法检测HPV16E6E7mRNA的生成,并将转染细胞接种小鼠皮下,观察成瘤情况.结果 酶切鉴定证实重组质粒中插入的目的基因片段及载体大小、方向和插入住点均正确,在转染的B16细胞中可见绿色荧光并检测到HPV16E6E7mRNA的生成,接种的转染细胞在小鼠皮下100%成瘤.结论 提示B16细胞转染E6E7后其致瘤性与转染空载体组和野生型B16细胞组无明显差异.     

Sequence variation of human papillomavirus type 16 E7 in preinvasive and invasive cervical neoplasias

Variation in the nucleotide sequence of the HPV 16 E7 gene in preinvasive cervical intra-epitherial neoplasia (CIN) and invasive cervical carcinoma specimens was analyzed. Direct DNA sequencing of PCR-amplified products with primers different from those used for PCR with 5-end labeling generated distinct sequence ladders with a low background, even in specimens containing relatively low copy numbers of HPV. Of 14 cervical neoplasias, 11 cases showed sequence diversity from prototype HPV16, and a total of 22 nucleotide exchanges were detected. Nine of these led to single amino acid exchanges: [Thr⁵] to [Lys⁵] in one case and [Asn²⁹] to [Ser²⁹] in eight cases. The [Ser²⁹] E7 was distributed uniformly among invasive carcinomas and precancerous legions, and was also found in a normal cervix. The [Lys⁵] E7 and [Ser²⁹] E7 had transforming potential similar to the prototype E7 assessed by cooperation with the activatedras gene in rat embryo fibroblasts.     

Activities of human papillomavirus 16 E6 natural variants in human keratinocytes

Genetic variations in the E6 oncogene have been associated with different risk for cancer progression. In the present study, the functional significance of human papillomavirus (HPV) polymorphism in the E6 oncogene was investigated. Ten HPV16 E6 variants containing amino acid substitutions in the N-terminal region of E6 were evaluated for different biological and biochemical activities in human keratinocytes, the target cells for HPV infection. Western blot analyses of primary foreskin human keratinocytes or immortalized human keratinocytes, stably transduced with the E6 variants, revealed reduced p53 and Bax levels in all E6 expressing cultures. The reduction induced by most E6 proteins was at similar levels and comparable to the reduction induced by the E6 prototype. The ability of the proteins to induce serum/calcium-differentiation resistant colonies in primary keratinocytes was more variable. Overall activities of the variants ranged between 0.24- and 2.18-fold of the E6 prototype activity. The I27R/L83V variant showed the lowest activity whereas the R8Q variant showed the highest activity. The L83V polymorphism previously associated with risk for cancer progression in some populations, showed significant activity, comparable to that of the E6 prototype, in reducing p53 and Bax levels. Furthermore, this variant showed enhancement in the ability to induce colonies resistant to serum/calcium-triggered differentiation, however, the difference from the prototype was not statistically significant. This, and augmentation of other described functions might result in differences in L83V pathogenicity.     

重组人乳头瘤病毒16型E6蛋白的表达、纯化和动物免疫效果分析

目的 诱导表达人乳头瘤病毒16型(HPV16)E6蛋白,制备可溶性蛋白,并通过动物免疫进行免疫效果评估.方法 采用IPTG诱导pQE30-HPV16E6/BL21(DE3)重组菌株,表达产物经SDS-PAGE和Western blot分析鉴定.提取包涵体进行变性处理,变性蛋白经Ni2+金属螯合层析纯化,将纯化产物用复性透析方法处理以获取可溶性蛋白.免疫Bal B/c小鼠,检测血清抗体、CD4+/CD8+和IFN-γ.结果 诱导后重组菌有相对分子量18 000蛋白质表达,以不溶性包涵体为主要表达方式.目的蛋白可与HPV16E6多克隆抗体识别,纯化和透析处理后得到可溶性的蛋白.小鼠免疫后血清抗体滴度升高,淋巴细胞CD4+/CD8+升高,IFN-γ未见升高.结论 成功表达了HPV16E6蛋白,同时处理包涵体并制备了可溶性目的蛋白.目的蛋白可刺激小鼠体内产生有效的免疫反应,该蛋白的成功制备为患者血清抗体的检测和肿瘤细胞杀伤的免疫研究奠定了坚实基础.     

Follow-up of antibody responses to human papillomavirus type 16 E7 in patients treated for cervical carcinoma

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.     

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.     

人乳头瘤病毒16型修饰后E6E7基因免疫原性增强

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。     

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.     

HPV16 E6 natural variants exhibit different activities in functional assays relevant to the carcinogenic potential of E6

Genetic studies have revealed natural amino acid variations within the human papillomavirus (HPV) type 16 E6 oncoprotein. To address the functional significance of E6 polymorphisms, 10 HPV16 E6 variants isolated from cervical lesions of Swedish women were evaluated for their activities in different in vitro and in vivo assays relevant to the carcinogenic potential of E6. Small differences between E6 prototype and variants, and among variants, were observed in transient expression assays that assessed p53 degradation, Bax degradation, and inhibition of p53 transactivation. More variable levels of activities were exhibited by the E6 proteins in assays that evaluated binding to the E6-binding protein (E6BP) or the human discs large protein (hDlg). The E6 prototype expressed moderate to high activity in the above assays. The L83V polymorphism, previously associated with risk for cancer progression in some populations, expressed similar levels of activity as that of the E6 prototype in most functional assays. On the other hand, L83V displayed more efficient degradation of Bax and binding to E6BP, but lower binding to hDlg. Results of this study indicate that naturally occurring amino acid variations in HPV16 E6 can alter activities of the protein important for its carcinogenic potential.