Generation and characterization of monoclonal antibodies against the E6 and E7 oncoproteins of HPV

Generation of three monoclonal antibodies (MAbs) to the major oncoproteins of human papillomavirus (HPV) was accomplished by an intense prime/boost regimen. Mice were primed with expression vectors expressing either the E6 or E7 oncoproteins of HPV-16 followed by boosting with a vaccinia virus construct and a replication-defective E1-deleted adenoviral recombinant of the human strain 5, and last, with baculovirus-derived HPV-16 E6 and E7 proteins in incomplete Freunds' adjuvant. Splenocytes were then fused with a myeloma cell line. The vaccination protocol generated one anti-E7 MAb of the IgM isotype and two anti-E6 MAbs of the IgG1 subisotype. The MAbs were tested for functionality in standard laboratory assays and found to detect the E6 and E7 proteins, respectively. The E7 MAb cross-reacted with the HPV-1a E7 oncoprotein. The binding sites of the MAbs were mapped to defined regions of each viral protein.     

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.     

Overexpression of aryl hydrocarbon receptor in human lung carcinomas

Exposure to polycylic aromatic hydrocarbons (PAH) has been associated with increased risk of lung cancer. Aryl hydrocarbon receptor (AhR) is known to play an essential role in PAH-induced toxicity. The objectives of this study were to identify and evaluate AhR expression in normal human lung tissues and in lung carcinomas. AhR protein and mRNA levels in human lung cell lines were evaluated with immunoblot and quantitative real-time RT-PCR assays, respectively. AhR protein expression was high in cytosol homogenates of adenocarcinoma (AD) cell lines and AhR mRNA levels corresponded well with AhR protein levels in these cell lines. AhR expression in human lung tissues and carcinomas were examined by means of immunohistochemical staining method. In normal lung tissues, immunostaining was found in the cytosol of bronchiolar epithelial cells. AhR immunostaining was more intense in AD than in squamous cell carcinomas. When AhR expression was compared with noral bronchiolar epithelial cells and neoplastic cells in the same specimens, the neoplastic cells, especially those of AD, demonstrated an increased staining. The upregulation of AhR mRNA expression was also demonstrated among 2 of 4 paired tissues with the quantitative real-time RT-PCR assay. Our data indicated that AhR expression was upregulated in lung AD and suggested that AhR and its expression might play an important role in the development of lung AD.     

HPV DNA、E6/E7 mRNA在各子宫颈病变及子宫颈鳞状细胞癌中的表达

目的 探讨HPV DNA和E6/E7 mRNA在各子宫颈病变及子宫颈鳞状细胞癌(cervical squamous cell carcinoma,CSCC)中的表达,并分析其与CSCC发生、发展的相关性。方法 应用PCR-反向杂交法和支链DNA技术对210例低级别鳞状上皮内病变(low-grade squamous intraepithelial lesion,LSIL)、200例高级别鳞状上皮内病变(high-grade squamous intraepithelial lesion,HSIL)及240例CSCC进行HPV DNA及E6/E7 mRNA的检测。结果 HSIL、CSCC中,HPV DNA百分率分别为100.00%、89.58%,呈递减趋势(P0.001);E6/E7 mRNA百分率分别为52.50%、95.83%,呈递增趋势(P0.001);HPV DNA+/mRNA+的百分率分别为52.50%、87.50%,呈递增趋势(P0.001);HPV DNA-/mRNA-的百分率分别为0、6.25%,呈递增趋势(P0.001)。LSIL、HSIL中,HPV DNA百分率均大于E6/E7 mRNA百分率,差异有统计学意义(P0.001);而CSCC中HPV DNA百分率均小于E6/E7 mRNA百分率,差异有统计学意义(P=0.008)。结论 HPV DNA在LSIL、HSIL中的表达较E6/E7 mRNA高;CSCC中HPV DNA表达较E6/E7 mRNA低,因此HPV DNA标志物可能与CSCC的发生过程关系较为紧密;E6/E7 mRNA标志物可能与CSCC的发展相关性更高。     

Induction of robust cellular immunity against HPV6 and HPV11 in mice by DNA vaccine encoding for E6/E7 antigen

Due to the strong relationship between the Human Papillomavirus (HPV) "high-risk" subtypes and cervical cancers, most HPV-related studies have been focusing on the "high-risk" HPV subtypes 16 and 18. However, it has been suggested that the "low-risk" subtypes of HPV, HPV6 and HPV11, are the major cause of recurrent respiratory papillomatosis and genital warts. In addition, HPV 6 and 11 are also associated with otolaryngologic malignancies, carcinoma of the lung, tonsil, larynx and low-grade cervical lesions. Therefore, development of HPV therapeutic vaccines targeting on subtypes 6 and 11 E6 or E7 are in great need. In this report, we describe two novel engineered DNA vaccines that encode HPV 6 and 11 consensus E6/E7 fusion proteins (p6E6E7 and p11E6E7) by utilizing a multi-phase strategy. Briefly, after generating consensus sequences, several modifications were performed to increase the expression of both constructs, including codon/RNA optimization, addition of a Kozak sequence and a highly efficient leader sequence. An endoproteolytic cleavage site was also introduced between E6 and E7 protein for proper protein folding and for better CTL processing. The expressions of both constructs were confirmed by western blot analysis and immunofluorescence assay. Vaccination with these DNA vaccines could elicit robust cellular immune responses. The epitope mapping assay was performed to further characterize the cellular immune responses induced by p6E6E7 and p11E6E7. The HPV 6 and 11 E6 or E7-specific immunodominant and subdominant epitopes were verified, respectively. The intracellular cytokine staining revealed that the magnitude of IFN-γ and TNF-α secretion in antigen-specific CD8(+) cells was significantly enhanced, indicating that the immune responses elicited by p6E6E7 and p11E6E7 was heavily skewed toward driving CD8(+) T cells. Such DNA immunogens are interesting candidates for further studies on HPV 6 and 11-associated diseases.     

芳香烃受体在体外高糖环境诱导心肌肥大过程中的表达

目的:观察高糖环境诱导心肌细胞肥大过程中芳香烃受体(AhR)的表达情况,并探讨其可能的作用机制。方法:以体外培养的大鼠心肌细胞H9c2为研究对象,实验分为正常糖浓度组、高糖组、DMSO组和白藜芦醇(AhR拮抗剂)组。免疫荧光染色观察AhR的表达情况,罗丹明标记的鬼笔环肽染色细胞骨架并计算细胞表面积,DCFH-DA法检测细胞内活性氧簇(ROS)的生成水平,实时荧光定量PCR及Western blot法检测AhR、CYP1A1、心钠素(ANP)和脑钠素(BNP)的表达情况。结果:正常糖浓度环境下,AhR的表达主要定位于细胞质,高糖刺激时转入细胞核内。高糖刺激可促使心肌细胞肥大、心肌细胞内ROS生成增加,白藜芦醇阻滞AhR后,心肌肥大得到明显改善,同时ROS生成水平明显减少。与正常糖浓度组及白藜芦醇组相比,高糖组的AhR、CYP1A1、ANP和BNP mRNA及蛋白表达水平明显升高,上述指标高糖组与DMSO组相比差异无统计学显著性,而白藜芦醇组明显低于DMSO组。结论:在高糖诱导的心肌肥大过程,心肌细胞的AhR表达增加可能参与维持正常糖代谢过程;高糖环境可激活AhR转入细胞核内,上调CYP1A1的表达,并促进ROS的生成,这可能是高糖诱导心肌肥大的重要机制之一。     

MicroRNA-25 regulates small cell lung cancer cell development and cell cycle through cyclin E2

Purpose: We intended to examine the underlying mechanism of microRNA-25 (miR-25) in regulating small cell lung cancer (SCLC). Methods: The miR-25 expression was measured by quantitative RT-PCR (qRT-PCR) in 5 SCLC cell lines and 9 human SCLC tissues. In SCLC cell line H510A cells, endogenous miR-25 was downregulated by stable transfection of antisense oligonucleotide of miR-25 (miR-25-as). Then the effects of miR-25 downregulation on SCLC growth, invasion and chemoresistance were assessed by MTT, migration and cisplatin assays, respectively. Furthermore, the effects of miR-25 downregulation on cancer cell cycle arrest, production of cell cycle proteins cyclin E2 and CDK2 were examined by cell cycle assay, western blot and luciferase assays, respectively. Finally, cyclin E2 was over-expressed in H510A cells to investigate its effect on miR-25 mediated SCLC regulation. Results: In both SCLC cells and human SCLC tumor tissues, miR-25 was overexpressed. Down-regulation of miR-25 in H510A cells significantly reduced cancer cell growth, invasive capability and resistance to cisplatin. Also, it induced G1 cell cycle arrest and downregulated cell cycle related proteins cyclin E2 and CDK2. Luciferase assay demonstrated cyclin E2 was directly targeted by miR-25. Overexpression of cyclin E2 in H510A cells reversed the cell cycle arrest and restored invasive capability impaired by miR-25 downregulation. Conclusions: Our study shows miR-25 is overexpressed in SCLC and acting as oncogenic regulator by regulating cyclin E2.     

Human papillomavirus type 16 E6 and E7 oncoproteins act synergistically to cause head and neck cancer in mice

High-risk human papillomaviruses (HPVs) contribute to cervical and other anogenital cancers, and they are also linked etiologically to a subset of head and neck squamous cell carcinomas (HNSCC). We previously established a model for HPV-associated HNSCC in which we treated transgenic mice expressing the papillomaviral oncoproteins with the chemical carcinogen 4-nitroquinoline-1-oxide (4-NQO). We found that the HPV-16 E7 oncoprotein was highly potent in causing HNSCC, and its dominance masked any potential oncogenic contribution of E6, a second papillomaviral oncoprotein commonly expressed in human cancers. In the current study, we shortened the duration of treatment with 4-NQO to reduce the incidence of cancers and discovered a striking synergy between E6 and E7 in causing HNSCC. Comparing the oncogenic properties of wild-type versus mutant E6 genes in this model for HNSCC uncovered a role for some but not other cellular targets of E6 previously shown to contribute to cervical cancer.     

Notch信号转导对小细胞肺癌的调控及其机制研究

目的 探讨Notch信号转导对小细胞肺癌的调控作用及可能机制.方法 应用重组质粒转染的方法,在小细胞肺癌细胞株NCI-H446中表达组成性活化的Notch1(NIC转染组),同时设立转染空质粒组和未转染组作为对照组,待筛选出稳定细胞株后,以MTT法检测细胞活力,应用半定量RT-PCR技术测定Notch1及其下游基因(HES1和hASH1)表达,并应用免疫细胞化学标记和Westernblot技术对神经内分泌标志物嗜铬粒素A(CgA)、神经元特异性烯醇化酶(NSE)的表达行半定量[阳性单位(PU)值]分析.结果 未转染组和转染空质粒组Notch1及其下游基因HES1表达不明显,而hASH1表达显著,转染组细胞Notch1及HES1表达升高,同时伴有hASH1表达明显降低.与两对照组比较,转染组细胞增殖速度显著降低,连续6 d测得的吸光度(A)值均小于未转染组和转染空质粒组(均P<0.05).免疫细胞化学染色显示,NIC转染组、转染空质粒组、未转染组的CgA染色的PU值分别为8.81±0.77、38.10±1.55、38.97±0.80,NSE染色的PU值分别为7.21±0.59、28.25±1.46、30.57±1.31,NIC转染组CgA和NSE的PU值均小于转染空质粒组和未转染组(均P<0.01).Western blot检测结果中,将未转染组的条带灰度值设为1.00,NIC转染组、转染空质粒组的CgA条带灰度值分别为0.54±0.03、0.99±0.05,NSE条带灰度值分别为0.43±0.02、1.07±0.09,NIC转染组cgA和NSE条带的灰度值均小于转染空质粒组和未转染组(均P<0.01).结论 Notch信号途径对小细胞肺癌的调控可能是通过靶基因HES1对分化效应基因hASH1的转录抑制来实现的;Notch信号途径可以抑制小细胞肺癌细胞的增殖,降低其神经内分泌标志物的表达.