A novel type of human papillomavirus associated with genital neoplasias

The role of human papillomaviruses (HPVs) in the development of genital neoplasias has been well documented. The genomes of two HPV types, HPV16 and HPV18, have been found to be associated with about 70% of invasive carcinomas of the uterine cervix. As, under non-stringent hybridization conditions, HPV DNA sequences have been detected in about 90% of cervical carcinomas, it seems likely that additional HPV types are associated with these tumours. Here we report the molecular cloning and characterization of a novel HPV type, tentatively named HPV33, whose sequences have been detected in 4-8% of biopsies of genital intra-epithelial neoplasias and cervical invasive carcinomas, usually as free monomeric or oligomeric molecules. However, in one specimen of vulvar Bowen's disease, HPV33 DNA sequences were integrated in the host-cell genome. Thus, HPV33 probably represents an additional type of potentially oncogenic genital HPV.     

宫颈癌中CALCA基因甲基化与HPV16-E7致癌蛋白表达的依存关系

 选择HPV16 阳性宫颈癌细胞和RNAi 技术,研究CALCA 基因甲基化与HPV16-E7 致癌蛋白表达的依存关系。构建慢病毒siRNA 重组表达载体,建立稳定表达HPV16-E7-siRNA 的RNAi 细胞模型。以SiHa 细胞和RNAi 细胞模型的基因组DNA 为对象,选择CALCA 基因启动子区富含CpG 岛屿的目标片段,使用亚硫酸氢盐测序法（bisulfite sequencing PCR,BSP）筛查分析,研究RNAi 抑制HPV16-E 7 表达后,CALCA 基因甲基化状态的可逆性程度。选出CALCA 基因启动子区富含CpG 位点的目标片段,其大小为365 bp,含有19 个CpG 岛屿,发现其中13 个CpG 位点的胞嘧啶在SiHa 细胞基因组DNA 中发生了甲基化（13/19）,而在表达HPV16-E7-siRNA 的RNAi 细胞模型中,所有CpG 位点的甲基化已发生逆转（0/19 位点）。本研究从细胞水平证明了宫颈癌细胞内的CALCA 基因启动子高甲基化对HPV16-E7 致癌蛋白表达有依赖性,为进一步研究E7 蛋白的作用及致癌机制奠定了重要的物质基础。     

Glucocorticoid-dependent oncogenic transformation by type 16 but not type 11 human papilloma virus DNA

Squamous cell carcinoma of the uterine cervix is one of the most common cancers among women. Correlation between human papilloma virus (HPV) infection of the uterine cervix and the development of cervical neoplasia has been established. More recent studies have shown the presence and expression of integrated HPV types 16 and 18 DNA sequences in 70-80% of cervical tumours and tumour cell lines. It has been suggested that, in addition to HPVs, other agents such as hormones and tobacco products act as cofactors in cervical neoplasia (for review see ref. 15). The presence and expression of a glucocorticoid-responsive element in HPV-16 has been reported. Here we provide evidence for the oncogenic transformation of primary cells with a combination of HPV-16 DNA, but not HPV-11 DNA, and the activated form of the human Ha-ras oncogene only in the presence of the glucocorticoid hormone dexamethasone.     

巨噬细胞增强宫颈癌细胞对SN-38的抗性

通过GDSC在线软件,分析了SN-38在数据库中8种宫颈癌细胞系对310多种化疗药的半抑制质量浓度(IC50)Z分数值,并选取HPV-18阳性HeLa细胞、HPV-16阳性SiHa和CaSki细胞以及HPV阴性C-33A细胞,经过SN-38处理24、48 h,通过CCK8试验检测了细胞活力来探究巨噬细胞是否介导宫颈癌细...     

mRNA transcripts related to full-length endogenous retroviral DNA in human cells

Mammalian cells contain multiple copies of endogenous type C retroviral DNA sequences. Among these sequences are complete, potentially infectious proviruses, proviral DNA that is expressed only in the form of viral antigens, retroviral segments that may contribute portions of envelope (env) genes during the generation of recombinant polytropic viruses, and many subgenomic viral DNA segments that may not be expressed at all. We have previously reported the identification and molecular cloning of type C retroviral sequences from human DNA and have shown that the partial nucleotide and deduced amino acid sequences of one of the clones obtained (lambda 51) are homologous to Moloney MuLV (MoMuLV) in the gag and pol regions. The lambda 51 clone as well as several others isolated from a human DNA library contained approximately 4.3 kilobases (kb) of retroviral sequences, were deleted in the env region, and were flanked by tandem repeats unlike the long terminal repeats (LTRs) typically found in proviral DNAs (P.E.S., in preparation). We describe here the characterization of a full-length human retroviral clone (lambda 4-1) containing LTR elements as well as a putative env region. DNA-RNA hybridization experiments reveal that human cells contain species of poly(A)+ RNA that anneal to segments of the full-length retroviral DNA clone.     

人乳头瘤病毒感染的宫颈癌组织的病理形态学观察

应用核酸杂交技术结合组织病理学的光镜和电镜观察、检测８０例子宫颈癌、４２例正常宫颈组织、３３例宫颈非典型增生组织和１０例宫颈尖锐湿疣组织中的ＨＰＶｓ基因组相关序列。结果表明：（１）４２例正常宫颈组织ＤＮＡ无一例检出ＨＰＶ，而在宫颈癌组织、宫颈非典型增生组织和宫颈尖锐湿疣组织中，ＨＰＶ检出率分别达４３．７５％、４５．４５％和８０％；（２）宫颈癌组织中阳性３５例，阳性率为４３．７５％；ＨＰＶ型别分布主要为ＨＰＶ１６型（１８例，５１．４３％）和ＨＰＶＲ型（８例，２２．８６％）；（３）１８例ＨＰＶ１６型和３例ＨＰＶ１１／６型均为鳞癌，８例ＨＰＶＲ型中有６例也为鳞癌。ＨＰＶ１６型的１８例中有１２例为低分化癌，ＨＰＶＲ型的８例中有５例也为低分化癌；（４）不同型别ＨＰＶ感染的宫颈癌组织，其病理形态学特征有所不同。研究表明，广西地区相当部分宫颈癌的发生与ＨＰＶ感染有关，其感染的宫颈癌组织具有一定的病理形态学特征。     

Novel submicroscopic extrachromosomal elements containing amplified genes in human cells

In previous work, several methotrexate (MTX)-resistant variants were isolated frm the human cell line HeLa BU25, which exhibited a high degree of dihydrofolate (DHFR) gene amplification (estimated to be 250- to 300-fold). These variants did not contain any chromosome with a homogeneously staining region (HSR) and exhibited only a small average number of minute chromosomes per cell: these two types of karyotypic abnormalities generally accompany selective gene amplification. We now report that structures containing amplified DHFR genes in one of these variants (HeLa BU25-10B3) can be isolated by pulsed-field gradient or field-inversion gel electrophoresis as homogeneous DNA molecules of approximately 650 kilobases (kb). Electron microscopy of metaphase spreads from these cells reveals chromatin fibres with a similar DNA content, which are probably related to the above elements. These represent a novel type of extrachromosomal structures in mammalian cells.     

Specific interaction between HPV-16 E1-E4 and cytokeratins results in collapse of the epithelial cell intermediate filament network.

The human papillomaviruses (HPV) are associated specifically with epithelial lesions, ranging from benign warts to invasive carcinoma. The virus encodes three late proteins, which are produced only in terminally differentiating keratinocytes, two of which are structural components of the virion. The third, E1-E4, is derived primarily from the E4 open reading frame, which represents a region of maximal divergence between different HPV types. E1-E4 does not seem to be a component of the virus particle or to be needed for transformation in vitro, but accumulates in the cytoplasm, where in certain benign lesions it can comprise 20-30% of total cell protein. We show here that expression of the HPV-16 E1-E4 protein in human keratinocytes (the natural host cell for HPV infection) results in the total collapse of the cytokeratin matrix. Tubulin and actin networks are unaffected by E1-E4, as are the nuclear lamins.     

A truncated human chromosome 16 associated with alpha thalassaemia is stabilized by addition of telomeric repeat (TTAGGG)n

The instability of chromosomes with breaks induced by X-irradiation led to the proposal that the natural ends of chromosomes are capped by a specialized structure, the telomere. Telomeres prevent end-to-end fusions and exonucleolytic degradation, enable the end of the linear DNA molecule to replicate, and function in cell division. Human telomeric DNA comprises approximately 2-20 kilobases (kb) of the tandemly repeated sequence (TTAGGG)n oriented 5'----3' in towards the end of the chromosome, interspersed with variant repeats in the proximal region. Immediately subtelomeric lie families of unrelated repeat motifs (telomere-associated sequences) whose function, if any, is unknown. In lower eukaryotes the formation and maintenance of telomeres may be mediated enzymatically (by telomerase) or by recombination; in man the mechanisms are poorly understood, although telomerase has been identified in HeLa cells. Here we describe an alpha thalassaemia mutation associated with terminal truncation of the short arm of chromosome 16 (within band 16p13-3) to a site 50 kb distal to the alpha globin genes, and show that (TTAGGG)n has been added directly to the site of the break. The mutation is stably inherited, proving that telomeric DNA alone is sufficient to stabilize the broken chromosome end. This mechanism may occur in any genetic disease associated with chromosome truncation.     

双特异性酪氨酸磷酸化调节激酶在宫颈病变组织和癌细胞中的表达

目的探讨双特异性酪氨酸磷酸化调节激酶1b(DYRK1b)在宫颈病变组织和癌细胞中的表达情况及其作用.方法选取宫颈癌患者127例为研究组,同期确诊为慢性宫颈炎患者32例为对照组,收集上述患者石蜡组织标本,免疫组化SP法检测DYRK1b蛋白表达情况.依据宫颈癌细胞系He La 229和Si Ha细胞将标本分为实验组(加DYRK1b抑制剂Az191)、对照组(不加Az191),Western blod法检测细胞中DYRK1蛋白表达情况,MTT法检测细胞增殖情况,流式细胞仪检测细胞凋亡情况.结果子宫颈鳞癌阳性表达率明显高于慢性宫颈炎、低级别鳞状上皮内病变、高级别鳞状上皮内病变,差异均具有统计学意义(P0.01);高级别鳞状上皮内病变明显高于慢性宫颈炎、低级别鳞状上皮内病变,差异具有统计学意义(P0.05).在He La 229和Si Ha细胞中,DYRK1b蛋白表达量随Az191剂量增加而减少,且均明显低于对照组,差异具有统计学意义(P0.01).研究组He La 229和Si Ha细胞抑制率随Az191浓度增加而提升,均明显高于对照组,差异具有统计学意义(P0.05).10μmol/L的Az191作用后,研究组He La 229和Si Ha细胞凋亡率明显高于对照组,差异具有统计学意义(P0.05).结论宫颈病变过程中DYRK1b蛋白表达水平逐渐提升,宫颈癌组织和细胞中均呈高表达;下调DYRK1b蛋白表达后可抑制宫颈癌细胞增殖,促进凋亡.     

东方蝼蛄提取物对3株人宫颈癌细胞的细胞毒性研究

采用乙醇冷浸法对东方蝼蛄的干燥品粉碎物进行提取,采用聚酰胺柱层析分离划段得到东方蝼蛄乙醇提取物的不同部位,所得部位通过四甲基偶氮唑盐微量酶反应比色法(MTT法)对3种人宫颈癌细胞株(He La、Caski、C-33A)进行了体外肿瘤细胞毒性测试.结果表明,东方蝼蛄提取物中有1个分离样品的半数抑制浓度(IC50)接近10.0μg/mL,对3种人宫颈癌细胞株均具有明显的细胞毒性.东方蝼蛄乙醇提取物中存在抑制肿瘤细胞生长的物质,值得对其进一步深入研究.     

云芝多糖影响人宫颈癌HeLa细胞的增殖和凋亡的研究

研究云芝多糖(CVP)对宫颈癌HeLa细胞系的增殖、凋亡及产生途径。采用MTT法测定了CVP对HeLa细胞增殖的体外抑制作用,流式细胞仪检测凋亡细胞的比例,qRT-PCR检测P53、Bcl-2和Fas基因在细胞处理前后表达量的变化。结果显示：CVP以时间和剂量依赖的方式抑制HeLa细胞的生长。作用时间在24 h,48 h和72 h时对细胞抑制的抑制率分别为92%、95%和98%;当CVP浓度达到1.25 mg/L时,作用24 h、48 h和72 h时的IC50值分别为0.2507±0.01 mg/L、0.2720±0.04 mg/L和0.2736±0.03 mg/L;当CVP浓度为0.5 mg/L时,作用24 h和48 h后细胞凋亡率分别为26.36% 和63.81%;CVP处理HeLa细胞24 h后,Bcl-2基因的表达被显著下调。研究表明：CVP促进HeLa细胞凋亡,其作用机制与Bcl-2基因的表达下调有关,推测是通过Bcl-2介导的途径促进细胞凋亡。图4表1参30     

Expression of the intact C<Subscript>4</Subscript> type<Emphasis Type="Italic">pepc</Emphasis> gene cloned from maize in transgenic winter wheat

Maize intact C4-pepc gene was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as pBAC214 (12 kb), which contains CaMV 35S promoter driven bar gene as selection marker. Comparing the cloned DNA sequences (6.7 kb) with published maize C4-pepc gene (GenBank accession E17154) sequences, the identity of DNA sequence alignment is 98.96%. There are only 49 differences between these two intact DNA sequences, of which 13 occur in the region of promoter, 18 in introns, and 18 in exons. The homology of mRNA sequence alignment is 99.38%, and the putative amino acids sequence identity is 99.38%. There are only 15 differences between these two mRNA, and these differences bring 4 sites mutant on the putative amino acids of PEPC protein. Through biolistic bombardment of PDS1000/He system, expression vector pBAC214 has been transformed into winter wheat. Southern blotting results show that the intact C4-pepc gene has been integrated into genome of winter wheat. SDS-PAGE analysis of leaf soluble protein in transgenic wheat showed that the intact C4opepc gene was well transcribed, spliced and translated as in maize. The enzyme activity of leaf PEPC in transgenic wheat has been detected. The activities of leaf PEPC increased over 3-5 times in some transgenic plants. The data of photosynthesis rate and transpiration rate of transgenic wheat flag leaves showed that the C4-pepc gene can increase the photosynthesis rate and transpiration rate of transgenic wheat.     

宫颈HPV感染与宫颈瘤样病变的相关性

为了研究湘西地区妇女宫颈人乳头瘤病毒（HPV）感染与宫颈瘤样病变的相关性,为宫颈癌的早期预防提供科学参考,选取692例湘西籍患者为研究对象,所有患者均行宫颈HPV检测,以其中HPV检查结果阳性的234例患者作为观察组,阴性的234例患者作为对照组,均在阴道镜监视下进行宫颈多点活检及宫颈管搔刮病理学检查,病理检测结果分为癌前病变（CINⅠ,Ⅱ,Ⅲ）、宫颈癌、慢性宫颈炎.结果显示：湘西地区女性中HPV感染以HPV16（25.64%）类型最多,其余依次为HPV52,HPV51,HPV58,HPV18,HPV53为常见;CIN的加重程度随HPV感染率升高,CINⅠ,Ⅱ,Ⅲ两两比较差异具有统计意义;HPV阳性率为33.81%,HR-HPV（+）31.74%,其中单一感染HR-HPV（+）21.97%,双重或多重感染11.99%,LR-HPV（+）感染1.88%.说明在宫颈癌的筛查中高危HPV检查具有临床价值,多途径切断高危HPV感染对本地区妇女宫颈癌的一级预防具有重要意义.