转录因子YY1对口腔鳞癌细胞ITGB6基因表达的影响

 目的：研究肿瘤相关转录因子YY1 对口腔鳞癌细胞整合素β6（integrin β6, ITGB6）基因表达调控的影响。方法：用生物信息学方法预测分布在ITGB6启动子区域的转录因子YY1潜在的结合位点,构建萤光素酶报告基因质粒,利用双萤光素酶报告基因系统检测ITGB6启动子片段的转录活性;利用染色质免疫沉淀技术检测在天然染色质条件下转录因子YY1与ITGB6启动子的结合情况;采用定点突变方法检测YY1结合位点对ITGB6启动子活性的影响;利用RT-PCR方法检测过表达转录因子YY1对口腔鳞癌细胞ITGB6 mRNA表达水平的影响。结果：ITGB6启动子-421~-150 nt区域存在多个转录因子YY1潜在的结合位点。在口腔鳞癌细胞的天然染色质中,转录因子YY1结合于ITGB6启动子-421~-150 nt区域;定点突变YY1潜在结合位点对口腔鳞癌细胞中ITGB6启动子活性无显著影响。另外,过表达的YY1对口腔鳞癌细胞ITGB6 mRNA的表达水平也无影响。结论:转录因子YY1在口腔鳞癌细胞中结合于ITGB6基因启动子-421~-150 nt区域,但对ITGB6基因的基础转录水平无影响。     

细胞转录调节因子YY1及其对人乳头瘤病毒16型早期？…

目的 为了检测ＹＹ１在不同宫颈癌细胞及人乳头瘤病毒易感细胞中的表达、功能状态和ＹＹ１位点破坏所诱导的Ｐ９７活性增强效应。方法 提取了４种宫颈癌细胞和２种人角源细胞胞核蛋白，检测其内源性ＹＹ１蛋白。同时将带有ＨＰＶ１６标准ＬＣＲ和ＹＹ１位点突变ＬＣＲ序列的荧光素酶质粒短暂转染上述细胞以检测Ｐ９７活性。结果 所有被检测细胞均含有良好生物学活性的ＹＹ１蛋白，其蛋白量在各细胞间无明显差别。ＨＰＶ１６ＬＣＲ     

细胞转录调节因子YY1及其对人乳头瘤病毒16型早期启动子P97抑制效应广泛存在于人上皮细胞

目的为了检测ＹＹ１在不同宫颈癌细胞及人乳头瘤病毒易感细胞中的表达、功能状态和ＹＹ１位点破坏所诱导的Ｐ９７活性增强效应。方法提取了４种宫颈癌细胞和２种人角源细胞胞核蛋白，检测其内源性ＹＹ１蛋白。同时将带有ＨＰＶ１６标准ＬＣＲ和ＹＹ１位点突变ＬＣＲ序列的荧光素酶质粒短暂转染上述细胞以检测Ｐ９７活性。结果所有被检细胞均含有良好生物学活性的ＹＹ１蛋白，其蛋白含量在各细胞系间无明显差别。ＨＰＶ１６ＬＣＲ上ＹＹ１位点的破坏可在多种细胞，包括人类原代角源细胞中诱导Ｐ９７活性增强。结论表明ＹＹ１蛋白调节系统广泛存于ＨＰＶ易感细胞系内。同时我们还发现转录激活因子ＮＦ１在Ｃ３３ａ细胞中的含量明显高于ＨＴ３细胞，并影响ＹＹ１位点改变所致的Ｐ９７活性增强效应。这提示在不同的细胞系中活性蛋白的表达和含量可能不同     

Cataracts in transgenic mice caused by a human papillomavirus type 18 E7 oncogene driven by KRT1-14

Human papillomavirus type 18 (HPV18) is a common cause of cervical cancer. To create a mouse model for this common neoplastic disease, we used a human keratin 14 promoter to drive the HPV18 E7 oncogene to create transgenic mice. No mice up to a year of age developed cervical cancer. However, all transgenic mice and none of the controls developed progressive bilateral cortical cataracts. By 6 months of age, the cortex liquefied leaving the lens nucleus. Proliferation of lens epithelium formed multifocal nodules and free floating lens epithelial cells within the liquefied cortex. These cells were hyperplastic not neoplastic. Other HPV transgenic stocks develop cataracts suggesting this virus may have a broad cellular tropism.     

Role of the cellular transcription factor YY1 in the latent-lytic switch of Kaposi's sarcoma-associated herpesvirus

Lytic cycle reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) is initiated by expression of the ORF50 gene. Here we show that YY1 protein specifically binds to the ORF50 promoter (ORF50p) region in vitro and in vivo. After treatment with chemical inducers, including sodium butyrate (SB) and TPA, the levels of YY1 protein are inversely correlated with the lytic induction of KSHV in cells. Overexpression of YY1 completely blocks the ORF50p activation in transient reporter assays, while mutation at the YY1 site in the ORF50p or knockdown of YY1 protein confers an enhancement of the ORF50p activation induced by SB and TPA. YY1 overexpression in a stable cell clone HH-B2(Dox-YY1) also inhibits expression of the ORF50 and its downstream lytic genes. On the other hand, a chimeric YY1 construct that links to its coactivator E1A can disrupt viral latency. These results imply that YY1 is involved in the regulation of KSHV reactivation.     

Presence and expression of human papillomavirus sequences in human cervical carcinoma cell lines.

A series of human carcinoma cell lines was examined for human papillomavirus (HPV) DNA sequences with the use of HPV-6, HPV-11, HPV-16, and HPV-18 DNA probes. Six of eight cell lines derived from human cervical carcinomas were shown to contain integrated HPV DNA sequences. In five of these six lines, HPV-specific polyadenylated RNA species could also be identified. The expression of HPV sequences was detected in three lines with a HPV-18 DNA probe and in two lines with a HPV-16 DNA probe. Of the two lines which contained HPV-16 specific RNA, one contained HPV DNA sequences which hybridized only to an HPV-16 probe, and the other contained HPV DNA sequences which hybridized to both HPV-16 and HPV-18 DNA probes. Six cell lines established from human squamous-cell carcinomas of the bladder, pharynx, lung, esophagus, and vulva were negative for HPV-6, HPV-11, HPV-16, and HPV-18 DNA sequences under stringent hybridization conditions.     

Enhanced human papillomavirus type 8 oncogene expression levels are crucial for skin tumorigenesis in transgenic mice

Human papillomavirus 8 (HPV8) is involved in skin cancer development in epidermodysplasia verruciformis patients. Transgenic mice expressing HPV8 early genes (HPV8-CER) developed papillomas, dysplasias and squamous cell carcinomas. UVA/B-irradiation and mechanical wounding of HPV8-CER mouse skin led to prompt papilloma induction in about 3 weeks. The aim of this study was to analyze the kinetics and level of transgene expression in response to skin irritations. Transgene expression was already enhanced 1 to 2 days after UVA/B-irradiation or tape-stripping and maintained during papilloma development. The enhanced transgene expression could be assigned to UVB and not to UVA. Papilloma development was thus always paralleled by an increased transgene expression irrespective of the type of skin irritation. A knock-down of E6 mRNA by tattooing HPV8-E6-specific siRNA led to a delay and a lower incidence of papilloma development. This indicates that the early increase of viral oncogene expression is crucial for induction of papillomatosis.     

Transcriptional control of MHC class II antigen expression on mouse T cell lines

We have analysed the factors which regulate MHC class II expression in mouse T cell lines. Two such lines, BW 5147 and PLT-24.2, were used in this study. Using 5-azacytidine (5 AzaC) we have shown that hypomethylation of DNA can induce class II antigen synthesis in BW 5147. The expression of class II in PLT-24.2 cells seems to be under a different control mechanism. Southern blot analysis of I-A beta gene in PLT-24.2 suggests that the expression of class II in this cell line is probably the outcome of a gene rearrangement. We hypothesise that insertion of viral long terminal repeats (LTR) next to the class II genes in transformed T cell lines can act as a promoter for the expression of class II antigens.     

Karyotypic analysis of two related cervical carcinoma cell lines that contain human papillomavirus type 18 DNA and express divergent differentiation

The cell lines C-4I and C-4II were established in culture from a nonkeratinizing squamous carcinoma of the uterine cervix. Both lines contain human papillomavirus (HPV) type 18 DNA (Brandt et al., Cold Spring Harbor Laboratories, 5:179, 1987) and both are hypodiploid with similar, but not identical, karyotypes. Each line expresses multiple characteristics of ectocervical epithelial differentiation, but the characteristics differ between the lines. In the present study, G banding of the lines showed that cells of both lines have two normal chromosomes 1-5, 8-10, 13, 16, and 17, one normal chromosome 12 and 14, and no normal chromosomes 15 and 18. The lines share three abnormal chromosomes, der(8)t(8q;12q), der(18)t(18q;?), and i(5p). There are specific differences between the lines. C-4I has two normal chromosomes 6, while C-4II has one; C-4II has two chromosomes 11 and der(18)t(18q;?), while C-4I lacks both chromosomes 11 and has one der(18)t(18q;?). Each line has unique markers that include del(11)(p11), del(22)(q12), and del(21)(q21) in C-4I and i(15q), der(X)t(Xq;9p), der(6)t(6p;14q), and del(4)(q21) in C-4II. The results show that these phenotypically distinct lines are derived from the same clone and that the 8q arm (the site of HPV 18 integration) is present in three copies in both lines. They also define several chromosome rearrangements that are compatible with the expression of specific differentiation markers.     

人乳头瘤病毒16型中国株L1基因的体外真核表达

目的 构建人类乳头瘤病毒16型(HPV16)中国株晚期基因L1的真核表达系统.方法 将HPV16中国株L1基因从pCR2.1/HPV16L1定向亚克隆至pTacer-CMV载体,构建重组质粒pTaeer-CMV/HPV16L1,将重组质粒用脂质体转染N1H3T3、HeLa细胞,用透射电镜、SDS-PAGE、蛋白印迹等方法观察HPV16 L1蛋白的表达.结果 NIH3T3、HeLa细胞经pTacer-CMV/HPV16L1转染后,SDS-PAGE、蛋白印迹等实验显示可表达HPV16 L1蛋白,电镜下可见病毒样颗粒(VLP).结论 pTacer-CMV/HPV16L1构建成功,可在体外表达HPV16中国株L1蛋白.     

HPV 16 YY1位点突变株诱导的永生化细胞系具有部分转化细胞特征

目的 检测ＨＰＶ １６ ＹＹ１位点突变株诱导的包皮角原细胞永生化细胞系的生物学特性。方法 提取永生化细胞株蛋白,以Ｗｅｓｔｅｒｎ ｂｌｏｔ检测细胞内源性ｐ５３蛋白含量;以ＴＲＡＰ法检测细胞中端粒体酶活性;将永生化细胞系接种于含有１０％ＦＣＳ的软琼脂培养基,观测细胞的软琼脂生长能力。结果 Ｗｅｓｔｅｒｎ ｂｌｏｔ检测结果显示４株永生化细胞系中ｐ５３均为阴性;端粒体酶活性均为阳性,并且随着细胞传代活性