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     

High levels of p105 (NFKB1) and p100 (NFKB2) proteins in HPV16-transformed keratinocytes: role of E6 and E7 oncoproteins

We have previously shown that functional components of the NF-kappaB signaling pathway are up-regulated and sequestered in the cytoplasm of human papillomavirus 16 (HPV16)-transformed cell lines leading to a reduced activity of NF-kappaB. In this study, we examined the expression of the NF-kappaB precursors p100 and p105 in keratinocytes transformed or not by HPV16. Western immunoblotting experiments demonstrated high levels of p100 and p105 proteins not only in HPV16+ cervical carcinoma-derived keratinocytes but also in keratinocytes stably transfected by HPV16 E6 or E7 oncogenes. Moreover, p100 and p105 proteins were predominantly cytoplasmic and nuclear in keratinocytes expressing E7 and E6, respectively. A predominantly cytoplasmic localization of E7 protein was also detected in all keratinocytes expressing E7. Our results suggest that HPV16 E6 and E7 proteins modulate the expression and the subcellular localization of p100 and p105 NF-kappaB precursors.     

Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation

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.     

Binding of PDZ proteins to HPV E6 proteins does neither correlate with epidemiological risk classification nor with the immortalization of foreskin keratinocytes

There is compelling evidence that high-risk human papillomaviruses (HPV) can cause cervical cancer. Strikingly, HPV16 and 18 account for ∼ 70% of all cervical cancers, whereas phylogenetically related types are found at much lower frequencies. Most likely, differences in the activities of the viral E6 and E7 oncoproteins account for the in vivo carcinogenicity. We demonstrate here that E6 proteins from low-risk HPV70 and possibly high-risk HPV82 interact and degrade PDZ proteins hDlg and Magi1 identical to HPV16E6 and HPV18E6. In contrast high-risk HPV66E6 did not bind or degrade hDlg or Magi1. We also show that low-risk HPV70 E6/E7 immortalizes normal human keratinocytes. Together with our previous analysis concerning p53 degradation, this shows that neither binding of E6 to p53, to E6AP, to Magi1 and hDlg, the degradation of hDlg and Magi1, nor immortalization of normal human keratinocytes seems to be a reliable predictor for carcinogenic behavior of HPV in the cervix.     

腺病毒E1A蛋白抑制大鼠肺泡上皮细胞γ-谷氨酰半胱氨酸合成酶催化亚单位表达的机制研究

目的： 研究腺病毒E1A蛋白抑制大鼠肺泡上皮细胞γ-谷氨酰半胱氨酸合成酶催化亚单位（GCLC）表达的机制。方法: 构建稳定表达腺病毒E1A蛋白的大鼠肺泡上皮细胞，将GCLC基因5’-上游调控序列不同长度的缺失体-萤火虫荧光素酶报道系统转染后分析转录活性的变化；检测AP-1、NF-κB、USF与GCLC基因调控序列的结合活性。结果: GCLC基因5’-上游调控序列报道系统检测结果显示大部分（9/11）缺失体的转录活性抑制，AP-1、NF-κB、USF与GCLC基因的结合活性增强，而对应的功能元件的转录活性降低。结论: 腺病毒E1A蛋白通过抑制GCLC基因 5’-上游调控序列的转录活性，扩大大鼠肺泡上皮细胞氧化应激时的氧化/抗氧化失衡,其机制可能涉及E1A对辅助转录因子的抑制。     

E6 and E7 oncoproteins from human papillomavirus type 16 induce activation of human transforming growth factor beta1 promoter throughout Sp1 recognition sequence

Human Papillomavirus (HPV) infection is the main etiologic agent of cervical cancer and HPV E6 and E7 oncogenes trans-regulate many cellular genes. An association between TGF-beta1 gene expression and cervical cancer development has been suggested; however, the mechanisms by which HPV influences TGF-beta1 expression remain unclear. In the present study we analyzed the mechanism through which HPV-16 E6 and E7 oncoproteins regulate the TGF-beta1 promoter in cervical tumor cells. Our results showed that E6 and E7 increased TGF-beta1 promoter activity. Furthermore, we identified a specific DNA sequence motif in the TGF-beta1 core promoter that is responsible for trans-activation and that corresponds to the Sp1e-binding site associated with HPV-16 E6 and E7 oncoproteins. Mutational analysis showed that the Sp1e recognition site abolished the trans-activation caused by E6 and E7. These results suggest a physical interaction and functional cooperation between viral oncoproteins and cellular regulatory elements of the TGF-beta1 promoter, and may explain the contribution of HPV-16 to TGF-beta1 gene expression in cervical cancer.     

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     

关于人乳头状瘤病毒16型启动子控制区域switch位点 …

目的 细胞转录调节因子ＹＹ１对人乳头状瘤病毒１６型（Ｈｕｍａｎ ｐａｐｉｌｌｏｍａｖｉｒｕｓ ｔｙｐｅ１６ ＨＰＶ１６）早期启动子Ｐ９７起抑制作用,而对ＨＰＶ１８早期启动子Ｐ１０５的调节作用受位于ＹＹ１特异性结合位点上游序列的ｓｗｉｔｃｈ位点的影响。本研究观测在ＨＰＶ１６调节区域有无类似的ｓｗｉｔｃｈ位点存在。