Cheng Ya-Wen, Wu Tzu-Chin, Chen Chih-Yi, Chou Ming-Chih, Ko Jiunn-Liang, Lee Huei
Institutes of Medicine, Chung Shan Medical University, Taichung, Taiwan, Republic of China.
Clin Cancer Res. 2008 Nov 15;14(22):7173-9. doi: 10.1158/1078-0432.CCR-08-0850.
Our recent report indicates that human papillomavirus (HPV)-16/18 E6 oncoprotein is expressed in lung tumors and is related to p53 inactivation. We further explored whether human telomerase reverse transcriptase (hTERT) transcription is up-regulated by E6 and contributes to lung tumor development.
Immunohistochemistry detected HPV-16 E6 oncoprotein in 135 lung tumors, and hTERT mRNA was evaluated by real-time reverse transcription-PCR and in situ hybridization, respectively. A small RNA interference (RNAi), Western blotting, and chromatin immunoprecipitation analysis were used to clarify whether hTERT transcription was regulated by c-Myc and Sp1. The telomerase activity and oncogenic potential of TL-1 with or without E6- or hTERT-RNAi was determined by real-time quantitative telomeric repeat amplification protocol analysis and soft-agar assay, respectively.
hTERT mRNA levels in E6-positive tumors, which were prevalent in females, nonsmokers, and adenocarcinomas, were significantly higher than in E6-negative tumors. In addition, hTERT mRNA levels in early tumors (stage I) were greater than levels in advanced tumors (stages II and III). Chromatin immunoprecipitation assay showed that Sp1 cooperated with c-Myc to activate hTERT transcription in TL-1 cells, which was similar to the SiHa cells. The telomerase activity of the TL-1 cells decreased concomitantly with the transfection of various doses of E6- or hTERT-RNAi. A soft-agar assay showed that the oncogenic potential of TL-1 cells was significantly reduced after being transfected with E6-RNAi. Moreover, a colony of TL-1 cells could not form after transfection with hTERT-RNAi.
Transcriptional activation of hTERT by E6 oncoprotein is required for HPV-16/18-infected lung tumorigenesis.
我们最近的报告表明，人乳头瘤病毒（HPV）-16/18 E6癌蛋白在肺肿瘤中表达，并与p53失活有关。我们进一步探究了人端粒酶逆转录酶（hTERT）转录是否受E6上调并促进肺肿瘤发展。
免疫组化检测135例肺肿瘤中的HPV-16 E6癌蛋白，分别通过实时逆转录-PCR和原位杂交评估hTERT mRNA。使用小RNA干扰（RNAi）、蛋白质免疫印迹和染色质免疫沉淀分析来阐明hTERT转录是否受c-Myc和Sp1调控。分别通过实时定量端粒重复序列扩增协议分析和软琼脂试验测定有或无E6-或hTERT-RNAi的TL-1细胞的端粒酶活性和致癌潜力。
E6阳性肿瘤（在女性、非吸烟者和腺癌中普遍存在）中的hTERT mRNA水平显著高于E6阴性肿瘤。此外，早期肿瘤（I期）中的hTERT mRNA水平高于晚期肿瘤（II期和III期）。染色质免疫沉淀试验表明，Sp1与c-Myc协同激活TL-1细胞中的hTERT转录，这与SiHa细胞相似。随着不同剂量的E6-或hTERT-RNAi转染，TL-1细胞的端粒酶活性随之降低。软琼脂试验表明，用E6-RNAi转染后，TL-1细胞的致癌潜力显著降低。此外，用hTERT-RNAi转染后，TL-1细胞无法形成集落。
HPV-16/18感染的肺肿瘤发生需要E6癌蛋白对hTERT进行转录激活。
