Activation of telomerase by human cytomegalovirus.
Activation of telomerase by human cytomegalovirus.
复制标题
DOI：
10.1093/jnci/djp031
复制
发表时间：
2009-04
期刊：
影响因子：
--
通讯作者：
K. Strååt;Cheng Liu;A. Rahbar;Qingjun Zhu;Li Liu;Nina Wolmer-Solberg;F. Lou;Zhaoxu Liu;Jie Shen;J. Jia;S. Kyo;M. Björkholm;J. Sjöberg;C. Söderberg-Nauclér;Dawei Xu
中科院分区：
文献类型：
--
作者：
K. Strååt;Cheng Liu;A. Rahbar;Qingjun Zhu;Li Liu;Nina Wolmer-Solberg;F. Lou;Zhaoxu Liu;Jie Shen;J. Jia;S. Kyo;M. Björkholm;J. Sjöberg;C. Söderberg-Nauclér;Dawei Xu
背景人巨细胞病毒（HCMV）刺激肿瘤发生的机制尚不清楚。由于细胞永生化和转化需要端粒酶逆转录酶（hTERT）基因的表达端粒酶激活，我们研究了HCMV在端粒酶激活中的作用。方法用HCMV感染正常人二倍体成纤维细胞（HDF）和人恶性胶质瘤（MG）细胞系，或用HCMV立即早期（IE）抗原72或86的表达载体转染细胞系。分别采用逆转录-聚合酶链反应、荧光素酶报告基因检测和端粒重复序列扩增技术检测hTERT表达、启动子活性和端粒酶活性。通过染色质免疫沉淀评估转录因子Sp1、IE抗原和组蛋白脱乙酰酶（HDAC）对hTERT启动子的占用。免疫组化法检测人原发性多形性胶质母细胞瘤（GBM）中hTERT和IE蛋白的表达。所有统计学检验均为双侧检验。结果在端粒酶和hTERT阴性的HDFs中，HCMV感染诱导了hTERT的组成性表达和端粒酶的激活。HCMV可显著增强HDFs和MG细胞中hTERT启动子的活性，并呈剂量依赖性（对照HDF和感染HCMV的HDF中的平均荧光素酶活性[任意单位]，感染复数[MOI]分别为6和521，差异= 515，95%CI = 178至850; MOI为1和10时的平均活性分别为8828和59，923;在所有MOI下，对照与HCMV感染的细胞相比，P <0.001）。HCMVIE-72蛋白的异位表达也刺激HDFs中hTERT启动子的活性。HCMV介导的hTERT基因的反式激活依赖于hTERT启动子中Sp1结合位点的存在，并伴随着Sp1结合的增加、组蛋白H3的乙酰化和HDAC结合在核心启动子处的减少。在GBM标本中，HCMV IE和hTERT蛋白在恶性细胞中共定位，且表达水平相互重叠。结论HCMV对HDFs及恶性肿瘤细胞端粒酶活性均有激活作用。这些发现开始揭示了HCMV感染可能通过端粒酶激活与肿瘤发生相关或调节肿瘤发生的新机制。
BACKGROUND The mechanism by which human cytomegalovirus (HCMV) stimulates oncogenesis is unclear. Because cellular immortalization and transformation require telomerase activation by expression of the telomerase reverse transcriptase (hTERT) gene, we examined the role of HCMV in telomerase activation. METHODS Normal human diploid fibroblasts (HDFs) and human malignant glioma (MG) cell lines were infected with HCMV or transfected with expression vectors encoding HCMV immediate early (IE) antigen 72 or 86. hTERT expression and promoter activity and telomerase activity were evaluated using reverse transcription-polymerase chain reaction, a luciferase reporter assay, and a telomeric repeat amplification protocol, respectively. hTERT promoter occupancy by the transcription factor Sp1, IE antigens, and histone deacetylases (HDACs) was assessed by chromatin immunoprecipitation. hTERT and IE protein expression in human primary glioblastoma multiforme (GBM) was determined immunohistochemically. All statistical tests were two-sided. RESULTS In telomerase and hTERT-negative HDFs, HCMV infection induced constitutive hTERT expression and telomerase activation. The hTERT promoter activity in HDFs and MG cell lines was statistically significantly enhanced by HCMV in a dose-dependent manner (mean luciferase activity [arbitrary units] in control HDFs and in HDFs infected with HCMV at multiplicities of infection [MOIs] of 0.1 = 6 and 521, respectively, difference = 515, 95% CI = 178 to 850; mean activity at MOI of 1 and 10 = 8828 and 59,923, respectively; P < .001 comparing control with HCMV-infected cells at all MOIs). Ectopic expression of HCMV IE-72 protein also stimulated hTERT promoter activity in HDFs. HCMV-mediated transactivation of the hTERT gene was dependent on the presence of Sp1-binding sites in the hTERT promoter and was accompanied by increases in Sp1 binding, acetylation of histone H3, and a reduction in HDAC binding at the core promoter. In specimens of GBM, HCMV IE and hTERT proteins were colocalized in malignant cells and their levels paralleled each other. CONCLUSIONS HCMV activates telomerase in both HDFs and malignant cells. These findings begin to reveal a novel mechanism by which HCMV infection may be linked to or modulate oncogenesis through telomerase activation.