端粒酶催化亚基hTERT在肿瘤治疗中的研究进展

人端粒酶催化亚基hTERT基因转录的从头激活是端粒酶活化的限速步骤，受到多种癌基因和抑癌基因产物的精密调控。构建hTERT启动子调控抗肿瘤基因的逆转录病毒载体，使抗肿瘤基因在端粒酶阳性的肿瘤细胞内定向表达，有望用于肿瘤基因治疗。本文首先简单介绍了hTERT蛋白的结构和表达调控机制，然后对其在肿瘤发生发展中的生物治疗的功能作一简要综述。     

端粒、端粒酶及端粒酶催化亚基在大肠癌中的作用

目的:探讨端粒、端粒酶活性及端粒酶催化亚基蛋白(hTERT)在大肠癌发生发展、侵袭转移中的作用。方法:应用Southern blot、端粒重复序列扩增(TRAP)和免疫组织化学方法检测端粒长度(terminal restrictionfragments,TRFs)、端粒酶活性及hTERT表达水平。结果:大肠癌TRFs明显缩短,且随大肠癌Dukes分期的进展进一步缩短;端粒酶活性及hTERT在大肠癌组织中的阳性表达率分别为83.33%及76.67%,显著高于其他组织(P<0.05);大肠癌组织端粒酶与淋巴结转移关系密切,伴淋巴结转移大肠癌组织中的端粒酶活性及hTERT阳性表达率为80%和70%,显著高于无淋巴结转移者的0%和5%(P<0.05);相关分析显示端粒酶活性与hTERT蛋白表达存在显著相关性(P<0.05)。结论:端粒的短缩及端粒酶的活化与大肠癌的发生发展密切相关,hTERT的表达对端粒酶的激活可能起着重要作用。     

Hypermethylation of the human telomerase catalytic subunit (hTERT) gene correlates with telomerase activity

DNA methylation is an epigenetic process involved in embryonic development, differentiation and aging. It is 1 of the mechanisms resulting in gene silencing in carcinogenesis, especially in tumor suppressor genes (e.g., p16, Rb). Telomerase, the DNA polymerase adding TTAGGG repeats to the chromosome end, is involved in the regulation of the replicative life span by maintaining telomere length. This enzyme is activated in germ and stem cells, repressed in normal somatic cells and reactivated in a large majority of tumor cells. The promoter region of the hTERT gene, encoding for the catalytic subunit of human telomerase, has been located in a CpG island and may therefore be regulated at least in part by DNA methylation. We analyzed the methylation status of 27 CpG sites within the hTERT promoter core region by methylation-sensitive single-strand conformation analysis (MS-SSCA) and direct sequencing using bisulfite-modified DNA in 56 human tumor cell lines, as well as tumor and normal tissues from different organs. A positive correlation was observed among hypermethylation of the hTERT promoter, hTERT mRNA expression and telomerase activity (p < 0.00001). Furthermore, this correlation was confirmed in normal tissues where hypermethylation of the hTERT promoter was found exclusively in hTERT-expressing telomerase-positive samples and was absent in telomerase-negative samples (p < 0.00002). Since tumor tissues contain also nonneoplastic stromal elements, we performed microdissection to allow confirmation that the hTERT promoter methylation truly occurred in tumor cells. Our results suggest that methylation may be involved in the regulation of hTERT gene expression. To our knowledge, this is the first gene in which methylation of its promoter sequence has been found to be positively correlated with gene expression.     

hTERT methylation is necessary but not sufficient for telomerase activity in colorectal cells

Colorectal cancers exhibit a high telomerase activity, usually correlated with the hypermethylation of the promoter of its hTERT catalytic subunit. Although telomerase is not expressed in normal tissue, certain proliferative somatic cells such as intestinal crypt cells have demonstrated telomerase activity. The aim of this study was to determine whether a correlation exists between telomerase activity, levels of hTERT methylation and telomere length in tumoral and normal colorectal tissues. Tumor, transitional and normal tissues were obtained from 11 patients with a colorectal cancer. After bisulfite modification of genomic DNA, hTERT promoter methylation was analyzed by methylation-sensitive single-strand conformation analysis (MS-SSCA). Telomerase activity and telomere length were measured by a fluorescent-telomeric repeat amplification protocol assay and by Southern blotting, respectively. A significant increase of hTERT methylation and telomerase activity, and a reduction of the mean telomere length were observed in the tumor tissues compared to the transitional and normal mucosa. In the transitional and normal mucosa, telomerase activity was significantly lower than that in tumor tissues, even with high levels of hTERT methylation. Nevertheless, hTERT promoter methylation was not linearly correlated to telomerase activity. These data indicate that hTERT promoter methylation is a necessary event for hTERT expression, as is telomerase activity. However, methylation is not sufficient for hTERT activation, particularly in normal colorectal cells.     

骨巨细胞瘤组织中端粒酶hTERT基因表达及其意义

目的 探讨骨巨细胞瘤组织中端粒酶hTERT基因的表达及意义.方法 应用原位分子杂交技术,对4l例骨巨细胞瘤组织、17例骨质增生骨组织、11例异位骨化组织和10例正常骨组织中端粒酶hTERT基因进行检测和定位,并运用图像分析系统对hTERT基因表达水平进行研究.结果 端粒酶hTERT基因在骨巨细胞瘤中表达阳性率为41.5%(17/41),表达强度与骨巨细胞瘤的分化程度明显相关(P＜0.05):低分化肿瘤＞中分化肿瘤＞高分化肿瘤,端粒酶hTERT细胞表达水平与肿瘤细胞的分布定位一致.17例骨质增生骨组织、11例异位骨化组织和10例正常骨组织中端粒酶hTERT基因的表达均为阴性.结论 原位分子杂交是检测端粒酶亚单位hTERT的有效方法,在骨巨细胞瘤细胞水平检测端粒酶hTERT基因的定位诊断具有重要意义.在骨巨细胞瘤发展和维持中端粒酶可能起到重要的作用,同时还可能存在端粒酶以外的机制导致肿瘤细胞永生化.     

端粒酶hTERT在子宫内膜癌中的表达及意义探讨

目的　探讨端粒酶hTERT基因蛋白表达与子宫内膜癌发生发展的关系及对内膜癌诊断、预后的临床意义。方法　采用免疫组化方法对 33例子宫内膜癌、36例绝经期子宫内膜及 36例子宫内膜增生过长标本进行端粒酶hTERT、雌激素受体ER及孕激素受体PR检测 ,同时与正常增殖期 ,分泌期子宫内膜进行对照。结果　hTERT在子宫内膜癌中的表达强度明显高于其他内膜病变 ,有显著性差异 (P<0 .0 5 ) ;hTERT在子宫内膜癌中的表达与肌层浸润及临床分期呈正相关 ;与病理分级及雌、孕激素受体ER ,PR呈负相关。结论　上述结果提示端粒酶hTERT在子宫内膜癌的发生发展中可能起重要作用 ,端粒酶hTERT与ER ,PR的联合检测可能成为子宫内膜癌判断预后的一个指标。