Study of telomerase as a useful target for diagnosis and therapy of neuroblastoma with the aim of clinical application

我们采用TRAP法和RT-PCR法检测了241例冷冻神经母细胞瘤组织的端粒长度、端粒酶活性以及端粒酶成分(HTR)、人端粒酶逆转录酶(HTERT)和人端粒酶相关蛋白1(HTEP1)的表达水平。根据端粒酶活性和hTERT的表达水平，可以区分恶性和良性神经气肿。一些端粒酶活性低或检测不到的肿瘤出现了hTERT的选择性剪接片段，虽然全长片段的数量一般与端粒酶活性有关，但考虑到选择性剪接的敏感性和特异性并不理想，于是我们尝试了hTERT的原位杂交和免疫组织化学方法检测hTERT的表达。通过这些方法，在良性肿瘤中，这些表达水平很低，并且有一些t…更多的乌莫尔细胞缺乏这些信号。另一方面，在高端粒酶活性的肿瘤中，几乎所有的肿瘤细胞都显示出hTR和hTERT的强信号，在端粒酶活性中等的肿瘤中，少数肿瘤细胞显示出hTR和hTERT的高表达。在12例端粒酶高表达和hTERT高表达的肿瘤中，有10例端粒缩短，这被认为是适合进行抗端粒酶治疗的病例。然后，我们在培养的端粒酶活性较高的神经母细胞瘤细胞中尝试了Risozyme治疗hTR、反义hTERT RNA治疗和抗hTERT抗体治疗。结果提示，人端粒酶的定量和原位分析可作为神经母细胞瘤恶性程度评估和抗端粒酶治疗指征的有用指标。较少

We examined telomere length, telomerase activity, and the expression levels of several human telomerase components such as RNA component(hTR), human telomerase reverse transcriptase (hTERT), and human telomerase associated protein 1 (hTEP1) in 241 frozen neuroblastoma tissues by the TRAP assay and RT-PCR using fluorescent thermal cycler. Unfavorable neurobalstomas were distinguishable from favorable ones by the levels of telomerase activity and hTERT expression. Some tumors with low or undetectable telomerase activity showed alternatively spliced fragments of hTERT.While the amount of the full-length fragment was in relation to the telomerase activity in general, the sensitivity and the specificity of quantitative RT-PCR in consideration of alternative splicing were not satisfactory.Then we tried in situ hybridization of hTR and immunohistochemical detection of hTERT expression using anti-hTERT antibody. By these methods, in favorable tumors, these expression levels were low and some t … More umor cells lacked these signals. On the other hand, in the tumors with high telomerase activity, almost all tumors cells exhibits strong signals of hTR and hTERT.In the tumors with mnoderate levels of telomerase activity, small amounts of tumor cells showed high expressions of hTR and hTERT.Among 12 tumors with high telomerase and high hTERT expressions, shortened telomere was detected in 10, which were considered as adequate cases for antitelomerase therapy. Then we tried risozyme treatment for hTR, antisense hTERT RNA treatment, and anti hTERT antibody treatment in the cultured neuroblastoma cells with high telomerase activity. In these antitelomerase treatments brought the growth inhibition and the induction of apoptosis, suggesting the correlation between telomerase reduciton without telomerase activity and apoptosis.These results suggested that the quatitative and in situ analyses of human telomerase are useful markers for evaluation of malignant grades of neuroblastoma and for indication of antitelomerase therapies. Less