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

利用荧光热循环仪和RT-PCR检测了241例冷冻神经母细胞瘤组织的端粒长度、端粒酶活性和几种人端粒酶组分如RNA组分（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。通过端粒酶活性和hTERT的表达水平，可以区分不利的神经母细胞瘤和有利的神经母细胞瘤。一些端粒酶活性低或检测不到的肿瘤显示有选择性剪接的hTERT片段。虽然全长片段的数量一般与端粒酶活性有关，但考虑选择性剪接的定量RT-PCR的灵敏度和特异性并不令人满意。然后尝试hTR原位杂交和抗hTERT抗体免疫组化检测hTERT表达。通过这些方法，在有利的肿瘤中，这些表达水平较低，一些肿瘤细胞缺乏这些信号。另一方面，在端粒酶活性高的肿瘤中，几乎所有肿瘤细胞都表现出强烈的hTR和hTERT信号。在端粒酶活性中等的肿瘤中，少量肿瘤细胞表现出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例检测到端粒缩短，认为适合抗端粒酶治疗。然后在培养的端粒酶活性较高的神经母细胞瘤细胞中进行了hTR的利菌酶治疗、反义hTERT RNA治疗和抗hTERT抗体治疗。在这些抗端粒酶的作用下，细胞的生长受到抑制，并诱导细胞凋亡，提示端粒酶的还原与细胞凋亡之间存在相关性。这些结果表明，人端粒酶的定量和原位分析是评价神经母细胞瘤恶性程度和抗端粒酶治疗指示的有用标记。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