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

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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