Telomere maintenance in childhood primitive neuroectodermal brain tumors

Abstract

Primitive neuroectodermal tumors (PNETs), including medulloblastoma (PNET/MB) and supratentorial PNET (sPNET), are the most common malignant brain tumors of childhood. The stabilization of telomere lengths by telomerase activation is an important step in carcinogenesis and cell immortalization. Epigallocatechin gallate (EGCG), the major polyphenol in green tea, is a telomerase inhibitor with antiproliferative and anticarcinogenic effects against different types of cancer. In this study, we used real-time reverse transcriptase-polymerase chain reaction to measure the mRNA expression of the human telomerase reverse transcriptase (hTERT) in 50 primary PNET samples (43 PNET/MB, 7 sPNET), 14 normal human brain samples, and 6 human PNET cell lines. Compared to normal human cerebellum, 38/50 (76%) primary PNET samples had у5-fold upregulated hTERT mRNA expression. We then examined PNET cell lines for telomerase activity using a quantitative telomeric repeat amplification protocol (TRAP), and for telomere length 2 These authors contributed equally to this work.3 Send correspondence to Michael Grotzer, University Children's Hospital, Steinwiesstrasse 75, Switzerland (Michael.Grotzer@kispi.unizh.ch). 4 Abbreviations used are as follows: ALT, alternative lengthening of telomeres; DIG, digoxigenin; EGCG, epigallocatechin gallate; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; hTERT, human telomerase reverse transcriptase; MB, medulloblastoma; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt; PCR, polymerase chain reaction; PNET, primitive neuroectodermal tumor; RT, reverse transcriptase; sPNET, supratentorial PNET; TRAP, telomeric repeat amplification protocol; TRF, terminal restriction fragment.using terminal restriction fragment analysis. While a positive correlation between hTERT mRNA expression and telomerase activity was detected in PNET cell lines, no correlation was found between telomerase activity and telomere length. Treatment of PNET cell lines with EGCG resulted in a dose-dependent inhibition of telomerase activity at micromolar levels. Although EGCG displayed strong proliferation inhibitory effects against TRAP-positive PNET cell lines, it had no significant effect against TRAP-negative D425 cells. These results provide evidence for a possible role of telomerase in the pathogenesis of most PNETs and indicate that subsets of PNETs maintain telomere length by alternative mechanisms. Inhibition of telomerase function represents a novel experimental therapeutic strategy in childhood PNETs that warrants further investigation. Neuro-Oncology 6, 1-8, 2004 (Posted to Neuro-Oncology [serial online], Doc. 03-017, November 19, 2003 DOI: 10.1215 P rimitive neuroectodermal tumors (PNETs) 4 of the cerebellum, also termed medulloblastomas (PNET/ MBs), and supratentorial PNETs (sPNETs) are the most common malignant pediatric neoplasms of the central nervous system (Gurney et al., 1999). PNETs constitute more than 20% of all pediatric brain tumors a...