Harada K, Kurisu K, Tahara H, Tahara E, Ide T, Tahara E
Department of Neurosurgery, Kanbara Hospital, Hiroshima, Fukuyama-City, Japan.
J Neurosurg. 2000 Oct;93(4):618-25. doi: 10.3171/jns.2000.93.4.0618.
Telomerase activity is responsible for cell immortality. To examine the role of telomerase in the carcinogenesis of human glioblastomas multiforme (GBMs), the authors studied telomerase activity, telomerase component expression, and telomere lengths in 42 GBM samples.
In all samples, EGFR and MDM2 amplifications and overexpressions were examined using Southern and Northern blot analyses. The p53 mutation was analyzed using polymerase chain reaction-single strand conformational polymorphism and by direct sequence analysis. Specimens of tissues were immunostained with p53, EGFR, and MDM2 antibodies. Allelic loss on chromosomes 17p and 10 was assessed by loss of heterozygosity (LOH) assays. Telomerase activity, expression of its components (human telomerase reverse transcriptase [hTERT], human telomerase RNA component [hTERC], and telomerase-associated protein [TEP1]), and telomere lengths were analyzed using the telomeric repeat amplification protocol (TRAP)-hybridization protection assay, reverse transcription-polymerase chain reaction, and Southern blot analysis. According to the results of assessments of EGFR and MDM2 amplifications, p53 mutation, LOHs in chromosomes 17p and 10, and the clinical course of the disease, the 42 samples were classified into 22 primary and 20 secondary glioblastomas. Twenty-six (61.9%) of all 42 samples demonstrated detectable telomerase activity during the TRAP assay. Secondary GBMs displayed significantly higher levels of telomerase activity and hTERT expression than primary GBMs. Tumors with a p53 gene mutation demonstrated significantly higher telomerase activity than those without a p53 mutation. Four samples with a codon 175 mutation demonstrated an exceptionally high amount of telomerase activity. In secondary GBMs, the increase in telomerase activity and the hTERT expression level correlated with the increased frequency of p53 mutations. There was no significant difference in telomere length between primary and secondary GBMs.
These results suggest that telomerase activity and p53 mutations both play important roles in the multistep carcinogenesis of GBMs. Telomerase activity and hTERT expression may be considered as novel distinctive factors in human GBMs.
端粒酶活性与细胞永生有关。为研究端粒酶在多形性胶质母细胞瘤（GBM）发生中的作用，作者对42例GBM样本中的端粒酶活性、端粒酶成分表达及端粒长度进行了研究。
对所有样本，采用Southern和Northern印迹分析检测表皮生长因子受体（EGFR）和鼠双微体2（MDM2）的扩增及过表达情况。采用聚合酶链反应-单链构象多态性分析及直接测序分析法分析p53基因突变情况。用p53、EGFR和MDM2抗体对组织标本进行免疫染色。通过杂合性缺失（LOH）分析评估17号染色体短臂（17p）和10号染色体上的等位基因缺失情况。采用端粒重复序列扩增法（TRAP）-杂交保护分析、逆转录-聚合酶链反应及Southern印迹分析检测端粒酶活性、其成分（人端粒酶逆转录酶[hTERT]、人端粒酶RNA成分[hTERC]及端粒酶相关蛋白[TEP1]）的表达及端粒长度。根据EGFR和MDM2扩增、p53突变、17p和10号染色体上的LOH评估结果及疾病临床进程，将42例样本分为22例原发性和20例继发性胶质母细胞瘤。42例样本中有26例（61.9%）在TRAP分析中显示可检测到的端粒酶活性。继发性GBM的端粒酶活性和hTERT表达水平显著高于原发性GBM。有p53基因突变的肿瘤的端粒酶活性显著高于无p53突变的肿瘤。4例密码子175突变的样本显示出极高的端粒酶活性。在继发性GBM中，端粒酶活性增加和hTERT表达水平升高与p53突变频率增加相关。原发性和继发性GBM的端粒长度无显著差异。
这些结果表明，端粒酶活性和p53突变在GBM的多步骤致癌过程中均起重要作用。端粒酶活性和hTERT表达可被视为人类GBM中的新的独特因素。
