Kitagawa Y, Kyo S, Takakura M, Kanaya T, Koshida K, Namiki M, Inoue M
Department of Urology, Kanazawa University, School of Medicine, Ishikawa, Japan.
Clin Cancer Res. 2000 Jul;6(7):2868-75.
Telomerase activation is thought to be a critical step in cellular immortality and oncogenesis. Several reagents including differentiation-inducing and antineoplastic agents are known to inhibit telomerase activity, although the molecular mechanisms through which they inhibit telomerase activity remain unclear. Demethylating reagents have recently been used as potential antineoplastic drugs for some types of cancers including those of the prostate. In the present study, we examined the effect of the demethylating reagent 5-azacytidine (5-aza-CR) on telomerase activity using cells of two prostate cancer cell lines, DU-145 and TSU-PR1. 5-aza-CR treatment significantly reduced telomerase activity in TSU-PR1 cells, but not in DU-145 cells, although growth inhibition was observed to a similar extent in both cell lines. Reverse transcription-PCR analyses revealed that inhibition of telomerase activity was accompanied by down-regulation of telomerase catalytic subunit (hTERT) mRNA expression. Transient expression assays showed that 5-aza-CR repressed the transcriptional activity of the hTERT promoter and that the E-box within the core promoter was responsible for this down-regulation. Western blot analyses revealed that 5-aza-CR reactivated p16 expression and repressed c-Myc expression in TSU-PR1 cells but not in DU-145 cells. Overexpression of p16 in TSU-PR1 cells led to significant repression of c-Myc transcription. These findings suggest that 5-aza-CR inhibits telomerase activity via transcriptional repression of hTERT, in which p16 and c-Myc may play a key role.
端粒酶激活被认为是细胞永生化和肿瘤发生的关键步骤。已知包括分化诱导剂和抗肿瘤剂在内的几种试剂可抑制端粒酶活性，尽管它们抑制端粒酶活性的分子机制尚不清楚。去甲基化试剂最近已被用作某些类型癌症（包括前列腺癌）的潜在抗肿瘤药物。在本研究中，我们使用两种前列腺癌细胞系DU-145和TSU-PR1的细胞，研究了去甲基化试剂5-氮杂胞苷（5-aza-CR）对端粒酶活性的影响。5-aza-CR处理显著降低了TSU-PR1细胞中的端粒酶活性，但对DU-145细胞没有影响，尽管在两种细胞系中观察到了相似程度的生长抑制。逆转录聚合酶链反应分析表明，端粒酶活性的抑制伴随着端粒酶催化亚基（hTERT）mRNA表达的下调。瞬时表达分析表明，5-aza-CR抑制了hTERT启动子的转录活性，并且核心启动子内的E盒负责这种下调。蛋白质印迹分析表明，5-aza-CR在TSU-PR1细胞中重新激活了p16表达并抑制了c-Myc表达，但在DU-145细胞中没有。TSU-PR1细胞中p16的过表达导致c-Myc转录的显著抑制。这些发现表明，5-aza-CR通过对hTERT的转录抑制来抑制端粒酶活性，其中p16和c-Myc可能起关键作用。
