Kanaya T, Kyo S, Hamada K, Takakura M, Kitagawa Y, Harada H, Inoue M
Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, Ishikawa, Japan.
Clin Cancer Res. 2000 Apr;6(4):1239-47.
Telomerase activation is a critical step in cellular immortality and oncogenesis. The activity of telomerase is known to be correlated with cell proliferation, but its regulation by cell cycle regulators is not well understood. In the present study, we examined the effects of p53 on telomerase activity. Wild-type p53 was introduced into SiHa cells via a recombinant adenoviral vector, Ad5CMV-p53, and change in telomerase activity was examined by quantitative telomerase assay. Telomerase activity in the Ad5CMV-p53-infected cells was significantly repressed 36 h after infection following down-regulation of human telomerase catalytic subunit [human telomerase reverse transcriptase (hTERT)] mRNA expression, whereas no change in telomerase activity was observed in the cells infected with control vector AdSCMV-beta-gal. Interestingly, repression of telomerase activity was an early event that preceded cell growth inhibition or apoptosis induced by p53 overexpression, suggesting that p53 directly regulates telomerase activity. Transient expression assays using hTERT-promoter reporter constructs revealed that overexpression of p53 significantly repressed promoter activity of hTERT. 5'-Truncation of the promoter sequences revealed that the proximal core promoter region containing multiple binding sites for transcription factor Spl was responsible for p53-mediated transcriptional repression. Mutations in these binding sites for Spl led to failure of p53 to repress transcription. These findings suggest that p53 repressed telomerase activity through down-regulation of hTERT transcription and that interaction of p53 with Sp1 or other transcription factors may be involved in this regulation.
端粒酶激活是细胞永生化和肿瘤发生的关键步骤。已知端粒酶活性与细胞增殖相关,但其受细胞周期调节因子的调控机制尚不清楚。在本研究中,我们检测了p53对端粒酶活性的影响。通过重组腺病毒载体Ad5CMV-p53将野生型p53导入SiHa细胞,并通过定量端粒酶检测法检测端粒酶活性的变化。Ad5CMV-p53感染的细胞在感染后36小时,随着人端粒酶催化亚基[人端粒酶逆转录酶(hTERT)]mRNA表达的下调,端粒酶活性显著受到抑制,而感染对照载体AdSCMV-β-gal的细胞中端粒酶活性未观察到变化。有趣的是,端粒酶活性的抑制是p53过表达诱导的细胞生长抑制或凋亡之前的早期事件,这表明p53直接调节端粒酶活性。使用hTERT启动子报告构建体的瞬时表达分析表明,p53的过表达显著抑制了hTERT的启动子活性。启动子序列的5'端截短显示,包含转录因子Spl多个结合位点的近端核心启动子区域负责p53介导的转录抑制。这些Spl结合位点的突变导致p53无法抑制转录。这些发现表明,p53通过下调hTERT转录来抑制端粒酶活性,并且p53与Sp1或其他转录因子的相互作用可能参与了这种调控。