Ectopic expression of c-myc fails to overcome downregulation of telomerase activity induced by herbimycin A, but ectopic hTERT expression overcomes it.

Telomerase plays a key role in the maintenance of chromosomal stability in tumors, but the mechanism regulating telomerase activity is still unclear. Recent studies have suggested that c-myc may be vital for regulation of hTERT mRNA expression and telomerase activity. In this study, we investigated the changes of telomerase activity and telomerase-related genes induced by herbimycin A in K562 human chronic myelogeous leukemic cells. Telomerase activity showed a biphasic pattern in herbimycin A-treated K562 cells. Initially, the telomerase activity decreased along with the decline of cells in S and G2/M phases, but it recovered slightly at the end of treatment. Expression of mRNA for the telomerase catalytic subunit (hTERT) was decreased before the decline of telomerase activity, and increased slightly before the reactivation of telomerase activity. During herbimycin A treatment, both c-myc and cyclin D1 mRNA showed transient downregulation before the increase of G1 cells. Herbimycin A treatment caused the downregulation of both telomerase activity and hTERT mRNA in cyclin D1-transfected K562 cells, while telomerase activity was partially restored in c-Myc-transfected cells. In contrast, hTERT-transfected K562 cells maintained a high level of telomerase activity during herbimycin A treatment. Neither the template RNA component of telomerase (hTERC) nor telomerase-associated protein (TEP-1) were altered in any of the transfected K562 cells. These results indicate that telomerase activity is mainly regulated by hTERT, and that c-Myc protein is one of the positive regulators of hTERT in leukemic cells but is not enough to counteract the downregulation of telomerase activity by herbimycin A completely.     

雌、孕激素对子宫内膜癌细胞端粒酶活性及细胞周期的影响

 目的　研究雌、孕激素对子宫内膜癌HHUA细胞端粒酶活性及细胞周期的影响。方法　用TRAP ELISA法和实时荧光定量PCR技术检测雌、孕激素作用前后HHUA子宫内膜腺癌细胞系端粒酶活性及hTERT RNA的表达 ,流式细胞仪检测细胞周期及细胞凋亡的变化。结果　雌激素对HHUA细胞端粒酶活性和hTERT的表达有明显增强作用 (P <0 .0 5 ) ,而孕激素对其影响不显著。孕激素作用下 ,G1期细胞比例明显增高 ,S期及G2 ～M期细胞比例明显降低 (P <0 .0 5 ) ;细胞的分裂、增殖受到明显抑制 ;凋亡细胞比例明显增加。雌激素的作用则相反。结论　雌激素能增强子宫内膜癌细胞端粒酶活性和hTERT的表达 ,促进其分裂和增殖 ,降低凋亡细胞比例 ,孕激素的作用相反     

全反式维甲酸对HL-60细胞hTERT基因表达和端粒酶活性的影响

目的 探讨全反式维甲(ATRA)诱导HL-60细胞分化过程中人类端粒酶逆转录酶(hTERT)蛋白表达和端粒酶活性的改变。 方法 应用间接免疫荧光标记法通过流式细胞仪检测hTERT蛋白含量的变化;采用多聚酶链反应-酶联免疫反应(PCR-ELISA)方法检测ATRA处理HL-60细胞前后端粒酶活性的改变,用碘化丙锭染色经流式细胞仪检测细胞周期变化。 结果 1μmol/L ATRA作用HL-60细胞24、48、72h,hTERT蛋白平均荧光强度分别为61.87±4.36、37.47±2.85、33.45±2.37,与空白对照组相比,具有显著性差异,P<0.05。1μmol/L ATRA作用48h后,HL-60细胞的端粒酶活性即出现下降,作用72h,端粒酶的活性明显受到抑制。 结论 在HL-60细胞分化过程中,ATRA能抑制HL60细胞的hTERT基因的表达和端粒酶活性。     

5-Aza-C对人鼻咽癌细胞hTERT基因表达及端粒酶活性的影响

目的 研究去甲基化药物5-氮杂胞嘧啶核苷（5-Azacytidine, 5-Aza-C）对鼻咽癌细胞亚端粒区D4Z4甲基化状态、hTERT基因表达及端粒酶活性的影响。方法 常规培养鼻咽癌CNE, CNE1,CNE2及5-8F细胞系。5-Aza-C处理鼻咽癌细胞后,甲基化测序聚合酶链反应（MSP）法检测亚端粒区D4Z4甲基化;RT-PCR检测hTERT mRNA水平;端粒重复序列扩增法（telomeric repeat amplification protocol,TRAP）法检测端粒酶活性。结果 5-Aza-C处理后,四种鼻咽癌细胞系的亚端粒区D4Z4序列的甲基化水平较处理前明显下降。2.5 μmol/L 5-Aza-C处理后,四种鼻咽癌细胞hTERT mRNA的表达明显下调,端粒酶活性显著抑制。 结论 在鼻咽癌的发生中,亚端粒区的DNA甲基化水平紊乱起了一定的作用,去甲基化药物5-Aza-C能下调hTERT表达,抑制端粒酶活性。探讨鼻咽癌细胞中hTERT表达与亚端粒区CpG岛甲基化状态之间的关系,可能为亚端粒区染色体结构异常在鼻咽癌发生中的作用提供深刻见解。     

反义寡核苷酸对K562细胞增殖和端粒酶活性的影响

 目的　研究靶向封闭端粒酶反转录酶（hTERT）mRNA的反义硫代寡核苷酸（ASPSODN）对K562细胞目的基因的抑制及其对端粒酶活性及细胞增殖周期和凋亡的影响 。方法　ASPSODN转染到人类红白血病细胞株K562,采用MTT法、酶联免疫吸附（ELISA）法和流式细胞术（FCM）检测K562细胞的增殖、端粒酶活性、细胞凋亡和细胞周期的改变,RT-PCR检测hTERT mRNA的表达。结果　0.6 μmol／L的ASPSODN（0.42±0.16）能明显下调hTERT表达（P＜0.05）,端粒酶相对活性降至52 %;MTT法检测显示明显抑制K562细胞增殖活性;FCM显示细胞凋亡率为10.31 %,PI染色显示细胞被阻止在G1／G0期,S期及G2／M期的细胞减少,但无特征性的凋亡峰。结论　ASPSODN靶向 hTERT 能特异性抑制K562细胞hTERT mRNA的表达,明显下调端粒酶活性,抑制K562细胞增殖,并通过降低细胞的端粒酶活性而诱发细胞凋亡。     

Telomerase activity in myeloma cells is closely related to cell cycle status, but not to apoptotic signals induced by interferon-alpha.

Telomeres, G-rich structures at the ends of chromosomes are essential for maintaining chromosomal integrity. Most tumor cells contain telomerase, a ribonucleoprotein that elongates telomeric repeats, and it plays an essential role in indefinite proliferation. To better understand regulatory mechanisms of telomerase, in relationship with apoptosis and the cell cycle, we examined telomerase activity in PCM6, an interleukin-6 (IL-6)-responsive, interferon-alpha (IFN-alpha)-sensitive multiple myeloma cell line, using a PCR-based assay. When PCM6 cells were cultured in serum-free media, the addition of IFN-alpha resulted in apoptosis of the cells, but with no influence on telomerase activity. When IFN-alpha was added to the culture with serum plus rIL-6 after serum deprivation, G1-S transition was inhibited and telomerase activity was lower compare to findings in culture with no IFN-alpha. Dose response experiments of rIL-6 and IFN-alpha, and the measurement of telomerase activity of sorted cells in S-phase using CD71, demonstrated a higher activity of telomerase in the samples which contained a larger proportion of cells in S-phase. These data indicate that regulation of telomerase activity is closely related to cell cycle status, in particular cells in S-phase have an high telomerase activity. While telomeres play an important role in cellular senescence, the regulation of telomerase is independent from apoptotic signals induced by IFN-alpha in myeloma cells.     

长春瑞滨对人肺腺癌Anip973细胞凋亡和端粒酶表达的影响

目的 探讨长春瑞滨(NVB)对人肺腺癌Anip973细胞的凋亡、端粒酶活性以及人端粒酶逆转录酶mRNA(hTERT mRNA)表达的影响.方法 以不同浓度的NVB作用于Anip973细胞,在不同时间内收集细胞,采用四甲基偶氮唑蓝(MTT)法观察NVB对Anip973细胞的生长抑制作用;通过流式细胞术观察Anip973细胞凋亡率;用倒置显微镜和电镜观察细胞形态学变化;应用以聚合酶链反应(PCR)为基础的端粒重复序列扩增(TRAP-PCR)银染法检测端粒酶活性;采用逆转录聚合酶链反应(RT-PCR)检测hTERT mRNA表达.结果 不同浓度的NVB可诱导Anip973细胞凋亡,而且可降低Anip973细胞中端粒酶活性和hTERT mRNA的表达,并呈时间-剂量依赖性.0.08μg/ml NVB作用Anip973细胞24 h,细胞增殖被抑制,细胞凋亡率为(7.37±0.35)%,hTERT mRNA表达为57.01±1.71,与对照组差异均有统计学意义(P＜0.01);端粒酶活性值为6.36±0.06,与对照组差异无统计学意义(P＞0.05);hTERT mRNA表达的改变比端粒酶活性更敏感.0.4μg/ml NVB组和2.0μg/mlNVB组各时间点的细胞凋亡率、端粒酶活性和hTERT mRNA表达与对照组比较,差异有统计学意义(P＜0.01).2.0μg/ml NVB作用Anip973细胞72 h时,端粒酶活性为1.36±0.27,而细胞凋亡率为(74.87±1.88)%,表明细胞凋亡率的增加与细胞hTERT mRNA表达呈负相关(r=-0.96046,P＜0.01).结论 NVB的作用机制与端粒酶有关,诱导凋亡是其发挥抗癌作用的机制之一.检测端粒酶活性和hTERT mRNA的表达,有助于判定NVB诱导肺癌细胞凋亡的敏感性.