Synthesis,telomerase inhibitory and anticancer activity of new 2-phenyl-4H-chromone derivatives containing 1,3,4-oxadiazole moiety

Based on previous studies, 66 2-phenyl-4H-chromone derivatives containing amide and 1,3,4-oxadiazole moieties were prepared as potential telomerase inhibitors. The results showed most of the title compounds exhibited significantly inhibitory activity on telomerase. Among them, some compounds demonstrated the most potent telomerase inhibitory activity (IC₅₀ < 1 µM), which was significantly superior to the staurosporine (IC₅₀ = 6.41 µM). In addition, clear structure–activity relationships were summarised, indicating that the substitution of the methoxy group and the position, type and number of the substituents on the phenyl ring had significant effects on telomerase activity. Among them, compound A33 showed considerable inhibition against telomerase. Flow cytometric analysis showed that compound A33 could arrest MGC-803 cell cycle at G2/M phase and induce apoptosis in a concentration-dependent way. Meanwhile, Western blotting revealed that this compound could reduce the expression of dyskerin, which is a fragment of telomerase.     

INHIBITION OF HUMAN TELOMERASE BY L-ENANTIOMERS OF NATURAL 2′-DEOXYRIBONUCLEOSIDE 5′-TRIPHOSPHATES

In order to clarify whether L-enantiomers of natural 2′-deoxyribonucleoside 5′-triphosphates (dNTPs) are recognized by human telomerase, a quantitative telomerase assay based on the ‘stretch PCR’ method was developed and used for kinetic analysis. Among the four L-dNTPs, L-dTTP and L-dGTP inhibited telomerase activity and the others showed slight or no inhibitory effect. Lineweaver-Burk plot analysis showed that the inhibition mode L-dGTP was competitive with dGTP.     

23-Hydroxybetulinic acid-mediated apoptosis is accompanied by decreases in bcl-2 expression and telomerase activity in HL-60 Cells

23-Hydroxybetulinic acid, a derivative of betulinic acid, was investigated for its apoptotic effect and the associated telomerase activity in human leukemia HL-60 cells. Apoptosis and bcl-2 were determined by flow cytometry analysis. A PCR-based telomeric repeat amplification protocol assay was used to detect telomerase activity. Results showed that 23-hydroxybetulinic acid induced growth arrest and apoptotic cell death in HL-60 cells. The apoptotic events were associated with concurrent down-regulation of bcl-2 and the telomerase activity. Our data suggest that 23-hydroxybetulinic acid may be a potential cytotoxic agent for treatment of cancer.     

Analysis of the structure of Tetrahymena nuclear RNAs in vivo: telomerase RNA, the self-splicing rRNA intron, and U2 snRNA.

Dimethyl sulfate modification of RNA in living Tetrahymena thermophila allowed assessment of RNA secondary structure and protein association. The self-splicing rRNA intron had the same methylation pattern in vivo as in vitro, indicating that the structures are equivalent and suggesting that this RNA is not stably associated with protein in the nucleolus. Methylation was consistent with the current secondary structure model. Much of telomerase RNA was protected from methylation in vivo, but the A's and C's in the template region were very reactive. Thus, most telomerase is not base paired to telomeres in vivo. Protein-free telomerase RNA adopts a structure different from that in vivo, especially in the template and pseudoknot regions. The U2 snRNA showed methylation protection at the Sm protein-binding sequence and the mRNA branch site recognition sequence. For both telomerase RNA and U2 snRNA, the in vivo methylation pattern corresponded much better to the structure determined by comparative sequence analysis than did the in vitro methylation pattern. Thus, as expected, comparative analysis gives the structure of the RNA in vivo.     

端粒酶反义cDNA对乳腺癌细胞损伤修复能力的影响

 利用反义核酸技术将端粒酶RNA的全长cDNA反向导入乳腺癌MCF 7细胞基因组中 .通过单细胞凝胶电泳实验发现 ,其DNA受H2 O2 损伤后的修复能力下降 .但端粒酶活性抑制为何引起其DNA损伤修复能力下降的原因尚待进一步研究 .     

Telomeric length and telomerase activity vary with age in peripheral blood cells obtained from normal individuals

The telomerase activity and length of telomeres of peripheral blood mononuclear cells obtained from 124 healthy individuals aged 4–95years was measured. Telomerase activity level was semiquantitatively assessed by a fluorescent-telomeric repeat amplification protocol (fluorescent-TRAP) using an internal telomerase assay standard, fluorescent primers and an automated laser fluorescent DNA sequencer. Telomeric length, measured by assay of terminal restriction fragments (TRFs), was determined in HinfI-digested DNA by Southern blot analysis using a (TTAGGG)₄ probe. TRF length was determined in 80 individuals and age-related progressive reduction of size was observed. TRF length in peripheral blood mononuclear cells obtained from normal individuals (aged 4–39years) decreased by approximately 84bp per year, while in individuals aged ≥40years it decreased by 41bp per year. In contrast, telomerase activity showed an apparent biphasic pattern with aging. Individuals aged 4–39years showed a progressive decrease in telomerase activity, whereas 65% of those aged ≥40years showed relatively stable but very low telomerase activity, and the remaining individuals aged ≥40years had no detectable telomerase activity. These data obtained from normal individuals might in the future be of value to help risk stratify and manage the care of patients with leukemia. Received: 18 August 1997 / Accepted: 10 December 1997     

Divalent hammerhead ribozyme targeting template region of human telomerase RNA has potent cleavage activity,but less inhibitory activity on telomerase

The template region of human telomerase RNA is a crucial area for regulating telomerase activity and would be a good target for ribozymes. In fact, potent telomerase inhibitory activity of the ribozyme targeting the GUC sequence of the 5(') end of this region (36-ribosome) has been well demonstrated. To search for a more potent ribozyme, we designed a divalent ribozyme to cleave both the phosphodiester bonds following the GUC and the 23 nucleotides downstream of GUA. An in vitro cleavage study showed that this divalent ribozyme cleaved telomerase RNA more efficiently than the 36-ribozyme or the 59-ribozyme to target the GUA. When this ribozyme was introduced into the carcinoma cells, its inhibitory effect on telomerase activity was less than that of the 36-ribozyme. The 59-ribozyme showed minimum activity on telomerase. This implies that, although the divalent ribozyme possesses a potent cleavage activity on hTR in vitro, the 36-ribozyme is most potent to suppress telomerase activity.     

Role of ceramide in mediating the inhibition of telomerase activity in A549 human lung adenocarcinoma cells

This study was designed to analyze whether ceramide, a bioeffector of growth suppression, plays a role in the regulation of telomerase activity in A549 cells. Telomerase activity was inhibited significantly by exogenous C(6)-ceramide, but not by the biologically inactive analog dihydro-C(6)-ceramide, in a time- and dose-dependent manner, with 85% inhibition produced by 20 microm C(6)-ceramide at 24 h. Moreover, analysis of phosphatidylserine translocation from the inner to the outer plasma membrane by flow cytometry and of poly(ADP-ribose) polymerase degradation by Western blotting showed that ceramide treatment (20 microm for 24 h) had no apoptotic effects. Trypan blue exclusion, [(3)H]thymidine incorporation, and cell cycle analyses, coupled with clonogenic cell survival assay on soft agar, showed that ceramide treatment with a 20 microm concentration at 24 h resulted in the cell cycle arrest of the majority of the cell population at G(0)/G(1) with no detectable cell death. These results suggest that the inhibition of telomerase by ceramide is not a consequence of cell death but is correlated with growth arrest. Next, to determine the role of endogenous ceramide in telomerase modulation, A549 cells were transiently transfected with an expression vector containing the full-length bacterial sphingomyelinase cDNA (b-SMase). The overexpression of b-SMase, but not exogenously applied purified b-SMase enzyme, resulted in significantly decreased telomerase activity compared with controls, showing that the increased endogenous ceramide is sufficient for telomerase inhibition. Moreover, treatment of A549 cells with daunorubicin at 1 microm for 6 h resulted in the inhibition of telomerase, which correlated with the elevation of endogenous ceramide levels and growth arrest. Finally, stable overexpression of human glucosylceramide synthase, which attenuates ceramide levels by converting ceramide to glucosylceramide, prevented the inhibitory effects of C(6)-ceramide and daunorubicin on telomerase. Therefore, these results provide novel data showing for the first time that ceramide is a candidate upstream regulator of telomerase.     

A triple helix within a pseudoknot is a conserved and essential element of telomerase RNA

Telomerase copies a short template within its integral telomerase RNA onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Telomerase action extends the proliferative potential of cells, and thus it is implicated in cancer and aging. Nontemplate regions of telomerase RNA are also crucial for telomerase function. However, they are highly divergent in sequence among species, and their roles are largely unclear. Using in silico three-dimensional modeling, constrained by mutational analysis, we propose a three-dimensional model for a pseudoknot in telomerase RNA of the budding yeast Kluyveromyces lactis. Interestingly, this structure includes a U-A.U major-groove triple helix. We confirmed the triple-helix formation in vitro using oligoribonucleotides and showed that it is essential for telomerase function in vivo. While triplex-disrupting mutations abolished telomerase function, triple compensatory mutations that formed pH-dependent G-C.C(+) triples restored the pseudoknot structure in a pH-dependent manner and partly restored telomerase function in vivo. In addition, we identified a novel type of triple helix that is formed by G-C.U triples, which also partly restored the pseudoknot structure and function. We propose that this unusual structure, so far found only in telomerase RNA, provides an essential and conserved telomerase-specific function.     

水稻端粒酶活性定量分析研究

摘要：端粒酶活性直接与细胞的分生能力、生活力、生命力有着密不可分的关系, 因此对其活性测定有着重要意义。本文参照人类端粒酶体外检测的原理和方法,设计了特别的先导引物和反向引物,采用不同的退火温度将PCR循环分成两步进行,再结合DNA凝胶电泳成像定量分析系统,以模式植物水稻为研究对象,对端粒酶活性定量检测方法和反应条件进行了探索。结果显示,水稻端粒酶活性的最佳反应条件为：温度19℃,反应时间13分钟,总蛋白浓度0.28μg/μl,与人端粒酶最佳反应条件有明显差异,建立了一种有效测定植物端粒酶活性的定量检测方法。应用该方法对6个水稻品种的根、幼叶及幼穗三个不同组织器官的端粒酶活性进行了定量测定,结果显示幼穗最高,其次为幼叶,根最弱。说明植物端粒酶活性与细胞、组织的生活力有着密切关系。