荧光定量PCR 法检测肿瘤细胞端粒酶活性的变化

目的：利用荧光定量PCR法检测端粒酶抑制剂作用于人肝癌细胞SMMC-7721后端粒酶活性的变化,探讨其抑制端粒酶活性的可能机制,为端粒酶抑制剂的临床应用提供理论依据。方法：利用荧光染料SYBR—Green I建立一种新的端粒酶活性检测方法：FQ—TRAP法。利用FQ—TRAP法检测端粒酶抑制剂作用后肿瘤细胞端粒酶活性变化。结果：端粒酶抑制剂作用后,肝癌细胞端粒酶活性都有变化,其中以ASODN,EGCG,AZT抑制效果较明显。结论：端粒酶FQ—TRAP法是一种特异性、灵敏度、重复性都较好,可快速、简便及定量检测人端粒酶活性的方法,端粒酶抑制剂作用后癌细胞端粒酶活性的变化,为端粒酶抑制剂的临床应用提供理论依据。     

端粒酶活性调节的分子机制

人端粒酶由RNA亚基、hTERT催化亚基和hTEP1调节蛋白等组成。端粒酶对端粒结构的稳定起着重要的作用,而端粒结构和端粒结合蛋白也影响着端粒酶活性。某些化疗药物通过破坏端粒结构下调端粒酶活性。端粒酶的激活需要hTERT基因的从头转录和各个蛋白亚基正确装配为端粒酶全酶。端粒酶活性调节的分子机制包括：（1）TERT基因的表达和转录是决定端粒酶活性的重要环节,受多种因素调控;（2）蛋白激酶Cα和蛋白激酶B磷酸化端粒酶蛋白而激活端粒酶,蛋白磷酸酯酶2A（PP2A）可逆转这一过程,下调端粒酶活性;（3）多种癌基因和抑癌基因及其编码的蛋白质也直接或间接与端粒蛋白、端粒酶蛋白反应,参与端粒酶活性的调控。     

Determinants in mammalian telomerase RNA that mediate enzyme processivity and cross-species incompatibility

Telomerase contains two essential components: an RNA molecule that templates telomeric repeat synthesis and a catalytic protein component. Human telomerase is processive, while the mouse enzyme has much lower processivity. We have identified nucleotide determinants in the telomerase RNA that are responsible for this difference in processivity. Mutations adjacent to the template region of human and mouse telomerase RNA significantly altered telomerase processivity both in vitro and in vivo. We also identified functionally important nucleotides in the pseudoknot domain of telomerase RNA that potentially mediate the incompatibility between human TERT and mouse telomerase RNA. These experiments identify essential residues of the telomerase RNA that regulate telomerase activity and processivity.     

Clonal heterogeneity in telomerase activity and telomere length in tumor-derived cell lines

The ribonucleoprotein, telomerase, is responsible for the maintenance of telomere length in most immortal and cancer cells. Telomerase appears to be a marker of human malignancy with at least 85% of human cancers expressing its activity. In the present study, we examined a series of tumor-derived and in vitro immortalized cell lines for telomerase activity levels, telomere lengths, and expression levels of the RNA and catalytic components of telomerase. We found significant variability in both telomere lengths and telomerase activity in clones from tumor cells. In addition, the levels of telomerase components or telomerase activity were not predictive of telomere length. Data from clonally derived cells suggest that critically shortened telomeres in these tumor-derived cell lines may signal activation of telomerase activity through an increase in the expression of the catalytic subunit of telomerase. Although clones with low telomerase shorten their telomeres over time, their subclones all have high levels of telomerase activity with no telomere shortening. In addition, analysis of early clones for telomerase activity indicates substantial variability, which suggests that activity levels fluctuate in individual cells. Our data imply that cell populations exhibit a cyclic expression of telomerase activity, which may be partially regulated by telomere shortening.     

Telomerase regulation

The intimate connection between telomerase regulation and human disease is now well established. The molecular basis for telomerase regulation is highly complex and entails multiple layers of control. While the major target of enzyme regulation is the catalytic subunit TERT, the RNA subunit of telomerase is also implicated in telomerase control. In addition, alterations in gene dosage and alternative isoforms of core telomerase components have been described. Finally, telomerase localization, recruitment to the telomere and enzymology at the chromosome terminus are all subject to modulation. In this review we summarize recent advances in understanding fundamental mechanisms of telomerase regulation.     

Calcium-mediated telomerase activity in ovarian epithelial cells

Though the potential of telomerase as an anti-cancer target is evident, information about regulation of telomerase remains fragmentary. In the present study, we examined the role of calcium, an essential cellular signaling molecule, in the regulation of telomerase. We found that calcium induced de novo telomerase activity in telomerase-negative ovarian surface epithelial (OSE) cell lines but not in primary cultures of OSE. In addition, we showed that calcium elevated endogenous telomerase levels in a telomerase-positive ovarian cancer cell line. The use of calcium channel blockers or calcium chelators inhibited this calcium-mediated induction of telomerase activity. Furthermore, cadmium and chromium appeared to cause a moderate induction of telomerase activity while several other metal salts did not. Our data provide the first example of calcium-induced telomerase activity in human cell lines, provide a novel avenue for possible intervention of telomerase, and permit development of therapeutic agents for adjunctive chemotherapy.     

Enzymatic activity of endogenous telomerase associated with intact nuclei from human leukemia CEM cells.

Telomerase, a telomere-specific DNA polymerase and novel target for chemotherapeutic intervention, is found in many types of cancers. Telomerase activity is typically assayed using an exogenous primer and cellular extracts as the source of enzyme. Since the nuclear organization might affect telomerase function, we developed a system in which telomerase in intact nuclei catalyzes primer extension. Telomerase activity in isotonically isolated nuclei from human CEM cells shows low processivity (addition of up to four TTAGGG repeats). In contrast, telomerase activity which leaks into a 500 g postnuclear supernatant and the activity in a CHAPS extract are highly processive. The nucleotide inhibitor, 7-deaza-dGTP, seems to be more inhibitory against the nuclei-associated enzyme compared to telomerase from cytoplasmic extracts. However, 7-deaza-dATP and ddGTP are less inhibitory against nuclei-associated telomerase. The results suggest that the association of telomerase with the nuclear chromatin affects telomerase activity. Examination of telomerase activity in a more natural nuclear environment may shed new light on the telomerase function and provide a useful system for the evaluation of new telomerase inhibitors.     

Processive and distributive extension of human telomeres by telomerase under homeostatic and nonequilibrium conditions

Specific information about how telomerase acts in vivo is necessary for understanding telomere dynamics in human tumor cells. Our results imply that, under homeostatic telomere length-maintenance conditions, only one molecule of telomerase acts at each telomere during every cell division and processively adds ～60 nt to each end. In contrast, multiple molecules of telomerase act at each telomere when telomeres are elongating (nonequilibrium conditions). Telomerase extension is less processive during the first few weeks following the reversal of long-term treatment with the telomerase inhibitor Imetelstat (GRN163L), a time when Cajal bodies fail to deliver telomerase RNA to telomeres. This result implies that processing of telomerase by Cajal bodies may affect its processivity. Overexpressed telomerase is also less processive than the endogenously expressed telomerase. These findings reveal two major distinct extension modes adopted by telomerase in vivo.     

One-step affinity purification protocol for human telomerase.

Human telomerase is a ribonucleoprotein (RNP) enzyme, comprising protein components and an RNA template that catalyses telomere elongation through the addition of TTAGGG repeats. Telomerase function has been implicated in aging and cancer cell immortalization. We report a rapid and efficient one-step purification protocol to obtain highly active telomerase from human cells. The purification is based on affinity chromatography of nuclear extracts with antisense oligonucleotides complementary to the template region of the human telomerase RNA component. Bound telomerase is eluted with a displacement oligonucleotide under mild conditions. The resulting affinity-purified telomerase is active in PCR-amplified telomerase assays. The purified telomerase complex has a molecular mass of approximately 550 kDa compared to the approximately 1000 kDa determined for the telomerase RNP in unfractionated nuclear extracts. The purification protocol provides a rapid and efficient tool for functional and structural studies of human telomerase.