核酶对食管癌EC9706细胞端粒酶活性及凋亡的影响

目的 :观察针对人端粒酶RNA模板区的核酶对食管癌细胞端粒酶活性和细胞凋亡的影响。方法 :利用脂质体Lipofectamine介导 ,将已构建好的带有端粒酶核酶基因的重组质粒pBBS2 12Rz及空载质粒pBBS2 12转染食管癌EC970 6细胞 ,采用TRAP ELISA法检测端粒酶活性 ,用倒置相差显微镜及流式细胞仪观察细胞生长和凋亡情况。结果 :重组质粒pBBS2 12Rz转染的食管癌EC970 6细胞的端粒酶活性明显下降 ,细胞生长速度明显变慢 ,凋亡加速。结论 :端粒酶核酶对食管癌细胞端粒酶活性和细胞生长有抑制作用 ,可望成为食管癌基因治疗的新方法     

Attenuation of Telomerase Activity by siRNA Targeted Telomerase RNA Leads to Apoptosis and Inhibition of Proliferation in Human Renal Carcinoma Cells

OBJECTIVE Telomerase is an attractive molecular target for cancer therapy because the activation of telomerase is one of the key steps in cell immortalization and carcinogenesis. RNA interference using small-interfering RNA (siRNA) has been demonstrated to be an effective method for inhibiting the expression of a given gene in human cells. The aim of the present study was to investigate whether inhibition of telomerase activity by siRNA targeted against human telomerase RNA (hTR) can inhibit proliferation and induce apoptotic cell death in human renal carcinoma cells (HRCCs). METHODS The siRNA duplexes for hTR were synthesized and 786-0 HRCCs were transfected with different concentrations of hTR-siRNA. The influence on the hTR mRNA level, telomerase activity, as well as the effect on cell proliferation and apoptosis was examined. RESULTS Anti-hTR siRNA treatment of HRCCs resulted in specific reduction of hTR mRNA and inhibition of telomerase activity. Additionally, significant inhibition of proliferation and induction of apoptosis were observed. CONCLUSION siRNA against the hTR gene can inhibit proliferation and induce apoptosis by blocking telomerase activity of HRCCs. Specific hTR inhibition by siRNA represents a promising new option for renal cancer treatment.     

Antitumor activity of systemically delivered ribozymes targeting murine telomerase RNA.

PURPOSE: To test ribozymes targeting mouse telomerase RNA (mTER) for suppression of the progression of B16-F10 murine melanoma metastases in vivo. EXPERIMENTAL DESIGN: Hammerhead ribozymes were designed to target mTER. The ribozyme sequences were cloned into a plasmid expression vector containing EBV genomic elements that substantially prolong expression of genes delivered in vivo. The activity of various antitelomerase ribozymes or control constructs was examined after i.v. injection of cationic liposome:DNA complexes containing control or ribozyme constructs. Expression of ribozymes and mTER at various time points were evaluated by quantitative real-time PCR. Telomerase activity was examined using the telomeric repeat amplification protocol. RESULTS: Systemic administration of cationic liposome:DNA complexes containing a plasmid-expressed ribozyme specifically targeting a cleavage site at mTER nucleotide 180 significantly reduced the metastatic progression of B16-F10 murine melanoma. The antitumor activity of the anti-TER 180 ribozyme in mice was abolished by a single inactivating base mutation in the ribozyme catalytic core. The EBV-based expression plasmid produced sustained levels of ribozyme expression for the full duration of the antitumor studies. In addition to antitumor activity, cationic liposome:DNA complex-based ribozyme treatment also produced reductions in both TER levels and telomerase enzymatic activity in tumor-bearing mice. CONCLUSIONS: Systemic, plasmid-based ribozymes specifically targeting TER can reduce both telomerase activity and metastatic progression in tumor-bearing hosts. The work reported here demonstrates the potential utility of plasmid-based anti-TER ribozymes in the therapy of melanoma metastasis.     

端粒酶特异性核酶抑制宫颈癌细胞的作用

目的 :研究端粒酶RNA特异性核酶对宫颈癌细胞活性的影响。方法 :通过脂质体将核酶转染至宫颈癌Hela细胞 ,以G4 18筛选细胞 ,经斑点杂交提示转染成功后 ,检测细胞活力及细胞周期变化 ,测定细胞端粒酶活性 ,观察细胞的增殖情况。结果 :试验组细胞的活力较对照组明显减弱。转染后细胞受阻于G1期的细胞明显增多 ,S期细胞比例明显降低 ,P <0 0 1。转核酶细胞端粒酶活性较对照组明显减低 ,转染空载体的细胞端粒酶活性与未转染细胞比较则无明显变化。结论 :端粒酶RNA特异性核酶可抑制宫颈癌细胞端粒酶活性 ,抑制细胞增殖 ,为核酶用于恶性肿瘤基因治疗提供了试验依据     

Delivering antisense telomerase RNA by a hybrid adenovirus/ adeno-associated virus significantly suppresses the malignant phenotype and enhances cell apoptosis of human breast cancer cells

Activated telomerase is frequently detected in cancer cells and is able to maintain and stabilize the integrity of telomeres; it also contributes to unlimited divisions in cancer cells. Recently, a new generation of selective anticancer strategies is under development targeting the blockage of telomerase activity either at the protein level or telomerase RNA. Here, we report suppression of the malignant phenotype by the expression of the full-length antisense human telomerase RNA (hTR) delivered by a novel hybrid vector recombining adenovirus and adeno-associated virus (vAd-AAV). The hybrid vector vAd-AAV retained the unique traits from two parental viruses, such as high efficiency of gene transfer in mammalian cells and the ability to integrate into the genomic DNA of host cells. The stable expression of antisense hTR in MCF-7 cells significantly suppressed telomerase activity and progressively shortened telomere length for 30 population doublings (PD30). Expression of antisense hTR leads to a telomere-based growth arrest and the induction of spontaneous apoptosis. Antisense hTR decreased soft agar colony formation and reduced the cell proliferation, leading to exit from the cell cycle at G1 at PD15. The expression of antisense hTR also sensitized MCF-7 cells to apoptosis induced by sodium butyrate or serum starvation. Our study demonstrates that delivering antisense hTR by the hybrid Ad/AAV vector is an effective antineoplastic gene therapeutic strategy, which significantly suppresses the malignant phenotype and enhances apoptosis of human breast cancer cells.     

Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase

Telomerase activity is necessary and sufficient for immortality in many cells and hence represents a prime target for antitumor strategies. Here, we show that a hammerhead ribozyme cleaves human telomerase (hTERT) mRNA in vitro. Stable transfection in clones of the human breast tumor line MCF-7 and the immortal breast cell line HBL-100 results in expression of the ribozyme, diminishes the abundance of hTERT mRNA, and inhibits telomerase activity. This led to shortened telomeres, inhibition of net growth, and induction of apoptosis. In HBL-100 mass cultures infected with a ribozyme-expressing adenovirus diminution of hTERT mRNA, attenuation of telomerase activity, inhibition of net growth, and induction of apoptosis was found as well. Attenuation of telomerase activity increased the sensitivity of HBL-100 and MCF-7 clones specifically to inhibitors of topoisomerase. Likewise, expression of exogenous telomerase in originally telomerase-negative human fibroblasts decreased their sensitivity to topoisomerase poisons but not to a number of other cytotoxic drugs. The data validate a ribozyme approach for telomerase inhibition therapy in cancer and suggest that it might be combined advantageously with topoisomerase-directed chemotherapy.     

Ribozyme-mediated cleavage of the human survivin mRNA and inhibition of antiapoptotic function of survivin in MCF-7 cells

Survivin is a new member of the inhibitor of apoptosis protein (IAP) family that is implicated in the control of cell proliferation and the regulation of cell life span. This protein is selectively expressed in most human carcinomas but not in normal adult tissues. To down-regulate a human survivin expression as a strategy for cancer gene therapy, we designed two hammerhead ribozymes (RZ-1, RZ-2) targeting human survivin mRNA. RZ-1 and RZ-2 efficiently cleaved the human survivin mRNA at nucleotide positions +279 and +289, which was identified by in vitro cleavage assay using in vitro transcribed ribozymes and truncated survivin mRNA substrate. To investigate the function of the ribozymes in cells, the sequences of the ribozymes were cloned into replication-deficient adenoviral vector and transferred to breast cancer cell, MCF-7. The infection with adenovirus encoding the ribozymes resulted in a significant reduction of survivin mRNA (74% and 73%, respectively) and protein. As revealed by nuclear condensation/ fragmentation and flow cytometry analysis, inhibition of survivin gene by ribozymes increased apoptosis and sensitivity induced by etoposide or serum starvation. Our results suggest that the designed hammerhead ribozymes against survivin mRNA are good candidates for feasible gene therapy in the treatment of cancer.     

Attenuation of telomerase activity does not increase sensitivity of human melanoma cells to anticancer agents

In tumour cells, replicative immortality is attained through stabilisation of telomeres by telomerase. Recent evidence suggests that telomerase plays an anti-apoptotic role. Since apoptosis is the primary mode of cell death induced by several drugs, telomerase could be involved in determining the chemosensitivity profile of tumour cells. We investigated whether inhibition of telomerase activity through a hammerhead ribozyme targeting the RNA template of telomerase influences the susceptibility of human melanoma cells to a variety of anticancer agents (platinum compounds, taxanes, topoisomerase I inhibitors). The ribozyme sequence was inserted into an expression vector and the JR8 human melanoma cell line was transfected with it. The cell clones obtained showed a reduced telomerase activity. Growth inhibition curves generated after exposure of ribozyme-transfectant clones to individual drugs were superimposable to those obtained from parental cells. Moreover, telomerase inhibition did not promote apoptosis as a cellular response to drug treatment. Overall, our results indicate that downregulation of telomerase activity does not increase the sensitivity of melanoma cells to anticancer drugs.     

RNA干扰对肾癌细胞端粒酶活性及增殖、凋亡的影响

目的 探讨针对人端粒酶RNA（hTR）及其催化亚基（hTERT）的小干扰RNA（siRNA）对肾癌细胞端粒酶活性及其增殖、凋亡的影响。方法 将hTR-siRNA、hTERT-siRNA（100nmol／L）单独或联合转染人肾癌786—0细胞,采用RT-PCR法检测hTR、hTERT mRNA表达,TRAP-ELISA法检测端粒酶活性,MTT法检测细胞增殖,免疫组化TUNEL法检测细胞凋亡。结果 （1）hTR-siRNA可显著降低786—0细胞hTR mRNA表达（P〈0．01）,hTERT-siRNA可显著降低hTERT mRNA表达（P〈0．01）,但彼此互不影响。（2）二者均能显著抑制端粒酶活性（P〈0．01,P〈0．01）,并增加786—0细胞增殖抑制率及凋亡细胞阳性率（P〈0．01,P〈0．01）。二者联合应用与单独应用差异亦无显著性（P〉0．05）。结论 hTR、hTERT siRNA通过抑制各自基因表达,抑制人肾癌细胞端粒酶活性,进而抑制增殖、促进凋亡。     

抗VEGF165核酶对人肺腺癌细胞生物学特性的影响

目的 探讨抗血管内皮生长冈子165(VEGF165)核酶对人肺腺癌细胞生物学特性的影响。方法设计合成针对VEGF165 212位点的锤头状核酶(vascular endothelial growth factor ribozyme，VRZ)及其突变体(mutant VRz，mVRz)，体外检测核酶的剪切活性。采用亚克隆技术，构建腺病毒介导的抗VEGF165核酶真核表达载体pAdVRz，用重组腺病毒感染人肺腺癌细胞A549，分别采用Northern blot印迹杂交、流式细胞仪、透射电镜和激光共聚焦等技术，观察感染前后A549细胞的生物学性状的改变。结果成功地合成了具有明显剪切活性的抗VEGF165核酶VRz及其重组腺病毒表达载体rpAdVRz，并在A549细胞中获得表达。重组腺病毒感染细胞的VEGF165表达减少了87％，而生物学性状不受外源基因表达的影响。结论抗VEGF165核酶能够显著抑制人肺腺癌细胞内VEGF165的表达，为进一步进行肺癌的抗血管治疗提供了实验基础。     

抑制VEGF表达的锤头状核酶系统

目的:建立和评估抑制血管内皮生长因子(VEGF)表达的锤头状核酶(hammerhead ribozyme)技术系统.方法:对VEGF121基因RNA序列进行二级结构分析,选择靶点;设计并构建针对VEGF的分泌肽RNA的可表达锤头状核酶(14)载体系统和VEGF-荧光色素酶融合基因报告质粒;通过试管内切割实验等方法评估核酶对于试管内转录得到的VEGF RNA切割特异性和效率;通过瞬时共转染实验和稳定转染实验评估核酶在细胞内对于VEGF RNA的切割效率.结果:设计和构建了对VEGF RNA二级结构水平上的暴露区( 8, 36和 71位点:核酶1,3和4)和非暴露区( 17位点:核酶2)4个锤头状核酶(14)的两套质粒;试管内切割检测表明,针对 8, 36和 71位点的核酶(1,3和4)可以有效地在试管内对VEGFRNA进行特异性切割,使其水平分别降至对照的61.7%,27.6%和44.8%(荧光色素酶活性)或66.3%,27.0%和30.0%(蛋白质水平);将核酶表达质粒与VEGF-LUC模板质粒瞬时共转染人SMMC-7721肝癌细胞,核酶1,3和4 VEGF-LUC水平分别降低到对照的81.4%,56.6%和69.1%;稳定表达针对VEGF的核酶1,3或4的SMMC-7721细胞株中,内源性VEGF RNA的水平降至对照水平的5%以下.对照未转染的、转染空载体的和转染了核酶2( 17)的SMMC-7721细胞.结论:分别针对VEGF 8, 36和 71位点锤头状核酶可有效地抑制VEGF的RNA水平.     

Effects of a novel telomerase inhibitor, GRN163L, in human breast cancer

Summary Telomerase activity is undetectable in most normal tissues but the vast majorities of cancers express active telomerase. Therefore, telomerase serves as an attractive target for the treatment of cancers. GRN163L is a lipid-modified oligonucleotide N3′→P5′ thio-phosphoramidate complementary to the RNA template region of human telomerase. The anti-telomerase activity of GRN163L was evaluated using MDA-MB-231 and MDA-MB-435 human breast adenocarcinoma cell lines. Twice weekly administration of GRN163L resulted in the inhibition of telomerase activity and progressive telomere shortening. Cells treated with GRN163L did not demonstrate decreased cell proliferation for up to 2 weeks. However, after additional treatment, cell proliferation gradually decreased in GRN163L-treated cells compared to untreated or mismatch control oligoncleotide treated cells. Furthermore, anti-tumorigenic effects were seen in cells treated with GRN163L, as cells lose their ability to form colonies in soft agar and were unable to form colonies in the clonal efficiency assay upon incubation with GRN163L. Moreover, breast cancer cells that were treated with GRN163L for only 1 week prior to plating in invasion chambers, and when bulk telomere are still long, exhibit significantly diminished invasive potential. These results reveal critical information regarding the effectiveness of GRN163L as a potential therapeutic agent for the treatment of human breast cancer.     

Inhibition of cell growth and telomerase activity of breast cancer cells in vitro by retinoic acids

The effects of retinoic acid (RA) and its analogs, all-trans RA, 9-cis RA and 13-cis RA, were investigated in human breast cancer MCF-7 cells and immortalized breast epithelial cell line MCF-10A. RA inhibited the telomerase activity of MCF-7 cells in a wide range of concentrations. RA at 10 microM also inhibited the growth of MCF-7 cells in a time-dependent manner. However, no significant growth inhibition was found between untreated control and RA-treated MCF-10A cells. Moreover, a marked inhibition of telomerase activity by RA was detected early in MCF-7 cells (after 24 h of RA treatment), which was preceded by a reduction of hTERT mRNA expression (after 12 h of RA treatment). However, MCF-10A cells showed a reduction of telomerase activity and down-regulation of hTERT after 4 days of RA treatment. Simultaneous changes in hTERT mRNA expression and telomerase activity were found for MCF-10A cells. The expressions of hTR and hTEP1 telomerase component genes were not changed after RA treatment. These results indicate that the anti-breast cancer activity of RA could be mediated by its ability to down-regulate the expression of hTERT telomerase gene.     

Curcumin inhibits telomerase activity through human telomerase reverse transcritpase in MCF-7 breast cancer cell line

The inhibitory effect of curcumin, the yellow-colored pigment from turmeric, on telomerase activity was analyzed in human mammary epithelial (MCF-10A) and breast cancer (MCF-7) cells. Telomerase activity in MCF-7 cells is 6.9-fold higher than that of human mammary epithelial cells. In MCF-7 cells, telomerase activity decreased with increasing concentrations of curcumin, inhibiting about 93.4% activity at 100 microM concentration. The inhibition of telomerase activity in MCF-7 cells may be due to down-regulation of hTERT expression. Increasing concentrations of curcumin caused a steady decrease in the level of hTERT mRNA in MCF-7 cells whereas the level of hTER and c-myc mRNAs remained the same. Our results suggest that curcumin inhibits telomerase activity by down-regulating hTERT expression in breast cancer cells and this down-regulation is not through the c-myc pathway.     

Inhibition of telomerase activity by geldanamycin and 17-allylamino, 17-demethoxygeldanamycin in human melanoma cells

As it has been demonstrated that the heat shock protein 90 (HSP90) is required for the assembly and activation of telomerase in human cells, we investigated the effect exerted by the ansamycin antibiotics geldanamycin (GA) and 17-allylamino,17-demethoxygeldanamycin (17-AAG), two well-known inhibitors of the HSP90 chaperone function, on telomerase activity in JR8 human melanoma cells. Using an antibody to HSP90, we precipitated the telomerase activity associated with the molecular chaperone. The results of TRAP (telomeric repeat amplification protocol) experiments carried out on HSP90 immunoprecipitates showed that exposure to 100 ng/ml GA and 17-AAG induced a significant (P < 0.01) inhibition of telomerase activity, which was observed at earlier time points than drug-induced inhibition of cell proliferation. Superimposable results were obtained from TRAP experiments carried out on total JR8 protein extracts. To investigate whether the basal level of telomerase activity of the tumour cell system plays a role in determining the cellular response to 17-AAG, we compared the cytotoxic activity of the drug in JR8 cells and in two JR8-derived clones that were stably transfected with a hammerhead ribozyme targeting the RNA template of telomerase and were characterized by a markedly lower telomerase activity than the parental cells. The cytotoxicity results indicated that both ribozyme-transfectant clones were almost 2-fold more sensitive to 72 h 17-AAG exposure than JR8 cells as a consequence of a more than double apoptotic response [in terms of the percentage of apoptotic nuclei in cells stained with propidium iodide and the percentage of Tdt-mediated dUTP nick-end labelling (TUNEL)-positive cells]. In summary, our results suggest that (i) telomerase is a target of GA and 17-AAG action and its inhibition may contribute to the cytotoxic activity of the drugs, (ii) the basal level of telomerase activity of the tumour cell system may also have a role in influencing 17-AAG cytotoxicity.