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.     

端粒酶反义核酸对BEL-7402肝癌细胞生长和活性的影响

目的 研究人类端粒酶RNA（hTR)基因的反义寡核苷酸（AS－ODN）对BEL－7402肝癌细胞生长和活性的影响。方法 将AS－ODN作用于BEL－7402细胞，观察细胞生长状况，PCR－ELISA法检测端粒酶活性，流式细胞仪和细胞DNA电泳观察细胞的凋亡情况。结果 AS－ODN能抑制BEL－7402细胞的生长，降低其端粒酶活性并诱导细胞凋亡。结论 针对hTR的AS－ODN对治疗肝癌有潜在意义。     

端粒酶反义RNA对人肾癌细胞系GRC—1生长的影响

目的 探讨抗端粒酶在肾细胞癌治疗中的意义。方法 采用脂质体介质导法将端粒酶反义RNA真核表达载体（pBBS212-hTR)导入人肾癌GRC－1细胞系中,采用TRAR法测定端粒酶活性,采用MTT法检测细胞生长动力,电镜下观察细胞增殖情况及端粒酶反义RNA对细胞凋亡的影响。结果 端粒酶反应RNA能显著抑制细胞的端粒酶活性,抑制细胞增殖并促进其凋亡。结论 应用端粒酶反义RNA抑制肾癌端粒酶活性是治疗肾癌的潜在途径。     

Simultaneous targeting of telomeres and telomerase as a cancer therapeutic approach

Telomeres, which are important for maintaining chromosome integrity and functions, shorten with each cell division. Telomerase, responsible for telomere synthesis, is expressed in approximately 90% of human tumor cells but seldom in normal somatic cells. This study evaluated the hypothesis that simultaneous shortening of telomeres and inhibition of telomerase results in synergistic and tumor-selective cytotoxicity. In telomerase-positive human pharynx FaDu tumor cells, paclitaxel caused telomere erosion (first detected at 1 h) and apoptosis. Expression of antisense to the RNA component of human telomerase (hTR) inhibited telomerase activity, shortened telomere length, reduced cell growth rate, and resulted in a significant higher sensitivity to paclitaxel. Another telomerase inhibitor, 3'-azido-3'-deoxythymidine (AZT), at a concentration that produced little or no cell detachment or apoptosis, inhibited the telomerase activity and enhanced the paclitaxel-induced cell detachment and apoptosis. AZT also enhanced the activity of paclitaxel in mice bearing well-established s.c. FaDu xenograft tumors (i.e., reduced residual tumor size, enhanced apoptotic cell fraction, and prolonged survival time), without enhancing host toxicity. In contrast, AZT did not enhance the paclitaxel activity in the telomerase-negative osteosarcoma Saos-2 cells nor in FaDu cells where telomerase was already suppressed by antisense hTR, confirming that the AZT effect in parent FaDu cells is mediated through telomerase inhibition. These results demonstrate that combined use of agents targeting both telomere and telomerase yielded synergistic activity selective for tumors that depend on telomerase for telomere maintenance.     

Treatment of prostate cancer in vitro and in vivo with 2-5A-anti-telomerase RNA component

Prostate cancer is the most common malignancy of elderly men in the United States. Since there is no curative treatment for advanced prostate cancer, exploration of novel modalities of treatment is essential. Telomerase, a ribonucleoprotein, is detected in the vast majority of prostate cancer, but not in normal or benign prostatic hyperplasia tissues. Thus, telomerase is expected to be a very strong candidate for targeted therapy of prostate cancer. In this study, we synthesized a 19-mer antisense oligonucleotide against the RNA component of human telomerase (hTR) linked to a 2-5A molecule (2-5A-anti-hTR) and examined its cytotoxic effect on prostate cancer cells. The 2-5A antisense strategy relies on the recruitment and activation of RNase L at the site of targeted RNA sequence. We here show that treatment with 2-5A-anti-hTR in the presence of a cationic liposome reduced cell viability of tumor cell lines tested to 9-18% within 6 days. In contrast, normal fibroblast cells were resistant to the treatment. Its effect was mainly due to induction of apoptosis by activated caspase family members. Furthermore, treatment of subcutaneous tumors in nude mice with 2-5A-anti-hTR significantly suppressed the tumor growth through induction of apoptosis (P<0.001). The treatment with 2-5A-anti-hTR may be a promising strategy for the treatment modality of prostate cancer with telomerase activity.     

2-5A antisense therapy directed against human telomerase RNA inhibits telomerase activity and induces apoptosis without telomere impairment in cervical cancer cells

Human telomerase RNA (hTR), an important component of telomerase, is a possible target of telomerase-based cancer gene therapy. The present study was undertaken to assess the efficacy of antisense hTR therapy using newly developed 2-5A (5'-phosphorylated 2'-5'-linked oligoadenylate)-linked oligonucleotides against cervical cancer cells. ME180 and SiHa cells were treated with 2-5A-linked antisense hTR designed to complement the region of hTR between residues 76 and 94. The hTR expression, telomerase activity, cell viability, and apoptosis were then examined. The 2-5A anti-hTR effectively degraded hTR and inhibited telomerase activity. The 2-5A mutant anti-hTR and the anti-hTR without 2-5A were not capable of inhibiting telomerase activity. Inhibition of telomerase by 2-5A anti-hTR rapidly decreased cell viability only in telomerase-positive cells within 3-6 days after the treatment, when telomere length has not yet been shortened. This inhibition was associated with apoptosis, possibly through activation of caspase family members. These findings suggest that 2-5A-linked antisense-hTR therapy has a potent telomerase-inhibitory effect associated with a cytocidal effect from caspase-induced apoptosis, and may therefore be a potential tool in telomerase-based gene therapy against cervical cancers.     

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.     

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。     

端粒酶反义RNA 对人喉癌细胞系Hep-2 生长的影晌

 目的　探讨抗端粒酶在喉癌细胞治疗中的意义。方法　用脂质体介导法将 pBBS2 12 hTR(反义端粒酶RNA真核表达载体 )转染入人喉癌Hep 2细胞系中 ,采用TRAP法测定端粒酶活性 ,细胞动力学检测 ,电镜下观察等方法研究其对Hep 2细胞的端粒酶活性、细胞增殖及细胞凋亡影响。 结果　端粒酶反义RNA能显著抑制细胞的端粒酶活性 ,抑制细胞增殖并促进其凋亡。结论　以端粒酶反义RNA抑制端粒酶活性是治疗喉癌的潜在途径。     

反义端粒酶RNA对人胃癌细胞端粒长度及端粒酶活性调控的研究

 目的　探讨反义端粒酶RNA(hTR)对人胃癌细胞 端粒长度(TRF)及端粒酶活性的调控作 用。方法　应用反义hTR真核表达载体经脂质体介导转染胃癌细胞SGC7901 ,通过分子杂交及端粒重复扩增PCR方法检测hTR表达及端粒、端粒酶活性变化。结 果　经HYR筛选,转染细胞形成稳定克隆,反义hTR表达增强、正义hTR表达下调,平 均TRF缩短、端粒酶活性受抑。结论　反义hTR对胃癌细胞TRF及端粒酶活性 的调控可能是通过其对hTR模板的“封闭”而发挥作用的,反义hTR可以作为胃癌基因治疗的 一种新靶点。     

端粒酶反义RNA 对HL-60 细胞端粒酶活性及细胞增殖的影响

 目的　探讨端粒酶反义 RNA对 HL- 60细胞端粒酶活性及细胞增殖的影响。方法　用脂质体介导法将 p BBS2 1 2 - h TR(反义端粒酶 RNA真核表达载体 )导入人髓性白血病细胞系 HL-60中 ,采用 TRAP法测定端粒酶活性 ,细胞生长动力学检测 ,FCM分析等方法观察其对 HL- 60细胞的端粒酶活性及细胞增殖影响。结果　端粒酶反义 RNA能显著抑制 HL- 60细胞的端粒酶活性 ,抑制 HL- 60细胞的增殖并诱导其凋亡。结论　以端粒酶反义 RNA抑制端粒酶活性是治疗白血病的潜在途径。     

抑制端粒酶活性对人肾癌细胞生长的抑制作用

目的 :探讨抗端粒酶在肾细胞癌治疗中的意义。方法 :采用脂质体介导法将pBBS2 12 hTR(反义端粒酶RNA真核表达载体 )导入人肾癌GRC 1细胞系中 ,采用TRAP法测定端粒酶活性 ,通过MTT法检测细胞动力学 ,观察细胞的增殖 ,电镜下观察其对细胞凋亡的影响。结果 :端粒酶反义RNA能显著抑制细胞的端粒酶活性 ,抑制细胞增殖并促进其凋亡。结论 :以端粒酶反义RNA抑制端粒酶活性是治疗肾癌的潜在途径     

Attenuation of telomerase activity by hammerhead ribozyme targeting human telomerase RNA induces growth retardation and apoptosis in human breast tumor cells

Ribozyme possesses specific endoribonuclease activity and catalyzes the hydrolysis of specific phosphodiester bonds, which results in the cleavage of target RNA sequences. Here, we evaluated the ability of hammerhead ribozymes targeting human telomerase RNA (hTR) to inhibit the catalytic activity of telomerase and the proliferation of cancer cells. Hammerhead ribozymes were designed against 7 NUX sequences located in open loops of the hTR secondary structure. We verified the ribozyme specificity by in vitro cleavage assay by using a synthetic RNA substrate. Subsequently, we introduced ribozyme expression vector into human breast tumor MCF-7 cells and assessed the biologic effects of ribozyme. Hammerhead ribozyme R1 targeting the template region of hTR efficiently cleaved hTR in vitro, and stable transfectants of this ribozyme induced the degradation of target hTR RNA and attenuated telomerase activity in MCF-7 cells. Moreover, the ribozyme R1 transfectant displayed a significant telomere shortening and a lower proliferation rate than parental cells. Clones with reduced proliferation capacity showed enlarged senescence-like shapes or highly differentiated dendritic morphologies of apoptosis. In conclusion, the inhibition of telomerase activity by hammerhead ribozyme targeting the template region of the hTR presents a promising strategy for inhibiting the growth of human breast cancer cells.     

hTR基因反义核酸对K562细胞端粒酶活性和凋亡的影响

目的: 研究人类端粒酶(hTR)基因的反义核酸对K562细胞端粒酶活性和细胞凋亡的影响.方法:采用TRAP法检测K562细胞在反义核酸处理前后端粒酶活性的变化,以及采用TUNEL法观察反义核酸处理前后细胞凋亡的变化.结果:hTR基因反义核酸能有效抑制K562细胞的端粒酶活性,并且诱导K562细胞发生凋亡.结论:hTR基因反义核酸能显著抑制肿瘤细胞的端粒酶活性,为恶性肿瘤治疗提供了一条新的有效的途径.     

Resistance to telomerase inhibition by human squamous cell carcinoma cell lines

Telomeres are nucleoprotein structures at the ends of chromosomes that are composed of a repetitive G rich sequence and telomeric binding proteins. Telomeres prevent the degradation of chromosomal ends and protect against inappropriate recombination. Telomere attrition involves a tumor suppressor pathway that limits the replication of premalignant cells. The loss of telomeric DNA with each round of replication leads to growth arrest accompanied by senescence or apoptosis. Many tumor cells activate the telomerase gene to bypass senescence. Telomerase is a multisubunit ribonucleoprotein that uses an RNA template to catalyze the addition of telomeric DNA to chromosomal ends. Overexpression of the TERT subunit leads to telomere lengthening and extension of the replicative lifespan. Dominant-negative telomerase has been shown to inhibit telomerase activity in many tumor cell lines, and this is associated with telomere shortening and apoptosis. Additionally, pharmacological telomerase inhibitors have been developed which lead to progressive telomere shortening and programmed cell death. In this study, we report a series of human squamous cell carcinoma cell lines that have high telomerase activity and short telomeres. Dominant-negative telomerase expression and pharmacological telomerase inhibition failed to completely inhibit enzymatic activity which was accompanied by the lack of telomere shortening. These cells continued to proliferate and demonstrated fewer responsive genes when treated with a pharmacological telomerase inhibitor. We concluded that some human squamous cell carcinoma cell lines are resistant to telomerase inhibition.     

Decreased telomerase activity is not a reliable indicator of chemosensitivity in testicular cancer cell lines

Telomere stabilisation is a critical step in tumorigenesis and telomerase, an enzyme which counteracts telomeric DNA loss, is active in most tumours. Conflicting evidence has been published concerning the potential use of telomerase activity as a measurement of drug-induced tumour cell killing. In this study, the time courses of telomerase loss and induction of apoptosis were investigated in two testicular cell lines, Susa CP and 833 K, following 4-h exposure to cisplatin, melphalan or doxorubicin. Telomerase activity was only affected in both cell lines at 20 h following exposure to high concentrations of cisplatin (100x the drug concentrations causing 50% growth inhibition (IC(50) values)). The time course of melphalan-induced telomerase loss, which was again only apparent at 100x IC(50) concentrations, varied between the cell lines and doxorubicin (100x IC(50)) did not induce telomerase loss in either of the cell lines. Importantly, the levels and rates of appearance of apoptotic cells (nuclear morphology and annexin V staining) were similar for all three drugs in both cell lines; i.e. cisplatin, melphalan and doxorubicin (100x IC(50)) caused similar frequencies of apoptosis in Susa CP cells at 24 h whereas telomerase activities were 65, 123 and 96% of the control, respectively. The possibility that telomerase activity was lost following cisplatin treatment through a direct interaction of cisplatin with telomerase was discounted. Additionally, the relative levels of the RNA component of telomerase (hTR) and mRNA for the telomerase catalytic subunit (hTERT) were not related to the observed decreases in telomerase activity. These data indicate that telomerase activity is not a reliable indicator of chemosensitivity in human testicular cancer cells. Furthermore, cisplatin-induced loss of telomerase activity is not due to a direct reaction with the enzyme or decreased hTR levels.     

端粒酶RNA的反义寡核苷酸抑制裸鼠移植瘤生长的研究

背景与目的许多研究已证明:端粒酶的激活在肺癌的发生、发展中具有重要作用,因而成为肺癌基因治疗的重要靶点之一。本研究旨在探讨针对人端粒酶RNA成分的反义寡核苷酸对裸鼠移植瘤的生长抑制作用。方法应用人肺腺癌细胞株A549细胞构建裸鼠皮下移植瘤模型,18只荷瘤裸鼠随机分为反义寡核苷酸组(Antisense Oligodeoxynucleotide Group,Group ASODN)、正义寡核苷酸组(Sense Oligodeoxynucleotide Group,Group SODN)和生理盐水对照组(Normal Saline Group,Group NS),每组6只,瘤体内分别注射反义寡核苷酸/阳离子脂质体复合物、正义寡核苷酸/阳离子脂质体复合物和生理盐水,均为每日1次,共14次,观察移植瘤的生长情况。结果反义寡核苷酸组、正义寡核苷酸组的体积抑瘤率分别是43.94%、6.91%,统计学检验有非常显著性差异(t=6.17,P<0.001)。治疗过程中裸鼠对药物耐受良好,无恶心、呕吐等消化道症状,未见皮下出血,治疗结束时各组裸鼠的体重较治疗开始时略有增加。结论瘤体内注射脂质体包封的端粒酶RNA的反义寡核苷酸能够明显抑制裸鼠体内移植瘤的生长。     

hTR反义PS-ODN联合顺铂对人胃癌裸鼠皮下移植瘤作用的体内研究

目的 探讨hTR反义寡脱氧核苷酸（ASODN）联合顺铂对人胃癌裸鼠皮下移植瘤的治疗作用。方法 30只裸鼠建立胃癌皮下移植瘤模型后，随机分成5组，予以不同条件处理，对照组（瘤周注射RPMI1640培养液）、ASODN组（瘤周注射反义寡脱氧核苷酸）、RODN组（瘤周注射随机寡脱氧核苷酸）、DDP组（瘤周注射化疗药物顺铂）、ASODN＋DDP组（瘤周注射反义寡脱氧核苷酸和顺铂）。治疗后定期观察肿瘤体积，计算肿瘤抑制率。采用TRAPPCR—ELISA法检测移植瘤的端粒酶活性。结果 ASODN＋DDP组、ASODN组、DDP组和RODN组的最高肿瘤抑制率分别为94.2％、84．3％，92．8％和26．9％。在端粒酶活性检测中，4个治疗组间均有统计学意义。结论 hTR反义PSODN对人胃癌裸鼠皮下移植瘤生长及端粒酶活性有一定抑制作用，且可增强DDP的上述作用。