Antisense hTR Research Articles

Inhibition of tankyrase 1 in human gastric cancer cells enhances telomere shortening by telomerase inhibitors

Telomere stability is believed to be related to aging and tumorigenesis. Besides telomerase, telomere length is also regulated by several telomere-specific binding proteins. Tankyrase 1, a telomeric poly(ADP-ribose) polymerase (PARP), elongates telomere length by inhibiting TRF1 binding to telomeres. In order to study the synergistic action of tankyrase 1 and telomerase in the maintenance of telomere length in mammalian cells, we constructed anti-sense tankyrase 1 (aTNKS) eukaryotic expression vectors and then transfected them into the SGC-7901 human gastric cancer cell line, as well as SGC-7901 cells that had been transfected with antisense hTR (7901-ahTR) and antisense hTERT (7901-ahTERT) with DOTAP liposomes. The activity of telomerase, telomere length and telomerase-associated protein activities were measure by TRAP-ELISA, Southern blot and western blot analysis, respectively, in aTNKS transfected and untransfected cells. The results demonstrated that telomere length was significantly shorter in cells with concomitant tankyrase 1 and telomerase inhibition than by either tankyrase 1 or telomerase inhibition alone, in SGC-7901 cells. We also found that aTNKS had no effect on telomerase activity. These results reveal that inhibition of tankyrase 1 could shorten telomere length and play a synergistic role with telomerase inhibitors in telomere length shortening in the SGC-7901 gastric cancer cell line. Co-inhibition of tankyrase 1 and telomerase activity may be a rational strategy for telomere-directed gastric cancer therapeutics.

Inhibition of tankyrase 1 in human gastric cancer cells enhances telomere shortening by telomerase inhibitors

Telomere stability is believed to be related to aging and tumorigenesis. Besides telomerase, telomere length is also regulated by several telomere-specific binding proteins. Tankyrase 1, a telomeric poly(ADP-ribose) polymerase (PARP), elongates telomere length by inhibiting TRF1 binding to telomeres. In order to study the synergistic action of tankyrase 1 and telomerase in the maintenance of telomere length in mammalian cells, we constructed anti-sense tankyrase 1 (aTNKS) eukaryotic expression vectors and then transfected them into the SGC-7901 human gastric cancer cell line, as well as SGC-7901 cells that had been transfected with antisense hTR (7901-ahTR) and antisense hTERT (7901-ahTERT) with DOTAP liposomes. The activity of telomerase, telomere length and telomerase-associated protein activities were measure by TRAP-ELISA, Southern blot and western blot analysis, respectively, in aTNKS transfected and untransfected cells. The results demonstrated that telomere length was significantly shorter in cells with concomitant tankyrase 1 and telomerase inhibition than by either tankyrase 1 or telomerase inhibition alone, in SGC-7901 cells. We also found that aTNKS had no effect on telomerase activity. These results reveal that inhibition of tankyrase 1 could shorten telomere length and play a synergistic role with telomerase inhibitors in telomere length shortening in the SGC-7901 gastric cancer cell line. Co-inhibition of tankyrase 1 and telomerase activity may be a rational strategy for telomere-directed gastric cancer therapeutics.

Combination of tamoxifen and antisense human telomerase RNA inhibits glioma cell proliferation and anti-apoptosis via suppression of telomerase activity

Accumulating evidence indicates that telomerase activity and human telomerase RNA (hTR) play a potentially crucial role in maintaining the malignant progression of human gliomas. Tamoxifen (TAM) induces cell growth inhibition by modulating several cellular activities, including signaling pathways and the cell cycle. In this study, we aimed to evaluate the effect of combination therapy with TAM and antisense hTR on malignant glioma growth in vitro. TAM treatment and the down-regulation of hTR expression by antisense hTR resulted in a significant suppression of cell growth and the induction of cell apoptosis through the inhibition of telomerase activity. The antitumor effect of treatment with TAM alone was found to be mediated in part by the down-regulation of telomerase activity and of PKC and IGF-1 expression. Our results indicate that TAM and antisense hTR may serve as a novel strategy for glioma therapy.

The Retrovirus-mediated Antisense Human Telomerase RNA (hTR) Gene Limits the Growth of Hepatocellular Carcinoma Growth in Cell Culture and Animals

To investigate the inhibitory effect of retrovirus-mediated antisense human telomerase RNA (hTR) gene therapy on hepatocelluar carcinoma. We first constructed the sense and antisense hTR vectors and then transfected these into HepG2 cells. Telomerase activity, cell growth curves, proliferating cell nuclear antigen expression (PCNA), cell cycle distribution, and cell apoptosis were detected by the means of telemere repeat amplification protocol (TRAP), MTT assay, immunofluorescence, flow cytometric analysis, and transferase-mediated nick end labeling (TUNEL), respectively. In order to further confirm the therapeutic effect of this gene therapy, we developed an experimental line of HepG2 tumor-bearing nude mice by and directly injected these with retrovirus expressing the antisense hTR gene. Tumor growth was determined by tumor volume, and cell apoptosis was analyzed by TUNEL. The antisense hTR gene was shown to be successfully integrated into the target cells' genome. HepG2 cells transfected with the antisense hTR gene showed down-regulated telomerase activity, inhibited cell growth, decreased PCNA expression, and increased apoptotic rate. Moreover, flow cytometry revealed a decrease of cells in the S phase with cell cycle arrest at the G2/M phase. In the antisense hTR-treated group, tumor growth was significantly reduced and showed an increase of apoptotic cells. The results indicate that the specific inhibitor of the hTR template is likely to be a very efficient tool for hepatocellular carcinoma research and may possess potential therapeutic significance in the future clinical practice.

Antisense telomerase RNA inhibits the growth of human glioma cells in vitro and in vivo

Telomerase is implicated in the development of cellular immortality and oncogenesis. It has been shown that telomerase activity is considerably higher in the tissue of many different cancers than in normal tissue, and that the inhibition or downregulation of telomerase activity can prevent the malignant proliferation of tumor cells. Antisense oligonucleotides have been widely used in suppressing the expression of genes and, therefore, in the present research, we evaluated the effect of antisense human telomerase RNA (hTR) on glioma cell growth in vitro and in vivo. We showed that antisense hTR cDNA significantly inhibited TJ905 human glioma cell proliferation in vitro and tumor growth in vivo, as determined by MTT assay and by measuring the volume of glioma in nude mice. Consistent with these results, we found that telomerase activity and the mRNA levels of hTR and hTERT (human telomerase reverse transcriptase) expression were markedly decreased in tumor cells treated with antisense hTR cDNA, as assessed by TRAP (telomeric repeat amplification protocol) assay and RT-PCR (reverse transcription-polymerase chain reaction) analysis. Our study conclusively demonstrates that antisense hTR effectively inhibits the growth of human glioma cells in vitro and in vivo and, thus, may be potentially used for gene therapy of malignant gliomas and other cancers.

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.

Relationship between telomerase activity and its subunit expression and inhibitory effect of antisense hTR on pancreatic carcinoma.

To directly investigate the relationship between telomerase activity and its subunit expression and the inhibitory effect of antisense hTR on pancreatic carcinogenesis. We examined the telomerase activity and its subunit expression by cell culture, polymerase chain reaction (PCR), PCR-silver staining, PCR-ELISA, DNA sequencing, MTT and flow cytometry methods. PCR-silver staining and PCR-ELISA methods had the same specificity and sensitivity as the TRAP method. Telomerase activity was detected in the extract of the 10(th),20(th) and 30(th) passages of P3 cells,while it was absent in fibroblasts. Furthermore, after the 30th generation, the proliferation period of fibroblast cells was significantly prolonged. Telomerase activity and hTERTmRNA were detected in two pancreatic carcinoma cell lines, but were found to be negative in human fibroblast cells. Telomerase activity and hTERTmRNA were tested in pancreatic carcinoma specimens of 24 cases. The telomerase activity was positive in 21 of the 24 cases (87.5 %), and the hTERTmRNA in 20 cases (83.3 %). In adjacent normal tissues positive rates were both 12.5 %. There was a significant difference between the two groups. This indicated a significant correlation between the expression level of telomerase activity and histologic differentiation, metastasis and advanced clinical stage of pancreatic carcinoma. Our findings showed that the expressions of hTR and TP1mRNA were not correlated with the activity of telomerase but the expression of hTERTmRNA was. After treatment with PS-ODNs, telomerase activity in P(3) cells weakened and the inhibiting effect became stronger with an increase in PS-ODNs concentration. There was a significant difference between different PS-ODN groups (P<0.05). Inhibition of telomerase activity occurred most significant with PS-ODN1. The results of the FCM test of pancreatic cancer P(3) cells showed an increase in the apoptotic rate with increasing PS-ODN1 and PS-ODN2 concentrations. The expression of telomerase activity has a significant relationship to carcinogenesis. A strong correlation exists between telomerase activity and hTERTmRNA expression. The up-regulation of hTERTmRNA expression may play a critical role in human carcinogenesis. The expression of telomerase activity and its subunit level in pancreatic carcinoma significantly correlate with the clinical stage of pancreatic carcinoma and hence, may be helpful in its diagnosis and prognosis. The anti-hTR complementary to the template region of hTR is sufficient to inhibit P3 cell telomerase activity and cell proliferation in vitro, and can lead to a profound induction of programmed cell death.

Telomere maintenance in telomerase-positive human ovarian SKOV-3 cells cannot be retarded by complete inhibition of telomerase

The two known mechanisms for telomere maintenance in eukaryocytes are telomerase in telomerase-positive cells and alternative lengthening of telomeres (ALT) in telomerase-negative cells. We report here that telomere maintenance in the telomerase-positive human ovarian SKOV-3 cells was not affected by inhibition of telomerase. For comparison, the effect of telomerase inhibitors on telomere maintenance in another telomerase-positive cell line (i.e. human pharynx FaDu cells) and the telomerase-negative human osteosarcoma Saos-2 cells was examined. Telomerase activity was measured using a modified telomeric repeat amplification protocol and telomere length was measured using a solution hybridization-based method and fluorescence in situ hybridization. A reverse transcriptase inhibitor (3′-azido-deoxythymidine or AZT) and an antisense against a component of human telomerase RNA (antisense hTR) were used to inhibit telomerase. FaDu and SKOV-3 cells showed comparable baseline telomerase activity. Telomerase activity in both cells was inhibited about equally by AZT (maximal inhibition of ∼80%) and by expression of antisense hTR (complete inhibition in SKOV-3 cells and maximal inhibition of ∼80% in FaDu cells). However, treatment with telomerase inhibitors resulted in ∼50% telomere shortening in FaDu cells but had no effect on SKOV-3 nor Saos-2 cells. SKOV-3 cells did not show the characteristic features of ALT (i.e. heterogeneous telomere length and promyelocytic leukemia bodies), whereas these ALT features were observed in Saos-2 cells. Collectively, these results suggest the existence of a telomerase-independent mechanism of telomere maintenance in the telomerase-positive SKOV-3 cells.

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.

Inhibition of human telomerase in MKN-45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest.

To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.

Expression of the RNA component of human telemorase (hTR) in ThinPrep preparations from bladder washings.

The enzyme telomerase is associated with cellular immortality and is expressed in the vast majority of human neoplasms. The expression of the RNA component of human telomerase (hTR) shows excellent concordance with enzyme activity. In this study, hTR expression was analyzed in a series of 18 perioperative bladder washings and compared with histologic diagnoses from material obtained in the same setting. The hTR expression analysis used an 35S-based in-situ hybridization assay. ThinPrep preparations fixed in PreservCyt solution (Cytyc Corporation, Boxborough, MA) were hybridized with sense and antisense hTR probes. A 1-4+ grading scheme was used, with appropriate positive and negative controls. Five of six (83%) lesions with benign histology had hTR expression that was 2+ or less in the exfoliated urothelial cells. In contrast, 11 of 12 (93%) lesions with malignant histology had an hTR expression that was focally 3+ or more, with 7 of 12 (58%) lesions having 4+ hTR expression in at least some urothelial clusters. Although increased hTR expression was present in smears with malignant urothelial cells, a similar trend was not seen with muscularis propria invasion or higher grades of TCC on subsequent histology. The use of in situ hybridization technique bypasses the need for stringent specimen processing and allows identification of the specific cell type that expresses telomerase. Cancer (Cancer Cytopathol)

Antisense Human Telomerase RNA Mediate d Inhibition of Telomere and Telomerase Activity in Human Gastric Cancer Cells

Objectivc To observe effects of antisense human telomerase RNA(hTR) on telomeric length (TRF) and telomerease activity of human gastric cancer cell SGC7901.Methods SGC7901 cell line was transfected with antisense hTR expression vector(pBBS-hTR) by lipofectamine,and expressions of hTR,telomere and telomerase activity in the gene transfected cells were detected by hybridization of nucleic acids and a telomeric repeat amplification protocol(TRAP).Results Clones containing antisense or control plasmids were selected 3 weeks after transfection by HyR selection and either antisense hTR expression had been enhanced or sense hTR expression had been inhibited 72h after transfection by specific stained RNA probe.Telomerase activity and mean TRF length were general low in the gene transfected cells in comparision with those in the control plasmid transfected cells.Conclusion Antisense hTR may either control TRF length by blocking the template region of hTR or serve as a effective target for treating gastric cancer by inhibiting telomerase.

Effect of Antisense Human Telomerase RNA Transfection on the Growth of Human Gastric Cancer Cell Lines

The majority of gastric cancers express high levels of human telomerase template RNA (hTR) that is essential for cellular survival. In this study, we examined whether antisense hTR (ahTR) had a growth inhibitory effect on three gastric cancer cell lines, MKN-1, MKN-28, and TMK-1, through transfection via an ahTR expression vector. Both the ahTR transfected MKN-1 and TMK-1 cells changed morphologically into multinucleate giant cells, and subsequently underwent cell death. Conversely, the ahTR transfected MKN-28 cells survived over 50 PDs in spite of telomere shortening. Surprisingly, high levels of telomerase activity were observed in the telomere-reduced cells. Furthermore, the expression of mRNAs for p21/Waf1/Cip1/Sdi1, IRF-1 and IFN inducible 6-16 was higher in the telomere-reduced cells than in the parental cells. These results suggest overall that the ahTR expression may bring about telomere shorting, leading to cell death or cellular senescence in gastric cancer cells.

The RNA component of human telomerase.

Eukaryotic chromosomes are capped with repetitive telomere sequences that protect the ends from damage and rearrangements. Telomere repeats are synthesized by telomerase, a ribonucleic acid (RNA)-protein complex. Here, the cloning of the RNA component of human telomerase, termed hTR, is described. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (TTAGGG)n. Germline tissues and tumor cell lines expressed more hTR than normal somatic cells and tissues, which have no detectable telomerase activity. Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity. HeLa cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings. Thus, human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells.

Telomere loss, telomerase, and cancer

AbstractTelomeric DNA safeguards chromosomes from genetic damage and rearrangement. Normal somatic cells lose telomeric DNA with each division and during normal human aging. Immortal cancer cells typically express the enzyme telomerase, a ribonucleoprotein polymerase that adds new telomere repeats onto the chromosome ends. To study the role of telomerase in cancer cells, the RNA component of human telomerase (hTR) was recently cloned. HeLa cells expressing antisense hTR exhibited reduced telomerase activity, gradual loss of telomeric DNA, and cell death after 23–26 doublings. These results demonstrate that telomerase is a critical enzyme for the continued proliferation of metastatic tumor cells. Whereas telomere loss occurs in both normal and precancerous cells, telomere shortening with age can lead to genetic damage, which may underlie the age‐related increases in cancer rates. © 1995 Wiley‐Liss, Inc.