Inhibition of telomerase activity and human telomerase reverse transcriptase gene expression by histone deacetylase inhibitor in human brain cancer cells

The aim of the present study is to investigate the effect of histone deacetylase inhibitor, trichostatin A (TSA) on the cell growth, apoptosis, genomic DNA damage and the expression of telomerase and associated factors in human normal and brain cancer cells. Here, human normal un-transformed fibroblasts (MRC-5), human normal hTERT-immortalised fibroblasts (hTERT-BJ1) and human brain cancer cell lines (glioblastoma cell line, A-172 and medulloblastoma cell line, ONS-76) were treated with 0.5–3.0 μM TSA for 24 h. Exposure to TSA resulted in apoptosis in a dose-dependent manner in the brain cancer cells. Glioblastoma cell line (A-172) displayed higher sensitivity to TSA-induced cell killing effect and apoptosis than the medulloblastoma cell line (ONS-76). The brain cancer cell lines and hTERT-BJ1 cell line displayed significant inhibition in telomerase activity and hTERT mRNA level after 2 μM TSA treatment. Elevated expressions of p53 and p21 with a decrease in cyclin-D level supported the observation on cell cycle arrest following TSA treatment. Upregulation of Bax and cytochrome c correlated with the apoptotic events in TSA-treated cells. This study suggests that telomerase and hTERT might be the primary targets of TSA which may have the potential to be used as a telomerase inhibitor in cancer therapy.     

Inhibition of human telomerase by PNA-cationic peptide conjugates.

The inhibition of human telomerase has been explored using peptide conjugated derivatives of a PNA pentamer directed at the RNA template of telomerase. It is demonstrated that the presence of cationic peptides at the N-terminus of the PNA results in enhanced inhibition of telomerase activity. Furthermore, inhibition depended on the specificity of PNA recognition.     

Inhibition of telomerase in tumor cells by ribozyme targeting telomerase RNA component

Telomerase plays an important role in cell proliferation and carcinogenesis and is believed to be a good target for anti-cancer drugs. Elimination of template function of telomerase RNA may repress the telomerase activity. A hammer-headed ribozyme (telomerase ribozyme, teloRZ) directed against the RNA component of human telomerase (hTR) was designed and synthesized. TeloRZ showed a specific cleavage activity against the hTR. The cleavage efficacy reached 60%. A eukaryotic expression plasmid containing teloRZ gene was inducted into HeLa cells by lipofectamine, the telomerase activity in HeLa cells expressing teloRZ decreased to one eighth of that in the control cells. The doubling time increased significantly and the apoptosis ratio was elevated with increasing population doublings (PDS). After 19–20 PDS 95% cells were apoptotic. To further investigate the effect of teloRZ on tumor growth, the eukaryotic expression plasmid containing teloRZ was injected into transplanted tumor of nude mouse. The teloRZ effectively inhibited the telomerase activity in transplanted tumor, promoted apoptosis of the transplanted tumor cells, and decreased the tumor size significantly. These results indicate that teloRZ can effectively inhibit telomerase activity and growth of tumor cells, and suggest the potential use of this ribozyme in anti-cancer therapy.     

Inhibition of telomerase in tumor cells by ribozyme targeting telomerase RNA component

Telomerase plays an important role in cell proliferation and carcinogenesis and is believed to be a good target for anti-cancer drugs. Elimination of template function of telomerase RNA may repress the telomerase activity. A hammer-headed ribozyme (telomerase ribozyme, te-loRZ) directed against the RNA component of human telomerase (hTR) was designed and synthesized. TeloRZ showed a specific cleavage activity against the hTR. The cleavage efficacy reached 60%. A eukaryotic expression plasmid containing teloRZ gene was inducted into HeLa cells by lipofectamine, the telomerase activity in HeLa cells expressing teloRZ decreased to one eighth of that in the control cells. The doubling time increased significantly and the apoptosis ratio was elevated with increasing population doublings (PDS). After 19-20 PDS 95% cells were apoptotic. To further investigate the effect of teloRZ on tumor growth, the eukaryotic expression plasmid containing teloRZ was injected into transplanted tumor of nude mouse. The teloRZ e     

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.     

Inhibition of telomerase activity by a cell-penetrating peptide nucleic acid construct in human melanoma cells

We investigated the effect of two peptide nucleic acids (PNAs), which are complementary to the RNA component of human telomerase, on the catalytic activity of the enzyme. PNAs induced a dose-dependent reduction of telomerase activity in cell extracts from human melanoma cell lines and surgical specimens. To down-regulate telomerase in intact cells, we generated a chimeric molecule synthesized by coupling the 13-mer PNA to the Antennapedia peptide. The PNA construct induced a dose- and time-dependent inhibition of telomerase activity. However, a 20-day exposure to the PNA construct only caused a slight increase in melanoma cell doubling time and failed to induce any telomere shortening.     

Targeting human telomerase for cancer therapeutics

The enzyme telomerase is involved in the replication of telomeres, specialized structures that cap and protect the ends of chromosomes. Its activity is required for maintenance of telomeres and for unlimited lifespan, a hallmark of cancer cells. Telomerase is overexpressed in the vast majority of human cancer cells and therefore represents an attractive target for therapy. Several approaches have been developed to inhibit this enzyme through the targeting of its RNA or catalytic components as well as its DNA substrate, the single-stranded 3′-telomeric overhang. Telomerase inhibitors are chemically diverse and include modified oligonucleotides as well as small diffusable molecules, both natural and synthetic. This review presents an update of recent investigations pertaining to these agents and discusses their biological properties in the context of the initial paradigm that the exposure of cancer cells to these agents should lead to progressive telomere shortening followed by a delayed growth arrest response.     

A single nucleotide incorporation step limits human telomerase repeat addition activity

Human telomerase synthesizes telomeric DNA repeats (GGTTAG)_n onto chromosome ends using a short template from its integral telomerase RNA (hTR). However, telomerase is markedly slow for processive DNA synthesis among DNA polymerases. We report here that the unique template‐embedded pause signal restricts the first nucleotide incorporation for each repeat synthesized, imparting a significantly greater K_M. This slow nucleotide incorporation step drastically limits repeat addition processivity and rate under physiological conditions, which is alleviated with augmented concentrations of dGTP or dGDP, and not with dGMP nor other nucleotides. The activity stimulation by dGDP is due to nucleoside diphosphates functioning as substrates for telomerase. Converting the first nucleotide of the repeat synthesized from dG to dA through the telomerase template mutation, hTR‐51U, correspondingly shifts telomerase repeat addition activity stimulation to dATP‐dependent. In accordance, telomerase without the pause signal synthesizes DNA repeats with extremely high efficiency under low dGTP concentrations and lacks dGTP stimulation. Thus, the first nucleotide incorporation step of the telomerase catalytic cycle is a potential target for therapeutic enhancement of telomerase activity.     

Inhibition of plasminogen activators and the growth of cultured human tumour cells

1. Inhibition of a human fibroblast cell-surface proteinase with alpha-1-antitrypsin also inhibited cell proliferation, confirming earlier observations. 2. Although a similar proteinase inhibition could be observed with human tumour cells, the growth of these cells was unaffected. 3. Plasminogen activators on the cell surface and in culture media could be inhibited with specific antibodies, but there was no corresponding effect on the growth of any of the cell types tested.     

The influence of compound aITEL1296 on telomerase activity and growth of cancer cells

Telomerase is a ribonucleoprotein complex, which synthesizes telomeric repeats and which has been identified as a promising target for anticancer therapy. Here we have investigated the effect of a new compound aITEL1296 on telomerase activity. aITEL1296 effectively inhibited telomerase activity; its inhibitory activity was a bit higher (IC₅₀ = 0.19 ± 0.02 ng/mL) than that of BIBR1532, one of the most potent telomerase inhibitors known to date. In addition to telomerase inhibition aITEL1296 activated apoptotic mechanisms and effectively suppressed proliferation of tumor cell lines (GI₅₀ = 5.0 ± 0.2 ng/mL for most sensitive cell line LnCap) but not normal fibroblast cell line.     

Cellular and gene expression responses involved in the rapid growth inhibition of human cancer cells by RNA interference-mediated depletion of telomerase RNA

Inhibition of the up-regulated telomerase activity in cancer cells has previously been shown to slow cell growth but only after prior telomere shortening. Previously, we have reported that, unexpectedly, a hairpin short interfering RNA specifically targeting human telomerase RNA rapidly inhibits the growth of human cancer cells independently of p53 or telomere length and without bulk telomere shortening (Li, S., Rosenberg, J. E., Donjacour, A. A., Botchkina, I. L., Hom, Y. K., Cunha, G. R., and Blackburn, E. H. (2004) Cancer Res. 64, 4833-4840). Here we have demonstrated that such telomerase RNA knockdown in cancer cells does not cause telomere uncapping but rather induces changes in the global gene expression profile indicative of a novel response pathway, which includes suppression of specific genes implicated in angiogenesis and metastasis, and is distinct from the expression profile changes induced by telomere-uncapping mutant template telomerase RNAs. These cellular responses to depleting telomerase in human cancer cells together suggest that cancer cells are "telomerase-addicted" and uncover functions of telomerase in tumor growth and progression in addition to telomere maintenance.     

Suramin suppresses growth,alkaline-phosphatase and telomerase activity of human osteosarcoma cells in vitro

Neoadjuvant chemotherapy in osteosarcoma improves the survival dramatically, but there is currents drug resistance in about 25% of patients, leading researchers to investigate alternative therapy forms. Suramin has in the last two decades been used as salvage therapy in some cancers. This study was undertaken to investigate suramin as a possible salvage therapy in osteosarcoma. The effect of suramin on three human osteosarcoma cell lines (MG-63, HOS and SaOS-2) and three primary osteosarcoma cell lines isolated from biopsies was investigated. Suramin significantly inhibited cell proliferation, determined by 3H-thymidine incorporation, of osteosarcoma cells at a dose ranging from 250 to 500 microg/ml. Suramin decreased the secretion of alkaline-phosphatase after stimulation by 1,25-dihydroxy-Vitamin D(3) up to 50% and decreased telomerase activity by up to 40%. The data demonstrate that suramin has marked in vitro effects on human osteosarcoma cells supporting further clinical investigation.