Inhibition of human telomerase activity by antisense phosphorothioate oligonucleotides encapsulated with the transfection reagent, FuGENE6, in HeLa cells

Telomerase, a ribonucleoprotein, synthesizes telomeric repeats (TTAGGG) onto the ends of chromosomes to maintain the constant length of the telomere DNA, and its activity is detectable in approximately 85%-90% of primary human cancers. Thus, it is postulated that human telomerase might be associated with malignant tumor development and could be a highly selective target for antitumor drug design. Antisense phosphorothioate oligonucleotides (S-ODN) were investigated for their abilities to inhibit telomerase activity in the HeLa cell line. The S-ODN were designed to be complementary to nucleotides within the RNA active site of telomerase. As a transfection reagent, FuGENE6 (Boehringer Mannheim, Mannheim, Germany) was used to enhance the cellular uptake of the oligonucleotides in cell cultures. The S-ODN encapsulated with FuGENE6 clearly inhibited telomerase activity in HeLa cells and showed sequence-specific inhibition. The encapsulated S-ODN-3 with a 19-nucleotide, (nt) chain length had inhibitory effects similar to those of the 21-mer and 23-mer S-ODN sequences (S-ODN-4 and 5), but the 15-mer and 17-mer S-ODN sequences (S-ODN-1 and 2) failed to satisfactorily prevent telomerase activity. However, apoptotic HeLa cell death was not associated with telomerase inhibition. Furthermore, the encapsulated S-ODN did not appear to be cytotoxic in terms of the cell growth rate. The oligonucleotides encapsulated with the transfection reagent had enhanced cellular uptake, and cytoplasmic and nuclear localizations were observed. However, weak fluorescent signals were observed within the cytoplasms of HeLa cells treated with the free S-ODN-3. Thus, the activities of the S-ODN were effectively enhanced by using the transfection reagent. The transfection reagent, FuGENE6, may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes and is appropriate for use in vitro and in vivo.     

Protein kinase CK1 is a p53-threonine 18 kinase which requires prior phosphorylation of serine 15

Human telomerase might be associated with malignant tumor development and could be a highly selective target for antitumor drug design. Antisense phosphodiester (ODNs) and phosphorothioate (S-ODNs) oligonucleotides were investigated for their abilities to inhibit telomerase activity in the HeLa cell line. The ODNs and S-ODNs were designed to be complementary to nucleotides within the RNA active site of telomerase. As a transfection reagent, FuGENE6 was used to enhance the cellular uptake of oligonucleotides in cell cultures. The results showed that S-ODN-3 (19-mer) encapsulated with FuGENE6 clearly inhibited the telomerase activity in HeLa cells, and the inhibitory efficiency increased with an increase in the S-ODN-3. However, free S-ODN-3 showed no inhibitory activity. On the other hand, ODN-3 encapsulated with FuGENE6 had no detectable inhibitory activity. The encapsulated S-ODNs exhibited higher inhibitory activities than the free S-ODNs, and showed sequence specific inhibition. Thus, the activities of the S-ODNs were effectively enhanced by using the transfection reagent. The transfection reagent, FuGENE6, may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, and is appropriate for use in vitro and in vivo.     

Implications of high-affinity hybridization by locked nucleic acid oligomers for inhibition of human telomerase

Oligonucleotides that contain locked nucleic acid (LNA) bases have remarkably high affinity for complementary RNA and DNA sequences. This increased affinity may facilitate the recognition of nucleic acid targets inside cells and thus improve our ability to use synthetic oligonucleotides for controlling cellular processes. Here we test the hypothesis that LNAs offer advantages for inhibiting human telomerase, a ribonucleoprotein that is critical for tumor cell proliferation. We observe that LNAs complementary to the telomerase RNA template are potent and selective inhibitors of human telomerase. LNAs can be introduced into cultured tumor cells using cationic lipid, with diffuse uptake throughout the cell. Transfected LNAs effectively inhibited intracellular telomerase activity up to 40 h post-transfection. Shorter LNAs of eight bases in length are also effective inhibitors of human telomerase. The melting temperatures of these LNAs for complementary sequences are superior to those of analogous peptide nucleic acid oligomers, emphasizing the value of LNA bases for high-affinity recognition. These results demonstrate that high-affinity binding by LNAs can be exploited for superior recognition of an intracellular target.     

Cancer treatment by telomerase inhibitors: predictions by a kinetic model

The inhibition of telomerase activity in actively dividing cells leads to shortening of their telomeres and suppression of cell growth when the telomere lengths become smaller than a certain threshold value (typically about 1-2 kb of DNA). We evaluated the time (efficacy delay) required to reach the threshold telomeric DNA size after initiation of treatment, which is of critical importance for the efficacy of telomerase inhibitors. A model based on the solution of a system of differential equations was developed to analyze the efficacy delay and dynamics of tumor growth. The efficacy delay was strongly dependent on the size distribution of telomere lengths at the treatment initiation. An increase in the heterogeneity of telomere size resulted in shortening of the delay. However, the long-term dynamics of tumors with homogeneous populations of telomeres were more significantly affected by telomerase inhibitors compared to tumors with heterogeneous size distribution of telomeres. Size distribution of telomeres and tumor doubling times are of critical importance for the dynamics of tumor growth in presence of telomerase inhibitors.     

Utilization of ribonucleotides and RNA primers by Tetrahymena telomerase.

Telomerase is a ribonucleoprotein (RNP) DNA polymerase involved in telomere synthesis. A short sequence within the telomerase RNA component provides a template for de novo addition of the G-rich strand of a telomeric simple sequence repeat onto chromosome termini. In vitro, telomerase can elongate single-stranded DNA primers processively: one primer can be extended by multiple rounds of template copying before product dissociation. Telomerase will incorporate dNTPs or ddNTPs and will elongate any G-rich, single-stranded primer DNA. In this report, we show that Tetrahymena telomerase was able to incorporate a ribonucleotide, rGTP, into product polynucleotide. Synthesis of the product [d(TT)r(GGGG)]n was processive, suggesting that the chimeric product remained associated with the enzyme both at the active site and at a second, previously characterized, template-independent product binding site. As predicted by this finding, RNA-containing oligonucleotides served as primers for elongation. More than 3 nt of RNA at a primer 3' end decreased the quantity of product synthesis but increased the affinity of the primer for telomerase. Thus, RNA-containing primers were effective as competitive inhibitors of DNA primer elongation by telomerase. These results support the possible evolutionary origin of telomerase as an RNA-dependent RNA polymerase.     

Telomeric profiles and telomerase activity in turkey satellite cell clones with different in vitro growth characteristics

The satellite cell population in postnatal skeletal muscle is heterogeneous because individual satellite cells isolated from a single muscle have differing abilities to proliferate under the same in vitro conditions. Telomeres are structures found at the ends of all eukaryotic chromosomes that are characterized by repetitive DNA sequences, and they are important in determining cellular proliferation potential. The relationship between satellite cell proliferative heterogeneity and telomeric DNA was examined by digesting genomic DNA from large-colony-forming and small-colony-forming turkey satellite cell clones with HinfI, separating the restriction fragments on an agarose gel, and hybridizing the gels with an oligonucleotide probe specific for telomeric DNA. Turkey satellite cells generated telomeric restriction fragments up to approximately 180 kB. The large-colony-forming satellite cell clones had a larger proportion (P<0.05) of total telomeric restriction fragments below 33 kB than the small-colony-forming satellite cell clones. However, telomerase expression was detected in cultures from large-colony-forming and small-colony-forming turkey satellite cells suggesting that the differences in telomeric restriction fragments may not be related to the differences in in vitro proliferative behavior and that telomerase may contribute to the high in vitro growth capacity of turkey satellite cells.     

应用mTR-/-鼠模型系统研究肿瘤发生机制的进展

端粒酶是真核细胞体内的一种核糖核酸蛋白质复合体,是一种特殊的DNA聚合酶,具有延伸DNA末端的功能,能够维持端粒长度和功能,TERT具有反转录酶活性。在大多数体细胞和原代细胞中,端粒酶活性很低,通常检测不到,但在肿瘤细胞中,端粒酶则被广泛激活,因此认为端粒酶与肿瘤的发生具有密切的关系,本介绍了应用端粒酶阴性鼠研究端粒酶与G链悬垂和P53在肿瘤的发生过程中的相互关系。     

Real-time determination of telomerase activity in cell extracts using an optical biosensor

A biosensoric approach has been developed to determine the activity of telomerase in tumor cell lysates. An optical sensor, the grating coupler, was used to monitor the association and dissociation of unlabeled compounds on the sensor surface in real time, by virtue of an evanescent field. An oligonucleotide was immobilized on the surface of the optical biosensor and linked with two other oligonucleotides by complementary sequences in an overlapping manner. The 3'-end of the last one carried the sequence of the telomeric substrate (TS) primer used for elongation by telomerase in the telomeric repeat amplification protocol (TRAP) assay. This primer sequence was phosphorothioate (PS)-modified, which is known to strongly increase the affinity to the primer binding site of telomerase protein and consequently the velocity of the telomerase reaction. We show that the PS primer binds to the modified biosensor and is elongated effectively by the telomerase from HL-60 cell lysates. A synthesis rate of 1 nucleotide/min was determined. The inhibitory effect of peptide nucleic acid (PNA) was shown by using immobilized TS. The velocity of the telomerase reaction was slowed down and the signal intensity was below the signal-to-noise ratio. Most nucleic acid detection systems use amplification steps such as polymerase chain reaction (PCR) to increase the amount of the probe. Since telomerase is a polymerase itself amplification of DNA by PCR is not required. Furthermore, no purification steps were required since all measurements were performed with crude cell extract.     

Dynamics of telomere erosion in transformed and non-transformed avian cells in vitro

Although vertebrate telomeres are highly conserved, telomere dynamics and telomerase profiles vary among species. The objective of the present study was to examine telomerase activity and telomere length profiles of transformed and non-transformed avian cells in vitro. Non-transformed chicken embryo fibroblasts (CEFs) showed little or no telomerase activity from the earliest passages through senescence. Unexpectedly, a single culture of particularly long-lived senescent CEFs showed telomerase activity after over 250 days in culture. Transformed avian lines (six chicken, two quail and one turkey) and tumor samples (two chicken) exhibited telomerase activity. Telomere length profiles of non-transformed CEF cultures derived from individual embryos of an inbred line (UCD 003) exhibited cycles of shortening and lengthening with a substantial net loss of telomeric DNA by senescence. The telomere length profiles of several transformed cell lines resembled telomere length profiles of senescent CEFs in that they exhibited little of the typical smear of terminal restriction fragments (TRFs) suggesting that these transformed cells may possess a reduced amount of telomeric DNA. These results show that avian telomerase activity profiles are consistent with the telomerase activity profiles of human primary and transformed cells. Further, monitoring of telomere lengths of primary cells provides evidence for a dynamic series of changes over the lifespan of any specific cell culture ultimately resulting in net telomeric DNA loss by senescence.     

The La antigen associates with the human telomerase ribonucleoprotein and influences telomere length in vivo

La is an important component of ribonucleoprotein complexes and telomerase is a ribonucleoprotein that compensates for the shortening of the ends of linear DNA by adding telomeric repeats onto the ends of chromosomes by using an integral RNA as the template. We have identified a direct and specific interaction between La and the RNA component of human telomerase. Antibodies specific to La precipitate the human telomerase ribonucleoprotein complex derived from tumor cells, telomerase immortalized normal cells, and in vitro transformed cells. Overexpression of La in both experimentally immortalized human cells and prostate cancer cells results in gradual telomere shortening. Our results demonstrate that La can associate with telomerase and its expression level can influence telomere homeostasis in vivo.     

Cancer cell dynamics in presence of telomerase inhibitors: analysis of in vitro data

The inhibition of telomerase activity in actively dividing cells leads to suppression of cell growth after a time delay (inhibitory delay) required to reach a threshold telomeric DNA size. We developed a mathematical model of the dynamics of telomere size distribution and cell growth in the presence of telomere inhibitors that allowed quantification of the inhibitory delay. The model based on the solution of a system of differential equations described quantitatively recent experimental data on dynamics of cultured cells in presence of telomerase inhibitors. The analysis of the data by this model suggested the existence of at least two distinct subpopulations of cells with different proliferative activity. Size distribution of telomeres, fraction of proliferating cells, and tumor doubling times are of critical importance for the dynamics of cancer cells growth in presence of telomerase inhibitors. Rapidly growing cells with large telomeric DNA heterogeneity and small proliferating fractions as well as those with very short homogeneous telomeres would be the most sensitive to telomerase inhibitors.     

Pharmacological intervention strategies for affecting telomerase activity: future prospects to treat cancer and degenerative disease

Telomerase enzyme is a ribonucleoprotein maintaining the length of the telomeres by adding G-rich repeats to the end of the eukaryotic chromosomes. Normal human somatic cells, cultured in vitro, have a strictly limited proliferative potential undergoing senescence after about 50-70 population doublings. In contrast, most of the tumor cells have unlimited replicative potential. Although the mechanisms of immortalization are not understood completely at a genetic level, the key role of the telomere/telomerase system in the process is clear. The DNA replication machinery is not able to replicate fully the DNA at the very end of the chromosomes; therefore, about 50-200 nucleotides are lost during each of the replication cycles resulting in a gradual decrease of telomere length. Critically short telomere induces senescence, subsequent crisis and cell death. In tumor cells, however, the telomerase enzyme prevents the formation of critically short telomeres, adding GGTTAG repeats to the 3' end of the chromosomes immortalizing the cells. Immortality is one of the hallmarks of cancer. Besides the catalytic activity dependent telomere maintenance, catalytic activity-independent effects of telomerase may also be involved in the regulation of cell cycle. The telomere/telomerase system offers two possibilities to intervene the proliferative activity of the cell: (1) inhibition the telomere maintenance by inhibiting the telomerase activity; (2) activating the residual telomerase enzyme or inducing telomerase expression. Whilst the former approach could abolish the limitless replicative potential of malignant cells, the activation of telomerase might be utilized for treating degenerative diseases. Here, we review the current status of telomerase therapeutics, summarizing the activities of those pharmacological agents which either inhibit or activate the enzyme. We also discuss the future opportunities and challenges of research on pharmacological intervention of telomerase activity.     

The induction of growth arrest in fibroblasts by SV40 T antigen

DNA tumor viruses such as SV40, Ras and papillomaviruses are the most commonly used agents in immortalization of non-hematopoietic cells, but the results are quite different. Some of them even lead instead to a senescence-like state. To verify the potential of SV40 T antigen-mediated immortalization or properties and functions of it to regulate cell growth, human dermal fibroblasts were cultured and then transfected with eukaryotic expressing plasmid psv3-neo which containing SV40 T DNA. We found that expression of oncogenic SV40 T in human dermal fibroblasts resulted in growth, arrest, earlier than the occurrence of control cell senescence, although telomerase was positive and cells grew faster than control ones in early stage following transfection. These observations suggest that SV40 T antigen can activate growth arrest in human dermal fibroblasts under normal growth condition instead of always prolonging the lifespan of fibroblasts. Moreover, high rate of cell division in early stage after transfection may be associated with the expression of telomerase activity.     

BRACO19 analog dimers with improved inhibition of telomerase and hPot 1

Human chromosomes terminate with telomeres, which contain double-stranded G-rich, repetitive DNA followed by a single-stranded overhang of the G-rich sequence. Single-stranded oligonucleotides containing G-rich telomeric repeats have been observed in vitro to fold into a variety of G-quadruplex topologies depending on the solution conditions. G-quadruplex structures are notable in part because G-quadruplex ligands inhibit both the enzyme telomerase and other telomere-binding proteins. Because telomerase is required for growth by the majority of cancers, G-quadruplex-stabilizing ligands have become an attractive platform for anticancer drug discovery. Here, we present the preparation and biochemical activities of a novel series of 3,6-disubstituted acridine dimers modeled after the known G-quadruplex ligand BRACO19. These BRACO19 Analog Dimer (BAD) ligands were shown to bind to human telomeric DNA and promote the formation of intramolecular G-quadruplexes in the absence of monovalent cations. As expected, the BAD ligands bound to telomeric DNA with a 1:1 stoichiometry, whereas the parent compound BRACO19, a monomer, bound with a 2:1 stoichiometry. The BAD ligands exhibited potent inhibition of human telomerase with IC₅₀ values similar to or lower than those of BRACO19. Furthermore, the BAD ligands displayed greater potency in the inhibition of hPot1 and increased selectivity for G-quadruplex DNA when compared to BRACO19. Collectively, these experiments support the hypothesis that there is an increased potency and selectivity to be gained in the design of G-quadruplex-stabilizing agents that incorporate multiple interactions.