Inhibition of human telomerase activity by peptide nucleic acids

We report the inhibition of human telomerase activity by peptide nucleic acids (PNAs). PNAs recognize the RNA component of human telomerase (hTR) and inhibit activity of the enzyme with IC50 values in the picomolar to nanomolar range. Inhibition depends on targeting exact functional boundaries of the hTR template and is 10- to 50-fold more efficient than inhibition by analogous phosphorothioate (PS) oligomers. In contrast to high selectivity of inhibition by PNAs, PS oligomers inhibit telomerase in a non-sequence-selective fashion. These results demonstrate that PNAs can control the enzymatic activity of ribonucleoproteins and possess important advantages relative to PS oligomers in both the affinity and the specificity of their recognition. These observations should facilitate the development of effective inhibitors of telomerase activity and affinity probes of telomerase structure.     

9-Hydroxyellipticine inhibits telomerase activity in human pancreatic cancer cells

There is increasing interest in identifying potent inhibitors of telomerase because the enzyme plays a crucial role in the development of cellular immortality and carcinogenesis. We hypothesized that 9-hydroxyellipticine (9-HE), an antitumor alkaloid, would inhibit telomerase activity because the drug has a unique mechanism of inhibiting phosphorylation of mutant p53 protein via inhibition of protein kinases, thereby restoring wild-type p53 function. This study was conducted to examine the effect of 9-HE on telomerase activity in human pancreatic cancer cells with differing p53 gene status. 9-HE treatment at relatively high concentrations resulted in rapid, complete inhibition of telomerase activity, irrespective of the p53 status. We conclude that 9-HE may exert a strong inhibitory effect on telomerase activity possibly through inhibition of protein kinases rather than through restoration of functional wild-type p53.     

Comparison of human telomerase RNA and telomerase activity in urine for diagnosis of bladder cancer

Currently the demand for transplant organs far outstrips the supply in the UK. This problem is even more severe for the Asian population, who have been shown to be disproportionately over-represented on transplant waiting lists in some regions of the UK. Several commentators have suggested that religious and cultural traditions may be the major determinant preventing Asians from donating organs. An exploratory qualitative study was undertaken with the aim of examining the influence of religious beliefs, amongst other things, on the extent and direction of public attitudes towards organ donation in a cross-section of the Asian population in Luton. This study indicates that, in the population studied, culture and religion play a much less prohibitive part in determining the level of organ donation than previously suggested. However, there is a desire to be aware of the religious stances so that people can make a more informed decision. The emphasis should clearly been a reconsideration of the presently inadequate approaches to organ procurement and on devising and supplementing these with more appropriate ones. An example of the failure to inform effectively the relevant populations about important developments is that only two of the 32 Muslims in the survey had heard of the 'fatwa' by the Muslim Legislative Council permitting organ donation.     

Induction of telomerase activity by UV-irradiation in Chinese hamster cells

Telomerase is a ribonucleoprotein whose activity has been detected in germline cells and in neoplastic and immortal cells. Telomerase compensates the telomere loss arising by the end replication problem by synthesizing telomeric repeats at the 3' end of the eukaryotic chromosomes. Telomerase is reactivated during cancer progression in human and mice. In order to determine whether the telomerase activity can be upregulated in vitro in response to DNA damaging agents, we examined the telomerase activity in five Chinese hamster cell lines following exposure to 5 J/m2 or 40 J/m2 UV-C radiation. All the cell lines tested showed an increase in telomerase activity in the PCR-based telomeric repeat amplification protocol (TRAP) in a dose dependent manner. This increase in telomerase activity correlated well with the number of cells being in the S and G2/M phase after UV exposure. However, in unirradiated control cells, similar levels of telomerase activity were observed in different phases of the cell cycle. Furthermore, telomeric signals were clustered in one or more parts of the disintegrating nuclear particles of the apoptotic cell as detected by fluorescence in situ hybridization (FISH). This is the first study to demonstrate the induction of telomerase activity following exposure to DNA-damaging agents like UV radiation in Chinese hamster cells in vitro.     

Inhibition of human telomerase by a retrovirus expressing telomeric antisense RNA

Human telomerase, the RNA-dependent DNA polymerase that adds TTAGGG repeats to chromosome ends, is selectively expressed in immortalised cells and most tumours, suggesting a potential role for telomerase inhibitors in cancer therapy. Replication-deficient retroviruses were used to determine whether mRNA containing UUAGGG, the complementary sequence to the template region of the hTR telomerase RNA, is sufficient to inhibit telomerase activity. Telomerase activities measured by the telomeric repeat amplification protocol (TRAP) assay in extracts prepared from immortalised mouse fibroblasts, human HeLa cells and human kidney carcinoma cells were inhibited by 75% or greater in 26 of 56 cell clones expressing UUAGGG. Telomerase activity was not inhibited by expression of mRNA containing a transposed sequence, GGGAUU. Telomerase activities in vivo were inferred from changes in cellular morphology, proliferation capacity, growth rate and measurement of the content of telomere DNA. Giant senescent-like cells emerged shortly after cloning mouse PA317 and human HeLa cells expressing UUAGGG. The fraction of giant cells varied from 100% at the fifth population doubling (PD) in one culture to 2-6% at 50 PD in several other cultures. Giant cells were absent in all parental cells and clones expressing GGGAUU. The average cellular content of telomere DNA was independent of telomerase activity over 50 PD. The results indicate that expression of RNA complementary to the template region of hTR is sufficient to inhibit telomerase in vitro and in vivo, but that the effect of inhibition on individual cells is highly variable.     

Expression of telomerase activity in human chorion

Telomerase activation is required for cellular immortalization and is found in most malignant tumors. Normal somatic cells are generally telomerase-negative, except for stem cells in renewing tissues. During pregnancy, human trophoblast continues to proliferate and acts as proliferating stem cells for the development of chorion and the formation of placenta. In the present study, a total of 105 chorions from placentas at various weeks of gestation were examined for telomerase activity using the telomeric repeat amplification protocol (TRAP) assay. Twenty-five of 33 (76%) normal early chorions at 5 to 9 weeks gestation were telomerase-positive. Chorions from early spontaneous abortions also exhibited telomerase activity but at a low level. In contrast, only 2 (4%) late chorions at 34 to 41 weeks gestation expressed telomerase activity. Significant telomerase activity was observed in trophoblast cell fractions of chorion, demonstrating trophoblast to be the source of the activity. Expression of human telomerase catalytic subunit (hTRT) was observed in early chorions, but not in late placenta, and there was a close correlation between telomerase activity and hTRT expression. In contrast, expression of human telomerase RNA component (hTR) was observed in both early and late chorions and was not liked to telomerase activity. These findings suggest that telomerase activity in chorion is critically regulated over the course of gestation, associated with hTRT expression. The findings of the present study also appear to support the emerging concept that normal somatic cells with stem cell-like characteristics can express telomerase activity.     

Inhibition of telomerase increases the susceptibility of human malignant glioblastoma cells to cisplatin-induced apoptosis

Malignant glioblastomas grow very rapidly and are generally resistant to either DNA-damaging drugs or gamma-irradiation. If tumor cells could be made more susceptible to cell death with treatments, this would clearly represent a significant improvement in the success of treatment. Recently, telomerase has become a focus of interest among oncologists as a target for treating cancer cells. Telomerase elongates telomeric DNA repeats (TTAGGG)n and is important in protecting and replicating DNA. The vast majority of tumor cells, indeed, express telomerase activity whereas normal somatic cells, except for a few cells, do not. Since telomerase is essential for protecting DNA, we may be able to make tumors more sensitive to treatments with DNA-damaging drugs by inhibiting telomerase activity. In this study, we used cis-diamminedichloroplatinum (cisplatin)-sensitive U87-MG cells and cisplatin-resistant U251-MG of human malignant glioblastoma cell lines. U87-MG cells did not express telomerase activity, whereas telomerase was highly detected in U251-MG cells. Interestingly, inhibition of telomerase with an antisense telomerase expression vector not only decreased telomerase activity but also increased susceptibility to cisplatin-induced apoptotic cell death in U251-MG cells. These findings suggest that treatment with antisense telomerase may represent a new chemosensitisation for tumors resistant to anticancer drugs.     

Inhibition of human caspases by peptide-based and macromolecular inhibitors

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes, with dissociation constants ranging from 75 pM to >10 microM. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order inactivation rates that range from 2.9 x 10(2) M-1 s-1 for caspase-2 to 2.8 x 10(5) M-1 s-1 for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (Ki < 20 nM) and selective inhibitor of Group I caspases (caspase-1, -4, and -5) and most Group III caspases (caspase-8, -9, and -10), suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response.     

Differential expression of telomerase activity in human cervical cancer and cervical intraepithelial neoplasia lesions

PURPOSE: Telomeres are tandem arrays of repeated DNA sequences located at the ends of eukaryotic chromosomes, and are synthesized by the enzyme telomerase. Loss of telomeric DNA may play an important role in the development of human cancers. However, very little is known about the status of telomerase during human cervical cancer development. PATIENTS AND METHODS: Telomerase activity was measured by telomere repeat amplification protocol (TRAP) assay in 24 cervical cancers, one carcinoma in situ (CIS), and 20 cervical intraepithelial neoplasia (CIN) lesions. Adjacent nontumor cervical tissue from the same 24 cervical cancer patients and normal cervical tissues from 11 control individuals also were examined for the presence of telomerase activity. RESULTS: Twenty two of the 24 (91.7%) cervical cancer specimens and the single CIS tissue were strongly positive for telomerase activity. Relatively weak but distinctive telomerase activity also was detectable in one of four CIN-I (25%), two of eight CIN-II (25%), and two of eight CIN-III (25%), respectively. However, telomerase activity was not found in the 24 corresponding nontumor cervical tissues from the same cervical cancer patients and the 11 normal cervical tissues from control individuals. CONCLUSION: The majority of cervical cancers contain strong telomerase activity. Significant proportions of noncancerous CIN tissues also contain telomerase activity, although weaker than that in cervical cancer. It seems that there is a progressive increase of telomerase activity in association with an increased degree of cervical malignancy. These results seem to suggest that the expression of telomerase may play a crucial role in cervical cancer carcinogenesis.     

Telomerase activity and metastasis: expansion of cells having higher telomerase activity within culture lines and tumor tissues

Tumor cells with metastatic potential may have a high telomerase activity that augments telomeric DNA repeats, allowing the cells to escape from the inhibition of cell proliferation due to shortened telomeres. We examined the expression level of telomerase activity using the telomeric repeat amplification protocol among a series of cell lines obtained by repeated transplantation of a mouse fibrosarcoma. The lines could be grouped into three; one has no metastatic potential, and the other two show metastatic abilities after intravenous or subcutaneous injection. Comparison of their telomerase activity indicated that more malignant lines had higher activity. A similar relation was seen in metastatic nodules formed through clonal expansion from the heterogeneous population of inoculated cells; clonality was monitored in terms of variable patterns of subtelomeric repeats. The results suggest that a high level of telomerase activity may not be requisite for metastasis, but may confer a propensity to dominate in a tumor tissue.     

Telomerase activity in human acute myelogenous leukemia: inhibition of telomerase activity by differentiation-inducing agents

The terminal regions of human chromosomes, the telomeres, shorten with each cell division in most normal somatic cells. Telomerase, a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends, is activated in germline cells and almost all tumor cells. Telomerase activity maintains the stability of telomere length, resulting in indefinite cellular proliferation (immortality). In the present study, telomerase activity was analyzed in leukemic mononuclear blood cells obtained from 56 patients with acute myelogenous leukemia (AML) with known cytogenetic alterations. Heterogenous levels of telomerase activity were observed and generally correlated with cytogenetic status. Patients with 11q abnormalities and -5/-7 (unfavorable cytogenetics) tended to have high telomerase activity compared with cells obtained from AML patients with other types of cytogenetics. Additional studies with a larger cohort of patients will determine whether these differences are statistically significant. Chemotherapy agents that result in differentiation of leukemic cells also resulted in inhibition of telomerase activity. Knowledge of telomerase activity in patients with AML, before and throughout therapy, may have clinical utility for following disease progression and may predict early cancer relapse.     

The direct effect of anticancer agents on the telomerase activity in human cancer cell lines

Telomerase, a ribonucleoprotein that maintains the ends of linear eukaryotic chromosomes, is expressed in the majority of malignant cells, while most normal somatic cells have no telomerase activity except germline and stem cells. Therefore, telomerase activity is considered one of important characteristics of tumors. In reviewing the possibility that conventional anticancer agents can partly perform their functions through modulation of telomerase. Several data including ours suggest that the down-modulation of telomerase activity along with inhibition by agents is the secondary event associated with cell death. Moreover, cells growth arrested at specific phase of cell cycle by agents still show the high level of telomerase activity. Since telomerase activity well correlates to the cell viability of treated cells, to study telomerase provides us a new implement to examine the sensitivity of cancer cells to agents.     

Extension of life-span by introduction of telomerase into normal human cells

Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.     

Attenuation of telomerase activity by a hammerhead ribozyme targeting the template region of telomerase RNA in endometrial carcinoma cells

Telomerase activity is found in almost all carcinoma cells but not in most somatic cells, suggesting that telomerase is an excellent target for cancer therapy. We designed hammerhead ribozymes against human telomerase RNA and studied their possible use as a tool for cancer therapy. Three ribozymes targeting the 3' end of the GUC sequence at 33-35 (the template region), 168-170, and 313-315 from the 5' end of telomerase RNA were designed. In a cell-free system, these three hammerhead ribozymes efficiently cleaved the RNA substrate. When these ribozyme RNAs were introduced into Ishikawa cells, which are endometrial carcinoma cells, only a ribozyme targeting the RNA template region could diminish the telomerase activity. Next we subcloned the ribozyme sequence into an expression vector and introduced this into AN3CA cells, which are endometrial carcinoma cells. The clones that were obtained showed reduced telomerase activity and telomerase RNA with expression of the ribozyme. These data suggest that the ribozyme against the RNA template region is a good tool to repress telomerase activity in cancer cells.     

Inhibition of human smooth muscle cell proliferation in culture by farnesyl pyrophosphate analogues, inhibitors of in vitro protein: farnesyl transferase

In this study, it was investigated whether and how inhibitors of protein:farnesyl transferase (PFT) can inhibit the proliferation of human smooth muscle cells (HSMC) in culture. Several farnesyl pyrophosphate (FPP) analogues were synthesized and tested in vitro for their specificity in inhibiting squalene synthase (SS), PFT, or protein:geranylgeranyl transferase-1 (PGGT-1) activities (the latter was determined using a newly designed assay). One of these compounds appeared to be a strong PFT inhibitor (IC50 value: 340 nM) and a weak inhibitor in the other two enzyme assays. This compound (designated as TR006) inhibited the farnesylation of Ras in a Ha-ras transfected cell line (Cohen et al., Biochem. Phamacol. 49: 839-845, 1995) and concomitantly slowed down the growth of these cells. Twenty-five microM of TR006 inhibited the proliferation of HSMC isolated from left internal mammary artery, as measured by counting the cells over a period of three cell cycles (10 days). A structurally related compound (TR007), a specific SS inhibitor, did not influence HSMC proliferation under the same conditions. The inhibition by TR006 was concentration-dependent. In HSMC, synchronized by serum depletion, platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF)-induced DNA synthesis was decreased by a 29-hr pretreatment with 100 microM of TR006, indicating that this inhibitor acted in an early phase of the cell cycle, probably by preventing protein isoprenylation. Some other FPP analogues with comparable IC50 values in the in vitro PFT assay were also able to decrease bFGF-induced DNA synthesis without affecting cell viability. A more negatively charged member of this group, TR018, did not influence the growth factor-induced DNA synthesis, probably due to an impaired uptake into the cells. However, the pivaloyloxomethyl derivative of this compound, which is uncharged, and is thought to be converted into TR018 within the cells, showed a strong decrease in bFGF-induced DNA synthesis in HSMC. These data suggest that the compounds investigated may be developed further for treatment of conditions in which undesirable proliferation of smooth muscle cells plays an important role.     

Differential inhibition of telomerase activity during induction of differentiation in hematopoietic, melanoma, and glioma cells in culture

Accumulating evidence indicates that telomerase activity is stringently repressed in normal human somatic cells but reactivated in cancers and immortal cells, suggesting that activation of telomerase activity may play a role in carcinogenesis and immortalization. Recently, down-regulation of telomerase activity by induction of differentiation has been reported for cells of pre-myelocytic and myelocytic leukemia as well as embryonic carcinoma. To gain further insight about the regulation of telomerase activity following induction of differentiation, telomerase activity was examined in a human hematopoietic progenitor cell line (D2), a melanoma cell line (CM73-36) and a glioma cell line (Ast812) before and after addition of differentiation inducing agents. The state of differentiation was assessed by growth inhibition and cell morphological maturation. Telomerase activity was assayed by a PCR-based telomeric repeat amplification protocol (TRAP). Our data show that telomerase activity was inhibited only in differentiation-induced D2 cells but not in differentiation-induced melanoma and glioma cells. A model for the differential inhibition of telomerase activity following induction of differentiation in different cancer cells will be presented.