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Telomerase: An Unusual Target for Cytotoxic Agents David R. Corey Departments of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390 Received April 17, 2000
Telomeres and the End Replication Problem. To prevent the loss of essential genes, linear chromosomes are capped by telomeres consisting of variable numbers of nucleotide repeats, TTAGGG in humans. Replication of linear chromosomes poses a special dilemma, termed the end replication problem, because DNA polymerase cannot fully replicate the extreme 3′ end of the chromosome during lagging strand synthesis. As a result, in the absence of mechanisms for maintaining telomere length, telomeres will steadily shorten at a rate of approximately 50-100 bases per cell division until erosion causes a growth arrest. Telomerase (1, 2) provides a solution to the end replication problem in germ cells, proliferative descendents of some stem cells, almost all immortal cell lines, and human tumors. Telomerase. Human telomerase consists of two core subunits, an RNA domain that acts as a template for replication (hTR) and a protein domain that catalyzes nucleotide polymerization (hTERT). The RNA component of human telomerase is 451 nucleotides long (3), and within this RNA, nucleotides 46-56 (CTAACCCTAAC) serve as a binding site for telomere ends and as a template for the addition of telomeric repeats. hTERT, the polymerase domain, is homologous to reverse transcriptase (4, 5) and mutations made at conserved residues within motifs conserved between hTERT and various reverse transcriptases abolish or reduce telomerase activity. hTERT and hTR can be expressed separately in vitro and reconstituted to yield high levels of telomerase activity (6), demonstrating that hTR and hTERT are both necessary and sufficient for function in vitro, although other protein or nucleic acid subunits are likely to influence telomere length regulation within cells. Ectopically expressed hTERT is sufficient to convert telomerase negative cells to telomerase positive cells, maintaining telomeres and functionally immortalizing cells (7). Telomerase and Cancer. Telomerase activity has been found in most types of human tumors, but not in most adjacent normal cells (8, 9). This correlation has led to the hypothesis that reactivation of telomerase is necessary for the sustained cell proliferation that characterizes cancer, and that telomerase is a promising target for a class of chemotherapeutic agents that act by a novel mechanism. Supporting this hypothesis is the observation that early stage neuroblastomas have low telomerase activity and this generally correlates with a favorable outcome, while the late stage disease exhibits high telomerase activity and a poor outcome (10). A similar correlation between telomerase activity and poor clinical outcome has been reported for ordinary menigioma (11), and other studies have suggested that telomerase activity is correlated with pathologic stage (1214) or tumor aggressiveness (13, 14).
Telomerase: Inhibition and Cell Proliferation. Studies of mice that lack the mouse RNA component (mTR) support the conclusion that telomerase is essential for sustained cell proliferation. These mice survived for six generations with few detectable phenotypic changes (15), but by the seventh generation, highly proliferative organ systems such as the testis, bone marrow, and spleen (16) appeared abnormal and the mice were no longer able to reproduce. Further studies of these mice revealed shortened life spans and an increased incidence of chromosomal abnormalities and spontaneous malignancies (17). The mice used in these experiments possessed much longer telomeres than those found in most cancerous cells, and inhibition of telomerase in human cancer would be expected to yield faster effects. Nevertheless, the long lag is a clear warning that development of telomerase inhibitors will offer unusual challenges during drug discovery. Potential for Toxic Effects from Telomerase Addition. The evidence described above supports the hypothesis that telomerase activity is essential for sustained tumor growth and that telomerase inhibitors will exert an antiproliferative effect. However, some immortal cell lines lack detectable telomerase activity, suggesting that anti-telomerase therapy may not always be effective (18). Furthermore, inhibitor resistance may arise through selection for cells that cannot take up inhibitors or which rapidly remove them, events that are often observed during use of other chemotherapeutic agents. In addition to problems that may arise from development of drug resistance, telomerase activity has been found in proliferative stem cells (T and B cells as examples), leading to the suggestion that inhibitors may cause serious side effects. However, cancer cells generally have shorter telomeres than stem cells and divide more rapidly, leading to the expectation that they might be exceptionally susceptible to telomerase inhibitors. Design of Molecules That Inhibit Telomerase. The controversy over the potential antiproliferative effects of telomerase inhibitors is a fascinating intellectual pursuit. However, no arguments can change the fact that the potential of telomerase as a target for clinical testing of drugs for humans will remain unknown until highly potent and selective inhibitors of human telomerase are discovered and rigorously tested. To confirm action through a telomerase-dependent mechanism, inhibitors of human telomerase should meet the following criteria: (i) addition of inhibitors selectively reduces telomerase activity; (ii) addition of inhibitors eventually leads to progressive shortening of telomeres; (iii) addition of inhibitors causes cell proliferation to decrease, and the time necessary to observe decreased proliferation varies with initial telomere length; and (iv) chemically related
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Figure 1. Chemical structures of oligonucleotide and peptide nucleic acid telomerase inhibitors.
molecules that do not inhibit telomerase do not cause decreased cell proliferation or telomere shortening. Studies of the results of inhibition by exogenously added inhibitors have produced contradictory results, ranging from immediate cell death, to decreased cell proliferation after a long delay, to no change in proliferation (19). Few of these studies have provided evidence for decreased telomere length. Advantages of Oligonucleotides as Telomerase Inhibitors. To resolve these contradictions and test the link between telomerase inhibition and decreased cell proliferation, we have developed oligonucleotides as telomerase inhibitors. Telomerase is an ideal target for oligonucleotides because its RNA template is essential for activity and is intrinsically accessible to binding by nucleic acids because of the need for the template to bind to the telomere. Oligonucleotides are being tested in 12 ongoing clinical trials, and one oligonucleotide, Fomivirsen, has recently been approved for the treatment of cytomegalovirus (CMV)1 retinitis. 2′-O-MeRNA and other 2′-O-alkyl-derivatized oligomers bind more tightly to complementary RNA sequences than do analogous DNA oligomers and have improved resistance to degradation by nucleases, reducing the need for phosphorothioate linkages and improving the selectivity of antisense effects (20). 2′-O-Alkyl-RNA is currently being used in two phase II clinical trials, including one trial directed against the R1-R subunit of protein kinase A, an application similar to use of telomerase inhibitors to prevent tumor regrowth. Recently, a phase III trial for antisense inhibition of ICAM-1 as a therapy for Crohn’s disease failed to yield positive evidence for efficacy. While this result was a setback for the development of antisense therapeutics, the trial did show that intravenous administration of oligonucleotide could be achieved without substantial toxic effects. Furthermore, the trial utilized a firstgeneration oligonucleotide chemistry, fully phosphorothioate-substituted DNA, whereas subsequent trials use more advanced chemistries chosen to maximize in vivo potency while minimizing the potential for nonsequenceselective toxicity. We have inhibited telomerase with peptide nucleic acid (PNA), 2′-O-meRNA, 2′-methoxyethoxy-RNA, and DNA 1Abbreviations: CMV, cytomegalovirus; TRF, terminal restriction fragment; PNAs, peptide nucleic acid oligomers.
Figure 2. Development cycle for inhibitors of human telomerase.
oligomers. The 2′-modified RNA and DNA oligomers have included varied degrees of phosphorothioate substitution to improve cellular stability and pharmacokinetic properties. Structures are shown in Figure 1. Our approach has been to follow an inhibitor development cycle that involves inhibitor design and synthesis and subsequent testing in cell extracts, cell cultures, and animals (Figure 2). Our progress through the first four stages of the cycle is summarized below and is described in detail in our published work (21, 22). Animal testing is ongoing. Given that chemically similar oligonucleotides are being used in clinical trials, phase I testing of telomerase inhibitors may be possible in the near term. End points for animal or human testing will include demonstration of delivery of the inhibitor to tumor cells, telomerase inhibition, telomere shortening, and decreased tumor growth. Effect on Cell Proliferation and Telomere Length of Inhibition by Oligonucleotides. We introduced 2′O-meRNA complementary to the template region of hTR into the immortalized human cell lines HME50-5E (breast epithelial) and DU145 (prostate tumor) (21). We also introduced 2′-O-meRNA into HME50-hTERT cells, a cell line generated by infecting preimmortal HME50 cells with the gene encoding the telomerase protein component (hTERT). A 2′-O-meRNA containing mismatched bases was employed as a control for the sequence specificity of inhibitor action. More than 95% of the telomerase activity 1 day after transfection and more
Forum: Mechanisms of Action of Cytotoxic Agents
than 70% of the activity was inhibited 3 days after transfection. To determine whether inhibition of telomerase would ultimately limit proliferation, we transfected HME50-5E, DU145, and HME50-hTERT cells with 2′-O-meRNA oligomers at 3-4 day intervals for 120 days. After 1525 days, proliferation of HME50-5E cells treated with the complementary oligomer began to slow, and after 110 days, no treated cells remained. The growth of cells treated with the control oligomer containing mismatch bases was not affected. To confirm that the effects of inhibitor addition were reproducible, treatment of HME505E cells with 2′-O-meRNA was repeated and a similar decrease in proliferation was observed. Addition of complementary 2′-O-meRNA to DU145 cells caused proliferation of cells to begin to slow after 60 days. By the end of the experiment (120 days), these cells had undergone 20 fewer population doublings than had cells treated with the mismatch-containing oligomer. HME50-5E cells have a mean terminal restriction fragment (TRF) length of 2000 base pairs, while DU145 cells have a mean length of 3600 base pairs. TRF length appears to correspond quantitatively to telomere length. Therefore, the finding that proliferation of treated HME505E cells decreases more dramatically than cell proliferation of treated DU145 cells may be due to HME50-5E cells possessing shorter telomeres. Consistent with this hypothesis, HME50-hTERT cells have a much longer mean TRF length, 7.6 kb, than either HME50-5E or DU145 cells, and we observed no significant reduction in their growth rates during the 120 day treatment period. We observed that the mean telomere length of all three cell lines was reduced by addition of complementary, but not noncomplementary, oligomers. Similar effects on cell proliferation and telomere length have also been observed upon treatment of cells with PNAs (22), reinforcing the conclusion that the effects we observe are due to interaction of oligomers with the RNA template of telomerase. Effects of Terminating Inhibitor Addition. An advantage of using synthetic inhibitors to reduce the level of gene expression is that the effects of regaining function can be evaluated by terminating inhibitor addition. After 76 days, we stopped adding inhibitor to DU145 cells that had been treated with complementary or mismatchcontaining oligomers. We noted that the eroded telomeres of cells treated with fully complementary oligomers had returned to approximately their initial lengths within 3 weeks. We also evaluated the effect of inhibitor withdrawal on proliferation of HME50-5E cells that had been treated with the fully complementary 2′-O-meRNA until population growth was static. We observed that, within 2 weeks of terminating inhibitor treatment, previously static HME50-5E cells regained the ability to grow at the same rate as cells treated with mismatch-containing 2′O-meRNA. Prospects for an Anti-Telomerase Strategy for Chemotherapy. Like any other drug, anti-telomerase therapeutics will need to combine efficacy with acceptable levels of toxicity. The oligonucleotide inhibitors that we have identified function by causing the steady erosion of telomeres. As a result, decreased cell proliferation does not occur immediately; instead, it may require weeks or months before an effect is observed. The length of treatment will depend on the initial telomere length of the tumor, which varies from tumor type to tumor type
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and from patient to patient, suggesting that it may be necessary to tailor anti-telomerase therapy to the telomere length of individual patients. To that end, one important area of future research will be to determine if the shortest telomere dictates the length of time required for therapy to be effective, or whether mean telomere length or some other factor is critical. The likelihood of a long lag phase before cell proliferation is decreased also suggests that anti-telomerase agents will not be an effective primary therapy for cancer; it is likely that the patient would be dead before telomeres had shortened to a critical point. Rather, to allow additional time for telomerase inhibitors to exert an effect, it is more likely that anti-telomerase agents will be used to treat minimal residual disease after initial chemotherapy or surgery. It is also likely that telomerase inhibitors will be used in combination with other antiproliferative agents. In this regard, a combination of telomerase inhibitors and antiangiogenesis agents is particularly attractive since antiangiogenesis agents would keep the tumor burden low while anti-telomerase agents would promote the death of residual tumor cells. One advantage conferred by the lag phase prior to decreased cell proliferation is that toxicity due to inhibition of telomerase in stem cells is likely to be low. In general, telomeres in stem cells are longer than telomeres in cancer cells, suggesting that the cancer cells will succumb to treatment first. This will be especially true if patients are chosen for possession of tumor cells containing short telomeres. Furthermore, our observation that telomeres regrow after inhibitor addition is stopped suggests that stem cells may recover after anti-telomerase therapy is discontinued. Examination of the available evidence suggests that anti-telomerase agents may be able to exert a therapeutic effect with minimal toxic effects, but that this outcome is far from certain. It is also conceivable that telomerase can be exploited as target for agents that promote immediate cell death. For example, application of agents that covalently link telomerase to the telomere or that cause telomere to incorporate unnatural nucleotides would be likely to effectively curb cell proliferation immediately. Development of such fast-acting agents represents an exciting area of research. However, while these agents may be more effective antiproliferative compounds than molecules that act by the proven mechanism of gradual telomere erosion, they will need to overcome the potential for toxic side effects due to destruction of stem cells and other telomerase-expressing proliferative tissues. It is also interesting to speculate that anti-telomerase agents might increase the susceptibility of cells to other anti-cancer agents before telomeres shorten to a critical length. Summary. Despite an impressive body of basic knowledge, the value of telomerase as a therapeutic target remains unknown. It is clear that development of antitelomerase agents will offer unusual challenges, but with cancer remaining a major cause of morbidity and mortality, these challenges are worth confronting. Oligonucleotides represent an excellent option for animal studies aimed at better characterizing the efficacy and toxicity of anti-telomerase agents. Favorable results in these studies would pave the way for clinical trials that will offer the only definitive means for accessing the telomerase-cancer connection.
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Acknowledgment. This work was supported by grants from the Robert A. Welch Foundation (Grant I-1244) and the National Institutes of Health (Grant CA74908).
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