Telomerase inhibition potentiates the effects of genotoxic agents in breast and colorectal cancer cells in a cell cycle-specific manner.

Previous studies have shown that telomerase facilitates DNA-damage repair and cell survival following stress. It is not clear how telomerase promotes DNA repair, or whether short-term telomerase inhibition, combined with genotoxic stress, can be exploited for cancer therapy. Here, we show that transient inhibition of telomerase activity by the specific inhibitor, GRN163L, increases the cytotoxicity of some, but not all, DNA-damaging agents. Such synergistic inhibition of growth requires the use of DNA-damaging agents that are toxic in the S/G(2) phase of the cell cycle. Notably, inhibition of Ataxia Telangiectasia Mutated (ATM) kinase, together with telomerase inhibition, synergistically increases the cytotoxicity induced by the G(2)-specific topoisomerase II inhibitor etoposide. By varying the timing of telomerase inhibition, relative to the timing of DNA damage, it is apparent that the prosurvival functions of telomerase occur at early stages of DNA damage recognition and repair. Our results suggest that the protective role of telomerase in cell cycle-restricted DNA damage repair could be exploited for combined anticancer chemotherapy.


Introduction
Telomerase is a specialized reverse transcriptase that maintains telomeres, nucleoprotein structures that cap the ends of linear chromosomes.Somatic inhibition of telomerase activity functions as a tumor suppression mechanism important in long-lived organisms, limiting indefinite proliferation and thus decreasing the likelihood of accumulating multiple mutations essential for carcinogenesis (1,2).Surveys of cancer-patient samples established that more than 85% of human tumors have high telomerase activity, primarily through the transcriptional up-regulation of the telomerase reverse transcriptase (hTERT) gene (3).Solid tumors reactivate telomerase in the late initiation phase of tumorigenesis (4).Accordingly, telomerase activity and in situ hTERT detection have been used as diagnostic and prognostic markers in tumor pathology studies (5)(6)(7).
The prevalence of telomerase activation in human cancers makes the targeting of telomerase an appealing therapeutic option.Current rationale for the use of telomerase inhibitors in anticancer therapies is based on the knowledge that these agents disrupt telomere length homeostasis in cancer cells, eventually leading to telomere depletion after a sufficient lag time (8).Previously, it was shown that faster cancer cell death resulted from combining other cytotoxic agents with telomerase inhibition, by chemical inhibitors or genetic manipulations such as RNA interference or expression of dominant-negative hTERT (9,10).These reports concluded that telomere attrition is essential for treatment efficacy (9), but this conclusion may have been influenced by the lag time required to genetically manipulate and generate stable cell lines to achieve telomerase inhibition.GRN163L is a thiophosphoamidate oligomer with 13 bases complementing the template region of telomerase RNA.GRN163L competitively inhibits the binding of the telomerase holoenzyme to its substrate (11,12).GRN163L-mediated telomerase inhibition has a synergistic effect when combined with breast cancer treatments fold, with a significant p-value at 0.005 (unpaired t-tests, GraphPad Prism. Figure 2A).The parallel shift of the dose-response curves suggests a conserved cytotoxic mechanism.Next, we measured the LD 50 of etoposide in the colorectal cancer cell line HT-29.Consistent with our observations in MCF-7 cells, the addition of GRN163L telomerase inhibition significantly increased the cytotoxicity of etoposide in HT-29 cells (Figure 2B).Adding telomerase inhibition reduced the lethal dose of etoposide by 2.5-fold in HT-29 colorectal cancer cells, with a significant p-value of 0.006.
To corroborate this observation, we combined telomerase inhibition with another cell-cycle dependent DNA-damaging agent.Irinotecan is converted to its active metabolite SN-38 by intracellular hydrolysis, resulting in the inhibition of topoisomerase I. Inhibition of topoisomerase I by these compounds results in the generation of DNA nicks in replication-and transcriptionactive cells (27).These single-stranded DNA nicks are converted into double-strand breaks by DNA replication machinery as they pass through the unrepaired loci during the DNA synthesis phase of the cell cycle (28).Co-administration of irinotecan with telomerase inhibitor GRN163L in breast cancer cell line MCF-7 resulted in an increased cytotoxicity of the DNA-damaging agent (Figure 2C).The addition of telomerase inhibitor reduced the lethal dose of irinotecan in MCF-7 cells by 2.1-fold, with a significant p-value at 0.02.A similar increase in the cytotoxicity Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Copyright © 2010 American Association for Cancer Research of irinotecan by the coadministration of telomerase inhibitor GRN163L was observed in the colorectal cell line HT-29 (Figure 2D).We observed a 1.7-fold increase in the cytotoxicity of irinotecan in HT-29 cells concurrently treated with the telomerase inhibitor GRN163L.This difference in LD50 is significant with a p-value of 0.02.Notably, the combination of irinotecan with GRN163L is less effective than etoposide with GRN163L.
These experiments indicate that telomerase activity conferred a survival advantage in DNAdamaged human cancer cells.Transient loss of telomerase activity induced by a specific enzyme inhibitor is sufficient to reverse this protection.Administration of telomerase inhibition at the time of DNA-damage induction resulted in synergistic increases of cytotoxicity.

TELOMERASE INHIBITION BY GRN163L HAS NO EFFECTS ON THE CYTOTOXICITY OF CELL CYCLE NON-SPECIFIC DNA-DAMAGING AGENTS
Telomerase activity in human cells is regulated in a manner that is specific to the cell cycle (19,29,30).Since both etoposide and irinotecan are cytotoxic at the S and G2 phases of the cell cycle, we next tested whether the synergy between telomerase inhibition and DNA-damaging agents was dependent on the cell cycle timing of the genotoxic agent action.
We treated MCF-7 breast cancer cells with the double-strand DNA-damaging agent bleomycin.
Bleomycin's cytotoxicity is attributed to its ability to cleave nucleic acids, leaving lesions that are difficult to repair (31).Bleomycin cytotoxicity is not cell-cycle dependent, although similar to other DNA-damaging agents, actively cycling cells exhibit increased sensitivity to bleomycin treatment.Unlike cell-cycle stage specific DNA-damaging agents, MCF-7 cells did not exhibit any increased sensitivity to bleomycin in the presence of telomerase inhibitor GRN163L (Figure 3A, p=0.90).In parallel, addition of GRN163L slightly increased the cytotoxicity of bleomycin in Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Copyright © 2010 American Association for Cancer Research associated proliferative immortality, we and others have proposed that high telomerase activity confers an additional growth advantage to cancer cells by promoting survival in the presence of cellular stresses (reviewed in (35) and ( 36)).A prior report, using short hairpin RNA knockdown of telomerase expression in primary fibroblasts ( 16), provided us with the impetus to inhibit this pro-survival function of telomerase in cancer cells as an additional anti-cancer therapeutic application for telomerase inhibition.Unlike long-term telomerase inhibition for the goal of telomere attrition, telomerase inhibition to reduce cell survival in conditions of DNA damage could be used as a short-term therapeutic application.
Short-termed, concurrent administration of the telomerase inhibitor GRN163L sensitized breast and colorectal cancer cells to the cytotoxic effects of cell-cycle stage specific DNA-damaging agents, but it showed minimal potentiation of other non-cell-cycle stage specific agents.It remains possible that GRN163L administration can contribute to the cytotoxic mechanism of most DNA-damaging agents, including the bleomycins and the platinum compounds under some conditions.However, the potentiation effect of concurrent GRN163L administration seems to be confined to the subpopulation of cells in S/G2 phase, thereby reducing the contribution of this mechanism's effect in the overall cytotoxicity of the non-cell cycle specific DNA-damaging agents.The small contribution of increased cytotoxicity in S/G2 cells may not be sufficient to increase cytotoxicity in the overall cell population.While off-target effects have been reported with therapeutic oligonucleotides (37), GRN163L treatment in telomerasenegative human cells, and mismatched control oligomer in cancer cells, produced minimal toxicities (38,39).Accordingly, we did not detect any significant loss of CFU growth in the three cancer cell models treated with short-term GRN163L, either alone or in combination with noncell cycle specific genotoxic agents.GRN163L is currently in multiple phase I and phase II clinical trials in breast cancer patients, to assess its efficacy and toxicity.(12). Research. on

Figure 1 .
Figure 1.GRN163L inhibition of telomerase activity in breast and colorectal cancer cells is dose dependent.TRAP activity profiles showing the inhibition of telomerase activities by GRN163L in (A) MCF-7, (B) HT29 and (C) LS180 cells.Addition of standard levels of internal control DNA to the TRAP activity assay obstructed detection of residual telomerase activity (data not shown).Smaller quantities of internal control DNA were used for these samples, to allow accurate detection of residual telomerase activity in GRN163L treated extracts.

Figure 2 .
Figure 2. Telomerase inhibition by GRN163L increases the cytotoxicity of the S/G2-specific DNA-damaging agents etoposide and irinotecan, in MCF-7 breast cancer and HT29 colorectal cancer cells.Dose-response curves show the loss of colony forming units in treated cells.Etoposide combination treatments were tested in (A) MCF-7 and (B) HT29 cells; irinotecan combination treatments were tested in (C) MCF-7 and (D) HT29 cells.E = etoposide, G = GRN163L, I = irinotecan.

Figure 3 .
Figure 3. Telomerase inhibition by GRN163L has no additional effect on the cytotoxicity of cellcycle non-specific DNA damaging agents bleomycin (A and B) and oxaliplatin (C and D), in MCF-7 (A and C) and HT29 (B and D) cells.None of the comparison groups showed a significant difference between the LD 50 s of the single agent and LD 50 s obtained from cells treated with GRN163L combinations.B = bleomycin, G = GRN163L, O = oxaliplatin.

Figure 4 .
Figure 4. Addition of ATM kinase inhibitor KU55933 further increased the cytotoxicity of etoposide and GRN163L combinations in (A) MCF-7 cells.To confirm that ATM inhibition by KU55933 was effective, we examined the stability of p53 following DNA damage induction.