Purpose: The aim of this study was to test the efficacy of telomestatin, an intramolecular G-quadruplex intercalating drug with specificity for telomeric sequences, as a potential therapeutic agent for multiple myeloma.
Experimental Design: We treated ARD, ARP, and MM1S myeloma cells with various concentrations of telomestatin for 7 days and evaluated for telomerase activity. Myeloma cells were treated with the minimal effective telomestatin concentration for 3–5 weeks. Every 7th day the fraction of live cells was determined by trypan blue exclusion, aliquots of cells were removed for various molecular assays, and the remaining cells were replated at the same cell number and at the same concentration of telomestatin. Telomere length, apoptosis, and gene expression changes were monitored as described in detail in “Materials and Methods.”
Results: Telomestatin treatment led to inhibition of telomerase activity, reduction in telomere length, and apoptotic cell death in ARD, MM1S, and ARP myeloma cells. Gene expression profile after 1 and 7 days of telomestatin treatment revealed ≥2-fold change in only 6 (0.027%) and 51 (0.23%) of 33,000 genes surveyed, respectively. No changes were seen in expression of genes involved in cell cycle, apoptosis, DNA repair, or recombination.
Conclusions: These results demonstrate that telomestatin exerts its antiproliferative and proapoptotic effects in myeloma cells via inhibition of telomerase and subsequent reduction in telomere length. We conclude that telomerase is an important potential therapeutic target for multiple myeloma therapy, and G-quadruplex interacting agents with specificity for binding to telomeric sequences can be important agents for additional evaluation.
Telomeres are DNA protein complexes at the ends of eukaryotic chromosomes that provide at least two essential functions, protecting of linear DNA ends from erosion over multiple replication cycles (1 , 2) , and protection of chromosome ends from recognition as double-strand DNA breaks and subsequent “repair” by exonucleolytic trimming and end-to-end fusion (3 , 4) . Telomeres also play a significant role in chromosomal replication during mitosis and meiosis (5) , as alignment of homologous chromosomes in meiosis is initiated by telomeres (6) . In all vertebrates, telomeric DNA consists of repeats of “TTAGGG” sequences (7, 8, 9) . As DNA replication mechanisms are unable to replicate telomeric sequences distal to the last primase site, approximately 50–100 bp of telomeric DNA is therefore lost with each cell division (10) . Telomerase, a ribonucleoprotein with reverse transcriptase activity, uses its RNA component as a template for the addition of G-rich repeats (TTAGGG) to the 3′ end of telomeric DNA (11) , thereby compensating for the telomere loss associated with cell division.
Telomerase is expressed in primitive human hematopoietic cells and germ-line cells, but is undetectable in most other somatic tissues (12) . In normal human diploid cells lacking telomerase, progressive telomere shortening leads to an early crisis in which short dysfunctional telomeres are recognized as DNA damage and may cause p53-mediated apoptosis. This may account for the limited proliferative potential of normal diploid cells (13) ; however, if p53 is mutated or lost, cells continue to divide and enter a late “crisis” with severe reduction in telomeres and chromosomal instability (14) .
Because telomerase activity is present in most immortal and neoplastic cells (15, 16, 17, 18) and is low or absent in somatic cells (12 , 15 , 19) , inhibitors of telomerase activity hold promise as antiproliferative agents with specificity for tumor cells, while sparing most or all normal tissues. We have demonstrated previously that introduction of peptide nucleic acid complementary to telomerase RNA, an exogenous telomerase inhibitor, results in loss of telomerase activity, telomere shortening, and cell death in transformed human cells (20) .
In this study we have examined the effects of telomestatin, a natural G-quadruplex-intercalating agent isolated from Streptomyces anulatus 3533-SV4, which specifically interacts and stabilizes intramolecular G-quadruplex structures, especially those formed by human telomeric DNA (TTAGGG)n (21) . It is a more potent and specific telomerase inhibitor than previously described G-quadruplex intercalating agents (21) . After treatment of multiple myeloma (MM) cells with telomestatin, we report inhibition of telomerase activity, reduction in telomere length, followed by cell growth inhibition, with minimal associated changes observed on gene microarray profiling.
MATERIALS AND METHODS
Telomestatin, isolated from Streptomyces anulatus3533-SV4, was dissolved in DMSO and used at 1–10 μm concentrations (21) .
Myeloma cell lines ARD and ARP were kindly provided by Dr. Joshua Epstein (University of Arkansas for Medical Sciences, Little Rock, AR). The MM1S cell line was provided by Dr. Steven Rosen (Northwestern University, Chicago, IL). All of the MM cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (HyClone, South Logan, UT).
MM cell lines (ARD, ARP, and MM1S) were treated with telomestatin at 1, 5, or 10 μm concentrations (in RPMI 1640 plus 10% fetal bovine serum) in a relatively dark room to reduce exposure of telomestatin to light. On day 7, cells were harvested and evaluated for telomerase activity to determine the minimal telomestatin concentration required for inhibition of >80% enzyme activity. This minimal effective telomestatin concentration was used in subsequent experiments. Constant numbers of cells were plated in multiple 100-mm dishes (5 × 105 cells/dish) and treated with telomestatin. The fraction of live cells was determined weekly by trypan blue exclusion, aliquots of cells were harvested for molecular assays, and the remaining cells were replated at the same cell number (5 × 105 cells/dish) and at the same concentration of the inhibitor. Cells aliquoted for telomere length assay were stored at −150°C, whereas those aliquoted for analyses of gene expression changes and apoptosis were processed immediately.
Assay of Telomerase Activity.
Telomerase activity was assayed using a fluorescence-based TRAPezeR XL telomerase detection kit (Intergen, Purchase, NY). Lysates (1000 cell-equivalents) were mixed with TRAPezeR XL reaction mix containing Amplifuor primers and incubated at 30°C for 30 min. Amplified telomerase products were quantitated with a fluorescence plate reader. Telomerase activity (in total product generated units) was calculated by comparing the ratio of telomerase products to an internal standard for each lysate, as described by the manufacturer.
Estimation of Telomere Length.
Genomic DNA was isolated from telomestatin-treated cells using “Puregene” DNA isolation kits (Gentra Systems, Minneapolis, MN), and telomere length was estimated using the TeloTAGGG Telomere Length Assay (Roche Diagnostics Corporation, Roche Applied Science, Indianapolis, IN). In brief, 6 μg of genomic DNA was digested with a 6-fold excess of restriction enzymes HinfI and RsaI. Digested DNA, along with a size standard (1-kb DNA ladder), was electrophoresed on 0.8% agarose gel and transferred to Hybond-n + nylon membrane (Amersham Biosciences Corp, Piscataway, NJ) as described by the manufacturer. Membranes were then hybridized to a digoxigenin-labeled telomere-specific probe, and telomeric DNA was detected by incubation with alkaline phosphatase coupled to antidigoxigenin antibody, followed by washing and reaction with a chromogenic substrate of alkaline phosphatase.
Apoptotic MM cells were detected using an Annexin V-Biotin Apoptosis Detection kit (Oncogene Research Products, San Diego, CA). Telomestatin-treated myeloma cells (1 × 106 cells/ml) were mixed with annexin V-biotin and medium-binding reagent, and incubated in the dark for 15 min at room temperature. Cells were then centrifuged and medium was replaced with 1 × Binding Buffer (Oncogene Research Products) containing FITC-streptavidin (Amersham). Propidium iodide was added to discriminate early apoptotic cells from late apoptotic or necrotic cells. A portion of cell suspension (50 μl) was placed onto a glass slide, covered with a coverslip, and viewed immediately using a fluorescence microscope equipped with FITC (green) and propidium iodide (red) filters. Two hundred cells, representing at least five distinct microscopic fields, were analyzed to assess the fraction of FITC and propidium iodide-labeled cells for each sample.
Gene Expression Analysis.
MM1S cells treated with 1 μm telomestatin were harvested on days 1 and 7, and total RNA was isolated using an RNeasy kit (Qiagen Inc., Valencia, CA) as described by the manufacturer. Total RNA (10–15 μg) was reverse transcribed to get cDNA using the Superscript II RT kit (Invitrogen Life Technologies, Inc., Carlsbad, CA). cDNA was used in an in vitro transcription reaction to synthesize biotin-labeled cRNA using ENZO RNA labeling kit (Enzo Diagnostics, Inc., Farmingdale, NY). Labeled cRNA was purified with the RNeasy Mini-kit (Qiagen Inc.) and quantitated. Purified cRNA (15 μg) was hybridized to Human Genome U133 (HG-U133) GeneChip arrays (Affymetrix) according to the manufacturer’s protocol. The HG-U133 set consists of two GeneChip arrays representing ∼33,000 human genes. GeneChip arrays were scanned on a GeneArray Scanner (Affymetrix, Inc., Santa Clara, CA).
Microarray Data Analysis.
Normalization of arrays and calculation of expression values was performed using the DNA-Chip Analyzer program (22) . Arrays were normalized based on relative signal produced for an invariant subset of genes. This model-based method was used for probe selection and computing expression values (22 , 23) . By pooling hybridization information across multiple arrays, it is possible to assess standard errors for the expression level indexes. This approach also allows automatic probe selection in the analysis stage to reduce errors due to cross-hybridizing probes and image contamination. We also used several high-level analysis functions in DNA-Chip Analyzer for comparative analysis and hierarchical clustering.
Inhibition of Telomerase Activity in MM Cells by Telomestatin.
Telomestatin is a relatively specific and more potent telomerase inhibitor than previously described G-quadruplex intercalators (21) . We evaluated the effect of telomestatin at 1, 5, or 10 μm for 7 days on telomerase activity in human MM cell lines, ARD, MM1S, and ARP. Telomestatin at 1 μm was sufficient to inhibit >90% of telomerase activity in ARD and MM1S cells (Fig. 1, A and B)⇓ . With their higher telomerase activity, however, ARP cells required 10 μm telomestatin for inhibition of >80% telomerase activity (Fig. 1C)⇓ . A time course (Fig. 1D)⇓ of telomestatin treatment (1 μm) in ARD MM cells showed no inhibition at 3 days, but similar levels of inhibition of telomerase activity on days 5 and 7.
Growth Inhibition After Telomestatin Treatment of MM Cells.
ARD, MM1S, and ARP cells were cultured in the presence or absence of telomestatin, and the viable cell fraction was determined by trypan blue exclusion weekly. Viable cell numbers declined over 3–5 weeks to 2–20% of the starting cell number. Treatment with telomestatin at 1 μm for 3 weeks resulted in cell death in 81% ± 6% ARD cells, 97% ± 3% MM1S cells, and 42% ± 2% ARP cells, respectively (Fig. 2, A–C)⇓ . Complete cell death was observed after exposure to telomestatin at 2 μm (ARD and MM1S cells) and 10 μm (ARP cells) concentrations (Fig. 2)⇓ .
The Nature of Cell Death After Telomerase Inhibition.
We analyzed MM cells for apoptosis after telomestatin treatment. After 2-week exposure to telomestatin, 61% of ARD cells, 90% of MM1S cells, and 69% of ARP cells stained positive for annexin V (Fig. 3B)⇓ , whereas untreated ARD, MM1S, and ARP cells were each <1% annexin V positive (Fig. 3A)⇓ .
Effect of Telomerase Inhibitors on Telomere Length.
Genomic DNA was isolated from MM cells treated with telomestatin for 2–3 weeks, and median (50th percentile) telomere length was estimated. Telomestatin treatment led to reduction in median telomere length by 1.6 kbp in ARD cells after 2 weeks (Fig. 4⇓ , Lane 1) but only by 0.4 or 0.5 kbp in MM1S cells after 2 or 3 weeks, respectively (Fig. 4⇓ , Lanes 2 and 3). In ARP cells, telomestatin treatment led to >3.1 kbp reduction in median telomere length (Fig. 4⇓ , Lane 4).
Gene Expression Profiles After Telomestatin Treatment.
We analyzed gene expression profiles of MM1S myeloma cells after 1 and 7 days of telomestatin treatment (Table 1)⇓ using HG-U133 GeneChip arrays (Affymetrix) representing ∼33,000 genes. However, no significant change was observed in the expression of genes involved in cell proliferation or cell cycle control, apoptosis, telomere maintenance, or DNA repair and recombination. Expression of nucleases and mismatch repair genes was also unaltered. Hypoxia-inducible factor 1-responsive gene (HIF-1), multiple endocrine neoplasia I, SEC63, and nuclear factor (erythroid-derived 2) -like 1 were among the genes with ≥2-fold up-regulation after both 1 and 7 days of exposure to telomestatin (Table 1)⇓ . Among those genes altered only after 7 days of treatment with telomestatin, the most noted were nuclear receptor corepressor (a transcriptional repressor), scaffold attachment factor B involved in binding of chromatin to the nuclear matrix or scaffold, RNA helicase-like protein involved in RNA processing and ribosome assembly, G2 and S phase expressed 1 (a cell cycle associated protein), and Yin Yang 1 associated protein (a transcription factor). Majority of the genes with changed expression were of unknown function and importance.
Telomerase activation and subsequent maintenance of telomeres are required for survival of proliferating tumor cells. Higher levels of telomerase activity are detected in MM cells compared with normal plasma cells.6 On the basis of earlier observations that oligonucleotides complementary to the RNA component of telomerase inhibit telomerase activity and limit proliferative potential of transformed human cell lines (20) , we have evaluated telomestatin, a novel telomerase inhibitor, as a potential therapeutic in MM. Telomestatin specifically interacts with and stabilizes intramolecular G-quadruplex structures (TTAGGG)n (21) . Inhibition of telomerase activity is mediated by binding of two telomestatin molecules to each intramolecular G-quadruplex structure, especially telomere-related G-quadruplex structures, making it an attractive agent, compared with the less specific G-quadruplex-intercalating agents such as TMPyP4 (24 , 25) .
The effective dose of telomestatin appears to be associated with the level of telomerase activity. MM cells with low levels of telomerase activity (ARD and MM1S) were almost completely inhibited by 1 μm telomestatin, whereas the cells with high telomerase activity (ARP) required 10 μm telomestatin for a similar level of inhibition. These results also parallel our observations with other G-quadruplex inhibitors (26) . Because telomeric structure is maintained by telomerase (27) , higher levels of the enzyme may preserve telomere structure even in the presence of low doses of telomestatin. Although the concentration of telomestatin required for inhibition of telomerase activity in vitro is 5 nm only (28) , the dose required in vivo is much higher and varies from cell line to cell line. Tauchi et al. (29) have observed that 1 μm telomestatin inhibited only ∼25% and 45% of telomerase activity at 48 h in leukemia cell lines K562 and OM9;22, respectively, whereas a dose of 10 μm telomestatin was required to observe a nearly complete inhibition of telomerase activity in these cell lines at 48 h. Because telomeric DNA does not exist as G-quadruplex under physiological salt and temperature conditions (30) , formation and stabilization of such a structure in vivo may depend on intracellular levels of appropriate cations, RecQ family of DNA helicases, and telomerase. Whereas certain cations promote formation of G-quadruplex structure, RecQ helicases unfold them into double-stranded DNA (31) . These factors, which differ among various cancer cell lines, may contribute to the higher concentration of telomestatin required for telomerase inhibition in vivo.
We determined the minimal concentration of telomestatin required by incubating the cells in the presence of various doses of drug for 7 days. At the minimal concentrations used, telomestatin and other G-quadruplex interactive agents including TMPyP4 (26) and QQ987 required 5–7 days for complete inhibition of telomerase activity in human cells. Because G-rich sequences do not usually exist as G-quadruplex structures (30) and G-quadruplexes may be adversely affected by intracellular levels of telomerase and RecQ helicases, the delayed inhibition of telomerase by telomestatin in myeloma cells may reflect the time required for conversion of all of the telomeric DNA into G-quadruplexes and their subsequent stabilization by minimal amount of telomestatin used.
In all three of the cell lines, telomestatin inhibition of telomerase activity resulted in >80% cell death after a lag period of 7–14 days. In contrast, prolonged treatment of MM cell lines with low concentration of telomestatin (0.01 μm) was not associated with cell death (data not shown). A lag phase of 7–14 days followed by abrupt cell death suggests that gradual telomere shortening leads to a critical telomere length threshold followed by apoptotic cell death.
Telomestatin-mediated growth arrest of MM cells is associated with a marked reduction in telomere length. Telomere length in MM1S was reduced by only 400 bp. Because median telomere length of MM1S cells is only 2.2 kbp, which is close to the putative minimum telomere length for survival of human cells (10) , the observed small reduction in telomeres can be a cell lethal event. The relative larger observed reduction in telomere length in ARP and ARD cells treated with telomestatin may both reflect their greater initial telomere length and a consequence of their higher proliferation rates. Effects of telomestatin on alternative mechanisms of telomere maintenance may also account for different levels of reduction in telomere length in these cell lines.
Although normal diploid cells (lacking telomerase) lose 50–100 bp of telomeric DNA per cell division, telomere loss after exogenous inhibition of telomerase in cancer and immortal cells does not seem to proceed with the usual rate, probably because of the existence of various genetic and epigenetic factors, including intracellular levels of nucleases, in these cells. Tauchi et al. (29) have also observed that telomestatin treatment of leukemia cells K562, with a heterogeneous population of telomeres and an average telomere length of 5.2 kbp, led to reduction in telomere length by ∼ 2200 bp after only 10 population doublings. Average telomere length reduction in K562 and OM9;22 cell lines at 20 population doublings was 2800 bp and 500 bp, respectively. Consistent with our data, these leukemia cell lines also showed a large variation in the reduction of telomere length after exposure to telomestatin.
A number of other G-rich regions in the human genome have the potential to form four-stranded G-quadruplex structures. These sequences are widely dispersed in the genome, especially in the promoter regions of many genes and at recombination hotspots (25 , 32) . Although telomestatin is an intramolecular G-quadruplex-interactive compound and has relatively high specificity for telomeric G-quadruplex structures (20) , it may affect expression of other genes, which can account for the antiproliferative activity of this agent. Therefore, we evaluated the effects of telomestatin treatment on the gene expression profile of MM cells using the HG-U133 gene array chips (Affymetrix) representing ∼33,000 genes. At the drug concentrations used, at least 2-fold change in expression were observed in only 6 genes after 1 day, and 51 genes after 7 days. None of these genes plays any significant role in DNA recombination, repair, cell cycle control, and apoptosis. Importantly, change in expression of telomerase or associated genes were not observed.
These data confirm that telomestatin induces apoptotic MM cell death predominantly through its effect on telomerase function, impairing its ability to extend telomeres.
Grant Support: Multiple Myeloma Research Foundation (to M. A. S.), the Department of Veterans Affairs (Merit Review Award and REAP; R. J. S. R.), The VA Merit Review Grant, the National Institutes of Health Grant P50-100707 and P01-78378 and Leukemia and Lymphoma Society Scholar in Translational Research Award (to N. C. M.); as well as National Institutes of Health Grants (R01-50947; P50-100707; and P01-78378), Doris Duke Distinguished Clinical Research Scientist Award, and the Cure for Myeloma Fund (to K. C. A.).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Nikhil C. Munshi, Dana-Farber Cancer Institute, 44 Binney Street, M557, Boston, MA 02115. E-mail: Nikhil [email protected] Phone: (617) 632-5607; Fax: (617) 632-4862; E-mail:
↵6 Unpublished observations.
↵7 M. A. Shammas, H. Koley, D. G. Beer, C. Li, R. K. Goyal, and N. C. Munshi. Growth arrest, apoptosis, and telomere shortening of Barrett’s associated adenocarcinoma cells by a telomerase inhibitor. Gastroenterology, in revision, 2003.
- Received May 19, 2003.
- Revision received October 3, 2003.
- Accepted October 7, 2003.