Abstract
Telomeres are specialized nucleoprotein complexes that protect against fusion and degradation of linear chromosomes. Critical shortening of telomeres leads to irreversible cessation of cell division, whereas telomerase elongates telomere sequences to compensate for losses that occur with each round of DNA replication. Continued proliferation of tumor cells requires this enzyme to maintain chromosomal stability and to counteract the cellular mitotic clock. In this study, we evaluated the effect of oligonucleotide N3′→P5′ thio-phosphoramidate (NP), which targets template RNA component, in human multiple myeloma (MM) cell lines and patient MM cells. Fluorescein staining at 24 h confirmed NP uptake in 84.7 and 86.1% of MM.1S cells and MM patient cells, respectively, without any transfection enhancer. High transfection efficiency was observed into both CD138+ and CD138− MM patient cells. Match NP (7S), but not mismatch NP (30S), inhibited telomerase activity in MM.1S cells, U266 cells, and RPMI 8226 cells, as well as in patient MM cells. Moreover, 7S inhibited cytokine-induced telomerase activity in MM.1S cells. 7S treatment-induced progressive telomere shortening was associated with growth inhibition and cell death in MM.1S cells with short telomeres (2.5 kb), but not in U266 cells with long telomeres (9.0 kb), at 56 days of culture. Progressive telomere shortening leading to growth inhibition and cell death in MM.1S cells was associated with up-regulation of p21 and phosphorylation of p53 (Ser-15). These studies, therefore, identify the molecular sequelae of NP oligonucleotide (GRN163) against human telomerase RNA component as a telomerase inhibitor and provide the rationale for the development of telomerase-targeted therapies to improve patient outcome in MM.
INTRODUCTION
Telomeres are specialized nucleoprotein complexes that protect against fusion and degradation of linear chromosomes (1) . Moreover, telomeres also regulate mitosis, because a critical shortening of telomeres leads to cessation of irreversible cell division (2) . Telomerase is a ribonucleoprotein DNA polymerase that elongates the telomeres of chromosomes to compensate for losses that occur with each round of DNA replication (3) , and continued proliferation of tumor cells requires this enzyme to maintain telomere stability and to counteract the cellular mitotic clock. Conversely, inhibition of telomerase by antisense oligonucleotides (peptide nucleic acid and 2′-O-methyl-RNA (2′-O-MeRNA); Ref. 4 ) and dominant-negative hTERT 3 constructs (5 , 6) leads to telomere shortening, growth arrest, and cell death in several human tumor cells.
MM is characterized by the expansion of monoclonal plasma cells in the bone marrow. MM remains incurable, despite the use of high-dose chemotherapy with autologous hematopoietic stem cell transplantation, and biologically based therapies are urgently needed. Activation of telomerase plays an important role in the evolution from monoclonal gammopathy of undetermined significance to MM (7) ; moreover, MM patients with high levels of telomerase activity have a poor prognosis (8) . We have shown that IGF-1 (9) and IL-6 (10, 11, 12) , known MM growth and survival factors, activate telomerase via the phosphatidylinositol 3-kinase/Akt/nuclear factor-κB signaling cascade (13) . Telomerase may, therefore, be a novel therapeutic target in MM.
Recently, Herbert et al. (14) have reported that NP targeting hTR inhibits telomerase activity in human breast epithelial HME50-5E cells. NP oligonucleotides form stable duplexes with single-stranded RNA, are resistant to nuclease degradation, and have both a high affinity and specificity for DNA and RNA targets, as well as a relatively low affinity for proteins (15) . In MM, however, the effect of NP on telomerase activity and telomere length, as well as the molecular mechanism whereby it modulates tumor cell growth, is undefined.
In this study, we demonstrate that NP (oligonucleotide GRN163, abbreviated as 7S throughout this article) targeting hTR inhibits telomerase activity in MM cell lines (MM.1S, U266, and RPMI 8226) as well as in MM patient cells, without any transfection enhancer. NP induces progressive telomere shortening in both MM.1S cells and U266 cells. However, progressive telomere shortening was associated with growth inhibition and cell death in MM.1S cells with short telomeres (2.5 kb), but not in U266 cells with long telomeres (9.0 kb), at 56 days of culture. Comprehensive and sequential gene expression analysis of 7S-treated MM.1S cells revealed modulation of several genes encoding for cell cycle-regulating proteins, including p21, MAD2, replication factor C, Cdc27, and cyclin E binding protein. Moreover, we confirmed increased phosphorylated p53 (Ser-15) and p21 protein expression in 7S-treated MM.1S cells. These studies, therefore, identify molecular sequelae of NP oligonucleotide against hTR as a telomerase inhibitor and provide the rationale for the development of telomerase-targeted therapies to improve patient outcome in MM.
MATERIALS AND METHODS
Cell Lines and Patient Cells.
Human MM cell lines U266 and RPMI 8226 were obtained from American Type Culture Collection (Rockville, MD). MM.1S cells were kindly provided by Dr. Steven Rosen (Northwestern University, Chicago, IL). Patient MM cells were purified from bone marrow samples by Ficoll-Hypaque density gradient centrifugation. MM cells (CD138+, >95.0%) were isolated using CD138 (Syndecan-1) Micro Beads and the auto MACS magnetic cell sorter, according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Informed consent was obtained from patients on an Institutional Review Board-approved protocol and in accordance with the Helsinki protocol. Cells were cultured in RPMI-1640 (Mediatech, Herndon, VA) with 10% fetal bovine serum (Harlan, Indianapolis, IN), containing 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Inc., Grand Island, NY).
Treatment of Cells.
NP targeting hTR (14 , 15) was obtained from Geron Co. (Menlo Park, CA). 7S (GRN163) is a 13-mer 2′-deoxyoligonucleotide N3′→P5′ thio-phosphoramidate complementary to hTR. 30S (GRN137227), a mismatch 13-mer oligonucleotide, was used as a negative control. In long-term MM cell cultures, NP-containing medium was replaced every 4 days. Viable cells were enumerated by trypan blue dye exclusion.
Analysis of Telomere Length.
Analysis of telomere length was performed by Southern blotting, as described previously (16) . Briefly, high molecular weight DNA was obtained by standard phenol/chloroform extraction. Then, 6 μg of HinfI/RsaI-digested DNA was size-fractionated by 0.8% agarose gel electrophoresis. After electrophoresis, the size-separated DNA was transferred to a Hybond N filter (Amersham Pharmacia Biotech, Piscataway, NJ) and cross-linked with the filter by exposure to UV light. Filters were hybridized with digoxigenin-labeled telomeric probe (TTAGGG)4, according to the manufacturer’s instructions (Boehringer Mannheim, Indianapolis, IN). Hybridization was carried out at 68°C for 12 h in the hybridization solution: 5 × SSC, 0.02% SDS, 0.1% sodium lauroylsarcosine, and 1% blocking reagent (Boehringer Mannheim). The filter was washed twice at 50°C in the washing solution (0.1× SSC, 0.1% SDS) and then rinsed in the recommended blocking reagent. The hybridized probe was detected by the chemiluminescence method, according to the manufacturer’s recommendations (Roche Molecular Biochemicals, Indianapolis, IN). Filters were exposed with BioMax film (Eastman Kodak, Rochester, NY). The films were photographed using a digital camera (Alpha Innotech Corporation, San Leandro, CA), and the average telomere length was analyzed using NIH image software. The strongest density peak was the mean telomere length.
Assessment of NP Transfection in MM Cells.
The uptake efficiency of 1 μm fluorescein-labeled NP at 12 and 24 h, without any transfection enhancer, was analyzed using flow cytometry in MM.1S cells and MM patient cells. Intracellular localization of fluorescein-labeled NP was analyzed using fluorescent microscopy (Zeiss, Oberkochen, Germany).
Analysis of Cell Cycle.
MM.1S cells (1 × 106) cultured with 7S or 30S were harvested, washed with PBS, fixed with 70% ethanol, and pretreated with 10 μg/ml of RNase (Sigma). The cell cycle profile was determined using PI staining and flow cytometry (Coulter Immunology, Hialeah, FL).
Analysis of Cell Viability.
Analysis of cell viability was performed by flow cytometry (Coulter Immunology) using Annexin-V-FLUOS staining kit (Boehringer Mannheim), according to the manufacturer’s instruction. Experiments were performed three times to determine the mean ± SD percentage of cell death.
Telomerase Assay.
The telomerase assay was performed using a TRAPEZE Telomerase Detection kit (Intergen, Purchase, NY). PCR amplification was performed with 30 cycles at 94°C for 30 s, at 58°C for 30 s, and at 72°C for 60 s. The PCR products were analyzed by electrophoresis on 12% polyacrylamide nondenaturating gels and stained with SYBR Green I (Molecular Probes, Eugene, OR). Telomerase activity was assessed by determining the ratio of the entire telomerase ladder to that of the internal control, using NIH image analysis software.
Cytoplasmic and Nuclear Fractionation.
The preparation of cytoplasmic and nuclear extracts was performed using the Nuclear Extract kit (Active Motif, Carlsbad, CA).
RNA Isolation and RT-PCR.
Total RNA was isolated using an Isogen RNA extraction kit (Nippongene, Toyama, Japan). The first-strand cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Life Technologies). Briefly, 4 μg of total RNA were transcribed using a random hexamer primer (Amersham Biosciences) in a reaction mixture with a total volume of 30 μl. PCR was done using 1.0 μl of first-strand cDNA. The reaction sequence was at 94°C for 30 s, at 60°C for 30 s, and at 72°C for 60 s (30 cycles for hTERT, 22 cycles for c-myc, and 21 cycles for β-actin). The primer sequences were as follows: for hTERT, (sense) 5′-TGAACTTGCGGAAGACAGTGG-3′ and (antisense) 5′-ATGCGTGAAACCTGTACGCCT-3′; for c-myc, (sense) 5′-AAGACTCCAGCGCCTTCTCTC-3′ and (antisense) 5′-GTTTTCCAACTCCGGGATCTG-3′; and for β-actin, (sense) 5′-GTGGGGCGCCCCAGGCACCA-3′ and (antisense) 5′-CTCCTTAATGTCACGCACGATTTC-3′.
Preparation of Biotinylated Probes and Hybridization on Microarrays.
Human Genome U133 arrays (HG-U133; Affymetrix, Santa Clara, CA) were used for mRNA expression profiling. Biotinylated RNA was synthesized using the Bioarray RNA transcript labeling kit (Enzo, Farmingdale, NY) with biotin-11-CTP and biotin-16-UTP for 5 h at 37°C. In vivo transcription products were purified using RNeasy columns (Qiagen, Valenica, CA). Biotinylated RNA was treated for 35 min at 94°C in a buffer containing 200 mm Tris acetate (pH 8.1), 500 mm potassium acetate, and 150 mm magnesium acetate. HG-U133 arrays were hybridized with biotinylated in vivo transcription products (15 μg/Chip) according to the manufacturer’s protocol. Fluidic station 400 (Affymetrix) was used for washing and staining the arrays. A three-step protocol was used to enhance detection of the hybridized biotinylated RNA: incubation with streptavidin-phycoerythrin conjugate, labeling with anti-streptavidin goat biotinylated Ab (Vector Laboratories, Burlingame, CA), and staining again with streptavidin-phycoerythrin conjugate. DNA chips were analyzed using the Gene Array Scanner (Affymetrix). Digitized image data were processed using version 4.0 GeneChip software (Affymetrix), and image data files were processed using Microarray Suite v4.0 (Affymetrix).
Immunoblotting.
Cells were harvested, washed with ice-cold PBS, and lysed with buffer containing 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1% NP40, 2 mm sodium orthovanadate, and protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN). An equal amount (50 μg) of the samples was separated on SDS-polyacrylamide gel and then transferred onto nitrocellulose filters (Bio-Rad, Hercules, CA). The membranes were immunoblotted with Abs against hTERT (Santa Cruz Biotechnology), phospho-p53 (Ser-15; Cell Signaling Technology, Beverly, MA), p53 (Santa Cruz Biotechnology), p27 (Santa Cruz Biotechnology), p21 (Santa Cruz Biotechnology), p16 (BD PharMingen, San Diego, CA), nucleolin (Santa Cruz Biotechnology), and α-tubulin (Sigma Chemical). The immunoblots were detected by ECL chemiluminescence (Pharmacia, Uppsala, Sweden).
Statistical Analysis.
A paired t test was performed by using Stat View4.5 software (Abacus Concept, Inc., Berkeley, CA). The minimal level of significance was P < 0.05.
RESULTS
Evaluation of NP Uptake in a MM Cell Line and MM Patient Cells.
We first used flow cytometry to evaluate the uptake of 1 μm fluorescein-labeled NP, without any transfection enhancer, in MM.1S cells and MM patient cells. Uptake of fluorescein-labeled NP at 12 and 24 h was 79.5 and 84.7% in MM.1S cells and 74.9 and 86.1% in MM patient 1 cells (CD138+), respectively (Fig. 1A) ⇓ . These high transfection rates were maintained for 4 days (data not shown). Moreover, we observed uptake of fluorescein-labeled NP at 24 h in both CD138+ and CD138− cells of MM patients 2 and 3 (Fig. 1B) ⇓ . Uptake of fluorescein-labeled NP at 24 h was 70.2 and 66.5% in CD138+ and CD138− cells of MM patient 2 and 99.3 and 96.5% in CD138+ and CD138− cells of MM patient 3, respectively. We next studied the intracellular localization of fluorescein-labeled NP using fluorescent microscopy. We demonstrated that fluorescein-labeled NP is present in both the cytoplasm and nucleus in MM.1S cells (Fig. 1C) ⇓ .
Expression of transfected fluorescein-labeled NP in MM cells. A, the uptake of 1 μm fluorescein-labeled NP, in the absence of transfection enhancer, into MM.1S cells and MM patient 1 (CD138+) cells at 12 and 24 h were analyzed by flow cytometry. B, the uptake of 1 μm fluorescein-labeled NP, in the absence of transfection enhancer, into CD138+ cells and CD138− cells of MM patients 2 and 3 at 24 h were analyzed by flow cytometry. C, the intracellular localization of fluorescein-labeled NP was analyzed using fluorescent microscopy.
Effect of NP on Telomerase Activity in MM Cell Lines and MM Patient Cells.
We next studied the effect of NP at various concentrations on telomerase activity of MM cell lines (MM.1S, RPMI 8226, and U266), as well as MM patient cells. We found that 0.1–10 μm 7S (match) inhibited telomerase activity in MM.1S cells at 24 h (NP concentration that inhibited 50% of the telomerase activity in the control (IC50) of 0.075 μm), whereas 1–10 μm 30S (mismatch control) did not (Fig. 2A) ⇓ . Consistent with Fig. 1C ⇓ , 1 μm 7S inhibits telomerase activity in both the cytoplasmic and nuclear fractions in MM.1S cells (Fig. 2B) ⇓ . We have reported recently that IL-6 and IGF-1 induce phosphatidylinositol 3-kinase/Akt activation, phosphorylation of hTERT, and increased telomerase activity in MM cells (13) . Importantly, 7S but not 30S inhibited cytokine-induced telomerase activity (Fig. 2C) ⇓ . Moreover, telomerase activity at 24 h was also inhibited by 7S in RPMI-8226 cells (IC50, 0.70 μm) and U266 cells (IC50:, 0.027 μm) but not by 30S (Fig. 2, D and E) ⇓ . Having defined these effects of NP on MM cell lines, we next examined freshly isolated patient MM cells. As can be seen in Fig. 2F ⇓ , telomerase activity in MM patients 1 and 4 decreased in 24-h cultures with 7S but not with 30S. Because NP directly interacts with hTR, inhibition of telomerase activity by NP was not associated with down-regulation of either hTERT or c-myc mRNA, a positive regulator of hTERT transcription (17) , in MM.1S, RPMI 8226, and U266 cells (Fig. 2G) ⇓ .
Effect of NP targeting hTR on telomerase activity in MM cells. A, MM.1S cells were cultured with various concentrations of match (7S) NP and mismatch (30S) for 24 h. B, MM.1S cells cultured with or without 1 μm 7S for 24 h, and lysates of cytoplasmic (C) and nuclear fractions (N) were prepared. C, MM.1S cells were cultured with 1 μm 7S and or 30S in the presence or absence of IL-6 and IGF-1 for 24 h. D, RPMI8226 cells were cultured with various concentrations of 7S or 30S NP for 24 h. E, U266 cells were cultured with various concentrations of 7S or 30S NP for 24 h. F, freshly isolated MM cells from two MM patients were cultured with and without NP for 24 h. Telomerase activity was assayed by TRAPeze Telomerase Detection kit. G, MM.1S, RPMI8226, and U266 cells were cultured with 7S NP and 30S for 24 h. hTERT, c-myc and β-actin were analyzed by RT-PCR.
Effect of Long-Term Culture with NP on Telomere Length and Telomerase Activity in MM.1S Cells and U266 Cells.
We next evaluated the effect of NP on telomere length in MM.1S cells and U266 cells. Treatment with 7S (1 μm), but not with 30S, reduced telomere length in MM.1S cells and U266 cells (Fig. 3, A and B) ⇓ . The mean telomere lengths of 7S-treated MM.1S cells were as follows: day 0, 2.5 kb; day 14, 2.1 kb; day 28, 1.8 kb; day 35, 1.8 kb; day 42, 1.8 kb; and day 49, 1.7 kb. The mean telomere lengths of 7S-treated U266 cells were as follows: day 0, 9.0 kb; day 14, 8.1 kb; day 28, 7.8 kb; and day 49, 7.0 kb. We confirmed that telomerase activity was also inhibited at these intervals in 7S-treated, but not 30S-treated, MM.1S cells and U266 cells, without associated changes in expression of hTERT mRNA and protein (Fig. 3C) ⇓ .
Effect of long-term culture with NP on telomere length and telomerase activity in MM.1S cells and U266 cells. A, MM.1S cells and U266 cells were cultured with 7S (1 μm) or 30S (1 μm), and telomere length was analyzed by Southern blotting. B, The strongest density peak detected by NIH image software was the mean telomere length. C, telomerase activity was analyzed by TRAP assay. The expression of hTERT and β-actin mRNA was analyzed by RT-PCR. Expression of hTERT and α-tubulin protein was analyzed by immunoblotting with Abs against hTERT and α-tubulin.
Effect of NP on Cell Growth, Cell Cycle, and Cell Viability in MM.1S Cells and U266 Cells.
Culture with 7S inhibited the growth of MM.1S cells with initially short telomeres (2.5 kb) after day 28, whereas 30S did not (Fig. 4A) ⇓ . We found that growth inhibition induced by 7S was associated with increased sub-G1 and G1, with decreased S and G2-M, cells at day 49; these changes were not observed in cultures with 30S (Fig. 4B) ⇓ . We next evaluated the effect of NP on cell viability by flow cytometry using Annexin-V-fluorescein and PI staining. Cell death (Annexin-V+PI+ and Annexin-V+PI−) in MM.1S was increased at day 49, but not at day 14 or day 28, in cultures with 7S compared with untreated controls (P = 0.01) and 30S-treated cells (P = 0.01; Fig. 4C ⇓ ). The percentages of cell death at day 49 were 12.1 ± 1.0%, 25.2 ± 2.7%, and 13.5 ± 1.0% in MM.1S cells, 7S (1 μm)-treated MM.1S cells, and 30S (1 μm)-treated MM.1S cells, respectively (Fig. 4C) ⇓ . In contrast, treatment with 7S for 56 days had no effects on growth (Fig. 4A) ⇓ , cell cycle (data not shown), and cell death (data not shown) in U266 cells with initially long telomeres (9.0 kb).
Effect of long-term culture with NP on growth, cell cycle, and viability. A, MM.1S cells and U266 cells were cultured with match (7S; 1 μm) and mismatch (30S; 1 μm) NP. Harvested cells were counted, and the number of population doublings was calculated. B, cell cycle analysis of MM.1S cells was performed by PI staining and flow cytometry. C, quantitative cell death analysis of MM.1S cells at days 14, 28, and 49 was performed by Annexin V and PI staining followed by flow cytometry. Results are mean ± SD of percentage cell death (Annexin V+, PI− and Annexin V+, PI+), compared with untreated cells. ∗, P = 0.01 versus untreated (C) and 30S-treated cells.
Effect of NP on Cell Cycle Regulatory Proteins in MM.1S Cells.
We next determined the effect of NP on cell cycle regulatory proteins. Significantly increased expression of p21 protein was observed at days 21 and 28 in 7S-treated MM.1S cells (P = 0.05) compared with 30S-treated MM.1S cells (Fig. 5A) ⇓ . There were no associated changes in p53, p27, and p16 protein expression.
Effect of NP on cell cycle regulatory molecules in MM.1S cells. A, MM.1S cells were treated with 1 μm 7S or 30S for 0, 7, 14, 21, and 28 days and subjected to protein analysis (p53, p27, p21, p16, and α-tubulin). Results are mean ± SD of relative protein expression, compared with 30S-treated MM.1S cells. ∗, P < 0.05 versus untreated cells. B, MM.1S cells were treated with 1 μm 7S for 0, 7, 14, 21, 28, 35, and 42 days and subjected to gene microarray profiling analysis. The ratios measure relative gene expression (−3.0. blue to +3.0. red) at each time point.
Having defined 7S-induced effects on cell cycle regulatory protein expression, we next used DNA microarray analysis to delineate sequential changes in gene transcript levels induced by 7S treatment. Total RNA was prepared from MM.1S cells exposed to 7S for 0, 7, 14, 21, 28, 35, and 42 days. As can be seen in Fig. 5B ⇓ , we observed modulation of genes encoding for cell cycle regulatory proteins: up-regulation of p21, MAD2, replication factor C, and cyclin E binding protein as well as down-regulation of Cdc27.
We also found that telomere shortening induced by NP was associated with up-regulation of phosphorylated p53 (Ser-15) in the nucleus, as well as up-regulation of p21 in both the cytoplasm and nucleus after day 14 (Fig. 6) ⇓ .
Effect of NP on cellular localization of cell cycle regulatory proteins in MM.1S cells. MM.1S cells were treated with 1 μm 7S or 30S for 0, 14, 21, and 28 days and subjected to protein analysis [phosphorylated p53 (Ser-15), p53, p27, p21, nucleolin, and α-tubulin] in both cytoplasmic and nuclear fractions.
DISCUSSION
Sustained proliferation in MM cells requires telomerase to maintain telomere stability and counteract the cellular mitotic clock. Several strategies to inhibit telomerase activity have been reported: peptide nucleic acids, 2′-O-methyl-RNA (2′-O-MeRNA) oligonucleotides directed toward hTR (4) , and dominant-negative hTERT (5 , 6) . Although very effective and selective in vitro, these gene therapies require pretreatment with cytotoxic transfection enhancers and therefore may not be readily translated to the clinic. Telomerase inhibitors do not initially affect tumor cell growth rate but induce progressive telomere shortening. Therefore, there is a lag phase between telomere shortening attributable to telomerase inhibition and resultant growth arrest and cell death, which occurs only after telomeres reach a critical length (18) . For example, NP oligonucleotide targeting hTR inhibits telomerase activity in breast epithelial cells at day 1, in the presence or absence of transfection enhancer, resulting in progressive telomere shortening and reduced cell growth with apoptosis at day 115 (14) . These NP oligonucleotides form very stable duplexes with single-stranded RNA, are resistant to nuclease degradation, and have high specificity for RNA and DNA targets (15) .
In this study, we characterized the effect of NP on human MM cell lines, as well as patient MM cells. We demonstrated that NP oligonucleotides were easily and stably transfected into MM cell lines and into patient MM cells in the absence of transfection enhancer. Moreover, the high transfection rate of NP was maintained for 4 days, with low cytotoxicity. Fluorescent microscopic analysis confirmed that NP was localized in both cytoplasmic and nuclear fractions of MM cells. We next demonstrated that NP inhibits both constitutive and cytokine-induced telomerase activity in MM cells. These results suggest that NP therapy targeting hTR may be therapeutically useful for MM, because IL-6 and IGF-1, which are growth and survival factors for MM cells, activate telomerase activity via phosphorylation of hTERT protein (13) .
In this study, we showed that match NP (7S), but not mismatch NP (30S), induced progressive telomere shortening and growth inhibition in MM.1S cells. Critical shortening of telomeres (1.8 kb) occurred by day 28, and significant growth inhibition and cell death occurred later (day 49) in MM.1S cells with short telomeres (2.5 kb). In contrast, treatment with NP for 56 days induced 2.0-kb telomere shortening, without associated growth inhibition and cell death in U266 cells with initially long telomeres (9.0 kb). These results therefore suggest that the effects of NP as a telomerase inhibitor on growth and cell death are dependent on initial telomere length.
Next, we elucidated the molecular sequelae of progressive telomere shortening induced by this telomerase inhibitor. Because telomere shortening induced by NP decreased MM.1S cells in S and G2-M, with increased cells in G1 and sub-G1 (apoptosis), we determined how critical telomere shortening modulates expression of cell cycle regulatory proteins. Progressive telomere shortening induced by NP triggered up-regulation of p21 protein after 21 and 28 days, without associated changes in p53, p27, and p16 protein expression. We also performed comprehensive and sequential gene microarray expression profiling of 7S-treated MM.1S cells to define effects at the transcriptional level and showed that progressive telomere shortening induced by 7S modulated expression of genes encoding for cell cycle regulatory proteins p21, MAD2, replication factor C, cyclin E binding protein, and Cdc27. p21, a cyclin-dependent kinase inhibitor that leads to cell growth arrest, is induced during replicative senescence triggered by telomerase inhibitor (19) . MAD2 (20, 21, 22) and Cdc27 (20) play critical roles in mitosis. Specifically, MAD2 is a key component of the mitotic checkpoint, which ensures accurate chromosome segregation (22) . Critical shortening of telomeres causes chromosomal instability (23) , and induction of MAD2 may therefore block segregation of aberrant chromosomes resulting from telomere dysfunction. Replication factor C is not only a mitotic checkpoint regulator (24) but also binds telomere sequences and inhibits telomerase activity (25) .
We next investigated the expression of cell cycle regulators in the cytoplasmic and nuclear fractions of 7S-treated MM.1S cells, because these molecules translocate from the cytoplasm to the nucleus to modulate gene transcription. We demonstrated phosphorylation of p53 (Ser-15) and up-regulation of p21 in the nucleus of 7S-treated MM.1S cells at days 14, 21, and 28. p53 plays a central role in sensing and executing appropriate cellular responses to DNA damage (26) . Critical telomere shortening (telomere dysfunction) stabilizes and activates p53 (27) , and activation of p53 by DNA damage and telomere dysfunction induces either cell cycle arrest or apoptotic cell death. Recent studies revealed that the cytostatic effect of p53 is mediated by transcriptional activation of the cyclin-dependent kinase inhibitor p21 and induction of G1 or G2 cell cycle arrest, whereas its apoptotic effect is mediated by transcriptional activation of mediators including PUMA (p53 up-regulated mediator of apoptosis) and PIG3 (p53-induced gene 3; 27, 28, 29, 30 ). Further studies will determine the mechanism of growth arrest and apoptosis in replicative senescence induced by telomerase inhibitor.
Recently, it has been reported that the mean telomere length of MM cells was markedly shorter than normal peripheral blood granulocytes and lymphocytes from the same patients (31) . We demonstrated a high transfection rate for NP in both MM (CD138+) cells and normal (CD138−) cells. These results suggest that the effect of NP as a telomerase inhibitor may be observed in MM cells, rather than normal blood cells. We have demonstrated that NP targeting hTR in MM cells inhibits telomerase activity and induces progressive telomere shortening, leading to growth inhibition and cell death in MM cells with short telomeres. These studies therefore identify the molecular mechanism of telomerase inhibitor-induced replicative senescence and provide the rationale for the development of telomerase-targeted therapies to improve patient outcome in MM.
Footnotes
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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.
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↵1 Supported by NIH Grants RO-1 50947 and PO-1 78378, the Multiple Myeloma Research Foundation (to T. Hi., T. Ha., and Y-T. T.), VA Merit Review and Leukemia and Lymphoma Society Scholar in translational research award (to N. C. M.), the Cure Myeloma Fund, the Myeloma Research Fund, and the Doris Duke Distinguished Clinical Research Scientist Award (to K. C. A.).
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↵2 To whom requests for reprints should be addressed, at Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. Phone: (617) 632-2144; Fax: (617) 632-2140; E-mail: kenneth_anderson{at}dfci.harvard.edu
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↵3 The abbreviations used are: hTERT, human telomerase reverse transcriptase; MM, multiple myeloma; IGF-1, insulin-like growth factor-1; IL, interleukin; TRAP, telomerase repeat amplification protocol; hTR, human telomerase RNA component; NP, oligonucleotide N3′→P5′ thio-phosphoramidate; PI, propidium iodide; Ab, antibody; MAD2, mitotic arrest-deficient 2; Cdc27, cell division cycle 27; RT-PCR, reverse transcription-PCR.
- Received February 24, 2003.
- Revision received July 7, 2003.
- Accepted July 21, 2003.
- ©2003 American Association for Cancer Research.
References
Volume 63, Issue 19
