Inhibition of Human Telomerase Enhances the Effect of the Tyrosine Kinase Inhibitor, Imatinib, in BCR-ABL-Positive Leukemia Cells1

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Vol. 8, 3341–3347, November 2002 Clinical Cancer Research 3341

Advances in Brief Inhibition of Human Telomerase Enhances the Effect of the Tyrosine Kinase Inhibitor, Imatinib, in BCR-ABL-positive Leukemia Cells1

Tetsuzo Tauchi,2 Akihiro Nakajima, by only two amino acids, exhibited normal morphology. The Goro Sashida, Takashi Shimamoto, evidence of apoptosis in the telomerase-inhibited cells was Junko H. Ohyashiki, Kenji Abe, determined by flow cytometric analysis with APO2.7 monoclonal antibody. We also observed enhanced induction of Kohtaro Yamamoto, and Kazuma Ohyashiki apoptosis by imatinib seen in DN-hTERT-expressing K562 First Department of Internal Medicine, Tokyo Medical University, cells, as compared with WT-hTERT-expressing cells. Tokyo, 160-0023 [T. T., A. N., G. S., T. S., K. O.]; Intractable Conclusions: These results demonstrate that disruption Disease Research Center, Tokyo Medical University, Shinjuku-ku, Tokyo 160-0023 [J. H. O.]; and Department of Virology, Medical of telomere maintenance limits the cellular life span of leu- Research Institute, Tokyo Medical and Dental University, Tokyo, kemia cells and show that the combined use of imatinib and 113-8519 [K. A., K. Y.], Japan telomere maintenance inhibition may be effective in the treatment of BCR-ABL-positive leukemia. Abstract Purpose: Telomerase is a ribonucleoprotein enzyme Introduction that maintains protective structures at the ends of eukary- BCR-ABL is a chimeric oncoprotein generated by recip- otic chromosomes. Earlier findings have supported an rocal translocation between chromosomes 9 and 22 and impli- association between progressive telomere shortening in cated in the pathogenesis of Ph3-positive leukemia (1). BCR- the chronic phase of chronic myelogenous leukemia and the ABL fusion proteins exhibit elevated tyrosine kinase activity up-regulation of telomerase activity occurring late in and transforming properties (2, 3). The 2-phenylaminopyrimi- the evolution of the disease. We examined the impact of dine derivative imatinib is a recently designed tyrosine kinase telomerase inhibition by dominant negative-human telomer- inhibitor that competitively inhibits ATP binding in the kinase ase reverse transcriptase (DN-hTERT) on the biological domains of both c-ABL and BCR-ABL (4). Imatinib is specif- features of BCR-ABL-transformed cells. ically active both in vitro and in vivo against a variety of Experimental Design: We introduced vectors encoding BCR-ABL-transformed cells (4–7). Imatinib has shown prom- DN-hTERT, wild-type (WT)-hTERT, or a control vector ise in human clinical trials of Ph-positive leukemia, but the expressing only a drug-resistant marker into Philadelphia emergence of imatinib resistance in patients with acute forms of chromosome-positive K562 cells and OM9;22 cells and as- Ph-positive leukemia highlights the need for combination chem- sessed the biological effect of telomerase inhibition on cellu- otherapy to eradicate disease (6, 8). lar immortality. Telomerase is a cellular RNA-dependent DNA polymerase Results: Ectopic expression of DN-hTERT resulted in that serves to maintain the tandem arrays of telomeric TTAGGG complete inhibition of telomerase activity and reduction of repeats at eukaryotic chromosome ends (9). Telomeres are telomere length. The entire population of telomerase-inhib- highly conserved in organisms ranging from unicellular eu- ited K562 cells exhibited cytoplasmic blebbling and chroma- karyocytes to mammals, indicating a strong role of their pro- tin condensation, features of apoptosis. In contrast, K562 tective mechanisms in preventing chromosomal ends from un- cells expressing WT-hTERT, which differ from the mutants dergoing degradation and ligation with other chromosomes (9). Without telomeric caps, human chromosomes would undergo end-to-end fusions, with formation of dicentric and multicentric chromosomes (10). These abnormal chromosomes would break Received 4/1/02; revised 7/22/02; accepted 7/23/02. during mitosis, resulting in severe damage to the genome and The costs of publication of this article were defrayed in part by the activation of DNA damage checkpoints, leading to cell senes- payment of page charges. This article must therefore be hereby marked cence or initiation of the apoptosis cell death pathway (11). advertisement in accordance with 18 U.S.C. Section 1734 solely to Indeed, it has been proposed that telomere length specifies the indicate this fact. 1 Supported by a grant-in-aid from the Ministry of Education, Science number of cell divisions a cell can undergo before senescence and Culture of Japan (to T. T.), a Grant-in-Aid for the Second Term (12). Telomerase activity is up-regulated in the vast majority of Comprehensive 10-year Strategy for Cancer Control from the Ministry human tumors, as compared with normal somatic tissues. Ex- of Health Labor and Welfare, Japan (to K. O.), the Promotion and Mutual Aid Corporation for Private School of Japan (to K. O.), and the high-tech research center for intractable disease of Tokyo Medical University from the Ministry of Education, Culture, Sports, Science, and Technology in Japan (to K. O.). 3 The abbreviations used are: Ph, Philadelphia chromosome; hTERT, 2 To whom requests for reprints should be addressed, at First Depart- human telomerase reverse transcriptase; DN, dominant negative; WT, ment of Internal Medicine Tokyo Medical University 6-7-1 Nishishin- wild type; PD, population doubling; TRF, telomere restriction fragment; juku, Shinjuku-ku Tokyo 160-0023, Japan. Phone: 81-3-3342-6111; mAb, monoclonal antibody; TRAP, telomere repeat amplification pro- Fax: 81-3-5381-6651; E-mail: [email protected]. tocol; ALT, alternative lengthening of telomere. Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. 3342 Telomerase Inhibition and Imatinib in Ph Leukemia

pression of the catalytic subunit of telomerase, hTERT, in determined by flow cytometric analysis with the FITC-conju-

cultured human primary cells reconstitutes telomerase activity gated APO2.7 mAb (clone 2.7), which was raised against the Mr and allows immortal growth (13–16). TERT-mediated telomer- 38,000 mitochondrial membrane protein (7A6 antigen) and is ase activation is able to cooperate with oncogenes in transform- expressed by cells undergoing apoptosis (25). ing cultured primary cells into neoplastic cells (17). In addition, Statistical Analysis. Comparisons between groups were it has been shown that TERT-derived cell proliferation results in analyzed by the Mann-Whitney t test. Values of P Ͻ 0.05 were activation of the c-myc oncogene (18). These findings in cul- considered to show statistical significance. The statistical tests tured cells have opened up the possibility that telomerase up- were performed with the Microsoft Word/Excel (Brain Power, regulation may contribute actively to tumor growth (19). As a Inc., Calabashes, CA) software package for the Macintosh per- corollary to this hypothesis, the inhibition of telomerase in sonal computer. tumor cells should disrupt telomere maintenance and turn ma- lignant cells toward proliferative crisis, followed by senescence or cell death. Genetic experiments using a DN-hTERT have Results demonstrated that telomerase inhibition can result in telomere Effects of DN-hTERT on Telomerase Activity and Te- shortening followed by proliferation arrest and cell death by lomere Length. Previous reports have identified several point apoptosis (20, 21). This makes telomerase a target not only for mutants in reverse transcriptase motifs of the telomerase cata- cancer diagnosis but also for the development of novel thera- lytic subunit hTERT that exhibit dramatically reduced telomer- peutic agents. We examined the impact of telomerase inhibition ase activity (20, 21). To study the effects of long-term expres- by DN-hTERT on the biological features of BCR-ABL-trans- sion of DN-hTERT on BCR-ABL transformed cells, we formed cells. We found that inhibition of telomerase activity by introduced DN-hTERT, WT-hTERT, or a control vector ex- DN-hTERT induced sensitivity to imatinib. pressing only a puromycin-resistant marker into BCR-ABL- positive K562 and OM9;22 cells. After puromycin selection, successfully transfected cells were clonally isolated. We se- Materials and Methods lected clones expressing DN-hTERT (clones 3, 6, 7), WT- Generation of Stable Clones Expressing WT-hTERT hTERT (clones 1, 2), or a control vector (clones 1, 2) for the and DN-hTERT Mutants. K562 cell line was obtained from TRAP assay (Fig. 1A). For the OM9;22 cells, we also selected the American Type Culture Collection (Manassas, VA). clones expressing DN-hTERT (clones 1, 2) or a control vector OM9;22 cell line, a human leukemia cell line derived from a (clones 1, 2; Fig. 1A). We investigated telomerase activity in Ph-positive acute lymphoblastic leukemia patient, was derived these clones. Expression of DN-hTERT in K562 (clones 3, 6, 7) as described previously (22). DN-hTERT was created by sub- and OM9;22 (clones 1, 2) dramatically reduced telomerase stituting the aspartic acid and valine residues at positions 710 activity (Fig. 1A). In contrast, expression of WT-hTERT (clones and 711 with alanine and isoleucine residues by site-directed 1, 2) increased almost 2-fold the overall telomerase activity in mutagenesis, as described previously (20). The resulting mutant K562 cells (Fig. 1A). Thus, the expression of a catalytically was completely sequenced and subcloned into the vector inactive hTERT mutant results in the disruption of telomerase pBABE-puro (20). K562 cells and OM9;22 cells were trans- activity. We next determined whether inhibition of telomerase fected with the expression vectors pBABE-puro, pBABE-puro- activity influenced telomere length in K562 cell lines (Fig. 1B). WT-hTERT, or pBABE-puro-DN-hTERT by electroporation We assessed peak telomere length in the K562 cell clones (23). Beginning 48 h after electroporation, cells were selected expressing either DN-hTERT (clones 3, 7), WT-hTERT (clone with 2 ␮g/ml puromycin and cloned by limiting dilution. PD0 1), or a control vector (clone 1; Fig. 1B). The telomeres in the was defined as the time at which cultures reached confluence in parental K562 cells and K562 cells expressing control vector 10-cm culture dishes. (clone 1) were maintained at 5.2 kb in length. As the K562 cell Telomerase Assay and Measurement of TRF. Telom- clones (clones 3, 7) expressing DN-hTERT were passaged,

erase activity was examined by TRAP assay with a TRAPEzE gradual telomere shortening was observed (Fig. 1B). Whereas telomerase detection kit (Oncor, Gaithersburg, MD; Ref. 24). all WT-hTERT- (clones 1, 2, 3) or control vector- (clones 1, 2, The PCR products were subjected to 12% acrylamide denatur- 3) expressing cells grew successfully in culture, DN-hTERT- ing electrophoresis in an automated laser fluorescence DNA expressing cells (clones 3, 6, 7) showed a loss of viability (see sequencer II (Pharmacia LKB Biotechnology, AB) and analyzed below). Therefore, we harvested DN-hTERT-expressing cells by the Fragment Manager program (Pharmacia LKB Biotech- on PD22 (clone 3) and PD30 (clone 7). We estimated that nology; Ref. 24). Activity in the extract-based PCR TRAP assay DN-hTERT-expressing cells on PD22 (clone 3) and PD30 was detected as a periodic 6-bp peak of telomerase products and, (clone 7) lost 1.7 and 2.3 kb in telomere length from the time of in each sample, relative telomerase activity was calculated semi- transfection (Fig. 1B). As the process of isolating clones ex- quantitatively in comparison with a 36-bp internal standard (24). pends ϳ20 population doublings, the loss of telomere sequence To measure TRF, genomic DNA was digested with the restric- per population doubling in DN-hTERT-expressing clone 3 and tion enzymes, HinfI and RsaI, fractionated on 0.7% agarose clone 7 were 38 and 46 bp, respectively. In contrast, cells gels, and transferred to nylon membranes. Hybridization was expressing WT-hTERT (clone 1) showed a telomere length of performed by using the Telo TTAGGG telomere length assay 8.0 kb (Fig. 1B). kit (Roche Molecular Biochemicals, Mannheim, Germany). Effects of DN-hTERT on Cell Proliferation and Apo- Apoptosis Assay. Imatinib was kindly provided by No- ptosis. We characterized the growth properties of cells ex- vartis, Inc. (Basel, Switzerland). The incidence of apoptosis was pressing either DN-hTERT (clones 3, 6, 7) or WT-hTERT

Fig. 2 Effects of DN-hTERT on cell proliferation. Clonal isolates analyzed are from the indicated cell lines. Top panel: for each K562 cell line, clones are shown expressing vectors C1(F), WT-hTERT C1 (Ⅺ)or DN-hTERT C3, C6, and C7 (Œ, ‚, f), respectively. Bottom panel: for each OM9;22 cell line, clones are shown expressing vectors C1, C2 (F, Ⅺ), or DN-hTERT C1, C2 (‚, Œ), respectively.

liferating on PD20 (clone 1) and PD18 (clone 2; Fig. 2). DN- hTERT-expressing K562 clones (clone 3, 6, 7) resulted in the Fig. 1 Effects of WT-hTERT and DN-hTERT on telomerase activity and telomere length. A, telomerase activity was examined by a telomere disruption of telomerase activity (Fig. 1A), however, clone 6 underwent a growth arrest more quickly than clone 3 and clone repeat amplification protocol assay by using a TRAPEzE telomerase detection kit (Oncor; Ref. 24). Each set of peaks corresponds to telo- 7 (Fig. 2). The response to DNA damage induced by telomere meric repeats that were synthesized by telomerase in cell extracts dysfunction might reflect this variation between DN-hTERT- obtained from K562 cells (Vector, C1 and C2; WT-hTERT, C1 and C2; expressing clones. The telomerase-inhibited K562 cells (clone DN-hTERT C3, C6, and C7) and OM9;22 (Vector, C1 and C2; DN- hTERT, C1 and C2). Similar results were obtained in two independent 3) exhibited morphological changes accompanied by apparent experiments. B, total genomic DNA from K562 cells was assessed for cell death over PD22 (data not shown). The entire population of telomere restriction fragment size by Southern blot analysis with a DN-hTERT-expressing K562 cells (clone 3) exhibited cytoplas- telomeric probe. Vector, C1; WT-hTERT, C1; DN-hTERT, C3; DN- mic blebbing and chromatin condensation, morphological fea- hTERT, C7, respectively; left margin, molecular-sized markers (kb). PD0 was defined as the time at which cultures reached confluence in tures of apoptosis (data not shown). In contrast, K562 cells 10-cm culture dishes. expressing WT-hTERT (clone 1), which differed from the mutant by only two amino acids, exhibited morphology resembling that of original K562 cells (data not shown). To determine whether DN-hTERT-expressing K562 cells underwent apopto- (clone 1) or control vector (clone 1; Fig. 2). The growth kinetics sis, we used flow cytometry analysis of living cells with a of K562 cells expressing WT-hTERT (clone 1) did not differ FITC-conjugated APO2.7 mAb (clone 2.7; Fig. 3; Ref. 23). substantially from those of cells carrying a control vector (clone After 40 days, 61.4 and 56.6% of DN-hTERT-transfected K562 1; Fig. 2). K562 cells expressing DN-hTERT (clones 3, 6, 7) cells (clone 3 and clone 6) were observed to undergo apoptosis showed slow growth and eventually stopped proliferating on (Fig. 3). In comparison, WT-hTERT- (clone 1) and control PD14 (clone 6), PD22 (clone 3), and PD30 (clone 7; Fig. 2). vector- (clone 1) transfected K562 cells underwent apoptosis at OM9;22 cells expressing DN-hTERT (clone 1, 2) stopped pro- rates of 5–15% throughout the entire experimental period (Fig.

Fig. 3 Expression of DN-hTERT induces apoptosis in K562 cells. The incidence of apoptosis was determined by flow cytometric analysis with the FITC-conjugated APO2.7 mAb (clone 2.7). K562 cells expressing either WT-hTERT (C1, C2) or control vector (C1, C2) were analyzed in PD36. K562 cells expressing DN-hTERT (C3, C6) were analyzed in PD22 and PD15, respectively. Each number represents the percentage of APO2.7 mAb-positive cells. Fig. 4 Enhancement of sensitivity to imatinib in DN-hTERT-expressing K562 cells. A, either DN-hTERT- (C3, C6, C7) or WT-hTERT- (C1, C2, C3) expressing K562 cells at PD10 were cultured with the indicated concentration of imatinib for 48 h. The incidence of apoptosis was determined by flow cytometric analysis with APO2.7 mAb. Values are 3). These results demonstrate that the expression of DN-hTERT shown as mean Ϯ SD of triplicates. Similar results were obtained in two in K562 cells inhibits telomerase activity, resulting in additional independent experiments. B, K562 cells were incubated with the indi- telomere shortening, which may lead to chromatin damage and cated concentrations of imatinib for 48 h, then telomerase activity was examined by a telomere repeat amplification protocol assay. C, K562 the subsequent induction of apoptosis. cells were cultured with the indicated concentrations of imatinib for 10 Enhancement of Sensitivity to Imatinib in DN-hTERT- days, then total genomic DNA from the indicated cells was assessed for expressing K562 Cells. To assess the effects of telomerase telomere restriction fragment size by Southern blot analysis with a inhibition in modulating responses to imatinib, which is a se- telomeric probe. lective inhibitor of BCR-ABL tyrosine kinase, experiments have focused on early passaged K562 cells (PD10) expressing DN- hTERT (clone 3, 6, 7) and WT-hTERT (clones 1, 2, 3; Fig. 4A). DN-hTERT-expressing K562 cells at PD10 showed no differ- or 1 ␮M imatinib (Fig. 4A). Although K562 cells expressing ence in the morphology (data not shown). In a series of exper- DN-hTERT at PD20 showed features of apoptosis spontane- iments, pools of K562 cells that expressed DN-hTERT (clones ously (Fig. 3), DN-hTERT-expressing K562 cells at PD10 3, 6, 7) or WT-hTERT (clones 1, 2, 3) were cultured with showed no difference in the features of apoptosis without ima- imatinib for 48 h to exclude the variation between each clones tinib (Fig. 3). These results suggest that there is a cytotoxic (Fig. 4A). The incidence of apoptosis was determined by flow synergy between telomerase inhibition and imatinib. To eluci- cytometric analysis with APO2.7 mAb (Fig. 4A). DN-hTERT- date the mechanism of a cytotoxic synergy between telomerase expressing K562 cells showed enhanced induction of apoptosis inhibition and imatinib, we examined the effect of imatinib on compared with WT-hTERT-expressing cells on exposure to 0.5 telomerase activity and telomere length in K562 cells (Fig. 4, B

and C). K562 cells were incubated with imatinib for 48 h, then existence of one or more nontelomerase mechanisms for te- we investigated the telomerase activity in these cells by TRAP lomere maintenance that have been termed ALT (36, 37). ALT assay (Fig. 4B). Imatinib (0.2 or 0.5 ␮M) had no effect on has been detected in a variety of human tumors, including short-term cell viability. Imatinib dramatically reduced the te- sarcomas, glioblastomas, and cancers of the lung, kidney, adre- lomerase activity in K562 cells (Fig. 4B). To examine the effect nal, breast, and ovary (38). An implication of the existence of of imatinib on telomere length in K562 cells, K562 cells were ALT is that tumors using this telomere maintenance mechanism incubated with the indicated concentration of imatinib for 10 (including mixed telomerase-positive/ALT-positive tumors) days, then telomere length was examined by Southern blotting will be resistant to telomerase inhibitors. Also, telomerase in- (Fig. 4C). The average telomere restriction fragment size from hibitors will put tumors that are initially telomerase-positive K562 cells was shortened progressively (Fig. 4C). These results under strong selection pressure for activation of ALT. There- suggest that imatinib regulates the telomerase activity and te- fore, combination therapy using ALT and telomerase inhibitors lomere length in BCR-ABL-transformed cells. may help prevent the emergence of drug resistance. In this context, a novel telomerase inhibitor, telomestatin, is a clinical candidate as a dual inhibitor of ALT and telomerase (39). Discussion Lee et al. (40) reported that neoplastic cells from telomer- Direct genetic manipulations have shown that telomerase- ase RNA null mice (mTERCϪ/Ϫ) showed enhanced chemo- mediated telomere elongation plays a key role in determining sensitivity to doxorubicin or related double strand DNA break- cellular replicative capacity and senescence (9). The mecha- inducing agents. Telomere dysfunction, rather than telomere nisms regulating the production of active telomerase enzyme are inhibition, is proposed to be the principal determinant governing still predominantly unknown, but roles for transcriptional con- chemosensitivity specifically to double strand DNA break- trol of hTERT or alternative-splicing of hTERT have been inducing agents (40). We observed enhanced induction of advocated (26–30). Several alternatively spliced variants of apoptosis by imatinib in DN-hTERT-expressing K562 cells hTERT have been identified, including two deletions (␣ and ␤) compared with WT-hTERT-expressing cells (Fig. 4A). DN- and four insertions of intron sequences into hTERT mRNA (29, hTERT-expressing K562 cells also showed enhanced induction 30). Although all known splice variants of hTERT are inactive, of apoptosis compared with WT-hTERT-expressing cells after the observation that hTERT-␣ is a dominant-negative inhibitor exposure to daunorubicin and vincristine, whereas significant of telomerase indicates that splicing may indeed represent a chemosensitivity was not observed in cells exposed to 6- means for cells to fine-tune their levels of active enzyme by mercaptopurine and methotrexate; 4-amino-10-methylpteroyl- differentially shifting the balance between the hTERT-␣ form glutamic acid (data not shown). We have not determined the and the full-length transcript (29, 30). Point mutations of telomere dysfunction in K562 cells in this study, however, hTERT protein have emerged as an additional mechanism of DN-hTERT-expressing K562 cells at PD10 (TRF 4.8 kb) is regulating telomerase activity (31). Telomerase activity can be enough to show the cytotoxic synergy with imatinib. abolished by replacement of any of the three conserved aspartic The enhanced sensitivity to imatinib may imply that there acid residues of amino acid motifs A and C to the hTERT is cytotoxic synergy between telomerase inhibition and imatinib. subunits in budding yeast and in humans (31). Ectopic expres- Recently, Maida et al. (41) reported that epidermal growth sion of these hTERT mutants in human cancer cells results in factor activates telomerase through Ras/MEK/ERK pathways. telomerase inhibition, telomere shortening, and apoptosis (20, In this study, we observed that imatinib suppressed telomerase 21). Therefore, these mutant hTERT subunits act as dominant activity and shortened TRF in K562 cells (Fig. 4, B and C). In negatives of wild-type hTERT and appear to be effective tools this regard, imatinib may suppress telomerase activity via inhi- for studying the characteristics of telomerase. bition of Ras pathways. It has been shown that activation of the In our study, we observed that inhibition of telomerase by nuclear c-Abl protein can contribute to the induction of apo- DN-hTERT reproducibly results in not only telomere shortening ptosis (42). Actually, Kharbanda et al. (43) have reported that but also induction of apoptosis in human leukemia cells (Figs. c-Abl protein is directly associated with hTERT and inhibits 1–3). These results suggest that telomerase activity is the dom- telomerase activity. Because imatinib stimulates nuclear import inant mechanism providing telomere maintenance and long- of BCR-ABL by combining with leptomycin B (44), imatinib term viability to human leukemia cells. Earlier findings support may affect subcellular localization of BCR-ABL in the situation an association between progressive telomere shortening in the of telomere dysfunction. Although the exact mechanism of this chronic myelogenous leukemia-chronic phase and the up-regu- apoptotic effect in DN-hTERT-expressing cells requires addi- lation of telomerase activity occurring late in the evolution of tional elucidation, our observation suggests that certain antine- the disease (32–34). Thus, specific inhibitors of the hTERT oplastic agents may enhance the telomere position effect in enzyme seem to be very effective in limiting the growth of neoplastic cells (45). We observed only the enhanced induction leukemia cells. We have not identified cells that survive the of apoptosis by imatinib in K562 cells expressing DN-hTERT period of crisis induced by DN-hTERT, however, Sachsinger et (Fig. 4), but inhibitors that are combined with the use of ima- al. (35) demonstrated that telomerase activity was efficiently tinib seem to be very useful for BCR-ABL-positive leukemia. reactivated in murine RenCa cells expressing DN-mTERT and There have also been concerns that inhibiting telomerase suggested the fundamental differences in telomere regulation might lead to an increase in malignancy by enhancing the between human and murine cells. genomic instability of cells (46). These concerns have arisen Some human tumor cells without any telomerase activity from observations of mTRϪ/Ϫ knockout mice, which have an are able to maintain the length of their telomeres, indicating the increased incidence of malignancies in both early and late

generations, particularly in the setting of p53 mutant tumors telomerase activity are required to immortalize human epithelial cells. (46). There is no evidence to suggest that this would be the case Nature (Lond.), 396: 84–88, 1998. in humans (47). Mouse telomeres are much longer than even the 15. Jiang, X. R., Jimenetz, G., Chang, E., Frolkis, M., Kusler, B., Sage, longest human telomeres and do not appear to have a role in M., Beeche, M., Bodnar, A. G., Wahl, G. M., Tlsty, T. D., and Chiu, C. P. Telomerase expression in human somatic cells does not induce signaling senescence. Although a critical and careful evaluation changes associated with a transformed phenotype. Nat. Genet., 21: of telomerase inhibitors in clinical development is certainly 111–114, 1999. required, inhibition of telomerase activity appears to be a po- 16. Morales, C. P., Holt, S. E., Ouellette, M., Kaur, K. J., Yan, Y., tential approach for the treatment of leukemia. This work sug- Wilson, K. S., White, M. 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Tetsuzo Tauchi, Akihiro Nakajima, Goro Sashida, et al.

Clin Cancer Res 2002;8:3341-3347.

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