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

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