[CANCER RESEARCH 61, 5796 –5802, August 1, 2001] Treatment of Malignant Glioma Cells with the Transfer of Constitutively Active Caspase-6 Using the Human Telomerase Catalytic Subunit (Human Telomerase Reverse Transcriptase) Gene Promoter 1 Tadashi Komata, Yasuko Kondo, Takao Kanzawa, Satoshi Hirohata, Shoji Koga, Hideaki Sumiyoshi, Srinivasa M. Srinivasula, Barbara P. Barna, Isabelle M. Germano, Masahiro Takakura, Masaki Inoue, Emad S. Alnemri, Jerry W. Shay, Satoru Kyo, and Seiji Kondo 2 Center for Surgery Research [T. Ko., Y. K., S. Kog., S. Kon.], Departments of Neurosurgery [T. Ko., S. Kon.] and Biomedical Engineering [S. H.], The Cleveland Clinic Foundation, Cleveland, Ohio 44195; Departments of Neurosurgery [T. Ko., Y. K., T. Ka., I. M. G., S. Kon.] and Biochemistry and Molecular Biology [H. S.], The Mount Sinai School of Medicine, New York, New York 10029; Center for Apoptosis Research and Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [S. M. S., E. S. A.]; RammelKamp Center for Education and Research, MetroHealth Medical Center, Cleveland, Ohio 44109 [B. P. B.]; Department of Obstetrics and Gynecology, Kanazawa University, School of Medicine, Kanazawa, Ishikawa 920-0934, Japan [M. T., M. I., S. Ky.]; and Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9039 [J. W. S.] ABSTRACT Because the apoptotic pathway is often disrupted in tumor cells, its genetic restoration is a very attractive approach for the treatment of tumors. To treat malignant gliomas with this approach, it would be preferred to restrict induction of apoptosis to tumor cells by establishing a tumor-specific expression system. Telomerase is an attractive target because the vast majority of malignant gliomas have telomerase activity whereas normal brain cells do not. Activation of telomerase is tightly regulated at the transcriptional level of the telomerase catalytic subunit [human telomerase reverse transcriptase, (hTERT)]. Therefore, we hy- pothesized that using a hTERT promoter-driven vector system, an apo- ptosis-inducible gene may be preferentially restricted to telomerase- or hTERT-positive tumor cells. In this study, we constructed an expression vector consisting of the constitutively active caspase-6 (rev-caspase-6) under the hTERT promoter (hTERT/rev-caspase-6) and then investigated its antitumor effect on malignant glioma cells. The rationale for using the rev-caspase-6 gene is because it induces apoptosis independent of the initiator caspases. We demonstrated that the hTERT/rev-caspase-6 con- struct induced apoptosis in hTERT-positive malignant glioma cells, but not in hTERT-negative astrocytes, fibroblasts, and alternative lengthening of telomeres cells. In addition, the growth of s.c. tumors in nude mice was significantly suppressed by the treatment with hTERT/rev-caspase-6 con- struct. The present results strongly suggest that the telomerase-specific transfer of the rev-caspase-6 gene under the hTERT promoter is a novel targeting approach for the treatment of malignant gliomas. INTRODUCTION Malignant gliomas are the most common tumors in the central nervous system. When treated with conventional therapy such as surgery, -irradiation, or chemotherapy, the average life expectancy is usually less than 1 year (1, 2). Clearly, novel therapeutic strategies are necessary. Generally, malignancy results not only from unregulated cell pro- liferation but also from decreased sensitivity to physiological pro- grammed cell death (apoptosis) signals (3). Apoptosis is a process in which cells actively participate in their own death (4); however, this pathway is often disrupted in tumor cells (5). Therefore, a genetic restoration of the apoptotic pathway or introduction of proapoptotic molecules is very attractive for the treatment of tumors including malignant gliomas. Caspases play a major role in the transduction of apoptotic signals and the execution of apoptosis in mammalian cells (6, 7). Currently, the caspase family consists of more than 12 members (8), and imple- mentation of the apoptotic program requires the participation of at least two classes of caspases, the initiators such as caspase-2, -8, -9, or -10 and the executioners including caspase-3, -6, or -7 (8, 9). Rev-caspases-3 and -6 3 have been described recently (10). This was achieved by making contiguous precursor caspases-3 and -6 mole- cules that have their small subunits preceding their large subunits. Unlike their wild-type counterparts, these recombinant molecules are capable of autocatalytic processing and inducing apoptosis independ- ent of the upstream, initiator caspases. Because caspases-3 and -6 are the most downstream executioners of apoptosis, the constitutively active versions of these caspases could be used at very low concen- trations to induce apoptosis in target tumors (10). We have demon- strated recently (11, 12) that the introduction of caspase genes inhib- ited the growth of malignant glioma cells in vitro and in vivo through induction of apoptosis, indicating the potential therapeutic usefulness of this approach for malignant gliomas. However, if caspases are transduced to normal brain cells, they would be predicted to also undergo apoptosis, resulting in undesirable brain damage. To restrict induction of apoptosis to tumor cells and increase the safety of this approach, we needed to establish a tumor-specific caspase expression system. Utilization of a promoter that is predominantly active in tumor cells would be an ideal system to restrict the cytotoxic caspase expression. Telomerase, a ribonucleoprotein enzyme, is a particularly attractive target for specifying tumors, because approximately 90% of tumors have telomerase activity, whereas most normal cells do not express telomerase (13). In malignant gliomas, telomerase activity is detected in 10 to 45% of anaplastic astrocytoma (WHO grade III) and in 75 to 89% of glioblastoma multiforme (grade IV; Refs. 14, 15). In contrast, normal brain tissues do not express telomerase activity (15–17). Therefore, telomerase is expected to represent a very good candidate for the targeted therapy of malignant gliomas. The main components of the human telomerase enzyme are the functional or template RNA component (hTER; Ref. 18) and the telomerase catalytic protein subunit or hTERT (19, 20). Although hTER and hTERT are both necessary for telomerase activity, the expression of hTERT is present specifically in tumor cells whereas hTER is present in both normal Received 5/8/00; accepted 6/1/01. 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. 1 Supported in part by Cleveland Clinic Foundation Research Fund No. 5928 (to S. Kon.), the John Gagliarducci Fund (to S. Kon.), and the NIH Grants CA88936 (to S. Kon.) and AG13487 (to E. S. A.). 2 To whom requests for reprints should be addressed, at Department of Neurosurgery, The Mount Sinai Medical Center, One Gustave L. Levy Place, Box 1136, New York, NY 10029-6574. Phone: (212) 241-6503; Fax: (212) 831-3324; E-mail: Seiji.Kondo@ mssm.edu. 3 The abbreviations used are: rev-caspase, constitutively active recombinant caspase; hTERT, human telomerase reverse transcriptase; hTER, human telomerase RNA compo- nent; ALT, alternative lengthening of telomeres; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CMV, cytomegalovirus; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; GFP, green fluores- cence protein. 5796 Research. on February 12, 2016. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from