Human Telomerase Reverse Transcriptase (hTERT): A Target Molecule for the Treatment of Cisplatin-resistant Tumors

Yuk Pheel Park; Kwang Dong Kim; Seong Ho Kang; Do-Young Yoon; Joo Won Park; Jong Wan Kim; Hee Gu Lee

doi:10.3343/kjlm.2008.28.6.430

Journal List > Korean J Lab Med > v.28(6) > 1011503

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Park, Kim, Kang, Yoon, Park, Kim, and Lee: Human Telomerase Reverse Transcriptase (hTERT): A Target Molecule for the Treatment of Cisplatin-resistant Tumors

Original Article

Korean J Leg Med 2008;28(6):430-437.

Published online: 14 February 2008

DOI: https://doi.org/10.3343/kjlm.2008.28.6.430

Human Telomerase Reverse Transcriptase (hTERT): A Target Molecule for the Treatment of Cisplatin-resistant Tumors

Yuk Pheel Park, Ph.D.¹, Kwang Dong Kim, Ph.D.², Seong Ho Kang, Ph.D.³, Do-Young Yoon, Ph.D.⁴, Joo Won Park, M.D.⁵, Jong Wan Kim, M.D.⁵, Hee Gu Lee, Ph.D.^1,

¹Medical Genomic Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea

²Division of Applied Life Science, Gyeongsang National University, Jinju, Korea

³Department of Chemistry and Basic Science Research Institute, Chonbuk National University, Jeonju, Korea

⁴Department of Bioscience and Biotechnology, Konkuk University, Seoul, Korea

⁵Department of Laboratory Medicine, College of Medicine Dankook University, Cheonan, Korea

Corresponding author: Hee Gu Lee, Ph.D. Medical Genomic Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), P.O. Box 115, Daejeon 305-333, Korea Tel: +82-42-860-4182, Fax: +82-42-860-4593 E-mail: hglee@kribb.re.kr

^∗This work was supported by FG08-12-01 of the 21C Frontier Functional Human Genome Project from the Ministry of Science & Technology in Korea and by the KRIBB Research Initiative Program.

Received 23 June 200822 October 2008 Accepted 30 October 2008

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance.

Methods

To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells.

Results

hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity.

Conclusions

These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy.

Keywords: Cisplatin, hTERT, Apoptosis, Bladder cancer, Caspase

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Fig. 1.

TA and hTERT expression in cisplatin-resistant T24/R2 cells. (A) T24 and T24/R2 cells were treated with cisplatin (0.1-10 μg/mL) for 24 hr. Cell cytotoxicity (%) was measured by an MTS assay. The optical density (OD) values are mean ±SD of triplicates. ^∗, P<0.01 by t-test. (B) Exponentially growing T24 and T24/R2 cells were treated with 10 μg/mL of cisplatin for 24 hr. Telomerase activity was analyzed with a PCR-based TRAPeze kit (Intergen, Purchase, NY, USA) in the presence of cell extracts following the protocols provided by the manufacturer. (C) T24 and T24/R2 cells were treated with cisplatin (10 μg/mL) for the periods indicated. Forty μg aliquots of cell lysate were immunoblotted with anti-hTERT, bcl-2, bcl-xL, bax, bak, and alpha-tubulin antibodies.

Abbreviations: TA, telomerase activity; hTERT, human telomerase reverse transcriptase; TRAP, a polymerase chain reaction-based telomere repeat amplification protocol assay.

Fig. 2.

Effects of hTERT knock down on bax translocation and cytochrome C release. (A) T24/R2 cells were transfected with hTERT siRNA using Lipofectamine. After 24 hr, the cells were treated with cisplatin for an additional 24 hr and western blot analysis was performed. (B) T24/R2 cells transfected with hTERT siRNA were separated into cytosolic and membrane fractions. The extracts were subjected to 15% SDS-PAGE and immunoblotted with antibax, cytochrome C, F1F0 ATPase, and alpha-tubulin antibodies. Abbreviations: hTERT, See Fig. 1.

Fig. 3.

Caspase inhibitors block the effects of hTERT knock down in cisplatin-induced apoptosis of T24/R2 cells. (A) T24/R2 cells were transfected with hTERT siRNA and pre-treated with caspase inhibitors before cisplatin treatment. After further incubation for 24 hr, the cells were fractionated and the locations of Bax and cytochrome C were evaluated by western blotting. (B) Treatment as in (A). Cells were fixed with ethanol and stained with PI solution in the dark for 30 min. Apoptotic cell death was determined by flow cytometry. Abbreviations: hTERT, See Fig. 1; PI, propidium iodide.

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