Korean J Lab Med.  2008 Dec;28(6):430-437. 10.3343/kjlm.2008.28.6.430.

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

• Park YP
• Kim KD
• Kang SH
• Yoon DY
• Park JW
• Kim JW
• Lee HG

• KMID: 1781577
• DOI: http://doi.org/10.3343/kjlm.2008.28.6.430

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.

Keyword

Cisplatin; hTERT; Apoptosis; Bladder cancer; Caspase

MeSH Terms

Amino Acid Chloromethyl Ketones/pharmacology
Antineoplastic Agents/*pharmacology
Caspases/antagonists & inhibitors/metabolism
Cell Line, Tumor
Cisplatin/*pharmacology
Cysteine Proteinase Inhibitors/pharmacology
Cytochrome c Group/metabolism
Drug Resistance, Neoplasm/genetics
Humans
Neoplasms/therapy
RNA, Small Interfering
Telomerase/*antagonists & inhibitors/genetics/metabolism
bcl-2-Associated X Protein/metabolism

Figure

• 

  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.

• 

  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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