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Volume 9, Issue 5 (September 2007) 9, 697–704; 10.1111/j.1745-7262.2007.00297.x
Inhibition of telomerase with human telomerase reverse transcriptase antisense increases the sensitivity of tumor necrosis factor--induced apoptosis in prostate cancer cells
Xiao-Dong Gao and Yi-Rong Chen
1.Department of Urology, Lanzhou University, Lanzhou 730000, China
2.Department of Urology, People's Hospital of Gansu Province, Lanzhou 730000, China
Correspondence: Dr Xiao-Dong Gao, Lanzhou University, 9 Qingyang Road, Zhaoyin Mansion, Chengguan District, Lanzhou 730000, China. E-mail: gxd58038@yahoo.com.cn
Received 23 November 2006; Accepted 5 April 2007.
| Abstract |
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Aim: To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase (hTERT) antisense on tumor necrosis factor- (TNF-)-induced apoptosis in prostate cancer cells (PC3).
Methods: Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured using the telomeric repeat amplification protocol (TRAP) and polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA was measured by reverse transcription PCR (RT-PCR) assay and gel-image system. hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by morphological method and determined by flow cytometry.
Results: The telomerase activity decreased with time after hTERT AS PS-ODN treatment. The levels of hTERT mRNA decreased with time after hTERT AS PS-ODN treatment, which appeared before the decline of the telomerase activity. The percentage of positive cells of hTERT protein declined with time after hTERT AS PS-ODN treatment, which appeared after the decline of hTERT mRNA. There was no difference in telomerase activity, hTERT mRNA and protein levels between hTERT sense phosphorothioate oligodeoxynucleotide (S PS-ODN) and the control group. The cell viability decreased with time after hTERT AS PS-ODN combined with TNF- treatment. The percentage of apoptosis increased with time after hTERT AS PS-ODN combined with TNF- treatment. There was no difference in cell viability and the percentage of apoptosis between hTERT S PS-ODN and the control group.
Conclusion: hTERT AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNF--induced apoptosis of PC3 cells.
Keywords: human telomerase reverse transcriptase, antisense phosphorothioate oligodeoxynucleotide, telomerase, prostate cancer cells, tumor necrosis factor-
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