Inhibition of telomerase with human telomerase reverse transcriptase antisense enhances tumor necrosis factor-alpha-induced apoptosis in bladder cancer cells.
BACKGROUND
Telomerase activity is found in 85%-90% of all human cancers but not in their adjacent normal cells. Human telomerase reverse transcriptase (hTERT) is an essential component in the telomerase complex that plays an important role in telomerase activity. This study investigated the effect of the telomerase inhibition with an hTERT antisense oligodeoxynucleotide (ODN) in bladder cancer cells (T24) on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis.


METHODS
Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA expression was measured by reverse transcription polymerase chain reaction (RT-PCR) assay and a gel-image system. hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by a morphological method and determined by flow cytometry.


RESULTS
AS PS-ODN significantly inhibited telomerase activity and decreased the levels of hTERT mRNA which preceded the decline in the telomerase activity. AS PS-ODN significantly reduced the percentage of positive cells expressing hTERT protein following the decline of hTERT mRNA levels. There was no difference seen in the telomerase activity, hTERT mRNA expression or the protein levels between the sense phosphorothioate oligodeoxynucleotide (SPS-ODN) and the control group. AS PS-ODN treatment significantly decreased the cell viability and enhanced the apoptotic rate of T24 cells in response to TNF-alpha while there was no difference in cell viability and apoptotic rate between the S PS-ODN and the control group.


CONCLUSIONS
AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression. Treatment with AS PS-ODN may be a potential and most promising strategy for bladder cancer with telomerase activity.
Gao
Xiao-dong
XD
Department of Tumor Surgery, Clinical Medical College of Lanzhou University, and Department of Urology, People's Hospital of Gansu Province, Lanzhou 730000, China. gxd58038@yahoo.com.cn
Chen
Yi-rong
YR
D017209:Apoptosis; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000032:analysis; D019098:Telomerase / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000235:genetics; D013873:Thionucleotides / Q000627:therapeutic use*; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D001749:Urinary Bladder Neoplasms / Q000201:enzymology / Q000473:pathology / Q000628:therapy*
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013873:Thionucleotides; D014409:Tumor Necrosis Factor-alpha; C509186:TERT protein, human; D019098:Telomerase