Effect Of Inhibition Of Telomerase With HTERT Antisense Enhances TNF-α Induced Apoptosis In Bladder Cancer Cells (T24)

Objective Telomeres are specialized heterochromatin structures that protect the ends of chromosomes. Previous studies have shown that telomerase activity is found in 85-90% of all human tumors, but not in their adjacent normal cells. This makes telomerase a good target not only for cancer diagnosis, but also for the development of novel therapeutic agents. Telomerase is composed of at least three subunits. The RNA subunit and the catalytic subunit are the essential components for telomerase activity. The RNA subunit of telomerase serves as the template for addition of short sequence repeats to the chromosome 3' ends. The catalytic subunit, telomerase reverse transcriptase (TERT), is the most important component in telomerase complex, which is responsible of catalytic activity of telomerase. The expression of TERT correlates with the presence of telomerase activity. The experiments investigated the effect of inhibition of telomerase with hTERT antisense on bladder cancer cells (T24) to TNF-α-induced apoptosis.Methods Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by telomeric repeat amplification protocol (TRAP) and telomerase PCR-ELISA kit. hTERT mRNA expression was measured by reverse transcription polymerase chain reaction (RT-PCR) assay and a gel-image system. hTERT protein was measured by immunochemistry and flow cytometry. Cell viability was determined by trypan blue dye exclusion assay and cell apoptosis was observed by morphological method and determined by flow cytometry.Result The experiments showed that after the telomerase activity of T24 cells was inhibited with 10μmol/L AS PS-ODN for 48 h, the TRAP products ladders decreased significantly, the telomerase activity was significantly repressed after 48h, sense oligonucleotides and control had little effect on telomerase activity(P<0.05) ;the levels of hTERT mRNA were significantly decreased after T24 cells were treated with AS PS-ODN for 24h, which appeared before the decline of telomerase activity, sense oligonucleotides had no effect on hTERT mRNA levels(P<0.05 ); after treated with AS PS-ODN for 48 h, the percentage of positive cells of hTERT protein was significantly declined than that of control and sense oligonucleotides, which appeared after the decline of hTERT mRNA (P<0.05); after the telomerase activity of T24 cells was inhibited with 10μmol/L AS PS-ODN for 24 h, 4μg/ml TNF-αwas incubated with T24 cells for more than 48 h, there was significant inhibition rate of T24 cells for TNF-α/AS PS-ODN as compared with TNF-α/S PS-ODN or TNF-α,with time increased, the inhibition rate of T24 cells increased significantly(P<0.01); and then the cells were observed under a microscope, many apoptotic cells in TNF-α/AS PS-ODN were discovered; at the same time, hTERT AS PS-ODN could increase the apoptotic rate of T24 cells(P<0.05).Conclusion hTERT antisense can inhibit telomerase activity of bladder cancer cells by down regulating the hTERT mRNA and protein expression and enhance TNF-α-induced apoptosis of T24 cells.