| In the present study, the possibility of controlling telomerase activity in hepatoma cells and its importance for tumor biology were probed. Results showed that antisense oligonucletides against template region of telomerase RNA component and dominant-negative form of telomerase reverse transcriptase subunit could effectively inhibit telomerase activity and the growth of hepatoma cells. Laboratory basis of using telomerase activity as the target for novel anti-hepatoma drug screening was also investigated.As the first step of the whole work, a non-radioisotopic,quantitative TRAP(telomere repeat amplification protocal) - based telomerase activity assay was established. In comparison with the conventional radioisotopic incorporating method, this novel method can offer reliable and quantitative result. Moreover, it can avoid environment pollution and harm to handler. Comparing with radioisotopic labelling quantitative method, it was better in reproducibility and accuracy, while its sensitivity was about at the same degree as the radioisotopic labelling method. Using this method, we found telomerase activities were absent in normal human liver cells, while present in all of four human hepatoma cell lines (BEL-7404, SMMC-7721, QGY-7903 and HCCM). The results indicated that telomerase activity was important for diagonosis,therapy andprevention of hepatocellular carcinoma.Telomerase activity of BEL-7404 human hepatoma cells was inhibited effectively by antisense oligonucleotides against template region of telomerase RNA component, whereas no effects of inhibition were observed in control group. After treating BEL-7404 human hepatoma cells 96 hours continuously with the antisense oligomers at the final concentration of 5μmol/L, cell shranked greatly with abnormal morphology, and the ratio of floating cells increased. TUNEL assay showed that treated cells were in the course of apoptosis, and FACS analysis showed that the apoptotic rate reached up to 42.58%. Cell cycle of attached BEL-7404 human hepatoma cells was mainly arrested at G2/M phase with the treatment of antisense oligomers, while sense and missense oligomers had no effects on cell cycle compared with control cell. The expression of a dominant-negative form of human telomerase reverse transcriptase (DN-hTERT) also resulted in the inhibition of telomerase activity and decrease in mean telomere length of BEL-7404 human hepatoma cells, whereas the expression of wild-type human telomerase reverse transcriptase (WT-hTERT) and control vector had no such effects. Cell growth was inhibited by this mutant (DN-hTERT), which was consistent with the changes of telomerase level. Flattened large cells were found in their late generations with the treatment of DN-hTERT. When mean telomere length of the DN-hTERT transfected cells reached to a critical length (about 1.7 Kb), the apoptosis was induced. Tumorigenicity of DN-hTERT expression cells was eliminated in vivo. These data indicated that hTERT was essential for the growth of hepatoma cells. To study the effect of clinical chemotherapy agents on telomerase activity of hepatocellular carcinoma,telomerase activity was tested in BEL-7404 human hepatoma cells treated with 5-fluorouracil, doxorobincin, cisplatin and mitomycin with their IC50 concentrations. Results showed that telomerase activity of BEL-7404 human hepatoma cells was inhibited effectively by doxorobicin and cisplatin, while 5-fluorouracil and mitomycin had no effects on its telomerase activity. Further study showed that telomerase activity was inhibited in a dose and time-dependent manner with the treatment of doxorobicin and cisplatin. There were no changes in expression pattern of three telomerase subunits, its reverse transcriptase subunit (hTERT), its RNA component (hTR) or the associated protein 1 subunit(TP1),after doxorobincin or cisplatin treatment for 72h with indicated concentrations. Mean telomere length (terminal restriction fragment) of BEL-7404 human hepatoma cells was decreased by doxorobicn and cisplain treatmen...
|
PDF Full Text Request