New Strategy Of Tumor Gene Therapy: Chronobiological Aspect Of Tumor And Inhibition Of Telomerase By Ribozyme

OBJECTIVE:Telomerase plays an important role in cell proliferation and carcinogenesis and is believed to be a good target for anti-cancer drugs. To evaluate the possibility of using ribozyme technology for telomerase inhibition and cancer therapy, to investigate chronobiological characteristic of telomerase and chronotherapy for tumor.Methods and Resultes:1 .A hammer head ribozyme (telomerase ribozyme, teloRZ) directed against the RNA component of human telomerase (hTR) was designed and synthesized to serve as a telomerase inhibitor. An in vitro transcription plasmid and a eukaryotic expression plasmid containing teloRZ gene were constructed. In vitro cleavage reaction was carried out by mixing the ribozyme RNA with DIG-labeled-hTR in different reaction conditions. Cleavage bands were detected by digoxin chemiluminescent assay. The eukaryotic expression plasmid was inducted into HeLa cells by lipofectamine; the telomerase activitiesand bio-characteristics of HeLa cells were detected continuously. Through the study we find that, teloRZ showed a specific cleavage activity against the telomerase RNA component used as template. The in vitro cleavage ratio reached about 60%. The telomerase activities of cells expressing teloRZ dropped to eight times; the doubling times became longer and apoptosis ratios became higher with increasing population doublings (PDS); at 19-20 PDS 95% cells showed apoptosis.2. A hammer head ribozyme directed against the hTERT mRNA (hTERT-2239RZ) was designed and synthesized to serve as a telomerase inhibitor. In order to test its in vitro cleavage activity, two in vitro transcription plasmids containing hTERT-2239RZ and hTERT gene respectively were constructed. Ribozyme RNA and DIG-Labeled-hTERT were synthesized by in vitro transcription. In vitro cleavage reactions were carried out by mixing the hTERT-2239RZ with DIG-labeled-hTERT under different reaction conditions, and cleavage bands were detected by digoxin chemiluminescent assay. HTERT-2239RZ showed a specific cleavage activity against the hTERT used as template. To investigate its in vivo effect of telomerase inhibition in tumor cells, a eukaryotic expression plasmid containing the hTERT ribozyme gene was introduced into HeLa cells and hepatoma cells by using LipofectAMINE. In the transfectants, the level of intact hTERT mRNA and the telomerase activity were clearly reduced, and the telomere length of these clones was apparently shortened at the beginning period, then kept a fixed value without further shortening. All the transfectants with ribozymegrew clearly more slowly than the parental cell line. The doubling time of the tansfectants prolonged compared to the negative control, but no apparent apoptosis was shown even at their 37th passage.3. A hammer head ribozyme targeted to hTERT-5'end (hTERT-5'RZ) was synthesized and cloned into pCDNA3.1(+), the ribozyme was produced by in vitro transcription. The teloRZ ribozyme was produced in the same way by in vitro transcription of pSPT19-teloRZ which we constructed before. The ribozymes were transiently transfected into Hela cells by liposome every 24 hours. After 72 hours, the cells were collected and their telomerase activities were assayed. We found that, the ribozyme targeting the 5'-end of hTERT mRNA exhibited a very strong telomerase-inhibitory activity, the combining use of hTERT-5'RZ and teloRZ also showed clear inhibitory activity, but the inhibitory efficiency of teloRZ only was not so strong. These observations suggest that the use of hTERT-5'RZ and the combining use of hTERT-5'RZ and teloRZ are more effective in telomerase inhibition than the use of teloRZ only. They may possess potential for cancer therapy.4. To study the Circadian rhythms of DNA synthesis and telomerase expression in hepatic cancer nude mice transplanted tumor. Sixteen BALB/C mice were synchronized with an alternative lighting regimen with 12 hours for light and 12 hours for dark (12:12 LD) for 4 weeks. Hepatic cancer cells (SMMC-7721) were implanted into b...