Effect Of HTERT Antisense On Chemotherapeutic Drugs-Sensitivity Of Acute Lymphoblastic Leukemic Cells

Object: To investigate the effect of human telomerase reverse transcriptase(hTERT) antisense phosphorothioate oligodeoxynucleotide(AS PS-ODN) on telomerase activity and apoptosis induced by chemotherapeutic drugs in Raji, Jurkat, CEM cell lines and acute lymphoblastic leukemic cells.Method: Polymerase chain reaction -enzyme linked immunoassay (PCR-ELISA) was used to assay telomerase activity. The expression levels of hTERT mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence-based flow cytometry assay respectively. The survival rates of cells were measured by trypan blue exclusion. Apoptosis was assayed by morphological observation, DNA gel electrophoresis and flow cytometry analysis technology. Result: Raji, Jurkat and CEM cells all expressed high level of telomerase activity, which decreased as the cells treated by hTERT AS PS-ODN. For Raji and CEM cells, the levels of both mRNA and protein of hTERT gene decreased under influences of hTERT AS PS-ODN. However, for Jurkat cell, the level of hTERT protein declined only, level of hTERT mRNA was not changed significantly comparing with the control or S PS-ODN. It was found that there was no significant difference in survival rate between cells treated with hTERT AS PS-ODN added Daunorubicin (DNR), Vincristin (VCR) and Etoposide (VP16) respectively 24h later and DNR, VCR and VP16 respectively. The survival rate of Raja , Junket and CEM cells cultured with cist-diamminedichicloroplatinum(DDP) added 24h later was higher than that cultured with hTERT AS PS-ODN plus DDP added 24h later. It was more obvious at 48h between 48h and 72h. There was no significant difference between cells added DDP and cells added DDP/ hTERT S PS-ODN. In morphological observation of apoptotic cells using Giemsa staining and Hoechst33258 and PI double staining techniques, cells displayed classic apoptotic changes treated with DDP alone and DDP plus hTERT AS PS-ODN or S PS-ODN at 48h. In this part, the number of apoptotic cells treated with DDP and hTERT AS PS-ODN together is obviously more than that treated with DDP alone and DDP combined with hTERT S PS-ODN, and it was similar in cells treated with DDP alone and DDP combined with hTERT S PS-ODN. Genomic DNA of Raji, Jurkat and CEM cells treated with hTERT AS PS-ODN and DDP(5?mol/L) added 24h later showed typical DNA "ladder" through agarose gel electrophoresis in 48h; Neither did genomic DNA from cells treated with DDP /S PS-ODN nor DDP alone. Apoptotic rates of Raj ,Jurkat and CEM cells were detected by flow cytometry assay. Apoptotic rates of cells added DDP (2.5?mol/L) /AS PS-ODN were higher than that of cells added DDP (2.5?mol/L) only (P<0.05). It was similar in cells added DDP (2.5?mol/L)/ S PS-ODN and DDP (2.5?mol/L) only (P>0.05). To primary acute lymphoblastic leukemic cells, the effects of AS PS-ODN including the survival rates, the expression of hTERT protein and apoptosis ,were similar with those observed in Raji, Jurkat and CEM cell lines. Conclusion: hTERT AS PS-ODN could inhibit telomerase activity of Raji, Jurkat, CEM cells and primary acute lymphoblastic leukemic cells, and increased the DDP-induced apoptosis and enhanced DDP-sensitivity.