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Background & Objective: Antisense oligodeoxynucleotides (ASODN) targeting bcl-2 have in vitro and in vivo antitumor activities. We have identified a novel antisense sequence in the coding region of bcl-2 mRNA that could enhance chemosensitivity and radiosensitivity of leukemia cells and lung carcinoma cells. This study was designed to investigate inhibitory effects of bcl-2 ASODN on development and growth ability of human lung carcinoma NCI-H460 cells xenograft in nude mice., Methods: bcl-2 ASODN was added to NCI-H460 cells. Inhibitory rate of cell growth was assayed by MTT assay. bcl-2 ASODN-treated NCI-H460 cells were implanted subcutaneously into nude mice to observe tumor growth and calculate tumor inhibitory rate. Seven days after subcutaneous implantation of untreated NCI-H460 cells, the tumor-bearing mice were randomized into 3 groups: control group, ASODN group, and nonsense oligodeoxynucleotides (NSODN) group. bcl-2 ASODN, bcl-2 NSODN, and saline were separately injected into the tumor bodies of mice in relevant groups for 3 week. The tumor weight and volume were measured, and the morphology of tumor cells was observed under microscope., Results: bcl-2 ASODN reduced the growth of NCI-H460 cells in a time-dependent manner. Inhibitory rate of NCI-H460 cells was significantly higher in bcl-2 ASODN group than in bcl-2 NSODN group (P<0.05). The tumorigenic ability of NCI-H460 cells was reduced by bcl-2 ASODN, with the maximum tumor growth inhibitory rate of 87.5% (P<0.01). The tumor formation time was prolonged to 12.6 days. Tumor growth was significantly inhibited in bcl-2 ASODN group as compared with that in NSODN group and control group(P<0.01). The tumor weight was significantly lighter in bcl-2 ASODN group than in bcl-2 NSODN group and control group (P<0.01). The growth inhibitory rate was 71.0% in bcl-2 ASODN group. Serious necrosis foci and inflammatory cell infiltration were observed in tumor tissues of bcl-2 ASODN group, while cancer cells were diffused in tumor tissues of bcl-2 NSODN group and control group., Conclusion: bcl-2 ASODN can suppress tumorigenic ability of NCI-H460 cells and inhibit tumor growth in nude mice.

To explore the effect of antisense phosphorothioate oligodeoxynucleotide (ASODN) of human telomerase reverse transcriptase (hTERT) gene on telomerase activity in CEM cells, PCR enzyme-linked immunoassay was used to determine telomerase activity. The expression levels of hTERT mRNA and protein were assayed by RT-PCR and immunofluorescence assay using fluoresce isothiocyanate label respectively. The results showed that the expression levels of hTERT mRNA and protein in CEM cells decreased with time after hTERT ASODN treatment. There was no difference in hTERT mRNA and protein levels between control and sense oligodeoxynucleotide-treated cells. Telomerase activity decreased when CEM cells were treated with ASODN for 48 hours. Telomerase activity of CEM cells was significantly inhibited when treated with ASODN for 72 hours. There was no difference in telomerase activity levels between control and hTERT sense oligodeoxynucleotide-treated cells. These results suggested that hTERT ASODN inhibited telomerase activity of CEM cells.

Background & Objective: The previous study has identified two novel antisense oligonucleotides (AS-ODN) of new target point in the translation initiation and the coding region of bcl-2 mRNA that could increase the sensitivity of HL-60 and K562 cells lines to etoposide, daunorubicin, and araninosyl cytosine (Ara-C). This study was designed to investigate whether the two novel bcl-2 AS-ODNs could increase Ara-C-induced apoptosis of cultured primary acute leukemia (AL) and chronic lymphocytic leukemia (CLL) Cells., Methods: Cell surviving fraction was determined using the trypan blue dye exclusion assay. The expression levels of bcl-2 protein were assayed by immunofluorescence using fluorescein isothiocyanate label. Apoptosis was detected by Giemsa staining and flow cytometry., Results: Treatment with the two AS-ODNs/Ara-C combination respectively for 48 h significantly inhibited the growth of AL and CLL cells. There was significant difference on AL and CLL cells survival between nonsense oligodeoxynucleotide(NS-ODN)/Ara-C combination and Ara-C treated cells alone (P < 0.05). It was found that the two AS-ODNs respectively combined with Ara-C could significantly downregulate bcl-2 protein expression (P < 0.05). There was a significant increase in apoptotic rates of AL or CLL cells after treatment with the two AS-ODNs respectively combined with Ara-C as respectively compared with the NS-ODN/Ara-C combination and Ara-C treated cells alone; and the effect had statistical difference (P < 0.05). Compared to the AS-ODN directed against the translation initiation of bcl-2 mRNA, the AS-ODN directed against the coding region showed stronger effect in the aspects of increasing the sensitivity of AL cells to Ara-C (P < 0.05)., Conclusions: The two novel AS-ODNs of new target point in the translation initiation and the coding region of bcl-2 mRNA could enhance Ara-C induced apoptosis of AL and CLL cells.

To explore the effect of human telomerase reverse transcriptase (hTERT) gene antisense oligodeoxynucleotide (ASODN) on telomerase activity in primary acute myeloid leukemia (AML) cells and chronic myeloid leukemia (CML) cells, polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) was used to determine telomerase activity. The expression levels of hTERT protein were assayed by flow cytometry using immunofluorescence labeling. Immunofluorescence assay showed that the expression levels of hTERT protein in AML and CML cells decreased with time after hTERT ASODN treatment. There was no difference in hTERT protein levels between control and sense oligodeoxynucleotide (SODN)-treated cells. Telomerase activity decreased when AML and CML cells were treated with ASODN for 48 h. Telomerase activity of AML and CML cells was significantly inhibited when the cells treated with ASODN for 72 h. There was no difference in telomerase activity levels between control and hTERT SODN-treated cells. It was concluded that hTERT ASODN could inhibit hTERT protein expression level, thus decreasing the telomerase activity in primary AML and CML cells.