[Caspase-3 antisense oligodeoxynucleotides inhibit caspase-3 expression and apoptosis of gamma-irradiated HL-60 cells].
It has been shown that gamma -irradiation induces apoptosis of the human promyeloid leukemia cell line HL-60, but the mechanism remains unclear. To explore the effect of caspase-3 in this apoptotic model, antisense oligodeoxynucleotides (ASODNs) targeting 5'-noncoding region (ASODN-1) and initial translation region (ASODN-2) of caspase-3 mRNA were designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by gamma-irradiation in the present study. The TUNEL assay was used for morphological analysis of HL-60 cell apoptosis. Immunocytochemical staining, Western blotting and RT-PCR were, respectively, performed for detecting expression of caspase-3 and its mRNA. HL-60 cells transfected with mismatched oligodeoxynucleotide (MODN) or untransfected were taken as the control groups. The TUNEL assay showed that the percentages of HL-60 cell apoptosis induced by gamma-Irradiation in both ASODN-1 and ASODN-2 groups were significantly reduced compared with those in the control group (P<0.01) when the final transfection concentration was > or = 3 micromol/L. Immunocytochemistry demonstrated that in the ASODN-1 and ASODN-2 groups, caspase-3 positive cell percentages were reduced and average gray values of the positive cells increased significantly compared with those in the control group (P<0.01). Western blotting found that procaspase-3 expression in HL-60 cells of the ASODNs groups was decreased,and it was lower in the ASODN-1 group than in the ASODN-2 group. RT-PCR revealed marked expression of caspase-3 mRNA in HL-60 cells of the control group. Expression of caspase-3 mRNA was decreased after ASODNs transfection. Furthermore, ASODN-1 was more effective in inhibiting HL-60 cell apoptosis (P<0.05) and caspase-3 expression (P<0.01) than ASODN-2. These results indicate that caspase-3 mRNA ASODNs prevent HL-60 cells from apoptosis induced by gamma-radiation,and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.
Zhang
Xiao Tian
XT
Environment and Genes Related to Diseases Key Laboratory of Education Ministry, Xi'an Jiaotong University School of Medicine.
Song
Tian Bao
TB
Li
Dong Min
DM
Wu
Xiao Lin
XL
D017209:Apoptosis / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D015153:Blotting, Western; D053148:Caspase 3 / Q000235:genetics* / Q000378:metabolism; D005720:Gamma Rays; D018922:HL-60 Cells; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D020319:Oligodeoxyribonucleotides, Antisense; D053148:Caspase 3