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[Antisense Oligodeoxynucleotide Directed to NF-kappaB-RelA Down-Regulates bcl-x(L) mRNA in Drug-Resistant Leukemia
Wen-Jing Cao1, Yao-Zhen Zhang, Dong-Hua Zhang
1Department of Hemotology, The Affiliated Tongji Hospital of Tongji Medical College, Huazhong Science and Technology University, Wuhan 430030, China; Institute of Hematology, The Affiliated Union Hospital of Tongji Medical College, Huazhong Science and Technology University, Wuhan 430022, China.
Abstract:
In order to explore the effect of nuclear factor-kappa B (NF-kappaB) on bcl-x gene transcrtiption in drug-resistant leukemia cell line HL-60/E6, first of all, drug-resistant subline HL-60/E6 was derived by intermittently exposing HL-60 cells to 6 ng/ml epirubicin, and then bcl-x(L) mRNA levels were detected by RT-PCR after exposing HL-60/E6 cells to 5 micro mol/L AS-PS-ODN-RelA at different times. Morever, indirect immuno-fluorescence and FCM were used to demonstrate the location of NF-kappaB-RelA in HL-60/E6 cells and the efficiency of liposome-mediated ODN transfection. The results showed that RelA kept active and located at the nuclei of HL-60/E6 cells, and the efficiency of liposome-mediated ODN transfection was significantly higher than that of null ODN (P < 0.01 in 4, 6 and 12 h). Exposure of HL-60/E6 cells to 5 micro mol/L AS-PS-ODN-RelA led to a maximal 40% decline of bcl-x(L) mRNA levels. No significant change of bcl-x(L) mRNA level occurred in control group. It was concluded that NF-kappaB was involved in regulating bcl-x transcription. Suppressing NF-kappaB-RelA by antisense technology may down-regulate level of bcl-x(L) mRNA.
Insights
Nuclear factor-kappa B (NF-kappaB) regulates bcl-x gene transcription in drug-resistant leukemia cells. Suppressing NF-kappaB-RelA with antisense technology reduced bcl-x(L) mRNA levels, indicating its role in leukemia drug resistance.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- Drug resistance in leukemia, specifically the HL-60/E6 cell line, is a significant clinical challenge.
- The bcl-x gene plays a crucial role in cell survival and is often implicated in cancer progression and drug resistance.
Purpose of the Study:
- To investigate the role of nuclear factor-kappa B (NF-kappaB) in regulating the transcription of the bcl-x gene in the drug-resistant leukemia cell line HL-60/E6.
- To determine if suppressing NF-kappaB activity can modulate bcl-x(L) mRNA levels.
Main Methods:
- Development of a drug-resistant HL-60/E6 subline by exposure to epirubicin.
- Quantification of bcl-x(L) mRNA levels using RT-PCR after treatment with AS-PS-ODN-RelA.
- Assessment of NF-kappaB-RelA localization and liposome-mediated ODN transfection efficiency via indirect immunofluorescence and flow cytometry (FCM).
Main Results:
- NF-kappaB-RelA was found to be active and localized in the nuclei of HL-60/E6 cells.
- Liposome-mediated transfection of AS-PS-ODN-RelA was highly efficient.
- Treatment with AS-PS-ODN-RelA resulted in a significant decrease (up to 40%) in bcl-x(L) mRNA levels compared to controls.
Conclusions:
- NF-kappaB is involved in the regulation of bcl-x transcription in drug-resistant leukemia cells.
- Targeting NF-kappaB-RelA using antisense technology offers a potential strategy to down-regulate bcl-x(L) mRNA levels and overcome drug resistance.
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