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Induction of apoptosis in K562 cells by dominant negative c-myb
Ho Keun Yi1, Sang Yun Nam, Jae Cheol Kim
1Department of Pediatrics, Chonbuk National University Medical School, Jeonju, South Korea.
Objective:
The aberrant expression of c-myb in leukemic cells suggests that c-myb may play an important role in leukemogenesis. Therefore, disrupting c-myb function might provide a strategy for controlling leukemic cell growth. Use of dominant negative mutants as a strategy for inhibiting oncogene function has attracted considerable attention. The aim of this study was to induce apoptosis in K562 cells by dominant negative c-myb (DN-myb).
Materials And Methods:
We constructed a DN-myb plasmid containing the DNA-binding domain of c-myb and transfected the dominant negative mutant, like its wild-type (WT) counterpart, into K562 cells. Consequently, cell viability and induction of apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nuclear condensation, DNA fragmentation, and Western hybridization analysis for expression of poly(ADP-ribose) polymerase. In addition, the effect of DN-myb on bcl-2 promoter activity and expression of bcl-2 and bcr-abl was studied.
Results:
We observed that DN-myb, cotransfected with WT c-myb and a chloramphenicol acetyltransferase reporter construct containing the bcl-2 promoter, bound competitively to the bcl-2 promoter and significantly decreased the activation of chloramphenicol acetyltransferase induced by WT c-myb. Moreover, the inactivation of transcription induced by DN-myb reduced not only the expression of bcl-2 but also the expression of bcr-abl. Further functional studies focused on the effect of DN-myb on the induction of apoptosis in K562 cells. Transfection of DN-myb into K562 cells caused a significant reduction in cell proliferation when cells were exposed to low concentrations of DNA-damaging agents (approximately 30% of control) and remarkably increased apoptosis.
Conclusions:
Our data demonstrate that disruption of c-myb function by dominant negative c-myb is an effective strategy to induce apoptosis of leukemic cells. The results of these studies support the thesis that dominant negative c-myb gene therapy may be useful for treatment of leukemia patients.
Insights
Dominant negative c-myb (DN-myb) induces apoptosis in K562 leukemic cells by disrupting c-myb function. This approach shows promise for leukemia gene therapy by reducing cell proliferation and increasing apoptosis.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Aberrant c-myb expression is implicated in leukemogenesis.
- Targeting oncogene function with dominant-negative mutants is a promising strategy.
- Dominant-negative c-myb (DN-myb) aims to inhibit oncogenic c-myb activity.
Purpose of the Study:
- To investigate the induction of apoptosis in K562 cells using DN-myb.
- To assess the impact of DN-myb on leukemic cell viability and apoptosis.
- To explore DN-myb's effect on bcl-2 and bcr-abl expression.
Main Methods:
- Constructed a DN-myb plasmid and transfected it into K562 cells.
- Assessed cell viability and apoptosis using MTT assays, nuclear condensation, and DNA fragmentation.
- Analyzed bcl-2 promoter activity and expression of bcl-2 and bcr-abl via Western hybridization.
Main Results:
- DN-myb competitively bound to the bcl-2 promoter, decreasing WT c-myb-induced activation.
- DN-myb transfection significantly reduced K562 cell proliferation and increased apoptosis.
- DN-myb suppressed the expression of both bcl-2 and bcr-abl.
Conclusions:
- Disrupting c-myb function with DN-myb effectively induces leukemic cell apoptosis.
- DN-myb demonstrates potential as a therapeutic agent for leukemia.
- Dominant-negative c-myb gene therapy may offer a novel treatment strategy for leukemia patients.