Induction of apoptosis in K562 cells by dominant negative c-myb.
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).


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.
Yi
Ho Keun
HK
Department of Pediatrics, Chonbuk National University Medical School, Jeonju, South Korea.
Nam
Sang Yun
SY
Kim
Jae Cheol
JC
Kim
Jung Soo
JS
Lee
Dae Yeol
DY
Hwang
Pyoung Han
PH
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D002467:Cell Nucleus / Q000502:physiology; D002470:Cell Survival / Q000502:physiology*; D015500:Chloramphenicol O-Acetyltransferase; D053938:DNA Fragmentation; D017931:DNA Primers; D017353:Gene Deletion; D005799:Genes, Dominant; D017930:Genes, Reporter; D020570:Genes, myb / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D051379:Mice; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D014162:Transfection
D017931:DNA Primers; D019253:Proto-Oncogene Proteins c-bcl-2; D015500:Chloramphenicol O-Acetyltransferase