title: Induction of apoptosis in K562 cells by dominant negative c-myb.
abstract: 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.
authors.affiliation: Department of Pediatrics, Chonbuk National University Medical School, Jeonju, South Korea.
authors.forename: Ho Keun
authors.initials: HK
authors.lastname: Yi
authors.forename: Sang Yun
authors.initials: SY
authors.lastname: Nam
authors.forename: Jae Cheol
authors.initials: JC
authors.lastname: Kim
authors.forename: Jung Soo
authors.initials: JS
authors.lastname: Kim
authors.forename: Dae Yeol
authors.initials: DY
authors.lastname: Lee
authors.forename: Pyoung Han
authors.initials: PH
authors.lastname: Hwang
mesh_terms: 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
chemical_list: D017931:DNA Primers; D019253:Proto-Oncogene Proteins c-bcl-2; D015500:Chloramphenicol O-Acetyltransferase