Functional knock-out of c-myb by an intracellular anti-c-Myb single-chain antibody.
Aberrant expression of the c-myb proto-oncogene is a key factor in the development of the neoplastic phenotype in a variety of contexts. On this basis, it has been proposed that ablation of c-myb function might be an effective approach for therapy. To this end, we have employed an intracellular single-chain antibody (sFv) approach to achieve the functional knock-out of the c-Myb onco-protein. We derived an anti-c-Myb sFv, which was configured into eukaryotic expression plasmids. We confirmed the expression of the cytoplasmic and nuclear forms of the sFvs in the correct subcellular compartments by immunofluorescent staining. Importantly, the anti-c-Myb sFvs strongly inhibited the transactivation activity of c-Myb. Furthermore, cytotoxic effect of the sFv was observed only in the c-Myb positive cell line K562. These results suggest that anti-c-Myb sFv is a valuable tool for understanding the molecular mechanisms of c-myb induced transformation. In addition, this approach may have potential utility in the gene therapy for c-myb-dependent malignant diseases.
Kasono
K
K
Gene Therapy Program, University of Alabama at Birmingham, 1824 6th Avenue South, WTI620, Birmingham, Alabama, 35294, USA.
Piché
A
A
Xiang
J
J
Kim
H G
HG
Bilbao
G
G
Johanning
F
F
Nawrath
M
M
Moelling
K
K
Curiel
D T
DT
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002999:Clone Cells; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D005822:Genetic Vectors / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D007424:Intracellular Fluid / Q000276:immunology*; D020014:K562 Cells; D008969:Molecular Sequence Data; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000502:physiology; D020598:Proto-Oncogene Proteins c-myb; D013997:Time Factors; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000502:physiology
D004268:DNA-Binding Proteins; D007135:Immunoglobulin Variable Region; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D015534:Trans-Activators