Transcriptional activity of RUNX1 isoforms p48, p38a, p30, and p24 on the CD56 promoter. (A) Upper panel: HEK293 cells were cotransfected with a CD56 promoter/luciferase reporter gene and the RUNX1 isoforms. RUNX1 p48 strongly up-regulates CD56 expression. RUNX1 p48-induced expression of CD56 is inhibited by p38, p30, and p24 (columns 7-9), suggesting a dominant negative effect of the latter isoforms. Controls are NCAM/CD56 promoter/luciferase reporter gene only and NCAM/CD56 promoter/luciferase reporter gene plus “empty” RSV expression vector (columns 1 and 2). (Lower panel) Confirmation of RUNX1 expression by Western blot. Similar amounts of the various recombinant RUNX1 isoforms were detected in the transfected cells by a RUNT domain antibody. GAPDH, protein loading control. (B) Expression of RUNX1 target genes in acute myeloid leukemia cell lines. CD56, granzyme B, p21, interleukin-6, and granulocyte-macrophage colony-stimulating factor were detected by reverse transcriptase–polymerase chain reaction in human acute myeloid leukemia cell lines K562, HL60, and U937 transfected with RUNX1 isoforms p48, p38a, p30, and p24. All target genes were strongly induced by p48; p30 had approximately no effect on CD56 and minor inducing effects on the other targets. p38a and p24 inhibited expression of all target genes relative to endogenous expression in vector control (VC) or nontransfected cells (–). Data are means plus or minus SD.