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RUNX1 transactivated and interacted with KLF4
tion, migration, and survival in AML.20 Loss of Runx1 down-regulated p19ARF to accelerate the development of MLL-ENL leukemia.21 Besides, RUNX1 co-operated with PU.1 to activate hematopoietic differentiation genes, such
as MCSF, and contributed to the downregulation of the erythroid gene expression program by repressing MIR451 transcription.22,23 Likewise, KLF4 overexpression inhibited cell proliferation of AML cell lines through regulation of
A
BCD
E
Figure 7. P57 is involved in “RUNX1-KLF4-P57” transcriptional activation cascade, which promotes cell proliferation inhibition and apoptosis induction of t(8;21) leukemia cells. (A) Schematic representation of P57 promoter fragments fused to a pGL3-Basic vector. The putative KLF4 binding sites are indicated by yellow boxes. Transcription start site (TSS) is indicated by an arrow. Numbers represent base pairs relative to TSS. (B) pGL3-P57 promoter reporter plasmid (pGL3-P57) was co- transfected with increasing doses of pCMV5-KLF4 into CV-1 cells. At 48 hours (h) after transfection, the luciferase transcriptional activity of pGL3-P57 was measured and normalized to that of Renilla luciferase. (C and D) Kasumi-1 cells were infected with pCDH lentivirus over-expressing KLF4 (C) and RUNX1 (D), respectively. At 72 h after infection, the protein levels of putative downstream genes P57 and KLF4 were evaluated by western blot assay. (E) Overexpression of P57 in Kasumi-1 cells was mediated by a pCDH lentivirus system. At 72 h after infection, the infected cells were sorted for GFP+ population and the overexpression efficiency was con- firmed by quantitative real-time polymerase chain reaction and western blot assay, respectively. Then, the biological effects of P57 on cell proliferation, cell cycle dis- tribution, cell apoptosis and differentiation were evaluated by MTS assay, flow cytometry, and cell morphological analysis (see Figure 6).
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