Down-regulation of Pten by BCR-ABL. (A) Total mRNA was isolated from parental ENU and ENU-BCR-ABL cells for DNA microarray analysis. The level of Pten mRNA was lower in BCR-ABL–expressing ENU cells than in parental ENU cells. (B) Pten protein level was also lower in Ba/F3-BCR-ABL cells than in parental Ba/F3 cells. Parental Ba/F3 and Ba/F3-BCR-ABL cells were treated with imatinib (IM; 1μM) for 48 and 72 hours, respectively. Protein lysates were analyzed by Western blot by the use of antibodies indicated. Independent experiments were repeated 3 times. (C) The mouse Pten promoter contains only one p53 binding site. (Top) Schematic representation of the mouse Pten genomics locus (GenBank accession number NM_008960). The exons are indicated by the black bars 1 to 9. (Middle) Region directly upstream of the Pten translation start site. The positions of oligonucleotide probes used for mapping the transcription stat site by PCR are indicated. (Bottom) Nucleotide sequence of the p53 binding site identified based on human p53 binding sequence.27 (D) p53 binds to Pten promoter directly. Chip was performed in BaF3 cells to show the binding of p53 to the Pten promoter as described in the Methods section.
