Abstract
Transformation by the Bcr-Abl oncoprotein of CML is mediated by the activation of a variety of signalling pathways, leading to transcriptional regulation of genes conferring the malignant phenotype of increased proliferation, altered adhesion and inhibition of apoptosis. We previously reported that expression of the BACH2 gene is downregulated by Bcr-Abl. Bach2 is a B-lymphoid specific transcription factor, which regulates somatic hypermutation and class switch recombination of Ig genes. It is also a pro-apoptotic factor, coupling oxidative stress to transcription repression. It is possible that in an environment of increased genomic instability, Bcr-Abl transformed cells may repress pro-apoptotic signals by suppressing BACH2 transcription. To determine the direct association between Bcr-Abl and decreased BACH2 transcription, we infected human B-lymphoid cells with a retroviral vector expressing both p210Bcr-Abl and eGFP genes. Infected cells were treated with imatinib, an Abl tyrosine kinase inhibitor, prior to quantification of BACH2 transcripts by Real Time RT/PCR. Ectopic expression of BCR-ABL significantly decreased BACH2 mRNA levels, and this effect was completely abolished by imatinib. To investigate whether this regulation was exerted at the transcriptional level, we identified the BACH2 transcription initiation site (TIS), and then cloned and characterised a 3.9 Kb genomic DNA fragment including the BACH2 promoter region. By generating luciferase reporter constructs of various lengths of the BACH2 promoter we found that a region of 725 bp upstream the TIS conferred maximum promoter activity in human B-lymphoid cells. The effect of Bcr-Abl on promoter activity was demonstrated by co-transfection of the reporter and p210Bcr-Abl constructs. BACH2-promoter activity was reduced up to 60% in the presence of Bcr-Abl. Furthermore, when co-transfected cells were incubated with different concentrations of imatinib, the Bcr-Abl-mediated promoter repression was abrogated in a dose dependent manner, confirming the dependence of the effect on the tyrosine kinase activity of the oncoprotein. In support of these data, no effect on promoter activity was seen when the BACH2 promoter was co-transfected with a kinase-dead BCR-ABL construct. Moreover, treatment with imatinib of the BCR-ABL+ cell line BV173 transfected with the reporter induced a nearly 2-fold upregulation in its activity. Bioinformatics inspection of the promoter sequence revealed potential sites for the Pax5 B-cell differentiation factor and the Foxo3a transcription factor, a regulator of pro-apoptotic genes. In co-transfection experiments of either factor with the BACH2 promoter, both demonstrated a significant inducing effect on its activity. Gel shift and chromatin immunoprecipitation showed direct binding of Pax5 within the BACH2 promoter in vitro and in vivo. Moreover, Western analysis showed elevated Pax5 levels in BCR-ABL+ cell lines after imatinib treatment, indicating that inhibition of Bach2 expression by Bcr-Abl is mediated at least in part by Pax5. As to Foxo3a, it has been reported to be constitutively phosphorylated and inactivated in BCR-ABL+ cells, processes which prevent its translocation to the cell nucleus. Altogether, our data suggest that Bcr-Abl transcriptional repression of Bach2 via Pax5 could lead to a differentiation arrest in transformed B-cells, and that Foxo3a may induce imatinib-mediated apoptosis through up-regulation of the Bach2 apoptotic function.
The P190, P210, and P230 Forms of the BCR/ABL Oncogene Induce a Similar Chronic Myeloid Leukemia–like Syndrome in Mice but Have Different Lymphoid Leukemogenic Activity
