The aim of this study was to test the ability of the BP1 homeobox protein to directly regulate transcription of bcl-2 in breast cancer cells. We have previously shown that BP1 mRNA was overexpressed in 80% of invasive ductal carcinomas. We hypothesize that BP1 is a potential modulator of cell survival. Recently, it was demonstrated that MCF-7 cells generated to stably overexpress BP1 showed enhanced viability in the presence of TNFα, an inducer of cell death. Overexpression of BP1 also resulted in increased mRNA and protein levels of bcl-2, an anti-apoptotic gene. BP1 protein binds in vitro to a consensus binding sequence located near the bcl-2 P1 promoter region at -2539. To test whether this binding has a functional significance, transient assays were performed to determine if BP1 could directly activate bcl-2 expression. Two constructs containing the bcl-2 promoter linked to the luciferase reporter gene, LB170 or LB322, were separately transfected into MCF-7 cell lines constitutively expressing BP1 or containing an empty vector. Both constructs carry the BP1 binding site. LB170 spans the P1 promoter and surrounding sequence, whereas LB322 additionally contains the downstream P2 promoter. BP1 overexpressing cell lines consistently exhibited up to a 2.5-fold activation of the bcl-2 P1 promoter. Upon mutating a 7-nucleotide region of the BP1 binding site, we observed loss of binding of BP1 protein to bcl-2 as revealed by electrophoretic mobility shift assays. Using LB170 as template, site-directed mutagenesis was performed to generate a construct lacking the BP1 binding site. Deletion of this site decreased activation of the bcl-2 promoter by approximately 2-fold. Thus, these results demonstrate that BP1 protein modulates upregulation of bcl-2 expression through binding. These data are consistent with the protective effect of constitutive BP1 in MCF-7 cells challenged with TNFα.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]