BP1 transcriptionally activates bcl-2 and inhibits TNFalpha-induced cell death in MCF7 breast cancer cells.
BACKGROUND
We have previously shown that the Beta Protein 1 (BP1) homeodomain protein is expressed in 81% of invasive ductal breast carcinomas, and that increased BP1 expression correlates with tumor progression. The purpose of our current investigation was to determine whether elevated levels of BP1 in breast cancer cells are associated with increased cell survival.


METHODS
Effects on cell viability and apoptosis of MCF7 cells stably overexpressing BP1 were determined using MTT and Annexin V assays, and through examination of caspase activation. TNFalpha was used to induce apoptosis. The potential regulation of apoptosis-associated genes by BP1 was studied using real-time PCR and western blot analyses. Electrophoretic mobility shift assays, site-directed mutagenesis, and transient assays were performed to specifically characterize the interaction of BP1 with the promoter of the bcl-2 gene.


RESULTS
Stable overexpression of BP1 led to inhibition of apoptosis in MCF7 breast cancer cells challenged with TNFalpha. Increased BP1 resulted in reduced processing and activation of caspase-7, caspase-8, and caspase-9, and inactivation of the caspase substrate Poly(ADP-Ribose) Polymerase (PARP). Increased levels of full-length PARP and a decrease in procaspase-8 were also associated with BP1 overexpression. The bcl-2 gene is a direct target of BP1 since: (i) BP1 protein bound to a consensus binding sequence upstream of the bcl-2 P1 promoter in vitro. (ii) MCF7 cells overexpressing BP1 showed increased levels of bcl-2 mRNA and protein. (iii) Transient assays indicated that increased bcl-2 promoter activity is due to direct binding and modulation by BP1 protein. BP1 expression also prevented TNFalpha-mediated downregulation of bcl-2 mRNA and protein.


CONCLUSIONS
These findings suggest mechanisms by which increased BP1 may impart a survival advantage to breast cancer cells, which could lead to increased resistance to therapeutic agents in patients.
Stevenson
Holly S
HS
Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, MD 20892, USA.
Fu
Sidney W
SW
Pinzone
Joseph J
JJ
Rheey
Jinguen
J
Simmens
Samuel J
SJ
Berg
Patricia E
PE
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D017304:Annexin A5; C105304:DLX4 protein, human; D018398:Homeodomain Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases