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Carcinogenesis Advance Access first published online on September 8, 2005
This version published online on September 12, 2005
Carcinogenesis, doi:10.1093/carcin/bgi225
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oupjournals.org
Received May 2, 2005
Revised August 9, 2005
Accepted September 5, 2005

CANCER BIOLOGY

7,12-Dimethylbenzanthracene-dependent transcriptional regulation of adenomatous polyposis coli (APC) gene expression in normal breast epithelial cells is mediated by GC-box binding protein Sp3

Aruna S. Jaiswal 1 2, Ramesh Balusu 1 2, and Satya Narayan 1*

1 Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, FL 32610

* To whom correspondence should be addressed.
Satya Narayan, E-mail: SNarayan{at}ufscc.ufl.edu


  Abstract

In the present investigation, we have examined the transcriptional regulation of adenomatous polyposis coli (APC) gene expression in the spontaneously immortalized human normal breast epithelial cell line, MCF10A, in response to carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) treatment. The APC mRNA levels and the APC gene's promoter (pAPCP) activity were increased in MCF10A cells after treatment with DMBA. A sequential deletion analysis and site-directed mutagenesis of the pAPCP promoter revealed that the DMBA response is mediated through a GC-box element. Also, the GC-box binding agent mithramycin A, which prevents binding of proteins to the GC-box region, abolished DMBA-mediated increase of the pAPCP promoter activity. The specificity of the proteins binding to the GC-box region was characterized by gel-shift analysis. An increased binding of the GC-box binding proteins was observed in the gel-shift analysis with nuclear extracts from DMBA-treated MCF10A cells, which corresponded to the increased levels of Sp1 and Sp3 proteins. However, a super-shift of the DNA-protein complexes was observed with only anti-Sp3 antibody. Based on the ChIP assay results, the Sp3 appeared to be a genuine protein binding to the GC-box site of the pAPCP promoter. In RNA interference experiments, in which the Sp3 expression was knocked down, the DMBA response on the pAPCP promoter activity was reduced, suggesting that the binding of Sp3 to the GC-box site is critical for DMBA-induced pAPCP promoter activity. From these results we conclude that the increased pAPCP promoter activity in the MCF10A cell line in response to DMBA treatment is mediated by Sp3.

Keywords: Adenomatous polyposis coli; chromatin-immunoprecipitation assay; DMBA; EMSA; GC-box-binding proteins; normal breast epithelial cells; SiRNA; transcriptional regulation.

2 These authors contributed equally to this manuscript


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