C/EBPbeta-mediated transcriptional regulation of bcl-xl gene expression in human breast epithelial cells in response to cigarette smoke condensate.
In earlier studies, we have shown that cigarette smoke condensate (CSC), a surrogate for cigarette smoke, is capable of transforming the spontaneously immortalized human breast epithelial cell line, MCF10A. These transformed cells displayed upregulation of the anti-apoptotic gene, bcl-xl. Upregulation of this gene may impede the apoptotic pathway and allow the accumulation of DNA damage that can lead to cell transformation and carcinogenesis. In the present study, we have determined the mechanism of CSC-mediated transcriptional upregulation of bcl-xl gene expression in MCF10A cells. We cloned the human bcl-xl promoter (pBcl-xLP) and identified putative transcription factor binding sites. Sequential deletion constructs that removed the putative cis-elements were constructed and transfected into MCF10A cells to determine the CSC-responsive cis-element(s) on the pBcl-xLP. Gel-shift, super-shift and chromatin immunoprecipitation analysis confirmed that CCAAT/enhancer-binding protein (C/EBPbeta) specifically bound to a C/EBP-binding site on the pBcl-xLP in vitro and in vivo. Additionally, overexpression of C/EBPbeta-LAP2 stimulated pBcl-xLP activity and Bcl-xL protein levels, which mimicked the conditions of CSC treatment. Our results indicate that C/EBPbeta regulates bcl-xl gene expression in MCF10A cells in response to CSC treatment; therefore, making it a potential target for chemotherapeutic intervention of cigarette smoke-induced breast carcinogenesis.
Connors
S K
SK
Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, FL 32610, USA.
Balusu
R
R
Kundu
C N
CN
Jaiswal
A S
AS
Gairola
C G
CG
Narayan
S
S
D001483:Base Sequence; D001665:Binding Sites; D001940:Breast / Q000378:metabolism*; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D045744:Cell Line, Tumor; D004249:DNA Damage; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012906:Smoke; D012907:Smoking; D051020:bcl-X Protein / Q000096:biosynthesis*
D022782:CCAAT-Enhancer-Binding Protein-beta; D012906:Smoke; D051020:bcl-X Protein