Abstract
The helix-loop-helix protein Id-1 is a dominant negative regulator of basic helix-loop-helix transcription factors, and plays a key role in the control of breast epithelial cell growth, invasion and differentiation. Previous investigations in our laboratory have shown that Id-1 mRNA was constitutively expressed in highly aggressive and invasive human breast cancer cells in comparison to non-transformed or non-aggressive cancerous cells, and that this loss of regulation is mediated by a 2.2-kb region of the human Id-1 promoter. Here we show that a 31 bp sequence within this 2.2-kb promoter, located 200 bp upstream of the initiation of transcription, is responsible for the constitutive expression of Id-1 in metastatic human breast cancer cells. Using gel shift experiments, we identified a high molecular weight complex present only in non-aggressive breast cancer cells cultured in serum-free medium and which appear to be necessary for proper Id-1 repression. In contrast, nuclear extracts from highly aggressive and metastatic cell lines do not contain this large molecular weight complex. Using DNA affinity precipitation assays (DAPA), we show that this complex contains SP-1, NF-1, Rb and HDAC-1 proteins. On the basis of these findings, we propose a mechanism for the loss of regulation of Id-1 promoter in invasive and metastatic human breast cancer cells.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Binding Sites
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Blotting, Western
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Breast Neoplasms / genetics*
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Breast Neoplasms / metabolism
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DNA Primers / chemistry
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DNA, Neoplasm / genetics
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DNA, Neoplasm / metabolism
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DNA-Binding Proteins / genetics*
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DNA-Binding Proteins / metabolism
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Electrophoretic Mobility Shift Assay
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Epithelial Cells / cytology
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Epithelial Cells / metabolism
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Female
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Gene Deletion
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Gene Expression Regulation, Neoplastic
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Helix-Loop-Helix Motifs / genetics
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Histone Deacetylase 1
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Histone Deacetylases / genetics*
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Histone Deacetylases / metabolism
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Humans
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Inhibitor of Differentiation Protein 1
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Mutation / genetics
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Neoplasm Invasiveness
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Neurofibromin 1 / genetics*
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Neurofibromin 1 / metabolism
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Plasmids
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Podophyllin / analogs & derivatives*
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Podophyllin / genetics
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Podophyllin / metabolism
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Podophyllotoxin / analogs & derivatives
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Precipitin Tests
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Promoter Regions, Genetic / genetics*
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Repressor Proteins*
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Retinoblastoma Protein / genetics*
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Retinoblastoma Protein / metabolism
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Transcription Factors / genetics*
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Transcription Factors / metabolism
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Transfection
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Tumor Cells, Cultured
Substances
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DNA Primers
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DNA, Neoplasm
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DNA-Binding Proteins
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ID1 protein, human
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Inhibitor of Differentiation Protein 1
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Neurofibromin 1
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Repressor Proteins
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Retinoblastoma Protein
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Transcription Factors
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mitopodozide
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Podophyllin
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HDAC1 protein, human
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Histone Deacetylase 1
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Histone Deacetylases
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Podophyllotoxin