mitopodozide and Breast-Neoplasms

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.

Topics: Binding Sites; Blotting, Western; Breast Neoplasms; DNA Primers; DNA-Binding Proteins; DNA, Neoplasm; Electrophoretic Mobility Shift Assay; Epithelial Cells; Female; Gene Deletion; Gene Expression Regulation, Neoplastic; Helix-Loop-Helix Motifs; Histone Deacetylase 1; Histone Deacetylases; Humans; Inhibitor of Differentiation Protein 1; Mutation; Neoplasm Invasiveness; Neurofibromin 1; Plasmids; Podophyllin; Podophyllotoxin; Precipitin Tests; Promoter Regions, Genetic; Repressor Proteins; Retinoblastoma Protein; Transcription Factors; Transfection; Tumor Cells, Cultured