Constitutive expression of the Id-1 promoter in human metastatic breast cancer cells is linked with the loss of NF-1/Rb/HDAC-1 transcription repressor complex.
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.
Singh
Jarnail
J
Geraldine Brush Cancer Research Institute, California Pacific Medical Center, San Francisco, California, CA 94115, USA.
Murata
Kenji
K
Itahana
Yoko
Y
Desprez
Pierre-Yves
PY
D001665:Binding Sites; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D056284:Histone Deacetylase 1; D006655:Histone Deacetylases / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1; D009154:Mutation / Q000235:genetics; D009361:Neoplasm Invasiveness; D025542:Neurofibromin 1 / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011033:Podophyllin / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D011034:Podophyllotoxin / Q000031:analogs & derivatives; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012097:Repressor Proteins; D016160:Retinoblastoma Protein / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C497453:ID1 protein, human; D051798:Inhibitor of Differentiation Protein 1; D025542:Neurofibromin 1; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D014157:Transcription Factors; C003170:mitopodozide; D011033:Podophyllin; C110550:HDAC1 protein, human; D056284:Histone Deacetylase 1; D006655:Histone Deacetylases; D011034:Podophyllotoxin