An Sp1 binding site and the minimal promoter contribute to overexpression of the cytokeratin 18 gene in tumorigenic clones relative to that in nontumorigenic clones of a human carcinoma cell line.
Clones of cells tumorigenic or nontumorigenic in nude mice have been previously isolated from the SW613-S human colon carcinoma cell line. We have already reported that tumorigenic cells overexpress the cytokeratin 18 (K18) gene in comparison with nontumorigenic cells and that this difference is mainly due to a transcriptional regulation. We now report that a 2,532-bp cloned human K18 gene promoter drives the differential expression of a reporter gene in a transient assay. A 62-bp minimal K18 promoter (TATA box and initiation site) has a low but differential activity. Analysis of deletion and substitution mutants as well as hybrid SV40-K18 promoters and reconstructed K18 promoters indicated that an important element for the activity of the K18 promoter is a high-affinity binding site for transcription factor Sp1 located just upstream of the TATA box. This Sp1 binding element, as well as the intron 1 enhancer element, stimulates the basal activity of the minimal promoter through mechanisms that maintain the differential activity. Gel shift assays and the use of an anti-Sp1 antibody have shown that both tumorigenic and nontumorigenic SW613-S cells contain three factors able to bind to the Sp1 binding element site and that one of them is Sp1. A hybrid GAL4-Sp1 protein transactivated to comparable extents in tumorigenic and nontumorigenic cells a reconstructed K18 promoter containing GAL4 binding sites and therefore without altering its differential behavior. These results indicate that the Sp1 transcription factor is involved in the overexpression of the K18 gene in tumorigenic SW613-S cells through its interaction with a component of the basal transcription machinery.
Gunther
M
M
Laboratoire de Génétique Oncologique, URA 1967 CNRS, Institut Gustave Roussy, Villejuif, France.
Frebourg
T
T
Laithier
M
M
Fossar
N
N
Bouziane-Ouartini
M
M
Lavialle
C
C
Brison
O
O
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007633:Keratins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D016329:Sp1 Transcription Factor; D007633:Keratins