Abstract
Insulin-like growth factor-I (IGF-I) stimulates the growth of MCF-7 breast cancer cells. This study determined the transcriptional activity of an IGF-I-responsive region (IGFRE) of porcine P450 11A (P450scc) after transfection into MCF-7 cells. IGF-I induced transcriptional activity of a porcine P450scc core promoter luciferase construct containing the IGFRE transfected in MCF-7 cells. Electrophoretic mobility shift assay with nuclear protein extract from MCF-7 cells showed two transcription factors binding to the IGFRE. Supershift assay determined that one transcription factor was Sp1. Electrophoretic mobility shift assay and transfection experiments with selected mutations to the IGFRE found that binding of both transcription factors was necessary to confer an IGF-I response. The binding activity of both transcription factors was increased with IGF-I treatment. In conclusion, MCF-7 cells contain Sp1 and another unknown transcription factor, P2, that bind to a known IGFRE (porcine P450scc) and induce reporter gene transcriptional activity with IGF-I treatment. Because Sp1 is a ubiquitous transcription factor, determining the identity of P2 may lead to cell-specific methods to impair breast cancer cell growth as mediated by IGF-I.
Highlights
Studies in human breast cancer cells show that insulin-like growth factors (IGFs)1 act through cell membrane receptors [1, 2] to stimulate cell growth [3, 4]
insulin-like growth factor-I (IGF-I)-stimulated Gene Expression in MCF-7 Cells—The Ϫ2320 porcine P450scc construct, containing the porcine IGFI-responsive regions (IGFREs), showed a significant increase in luciferase activity when transfected into MCF-7 cells with a control plasmid and treated with IGF-I (Fig. 1)
MCF-7 Transcription Factors Binding to the Porcine IGFRE—electrophoretic mobility shift assay (EMSA) with MCF-7 nuclear extract protein and an oligonucleotide of the porcine IGFRE identified two transcription factors binding to the IGFRE (Fig. 2)
Summary
Studies in human breast cancer cells show that insulin-like growth factors (IGFs)1 act through cell membrane receptors [1, 2] to stimulate cell growth [3, 4]. We determined that the porcine P450scc IGFRE could induce IGF-I-stimulated gene expression in MCF-7 cells. We showed that Sp1 and a another unknown transcription factor, P2, bound to the IGFRE and mediated IGF-I-induced gene expression.
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