Small inhibitory RNA duplexes for Sp1 mRNA block basal and estrogen-induced gene expression and cell cycle progression in MCF-7 breast cancer cells.
Small interfering RNA duplexes containing 21-22 nucleotides that mediate sequence-specific mRNA degradation and inhibitory RNA (iRNA) for Sp1 mRNA were used in this study to investigate the role of Sp1 on basal and hormone-induced growth and transactivation in MCF-7 and ZR-75 human breast cancer cells. Transfection of Sp1 iRNA in MCF-7 or ZR-75 cells for 36-44 h decreased Sp1 protein (50-70%) in nuclear extracts, and immunohistochemical analysis showed that the Sp1 protein in transfected MCF-7 cells was barely detectable. In cell cycle progression studies in MCF-7 cells, decreased Sp1 protein was accompanied by a decrease in cells in the S phase and an increase in cells in G(0)/G(1), and estrogen-induced G(0)/G(1) --> S phase progression was inhibited in cells treated with iRNA for Sp1. Sp1 iRNA also specifically blocked basal and estrogen-induced transactivation in cells transfected with a GC-rich construct linked to a luciferase reporter gene (pSp1(3)), and this was accompanied by decreased Sp1 binding to this GC-rich promoter as determined in gel mobility shift and chromatin immunoprecipitation assays. These results clearly demonstrate the key role of the Sp1 protein in basal and estrogen-induced growth and gene expression in breast cancer cells.
Abdelrahim
Maen
M
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466, USA.
Samudio
Ismael
I
Smith
Roger
R
Burghardt
Robert
R
Safe
Stephen
S
D001943:Breast Neoplasms / Q000378:metabolism*; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D008156:Luciferases / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013997:Time Factors; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D002843:Chromatin; D004967:Estrogens; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D008156:Luciferases