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Cell Growth & Differentiation Vol. 11, 541-550, October 2000
© 2000 American Association for Cancer Research

Articles

Okadaic Acid-mediated Induction of the c-fos Gene in Estrogen Receptor-negative Human Breast Carcinoma Cells Utilized, in Part, Posttranscriptional Mechanisms Involving Adenosine-Uridine-rich Elements1

Lulu Farhana, Madanamohan Boyanapalli, Sheng-Hung R. Tschang, Rong-Juan Sun, C. K. Alex Hsu, Yu-Xiang Zhang, Joseph A. Fontana and Arun K. Rishi2

John D. Dingell Veterans Affairs Medical Center, Department of Internal Medicine and Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201

Abstract

Signal transduction via modulation of phosphorylation after selective inhibition of protein phosphatase (PP) 1 and/or PP2A appears to play a role in okadaic acid (OA)-mediated effects. Treatment of several estrogen receptor-negative human breast carcinoma (HBC) cells with 100 nM OA resulted in induction of c-fos, c-myc, and cyclin-dependent kinase inhibitor p21WAF1/CIP1 genes. Transfections of various luciferase reporter constructs in HBC cells revealed involvement of activator protein-1-dependent as well as -independent pathways in induction of the c-fos gene by OA. MDA-MB-468 HBC cells were stably transfected with plasmids expressing luciferase, chimeric luciferase- c-fos 3' untranslated region (3'UTR), or chimeric luciferase-p21WAF1/CIP1 3'UTR mRNAs. Expression of chimeric luciferase-c-fos and luciferase-p21WAF1/CIP1 mRNAs was elevated by OA in several independent sublines. Actinomycin D chase experiments revealed an enhanced rate of decay of luciferase-c-fos mRNA, whereas treatment with OA caused ~3.5-fold enhanced stability of the chimeric luciferase-c-fos mRNA only. By transfecting different plasmids containing deletions of c-fos 3'UTR, OA-responsive sequences were mapped to an 86-nucleotide, AU-rich region. UV cross-linking experiments using HBC cell cytosolic proteins showed multiple complexes with the AU-rich region subfragments of c-fos, as well as c-myc and p21WAF1/CIP1 mRNAs. OA enhanced binding of a novel Mr ~75,000 protein present in the cytosolic extracts of HBC cells to the AU-rich RNA probes of all of the above three genes. Taken together, OA regulation of HBC cell gene expression involves the activator protein-1 pathway, as well as enhanced binding of a novel Mr ~75,000 protein to an AU-rich region of the 3'UTRs of the target genes.




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