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 Elements

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 p21^WAF1/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-p21^WAF1/CIP1 3*UTR mRNAs. Expression of chimeric luciferase-c-fos and luciferasep21^{WAF1/ 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 crosslinking experiments using HBC cell cytosolic proteins showed multiple complexes with the AU-rich region subfragments of c-fos, as well as c-myc and p21^WAF1/CIP1 mRNAs. OA enhanced binding of a novel M_r ~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 M_r ~75,000 protein to an AU-rich region of the 3'UTRs of the target genes.