Farhana L, Boyanapalli M, Tschang S H, Sun R J, Hsu C K, Zhang Y X, Fontana J A, Rishi A K
Department of Internal Medicine and Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201, USA.
Cell Growth Differ. 2000 Oct;11(10):541-50.
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/CIP 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 approximately 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 approximately 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 approximately 75,000 protein to an AU-rich region of the 3'UTRs of the target genes.
在选择性抑制蛋白磷酸酶（PP）1和/或PP2A后，通过磷酸化调节进行的信号转导似乎在冈田酸（OA）介导的效应中发挥作用。用100 nM OA处理几种雌激素受体阴性的人乳腺癌（HBC）细胞，导致c-fos、c-myc和细胞周期蛋白依赖性激酶抑制剂p21WAF1/CIP1基因的诱导。在HBC细胞中对各种荧光素酶报告构建体进行转染，揭示了OA诱导c-fos基因涉及激活蛋白-1依赖性和非依赖性途径。MDA-MB-468 HBC细胞用表达荧光素酶、嵌合荧光素酶-c-fos 3'非翻译区（3'UTR）或嵌合荧光素酶-p21WAF1/CIP 3'UTR mRNA的质粒进行稳定转染。在几个独立的亚系中，OA提高了嵌合荧光素酶-c-fos和荧光素酶-p21WAF1/CIP1 mRNA的表达。放线菌素D追踪实验显示荧光素酶-c-fos mRNA的衰减速率加快，而用OA处理仅使嵌合荧光素酶-c-fos mRNA的稳定性提高约3.5倍。通过转染含有c-fos 3'UTR缺失的不同质粒，OA反应序列被定位到一个86个核苷酸的富含AU的区域。使用HBC细胞胞质蛋白进行的紫外线交联实验显示，与c-fos、c-myc和p21WAF1/CIP1 mRNA的富含AU区域亚片段形成多个复合物。OA增强了HBC细胞胞质提取物中一种新的约75,000 Mr蛋白与上述所有三个基因的富含AU的RNA探针的结合。综上所述，OA对HBC细胞基因表达的调节涉及激活蛋白-1途径，以及一种新的约75,000 Mr蛋白与靶基因3'UTR富含AU区域的增强结合。
