Down-regulation of cyclin D1 expression by prostaglandin A(2) is mediated by enhanced cyclin D1 mRNA turnover

Prostaglandin A(2) (PGA(2)), an experimental chemotherapeutic agent, causes growth arrest associated with decreased cyclin D1 expression in several ca ncer cell lines. Here, using human non-small-cell lung carcinoma H1299 cell s, we investigated the mechanisms whereby PGA(2) down-regulates cyclin D1 e xpression. Transcription rates of the cyclin D1 gene, studied using a cycli n D1 promoter-luciferase construct and nuclear run-on assays, were not affe cted by PGA(2) treatment. Instead, the cyclin D1 mRNA was rendered unstable after exposure to PGA(2). Since the stability of labile mRNA is modulated through binding of proteins to specific mRNA sequences, we sought to identi fy protein(s) recognizing the cyclin D1 mRNA. In electrophoretic mobility-s hift assays using radiolabeled RNA probes derived from different regions of cyclin D1 mRNA,we observed that (i) lysates prepared from PGA(2)-treated c ells exhibited enhanced protein-cyclin D1 RNA complex formation; (ii) the k inetics of complex formation correlated closely,vith that of cyclin D1 mRNA loss; and (iii) binding occurred within a 390-base cyclin D1 3' untranslat ed region (UTR) (K12). This binding activity could be cross-linked, reveali ng proteins ranging from 30 to 37 kDa. The RNA-binding protein AUF1, previo usly associated with the degradation of target mRNAs, bound cyclin D1 mRNA, because anti-AUF1 antibodies were capable of supershifting or immunoprecip itating cyclin D1 mRNA protein complexes. Finally, insertion of K12 in the 3'UTR of reporter genes markedly reduced the expression and half-life of th e resulting chimeric mRNAs in transfected, PGA(2)-treated cells. Our data d emonstrate that PGA(2) down-regulates cyclin D1 expression by decreasing cy clin D1 mRNA stability and implicates a 390-base element in the 3'UTR in th is regulation.