Molecular mechanisms of zearalenone-induced G1/S phase transition in MCF-7 breast carcinoma cells

Zearalenone is a naturally occurring estrogenic contaminant of moldy feeds and is present in high concentrations m dairy products and cereals Zearalenone has been postulated to contribute to the overall estrogen load of women,however,the mechanisms of its action are not known We demonstrate the ability of zearalenone to stimulate growth of the estrogen-receptor positive human breast carcinoma cell line, MCF-7 Zearalenone has multiple effects in the regulation of the MCF-7 cell cycle Treatment of these cells with zearalenone induced cell cycle transit following increases in expression of Myc and the cyclins D1,A,and Bl,as well as downregulation of p27G1/G2 phase kmase activity and phosphorylation of the retinoblastoma gene product(Rb) was evident as well Flow cytometric analysis demonstrated entry of cells into S and G2/M phases of the cell cycle, and phosphorylation of Histone H3(HH3) occurred by 36 hr of zearalenone treatment. We further examined how zearalenone treatment induces cyclin E-associated kmase activity within 9-12 hr This increase m activity could be due to the sequestration of p27 by newly formed cyclm Dl/Cdk4 complexes and or due to zearalenone-induced downregulation ofp27 The Activity of cyclm E/Cdk2 complexes from zearalenone treated lysates could be inhibited in a dose dependent manner by His-tagged p27 This inhibition was relieved by addition of increasing levels of recombinant cyclm Dl/Cdk4 complexes Boiling released latent inhibitory activity from lysates of cells treated with zearalenone, and could be depleted by anti-p27 and anti-p21 antibodiesOverexpression ofthe Cdk4/6 specific inhibitor, pl6, was associated with increased association ofp27 with Cdk2 concomitant with disruption of D cyclm Cdk4 complexes Nevertheless, in pi6-arrested cells downregulation of p27 by zearalenone was present and resulted in cells entering S phase after a lag of 18-24 hr These data suggest that sequestration of CKI by cyclm Dl/Cdk4 complexes as well as downregulation of p27 protein play major roles m the induction of cyclin E-associated kmase activity and in S phase entry m zearalenone-treated MCF-7 cells