Removal of Cdk inhibitors through both sequestration and downregulation in zearalenone‐treated MCF‐7 breast cancer cells

Treatment of MCF 7 cells with the fungal estrogen zearalenone induced cyclin E–associated kinase activity transiently within 9–12 h; total cyclin‐dependent kinase (Cdk) 2 activity was elevated for 24 h and beyond. This increased cyclin E/Cdk2 activity was associated with sequestration of the Cdk inhibitor p27 Cdk inhibitor 1B (p27 KIP1 ) by newly formed cyclin D1/Cdk4 complexes and with downregulation of p27 KIP1 expression. The activation of cyclin A/Cdk2 activity corresponded with virtual elimination of p27 KIP1 . The activity of cyclin E/Cdk2 complexes from zearalenone‐treated lysates was inhibited in vitro by recombinant p27 KIP1 , and this inhibition was relieved by the addition of recombinant cyclin D1/Cdk4 complexes. Thus, sequestration of p27 KIP1 by cyclin D1/Cdk4 resulted in activation of Cdk2 in vitro. Cdk inhibitory activity in lysates of zearalenone‐treated cells was depleted by anti‐p27 KIP1 and anti‐Cdc2 interacting protein (p21 CIP1 ) antibodies. Overexpression of the Cdk4/6‐specific Cdk inhibitor of Cdk4 p16 INK4A was associated with increased association of p27 KIP1 with Cdk2, concomitant with disruption of D cyclin/Cdk4 complexes. The proteasome inhibitor 2‐leu‐leu‐leu‐H aldehyde (MG‐132) was relatively ineffective in inhibiting the initial, sequestration‐dependent activation of cyclin E/Cdk2 yet was as effective as p16 INK4A in inhibiting activation of cyclin A/Cdk2 later in G 1 . Downregulation of p27 KIP1 proceeded in p16 INK4A ‐expressing cells after zearalenone treatment, and G 1 arrest afforded by p16 INK4A expression was reversible upon prolonged treatment with zearalenone. Zearalenone treatment of MCF‐7 cells elicited expression of F‐box protein S phase kinase–associated protein 2 (p45 SKP2 ), a substrate‐specific component of the ubiquitin‐ligase complex that targets p27 KIP1 for degradation in the proteasome. These studies suggest that both sequestration of Cdk inhibitors by cyclin D1/Cdk4 complexes and downregulation of p27 KIP1 play major roles in the induction of Cdk2 activity and S phase entry elicited by estrogens in MCF‐7 cells. © 2002 Wiley‐Liss, Inc.