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.