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Molecular and Cellular Biology, February 2001, p. 794-810, Vol. 21, No. 3
Department of Obstetrics and Gynecology,
Graduate School of Medicine, University of Tennessee Medical Center,
Knoxville, Tennessee 37920,1 The
Medicine Branch, National Cancer Institute, National Institutes of
Health, Bethesda, Maryland 208922
Received 18 July 2000/Returned for modification 25 August
2000/Accepted 9 November 2000
Estrogens induce proliferation of estrogen receptor (ER)-positive
MCF-7 breast cancer cells by stimulating G1/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the
retinoblastoma protein (pRb). We have utilized blockade of cyclin
D1-Cdk4 complex formation through adenovirus-mediated expression of
p16INK4a to demonstrate that estrogen regulates Cdk
inhibitor expression and expression of the Cdk-activating phosphatase
Cdc25A independent of cyclin D1-Cdk4 function and cell cycle
progression. Expression of p16INK4a inhibited
G1/S transition induced in MCF-7 cells by 17-
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.3.794-810.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Multifaceted Regulation of Cell Cycle Progression
by Estrogen: Regulation of Cdk Inhibitors and Cdc25A Independent of
Cyclin D1-Cdk4 Function
-estradiol (E2) with associated inhibition of both Cdk4- and
Cdk2-associated kinase activities. Inhibition of Cdk2 activity was
associated with delayed removal of Cdk-inhibitory activity in early
G1 and decreased cyclin A expression. Cdk-inhibitory
activity and expression of both p21Cip1 and
p27Kip1 was decreased, however, in both control and
p16INK4a-expressing cells 20 h after estrogen
treatment. Expression of Cdc25A mRNA and protein was induced by
E2 in control and p16INK4a-expressing MCF-7
cells; however, functional activity of Cdc25A was inhibited in cells
expressing p16INK4a. Inhibition of Cdc25A activity in
p16INK4a-expressing cells was associated with depressed
Cdk2 activity and was reversed in vivo and in vitro by active Cdk2.
Transfection of MCF-7 cells with a dominant-negative Cdk2 construct
inhibited the E2-dependent activation of ectopic Cdc25A.
Supporting a role for Cdc25A in estrogen action, antisense
CDC25A oligonucleotides inhibited estrogen-induced Cdk2
activation and DNA synthesis. In addition, inactive cyclin E-Cdk2
complexes from p16INK4a-expressing, estrogen-treated cells
were activated in vitro by treatment with recombinant Cdc25A and in
vivo in cells overexpressing Cdc25A. The results demonstrate that
functional association of cyclin D1-Cdk4 complexes is required for Cdk2
activation in MCF-7 cells and that Cdk2 activity is, in turn, required
for the in vivo activation of Cdc25A. These studies establish Cdc25A as
a growth-promoting target of estrogen action and further indicate that
estrogens independently regulate multiple components of the cell cycle
machinery, including expression of p21Cip1 and
p27Kip1.
*
Corresponding author. Mailing address: Department of
Obstetrics and Gynecology, University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN 37920. Phone: (865) 544-8960. Fax: (423)
544-6863. E-mail: mcf7{at}msn.com.
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