Multifaceted Regulation of Cell Cycle Progression by Estrogen: Regulation of Cdk Inhibitors and Cdc25A Independent of Cyclin D1-Cdk4 Function

Department of Obstetrics and Gynecology, Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, Tennessee 37920, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 03/2001; 21(3):794-810. DOI: 10.1128/MCB.21.3.794-810.2001
Source: PubMed


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-β-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, antisenseCDC25A 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.

Download full-text


Available from: James S Foster, Jan 05, 2015
  • Source
    • "For Western blot analysis [21], cells growing at ~80% confluence in 100 mm dishes were washed in cold PBS and lysed in RIPA buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 0.5% Triton X-100, 0.2% sodium deoxycholate, 0.15% SDS, 1mM sodium orthovanadate, 5 mM sodium fluoride, 5 mM β-glycerophosphate and 0.5 mM PMSF) followed by centrifugation at 15,000 × g for 20 min at 4°C. Lysate protein concentrations were determined by BCA protein assay (Thermo-Fisher-Pierce, Rockwood, IL) and equal 50-100 μg amounts (control vs. ODAM-expressing cultures) were electrophoresed in 10% Bis-Tris gels (Invitrogen) and blotted to PVDF membranes. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background The Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity. Herein we have extended these studies to the effects of ODAM on cultured melanoma cell lines. Methods The A375 and C8161 melanoma cell lines were stably transfected with ODAM and assayed for properties associated with tumorigenicity including cell growth, motility, and extracellular matrix adhesion. In addition, ODAM–transfected cells were assayed for signal transduction via AKT which promotes cell proliferation and survival in many neoplasms. Results ODAM expression in A375 and C8161 cells strongly inhibited cell growth and motility in vitro, increased cell adhesion to extracellular matrix, and yielded significant cytoskeletal/morphologic rearrangement. Furthermore, AKT activity was downregulated by ODAM expression while an increase was noted in expression of the PTEN (phosphatase and tensin homolog on chromosome 10) tumor suppressor gene, an antagonist of AKT activation. Increased PTEN in ODAM-expressing cells was associated with increases in PTEN mRNA levels and de novo protein synthesis. Silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing melanoma cells. Similar PTEN elevation and inhibition of AKT by ODAM was observed in MDA-MB-231 breast cancer cells while ODAM expression had no effect in PTEN-deficient BT-549 breast cancer cells. Conclusions The apparent anti-neoplastic effects of ODAM in cultured melanoma and breast cancer cells are associated with increased PTEN expression, and suppression of AKT activity. This association should serve to clarify the clinical import of ODAM expression and any role it may serve as an indicator of tumor behavior.
    Full-text · Article · May 2013 · BMC Cancer
  • Source
    • "In ER-positive breast cancer treatment, ER antagonists are effective at stopping cell division, indicating that such tumors are dependent upon estrogen for proliferation and survival (Musgrove and Sutherland, 2009). It was further shown that estrogen inhibition results in cell cycle arrest in the G0/ G1 phase of the cell cycle through attenuation of CDK/cyclin complexes at multiple levels (Foster et al., 2001). In particular, cyclin D1 is a direct transcriptional target of ER signalling (Eeckhoute et al., 2006). "

    Full-text · Chapter · Mar 2012
  • Source
    • "ICI 182 780 or Tamoxifen) results in an arrest in the G0/G1 phase of the cell cycle (Watts et al. 1995, Carroll et al. 2000, Foster et al. 2001, Markey et al. 2007). In this context, reduced ER signaling leads to the attenuation of CDK/cyclin complexes at multiple levels (Watts et al. 1995, Carroll et al. 2000, Foster et al. 2001). Most dramatically, cyclin D1 is a known and direct transcriptional target of the ER signaling network (Watts et al. 1994, Eeckhoute et al. 2006). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The majority of estrogen receptor (ER)-positive breast cancers are treated with endocrine therapy. While this is effective, acquired resistance to therapies targeted against ER is a major clinical challenge. Here, model systems of ER-positive breast cancers with differential susceptibility to endocrine therapy were employed to define common nodes for new therapeutic interventions. These analyses revealed that cell cycle progression is effectively uncoupled from the activity and functional state of ER in these models. In this context, cyclin D1 expression and retinoblastoma tumor suppressor protein (RB) phosphorylation are maintained even with efficient ablation of ER with pure antagonists. These therapy-resistant models recapitulate a key feature of deregulated RB/E2F transcriptional control. Correspondingly, a gene expression signature of RB-dysfunction is associated with luminal B breast cancer, which exhibits a relatively poor response to endocrine therapy. These collective findings suggest that suppression of cyclin D-supported kinase activity and restoration of RB-mediated transcriptional repression could represent a viable therapeutic option in tumors that fail to respond to hormone-based therapies. Consistent with this hypothesis, a highly selective CDK4/6 inhibitor, PD-0332991, was effective at suppressing the proliferation of all hormone refractory models analyzed. Importantly, PD-0332991 led to a stable cell cycle arrest that was fundamentally distinct from those elicited by ER antagonists, and was capable of inducing aspects of cellular senescence in hormone therapy refractory cell populations. These findings underscore the clinical utility of downstream cytostatic therapies in treating tumors that have experienced failure of endocrine therapy.
    Full-text · Article · Mar 2011 · Endocrine Related Cancer
Show more