Characterization of stably transfected fusion protein GFP-estrogen receptor-alpha in MCF-7 human breast cancer cells

Tagging hormone receptors with the green fluorescent protein (GFP) has increased our knowledge of ligand dependent sub-cellular trafficking of hormone receptors. However, the effect of the tagged hormone receptor expression on the corresponding wild type hormone receptor and endogenous gene expression has not been investigated. In this study, we constructed a MCF-7 cell line stably expressing GFP-tagged human estrogen receptor-alpha (ER) under control of the tetracycline-on system to determine the effect of GFP-ER expression on cell proliferation and expression of endogenous ER and hormone-responsive genes. Further, the inducible system was applied to determine the ligand dependent turnover rates of GFP-ER protein and mRNA. Our results demonstrate that GFP-ER expression did not affect cell cycling. Independent of ligand, GFP-ER markedly reduced the level of endogenous ER mRNA and protein, suggesting that ER negatively autoregulates its expression. Cisplatin cross-linking studies showed that GFP-ER is associated with nuclear DNA in situ, suggesting that GFP-ER is partially replacing ER at estrogen response elements. Furthermore, GFP-ER expression did not affect the estradiol induced temporal expression of hormone responsive genes c-myc and pS2.     

Insulin/insulin-like growth factor-I and estrogen cooperate to stimulate cyclin E-Cdk2 activation and cell Cycle progression in MCF-7 breast cancer cells through differential regulation of cyclin E and p21(WAF1/Cip1)

Estrogens and insulin/insulin-like growth factor-I (IGF-I) are potent mitogens for breast epithelial cells and, when co-administered, induce synergistic stimulation of cell proliferation. To investigate the molecular basis of this effect, a MCF-7 breast cancer cell model was established where serum deprivation and concurrent treatment with the pure estrogen antagonist, ICI 182780, inhibited growth factor and estrogen action and arrested cells in G(0)/G(1) phase. Subsequent stimulation with insulin or IGF-I alone failed to induce significant S-phase entry. However, these treatments increased cyclin D1, cyclin E, and p21 gene expression and induced the formation of active Cdk4 complexes but resulted in only minor increases in cyclin E-Cdk2 activity, likely due to recruitment of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1/Cip1) into these complexes. Treatment with estradiol alone resulted in a greater increase in cyclin D1 gene expression but markedly decreased p21 expression, with a concurrent increase in Cdk4 and Cdk2 activity and subsequent synchronous entry of cells into S phase. Co-administration of insulin/IGF-I and estrogen induced synergistic stimulation of S-phase entry coincident with synergistic activation of high molecular mass (approximately 350 kDa) cyclin E-Cdk2 complexes lacking p21. To determine if the ability of estrogen to deplete p21 was central to these effects, cells stimulated with insulin and estradiol were infected with an adenovirus expressing p21. Induction of p21 to levels equivalent to those following treatment with insulin alone markedly inhibited the synergism between estradiol and insulin on S-phase entry. Thus the ability of estradiol to antagonize the insulin-induced increase in p21 gene expression, with consequent activation of cyclin E-Cdk2, is a central component of the synergistic stimulation of breast epithelial cell proliferation induced by simultaneous activation of the estrogen and insulin/IGF-I signaling pathways.     

Multifaceted regulation of cell cycle progression by estrogen: regulation of Cdk inhibitors and Cdc25A independent of cyclin D1-Cdk4 function

Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G(1)/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 p16(INK4a) 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 p16(INK4a) inhibited G(1)/S transition induced in MCF-7 cells by 17-beta-estradiol (E(2)) 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 G(1) and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21(Cip1) and p27(Kip1) was decreased, however, in both control and p16(INK4a)-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E(2) in control and p16(INK4a)-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16(INK4a). Inhibition of Cdc25A activity in p16(INK4a)-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 E(2)-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 p16(INK4a)-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 p21(Cip1) and p27(Kip1).     

Cyclin A but not cyclin D1 is essential for c-myc-modulated cell-cycle progression

The proto-oncogene c-myc is a key player in cell-cycle regulation and is deregulated in a broad range of human cancers and cell proliferation disorders. Here we reported that overexpression of c-myc in human embryonic lung fibroblasts (HEL) that have low endogenous c-myc enriched S phase cells with increased expression of cyclin D3, E, A, Cdk2, and Cdk4, and decreased expression of p21 and p27. To the opposite, using RNAi to downregulate c-myc expression in A549 cells that have high endogenous c-myc enriched G1 phase cells with decreased expression of cyclin D3, E, A, Cdk2, Cdk4, and increased expression of p21 and p27. We found that cyclin A expression was the most susceptive to changes in c-myc levels and essential in c-myc-modulated cell cycle pathway via co-transfection, however, cyclin D1 showed no change between treated and control groups in either HEL or A549 cells. Our results indicated that upregulation of c-myc expression promotes cell cycling in HEL cells, whereas downregulation of c-myc expression causes G1 phase arrest in A549 cells, and the c-myc-mediated cell-cycle regulation pathway was dependent on cyclin A and involved cyclin D3, E, Cdk2, Cdk4, p21, and p27, but not cyclin D1.     

Estrogen receptor activation at serine 305 is sufficient to upregulate cyclin D1 in breast cancer cells

Recent studies have shown that p21-activated kinase 1 (Pak1) phosphorylates estrogen receptor-alpha (ER alpha) at Ser 305 and also promotes its transactivation function. Here, we sought to investigate whether substitution of serine 305 in ER with glutamic acid (ER alpha-S305E), which mimics the phosphorylation state, would influence the status of ER-target genes. To explore this possibility, we generated clones overexpressing ER alpha-S305E in ER-negative MDA-MB-231 cells and analyzed the status of ER-regulated genes using a gene array. Results indicated that the expression of ER alpha-S305E is sufficient to upregulate the expression of a few but not all ER-regulated genes, i.e., cyclin D1 and zinc finger protein 147 (estrogen-responsive finger protein), while there was no significant change in the expression of remaining genes on the array. In addition, we found an increased expression as well as nuclear accumulation of cyclin D1 protein in MDA-MB-231 cells expressing ER alpha-S305E as compared to the level of cyclin D1 in MDA-MB-231 cells expressing WT-ER alpha or pcDNA. Furthermore, ER alpha-S305E, but not mutation of ER alpha-S305 to alanine, enhanced the cyclin D1 promoter activity. These findings suggest that ER alpha activation at S305 is sufficient to upregulate the expression of cyclin D1, an ER-regulated gene that is implicated in the progression of breast cancer. Phosphorylation of ER alpha by Pak1 or its upstream regulators could upregulate the expression of a subset of ER-target genes in a ligand-independent manner and hence, might contribute toward the development of hormone independence in breast cancer cells.     

Functional ablation of pRb activates Cdk2 and causes antiestrogen resistance in human breast cancer cells

Estrogens are required for the proliferation of hormone dependent breast cancer cells, making estrogen receptor (ER) positive tumors amenable to endocrine therapies such as antiestrogens. However, resistance to these agents remains a significant cause of treatment failure. We previously demonstrated that inactivation of the retinoblastoma protein (pRb) family tumor suppressors causes antiestrogen resistance in MCF-7 cells, a widely studied model of estrogen responsive human breast cancers. In this study, we investigate the mechanism by which pRb inactivation leads to antiestrogen resistance. Cdk4 and cdk2 are two key cell cycle regulators that can phosphorylate and inactivate pRb, therefore we tested whether these kinases are required in cells lacking pRb function. pRb family members were inactivated in MCF-7 cells by expressing polyomavirus large tumor antigen (PyLT), and cdk activity was inhibited using the cdk inhibitors p16(INK4A) and p21(Waf1/Cip1). Cdk4 activity was no longer required in cells lacking functional pRb, while cdk2 activity was required for proliferation in both the presence and absence of pRb function. Using inducible PyLT cell lines, we further demonstrated that pRb inactivation leads to increased cyclin A expression, cdk2 activation and proliferation in antiestrogen arrested cells. These results demonstrate that antiestrogens do not inhibit cdk2 activity or proliferation of MCF-7 cells in the absence of pRb family function, and suggest that antiestrogen resistant breast cancer cells resulting from pRb pathway inactivation would be susceptible to therapies that target cdk2.     

A pure estrogen antagonist inhibits cyclin E-Cdk2 activity in MCF-7 breast cancer cells and induces accumulation of p130-E2F4 complexes characteristic of quiescence

Estrogen antagonists inhibit cell cycle progression in estrogen-responsive cells, but the molecular mechanisms are not fully defined. Antiestrogen-mediated G(0)/G(1) arrest is associated with decreased cyclin D1 gene expression, inactivation of cyclin D1-cyclin dependent kinase (Cdk) 4 complexes, and decreased phosphorylation of the retinoblastoma protein (pRb). We now show that treatment of MCF-7 breast cancer cells with the pure estrogen antagonist ICI 182780 results in inhibition of cyclin E-Cdk2 activity prior to a decrease in the G(1) to S phase transition. This decrease was dependent on p21(WAF1/Cip1) since treatment with antisense oligonucleotides to p21 attenuated the effect. Recruitment of p21 to cyclin E-Cdk2 complexes was in turn dependent on decreased cyclin D1 expression since it was apparent following treatment with antisense cyclin D1 oligonucleotides. To define where within the G(0) to S phase continuum antiestrogen-treated cells arrested, we assessed the relative abundance and phosphorylation state of pocket protein-E2F complexes. While both pRb and p107 levels were significantly decreased, p130 was increased 4-fold and was accompanied by the formation of p130.E2F4 complexes and the accumulation of hyperphophorylated E2F4, putative markers of cellular quiescence. Thus, ICI 182780 inhibits both cyclin D1-Cdk4 and cyclin E-Cdk2 activity, resulting in the arrest of MCF-7 cells in a state with characteristics of quiescence (G(0)), as opposed to G(1) arrest.     

The cyclin-dependent kinase inhibitor p21WAF1/Cip1 is an antiestrogen-regulated inhibitor of Cdk4 in human breast cancer cells.

The MCF-7 cell line is a model of estrogen-dependent, antiestrogen-sensitive human breast cancer. Antiestrogen treatment of MCF-7 cells causes dramatic decreases in both Cdk4 and Cdk2 activities, which leads to a G(1) phase cell cycle arrest. In this report, we investigate the mechanism(s) by which Cdk4 activity is regulated in MCF-7 cells. Through time course analysis, we demonstrate that changes in Cdk4 activity in response to estrogen or antiestrogen treatment do not correlate directly with cyclin D1 protein levels or association. In contrast, Cdk4 activity does correlate with changes in the level of the Cdk inhibitor p21(WAF1/Cip1). Furthermore, we show that extracts of antiestrogen-treated cells contain a factor capable of inhibiting the Cdk4 activity present in extracts of estrogen-treated cells, and immunodepletion experiments identify this factor as p21(WAF1/Cip1). These results identify p21(WAF1/Cip1) as an important physiological regulator of Cdk4 complexes in human breast cancer cells.     

p21Waf1/Cip1 Inhibition of Cyclin E/Cdk2 Activity Prevents Endoreduplication after Mitotic Spindle Disruption

During a normal cell cycle, entry into S phase is dependent on completion of mitosis and subsequent activation of cyclin-dependent kinases (Cdks) in G₁. These events are monitored by checkpoint pathways. Recent studies and data presented herein show that after treatment with microtubule inhibitors (MTIs), cells deficient in the Cdk inhibitor p21^Waf1/Cip1 enter S phase with a ≥4N DNA content, a process known as endoreduplication, which results in polyploidy. To determine how p21 prevents MTI-induced endoreduplication, the G₁/S and G₂/M checkpoint pathways were examined in two isogenic cell systems: HCT116 p21^+/+ and p21^−/− cells and H1299 cells containing an inducible p21 expression vector (HIp21). Both HCT116 p21^−/− cells and noninduced HIp21 cells endoreduplicated after MTI treatment. Analysis of G₁-phase Cdk activities demonstrated that the induction of p21 inhibited endoreduplication through direct cyclin E/Cdk2 regulation. The kinetics of p21 inhibition of cyclin E/Cdk2 activity and binding to proliferating-cell nuclear antigen in HCT116 p21^+/+ cells paralleled the onset of endoreduplication in HCT116 p21^−/− cells. In contrast, loss of p21 did not lead to deregulated cyclin D1-dependent kinase activities, nor did p21 directly regulate cyclin B1/Cdc2 activity. Furthermore, we show that MTI-induced endoreduplication in p53-deficient HIp21 cells was due to levels of p21 protein below a threshold required for negative regulation of cyclin E/Cdk2, since ectopic expression of p21 restored cyclin E/Cdk2 regulation and prevented endoreduplication. Based on these findings, we propose that p21 plays an integral role in the checkpoint pathways that restrain normal cells from entering S phase after aberrant mitotic exit due to defects in microtubule dynamics.     

Induction of G1 phase arrest and apoptosis in MDA-MB-231 breast cancer cells by troglitazone, a synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) ligand

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands inhibit cell proliferation and induce apoptosis in cancer cells. Here we wished to determine whether the PPARgamma ligand induces apoptosis and cell cycle arrest of the MDA-MB-231 cell, an estrogen receptor alpha negative breast cancer cell line. The treatment of MDA-MB-231 cell with PPARgamma ligands was shown to induce inhibition of cell growth in a dose-dependent manner as determined by MTT assay. Cell cycle analysis showed a G1 arrest in MDA-MB-231 cells exposed to troglitazone. An apoptotic effect by troglitazone demonstrated that apoptotic cells elevated by 2.5-fold from the control level at 10 microM, to 3.1-fold at 50 microM and to 3.5-fold at 75 microM. Moreover, troglitazone treatment, applied in a dose-dependent manner, caused a marked decrease in pRb, cyclin D1, cyclin D2, cyclin D3, Cdk2, Cdk4 and Cdk6 expression as well as a significant increase in p21 and p27 expression. These results indicate that troglitazone causes growth inhibition, G1 arrest and apoptotic death of MDA-MB-231 cells.     

c-Myc or Cyclin D1 Mimics Estrogen Effects on Cyclin E-Cdk2 Activation and Cell Cycle Reentry

Estrogen-induced progression through G₁ phase of the cell cycle is preceded by increased expression of the G₁-phase regulatory proteins c-Myc and cyclin D1. To investigate the potential contribution of these proteins to estrogen action, we derived clonal MCF-7 breast cancer cell lines in which c-Myc or cyclin D1 was expressed under the control of the metal-inducible metallothionein promoter. Inducible expression of either c-Myc or cyclin D1 was sufficient for S-phase entry in cells previously arrested in G₁ phase by pretreatment with ICI 182780, a potent estrogen antagonist. c-Myc expression was not accompanied by increased cyclin D1 expression or Cdk4 activation, nor was cyclin D1 induction accompanied by increases in c-Myc. Expression of c-Myc or cyclin D1 was sufficient to activate cyclin E-Cdk2 by promoting the formation of high-molecular-weight complexes lacking the cyclin-dependent kinase inhibitor p21, as has been described, following estrogen treatment. Interestingly, this was accompanied by an association between active cyclin E-Cdk2 complexes and hyperphosphorylated p130, identifying a previously undefined role for p130 in estrogen action. These data provide evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis which converge on or prior to the formation of active cyclin E-Cdk2-p130 complexes and loss of inactive cyclin E-Cdk2-p21 complexes, indicating a physiologically relevant role for the cyclin E binding motifs shared by p130 and p21.     

Biological effects of G1 phase arrest compound,sesquicillin, in human breast cancer cell lines

Sesquicillin, isolated from fungal fermentation broth, strongly induced G1 phase arrest in human breast cancer cells. During G1 phase arrest, the expression level of cyclin D1, cyclin A, and cyclin E was decreased, and the expression of CDK (cyclin-dependent-kinase) inhibitor, protein p21(Waf1/Cip1), was increased in a time-dependent manner in a breast cancer cell MCF-7. Interestingly, the G1 phase arrest induced by sesquicillin also occurred independently of the tumor suppressor protein, p53. Sesquicillin inhibits the proliferation of MCF-7 via G1 phase arrest in association with the induction of CDK inhibitor protein, p21(Waf1/Cip1), and the reduction of G1 phase related-cyclin proteins.     

The over-expression of p53 H179Y residue mutation causes the increase of cyclin A1 and Cdk4 expression in HELF cells

Down-regulation of p53 expression has been found in a broad range of human cancers and cell proliferation disorders, indicating that p53 plays a key role in cell cycle regulation and tumor suppression. In our current study, we transfected human embryonic lung fibroblast (HELF) cells with pcDNA3-wild-type p53 (pcDNA3-wtp53) plasmid, or pcDNA3-H179Y-mutated p53 (pcDNA3-mtp53) plasmid that mimics the mutation found in some human lung tumors, and further studied the role of p53 in the regulation of cell proliferation. Over expression of wild-type p53 caused cell cycle arrest at G1 phase with reduced cell size, decreased expression of cyclin D3, cyclin E, Cdk2 and Cdk4, and increased expression of p21. In contrast, over expression of H179Y-mutant p53 promoted G1 to S phase transition with enlarged cell size and increased cyclin A1 and Cdk4 expression in HELF cells. These results indicate that mutation at the p53 H179Y residue up-regulates cyclin A1 and Cdk4 expression, and promotes HELF cell proliferation.     

p14ARF Post-Transcriptional Regulation of Nuclear Cyclin D1 in MCF-7 Breast Cancer Cells: Discrimination between a Good and Bad Prognosis?

As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy.     

Impact of cyclin E overexpression on Smad3 activity in breast cancer cell lines

Smad3, a component of the TGFβ signaling pathway, contributes to G₁ arrest in breast cancer cells. Overexpression of the cell cycle mitogen, cyclin E, is associated with poor prognosis in breast cancer, and cyclin E/CDK2 mediated phosphorylation of Smad3 has been linked with inhibition of Smad3 activity. We hypothesized that the biological aggressiveness of cyclin E overexpressing breast cancer cells would be associated with CDK2 phosphorylation and inhibition of the tumor suppressant action of Smad3. Expression constructs containing empty vector, wild-type (WT) Smad3 or Smad3 with CDK phosphorylation site mutations were co-transfected with a Smad3-responsive reporter construct into parental, vector control (A1) or cyclin E overexpressing (EL1) MCF7 cells. Smad3 function was evaluated by luciferase reporter assay and mRNA analysis. The impact of a Cdk2 inhibitor and cdk2 siRNA on Smad3 activity was also assessed. Cells expressing Smad3 containing mutations of the CDK phosphorylation sites had higher p15 and p21 and lower c-myc mRNA levels, as well as higher Smad3-responsive reporter activity, compared with controls or cells expressing WT Smad3. Transfection of cdk2 siRNA resulted in a significant increase in Smad3-responsive reporter activity compared with control siRNA; reporter activity was also increased after the treatment with a Cdk2 inhibitor. Thus, cyclin E-mediated inhibition of Smad3 is regulated by CDK2 phosphorylation of the Smad3 protein in MCF7 cells. Inhibition of CDK2 may lead to restoration of Smad3 tumor suppressor activity in breast cancer cells, and may represent a potential treatment approach for cyclin E overexpressing breast cancers.Key words: Smad3, breast cancer, cyclin E, CDK2, TGFβ     

Dna damage-induced G(1) arrest in hematopoietic cells is overridden following phosphatidylinositol 3-kinase-dependent activation of cyclin-dependent kinase 2

Exposure of hematopoietic cells to DNA-damaging agents induces p53-independent cell cycle arrest at a G(1) checkpoint. Previously, we have shown that this growth arrest can be overridden by cytokine growth factors, such as erythropoietin or interleukin-3, through activation of a phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-dependent signaling pathway. Here, we show that gamma-irradiated murine myeloid 32D cells arrest in G(1) with active cyclin D-cyclin-dependent kinase 4 (Cdk4) but with inactive cyclin E-Cdk2 kinases. The arrest was associated with elevated levels of the Cdk inhibitors p21(Cip1) and p27(Kip1), yet neither was associated with Cdk2. Instead, irradiation-induced inhibition of cyclin E-Cdk2 correlated with absence of the activating threonine-160 phosphorylation on Cdk2. Cytokine treatment of irradiated cells induced Cdk2 phosphorylation and activation, and cells entered into S phase despite sustained high-level expression of p21 and p27. Notably, the PI 3-kinase inhibitor, LY294002, completely blocked cytokine-induced Cdk2 activation and cell growth in irradiated 32D cells but not in nonirradiated cells. Together, these findings demonstrate a novel mechanism underlying the DNA damage-induced G(1) arrest of hematopoietic cells, that is, inhibition of Cdk2 phosphorylation and activation. These observations link PI 3-kinase signaling pathways with the regulation of Cdk2 activity.