scholarly journals Differential effects of 16α-hydroxyestrone and 2-methoxyestradiol on cyclin D1 involving the transcription factor ATF-2 in MCF-7 breast cancer cells

2005 ◽  
Vol 34 (1) ◽  
pp. 91-105 ◽  
Author(s):  
Joan S Lewis ◽  
T J Thomas ◽  
Richard G Pestell ◽  
Chris Albanese ◽  
Michael A Gallo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Dna Synthesis ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
D1 Protein ◽  
Fold Increase ◽  
Cycle Progression ◽  
Cyclin D1 Protein ◽  
Mcf 7

We studied the effects of 2-methoxyestradiol (2-ME2) and 16α-hydroxyestrone (16α-OHE1), two metabolites of estradiol (E2), on DNA synthesis, cell cycle progression and cyclin D1 protein levels in estrogen receptor-positive MCF-7 cells. E2 and 16α-OHE1 stimulated DNA synthesis, and 2-ME2 inhibited the stimulatory effects of these agents. E2 and 16α-OHE1 stimulated the progression of cells from G1 to S phase and this effect was attenuated by 2-ME2. Western blot analysis showed that E2 and 16α-OHE1 increased cyclin D1 protein level by about fourfold compared with control. 2-ME2 had no significant effect on cyclin D1; however, it prevented the accumulation of cyclin D1 in the presence of E2 and 16α-OHE1. Cells transfected with a cyclin D1 reporter gene and treated with E2 or 16α-OHE1 showed 7- and 9.5-fold increase respectively in promoter activity compared with control. This activity was significantly inhibited by 2-ME2. Cyclin D1 transactivation was mediated by the cAMP response element (CRE) region, which binds activating transcription factor 2 (ATF-2). DNA affinity assay showed 2.5- and 3.5-fold increases in ATF-2 binding to CRE in the presence of E2 and 16α-OHE1 respectively. The binding of ATF-2 was inhibited by the presence of 2-ME2. These results show that 2-ME2 can downregulate cyclin D1 and thereby cell cycle progression by a mechanism involving the disruption of ATF-2 binding to cyclin D1 promoter.

scholarly journals Effects of Auraptene on IGF-1 Stimulated Cell Cycle Progression in the Human Breast Cancer Cell Line, MCF-7

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Prasad Krishnan ◽  
Heather Kleiner-Hancock
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
D1 Protein ◽  
Cancer Cell Line ◽  
S Phase ◽  
Human Breast ◽  
Cycle Progression ◽  
Cyclin D1 Protein ◽  
Mcf 7

Auraptene is being investigated for its chemopreventive effects in many models of cancer including skin, colon, prostate, and breast. Many mechanisms of action including anti-inflammatory, antiproliferative, and antiapoptotic effects are being suggested for the chemopreventive properties of auraptene. We have previously shown in theN-methylnitrosourea induced mammary carcinogenesis model that dietary auraptene (500 ppm) significantly delayed tumor latency. The delay in time to tumor corresponded with a significant reduction in cyclin D1 protein expression in the tumors. Since cyclin D1 is a major regulator of cell cycle, we further studied the effects of auraptene on cell cycle and the genes related to cell cycle in MCF-7 cells. Here we show that auraptene significantly inhibited IGF-1 stimulated S phase of cell cycle in MCF-7 cells and significantly changed the transcription of many genes involved in cell cycle.

scholarly journals Regulation of cell cycle and cyclins by 16alpha-hydroxyestrone in MCF-7 breast cancer cells

2001 ◽  
Vol 27 (3) ◽  
pp. 293-307 ◽  
Author(s):  
JS Lewis ◽  
TJ Thomas ◽  
CM Klinge ◽  
MA Gallo ◽  
T Thomas
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Dna Synthesis ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Cell Cycle Progression ◽  
Fold Increase ◽  
Cycle Progression ◽  
Synchronized Cells ◽  
Mcf 7

It has been suggested that alterations in estradiol (E(2)) metabolism, resulting in increased production of 16alpha-hydroxyestrone (16alpha-OHE(1)), is associated with an increased risk of breast cancer. In the present study, we examined the effects of 16alpha-OHE(1)on DNA synthesis, cell cycle progression, and the expression of cell cycle regulatory genes in MCF-7 breast cancer cells. G(1) synchronized cells were treated with 1 to 25 nM 16alpha-OHE(1) for 24 and 48 h. [(3)H]Thymidine incorporation assay showed that 16alpha-OHE(1) caused an 8-fold increase in DNA synthesis compared with that of control cells, whereas E(2) caused a 4-fold increase. Flow cytometric analysis of cell cycle progression also demonstrated the potency of 16alpha-OHE(1) in stimulating cell growth. When G(1) synchronized cells were treated with 10 nM 16alpha-OHE(1) for 24 h, 62+/-3% of cells were in S phase compared with 14+/-3% and 52+/-2% of cells in the control and E(2)-treated groups respectively. In order to explore the role of 16alpha-OHE(1) in cell cycle regulation, we examined its effects on cyclins (D1, E, A, B1), cyclin dependent kinases (Cdk4, Cdk2), and retinoblastoma protein (pRB) using Western and Northern blot analysis. Treatment of cells with 10 nM 16alpha-OHE(1) resulted in 4- and 3-fold increases in cyclin D1 and cyclin A, respectively, at the protein level. There was also a significant increase in pRB phosphorylation and Cdk2 activation. In addition, transient transfection assay using an estrogen response element-driven luciferase reporter vector showed a 15-fold increase in estrogen receptor-mediated transactivation compared with control. These results show that 16alpha-OHE(1) is a potent estrogen capable of accelerating cell cycle kinetics and stimulating the expression of cell cycle regulatory proteins.

scholarly journals Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 Cells

1999 ◽  
Vol 19 (7) ◽  
pp. 4623-4632 ◽  
Author(s):  
Masahiro Hitomi ◽  
Dennis W. Stacey
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
Time Lapse ◽  
S Phase ◽  
3T3 Cells ◽  
Cycle Progression ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.

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

2001 ◽  
Vol 21 (3) ◽  
pp. 794-810 ◽  
Author(s):  
James S. Foster ◽  
Donald C. Henley ◽  
Antonin Bukovsky ◽  
Prem Seth ◽  
Jay Wimalasena
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Inhibitory Activity ◽  
Cell Cycle Progression ◽  
Estrogen Action ◽  
Cycle Progression ◽  
Cdk2 Activity ◽  
Mcf 7

ABSTRACT 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.

scholarly journals Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins.

1996 ◽  
Vol 16 (6) ◽  
pp. 2570-2578 ◽  
Author(s):  
A Lasorella ◽  
A Iavarone ◽  
M A Israel
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Cell Cycle Progression ◽  
D1 Protein ◽  
Protein Kinase Activity ◽  
Cycle Progression ◽  
Tumor Suppressor Proteins ◽  
Cycle Arrest

Cells which are highly proliferative typically lack expression of differentiated, lineage-specific characteristics. Id2, a member of the helix-loop-helix (HLH) protein family known to inhibit cell differentiation, binds to the retinoblastoma protein (pRb) and abolishes its growth-suppressing activity. We found that Id2 but not Id1 or Id3 was able to bind in vitro not only pRb but also the related proteins p107 and p130. Also, an association between Id2 and p107 or p130 was observed in vivo in transiently transfected Saos-2 cells. In agreement with these results, expression of Id1 or Id3 did not affect the block of cell cycle progression mediated by pRb. Conversely, expression of Id2 specifically reversed the cell cycle arrest induced by each of the three members of the pRb family. Furthermore, the growth-suppressive activities of cyclin-dependent kinase inhibitors p16 and p21 were efficiently antagonized by high levels of Id2 but not by Id1 Id3. Consistent with the role of p16 as a selective inhibitor of pRb and pRb-related protein kinase activity, p16-imposed cell cycle arrest was completely abolished by Id2. Only a partial reversal of p21-induced growth suppression was observed, which correlated with the presence of a functional pRb. We also documented decreased levels of cyclin D1 protein and mRNA and the loss of cyclin D1-cdk4 complexes in cells constitutively expressing Id2. These data provide evidence for important Id2-mediated alterations in cell cycle components normally involved in the regulatory events of cell cycle progression, and they highlight a specific role for Id2 as an antagonist of multiple tumor suppressor proteins.

scholarly journals Inhibition of Cyclin D1 Kinase Activity Is Associated with E2F-Mediated Inhibition of Cyclin D1 Promoter Activity through E2F and Sp1

1998 ◽  
Vol 18 (6) ◽  
pp. 3212-3222 ◽  
Author(s):  
Genichi Watanabe ◽  
Chris Albanese ◽  
Richard J. Lee ◽  
Anne Reutens ◽  
Gino Vairo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Promoter Activity ◽  
Cell Cycle Progression ◽  
Cellular Proliferation ◽  
Kinase Activity ◽  
D1 Protein ◽  
Binding Domain ◽  
Cycle Progression ◽  
Amino Terminal

ABSTRACT Coordinated interactions between cyclin-dependent kinases (Cdks), their target “pocket proteins” (the retinoblastoma protein [pRB], p107, and p130), the pocket protein binding E2F-DP complexes, and the Cdk inhibitors regulate orderly cell cycle progression. The cyclin D1 gene encodes a regulatory subunit of the Cdk holoenzymes, which phosphorylate the tumor suppressor pRB, leading to the release of free E2F-1. Overexpression of E2F-1 can induce apoptosis and may either promote or inhibit cellular proliferation, depending upon the cell type. In these studies overexpression of E2F-1 inhibited cyclin D1-dependent kinase activity, cyclin D1 protein levels, and promoter activity. The DNA binding domain, the pRB pocket binding region, and the amino-terminal Sp1 binding domain of E2F-1 were required for full repression of cyclin D1. Overexpression of pRB activated the cyclin D1 promoter, and a dominant interfering pRB mutant was defective in cyclin D1 promoter activation. Two regions of the cyclin D1 promoter were required for full E2F-1-dependent repression. The region proximal to the transcription initiation site at −127 bound Sp1, Sp3, and Sp4, and the distal region at −143 bound E2F-4–DP-1–p107. In contrast with E2F-1, E2F-4 induced cyclin D1 promoter activity. Differential regulation of the cyclin D1 promoter by E2F-1 and E2F-4 suggests that E2Fs may serve distinguishable functions during cell cycle progression. Inhibition of cyclin D1 abundance by E2F-1 may contribute to an autoregulatory feedback loop to reduce pRB phosphorylation and E2F-1 levels in the cell.

scholarly journals TGFβ and PTHrP Control Chondrocyte Proliferation by Activating Cyclin D1 Expression

2001 ◽  
Vol 12 (12) ◽  
pp. 3852-3863 ◽  
Author(s):  
Frank Beier ◽  
Zenobia Ali ◽  
Dereck Mok ◽  
Allison C. Taylor ◽  
Todd Leask ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
Bone Development ◽  
Chondrocyte Proliferation ◽  
Endochondral Bone ◽  
Cycle Progression ◽  
Related Transcription Factor

Exact coordination of growth plate chondrocyte proliferation is necessary for normal endochondral bone development and growth. Here we show that PTHrP and TGFβ control chondrocyte cell cycle progression and proliferation by stimulating signaling pathways that activate transcription from the cyclin D1 promoter. The TGFβ pathway activates the transcription factor ATF-2, whereas PTHrP uses the related transcription factor CREB, to stimulate cyclin D1 promoter activity via the CRE promoter element. Inhibition of cyclin D1 expression with antisense oligonucleotides causes a delay in progression of chondrocytes through the G1 phase of the cell cycle, reduced E2F activity, and decreased proliferation. Growth plates from cyclin D1–deficient mice display a smaller zone of proliferating chondrocytes, confirming the requirement for cyclin D1 in chondrocyte proliferation in vivo. These data identify the cyclin D1 gene as an essential component of chondrocyte proliferation as well as a fundamental target gene of TGFβ and PTHrP during skeletal growth.

scholarly journals Phosphorylation of Cyclin D1 Regulated by ATM or ATR Controls Cell Cycle Progression

2008 ◽  
Vol 28 (17) ◽  
pp. 5478-5493 ◽  
Author(s):  
Masahiro Hitomi ◽  
Ke Yang ◽  
Andrew W. Stacey ◽  
Dennis W. Stacey
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
Critical Role ◽  
Cycle Progression ◽  
Checkpoint Kinase ◽  
Checkpoint Kinases ◽  
Stressed Cells ◽  
Interfering Rna

ABSTRACT Cyclin D1 is required at high levels for passage through G1 phase but must be reduced to low levels during S phase to avoid the inhibition of DNA synthesis. This suppression requires the phosphorylation of Thr286, which is induced directly by DNA synthesis. Because the checkpoint kinase ATR is activated by normal replication as well as by DNA damage, its potential role in regulating cyclin D1 phosphorylation was tested. We found that ATR, activated by either UV irradiation or the topoisomerase IIβ binding protein 1 activator, promoted cyclin D1 phosphorylation. Small interfering RNA against ATR inhibited UV-induced Thr286 phosphorylation, together with that seen in normally cycling cells, indicating that ATR regulates cyclin D1 phosphorylation in normal as well as stressed cells. Following double-stranded DNA (dsDNA) breakage, the related checkpoint kinase ATM was also able to promote the phosphorylation of cyclin D1 Thr286. The relationship between these checkpoint kinases and cyclin D1 was extended when we found that normal cell cycle blockage in G1 phase observed following dsDNA damage was efficiently overcome when exogenous cyclin D1 was expressed within the cells. These results indicate that checkpoint kinases play a critical role in regulating cell cycle progression in normal and stressed cells by directing the phosphorylation of cyclin D1.

Sign in / Sign up

Export Citation Format

Share Document