scholarly journals ΔRaf-1:ER* Bypasses the Cyclic AMP Block of Extracellular Signal-Regulated Kinase 1 and 2 Activation but Not CDK2 Activation or Cell Cycle Reentry

2003 ◽  
Vol 23 (24) ◽  
pp. 9303-9317 ◽  
Author(s):  
Kathryn Balmanno ◽  
Tracy Millar ◽  
Martin McMahon ◽  
Simon J. Cook
Keyword(s):  
Cell Cycle ◽  
Cyclic Amp ◽  
Dna Synthesis ◽  
Cyclin D1 ◽  
Cyclin A ◽  
3T3 Cells ◽  
Cell Cycle Reentry ◽  
Extracellular Signal ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT Elevation of cellular cyclic AMP (cAMP) levels inhibits cell cycle reentry in a variety of cell types. While cAMP can prevent the activation of Raf-1 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) by growth factors, we now show that activation of ERK1/2 by ΔRaf-1:ER is insensitive to cAMP. Despite this, ΔRaf-1:ER-stimulated DNA synthesis is still inhibited by cAMP, indicating a cAMP-sensitive step downstream of ERK1/2. Although cyclin D1 expression has been proposed as an alternative target for cAMP, we found that cAMP could inhibit ΔRaf-1:ER-induced cyclin D1 expression only in Rat-1 cells, not in CCl39 or NIH 3T3 cells. ΔRaf-1:ER-stimulated activation of CDK2 was strongly inhibited by cAMP in all three cell lines, but cAMP had no effect on the induction of p21CIP1. cAMP blocked the fetal bovine serum (FBS)-induced degradation of p27KIP1; however, loss of p27KIP1 in response to ΔRaf-1:ER was less sensitive in CCl39 and Rat-1 cells and was completely independent of cAMP in NIH 3T3 cells. The most consistent effect of cAMP was to block both FBS- and ΔRaf-1:ER-induced expression of Cdc25A and cyclin A, two important activators of CDK2. When CDK2 activity was bypassed by activation of the ER-E2F1 fusion protein, cAMP no longer inhibited expression of Cdc25A or cyclin A but still inhibited DNA synthesis. These studies reveal multiple points of cAMP sensitivity during cell cycle reentry. Inhibition of Raf-1 and ERK1/2 activation may operate early in G1, but when this early block is bypassed by ΔRaf-1:ER, cells still fail to enter S phase due to inhibition of CDK2 or targets downstream of E2F1.

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 Anchorage-dependent transcription of the cyclin A gene.

1996 ◽  
Vol 16 (9) ◽  
pp. 4632-4638 ◽  
Author(s):  
A Schulze ◽  
K Zerfass-Thome ◽  
J Bergès ◽  
S Middendorp ◽  
P Jansen-Dürr ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Promoter Activity ◽  
Kinase Inhibitor ◽  
Ectopic Expression ◽  
Cyclin A ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Suspended Cells ◽  
Nih 3T3 Cells

NIH 3T3 cells cultured in suspension fail to express cyclin A and hence cannot enter S phase and divide. We show that loss of cell adhesion to substratum abrogates cyclin A gene expression by blocking its promoter activity through the E2F site that mediates its cell cycle regulation in adherent cells. In suspended cells, G0-specific E2F complexes remain bound to the cyclin A promoter. Overexpression of cyclin D1 restores cyclin A transcription in suspended cells and rescues them from cell cycle arrest. In suspended cells, cyclin D1 and cyclin E accumulate normally upon serum stimulation, but their associated kinases remain inactive; their substrates, pRb and p107, are not hyperphosphorylated. Concomitantly, the cyclin-dependent kinase inhibitor, p27KIP1, is stabilized. Ectopic expression of p27KIP1 blocks cyclin A promoter activity through its EN binding site. These data suggest that the block to cyclin A transcription in nonadherent NIH 3T3 cells results from stabilization of p27KIP1 and subsequent inactivation of the specific E2F moiety required for its induction.

scholarly journals Cyclic AMP Blocks Cell Growth through Raf-1-Dependent and Raf-1-Independent Mechanisms

2002 ◽  
Vol 22 (11) ◽  
pp. 3717-3728 ◽  
Author(s):  
Nicolas Dumaz ◽  
Yvonne Light ◽  
Richard Marais
Keyword(s):  
Protein Kinase ◽  
Cell Growth ◽  
Cyclic Amp ◽  
3T3 Cells ◽  
Extracellular Signal ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT It is widely accepted that cyclic AMP (cAMP) can block cell growth by phosphorylating Raf-1 on serine 43 and inhibiting signaling to extracellular signal-regulated protein kinase. We show that the suppression of Raf-1 by cAMP is considerably more complex than previously reported. When cellular cAMP is elevated, Raf-1 is phosphorylated on three residues (S43, S233, and S259), which work independently to block Raf-1. Both Ras-dependent and Ras-independent processes are disrupted. However, when cAMP-insensitive versions of Raf-1 are expressed in NIH 3T3 cells, their growth is still strongly suppressed when cAMP is elevated. Thus, although Raf-1 appears to be an important cAMP target, other pathways are also targeted by cAMP, providing alternative mechanisms that lead to suppression of cell growth.

scholarly journals Ras Signals to the Cell Cycle Machinery via Multiple Pathways To Induce Anchorage-Independent Growth

1998 ◽  
Vol 18 (5) ◽  
pp. 2586-2595 ◽  
Author(s):  
Jaw-Ji Yang ◽  
Jong-Sun Kang ◽  
Robert S. Krauss
Keyword(s):  
Cell Cycle ◽  
Colony Formation ◽  
Cyclin A ◽  
Soft Agar ◽  
Specific Cell ◽  
3T3 Cells ◽  
Anchorage Independent Growth ◽  
Nih 3T3 ◽  
Cell Cycle Machinery ◽  
Nih 3T3 Cells

ABSTRACT Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells, and the ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins, a synergistic combination of which may be required for the highly altered phenotype of fully transformed cells. We describe here studies on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) was defective in producing this response. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations were complementary in Rat 6 cells. Each individual effector loop mutant partially relieved adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3, but not Rat 6, cells. The pairwise combinations of effector loop mutants that were synergistic in producing anchorage-independent growth in Rat 6 cells also led to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities was not as clear in NIH 3T3 cells that expressed pairs of mutants, implying the existence of either thresholds for these activities or additional requirements in the induction of anchorage-independent growth. Ectopic expression of cyclin D1, E, or A synergized with individual effector loop mutants to induce soft agar colony formation in NIH 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways to signal to the cell cycle machinery and that these pathways synergize to supplant the adhesion requirements of specific cell cycle events, leading to anchorage-independent growth.

scholarly journals Induction of β3-Integrin Gene Expression by Sustained Activation of the Ras-Regulated Raf–MEK–Extracellular Signal-Regulated Kinase Signaling Pathway

2001 ◽  
Vol 21 (9) ◽  
pp. 3192-3205 ◽  
Author(s):  
Douglas Woods ◽  
Holly Cherwinski ◽  
Eleni Venetsanakos ◽  
Arun Bhat ◽  
Stephan Gysin ◽  
...  
Keyword(s):  
Cell Surface ◽  
Signaling Pathway ◽  
Erk Signaling ◽  
Integrin Expression ◽  
3T3 Cells ◽  
Extracellular Signal ◽  
Integrin Gene ◽  
Β3 Integrin ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT Alterations in the expression of integrin receptors for extracellular matrix (ECM) proteins are strongly associated with the acquisition of invasive and/or metastatic properties by human cancer cells. Despite this, comparatively little is known of the biochemical mechanisms that regulate the expression of integrin genes in cells. Here we demonstrate that the Ras-activated Raf–MEK–extracellular signal-regulated kinase (ERK) signaling pathway can specifically control the expression of individual integrin subunits in a variety of human and mouse cell lines. Pharmacological inhibition of MEK1 in a number of human melanoma and pancreatic carcinoma cell lines led to reduced cell surface expression of α6- and β3-integrin. Consistent with this, conditional activation of the Raf-MEK-ERK pathway in NIH 3T3 cells led to a 5 to 20-fold induction of cell surface α6- and β3-integrin expression. Induced β3-integrin was expressed on the cell surface as a heterodimer with αv-integrin; however, the overall level of αv-integrin expression was not altered by Ras or Raf. Raf-induced β3-integrin was observed in primary and established mouse fibroblast lines and in mouse and human endothelial cells. Consistent with previous reports of the ability of the Raf-MEK-ERK signaling pathway to induce β3-integrin gene transcription in human K-562 erythroleukemia cells, Raf activation in NIH 3T3 cells led to elevated β3-integrin mRNA. However, unlike immediate-early Raf targets such as heparin binding epidermal growth factor and Mdm2, β3-integrin mRNA was induced by Raf in a manner that was cycloheximide sensitive. Surprisingly, activation of the Raf-MEK-ERK signaling pathway by growth factors and mitogens had little or no effect on β3-integrin expression, suggesting that the expression of this gene requires sustained activation of this signaling pathway. In addition, despite the robust induction of cell surface αvβ3-integrin expression by Raf in NIH 3T3 cells, such cells display decreased spreading and adhesion, with a loss of focal adhesions and actin stress fibers. These data suggest that oncogene-induced alterations in integrin gene expression may participate in the changes in cell adhesion and migration that accompany the process of oncogenic transformation.

Cell Cycle-Related Differences in Susceptibility of NIH/3T3 Cells to Ribonucleases

1999 ◽  
Vol 247 (1) ◽  
pp. 220-232 ◽  
Author(s):  
Mark R. Smith ◽  
Dianne L. Newton ◽  
Stanley M. Mikulski ◽  
Susanna M. Rybak
Keyword(s):  
Cell Cycle ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals Cell Cycle Progression Regulates Biogenesis and Cellular Localization of Lipid Droplets

2019 ◽  
Vol 39 (9) ◽  
Author(s):  
André L. S. Cruz ◽  
Nina Carrossini ◽  
Leonardo K. Teixeira ◽  
Luis F. Ribeiro-Pinto ◽  
Patricia T. Bozza ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Lipid Accumulation ◽  
Lipid Droplets ◽  
Cell Cycle Progression ◽  
Cellular Transformation ◽  
3T3 Cells ◽  
Cycle Progression ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACTIntracellular lipid accumulation has been associated with a poor prognosis in cancer. We have previously reported the involvement of lipid droplets in cell proliferation in colon cancer cells, suggesting a role for these organelles in cancer development. In this study, we evaluate the role of lipid droplets in cell cycle regulation and cellular transformation. Cell cycle synchronization of NIH 3T3 cells revealed increased numbers and dispersed distribution of lipid droplets specifically during S phase. Also, the transformed cell lineage NIH 3T3-H-rasV12showed an accumulation of both lipid droplets and PLIN2 protein above the levels in NIH 3T3 cells.PLIN2gene overexpression, however, was not able to induce NIH 3T3 cell transformation, disproving the hypothesis thatPLIN2is an oncogene. Furthermore, positive PLIN2 staining was strongly associated with highly proliferative Ki-67-positive areas in human colon adenocarcinoma tissue samples. Taken together, these results indicate that cell cycle progression is associated with tight regulation of lipid droplets, a process that is altered in transformed cells, suggesting the existence of a mechanism that connects cell cycle progression and cell proliferation with lipid accumulation.

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