scholarly journals A dominant-negative mutant of c-Jun inhibits cell cycle progression during the transition of CD4–CD8– to CD4+CD8+ thymocytes

1999 ◽  
Vol 11 (8) ◽  
pp. 1203-1216 ◽  
Author(s):  
Leslie B. King ◽  
Eva Tolosa ◽  
Joi M. Lenczowski ◽  
Frank Lu ◽  
Evan F. Lind ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cycle Progression ◽  
Negative Mutant

scholarly journals Expression of dominant-negative mutant DP-1 blocks cell cycle progression in G1.

1996 ◽  
Vol 16 (7) ◽  
pp. 3698-3706 ◽  
Author(s):  
C L Wu ◽  
M Classon ◽  
N Dyson ◽  
E Harlow
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Dominant Negative ◽  
Functional Domains ◽  
Dominant Negative Mutant ◽  
G1 Arrest ◽  
Wild Type ◽  
Cycle Progression

Unregulated expression of the transcription factor E2F promotes the G1-to-S phase transition in cultured mammalian cells. However, there has been no direct evidence for an E2F requirement in this process. To demonstrate that E2F is obligatory for cell cycle progression, we attempted to inactivate E2F by overexpressing dominant-negative forms of one of its heterodimeric partners, DP-1. We dissected the functional domains of DP-1 and separated the region that facilitate heterodimer DNA binding from the E2F dimerization domain. Various DP-1 mutants were introduced into cells via transfection, and the cell cycle profile of the transfected cells was analyzed by flow cytometry. Expression of wild-type DP-1 or DP-1 mutants that bind to both DNA and E2F drove cells into S phase. In contrast, DP-1 mutants that retained E2F binding but lost DNA binding arrested cells in the G1 phase of the cell cycle. The DP-1 mutants that were unable to bind DNA resulted in transcriptionally inactive E2F complexes, suggesting that the G1 arrest is caused by formation of defective E2F heterodimers. Furthermore, the G1 arrest instigated by these DP-1 mutants could be rescued by coexpression of wild-type E2F or DP protein. These experiments define functional domains of DP and demonstrate a requirement for active E2F complexes in cell cycle progression.

scholarly journals A Dominant-Negative PPARγMutant Promotes Cell Cycle Progression and Cell Growth in Vascular Smooth Muscle Cells

PPAR Research ◽  
2009 ◽  
Vol 2009 ◽  
pp. 1-10 ◽  
Author(s):  
Joey Z. Liu ◽  
Christopher J. Lyon ◽  
Willa A. Hsueh ◽  
Ronald E. Law
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cell Cycle Progression ◽  
Muscle Cells ◽  
Dominant Negative ◽  
Wild Type ◽  
Cycle Progression ◽  
Positive Regulators

PPARγligands have been shown to have antiproliferative effects on many cell types. We herein report that a synthetic dominant-negative (DN) PPARγmutant functions like a growth factor to promote cell cycle progression and cell proliferation in human coronary artery smooth muscle cells (CASMCs). In quiescent CASMCs, adenovirus-expressed DN-PPARγpromoted G1→S cell cycle progression, enhanced BrdU incorporation, and increased cell proliferation. DN-PPARγexpression also markedly enhanced positive regulators of the cell cycle, increasing Rb and CDC2 phosphorylation and the expression of cyclin A, B1, D1, and MCM7. Conversely, overexpression of wild-type (WT) or constitutively-active (CA) PPARγinhibited cell cycle progression and the activity and expression of positive regulators of the cell cycle. DN-PPARγexpression, however, did not up-regulate positive cell cycle regulators in PPARγ-deficient cells, strongly suggesting that DN-PPARγeffects on cell cycle result from blocking the function of endogenous wild-type PPARγ. DN-PPARγexpression enhanced phosphorylation of ERK MAPKs. Furthermore, the ERK specific-inhibitor PD98059 blocked DN-PPARγ-induced phosphorylation of Rb and expression of cyclin A and MCM7. Our data thus suggest that DN-PPARγpromotes cell cycle progression and cell growth in CASMCs by modulating fundamental cell cycle regulatory proteins and MAPK mitogenic signaling pathways in vascular smooth muscle cells (VSMCs).

Pivotal Role of Survivin in Leukemogenesis by E2A-HLF Chimeric Transcription Factor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2988-2988
Author(s):  
Mayuko Okuya ◽  
Hidemitsu Kurosawa ◽  
Takayuki Matsunaga ◽  
Mitsuoki Eguchi ◽  
Yusuke Furukawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Survivin Expression ◽  
Small Population ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Transcriptional Level ◽  
Survivin Promoter ◽  
M Phase ◽  
Negative Mutant

Abstract The E2A-HLF fusion transcription factor generated by the t(17;19)(q22;p13) translocation is found in a small population of pro-B cell ALL. Patients associated with this chimera share distinct clinical features such as hypercalcemia, coagulopathy and very poor prognosis due to resistance to intensive chemotherapy including aggressive conditioning for BMT, all of which are unusual for this type of ALL. We have previously demonstrated that inhibition of the trans-activation potential of the E2A-HLF chimera by the dominant negative mutant results in apoptosis in t(17;19)+ ALL cells but does not affect cell cycle. Moreover, E2A-HLF blocks apoptosis induced by cytokine deprivation in IL-3-dependent cells, suggesting that this fusion protein contributes to leukemogenesis by substituting for the anti-apoptotic function of cytokines. The present study shows that survivin is a downstream target molecule of E2A-HLF. Four t(17;19)+ ALL cell lines expressed survivin at high levels and down-regulation of E2A-HLF function by the dominant negative mutant suppressed survivin expression. In addition, forced expression of E2A-HLF in Nalm-6, a t(17;19)− ALL cell line, up-regulated survivin expression. Survivin is known to be expressed predominantly in the G2/M phase. Indeed, separation of the fractions enriched for in each phase of the cell cycle using a counterflow centrifugal elutriator revealed G2/M phase-dominant survivin expression in t(17;19) − ALL cells including Nalm-6. In t(17;19)+ ALL cells, however, survivin was expressed throughout the cell cycle. Moreover, Nalm-6 cells forced to express E2A-HLF showed cell cycle-independent survivin expression. Reporter assay revealed that E2A-HLF induced luciferase activity by transfecting with each reporter construct containing the survivin promoter at a different length from the initial ATG, suggesting that E2A-HLF induces survivin expression at the transcriptional level, but not by direct binding of E2A-HLF to the survivin promoter. To test whether survivin plays anti-apoptotic roles in t(17:19)+ cells, we used a survivin mutant lacking a phosphorylation site (T34A-survivin) and considered to inhibit survivin function in a dominant negative manner. T34A-survivin induced massive apoptosis throughout the cell cycle in t(17;19)+ cells. In contrast, T34A-survivin in t(17;19) − cells induced cell death in only a small population in G2/M phase. In addition to caspase-dependent pathways, T34A-survivin induced apoptosis in t(17;19)+ ALL cells through caspase-independent pathways, in which apoptosis-inducing factor (AIF) translocated from cytoplasm to the nucleus. These results indicate that cell cycle-independent up-regulation of survivin by the E2A-HLF chimera is indispensable for the survival of t(17;19)+ ALL cells, and that inhibition of survivin may offer an effective therapeutic strategy against this refractory ALL.

scholarly journals Chloroplast division checkpoint in eukaryotic algae

2016 ◽  
Vol 113 (47) ◽  
pp. E7629-E7638 ◽  
Author(s):  
Nobuko Sumiya ◽  
Takayuki Fujiwara ◽  
Atsuko Era ◽  
Shin-ya Miyagishima
Keyword(s):  
Cell Cycle ◽  
Host Cell ◽  
Cell Cycle Progression ◽  
Chloroplast Division ◽  
Dominant Negative ◽  
Cyanidioschyzon Merolae ◽  
Temperature Sensitive ◽  
Specific Expression ◽  
Cycle Progression ◽  
Division Site

Chloroplasts evolved from a cyanobacterial endosymbiont. It is believed that the synchronization of endosymbiotic and host cell division, as is commonly seen in existing algae, was a critical step in establishing the permanent organelle. Algal cells typically contain one or only a small number of chloroplasts that divide once per host cell cycle. This division is based partly on the S-phase–specific expression of nucleus-encoded proteins that constitute the chloroplast-division machinery. In this study, using the red alga Cyanidioschyzon merolae, we show that cell-cycle progression is arrested at the prophase when chloroplast division is blocked before the formation of the chloroplast-division machinery by the overexpression of Filamenting temperature-sensitive (Fts) Z2-1 (Fts72-1), but the cell cycle progresses when chloroplast division is blocked during division-site constriction by the overexpression of either FtsZ2-1 or a dominant-negative form of dynamin-related protein 5B (DRP5B). In the cells arrested in the prophase, the increase in the cyclin B level and the migration of cyclin-dependent kinase B (CDKB) were blocked. These results suggest that chloroplast division restricts host cell-cycle progression so that the cell cycle progresses to the metaphase only when chloroplast division has commenced. Thus, chloroplast division and host cell-cycle progression are synchronized by an interactive restriction that takes place between the nucleus and the chloroplast. In addition, we observed a similar pattern of cell-cycle arrest upon the blockage of chloroplast division in the glaucophyte alga Cyanophora paradoxa, raising the possibility that the chloroplast division checkpoint contributed to the establishment of the permanent organelle.

scholarly journals BCL-2 Is Phosphorylated and Inactivated by an ASK1/Jun N-Terminal Protein Kinase Pathway Normally Activated at G2/M

1999 ◽  
Vol 19 (12) ◽  
pp. 8469-8478 ◽  
Author(s):  
Kazuhito Yamamoto ◽  
Hidenori Ichijo ◽  
Stanley J. Korsmeyer
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Cell Cycle Progression ◽  
Peptide Mapping ◽  
Dominant Negative ◽  
Terminal Protein ◽  
Cycle Progression ◽  
M Phase ◽  
Death Signals

ABSTRACT Multiple signal transduction pathways are capable of modifying BCL-2 family members to reset susceptibility to apoptosis. We used two-dimensional peptide mapping and sequencing to identify three residues (Ser70, Ser87, and Thr69) within the unstructured loop of BCL-2 that were phosphorylated in response to microtubule-damaging agents, which also arrest cells at G2/M. Changing these sites to alanine conferred more antiapoptotic activity on BCL-2 following physiologic death signals as well as paclitaxel, indicating that phosphorylation is inactivating. An examination of cycling cells enriched by elutriation for distinct phases of the cell cycle revealed that BCL-2 was phosphorylated at the G2/M phase of the cell cycle. G2/M-phase cells proved more susceptible to death signals, and phosphorylation of BCL-2 appeared to be responsible, as a Ser70Ala substitution restored resistance to apoptosis. We noted that ASK1 and JNK1 were normally activated at G2/M phase, and JNK was capable of phosphorylating BCL-2. Expression of a series of wild-type and dominant-negative kinases indicated an ASK1/Jun N-terminal protein kinase 1 (JNK1) pathway phosphorylated BCL-2 in vivo. Moreover, the combination of dominant negative ASK1, (dnASK1), dnMKK7, and dnJNK1 inhibited paclitaxel-induced BCL-2 phosphorylation. Thus, stress response kinases phosphorylate BCL-2 during cell cycle progression as a normal physiologic process to inactivate BCL-2 at G2/M.

scholarly journals Scatter Factor/Hepatocyte Growth Factor Stimulation of Glioblastoma Cell Cycle Progression through G1 Is c-Myc Dependent and Independent of p27 Suppression, Cdk2 Activation, or E2F1-Dependent Transcription

2002 ◽  
Vol 22 (8) ◽  
pp. 2703-2715 ◽  
Author(s):  
Kevin A. Walter ◽  
Mir Ahamed Hossain ◽  
Carey Luddy ◽  
Nidhi Goel ◽  
Thomas E. Reznik ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Glioma Cell ◽  
Cell Cycle Progression ◽  
Dominant Negative ◽  
Wild Type ◽  
Scatter Factor ◽  
Cycle Progression ◽  
Glioma Cell Lines ◽  
Hepatocyte Growth

ABSTRACT Scatter factor/hepatocyte growth factor (SF/HGF) expression has been linked to malignant progression in glial neoplasms. Using two glioma cell lines, U373MG and SNB-19, we have demonstrated that SF/HGF stimulation allows cells to escape G1/G0 arrest induced by contact inhibition or serum withdrawal. SF/HGF induced effects on two mechanisms of cell cycle regulation: suppression of the cyclin-dependent kinase inhibitor p27 and induction of the transcription factor c-Myc. Regulation of p27 by SF/HGF was posttranslational and is associated with p27 nuclear export. Transient transfections of U373MG and SNB-19 with wild-type p27 and a degradation-resistant p27T187A mutant were insufficient to induce cell cycle arrest, and SF/HGF downregulation of p27 was not necessary for cell cycle reentry. Analysis of Cdk2 kinase activity and p27 binding to cyclin E complexes in the presence of exogenous wild-type p27 or p27T187A demonstrated that Cdk2 activity was not necessary for SF/HGF-mediated G1/S transition. Similarly, overexpression of dominant-negative forms of Cdk2 did not block SF/HGF-triggered cell cycle progression. In contrast, SF/HGF transcriptionally upregulated c-Myc, and overexpression of c-Myc was able to prevent G1/G0 arrest in the absence of SF/HGF. Transient overexpression of MadMyc, a dominant-negative chimera for c-Myc, caused G1/G0 arrest in logarithmically growing cells and blocked SF/HGF-mediated G1/S transition. c-Myc did not exert its effects through p27 downregulation in these cell lines. SF/HGF induced E2F1-dependent transcription, the inhibition of which did not block SF/HGF-induced cell cycle progression. We conclude that SF/HGF prevents G1/G0 arrest in glioma cell lines by a c-myc-dependent mechanism that is independent of p27, Cdk2, or E2F1.

scholarly journals Blocking the transcription factor E2F/DP by dominant-negative mutants in a normal breast epithelial cell line efficiently inhibits apoptosis and induces tumor growth in SCID mice.

1996 ◽  
Vol 183 (3) ◽  
pp. 1205-1213 ◽  
Author(s):  
R C Bargou ◽  
C Wagener ◽  
K Bommert ◽  
W Arnold ◽  
P T Daniel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Tumor Growth ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Dominant Negative ◽  
Serum Starvation ◽  
Cycle Progression ◽  
Transcription Factor E2f

The transcription factor E2F is regulated during the cell cycle through interactions with the product of the retinoblastoma susceptibility gene and related proteins. It is thought that E2F-mediated gene regulation at the G1/S boundary and during S phase may be one of the rate-limiting steps in cell proliferation. It was reported that in vivo overexpression of E2F-1 in fibroblasts induces S phase entry and leads to apoptosis. This observation suggests that E2F plays a role in both cell cycle regulation and apoptosis. To further understand the role of E2F in cell cycle progression, cell death, and tumor development, we have blocked endogenous E2F activity in HBL-100 cells, derived from nonmalignant human breast epithelium, using dominant-negative mutants under the control of a tetracycline-dependent expression system. We have shown here that induction of dominant-negative mutants led to strong downregulation of transiently transfected E2F-dependent chloramphenicol acetyl transferase reporter constructs and of endogenous c-myc, which has been described as a target gene of the transcription factor E2F/DP. In addition, we have shown that blocking of E2F could efficiently protect from apoptosis induced by serum starvation within a period of 10 d, whereas control cells started to die after 24 h. Surprisingly, blocking of E2F did not alter the rate of proliferation or of DNA synthesis of these cells; this finding indicates that cell-cycle progression could be driven in an E2F-independent manner. In addition, we have been able to show that blocking of endogenous E2F in HBL-100 cells led to rapid induction of tumor growth in severe combined immunodeficiency mice. No tumor growth could be observed in mice that received mock-transfected clones or tetracycline to block expression of the E2F mutant constructs in vivo. Thus, it appears that E2F has a potential tumor-suppressive function under certain circumstances. Furthermore, we provide evidence that dysregulation of apoptosis may be an important step in tumorigenesis.

scholarly journals Atypical protein kinase Cλ binds and regulates p70 S6 kinase

1998 ◽  
Vol 335 (2) ◽  
pp. 417-424 ◽  
Author(s):  
Kazunori AKIMOTO ◽  
Masaaki NAKAYA ◽  
Tomoyuki YAMANAKA ◽  
Junpei TANAKA ◽  
Shin-ichi MATSUDA ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Cell Cycle Progression ◽  
Active Form ◽  
Dominant Negative ◽  
Cycle Progression ◽  
S6 Kinase ◽  
P70 S6 Kinase ◽  
Atypical Protein Kinase ◽  
P70 S6k

p70 S6 kinase (p70 S6K) has been implicated in the regulation of cell cycle progression. However, the mechanism of its activation is not fully understood. In the present work, evidence is provided that an atypical protein kinase C (PKC) isotype, PKCλ, is indispensable, but not sufficient, for the activation of p70 S6K. Both the regulatory and kinase domains of PKCλ associate directly with p70 S6K. Overexpression of the kinase domain without kinase activity or the regulatory domain of PKCλ results in the suppression of the serum-induced activation of p70 S6K. In addition, two types of dominant-negative mutants of PKCλ, as well as a kinase-deficient mutant of p70 S6K, suppress serum-induced DNA synthesis and E2F activation. The overexpresion of the active form of PKCλ, however, fails to activate p70 S6K. These results suggest that PKCλ is a mediator in the regulation of p70 S6K activity and plays an important role in cell cycle progression.

scholarly journals Transcriptional Activation of Cyclin D1 Promoter by FAK Contributes to Cell Cycle Progression

2001 ◽  
Vol 12 (12) ◽  
pp. 4066-4077 ◽  
Author(s):  
Jihe Zhao ◽  
Richard Pestell ◽  
Jun-Lin Guan
Keyword(s):  
Cell Cycle ◽  
Cell Adhesion ◽  
Cyclin D1 ◽  
Signaling Pathways ◽  
Transcriptional Activation ◽  
Cell Cycle Progression ◽  
Dominant Negative ◽  
Transcriptional Level ◽  
Cycle Progression ◽  
Regulation Of Cell Cycle

Integrin-mediated cell adhesion to the extracellular matrix is required for normal cell growth. Cyclin D1 is a key regulator of G1-to-S phase progression of the cell cycle. Our previous studies have demonstrated that integrin signaling through focal adhesion kinase (FAK) plays a role in the regulation of cell cycle progression, which correlates with changes in the expression of cyclin D1 and the cdk inhibitor, p21, induced by FAK. In this report, we first investigated the roles of both cyclin D1 and p21 in the regulation of cell cycle progression by FAK. We found that overexpression of a dominant-negative FAK mutant ΔC14 suppressed cell cycle progression in p21−/− cells as effectively as in the control p21+/+ cells. Furthermore, we found that overexpression of ectopic cyclin D1 could rescue cell cycle inhibition by ΔC14. These results suggested that cyclin D1, but not p21, was the primary functional target of FAK signaling pathways in cell cycle regulation. We then investigated the mechanisms underlying the regulation of cyclin D1 expression by FAK signaling. Using Northern blotting and cyclin D1 promoter/luciferase assays, we showed that FAK signaling regulated cyclin D1 expression at the transcriptional level. Using a series of cyclin D1 promoter mutants in luciferase assays as well as electrophoretic mobility shift assays (EMSA), we showed that the EtsB binding site mediated cyclin D1 promoter regulation by FAK. Finally, we showed that FAK regulation of cyclin D1 depends on integrin-mediated cell adhesion and is likely through its activation of the Erk signaling pathway. Together, these studies demonstrate that transcriptional regulation of cyclin D1 by FAK signaling pathways contributes to the regulation of cell cycle progression in cell adhesion.

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