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 The p110 Isoform of the CDP/Cux Transcription Factor Accelerates Entry into S Phase

2006 ◽  
Vol 26 (6) ◽  
pp. 2441-2455 ◽  
Author(s):  
Laurent Sansregret ◽  
Brigitte Goulet ◽  
Ryoko Harada ◽  
Brian Wilson ◽  
Lam Leduy ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Proteolytic Processing ◽  
Experimental Conditions ◽  
Division Rate ◽  
Cycle Progression ◽  
Processing Event

ABSTRACT The CDP/Cux transcription factor was previously found to acquire distinct DNA binding and transcriptional properties following a proteolytic processing event that takes place at the G1/S transition of the cell cycle. In the present study, we have investigated the role of the CDP/Cux processed isoform, p110, in cell cycle progression. Populations of cells stably expressing p110 CDP/Cux displayed a faster division rate and reached higher saturation density than control cells carrying the empty vector. p110 CDP/Cux cells reached the next S phase faster than control cells under various experimental conditions: following cell synchronization in G0 by growth factor deprivation, synchronization in S phase by double thymidine block treatment, or enrichment in G2 by centrifugal elutriation. In each case, duration of the G1 phase was shortened by 2 to 4 h. Gene inactivation confirmed the role of CDP/Cux as an accelerator of cell cycle progression, since mouse embryo fibroblasts obtained from Cutl1z/z mutant mice displayed a longer G1 phase and proliferated more slowly than their wild-type counterparts. The delay to enter S phase persisted following immortalization by the 3T3 protocol and transformation with H-RasV12. Moreover, CDP/Cux inactivation hindered both the formation of foci on a monolayer and tumor growth in mice. At the molecular level, expression of both cyclin E2 and A2 was increased in the presence of p110 CDP/Cux and decreased in its absence. Overall, these results establish that p110 CDP/Cux functions as a cell cycle regulator that accelerates entry into S phase.

scholarly journals Lamina-associated polypeptide 2α regulates cell cycle progression and differentiation via the retinoblastoma–E2F pathway

2006 ◽  
Vol 173 (1) ◽  
pp. 83-93 ◽  
Author(s):  
Daniela Dorner ◽  
Sylvia Vlcek ◽  
Nicole Foeger ◽  
Andreas Gajewski ◽  
Christian Makolm ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Serum Starvation ◽  
Dependent Manner ◽  
Cycle Progression ◽  
Promoter Sequences ◽  
Delayed Entry ◽  
Accelerated Proliferation

Lamina-associated polypeptide (LAP) 2α is a nonmembrane-bound LAP2 isoform that forms complexes with nucleoplasmic A-type lamins. In this study, we show that the overexpression of LAP2α in fibroblasts reduced proliferation and delayed entry into the cell cycle from a G0 arrest. In contrast, stable down-regulation of LAP2α by RNA interference accelerated proliferation and interfered with cell cycle exit upon serum starvation. The LAP2α-linked cell cycle phenotype is mediated by the retinoblastoma (Rb) protein because the LAP2α COOH terminus directly bound Rb, and overexpressed LAP2α inhibited E2F/Rb-dependent reporter gene activity in G1 phase in an Rb-dependent manner. Furthermore, LAP2α associated with promoter sequences in endogenous E2F/Rb-dependent target genes in vivo and negatively affected their expression. In addition, the expression of LAP2α in proliferating preadipocytes caused the accumulation of hypophosphorylated Rb, which is reminiscent of noncycling cells, and initiated partial differentiation into adipocytes. The effects of LAP2α on cell cycle progression and differentiation may be highly relevant for the cell- and tissue-specific phenotypes observed in laminopathic diseases.

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 Cdc53p acts in concert with Cdc4p and Cdc34p to control the G1-to-S-phase transition and identifies a conserved family of proteins.

1996 ◽  
Vol 16 (12) ◽  
pp. 6634-6643 ◽  
Author(s):  
N Mathias ◽  
S L Johnson ◽  
M Winey ◽  
A E Adams ◽  
L Goetsch ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Large Family ◽  
S Phase ◽  
Genetic Interactions ◽  
Cycle Progression ◽  
Cyclin Dependent Kinases ◽  
Ubiquitin Conjugating Enzyme ◽  
Regulation Of Cell Cycle

Regulation of cell cycle progression occurs in part through the targeted degradation of both activating and inhibitory subunits of the cyclin-dependent kinases. During G1, CDC4, encoding a WD-40 repeat protein, and CDC34, encoding a ubiquitin-conjugating enzyme, are involved in the destruction of these regulators. Here we describe evidence indicating that CDC53 also is involved in this process. Mutations in CDC53 cause a phenotype indistinguishable from those of cdc4 and cdc34 mutations, numerous genetic interactions are seen between these genes, and the encoded proteins are found physically associated in vivo. Cdc53p defines a large family of proteins found in yeasts, nematodes, and humans whose molecular functions are uncharacterized. These results suggest a role for this family of proteins in regulating cell cycle proliferation through protein degradation.

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 TRPC1 promotes the genesis and progression of colorectal cancer via activating CaM-mediated PI3K/AKT signaling axis

Oncogenesis ◽  
2021 ◽  
Vol 10 (10) ◽  
Author(s):  
Yang Sun ◽  
Chen Ye ◽  
Wen Tian ◽  
Wen Ye ◽  
Yuan-Yuan Gao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Tumor Growth ◽  
Cell Cycle Progression ◽  
Akt Signaling ◽  
Cycle Progression ◽  
Colorectal Tumorigenesis ◽  
Signaling Axis

AbstractTransient receptor potential canonical (TRPC) channels are the most prominent nonselective cation channels involved in various diseases. However, the function, clinical significance, and molecular mechanism of TRPCs in colorectal cancer (CRC) progression remain unclear. In this study, we identified that TRPC1 was the major variant gene of the TRPC family in CRC patients. TRPC1 was upregulated in CRC tissues compared with adjacent normal tissues and high expression of TRPC1 was associated with more aggressive tumor progression and poor overall survival. TRPC1 knockdown inhibited cell proliferation, cell-cycle progression, invasion, and migration in vitro, as well as tumor growth in vivo; whereas TRPC1 overexpression promoted colorectal tumor growth and metastasis in vitro and in vivo. In addition, colorectal tumorigenesis was significantly attenuated in Trpc1-/- mice. Mechanistically, TRPC1 could enhance the interaction between calmodulin (CaM) and the PI3K p85 subunit by directly binding to CaM, which further activated the PI3K/AKT and its downstream signaling molecules implicated in cell cycle progression and epithelial-mesenchymal transition. Silencing of CaM attenuated the oncogenic effects of TRPC1. Taken together, these results provide evidence that TRPC1 plays a pivotal oncogenic role in colorectal tumorigenesis and tumor progression by activating CaM-mediated PI3K/AKT signaling axis. Targeting TRPC1 represents a novel and specific approach for CRC treatment.

Selective Inhibition of CDK4 and CDK6 Primes Chemoresistant MCL Cells for Killing by Proteasome Inhibitor Bortezomib by Activating Cell Cycle-Coupled Apoptosis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3624-3624
Author(s):  
Maurizio Di Li berto ◽  
Xiangao Huang ◽  
Amy Chadburn ◽  
Peter Martin ◽  
Ruben Niesvizky ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Growth ◽  
Cell Cycle Progression ◽  
Selective Inhibition ◽  
S Phase ◽  
Rational Approach ◽  
Effective Control ◽  
Cycle Progression ◽  
Selective Targeting ◽  
Phase Entry

Abstract Mantle Cell Lymphoma (MCL) remains generally incurable, suggesting that more effective control of unrestrained tumor growth is essential. Loss of cell cycle control is a hallmark of cancer, in particular of MCL where cell cycle progression through G1 is accelerated due to elevation of cyclin-dependent kinase 4 (CDK4) and constitutive cyclin D1 expression. Thus, one rational approach to improve MCL therapy is to target CDK4/6 in combination with cytotoxic killing. Although success in targeting the cell cycle in cancer with broad-spectrum CDK inhibitors has been modest, PD 0332991, the only known CDK4/6-specific inhibitor with oral bioavailability, has been shown to selectively and potently inhibit CDK4/6 in MCL cells ex vivo. Additionally, in a proof-of-mechanism study in patients with recurrent MCL, we have found that PD 0332991 is well tolerated, and effective in inhibiting CDK4 and CDK6 and suppressing tumor growth in vivo. Of note, 50% of the patients (8/16) have achieved a stable disease for greater then 40 weeks (Leonard et al, abstract submitted to ASH 2008). These findings suggest that selective targeting of CDK4 and CDK6 with PD 0332991 is a promising therapy for MCL. To advance targeting of the cell cycle in cancer, we have developed two novel approaches to both inhibit tumor cell proliferation and activate cell cycle-coupled apoptosis in MCL. We show in primary MCL tumor cells and MCL cell lines by BrdU pulse labeling and DNA content analysis that selective inhibition of CDK4/6 with PD 0332991 leads to a complete G1 arrest, despite high level of c-Myc expression and extensive chromosomal abnormality. As PD 0332991 acts reversibly, removal of PD 0332991 immediately releases the G1 block and induces synchronous (>90%) G1-S cell cycle progression and S phase entry. This sensitizes chemoresistant MCL cells to killing by suboptimal doses of cytotoxic agents such as bortezomib, through activating cell cycle-coupled apoptosis during S phase entry. Synergistic killing of MCL cells by induction of cell cycle synchronization with PD 0332991 in combination with bortezomib is mediated by induction of mitochondrial membrane depolarization and activation of caspase-9. In a complementary study, we have demonstrated that selective targeting of CDK4 and CDK6 by PD 0332991 similarly primes chemoresistant primary myeloma cells for cytotoxic killing by activating cell cycle-coupled apoptosis, and induces synergistic tumor suppression in animal models. Selective targeting of CDK4 and CDK6 by PD 0332991 in combination with cytotoxic killing, therefore, represents a promising new strategy for cell cycle-based therapy for MCL and other hematopoietic malignancies.

scholarly journals Caveolin-1 Expression Negatively Regulates Cell Cycle Progression by Inducing G0/G1 Arrest via a p53/p21WAF1/Cip1-dependent Mechanism

2001 ◽  
Vol 12 (8) ◽  
pp. 2229-2244 ◽  
Author(s):  
Ferruccio Galbiati ◽  
Daniela Volonte' ◽  
Jun Liu ◽  
Franco Capozza ◽  
Philippe G. Frank ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Recombinant Expression ◽  
Critical Role ◽  
Primary Cultures ◽  
Serum Starvation ◽  
G1 Arrest ◽  
Cycle Progression ◽  
Caveolin 1

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether caveolin-1 plays any role in regulating cell cycle progression. Here, we directly demonstrate that caveolin-1 expression arrests cells in the G0/G1 phase of the cell cycle. We show that serum starvation induces up-regulation of endogenous caveolin-1 and arrests cells in the G0/G1 phase of the cell cycle. Moreover, targeted down-regulation of caveolin-1 induces cells to exit the G0/G1 phase. Next, we constructed a green fluorescent protein-tagged caveolin-1 (Cav-1-GFP) to examine the effect of caveolin-1 expression on cell cycle regulation. We directly demonstrate that recombinant expression of Cav-1-GFP induces arrest in the G0/G1 phase of the cell cycle. To examine whether caveolin-1 expression is important for modulating cell cycle progression in vivo, we expressed wild-type caveolin-1 as a transgene in mice. Analysis of primary cultures of mouse embryonic fibroblasts from caveolin-1 transgenic mice reveals that caveolin-1 induces 1) cells to exit the S phase of the cell cycle with a concomitant increase in the G0/G1 population, 2) a reduction in cellular proliferation, and 3) a reduction in the DNA replication rate. Finally, we demonstrate that caveolin-1-mediated cell cycle arrest occurs through a p53/p21-dependent pathway. Taken together, our results provide the first evidence that caveolin-1 expression plays a critical role in the modulation of cell cycle progression in vivo.

Phospholipase Cδ1 regulates cell proliferation and cell-cycle progression from G1- to S-phase by control of cyclin E–CDK2 activity

2008 ◽  
Vol 415 (3) ◽  
pp. 439-448 ◽  
Author(s):  
Katherine A. Kaproth-Joslin ◽  
Xiangquan Li ◽  
Sarah E. Reks ◽  
Grant G. Kelley
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cellular Proliferation ◽  
Cyclin E ◽  
Critical Role ◽  
S Phase ◽  
G1 Phase ◽  
Serum Starvation ◽  
Cycle Progression ◽  
Cdk2 Activity

In the present study, we examined the role of PLCδ1 (phospholipase C δ1) in the regulation of cellular proliferation. We demonstrate that RNAi (RNA interference)-mediated knockdown of endogenous PLCδ1, but not PLCβ3 or PLCϵ, induces a proliferation defect in Rat-1 and NIH 3T3 fibroblasts. The decreased proliferation was not due to an induction of apoptosis or senescence, but was associated with an approx. 60% inhibition of [3H]thymidine incorporation. Analysis of the cell cycle with BrdU (bromodeoxyuridine)/propidium iodide-labelled FACS (fluorescence-activated cell sorting) demonstrated an accumulation of cells in G0/G1-phase and a corresponding decrease in cells in S-phase. Further examination of the cell cycle after synchronization by serum-starvation demonstrated normal movement through G1-phase but delayed entry into S-phase. Consistent with these findings, G1 cyclin (D2 and D3) and CDK4 (cyclin-dependent kinase 4) levels and associated kinase activity were not affected. However, cyclin E-associated CDK2 activity, responsible for G1-to-S-phase progression, was inhibited. This decreased activity was accompanied by unchanged CDK2 protein levels and paradoxically elevated cyclin E and cyclin E-associated CDK2 levels, suggesting inhibition of the cyclin E–CDK2 complex. This inhibition was not due to altered stimulatory or inhibitory phosphorylation of CDK2. However, p27, a Cip/Kip family CKI (CDK inhibitor)-binding partner, was elevated and showed increased association with CDK2 in PLCδ1-knockdown cells. The result of the present study demonstrate a novel and critical role for PLCδ1 in cell-cycle progression from G1-to-S-phase through regulation of cyclin E–CDK2 activity and p27 levels.

scholarly journals NPAT Expression Is Regulated by E2F and Is Essential for Cell Cycle Progression

2003 ◽  
Vol 23 (8) ◽  
pp. 2821-2833 ◽  
Author(s):  
Guang Gao ◽  
Adrian P. Bracken ◽  
Karina Burkard ◽  
Diego Pasini ◽  
Marie Classon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Histone Gene ◽  
Cycle Progression ◽  
Histone Gene Transcription ◽  
E2f Proteins ◽  
Phase Entry

ABSTRACT NPAT is an in vivo substrate of cyclin E-Cdk2 kinase and is thought to play a critical role in coordinated transcriptional activation of histone genes during the G1/S-phase transition and in S-phase entry in mammalian cells. Here we show that NPAT transcription is up-regulated at the G1/S-phase boundary in growth-stimulated cells and that the NPAT promoter responds to activation by E2F proteins. We demonstrate that endogenous E2F proteins interact with the promoter of the NPAT gene in vivo and that induced expression of E2F1 stimulates NPAT mRNA expression, supporting the idea that the expression of NPAT is regulated by E2F. Consistently, we find that the E2F sites in the NPAT promoter are required for its activation during the G1/S-phase transition. Moreover, we show that the expression of NPAT accelerates S-phase entry in cells released from quiescence. The inhibition of NPAT expression by small interfering RNA duplexes impedes cell cycle progression and histone gene expression in tissue culture cells. Thus, NPAT is an important E2F target that is required for cell cycle progression in mammalian cells. As NPAT is involved in the regulation of S-phase-specific histone gene transcription, our findings indicate that NPAT links E2F to the activation of S-phase-specific histone gene transcription.

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