scholarly journals G1 arrest and differentiation can occur independently of Rb family function

2010 ◽  
Vol 191 (4) ◽  
pp. 809-825 ◽  
Author(s):  
Stacey E. Wirt ◽  
Adam S. Adler ◽  
Véronique Gebala ◽  
James M. Weimann ◽  
Bethany E. Schaffer ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Regulatory Networks ◽  
Target Genes ◽  
Family Members ◽  
Mouse Embryos ◽  
G1 Arrest ◽  
Cell Cycle Exit ◽  
Family Function ◽  
Rb Pathway

The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9–11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway.

scholarly journals Target Gene Specificity of E2F and Pocket Protein Family Members in Living Cells

2000 ◽  
Vol 20 (16) ◽  
pp. 5797-5807 ◽  
Author(s):  
Julie Wells ◽  
Kathryn E. Boyd ◽  
Christopher J. Fry ◽  
Stephanie M. Bartley ◽  
Peggy J. Farnham
Keyword(s):  
Cell Cycle ◽  
Target Gene ◽  
Target Genes ◽  
Protein Complexes ◽  
Current Model ◽  
Family Members ◽  
Living Cells ◽  
Gene Promoters ◽  
Pocket Protein ◽  
Protein Dna Interactions

ABSTRACT E2F-mediated transcription is thought to involve binding of an E2F-pocket protein complex to promoters in the G0 phase of the cell cycle and release of the pocket protein in late G1, followed by release of E2F in S phase. We have tested this model by monitoring protein-DNA interactions in living cells using a formaldehyde cross-linking and immunoprecipitation assay. We find that E2F target genes are bound by distinct E2F-pocket protein complexes which change as cells progress through the cell cycle. We also find that certain E2F target gene promoters are bound by pocket proteins when such promoters are transcriptionally active. Our data indicate that the current model applies only to certain E2F target genes and suggest that Rb family members may regulate transcription in both G0 and S phases. Finally, we find that a given promoter can be bound by one of several different E2F-pocket protein complexes at a given time in the cell cycle, suggesting that cell cycle-regulated transcription is a stochastic, not a predetermined, process.

Repression of Mir-106b Family Members Does Not Alter Cell Cycle Progression in Hodgkin Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4722-4722
Author(s):  
Johan H Gibcus ◽  
Lu Ping Tan ◽  
Rikst Nynke Schakel ◽  
Geert Harms ◽  
Peter Moeller ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Cycle Arrest ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Target Genes ◽  
Family Members ◽  
Luciferase Reporter ◽  
P21 Protein ◽  
Cycle Arrest

Abstract MicroRNAs (miRNAs) are 19–25 nucleotide long RNA molecules derived from precursor genes that inhibit the expression of target genes by binding to their 3′ UTR region. Expression of miRNAs is often tissue specific and miRNA profiling has shown specific miRNA expression patterns in both B-cell development and lymphomagenesis. Hodgkin lymphoma is derived from pre-apoptotic germinal center B-cells, although a general loss of B cell phenotype is noted. Using quantitative RT-PCR and miRNA microarray, we determined the miRNA profile of HL and compared this with the profile of a panel of B-cell non-Hodgkin lymphomas (NHL). The two methods showed a very good correlation for the expression levels of the individual miRNAs. Using a large panel of cell lines, we confirmed differential expression between HL and other B-cell lymphoma derived cell lines for 27 miRNAs. The HL specific miRNAs included miR-155, miR-21 and miR-106b seed family members miR-17-5p, miR-20a, miR-93, miR-106a and miR- 106b. Next, we performed target gene validation of predicted target genes for miR-17-5p, which is highly expressed in HL. Using luciferase reporter assays with stabilized anti-sense miR17-5p oligonucleotides, we showed that GPR137B, RAB12 and RBJ are likely miR-17-5p target genes in two different HL cell lines. Previous publications indicated that miR-106b seed family members negatively regulate the cyclin-dependent kinase inhibitor 1A (p21/CIP1) resulting in cell cycle arrest at G1. Consistent with these findings, we show that the miR-106b family members are highly expressed in L428, whereas p21 is not. However, inhibition of the miR-106b seed family members in L428 does not result in elevated p21 protein expression. Furthermore, there is no cell cycle arrest, growth reduction or increase in cell death and apoptosis after inhibition of the miR-106b seed family members. Thus, we conclude that blocking of the miR-106b seed family members does not necessarily lead to indiction of p21 protein. This suggests an additional regulatory layer of p21 expression in L428 cells.

scholarly journals Hypoxia. Cross talk between oxygen sensing and the cell cycle machinery

2011 ◽  
Vol 301 (3) ◽  
pp. C550-C552 ◽  
Author(s):  
Gregg L. Semenza
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Hypoxia Inducible Factor ◽  
Physiological Property ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Transactivation Domain ◽  
Amino Terminal ◽  
Hypoxic Conditions ◽  
Mcm Proteins

A fundamental physiological property of mammalian cells is the regulation of proliferation according to O2 availability. Progression through the cell cycle is inhibited under hypoxic conditions in many, but not all, cell types, and this G1 arrest is dependent on hypoxia-inducible factor (HIF) 1α. Components of the hexameric MCM helicase, which binds to replication origins before the onset of DNA synthesis, are present in large excess in mammalian cells relative to origins, suggesting that they may have additional functions. Screens for HIF-1α interacting proteins revealed that MCM7 binds to the amino-terminal PER-SIM-ARNT (PAS) domain of HIF-1α and stimulates prolyl hydroxylation-dependent ubiquitination and degradation of HIF-1α, whereas MCM3 binds to the carboxyl terminus of HIF-1α and enhances asparaginyl hydroxylation-dependent inhibition of HIF-1α transactivation domain function. Thus MCM proteins inhibit HIF activity via two distinct O2-dependent mechanisms. Under prolonged hypoxic conditions, MCM mRNA expression is inhibited in a HIF-1α-dependent manner. Thus HIF and MCM proteins act in a mutually antagonistic manner, providing a novel molecular mechanism for homeostatic regulation of cell proliferation based on the relative levels of these proteins.

scholarly journals Dissection of the genetic programs of p53-mediated G1 growth arrest and apoptosis: blocking p53-induced apoptosis unmasks G1 arrest

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2691-2698 ◽  
Author(s):  
C Guillouf ◽  
X Grana ◽  
M Selvakumaran ◽  
A De Luca ◽  
A Giordano ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Target Genes ◽  
Ectopic Expression ◽  
Growth Arrest ◽  
Genetically Engineered ◽  
Temperature Sensitive ◽  
G1 Arrest ◽  
Induced Apoptosis ◽  
High Level ◽  
And Function

Employing the myeloblastic leukemia M1 cell line, which does not express endogenous p53, and genetically engineered variants, it was recently shown that activation of p53, using a p53 temperature-sensitive mutant transgene (p53ts), resulted in rapid apoptosis that was delayed by high level ectopic expression of bcl-2. In this report, advantage has been taken of these M1 variants to investigate the relationship between p53-mediated G1 arrest and apoptosis. Flow cytometric cell cycle analysis has provided evidence that activation of wild-type (wt) p53 function in M1 cells resulted in the induction of G1 growth arrest; this was clearly seen in the M1p53/bcl-2 cells because of the delay in apoptosis that unmasked p53-induced G1 growth arrest. This finding was further corroborated at the molecular level by analysis of the expression and function of key cell cycle regulatory genes in M1p53 versus M1p53/bcl-2 cells after the activation of wt p53 function; events that take place at early times during the p53-induced G1 arrest occur in both the M1p53 and the M1p53/bcl-2 cells, whereas later events occur only in the M1p53/bcl-2 cells, which undergo delayed apoptosis, thereby allowing the cells to complete G1 arrest. Finally, it was observed that a spectrum of p53 target genes implicated in p53-induced growth suppression and apoptosis were similarly regulated, either induced (gadd45, waf1, mdm2, and bax) or suppressed (c-myc and bcl-2), after activation of wt p53 function in M1p53 and M1p53/bcl-2 cells. Taken together, these findings show that wt p53 can simultaneously induce the genetic programs of both G1 growth arrest and apoptosis within the same cell type, in which the genetic program of cell death can proceed in either G1-arrested (M1p53/bcl-2) or cycling (M1p53) cells. These findings increase our understanding of the functions of p53 as a tumor suppressor and how alterations in these functions could contribute to malignancy.

scholarly journals Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4.

1995 ◽  
Vol 15 (5) ◽  
pp. 2682-2688 ◽  
Author(s):  
F K Chan ◽  
J Zhang ◽  
L Cheng ◽  
D N Shapiro ◽  
A Winoto
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Mammalian Cells ◽  
Cyclin E ◽  
G1 Arrest ◽  
Cdk Inhibitor ◽  
Cyclin D ◽  
Human And Mouse

The cell cycle in mammalian cells is regulated by a series of cyclins and cyclin-dependent kinases (CDKs). The G1/S checkpoint is mainly dictated by the kinase activities of the cyclin D-CDK4 and/or cyclin D-CDK6 complex and the cyclin E-CDK2 complex. These G1 kinases can in turn be regulated by cell cycle inhibitors, which may cause the cells to arrest at the G1 phase. In T-cell hybridomas, addition of anti-T-cell receptor antibody results not only in G1 arrest but also in apoptosis. In searching for a protein(s) which might interact with Nur77, an orphan steroid receptor required for activation-induced apoptosis of T-cell hybridomas, we have cloned a novel human and mouse CDK inhibitor, p19. The deduced p19 amino acid sequence consists of four ankyrin repeats with 48% identity to p16. The human p19 gene is located on chromosome 19p13, distinct from the positions of p18, p16, and p15. Its mRNA is expressed in all cell types examined. The p19 fusion protein can associate in vitro with CDK4 but not with CDK2, CDC2, or cyclin A, B, E, or D1 to D3. Addition of p19 protein can lead to inhibition of the in vitro kinase activity of cyclin D-CDK4 but not that of cyclin E-CDK2. In T-cell hybridoma DO11.10, p19 was found in association with CDK4 and CDK6 in vivo, although its association with Nur77 is not clear at this point. Thus, p19 is a novel CDK inhibitor which may play a role in the cell cycle regulation of T cells.

scholarly journals Nuclear Reorganization of Mammalian DNA Synthesis Prior to Cell Cycle Exit

2004 ◽  
Vol 24 (2) ◽  
pp. 595-607 ◽  
Author(s):  
David A. Barbie ◽  
Brian A. Kudlow ◽  
Richard Frock ◽  
Jiyong Zhao ◽  
Brett R. Johnson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Large Scale ◽  
Replicative Senescence ◽  
S Phase ◽  
Serum Starvation ◽  
Cell Cycle Exit ◽  
Replication Foci

ABSTRACT In primary mammalian cells, DNA replication initiates in a small number of perinucleolar, lamin A/C-associated foci. During S-phase progression in proliferating cells, replication foci distribute to hundreds of sites throughout the nucleus. In contrast, we find that the limited perinucleolar replication sites persist throughout S phase as cells prepare to exit the cell cycle in response to contact inhibition, serum starvation, or replicative senescence. Proteins known to be involved in DNA synthesis, such as PCNA and DNA polymerase δ, are concentrated in perinucleolar foci throughout S phase under these conditions. Moreover, chromosomal loci are redirected toward the nucleolus and overlap with the perinucleolar replication foci in cells poised to undergo cell cycle exit. These same loci remain in the periphery of the nucleus during replication under highly proliferative conditions. These results suggest that mammalian cells undergo a large-scale reorganization of chromatin during the rounds of DNA replication that precede cell cycle exit.

scholarly journals Mammalian WDR12 is a novel member of the Pes1–Bop1 complex and is required for ribosome biogenesis and cell proliferation

2005 ◽  
Vol 170 (3) ◽  
pp. 367-378 ◽  
Author(s):  
Michael Hölzel ◽  
Michaela Rohrmoser ◽  
Martin Schlee ◽  
Thomas Grimm ◽  
Thomas Harasim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Mammalian Cells ◽  
Ribosome Biogenesis ◽  
Target Genes ◽  
Dominant Negative ◽  
Stable Complex ◽  
Dominant Negative Mutant ◽  
Proliferating Cells ◽  
Independent Manner

Target genes of the protooncogene c-myc are implicated in cell cycle and growth control, yet the linkage of both is still unexplored. Here, we show that the products of the nucleolar target genes Pes1 and Bop1 form a stable complex with a novel member, WDR12 (PeBoW complex). Endogenous WDR12, a WD40 repeat protein, is crucial for processing of the 32S precursor ribosomal RNA (rRNA) and cell proliferation. Further, a conditionally expressed dominant-negative mutant of WDR12 also blocks rRNA processing and induces a reversible cell cycle arrest. Mutant WDR12 triggers accumulation of p53 in a p19ARF-independent manner in proliferating cells but not in quiescent cells. Interestingly, a potential homologous complex of Pes1–Bop1–WDR12 in yeast (Nop7p–Erb1p–Ytm1p) is involved in the control of ribosome biogenesis and S phase entry. In conclusion, the integrity of the PeBoW complex is required for ribosome biogenesis and cell proliferation in mammalian cells.

scholarly journals Only Akt1 Is Required for Proliferation, while Akt2 Promotes Cell Cycle Exit through p21 Binding

2006 ◽  
Vol 26 (22) ◽  
pp. 8267-8280 ◽  
Author(s):  
Lisa Héron-Milhavet ◽  
Celine Franckhauser ◽  
Vanessa Rana ◽  
Cyril Berthenet ◽  
Daniel Fisher ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Specific Interaction ◽  
Cyclin A ◽  
Cell Cycle Exit ◽  
Cycle Progression ◽  
Negative Effect

ABSTRACT Protein kinase B (PKB/Akt) is an important modulator of insulin signaling, cell proliferation, and survival. Using small interfering RNA duplexes in nontransformed mammalian cells, we show that only Akt1 is essential for cell proliferation, while Akt2 promotes cell cycle exit. Silencing Akt1 resulted in decreased cyclin A levels and inhibition of S-phase entry, effects not seen with Akt2 knockdown and specifically rescued by microinjection of Akt1, not Akt2. In differentiating myoblasts, Akt2 knockout prevented myoblasts from exiting the cell cycle and showed sustained cyclin A expression. In contrast, overexpression of Akt2 reduced cyclin A and hindered cell cycle progression in M-G1 with increased nuclear p21. p21 is a major target in the differential effects of Akt isoforms, with endogenous Akt2 and not Akt1 binding p21 in the nucleus and increasing its level. Accordingly, Akt2 knockdown cells, and not Akt1 knockdown cells, showed reduced levels of p21. A specific Akt2/p21 interaction can be reproduced in vitro, and the Akt2 binding site on p21 is similar to that in cyclin A spanning T145 to T155, since (i) prior incubation with cyclin A prevents Akt2 binding, (ii) T145 phosphorylation on p21 by Akt1 prevents Akt2 binding, and (iii) binding Akt2 prevents phosphorylation of p21 by Akt1. These data show that specific interaction of the Akt2 isoform with p21 is key to its negative effect on normal cell cycle progression.

scholarly journals Knockdown of the oncogene lncRNA NEAT1 restores the availability of miR-34c and improves the sensitivity to cisplatin in osteosarcoma

2018 ◽  
Vol 38 (3) ◽  
Author(s):  
Yuliang Hu ◽  
Qiuyong Yang ◽  
Long Wang ◽  
Shuo Wang ◽  
Fei Sun ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Tumor Growth ◽  
Cyclin D1 ◽  
Clinical Significance ◽  
Cell Lines ◽  
Target Genes ◽  
Tumor Regression ◽  
G1 Arrest ◽  
Ki 67

Aberrant expressions of long non-coding RNAs (lncRNAs) are the culprits of carcinogenesis via regulating the tumor suppressor or oncogene. LncRNA nuclear enriched abundant transcript 1 (NEAT1) has been identified to be an oncogene to promote tumor growth and metastasis of many cancers. However, the clinical significance and function of NEAT1 in osteosarcoma (OS) remain to be discovered. We here collected OS tissues (n=40) and adjacent non-tumor tissues (n=20) to determine the expression of NEAT1 and its clinical significance. NEAT1 was overexpressed in OS tissues, which positively correlated with tumor size, Enneking stage, and distant metastasis of OS patients. The elevated level of NEAT1 was confirmed in OS cell lines including MG63 and HOS in vitro. Knockdown of NEAT1 by two siRNAs induced impaired cell vitalities, promoted the apoptosis, and G0/G1 arrest in two cell lines, which was associated with inhibited anti-apoptosis signals BCL-2 pathway and cell cycle-related cyclin D1 (CCND1) signals. Moreover, the tumor suppressor miR-34c was negatively regulated and inhibited by NEAT1 in OS. Suppression of miR-34c could up-regulate the expressions of its target genes BCL-2 and CCND1 to antagonize the effects of NEAT1 knockdown. Furthermore, overexpressed NEAT1 reduced the sensitivity of cisplatin (DDP) and inhibited DDP-induced apoptosis and cell cycle arrest via miR-34c. The results in vivo also confirmed that knockdown of NEAT1 sensitized the OS cells to DPP-induced tumor regression, delayed the tumor growth with reduced levels of Ki-67, BCL-2, and cyclin D1 signals, suggesting that NEAT1 is an oncogene and chemotherapy resistant factor in OS.

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