scholarly journals Cyclin A expression is under negative transcriptional control during the cell cycle.

1996 ◽  
Vol 16 (7) ◽  
pp. 3789-3798 ◽  
Author(s):  
X Huet ◽  
J Rech ◽  
A Plet ◽  
A Vié ◽  
J M Blanchard
Keyword(s):  
Cell Cycle ◽  
Transcriptional Repression ◽  
Transcriptional Control ◽  
Cell Cycle Progression ◽  
Cyclin A ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Proliferating Cells

Transcription of the gene coding for cyclin A, a protein required for S-phase transit, is cell cycle regulated and is restricted to proliferating cells. To further explore transcriptional regulation linked to cell division cycle control, a genomic clone containing 5' flanking sequences of the murine cyclin A gene was isolated. When it was fused to a luciferase reporter gene, it was shown to function as a proliferation-regulated promoter in NIH 3T3 cells. Transcription of the mouse cyclin A gene is negatively regulated by arrest of cell proliferation. A mutation of a GC-rich sequence conserved between mice and humans is sufficient to relieve transcriptional repression, resulting in a promoter with constitutively high activity. In agreement with this result, in vivo footprinting reveals a protection of the cell cycle-responsive element in G0/early G1 cells which is not observed at later stages of the cell cycle. Moreover, the footprint is present in dimethyl sulfoxide-induced differentiating and not in proliferating Friend erythroleukemia cells. Conversely, two other sites, which in vitro bind ATF-1 and NF-Y, respectively, are constitutively occupied throughout cell cycle progression.

scholarly journals BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

1999 ◽  
Vol 19 (7) ◽  
pp. 4843-4854 ◽  
Author(s):  
Heinz Ruffner ◽  
Wei Jiang ◽  
A. Grey Craig ◽  
Tony Hunter ◽  
Inder M. Verma
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

scholarly journals Cyclin D3-CDK6 Complex Facilitates Tumorigenesis by Regulating the C-Myc/miR-15a/16 Axis in a Feedback Loop in Gastric Cancer

2020 ◽  
Author(s):  
Yeting Hong ◽  
Wei He ◽  
Jianbin Zhang ◽  
Lu Shen ◽  
Chong Yu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Feedback Loop ◽  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Cyclin D3 ◽  
Luciferase Reporter

Abstract Background: Cyclin D3-CDK6 complex is a component of the core cell cycle machinery that regulates cell proliferation. By using Human Protein Atlas database, a higher expression level of this complex was found in gastric cancer. However, the function of this complex in gastric cancer remain poorly understood. This study aims to determine the expression pattern of this complex in gastric cancer and to investigate its biological role during tumorigenesis.Methods: To demonstrate that Cyclin D3-CDK6 regulate the c-Myc/miR-15a/16 axis in a feedback loop in gastric cancer, a series of methods were conducted both in vitro and in vivo experiments, including qRT-PCR, western blot analysis, EdU assay, flow cytometry, luciferase reporter assay and immunohistochemical staining. SPSS and Graphpad prism software were used for data analysis.Results: In this study, we found that Cyclin D3 and CDK6 were significantly upregulated in gastric cancer and correlated with poorer overall survival. Further study proved that this complex significantly promoted cell proliferation and cell cycle progression in vitro and accelerated xenografted tumor growth in vivo. Furthermore, we explored the molecular mechanisms through which the complex mediated Rb phosphorylation and then promoted c-Myc expression in vitro, we also found c-Myc could suppress miR-15a/16 expression in gastric cancer cell. Finally, we found that miR-15a/16 can simultaneously regulate Cyclin D3 and CDK6 expression as direct target genes.Conclusions: Our findings uncover the Cyclin D3-CDK6/c-Myc/miR-15a/16 feedback loop axis as a pivotal role in the regulation of gastric cancer tumorigenesis, and this regulating axis may provide a potential therapeutic target for gastric cancer treatment.

scholarly journals LMNB2 Promotes the Progression of Colorectal Cancer by Silencing p21 Expression

2020 ◽  
Author(s):  
Chen-Hua Dong ◽  
Tao Jiang ◽  
Hang Yin ◽  
Hu Song ◽  
Yi Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Cycle Progression ◽  
Luciferase Reporter ◽  
Cycle Progression ◽  
Cancer Tissues ◽  
P21 Promoter

Abstract Background: Lamin B2 (LMNB2) is involved in chromatin remodelling and the rupture and reorganization of the nuclear membrane during mitosis, which is necessary for eukaryotic cell proliferation. However, there are few reports on the expression and function of LMNB2 in colorectal cancer.Methods: A tissue microarray (TAM) was used to detect the expression of LMNB2 in 226 colorectal cancer tissues and the corresponding adjacent tissues. The CCK-8 colorimetric assay, EdU incorporation analyses, colony formation assays and cell cycle experiments were used to evaluate the effect of LMNB2 on colorectal cancer cell proliferation in vitro, and a mouse tumorigenic model was used to study the effect of LMNB2 on colorectal cancer cells in vivo. The main pathways and genes regulated by LMNB2 were detected by RNA sequencing. Dual-luciferase reporter assays were conducted to test the direct binding between LMNB2 and p21, and ChIP analysis showed that LMNB2 promotes cell proliferation by regulating the p21 promoter.Results: The results showed that LMNB2 expression is increased in colorectal cancer tissues. Highly expressed LMNB2 is associated with tumour size and TNM stage. Multivariate Cox analysis showed that LMNB2 can be used as an independent prognostic factor in patients with colorectal cancer. Functional assays indicated that LMNB2 obviously enhanced cell proliferation by promoting cell cycle progression in vitro and in vivo. LMNB2 facilitates cell proliferation via regulating the p21 promoter, whereas LMNB2 had no effect on cell apoptosis in terms of mechanism.Conclusion: LMNB2 promotes the proliferation of colorectal cancer by regulating p21-mediated cell cycle progression, indicating the potential value of LMNB2 as a clinical prognostic marker and molecular therapeutic target.

scholarly journals Ectopic Methylation of a Single Persistently Unmethylated CpG in the Promoter of the Vitellogenin Gene Abolishes Its Inducibility by Estrogen through Attenuation of Upstream Stimulating Factor Binding

2019 ◽  
Vol 39 (23) ◽  
Author(s):  
Lia Kallenberger ◽  
Rachel Erb ◽  
Lucie Kralickova ◽  
Andrea Patrignani ◽  
Esther Stöckli ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Transcriptional Control ◽  
Dna Demethylation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Reporter System ◽  
E Box ◽  
Stimulating Factor

ABSTRACT The enhancer/promoter of the vitellogenin II gene (VTG) has been extensively studied as a model system of vertebrate transcriptional control. While deletion mutagenesis and in vivo footprinting identified the transcription factor (TF) binding sites governing its tissue specificity, DNase hypersensitivity and DNA methylation studies revealed the epigenetic changes accompanying its hormone-dependent activation. Moreover, upon induction with estrogen (E2), the region flanking the estrogen-responsive element (ERE) was reported to undergo active DNA demethylation. We now show that although the VTG ERE is methylated in embryonic chicken liver and in LMH/2A hepatocytes, its induction by E2 was not accompanied by extensive demethylation. In contrast, E2 failed to activate a VTG enhancer/promoter-controlled luciferase reporter gene methylated by SssI. Surprisingly, this inducibility difference could be traced not to the ERE but rather to a single CpG in an E-box (CACGTG) sequence upstream of the VTG TATA box, which is unmethylated in vivo but methylated by SssI. We demonstrate that this E-box binds the upstream stimulating factor USF1/2. Selective methylation of the CpG within this binding site with an E-box-specific DNA methyltransferase, Eco72IM, was sufficient to attenuate USF1/2 binding in vitro and abolish the hormone-induced transcription of the VTG gene in the reporter system.

scholarly journals Ectopic methylation of a single persistently-unmethylated CpG in the promoter of the vitellogenin gene abolishes its inducibility by estrogen through attenuation of USF binding

2019 ◽  
Author(s):  
Lia Kallenberger ◽  
Rachel Erb ◽  
Lucie Kralickova ◽  
Andrea Patrignani ◽  
Esther Stöckli ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Dna Demethylation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Reporter System ◽  
Active Dna Demethylation ◽  
E Box ◽  
Vitellogenin Gene

ABSTRACTThe enhancer/promoter of the vitellogenin II (VTG) gene has been extensively studied as a model system of vertebrate transcriptional control. While deletion mutagenesis and in vivo footprinting identified the transcription factor (TF) binding sites governing its tissue specificity, DNase hypersensitivity- and DNA methylation studies revealed the epigenetic changes accompanying its hormone-dependent activation. Moreover, upon induction with estrogen (E2), the region flanking the estrogen-responsive element (ERE) was reported to undergo active DNA demethylation. We now show that although the VTG ERE is methylated in embryonic chicken liver and in LMH/2A hepatocytes, its induction by E2 was not accompanied by extensive demethylation. In contrast, E2 failed to activate a VTG enhancer/promoter-controlled luciferase reporter gene methylated by SssI. Surprisingly, this inducibility difference could be traced not to the ERE, but rather to a single CpG in an E-box (CACGTG) sequence upstream of the VTG TATA box, which is unmethylated in vivo, but methylated by SssI. We demonstrate that this E-box binds the upstream stimulating factor USF1/2. Selective methylation of the CpG within this binding site with an E-box-specific DNA methyltranferase Eco72IM was sufficient to attenuate USF1/2 binding in vitro and abolish the hormone-induced transcription of the VTG gene in the reporter system.

scholarly journals LINC00346 Sponges miR-30c-2-3p to Promote the Development of Lung Adenocarcinoma by Targeting MYBL2 and Regulating CELL CYCLE Signaling Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Qian Xu ◽  
Zhenwu Xu ◽  
Kai Zhu ◽  
Jinlan Lin ◽  
Bo Ye
Keyword(s):  
Cell Cycle ◽  
Tumor Growth ◽  
Lung Adenocarcinoma ◽  
Signaling Pathway ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Cell Cycle Signaling

BackgroundLINC00346 has recently been reported to regulate the development of several cancer types, but its biological functions and underlying mechanisms in lung adenocarcinoma (LUAD) have not been elucidated. The purpose of this study was to investigate the molecular mechanism of LINC00346 in the progression of LUAD.MethodsBioinformatics was performed to find the target lncRNA, miRNA and mRNA, and the binding relationship between the target genes was verified by dual luciferase reporter gene and RIP assays. Fluorescence in situ hybridization was used to detect the location of LINC00346 in LUAD tissues. The expressions of LINC00346, miR-30c-2-3p and MYBL2 in each group were detected by qRT-PCR, and western blot was performed to detect expressions of MYBL2 and CELL CYCLE related proteins. Proliferation, metastasis, apoptosis and cell cycle of LUAD cells were detected by CCK-8, colony formation, Transwell and flow cytometry assays, respectively. Mouse xenograft models were established to further determine the effects of LINC00346 on LUAD tumor growth in vivo.ResultsLINC00346 was upregulated in LUAD tissues and cells and was mainly localized in the cytoplasm. Knockdown of LINC00346 inhibited tumor growth in vivo, proliferation, metastasis and cell cycle progression, while induced apoptosis. LINC00346 sponged miR-30c-2-3 by targeting MYBL2 and regulating CELL CYCLE signaling pathway. Inhibiting miR-30c-2-3p or overexpressing MYBL2 could reverse the inhibitory effect of LINC00346 knockdown on LUAD process.ConclusionsLINC00346 as a ceRNA played a carcinogenic role in the development of LUAD via miR-30c-2-3p/MYBL2 axis regulating the CELL CYCLE signaling pathway. The study generally elucidated the mechanism by which LINC00346 regulated the development of LUAD, providing new ideas for the diagnosis and treatment of LUAD guided by lncRNA.

scholarly journals Circular RNA hsa_circ_0061395 accelerates hepatocellular carcinoma progression via regulation of the miR-877-5p/PIK3R3 axis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yanhui Yu ◽  
Lijuan Bian ◽  
Renfei Liu ◽  
Yitong Wang ◽  
Xia Xiao
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Circular Rna ◽  
Cell Counting ◽  
Regulatory Subunit ◽  
Luciferase Reporter ◽  
Hcc Cells

Abstract Background Circular RNA hsa_circ_0061395 (circ_0061395) has been reported to accelerate the advancement of hepatocellular carcinoma (HCC). However, the regulatory mechanism by which circ_0061395 modulates the progression of HCC is unclear. Methods The morphology and size of exosomes were analyzed by transmission electron microscope (TEM) and nanoparticle-tracking analysis (NTA). Protein levels were detected by western blotting. Expression levels of circ_0061395, microRNA (miR)-877-5p, and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) mRNA were assessed by quantitative real time polymerase chain reaction (qRT-PCR). The proliferation, invasion, migration, cell cycle progression, and apoptosis were analyzed by cell counting kit-8 (CCK-8), plate clone, transwell, or flow cytometry assays. The targeting relationship between circ_0061395 or PIK3R3 and miR-877-5p was verified using the dual-luciferase reporter and/or RNA immunoprecipitation (RIP) assays. Xenograft assay was performed to confirm the biological function of circ_0061395 in HCC. Results Circ_0061395 was upregulated in HCC tissues, serum, cells, and serum-derived exosomes. Circ_0061395 silencing decreased tumor growth in vivo, and induced cell cycle arrest, apoptosis, repressed proliferation, invasion, and migration of HCC cells in vitro. MiR-877-5p was downregulated while PIK3R3 was upregulated in HCC. Circ_0061395 regulated PIK3R3 expression via competitively binding to miR-877-5p. MiR-877-5p inhibitor overturned circ_0061395 knockdown-mediated influence on malignant behaviors of HCC cells. PIK3R3 overexpression reversed the suppressive influence of miR-877-5p mimic on malignant behaviors of HCC cells. Conclusion Circ_0061395 facilitated HCC progression via regulating the miR-877-5p/PIK3R3 axis, providing a new perspective on the advancement of HCC.

Phosphorylation of MAP4 affects microtubule properties and cell cycle progression

2001 ◽  
Vol 114 (15) ◽  
pp. 2879-2887 ◽  
Author(s):  
Winston Chang ◽  
Dorota Gruber ◽  
Sripriya Chari ◽  
Hidefumi Kitazawa ◽  
Yuko Hamazumi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Binding Domain ◽  
Proliferating Cells ◽  
Cycle Progression ◽  
Microtubule Binding ◽  
Microtubule Binding Domain ◽  
In Cells

In human cells, MAP4, a microtubule-associated protein ubiquitously expressed in proliferating cells, has been shown to undergo in vivo phosphorylation. Two phosphorylation sites, serines 696 and 787, lie within the proline-rich region of its microtubule-binding domain. To test the hypothesis that phosphorylation at these sites influences microtubule properties or cell cycle progression, we prepared stable cell lines that inducibly express versions of MAP4 in which phosphorylation of these two serines was prevented by their replacement with alanine, lysine, or glutamate residues (AA-, KK-, or EE-MAP4). All non-phosphorylatable mutant forms of MAP4 expressed in mouse Ltk- cells were localized to MT arrays that were unremarkable in appearance. Expression of non-phosphorylatable mutants of MAP4 did not affect cell doubling time; however, expression of some mutants altered progression into or through cell division. Interactions of mutant MAP4 with MTs were examined in vitro. KK mutant MAP4 bound MTs more avidly than its wild-type counterpart, WT-MAP4. In vivo MT polymer also differed among the mutants: MTs in cells expressing the KK- and AA-MAP4 forms were more resistant to nocodazole depolymerization than those in cells expressing EE- or WT-MAP4 forms. Our results demonstrate that phosphorylation alters MAP4 properties and suggest a raison d'être for phosphorylation of the MAP4 microtubule-binding domain during cell cycle progression.

scholarly journals MiR-200-3p Is Potentially Involved in Cell Cycle Arrest by Regulating Cyclin A during Aestivation in Apostichopus japonicus

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 843 ◽  
Author(s):  
Shanshan Wang ◽  
Muyan Chen ◽  
Yingchao Yin ◽  
Kenneth B. Storey
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Marine Invertebrates ◽  
Apostichopus Japonicus ◽  
Model Organism ◽  
Cyclin A ◽  
Luciferase Reporter ◽  
Gene Transcript ◽  
Loss Of Function

The sea cucumber (Apostichopus japonicus) has become a good model organism for studying environmentally induced aestivation in marine invertebrates. We hypothesized that mechanisms that arrest energy-expensive cell cycle activity would contribute significantly to establishing the hypometabolic state during aestivation. Cyclin A is a core and particularly interesting cell cycle regulator that functions in both the S phase and in mitosis. In the present study, negative relationships between miR-200-3p and AjCA expressions were detected at both the transcriptional and the translational levels during aestivation in A. japonicus. Dual-luciferase reporter assays confirmed the targeted location of the miR-200-3p binding site within the AjCA gene transcript. Furthermore, gain- and loss-of-function experiments were conducted in vivo with sea cucumbers to verify the interaction between miR-200-3p and AjCA in intestine tissue by qRT-PCR and Western blotting. The results show that the overexpression of miR-200-3p mimics suppressed AjCA transcript levels and translated protein production, whereas transfection with a miR-200-3p inhibitor enhanced both AjCA mRNA and AjCA protein in A. japonicus intestine. Our findings suggested a potential mechanism that reversibly arrests cell cycle progression during aestivation, which may center on miR-200-3p inhibitory control over the translation of cyclin A mRNA transcripts.

scholarly journals A Conserved Cyclin-Binding Domain Determines Functional Interplay between Anaphase-Promoting Complex–Cdh1 and Cyclin A-Cdk2 during Cell Cycle Progression

2001 ◽  
Vol 21 (11) ◽  
pp. 3692-3703 ◽  
Author(s):  
Claus Storgaard Sørensen ◽  
Claudia Lukas ◽  
Edgar R. Kramer ◽  
Jan-Michael Peters ◽  
Jiri Bartek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Mechanistic Explanation ◽  
Cyclin A ◽  
Binding Motif ◽  
Cell Cycle Regulators ◽  
Anaphase Promoting Complex ◽  
Cycle Progression

ABSTRACT Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G1/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism of how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified a conserved cyclin-binding motif within the Cdh1 WD-40 domain and show that its disruption abolished the Cdh1–cyclin A-Cdk2 interaction, eliminated Cdh1-associated histone H1 kinase activity, and impaired Cdh1 phosphorylation by cyclin A-Cdk2 in vitro and in vivo. Overexpression of cyclin binding-deficient Cdh1 stabilized the APC-Cdh1 interaction and induced prolonged cell cycle arrest at the G1/S transition. Conversely, cyclin binding-deficient Cdh1 lost its capability to support APC-dependent proteolysis of cyclin A but not that of other APC substrates such as cyclin B and securin Pds1. Collectively, these data provide a mechanistic explanation for the mutual functional interplay between cyclin A-Cdk2 and APC-Cdh1 and the first evidence that Cdh1 may activate the APC by binding specific substrates.

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