RÉSUMÉ
Obesity is considered an important factor for many chronic diseases, including diabetes, cardiovascular disease and cancer. The expansion of adipose tissue in obesity is due to an increase in both adipocyte progenitor differentiation and mature adipocyte cell size. Adipocytes, however, are thought to be unable to divide or enter the cell cycle. We demonstrate that mature human adipocytes unexpectedly display a gene and protein signature indicative of an active cell cycle program. Adipocyte cell cycle progression associates with obesity and hyperinsulinemia, with a concomitant increase in cell size, nuclear size and nuclear DNA content. Chronic hyperinsulinemia in vitro or in humans, however, is associated with subsequent cell cycle exit, leading to a premature senescent transcriptomic and secretory profile in adipocytes. Premature senescence is rapidly becoming recognized as an important mediator of stress-induced tissue dysfunction. By demonstrating that adipocytes can activate a cell cycle program, we define a mechanism whereby mature human adipocytes senesce. We further show that by targeting the adipocyte cell cycle program using metformin, it is possible to influence adipocyte senescence and obesity-associated adipose tissue inflammation.
Sujet(s)
Adipocytes/métabolisme , Cycle cellulaire/physiologie , Vieillissement de la cellule/physiologie , Hyperinsulinisme/anatomopathologie , Obésité/anatomopathologie , Tissu adipeux/métabolisme , Différenciation cellulaire/physiologie , Cycline D1/métabolisme , Humains , Hypoglycémiants/pharmacologie , Metformine/pharmacologieRÉSUMÉ
Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2 phase, but why active Cyclin A2-CDK2 during the S phase does not trigger mitotic kinase activation remains unclear. Here, we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in the G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation.
Sujet(s)
Protéines du cycle cellulaire/métabolisme , Cycline A2/métabolisme , Cytoplasme/métabolisme , Phase G2/génétique , Protein-Serine-Threonine Kinases/métabolisme , Protéines proto-oncogènes/métabolisme , Phase S/génétique , Transduction du signal/génétique , Protéine-kinase CDC2/déficit , Protéine-kinase CDC2/génétique , Noyau de la cellule/métabolisme , Chromatine/métabolisme , Cycline A2/génétique , Kinase-2 cycline-dépendante/déficit , Kinase-2 cycline-dépendante/génétique , Altération de l'ADN/génétique , Activation enzymatique/génétique , Cellules HeLa , Humains , Mitose/génétique , Phosphorylation/génétique , Liaison aux protéines , Transfection ,RÉSUMÉ
In response to DNA damage, a cell can be forced to permanently exit the cell cycle and become senescent. Senescence provides an early barrier against tumor development by preventing proliferation of cells with damaged DNA. By studying single cells, we show that Cdk activity persists after DNA damage until terminal cell cycle exit. This low level of Cdk activity not only allows cell cycle progression, but also promotes cell cycle exit at a decision point in G2 phase. We find that residual Cdk1/2 activity is required for efficient p21 production, allowing for nuclear sequestration of Cyclin B1, subsequent APC/CCdh1 -dependent degradation of mitotic inducers and induction of senescence. We suggest that the same activity that triggers mitosis in an unperturbed cell cycle enforces senescence in the presence of DNA damage, ensuring a robust response when most needed.
Sujet(s)
Protéine-kinase CDC2/génétique , Vieillissement de la cellule/effets des médicaments et des substances chimiques , Kinase-2 cycline-dépendante/génétique , Étoposide/pharmacologie , Points de contrôle de la phase G2 du cycle cellulaire/effets des médicaments et des substances chimiques , Ostéoblastes/effets des médicaments et des substances chimiques , Antigènes CD , Protéine-kinase CDC2/antagonistes et inhibiteurs , Protéine-kinase CDC2/métabolisme , Cadhérines/génétique , Cadhérines/métabolisme , Lignée cellulaire , Lignée cellulaire tumorale , Taille de la cellule , Survie cellulaire/effets des médicaments et des substances chimiques , Cycline B1/génétique , Cycline B1/métabolisme , Kinase-2 cycline-dépendante/antagonistes et inhibiteurs , Kinase-2 cycline-dépendante/métabolisme , Inhibiteur p21 de kinase cycline-dépendante/génétique , Inhibiteur p21 de kinase cycline-dépendante/métabolisme , Altération de l'ADN , Cellules épithéliales/cytologie , Cellules épithéliales/effets des médicaments et des substances chimiques , Cellules épithéliales/enzymologie , Régulation de l'expression des gènes , Humains , Ostéoblastes/cytologie , Ostéoblastes/enzymologie , Ptéridines/pharmacologie , Purines/pharmacologie , Quinoléines/pharmacologie , Épithélium pigmentaire de la rétine/cytologie , Épithélium pigmentaire de la rétine/effets des médicaments et des substances chimiques , Épithélium pigmentaire de la rétine/enzymologie , Transduction du signal , Analyse sur cellule unique , Thiazoles/pharmacologieRÉSUMÉ
Notch signaling is an important regulator of stem cell differentiation. All canonical Notch signaling is transmitted through the DNA-binding protein CSL, and hyperactivated Notch signaling is associated with tumor development; thus it may be anticipated that CSL deficiency should reduce tumor growth. In contrast, we report that genetic removal of CSL in breast tumor cells caused accelerated growth of xenografted tumors. Loss of CSL unleashed a hypoxic response during normoxic conditions, manifested by stabilization of the HIF1α protein and acquisition of a polyploid giant-cell, cancer stem cell-like, phenotype. At the transcriptome level, loss of CSL upregulated more than 1,750 genes and less than 3% of those genes were part of the Notch transcriptional signature. Collectively, this suggests that CSL exerts functions beyond serving as the central node in the Notch signaling cascade and reveals a role for CSL in tumorigenesis and regulation of the cellular hypoxic response.
Sujet(s)
Tumeurs du sein/génétique , Sous-unité alpha du facteur-1 induit par l'hypoxie/génétique , Facteur de transcription CBF-1/génétique , Mitose/génétique , Animaux , Tumeurs du sein/anatomopathologie , Différenciation cellulaire/génétique , Hypoxie cellulaire/génétique , Lignée cellulaire tumorale , Femelle , Régulation de l'expression des gènes tumoraux , Humains , Souris , Cellules souches tumorales/métabolisme , Cellules souches tumorales/anatomopathologie , Récepteurs Notch/génétique , Transduction du signal/génétique , Transcriptome/génétique , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
Upon DNA damage, cell cycle progression is temporally blocked to avoid propagation of mutations. While transformed cells largely maintain the competence to recover from a cell cycle arrest, untransformed cells past the G1/S transition lose mitotic inducers, and thus the ability to resume cell division. This permanent cell cycle exit depends on p21, p53, and APC/C(Cdh1). However, when and how permanent cell cycle exit occurs remains unclear. Here, we have investigated the cell cycle response to DNA damage in single cells that express Cyclin B1 fused to eYFP at the endogenous locus. We find that upon DNA damage Cyclin B1-eYFP continues to accumulate up to a threshold level, which is reached only in G2 phase. Above this threshold, a p21 and p53-dependent nuclear translocation required for APC/C(Cdh1)-mediated Cyclin B1-eYFP degradation is initiated. Thus, cell cycle exit is decoupled from activation of the DNA damage response in a manner that correlates to Cyclin B1 levels, suggesting that G2 activities directly feed into the decision for cell cycle exit. Once Cyclin B1-eYFP nuclear translocation occurs, checkpoint inhibition can no longer promote mitotic entry or re-expression of mitotic inducers, suggesting that nuclear translocation of Cyclin B1 marks the restriction point for permanent cell cycle exit in G2 phase.
Sujet(s)
Points de contrôle du cycle cellulaire , Noyau de la cellule/métabolisme , Cycline B1/métabolisme , Phase G2 , Complexe promoteur de l'anaphase/métabolisme , Lignée cellulaire tumorale , Inhibiteur p21 de kinase cycline-dépendante/métabolisme , Altération de l'ADN , Ciblage de gène , Humains , Transport des protéines , Protéolyse , Protéine p53 suppresseur de tumeur/métabolismeRÉSUMÉ
During the cell cycle, DNA duplication in S phase must occur before a cell divides in mitosis. In the intervening G2 phase, mitotic inducers accumulate, which eventually leads to a switch-like rise in mitotic kinase activity that triggers mitotic entry. However, when and how activation of the signaling network that promotes the transition to mitosis occurs remains unclear. We have developed a system to reduce cell-cell variation and increase accuracy of fluorescence quantification in single cells. This allows us to use immunofluorescence of endogenous marker proteins to assess kinetics from fixed cells. We find that mitotic phosphorylations initially occur at the completion of S phase, showing that activation of the mitotic entry network does not depend on protein accumulation through G2. Our data show insights into how mitotic entry is linked to the completion of S phase and forms a quantitative resource for mathematical models of the human cell cycle.
Sujet(s)
Phase G2/génétique , Mitose/génétique , Phase S/génétique , Protéines bactériennes/composition chimique , Cycle cellulaire , Lignée cellulaire tumorale , Centrosome/métabolisme , Réplication de l'ADN , Fibronectines/composition chimique , Marqueurs génétiques , Humains , Traitement d'image par ordinateur , Cinétique , Kinétochores/composition chimique , Protéines luminescentes/composition chimique , Microscopie de fluorescence , Modèles théoriques , Phosphorylation , Petit ARN interférent/métabolisme , Facteurs tempsRÉSUMÉ
Förster resonance energy transfer (FRET)-based reporters(1) allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA(2), PKB(3), PKC(4), PKD(5), ERK(6), JNK(7), Cdk(18), Aurora B(9) and Plk1(9). Given the modular design, additional probes are likely to emerge in the near future(10). Progression through the cell cycle is affected by stress signaling pathways( 11). Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress(12).Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle(8,9,13,14). Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.