RESUMEN
Senescence is an irreversible withdrawal from cell proliferation that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 (also known as TP53) and retinoblastoma protein (RB, also known as RB1) family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the cyclin-dependent kinase (CDK) inhibitor p21 and the checkpoint kinase Chk1 controls cyclin D-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting cyclin D1 complexed with CDK2 or CDK4. The resulting G2 exit, which precedes the appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and reduction in the number of DNA damage foci. In p53/RB-proficient cancer cells, a compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to cyclin D1- and cyclin E1-CDK complexes and downregulating CDK6, whereas knockdown of the checkpoint kinase Chk2 enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting the onset of senescence induced by DNA damage in G2.
Asunto(s)
Ciclina D1 , Proteína p53 Supresora de Tumor , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo , Fosforilación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Although cyclin-dependent kinase 2 (Cdk2) controls the G1/S transition and promotes DNA replication, it is dispensable for cell cycle progression due to redundancy with Cdk1. Yet Cdk2 also has non-redundant functions that can be revealed in certain genetic backgrounds and it was reported to promote the G2/M DNA damage response checkpoint in TP53 (p53)-deficient cancer cells. However, in p53-proficient cells subjected to DNA damage, Cdk2 is inactivated by the CDK inhibitor p21. We therefore investigated whether Cdk2 differentially affects checkpoint responses in p53-proficient and deficient cell lines. We show that, independently of p53 status, Cdk2 stimulates the ATR/Chk1 pathway and is required for an efficient DNA replication checkpoint response. In contrast, Cdk2 is not required for a sustained DNA damage response and G2 arrest. Rather, eliminating Cdk2 delays S/G2 progression after DNA damage and accelerates appearance of early markers of cell cycle exit. Notably, Cdk2 knockdown leads to down-regulation of Cdk6, which we show is a non-redundant pRb kinase whose elimination compromises cell cycle progression. Our data reinforce the notion that Cdk2 is a key p21 target in the DNA damage response whose inactivation promotes exit from the cell cycle in G2.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/metabolismo , Daño del ADN , Puntos de Control de la Fase S del Ciclo Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células HCT116 , Humanos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The polyphenol resveratrol activates the deacetylase Sirt1, resulting in various antioxidant, chemoprotectant, neuroprotective, cardioprotective, and anti-inflammatory properties. We found that at high concentrations of resveratrol, human CD4+ T cells showed defective antigen receptor signaling and arrest at the G1 stage of the cell cycle, whereas at low concentrations, cells were readily activated and exhibited enhanced Sirt1 deacetylase activity. Nevertheless, low-dose resveratrol rapidly stimulated genotoxic stress in the T cells, which resulted in engagement of a DNA damage response pathway that depended on the kinase ATR [ataxia telangiectasia-mutated (ATM) and Rad3-related], but not ATM, and subsequently in premitotic cell cycle arrest. The concomitant activation of p53 was coupled to the expression of gene products that regulate cell metabolism, leading to a metabolic reprogramming that was characterized by decreased glycolysis, increased glutamine consumption, and a shift to oxidative phosphorylation. These alterations in the bioenergetic homeostasis of CD4+ T cells resulted in enhanced effector function, with both naïve and memory CD4+ T cells secreting increased amounts of the inflammatory cytokine interferon-γ. Thus, our data highlight the wide range of metabolic adaptations that CD4+ T lymphocytes undergo in response to genomic stress.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Daño del ADN , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Estilbenos/farmacología , Adulto , Antioxidantes/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Perfilación de la Expresión Génica/métodos , Glucólisis/efectos de los fármacos , Glucólisis/genética , Humanos , Fosforilación Oxidativa/efectos de los fármacos , Fosforilación/efectos de los fármacos , Resveratrol , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The cell proliferation antigen Ki-67 is widely used in cancer histopathology, but estimations of Ki-67 expression levels are inconsistent and understanding of its regulation is limited. Here we show that cell-cycle regulation underlies variable Ki-67 expression in all situations analyzed, including nontransformed human cells, normal mouse intestinal epithelia and adenomas, human cancer cell lines with or without drug treatments, and human breast and colon cancers. In normal cells, Ki-67 was a late marker of cell-cycle entry; Ki-67 mRNA oscillated with highest levels in G2 while protein levels increased throughout the cell cycle, peaking in mitosis. Inhibition of CDK4/CDK6 revealed proteasome-mediated Ki-67 degradation in G1 After cell-cycle exit, low-level Ki-67 expression persisted but was undetectable in fully quiescent differentiated cells or senescent cells. CDK4/CDK6 inhibition in vitro and in tumors in mice caused G1 cell-cycle arrest and eliminated Ki-67 mRNA in RB1-positive cells but had no effect in RB1-negative cells, which continued to proliferate and express Ki-67. Thus, Ki-67 expression varies due to cell-cycle regulation, but it remains a reliable readout for effects of CDK4/CDK6 inhibitors on cell proliferation. Cancer Res; 77(10); 2722-34. ©2017 AACR.
Asunto(s)
Ciclo Celular/genética , Expresión Génica , Antígeno Ki-67/genética , Animales , Antineoplásicos/farmacología , Biomarcadores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/genética , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Inmunohistoquímica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Antígeno Ki-67/metabolismo , Ratones , Ratones Noqueados , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Diurnal oscillations of gene expression controlled by the circadian clock and its connected feeding rhythm enable organisms to coordinate their physiologies with daily environmental cycles. While available techniques yielded crucial insights into regulation at the transcriptional level, much less is known about temporally controlled functions within the nucleus and their regulation at the protein level. Here, we quantified the temporal nuclear accumulation of proteins and phosphoproteins from mouse liver by SILAC proteomics. We identified around 5,000 nuclear proteins, over 500 of which showed a diurnal accumulation. Parallel analysis of the nuclear phosphoproteome enabled the inference of the temporal activity of kinases accounting for rhythmic phosphorylation. Many identified rhythmic proteins were parts of nuclear complexes involved in transcriptional regulation, ribosome biogenesis, DNA repair, and the cell cycle and its potentially associated diurnal rhythm of hepatocyte polyploidy. Taken together, these findings provide unprecedented insights into the diurnal regulatory landscape of the mouse liver nucleus.
Asunto(s)
Núcleo Celular/metabolismo , Ritmo Circadiano , Hígado/metabolismo , Proteómica/métodos , Animales , Relojes Circadianos/genética , Ritmo Circadiano/genética , Reparación del ADN , Regulación de la Expresión Génica , Marcaje Isotópico , Espectrometría de Masas , Ratones , Ratones Noqueados , Proteínas Nucleares/metabolismo , Biogénesis de Organelos , Fosfoproteínas/metabolismo , Fosforilación , Poliploidía , Proteínas Quinasas/metabolismo , Proteoma/metabolismo , Ribosomas/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.
Asunto(s)
Proliferación Celular , Heterocromatina/metabolismo , Heterocromatina/ultraestructura , Antígeno Ki-67/metabolismo , Animales , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , XenopusRESUMEN
Senescence was classically defined as an irreversible cell cycle arrest in G1 phase (G1 exit) triggered by eroded telomeres in aged primary cells. The molecular basis of this G1 arrest is thought to be due to a DNA damage response, resulting in accumulation of the cyclin dependent kinase (Cdk) inhibitors p21 and p16 that block the inactivating phosphorylation of the retinoblastoma tumor suppressor pRb, thereby preventing DNA replication. More than a decade ago, several studies showed that p21 also mediates permanent DNA damage-induced cell cycle arrest in G2 (G2 exit) by inhibiting mitotic Cdk complexes and pRb phosphorylation. The idea that the senescence program can also be launched after G2 arrest has gained support from several recent publications, including evidence for its existence in vivo.
Asunto(s)
Senescencia Celular , Puntos de Control de la Fase G2 del Ciclo Celular , Animales , Proliferación Celular , Fase G1 , Humanos , Modelos Biológicos , Transducción de SeñalRESUMEN
Since the mid 1970s, cancer has been described as a process of Darwinian evolution, with somatic cellular selection and evolution being the fundamental processes leading to malignancy and its many manifestations (neoangiogenesis, evasion of the immune system, metastasis, and resistance to therapies). Historically, little attention has been placed on applications of evolutionary biology to understanding and controlling neoplastic progression and to prevent therapeutic failures. This is now beginning to change, and there is a growing international interest in the interface between cancer and evolutionary biology. The objective of this introduction is first to describe the basic ideas and concepts linking evolutionary biology to cancer. We then present four major fronts where the evolutionary perspective is most developed, namely laboratory and clinical models, mathematical models, databases, and techniques and assays. Finally, we discuss several of the most promising challenges and future prospects in this interdisciplinary research direction in the war against cancer.
RESUMEN
The senescence program is activated in response to diverse stress stimuli potentially compromising genetic stability and leads to an irreversible cell cycle arrest. The mTOR pathway plays a crucial role in the regulation of cell metabolism and cellular growth. The goal of this chapter is to present evidence linking these two processes, which have one common regulator-the tumor suppressor p53. While the role of mTOR in senescence is still controversial, recent papers have shed new light onto this issue. This review, far from being exhaustive given the complexity of the field, will hopefully stimulate further research in this domain, whose relevance for ageing is becoming increasingly documented.
Asunto(s)
Senescencia Celular , Serina-Treonina Quinasas TOR/metabolismo , Animales , Puntos de Control del Ciclo Celular , Daño del ADN , Humanos , Imidazoles/metabolismo , Piperazinas/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Cellular senescence is, essentially, a permanent proliferation arrest induced by various cellular stresses or inappropriate stimuli. This arrest, which is associated with dramatic changes in cell morphology, metabolism and gene expression, involves a complex signalling network aiming at stable inactivation of CDKs, major cell cycle regulators. Notably, several tumour suppressors, such as p53, pRb or p16(Ink4a), play key roles both in the initiation of the senescence program and in its maintenance, which often involves epigenetic changes. While having widely recognized roles in tumour suppression and wound healing, senescence, like the roman god Janus, recently revealed another darker face. Mostly due to altered secretion phenotype favouring inflammation, senescent cells strongly influence surrounding tissue contributing to the development of age-related pathologies, including cancer.
Asunto(s)
Senescencia Celular , Animales , Autofagia , Ciclo Celular/fisiología , Transformación Celular Neoplásica , Células/metabolismo , Senescencia Celular/fisiología , Ensamble y Desensamble de Cromatina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/fisiología , Citocinas/metabolismo , Replicación del ADN , Humanos , Inflamación , Ratones , MicroARNs/fisiología , Mitocondrias/fisiología , Modelos Biológicos , Fenotipo , Proteína de Retinoblastoma/fisiología , Transducción de Señal , Serina-Treonina Quinasas TOR/fisiología , Homeostasis del Telómero , Proteína p53 Supresora de Tumor/fisiologíaAsunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Sustitución de Aminoácidos , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Fase G1 , Humanos , Mutagénesis Sitio-Dirigida , Proteína de Retinoblastoma/metabolismoRESUMEN
Tissue homeostasis requires precise control of cell proliferation and arrest in response to environmental cues. In situation such as wound healing, injured cells are stimulated to divide, but as soon as confluence is reached proliferation must be blocked. Such reversible cell cycle exit occurs in G(1), requires pRb family members, and is driven by p27(Kip1)-dependent Cdk inactivation. This implies that, while dividing, cells should simultaneously prepare the exit once mitosis is accomplished. For a long time, the decision to cycle or not was presumed to occur in G(1), prior to the restriction point, beyond which the cells were bound to divide even in the absence of mitogens, before finally arresting after mitosis. However, more recent reports suggested that the commitment to cycle in response to serum occurs already in G(2) phase and requires the Ras-dependent induction of cyclin D1, which promotes following G(1)/S transition. To test whether this hypothesis applies to arrest induced by contact inhibition, we used an in vitro wounding model where quiescent human dermal fibroblasts, stimulated to proliferate by mechanical injury, synchronously exit cell cycle after mitosis due to renewed confluence. We show that this exit is preceded by p27-dependent inhibition of cyclin A-Cdk1/2, cyclin D1 downregulation and reduced pre-mitotic pRb pocket protein phosphorylation. Overexpression of cyclin D1 but not p27 depletion reversed this phenotype and compromised confluence-driven cell cycle exit. Thus, a balance between cyclin D1 and p27 may provide sensitive responses to variations in proliferative cues operating throughout the cell cycle.
Asunto(s)
Ciclo Celular/fisiología , Ciclina D1/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Fibroblastos/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Regulación hacia Abajo/fisiología , Fase G2 , Humanos , Mitosis , Fosforilación , Cicatrización de Heridas/fisiologíaRESUMEN
D-type cyclins are key regulators of the cell division cycle. In association with Cyclin Dependent Kinases (CDK) 2/4/6, they control the G1/S-phase transition in part by phosphorylation and inactivation of tumor suppressor of retinoblastoma family. Defective regulation of the G1/S transition is a well-known cause of cancer, making the cyclin D1-CDK4/6 complex a promising therapeutic target. Our objective is to develop inhibitors that would block the formation or the activation of the cyclin D1-CDK4/6 complex, using in silico docking experiments on a structural homology model of the cyclin D1-CDK4/6 complex. To this end we focused on the cyclin subunit in three different ways: (1) targeting the part of the cyclin D1 facing the N-terminal domain of CDK4/6, in order to prevent the dimer formation; (2) targeting the part of the cyclin D1 facing the C-terminal domain of CDK4/6, in order to prevent the activation of CDK4/6 by blocking the T-loop in an inactive conformation, and also to destabilize the dimer; (3) targeting the groove of cyclin D1 where p21 binds, in order to mimic its inhibition mode by preventing binding of cyclin D1-CDK4/6 complex to its targets. Our strategy, and the tools we developed, will provide a computational basis to design lead compounds for novel cancer therapeutics, targeting a broad range of proteins involved in the regulation of the cell cycle.
Asunto(s)
Ciclina D1/química , Ciclina D1/metabolismo , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Secuencia de Aminoácidos , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Estructura Secundaria de Proteína , Alineación de Secuencia , Electricidad Estática , Homología Estructural de Proteína , Relación Estructura-ActividadRESUMEN
G2 arrest of cells suffering DNA damage in S phase is crucial to avoid their entry into mitosis, with the concomitant risks of oncogenic transformation. According to the current model, signals elicited by DNA damage prevent mitosis by inhibiting both activation and nuclear import of cyclin B1-Cdk1, a master mitotic regulator. We now show that normal human fibroblasts use additional mechanisms to block activation of cyclin B1-Cdk1. In these cells, exposure to nonrepairable DNA damage leads to nuclear accumulation of inactive cyclin B1-Cdk1 complexes. This nuclear retention, which strictly depends on association with endogenous p21, prevents activation of cyclin B1-Cdk1 by Cdc25 and Cdk-activating kinase as well as its recruitment to the centrosome. In p21-deficient normal human fibroblasts and immortal cell lines, cyclin B1 fails to accumulate in the nucleus and could be readily detected at the centrosome in response to DNA damage. Therefore, in normal cells, p21 exerts a dual role in mediating DNA damage-induced cell cycle arrest and exit before mitosis. In addition to blocking pRb phosphorylation, p21 directly prevents mitosis by inactivating and maintaining the inactive state of mitotic cyclin-Cdk complexes. This, with subsequent degradation of mitotic cyclins, further contributes to the establishment of a permanent G2 arrest.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Ciclina B/metabolismo , Daño del ADN/fisiología , Proteína Quinasa CDC2/química , Ciclo Celular , Células Cultivadas , Ciclina B/química , Ciclina B1 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Activación Enzimática , Humanos , Mitosis , Modelos Biológicos , Complejos Multiproteicos , Fosforilación , Unión Proteica , Fosfatasas cdc25/metabolismoRESUMEN
Telomere shortening in normal human cells causes replicative senescence, a p53-dependent growth arrest state, which is thought to represent an innate defence against tumour progression. However, although it has been postulated that critical telomere loss generates a 'DNA damage' signal, the signalling pathway(s) that alerts cells to short dysfunctional telomeres remains only partially defined. We show that senescence in human fibroblasts is associated with focal accumulation of gamma-H2AX and phosphorylation of Chk2, known mediators of the ataxia-telangiectasia mutated regulated signalling pathway activated by DNA double-strand breaks. Both these responses increased in cells grown beyond senescence through inactivation of p53 and pRb, indicating that they are driven by continued cell division and not a consequence of senescence. gamma-H2AX (though not Chk2) was shown to associate directly with telomeric DNA. Furthermore, inactivation of Chk2 in human fibroblasts led to a fall in p21(waf1) expression and an extension of proliferative lifespan, consistent with failure to activate p53. Thus, Chk2 forms an essential component of a common pathway signalling cell cycle arrest in response to both telomere erosion and DNA damage.
Asunto(s)
Ciclo Celular , Senescencia Celular , Daño del ADN , Replicación del ADN , ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proliferación Celular , Quinasa de Punto de Control 2 , Inmunoprecipitación de Cromatina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes , Genes Supresores de Tumor , Histonas/metabolismo , Humanos , Pulmón/citología , Masculino , Microscopía Confocal , Modelos Biológicos , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/metabolismo , Proteína de Retinoblastoma/metabolismo , Retroviridae/genética , Telomerasa/metabolismo , Telómero/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , XantenosRESUMEN
Although the Cdk inhibitor p21(Waf1/Cip1), one of the transcriptional targets of p53, has been implicated in the maintenance of G(2) arrest after DNA damage, its function at this stage of the cell cycle is not really understood. Here, we show that the exposure of normal human fibroblasts (NHFs) to genotoxic agents provokes permanent cell cycle exit in G(2) phase, whereas mouse embryo fibroblasts and transformed human cells progress through mitosis and arrest in G(1) without intervening cytokinesis. p21(Waf1/Cip1) exerts a key role in driving this G(2) exit both by inhibiting cyclin B1-Cdk1 and cyclin A-Cdk1/2 complexes, which control G(2)/M progression, and by blocking the phosphorylation of pRb family proteins. NHFs with compromised pRb proteins could still efficiently arrest in G(2) but were unable to exit the cell cycle, resulting in cell death. Our experiments show that, when under continuous genotoxic stress, normal cells can reverse their commitment to mitotic progression due to passage through the restriction point and that mechanisms involving p21(Waf1/Cip1) and pocket proteins can induce exit in G(2) and G(1).