RESUMO
Proteins disabled in Fanconi anemia (FA) are necessary for the maintenance of genome stability during cell proliferation. Upon replication stress signaling by ATR, the FA core complex monoubiquitinates FANCD2 and FANCI in order to activate DNA repair. Here, we identified FANCD2 and FANCI in a proteomic screen of replisome-associated factors bound to nascent DNA in response to replication arrest. We found that FANCD2 can interact directly with minichromosome maintenance (MCM) proteins. ATR signaling promoted the transient association of endogenous FANCD2 with the MCM2-MCM7 replicative helicase independently of FANCD2 monoubiquitination. FANCD2 was necessary for human primary cells to restrain DNA synthesis in the presence of a reduced pool of nucleotides and prevented the accumulation of single-stranded DNA, the induction of p21, and the entry of cells into senescence. These data reveal that FANCD2 is an effector of ATR signaling implicated in a general replisome surveillance mechanism that is necessary for sustaining cell proliferation and attenuating carcinogenesis.
Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proliferação de Células , Células Cultivadas , Senescência Celular , Replicação do DNA , Humanos , Componente 2 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Transdução de Sinais/genéticaRESUMO
Candida albicans is an opportunistic fungal pathogen. In immunocompromised individuals, it can cause bloodstream infections with high mortality rates. The ability to switch between yeast and hyphal morphologies is a critical virulence factor of C. albicans. In response to diverse environmental cues, several signaling pathways are activated resulting in filamentous growth. Interestingly, cell cycle arrest can also trigger filamentous growth although the pathways involved are not well-understood. Here, we demonstrate that the cAMP-PKA pathway is involved in the filamentous growth caused by G1 arrest due to the depletion of the G1 cyclin Cln3 and S phase arrest due to hydroxyurea treatment. The downstream mechanisms involved in filamentation are different between the two cell cycle arrest phenomena. Cln3-depleted cells require HGC1 and UME6 for filamentous growth, but hydroxyurea-induced filamentation does not. Also, the hyphal repressor Nrg1 is not involved in the suppression of Cln3-depletion and hydroxyurea-induced filamentous growth. The findings highlight the complexity of the signaling networks that control filamentous growth in which different mechanisms downstream of the cAMP-PKA pathway are activated based on the nature of the inducing signals.
Assuntos
Candida albicans/crescimento & desenvolvimento , Ciclinas/metabolismo , Proteínas Fúngicas/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Hifas/crescimento & desenvolvimento , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclinas/genética , Proteínas Fúngicas/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Deleção de Genes , Humanos , Hidroxiureia/farmacologia , Hifas/efeitos dos fármacos , Plasmídeos , Proteínas Repressoras/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacosRESUMO
Polo-like kinase 1 (PLK1) is a serine/threonine kinase involved in several stages of the cell cycle, including the entry and exit from mitosis, and cytokinesis. Furthermore, it has an essential role in the regulation of DNA replication. Together with cyclin A, PLK1 also promotes CDH1 phosphorylation to trigger its ubiquitination and degradation, allowing cell cycle progression. The PLK1 levels in different type of tumors are very high compared to normal tissues, which is consistent with its role in promoting proliferation. Therefore, several PLK1 inhibitors have been developed and tested for the treatment of cancer. Here, we further analyzed PLK1 degradation and found that cytoplasmic PLK1 is ubiquitinated and subsequently degraded by the SCFßTrCP/proteasome. This procedure is triggered when heat shock protein (HSP) 90 is inhibited with geldanamycin, which results in misfolding of PLK1. We also identified CDK1 as the major kinase involved in this degradation. Our work shows for the first time that HSP90 inhibition arrests cell cycle progression at the G1/S transition. This novel mechanism inhibits CDH1 degradation through CDK1-dependent PLK1 destruction by the SCFßTrCP/proteasome. In these conditions, CDH1 substrates do not accumulate and cell cycle arrests, providing a novel pathway for regulation of the cell cycle at the G1-to-S boundary.-Giráldez, S., Galindo-Moreno, M., Limón-Mortés, M. C., Rivas, A. C., Herrero-Ruiz, J., Mora-Santos, M., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. G1/S phase progression is regulated by PLK1 degradation through the CDK1/ßTrCP axis.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Animais , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Plasmídeos , Mutação Puntual , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas Contendo Repetições de beta-Transducina/genética , Quinase 1 Polo-LikeRESUMO
CONTEXT: Dicranopteris linearis (Burm.f.) Underw. (Gleicheniaceae) has been scientifically proven to exert various pharmacological activities. Nevertheless, its anti-proliferative potential has not been extensively investigated. OBJECTIVE: To investigate the anti-proliferative potential of D. linearis leaves and determine possible mechanistic pathways. MATERIALS AND METHODS: MTT assay was used to determine the cytotoxic effects of D. linearis methanol (MEDL) and petroleum ether (PEEDL) extracts at concentrations of 100, 50, 25, 12.5, 6.25 and 3.125 µg/mL against a panel of cancer cell lines (breast [MCF-7 and MDA-MB-231], cervical [HeLa], colon [HT-29], hepatocellular [HepG2] and lung [A549]), as compared to negative (untreated) and positive [5-fluorouracil (5-FU)-treated] control groups. Mouse fibroblast cells (3T3) were used as normal cells. The mode of cell death was examined using morphological analysis via acridine orange (AO) and propidium iodide (PI) double staining. Cell cycle arrest was determined using flow cytometer, followed by annexin V-PI apoptosis detection kit. RESULTS: MEDL demonstrated the most significant growth inhibition against MDA-MB-231 cells (IC50 22.4 µg/mL). PEEDL showed no cytotoxic effect. Induction of apoptosis by MEDL was evidenced via morphological analysis and acridine orange propidium iodide staining. MEDL could induce S phase cell cycle arrest after 72 h of incubation. Early apoptosis induction in MDA-MB-231 cells was confirmed by annexin V-FITC and PI staining. Significant increase in apoptotic cells were detected after 24 h of treatment with 15.07% cells underwent apoptosis, and the amount escalated to 18.24% with prolonged 48 h incubation. CONCLUSIONS: MEDL has potential as a potent cytotoxic agent against MDA-MB-231 adenocarcinoma.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Extratos Vegetais/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Células 3T3 , Células A549 , Animais , Apiaceae , Apoptose/fisiologia , Neoplasias da Mama/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Camundongos , Extratos Vegetais/isolamento & purificação , Pontos de Checagem da Fase S do Ciclo Celular/fisiologiaRESUMO
Activating transcription factor 6 (ATF6), a sensor protein located in the endoplasmic reticulum (ER) membrane, is an important factor in the ER stress signaling pathway. ER stress is known to be involved in folliculogenesis, follicular growth, and ovulation; however, the physiological function of ATF6 in mouse granulosa cells remains largely unknown. The aim of this study was to assess the role of ATF6 in mouse granulosa cells with respect to apoptosis, the cell cycle, and steroid hormone production, as well as several key genes related to follicular development, via RNA interference, immunohistochemical staining, real-time quantitative PCR, Western blotting, flow cytometry, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) assay, and ELISA. Immunohistochemical staining revealed that ATF6 was extensively distributed in the granulosa cells of various ovarian follicles and oocytes in adult female mice. FSH or LH treatment significantly increased ATF6 protein levels in mouse granulosa cells. In the meantime, a recombinant plasmid was used to deplete ATF6 successfully using short hairpin RNA-mediated interference technology, which was verified at both the mRNA and protein levels. Flow cytometry and TUNEL assay analysis indicated that ATF6 depletion decreased apoptosis and arrested the S phase of the cell cycle in mouse granulosa cells. Consistent with these results, p53, caspase-3, B cell lymphoma 2 (Bcl-2)-associated X protein, CCAAT-enhancer-binding protein homologous protein, cyclin A1, cyclin B1, and cyclin D2 mRNA expression decreased, whereas Bcl-2 and glucose-regulated protein 78 kDa mRNA expression increased. Interestingly, ATF6 knockdown obviously increased progesterone and estradiol production in mouse granulosa cells. Cytochrome P450 1b1 (Cyp1b1) mRNA levels were downregulated, whereas Cyp11a1, steroidogenic acute regulatory, and Cyp19a1 mRNA levels were upregulated, in keeping with the changes in steroid hormones. Furthermore, ATF6 disruption remarkably increased insulin-like growth factor binding protein4 (Igfbp4) expression and decreased hyaluronan synthase 2 (Has2), prostaglandin-endoperoxide synthase 2 (Ptgs2), and prostaglandin F receptor (Ptgfr) expression in mouse granulosa cells, which are proteins crucial for follicular development. But, after treating with tunicamycin, the levels of Has2, Ptgs2, and Ptgfr increased relatively, whereas Igfbp4 expression decreased. Collectively, these results imply that ATF6, as a key player in ER stress signaling, may regulate apoptosis, the cell cycle, steroid hormone synthesis, and other modulators related to folliculogenesis in mouse granulosa cells, which may indirectly be involved in the development, ovulation, and atresia of ovarian follicles by affecting the physiological function of granulosa cells. The present study extends our understanding and provides new insights into the physiological significance of ATF6, a key signal transducer of ER stress, in ovarian granulosa cells.
Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Apoptose/fisiologia , Hormônios Esteroides Gonadais/metabolismo , Células da Granulosa/citologia , Células da Granulosa/fisiologia , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Animais , Células Cultivadas , Feminino , Técnicas de Silenciamento de Genes , CamundongosRESUMO
Cyclin-dependent kinases (Cdks) coordinate cell division, and their activities are tightly controlled. Phosphorylation of threonine 14 (T14) and tyrosine 15 (Y15) inhibits Cdks and regulates their activities in numerous physiologic contexts. Although the roles of Cdk1 inhibitory phosphorylation during mitosis are well described, studies of Cdk2 inhibitory phosphorylation during S phrase have largely been indirect. To specifically study the functions of Cdk2 inhibitory phosphorylation, we used gene targeting to make an endogenous Cdk2 knockin allele in human cells, termed Cdk2AF, which prevents Cdk2 T14 and Y15 phosphorylation. Cdk2AF caused premature S-phase entry, rapid cyclin E degradation, abnormal DNA replication, and genome instability. Cdk2AF cells also exhibited strikingly abnormal responses to replication stress, accumulated irreparable DNA damage, and permanently exited the cell cycle after transient exposure to S-phase inhibitors. Our results reveal the specific and essential roles of Cdk2 inhibitory phosphorylation in the successful execution of the replication stress checkpoint response and in maintaining genome integrity.
Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Replicação do DNA/fisiologia , Fase S/fisiologia , Transdução de Sinais/fisiologia , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/genética , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Campo Pulsado , Citometria de Fluxo , Técnicas de Introdução de Genes , Instabilidade Genômica/fisiologia , Humanos , Microfluídica , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle.
Assuntos
Proliferação de Células , Exossomos/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , MicroRNAs/biossíntese , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Toxoplasma/metabolismo , Toxoplasmose/patologia , Animais , Linhagem Celular , Citometria de Fluxo , Interações Hospedeiro-Parasita , Ratos , Toxoplasma/patogenicidade , Toxoplasmose/parasitologiaRESUMO
Inorganic pyrophosphatases are required for anabolism to take place in all living organisms. Defects in genes encoding these hydrolytic enzymes are considered inviable, although their exact nature has not been studied at the cellular and molecular physiology levels. Using a conditional mutant in IPP1, the Saccharomyces cerevisiae gene encoding the cytosolic soluble pyrophosphatase, we show that respiring cells arrest in S phase upon Ipp1p deficiency, but they remain viable and resume growth if accumulated pyrophosphate is removed. However, fermenting cells arrest in G1/G0 phase and suffer massive vacuolization and eventual cell death by autophagy. Impaired NAD(+) metabolism is a major determinant of cell death in this scenario because demise can be avoided under conditions favoring accumulation of the oxidized pyridine coenzyme. These results posit that the mechanisms related to excess pyrophosphate toxicity in eukaryotes are dependent on the energy metabolism of the cell.
Assuntos
Autofagia/fisiologia , Metabolismo Energético/fisiologia , Pirofosfatase Inorgânica/metabolismo , NAD/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Difosfatos/metabolismo , Pirofosfatase Inorgânica/genética , NAD/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Ras association domain family (RASSF) 6 is a member of the C-terminal RASSF proteins such as RASSF1A and RASSF3. RASSF6 is involved in apoptosis in various cells under miscellaneous conditions, but it remains to be clarified how RASSF6 exerts tumor-suppressive roles. We reported previously that RASSF3 facilitates the degradation of MDM2, a major E3 ligase of p53, and stabilizes p53 to function as a tumor suppressor. In this study, we demonstrate that RASSF6 overexpression induces G1/S arrest in p53-positive cells. Its depletion prevents UV- and VP-16-induced apoptosis and G1/S arrest in HCT116 and U2OS cells. RASSF6-induced apoptosis partially depends on p53. RASSF6 binds MDM2 and facilitates its ubiquitination. RASSF6 depletion blocks the increase of p53 in response to UV exposure and up-regulation of p53 target genes. RASSF6 depletion delays DNA repair in UV- and VP-16-treated cells and increases polyploid cells after VP-16 treatment. These findings indicate that RASSF6 stabilizes p53, regulates apoptosis and the cell cycle, and functions as a tumor suppressor. Together with the previous reports regarding RASSF1A and RASSF3, the stabilization of p53 may be the common function of the C-terminal RASSF proteins.
Assuntos
Apoptose/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/fisiologia , Reparo do DNA/efeitos da radiação , Etoposídeo/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos da radiação , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/efeitos da radiação , Proteína Supressora de Tumor p53/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/fisiologia , Ubiquitinação/efeitos da radiação , Raios Ultravioleta , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Regulação para Cima/efeitos da radiaçãoRESUMO
Activation of a host DNA damage response (DDR) is essential for DNA replication of minute virus of canines (MVC), a member of the genus Bocavirus of the Parvoviridae family; however, the mechanism by which DDR contributes to viral DNA replication is unknown. In the current study, we demonstrate that MVC infection triggers the intra-S-phase arrest to slow down host cellular DNA replication and to recruit cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is regulated by ATM (ataxia telangiectasia-mutated kinase) signaling in a p53-independent manner. Moreover, we demonstrate that SMC1 (structural maintenance of chromosomes 1) is the key regulator of the intra-S-phase arrest induced during infection. Either knockdown of SMC1 or complementation with a dominant negative SMC1 mutant blocks both the intra-S-phase arrest and viral DNA replication. Finally, we show that the intra-S-phase arrest induced during MVC infection was caused neither by damaged host cellular DNA nor by viral proteins but by replicating viral genomes physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. In conclusion, the feedback loop between MVC DNA replication and the intra-S-phase arrest is mediated by ATM-SMC1 signaling and plays a critical role in MVC DNA replication. Thus, our findings unravel the mechanism underlying DDR signaling-facilitated MVC DNA replication and demonstrate a novel strategy of DNA virus-host interaction.
Assuntos
Bocavirus/genética , Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Replicação do DNA/fisiologia , Infecções por Parvoviridae/fisiopatologia , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Southern Blotting , Bromodesoxiuridina , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Ensaio Cometa , Proteínas de Ligação a DNA/metabolismo , Cães , Citometria de Fluxo , Imunofluorescência , Interações Hospedeiro-Patógeno , Immunoblotting , Plasmídeos/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Maintenance of genomic stability in proliferating cells depends on a network of proteins that coordinate chromosomal replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of replication protein A (RPA) recruited to chromatin. We show using purified proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from replication stress.
Assuntos
Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Proteína de Replicação A/metabolismo , Estresse Fisiológico/fisiologia , Sequência de Aminoácidos , Cromossomos/fisiologia , DNA Helicases/química , DNA Helicases/genética , Células HCT116 , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Osteossarcoma , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteína de Replicação A/química , Proteína de Replicação A/genética , Pontos de Checagem da Fase S do Ciclo Celular/fisiologiaRESUMO
BACKGROUND: Secondary pulmonary alveolar septal formation requires platelet derived growth factor (PDGF-A) and platelet derived growth factor receptor-alpha (PDGFRα), and their regulation influences alveolar septal areal density and thickness. Insufficient PDGFRα expression in lung fibroblasts (LF) results in failed septation. METHODS: Mice in which the endogenous PDGFRα-gene regulates expression of the green fluorescent protein were used to temporally and spatially track PDGFRα-signaling. Transition from the G1/G0 to the S-phase of the cell cycle was compared in PDGFRα-expressing and non-expressing LF using flow cytometry. Laser scanning confocal microscopy was used to quantify p27(kip1) and forkhead box "other" 3a (FoxO3a) in the nuclei of alveolar cells from mice bearing the PDGFRα-GFP knock-in, and p27(kip1) in mice with a conditional deletion of PDGFRα-gene function. The effects of PDGF-A on the phosphorylation and the intracellular location of FoxO3a were examined using Western immuoblotting and immunocytochemistry. RESULTS: In neonatal mouse lungs, entry of the PDGFRα-expressing LF subpopulation into the S-phase of the cell cycle diminished sooner than in their non-expressing LF counterparts. This preferential diminution was influenced by PDGFRα-mediated signaling, which phosphorylates and promotes cytoplasmic localization of FoxO3a. Comparative observations of LF at different ages during secondary septation and in mice that lack PDGFRα in alveolar LF demonstrated that nuclear localization of the G1 cyclin-dependent kinase inhibitor p27(kip1) correlated with reduced LF entry into S-phase. CONCLUSIONS: Nuclear localization of FoxO3a, an important regulator of p27(kip1) gene-expression, correlates with diminished proliferation of the PDGFRα-expressing LF subpopulation. These mechanisms for diminishing the effects of PDGFRα-mediated signaling likely regulate secondary septal formation and their derangement may contribute to imbalanced fibroblast cell kinetics in parenchymal lung diseases.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Proteína Forkhead Box O3 , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Transgênicos , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Tissue microarchitecture imposes physical constraints to the migration of individual cells. Especially in cancer metastasis, three-dimensional structural barriers within the extracellular matrix are known to affect the migratory behavior of cells, regulating the pathological state of the cells. Here, we employed a culture platform with micropillar arrays of 2 µm diameter and 16 µm pitch (2.16 micropillar) as a mechanical stimulant. Using this platform, we investigated how a long-term culture of A549 human lung carcinoma cells on the (2.16) micropillar-embossed dishes would influence the pathological state of the cell. A549 cells grown on the (2.16) micropillar array with 10 µm height exhibited a significantly elongated morphology and enhanced migration even after the detachment and reattachment, as evidenced in the conventional wound-healing assay, single-cell tracking analysis, and in vivo tumor colonization assays. Moreover, the pillar-induced morphological deformation in nuclei was accompanied by cell-cycle arrest in the S phase, leading to suppressed proliferation. While these marked traits of morphology-migration-proliferation support more aggressive characteristics of metastatic cancer cells, typical indices of epithelial-mesenchymal transition were not found, but instead, remarkable traces of amoeboidal transition were confirmed. Our study also emphasizes the importance of mechanical stimuli from the microenvironment during pathogenesis and how gained traits can be passed onto subsequent generations, ultimately affecting their pathophysiological behavior. Furthermore, this study highlights the potential use of pillar-based mechanical stimuli as an in vitro cell culture strategy to induce more aggressive tumorigenic cancer cell models.
Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias Pulmonares/metabolismo , Células A549 , Animais , Técnicas de Cultura de Células/instrumentação , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Ácidos Graxos/metabolismo , Feminino , Humanos , Fenômenos Mecânicos , Metabolômica , Camundongos Endogâmicos BALB C , Camundongos Nus , Pontos de Checagem da Fase S do Ciclo Celular/fisiologiaRESUMO
BACKGROUND: Dlx5 and Dlx6 stimulate differentiation of diverse progenitors during embryonic development. Their actions as pro-differentiation transcription factors includes the up-regulation of differentiation markers but the extent to which differentiation may also be stimulated by regulation of the cell cycle has not been addressed. RESULTS: We document that expression of Dlx5 and Dlx6 antagonizes cell proliferation in a variety of cell types without inducing apoptosis or promoting cell cycle exit. Rather, a variety of evidence indicates that elevated Dlx5 and Dlx6 expression reduces the proportion of cells in S phase and affects the length of the cell cycle. CONCLUSIONS: Antagonism of S-phase entry by Dlx5 and Dlx6 proteins likely represents a lineage-independent function to effect Dlx-mediated differentiation in multiple progenitor cell types.
Assuntos
Divisão Celular/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Proteínas de Homeodomínio/genética , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Fatores de Transcrição/genética , Animais , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos , Plasmídeos/genética , TransfecçãoRESUMO
Glioblastoma is the most aggressive brain tumor. Although miR-141 has been demonstrated to primarily function as a tumor suppressor in numerous malignancies, including glioblastoma, the mechanisms involved remain poorly understood. Here, it is shown that miR-141 is downregulated in glioblastoma cell lines and tissues and may exert its biological function via directly targeting myelin transcription factor 1-like (MYT1L). Using two glioblastoma cell lines that differ from each other by the functionality of DNA-dependent protein kinase (DNAPK), a functional involvement of DNAPK in the miR-141 tumor suppression network was observed. In M059K cells with a normal function of DNAPK, the enforced expression of miR-141 attenuated MYT1L expression and suppressed cell proliferation. Conversely, the inhibition of miR-141 expression promoted cell proliferation; however, in M059J cells with a loss-of-function DNAPK, miR-141 constitutively inhibited cell proliferation upon ectopic overexpression or inhibition. An overexpression of miR-141 suppressed M059J cell migration, while it had no effect on M059K. Furthermore, the ectopic expression of miR-141 induced an S-phase arrest in both cell lines, whereas the inhibition of miR-141 caused a G1 arrest in M059J and accelerated the S phase in M059K. An overexpression and suppression of miR-141 resulted in an aberrant expression of cell-cycle proteins, including p21. Moreover, MYT1L may be a transcription factor of p21 in p53-mutant cells, whereas DNAPK may function as a repressor of MYT1L. The findings revealed the crucial role of DNAPK in miR-141-mediated suppression of gliomagenesis and demonstrated that it may be a target molecule in miR-141-associated therapeutic interventions for glioblastoma.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Glioblastoma/patologia , MicroRNAs/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas de Ligação a DNA/metabolismo , Genes Supressores de Tumor/fisiologia , Glioblastoma/genética , Glioblastoma/terapia , Humanos , Pontos de Checagem da Fase S do Ciclo Celular/fisiologiaRESUMO
Bone morphogenetic protein 4 (BMP4) has been reported to regulate adipose development, but its role in preadipocyte proliferation has not been explored in vitro. Here, we investigated the effect of BMP4 on chicken preadipocyte proliferation using immortalized chicken preadipocytes (ICP1 cells) as a cell model. We report that BMP4 expression increases during preadipocyte proliferation. Overexpression and knockdown of BMP4 promotes and inhibits preadipocyte proliferation, respectively. In addition, overexpression of BMP4 decreased the number of preadipocytes at the G0/G1 phase of the cell cycle, and increased the proportion of cells at S phase. In contrast, knockdown of BMP4 increased the number of preadipocytes at the G0/G1 phase of the cell cycle, and decreased the proportion of cells at the S and G2 phases. Furthermore, overexpression of BMP4 promoted the expression of proliferating cell nuclear antigen (PCNA), Id2, cyclin E, and cyclin-dependent kinase 2 (CDK2), while knockdown of BMP4 inhibited the expression of Id2, cyclin E, and CDK2. Finally, neither BMP4 overexpression nor BMP4 knockdown affected cell apoptosis. Taken together, our results suggest that BMP4 may promote proliferation of ICP1 cells by driving cell cycle transition from G1 to S phase.
Assuntos
Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Proliferação de Células/fisiologia , Galinhas , Fibroblastos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Animais , Apoptose , Linhagem Celular , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Técnicas de Silenciamento de Genes , Proteína 2 Inibidora de Diferenciação/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transfecção , Regulação para CimaRESUMO
The precise coordination of growth and proliferation has a universal prevalence in cell homeostasis. As a prominent property, cell size is modulated by the coordination between these processes in bacterial, yeast, and mammalian cells, but the underlying molecular mechanisms are largely unknown. Here, we show that multifunctional chaperone systems play a concerted and limiting role in cell-cycle entry, specifically driving nuclear accumulation of the G1 Cdk-cyclin complex. Based on these findings, we establish and test a molecular competition model that recapitulates cell-cycle-entry dependence on growth rate. As key predictions at a single-cell level, we show that availability of the Ydj1 chaperone and nuclear accumulation of the G1 cyclin Cln3 are inversely dependent on growth rate and readily respond to changes in protein synthesis and stress conditions that alter protein folding requirements. Thus, chaperone workload would subordinate Start to the biosynthetic machinery and dynamically adjust proliferation to the growth potential of the cell.
Assuntos
Crescimento Celular , Tamanho Celular , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Resposta ao Choque Térmico/fisiologia , Chaperonas Moleculares/metabolismo , Estresse Salino/fisiologia , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Nucléolo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Modelos Moleculares , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Human CTC1-STN1-TEN1 (CST) is an RPA-like single-stranded DNA-binding protein that interacts with DNA polymerase α-primase (pol α) and functions in telomere replication. Previous studies suggest that CST also promotes replication restart after fork stalling. However, the precise role of CST in genome-wide replication remains unclear. In this study, we sought to understand whether CST alters origin licensing and activation. Replication origins are licensed by loading of the minichromosome maintenance 2-7 (MCM) complex in G1 followed by replisome assembly and origin firing in S-phase. We find that CST directly interacts with the MCM complex and disrupts binding of CDT1 to MCM, leading to decreased origin licensing. We also show that CST enhances replisome assembly by promoting AND-1/pol α chromatin association. Moreover, these interactions are not dependent on exogenous replication stress, suggesting that CST acts as a specialized replication factor during normal replication. Overall, our findings implicate CST as a novel regulator of origin licensing and replisome assembly/fork progression through interactions with MCM, AND-1, and pol α.
Assuntos
Cromatina/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA Polimerase I/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Técnicas de Silenciamento de Genes , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Proteínas de Manutenção de Minicromossomo/metabolismo , RNA Interferente Pequeno/genética , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genéticaRESUMO
Budding yeast treated with hydroxyurea (HU) activate the S phase checkpoint kinase Rad53, which prevents DNA replication forks from undergoing aberrant structural transitions and nuclease processing. Rad53 is also required to prevent premature extension of the mitotic spindle that assembles during a HU-extended S phase. Here we present evidence that checkpoint restraint of spindle extension is directly coupled to Rad53 control of replication fork stability. In budding yeast, centromeres are flanked by replication origins that fire in early S phase. Mutations affecting the Zn2+-finger of Dbf4, an origin activator, preferentially reduce centromere-proximal origin firing in HU, corresponding with suppression of rad53 spindle extension. Inactivating Exo1 nuclease or displacing centromeres from origins provides a similar suppression. Conversely, short-circuiting Rad53 targeting of Dbf4, Sld3, and Dun1, substrates contributing to fork stability, induces spindle extension. These results reveal spindle extension in HU-treated rad53 mutants is a consequence of replication fork catastrophes at centromeres. When such catastrophes occur, centromeres become susceptible to nucleases, disrupting kinetochore function and spindle force balancing mechanisms. At the same time, our data indicate centromere duplication is not required to stabilize S phase spindle structure, leading us to propose a model for how monopolar kinetochore-spindle attachments may contribute to spindle force balance in HU.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Replicação do DNA/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Centrômero/genética , Centrômero/metabolismo , Quinase do Ponto de Checagem 2/genética , Segregação de Cromossomos/efeitos dos fármacos , Estruturas Cromossômicas/metabolismo , Dano ao DNA/genética , Replicação do DNA/genética , DNA Fúngico/genética , Cinetocoros/metabolismo , Origem de Replicação , Fase S/fisiologia , Pontos de Checagem da Fase S do Ciclo Celular/genética , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Cell growth and survival depend on a delicate balance between energy production and synthesis of metabolites. Here, we provide evidence that an alternative mitochondrial complex II (CII) assembly, designated as CIIlow, serves as a checkpoint for metabolite biosynthesis under bioenergetic stress, with cells suppressing their energy utilization by modulating DNA synthesis and cell cycle progression. Depletion of CIIlow leads to an imbalance in energy utilization and metabolite synthesis, as evidenced by recovery of the de novo pyrimidine pathway and unlocking cell cycle arrest from the S-phase. In vitro experiments are further corroborated by analysis of paraganglioma tissues from patients with sporadic, SDHA and SDHB mutations. These findings suggest that CIIlow is a core complex inside mitochondria that provides homeostatic control of cellular metabolism depending on the availability of energy.