RESUMO
The study of the physiological and pathophysiological processes under extreme conditions facilitates a better understanding of the state of a healthy organism and can also shed light on the pathogenesis of diseases. In recent years, it has become evident that gravitational stress affects both the whole organism and individual cells. We have previously demonstrated that simulated microgravity inhibits proliferation, induces apoptosis, changes morphology, and alters the surface marker expression of megakaryoblast cell line MEG-01. In the present work, we investigate the expression of cell cycle cyclins in MEG-01 cells. We performed several experiments for 24 h, 72 h, 96 h and 168 h. Flow cytometry and Western blot analysis demonstrated that the main change in the levels of cyclins expression occurs under conditions of simulated microgravity after 96 h. Thus, the level of cyclin A expression showed an increase in the RPM group during the first 4 days, followed by a decrease, which, together with the peak of cyclin D, may indicate inhibition of the cell cycle in the G2 phase, before mitosis. In addition, based on the data obtained by PCR analysis, we were also able to see that both cyclin A and cyclin B expression showed a peak at 72 h, followed by a gradual decrease at 96 h. STED microscopy data also confirmed that the main change in cyclin expression of MEG-01 cells occurs at 96 h, under simulated microgravity conditions, compared to static control. These results suggested that the cell cycle disruption induced by RPM-simulated microgravity in MEG-01 cells may be associated with the altered expression of the main regulators of the cell cycle. Thus, these data implicate the development of cellular stress in MEG-01 cells, which may be important for proliferating human cells exposed to microgravity in real space.
Assuntos
Ciclo Celular , Ciclinas , Simulação de Ausência de Peso , Humanos , Linhagem Celular , Ciclinas/metabolismo , Ciclinas/genética , Células Progenitoras de Megacariócitos/metabolismo , Células Progenitoras de Megacariócitos/citologia , Ciclina A/metabolismo , Ciclina A/genética , Proliferação de Células , Ciclina B/metabolismo , Ciclina B/genéticaRESUMO
Skp2, the substrate recognition component of the SCFSkp2 ubiquitin ligase, has been implicated in the targeted destruction of a number of key cell cycle regulators and the promotion of S-phase. One of its critical targets is the Cyclin dependent kinase (Cdk) inhibitor p27, and indeed the overexpression of Skp2 in a number of cancers is directly correlated with the premature degradation of p27. Skp2 was first identified as a protein that interacts with Cyclin A in transformed cells, but its role in this complex has remained unclear. In this paper, we demonstrate that Skp2 interacts with Cyclin A in Drosophila and is required to maintain Cyclin A levels and permit mitotic entry. Failure of mitotic entry in Skp2 mutant cells results in polyploidy. If these cells enter mitosis again they are unable to properly segregate their chromosomes, leading to checkpoint dependent cell cycle arrest or apoptosis. Thus, Skp2 is required for mitosis and for maintaining diploidy and genome stability.
Assuntos
Proteínas de Ciclo Celular , Ciclina A , Diploide , Mitose , Animais , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclina A/genética , Ciclina A/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismoRESUMO
The stem cell pools at the shoot apex and root tip give rise to all the above- and below-ground tissues of a plant. Previous studies in Arabidopsis identified a TSO1-MYB3R1 transcriptional module that controls the number and size of the stem cell pools at the shoot apex and root tip. As TSO1 and MYB3R1 are homologous to components of an animal cell cycle regulatory complex, DREAM, Arabidopsis mutants of TSO1 and MYB3R1 provide valuable tools for investigations into the link between cell cycle regulation and stem cell maintenance in plants. In this study, an Arabidopsis cyclin A gene, CYCA3;4, was identified as a member of the TSO1-MYB3R1 regulatory module and cyca3;4 mutations suppressed the tso1-1 mutant phenotype specifically in the shoot. The work reveals how the TSO1-MYB3R1 module is integrated with the cell cycle machinery to control cell division at the shoot meristem.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Animais , Arabidopsis/genética , Meristema/metabolismo , Proteínas de Arabidopsis/metabolismo , Ciclina A/genética , Ciclina A/metabolismo , Mutação , Fertilidade , Regulação da Expressão Gênica de Plantas , Brotos de Planta/metabolismoRESUMO
DENR and MCTS1 have been identified as oncogenes in several different tumor entities. The heterodimeric DENR·MCTS1 protein complex promotes translation of mRNAs containing upstream Open Reading Frames (uORFs). We show here that DENR is phosphorylated on Serine 73 by Cyclin B/CDK1 and Cyclin A/CDK2 at the onset of mitosis, and then dephosphorylated as cells exit mitosis. Phosphorylation of Ser73 promotes mitotic stability of DENR protein and prevents its cleavage at Asp26. This leads to enhanced translation of mRNAs involved in mitosis. Indeed, we find that roughly 40% of all mRNAs with elevated translation in mitosis are DENR targets. In the absence of DENR or of Ser73 phosphorylation, cells display elevated levels of aberrant mitoses and cell death. This provides a mechanism how the cell cycle regulates translation of a subset of mitotically relevant mRNAs during mitosis.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Western Blotting , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Linhagem Celular Tumoral , Ciclina A/genética , Ciclina B/genética , Quinase 2 Dependente de Ciclina/genética , Fatores de Iniciação em Eucariotos/genética , Células HeLa , Humanos , Células MCF-7 , Mitose/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fases de Leitura Aberta/genética , Fosforilação , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina/genética , Serina/metabolismoRESUMO
Cyclin A promotes Cdk activity in a cell cycle-dependent manner to facilitate specific cell cycle events and transitions with an established role for DNA replication in S phase. Recent evidence demonstrates that cyclin A also activates Cdk during early mitosis to promote faithful chromosome segregation by regulating the stability of kinetochore-microtubule (k-MT) attachments. Here we describe a methodology to identify protein substrates of cyclin A/Cdk during mitosis in human cells. The method combines selective cell cycle synchrony in mitosis with stable isotope labeling of amino acids in cell culture (SILAC) coupled to mass spectrometry. This strategy identified a catalogue of potential cyclin A/Cdk substrates in mitosis, as well as unveiled potential intersections between signaling regulated by Aurora, Polo-like, and Cdk mitotic kinases.
Assuntos
Ciclina A , Mitose , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Ciclina A/genética , Ciclina A/metabolismo , Humanos , Microtúbulos/metabolismo , FosforilaçãoRESUMO
The cell cycle is strictly programmed with control mechanisms that dictate order in cell cycle progression to ensure faithful DNA replication, whose deviance may lead to cancer. Checkpoint control at the G1/S, S/G2 and G2/M portals have been defined but no statutory time-programmed control for securing orderly transition through S phase has so far been identified. Here we report that in normal cells DNA synthesis is controlled by a checkpoint sited within the early part of S phase, enforced by the ßGBP cytokine an antiproliferative molecule otherwise known for its oncosuppressor properties that normal cells constitutively produce for self-regulation. Suppression of active Ras and active MAPK, block of cyclin A gene expression and suppression of CDK2-cyclin A activity are events which while specific to the control of a cell cycle phase in normal cells are part of the apoptotic network in cancer cells.
Assuntos
Quinases Ciclina-Dependentes , Pontos de Checagem da Fase S do Ciclo Celular , Ciclo Celular , Ciclina A/genética , Ciclina A/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Citocinas , Humanos , Fase SRESUMO
Macrobrachium nipponense has the characteristics of fast ovarian development cycle, which leads to the coexistence of multiple generations, the reduction of commodity specifications and the low economic benefit. Therefore, the study on the mechanism of ovarian development is of great significance to the development of industry. Cyclin A (CycA)is a key gene regulating ovarian development in vertebrates, but little information was available for its function in crustaceans. In this study, the full-length cDNA of Mn-CycA was obtained from the ovary. The full-length cDNA (2033 bp) with an open reading frame of 1368 bp, encoded a 456-amino acid protein. qRT-PCR revealed tissue-specific expression pattern of Mn-CycA, with abundant expression in the ovary. Results in different developmental stages of ovary indicated that Mn-CycA expression is positively correlated with ovarian maturation. qRT-PCR In different developmental stages, the expression of Mn-CycA mRNA gradually increased during the embryonic stage and decreased significantly on the first day of the hatching stage. At the 25th day of the metamorphosis stage, the expression level of Mn-CycAmRNA in female shrimp was 3.5 times higher than that in male shrimp, which may be related to the proliferation of oogonia and the formation of oocytes. In situ hybridization (ISH) of ovary showed Mn-CycA was examined in all stages and was mainly located in oogonia and oocytes. Compared with the control group, the obvious change of gonad somatic index (GSI) proved that injection of Mn-CycA dsRNA could delay the ovarian development cycle, which provided strong evidence for the involvement of Mn-CycA in ovarian maturation and oogenesis, and expanded a new perspective for studying the fast ovarian development cycle in M. nipponense.
Assuntos
Ciclina A/genética , Ciclina A/metabolismo , Perfilação da Expressão Gênica/métodos , Palaemonidae/crescimento & desenvolvimento , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Sequência de Bases , Clonagem Molecular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Oogônios/crescimento & desenvolvimento , Oogônios/metabolismo , Fases de Leitura Aberta , Especificidade de Órgãos , Palaemonidae/genética , Palaemonidae/metabolismo , FilogeniaRESUMO
The initiation of DNA replication involves cell cycle-dependent assembly and disassembly of protein complexes, including the origin recognition complex (ORC) and CDC6 AAA+ ATPases. We report that multiple short linear protein motifs (SLiMs) within intrinsically disordered regions (IDRs) in ORC1 and CDC6 mediate cyclin-CDK-dependent and independent protein-protein interactions, conditional on the cell cycle phase. A domain within the ORC1 IDR is required for interaction between the ORC1 and CDC6 AAA+ domains in G1, whereas the same domain prevents CDC6-ORC1 interaction during mitosis. Then, during late G1, this domain facilitates ORC1 destruction by a SKP2-cyclin A-CDK2-dependent mechanism. During G1, the CDC6 Cy motif cooperates with cyclin E-CDK2 to promote ORC1-CDC6 interactions. The CDC6 IDR regulates self-interaction by ORC1, thereby controlling ORC1 protein levels. Protein phosphatase 1 binds directly to a SLiM in the ORC1 IDR, causing ORC1 de-phosphorylation upon mitotic exit, increasing ORC1 protein, and promoting pre-RC assembly.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Replicação do DNA , Proteínas Intrinsicamente Desordenadas/metabolismo , Mitose , Proteínas Nucleares/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Domínio AAA , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Ciclina A/genética , Ciclina A/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Fase G1 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Nucleares/genética , Complexo de Reconhecimento de Origem/genética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Estabilidade Proteica , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismoRESUMO
Mammalian cyclin A-CDK (cyclin-dependent kinase) activity during mitotic exit is regulated by two redundant pathways, cyclin degradation and CDK inhibitors (CKIs). Ectopic expression of a destruction box-truncated (thereby stabilized) mutant of cyclin A in the mouse embryonic fibroblasts nullizygous for three CKIs (p21, p27, and p107) results in constitutive activation ("hyperactivation") of cyclin A-CDK and induces rapid tetraploidization, suggesting loss of the two redundant pathways causes genomic instability. To elucidate the mechanism underlying teraploidization by hyperactive cyclin A-CDK, we first examined if the induction of tetraploidization depends on specific cell cycle stage(s). Arresting the cell cycle at either S phase or M phase blocked the induction of tetraploidization, which was restored by subsequent release from the arrest. These results suggest that both S- and M-phase progressions are necessary for the tetraploidization by hyperactive cyclin A-CDK and that the tetraploidization is not caused by chromosome endoreduplication but by mitotic failure. We also observed that the induction of tetraploidization is associated with excessive duplication of centrosomes, which was suppressed by S-phase but not M-phase block, suggesting that hyperactive cyclin A-CDK promotes centrosome overduplication during S phase. Time-lapse microscopy revealed that hyperactive cyclin A-CDK can lead cells to bypass cell division and enter pseudo-G1 state. These observations implicate that hyperactive cyclin A-CDK causes centrosome overduplication, which leads to mitotic slippage and subsequent tetraploidization.
Assuntos
Centrossomo/metabolismo , Cromossomos de Mamíferos/metabolismo , Ciclina A/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Poliploidia , Animais , Proteínas de Ciclo Celular/metabolismo , Ciclina A/genética , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Mitose , Mutação/genética , Fase SRESUMO
OBJECTIVE: Endothelial dysfunction and denudation are considered a first step in atherosclerosis. Endothelial proliferation is key for cellular repair. The effect of bazedoxifene on the vascular endothelium has not been explored. We investigated the effect of bazedoxifene on endothelial cell proliferation. METHODS: Primary cultures from human umbilical artery endothelial cells were used in dose-response experiments (0.1, 1.0, and 10.0 EC50 dose) with bazedoxifene, estradiol, raloxifene and a combination of bazedoxifene and estradiol. Proliferation was assessed with the XTT colorimetric cell-proliferation assay. The possible participation of cyclins A, B, D1 and p27Kip1 was analyzed by the measurement of their expression at both the protein and the gene levels. RESULTS: A significant increase of similar size for cell proliferation was obtained with bazedoxifene, estradiol and raloxifene, but no significant change was observed for the association of bazedoxifene and estradiol. The impact was detected at the first 0.1 EC50 dose and was not dose-dependent. Estradiol achieved a significant increase in the protein expression of cyclin A and p27Kip1, but no change was detected for the other compounds at either the gene or protein level. CONCLUSION: Bazedoxifene demonstrated a proliferative effect of similar size to estradiol in cultured human umbilical artery endothelial cells. The molecular mechanisms need further investigation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Indóis/farmacologia , Proliferação de Células/genética , Células Cultivadas , Ciclina A/genética , Ciclina A/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Recém-Nascido , Gravidez , Artérias Umbilicais/citologiaRESUMO
The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.
Assuntos
Ciclina A/química , Quinase 2 Dependente de Ciclina/química , Inibidor de Quinase Dependente de Ciclina p27/química , Pontos de Checagem da Fase G1 do Ciclo Celular , Proteínas Quinases Associadas a Fase S/química , Sítios de Ligação , Quinases relacionadas a CDC2 e CDC28/química , Quinases relacionadas a CDC2 e CDC28/genética , Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclina A/genética , Ciclina A/metabolismo , Ciclina E/química , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
The eukaryotic cell cycle involves a highly orchestrated series of events in which the cellular genome is replicated during a synthesis (S) phase and each of the two resulting copies are segregated properly during mitosis (M). Host cell factor-1 (HCF-1) is a transcriptional co-regulator that is essential for and has been implicated in basic cellular processes, such as transcriptional regulation and cell cycle progression. Although a series of HCF-1 transcriptional targets have been identified, few functional clues have been provided, especially for chromosome segregation. Our results showed that HCF-1 activated CDC42 expression by binding to the -881 to -575 region upstream of the CDC42 transcription start site, and the regulation of CDC42 expression by HCF-1 was correlated with cell cycle progression. The overexpression of a spontaneously cycling and constitutively active CDC42 mutant (CDC42F28L) rescued G1 phase delay and multinucleate defects in mitosis upon the loss of HCF-1. Therefore, these results establish that HCF-1 ensures proper cell cycle progression by regulating the expression of CDC42, which indicates a possible mechanism of cell cycle coordination and the regulation mode of typical Rho GTPases.
Assuntos
Fator C1 de Célula Hospedeira/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Ciclo Celular/fisiologia , Segregação de Cromossomos , Ciclina A/biossíntese , Ciclina A/genética , Progressão da Doença , Pontos de Checagem da Fase G1 do Ciclo Celular , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Fator C1 de Célula Hospedeira/genética , Humanos , Mitose , Regiões Promotoras Genéticas , Proteína cdc42 de Ligação ao GTP/biossíntese , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
Inactivation of Rb is a major event in the development of hepatocellular carcinoma (HCC). The activity of CDK4, determined by T172 phosphorylation, correlates with the onset of RB phosphorylation and G1/S cell cycle transition. However, the regulation of CDK4 activation and of the Rb pathway in HCC remain unclear. Here, we report that cyclin Y, a novel member of the cyclin family, is a potential regulator of the Rb pathway. We demonstrate that the Cyclin Y protein was overexpressed in human HCC tissues and that it was associated with poor patient prognosis. Cyclin Y could regulate the G1/S phase transition in human HCC cell lines. We found that CDK4 can bind to Cyclin Y in vitro. Furthermore, the accumulation of Cyclin Y could activate CDK4 through T172 phosphorylation of CDK4, inactivate Rb with increasing Rb phosphorylation, and enable the expression of E2F target genes such as CDK2 and Cyclin A. Thus, our findings suggest that Cyclin Y plays a role in the G1/S phase transition of HCC cells via Cyclin Y/CDK4/Rb signaling and that Cyclin Y could be used as a potential prognostic biomarker in HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Ciclinas/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Neoplasias Hepáticas/metabolismo , Proteína do Retinoblastoma/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Pontos de Checagem do Ciclo Celular/genética , Ciclina A/genética , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Fosforilação , Prognóstico , Proteína do Retinoblastoma/antagonistas & inibidores , Transdução de Sinais , Análise Serial de TecidosRESUMO
Herpesviruses encode conserved protein kinases (CHPKs) to stimulate phosphorylation-sensitive processes during infection. How CHPKs bind to cellular factors and how this impacts their regulatory functions is poorly understood. Here, we use quantitative proteomics to determine cellular interaction partners of human herpesvirus (HHV) CHPKs. We find that CHPKs can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a signature of ß-herpesvirus kinases. Cyclin A is recruited via RXL motifs that overlap with nuclear localization signals (NLS) in the non-catalytic N termini. This architecture is conserved in HHV6, HHV7 and rodent cytomegaloviruses. Cyclin A binding competes with NLS function, enabling dynamic changes in CHPK localization and substrate phosphorylation. The cytomegalovirus kinase M97 sequesters Cyclin A in the cytosol, which is essential for viral inhibition of cellular replication. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases.
Assuntos
Replicação do DNA/fisiologia , Infecções por Herpesviridae/metabolismo , Herpesviridae/metabolismo , Proteínas Quinases/metabolismo , Animais , Ciclina A/genética , Ciclina A/metabolismo , Citomegalovirus/genética , DNA/metabolismo , Células HEK293 , Herpesviridae/enzimologia , Herpesviridae/genética , Infecções por Herpesviridae/virologia , Humanos , Camundongos , Células NIH 3T3 , Sinais de Localização Nuclear/metabolismo , Fosforilação , Mapas de Interação de Proteínas , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
We studied the effects of berberine on the proliferation, apoptosis, and migration of skin melanoma A375 cells, as well as cell cycle-related miRNAs and their target genes, CDK1, CDK2, and cyclins D1 and A. The inhibitory effect of berberine on the growth of A375 cells was evaluated by MTT assay. Cell apoptosis was detected by trypan blue staining. Cell migration was assessed by the scratch test. Cell cycle phases were determined by flow cytometry. The levels of miRNA-582-5p and miRNA-188-5, and mRNA of their target genes encoding CDK1, CDK2, and cyclins D1 and A were measured by qRT-PCR. The expression of cell cycle-related proteins (CDK1, CDK2, and cyclins D1 and A) was determined by Western blotting. Berberine inhibited the proliferation of A375 cells in a time- and dose-dependent manner and significantly and dose-dependently enhanced cell apoptosis. Scratch assay showed an inhibitory effect of berberine on migration of A375 cells. Berberine in low concentrations (20 and 40 µM) caused cell cycle arrest in the S and G2/M phases, while treatment with high concentrations of berberine (60 and 80 µM) arrested cell-cycle in the G2/M phase. The increase in berberine concentration led to an increase in miRNA-582-5p and miRNA-188-5p expression and a decrease in the expression of mRNA for the corresponding target genes encoding CDK1, CDK2, and cyclins D1 and A. Western blotting also revealed reduced expression of CDK1, CDK2, and cyclins D1 and A. Thus, berberine suppressed the growth and migration of human melanoma cells and promoted their apoptosis. Berberine can increase the expression of cell cycle-related miRNAs and cause degradation of the corresponding target genes, thereby blocking the cell cycle progression and inhibiting the melanoma A375 cells.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Berberina/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanócitos/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina A/genética , Ciclina A/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Relação Dose-Resposta a Droga , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Transdução de SinaisRESUMO
Achieving complete and precise genome duplication requires that each genomic segment be replicated only once per cell division cycle. Protecting large eukaryotic genomes from re-replication requires an overlapping set of molecular mechanisms that prevent the first DNA replication step, the DNA loading of MCM helicase complexes to license replication origins, after S phase begins. Previous reports have defined many such origin licensing inhibition mechanisms, but the temporal relationships among them are not clear, particularly with respect to preventing re-replication in G2 and M phases. Using a combination of mutagenesis, biochemistry, and single cell analyses in human cells, we define a new mechanism that prevents re-replication through hyperphosphorylation of the essential MCM loading protein, Cdt1. We demonstrate that Cyclin A/CDK1 can hyperphosphorylate Cdt1 to inhibit MCM re-loading in G2 phase. The mechanism of inhibition is to block Cdt1 binding to MCM independently of other known Cdt1 inactivation mechanisms such as Cdt1 degradation during S phase or Geminin binding. Moreover, our findings suggest that Cdt1 dephosphorylation at the mitosis-to-G1 phase transition re-activates Cdt1. We propose that multiple distinct, non-redundant licensing inhibition mechanisms act in a series of sequential relays through each cell cycle phase to ensure precise genome duplication.
Assuntos
Replicação do DNA/genética , Genoma Humano/genética , Origem de Replicação/genética , Duplicações Segmentares Genômicas/genética , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Ciclina A/genética , Fase G2/genética , Geminina/genética , Genes Duplicados/genética , Células HEK293 , Humanos , Proteínas de Manutenção de Minicromossomo/genética , Fosforilação/genética , Fase S/genéticaRESUMO
This study aimed to investigate the expression and prognosis of CyclinA and CDK2 in patients with advanced cervical cancer after chemotherapy. The patient history of 108 patients with advanced cervical cancer admitted to our hospital from December 2013 to January 2016 was selected as a cervical cancer group. 54 normal healthy people admitted to our hospital for physical examination in the same period were selected as the control group. Western blotting and RT-PCR were used to detect the difference between CyclinA and CDK2 proteins and mRNA expression between the two groups and the correlation between them was analyzed. The expressions of CyclinA and CDK2 in serum and the changes in detection index level of squamous cell carcinoma antigen (SCCA), carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF) were observed in cervical cancer group at different stages of treatment. The correlation between the two indexes and SCCA, CEA, VEGF and the 3-year survival and prognostic significance of cervical cancer patients with different CyclinA and CDK2 expressions were analyzed. The relative expressions of CyclinA and CDK2 proteins and mRNA in the cervical cancer group were significantly higher than those in the control group (P< 0.05). Pearson correlation analysis showed a positive correlation between CyclinA and CDK2 proteins and mRNA expressions. After treatment, the expressions of CyclinA, CDK2 mRNA and SCCA, CEA and VEGF were significantly lower than those before treatment (P< 0.05). The 3-year survival rate of CyclinA and CDK2 in the high expression group was significantly lower than that of the low expression group. CyclinA and CDK2 are highly expressed in advanced cervical cancer. The expression is decreased after chemotherapy. The prognosis of both low expressions is higher and the expression is good. It can be used to predict the efficacy and prognosis of cervical cancer in the clinic.
Assuntos
Ciclina A/genética , Quinase 2 Dependente de Ciclina/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Adulto , Antígenos de Neoplasias/sangue , Antígeno Carcinoembrionário/sangue , Ciclina A/sangue , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/sangue , Quinase 2 Dependente de Ciclina/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serpinas/sangue , Análise de Sobrevida , Neoplasias do Colo do Útero/sangue , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
Two mitotic cyclin types, cyclin A and B, exist in higher eukaryotes, but their specialised functions in mitosis are incompletely understood. Using degron tags for rapid inducible protein removal, we analyse how acute depletion of these proteins affects mitosis. Loss of cyclin A in G2-phase prevents mitotic entry. Cells lacking cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phosphatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown, which requires a decisive shift in the balance of cyclin-dependent kinase Cdk1 and PP2A:B55 activity. Beyond this point, cyclin B/Cdk1 is essential for phosphorylation of a distinct subset of mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how cyclin A, cyclin B and Greatwall kinase coordinate mitotic progression by increasing levels of Cdk1-dependent substrate phosphorylation.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Mitose , Proteína Fosfatase 2/metabolismo , Proteína Quinase CDC2/genética , Linhagem Celular , Ciclina A/genética , Ciclina B/genética , Humanos , Proteína Fosfatase 2/genéticaRESUMO
BACKGROUND: The cancer risk in Barrett's oesophagus (BO) is difficult to estimate. Histologic dysplasia has strong predictive power, but can be missed by random biopsies. Other clinical parameters have limited utility for risk stratification. We aimed to assess whether a molecular biomarker panel on targeted biopsies can predict neoplastic progression of BO. METHODS: 203 patients with BO were tested at index endoscopy for 9 biomarkers (p53 and cyclin A expression; aneuploidy and tetraploidy; CDKN2A (p16), RUNX3 and HPP1 hypermethylation; 9p and 17p loss of heterozygosity) on autofluorescence-targeted biopsies and followed-up prospectively. Data comparing progressors to non-progressors were evaluated by univariate and multivariate analyses using survival curves, Cox-proportional hazards and logistic regression models. FINDINGS: 127 patients without high-grade dysplasia (HGD) or oesophageal adenocarcinoma (OAC) at index endoscopy were included, of which 42 had evidence of any histologic progression over time. Aneuploidy was the only predictor of progression from non-dysplastic BO (NDBO) to any grade of neoplasia (p = 0.013) and HGD/OAC (p = 0.002). Aberrant p53 expression correlated with risk of short-term progression within 12 months, with an odds ratio of 6.0 (95% CI: 3.1-11.2). A panel comprising aneuploidy and p53 had an area under the receiving operator characteristics curve of 0.68 (95% CI: 0.59-0.77) for prediction of any progression. INTERPRETATION: Aneuploidy is the only biomarker that predicts neoplastic progression of NDBO. Aberrant p53 expression suggests prevalent dysplasia, which might have been missed by random biopsies, and warrants early follow up.
Assuntos
Adenocarcinoma/genética , Aneuploidia , Esôfago de Barrett/patologia , Neoplasias Esofágicas/genética , Marcadores Genéticos , Idoso , Idoso de 80 Anos ou mais , Esôfago de Barrett/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Ciclina A/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Progressão da Doença , Endoscopia , Feminino , Humanos , Modelos Logísticos , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Estudos Prospectivos , Proteína Supressora de Tumor p53/genéticaRESUMO
The adult mammalian cardiomyocyte has a very limited capacity to reenter the cell cycle and advance into mitosis. Therefore, diseases characterized by lost contractile tissue usually evolve into myocardial remodeling and heart failure. Analyzing the cardiac transcriptome at different developmental stages in a large mammal closer to the human than laboratory rodents may serve to disclose positive and negative cardiomyocyte cell cycle regulators potentially targetable to induce cardiac regeneration in the clinical setting. Thus we aimed at characterizing the transcriptomic profiles of the early fetal, late fetal, and adult sheep heart by employing RNA-seq technique and bioinformatic analysis to detect protein-encoding genes that in some of the stages were turned off, turned on, or differentially expressed. Genes earlier proposed as positive cell cycle regulators such as cyclin A, cdk2, meis2, meis3, and PCNA showed higher expression in fetal hearts and lower in AH, as expected. In contrast, genes previously proposed as cell cycle inhibitors, such as meis1, p16, and sav1, tended to be higher in fetal than in adult hearts, suggesting that these genes are involved in cell processes other than cell cycle regulation. Additionally, we described Gene Ontology (GO) enrichment of different sets of genes. GO analysis revealed that differentially expressed gene sets were mainly associated with metabolic and cellular processes. The cell cycle-related genes fam64a, cdc20, and cdk1, and the metabolism-related genes pitx and adipoq showed strong differential expression between fetal and adult hearts, thus being potent candidates to be targeted in human cardiac regeneration strategies.NEW & NOTEWORTHY We characterized the transcriptomic profiles of the fetal and adult sheep hearts employing RNAseq technique and bioinformatic analyses to provide sets of transcripts whose variation in expression level may link them to a specific role in cell cycle regulation. It is important to remark that this study was performed in a large mammal closer to humans than laboratory rodents. In consequence, the results can be used for further translational studies in cardiac regeneration.