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1.
Biomed Pharmacother ; 141: 111795, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34098217

RESUMO

Cancer recurrence poses a significant challenge. At the cellular level, recurrence takes place as a result of reactivation of dormant cancer cells residing at G0 phase. The aim of the study was to identify compounds that can trap prostate and lung cancer cells in G0 phase from a new Chinese herb recipe, Astringent recipe, consisting of Radix Paeoniae Alba, Agrimonia pilosa Ledeb, Fructus Mume, Fritillaria thunbergii Miq., Ganoderma Lucidum Karst, and Astragalus membranaceus (Fisch.) Bunge. Astringent recipe impeded cell cycle progression in prostate and lung cancer cells by rounding them up at G0 phase by flow cytometric analysis of cancer cells stained with Hoechst 33342 and Pyronin Y, respectively, for DNA and RNA. The anti-cancer efficacy of the recipe was found to be attributable to Agrimonia pilosa Ledeb. Further study established that agrimol B, a polyphenol derived from Agrimonia pilosa Ledeb, contributed to the activity of the herb. The action of agrimol B on the cancer cells was likely derived from its effect on c-MYC, SKP2 and p27 by immunoblotting and immunofluorescence. Oral administration of Agrimonia pilosa Ledeb or agrimol B reduced growth of prostate cancer cell xenograft in animal. In conclusion, Agrimol B can enrich for prostate and lung cancer cells in G0 state and influence key regulators that govern G0 status.


Assuntos
Agrimonia , Antineoplásicos Fitogênicos/farmacologia , Butanonas/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Fenóis/farmacologia , Extratos Vegetais/farmacologia , Carga Tumoral/efeitos dos fármacos , Células A549 , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Butanonas/isolamento & purificação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/fisiologia , Relação Dose-Resposta a Droga , Ácido Elágico/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fenóis/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Carga Tumoral/fisiologia
2.
Neurochem Res ; 46(8): 2046-2055, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34003417

RESUMO

Mild intrauterine hypoperfusion (MIUH) can induce placental dysfunction and lead to long-term changes during the process of brain development. A better understanding of the mechanism of MIUH will help in the development of new neuroprotective strategies for the placental chamber. To better understand the mechanism of the effect of MIUH on the neural development of offspring, we constructed a model of MIUH in pregnant rats. The proliferation, apoptosis, and autophagy of hippocampal neurons in fetal rats were studied via flow cytometry, immunofluorescence staining, JC-1 staining, western blotting, and real-time polymerase chain reaction at different time points (6, 24, 48, and 72 h). The results showed that MIUH significantly inhibited the proliferation of hippocampal neurons and promoted their apoptosis and autophagy. Simultaneously, MIUH could promote PTEN expression and affect the PTEN signaling pathway. bpV, an inhibitor of PTEN, could restore the inhibition of hippocampal nerve cell growth caused by MIUH. MIUH may inhibit neuronal proliferation and promote neuronal apoptosis and autophagy by regulating the PTEN signaling pathway.


Assuntos
Proliferação de Células/fisiologia , Hipóxia Fetal/fisiopatologia , Transtornos do Neurodesenvolvimento/fisiopatologia , Neurônios/metabolismo , Circulação Placentária/fisiologia , Transdução de Sinais/fisiologia , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Constrição Patológica , Feminino , Retardo do Crescimento Fetal/fisiopatologia , Feto , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Hipocampo/patologia , Neurônios/patologia , Ovário/irrigação sanguínea , Ovário/patologia , PTEN Fosfo-Hidrolase/metabolismo , Gravidez , Ratos Sprague-Dawley , Artéria Uterina/patologia
3.
Biochem J ; 478(1): 179-196, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33346336

RESUMO

Human body temperature limits below 40°C during heat stroke or fever. The implications of prolonged exposure to the physiologically relevant temperature (40°C) on cellular mechanobiology is poorly understood. Here, we have examined the effects of heat stress (40°C for 72 h incubation) in human lung adenocarcinoma (A549), mouse melanoma (B16F10), and non-cancerous mouse origin adipose tissue cells (L929). Hyperthermia increased the level of ROS, γ-H2AX and HSP70 and decreased mitochondrial membrane potential in the cells. Heat stress impaired cell division, caused G1 arrest, induced cellular senescence, and apoptosis in all the tested cell lines. The cells incubated at 40°C for 72 h displayed a significant decrease in the f-actin level and cellular traction as compared with cells incubated at 37°C. Also, the cells showed a larger focal adhesion area and stronger adhesion at 40°C than at 37°C. The mitotic cells at 40°C were unable to round up properly and displayed retracting actin stress fibers. Hyperthermia down-regulated HDAC6, increased the acetylation level of microtubules, and perturbed the chromosome alignment in the mitotic cells at 40°C. Overexpression of HDAC6 rescued the cells from the G1 arrest and reduced the delay in cell rounding at 40°C suggesting a crucial role of HDAC6 in hyperthermia mediated responses. This study elucidates the significant role of cellular traction, focal adhesions, and cytoskeletal networks in mitotic cell rounding and chromosomal misalignment. It also highlights the significance of HDAC6 in heat-evoked senile cellular responses.


Assuntos
Senescência Celular/fisiologia , Citoesqueleto/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Desacetilase 6 de Histona/metabolismo , Hipertermia/metabolismo , Fibras de Estresse/metabolismo , Acetilação , Actinas/metabolismo , Apoptose/fisiologia , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Forma Celular , Tamanho Celular , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Desacetilase 6 de Histona/genética , Humanos , Hipertermia/genética , Potencial da Membrana Mitocondrial/fisiologia , Microtúbulos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
4.
Viruses ; 12(8)2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32731335

RESUMO

Non-structural protein 1 (nsp1) is only characterized in alphacoronaviruses (α-CoVs) and betacoronaviruses (ß-CoVs). There have been extensive researches on how the ß-CoVs nsp1 regulates viral virulence by inhibiting host protein synthesis, but the regulatory mechanism of the α-CoVs nsp1 is still unclear. Here, we report the 2.1-Å full-length crystal structure of nsp1 in emerging porcine SADS-CoV and the 1.8-Å full-length crystal structure of nsp1 in the highly lethal cat FIPV. Although they belong to different subtypes of α-CoVs, these viruses all have a bucket-shaped fold composed of six ß-sheets, similar to the crystal structure of PEDV and TGEV nsp1. Comparing the above four structures, we found that the structure of α-CoVs nsp1 in the same subtype was more conserved. We then selected mammalian cells that were treated with SADS-CoV and FIPV nsp1 for RNA sequencing analysis and found that nsp1 had a specific inhibitory effect on interferon (IFN) and cell cycle genes. Using the Renilla luciferase (Rluc) assay and Western blotting, we confirmed that seven representative α-CoVs nsp1s could significantly inhibit the phosphorylation of STAT1-S727 and interfere with the effect of IFN-I. Moreover, the cell cycle experiment confirmed that α-CoVs nsp1 could encourage host cells to stay in the G0/G1 phase. Based on these findings, we not only greatly improved the crystal structure data on α-CoVs nsp1, but we also speculated that α-CoVs nsp1 regulated host proliferation and immune evasion-related biological functions by inhibiting the synthesis of host proteins, thus creating an environment conducive to the virus.


Assuntos
Alphacoronavirus/imunologia , Alphacoronavirus/fisiologia , Evasão da Resposta Imune/imunologia , Interferon Tipo I/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Alphacoronavirus/genética , Sequência de Aminoácidos , Animais , Gatos , Linhagem Celular , Cristalografia por Raios X , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Fosforilação , Estrutura Terciária de Proteína , Fator de Transcrição STAT1/metabolismo , Homologia de Sequência , Suínos , Proteínas não Estruturais Virais/genética , Replicação Viral/genética
5.
Int J Nanomedicine ; 15: 1997-2010, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32273698

RESUMO

BACKGROUND: As one of the most widely produced engineered nanomaterials, titanium dioxide nanoparticles (nano-TiO2) are used in biomedicine and healthcare products, and as implant scaffolds; therefore, the toxic mechanism of nano-TiO2 has been extensively investigated with a view to guiding application. Three-dimensional (3D) spheroid models can simplify the complex physiological environment and mimic the in vivo architecture of tissues, which is optimal for the assessment of nano-TiO2 toxicity under ultraviolet A (UVA) irradiation. METHODS AND RESULTS: In the present study, the toxicity of nano-TiO2 under UVA irradiation was investigated in 3D H22 spheroids cultured in fibrin gels. A significant reduction of approximately 25% in spheroid diameter was observed following treatment with 100 µg/mL nano-TiO2 under UVA irradiation after seven days of culture. Nano-TiO2 under UVA irradiation triggered the initiation of the TGF-ß/Smad signaling pathway, increasing the expression levels of TGF-ß1, Smad3, Cdkn1a, and Cdkn2b at both the mRNA and protein level, which resulted in cell cycle arrest in the G1 phase. In addition, nano-TiO2 under UVA irradiation also triggered the production of reactive oxygen species (ROS), which were shown to be involved in cell cycle regulation and the induction of TGF-ß1 expression. CONCLUSION: Nano-TiO2 under UVA irradiation induced cell cycle arrest in the G1 phase and the formation of smaller spheroids, which were associated with TGF-ß/Smad signaling pathway activation and ROS generation. These results reveal the toxic mechanism of nano-TiO2 under UVA irradiation, providing the possibility for 3D spheroid models to be used in nanotoxicology studies.


Assuntos
Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Nanopartículas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Titânio/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos da radiação , Camundongos , Nanopartículas/química , Proteína Smad3/genética , Proteína Smad3/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/efeitos da radiação , Raios Ultravioleta
6.
Biosystems ; 191-192: 104128, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32165312

RESUMO

Biological systems are difficult to understand complex systems. Scientists continue to create models to simulate biological systems but these models are complex too; for this reason, new reduction methods to simplify complex biological models into simpler ones are increasingly needed. In this paper, we present a way of reducing complex quantitative (continuous) models into logical models based on time windows of system activity and logical (Boolean) models. Time windows were used to define slow and fast activity areas. We use the proposed approach to reduce a continuous ODE model into a logical model describing the G1/S checkpoint with and without DNA damage as a case study. We show that the temporal unfolding of this signalling system can be broken down into three time windows where only two display high level of activity and the other shows little or no activity. The two active windows represent a cell committing to cell cycle and making the G1/S transition, respectively, the two most important high level functions of cell cycle in the G1 phase. Therefore, we developed two models to represent these time windows to reduce time complexity and used Boolean approach to reduce interaction complexity in the ODE model in the respective time windows. The developed reduced models correctly produced the commitment to cell cycle and G1/S transfer through the expected behavior of signalling molecules involved in these processes. As most biological models have a large number of fast reactions and a relatively smaller number of slow reactions, we believe that the proposed approach could be suitable for representing many, if not all biological signalling networks. The approach presented in this study greatly helps in simplifying complex continuous models (ODE models) into simpler models. Moreover, it will also assist scientists build models concentrating on understanding and representing system behavior rather than setting values for a large number of kinetic parameters.


Assuntos
Algoritmos , Dano ao DNA , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Modelos Biológicos , Transdução de Sinais/fisiologia , Simulação por Computador , Fase G1/genética , Fase G1/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Redes Reguladoras de Genes , Mapas de Interação de Proteínas , Fase S/genética , Fase S/fisiologia , Transdução de Sinais/genética , Fatores de Tempo
7.
Artigo em Inglês | MEDLINE | ID: mdl-31385777

RESUMO

BACKGROUND: Nowadays, the potential therapeutic role of various bioflavonoids including Curcumin, Luteolin and Resveratrol has currently been well-documented in a vast range of fatal complications including synaptic failure and cancers. These bioflavonoids are widely being implemented for the treatment of various cancers as they possess anti-cancerous, anti-oxidant and anti-inflammatory properties. Moreover, they are also used as a better alternative to conventional therapies since; these are non-toxic to cells and having no or least side effects. Notably, the pertinent therapeutic role of Rutin in cervical cancer is still unsettled however, its anti-cancerous role has already been reported in other cancers including prostate and colon cancer. Rutin (Vitamin P or Rutoside) is a polyphenolics flavonoid exhibiting multi-beneficial roles against several carcinomas. OBJECTIVE: Despite the evidence for its several biological activities, the anticancer effects of Rutin on human cervical cancer (C33A) cells remain to be explored. In this study, the anticancer potential of Rutin was investigated by employing the key biomarkers such as nuclear condensation reactive oxygen species (ROS), apoptosis, and changes in mitochondrial membrane potential (MMP). RESULTS: Our findings showed that Rutin treatment reduced the cell viability, induced significant increase in ROS production and nuclear condensation in dose-dependent manner. Moreover, Rutin provoked apoptosis by inducing decrease in MMP and activation of caspase-3. Cell cycle analysis further confirmed the efficacy of Rutin by showing cell cycle arrest at G0/G1 phase. CONCLUSION: Thus, our study is envisaged to open up interests for elucidating Rutin as an anticancerous agent against cervical cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Rutina/farmacologia , Neoplasias do Colo do Útero/fisiopatologia , Alphapapillomavirus , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/fisiologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Células HEK293 , Humanos , Fase de Repouso do Ciclo Celular/fisiologia , Rutina/uso terapêutico , Neoplasias do Colo do Útero/tratamento farmacológico
8.
PLoS Biol ; 17(8): e3000203, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31430272

RESUMO

Zebrafish dorsal forerunner cells (DFCs) undergo vigorous proliferation during epiboly and then exit the cell cycle to generate Kupffer's vesicle (KV), a ciliated organ necessary for establishing left-right (L-R) asymmetry. DFC proliferation defects are often accompanied by impaired cilia elongation in KV, but the functional and molecular interaction between cell-cycle progression and cilia formation remains unknown. Here, we show that chemokine receptor Cxcr4a is required for L-R laterality by controlling DFC proliferation and KV ciliogenesis. Functional analysis revealed that Cxcr4a accelerates G1/S transition in DFCs and stabilizes forkhead box j1a (Foxj1a), a master regulator of motile cilia, by stimulating Cyclin D1 expression through extracellular regulated MAP kinase (ERK) 1/2 signaling. Mechanistically, Cyclin D1-cyclin-dependent kinase (CDK) 4/6 drives G1/S transition during DFC proliferation and phosphorylates Foxj1a, thereby disrupting its association with proteasome 26S subunit, non-ATPase 4b (Psmd4b), a 19S regulatory subunit. This prevents the ubiquitin (Ub)-independent proteasomal degradation of Foxj1a. Our study uncovers a role for Cxcr4 signaling in L-R patterning and provides fundamental insights into the molecular linkage between cell-cycle progression and ciliogenesis.


Assuntos
Cílios/metabolismo , Fatores de Determinação Direita-Esquerda/metabolismo , Receptores CXCR4/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Padronização Corporal/genética , Ciclo Celular/fisiologia , Divisão Celular , Proliferação de Células , Quimiocinas/metabolismo , Embrião não Mamífero/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Morfogênese , Receptores CXCR4/fisiologia , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
9.
EMBO Rep ; 20(9): e47592, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31347271

RESUMO

CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog-sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA-seq and ChIP-seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3'ends of predominantly long, poly(A)-signal-rich genes. CDK12 inhibition does not globally reduce levels of RNAPII-Ser2 phosphorylation. However, individual CDK12-dependent genes show a shift of P-Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/genética , Reparo do DNA/genética , Reparo do DNA/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Células HCT116 , Humanos , Fosforilação , RNA Polimerase II/genética , RNA Polimerase II/metabolismo
10.
Nat Commun ; 10(1): 2110, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068593

RESUMO

Ribosome biogenesis is a canonical hallmark of cell growth and proliferation. Here we show that execution of Epithelial-to-Mesenchymal Transition (EMT), a migratory cellular program associated with development and tumor metastasis, is fueled by upregulation of ribosome biogenesis during G1/S arrest. This unexpected EMT feature is independent of species and initiating signal, and is accompanied by release of the repressive nucleolar chromatin remodeling complex (NoRC) from rDNA, together with recruitment of the EMT-driving transcription factor Snai1 (Snail1), RNA Polymerase I (Pol I) and the Upstream Binding Factor (UBF). EMT-associated ribosome biogenesis is also coincident with increased nucleolar recruitment of Rictor, an essential component of the EMT-promoting mammalian target of rapamycin complex 2 (mTORC2). Inhibition of rRNA synthesis in vivo differentiates primary tumors to a benign, Estrogen Receptor-alpha (ERα) positive, Rictor-negative phenotype and reduces metastasis. These findings implicate the EMT-associated ribosome biogenesis program with cellular plasticity, de-differentiation, cancer progression and metastatic disease.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Ribossomos/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral/transplante , Movimento Celular/fisiologia , Nucléolo Celular/metabolismo , Embrião de Galinha , Proteínas Cromossômicas não Histona/metabolismo , DNA Ribossômico/metabolismo , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Ribossômico/metabolismo , Ribossomos/genética
11.
Sci Signal ; 12(579)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040260

RESUMO

The role of the mitochondrial Ca2+ uniporter (MCU) in physiologic cell proliferation remains to be defined. Here, we demonstrated that the MCU was required to match mitochondrial function to metabolic demands during the cell cycle. During the G1-S transition (the cycle phase with the highest mitochondrial ATP output), mitochondrial fusion, oxygen consumption, and Ca2+ uptake increased in wild-type cells but not in cells lacking MCU. In proliferating wild-type control cells, the addition of the growth factors promoted the activation of the Ca2+/calmodulin-dependent kinase II (CaMKII) and the phosphorylation of the mitochondrial fission factor Drp1 at Ser616 The lack of the MCU was associated with baseline activation of CaMKII, mitochondrial fragmentation due to increased Drp1 phosphorylation, and impaired mitochondrial respiration and glycolysis. The mitochondrial fission/fusion ratio and proliferation in MCU-deficient cells recovered after MCU restoration or inhibition of mitochondrial fragmentation or of CaMKII in the cytosol. Our data highlight a key function for the MCU in mitochondrial adaptation to the metabolic demands during cell cycle progression. Cytosolic CaMKII and the MCU participate in a regulatory circuit, whereby mitochondrial Ca2+ uptake affects cell proliferation through Drp1.


Assuntos
Canais de Cálcio/metabolismo , Proliferação de Células/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Dinâmica Mitocondrial/fisiologia , Miócitos de Músculo Liso/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proliferação de Células/genética , Células Cultivadas , Dinaminas/metabolismo , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Masculino , Camundongos Knockout , Dinâmica Mitocondrial/genética , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Fosforilação
12.
BMC Mol Cell Biol ; 20(1): 8, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-31041891

RESUMO

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 , Transfecção
13.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30979824

RESUMO

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/genética
14.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30988162

RESUMO

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/metabolismo
15.
FEBS Open Bio ; 9(6): 1109-1118, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30972973

RESUMO

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 Cima
16.
J Exp Clin Cancer Res ; 38(1): 50, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30717766

RESUMO

BACKGROUND: Altered glucose metabolism endows tumor cells with metabolic flexibility for biosynthesis requirements. Phosphoenolpyruvate carboxykinase 1 (PCK1), a key enzyme in the gluconeogenesis pathway, is downregulated in hepatocellular carcinoma (HCC) and predicts poor prognosis. Overexpression of PCK1 has been shown to suppress liver tumor growth, but the underlying mechanism remains unclear. METHODS: mRNA and protein expression patterns of PCK1, AMPK, pAMPK, and the CDK/Rb/E2F pathway were determined using qRT-PCR and western blotting. Cell proliferation ability and cell cycle were assessed by MTS assay and flow cytometric analysis. The effect of PCK1 on tumor growth was examined in xenograft implantation models. RESULTS: Both gain and loss-of-function experiments demonstrated that PCK1 deficiency promotes hepatoma cell proliferation through inactivation of AMPK, suppression of p27Kip1 expression, and stimulation of the CDK/Rb/E2F pathway, thereby accelerating cell cycle transition from the G1 to S phase under glucose-starved conditions. Overexpression of PCK1 reduced cellular ATP levels and enhanced AMPK phosphorylation and p27Kip1 expression but decreased Rb phosphorylation, leading to cell cycle arrest at G1. AMPK knockdown significantly reversed G1-phase arrest and growth inhibition of PCK1-expressing SK-Hep1 cells. In addition, the AMPK activator metformin remarkably suppressed the growth of PCK1-knockout PLC/PRF/5 cells and inhibited tumor growth in an orthotropic HCC mouse model. CONCLUSION: This study revealed that PCK1 negatively regulates cell cycle progression and hepatoma cell proliferation via the AMPK/p27Kip1 axis and supports a potential therapeutic and protective effect of metformin on HCC.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Hepatocelular/patologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/patologia , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Regulação para Baixo , Fatores de Transcrição E2F/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Humanos , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Biochim Biophys Acta Mol Cell Res ; 1866(3): 409-417, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30315834

RESUMO

Joint regulation of growth rate and cell division rate determines cell size. Here we discuss how animal cells achieve cell size homeostasis potentially involving multiple signaling pathways converging at metabolic regulation of growth rate and cell cycle progression. While several models have been developed to explain cell size control, comparison of the two predominant models shows that size homeostasis is dependent on the ability to adjust cellular growth rate based on cell size. Consequently, maintenance of size homeostasis requires that larger cells can grow slower than small cells in relative terms. We review recent experimental evidence showing that such size adjustment occurs primarily at or immediately before the G1/S transition of the cell cycle. We further propose that bidirectional feedback between growth rate and size results in cell size sensing and discuss potential mechanisms how this may be accomplished.


Assuntos
Divisão Celular/fisiologia , Proliferação de Células/fisiologia , Homeostase/fisiologia , Animais , Ciclo Celular/fisiologia , Tamanho Celular , Células/citologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Crescimento/fisiologia , Humanos , Modelos Biológicos , Transdução de Sinais/fisiologia
18.
Cancer Biol Ther ; 20(5): 608-616, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30404567

RESUMO

C-X-C motif chemokine ligand 5 (CXCL5) is initially identified to recruit neutrophils by interacting with its receptor, C-X-C motif chemokine receptor 2 (CXCR2). Our prior work demonstrated that the expression levels of CXCL5 and CXCR2 were higher in the papillary thyroid carcinoma (PTC) tumors than that in the non-tumors. This study was performed to further investigate how this axis regulates the growth of PTC cells. B-CPAP cells (BRAFV600E) and TPC-1 cells (RET/PTC rearrangement) expressing CXCR-2 were used as in vitro cell models. Our results showed that the recombinant human CXCL5 (rhCXCL5) promoted the proliferation of PTC cells. rhCXCL5 accelerated the G1/S transition, upregulated the expression of a group of S (DNA synthesis) or M (mitosis)-promoting cyclins and cyclin-dependent kinases (CDKs), and downregulated CDK inhibitors in PTC cells. The CDS region of homo sapiens CXCL5 gene was inserted into an eukaryotic expression vector to mediate the overexpression of CXCL5 in PTC cells. The phosphorylation of c-Jun N-terminal kinases (JNK) and p38, and the nuclear translocation of c-Jun were enhanced by CXCL5 overexpression, whereas attenuated by CXCR2 antagonist SB225002. Additionally, CXCL5/CXCR2 axis, JNK and p38 pathway inhibitors, SB225002, SP600125 and SB203580, suppressed the growth of PTC cells overexpressing CXCL5 in nude mice, respectively. Collectively, our study demonstrates a growth-promoting effect of CXCL5-CXCR2 axis in PTC cells in vitro and in vivo.


Assuntos
Quimiocina CXCL5/metabolismo , Receptores de Interleucina-8B/metabolismo , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Animais , Antracenos/farmacologia , Antracenos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL5/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Nus , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Piridinas/uso terapêutico , Receptores de Interleucina-8B/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Câncer Papilífero da Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Int J Mol Med ; 43(2): 739-748, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30569159

RESUMO

Biliary atresia (BA) is the most common cause of chronic cholestasis in children. The long non­coding RNA (lncRNA) Annexin A2 pseudogene 3 (ANXA2P3) and Annexin A2 (ANXA2) have been suggested to serve pivotal roles in BA; however, the clinical significance and biological roles of ANXA2P3 and ANXA2 in BA remain to be elucidated. The present study aimed to elucidate the function of ANAX2P3 and ANXA2 in BA­induced liver injury using a human liver cell line and liver tissues from patients with BA. Reverse transcription­quantitative polymerase chain reaction, western blotting and immunohistochemistry were conducted to determine the expression levels of ANXA2 and ANXA2P3 in liver tissues from patients with BA. Classification of fibrosis was analyzed by Masson staining. The functional roles of ANXA2 and ANXA2P3 in liver cells were determined by Cell Counting kit­8 assay, and flow cytometric and cell cycle analyses. Activation of the ANXA2/ANXA2P3 signaling pathway in liver cells was evaluated by western blot analysis. According to the present results, the expression levels of ANXA2 and ANXA2P3 were significantly increased in liver tissues from patients with BA. In addition, knocking down the expression of ANXA2P3 and ANXA2 may result in reduced liver cell proliferation, cell cycle arrest in G1 phase and increased apoptosis of liver cells in vitro. Furthermore, in cells in which ANXA2 and ANXA2P3 were overexpressed, cell apoptosis was reduced and cell cycle arrest in G2 phase. Taken together, these results indicated that ANXA2P3 and ANXA2 may have protective effects against liver injury progression and may be considered biomarkers in patients with BA.


Assuntos
Anexina A2/fisiologia , Atresia Biliar/metabolismo , Hepatoblastoma/metabolismo , Neoplasias Hepáticas/metabolismo , RNA Longo não Codificante/fisiologia , Transdução de Sinais/fisiologia , Apoptose/fisiologia , Linhagem Celular , Pré-Escolar , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Humanos , Lactente , Pseudogenes/fisiologia
20.
Insect Biochem Mol Biol ; 102: 75-83, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30287269

RESUMO

Insects enter diapause to synchronize their life cycle with biotic and abiotic conditions favorable for their development, reproduction, and survival. Adult females of the band-legged ground cricket Dianemobius nigrofasciatus (Orthoptera, Glyllidae) respond to environmental factors in autumn and lay diapause-destined eggs. The eggs arrest their development and enter diapause at a very early embryonic stage, specifically the cellular blastoderm. To elucidate the physiological mechanisms underlying this very early stage programmed developmental arrest, we investigated the cell division cycle as well as the expression of cell cycle regulators, small silencing RNAs, and segment patterning genes. The diapause embryo arrests its cell cycle predominantly at the G0/G1 phase. The proportion of cells in the S phase of the cell cycle abruptly decreased at the time of developmental arrest, but further changes of the G0/G1 and G2/M were later observed. Thus, cell cycle arrest in the diapause embryo is not an immediate event, but it takes longer to reach the steady state. We further elucidated molecular events possibly involved in diapause preparation and entry. Downregulation of Proliferating cellular antigen (PCNA; a cell cycle regulator), caudal and pumilio (cad and pum; early segmentation genes) as well as P-element induced wimpy testis (piwi) (a small silencing RNA) prior to the onset of developmental arrest was notable. The downregulation of PCNA, cad and pum continued even after entry into developmental arrest. In contrast to upregulation in non-diapause eggs, Cyclin D (another cell cycle regulator) and hunchback, Krüppel, and runt (gap and pair-rule genes) were downregulated in diapause eggs. These molecular events may contribute to embryonic diapause of D. nigrofasciatus.


Assuntos
Blastoderma/embriologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Gryllidae/embriologia , RNA Interferente Pequeno/biossíntese , Fase de Repouso do Ciclo Celular/fisiologia , Animais , Gryllidae/genética , RNA Interferente Pequeno/genética
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