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1.
Gene ; 766: 145134, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32898605

RESUMO

BACKGROUND: Artesunate (ART) has been used extensively as anti-malarial drugs worldwide. Besides, it has also been reported to have anti-cancer activities. This study was aimed to explore the anti-cancer activity of ART in combination with cisplatin (CIS) on A549 cells. METHODS: Cells were cultured with different concentrations of ART and/or CIS for 24, 48, or 72 h to test the anti-proliferative effects by CCK-8 assay. Colony formation assay and EdU staining were also performed. TUNEL staining was used to illustrate the morphologic changes. Cell cycle and apoptosis were determined by flow cytometry assay, and Western blot analysis was conducted to detect the expression of apoptosis- and proliferation-related proteins. Caspase activities were determined by colorimetric assay kit. Moreover, the synergistic effect of ART with CIS in A549 cell xenograft model was also determined. RESULTS: ART significantly inhibited cell proliferation in dose- and time-dependent manners. Collectively, the combination treatment remarkably decreased colony formation rates and increased the rates of TUNEL-positive cells compared with mono-treatment. Mechanistically, the combination treatment influenced the expression of Bcl-2, Bax, p-P53, Caspase-3/7, Caspase-9, CyclinB1, P34, P21, and synergistically regulated the activity of P38/JNK/ERK1/2 MAPK pathway. In mice A549 xenograft tumors, the combination strategy significantly increased the anti-cancer efficacy of ART and CIS alone, consistent with the in vitro observations. CONCLUSIONS: ART exhibited significant anti-tumor effect on A549 cells and this efficiency could be enhanced by combination with CIS.


Assuntos
Antineoplásicos/farmacologia , Artesunato/farmacologia , Cisplatino/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células A549 , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia
2.
Nat Commun ; 11(1): 5466, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33122719

RESUMO

Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure-function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Nucleares , Sítios de Ligação , Ciclo Celular , Cristalografia por Raios X , DNA/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Ligação Proteica , Domínios Proteicos/fisiologia , Pontos de Checagem da Fase S do Ciclo Celular , Espectrometria de Fluorescência , Fatores de Transcrição/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/metabolismo
3.
Anticancer Res ; 40(11): 6051-6062, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109543

RESUMO

BACKGROUND/AIM: Chemoresistance is a major obstacle in the treatment of prostate cancer (PCa). It is imperative to develop novel strategies for overcoming chemoresistance and improving clinical outcomes. We evaluated the in vitro activity and mechanism of action of dihydroergocristine (DHECS), an ergot alkaloid approved for the treatment of dementia, in PCa cells. MATERIALS AND METHODS: The in vitro effects of DHECS on PCa cell cycle and viability were determined by flow cytometry and colorimetric assay. The effects of DHECS on PCa cell signaling were evaluated by quantitative PCR, western blot analysis and reporter assay. RESULTS: DHECS was effective in inducing cell cycle arrest and apoptosis in human PCa cells. Of particular interest, DHECS demonstrated high potency against chemoresistant PCa cells. At the molecular level, DHECS affected multiple factors implicated in the regulation of cancer cell cycle and programmed cell death, including p53, mouse double minute 2 homolog (MDM2), retinoblastoma protein (RB), p21, E2F transcription factor 1 (E2F1), survivin, myeloid cell leukemia 1 (Mcl-1) and poly ADP ribose polymerase (PARP). Furthermore, DHECS may function through dopamine receptor-mediated effects on 5'-AMP-activated protein kinase (AMPK) and nuclear factor kappa B (NF-ĸB). CONCLUSION: DHECS has the potential to be repurposed as a novel anticancer agent for the management of chemoresistant PCa.


Assuntos
Di-Hidroergocristina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias da Próstata/patologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Masculino , Neoplasias da Próstata/genética , Receptores Dopaminérgicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Survivina/genética , Survivina/metabolismo , Transcrição Genética/efeitos dos fármacos
4.
Anticancer Res ; 40(11): 6151-6158, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33109552

RESUMO

BACKGROUND/AIM: Glioma is the most malignant tumour of the human brain still lacking effective treatment modalities. Betulin, a pentacyclic triterpene abundantly found in the birch bark, has been shown to demonstrate interesting anti-cancer activity towards many cancer cells. We determined the effects of acetylenic synthetic betulin derivatives (ASBDs) as anti-tumour agents on glioma cells in vitro. MATERIALS AND METHODS: T98G and C6 glioma cell viability and proliferation were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and BrdU (bromo deoxyuridine) test, respectively. Cell-cycle progression and induction of apoptosis were investigated with flow cytometry. RESULTS: ASBDs significantly decreased glioma cell viability/survival and inhibited proliferation in a dose-dependent manner in vitro. Moreover, ASBDs were more cytotoxic than clinically used chemotherapeutics - temozolomide and cisplatin. CONCLUSION: ASBDs may be considered for further study as potent anti-tumour agents in glioma treatment.


Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Triterpenos/farmacologia , Acetileno/química , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Humanos , Ratos , Temozolomida/farmacologia , Triterpenos/química
5.
Nucleic Acids Res ; 48(19): e111, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33010172

RESUMO

Ionizing radiation (IR) is environmentally prevalent and, depending on dose and linear energy transfer (LET), can elicit serious health effects by damaging DNA. Relative to low LET photon radiation (X-rays, gamma rays), higher LET particle radiation produces more disease causing, complex DNA damage that is substantially more challenging to resolve quickly or accurately. Despite the majority of human lifetime IR exposure involving long-term, repetitive, low doses of high LET alpha particles (e.g. radon gas inhalation), technological limitations to deliver alpha particles in the laboratory conveniently, repeatedly, over a prolonged period, in low doses and in an affordable, high-throughput manner have constrained DNA damage and repair research on this topic. To resolve this, we developed an inexpensive, high capacity, 96-well plate-compatible alpha particle irradiator capable of delivering adjustable, low mGy/s particle radiation doses in multiple model systems and on the benchtop of a standard laboratory. The system enables monitoring alpha particle effects on DNA damage repair and signalling, genome stability pathways, oxidative stress, cell cycle phase distribution, cell viability and clonogenic survival using numerous microscopy-based and physical techniques. Most importantly, this method is foundational for high-throughput genetic screening and small molecule testing in mammalian and yeast cells.


Assuntos
Partículas alfa/efeitos adversos , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Radiogenética/instrumentação , Células A549 , Ciclo Celular/efeitos da radiação , Células HeLa , Humanos , Estresse Oxidativo/efeitos da radiação , Saccharomyces cerevisiae , Transdução de Sinais/efeitos da radiação
6.
Cell Prolif ; 53(11): e12920, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33029857

RESUMO

OBJECTIVES: The level of histone H3 lysine 79 methylation is regulated by the cell cycle and involved in cell proliferation. KDM2B is an H3K79 demethylase. Proliferating cell nuclear antigen (PCNA) is a component of the DNA replication machinery. This study aimed at elucidating a molecular link between H3K79me recognition of PCNA and cell cycle control. MATERIALS AND METHODS: We generated KDM2B-depleted 293T cells and histone H3-K79R mutant-expressing 293T cells. Western blots were primarily utilized to examine the H3K79me level and its effect on subsequent PCNA dissociation from chromatin. We applied IP, peptide pull-down, isothermal titration calorimetry (ITC) and ChIP experiments to show the PCNA binding towards methylated H3K79 and DNA replication origins. Flow cytometry, MTT, iPOND and DNA fibre assays were used to assess the necessity of KDM2B for DNA replication and cell proliferation. RESULTS: We revealed that KDM2B-mediated H3K79 demethylation regulated cell cycle progression. We found that PCNA bound chromatin in an H3K79me-dependent manner during S phase. KDM2B was responsible for the timely dissociation of PCNA from chromatin, allowing to efficient DNA replication. Depletion of KDM2B aberrantly enriched chromatin with PCNA and caused slow dissociation of residual PCNA, leading to a negative effect on cell proliferation. CONCLUSIONS: We suggested a novel interaction between PCNA and H3K79me. Thus, our findings provide a new mechanism of KDM2B in regulation of DNA replication and cell proliferation.


Assuntos
Replicação do DNA , Proteínas F-Box/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ciclo Celular , Proliferação de Células , Cromatina , Desmetilação , Células HEK293 , Humanos , Fase S
7.
Nat Commun ; 11(1): 5239, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067475

RESUMO

The alternative non-homologous end-joining (NHEJ) pathway promotes DNA double-strand break (DSB) repair in cells deficient for NHEJ or homologous recombination, suggesting that it operates at all stages of the cell cycle. Here, we use an approach in which DNA breaks can be induced in G1 cells and their repair tracked, enabling us to show that joining of DSBs is not functional in G1-arrested XRCC4-deficient cells. Cell cycle entry into S-G2/M restores DSB repair by Pol θ-dependent and PARP1-independent alternative NHEJ with repair products bearing kilo-base long DNA end resection, micro-homologies and chromosome translocations. We identify a synthetic lethal interaction between XRCC4 and Pol θ under conditions of G1 DSBs, associated with accumulation of unresolved DNA ends in S-G2/M. Collectively, our results support the conclusion that the repair of G1 DSBs progressing to S-G2/M by alternative NHEJ drives genomic instability and represent an attractive target for future DNA repair-based cancer therapies.


Assuntos
Ciclo Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fase G1 , Camundongos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo
8.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 1432-1435, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018259

RESUMO

The progression of cells through the cell cycle is a tightly regulated process and is known to be key in maintaining normal tissue architecture and function. Disruption of these orchestrated phases will result in alterations that can lead to many diseases including cancer. Regrettably, reliable automatic tools to evaluate the cell cycle stage of individual cells are still lacking, in particular at interphase. Therefore, the development of new tools for a proper classification are urgently needed and will be of critical importance for cancer prognosis and predictive therapeutic purposes. Thus, in this work, we aimed to investigate three deep learning approaches for interphase cell cycle staging in microscopy images: 1) joint detection and cell cycle classification of nuclei patches; 2) detection of cell nuclei patches followed by classification of the cycle stage; 3) detection and segmentation of cell nuclei followed by classification of cell cycle staging. Our methods were applied to a dataset of microscopy images of nuclei stained with DAPI. The best results (0.908 F1-Score) were obtained with approach 3 in which the segmentation step allows for an intensity normalization that takes into account the intensities of all nuclei in a given image. These results show that for a correct cell cycle staging it is important to consider the relative intensities of the nuclei. Herein, we have developed a new deep learning method for interphase cell cycle staging at single cell level with potential implications in cancer prognosis and therapeutic strategies.


Assuntos
Núcleo Celular , Aprendizado Profundo , Ciclo Celular , Divisão Celular , Interfase
9.
Folia Biol (Praha) ; 66(3): 91-103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33069188

RESUMO

The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.


Assuntos
Candida albicans/fisiologia , Proteínas Fúngicas/fisiologia , Genes Fúngicos , Acetiltransferases/deficiência , Acetiltransferases/genética , Acetiltransferases/fisiologia , Antifúngicos/farmacologia , Sistemas CRISPR-Cas , Cálcio/fisiologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/patogenicidade , Cátions/farmacologia , Adesão Celular , Ciclo Celular , Parede Celular/efeitos dos fármacos , Quitinases/farmacologia , Dano ao DNA , Proteínas Fúngicas/genética , Deleção de Genes , Glucana Endo-1,3-beta-D-Glucosidase/farmacologia , Hifas/crescimento & desenvolvimento , Isocitrato Desidrogenase/deficiência , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/fisiologia , Fases de Leitura Aberta , Reprodução Assexuada , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/fisiologia , Virulência/genética
10.
Wei Sheng Yan Jiu ; 49(5): 780-784, 2020 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-33070824

RESUMO

OBJECTIVE: Investigate the estrogen receptor expression in human thyroid squamous cell carcinoma SW579 and the effects of genistein on the apoptosis and cycle of SW579 and its mechnism. METHODS: The real-time PCR was applied to detect the expression of estrogen receptor(ER)α、ERß and G protein-coupled receptor(GPR)30 in human thyroid squamous cell line SW579; MTT was used to test the effect of genistein on cell proliferation in the SW579 cells before and after blocking GPR30; flow cytometry was explorited to measure the effect of genistein on the cell cycle and apoptosis in the SW579 was detected before and after blocking GPR30. RESULTS: The high concentration of genistein promoted the expression of ERß and GPR30 in the SW579 cells, but ERα was not expressed. The specific blocking of GPR30, the cell proliferation was aboviously inhibited by genistein in the SW579 cells and the cell apoptosis was markedly promoted after the GPR30 was blocked; The cell cycle was mainly blocked in G_2/M phase. CONCLUSION: Genistein can obiviously promote the cell proliferation in the SW579 cells, which may be related to the action of GPR30.


Assuntos
Genisteína , Neoplasias da Glândula Tireoide , Apoptose , Ciclo Celular , Divisão Celular , Linhagem Celular Tumoral , Células Epiteliais , Genisteína/farmacologia , Humanos
11.
Nat Commun ; 11(1): 5007, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024116

RESUMO

p50, the mature product of NFKB1, is constitutively produced from its precursor, p105. Here, we identify BARD1 as a p50-interacting factor. p50 directly associates with the BARD1 BRCT domains via a C-terminal phospho-serine motif. This interaction is induced by ATR and results in mono-ubiquitination of p50 by the BARD1/BRCA1 complex. During the cell cycle, p50 is mono-ubiquitinated in S phase and loss of this post-translational modification increases S phase progression and chromosomal breakage. Genome-wide studies reveal a substantial decrease in p50 chromatin enrichment in S phase and Cycln E is identified as a factor regulated by p50 during the G1 to S transition. Functionally, interaction with BARD1 promotes p50 protein stability and consistent with this, in human cancer specimens, low nuclear BARD1 protein strongly correlates with low nuclear p50. These data indicate that p50 mono-ubiquitination by BARD1/BRCA1 during the cell cycle regulates S phase progression to maintain genome integrity.


Assuntos
Neoplasias da Mama/metabolismo , Ciclo Celular/fisiologia , Instabilidade Genômica , Subunidade p50 de NF-kappa B/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sítios de Ligação , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Feminino , Fibroblastos , Humanos , Lisina/metabolismo , Camundongos , Subunidade p50 de NF-kappa B/genética , Neuroblastoma/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Serina/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
12.
Zhonghua Gan Zang Bing Za Zhi ; 28(10): 861-867, 2020 Oct 20.
Artigo em Chinês | MEDLINE | ID: mdl-33105932

RESUMO

Objective: Aldo-keto reductase family 1 member B10 (AKR1B10) pathogenesis, early diagnosis and prognosis are closely related with hepatoma. Therefore, this study explores the effect and mechanism of AKR1B10 on cell cycle in hepatoma cells. Methods: HepG2 cells were infected with lentivirus LV-AKR1B10-shRNA or treated with epalrestat, an AKR1B10 inhibitor. The expression level of AKR1B10 was detected by Western blot assay and real-time fluorescence quantitative PCR (RT-qPCR). Decreased AKR1B10 activity was detected by reduced coenzyme II (NADPH) absorbance at 340 nm. The low expression of AKR1B10 and the effect of different concentrations of epalrestat on cell proliferation and cell cycle were detected by CCK-8 method and flow cytometry. The protein expression levels of p-rb, cyclin D1, E1, p27 in HepG2 cells were detected by Western blot. The mean of the two samples was tested using independent sample t-test. Results: AKR1B10 expression level in hepatoma cells was significantly increased compared to normal liver cells, and the relative expression level of AKR1B10 protein in HepG2 cells was 6.71 ± 1.11 (P = 0.012). Epalrestat was significantly inhibited with the enzymatic activity of AKR1B10 in a dose-dependent manner. AKR1B10 gene in HepG2 cells was effectively silenced. HepG2 cells treated with different concentrations of epalrestat (AKR1B10 inhibitor) for 24, 48 and 72 h had inhibited cell proliferation, promoted G0/G1 cell cycle arrest, reduced the expression of p-Rb, cyclin D1, and cyclin E1 and increased the expression of cyclin dependent kinase inhibitor p27 expression. Conclusion: AKR1B10 inhibitory expression and activity can promote G0/G1 cell cycle arrest in HepG2 cells through the p27 / p-Rb pathway.


Assuntos
Aldo-Ceto Redutases/metabolismo , Carcinoma Hepatocelular/metabolismo , Ciclo Celular , Neoplasias Hepáticas/metabolismo , Transdução de Sinais , Aldo-Ceto Redutases/genética , Carcinoma Hepatocelular/genética , Pontos de Checagem do Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inativação Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Proteína do Retinoblastoma
13.
An Acad Bras Cienc ; 92(suppl 2): e20181010, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33084751

RESUMO

Although the effects of nickel chloride (NiCl2) on the immune system have long been recognized, little is known about the effects of nickel (II) on the cell cycle and related signaling events in immune organs, such as cecal tonsil, a key immune organ of chicken. In the present study, we investigated the effect of NiCl2 on the cell cycle of cecal tonsil. The cell cycle was detected by the methods of flow cytometry (FCM), quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). The results showed that dietary NiCl2 in excess of 300 mg/kg caused the G2/M cell cycle arrest and the reduction of cell proportion at S phase of the cecal tonsil. The G2/M cell cycle arrest was accompanied by the up-regulation of p53, p21 protein expression and mRNA expression, and down-regulation of cyclinB and proliferating cell nuclear antigen (PCNA) protein expression and mRNA expression. The data suggested that the cells' (mainly the T lymphocytes) proliferation in the cecal tonsil was inhibited by the high dietary NiCl2.


Assuntos
Galinhas , Tonsila Palatina , Animais , Ceco , Ciclo Celular , Transdução de Sinais
14.
PLoS Genet ; 16(9): e1008744, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32956370

RESUMO

Qsp1 is a secreted quorum sensing peptide required for virulence of the fungal meningitis pathogen Cryptococcus neoformans. Qsp1 functions to control cell wall integrity in vegetatively growing cells and also functions in mating. Rather than acting on a cell surface receptor, Qsp1 is imported to act intracellularly via the predicted oligopeptide transporter Opt1. Here, we identify a transcription factor network as a target of Qsp1. Using whole-genome chromatin immunoprecipitation, we find Qsp1 controls the genomic associations of three transcription factors to genes whose outputs are regulated by Qsp1. One of these transcription factors, Cqs2, is also required for the action of Qsp1 during mating, indicating that it might be a shared proximal target of Qsp1. Consistent with this hypothesis, deletion of CQS2 impacts the binding of other network transcription factors specifically to Qsp1-regulated genes. These genetic and genomic studies illuminate mechanisms by which an imported peptide acts to modulate eukaryotic gene expression.


Assuntos
Cryptococcus neoformans/genética , Percepção de Quorum/genética , Fatores de Transcrição/genética , Ciclo Celular/genética , Parede Celular/metabolismo , Criptococose/microbiologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Genômica , Meningite Fúngica/genética , Peptídeos/genética , Fatores de Transcrição/metabolismo , Virulência/genética , Fatores de Virulência/genética
15.
PLoS Genet ; 16(9): e1009011, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32986715

RESUMO

Neuronal precursor cells undergo self-renewing and non-self-renewing asymmetric divisions to generate a large number of neurons of distinct identities. In Drosophila, primary precursor neuroblasts undergo a varying number of self-renewing asymmetric divisions, with one known exception, the MP2 lineage, which undergoes just one terminal asymmetric division similar to the secondary precursor cells. The mechanism and the genes that regulate the transition from self-renewing to non-self-renewing asymmetric division or the number of times a precursor divides is unknown. Here, we show that the T-box transcription factor, Midline (Mid), couples these events. We find that in mid loss of function mutants, MP2 undergoes additional self-renewing asymmetric divisions, the identity of progeny neurons generated dependent upon Numb localization in the parent MP2. MP2 expresses Mid transiently and an over-expression of mid in MP2 can block its division. The mechanism which directs the self-renewing asymmetric division of MP2 in mid involves an upregulation of Cyclin E. Our results indicate that Mid inhibits cyclin E gene expression by binding to a variant Mid-binding site in the cyclin E promoter and represses its expression without entirely abolishing it. Consistent with this, over-expression of cyclin E in MP2 causes its multiple self-renewing asymmetric division. These results reveal a Mid-regulated pathway that restricts the self-renewing asymmetric division potential of cells via inhibiting cyclin E and facilitating their exit from cell cycle.


Assuntos
Divisão Celular/genética , Sistema Nervoso Central/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Ciclo Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Hormônios Juvenis/genética , Hormônios Juvenis/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Proteínas com Domínio T/genética , Fatores de Transcrição/genética
16.
Int J Nanomedicine ; 15: 6201-6209, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32884270

RESUMO

Background: Unique properties of graphene and its derivatives make them attractive in the field of nanomedicine. However, the mass application of graphene might lead to side effects, which has not been properly addressed in previous studies, especially with regard to its effect on the cell cycle. Methods: The effect of two concentrations (100 and 200 µg/mL) of nano- and microsized graphene oxide (nGO and mGO) on apoptosis, cell cycle, and ROS generation was studied. The effect of both sizes on viability and genotoxicity of the embryonic fibroblast cell cycle was evaluated. MTT and flow cytometry were applied to evaluate the effects of graphene oxide (GO) nanosheets on viability of cells. Apoptosis and cell cycle were analyzed by flow cytometry. Results: The results of this study showed that GO disturbed the cell cycle and nGO impaired cell viability by inducing cell apoptosis. Interestingly, both nGO and mGO blocked the cell cycle in the S phase, which is a critical phase of the cell cycle. Upregulation of TP53-gene transcripts was also detected in both nGO- and mGO-treated cells compared to the control, especially at 200 µg/mL. DNA content of the treated cells increased; however, because of DNA degradation, its quality was decreased. Conclusion: In conclusion, graphene oxide at both nano- and micro-scale damages cell physiology and increases cell population in the S phase of the cell cycle.


Assuntos
Ciclo Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Grafite/farmacologia , Nanoestruturas/toxicidade , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Grafite/toxicidade , Camundongos , Testes de Mutagenicidade , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genética
17.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 36(3): 193-196, 2020 May.
Artigo em Chinês | MEDLINE | ID: mdl-32981270

RESUMO

Objective: To investigate the protective effect of spermine (Sp) on diabetic cardiomyopathy (DCM) and high glucose-induced cardiac fibroblasts (CFs), and to explore its mechanism.Methods: ①Animal experiments: 24 male Wistar rats were randomly divided into control group, type 1 diabetes group (TID) and spermine group (TID+Sp, each group n=8). TID rats were induced by streptozocin (STZ, 60 mg/kg), and TID+Sp rat were pretreated with spermine (Sp, 5 mg/(kg·d)) for 2 weeks before STZ injection. After 12 weeks of modeling, blood glucose, insulin levels, ejection fraction (EF) and shortening fraction (FS) were measured, and Masson staining and Sirius red staining were performed in the rat cardiac tissues. ②Cell experiments: primary CFs were extracted from newborn (1-3 d) Wistar rat hearts, and were randomly divided into control group, high-glucose group (HG) and HG+Sp group (n=6 per group). HG group was treated with 40 mmol/L glucose, and the HG+Sp group was pretreated with 5 µmol/L Sp for 30 min before HG treatment. The cell viability of CFs was detected by CCK8, the content of collagen in culture medium was analyzed by ELISA, and protein expressions of cell cycle related proteins (PCNA, CyclinD1 and P27) were detected by Western blot. Results: Compared with control group, the blood glucose and collagen content were increased, and the insulin level and heart function were decreased in the T1D group. Meanwhile, HG induced an increasing of the cell viability, the collagen content in the medium and the expressions of PCNA and CyclinD1, while the expression of P27 was down-regulated. Spermine could reduce the above changes, manifested as improving the cardiac function, regulating the expression of cyclin and reducing the level of myocardial fibrosis. Conclusion: Spermine can alleviate myocardial fibrosis in diabetic cardiomyopathy, which mechanism is related to the regulation of cell cycle.


Assuntos
Ciclo Celular , Complicações do Diabetes , Cardiomiopatias Diabéticas , Fibrose , Espermina , Animais , Glicemia/análise , Glicemia/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Complicações do Diabetes/induzido quimicamente , Complicações do Diabetes/tratamento farmacológico , Cardiomiopatias Diabéticas/induzido quimicamente , Cardiomiopatias Diabéticas/complicações , Cardiomiopatias Diabéticas/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Fibrose/tratamento farmacológico , Fibrose/etiologia , Glucose/toxicidade , Coração/efeitos dos fármacos , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar , Espermina/farmacologia , Espermina/uso terapêutico
18.
J Environ Pathol Toxicol Oncol ; 39(3): 281-290, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32865918

RESUMO

Objective-To investigate cystathionine ß synthase (CBS)/hydrogen sulfide (H2S) signaling in multiple myeloma (MM) patients and to identify its effect on the proliferation of U266 cells. Methods-Bone marrow samples of 19 MM patients and 23 healthy donors were collected. qRT-PCR was performed to measure the mRNA expression levels of H2S synthases, cystathionine ß synthase, and cystathionine γ lyase. ELISA assays quantified the amount of H2S produced by the two enzymes CBS and CSE. CCK-8 experiment was used to investigate the influence of the CBS inhibitor amino oxyacetic acid and the CSE inhibitor propargylglycine on the proliferation of U266 cells. Flow cytometry and western blotting were performed to determine the effects of AOAA, PAG, and NaHS on cell cycle distribution as well as Caspase-3 and Bcl-2 expression. Results-Patients with MM had higher level of CBS compared with healthy donors. AOAA significantly inhibited cell proliferation in both a time and concentration dependent characteristic, whereas PAG does not. After 24 hours of treatment, AOAA significantly elevated the G0/G1 phase proportion of cells, and reduced the cell distribution in both S and G2/M phases, while NaHS accelerated cell cycle progression by reducing the relative number of cells in G0/G1 phase and increasing the proportion of cells in the G2/M phase. Moreover, AOAA abolished the impact of NaHS on cell cycle progression of U266 cells. AOAA treatment also led to a significant decrease in Bcl-2 expression and dramatic increase in Caspase-3 expression, though NaHS reversed these effects. Conclusion-CBS/H2S system might have a certain effect on the proliferation and apoptosis of MM cells.


Assuntos
Apoptose , Proliferação de Células , Cistationina beta-Sintase/metabolismo , Sulfeto de Hidrogênio/metabolismo , Mieloma Múltiplo/metabolismo , Adulto , Idoso , Alquinos/farmacologia , Ácido Amino-Oxiacético/farmacologia , Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Estudos de Casos e Controles , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Transdução de Sinais
19.
Zhonghua Zhong Liu Za Zhi ; 42(8): 629-634, 2020 Aug 23.
Artigo em Chinês | MEDLINE | ID: mdl-32867453

RESUMO

Objective: To investigate the effect of esculin on the proliferation of triple negative breast cancer cells and its molecular mechanism. Methods: MDA-MB-231 cells were treated with 28, 56, 112, 225, 450 and 900 µmol/L of esculin for 24, 48 and 72 h, respectively, and the cell viability was detected by cell counting kit 8 (CCK-8) assay. In addition, MDA-MB-231 cells were treated with 0, 225, 450 and 900 µmol/L of esculin for 48 h. And then the changes in cell morphology were observed by inverted microscope. The clone-forming ability was detected by colony formation assay. The mRNA expression levels of FBI-1, p53 and p21 were detected using real-time fluorescence quantitative polymerase chain reaction. The protein expression levels of FBI-1, p53, p21 and Ki67 were detected by western blot. Results: Compared with the blank control group, the cell viability of MDA-MB-231 cells that treated with esculin significantly decreased in a dose-dependent and time-dependent manners. After treatment with esculin, MDA-MB-231 cells shrunk, flattened, adhered poorly to the culture dish and the cell spacing became larger. Meanwhile, shedding and incomplete cells appeared, of which 900 µmol/L of esculin treatment group showed the most dramatic changes. In addition, the colony formation ratios were decreased to (77.18±5.13)%, (65.94±4.98)% and (45.92±3.70)% in the 225, 450 and 900 µmol/L of esculin treatment groups compared with blank control, respectively (P<0.01). Furthermore, the mRNA and protein expressions of FBI-1 increased, while the levels of p53 and p21 mRNA and protein, as well as the protein expression of Ki67 decreased in a concentration-dependent manner (P<0.01). Conclusion: Esculin may regulate cell cycle-related p53-p21 pathway via FBI-1 mediated DNA replication, thus inhibit the proliferation of triple negative breast cancer cells.


Assuntos
Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Esculina/farmacologia , RNA Mensageiro/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias da Mama/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Fatores de Transcrição , Neoplasias de Mama Triplo Negativas/patologia
20.
PLoS Biol ; 18(9): e3000852, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32931487

RESUMO

Olfaction in most animals is mediated by neurons bearing cilia that are accessible to the environment. Olfactory sensory neurons (OSNs) in chordates usually have multiple cilia, each with a centriole at its base. OSNs differentiate from stem cells in the olfactory epithelium, and how the epithelium generates cells with many centrioles is not yet understood. We show that centrioles are amplified via centriole rosette formation in both embryonic development and turnover of the olfactory epithelium in adult mice, and rosette-bearing cells often have free centrioles in addition. Cells with amplified centrioles can go on to divide, with centrioles clustered at each pole. Additionally, we found that centrioles are amplified in immediate neuronal precursors (INPs) concomitant with elevation of mRNA for polo-like kinase 4 (Plk4) and SCL/Tal1-interrupting locus gene (Stil), key regulators of centriole duplication. These results support a model in which centriole amplification occurs during a transient state characterized by elevated Plk4 and Stil in early INP cells. These cells then go on to divide at least once to become OSNs, demonstrating that cell division with amplified centrioles, known to be tolerated in disease states, can occur as part of a normal developmental program.


Assuntos
Divisão Celular/fisiologia , Centríolos/fisiologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Envelhecimento/fisiologia , Animais , Ciclo Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos , Desenvolvimento Embrionário/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Córtex Olfatório/citologia , Córtex Olfatório/embriologia , Mucosa Olfatória/citologia , Mucosa Olfatória/embriologia , Mucosa Olfatória/ultraestrutura , Neurônios Receptores Olfatórios/citologia , Neurônios Receptores Olfatórios/ultraestrutura
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