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1.
Gene ; 725: 144159, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31629818

RESUMO

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide due to its frequent metastasis, tumor recurrence, and lack of curative treatment. However, the underlying molecular mechanisms involved in HCC progression remain unclear. Here, we analyzed the global gene expression of spontaneous liver tumor tissue from CBA/CaJ mice by RNA-Seq and identified 10,706 and 10,374 genes in the normal and liver tumor groups, respectively. Only 9793 genes were expressed in both, 913 genes were identified in only the liver tumor group, and 581 genes were found in normal liver tissues. There were 2054 differentially expressed genes (DEGs), with 975 down-regulated genes and 1079 up-regulated genes. Gene ontology (GO) term enrichment analysis showed that 43 up-regulated genes were significantly associated with cell cycle regulation and hundreds of up-regulated genes were related to cell migration, adhesion, or metabolic processes. KEGG pathway enrichment also demonstrated that some DEGs were tightly associated with the cell cycle, extracellular matrix (ECM)-receptor interactions, as well as protein digestion and absorption pathways, indicating that the activation of these oncogenic cascades was closely related to tumor liver progression in CBA/CaJ mice. Ninety-three genes with elevated expression levels preferentially localized in microtubules, kinetochores, and spindles play an important role during mitosis and meiosis and are associated with the reorganization of the cytoskeleton in cancer cells during migration and invasion. Some ECM-related genes were significantly different in the tumor group, including collagen types I, III, IV, V, and VI, non-collagenous glycoproteins, laminin, and fibronectin. We further validated the functions of upregulated genes, such as cyclin-dependent kinase 1 (CDK1) and polo-like kinase 1 (PLK1), with regards to cell cycle regulation, apoptosis, and proliferation in normal human liver or liver tumor-derived cell lines. Our results indicated that the cell cycle dysregulation, ECM-receptor interaction, and cytoskeleton-associated genes in mouse livers may promote HCC progression and deciphering the function of the genes will help investigators understand the underlying molecular mechanism of HCC.


Assuntos
Neoplasias Hepáticas Experimentais/genética , Animais , Apoptose/genética , Proteína Quinase CDC2/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos CBA , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transcrição Genética , Transcriptoma
2.
EMBO J ; 38(19): e101704, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31429971

RESUMO

The TRAnsport Protein Particle (TRAPP) complex controls multiple membrane trafficking steps and is strategically positioned to mediate cell adaptation to diverse environmental conditions, including acute stress. We have identified the TRAPP complex as a component of a branch of the integrated stress response that impinges on the early secretory pathway. The TRAPP complex associates with and drives the recruitment of the COPII coat to stress granules (SGs) leading to vesiculation of the Golgi complex and arrest of ER export. The relocation of the TRAPP complex and COPII to SGs only occurs in cycling cells and is CDK1/2-dependent, being driven by the interaction of TRAPP with hnRNPK, a CDK substrate that associates with SGs when phosphorylated. In addition, CDK1/2 inhibition impairs TRAPP complex/COPII relocation to SGs while stabilizing them at ER exit sites. Importantly, the TRAPP complex controls the maturation of SGs. SGs that assemble in TRAPP-depleted cells are smaller and are no longer able to recruit RACK1 and Raptor, two TRAPP-interactive signaling proteins, sensitizing cells to stress-induced apoptosis.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Estresse Fisiológico , Animais , Proteína Quinase CDC2/metabolismo , Linhagem Celular , Quinase 2 Dependente de Ciclina/metabolismo , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Ratos
3.
Genes (Basel) ; 10(8)2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31430963

RESUMO

Recently, we reported a novel therapeutic probiotic-derived protein, p8, which has anti-colorectal cancer (anti-CRC) properties. In vitro experiments using a CRC cell line (DLD-1), anti-proliferation activity (about 20%) did not improve after increasing the dose of recombinant-p8 (r-p8) to >10 µM. Here, we show that this was due to the low penetrative efficiency of r-p8 exogenous treatment. Furthermore, we found that r-p8 entered the cytosol through endocytosis, which might be a reason for the low penetration efficiency. Therefore, to improve the therapeutic efficacy of p8, we tried to improve delivery to CRC cells. This resulted in endogenous expression of p8 and increased the anti-proliferative effects by up to 2-fold compared with the exogenous treatment (40 µM). Anti-migration activity also increased markedly. Furthermore, we found that the anti-proliferation activity of p8 was mediated by inhibition of the p53-p21-Cyclin B1/Cdk1 signal pathway, resulting in growth arrest at the G2 phase of the cell cycle. Taken together, these results suggest that p8 is toxic to cancer cells, shows stable expression within cells, and shows strong cancer suppressive activity by inducing cell cycle arrest. Therefore, p8 is a strong candidate for gene therapy if it can be loaded onto cancer-specific viruses.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Bactérias/farmacologia , Neoplasias Colorretais/metabolismo , Lactobacillus rhamnosus/metabolismo , Probióticos/metabolismo , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina B1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Endocitose , Fase G2 , Humanos , Lactobacillus rhamnosus/química , Probióticos/química , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
4.
Biomed Pharmacother ; 117: 109076, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31203132

RESUMO

PURPOSE: Colorectal cancer (CRC) caused more than 65,000 mortalities worldwide per year. It is a result of one or a combination of chromosomal instability, CpG island methylator phenotype, and microsatellite instability. SNRPA1 (small nuclear ribonucleoprotein polypeptide A) is a subunit of spliceosome complex that is involved in the RNA processing. Overexpression of SNRPA1 has been implicated in a variety of cancers including CRC. Besides from its role in mediating the RNA processing, the other aspects regarding its function in the progression of colorectal cancer have not been revealed. METHODS: Herein, we combined regular gene overexpression or knock down in vitro and in vivo and microarray gene profiling analysis to decipher the unknow regulatory role of SNRPA1 in CRC. RESULTS: We found SNRPA1 widely expression in many representative CRC cell lines. Knocking down expression of SNRPA1 by shRNA lentivirus inhibited the cell proliferation in vitro and impaired tumor formation from implanted CRC cells transduced with SNRPA1 silencing shRNA lentivirus in nude mice. It also promoted the cell apoptosis by upregulating the caspase 3/7 activity. Additional microarray gene profiling analysis uncovered the gene interaction network of SNRPA1, special focus was placed on its association with tumor suppressor or oncogenes. CONCLUSIONS: According to the results of gene interaction network as well as qRT-PCR verification, it revealed that SNPRA1 regulates PIK3R1, VEGFC, MKI67, CDK1 in CRC. These novel findings identified new roles played by SNRPA1 in the progression of CRC and it may become a potential therapeutic target in the treatment of CRC.


Assuntos
Proteína Quinase CDC2/metabolismo , Carcinogênese/genética , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Neoplasias Colorretais/genética , Genes Neoplásicos , Antígeno Ki-67/metabolismo , Ribonucleoproteína Nuclear Pequena U1/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose/genética , Proteína Quinase CDC2/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mapas de Interação de Proteínas
5.
Cells ; 8(6)2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31174389

RESUMO

Williams-Beuren syndrome (WBS) is caused by microdeletions of 28 genes and is characterized by cognitive disorder and hypotrophic corpus callosum (CC). Nsun5 gene, which encodes cytosine-5 RNA methyltransferase, is located in the deletion loci of WBS. We have reported that single-gene knockout of Nsun5 (Nsun5-KO) in mice impairs spatial cognition. Herein, we report that postnatal day (PND) 60 Nsun5-KO mice showed the volumetric reduction of CC with a decline in the number of myelinated axons and loose myelin sheath. Nsun5 was highly expressed in callosal oligodendrocyte precursor cells (OPCs) and oligodendrocytes (OLs) from PND7 to PND28. The numbers of OPCs and OLs in CC of PND7-28 Nsun5-KO mice were significantly reduced compared to wild-type littermates. Immunohistochemistry and Western blot analyses of myelin basic protein (MBP) showed the hypomyelination in the CC of PND28 Nsun5-KO mice. The Nsun5 deletion suppressed the proliferation of OPCs but did not affect transition of radial glial cells into OPCs or cell cycle exit of OPCs. The protein levels, rather than transcriptional levels, of CDK1, CDK2 and Cdc42 in the CC of PND7 and PND14 Nsun5-KO mice were reduced. These findings point to the involvement of Nsun5 deletion in agenesis of CC observed in WBS.


Assuntos
Corpo Caloso/metabolismo , Metiltransferases/genética , Bainha de Mielina/metabolismo , Agenesia do Corpo Caloso/metabolismo , Agenesia do Corpo Caloso/patologia , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proliferação de Células , Corpo Caloso/crescimento & desenvolvimento , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Metiltransferases/deficiência , Camundongos , Camundongos Knockout , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Síndrome de Williams/metabolismo , Síndrome de Williams/patologia
6.
Med Sci Monit ; 25: 4059-4067, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31150370

RESUMO

BACKGROUND Previous studies have shown that exocyst complex is located at polarized growth sites at different cell cycle stages in budding yeast. But how cell cycle and the cyclin-dependent kinase, Cdk1, regulate the distribution of exocyst complex on the plasma membrane and the protein level of each exocyst subunit is not clear. MATERIAL AND METHODS Using budding yeast as a research material, regulation of cell cycle and Cdk1 on exocyst localization on the plasma membrane and on level of each exocyst subunit were examined by methods of cell biology and molecular biology. RESULTS Exocyst complex is located at growth sites on the plasma membrane in both budding and non-budding stages. Cdk1 activity is required for polarized distribution of exocyst complex in late G1, S and M phases, but not in cytokinesis stage. Cdk1 is not required for the assembly and localization of exocyst complex on plasma membrane. The protein level of Sec3 but not other exocyst subunits is regulated by the cell cycle. CONCLUSIONS Cdk1 activity is required for exocyst polarization before cytokinesis during the cell cycle progression, but not for its assembly and localization on the plasma membrane. Dynamic localization and protein level of the complex subunits are regulated by the cell cycle.


Assuntos
Proteína Quinase CDC2/metabolismo , Membrana Celular/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Ciclo Celular , Divisão Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Proteínas de Transporte Vesicular/genética
7.
Life Sci ; 231: 116528, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31176784

RESUMO

AIMS: Lycorine is a kind of natural alkaloid with anti-cancer potential. It has been demonstrated that lycorine processes high activity and specificity against the progression of cancers. However, the underlying molecular mechanisms by which lycorine regulates the formation and development of non-small cell lung cancer (NSCLC) remain largely unknown. MAIN METHODS: The effects of lycorine on the growth of NSCLC cells were determined by the cell counting kit-8 (CCK-8) assay, colony formation and flow cytometry analysis. RT-qPCR was performed to detect the expression of microRNA with lycorine treatment. The binding of miRNA and target genes was confirmed by luciferase reporter assay. KEY FINDINGS: Lycorine significantly inhibited the proliferation and induced apoptosis of NSCLC cells. Mechanistically, lycorine up-regulated the expression of microRNA-186 in NSCLC cells. Depletion of miR-186 significantly reversed the suppressive effect of lycorine on the proliferation of NSCLC cells. Furthermore, the cyclin dependent kinase 1 (CDK1) was identified as one of the binding candidates of miR-186. Experimental analysis showed that miR-186 bound the 3'-untranslated region (3'-UTR) of CDK1 and suppressed the level of CDK1 in NSCLC cells. Consistently, exposure of lycorine significantly decreased the expression of CDK1. Restoration of CDK1 remarkably attenuated the inhibition of lycorine on the proliferation of NSCLC cells. SIGNIFICANCE: Our results uncovered the novel molecular mechanism of lycorine in suppressing the progression of NSCLC partially via regulating the miR-186/CDK1 axis.


Assuntos
Alcaloides de Amaryllidaceae/farmacologia , Proteína Quinase CDC2/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , MicroRNAs/metabolismo , Fenantridinas/farmacologia , Regiões 3' não Traduzidas , Apoptose/efeitos dos fármacos , Proteína Quinase CDC2/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , MicroRNAs/efeitos dos fármacos , MicroRNAs/genética , Regulação para Cima/efeitos dos fármacos
8.
Int J Mol Sci ; 20(9)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035650

RESUMO

Citrate is a key intermediate of the tricarboxylic acid cycle and acts as an allosteric signal to regulate the production of cellular ATP. An elevated cytosolic citrate concentration inhibits growth in several types of human cancer cells; however, the underlying mechanism by which citrate induces the growth arrest of cancer cells remains unclear. The results of this study showed that treatment of human pharyngeal squamous carcinoma (PSC) cells with a growth-suppressive concentration of citrate caused cell cycle arrest at the G2/M phase. A coimmunoprecipitation study demonstrated that citrate-induced cell cycle arrest in the G2/M phase was associated with stabilizing the formation of cyclin B1-phospho (p)-cyclin-dependent kinase 1 (CDK1) (Thr 161) complexes. The citrate-induced increased levels of cyclin B1 and G2/M phase arrest were suppressed by the caspase-3 inhibitor Ac-DEVD-CMK and caspase-3 cleavage of mutant p21 (D112N). Ectopic expression of the constitutively active form of protein kinase B (Akt1) could overcome the induction of p21 cleavage, cyclin B1-p-CDK1 (Thr 161) complexes, and G2/M phase arrest by citrate. p85α-phosphatase and tensin homolog deleted from chromosome 10 (PTEN) complex-mediated inactivation of Akt was required for citrate-induced G2/M phase cell cycle arrest because PTEN short hairpin RNA or a PTEN inhibitor (SF1670) blocked the suppression of Akt Ser 473 phosphorylation and the induction of cyclin B1-p-CDK1 (Thr 161) complexes and G2/M phase arrest by citrate. In conclusion, citrate induces G2/M phase arrest in PSC cells by inducing the formation of p85α-PTEN complexes to attenuate Akt-mediated signaling, thereby causing the formation of cyclin B1-p-CDK1 (Thr 161) complexes.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Ácido Cítrico/farmacologia , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias Faríngeas/metabolismo , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Ciclina B1/metabolismo , Humanos , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Neoplasias Faríngeas/genética , Fosforilação , Transdução de Sinais
9.
Mol Carcinog ; 58(8): 1438-1449, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31006917

RESUMO

Substantial evidence suggests that 7,12-dimethylbenzanthracene (DMBA)-induced mammary carcinogenesis in mice mimics human breast cancer (BC) in many respects. Therefore, it has been used extensively to evaluate preventive and therapeutic agents for human BC. Mammary carcinogenesis induced by DMBA administration in female SENsitive to CARcinogen (SENCAR) mice was characterized by histopathological analysis of the mammary glands and alterations to the phosphatidylinositol 3-kinase/protein kinase B/cyclin-dependent kinase 1 (PI3K/Akt/CDK1) pathway. We recently reported that 2'-hydroxyflavanone (2HF) is a promising diet-derived chemotherapeutic agent that suppresses BC growth in vitro and in vivo by targeting a 76 kDa ral-interacting protein (RLIP). The objective of the current study was to investigate the synergistic anticarcinogenic effects of RLIP inhibition/depletion and 2HF in an in vivo model of DMBA-induced mammary carcinogenesis in SENCAR mice. Mice were given 2HF (50 mg/kg, bw, orally on alternate days), RLIP antibody (Rab; 5 mg/kg, bw, ip weekly), RLIP antisense (RAS; 5 mg/kg, b.w., ip weekly), or a combination of 2HF + Rab + RAS. Animals were monitored daily, and 7 days after the first appearance of moribund behavior, tissues were harvested for morphological and immunohistological analysis. Western blot analyses were performed to determine the expression of anti- and proapoptotic proteins in the mammary glands. Our results reveal that 2HF, RAS, and Rab significantly prevented the carcinogenic effects of DMBA administration in the mammary glands and other organs. Further, mice treated with a combination of 2HF + RAS + Rab exhibited no carcinogenic effect of DMBA as compared to either or the single agent-treated mice. This study demonstrates for the first time the anticarcinogenic effects of 2HF and RLIP inhibition/depletion in vivo in a novel DMBA-induced model of BC in SENCAR mice and provides the rationale for further clinical investigation.


Assuntos
9,10-Dimetil-1,2-benzantraceno/toxicidade , Anticarcinógenos/farmacologia , Transformação Celular Neoplásica/patologia , Flavanonas/farmacologia , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Neoplasias Mamárias Experimentais/prevenção & controle , Animais , Proteína Quinase CDC2/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas Ativadoras de GTPase/genética , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos SENCAR , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
10.
Phytomedicine ; 61: 152846, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31035041

RESUMO

BACKGROUND: The use of plant essential oils as pharmaceuticals is a fast-growing market especially in China. Throughout the 20th century, a rapid increase took place in the use of many essential oil-derived products in the medicinal industry as nutraceuticals, medicinal supplements, and pharmaceuticals. PURPOSE: The objective of this study was to explore the chemical composition of Croton crassifolius essential oil as well as its potential anti-tumour properties and related anti-proliferative, autophagic, and apoptosis-inducing effects. METHODS: Supercritical CO2 fluid extraction technology was used to extract CCEO and the chemical constituents of the essential oil were identified by comparing the retention indices and mass spectra data taken from the NIST library with those calculated based on the C7-C40 n-alkanes standard. The cytotoxic activity and anti-proliferative effects of CCEO were evaluated against five cancer cell lines and one normal human cell line via CCK-8 assays. In addition, flow cytometry was used to detect cell cycle arrest. The efficacy of CCEO treatments in controlling cancer cell proliferation was assessed by cell cycle analysis, clonal formation assays, RT-qPCR, and western blot analysis. Autophagic and apoptosis-inducing effects of oils and the associated molecular mechanisms were assessed by flow cytometry, cell staining, reactive oxygen species assays, RT-qPCR, and western blot analysis. CONCLUSION: Forty compounds representing 92.90% of the total oil were identified in CCEO. The results showed that CCEO exerted a measurable selectivity for cancer cell lines, especially for A549 with the lowest IC50 value of 25.00 ± 1.62 µg/mL. Assessment of the anti-proliferative effects of CCEO on A549 cells showed that the oil inhibited cell proliferation and colony formation in a dose- and time-dependent manner. Investigation of the molecular mechanisms of cell cycle regulation confirmed that the oil arrested A549 cells in G2/M phase by decreasing the expression of cyclin B1-CDK1 and cyclin A-CDK1 and increasing the expression of cyclin-dependent kinase inhibitor (CKI) P21 at both the transcriptional and translational levels. Autophagy staining assays and western blot analysis revealed that CCEO promoted the formation of autophagic vacuoles in A549 cells and increased the expression of autophagy-related proteins beclin-1 and LC3-II in a dose-dependent manner. A series of apoptosis analyses indicated that CCEO induces apoptosis through a mitochondria-mediated intrinsic pathway. This study revealed that CCEO is a promising candidate for development into an anti-tumour drug of the future.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Cromatografia com Fluido Supercrítico/métodos , Croton/química , Óleos Voláteis/química , Células A549 , Antineoplásicos Fitogênicos/química , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Proteína Quinase CDC2/metabolismo , Dióxido de Carbono/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Óleos Voláteis/análise , Raízes de Plantas/química , Espécies Reativas de Oxigênio/metabolismo
11.
Food Chem Toxicol ; 127: 143-155, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30885713

RESUMO

Camptothecin (CPT) is a popular therapeutic agent that targets topoisomerase I. Our findings demonstrated that CPT-induced microtubule polymerization results in markedly increased histone H3 phosphorylation. CPT also enhanced interactions between the mitotic checkpoint proteins, Mad2 and Cdc20, and thereby increased mitotic arrest. Transient knockdown of Mad2 completely restored cell cycle progression from CPT-induced mitotic arrest, while simultaneously reduced cyclin B1 and Cdk1 expression. Moreover, we found that c-Jun N-terminal kinase (JNK) acts upstream of Sp1, which upregulates p21-mediated mitotic arrest in response to CPT; furthermore, knockdown of p21 restored cell cycle progression, while inhibition of Cdks completely restored cell cycle progression from CPT-induced mitotic arrest. We hypothesized that, during mitotic arrest in response to CPT, cell survival signaling blocks apoptosis, thereby enhancing mitotic arrest. As expected, a caspase-9 inhibitor, z-LEHD-FMK, and an autophagy inhibitor, 3-methyladenine (3 MA), significantly diminished CPT-induced mitotic arrest. On the other hand, when Mad2 was depleted, z-LEHD-FMK and 3 MA markedly increased apoptosis, and restored cell cycle progression. Taken together, these results suggest that CPT decodes the action of topoisomerase I-mediated tubulin targeting drugs, leading to mitotic arrest by upregulating Mad2 through the JNK-mediated Sp1 pathway and autophagy formation from tubulin polymerization.


Assuntos
Camptotecina/farmacologia , Proteínas Cdc20/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Mad2/metabolismo , Mitose/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo , Autofagia , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Ciclina B1/metabolismo , Humanos , Fosforilação , Polimerização , Tubulina (Proteína)/metabolismo , Regulação para Cima/efeitos dos fármacos
12.
Cell Mol Life Sci ; 76(18): 3601-3620, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30927017

RESUMO

Exit from mitosis and completion of cytokinesis require the inactivation of mitotic cyclin-dependent kinase (Cdk) activity. In budding yeast, Cdc14 phosphatase is a key mitotic regulator that is activated in anaphase to counteract Cdk activity. In metaphase, Cdc14 is kept inactive in the nucleolus, where it is sequestered by its inhibitor, Net1. At anaphase onset, downregulation of PP2ACdc55 phosphatase by separase and Zds1 protein promotes Net1 phosphorylation and, consequently, Cdc14 release from the nucleolus. The mechanism by which PP2ACdc55 activity is downregulated during anaphase remains to be elucidated. Here, we demonstrate that Cdc55 regulatory subunit is phosphorylated in anaphase in a Cdk1-Clb2-dependent manner. Interestingly, cdc55-ED phosphomimetic mutant inactivates PP2ACdc55 phosphatase activity towards Net1 and promotes Cdc14 activation. Separase and Zds1 facilitate Cdk-dependent Net1 phosphorylation and Cdc14 release from the nucleolus by modulating PP2ACdc55 activity via Cdc55 phosphorylation. In addition, human Cdk1-CyclinB1 phosphorylates human B55, indicating that the mechanism is conserved in higher eukaryotes.


Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Anáfase , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Núcleo Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Mitose , Fosfopeptídeos/análise , Fosforilação , Separase/metabolismo , Espectrometria de Massas em Tandem
13.
Biol Cell ; 111(8): 199-212, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30905068

RESUMO

Motile cilia of epithelial multiciliated cells transport vital fluids along organ lumens to promote essential respiratory, reproductive and brain functions. Progenitors of multiciliated cells undergo massive and coordinated organelle remodelling during their differentiation for subsequent motile ciliogenesis. Defects in multiciliated cell differentiation lead to severe cilia-related diseases by perturbing cilia-based flows. Recent work designated the machinery of mitosis as the orchestrator of the orderly progression of differentiation associated with multiple motile cilia formation. By examining the events leading to motile ciliogenesis with a methodological prism of mitosis, we contextualise and discuss the recent findings to broaden the spectrum of questions related to the differentiation of mammalian multiciliated cells.


Assuntos
Centríolos/metabolismo , Cílios/fisiologia , Células Epiteliais , Mitose/fisiologia , Organelas/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Linhagem Celular , Transformação Celular Neoplásica , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Camundongos , Leveduras/metabolismo
14.
Nat Commun ; 10(1): 981, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30816115

RESUMO

Animal cells undergo rapid rounding during mitosis, ensuring proper chromosome segregation, during which an outward rounding force abruptly increases upon prometaphase entry and is maintained at a constant level during metaphase. Initial cortical tension is generated by the actomyosin system to which both myosin motors and actin network architecture contribute. However, how cortical tension is maintained and its physiological significance remain unknown. We demonstrate here that Cdk1-mediated phosphorylation of DIAPH1 stably maintains cortical tension after rounding and inactivates the spindle assembly checkpoint (SAC). Cdk1 phosphorylates DIAPH1, preventing profilin1 binding to maintain cortical tension. Mutation of DIAPH1 phosphorylation sites promotes cortical F-actin accumulation, increases cortical tension, and delays anaphase onset due to SAC activation. Measurement of the intra-kinetochore length suggests that Cdk1-mediated cortex relaxation is indispensable for kinetochore stretching. We thus uncovered a previously unknown mechanism by which Cdk1 coordinates cortical tension maintenance and SAC inactivation at anaphase onset.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Quinase CDC2/metabolismo , Segregação de Cromossomos/fisiologia , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Anáfase/fisiologia , Ciclina B1/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Cinetocoros/metabolismo , Metáfase/fisiologia , Fosforilação , Profilinas/química , Profilinas/genética , Profilinas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
15.
Fertil Steril ; 111(3): 510-518, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30827523

RESUMO

OBJECTIVE: To investigate the genetic cause of fertilization failure or poor fertilization. DESIGN: Genetic analysis. SETTING: University-affiliated center. PATIENT(S): Twenty-four Chinese women who underwent assisted reproductive technology (ART) and had repeated fertilization failure or poor fertilization. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Twenty-four affected patients were subjected to whole-exome sequencing and candidate mutations were validated by Sanger sequencing. Single-cell reverse transcription was used to analyze the functional characterization of the splice-site mutation in vivo. Evolutionary conservation and molecular modeling analyses were used to predict the impact of missense mutations on secondary protein structure. Immunofluorescence was used to analyze the protein levels of WEE2 and phosphorylated CDC2. RESULT(S): Biallelic mutations in WEE2 were identified in 5 of 24 (20.8%) Chinese patients with fertilization failure or poor fertilization. Among these individuals we found a novel splice-site mutation, two novel missense mutations, and a previously reported frame-shift mutation. Splicing mutation c.1136-2A>G of WEE2 caused an alteration of the reading frame and introduced a premature stop codon (p.Gly379Glufs*6/p.Asp380Leufs*39). The missense mutations c.585G>C (p.Lys195Asn) and c.1228C>T (p.Arg410Trp) produced obvious changes in secondary protein structures. Immunostaining indicated that mutated WEE2 resulted in the loss of phosphorylated CDC2. The phenotypes of women carrying WEE2 mutations exhibited slight variability, from total fertilization failure to poor fertilization. CONCLUSION(S): Novel mutations in the known causative gene WEE2 were identified in 5 of 24 women with fertilization failure or poor fertilization, indicating a high prevalence of WEE2 mutations in Chinese women experiencing fertilization failure or poor fertilization.


Assuntos
Proteínas de Ciclo Celular/genética , Fertilidade/genética , Infertilidade Feminina/genética , Infertilidade Feminina/terapia , Mutação , Proteínas Tirosina Quinases/genética , Técnicas de Reprodução Assistida/efeitos adversos , Adulto , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , China , Análise Mutacional de DNA/métodos , Feminino , Predisposição Genética para Doença , Humanos , Infertilidade Feminina/enzimologia , Infertilidade Feminina/fisiopatologia , Modelos Moleculares , Taxa de Mutação , Fenótipo , Fosforilação , Gravidez , Estrutura Secundária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Relação Estrutura-Atividade , Falha de Tratamento , Sequenciamento Completo do Exoma
16.
Pharm Res ; 36(4): 57, 2019 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-30796530

RESUMO

PURPOSE: Since the molecular mechanism of the cell cycle was established, various theoretical models of this process have been developed. A recent study revealed significant variability in cell cycle duration between mother and daughter cells, but this observation has not been incorporated into the theoretical models. METHODS: We used fluorescent ubiquitination-based cell cycle indicator (FUCCI) systems and live-monitored the heterogeneity of cell cycle progression within daughter cells, which accounts for dephasing synchrony. To incorporate the variable cell cycle durations into a model, we modified a two-ordinary differential equation (ODE) model based on reciprocal activation between CDK1 and APC. RESULTS: Our model reproduced the experimental population profile, in which cell cycle synchrony dephased due to variability. Based on this model, we determined parameters for CDK1 and APC in the cell cycle profile after treatment with antimitotic drugs and associated the parameters with the drugs' mode of action as cell cycle inhibitors. CONCLUSION: This suggests that this model is useful for determining the mode of action of unknown small molecules on the cell cycle.


Assuntos
Antimitóticos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Mitose/efeitos dos fármacos , Modelos Biológicos , Neoplasias do Colo do Útero/tratamento farmacológico , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Técnicas Biossensoriais , Proteína Quinase CDC2/metabolismo , Simulação por Computador , Feminino , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Microscopia de Fluorescência , Análise Numérica Assistida por Computador , Processos Estocásticos , Fatores de Tempo , Imagem com Lapso de Tempo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
17.
Int Heart J ; 60(2): 374-383, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30745530

RESUMO

Atrial fibrillation has caused severe burden for people worldwide. Differentiation of fibroblasts into myofibroblasts, and consequent progress in atrial structural remodeling have been considered the basis for persistent atrial fibrillation, yet little is known about the molecular mechanisms underlying the process. Here, we show that cyclin-dependent kinase 1 (CDK1) is activated in atrial fibroblasts from patients with atrial fibrillation (AFPAF) and in platelet derived growth factor BB (PDGF-BB)-treated atrial fibroblasts from patients with sinus rhythm (AFPSR). We also demonstrate that inhibition of CDK1 suppresses fibroblast differentiation and focal adhesion (FA) complex formation. The FA protein paxillin is phosphorylated directly at Ser244 by CDK1. Importantly, transfection of a paxillin construct harboring a Ser to Ala mutation causes FA complex disassembly and greatly inhibits fibroblast activation. AFPSRs applied with a lentiviral vector carrying the shRNA sequence of paxillin dramatically prevents PDGF-BB induced functional activation. Taken together, all these results suggest that phosphorylation of paxillin at Ser244 by CDK1 is a key mechanism in fibroblast differentiation and could eventually assist atrial fibrosis.


Assuntos
Fibrilação Atrial , Proteína Quinase CDC2/metabolismo , Diferenciação Celular/fisiologia , Fibroblastos/fisiologia , Adesões Focais/fisiologia , Serina/metabolismo , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Remodelamento Atrial , Becaplermina , Adesão Celular , Agregação Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Fibrose , Humanos , Paxilina/metabolismo , Fosforilação , Transdução de Sinais
18.
Dev Cell ; 48(5): 672-684.e5, 2019 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-30745144

RESUMO

Successful mitosis requires that cyclin B1:CDK1 kinase activity remains high until chromosomes are correctly aligned on the mitotic spindle. It has therefore been unclear why, in mammalian oocyte meiosis, cyclin B1 destruction begins before chromosome alignment is complete. Here, we resolve this paradox and show that mouse oocytes exploit an imbalance in the ratio of cyclin B1 to CDK1 to control CDK1 activity; early cyclin B1 destruction reflects the loss of an excess of non-CDK1-bound cyclin B1 in late prometaphase, while CDK1-bound cyclin B1 is destroyed only during metaphase. The ordered destruction of the two forms of cyclin B1 is brought about by a previously unidentified motif that is accessible in free cyclin B1 but masked when cyclin B1 is in complex with CDK1. This protects the CDK1-bound fraction from destruction in prometaphase, ensuring a period of prolonged CDK1 activity sufficient to achieve optimal chromosome alignment and prevent aneuploidy.


Assuntos
Aneuploidia , Proteína Quinase CDC2/metabolismo , Ciclina B1/genética , Oócitos/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Feminino , Meiose/fisiologia , Camundongos , Mitose/fisiologia , Fuso Acromático/metabolismo
19.
Cells ; 8(2)2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30754676

RESUMO

The CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor that regulates cellular proliferation, differentiation, apoptosis and tumorigenesis. Although the pro-oncogenic roles of C/EBPß have been implicated in various human cancers, how it contributes to tumorigenesis or tumor progression has not been determined. Immunohistochemistry with human non-small cell lung cancer (NSCLC) tissues revealed that higher levels of C/EBPß protein were expressed compared to normal lung tissues. Knockdown of C/EBPß by siRNA reduced the proliferative capacity of NSCLC cells by delaying the G2/M transition in the cell cycle. In C/EBPß-knockdown cells, a prolonged increase in phosphorylation of cyclin dependent kinase 1 at tyrosine 15 (Y15-pCDK1) was displayed with simultaneously increased Wee1 and decreased Cdc25B expression. Chromatin immunoprecipitation (ChIP) analysis showed that C/EBPß bound to distal promoter regions of WEE1 and repressed WEE1 transcription through its interaction with histone deacetylase 2. Treatment of C/EBPß-knockdown cells with a Wee1 inhibitor induced a decrease in Y15-pCDK1 and recovered cells from G2/M arrest. In the xenograft tumors, the depletion of C/EBPß significantly reduced tumor growth. Taken together, these results indicate that Wee1 is a novel transcription target of C/EBPß that is required for the G2/M phase of cell cycle progression, ultimately regulating proliferation of NSCLC cells.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Fase G2 , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Fase G2/efeitos dos fármacos , Fase G2/genética , Histona Desacetilase 2/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Transcrição Genética/efeitos dos fármacos
20.
J Biol Chem ; 294(12): 4656-4666, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30700550

RESUMO

ß-Cell mitochondria play a central role in coupling glucose metabolism with insulin secretion. Here, we identified a metabolic function of cyclin-dependent kinase 1 (CDK1)/cyclin B1-the activation of mitochondrial respiratory complex I-that is active in quiescent adult ß-cells and hyperactive in ß-cells from obese (ob/ob) mice. In WT islets, respirometry revealed that 65% of complex I flux and 49% of state 3 respiration is sensitive to CDK1 inhibition. Islets from ob/ob mice expressed more cyclin B1 and exhibited a higher sensitivity to CDK1 blockade, which reduced complex I flux by 76% and state 3 respiration by 79%. The ensuing reduction in mitochondrial NADH utilization, measured with two-photon NAD(P)H fluorescence lifetime imaging (FLIM), was matched in the cytosol by a lag in citrate cycling, as shown with a FRET reporter targeted to ß-cells. Moreover, time-resolved measurements revealed that in ob/ob islets, where complex I flux dominates respiration, CDK1 inhibition is sufficient to restrict the duty cycle of ATP/ADP and calcium oscillations, the parameter that dynamically encodes ß-cell glucose sensing. Direct complex I inhibition with rotenone mimicked the restrictive effects of CDK1 inhibition on mitochondrial respiration, NADH turnover, ATP/ADP, and calcium influx. These findings identify complex I as a critical mediator of obesity-associated metabolic remodeling in ß-cells and implicate CDK1 as a regulator of complex I that enhances ß-cell glucose sensing.


Assuntos
Proteína Quinase CDC2/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Obesidade/metabolismo , Transdução de Sinais , Animais , Ciclo do Ácido Cítrico , Ciclina B1/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
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