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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.
Neoplasma ; 66(6): 946-953, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31607131

RESUMO

The aim of this study was to determine the expression levels of TTK in clear cell renal cell carcinoma (ccRCC) tissues and its possible link with the clinical pathologic characteristics and the prognosis of patients suffering this disease, and to further explore the potential role of TTK in the progression of ccRCC. Immunohistochemical (IHC) assays were performed to detect the expression levels of TTK in 112 samples of ccRCC tissues and corresponding non-tumor tissues. According to the results of IHC assays, patients were divided into TTK high expression and low expression group. Clinical analysis related to the clinical features (age, gender, T stage), and the potential link between TTK expression levels and clinical features were analyzed. In addition, the effects of TTK on the proliferation and invasion of ccRCC cells were detected through colony formation assay and transwell assays, respectively. The possible effects of TTK on tumor growth and metastasis were measured in mice. We found a high expression level of TTK in human ccRCC tissues from patients who received surgical treatment. We also found its expression level was obviously associated with clinical characteristics, such as T stage (p=0.008) and lymphatic metastasis (p=0.023). We further confirmed that knockdown of TTK suppressed cell proliferation and invasion in 2 types of ccRCC cells, HTB-47 and CRL-1932 cells. Furthermore, TTK contributes to tumor growth and metastasis of ccRCC in mice. We found that TTK affected the progression of ccRCC and further mechanically confirmed it could be a novel therapeutic target for ccRCC treatment.


Assuntos
Carcinoma de Células Renais/patologia , Proteínas de Ciclo Celular/metabolismo , Neoplasias Renais/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/metabolismo , Camundongos , Metástase Neoplásica , Prognóstico
3.
Nature ; 574(7779): 571-574, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645724

RESUMO

To safeguard genome integrity in response to DNA double-strand breaks (DSBs), mammalian cells mobilize the neighbouring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes1. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin-CST-POLα complex2. How this pathway reflects and influences the three-dimensional nuclear architecture is not known. Here we use super-resolution microscopy to show that 53BP1 and RIF1 form an autonomous functional module that stabilizes three-dimensional chromatin topology at sites of DNA breakage. This process is initiated by accumulation of 53BP1 at regions of compact chromatin that colocalize with topologically associating domain (TAD) sequences, followed by recruitment of RIF1 to the boundaries between such domains. The alternating distribution of 53BP1 and RIF1 stabilizes several neighbouring TAD-sized structures at a single DBS site into an ordered, circular arrangement. Depletion of 53BP1 or RIF1 (but not shieldin) disrupts this arrangement and leads to decompaction of DSB-flanking chromatin, reduction in interchromatin space, aberrant spreading of DNA repair proteins, and hyper-resection of DNA ends. Similar topological distortions are triggered by depletion of cohesin, which suggests that the maintenance of chromatin structure after DNA breakage involves basic mechanisms that shape three-dimensional nuclear organization. As topological stabilization of DSB-flanking chromatin is independent of DNA repair, we propose that, besides providing a structural scaffold to protect DNA ends against aberrant processing, 53BP1 and RIF1 safeguard epigenetic integrity at loci that are disrupted by DNA breakage.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Instabilidade Genômica , Conformação de Ácido Nucleico , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
4.
Nat Chem Biol ; 15(10): 992-1000, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31527837

RESUMO

Post-translational modifications of histone variant H2A.Z accompany gene transactivation, but its modifying enzymes still remain elusive. Here, we reveal a hitherto unknown function of human KAT2A (GCN5) as a histone acetyltransferase (HAT) of H2A.Z at the promoters of a set of transactivated genes. Expression of these genes also depends on the DNA repair complex XPC-RAD23-CEN2. We established that XPC-RAD23-CEN2 interacts both with H2A.Z and KAT2A to drive the recruitment of the HAT at promoters and license H2A.Z acetylation. KAT2A selectively acetylates H2A.Z.1 versus H2A.Z.2 in vitro on several well-defined lysines and we unveiled that alanine-14 in H2A.Z.2 is responsible for inhibiting the activity of KAT2A. Notably, the use of a nonacetylable H2A.Z.1 mutant shows that H2A.Z.1ac recruits the epigenetic reader BRD2 to promote RNA polymerase II recruitment. Our studies identify KAT2A as an H2A.Z.1 HAT in mammals and implicate XPC-RAD23-CEN2 as a transcriptional co-activator licensing the reshaping of the promoter epigenetic landscape.


Assuntos
Reparo do DNA/fisiologia , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Acetilação , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos , Regulação da Expressão Gênica , Humanos , Lisina Acetiltransferase 5
5.
Nat Cell Biol ; 21(9): 1127-1137, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481798

RESUMO

The inner centromere is a region on every mitotic chromosome that enables specific biochemical reactions that underlie properties, such as the maintenance of cohesion, the regulation of kinetochores and the assembly of specialized chromatin, that can resist microtubule pulling forces. The chromosomal passenger complex (CPC) is abundantly localized to the inner centromeres and it is unclear whether it is involved in non-kinase activities that contribute to the generation of these unique chromatin properties. We find that the borealin subunit of the CPC drives phase separation of the CPC in vitro at concentrations that are below those found on the inner centromere. We also provide strong evidence that the CPC exists in a phase-separated state at the inner centromere. CPC phase separation is required for its inner-centromere localization and function during mitosis. We suggest that the CPC combines phase separation, kinase and histone code-reading activities to enable the formation of a chromatin body with unique biochemical activities at the inner centromere.


Assuntos
Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Citoesqueleto/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose
6.
Genes Dev ; 33(17-18): 1175-1190, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31395742

RESUMO

The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. DNA double-strand breaks (DSBs) within rDNA induce both rDNA transcriptional repression and nucleolar segregation, but the link between the two events remains unclear. Here we found that DSBs induced on rDNA trigger transcriptional repression in a cohesin- and HUSH (human silencing hub) complex-dependent manner throughout the cell cycle. In S/G2 cells, transcriptional repression is further followed by extended resection within the interior of the nucleolus, DSB mobilization at the nucleolar periphery within nucleolar caps, and repair by homologous recombination. We showed that nuclear envelope invaginations frequently connect the nucleolus and that rDNA DSB mobilization, but not transcriptional repression, involves the nuclear envelope-associated LINC complex and the actin pathway. Altogether, our data indicate that rDNA break localization at the nucleolar periphery is not a direct consequence of transcriptional repression but rather is an active process that shares features with the mobilization of persistent DSB in active genes and heterochromatin.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA Ribossômico/genética , Regulação da Expressão Gênica/genética , RNA Longo não Codificante/metabolismo , Nucléolo Celular/metabolismo , Histonas/metabolismo , Recombinação Homóloga/genética , Membrana Nuclear/metabolismo
7.
Nat Commun ; 10(1): 3435, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387991

RESUMO

Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.


Assuntos
Histona Acetiltransferases/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Acetilação , Domínio Catalítico , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Histona Acetiltransferases/isolamento & purificação , Chaperonas de Histonas/isolamento & purificação , Histonas/isolamento & purificação , Lisina/metabolismo , Chaperonas Moleculares/isolamento & purificação , Chaperonas Moleculares/metabolismo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Proteínas de Xenopus/isolamento & purificação , Proteínas de Xenopus/metabolismo
8.
Cell Mol Life Sci ; 76(21): 4245-4273, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31317204

RESUMO

Molecular self-organziation, also regarded as pattern formation, is crucial for the correct distribution of cellular content. The processes leading to spatiotemporal patterns often involve a multitude of molecules interacting in complex networks, so that only very few cellular pattern-forming systems can be regarded as well understood. Due to its compositional simplicity, the Escherichia coli MinCDE system has, thus, become a paradigm for protein pattern formation. This biological reaction diffusion system spatiotemporally positions the division machinery in E. coli and is closely related to ParA-type ATPases involved in most aspects of spatiotemporal organization in bacteria. The ATPase MinD and the ATPase-activating protein MinE self-organize on the membrane as a reaction matrix. In vivo, these two proteins typically oscillate from pole-to-pole, while in vitro they can form a variety of distinct patterns. MinC is a passenger protein supposedly operating as a downstream cue of the system, coupling it to the division machinery. The MinCDE system has helped to extract not only the principles underlying intracellular patterns, but also how they are shaped by cellular boundaries. Moreover, it serves as a model to investigate how patterns can confer information through specific and non-specific interactions with other molecules. Here, we review how the three Min proteins self-organize to form patterns, their response to geometric boundaries, and how these patterns can in turn induce patterns of other molecules, focusing primarily on experimental approaches and developments.


Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Proteínas de Escherichia coli/fisiologia , Proteínas de Membrana/fisiologia , Transporte Proteico/fisiologia , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Multimerização Proteica/fisiologia , Transporte Proteico/genética
9.
Yi Chuan ; 41(6): 509-523, 2019 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-31257199

RESUMO

UDP-glucuronosyltransferases (UGTs) are an important family of phase 2 drug-metabolizing enzymes that catalyze the glucuronidation of numerous endogenous or exogenous small compounds. The aberrant expression of UGT isoforms causes many diseases, such as hyperbilirubinemia and affect drug efficacy or toxicity. Understanding mechanisms of UGT gene regulation will provide scientific foundations for disease prevention and personalized or precision medicine. Vertebrate UGT family genes can be divided into UGT1 and UGT2 subfamilies. Similar to the protocadherin, immunoglobulin, and T-cell receptor gene clusters and different from the UGT2 gene cluster, the UGT1 gene cluster is organized into variable and constant regions. The UGT1 variable region contains a tandem array of variable exons, each of which can be alternatively spliced to a single set of 4 downstream constant exons, generating at least nine UGT1 mRNAs that could be translated into different UGT1 glucuronyltransferase isoforms. Our previous work reveals that the relative orientations and locations of CTCF binding sites play a key role in the three-dimensional organization of the mammalian genomes in cell nuclei. Thus in order to study the transcriptional mechanisms of UGT1 gene cluster, the distributions and orientations of CTCF binding sites (CBSs) are analyzed and compared between human and mouse UGT1 gene clusters. We find that the CBSs in the UGT1 gene cluster are not conserved between human and mouse species. We show that CTCF and cohesin regulate the transcription of the UGT1 gene cluster by knocking down the CTCF or the cohesin subunit SMC3 in the human A549 cell line. By using CRISPR DNA-fragment editing, we deleted and inverted hCBS1. By RNA-seq experiments, we find that hCBS1 deletion results in a significant decrease of levels of the UGT1A6, UGT1A7, and UGT1A9 gene expression and that hCBS1 inversion results in a significant decrease of levels of the UGT1A7 gene expression. Our data suggest that the CTCF binding site hCBS1 plays an important regulatory role in the regulation of UGT1 gene expression, providing an experimental basis for further mechanistic studies of the 3D genome regulation of the UGT1 gene cluster.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Regulação da Expressão Gênica , Glucuronosiltransferase/genética , Família Multigênica , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Éxons , Humanos , Camundongos , RNA Mensageiro
10.
Nucleic Acids Res ; 47(15): 7825-7841, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31299083

RESUMO

The understanding of the multi-scale nature of molecular networks represents a major challenge. For example, regulation of a timely cell cycle must be coordinated with growth, during which changes in metabolism occur, and integrate information from the extracellular environment, e.g. signal transduction. Forkhead transcription factors are evolutionarily conserved among eukaryotes, and coordinate a timely cell cycle progression in budding yeast. Specifically, Fkh1 and Fkh2 are expressed during a lengthy window of the cell cycle, thus are potentially able to function as hubs in the multi-scale cellular environment that interlocks various biochemical networks. Here we report on a novel ChIP-exo dataset for Fkh1 and Fkh2 in both logarithmic and stationary phases, which is analyzed by novel and existing software tools. Our analysis confirms known Forkhead targets from available ChIP-chip studies and highlights novel ones involved in the cell cycle, metabolism and signal transduction. Target genes are analyzed with respect to their function, temporal expression during the cell cycle, correlation with Fkh1 and Fkh2 as well as signaling and metabolic pathways they occur in. Furthermore, differences in targets between Fkh1 and Fkh2 are presented. Our work highlights Forkhead transcription factors as hubs that integrate multi-scale networks to achieve proper timing of cell division in budding yeast.


Assuntos
Proteínas de Ciclo Celular/genética , DNA Fúngico/química , Fatores de Transcrição Forkhead/genética , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Imunoprecipitação da Cromatina , Replicação do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Ontologia Genética , Anotação de Sequência Molecular , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
11.
Nat Commun ; 10(1): 2908, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266948

RESUMO

Cohesin and CTCF are master regulators of genome topology. How these ubiquitous proteins contribute to cell-type specific genome structure is poorly understood. Here, we explore quantitative aspects of topologically associated domains (TAD) between pluripotent embryonic stem cells (ESC) and lineage-committed cells. ESCs exhibit permissive topological configurations which manifest themselves as increased inter- TAD interactions, weaker intra-TAD interactions, and a unique intra-TAD connectivity whereby one border makes pervasive interactions throughout the domain. Such 'stripe' domains are associated with both poised and active chromatin landscapes and transcription is not a key determinant of their structure. By tracking the developmental dynamics of stripe domains, we show that stripe formation is linked to the functional state of the cell through cohesin loading at lineage-specific enhancers and developmental control of CTCF binding site occupancy. We propose that the unique topological configuration of stripe domains represents a permissive landscape facilitating both productive and opportunistic gene regulation and is important for cellular identity.


Assuntos
Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Elementos Facilitadores Genéticos , Células-Tronco Pluripotentes/metabolismo , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Pluripotentes/química , Ligação Proteica , Domínios Proteicos , Especificidade da Espécie
12.
Nat Commun ; 10(1): 2910, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266951

RESUMO

PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA-intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc-mediated transcriptional repression of CDK18, while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recombination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensitizing GSCs. ATR inhibitor VE822 combined with PARPi extended survival of mice bearing GSC-derived orthotopic tumors, irrespective of PARPi-sensitivity. These studies identify a role of CDK18 in ATR-regulated HR. We propose that combined blockade of ATR and PARP is an effective strategy for GBM, even for low-Myc GSCs that do not respond to PARPi alone, and potentially other PARPi-refractory tumors.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Quinases Ciclina-Dependentes/genética , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/metabolismo , Recombinação Homóloga , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Camundongos , Camundongos SCID , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Células-Tronco Neoplásicas/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Medicine (Baltimore) ; 98(30): e16534, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31348270

RESUMO

BACKGROUND: High-grade prostate cancer (PCa) has a poor prognosis, and up to 15% of patients worldwide experience lymph node invasion (LNI). To further improve the prediction lymph node invasion in prostate cancer, we adopted risk scores of the genes expression based on the nomogram in guidelines. METHODS: We analyzed clinical data from 320 PCa patients from the Cancer Genome Atlas database. Weighted gene coexpression network analysis was used to identify the genes that were significantly associated with LNI in PCa (n = 390). Analyses using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were performed to identify the activated signaling pathways. Univariate and multivariate logistic regression analyses were performed to identify the independent risk factors for the presence of LNI. RESULTS: We found that patients with actual LNI and predicted LNI had the worst survival outcomes. The 7 most significant genes (CTNNAL1, ENSA, MAP6D1, MBD4, PRCC, SF3B2, TREML1) were selected for further analysis. Pathways in the cell cycle, DNA replication, oocyte meiosis, and 9 other pathways were dramatically activated during LNI in PCa. Multivariate analyses identified that the risk score (odds ratio [OR] = 1.05 for 1% increase, 95% confidence interval [CI]: 1.04-1.07, P < .001), serum PSA level, clinical stage, primary biopsy Gleason grade (OR = 2.52 for a grade increase, 95% CI: 1.27-5.22, P = .096), and secondary biopsy Gleason grade were independent predictors of LNI. A nomogram built using these predictive variables showed good calibration and a net clinical benefit, with an area under the curve (AUC) value of 90.2%. CONCLUSIONS: In clinical practice, the application of our nomogram might contribute significantly to the selection of patients who are good candidates for surgery with extended pelvic lymph node dissection.


Assuntos
Biomarcadores Tumorais/genética , Metástase Linfática/genética , Nomogramas , Neoplasias da Próstata/genética , Idoso , Área Sob a Curva , Proteínas de Ciclo Celular/metabolismo , Bases de Dados Genéticas , Endodesoxirribonucleases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Modelos Logísticos , Linfonodos/patologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Análise Multivariada , Gradação de Tumores , Proteínas de Neoplasias/metabolismo , Razão de Chances , Peptídeos/metabolismo , Valor Preditivo dos Testes , Neoplasias da Próstata/patologia , Fatores de Processamento de RNA/metabolismo , Receptores Imunológicos/metabolismo , Reprodutibilidade dos Testes , Fatores de Risco , alfa Catenina/metabolismo
14.
Oncol Rep ; 42(4): 1467-1474, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31322269

RESUMO

With the increasing use of poly(ADP­ribose) polymerase (PARP) inhibitors in cancer therapy, understanding their resistance is an urgent research quest. Additionally, CHFR is an E3 ubiquitin ligase, recruited to double­strand breaks (DSBs) by PAR. Furthermore, ALC1 is a new oncogene involved in the invasion and metastasis of breast cancer. Moreover, PARylated PARP1 activates ALC1 at sites of DNA damage, yet the underlying mechanism remains unclear. Mass spectrometric analysis, western blot analysis and immunoprecipitation were performed to confirm the interaction between CHFR and ALC1 in the physiological condition. Deletion mutants of CHFR and ALC1 were generated to map the interaction domain. PARP1/2 inhibitors were added to identify the ubiquitination of ALC1 by CHFR. ALC1 half­life was examined to compare the expression of ALC1 protein in the presence and absence of PARP1/2 inhibitors. The results revealed that the transcriptional level of ALC1 was not upregulated in breast cancer tissues. CHFR interacted with ALC1. The PBZ domain of CHFR, the PMD domain and the MACRO domain of ALC1 domain are the necessary regions for the interaction depending on PAR. Ubiquitination of ALC1 by CHFR was dependent on PARylation and resulted in the degradation of PARylated ALC1. PARP1/2 inhibitors decreased the ubiquitination of PAR­dependent ALC1, and the expression of ALC1 was upregulated by PARP1/2 inhibitors. Ubiquitination mediated by CHFR resulted in the degradation of ALC1. In conclusion, PARP1/2 inhibitors decrease the ubiquitination of ALC1 leading to the accumulation of ALC1, which affects the therapeutic effects of DNA damage response drugs in breast cancer treatment.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Poli(ADP-Ribose) Polimerase-1/metabolismo , Transcrição Genética , Ubiquitinação/efeitos dos fármacos
15.
Nat Commun ; 10(1): 2502, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175280

RESUMO

Accumulation of nucleotide building blocks prior to and during S phase facilitates DNA duplication. Herein, we find that the anaphase-promoting complex/cyclosome (APC/C) synchronizes ribose-5-phosphate levels and DNA synthesis during the cell cycle. In late G1 and S phases, transketolase-like 1 (TKTL1) is overexpressed and forms stable TKTL1-transketolase heterodimers that accumulate ribose-5-phosphate. This accumulation occurs by asymmetric production of ribose-5-phosphate from the non-oxidative pentose phosphate pathway and prevention of ribose-5-phosphate removal by depleting transketolase homodimers. In the G2 and M phases after DNA synthesis, expression of the APC/C adaptor CDH1 allows APC/CCDH1 to degrade D-box-containing TKTL1, abrogating ribose-5-phosphate accumulation by TKTL1. TKTL1-overexpressing cancer cells exhibit elevated ribose-5-phosphate levels. The low CDH1 or high TKTL1-induced accumulation of ribose-5-phosphate facilitates nucleotide and DNA synthesis as well as cell cycle progression in a ribose-5-phosphate-saturable manner. Here we reveal that the cell cycle control machinery regulates DNA synthesis by mediating ribose-5-phosphate sufficiency.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdh1/metabolismo , Ciclo Celular , Replicação do DNA , Ribosemonofosfatos/metabolismo , Transcetolase/metabolismo , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Fase G2 , Humanos , Via de Pentose Fosfato , Fase S
16.
Life Sci ; 231: 116563, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31200003

RESUMO

AIMS: In the present study, we investigated the roles of renin-angiotensin system (RAS) activation and imbalance of matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in cold-induced stroke during chronic hypertension, as well as the protective effects of captopril and recombinant human TIMP-1 (rhTIMP-1). MAIN METHODS: Rats were randomly assigned to sham; 2-kidney, 2-clip (2K-2C); 2K-2C + captopril, and 2K-2C + rhTIMP-1 groups. After blood pressure values had stabilized, each group was randomly divided into an acute cold exposure (ACE) group (12-h light at 22 °C/12-h dark at 4 °C) and a non-acute cold exposure (NACE) group (12-h light/12-h dark at 22 °C), each of which underwent three cycles of exposure. Captopril treatment was administered via gavage (50 mg/kg/d), while rhTIMP-1 treatment was administered via the tail vein (60 µg/kg/36 h). KEY FINDINGS: In the 2K-2C group, angiotensin II (AngII) and MMP-9 levels increased in both the plasma and cortex, while no such changes in TIMP-1 expression were observed. Cold exposure further upregulated AngII and MMP-9 levels and increased stroke incidence. Captopril and rhTIMP-1 treatment inhibited MMP-9 expression and activation and decreased stroke incidence in response to cold exposure. SIGNIFICANCE: The present study is the first to demonstrate that cold exposure exacerbates imbalance between MMP-9 and TIMP-1 by activating the RAS, which may be critical in the initiation of stroke during chronic hypertension. In addition, our results suggest that captopril and rhTIMP-1 exert protective effects against cold-induced stroke by ameliorating MMP-9/TIMP-1 imbalance.


Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Sistema Renina-Angiotensina/fisiologia , Acidente Vascular Cerebral/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Angiotensina II/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Captopril/metabolismo , Captopril/farmacologia , Proteínas de Ciclo Celular/metabolismo , Temperatura Baixa/efeitos adversos , Humanos , Rim/metabolismo , Masculino , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Sistema Renina-Angiotensina/genética , Acidente Vascular Cerebral/fisiopatologia , Inibidor Tecidual de Metaloproteinase-1/farmacologia , Inibidor Tecidual de Metaloproteinase-2
17.
Nat Commun ; 10(1): 2862, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253793

RESUMO

DNA double strand breaks (DSBs) pose a high risk for genome integrity. Cells repair DSBs through homologous recombination (HR) when a sister chromatid is available. HR is upregulated by the cycling dependent kinase (CDK) despite the paradox of telophase, where CDK is high but a sister chromatid is not nearby. Here we study in the budding yeast the response to DSBs in telophase, and find they activate the DNA damage checkpoint (DDC), leading to a telophase-to-G1 delay. Outstandingly, we observe a partial reversion of sister chromatid segregation, which includes approximation of segregated material, de novo formation of anaphase bridges, and coalescence between sister loci. We finally show that DSBs promote a massive change in the dynamics of telophase microtubules (MTs), together with dephosphorylation and relocalization of kinesin-5 Cin8. We propose that chromosome segregation is not irreversible and that DSB repair using the sister chromatid is possible in telophase.


Assuntos
Cromátides/metabolismo , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Fúngico/genética , Troca de Cromátide Irmã , Telófase/genética , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
Nat Commun ; 10(1): 2861, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253795

RESUMO

Centromeres provide a pivotal function for faithful chromosome segregation. They serve as a foundation for the assembly of the kinetochore complex and spindle connection, which is essential for chromosome biorientation. Cells lacking Polo-like kinase 1 (PLK1) activity suffer severe chromosome alignment defects, which is believed primarily due to unstable kinetochore-microtubule attachment. Here, we reveal a previously undescribed mechanism named 'centromere disintegration' that drives chromosome misalignment in PLK1-inactivated cells. We find that PLK1 inhibition does not necessarily compromise metaphase establishment, but instead its maintenance. We demonstrate that this is caused by unlawful unwinding of DNA by BLM helicase at a specific centromere domain underneath kinetochores. Under bipolar spindle pulling, the distorted centromeres are promptly decompacted into DNA threadlike molecules, leading to centromere rupture and whole-chromosome arm splitting. Consequently, chromosome alignment collapses. Our study unveils an unexpected role of PLK1 as a chromosome guardian to maintain centromere integrity for chromosome biorientation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/fisiologia , Linhagem Celular , Pareamento Cromossômico/fisiologia , Humanos , Cinetocoros , Interferência de RNA , Timidina/farmacologia
19.
Life Sci ; 232: 116583, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31226417

RESUMO

TP53 mutation is an indicator of poor prognostic in chronic lymphocytic leukemia (CLL). Worse still, CLL patients with TP53 mutation are associated with poor efficacy to current chemotherapeutic, such as Fludarabine. Here, we confirmed that high expression of HDAC1 in CLL patients with TP53 mutation, which is closely related to poor prognosis and drug-resistance. Subsequently, we demonstrated Entinostat (HDAC1 inhibitor) combination with Fludarabine significantly induced apoptosis in TP53 mutations CLL cells. Its mechanism was associated with up-regulation of the pro-apoptotic protein Bax and the down-regulation of HDAC1, HO-1 and BCL-2 proteins. More importantly, we also confirmed that upregulation of HDAC1 could resistant Entinostat-induced apoptosis in TP53 mutations CLL cells by activating the HDAC1/P38/HO-1 pathway. In vivo, we found that Entinostat combination with Fludarabine significantly induced tumor cells apoptosis and prolong survival time in xenograft mouse model. Finally, combining vitro and vivo experiments, we presented the first demonstration that Entinostat combination with Fludarabine had a synergistic effect on the induction of apoptosis in TP53 mutations CLL cells. In conclusion, we provide valuable pre-clinical experimental evidence for the treatment of CLL patients with poor prognosis, especially for TP53 mutations.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzamidas/farmacologia , Heme Oxigenase-1/metabolismo , Histona Desacetilase 1/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Piridinas/farmacologia , Proteína Supressora de Tumor p53/genética , Vidarabina/análogos & derivados , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Benzamidas/administração & dosagem , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Histona Desacetilase 1/biossíntese , Histona Desacetilase 1/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Piridinas/administração & dosagem , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Vidarabina/administração & dosagem , Vidarabina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
20.
Biochim Biophys Acta Proteins Proteom ; 1867(9): 813-820, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31226489

RESUMO

Cdc37 is a protein kinase-targeting molecular chaperone, which cooperates with Hsp90 to assist the folding, assembly and maturation of various signaling kinases. It consists of three distinct domains: the N-terminal, middle, and C-terminal domain. While the middle domain is an Hsp90-binding domain, the N-terminal domain is recognized as a kinase-interacting domain. The N-terminal domain contains a well-conserved Ser residue at position 13, and the phosphorylation at this site has been shown to be a prerequisite for the interaction between Cdc37 and signaling kinases. Although the phosphorylation of Ser13 might induce some conformational change in Cdc37 molecule, little is known about the structure of the N-terminal domain of Cdc37. We examined the structural and dynamic properties of several fragment proteins corresponding to the N-terminal region of Cdc37 by circular dichroism and solution NMR spectroscopy. We found that the N-terminal domain of Cdc37 exhibits highly dynamic structure, and it exists in the equilibrium between α-helical and more disordered structures. We also found that phosphorylation at Ser13 did not significantly change the overall structure of N-terminal fragment protein of Cdc37. The results suggested that more complicated mechanisms might be necessary to explain the phosphorylation-activated interaction of Cdc37 with various kinases.


Assuntos
Proteínas de Ciclo Celular/química , Chaperoninas/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Dicroísmo Circular , Humanos , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Domínios Proteicos , Estrutura Secundária de Proteína
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