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1.
Nat Commun ; 11(1): 4861, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978398

RESUMO

Advanced tumours are often heterogeneous, consisting of subclones with various genetic alterations and functional roles. The precise molecular features that characterize the contributions of multiscale intratumour heterogeneity to malignant progression, metastasis, and poor survival are largely unknown. Here, we address these challenges in breast cancer by defining the landscape of heterogeneous tumour subclones and their biological functions using radiogenomic signatures. Molecular heterogeneity is identified by a fully unsupervised deconvolution of gene expression data. Relative prevalence of two subclones associated with cell cycle and primary immunodeficiency pathways identifies patients with significantly different survival outcomes. Radiogenomic signatures of imaging scale heterogeneity are extracted and used to classify patients into groups with distinct subclone compositions. Prognostic value is confirmed by survival analysis accounting for clinical variables. These findings provide insight into how a radiogenomic analysis can identify the biological activities of specific subclones that predict prognosis in a noninvasive and clinically relevant manner.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/genética , Perfilação da Expressão Gênica/métodos , Heterogeneidade Genética , Biomarcadores Tumorais/genética , Mama , Ciclo Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Imageamento Tridimensional/métodos , Análise Multivariada , Prognóstico , Análise de Sobrevida , Transcriptoma
2.
PLoS One ; 15(8): e0236881, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745107

RESUMO

PIERCE1, p53 induced expression 1 in Rb null cells, is a novel p53 target involved in the DNA damage response and cell cycle in mice. These facts prompted us to study the function of PIERCE1 with respect to p53-associated pathophysiology of cancer in humans. Unexpectedly, PIERCE1 did not respond to overexpression and activation of p53 in humans. In this study, we swapped p53 protein expression in human and mouse cells to find the clue of this difference between species. Human p53 expression in mouse cells upregulated PIERCE1 expression, suggesting that p53-responsive elements on the PIERCE1 promoter are crucial, but not the p53 protein itself. Indeed, in silico analyses of PIERCE1 promoters revealed that p53-responsive elements identified in mice are not conserved in humans. Consistently, chromatin immunoprecipitation-sequencing (ChIP-seq) analyses confirmed p53 enrichment against the PIERCE1 promoter region in mice, not in human cells. To complement the p53 study in mice, further promoter analyses suggested that the human PIERCE1 promoter is more similar to guinea pigs, lemurs, and dogs than to rodents. Taken together, our results confirm the differential responsiveness of PIERCE1 expression to p53 due to species differences in PIERCE1 promoters. The results also show partial dissimilarity after p53 induction between mice and humans.


Assuntos
Proteínas de Ciclo Celular , Elementos de Resposta/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/genética , Humanos , Camundongos , Regiões Promotoras Genéticas , Transcrição Genética/fisiologia
3.
PLoS Biol ; 18(8): e3000836, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32804946

RESUMO

Pleiotropy-when a single mutation affects multiple traits-is a controversial topic with far-reaching implications. Pleiotropy plays a central role in debates about how complex traits evolve and whether biological systems are modular or are organized such that every gene has the potential to affect many traits. Pleiotropy is also critical to initiatives in evolutionary medicine that seek to trap infectious microbes or tumors by selecting for mutations that encourage growth in some conditions at the expense of others. Research in these fields, and others, would benefit from understanding the extent to which pleiotropy reflects inherent relationships among phenotypes that correlate no matter the perturbation (vertical pleiotropy). Alternatively, pleiotropy may result from genetic changes that impose correlations between otherwise independent traits (horizontal pleiotropy). We distinguish these possibilities by using clonal populations of yeast cells to quantify the inherent relationships between single-cell morphological features. Then, we demonstrate how often these relationships underlie vertical pleiotropy and how often these relationships are modified by genetic variants (quantitative trait loci [QTL]) acting via horizontal pleiotropy. Our comprehensive screen measures thousands of pairwise trait correlations across hundreds of thousands of yeast cells and reveals ample evidence of both vertical and horizontal pleiotropy. Additionally, we observe that the correlations between traits can change with the environment, genetic background, and cell-cycle position. These changing dependencies suggest a nuanced view of pleiotropy: biological systems demonstrate limited pleiotropy in any given context, but across contexts (e.g., across diverse environments and genetic backgrounds) each genetic change has the potential to influence a larger number of traits. Our method suggests that exploiting pleiotropy for applications in evolutionary medicine would benefit from focusing on traits with correlations that are less dependent on context.


Assuntos
Pleiotropia Genética , Modelos Genéticos , Herança Multifatorial , Locos de Características Quantitativas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Evolução Biológica , Ciclo Celular/genética , Células Clonais , Variação Genética , Ensaios de Triagem em Larga Escala , Mutação , Fenótipo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Célula Única
4.
Nucleic Acids Res ; 48(15): 8374-8392, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32619237

RESUMO

The core-promoter, a stretch of DNA surrounding the transcription start site (TSS), is a major integration-point for regulatory-signals controlling gene-transcription. Cellular differentiation is marked by divergence in transcriptional repertoire and cell-cycling behaviour between cells of different fates. The role promoter-associated gene-regulatory-networks play in development-associated transitions in cell-cycle-dynamics is poorly understood. This study demonstrates in a vertebrate embryo, how core-promoter variations define transcriptional output in cells transitioning from a proliferative to cell-lineage specifying phenotype. Assessment of cell proliferation across zebrafish embryo segmentation, using the FUCCI transgenic cell-cycle-phase marker, revealed a spatial and lineage-specific separation in cell-cycling behaviour. To investigate the role differential promoter usage plays in this process, cap-analysis-of-gene-expression (CAGE) was performed on cells segregated by cycling dynamics. This analysis revealed a dramatic increase in tissue-specific gene expression, concurrent with slowed cycling behaviour. We revealed a distinct sharpening in TSS utilization in genes upregulated in slowly cycling, differentiating tissues, associated with enhanced utilization of the TATA-box, in addition to Sp1 binding-sites. In contrast, genes upregulated in rapidly cycling cells carry broad distribution of TSS utilization, coupled with enrichment for the CCAAT-box. These promoter features appear to correspond to cell-cycle-dynamic rather than tissue/cell-lineage origin. Moreover, we observed genes with cell-cycle-dynamic-associated transitioning in TSS distribution and differential utilization of alternative promoters. These results demonstrate the regulatory role of core-promoters in cell-cycle-dependent transcription regulation, during embryo-development.


Assuntos
Redes Reguladoras de Genes/genética , Regiões Promotoras Genéticas/genética , Sítio de Iniciação de Transcrição , Transcrição Genética , Animais , Sítios de Ligação/genética , Ciclo Celular/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Desenvolvimento Embrionário/genética , Humanos , Morfogênese/genética , Fator de Transcrição Sp1/genética , TATA Box/genética , Peixe-Zebra/genética
5.
PLoS One ; 15(7): e0233755, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628677

RESUMO

Systems biology aims at holistically understanding the complexity of biological systems. In particular, nowadays with the broad availability of gene expression measurements, systems biology challenges the deciphering of the genetic cell machinery from them. In order to help researchers, reverse engineer the genetic cell machinery from these noisy datasets, interactive exploratory clustering methods, pipelines and gene clustering tools have to be specifically developed. Prior methods/tools for time series data, however, do not have the following four major ingredients in analytic and methodological view point: (i) principled time-series feature extraction methods, (ii) variety of manifold learning methods for capturing high-level view of the dataset, (iii) high-end automatic structure extraction, and (iv) friendliness to the biological user community. With a view to meet the requirements, we present AGCT (A Geometric Clustering Tool), a software package used to unravel the complex architecture of large-scale, non-necessarily synchronized time-series gene expression data. AGCT capture signals on exhaustive wavelet expansions of the data, which are then embedded on a low-dimensional non-linear map using manifold learning algorithms, where geometric proximity captures potential interactions. Post-processing techniques, including hard and soft information geometric clustering algorithms, facilitate the summarizing of the complete map as a smaller number of principal factors which can then be formally identified using embedded statistical inference techniques. Three-dimension interactive visualization and scenario recording over the processing helps to reproduce data analysis results without additional time. Analysis of the whole-cell Yeast Metabolic Cycle (YMC) moreover, Yeast Cell Cycle (YCC) datasets demonstrate AGCT's ability to accurately dissect all stages of metabolism and the cell cycle progression, independently of the time course and the number of patterns related to the signal. Analysis of Pentachlorophenol iduced dataset demonstrat how AGCT dissects data to identify two networks: Interferon signaling and NRF2-signaling networks.


Assuntos
Expressão Gênica , Software , Biologia de Sistemas/métodos , Análise de Ondaletas , Algoritmos , Animais , Ciclo Celular/genética , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cadeias de Markov , Camundongos , Pentaclorofenol/farmacologia , Pentaclorofenol/envenenamento , Distribuição Aleatória , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biologia de Sistemas/estatística & dados numéricos
6.
Arch Biochem Biophys ; 690: 108480, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32681832

RESUMO

Although a potentially preventable disease, cervical cancer (CC) is the second most commonly diagnosed gynaecological cancer with at least 530,000 new cases annually, and the prognosis with CC is still poor. Studies suggest that aberrant expression of microRNA (miRNA) contributes to the progression of CC. As a group of small non-coding RNA with 18-25 nucleotides, miRNA regulate about one-third of all human genes. They function by repressing translation or inducing mRNA cleavage or degradation, including genes involved in diverse and important cellular processes, including cell cycling, proliferation, differentiation, and apoptosis. Results showed that misexpression of miRNA is closely related to the onset and progression of CC. This review will provide an overview of the function of miRNA in CC and the mechanisms involved in cervical carcinogenesis.


Assuntos
MicroRNAs/metabolismo , Neoplasias do Colo do Útero/genética , Animais , Apoptose/genética , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Prognóstico , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/terapia
7.
BMC Evol Biol ; 20(1): 84, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32664907

RESUMO

BACKGROUND: Diverse architectures of nervous systems (NSs) such as a plexus in cnidarians or a more centralized nervous system (CNS) in insects and vertebrates are present across Metazoa, but it is unclear what selection pressures drove evolution and diversification of NSs. One underlying aspect of this diversity lies in the cellular and molecular mechanisms driving neurogenesis, i.e. generation of neurons from neural precursor cells (NPCs). In cnidarians, vertebrates, and arthropods, homologs of SoxB and bHLH proneural genes control different steps of neurogenesis, suggesting that some neurogenic mechanisms may be conserved. However, data are lacking for spiralian taxa. RESULTS: To that end, we characterized NPCs and their daughters at different stages of neurogenesis in the spiralian annelid Capitella teleta. We assessed cellular division patterns in the neuroectoderm using static and pulse-chase labeling with thymidine analogs (EdU and BrdU), which enabled identification of NPCs that underwent multiple rounds of division. Actively-dividing brain NPCs were found to be apically-localized, whereas actively-dividing NPCs for the ventral nerve cord (VNC) were found apically, basally, and closer to the ventral midline. We used lineage tracing to characterize the changing boundary of the trunk neuroectoderm. Finally, to start to generate a genetic hierarchy, we performed double-fluorescent in-situ hybridization (FISH) and single-FISH plus EdU labeling for neurogenic gene homologs. In the brain and VNC, Ct-soxB1 and Ct-neurogenin were expressed in a large proportion of apically-localized, EdU+ NPCs. In contrast, Ct-ash1 was expressed in a small subset of apically-localized, EdU+ NPCs and subsurface, EdU- cells, but not in Ct-neuroD+ or Ct-elav1+ cells, which also were subsurface. CONCLUSIONS: Our data suggest a putative genetic hierarchy with Ct-soxB1 and Ct-neurogenin at the top, followed by Ct-ash1, then Ct-neuroD, and finally Ct-elav1. Comparison of our data with that from Platynereis dumerilii revealed expression of neurogenin homologs in proliferating NPCs in annelids, which appears different than the expression of vertebrate neurogenin homologs in cells that are exiting the cell cycle. Furthermore, differences between neurogenesis in the head versus trunk of C. teleta suggest that these two tissues may be independent developmental modules, possibly with differing evolutionary trajectories.


Assuntos
Neurogênese/genética , Filogenia , Poliquetos/citologia , Poliquetos/genética , Animais , Encéfalo/citologia , Ciclo Celular/genética , Divisão Celular , Proliferação de Células/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Cinética , Modelos Biológicos , Placa Neural/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Fatores de Transcrição SOX/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(30): 17808-17819, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661168

RESUMO

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzamidas/farmacologia , Caspase 8/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Sinergismo Farmacológico , Regulação da Expressão Gênica , Humanos , Imidazóis/metabolismo , Modelos Biológicos , Piperazinas/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Piridinas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Supressora de Tumor p53/genética
9.
Cancer Sci ; 111(9): 3111-3121, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32639661

RESUMO

Cancer cells are often characterized by abnormalities in DNA damage response including defects in cell cycle checkpoints and/or DNA repair. Synthetic lethality between DNA damage repair (DDR) pathways has provided a paradigm for cancer therapy by targeting DDR. The successful example is that cancer cells with BRCA1/2 mutations are sensitized to poly(adenosine diphosphate [ADP]-ribose)polymerase (PARP) inhibitors. Beyond the narrow scope of defects in the BRCA pathway, "BRCAness" provides more opportunities for synthetic lethality strategy. In human pancreatic cancer, frequent mutations were found in cell cycle and DDR genes, including P16, P73, APC, MLH1, ATM, PALB2, and MGMT. Combined DDR inhibitors and chemotherapeutic agents are under preclinical or clinical trials. Promoter region methylation was found frequently in cell cycle and DDR genes. Epigenetics joins the Knudson's "hit" theory and "BRCAness." Aberrant epigenetic changes in cell cycle or DDR regulators may serve as a new avenue for synthetic lethality strategy in pancreatic cancer.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias Pancreáticas/etiologia , Mutações Sintéticas Letais , Animais , Ciclo Celular/genética , Quimiorradioterapia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Suscetibilidade a Doenças , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais
10.
Nat Commun ; 11(1): 3521, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665556

RESUMO

Microtubules (MTs) mediate mitosis, directional signaling, and are therapeutic targets in cancer. Yet in vivo analysis of cancer cell MT behavior within the tumor microenvironment remains challenging. Here we developed an imaging pipeline using plus-end tip tracking and intravital microscopy to quantify MT dynamics in live xenograft tumor models. Among analyzed features, cancer cells in vivo displayed higher coherent orientation of MT dynamics along their cell major axes compared with 2D in vitro cultures, and distinct from 3D collagen gel cultures. This in vivo MT phenotype was reproduced in vitro when cells were co-cultured with IL4-polarized MΦ. MΦ depletion, MT disruption, targeted kinase inhibition, and altered MΦ polarization via IL10R blockade all reduced MT coherence and/or tumor cell elongation. We show that MT coherence is a defining feature for in vivo tumor cell dynamics and migration, modulated by local signaling from pro-tumor macrophages.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Feminino , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Mitose/genética , Mitose/fisiologia , Análise de Componente Principal , Células RAW 264.7
11.
PLoS One ; 15(7): e0234795, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645018

RESUMO

Forkhead box L2 (FOXL2) is a single-exon gene encoding a forkhead transcription factor, which is mainly expressed in the ovary, eyelids and the pituitary gland. FOXL2 plays an essential role in ovarian development. To reveal the effects of FOXL2 on the biological process and gene expression of ovarian granulosa cells (GCs), we established stable FOXL2-knockdown GCs and then analysed them using transcriptome sequencing. It was observed that knocking down FOXL2 affected the biological processes of cell proliferation, DNA replication, and apoptosis and affected cell cycle progression. FOXL2 knockdown promoted cell proliferation and DNA replication, decreased cell apoptosis, and promoted mitosis. In addition, by comparing the transcriptome after FOXL2 knockdown, we found a series of DEGs (differentially expressed genes) and related pathways. These results indicated that, through mediating these genes and pathways, the FOXL2 might induce the cell proliferation, cycle, and DNA replication, and play a key role during ovarian development and maintenance.


Assuntos
Proteína Forkhead Box L2/genética , Proteína Forkhead Box L2/metabolismo , Ovário/metabolismo , Animais , Ciclo Celular/genética , Divisão Celular/genética , Proliferação de Células/genética , Galinhas/genética , Replicação do DNA/genética , Feminino , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/genética , Células da Granulosa/metabolismo , Folículo Ovariano/metabolismo , RNA Mensageiro/genética , Transcriptoma , Sequenciamento Completo do Exoma
12.
Medicine (Baltimore) ; 99(24): e19905, 2020 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-32541446

RESUMO

BACKGROUND: A group of differentially expressed long non-coding RNAs (lncRNAs) have been shown to play key roles in osteoarthritis (OA), although they represented only a small proportion of lncRNAs that may be biologically and physiologically relevant. Since our knowledge of regulatory functions of non-coding RNAs is still limited, it is important to gain better understanding of their relation to the pathogenesis of OA. METHODS: We performed mRNA and lncRNA microarray analysis to detect differentially expressed RNAs in chondrocytes from three OA patients compared with four healthy controls. Then, enrichment analysis of the differentially expressed mRNAs was carried out to define disease molecular networks, pathways and gene ontology (GO) function. Furthermore, target gene prediction based on the co-expression network was performed to reveal the potential relationships between lncRNAs and mRNAs, contributing an exploration of a role of lncRNAs in OA mechanism. Quantitative RT-PCR analyses were used to demonstrate the reliability of the experimental results. FINDINGS: Altogether 990 lncRNAs (666 up-regulated and 324 down-regulated) and 546 mRNAs (419 up-regulated and 127 down-regulated) were differentially expressed in OA samples compared with the normal ones. The enrichment analysis revealed a set of genes involved in cell cycle. In total, 854 pairs of mRNA and lncRNA were highly linked, and further target prediction appointed 12 genes specifically for their corresponding lncRNAs. The lncRNAs lncRNA-CTD-2184D3.4, ENST00000564198.1, and ENST00000520562.1 were predicted to regulate SPC24, GALM, and ZNF345 mRNA expressions in OA. INTERPRETATION: This study uncovered several novel genes potentially important in pathogenesis of OA, and forecast the potential function of lnc-CTD-2184D3.4, especially for the cell cycle in the chondrocytes. These findings may promote additional aspects in studies of OA.


Assuntos
Ciclo Celular/genética , Condrócitos/metabolismo , Osteoartrite/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Adulto , Grupo com Ancestrais do Continente Asiático/etnologia , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , Feminino , Ontologia Genética , Redes Reguladoras de Genes/genética , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/fisiopatologia , Reprodutibilidade dos Testes , Projetos de Pesquisa
13.
Proc Natl Acad Sci U S A ; 117(27): 15659-15665, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32581119

RESUMO

Cell size is believed to influence cell growth and metabolism. Consistently, several studies have revealed that large cells have lower mass accumulation rates per unit mass (i.e., growth efficiency) than intermediate-sized cells in the same population. Size-dependent growth is commonly attributed to transport limitations, such as increased diffusion timescales and decreased surface-to-volume ratio. However, separating cell size- and cell cycle-dependent growth is challenging. To address this, we monitored growth efficiency of pseudodiploid mouse lymphocytic leukemia cells during normal proliferation and polyploidization. This was enabled by the development of large-channel suspended microchannel resonators that allow us to monitor buoyant mass of single cells ranging from 40 pg (small pseudodiploid cell) to over 4,000 pg, with a resolution ranging from ∼1% to ∼0.05%. We find that cell growth efficiency increases, plateaus, and then decreases as cell cycle proceeds. This growth behavior repeats with every endomitotic cycle as cells grow into polyploidy. Overall, growth efficiency changes 33% throughout the cell cycle. In contrast, increasing cell mass by over 100-fold during polyploidization did not change growth efficiency, indicating exponential growth. Consistently, growth efficiency remained constant when cell cycle was arrested in G2 Thus, cell cycle is a primary determinant of growth efficiency. As growth remains exponential over large size scales, our work finds no evidence for transport limitations that would decrease growth efficiency.


Assuntos
Técnicas Biossensoriais , Crescimento Celular , Proliferação de Células/genética , Leucemia Linfoide/genética , Animais , Ciclo Celular/genética , Divisão Celular/genética , Linhagem Celular Tumoral , Humanos , Leucemia Linfoide/patologia , Camundongos , Técnicas Analíticas Microfluídicas , Poliploidia
14.
Nat Commun ; 11(1): 2919, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522980

RESUMO

Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. Centromeric transcription is required for stable incorporation of the centromere-specific histone dCENP-A in M/G1 phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes. Here we demonstrate that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and dCENP-A mutants carrying phosphomimetic residues alleviate this association. Retention of phosphomimetic dCENP-A mutants is reduced relative to wildtype, while non-phosphorylatable dCENP-A retention is increased and accumulates at the centromere. We conclude that Spt6 acts as a conserved CENP-A maintenance factor that ensures long-term stability of epigenetic centromere identity during transcription-mediated chromatin remodeling.


Assuntos
Proteína Centromérica A/metabolismo , Proteínas de Drosophila/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular , Proteína Centromérica A/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Drosophila , Proteínas de Drosophila/genética , Citometria de Fluxo , Imunofluorescência , Células HeLa , Humanos , Imunoprecipitação , Mitose/genética , Mitose/fisiologia , Fatores de Alongamento de Peptídeos/genética , Fatores de Transcrição/genética
15.
Anticancer Res ; 40(5): 2675-2685, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366412

RESUMO

BACKGROUND/AIM: To evaluate the anti-cancer mechanism of N-Farnesyl-norcantharimide (NC15). MATERIALS AND METHODS: The viability of NC15-treated human leukemic Jurkat T (JKT) cells was assessed using the Kit-8 cell counting method. Flow cytometry analysis, human apoptosis antibody array assay, and whole genome sequencing were adopted to investigate the mechanism underlying the anti-cancer activity of NC15 in JKT cells. RESULTS: The growth inhibition rates of NC15 in JKT cells were about 80% and 95% after treatment with 8 µmol/l NC15 for 24 and 48 h, respectively. The percentages of NC15-treated JKT cells in the sub-G1 phase at 24 and 48 h were 22.0% and 34.3%, respectively, in contrast to the 1.5% in the control. Next-generation sequencing showed that many tumor suppressor genes (TSG) were up-regulated, while many genes associated with steroid biosynthesis, metabolic pathways, and fatty acid metabolism were down-regulated. CONCLUSION: NC15 can reduce the cell viability and increase the percentage of JKT cells in the sub-G1 phase by up-regulating TSG and related genes, and down-regulating the genes for steroid biosynthesis, metabolic pathways and fatty acid metabolism, instead of through apoptosis.


Assuntos
Cantaridina/análogos & derivados , Regulação para Baixo/efeitos dos fármacos , Ácidos Graxos/metabolismo , Genes Supressores de Tumor , Redes e Vias Metabólicas/genética , Esteroides/biossíntese , Linfócitos T/citologia , Regulação para Cima/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Cantaridina/química , Cantaridina/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Regulação para Baixo/genética , Humanos , Células Jurkat , Redes e Vias Metabólicas/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Regulação para Cima/genética
16.
Toxicol Appl Pharmacol ; 399: 115054, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32422326

RESUMO

Radiation-induced rectal injury is a major side-effect observed in patients with pelvic malignancies who receive radiotherapy. MicroRNA (miRNA), involved in many cellular biological processes, can be disturbed by ionizing radiation (IR). In this study, we have investigated the function of microRNA-122-5p (miR-122-5p) in radiation-induced rectal injury. MiR-122-5p levels in the serum of rectal cancer patients or in the rectal tissues of C57BL/6 mice before and after IR were detected by quantitative real-time PCR (qRT-PCR). We found that the miR-122-5p levels were significantly up-regulated in patients' serum or in mice rectal tissues after IR. Elevation of miR-122-5p levels sensitized human intestinal epithelial crypt (HIEC) cells to IR both in vitro and in vivo. MiR-122-5p mimic was transfected to HIEC cells and the downstream targets were predicted by bioinformatic analysis. Two putative target sites of miR-122-5p in the 3'UTR of the cell cycle and apoptosis regulator 1 (CCAR1) mRNA were found and verified by luciferase reporter assay. Overexpression of miR-122-5p or silencing CCAR1 combined with IR significantly inhibited cell survival, enhanced radiosensitivity, and increased cell apoptosis compared to that in the negative control group in vitro. In vivo injection of miR-122-5p antagomir after IR significantly alleviated radiation-induced rectal injury in mice. These results suggest that miR-122-5p aggravates radiation-induced rectal injury through targeting CCAR1.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteínas de Ciclo Celular/genética , MicroRNAs/genética , Lesões por Radiação/genética , Tolerância a Radiação/genética , Reto/efeitos da radiação , Regiões 3' não Traduzidas/genética , Animais , Apoptose/genética , Ciclo Celular/genética , Linhagem Celular , Proliferação de Células/genética , Sobrevivência Celular/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , RNA Mensageiro/genética , Regulação para Cima/genética
17.
RNA ; 26(9): 1104-1117, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32393525

RESUMO

Noncoding RNA has a proven ability to direct and regulate chromatin modifications by acting as scaffolds between DNA and histone-modifying complexes. However, it is unknown if ncRNA plays any role in DNA replication and epigenome maintenance, including histone eviction and reinstallment of histone modifications after genome duplication. Isolation of nascent chromatin has identified a large number of RNA-binding proteins in addition to unknown components of the replication and epigenetic maintenance machinery. Here, we isolated and characterized long and short RNAs associated with nascent chromatin at active replication forks and track RNA composition during chromatin maturation across the cell cycle. Shortly after fork passage, GA-rich-, alpha- and TElomeric Repeat-containing RNAs (TERRA) are associated with replicated DNA. These repeat containing RNAs arise from loci undergoing replication, suggesting an interaction in cis. Post-replication during chromatin maturation, and even after mitosis in G1, the repeats remain enriched on DNA. This suggests that specific types of repeat RNAs are transcribed shortly after DNA replication and stably associate with their loci of origin throughout the cell cycle. The presented method and data enable studies of RNA interactions with replication forks and post-replicative chromatin and provide insights into how repeat RNAs and their engagement with chromatin are regulated with respect to DNA replication and across the cell cycle.


Assuntos
Replicação do DNA/genética , DNA/genética , Processamento de Proteína Pós-Traducional/genética , RNA/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Cromatina/genética , Células HeLa , Histonas/genética , Humanos
18.
Clin Sci (Lond) ; 134(11): 1279-1293, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32463458

RESUMO

Long non-coding RNAs (lncRNAs) play important roles in hematological malignancies. We have previously identified several differentially expressed lncRNAs in myelodysplastic syndromes (MDS) by microarray analysis. In the present study, we explored the regulatory circuitry, potential functions, clinical and prognostic relevance of these lncRNAs in MDS by developing a lncRNA regulation network. We identified a novel lncRNA, LOC101928834, which was significantly up-regulated in the bone marrow of patients with MDS and acute myeloid leukemia (AML). We further evaluated the clinical relevance of LOC101928834 in 89 MDS and 110 AML patients and found that higher level of LOC101928834 expression was associated with higher white blood cell count, higher blast percentage, the subtype of refractory cytopenia with excess blasts (RAEB) and shorter overall survival in MDS patients. Receiver operating characteristic (ROC) curve analysis showed that LOC101928834 expression could discriminate MDS-RAEB patients from control with an area under the receiver-operating curve (AUC) of 0.9048. Moreover, functional analysis showed that LOC101928834 promoted cell proliferation and cell cycle progression, and activated Wnt/ß-catenin signaling pathway in vitro. In conclusion, LOC101928834 expression is correlated with clinical and biological features of MDS and may serve as a novel diagnostic and prognostic biomarker.


Assuntos
Síndromes Mielodisplásicas/genética , RNA Longo não Codificante/metabolismo , Via de Sinalização Wnt/genética , Adulto , Medula Óssea/patologia , Ciclo Celular/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Feminino , Redes Reguladoras de Genes , Humanos , Células K562 , Masculino , Pessoa de Meia-Idade , RNA Longo não Codificante/genética , Células THP-1 , Resultado do Tratamento , Regulação para Cima/genética
19.
NPJ Syst Biol Appl ; 6(1): 11, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376972

RESUMO

Over the last 30 years, computational biologists have developed increasingly realistic mathematical models of the regulatory networks controlling the division of eukaryotic cells. These models capture data resulting from two complementary experimental approaches: low-throughput experiments aimed at extensively characterizing the functions of small numbers of genes, and large-scale genetic interaction screens that provide a systems-level perspective on the cell division process. The former is insufficient to capture the interconnectivity of the genetic control network, while the latter is fraught with irreproducibility issues. Here, we describe a hybrid approach in which the 630 genetic interactions between 36 cell-cycle genes are quantitatively estimated by high-throughput phenotyping with an unprecedented number of biological replicates. Using this approach, we identify a subset of high-confidence genetic interactions, which we use to refine a previously published mathematical model of the cell cycle. We also present a quantitative dataset of the growth rate of these mutants under six different media conditions in order to inform future cell cycle models.


Assuntos
Ciclo Celular/genética , Saccharomyces cerevisiae/genética , Divisão Celular/genética , Biologia Computacional/métodos , Epistasia Genética/genética , Regulação Fúngica da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Ensaios de Triagem em Larga Escala/métodos , Modelos Teóricos , Proteínas de Saccharomyces cerevisiae/genética
20.
Cancer Sci ; 111(7): 2400-2412, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32391593

RESUMO

Escape of cancer cells from chemotherapy is a problem in the management of cancer patients. Research on chemotherapy resistance has mainly focused on the heterogeneity of cancer cells, multiple gene mutations, and quiescence of malignant cancer cells. However, some studies have indicated that interactions between cancer cells and vascular cells promote resistance to chemotherapy. Here, we established mouse leukemia models using the cell lines THP-1 or MEG-1. These were derived from acute and chronic myeloid leukemias, respectively, and highly expressed DNA replication factor PSF1, a member of the GINS complex. We found that, after anti-cancer drug administration, surviving GFP-positive leukemia cells in the bone marrow were located adjacent to blood vessels, as previously reported in a subcutaneous solid tumor transplantation model. Treating THP-1 and MEG-1 cells with anti-cancer drugs in vitro revealed that those most strongly expressing PSF1 were most chemoresistant, suggesting that PSF1 induces not only cell cycle progression but also facilitates cell survival. Indeed, when PSF1 expression was suppressed by shRNA, the growth rate was reduced and cell death was enhanced in both cell lines. Furthermore, PSF1 knockdown in leukemia cells led to a change in their location at a distance from the blood vessels in a bone marrow transplantation model. These findings potentially reflect a mechanism of escape of leukemic cells from chemotherapy and suggest that PSF1 may be a possible therapeutic target to enhance the effect of chemotherapy.


Assuntos
Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Ciclo Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Leucemia/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Ensaios Antitumorais Modelo de Xenoenxerto
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