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1.
Immunity ; 54(11): 2465-2480.e5, 2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34706222

RESUMO

Epigenetic reprogramming underlies specification of immune cell lineages, but patterns that uniquely define immune cell types and the mechanisms by which they are established remain unclear. Here, we identified lineage-specific DNA methylation signatures of six immune cell types from human peripheral blood and determined their relationship to other epigenetic and transcriptomic patterns. Sites of lineage-specific hypomethylation were associated with distinct combinations of transcription factors in each cell type. By contrast, sites of lineage-specific hypermethylation were restricted mostly to adaptive immune cells. PU.1 binding sites were associated with lineage-specific hypo- and hypermethylation in different cell types, suggesting that it regulates DNA methylation in a context-dependent manner. These observations indicate that innate and adaptive immune lineages are specified by distinct epigenetic mechanisms via combinatorial and context-dependent use of key transcription factors. The cell-specific epigenomics and transcriptional patterns identified serve as a foundation for future studies on immune dysregulation in diseases and aging.


Assuntos
Metilação de DNA , Epigênese Genética , Epigenômica , Regulação da Expressão Gênica , Imunidade , Fatores de Transcrição/metabolismo , Transcriptoma , Epigenômica/métodos , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Fatores de Transcrição/genética
3.
Immunity ; 30(6): 912-25, 2009 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-19523850

RESUMO

Memory lymphocytes are characterized by their ability to exhibit a rapid response to the recall antigen, in which differential transcription is important, yet the underlying mechanism is not understood. We report here a genome-wide analysis of histone methylation on two histone H3 lysine residues (H3K4me3 and H3K27me3) and gene expression profiles in naive and memory CD8(+) T cells. We found that specific correlation exists between gene expression and the amounts of H3K4me3 (positive correlation) and H3K27me3 (negative correlation) across the gene body. These correlations displayed four distinct modes (repressive, active, poised, and bivalent), reflecting different functions of these genes in CD8(+) T cells. Furthermore, a permissive chromatin state of each gene was established by a combination of different histone modifications. Our findings reveal a complex regulation by histone methylation in differential gene expression and suggest that histone methylation may be responsible for memory CD8(+) T cell function.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/genética , Cromatina/metabolismo , Histonas/metabolismo , Memória Imunológica/genética , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/imunologia , Cromatina/imunologia , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Histonas/imunologia , Humanos , Metilação
4.
J Immunol ; 196(8): 3385-97, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26983789

RESUMO

B cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL(+)MHC class-I(Hi)CD86(Hi)B cells of unknown origin. In this article, we report that these cells, termed 4BL cells, are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. They induce expression and activation of 4-1BBL and IFN-γR1 on B1a cells to subsequently upregulate membrane TNF-α and CD86. As a result, activated B1a/4BL cells induce expression of granzyme B in CD8(+)T cells by targeting TNFR2 via membrane TNF-α and providing costimulation with CD86. Thus, for the first time, to our knowledge, these results indicate that aging affects the function of B1a cells. Upon aging, these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8(+)T cells.


Assuntos
Envelhecimento/imunologia , Subpopulações de Linfócitos B/citologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Ligante 4-1BB/metabolismo , Adulto , Idoso , Animais , Autoimunidade/imunologia , Subpopulações de Linfócitos B/imunologia , Antígeno B7-2/biossíntese , Antígeno B7-2/metabolismo , Células Cultivadas , Senescência Celular , Ativação Enzimática/imunologia , Feminino , Granzimas/biossíntese , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Receptores de Interferon/biossíntese , Receptores de Interferon/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Receptor de Interferon gama
5.
Nature ; 464(7290): 858-63, 2010 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-20336070

RESUMO

Exceptional genomic stability is one of the hallmarks of mouse embryonic stem (ES) cells. However, the genes contributing to this stability remain obscure. We previously identified Zscan4 as a specific marker for two-cell embryo and ES cells. Here we show that Zscan4 is involved in telomere maintenance and long-term genomic stability in ES cells. Only 5% of ES cells express Zscan4 at a given time, but nearly all ES cells activate Zscan4 at least once during nine passages. The transient Zscan4-positive state is associated with rapid telomere extension by telomere recombination and upregulation of meiosis-specific homologous recombination genes, which encode proteins that are colocalized with ZSCAN4 on telomeres. Furthermore, Zscan4 knockdown shortens telomeres, increases karyotype abnormalities and spontaneous sister chromatid exchange, and slows down cell proliferation until reaching crisis by passage eight. Together, our data show a unique mode of genome maintenance in ES cells.


Assuntos
Células-Tronco Embrionárias/metabolismo , Instabilidade Genômica , Telômero/genética , Telômero/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Aberrações Cromossômicas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/patologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Cariotipagem , Meiose/genética , Meiose/fisiologia , Camundongos , Transporte Proteico , Recombinação Genética/genética , Troca de Cromátide Irmã/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Regulação para Cima
6.
Mol Cell Proteomics ; 11(8): 303-16, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22493178

RESUMO

Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.


Assuntos
Separação Celular/métodos , Glicoproteínas/análise , Imunofenotipagem/métodos , Proteínas de Membrana/análise , Células-Tronco Pluripotentes/metabolismo , Proteômica/métodos , Animais , Células Cultivadas , Receptor gp130 de Citocina/análise , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/transplante , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Espectrometria de Massas , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Microscopia Confocal , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Teratoma/metabolismo , Teratoma/patologia
7.
Nucleic Acids Res ; 40(4): 1621-35, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22013166

RESUMO

DNA decatenation mediated by Topoisomerase II is required to separate the interlinked sister chromatids post-replication. SGS1, a yeast homolog of the human RecQ family of helicases interacts with Topoisomerase II and plays a role in chromosome segregation, but this functional interaction has yet to be identified in higher organisms. Here, we report a physical and functional interaction of Topoisomerase IIα with RECQL5, one of five mammalian RecQ helicases, during DNA replication. Direct interaction of RECQL5 with Topoisomerase IIα stimulates the decatenation activity of Topoisomerase IIα. Consistent with these observations, RECQL5 co-localizes with Topoisomerase IIα during S-phase of the cell cycle. Moreover, cells with stable depletions of RECQL5 display a slow proliferation rate, a G2/M cell cycle arrest and late S-phase cycling defects. Metaphase spreads generated from RECQL5-depleted cells exhibit undercondensed and entangled chromosomes. Further, RECQL5-depleted cells activate a G2/M checkpoint and undergo apoptosis. These phenotypes are similar to those observed when Topoisomerase II catalytic activity is inhibited. These results reveal an important role for RECQL5 in the maintenance of genomic stability and a new insight into the decatenation process.


Assuntos
Antígenos de Neoplasias/metabolismo , Ciclo Celular , DNA Topoisomerases Tipo II/metabolismo , DNA Catenado/metabolismo , Proteínas de Ligação a DNA/metabolismo , RecQ Helicases/metabolismo , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Proliferação de Células , Aberrações Cromossômicas , Exodesoxirribonucleases/metabolismo , Humanos , Metáfase/genética , RecQ Helicases/antagonistas & inibidores , Helicase da Síndrome de Werner
8.
J Pharmacol Exp Ther ; 343(1): 157-66, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22776956

RESUMO

Inhibition of cell proliferation by fenoterol and fenoterol derivatives in 1321N1 astrocytoma cells is consistent with ß(2)-adrenergic receptor (ß(2)-AR) stimulation. However, the events that result in fenoterol-mediated control of cell proliferation in other cell types are not clear. Here, we compare the effect of the ß(2)-AR agonists (R,R')-fenoterol (Fen) and (R,R')-4-methoxy-1-naphthylfenoterol (MNF) on signaling and cell proliferation in HepG2 hepatocarcinoma cells by using Western blotting and [(3)H]thymidine incorporation assays. Despite the expression of ß(2)-AR, no cAMP accumulation was observed when cells were stimulated with isoproterenol or Fen, although the treatment elicited both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt activation. Unexpectedly, isoproterenol and Fen promoted HepG2 cell growth, but MNF reduced proliferation together with increased apoptosis. The mitogenic responses of Fen were attenuated by 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118,551), a ß(2)-AR antagonist, whereas those of MNF were unaffected. Because of the coexpression of ß(2)-AR and cannabinoid receptors (CBRs) and their impact on HepG2 cell proliferation, these Gα(i)/Gα(o)-linked receptors may be implicated in MNF signaling. Cell treatment with (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a synthetic agonist of CB(1)R and CB(2)R, led to growth inhibition, whereas inverse agonists of these receptors blocked MNF mitogenic responses without affecting Fen signaling. MNF responses were sensitive to pertussis toxin. The ß(2)-AR-deficient U87MG cells were refractory to Fen, but responsive to the antiproliferative actions of MNF and WIN 55,212-2. The data indicate that the presence of the naphthyl moiety in MNF results in functional coupling to the CBR pathway, providing one of the first examples of a dually acting ß(2)-AR-CBR ligand.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Apoptose/fisiologia , Carcinoma Hepatocelular/metabolismo , Fenoterol/farmacologia , Neoplasias Hepáticas/metabolismo , Receptores de Canabinoides/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Fenoterol/química , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia
9.
FASEB J ; 25(10): 3634-45, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21685331

RESUMO

Krüppel-like factor 4 (KLF4), a transcription factor, plays a key role in the pluripotency of stem cells. We sought to determine the function of KLF4 in T-cell development and differentiation by using T-cell-specific Klf4-knockout (KO) mice. We found that KLF4 was highly expressed in thymocytes and mature T cells and was rapidly down-regulated in mature T cells after activation. In Klf4-KO mice, we observed a modest reduction of thymocytes (27%) due to the reduced proliferation of double-negative (DN) thymocytes. We demonstrated that a direct repression of Cdkn1b by KLF4 was a cause of decreased DN proliferation. During in vitro T-cell differentiation, we observed significant reduction of IL-17-expressing CD4(+) T cells (Th17; 24%) but not in other types of Th differentiation. The reduction of Th17 cells resulted in a significant attenuation of the severity (35%) of experimental autoimmune encephalomyelitis in vivo in Klf4-KO mice as compared with the Klf4 wild-type littermates. Finally, we demonstrated that KLF4 directly binds to the promoter of Il17a and positively regulates its expression. In summary, these findings identify KLF4 as a critical regulator in T-cell development and Th17 differentiation.


Assuntos
Interleucina-17/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Timo/citologia , Animais , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação da Expressão Gênica/fisiologia , Interleucina-17/genética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Knockout
10.
Elife ; 112022 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-35507394

RESUMO

Many age-associated changes in the human hematopoietic system have been reproduced in murine models; however, such changes have not been as robustly explored in rats despite the fact these larger rodents are more physiologically similar to humans. We examined peripheral blood of male F344 rats ranging from 3 to 27 months of age and found significant age-associated changes with distinct leukocyte population shifts. We report CD25+ CD4+ population frequency is a strong predictor of healthy aging, generate a model using blood parameters, and find rats with blood profiles that diverge from chronologic age indicate debility; thus, assessments of blood composition may be useful for non-lethal disease profiling or as a surrogate measure for efficacy of aging interventions. Importantly, blood parameters and DNA methylation alterations, defined distinct juncture points during aging, supporting a non-linear aging process. Our results suggest these inflection points are important considerations for aging interventions. Overall, we present rat blood aging metrics that can serve as a resource to evaluate health and the effects of interventions in a model system physiologically more reflective of humans.


Our blood contains many types of white blood cells, which play important roles in defending the body against infections and other threats to our health. The number of these cells changes with age, and this in turn contributes to many other alterations that happen in the body as we get older. For example, the immune system generally gets weaker at fighting infections and preventing other cells from developing into cancer. On top of that, the white blood cells themselves can become cancerous, resulting in several types of blood cancer that are more likely to happen in older people. Many previous studies have examined how the number of white blood cells changes with age in humans and mice. However, our understanding of this process in rats is still poor, despite the fact that the way the human body works has more in common with the rat body than the mouse body. Here, Yanai, Dunn et al. have studied samples of blood from rats between three to 27 months old. The experiments found that it is possible to accurately predict the age of healthy rats by measuring the frequency of populations of white blood cells, especially a certain type known as CD25+ CD4+ cells. If the animals had any form of illness, their predicted age deviated from their actual age. Furthermore, while some changes in the blood were gradual and continuous, others displayed distinct shifts when the rats reached specific ages. In the future, these findings may be used as a tool to help researchers diagnose illnesses in rats before the animals develop symptoms, or to more easily establish if a treatment is having a positive effect on the rats' health. The work of Yanai, Dunn et al. also provides new insights into aging that could potentially aid the design of new screening methods to predict cancer and intervene using a model system that is more similar to humans.


Assuntos
Envelhecimento , Leucócitos , Envelhecimento/genética , Animais , Metilação de DNA , Masculino , Camundongos , Dinâmica Populacional , Ratos , Ratos Endogâmicos F344
11.
Curr Biol ; 18(19): 1489-94, 2008 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-18818083

RESUMO

Removal of TRF2, a telomere shelterin protein, recapitulates key aspects of telomere attrition including the DNA-damage response and cell-cycle arrest [1]. Distinct from the response of proliferating cells to loss of TRF2 [2, 3], in rodent noncycling cells, TRF2 inhibition promotes differentiation and growth [4, 5]. However, the mechanism that couples telomere gene-silencing features [6-8] to differentiation programs has yet to be elucidated. Here we describe an extratelomeric function of TRF2 in the regulation of neuronal genes mediated by the interaction of TRF2 with repressor element 1-silencing transcription factor (REST), a master repressor of gene networks devoted to neuronal functions [9-12]. TRF2-REST complexes are readily detected by coimmunoprecipitation assays and are localized to aggregated PML-nuclear bodies in undifferentiated pluripotent human NTera2 stem cells. Inhibition of TRF2, either by a dominant-negative mutant or by RNA interference, dissociates TRF2-REST complexes resulting in ubiquitin-proteasomal degradation of REST. Consequentially, REST-targeted neural genes (L1CAM, beta3-tubulin, synaptophysin, and others) are derepressed, resulting in acquisition of neuronal phenotypes. Notably, selective damage to telomeres without affecting TRF2 levels causes neither REST degradation nor cell differentiation. Thus, in addition to protecting telomeres, TRF2 possesses a novel role in stabilization of REST thereby controlling neural tumor and stem cell fate.


Assuntos
Diferenciação Celular , Inativação Gênica , Neurônios/citologia , Proteínas Repressoras/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo
13.
Neuron ; 41(4): 549-61, 2004 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-14980204

RESUMO

Increasing evidence indicates that neurodegeneration involves the activation of the cell cycle machinery in postmitotic neurons. However, the purpose of these cell cycle-associated events in neuronal apoptosis remains unknown. Here we tested the hypothesis that cell cycle activation is a critical component of the DNA damage response in postmitotic neurons. Different genotoxic compounds (etoposide, methotrexate, and homocysteine) induced apoptosis accompanied by cell cycle reentry of terminally differentiated cortical neurons. In contrast, apoptosis initiated by stimuli that do not target DNA (staurosporine and colchicine) did not initiate cell cycle activation. Suppression of the function of ataxia telangiectasia mutated (ATM), a proximal component of DNA damage-induced cell cycle checkpoint pathways, attenuated both apoptosis and cell cycle reentry triggered by DNA damage but did not change the fate of neurons exposed to staurosporine and colchicine. Our data suggest that cell cycle activation is a critical element of the DNA damage response of postmitotic neurons leading to apoptosis.


Assuntos
Apoptose/genética , Ciclo Celular/genética , Dano ao DNA/genética , Degeneração Neural/genética , Neurônios/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular , Células Cultivadas , Colchicina/farmacologia , Dano ao DNA/efeitos dos fármacos , Proteínas de Ligação a DNA , Etoposídeo/farmacologia , Feminino , Homocisteína/farmacologia , Masculino , Metotrexato/farmacologia , Camundongos , Degeneração Neural/metabolismo , Neurônios/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Ratos , Estaurosporina/farmacologia , Proteínas Supressoras de Tumor
14.
Cell Tissue Res ; 331(1): 5-22, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18026755

RESUMO

Embryonic stem (ES) cells derived from pre-implantation embryos have the potential to differentiate into any cell type derived from the three germ layers of ectoderm (epidermal tissues and nerves), mesoderm (muscle, bone, blood), and endoderm (liver, pancreas, gastrointestinal tract, lungs), including fetal and adult cells. Alone, these cells do not develop into a viable fetus or adult animal because they do not retain the potential to contribute to extraembryonic tissue, and in vitro, they lack spatial and temporal signaling cues essential to normal in vivo development. The basis of pluripotentiality resides in conserved regulatory networks composed of numerous transcription factors and multiple signaling cascades. Together, these regulatory networks maintain ES cells in a pluripotent and undifferentiated form; however, alterations in the stoichiometry of these signals promote differentiation. By taking advantage of this differentiation capacity in vitro, ES cells have clearly been shown to possess the potential to generate multipotent stem and progenitor cells capable of differentiating into a limited number of cell fates. These latter types of cells may prove to be therapeutically viable, but perhaps more importantly, the studies of these cells have led to a greater understanding of mammalian development.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Animais , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Humanos , Fator Inibidor de Leucemia/farmacologia , Camundongos , Fosforilação/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Proteoma/metabolismo , Soro , Fatores de Transcrição/metabolismo
15.
Front Immunol ; 9: 549, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29632530

RESUMO

Transition from resting to cell cycle in response to antigenic stimulation is an essential step for naïve CD8+ T cells to differentiate to effector and memory cells. Leaving the resting state requires dramatic changes of chromatin status in the key cell cycle inhibitors but the details of these concerted events are not fully elucidated. Here, we showed that Ezh2, an enzymatic component of polycomb repressive complex 2 (PRC2) catalyzing the trimethylation of lysine 27 on histone 3 (H3K27me3), regulates activation induced naïve CD8+ T cells proliferation and apoptosis. Upon deletion of Ezh2 during thymocyte development (Ezh2fl/flCd4Cre+ mice), naive CD8+ T cells displayed impaired proliferation and increased apoptosis in response to antigen stimulation. However, naive CD8+ T cells only had impaired proliferation but no increase in apoptosis when Ezh2 was deleted after activation (Ezh2fl/flGzmBCre+ mice), suggesting cell cycle and apoptosis are temporally separable events controlled by Ezh2. We then showed that deletion of Ezh2 resulted in the increase in expression of cyclin-dependent kinase inhibitors Cdkn2a (p16 and Arf) and Cdkn1c (p57) in activated naïve CD8+ T cells as the consequence of reduced levels of H3K27me3 at these two gene loci. Finally, with real time imaging, we observed prolonged cell division times of naïve CD8+ T cells in the absence of Ezh2 post in vitro stimulation. Together, these findings reveal that repression of Cdkn1c and Cdkn2a by Ezh2 plays a critical role in execution of activation-induced CD8+ T cell proliferation.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Ciclo Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Inibidor de Quinase Dependente de Ciclina p57/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste/fisiologia , Animais , Antígenos/imunologia , Apoptose , Proliferação de Células , Listeria monocytogenes , Listeriose/imunologia , Camundongos Knockout , Ovalbumina/imunologia
16.
Clin Cancer Res ; 12(2): 405-10, 2006 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-16428479

RESUMO

PURPOSE: Most breast cancers have chromosomal instability that seems related to defective mitotic spindle checkpoints. Because the molecular basis of this defect is unknown, we evaluated breast cancer cell lines and tissues for possible defects involving the major mitotic checkpoint genes responsible for maintaining chromosomal stability. EXPERIMENTAL DESIGN: We analyzed sequences and expression levels (RNA and protein) of eight major spindle checkpoint genes (MAD1L1, MAD2L1, MAD2L2, BUB1, BUB1B, BUB3, CDC20, and TTK) in a panel of 12 breast cancer cell lines, most with established genetic instability and defective spindle damage checkpoint response. mRNA levels of these genes were also measured in primary tumor samples, and immunohistochemical staining was used to evaluate BUB1B protein levels in a panel of 270 additional cases of breast cancer. RESULTS: No functionally significant sequence variations were found for any of the eight genes in the breast cancer cell lines with chromosomal instability. More surprisingly, the mRNA and protein levels for these checkpoint genes are significantly higher in the genetically unstable breast cancer cell lines and in high-grade primary breast cancer tissues than in the stable (and checkpoint proficient) MCF-10A and normal mammary epithelial cells, or in normal breast tissues. In fact, overexpression of the BUB1B protein is a marker that recognizes nearly 80% of breast cancers in paraffin-embedded tissues. CONCLUSIONS: Defective mitotic spindle checkpoints in breast cancer are most likely not caused by low expression or mutations of these eight checkpoint genes. High levels of these particular transcripts could represent a cellular compensation for defects in other molecular components of the mitotic spindle damage checkpoint, and increased expression of these genes might be markers of breast cancers with chromosomal instability.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Proteínas Quinases/genética , Fuso Acromático/genética , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fragilidade Cromossômica , Feminino , Variação Genética , Humanos , Mitose/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
17.
Environ Mol Mutagen ; 58(2): 84-98, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28181292

RESUMO

Base excision repair (BER) is the major pathway for coping with most forms of endogenous DNA damage, and defects in the process have been associated with carcinogenesis. Apurinic/apyrimidinic endonuclease 1 (APE1) is a central participant in BER, functioning as a critical endonuclease in the processing of noncoding abasic sites in DNA. Evidence has suggested that APE1 missense mutants, as well as altered expression or localization of the protein, can contribute to disease manifestation. We report herein that the tumor-associated APE1 variant, R237C, shows reduced complementation efficiency of the methyl methanesulfonate hypersensitivity and impaired cell growth exhibited by APE1-deficient mouse embryonic fibroblasts. Overexpression of wild-type APE1 or the R237C variant in the nontransformed C127I mouse cell line had no effect on proliferation, cell cycle status, steady-state DNA damage levels, mitochondrial function, or cellular transformation. A human cell line heterozygous for an APE1 knockout allele had lower levels of endogenous APE1, increased cellular sensitivity to DNA-damaging agents, impaired proliferation with time, and a distinct global gene expression pattern consistent with a stress phenotype. Our results indicate that: (i) the tumor-associated R237C variant is a possible susceptibility factor, but not likely a driver of cancer cell phenotypes, (ii) overexpression of APE1 does not readily promote cellular transformation, and (iii) haploinsufficiency at the APE1 locus can have profound cellular consequences, consistent with BER playing a critical role in proliferating cells. Environ. Mol. Mutagen. 58:84-98, 2017. © 2017 Wiley Periodicals, Inc.


Assuntos
Transformação Celular Neoplásica/genética , Dano ao DNA/genética , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Teste de Complementação Genética , Células HCT116 , Humanos , Mesilatos/farmacologia , Camundongos Transgênicos , Tamoxifeno/farmacologia
18.
Cell Signal ; 36: 176-188, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28495590

RESUMO

Activation of ß2-adrenergic receptor (ß2AR) and deorphanized GPR55 has been shown to modulate cancer growth in diverse tumor types in vitro and in xenograft models in vivo. (R,R')-4'-methoxy-1-naphthylfenoterol [(R,R')-MNF] is a bivalent compound that agonizes ß2AR but inhibits GPR55-mediated pro-oncogenic responses. Here, we investigated the molecular mechanisms underlying the anti-tumorigenic effects of concurrent ß2AR activation and GPR55 blockade in C6 glioma cells using (R,R')-MNF as a marker ligand. Our data show that (R,R')-MNF elicited G1-phase cell cycle arrest and apoptosis, reduced serum-inducible cell motility, promoted the phosphorylation of PKA target proteins, and inhibited constitutive activation of ERK and AKT in the low nanomolar range, whereas high nanomolar levels of (R,R')-MNF were required to block GPR55-mediated cell motility. siRNA knockdown and pharmacological inhibition of ß2AR activity were accompanied by significant upregulation of AKT and ERK phosphorylation, and selective alteration in (R,R')-MNF responsiveness. The effects of agonist stimulation of GPR55 on various readouts, including cell motility assays, were suppressed by (R,R')-MNF. Lastly, a significant increase in phosphorylation-mediated inactivation of ß-catenin occurred with (R,R')-MNF, and we provided new evidence of (R,R')-MNF-mediated inhibition of oncogenic ß-catenin signaling in a C6 xenograft tumor model. Thus, simultaneous activation of ß2AR and blockade of GPR55 may represent a novel therapeutic approach to combat the progression of glioblastoma cancer.


Assuntos
Neoplasias Encefálicas/metabolismo , Carcinogênese/metabolismo , Glioma/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Canabinoides/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colforsina/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fenoterol/análogos & derivados , Fenoterol/farmacologia , Glioma/patologia , Humanos , Isoproterenol/farmacologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Soro , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Neuromolecular Med ; 8(3): 389-414, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16775390

RESUMO

The high-metabolic demand of neurons and their reliance on glucose as an energy source places them at risk for dysfunction and death under conditions of metabolic and oxidative stress. Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins implicated in the regulation of mitochondrial membrane potential (Deltapsim) and cellular energy metabolism. The authors cloned UCP4 cDNA from mouse and rat brain, and demonstrate that UCP4 mRNA is expressed abundantly in brain and at particularly high levels in populations of neurons believed to have high-energy requirements. Neural cells with increased levels of UCP4 exhibit decreased Deltapsim, reduced reactive oxygen species (ROS) production and decreased mitochondrial calcium accumulation. UCP4 expressing cells also exhibited changes of oxygen-consumption rate, GDP sensitivity, and response of Deltapsim to oligomycin that were consistent with mitochondrial uncoupling. UCP4 modulates neuronal energy metabolism by increasing glucose uptake and shifting the mode of ATP production from mitochondrial respiration to glycolysis, thereby maintaining cellular ATP levels. The UCP4-mediated shift in energy metabolism reduces ROS production and increases the resistance of neurons to oxidative and mitochondrial stress. Knockdown of UCP4 expression by RNA interference in primary hippocampal neurons results in mitochondrial calcium overload and cell death. UCP4-mRNA expression is increased in neurons exposed to cold temperatures and in brain cells of rats maintained on caloric restriction, suggesting a role for UCP4 in the previously reported antiageing and neuroprotective effects of caloric restriction. By shifting energy metabolism to reduce ROS production and cellular reliance on mitochondrial respiration, UCP4 can protect neurons against oxidative stress and calcium overload.


Assuntos
Metabolismo Energético , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Cálcio/metabolismo , Restrição Calórica , Células Cultivadas , Temperatura Baixa , Glucose/metabolismo , Humanos , Hibridização In Situ , Canais Iônicos/química , Canais Iônicos/genética , Ácido Láctico/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas de Desacoplamento Mitocondrial , Dados de Sequência Molecular , Neurônios/citologia , Oxigênio/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/metabolismo , Alinhamento de Sequência
20.
FASEB J ; 19(1): 109-11, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15489248

RESUMO

Activation of p38 mitogen-activated protein kinase (MAPK) plays a central role in cellular responses to a multitude of stress signals. In the heart, enhanced p38 MAPK signaling has been implicated in cardiac hypoxic and ischemic injury. However, the mechanism underlying hypoxia-induced p38 MAPK activation remains elusive. We investigated p38 MAPK activation during hypoxia in adult rat cardiomyocytes. Here, we reported that hypoxia leads to concurrent intracellular acidosis and activation of p38 MAPK and that the hypoxia-induced p38 MAPK signaling can be fully abolished by neutralizing intracellular pH, whereas intracellular acidosis (intracellular pH<7.0) per se overtly augments activation of p38 MAPK but not ERK1/2 and JNK. Furthermore, inhibition of p38 MAPK protects myocytes against hypoxic cell death, suggesting that acidosis-evoked p38 MAPK signaling plays an important role in hypoxic cell injury and cell death. These results demonstrate, for the first time, that intracellular acidosis constitutes a necessary and sufficient link responsible for hypoxia-activated p38 MAPK signaling and the subsequent hypoxic cardiomyocyte injury and death.


Assuntos
Acidose/enzimologia , Hipóxia/etnologia , Miócitos Cardíacos/química , Miócitos Cardíacos/enzimologia , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Acidose/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Ativação Enzimática/fisiologia , Ventrículos do Coração/citologia , Concentração de Íons de Hidrogênio , MAP Quinase Quinase 3/metabolismo , MAP Quinase Quinase 6/metabolismo , Miócitos Cardíacos/patologia , Fosforilação , Ratos
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