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1.
Methods ; 208: 9-18, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36229002

RESUMO

Cellular heterogeneity is fundamental to both developmental differentiation and disease establishment. Recent advances in high-throughput single-cell technology have been rapidly revolutionizing the resolution of our understanding of development and disease. However, while the study of single-cell transcriptomes is easily accessible, the analysis of single-cell proteomes is still in its infancy. In this study, we describe simultaneous profiling of multiple regulatory proteins at a single-cell level using mass cytometry or cytometry by time of flight. We develop mass cytometry reagents to study key transcription factors, signaling proteins and chromatin modifiers that regulate mouse embryonic stem cells. Our data reveal that the protein level of stem cell regulators significantly varies and that cell signaling pathways are extensively cross-activated across defined culture conditions of embryonic stem cells. In addition, the mass cytometry data enabled us to identify distinct multiple cell states of embryonic stem cells and determine their variation across culture conditions. We discuss the mass cytometry method, our results of the multi-protein analysis in embryonic stem cells and potential future perspectives for single-cell protein analysis.


Assuntos
Células-Tronco Pluripotentes , Animais , Camundongos , Células-Tronco Embrionárias , Análise de Célula Única/métodos , Diferenciação Celular/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Citometria de Fluxo/métodos
2.
Nucleic Acids Res ; 49(8): 4472-4492, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33836079

RESUMO

Skeletal muscle is a dynamic tissue the size of which can be remodeled through the concerted actions of various cues. Here, we investigated the skeletal muscle transcriptional program and identified key tissue-specific regulatory genetic elements. Our results show that Myod1 is bound to numerous skeletal muscle enhancers in collaboration with the glucocorticoid receptor (GR) to control gene expression. Remarkably, transcriptional activation controlled by these factors occurs through direct contacts with the promoter region of target genes, via the CpG-bound transcription factor Nrf1, and the formation of Ctcf-anchored chromatin loops, in a myofiber-specific manner. Moreover, we demonstrate that GR negatively controls muscle mass and strength in mice by down-regulating anabolic pathways. Taken together, our data establish Myod1, GR and Nrf1 as key players of muscle-specific enhancer-promoter communication that orchestrate myofiber size regulation.


Assuntos
Cromatina/metabolismo , Elementos Facilitadores Genéticos , Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , Regulação da Expressão Gênica/genética , Histonas/genética , Histonas/metabolismo , Masculino , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Força Muscular/genética , Músculo Esquelético/fisiologia , Proteína MyoD/genética , Mioblastos/metabolismo , Fator 1 Nuclear Respiratório/genética , Receptores de Glucocorticoides/genética , Proteínas Recombinantes
3.
Cell Rep ; 42(5): 112438, 2023 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-37126449

RESUMO

Embryonic stem cells (ESCs) are an attractive model to study the relationship between signaling and cell fates. Cultured mouse ESCs can exist in multiple states resembling distinct stages of early embryogenesis, such as totipotent, pluripotent, primed, and primitive endoderm. The signaling mechanisms regulating the totipotent state and coexistence of these states are poorly understood. Here we identify bone morphogenetic protein (BMP) signaling as an inducer of the totipotent state. However, we discover that BMP's role is constrained by the cross-activation of FGF, NODAL, and WNT pathways. We exploit this finding to enhance the proportion of totipotent cells by rationally inhibiting the cross-activated pathways. Single-cell mRNA sequencing reveals that induction of the totipotent state is accompanied by suppression of primed and primitive endoderm states. Furthermore, reprogrammed totipotent cells we generate in culture resemble totipotent cells of preimplantation embryo. Our findings reveal a BMP signaling mechanism regulating both the totipotent state and heterogeneity of ESCs.


Assuntos
Células-Tronco Embrionárias Murinas , Via de Sinalização Wnt , Animais , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Via de Sinalização Wnt/fisiologia , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Fator de Crescimento Transformador beta/metabolismo
4.
Sci Adv ; 8(29): eabo2295, 2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35867798

RESUMO

Prostate cancer (PCa) is a leading cause of cancer-related deaths. The slow evolution of precancerous lesions to malignant tumors provides a broad time frame for preventing PCa. To characterize prostatic intraepithelial neoplasia (PIN) progression, we conducted longitudinal studies on Pten(i)pe-/- mice that recapitulate prostate carcinogenesis in humans. We found that early PINs are hypoxic and that hypoxia-inducible factor 1 alpha (HIF1A) signaling is activated in luminal cells, thus enhancing malignant progression. Luminal HIF1A dampens immune surveillance and drives luminal plasticity, leading to the emergence of cells that overexpress Transglutaminase 2 (TGM2) and have impaired androgen signaling. Elevated TGM2 levels in patients with PCa are associated with shortened progression-free survival after prostatectomy. Last, we show that pharmacologically inhibiting HIF1A impairs cell proliferation and induces apoptosis in PINs. Therefore, our study demonstrates that HIF1A is a target for PCa prevention and that TGM2 is a promising prognostic biomarker of early relapse after prostatectomy.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasia Prostática Intraepitelial , Neoplasias da Próstata , Animais , Plasticidade Celular , Progressão da Doença , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos , Neoplasia Prostática Intraepitelial/genética , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia
5.
Cancers (Basel) ; 14(22)2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36428689

RESUMO

Pancreatic cancer (pancreatic ductal adenocarcinoma: PDAC) is one of the most aggressive neoplastic diseases. Metformin use has been associated with reduced pancreatic cancer incidence and better survival in diabetics. Metformin has been shown to inhibit PDAC cells growth and survival, both in vitro and in vivo. However, clinical trials using metformin have failed to reduce pancreatic cancer progression in patients, raising important questions about molecular mechanisms that protect tumor cells from the antineoplastic activities of metformin. We confirmed that metformin acts through inhibition of mitochondrial complex I, decreasing the NAD+/NADH ratio, and that NAD+/NADH homeostasis determines metformin sensitivity in several cancer cell lines. Metabolites that can restore the NAD+/NADH ratio caused PDAC cells to be resistant to metformin. In addition, metformin treatment of PDAC cell lines induced a compensatory NAMPT expression, increasing the pool of cellular NAD+. The NAMPT inhibitor FK866 sensitized PDAC cells to the antiproliferative effects of metformin in vitro and decreased the cellular NAD+ pool. Intriguingly, FK866 combined with metformin increased survival in mice bearing KP4 cell line xenografts, but not in mice with PANC-1 cell line xenografts. Transcriptome analysis revealed that the drug combination reactivated genes in the p53 pathway and oxidative stress, providing new insights about the mechanisms leading to cancer cell death.

6.
Sci Rep ; 11(1): 9854, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972583

RESUMO

We present the design and synthesis of a small library of substituted biguanidium salts and their capacity to inhibit the growth of pancreatic cancer cells. We first present their in vitro and membrane activity, before we address their mechanism of action in living cells and in vivo activity. We show that phenylethynyl biguanidium salts possess higher ability to cross hydrophobic barriers, improve mitochondrial accumulation and anticancer activity. Mechanistically, the most active compound, 1b, like metformin, activated AMPK, decreased the NAD+/NADH ratio and mitochondrial respiration, but at 800-fold lower concentration. In vivo studies show that compound 1b significantly inhibits the growth of pancreatic cancer xenografts in mice, while biguanides currently in clinical trials had little activity.


Assuntos
Biguanidas/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Biguanidas/química , Biguanidas/uso terapêutico , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral/transplante , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Fibroblastos , Humanos , Concentração Inibidora 50 , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/patologia
7.
Biochim Biophys Acta ; 1790(12): 1660-4, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19766701

RESUMO

BACKGROUND: Retinal dehydrogenases (RALDHs) catalyze the dehydrogenation of retinal into retinoic acids (RAs), which are required for embryogenesis and tissue differentiation. This study sought to determine the detailed kinetic properties of 2 mouse RALDHs, namely RALDH3 and 4, for retinal isomer substrates, to better define their specificities in RA isomer synthesis. METHODS: RALDH3 and 4 were expressed in Escherichia coli as His-tagged proteins and affinity-purified. Enzyme kinetics were performed with retinal isomer substrates. The enzymatic products were analyzed by high pressure liquid chromatography. RESULTS: RALDH3 oxidized all-trans retinal with high catalytic efficiency (Vmax/Km=77.9) but did not show activity for either 9-cis or 13-cis retinal substrates. On the other hand, RALDH4 was inactive for all-trans retinal substrate, exhibited high activity for 9-cis retinal oxidation (Vmax/Km=27.4), and oxidized 13-cis retinal with lower catalytic efficiency (Vmax/Km=8.24). beta-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis retinal oxidation competitively with inhibition constants of 0.60 and 0.32, respectively, but had no effect on RALDH3 activity. The divalent cation MgCl2 activated 13-cis retinal oxidation by RALDH4 by 3-fold, did not significantly influence 9-cis retinal oxidation, and slightly activated RALDH3 activity. CONCLUSIONS: These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates. GENERAL SIGNIFICANCE: The kinetic characterization of RALDHs should give useful information in determining amino acid residues that are involved in the specificity for retinal isomers and on the role of these enzymes in the synthesis of RAs in specific tissues.


Assuntos
Retinal Desidrogenase/metabolismo , Retinaldeído/análogos & derivados , Retinoides/farmacocinética , Animais , Relação Dose-Resposta a Droga , Ativação Enzimática , Técnicas In Vitro , Isomerismo , Cinética , Camundongos , Proteínas Recombinantes/metabolismo , Retinaldeído/farmacocinética , Especificidade por Substrato , Tretinoína/análogos & derivados , Tretinoína/farmacocinética
8.
Mol Cell Oncol ; 6(1): 1511205, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788414

RESUMO

We report that Pten (phosphatase and tensin homologue) ablation in prostatic epithelial cells of adult mice promotes cell proliferation to generate prostatic intraepithelial neoplasia. Moreover, our results demonstrate that proliferating Pten-deficient cells undergo replication stress and exhibit a DNA damage response, leading to cell senescence, as seen in oncogene-induced senescence.

9.
Aging Cell ; 18(2): e12889, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30614183

RESUMO

Most cancers arise in old individuals, which also accumulate senescent cells. Cellular senescence can be experimentally induced by expression of oncogenes or telomere shortening during serial passage in culture. In vivo, precursor lesions of several cancer types accumulate senescent cells, which are thought to represent a barrier to malignant progression and a response to the aberrant activation of growth signaling pathways by oncogenes (oncogene toxicity). Here, we sought to define gene expression changes associated with cells that bypass senescence induced by oncogenic RAS. In the context of pancreatic ductal adenocarcinoma (PDAC), oncogenic KRAS induces benign pancreatic intraepithelial neoplasias (PanINs), which exhibit features of oncogene-induced senescence. We found that the bypass of senescence in PanINs leads to malignant PDAC cells characterized by gene signatures of epithelial-mesenchymal transition, stem cells, and mitochondria. Stem cell properties were similarly acquired in PanIN cells treated with LPS, and in primary fibroblasts and mammary epithelial cells that bypassed Ras-induced senescence after reduction of ERK signaling. Intriguingly, maintenance of cells that circumvented senescence and acquired stem cell properties was blocked by metformin, an inhibitor of complex I of the electron transport chain or depletion of STAT3, a protein required for mitochondrial functions and stemness. Thus, our studies link bypass of senescence in premalignant lesions to loss of differentiation, acquisition of stemness features, and increased reliance on mitochondrial functions.


Assuntos
Senescência Celular/efeitos dos fármacos , Metformina/farmacologia , Células-Tronco/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Camundongos , Células-Tronco/citologia , Relação Estrutura-Atividade , Células Tumorais Cultivadas
10.
Biochim Biophys Acta ; 1770(11): 1548-56, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17916406

RESUMO

Retinal dehydrogenase type 1 (RALDH1) catalyzes the oxidation of all-trans and 9-cis retinal to the respective retinoic acids (RAs), whereas another member of the aldehyde dehydrogenase family, the phenobarbital-induced aldehyde dehydrogenase (PB-ALDH), is very poorly active. We have previously generated chimeras between these two enzymes that displayed selectivity for retinal isomers in crude bacterial extracts. To examine whether the selectivity of the recombinant enzymes is retained in intact cells, we first assessed whether retinoid-isomerizing activity is present in cultured eukaryotic cells. Our results demonstrate that the only RA isomers detected in RALDH1-expressing or non-expressing cells corresponded to the same steric conformation as the supplied retinoids, indicating a lack of measurable 9-cis/all-trans retinoid-isomerizing activity. Finally, HeLa cells transfected with RALDH1 derivatives that were retinal isomer-selective in vitro produced only the corresponding RA isomers, establishing these enzymes as useful tools to assess the respective roles of the two RA isomers in vivo.


Assuntos
Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Retinal Desidrogenase/biossíntese , Retinal Desidrogenase/genética , Tretinoína/química , Tretinoína/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Clonagem Molecular , Haplorrinos , Células HeLa , Humanos , Retinal Desidrogenase/classificação , Estereoisomerismo , Transfecção
11.
J Exp Med ; 215(6): 1749-1763, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29743291

RESUMO

Genetic ablation of the tumor suppressor PTEN in prostatic epithelial cells (PECs) induces cell senescence. However, unlike oncogene-induced senescence, no hyperproliferation phase and no signs of DNA damage response (DDR) were observed in PTEN-deficient PECs; PTEN loss-induced senescence (PICS) was reported to be a novel type of cellular senescence. Our study reveals that PTEN ablation in prostatic luminal epithelial cells of adult mice stimulates PEC proliferation, followed by a progressive growth arrest with characteristics of cell senescence. Importantly, we also show that proliferating PTEN-deficient PECs undergo replication stress and mount a DDR leading to p53 stabilization, which is however delayed by Mdm2-mediated p53 down-regulation. Thus, even though PTEN-deficiency induces cellular senescence that restrains tumor progression, as it involves replication stress, strategies promoting PTEN loss-induced senescence are at risk for cancer prevention and therapy.


Assuntos
Senescência Celular , Deleção de Genes , PTEN Fosfo-Hidrolase/genética , Próstata/metabolismo , Próstata/patologia , Estresse Fisiológico , Animais , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Dano ao DNA , Reparo do DNA , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Masculino , Camundongos , Células Mieloides/metabolismo , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/metabolismo , Fenótipo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
12.
Cell Metab ; 28(6): 817-832.e8, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30244971

RESUMO

There is increasing interest in therapeutically exploiting metabolic differences between normal and cancer cells. We show that kinase inhibitors (KIs) and biguanides synergistically and selectively target a variety of cancer cells. Synthesis of non-essential amino acids (NEAAs) aspartate, asparagine, and serine, as well as glutamine metabolism, are major determinants of the efficacy of KI/biguanide combinations. The mTORC1/4E-BP axis regulates aspartate, asparagine, and serine synthesis by modulating mRNA translation, while ablation of 4E-BP1/2 substantially decreases sensitivity of breast cancer and melanoma cells to KI/biguanide combinations. Efficacy of the KI/biguanide combinations is also determined by HIF-1α-dependent perturbations in glutamine metabolism, which were observed in VHL-deficient renal cancer cells. This suggests that cancer cells display metabolic plasticity by engaging non-redundant adaptive mechanisms, which allows them to survive therapeutic insults that target cancer metabolism.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aminoácidos/metabolismo , Animais , Biguanidas/farmacologia , Proteínas de Ciclo Celular , Fatores de Iniciação em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Células K562 , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Med Sci (Paris) ; 22(12): 1101-6, 2006 Dec.
Artigo em Francês | MEDLINE | ID: mdl-17156733

RESUMO

Retinoids play important roles in cell differentiation and apoptosis, notably in epithelial tissues. Their utility in cancer therapy has been demonstrated in specific cancer types. Use of retinoic acid (RA) in the treatment of acute promyelocytic leukemia was the first successful example of retinoid-based differentiation therapy. RA has since been evaluated for treatment of other cancers, revealing variable effectiveness. The observation that expression of enzymes involved in RA biosynthesis is suppressed during tumorigenesis suggests that intra-tumor depletion in RA levels may contribute to tumor development and argues for the use of retinoids in cancer treatment. However, the induction of RA-inactivating enzymes is one of the mechanisms that may limit the efficacy of retinoid therapy and contribute to acquired resistance to RA treatment, suggesting that retinoic acid metabolism blocking agents may be effective agents in differentiation therapy.


Assuntos
Neoplasias/tratamento farmacológico , Retinoides/metabolismo , Retinoides/uso terapêutico , Anticarcinógenos/uso terapêutico , Humanos , Absorção Intestinal , Receptores do Ácido Retinoico/fisiologia , Tretinoína/metabolismo , Vitamina A/metabolismo
14.
Mol Oncol ; 7(2): 190-205, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23481269

RESUMO

Despite major improvement in treatment of early stage localised prostate cancer, the distinction between indolent tumors and those that will become aggressive, as well as the lack of efficient therapies of advanced prostate cancer, remain major health problems. Genetically engineered mice (GEM) have been extensively used to investigate the molecular and cellular mechanisms underlying prostate tumor initiation and progression, and to evaluate new therapies. Moreover, the recent development of conditional somatic mutagenesis in the mouse prostate offers the possibility to generate new models that more faithfully reproduce the human disease, and thus should contribute to improve diagnosis and treatments. The strengths and weaknesses of various models will be discussed, as well as future opportunities.


Assuntos
Modelos Animais de Doenças , Engenharia Genética , Neoplasias da Próstata/patologia , Androgênios/metabolismo , Animais , Humanos , Masculino , Camundongos , Mutação/genética , Neoplasias da Próstata/metabolismo , Células-Tronco/metabolismo , Células-Tronco/patologia
16.
Biochem Cell Biol ; 84(5): 799-804, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17167544

RESUMO

Retinal dehydrogenase type 1 (RALDH1) catalyzes the oxidation of all-trans and 9-cis retinal to the respective retinoic acids (RAs), whereas another member of the aldehyde dehydrogenase (ALDH) family, the phenobarbital-induced aldehyde dehydrogenase (PB-ALDH), is very poorly active. We have previously generated chimeras between these 2 enzymes that displayed selectivity for retinal isomers in crude bacterial extracts. Here we have characterized the kinetic properties of the corresponding purified recombinant proteins. The all-trans selective chimera RALDH-131 converted all-trans retinal to all-trans RA with 2.9-fold lower efficiency than the wild-type RALDH1 and had only residual activity with 9-cis retinal. The converse chimera PB-131 was specific for 9-cis retinal, with no residual activity for all-trans retinal. MgCl2 inhibited the activities of RALDH1 and PB-131, but not of RALDH-131, suggesting that amino acids 132-510 in RALDH are necessary for inhibition by MgCl2. These data demonstrate that the chimeric enzymes act as retinal isomer-selective ALDHs, and suggest that these enzymes may be useful to study the roles of cis RA isomers in embryogenesis and differentiation in vivo.


Assuntos
Aldeído Desidrogenase/farmacocinética , Isoenzimas/farmacocinética , Proteínas Recombinantes de Fusão/farmacocinética , Retinal Desidrogenase/farmacocinética , Família Aldeído Desidrogenase 1 , Alitretinoína , Animais , Magnésio/farmacologia , Ratos , Tretinoína/metabolismo
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