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1.
Mol Cell ; 67(1): 55-70.e4, 2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28673543

RESUMO

Ribosomal protein (RP) expression in higher eukaryotes is regulated translationally through the 5'TOP sequence. This mechanism evolved to more rapidly produce RPs on demand in different tissues. Here we show that 40S ribosomes, in a complex with the mRNA binding protein LARP1, selectively stabilize 5'TOP mRNAs, with disruption of this complex leading to induction of the impaired ribosome biogenesis checkpoint (IRBC) and p53 stabilization. The importance of this mechanism is underscored in 5q− syndrome, a macrocytic anemia caused by a large monoallelic deletion, which we found to also encompass the LARP1 gene. Critically, depletion of LARP1 alone in human adult CD34+ bone marrow precursor cells leads to a reduction in 5'TOP mRNAs and the induction of p53. These studies identify a 40S ribosome function independent of those in translation that, with LARP1, mediates the autogenous control of 5'TOP mRNA stability, whose disruption is implicated in the pathophysiology of 5q− syndrome.


Assuntos
Autoantígenos/metabolismo , Biossíntese de Proteínas , Sequência de Oligopirimidina na Região 5' Terminal do RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Anemia Macrocítica/genética , Anemia Macrocítica/metabolismo , Autoantígenos/genética , Células da Medula Óssea/metabolismo , Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Cromossomos Humanos Par 5/metabolismo , Células HCT116 , Humanos , Complexos Multiproteicos , Ligação Proteica , Interferência de RNA , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Fatores de Tempo , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Antígeno SS-B
2.
EMBO J ; 39(13): e103838, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32484960

RESUMO

Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21-mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC-mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability.


Assuntos
Dano ao DNA , Pontos de Checagem da Fase G1 do Ciclo Celular , Nucleotídeos/metabolismo , Ribossomos/metabolismo , Células HCT116 , Humanos , Nucleotídeos/genética , Ribossomos/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
3.
Mol Cancer ; 15: 1, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26728659

RESUMO

BACKGROUND: Neuroblastoma is a malignant embryonal tumor occurring in young children, consisting of undifferentiated neuroectodermal cells derived from the neural crest. Current therapies for high-risk neuroblastoma are insufficient, resulting in high mortality rates and high incidence of relapse. With the intent to find new therapies for neuroblastomas, we investigated the efficacy of low-doses of actinomycin D, which at low concentrations preferentially inhibit RNA polymerase I-dependent rRNA trasncription and therefore, ribosome biogenesis. METHODS: Neuroblastoma cell lines with different p53 genetic background were employed to determine the response on cell viability and apoptosis of low-dose of actinomycin D. Subcutaneously-implanted SK-N-JD derived neuroblastoma tumors were used to assess the effect of low-doses of actinomycin D on tumor formation. RESULTS: Low-dose actinomycin D treatment causes a reduction of cell viability in neuroblastoma cell lines and that this effect is stronger in cells that are wild-type for p53. MYCN overexpression contributes to enhance this effect, confirming the importance of this oncogene in ribosome biogenesis. In the wild-type SK-N-JD cell line, apoptosis was the major mechanism responsible for the reduction in viability and we demonstrate that treatment with the MDM2 inhibitor Nutlin-3, had a similar effect to that of actinomycin D. Apoptosis was also detected in p53(-/-)deficient LA1-55n cells treated with actinomycin D, however, only a small recovery of cell viability was found when apoptosis was inhibited by a pan-caspase inhibitor, suggesting that the treatment could activate an apoptosis-independent cell death pathway in these cells. We also determined whether actinomycin D could increase the efficacy of the histone deacetylase inhibitor, SAHA, which is in being used in neuroblastoma clinical trials. We show that actinomycin D synergizes with SAHA in neuroblastoma cell lines. Moreover, on subcutaneously-implanted neuroblastoma tumors derived from SK-N-JD cells, actinomycin D led to tumor regression, an effect enhanced in combination with SAHA. CONCLUSIONS: The results presented in this work demonstrate that actinomycin D, at low concentrations, inhibits proliferation and induces cell death in vitro, as well as tumor regression in vivo. From this study, we propose that use of ribosome biogenesis inhibitors should be clinically considered as a potential therapy to treat neuroblastomas.


Assuntos
Dactinomicina/uso terapêutico , Neuroblastoma/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Ácidos Hidroxâmicos/farmacologia , Imidazóis/farmacologia , Camundongos , Neuroblastoma/patologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , Vorinostat
4.
Methods Mol Biol ; 2445: 117-125, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34972989

RESUMO

Chromosomal instability (CIN) is a hallmark of cancer, which is characterized by the gain or loss of chromosomes as well as the rearrangement of the genetic material during cell division. Detection of mitotic errors such as misaligned chromosomes or chromosomal bridges (also known as lagging chromosomes) is challenging as it requires the analysis and manual discrimination of chromosomal aberrations in mitotic cells by molecular techniques. In interphase cells, more frequent in the cell population than mitotic cells, two distinct nuclear phenotypes are associated with CIN: the micronucleus and the toroidal nucleus. Several methods are available for the detection of micronuclei, but none for toroidal nuclei. Here, we provide a method to quantify the presence of both nuclear biomarkers for the evaluation of CIN status in non-mitotic cells particularly suited for genotoxicity screens.


Assuntos
Instabilidade Cromossômica , Mitose , Biomarcadores , Núcleo Celular/genética , Cromossomos , Humanos , Mitose/genética
5.
Methods Mol Biol ; 2445: 127-137, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34972990

RESUMO

The detection of autophagic vesicles in interphase cells is well characterized with markers such as LC3, SQSTM1 (also known as p62) and LAMP2, which are commonly used in immunofluorescence and biochemistry assays to evaluate the status of autophagy in adherent cells. During mitosis, cells undergo important morphological changes which alter the position of the central plane, therefore the imaging of dividing cells has to be specifically designed. Here, we describe a method to label and image autophagic vesicles in mitotic cells to systematically analyze their number, morphology and distribution.


Assuntos
Autofagia , Mitose , Imunofluorescência , Proteína Sequestossoma-1
6.
Biochim Biophys Acta ; 1803(8): 912-8, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20447425

RESUMO

Multiple E2F1 phosphorylation sites have been described as targets of different kinases, yet their in vivo implication is uncertain. We previously reported that GSK3beta is able to phosphorylate E2F1 in vitro at Ser403 and Ser433. Recently, it has been shown that both residues are also direct targets of p38 MAP kinase. In order to determine whether Ser403 phosphorylation occurs in vivo and to elucidate its role in E2F1 transcription activity, we developed a phospho-E2F1(Ser403) antibody for use in in vivo detection studies. Our results demonstrate that endogenous E2F1 is phosphorylated in vivo on Ser403, however neither GSK3beta nor p38 MAP kinase are responsible for this event. E2F1 phosphorylation on Ser403 is induced after treatment with doxorubicin in a dose response manner. The transcriptional response of E2F1 to doxorubicin is lower in an E2F1 Ser/Ala403 mutated construct relative to the wild type, suggesting a role for Ser403 phosphorylation in DNA damage conditions. Comparative study between the expression of the bcl2 gene family induced by the wild type and E2F1 Ser/Ala403 mutant revealed a statistically different pattern between both conditions. These results suggest that phosphorylation of Ser403 could influence the selection and regulation of E2F1 target genes.


Assuntos
Anticorpos Fosfo-Específicos/metabolismo , Fator de Transcrição E2F1/metabolismo , Serina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Fator de Transcrição E2F1/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
7.
Autophagy ; 17(3): 796-813, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32573315

RESUMO

Lysosomes, as primary degradative organelles, are the endpoint of different converging pathways, including macroautophagy. To date, lysosome degradative function has been mainly studied in interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition also relies on lysosome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel SQSTM1-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent chromosomal instability.Abbreviations: 3D: three-dimensional; APC/C: anaphase-promoting complex; ARL8B: ADP ribosylation factor like GTPase 8B; ATG: autophagy-related; BORC: BLOC-one-related complex; CDK: cyclin-dependent kinase; CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin associated factor B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain containing M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H+-translocating ATPase; WAPL: WAPL cohesion release factor.


Assuntos
Autofagia/fisiologia , Instabilidade Cromossômica/fisiologia , Fibroblastos/metabolismo , Lisossomos/metabolismo , Animais , Células HeLa , Humanos , Mitose/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo
8.
Sci Adv ; 7(48): eabg9275, 2021 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-34818049

RESUMO

Ribosomes execute the transcriptional program in every cell. Critical to sustain nearly all cellular activities, ribosome biogenesis requires the translation of ~200 factors of which 80 are ribosomal proteins (RPs). As ribosome synthesis depends on RP mRNA translation, a priority within the translatome architecture should exist to ensure the preservation of ribosome biogenesis capacity, particularly under adverse growth conditions. Here, we show that under critical metabolic constraints characterized by mTOR inhibition, LARP1 complexed with the 40S subunit protects from ribophagy the mRNAs regulon for ribosome biogenesis and protein synthesis, acutely preparing the translatome to promptly resume ribosomes production after growth conditions return permissive. Characterizing the LARP1-protected translatome revealed a set of 5'TOP transcript isoforms other than RPs involved in energy production and in mitochondrial function, among other processes, indicating that the mTOR-LARP1-5'TOP axis acts at the translational level as a primary guardian of the cellular anabolic capacity.

9.
Biochim Biophys Acta ; 1783(1): 144-52, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17900713

RESUMO

Mitogenic response to growth factors is concomitant with the modulation they exert on the levels of Fructose 2,6-bisphosphate (Fru-2,6-P2), an essential activator of the glycolytic flux. In mammalian cells, decreased Fru-2,6-P2 concentration causes cell cycle delay, whereas high levels of Fru-2,6-P2 sensitize cells to apoptosis. In order to analyze the cell cycle consequences due to changes in Fru-2,6-P2 levels, the bisphosphatase-dead mutant (H258A) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme was over-expressed in Schizosaccharomyces pombe cells and the variation in cell phenotype was studied. The results obtained demonstrate that the increase in Fru-2,6-P2 levels results in a defective division of S. pombe, as revealed by an altered multisepted phenotype. The H258A-expressing cells showed impairment of cytokinesis, but normal nuclear division. In order to identify cellular mediators responsible for this effect, we transformed different S. pombe strains and observed that the cytokinetic defect was absent in cells defective for Wee1 kinase function. Therefore, in S. pombe, Wee1 integrates the metabolic signal emerging from changes in Fru-2,6-P2 content, thus coupling metabolism with cell proliferation. As the key regulators of the cell cycle checkpoints are conserved throughout evolution, these results may help to understand the experimental evidences obtained by manipulation of Fru-2,6-P2 levels in mammalian cells.


Assuntos
Frutosedifosfatos/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Divisão Celular , Regulação Fúngica da Expressão Gênica , Mutação/genética , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transativadores/genética , Transativadores/metabolismo
10.
iScience ; 20: 434-448, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31627130

RESUMO

Cancer cells rely on mTORC1 activity to coordinate mitogenic signaling with nutrients availability for growth. Based on the metabolic function of E2F1, we hypothesize that glucose catabolism driven by E2F1 could participate on mTORC1 activation. Here, we demonstrate that glucose potentiates E2F1-induced mTORC1 activation by promoting mTORC1 translocation to lysosomes, a process that occurs independently of AMPK activation. We showed that E2F1 regulates glucose metabolism by increasing aerobic glycolysis and identified the PFKFB3 regulatory enzyme as an E2F1-regulated gene important for mTORC1 activation. Furthermore, PFKFB3 and PFK1 were found associated to lysosomes and we demonstrated that modulation of PFKFB3 activity, either by substrate accessibility or expression, regulates the translocation of mTORC1 to lysosomes by direct interaction with Rag B and subsequent mTORC1 activity. Our results support a model whereby a glycolytic metabolon containing phosphofructokinases transiently interacts with the lysosome acting as a sensor platform for glucose catabolism toward mTORC1 activity.

11.
Cancer Res ; 79(17): 4348-4359, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31292158

RESUMO

The role of MYC in regulating p53 stability as a function of increased ribosome biogenesis is controversial. On the one hand, it was suggested that MYC drives the overexpression of ribosomal proteins (RP)L5 and RPL11, which bind and inhibit HDM2, stabilizing p53. On the other, it has been proposed that increased ribosome biogenesis leads the consumption of RPL5/RPL11 into nascent ribosomes, reducing p53 levels and enhancing tumorigenesis. Here, we show that the components that make up the recently described impaired ribosome biogenesis checkpoint (IRBC) complex, RPL5, RPL11, and 5S rRNA, are reduced following MYC silencing. This leads to a rapid reduction in p53 protein half-life in an HDM2-dependent manner. In contrast, MYC induction leads to increased ribosome biogenesis and p53 protein stabilization. Unexpectedly, there is no change in free RPL5/RPL11 levels, but there is a striking increase in IRBC complex bound to HDM2. Our data support a cell-intrinsic tumor-suppressor response to MYC expression, which is presently being exploited to treat cancer. SIGNIFICANCE: Oncogenic MYC induces the impaired ribosome biogenesis checkpoint, which could be potentially targeted for cancer treatment.


Assuntos
Proteínas Proto-Oncogênicas c-myc/genética , Ribossomos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Regulação da Expressão Gênica , Humanos , Biossíntese de Proteínas , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Ribossômico 5S/genética , RNA Ribossômico 5S/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Proteína Supressora de Tumor p53/genética
12.
Biochim Biophys Acta ; 1773(3): 375-82, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17050006

RESUMO

GSK3beta and E2F1 play an important role in the control of proliferation and apoptosis. Previous work has demonstrated that GSK3beta indirectly regulates E2F activity through modulation of cyclin D1 levels. In this work we show that GSK3beta phosphorylates human E2F1 in vitro at serine 403 and threonine 433, both residues localized at its transactivation domain. This phosphorylation was not detected in vivo. However, co-immunoprecipitation experiments do reveal in vivo binding of these proteins. Moreover, uninhibitable and catalitycally inactive GSK3beta forms inhibit the transcriptional activity of a fusion protein containing E2F1 transactivation domain. Both forms of GSK3beta inhibit E2F1 with similar efficiency. Interestingly the effect was independent of the mutation of serine 403 and threonine 433 to alanine. This suggests that this transcriptional modulation is independent of GSK3beta kinase activity and phosphorylation state of serine 403 and threonine 433. The re-targeting of these GSK3beta forms to the nucleus results in a higher capacity to regulate E2F1 transcriptional activity. Depletion of the levels of GSK3beta protein using siRNA activates E2F1 transcriptional activity. The data presented in this study offer a new mechanism of regulation of E2F1 by direct binding of GSK3beta to its transactivation domain.


Assuntos
Fator de Transcrição E2F1/metabolismo , Regulação Enzimológica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Transativadores/metabolismo , Linhagem Celular , Fator de Transcrição E2F1/genética , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Transativadores/genética
13.
Biochim Biophys Acta ; 1574(2): 131-6, 2002 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-11955621

RESUMO

The expression of F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is rapidly induced by growth factors. We report here that an AP-1 intragenic sequence located at position +612 of the F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase transcription initiation site is involved in the transcriptional activation of this gene by serum. We have demonstrated in vitro DNA-protein interaction on this AP-1 site of the F-promoter. Indeed, this element was recognized by c-Fos and JunD in vitro, and mutation or deletion of this element reduced the early response to serum stimulation by 60%. We conclude that the serum response of the F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene requires the co-ordinated function of ets, E2F and AP-1 binding sites.


Assuntos
Fosfofrutoquinase-2/genética , Fator de Transcrição AP-1/genética , Animais , Sangue , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Íntrons , Fosfofrutoquinase-2/biossíntese , Ratos , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional
14.
Cell Oncol (Dordr) ; 38(5): 341-52, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26306783

RESUMO

BACKGROUND: In the past, the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) has been shown to induce apoptosis in several human tumor types, including neuroblastomas. Amplification and over-expression of the MYCN oncogene is a diagnostic hallmark and a poor prognostic indicator in high-risk neuroblastomas. Here, we studied the relationship between MYCN amplification and over-expression and the anti-tumor effect of SAHA to assess whether this drug may serve as a treatment option for high-risk neuroblastomas. METHODS: Different human neuroblastoma cell lines, over-expressing or not over-expressing MYCN, were used in this study. Targeted knockdown and exogenous over-expression of MYCN were employed to examine correlations between MYCN expression levels and SAHA responses. After various time periods and concentration exposures to the drug, cell viability was measured by MTS assay, and variations in MYCN mRNA and protein levels were assessed by qPCR and Western blotting, respectively. RESULTS: We found that SAHA decreased cell viability in all cell lines tested through apoptosis induction, and that SAHA had a stronger effect on cell lines carrying an amplified MYCN gene. A decrease in MYCN mRNA and protein levels was observed in the SAHA treated cell lines. Subsequent silencing and exogenous over-expression of MYCN changed the proliferation rate of the cells, but did not have any significant impact on the effect of SAHA on the viability of the cells. We also found that SAHA blocked the expression of MYCN and, by doing so, reduced the effects mediated by this protein. CONCLUSIONS: Our results suggest that SAHA may be used as a single-drug treatment option for neuroblastomas with an amplified MYCN gene, and as an adjuvant treatment option for all neuroblastomas.


Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Relação Dose-Resposta a Droga , Inibidores de Histona Desacetilases/farmacologia , Humanos , Microscopia de Fluorescência , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Vorinostat
15.
Oncotarget ; 6(29): 28057-70, 2015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-26356814

RESUMO

In addition to being a master regulator of cell cycle progression, E2F1 regulates other associated biological processes, including growth and malignancy. Here, we uncover a regulatory network linking E2F1 to lysosomal trafficking and mTORC1 signaling that involves v-ATPase regulation. By immunofluorescence and time-lapse microscopy we found that E2F1 induces the movement of lysosomes to the cell periphery, and that this process is essential for E2F1-induced mTORC1 activation and repression of autophagy. Gain- and loss-of-function experiments reveal that E2F1 regulates v-ATPase activity and inhibition of v-ATPase activity repressed E2F1-induced lysosomal trafficking and mTORC1 activation. Immunoprecipitation experiments demonstrate that E2F1 induces the recruitment of v-ATPase to lysosomal RagB GTPase, suggesting that E2F1 regulates v-ATPase activity by enhancing the association of V0 and V1 v-ATPase complex. Analysis of v-ATPase subunit expression identified B subunit of V0 complex, ATP6V0B, as a transcriptional target of E2F1. Importantly, ATP6V0B ectopic-expression increased v-ATPase and mTORC1 activity, consistent with ATP6V0B being responsible for mediating the effects of E2F1 on both responses. Our findings on lysosomal trafficking, mTORC1 activation and autophagy suppression suggest that pharmacological intervention at the level of v-ATPase may be an efficacious avenue for the treatment of metastatic processes in tumors overexpressing E2F1.


Assuntos
Fator de Transcrição E2F1/metabolismo , Complexos Multiproteicos/metabolismo , Neoplasias/patologia , Transporte Proteico/fisiologia , Serina-Treonina Quinases TOR/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Autofagia/fisiologia , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Imunofluorescência , Humanos , Imunoprecipitação , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Transfecção
16.
J Clin Invest ; 125(7): 2736-47, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26075820

RESUMO

Type 2 diabetes mellitus (T2DM) is a worldwide heath problem that is characterized by insulin resistance and the eventual loss of ß cell function. As recent studies have shown that loss of ribosomal protein (RP) S6 kinase 1 (S6K1) increases systemic insulin sensitivity, S6K1 inhibitors are being pursued as potential agents for improving insulin resistance. Here we found that S6K1 deficiency in mice also leads to decreased ß cell growth, intrauterine growth restriction (IUGR), and impaired placental development. IUGR is a common complication of human pregnancy that limits the supply of oxygen and nutrients to the developing fetus, leading to diminished embryonic ß cell growth and the onset of T2DM later in life. However, restoration of placental development and the rescue of IUGR by tetraploid embryo complementation did not restore ß cell size or insulin levels in S6K1-/- embryos, suggesting that loss of S6K1 leads to an intrinsic ß cell lesion. Consistent with this hypothesis, reexpression of S6K1 in ß cells of S6K1-/- mice restored embryonic ß cell size, insulin levels, glucose tolerance, and RPS6 phosphorylation, without rescuing IUGR. Together, these data suggest that a nutrient-mediated reduction in intrinsic ß cell S6K1 signaling, rather than IUGR, during fetal development may underlie reduced ß cell growth and eventual development of T2DM later in life.


Assuntos
Retardo do Crescimento Fetal/enzimologia , Retardo do Crescimento Fetal/patologia , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/patologia , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Animais , Tamanho Celular , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/patologia , Feminino , Teste de Complementação Genética , Humanos , Insulina/metabolismo , Resistência à Insulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Placentação/genética , Placentação/fisiologia , Gravidez , Gravidez em Diabéticas/enzimologia , Gravidez em Diabéticas/patologia , Proteínas Quinases S6 Ribossômicas 90-kDa/deficiência , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Tetraploidia
17.
Cancer Lett ; 318(1): 42-52, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22186300

RESUMO

Neuroblastoma (NB) is the most common solid extracranial tumor in children. Here we showed that trichostatin A, a histone deacetylase inhibitor (HDACi), decreases cell viability in three NB cell lines of different phenotypes. The treatment leads to G2/M-phase arrest, apoptosis and autophagy. Autophagy induction accompanies apoptosis in the most proliferative, N-Myc overexpressing cells. In contrast, autophagy precedes apoptosis and acts as a protective mechanism in the less proliferative, non-N-Myc overexpressing cells. Therefore, the autophagy induction is a relevant event in the NB response to HDACis, and it should be considered in the design of new treatments for this malignancy.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Neuroblastoma/patologia , Acetilação/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real
18.
PLoS One ; 7(12): e51544, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23251571

RESUMO

In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3ß blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator.


Assuntos
Apoptose , Fator de Transcrição E2F1/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Feocromocitoma/metabolismo , Feocromocitoma/patologia , Espécies Reativas de Oxigênio/metabolismo , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/patologia , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Tumoral , Humanos , Mitocôndrias/metabolismo , Multimerização Proteica , Transporte Proteico , Ratos , Transdução de Sinais , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo
19.
PLoS One ; 6(1): e16163, 2011 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-21283628

RESUMO

During cell proliferation, growth must occur to maintain homeostatic cell size. Here we show that E2F1 is capable of inducing growth by regulating mTORC1 activity. The activation of cell growth and mTORC1 by E2F1 is dependent on both E2F1's ability to bind DNA and to regulate gene transcription, demonstrating that a gene induction expression program is required in this process. Unlike E2F1, E2F3 is unable to activate mTORC1, suggesting that growth activity could be restricted to individual E2F members. The effect of E2F1 on the activation of mTORC1 does not depend on Akt. Furthermore, over-expression of TSC2 does not interfere with the effect of E2F1, indicating that the E2F1-induced signal pathway can compensate for the inhibitory effect of TSC2 on Rheb. Immunolocalization studies demonstrate that E2F1 induces the translocation of mTORC1 to the late endosome vesicles, in a mechanism dependent of leucine. E2F1 and leucine, or insulin, together affect the activation of S6K stronger than alone suggesting that they are complementary in activating the signal pathway. From these studies, E2F1 emerges as a key protein that integrates cell division and growth, both of which are essential for cell proliferation.


Assuntos
Processos de Crescimento Celular , Fator de Transcrição E2F1/fisiologia , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Ciclo Celular , Divisão Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Insulina/farmacologia , Leucina/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Transporte Proteico , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR , Ativação Transcricional
20.
J Neurochem ; 102(6): 2020-2028, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17555552

RESUMO

Both E2F1 and GSK3beta have been described as essential targets in neuronal apoptosis. Previous studies have demonstrated that GSK3beta binds to E2F1 in vivo. We wanted to investigate whether these proteins could share a common apoptotic signal pathway in neuronal cells. With this intention, we developed a PC12 ER-E2F1 stable cell line in which E2F1 activity was dependent on the presence of 4-hydroxitamoxifen. E2F1 activation produced apoptosis in naive and post-mitotic cells; serum and nerve growth factor respectively protected them from E2F1 apoptotic stimuli. The presence of specific GSK3beta inhibitors SB216763 and LiCl completely protected cells from apoptosis induced by E2F1 activation. In addition, knocked down GSK3beta experiments by small interference RNAs have demonstrated that a reduction of GSK3beta protein levels can lower the apoptotic effect of E2F1. Finally, we demonstrated that the apoptotic effect of E2F1 is not due to the regulation of GSK3beta activity, and that the inhibitory effect of GSK3beta inhibitor SB216763 on E2F1 induced apoptosis could be due to an alteration in the E2F1-regulated transcription gene pattern. In summary, we have demonstrated that the apoptotic action of E2F1 requires GSK3beta activity.


Assuntos
Apoptose/fisiologia , Fator de Transcrição E2F1/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Animais , Apoptose/efeitos dos fármacos , Regulação para Baixo/genética , Inibidores Enzimáticos/farmacologia , Antagonistas de Estrogênios/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Neurônios/efeitos dos fármacos , Células PC12 , RNA Interferente Pequeno/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética
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