RESUMO
OBJECTIVES: Gastric cancer (GC) is an aggressive disease due to late diagnosis resulting from the lack of easy diagnostic tools, resistances toward immunotherapy (due to low PD-L1 expression), or chemotherapies (due to p53 mutations), and comorbidity factors, notably muscle atrophy. To improve our understanding of this complex pathology, we established patient-derived xenograft (PDX) models and characterized the tumor ecosystem using a morpho-functional approach combining high-resolution imaging with molecular analyses, regarding the expression of relevant therapeutic biomarkers and the presence of muscle atrophy. MATERIALS AND METHODS: GC tissues samples were implanted in nude mice. Established PDX, treated with cisplatin or not, were imaged by magnetic resonance imaging (MRI) and analyzed for the expression of relevant biomarkers (p53, PD-L1, PD-1, HER-2, CDX2, CAIX, CD31, a-SAM) and by transcriptomics. RESULTS: Three well-differentiated, one moderately and one poorly differentiated adenocarcinomas were established. All retained the architectural and histological features of their primary tumors. MRI allowed in-real-time evaluation of differences between PDX, in terms of substructure, post-therapeutic changes, and muscle atrophy. Immunohistochemistry showed differential expression of p53, HER-2, CDX2, a-SAM, PD-L1, PD-1, CAIX, and CD31 between models and upon cisplatin treatment. Transcriptomics revealed treatment-induced hypoxia and metabolic reprograming in the tumor microenvironment. CONCLUSION: Our PDX models are representative for the heterogeneity and complexity of human tumors, with differences in structure, histology, muscle atrophy, and the different biomarkers making them valuable for the analyses of the impact of platinum drugs or new therapies on the tumor and its microenvironment.
Assuntos
Sarcopenia , Neoplasias Gástricas , Animais , Camundongos , Humanos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Cisplatino , Antígeno B7-H1/metabolismo , Camundongos Nus , Receptor de Morte Celular Programada 1/metabolismo , Ecossistema , Xenoenxertos , Proteína Supressora de Tumor p53 , Microambiente TumoralRESUMO
Ruthenium-based compounds are developed for anticancer treatment, but their mode of action including their import mechanism and subcellular localization remains elusive. Here, we used the intrinsic luminescent properties of cytotoxic organoruthenium (Ru(II)) compounds obtained with an anionic cyclometalated 2-phenylpyridine chelate and neutral aromatic chelating ligands (e.g., phenanthrolines) to follow their behavior in living cells. We established that the difference in sensitivity between cancer cells and noncancerous cells toward one of the compounds correlates with its import kinetics and follows a balance between active and passive transport. The active-transport mechanism involves iron and amino-acid transporters, which are transcriptionally regulated by the drug. We also demonstrated a correlation between the accumulation of these compounds in specific compartments (endoplasmic reticulum, nucleus, mitochondria) and the activation of specific cytotoxic mechanisms such as the mitochondrial stress pathway. Our study pinpoints a novel and complex mechanism of accumulation of ruthenium drugs in cancer cells.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Antineoplásicos/farmacologia , Proteínas de Transporte de Cátions/metabolismo , Compostos Organometálicos/farmacologia , Rutênio/química , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ferro/metabolismo , Cinética , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
MDMX has been shown to modulate p53 in dividing cells after DNA damage. In this study, we investigated the role of MDMX in primary cultures of neurons undergoing cell death. We found that DNA damage, but also membrane-initiated apoptotic stresses (glutamate receptor; Amyloid beta precursor) or survival factor deprivation downregulated MDMX protein levels. Forced downregulation of murine double minute X (MDMX) by shRNA induced apoptosis suggesting that MDMX is required for survival in neurons. Protease inhibitors prevented the loss of MDMX after neurotoxic treatments, indicating a regulation of protein stability. Some, but not all, neurotoxic stresses induced phosphorylation of MDMX at serine 367, further supporting regulation at the protein level. Interestingly, we found that depending on the stimulus either p53 or E2F1 was induced, but overexpression of MDMX inhibited the transcriptional activity of both proapoptotic factors, and maintained neuronal viability upon neurotoxic stresses. Taken together, our data show that MDMX is an antiapoptotic factor in neurons, whose degradation is induced by various stresses and allows activation of p53 and E2F-1 during neuronal apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Precursor de Proteína beta-Amiloide/toxicidade , Animais , Inibidores de Caspase , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Fator de Transcrição E2F1/metabolismo , Inibidores Enzimáticos/farmacologia , Inativação Gênica/efeitos dos fármacos , Camundongos , Inibidores de Proteassoma , RNA Interferente Pequeno , Proteína Supressora de Tumor p53/metabolismoRESUMO
Sprouty (Spry) proteins are ligand-inducible inhibitors of receptor tyrosine kinases-dependent signaling pathways, which control various biological processes, including proliferation, differentiation and survival. Here, we investigated the regulation and the role of Spry2 in cells of the central nervous system (CNS). In primary cultures of immature neurons, the neurotrophic factor BDNF (brain-derived neurotrophic factor) regulates spry2 expression. We identified the transcription factors CREB and SP1 as important regulators of the BDNF activation of the spry2 promoter. In immature neurons, we show that overexpression of wild-type Spry2 blocks neurite formation and neurofilament light chain expression, whereas inhibition of Spry2 by a dominant-negative mutant or small interfering RNA favors sprouting of multiple neurites. In mature neurons that exhibit an extensive neurite network, spry2 expression is sustained by BDNF and is downregulated during neuronal apoptosis. Interestingly, in these differentiated neurons, overexpression of Spry2 induces neuronal cell death, whereas its inhibition favors neuronal survival. Together, our results imply that Spry2 is involved in the development of the CNS by inhibiting both neuronal differentiation and survival through a negative-feedback loop that downregulates neurotrophic factors-driven signaling pathways.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , Diferenciação Celular/fisiologia , Proteínas de Membrana/metabolismo , Neurônios/citologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Proliferação de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Camundongos , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismoRESUMO
Localized inflammation of a rat's hindpaw elicits an accumulation of beta-endorphin-(END) containing immune cells. We investigated the production, release, and antinociceptive effects of lymphocyte-derived END in relation to cell trafficking. In normal animals, END and proopiomelanocortin mRNA were less abundant in circulating lymphocytes than in those residing in lymph nodes (LN), suggesting that a finite cell population produces END and homes to LN. Inflammation increased proopiomelanocortin mRNA in cells from noninflamed and inflamed LN. However, END content was increased only in inflamed paw tissue and noninflamed LN-immune cells. Accordingly, corticotropin-releasing factor and IL-1beta released significantly more END from noninflamed than from inflamed LN-immune cells. This secretion was receptor specific, calcium dependent, and mimicked by potassium, consistent with vesicular release. Finally, both agents, injected into the inflamed paw, induced analgesia which was blocked by the co-administration of antiserum against END. Together, these findings suggest that END-producing lymphocytes home to inflamed tissue where they secrete END to reduce pain. Afterwards they migrate to the regional LN, depleted of the peptide. Consistent with this notion, immunofluorescence studies of cell suspensions revealed that END is contained predominantly within memory-type T cells. Thus, the immune system is important for the control of inflammatory pain. This has implications for the understanding of pain in immunosuppressed conditions like cancer or AIDS.
Assuntos
Inflamação/fisiopatologia , Dor/fisiopatologia , Linfócitos T/metabolismo , Transcrição Gênica , beta-Endorfina/biossíntese , Análise de Variância , Animais , Hormônio Liberador da Corticotropina/farmacologia , Adjuvante de Freund , Membro Posterior , Humanos , Interleucina-1/farmacologia , Linfonodos/metabolismo , Masculino , Dor/imunologia , Pró-Opiomelanocortina/biossíntese , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Análise de Regressão , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de TempoRESUMO
The p53 tumor suppressor protein, found mutated in over 50% of all human tumors, is a sequence-specific transcriptional activator. Recent studies have identified a p53 relative, termed p73. We were interested in determining the relative abilities of wild-type and mutant forms of p53 and p73alpha and -beta isoforms to transactivate various p53-responsive promoters. We show that both p73alpha and p73beta activate the transcription of reporters containing a number of p53-responsive promoters in the p53-null cell line H1299. However, a number of significant differences were observed between p53 and p73 and even between p73alpha and p73beta. Additionally, a Saccharomyces cerevisiae-based reporter assay revealed a broad array of transcriptional transactivation abilities by both p73 isoforms at 37 degreesC. Recent data have shown that p73 can associate with p53 by the yeast two-hybrid assay. When we examined complex formation in transfected mammalian cells, we found that p73alpha coprecipitates with mutant but not wild-type p53. Since many tumor-derived p53 mutants are capable of inhibiting transactivation by wild-type p53, we tested the effects of two representative hot-spot mutants (R175H and R248W) on p73. By cotransfecting p73alpha along with either p53 mutant and a p53-responsive reporter, we found that both R175H and R248W reduces the transcriptional activity of p73alpha. This decrease in transcriptional activity is correlated with the reduced ability of p73alpha to promote apoptosis in the presence of tumor-derived p53 mutants. Our data suggest the possibility that in some tumor cells, an outcome of the expression of mutant p53 protein may be to interfere with the endogenous p73 protein.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia , Apoptose/genética , Apoptose/fisiologia , Sequência de Bases , Linhagem Celular , Genes Reporter , Genes Supressores de Tumor , Humanos , Plasmídeos/genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Ativação Transcricional , Transfecção , Proteína Tumoral p73 , Proteínas Supressoras de TumorRESUMO
The p53 protein is related by sequence homology and function to the products of two other genes, p63 and p73, that each encode several isoforms. We and others have discovered previously that certain tumor-derived mutants of p53 can associate and inhibit transcriptional activation by the alpha and beta isoforms of p73. In this study we have extended these observations to show that in transfected cells a number of mutant p53 proteins could bind and down-regulate several isoforms not only of p73 (p73 alpha, -beta, -gamma, and -delta) but also of p63 (p63 alpha and -gamma; Delta Np63 alpha and -gamma). Moreover, a correlation existed between the efficiency of p53 binding and the inhibition of p63 or p73 function. We also found that wild-type p63 and p73 interact efficiently with each other when coexpressed in mammalian cells. The interaction between p53 mutants and p63 or p73 was confirmed in a physiological setting by examining tumor cell lines that endogenously express these proteins. We also demonstrated that purified p53 and p73 proteins interact directly and that the p53 core domain, but not the tetramerization domain, mediates this interaction. Using a monoclonal antibody (PAb240) that recognizes an epitope within the core domain of a subset of p53 mutants, we found a correlation between the ability of p53 proteins to be immunoprecipitated by this antibody and their ability to interact with p73 or p63 in vitro and in transfected cells. Based on these results and those of others, we propose that interactions between the members of the p53 family are likely to be widespread and may account in some cases for the ability of tumor-derived p53 mutants to promote tumorigenesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Genes p53/genética , Proteínas de Membrana , Mutação , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Transativadores , Proteína Supressora de Tumor p53/metabolismo , Anticorpos Monoclonais/metabolismo , Células Cultivadas , Genes Reporter , Genes Supressores de Tumor , Vetores Genéticos/metabolismo , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Luciferases/metabolismo , Neoplasias/metabolismo , Testes de Precipitina , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Fatores de Transcrição , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/química , Proteínas Supressoras de TumorRESUMO
To investigate the possible link between Ca2+ signalling and cell cycle control we analysed Ca2+/calmodulin kinases (CamK) interaction with the retinoblastoma susceptibility gene product/SP1 pathway. CamK II and IV activate c-fos transcription through a short promoter region (-99 to -53) containing the retinoblastoma control element (RCE) and a cAMP response element (CRE) related sequences. Deletion analysis revealed that the RCE is a major CamK responsive element and is sufficient to confer CamK and Ca2+ regulation to a minimal promoter. Direct interactions between SP1 and RCE were confirmed by gel shift experiments. Using transient transfection experiments, we show that CamK-dependent transcription is regulated by the retinoblastoma (Rb) susceptibility gene product and the p107 Rb related protein. However, the stimulatory effects of CamKs and Rb on c-fos are blocked by overexpression of both proteins. These effects appear to be directly mediated by SP1 as shown by the use of a Gal4/SP1 fusion proteins. In conclusion, CamK II and IV, two major Ca2+-dependent intracellular effectors, may represent a molecular link between this second messenger transduction pathway and effectors that control cell cycle progression through Rb/SP1 signalling pathway.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Neoplasias da Retina/genética , Retinoblastoma/genética , Transdução de Sinais/fisiologia , Fator de Transcrição Sp1/fisiologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Predisposição Genética para Doença , Camundongos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/genética , Ativação Transcricional , Células Tumorais CultivadasRESUMO
Pit-1, a POU domain-containing transcription factor, is involved in two functions in the pituitary: PRL and GH tissue-specific expression and somatolactotroph cells expansion. To analyze the molecular basis of the latter function, we tested whether Pit-1 can directly transactivate expression of an early marker of cell cycle initiation, the c-fos gene. We show that Pit-1 overexpression in PC12 cells, which do not express Pit-1, increases c-fos expression. Moreover, cAMP-induced c-fos promoter activity is decreased in the somatolactotroph cell line GH3 when Pit-1 expression is reduced by hybrid arrest with an antisense sequence complementary to Pit-1 cDNA. In contrast to hormonal genes regulation, where it has been shown that any Pit-1 phosphorylation site is involved, we show that the Pit-1 phosphorylation sites are required to allow increase of c-fos promoter activity by Pit-1. We further show, by gel shift analyses, that Pit-1 is able to specifically bind the serum response element sequence present within the c-fos promoter but with a lesser affinity than the Pit-1 response element. Taken together, these results demonstrate that the tissue-specific transcription factor Pit-1 is able to enhance expression of genes involved in cell cycle initiation, suggesting that this mechanism allows Pit-1 to increase somato-lactotroph cell proliferation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Elementos de Resposta/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Proteínas de Ligação a DNA/genética , Especificidade de Órgãos , Células PC12/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição Pit-1 , Fatores de Transcrição/genéticaRESUMO
Constitutively active mutations of the G protein alpha(S) subunit are detected at a high frequency in human pituitary adenomas that secrete GH or PRL. It seems possible that over-expression of the pituitary cell-specific transcription factor Pit-1/GHF-1 (Pit-1) gene in response to active alpha(S) subunits contributes to the formation of these adenomas. We have examined whether expression in pituitary cells of one of these constitutively active alpha(S) subunits, Q227L-alpha(S), stimulates expression directed by the Pit-1 promoter. Transient expression of Q227L-alpha(S) yielded a strong stimulation of a target Pit-1 promoter-chloramphenicol acetyl transferase (CAT) construct, (-200)Pit-1-CAT. Expression of wild-type alpha(S) or an inactive alpha(S) mutant yielded, respectively, reduced or no stimulation of CAT activity. A dominant inhibitor of protein kinase A (PKA), RAB, blocked almost completely either forskolin (FSK) or Q227L-alpha(S) stimulation of (-200)Pit-1-CAT expression, implying that PKA is required for the action of Q227L-alpha(S) on the Pit-1 promoter. The Pit-1 promoter contains a binding site for Pit-1 and two CREB binding sites. Mutation of the Pit-1 binding site reduced but did not eliminate either FSK or Q227L-alpha(S) stimulation of Pit-1 promoter activity, implying a partial but incomplete requirement for this element for a PKA-mediated response to Q227L-alpha(S). The CREB dominant inhibitor S133A-CREB yielded a partial reduction in either FSK or Q227L-alpha(S) stimulation of (-200)Pit-1-CAT expression, implying that one or both of the Pit-1 promoter adenosine 3'5'-monophosphate response element binding protein (CREB) binding sites is/are also required for a complete PKA-mediated response to Q227L-alpha(S). The observation that S133A-CREB completely blocked the response to FSK or Q227L-alpha(S) of a Pit-1 promoter containing a mutated site PitB1 implies that the binding sites for Pit-1 and CREB account for all of the response elements for FSK or alpha(S) in the Pit-1 promoter.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Hipófise/citologia , Hipófise/metabolismo , Ratos , Fator de Transcrição Pit-1RESUMO
Somatic mutations of the alpha-subunit of Gs (Gs alpha) have been detected previously at high frequency in human PRL- and/or GH-producing pituitary tumors. To test whether these mutants are responsible for the increased production of these hormones, we used transient cotransfection assays to analyze their genomic effects in GH3 rat pituitary cells. We first show that guanosine triphosphatase (GTPase)-deficient Gs alpha subunits (mutated at amino acid 201 or 227) stimulate transcription from a reporter construct bearing the consensus cAMP response element (CRE; TGACGTCA). Using GAL4-CRE-binding protein fusion constructs, we further show that this stimulatory effects of Gs alpha on the CRE is probably mediated by the transacting factor CRE-binding protein. Then, in experiments using a reporter gene driven by the human promoters for either the PRL (position -250 to 18) or GH (position -500 to 13) genes, we show that these mutant Gs alpha subunits stimulate expression driven by either the PRL or GH promoter. Finally, we show that a dominant inhibitory mutant of cAMP-dependent kinase (protein kinase A) completely blocks the ability of these Gs alpha mutants to stimulate the activity of either the PRL or GH promoter, implying that GTPase-deficient Gs alpha subunits stimulation of the activities of these promoters is mediated entirely via the cAMP/protein kinase A pathway. Taken together, these results imply that activation of this pathway by the GTPase-deficient mutants found in human pituitary tumors stimulates the expression of PRL and GH genes. The transcriptional effects exerted via this pathway may thus provide a basis for the secretory phenotype and endocrine disorders associated with these tumors.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Hormônio do Crescimento/genética , Neoplasias Hipofisárias/metabolismo , Prolactina/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Humanos , Mutação , Células Tumorais CultivadasRESUMO
In the corticotroph cell line AtT20, Ca2+ stimulates c-fos mRNA and nuclear c-fos protein accumulation. We show that Ca2+ stimulates transcription of a chloramphenicol-acetyl-transferase reporter gene driven by the c-fos promoter. This effect is mimicked by both constitutively active Ca2+/calmodulin-dependent protein kinase types II (CamK II) and IV (CamK IV). The effects of CamK IV are synergistic with those obtained by cAMP stimulation, whereas CamK II-dependent transcription is not further stimulated. Deletion of the distal promoter sequence revealed that CamK IV, and to a lesser extent CamK II, activate a cis-acting element(s) located between -99 and -53 bp prior to the transcription initiation site. Moreover, CamK II further stimulates transcription through the fos AP1-like element. CamK IV has a lower effect on this element but represses the serum response element. The latter observation might account for the lower transcriptional effects of CamK IV on the full length c-fos promoter.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Transcrição Gênica , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Camundongos , Sequências Reguladoras de Ácido Nucleico , Células Tumorais CultivadasRESUMO
The study of metal complexes for the treatment of cancer diseases has resulted in the identification of some unique properties of ruthenium-based compounds. Among these inorganic-based agents, two of them, namely the ruthenium(III) drugs NAMI-A and KP1019 have undertaken with some success the clinical evaluations of phase I and preliminary phase II trials in patients. Here we highlight the strategies that have led to the discovery of metal-based (NAMI-A and KP1019) and of organometallic (RM175, RAPTA-T, RDC11 and DW1/2) ruthenium-based complexes, and we report their main biological/pharmacological characteristics and expectations for further development.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Compostos Organometálicos/uso terapêutico , Rutênio/química , Animais , Antineoplásicos/química , Sobrevivência Celular/efeitos dos fármacos , Ensaios Clínicos como Assunto , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Neoplasias/patologia , Compostos Organometálicos/químicaRESUMO
The metallic compound cisplatin has been used for many years to treat various human cancers. Here, we describe the cytostatic and cytotoxic properties of a new class of organometallic compounds that contain a ruthenium (II) atom covalently linked to carbon and nitrogen atoms. We found that several ruthenium-derived compounds (RDCs) led to G1 arrest and induced apoptosis in tumor cell lines derived from glioblastomas, neuroblastomas, and lymphoid tumors at least as efficiently as cisplatin. We further analyzed the signaling pathways underlying these effects, and we showed that both RDCs and cisplatin induced p53 and p73 protein levels but with different intensities and kinetics. This accumulation of p53 and p73 proteins correlated with an increase in p21 and Bax expression, two p53 target genes linked to cell growth arrest and apoptosis. However, in contrast to cisplatin-induced apoptosis, overexpression of DeltaNp73, a p53 and p73 dominant-negative isoform, only partly reduced RDC-induced apoptosis, suggesting p53-dependent and p53-independent modes of action. This observation was further confirmed by the ability of RDC to induce apoptosis in p53-/- cells. Altogether, this study highlights key cellular and molecular features of RDCs and suggests that further development of this new class of compounds may contribute to improve future chemotherapeutic protocols.
Assuntos
Antineoplásicos/toxicidade , Compostos Organometálicos/toxicidade , Rutênio/toxicidade , Proteína Supressora de Tumor p53/metabolismo , Animais , Anexina A5/análise , Anexina A5/metabolismo , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , DNA de Neoplasias/análise , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Fase G1/efeitos dos fármacos , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Compostos Organometálicos/química , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de TumorRESUMO
Ref-1 is a multifunctional protein that stimulates DNA binding by a number of transcription factors and serves as the abasic (A/P) endonuclease in base excision repair. Ref-1 was discovered to be a potent activator of p53 DNA binding in vitro. To address the physiological significance of the effects of Ref-1 on p53, we have analyzed its role in regulating p53 function in vivo. We found that Ref-1 over-expression enhances the ability of p53 to transactivate a number of p53 target promoters and increases the ability of p53 to stimulate endogenous p21 and cyclin G expression. Additionally, it was observed that Ref-1 associates with p53 in vivo and in vitro. Importantly, downregulation of Ref-1 (by antisense) causes a marked reduction in p53 induction of p21 mRNA and protein, as well as diminished ability of p53 to transactivate the p21 and Bax promoters. Moreover, Ref-1 levels are correlated with the extent of apoptosis induced by p53. Finally, we observed that Ref-1 cooperates with a DNA-damaging compound, camptothecin, to stimulate the transcriptional activity of p53. Together these data indicate that Ref-1 is a key cellular regulator of p53.
Assuntos
Apoptose/genética , Apoptose/fisiologia , Carbono-Oxigênio Liases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Genes p53 , Sequência de Bases , Camptotecina/farmacologia , Carbono-Oxigênio Liases/antagonistas & inibidores , Carbono-Oxigênio Liases/genética , Linhagem Celular , Dano ao DNA , Primers do DNA/genética , Regulação para Baixo , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ativação Transcricional/efeitos dos fármacos , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, regulates survival and apoptosis of several neuronal populations. These effects are initiated by high-affinity membrane receptors displaying tyrosine kinase activity (trk). However, the intracellular pathways and genetic mechanisms associated with these receptors are largely unknown. Here we show that BDNF stimulates AP1 binding activity in primary cerebellar neurons. This binding corresponds to a functional complex as it is associated with the induction of AP1-dependent transactivation. Application of AP1 partner mRNAs shows an increase in levels of c-fos and c-jun mRNAs after BDNF treatment, resulting from an induction of their promoters. The cis-acting elements by which BDNF stimulates c-fos transcription were further studied. We show that BDNF impinges on multiple regulatory elements, including the serum-responsive element, Fos AP1-like element, and cyclic AMP (cAMP)-responsive element (CRE) sequences. The latter was stimulated without any detectable increase in cAMP or Ca2+ levels. To confirm that BDNF induces c-fos transcription independently of the protein kinase A/cAMP pathway, we transfected a dominant inhibitory mutant of the regulatory subunit of protein kinase A. The overexpression of this mutant does not affect the c-fos promoter transactivation by BDNF. In summary, we show that BDNF stimulates AP1- and CRE-dependent transcription through a mechanism that is distinct from the cAMP- and Ca(2+)-dependent pathways in CNS neurons.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Fator de Transcrição AP-1/genética , Animais , Fator Neurotrófico Derivado do Encéfalo , Cálcio/fisiologia , Sobrevivência Celular/genética , Sistema Nervoso Central/citologia , Cerebelo/citologia , AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Genes Precoces/genética , Genes fos/genética , Genes jun/genética , Neurônios/citologia , Neurônios/enzimologia , Regiões Promotoras Genéticas/genética , Ligação Proteica/fisiologia , Ratos , Sistemas do Segundo Mensageiro/fisiologia , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/genética , TransfecçãoRESUMO
To investigate the molecular mechanisms underlying brain-derived neurotrophic factor (BDNF)-controlled synaptic plasticity, we studied beta2-adrenergic receptor (beta2-AR) expression in cultured cerebellar granule cells. We show that, depending on the state of depolarization, BDNF exerts opposite effects on beta2-AR expression. In neurons maintained in low K+ medium (5 mM K+) that will enter apoptosis, BDNF increases beta2-AR and beta2-AR transcripts. In contrast, in depolarized neurons (high K+ medium, 25 mM K+) BDNF represses beta2-AR expression. The use of reporter genes (driven by the beta2-AR promoter or restricted regulatory elements) revealed that BDNF exerts its opposite effects at the transcriptional level by recruiting a cyclic AMP response element (CRE) and the trans-acting factor CRE binding protein. These results provide the first evidence that a neurotrophin, e.g., BDNF, may exert an opposite effect on receptor expression and function (beta2-AR) according to the depolarization status of the neuron. Based on this finding, we propose that BDNF not only mediates neuronal survival, but is also involved in the modulation of the general sensitivity of the neuron to external signals, thus maintaining its optimal functional integration within the neuronal network.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Cerebelo/fisiologia , Neurônios/fisiologia , Receptores Adrenérgicos beta 2/fisiologia , Transcrição Gênica/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Humanos , Isoproterenol/farmacologia , Cinética , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/efeitos dos fármacos , Potássio/farmacologia , Regiões Promotoras Genéticas , Ratos , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes de Fusão/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Somatic mutations of the alpha subunit of Gs (G alpha s) have been detected in a variety of endocrine tumors. To test whether G alpha s is an oncogene, we investigated the genomic effects of G alpha s protein in which the GTPase activity had been inactivated. Results from transient transfection studies show that such proteins increase 1) transcription of a reporter gene driven by the minimal cAMP-responsive element (TGACGTCA) and 2) c-fos transcription in several endocrine cell lines (GH3, AtT20, and PC12). By promoter deletion analyses and genetic inactivation of cAMP-dependent protein kinase, we show that this transcriptional stimulation by G alpha s impinges on several regulatory elements within the c-fos promoter and operates within the protein kinase A pathways. Stable PC12 cell lines were established to analyze long-term effects of constitutively active G alpha s. Cell lines expressing mutated G alpha s have elevated cAMP levels and increased AP1 binding activity. Transcription of a variety of genes, including c-fos, c-jun, and junB, is increased in these cells. The strong and permanent effects of G alpha s on early immediate genes, and c-fos in particular, may be responsible for the oncogenic potential of G alpha s in endocrine cells.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Genes fos , Proto-Oncogenes , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , Cricetinae , Cricetulus , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Células PC12 , Hipófise , Ratos , Proteínas Recombinantes/metabolismo , Deleção de SequênciaRESUMO
The proopiomelanocortin (POMC) gene expressed in corticotrophs of the anterior pituitary encodes several biologically active peptides and is primarily under the positive control of hypophysiotropic factors (e.g. corticotropin releasing hormone). Using AtT20 cells as a model, we show that these factors increase levels of POMC primary RNA transcripts representative of a transcriptional activation of the gene. This effect is mimicked by several activators of the cAMP signaling pathway. Inhibition of protein synthesis with cycloheximide did not modify the CRH-induced increase in POMC hnRNA suggesting that these early effects are mediated by preexisting transcription factors. Using a reporter gene containing 706 bp of the POMC promoter region, we observe transcriptional activation with the same compounds, their effects being abolished when protein kinase A (PKA) is inactivated by a dominant inhibitory mutant. Promoter deletion analyses mapped an essential cAMP inducible element within the first exon of the POMC gene. This element (PTRE: TGACTAA) located at nucleotides +41/+47 was shown to bind the cAMP responsive element binding protein (CREB) by gel shift analyses and confers strong transcriptional activation by an expression vector coding a CREB-VP16 activator domain fusion protein. Further, expression of a dominant inhibitory mutant of CREB reduced cAMP stimulated transcription of the full length POMC promoter and the PTRE. Taken together, these results show that the major hypophysiotropic factors stimulate POMC transcription through a signaling cascade that involves PKA and CREB.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Pró-Opiomelanocortina/genética , Animais , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cicloeximida/farmacologia , Genes Reporter , Camundongos , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) receptors were characterized and their function investigated in mouse pituitary neurointermediate lobe explants. We show that mouse neurointermediate lobes can be maintained for up to 1 month in defined medium. After 8 days in culture, these explants are devoid of any of the original tyrosine hydroxylase or glutamate decarboxylase immunoreactive fibers, which in situ innervate the melanotropes. Under these culture conditions, no mitotic activity is detectable in melanotropes and these cells remain sensitive to physiological regulation such as dopamine and corticotropin-releasing hormone. Using in situ hybridization and polymerase chain reaction, we show that in situ and in neurointermediate lobe explants, melanotropes express PACAP receptor type I isoforms that transduce through the cAMP and inositol phosphate pathways. In neurointermediate lobe explants, PACAP 27 and PACAP 38 (10(-8) M) stimulate cAMP accumulation whereas PACAP 38 but not PACAP 27 stimulates inositol phosphate breakdown. However, both ligands are potent stimulators of proopiomelanocortin (POMC)-derived peptides exocytosis and POMC gene transcription. In addition, stimulation of POMC gene transcription is mediated both by cAMP and by inositol phosphate pathways. Taken together, our data suggest that PACAP is a major regulator of melanotrope functions.