RESUMO
CASZ1 is a conserved transcription factor involved in neural development, blood vessel assembly and heart morphogenesis. CASZ1 has been implicated in cancer, either suppressing or promoting tumor development depending on the tissue. However, the impact of CASZ1 on hematological tumors remains unknown. Here, we show that the T-cell oncogenic transcription factor TAL1 is a direct positive regulator of CASZ1, that T-cell acute lymphoblastic leukemia (T-ALL) samples at diagnosis overexpress CASZ1b isoform, and that CASZ1b expression in patient samples correlates with PI3KAKT- mTOR signaling pathway activation. In agreement, overexpression of CASZ1b in both Ba/F3 and T-ALL cells leads to the activation of PI3K signaling pathway, which is required for CASZ1b-mediated transformation of Ba/F3 cells in vitro and malignant expansion in vivo. We further demonstrate that CASZ1b cooperates with activated NOTCH1 to promote T-ALL development in zebrafish, and that CASZ1b protects human T-ALL cells from serum deprivation and treatment with chemotherapeutic drugs. Taken together, our studies indicate that CASZ1b is a TAL1-regulated gene that promotes T-ALL development and resistance to chemotherapy.
RESUMO
Interleukin-7 receptor α (encoded by IL7R) is essential for lymphoid development. Whether acute lymphoblastic leukemia (ALL)-related IL7R gain-of-function mutations can trigger leukemogenesis remains unclear. Here, we demonstrate that lymphoid-restricted mutant IL7R, expressed at physiological levels in conditional knock-in mice, establishes a pre-leukemic stage in which B-cell precursors display self-renewal ability, initiating leukemia resembling PAX5 P80R or Ph-like human B-ALL. Full transformation associates with transcriptional upregulation of oncogenes such as Myc or Bcl2, downregulation of tumor suppressors such as Ikzf1 or Arid2, and major IL-7R signaling upregulation (involving JAK/STAT5 and PI3K/mTOR), required for leukemia cell viability. Accordingly, maximal signaling drives full penetrance and early leukemia onset in homozygous IL7R mutant animals. Notably, we identify 2 transcriptional subgroups in mouse and human Ph-like ALL, and show that dactolisib and sphingosine-kinase inhibitors are potential treatment avenues for IL-7R-related cases. Our model, a resource to explore the pathophysiology and therapeutic vulnerabilities of B-ALL, demonstrates that IL7R can initiate this malignancy.
Assuntos
Subunidade alfa de Receptor de Interleucina-7/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Mutação com Ganho de Função , Heterozigoto , Homozigoto , Humanos , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Camundongos , Penetrância , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Células Precursoras de Linfócitos B/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a dismal prognosis in patients with resistant or relapsed disease. Although NOTCH is a known driver in T-ALL, its clinical inhibition has significant limitations. Our previous studies suggested that NRARP, a negative regulator of Notch signaling, could have a suppressive role in T-ALL. Here, we report that NRARP levels are significantly increased in primary T-ALL cells suggesting that NRARP is not sufficient to block NOTCH oncogenic signals. Interestingly, although NRARP overexpression blocks NOTCH1 signaling and delays the proliferation of T-ALL cells that display high levels of Notch1 signaling, it promotes the expansion of T-ALL cells with lower levels of Notch1 activity. We found that NRARP interacts with lymphoid enhancer-binding factor 1 (LEF1) and potentiates Wnt signaling in T-ALL cells with low levels of Notch. Together these results indicate that NRARP plays a dual role in T-ALL pathogenesis, regulating both Notch and Wnt pathways, with opposite functional effects depending on Notch activity. Consistent with this hypothesis, mice transplanted with T-cells co-expressing NOTCH1 and NRARP develop leukemia later than mice transplanted with T-NOTCH1 cells. Importantly, mice transplanted with T-cells overexpressing NRARP alone developed leukemia with similar kinetics to those transplanted with T-NOTCH1 cells. Our findings uncover a role for NRARP in T-ALL pathogenesis and indicate that Notch inhibition may be detrimental for patients with low levels of Notch signaling, which would likely benefit from the use of Wnt signaling inhibitors. Importantly, our findings may extend to other cancers where Notch and Wnt play a role.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptores Notch/metabolismo , Via de Sinalização Wnt , Linhagem Celular Tumoral , Humanos , Regulação para CimaAssuntos
Transplante de Células-Tronco Hematopoéticas , Reação em Cadeia da Polimerase em Tempo Real , Quimeras de Transplante , Adulto , Deleção de Genes , Doença Enxerto-Hospedeiro/diagnóstico , Humanos , Mutação INDEL , Recidiva Local de Neoplasia/diagnóstico , Polimorfismo Genético , Células-Tronco/citologia , Fatores de Tempo , Transplante Homólogo , Resultado do TratamentoRESUMO
Phosphatase and tensin homolog (PTEN)-inactivating mutations and/or deletions are an independent risk factor for relapse of T-cell acute lymphoblastic leukemia (T-ALL) patients treated on Dutch Childhood Oncology Group or German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia protocols. Some monoallelic mutated or PTEN wild-type patients lack PTEN protein, implying that additional PTEN inactivation mechanisms exist. We show that PTEN is inactivated by small deletions affecting a few exons in 8% of pediatric T-ALL patients. These microdeletions were clonal in 3% and subclonal in 5% of patients. Conserved deletion breakpoints are flanked by cryptic recombination signal sequences (cRSSs) and frequently have non-template-derived nucleotides inserted in between breakpoints, pointing to an illegitimate RAG recombination-driven activity. Identified cRSSs drive RAG-dependent recombination in a reporter system as efficiently as bona fide RSSs that flank gene segments of the T-cell receptor locus. Remarkably, equivalent microdeletions were detected in thymocytes of healthy individuals. Microdeletions strongly associate with the TALLMO subtype characterized by TAL1 or LMO2 rearrangements. Primary and secondary xenotransplantation of TAL1-rearranged leukemia allowed development of leukemic subclones with newly acquired PTEN microdeletions. Ongoing RAG activity may therefore actively contribute to the acquisition of preleukemic hits, clonal diversification, and disease progression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Deleção de Genes , Proteínas de Homeodomínio/genética , Proteínas com Domínio LIM/genética , PTEN Fosfo-Hidrolase/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas/genética , Recombinação Genética/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Rearranjo Gênico , Humanos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Transplante HeterólogoRESUMO
Healthy radio-exposed individuals who received low levels of Cesium-137 radiation during the accident that occurred in Goiânia in 1987, their families and controls were tested for the detection of t(14;18)-rearranged B cells in peripheral blood by using a highly sensitive, real-time quantitative PCR method. The chromosomal translocation t(14;18)(q32;q21) is characteristic of follicular lymphoma and is a frequent abnormality observed in other types of non-Hodgkin's lymphoma. This translocation leads to constitutive activation of the BCL2 oncogene by the enhancers of the immunoglobulin heavy-chain locus. In healthy individuals, the same translocation may also be found in a small fraction of peripheral blood lymphocytes, and positive cells might serve as an indicator for environmental exposure to carcinogens and possibly correlate with the cumulative risk of developing t(14;18)- positive non-Hodgkin's lymphoma. Twenty healthy radio-exposed individuals, 10 relatives and 10 non-exposed healthy individuals were tested for the detection of this translocation. Only 1 non-exposed individual was positive for the chromosomal translocation, and healthy radio-exposed individuals presented lower levels of cells bearing the BCL2/J(H) rearrangement when compared to the levels of the patients with follicular lymphoma before treatment. However, evaluation of more cells would be required to confirm the total absence of circulating cells bearing BCL2/J(H) rearrangement.
Assuntos
Radioisótopos de Césio/efeitos adversos , Genes bcl-2 , Liberação Nociva de Radioativos , Translocação Genética/efeitos da radiação , Adulto , Linfócitos B/efeitos da radiação , Brasil , Linhagem Celular , Cromossomos Humanos Par 14/efeitos da radiação , Cromossomos Humanos Par 18/efeitos da radiação , Exposição Ambiental/efeitos adversos , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma Folicular/etiologia , Linfoma Folicular/genética , Linfoma não Hodgkin/etiologia , Linfoma não Hodgkin/genética , Neoplasias Induzidas por Radiação/etiologia , Neoplasias Induzidas por Radiação/genéticaRESUMO
The interaction of acute lymphoblastic leukemia (ALL) blasts with bone marrow (BM) stromal cells (BMSCs) has a positive impact on ALL resistance to chemotherapy. We investigated the modulation of a series of putative asparaginase-resistance/sensitivity genes in B-precursor ALL cells upon coculture with BMSCs. Coculture with stromal cells resulted in increased insulin-like growth factor (IGF)-binding protein 7 (IGFBP7) expression by ALL cells. Assays with IGFBP7 knockdown ALL and stromal cell lines, or with addition of recombinant rIGFBP7 (rIGFBP7) to the culture medium, showed that IGFBP7 acts as a positive regulator of ALL and stromal cells growth, and significantly enhances in-vitro resistance of ALL to asparaginase. In these assays, IGFBP7 function occurred mainly in an insulin- and stromal-dependent manner. ALL cells were found to contribute substantially to extracellular IGFBP7 levels in the conditioned coculture medium. Diagnostic BM plasma from children with ALL had higher levels of IGFBP7 than controls. IGFBP7, in an insulin/IGF-dependent manner, enhanced asparagine synthetase expression and asparagine secretion by BMSCs, thus providing a stromal-dependent mechanism by which IGFBP7 protects ALL cells against asparaginase in this coculture system. Importantly, higher IGFBP7 mRNA levels were associated with lower leukemia-free survival (Cox regression model, P=0.003) in precursor B-cell Ph(-) ALL patients (n=147) treated with a contemporary polychemotherapy protocol.
Assuntos
Asparaginase/farmacologia , Células da Medula Óssea/patologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Células Estromais/patologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Técnicas de Cocultura , Meios de Cultivo Condicionados , Resistencia a Medicamentos Antineoplásicos , Feminino , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Humanos , Lactente , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RNA Interferente Pequeno , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Interleukin 7 (IL-7) and its receptor, formed by IL-7Rα (encoded by IL7R) and γc, are essential for normal T-cell development and homeostasis. Here we show that IL7R is an oncogene mutated in T-cell acute lymphoblastic leukemia (T-ALL). We find that 9% of individuals with T-ALL have somatic gain-of-function IL7R exon 6 mutations. In most cases, these IL7R mutations introduce an unpaired cysteine in the extracellular juxtamembrane-transmembrane region and promote de novo formation of intermolecular disulfide bonds between mutant IL-7Rα subunits, thereby driving constitutive signaling via JAK1 and independently of IL-7, γc or JAK3. IL7R mutations induce a gene expression profile partially resembling that provoked by IL-7 and are enriched in the T-ALL subgroup comprising TLX3 rearranged and HOXA deregulated cases. Notably, IL7R mutations promote cell transformation and tumor formation. Overall, our findings indicate that IL7R mutational activation is involved in human T-cell leukemogenesis, paving the way for therapeutic targeting of IL-7R-mediated signaling in T-ALL.
Assuntos
Oncogenes , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptores de Interleucina-7/genética , Transdução de Sinais , Animais , Ciclo Celular , Linhagem Celular , Sobrevivência Celular , Criança , Cisteína/genética , Cisteína/metabolismo , Análise Mutacional de DNA , Éxons , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Interleucina-7/genética , Interleucina-7/metabolismo , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Camundongos , Camundongos Knockout , Mutação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Interleucina-7/metabolismo , Análise de Sequência de DNA , Linfócitos T/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
The transcription factor T-cell acute lymphocytic leukemia (TAL)-1 is a major T-cell oncogene associated with poor prognosis in T-cell acute lymphoblastic leukemia (T-ALL). TAL1 binds histone deacetylase 1 and incubation with histone deacetylase inhibitors (HDACis) promotes apoptosis of leukemia cells obtained from TAL1 transgenic mice. Here, we show for the first time that TAL1 protein expression is strikingly downregulated upon histone deacetylase inhibition in T-ALL cells. This is due to decreased TAL1 gene transcription in cells with native TAL1 promoter, and due to impaired TAL1 mRNA translation in cells that harbor the TAL1(d) microdeletion and consequently express TAL1 under the control of the SCL/TAL1 interrupting locus (SIL) promoter. Notably, HDACi-triggered apoptosis of T-ALL cells is significantly reversed by TAL1 forced overexpression. Our results indicate that the HDACi-mediated apoptotic program in T-ALL cells is partially dependent on their capacity to downregulate TAL1 and provide support for the therapeutic use of HDACi in T-ALL.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação para Baixo , Inibidores de Histona Desacetilases/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Regulação para Cima , Imunoprecipitação da Cromatina , Humanos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Células Tumorais CultivadasRESUMO
The importance of microenvironmental factors for driving progression in leukemia has been debated. Previous evidence has pointed to interleukin-7 (IL-7), a fundamental cytokine to normal T-cell development and homeostasis, as an important determinant of the viability and proliferation of T-cell acute lymphoblastic leukemia (T-ALL) cells in vitro. In this study, we report that IL-7 is also a critical determinant of T-ALL progression. T-ALL cell lines and primary T-ALL samples initiated leukemia more slowly when engrafted to immunocompromised Rag2(-/-)IL2rg(-/-) mice lacking IL-7. This effect was not related to reduced engraftment or homing of transplanted cells to the bone marrow. Instead, IL-7 deficiency diminished expansion of leukemia cells in the bone marrow and delayed leukemia-associated death of transplanted mice. Moreover, infiltration of different organs by T-ALL cells, which characterizes patients with advanced disease, was more heterogeneous and generally less efficient in IL-7-deficient mice. Leukemia progression was associated with increased Bcl-2 expression and cell viability, reduced p27(Kip1) expression, and decreased cell-cycle progression. Clinical measurements of IL-7 plasma levels and IL-7 receptor (IL-7R) expression in T-ALL patients versus healthy controls confirmed that IL-7 stimulates human leukemia cells. Our results establish that IL-7 contributes to the progression of human T-cell leukemia, and they offer preclinical validation of the concept that targeting IL-7/IL-7R signaling in the tumor microenvironment could elicit therapeutic effects in T-ALL.
Assuntos
Interleucina-7/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Animais , Processos de Crescimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Regulação para Baixo , Humanos , Interleucina-7/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina-7/biossíntese , Receptores de Interleucina-7/genética , Transplante Heterólogo , Microambiente Tumoral , Regulação para CimaRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) patients frequently display NOTCH1 activating mutations and Notch can transcriptionally down-regulate the tumor suppressor PTEN. However, it is not clear whether NOTCH1 mutations associate with decreased PTEN expression in primary T-ALL. Here, we compared patients with or without NOTCH1 mutations and report that the former presented higher MYC transcript levels and decreased PTEN mRNA expression. We recently showed that T-ALL cells frequently display CK2-mediated PTEN phosphorylation, resulting in PTEN protein stabilization and concomitant functional inactivation. Accordingly, the T-ALL samples analyzed, irrespectively of their NOTCH1 mutational status, expressed significantly higher PTEN protein levels than normal controls. To evaluate the integrated functional impact of Notch transcriptional and CK2 post-translational inactivation of PTEN, we treated T-ALL cells with both the gamma-secretase inhibitor DAPT and the CK2 inhibitors DRB/TBB. Our data suggest that combined use of gamma-secretase and CK2 inhibitors may have therapeutic potential in T-ALL.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , PTEN Fosfo-Hidrolase/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor Notch1/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Western Blotting , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Criança , Diclororribofuranosilbenzimidazol/farmacologia , Quimioterapia Combinada , Inibidores Enzimáticos/farmacologia , Regulação Leucêmica da Expressão Gênica , Humanos , Mutação/genética , PTEN Fosfo-Hidrolase/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Notch1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Triazóis/farmacologiaAssuntos
Mutação , PTEN Fosfo-Hidrolase/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Criança , Éxons , Humanos , Fosfatidilinositol 3-Quinases/fisiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidade , Prognóstico , Proteínas Proto-Oncogênicas c-akt/fisiologiaRESUMO
Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. Here we show that PTEN inactivation in human T cell acute lymphoblastic leukemia (T-ALL) cells is not always synonymous with PTEN gene lesions and diminished protein expression. Samples taken from patients with T-ALL at the time of diagnosis very frequently showed constitutive hyperactivation of the PI3K/Akt pathway. In contrast to immortalized cell lines, most primary T-ALL cells did not harbor PTEN gene alterations, displayed normal PTEN mRNA levels, and expressed higher PTEN protein levels than normal T cell precursors. However, PTEN overexpression was associated with decreased PTEN lipid phosphatase activity, resulting from casein kinase 2 (CK2) overexpression and hyperactivation. In addition, T-ALL cells had constitutively high levels of ROS, which can also downmodulate PTEN activity. Accordingly, both CK2 inhibitors and ROS scavengers restored PTEN activity and impaired PI3K/Akt signaling in T-ALL cells. Strikingly, inhibition of PI3K and/or CK2 promoted T-ALL cell death without affecting normal T cell precursors. Overall, our data indicate that T-ALL cells inactivate PTEN mostly in a nondeletional, posttranslational manner. Pharmacological manipulation of these mechanisms may open new avenues for T-ALL treatment.
Assuntos
PTEN Fosfo-Hidrolase/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Humanos , Modelos Biológicos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
Oxidative stress induced by microcystins was evaluated in an estuarine worm, Laeonereis acuta (Nereididae). Ten organisms were exposed to lyophilized cells of a toxic Microcystisaeruginosa strain RST9501 ( approximately 2 microg/mL microcystins, MC); 10 were exposed to lyophilized cells of a nontoxic Aphanotece sp. strain RSMan92 and 10 were maintained without cyanobacterial cells. Exposure time was 48 h. The enzymatic antioxidant defenses, as well as the oxidative damage, were analyzed. Toxic and nontoxic cyanobacteria lowered catalase activity with no changes in glutathione reductase and glutathione-S-transferase activities. This may have led to toxin intracellular accumulation, which should favor oxidative stress generation, observed by the high lipid peroxide and DNA-protein crosslink levels in the group exposed to MC.
Assuntos
Toxinas Marinhas/toxicidade , Microcistinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Poliquetos/efeitos dos fármacos , Animais , Microcystis/química , Poliquetos/enzimologiaRESUMO
The present study compares two computer models of the first part of glucose catabolism in different organisms in search of evolutionarily conserved characteristics of the glycolysis cycle and proposes the main parameters that define the stable steady-state or oscillatory behavior of the glycolytic system. It is suggested that in both human pancreatic beta-cells and Saccharomyces cerevisiae there are oscillations that, despite differences in wave form and period of oscillation, share the same robustness strategy: the oscillation is not controlled by only one but by at least two parameters that will have more or less control over the pathway flux depending on the initial state of the system as well as on extra-cellular conditions. This observation leads to two important interpretations: the first is that in both S. cerevisiae and human beta-cells, despite differences in enzyme kinetics and mechanism of feedback control, evolution seems to have kept an oscillatory behavior coupled to the glucose concentration outside the cytoplasm, and the second is that the development of drugs to regulate metabolic dysfunctions in more complex systems may require further study, not only determining which enzyme is controlling the flux of the system but also under which conditions and how its control is maintained by the enzyme or transferred to other enzymes in the pathway as the drug starts acting.
Assuntos
Glicólise , Células Secretoras de Insulina/metabolismo , Saccharomyces cerevisiae/metabolismo , Simulação por Computador , Ativação Enzimática , Glucoquinase/metabolismo , Glucose/metabolismo , Humanos , Células Secretoras de Insulina/enzimologia , Cinética , Modelos Biológicos , Oscilometria , Fosfofrutoquinases/metabolismo , Saccharomyces cerevisiae/enzimologiaRESUMO
The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (<10 US dollars) but equally accurate alternative to the Test arrays in determining biotinylated cRNA quality. Forty-one different cRNA samples were hybridized to HG-U133A microarrays from Affymetrix. Ten cRNA samples with a beta-actin 3'/5' ratio >6 were chosen and classified as degraded cRNAs, and 31 samples with a beta-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of beta-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r(2) = 0.6802) between the array and the RT-PCR beta-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean +/- SD, 0.34 +/- 0.09) and degraded (1.71 +/- 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Complementar/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Biotinilação , HumanosRESUMO
The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (
Assuntos
Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Complementar/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , BiotinilaçãoRESUMO
Microcystins produced by cyanobacteria are potent inhibitors of some protein phosphatases, but recent evidence also indicates its potential to generate oxidative stress. In the present study, the effects of microcystin raw extracts (Mic; 0.01 and 20microg/L) and purified okadaic acid (OA; 0.01 and 10microg/L) on short- and long-term memory alteration and antioxidant and oxidative damage were investigated in hippocampus of rats. The results showed an amnesic effect with 0.01 and 20microg/L Mic on retrieval and only with 0.01microg/L Mic on spatial learning. Parallel to these effects oxidative damage was observed as evidenced by augmented levels of lipid peroxides and DNA damage and the absence of antioxidant responses in terms of total oxyradical scavenging capacity. Phase II reactions catalyzed by glutathione-S-transferase were not modified after microcystins exposure. Overall this study showed physiological events (retrieval and spatial learning) that can be related to the classical toxic effects of microcystins (i.e., phosphatase inhibition). In addition, evidence of alternative toxicity mechanisms via oxidative stress generation was also obtained. The fact that organic anion transporter polypeptides (OATP) involved in microcystins uptake are expressed not only in liver but also in brain points to the environmental relevance of the observed effects.
Assuntos
Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Memória de Curto Prazo/efeitos dos fármacos , Memória/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Animais , Hipocampo/metabolismo , Memória/fisiologia , Memória de Curto Prazo/fisiologia , Microcistinas , Ácido Okadáico/farmacologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Microcystins are usually the predominant cyanotoxins present in both drinking and recreational waters after cyanobacterial blooms. Their classic toxic effect is hepatotoxicity through inhibition of serine/threonine phosphatases. However, recent studies also reported oxidative stress generation and disruption of ion regulation in aquatic organisms after microcystins exposure. In the present study, aqueous extracts of Microcystis aeruginosa were administered to the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura) by gavage in variable doses (from 34 to 860 microg kg(-1)) and exposure times (6, 12, and 72 h). A control group was exposed to saline solution. Analyzed variables included oxygen consumption, lipid peroxidation (LPO), enzyme activities (glutathione S-transferases or GST; alanine aminotransferase or ALT; aspartate aminotransferase or AST; and lactate dehydrogenase or LDH), glycogen, and microcystins content. Oxygen consumption increased in organisms exposed for 12h to 860 microg kg(-1) of microcystins and a similar result was observed after 72 h at doses equal to or higher than 34 microg kg(-1). LPO levels increased in doses equal to or higher than 34 microg kg(-1) after 72 h. GST and LDH activities increased after 12 h (at a dose of 860 microg kg(-1)), but ALT and AST activities remained unaltered in all experimental conditions. Glycogen content decreased after 72 h exposure at doses equal to or higher than 172 microg kg(-1). After 12h of exposure to 860 microg kg(-1) of microcystins, the concentration found in the hepatopancreas of C. granulatus was 13.17+/-0.56 microg kg(-1). In crabs exposed to doses higher than 172 microg kg(-1) during 72 h this value raised to 32.14+/-4.12 microg kg(-1). The obtained results indicated that microcystins exposure led the tissue to an oxidative stress condition (high LPO levels), at least in part favored by the augment of oxygen consumption, altering the glycogen metabolism. GST responses were only observed in the short-term experiment (12 h) and no effect on classical markers of vertebrate liver damage (ALT and AST) was observed. Although the hepatopancreas from C. granulatus accumulated a relatively low concentration of toxins, it was enough to induce physiological and biochemical disturbances.
Assuntos
Braquiúros , Microcistinas/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Braquiúros/efeitos dos fármacos , Braquiúros/enzimologia , Braquiúros/metabolismo , Braquiúros/fisiologia , Relação Dose-Resposta a Droga , Hepatopâncreas/efeitos dos fármacos , Hepatopâncreas/enzimologia , Hepatopâncreas/metabolismo , Peróxidos Lipídicos/metabolismo , Masculino , Microcistinas/farmacocinética , Estresse Oxidativo/efeitos dos fármacos , Poluentes Químicos da Água/farmacocinéticaRESUMO
The present study compares two computer models of the first part of glucose catabolism in different organisms in search of evolutionarily conserved characteristics of the glycolysis cycle and proposes the main parameters that define the stable steady-state or oscillatory behavior of the glycolytic system. It is suggested that in both human pancreatic b-cells and Saccharomyces cerevisiae there are oscillations that, despite differences in wave form and period of oscillation, share the same robustness strategy: the oscillation is not controlled by only one but by at least two parameters that will have more or less control over the pathway flux depending on the initial state of the system as well as on extra-cellular conditions. This observation leads to two important interpretations: the first is that in both S. cerevisiae and human b-cells, despite differences in enzyme kinetics and mechanism of feedback control, evolution seems to have kept an oscillatory behavior coupled to the glucose concentration outside the cytoplasm, and the second is that the development of drugs to regulate metabolic dysfunctions in more complex systems may require further study, not only determining which enzyme is controlling the flux of the system but also under which conditions and how its control is maintained by the enzyme or transferred to other enzymes in the pathway as the drug starts acting.