RESUMEN
1. An experiment was conducted to study the effect of supplementing higher concentrations (100% vs. 110%) of critical amino acids (CAA) on performance (body weight gain - BWG, feed efficiency - FE), slaughter variables and nitrogen retention in broiler chicken (1-6 weeks of age) fed graded levels of toasted guar meal (TGM) as a protein source in diets. 2. The TGM was included at five graded concentrations (0, 50, 100, 150 and 200 g/kg) in iso-caloric and iso-protein diets with either the recommended concentration (100%) of CAA (lysine, total sulphur amino acids, threonine, tryptophan and valine) or at 10% higher (110%) concentration. A metabolism trial of 3-day duration was conducted during 6th week of age to study nitrogen retention. 3. The TGM levels and CAA concentration at 21 or 42 d of age did not influence BWG, FI and FE. BWG was not affected with inclusion of TGM up to 100 g/kg in starter and overall production (1-42 d of age) phases. The FE improved with TGM supplementation during starter phase, while at the end of experiment (42 d), FE was depressed by inclusion of TGM in dose dependant manner. All performance variables improved with increase in concentration of CAA from 100% to 110%. 4. Breast meat weight improved and abdominal fat weight reduced with higher levels of CAA in diet. Retention of nitrogen reduced with increase in level of TGM in broiler diet. Increasing concentrations of CAA in diet improved nitrogen retention. 5. It was concluded that TGM could be incorporated up to 100 g/kg with 100% CAA and up to 150 g/kg with 110% CAA without affecting performance. Increasing CAA concentration (110%) in diets significantly improved BWG and FE (21 and 42 d), breast meat weight and nitrogen retention in broiler chicken.
Asunto(s)
Aminoácidos/farmacología , Pollos/metabolismo , Cyamopsis , Aditivos Alimentarios/farmacología , Nitrógeno/metabolismo , Aminoácidos/metabolismo , Alimentación Animal/análisis , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Peso Corporal , Cyamopsis/química , Digestión , Aditivos Alimentarios/análisis , Masculino , Nitrógeno/farmacología , Aumento de Peso/efectos de los fármacosRESUMEN
An experiment was conducted to study the effects of supplementing sprouts of pulses on performance, carcass variables, immune responses, and anti-oxidant variables in broiler chicken (day 1 to 6 weeks of age) reared during summer season in tropical region. Sprouts of black gram (BG, Vigna mungo), green gram (GG, Vigna radiata), and wild gram (WG, Vigna trilobata) were produced by soaking the pulses in water for 16 h and incubating at 37 °C for 24 h. Total phenolic content in sprouts of WG, BG, and GG was 102, 96.1, and 79.2 mg GAE/g, respectively, while the anti-radical activity in the sprouts was 61, 58, and 52%, respectively. A total of 200-day-old broiler male chicks were equally and randomly distributed in to 4 groups, each having 10 replicates of 5 chicks and housed in battery brooders in open-sided poultry house. Each of these groups was fed sprouts of BG, GG, or WG at 5% of feed intake, while the control group without feeding sprouts was kept for comparison. The trial was conducted during mid summer season (April and May, 2017). Feed conversion ratio (FCR) was reduced (P < 0.05) in broilers fed sprouted pulses compared to the control group at day 21. However, the body weight gain and FCR at 42 days of age, slaughter variables, and immune responses were not affected due to feeding of sprouted pulses. Feeding of sprouts significantly (P < 0.05) reduced lipid peroxidation and increased (P < 0.05) the activities of glutathione peroxidase, glutathione reductase, and superoxide dismutase in liver and spleen of broilers compared to the control group. Based on the results, it is concluded that oxidative stress in broiler chicken reared in tropical summer could be reduced by supplementing sprouted pulses without affecting performance, carcass variables, and immune responses.
Asunto(s)
Pollos/crecimiento & desarrollo , Dieta/veterinaria , Estrés Oxidativo , Plantones , Vigna , Alimentación Animal/análisis , Animales , Pollos/inmunología , Suplementos Dietéticos , Peroxidación de Lípido , Masculino , Distribución Aleatoria , Estaciones del Año , Clima TropicalRESUMEN
The present study was carried out to explore the altered lipid, lipoprotein and apoprotein abnormalities along with lipoprotein (a) in chronic kidney disease patients with stage I to V which were further divided into group 1 (stage I and II), group 2 (stage III and IV) and group 3 (stage V). 50 chronic kidney disease patients with stage I to V and 20 healthy normal subjects as controls were recruited for this study. Among the various parameters tested triglyceride levels were high in group 1 and 2, whereas VLDL cholesterol, Lp (a) and apo B levels were significantly high in all the groups when compared to controls (P<0.05). However, LDL cholesterol level was significantly low in group 3 only as compared to control group (P<0.05). Apoprotein AI values also showed significant decrease in all groups as compared to controls (P<0.05). Though total cholesterol levels in group 1 and LDL levels in group 1 and 2 were higher than controls, but the values attained not statistically significant (P>0.05). In conclusion high levels of VLDL cholesterol, Lp (a), apo B and low levels of apoprotein AI as reported in this study are the major lipid disorders in the development of cardiovascular complications at all the stages in these patients.
RESUMEN
Mantle cell lymphoma (MCL) is characterized by the uncontrolled overexpression of cyclin D1. Styryl sulfonyl compounds have shown potent antitumor activity against MCL by inducing cell-cycle arrest and apoptosis. However, the exact molecular mechanism by which these compounds function is yet to be elucidated. Here, we show that the prototypical styryl sulfonyl compound ON 01910.Na decreased cyclin D1 and c-Myc protein levels in MCL cells, whereas mRNA levels of cyclin D1 were minimally affected. Notably, ON 01910.Na suppressed eukaryotic translation initiation factor 4E (eIF4E)-mediated cyclin D1 mRNA translation, decreased levels of phosphorylated Akt, mammalian target of Rapamycin (mTOR) and eIF4E-binding protein (eIF4E-BP), lowered the cap site binding activity of eIF4E and directly inhibited activity of phosphatidylinositol-3 kinase (PI-3K). Analysis of apoptotic signaling pathways revealed that ON 01910.Na induced the release of cytochrome c from mitochondria, altered expression of Bcl-2 family of proteins and stimulated activation of caspases. Taken together, styryl sulfonyls can cause a rapid decrease of cyclin D1 by blocking cyclin D1 mRNA translation through inhibition of the PI-3K/Akt/mTOR/eIF4E-BP signaling pathway and triggering a cytochrome c-dependent apoptotic pathway in MCL cells.
Asunto(s)
Antineoplásicos/farmacología , Ciclina D1/genética , Glicina/análogos & derivados , Linfoma de Células del Manto/tratamiento farmacológico , Biosíntesis de Proteínas/efectos de los fármacos , Sulfonas/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Factor 4E Eucariótico de Iniciación/fisiología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Glicina/farmacología , Humanos , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/patología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteínas Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Quinasas raf/fisiologíaRESUMEN
The recent identification of tumor-initiating colorectal cancer (CRC) stem cells in the pathogenesis of CRC has provided a potential target for novel therapeutics. Many details about CRC stem cells, however, remain poorly understood. Several potential markers of CRC stem cells have been proposed, including CD133, CD44, and, recently, Lgr5. Attention also has been drawn to control of stem cell self-renewal, proliferation, and differentiation by the Wnt and transforming growth factor (TGF)-ß pathways. Disruption of Wnt signaling, via loss of APC (adenomatous polyposis coli), is among the earliest events in the multistage progression of CRC and likely occurs in basal crypt stem cells, generating a neoplastic cell population that then expands upward to occupy the rest of the crypt. TGF-ß signaling is a key tumor suppressor pathway, and mutations in the type II receptor and Smad4 are observed in CRC specimens and are associated with more aggressive disease in tumors with disrupted Wnt signaling. Loss of the TGF-ß adaptor protein ß(2)-spectrin is associated with loss of colonic cell polarity and architecture, and its expression parallels that of Smad4. This review suggests rational approaches to target CRC stem cells as a novel and effective way to treat advanced and difficult-to-treat CRC.
RESUMEN
Although the existence of cancer stem cells (CSCs) was first proposed over 40 years ago, only in the past decade have these cells been identified in hematological malignancies, and more recently in solid tumors that include liver, breast, prostate, brain, and colon. Constant proliferation of stem cells is a vital component in liver tissues. In these renewing tissues, mutations will most likely result in expansion of the altered stem cells, perpetuating and increasing the chances of additional mutations and tumor progression. However, many details about hepatocellular cancer stem cells that are important for early detection remain poorly understood, including the precise cell(s) of origin, molecular genetics, and the mechanisms responsible for the highly aggressive clinical picture of hepatocellular carcinoma (HCC). Exploration of the difference between CSCs from normal stem cells is crucial not only for the understanding of tumor biology but also for the development of specific therapies that effectively target these cells in patients. These ideas have drawn attention to control of stem cell proliferation by the transforming growth factor beta (TGF-beta), Notch, Wnt, and Hedgehog pathways. Recent evidence also suggests a key role for the TGF-beta signaling pathway in both hepatocellular cancer suppression and endoderm formation, suggesting a dual role for this pathway in tumor suppression as well as progression of differentiation from a stem or progenitor stage. This review provides a rationale for detecting and analyzing tumor stem cells as one of the most effective ways to treat cancers such as HCC.
Asunto(s)
Carcinoma Hepatocelular/terapia , Células Madre Neoplásicas/patología , Biomarcadores/análisis , Carcinoma Hepatocelular/patología , Linaje de la Célula , Células Madre Embrionarias/citología , Proteínas Hedgehog/fisiología , Humanos , Hígado/citología , Hígado/crecimiento & desarrollo , Regeneración Hepática/fisiología , Trasplante de Hígado , Donadores Vivos , Masculino , Células Madre Neoplásicas/fisiología , Pronóstico , Receptores Notch/fisiología , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/fisiología , Proteínas Wnt/fisiología , beta Catenina/fisiologíaRESUMEN
The pupose of this study was to evaluate the activity of ON 01910.Na, a mitotic inhibitor, in in vitro and in vivo models of pancreatic cancer and to discover biomarkers predictive of efficacy. Successive in vitro and in vivo models were used; these included cell line-derived and patient-derived tumors from our PancXenoBank, a live collection of freshly generated pancreatic cancer xenografts. ON 01910.Na showed equivalent activity to gemcitabine against pancreatic cancer cell lines in vitro. The activity of the agent correlated with suppression of phospho-CDC25C and cyclin B1. These markers were optimized for a fine-needle aspirate ex vivo rapid assay. Cyclin B1 mRNA evaluation yielded the most optimal combination of accuracy and reproducibility. Next, nine patient-derived tumors from the PancXenoBank were profiled using the assay developed in cell lines and treated with ON01910.Na for 28 days. Two cases were cataloged as potential responders and seven as resistants. There was a correlation between the ex vivo assay and sensitivity to the tested agent, as the two cases prospectively identified as sensitive met prespecified criteria for response. Of the seven tumors of predictive resistant, only one was found to be sensitive to ON 01910.Na. In addition, there was a good correlation between cyclin B1 downregulation ex vivo and changes in cyclin B1 protein post-treatment. The novel mitotic inhibitor, ON 01910.Na, showed activity in preclinical model of pancreatic cancer. A rapid assay was rationally developed that not only identified cases sensitive to ON 01910.Na, but also anticipated the pharmacodynamic events occurring after in vivo exposure.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Glicina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Sulfonas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antimetabolitos Antineoplásicos/uso terapéutico , Biopsia con Aguja Fina , Línea Celular Tumoral , Ciclina B/metabolismo , Ciclina B1 , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Femenino , Glicina/farmacología , Glicina/uso terapéutico , Humanos , Ratones , Ratones Desnudos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Valor Predictivo de las Pruebas , Sulfonas/uso terapéutico , Fosfatasas cdc25/metabolismo , GemcitabinaRESUMEN
Jun N-terminal kinases or JNKs play a critical role in death receptor-initiated extrinsic as well as mitochondrial intrinsic apoptotic pathways. JNKs activate apoptotic signaling by the upregulation of pro-apoptotic genes through the transactivation of specific transcription factors or by directly modulating the activities of mitochondrial pro- and antiapoptotic proteins through distinct phosphorylation events. This review analyses our present understanding of the role of JNK in apoptotic signaling and the various mechanisms by which JNK promotes apoptosis.
Asunto(s)
Apoptosis , MAP Quinasa Quinasa 4/metabolismo , Transducción de Señal , Apoptosis/genética , Núcleo Celular/enzimología , Humanos , Mitocondrias/enzimología , FosforilaciónRESUMEN
Cancer stem cells (CSCs) are critical for the initiation, propagation, and treatment resistance of multiple cancers. Yet functional interactions between specific signaling pathways in solid organ "cancer stem cells," such as those of the liver, remain elusive. We report that in regenerating human liver, two to four cells per 30,000-50,000 cells express stem cell proteins Stat3, Oct4, and Nanog, along with the prodifferentiation proteins TGF-beta-receptor type II (TBRII) and embryonic liver fodrin (ELF). Examination of human hepatocellular cancer (HCC) reveals cells that label with stem cell markers that have unexpectedly lost TBRII and ELF. elf(+/-) mice spontaneously develop HCC; expression analysis of these tumors highlighted the marked activation of the genes involved in the IL-6 signaling pathway, including IL-6 and Stat3, suggesting that HCC could arise from an IL-6-driven transformed stem cell with inactivated TGF-beta signaling. Similarly, suppression of IL-6 signaling, through the generation of mouse knockouts involving a positive regulator of IL-6, Inter-alpha-trypsin inhibitor-heavy chain-4 (ITIH4), resulted in reduction in HCC in elf(+/-) mice. This study reveals an unexpected functional link between IL-6, a major stem cell signaling pathway, and the TGF-beta signaling pathway in the modulation of mammalian HCC, a lethal cancer of the foregut. These experiments suggest an important therapeutic role for targeting IL-6 in HCCs lacking a functional TGF-beta pathway.
Asunto(s)
Interleucina-6/metabolismo , Neoplasias Hepáticas/metabolismo , Transducción de Señal , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Apoptosis , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Proliferación Celular , Separación Celular , Regulación hacia Abajo , Perfilación de la Expresión Génica , Glicoproteínas/deficiencia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Hígado/citología , Hígado/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Noqueados , Proteínas Inhibidoras de Proteinasas Secretoras , Factor de Transcripción STAT3/metabolismoRESUMEN
Cyclooxygenase-2 (COX-2) inhibitors are promising anticancer agents but their long-term use at high doses is associated with adverse cardiovascular events. The molecular mechanisms underlying the anticancer or toxic cardiovascular effects of COX-2 inhibitors remain unknown. Here we report that COX-2-selective celecoxib and a novel COX-2 inhibitor ON09310 upregulate death receptor 5 (DR5) and cooperate with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), the ligand for DR5, to induce apoptosis in COX-2-positive and -negative cancer cells. We also show that both agents engage GADD153/CHOP to transcriptionally upregulate DR5 expression; GADD153/CHOP is a C/EBP homologous transcription factor implicated in cellular stress response and apoptosis. Based on our results, we propose that (1) these agents appear to mediate their effects, at least in part, by engaging GADD153/CHOP to activate DR5-dependent apoptotic pathway and (2) their regulation of GADD153/CHOP and DR5 expression appears to occur independent of their COX-2 inhibitory effects. Our results also indicate that ON09310 is generally more potent than celecoxib and, at lower concentration, strongly cooperates with TRAIL to induce apoptosis. Taken together, our findings form the basis for future in-depth studies to further explore the utility of TRAIL and/or agonistic anti-DR5 antibodies in combination with low-dose COX-2 inhibitors as a rational approach for cancer prevention and treatment.
Asunto(s)
Inhibidores de la Ciclooxigenasa 2/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Indoles/farmacología , Proteínas de Neoplasias/metabolismo , Neoplasias/prevención & control , Pirazoles/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Sulfonamidas/farmacología , Factor de Transcripción CHOP/metabolismo , Apoptosis/efectos de los fármacos , Celecoxib , Línea Celular Tumoral , Inhibidores de la Ciclooxigenasa 2/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Indoles/uso terapéutico , Pirazoles/uso terapéutico , Sulfonamidas/uso terapéutico , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Hematopoiesis is the cumulative result of intricately regulated signaling pathways that are mediated by cytokines and their receptors. Proper culmination of these diverse pathways forms the basis for an orderly generation of different cell types. Recent studies conducted over the past 10-15 years have revealed that hematopoietic cytokine receptor signaling is largely mediated by a family of tyrosine kinases termed Janus kinases (JAKs) and their downstream transcription factors termed STATs (signal transducers and activators of transcription). Aberration in these pathways, such as that caused by the recently identified JAK2V617F mutation, is an underlying cause for diseases such as leukemias and other myeloproliferative disorders. This recent discovery, when coupled with the fact that STATs are activated by oncoproteins such as BCR-ABL, underscores the importance of the JAK-STAT pathway in both normal cellular development and disease states.
Asunto(s)
Citocinas/metabolismo , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/fisiología , Receptores de Citocinas/fisiología , Transducción de Señal/fisiología , Animales , Citocinas/fisiología , Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/metabolismo , HumanosRESUMEN
Transforming growth factor-beta (TGF-beta) signaling members, TGF-beta receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf(+/-) mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P<0.017). ELF and TBRII are also markedly decreased in human HCC cell lines SNU-398 and SNU-475. Restoration of ELF and TBRII in SNU-398 cells markedly decreases cyclin D1 as well as hyperphosphorylated-retinoblastoma (hyperphosphorylated-pRb). Thus, we show that TGF-beta signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.
Asunto(s)
Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/metabolismo , Proteínas Portadoras/fisiología , Ciclinas/metabolismo , Neoplasias Hepáticas Experimentales/etiología , Proteínas de Microfilamentos/fisiología , Transducción de Señal/fisiología , Espectrina/fisiología , Factor de Crecimiento Transformador beta2/antagonistas & inhibidores , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Ciclina D , Ciclinas/antagonistas & inhibidores , Humanos , Neoplasias Hepáticas Experimentales/metabolismo , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Fosforilación , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Retinoblastoma/metabolismo , Transducción de Señal/genética , Espectrina/deficiencia , Espectrina/genética , Factor de Crecimiento Transformador beta2/metabolismo , Factor de Crecimiento Transformador beta2/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Mitogen-activated protein kinases (MAPKs) regulate critical signaling pathways involved in cell proliferation, differentiation and apoptosis. Recent studies have shown that a novel class of scaffold proteins mediates the structural and functional organization of the three-tier MAPK module. By linking the MAP3K, MAP2K and MAPK into a multienzyme complex, these MAPK-specific scaffold proteins provide an insulated physical conduit through which signals from the respective MAPK can be transmitted to the appropriate spatiotemporal cellular loci. Scaffold proteins play a determinant role in modulating the signaling strength of their cognate MAPK module by regulating the signal amplitude and duration. The scaffold proteins themselves are finely regulated resulting in dynamic intra- and inter-molecular interactions that can modulate the signaling outputs of MAPK modules. This review focuses on defining the diverse mechanisms by which these scaffold proteins interact with their respective MAPK modules and the role of such interactions in the spatiotemporal organization as well as context-specific signaling of the different MAPK modules.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Proteínas Asociadas a Matriz Nuclear/fisiología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Humanos , Proteínas Quinasas Activadas por Mitógenos/química , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/metabolismoRESUMEN
Signature abnormalities in the cell cycle and apoptotic pathway have been identified in mantle cell lymphoma (MCL), affording the opportunity to develop targeted therapies. In this study, we tested a novel class of kinase inhibitors, styryl sulfones, which differ from prior cell cycle inhibitors in that they are not related to purines or pyrimidines. We observed that two closely related compounds, ON013100 and ON01370, altered the growth and cell cycle status of MCL lines and potently inhibited the expression of several important molecules, including cyclin-dependent kinase 4, p53, mouse double minute 2 (MDM2), and cyclin D as well as increased cyclin B expression. Using both terminal deoxy transferase uridine triphosphate nick end-labelling and poly ADP-ribose polymerase assays, we found that these compounds caused apoptosis in MCL cells. In addition, using molecular analyses, we observed the modulation of caspase-3 activity but not the expression of B-cell lymphoma family molecules. Next, we investigated the cytotoxicity of the MCL lines upon treatment with styryl sulfone compounds in combination with other currently used chemotherapeutic agents, such as doxorubicin (DOX) or vincristine (VCR). We found that the combination of DOX plus styryl sulfone or VCR plus styryl sulfone increased cytotoxicity by one log scale, compared with the single styryl sulfone compound. Thus, styryl sulfones alone, or in combination with chemotherapeutic agents, present attractive opportunities for new drug development in MCL.
Asunto(s)
Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Sulfonas/farmacología , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Western Blotting , Caspasa 3/metabolismo , Inhibidores de Caspasas , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Citometría de Flujo , Fase G2/efectos de los fármacos , Humanos , Etiquetado Corte-Fin in Situ , Linfoma de Células del Manto/metabolismo , Linfoma de Células del Manto/patología , Estructura Molecular , Estirenos/química , Estirenos/farmacología , Sulfonas/químicaRESUMEN
We previously demonstrated that Jak3 is a primary response gene for G-CSF and ectopic overexpression of Jak3 can accelerate granulocytic differentiation of normal mouse bone marrow cells induced by G-CSF and GM-CSF. To gain insight into the regulation of G-CSF-induced transcription of Jak3, we constructed deletion and linker scanning mutants of the Jak3 promoter sequences and performed luciferase reporter assays in the murine myeloid cell line 32Dcl3, with and without G-CSF stimulation. These experiments showed that mutation of a -67 to -85 element, which contained a putative Sp1 binding site, or mutation of a -44 to -53 GAS element resulted in a marked reduction of Jak3 promoter activity. Electrophoretic mobility shift assays revealed that Sp1 and Stat3 present in nuclear lysates of 32Dcl3 cells stimulated with G-CSF can bind to the -67 to -85 element and -44 to -53 GAS element, respectively. In addition, cotransfection of a constitutively active mutant of Stat3 along with a Jak3 promoter/luciferase reporter resulted in enhanced Jak3 promoter activity. Together, these results demonstrate that activation of Jak3 transcription during G-CSF- induced granulocytic differentiation is mediated by the combined action of Sp1 and Stat3, a mechanism also shown to be important in IL-6-induced monocytic differentiation.
Asunto(s)
Diferenciación Celular , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos/farmacología , Granulocitos/citología , Proteínas Tirosina Quinasas/genética , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción Sp1/metabolismo , Transcripción Genética , Animales , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Janus Quinasa 3 , Ratones , Proteínas Tirosina Quinasas/metabolismo , Regulación hacia ArribaRESUMEN
Inactivation of the transforming growth factor-beta (TGF-beta) pathway occurs often in malignancies of the gastrointestinal (GI) system. However, only a fraction of sporadic GI tumors exhibit inactivating mutations in early stages of cancer formation, suggesting that other mechanisms play a critical role in the inactivation of this pathway. Here, we show a wide range of GI tumors, including those of the stomach, liver and colon in elf+/- and elf+/- / Smad4+/- mutant mice. We found that embryonic liver fodrin (ELF), a beta-Spectrin originally identified in endodermal stem/progenitor cells committed to foregut lineage, possesses potent antioncogenic activity and is frequently inactivated in GI cancers. Specifically, E-cadherin accumulation at cell-cell contacts and E-cadherin-beta-catenin-dependent epithelial cell-cell adhesion is disrupted in elf+/- / Smad4+/- mutant gastric epithelial cells, and could be rescued by ectopic expression of full-length elf, but not Smad3 or Smad4. Subcellular fractionation revealed that E-cadherin is expressed mainly at the cell membrane after TGF-beta stimulation. In contrast, elf+/- / Smad4+/- mutant tissues showed abnormal distribution of E-cadherin that could be rescued by overexpression of ELF but not Smad3 or Smad4. Our results identify a group of common lethal malignancies in which inactivation of TGF-beta signaling, which is essential for tumor suppression, is disrupted by inactivation of the ELF adaptor protein.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas de Unión al ADN , Neoplasias Gastrointestinales/genética , Proteínas de Microfilamentos/biosíntesis , Factor de Crecimiento Transformador beta/fisiología , Animales , Cadherinas/fisiología , Proteínas Portadoras/fisiología , Adhesión Celular , Células Epiteliales/fisiología , Neoplasias Gastrointestinales/fisiopatología , Perfilación de la Expresión Génica , Ratones , Proteínas de Microfilamentos/fisiología , Transducción de Señal , Proteína Smad4/biosíntesis , Proteína Smad4/genética , beta Catenina/fisiologíaRESUMEN
To localize the regulatory elements in the human follicle-stimulating hormone receptor (FSH-R) promoter/enhancer and to determine the role of upstream stimulatingfactors (USFs) in these elements, we transiently transfected constructs of FSH-R promoter/enhancer in pGL3 luciferase reporter plasmids into Chinese hamster ovary cells and the activities were determined by measuring luciferase luminescence of the cell lysates. The 5'-flanking regions of the human FSH-R gene from nt -1485 to -1 with respect to the gene translation start site were amplified by polymerase chain reaction (PCR) and subcloned in pGL3. Deletion mutants were created using PCR or restriction enzyme digestion. Mutation in the E-box sequence from nt -124 to -119 (E-box 3), in the construct from -224 to nt -1 or in the Inr element, which encompasses the transcriptional start site at nt -99, resulted in a substantial reduction in the human FSH-R promoter/enhancer activity. Overexpression of upstream stimulating factor-1 (USF1) suppresses the activity of the human FSH-R promoter/enhancer via Inr and E-box elements. Upstream stimulating factor-2 (USF2) decreases FSH-R promoter/enhancer activity by acting on E-box 3. The results indicate that E-box 3 and the Inr element are important elements of the human FSH-R promoter/ enhancer. USF family members inhibit FSH-R gene activity by acting via these elements. USF1 and USF2 suppress human FSH-R promoter/enhancer activity by acting on E-box 3. USF1 also decreases activity by interacting with the Inr element.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Elementos E-Box/genética , Elementos de Facilitación Genéticos/genética , Regiones Promotoras Genéticas/genética , Receptores de HFE/genética , Factores de Transcripción/farmacología , Factores de Transcripción/fisiología , Animales , Células CHO , Clonación Molecular , Cricetinae , Proteínas de Unión al ADN/genética , Expresión Génica , Humanos , Luciferasas/genética , Mutagénesis , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Transfección , Factores Estimuladores hacia 5'RESUMEN
In this study, we have isolated the genomic clone of the murine GSTM2 gene and determined its sequence. Consistent with the class mu genefamily, the mGSTM2 gene consists of eight exons. The exon-intron boundaries and the distribution of coding sequences within the exons of the known GST class mu family members were found to follow a similar pattern suggesting that various members of this family have originated from a single primordial gene by duplication and the structure has been closely maintained through evolution. By primer extension, the start of transcription was determined to be 40 bp upstream of the initial AUG codon. To gain an understanding of the mGSTM2 regulation, we have also cloned and analyzed its promoter region. Maximal activity was observed in a 170 bp 5'-flanking region. The activity was decreased by 3-fold in a 402 bp 5'-flanking region suggesting the presence of repressor elements. While no TATA box was identified, the presence of an SP1 site at position -38 was noted. Deletion of this SP1 site completely abrogated promoter activity. The promoter contained eight putative Myb responsive elements and its transcriptional activity was upregulated by t-Myb but not by c-Myb.
Asunto(s)
Genes/genética , Glutatión Transferasa/genética , Regiones Promotoras Genéticas/genética , Animales , Secuencia de Bases , ADN/química , ADN/genética , ADN/aislamiento & purificación , Exones , Intrones , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Mutación , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
The AATYK gene encodes a tyrosine kinase whose expression is up-regulated during the apoptosis and differentiation of 32Dcl3 myeloblastic cells. Because high levels of AATYK mRNA have also been detected in the brain, and because these transcripts differ in size from that observed in the 32Dcl3 cell line, it was of interest to determine whether this gene encodes mRNAs that are alternatively spliced and whether these mRNAs are expressed in a tissue-specific manner. We have isolated a novel, alternatively spliced AATYK mRNA using cDNA library screening and RT-PCR, whose expression is readily detected in the brain but not myeloid cells. Western blot analysis revealed that the AATYK protein was expressed in virtually all regions of the adult rat brain in which neurons are present, including olfactory bulb, forebrain, cortex, midbrain, cerebellum and pons. Immunohistochemical labeling of adult brain sections showed the highest levels of AATYK expression in the cerebellum and olfactory bulb. Expression of AATYK was also up-regulated as a function of RA-induced neuronal differentiation of p19 embryonal carcinoma cells, supporting a role for this protein in mature neurons and neuronal differentiation.