RESUMEN
Hepatocellular carcinoma (HCC) is a major public health concern because of the absence of early diagnosis and effective treatments. Efficient diagnosis modalities and therapies to treat HCC are needed. Kruppel-like factor (KLF) family members, such as KLF6, are involved in cell proliferation and differentiation. KLF6 is inactivated in solid tumors, which may contribute to pathogenesis. However, KLF6 status in HCC is controversial. Thus, we undertook the characterization of KLF6 expression and function in HCC and HCC-derived cell lines. We found that HCC, HepG2 and HuH7 cells expressed KLF6 messenger ribonucleic acid and protein. Next, using RNA interference, we demonstrated that inhibiting KLF6 expression in vitro strongly impaired cell proliferation-induced G1-phase arrest, inhibited cyclin-dependent kinase 4 and cyclin D1 expression, and subsequent retinoblastoma phosphorylation. Finally, KLF6 silencing caused p53 upregulation and inhibited Bcl-xL expression, to induce cell death by apoptosis. Taken together, these data demonstrated that KLF6 is essential for HCC-derived cells to evade apoptosis.
Asunto(s)
Apoptosis/fisiología , Factores de Transcripción de Tipo Kruppel/fisiología , Apoptosis/genética , Secuencia de Bases , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular Tumoral , Proliferación Celular , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Datos de Secuencia Molecular , Mutación , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteína de Retinoblastoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido NucleicoRESUMEN
BACKGROUND: Peritoneal carcinomatosis from pancreatic cancer has a poor prognosis with a median survival of 3.1 months. This is mainly due to lack of effective treatment. Interleukin 12 (IL12) is a proinflammatory cytokine that has a potent antitumoral effect by stimulating innate and adoptive immunity. AIM: To examine the antitumoral effect and toxicity of intraperitoneal delivery of IL12 using an ex vivo gene therapy approach in a murine model of pancreatic peritoneal carcinomatosis. METHODS: Peritoneal carcinomatosis was generated by direct intraperitoneal inoculation of the pancreatic cancer cell line Capan-1 in athymic mice. Syngenic fibroblasts were genetically modified in vitro to secrete IL12 using a polycistronic TFG murine IL12 retroviral vector coding for both p35 and p40 murine IL12 subunits. Ex vivo gene therapy involved injection of the genetically modified fibroblasts intraperitoneally twice a week for 4 weeks. RESULTS: Treatment of pre-established peritoneal carcinomatosis with fibroblasts genetically modified to express IL12 induced a marked inhibition of tumour growth as measured by comparison of the weights of the intraperitoneal tumour nodules in the treated and control animals (3.52 (SD 0.47) v 0.93 (SD 0.21) g, p<0.05) and improved survival. This effect was associated with infiltration of the peritoneal tumour nodules with macrophages. Peritoneal lavage confirmed enhancement of the innate peritoneal inflammatory activity, with an increased number of activated macrophages and natural killer cells. Moreover, macrophages harvested from animals with peritoneal carcinomatosis and treated with IL12-expressing fibroblasts expressed an activated proinflammatory antitumoral M1 phenotype that included strongly enhanced reactive oxygen species and nitric oxide production. There was no treatment-related toxicity. CONCLUSION: Multiple injections of genetically modified fibroblasts to express IL12 is an effective and well-tolerated treatment for experimental murine pancreatic peritoneal carcinomatosis via activated innate immunity and in particular activated M1 macrophages.
Asunto(s)
Antineoplásicos/inmunología , Fibroblastos/inmunología , Terapia Genética/métodos , Interleucina-12/inmunología , Neoplasias Peritoneales/terapia , Animales , División Celular/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo/métodos , Inmunidad Innata/inmunología , Inmunohistoquímica/métodos , Inyecciones Intraperitoneales , Interleucina-12/administración & dosificación , Interleucina-12/genética , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Óxido Nítrico/biosíntesis , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/inmunología , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/inmunología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Pancreatic cancer is one of the most aggressive and devastating human malignancies. There is an urgent need for more effective therapy for patients with advanced disease. In this context, genetic therapy potentially represents a rational new approach to treating pancreatic cancer, which could provide an adjunct to conventional options. Because of the promise of recombinant SV40 vectors, we tested their ability to deliver a transgene, and to target a transcript, so as to inhibit pancreatic tumors growth in vivo. BxPC3 and Capan-1 cells were efficiently transduced using SV40 vectors without selection, as compared to synthetic vectors PEI. SV40 vectors were as efficient as adenoviral vectors, and provided long-term transgene expression. Next, we devised a SV40-derived, targeted gene therapy approach of pancreatic cancer, by combining hTR tumor-specific promoter with sst2 somatostatin receptor tumor-suppressor gene. In vitro cell proliferation was strongly impaired following administration of SV(hTR-sst2). SV40-derived sst2-mediated antiproliferative effect was dependent on the local production of somatostatin. In vivo, intratumoral gene transfer of sst2 using rSV40 vectors resulted in a marked inhibition of Capan-1 tumor progression, and proliferation. These results represent the initial steps toward a novel approach to the gene therapy of pancreatic cancer using SV40 as a vector.
Asunto(s)
Virus Defectuosos/fisiología , Técnicas de Transferencia de Gen , Neoplasias Pancreáticas/patología , Virus 40 de los Simios/fisiología , Replicación Viral , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Humanos , Reacción en Cadena de la Polimerasa , Transducción GenéticaRESUMEN
Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.
Asunto(s)
Apoptosis , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Receptores de Somatostatina/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Somatostatina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Ratones , Modelos Biológicos , Imitación Molecular/efectos de los fármacos , Células 3T3 NIH , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Transducción de Señal/efectos de los fármacos , Transfección , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Ornithine decarboxylase (ODC) is the ratelimiting enzyme in the biosynthesis of polyamines, which are required for optimal cell growth and proliferation. ODC is overexpressed in many tumors and, conversely, its overexpression induces transformation. We have previously reported that ODC mRNA alternative splicing relieves the translation repression normally imposed by a long and structured 5' untranslated region (UTR), and that the ODC 5' UTR contains an internal ribosome entry site (IRES). Here we show that ODC IRES activity is enhanced following inclusion of alternative sequences generated by splicing at cryptic acceptor sites. Furthermore, the alternative ODC IRES is more sensitive to cell-cycledependent changes in the rate of translation. These findings uncover a new biological property of differentially spliced transcripts. This is the first example of alternative splicing that modulates mRNA translation through the cell cycle in a cap-independent manner.
Asunto(s)
Empalme Alternativo , Ornitina Descarboxilasa/genética , Ornitina Descarboxilasa/metabolismo , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Regiones no Traducidas 5'/genética , Regiones no Traducidas 5'/metabolismo , Células HeLa , Humanos , ARN Mensajero/genéticaRESUMEN
We present evidence that gastrin, binding to a G protein-coupled receptor, activates the p38-mitogen-activated protein kinase (MAPK) pathway. Blockage of protein kinase C (PKC) by GF109203X, depletion of intracellular calcium by thapsigargin or inhibition of Src family kinases by PP2 prevented p38-MAPK activation and the Src kinase activity stimulated by gastrin. Inhibition of the PI 3-kinase by wortmannin or LY294002 did not affect these responses. In addition, the p38-MAPK inhibitor, SB203580, repressed gastrin-induced [(3)H]thymidine incorporation, indicating a major role of p38-MAPK in the growth-promoting effect of gastrin. Our results demonstrate that gastrin-induced DNA synthesis requires p38-MAPK activation through mechanisms that involve calcium mobilization, PKC and Src family kinases.
Asunto(s)
ADN/biosíntesis , Gastrinas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Quinasa C/metabolismo , Familia-src Quinasas/metabolismo , Androstadienos/farmacología , Animales , Células CHO , Calcio/metabolismo , Cromonas/farmacología , Cricetinae , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/metabolismo , Gastrinas/farmacología , Humanos , Líquido Intracelular/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Receptor de Colecistoquinina B , Receptores de Colecistoquinina/genética , Receptores de Colecistoquinina/metabolismo , Transducción de Señal/efectos de los fármacos , Tapsigargina , Transfección , Wortmanina , Proteínas Quinasas p38 Activadas por Mitógenos , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
The high molecular weight (HMW) fibroblast growth factor (FGF)-2 isoform of 210 amino acids initiated at a CUG start codon possesses a nuclear localization sequence and is not secreted. In contrast, the low molecular weight (LMW) isoform of 155 amino acids initiated at the AUG start codon can be secreted and activates the cell surface FGF receptors. The two isoforms possess different biological properties; however, little is known about the intracrine regulatory mechanisms involved in the biological effects of the HMW FGF-2 isoform. Using pancreatic cells stably transfected with cDNAs leading to the expression of either the HMW FGF-2 (A3 cells) or the LMW form (A5 cells), we provide evidence that the two FGF-2 isoforms differentially modulate PKC levels. The LMW FGF-2 up-regulated the PKC epsilon levels by 1.6-fold; by contrast the HMW isoform down-regulated the level of this PKC isotype by about 3-fold and increased the amount of PKC delta by 1.7-fold. PKC mRNAs were also modified, suggesting that PKC expression was regulated at a pretranslational level. Additionally, expression of different levels of the HMW FGF-2 with an inducible expression system confirmed the role of this isoform on PKC delta and epsilon expressions. Increased activation of ERK-1 and -2 was also observed in cells expressing the HMW FGF-2. By using different PKC inhibitors and a dominant negative PKC delta, it was found that ERK activation was PKC delta-dependent. These data indicate that expression of HMW FGF-2 can modify PKC levels by acting at the intracellular level and that the overexpression of PKC delta induces ERK-1/2 activation. The expression of a dominant negative FGFR1 did not reduce ERK-1/2 activation by the HMW FGF-2, suggesting that ERK activation does not require FGFR activity. The signaling cascade downstream of ERK might be involved in the known mitogenic effect exerted by this FGF-2 isoform.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , Isoenzimas/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Quinasa C/genética , Transcripción Genética/fisiología , Animales , Línea Celular , Doxiciclina/farmacología , Activación Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Isoenzimas/metabolismo , Cinética , Proteína Quinasa 3 Activada por Mitógenos , Peso Molecular , Páncreas , Fosforilación , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteína Quinasa C/metabolismo , Proteína Quinasa C-delta , Proteína Quinasa C-epsilon , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , TransfecciónRESUMEN
The eukaryotic mRNA 5' cap structure facilitates translation. However, cap-dependent translation is impaired at mitosis, suggesting a cap-independent mechanism for mRNAs translated during mitosis. Translation of ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, peaks twice during the cell cycle, at the G1/S transition and at G2/M. Here, we describe a cap-independent internal ribosome entry site (IRES) in the ODC mRNA that functions exclusively at G2/M. This ensures elevated levels of polyamines, which are implicated in mitotic spindle formation and chromatin condensation. c-myc mRNA also contains an IRES that functions during mitosis. Thus, IRES-dependent translation is likely to be a general mechanism to synthesize short-lived proteins even at mitosis, when cap-dependent translation is interdicted.
Asunto(s)
Regiones no Traducidas 5' , Interfase/fisiología , Ornitina Descarboxilasa/biosíntesis , Iniciación de la Cadena Peptídica Traduccional/genética , Caperuzas de ARN , Codón Iniciador , Inducción Enzimática/efectos de los fármacos , Fase G1/fisiología , Fase G2/fisiología , Células HeLa , Humanos , Picornaviridae/genética , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Sirolimus/farmacologíaRESUMEN
Among the most conserved regions in the G-protein-coupled receptors is the (N/D)PX(2-3)Y motif of the seventh transmembrane domain (X represents any amino acid). The mutation of the Asn/Asp residue of this motif in different G-protein-coupled receptors was shown to affect the activation of either adenylyl cyclase or phospholipase C. We have mutated the Asn residue (Asn-391) of the NPXXY motif in the CCKBR to Ala and determined the effects of the mutation on binding, signaling, and G-proteins coupling after expression of the mutated receptor in COS cells. The mutated receptor displayed similar expression levels and high affinity CCK binding compared with the wild type CCKBR. However, unlike the wild type CCKBR, the mutated receptor was completely unable to mediate activation of either phospholipase C and protein kinase C-dependent and -independent mitogen-activated protein kinase pathways, indicating an essential role of Asn-391 in CCKBR signaling. Coimmunoprecipitation experiments allowed us to show that the inactive mutant retains an intact capacity to form stable complexes with G(q)alpha subunits in response to CCK. These results indicate that the formation of high affinity CCK-receptor-G(q) protein complexes is not sufficient to activate G(q) and suggest that Asn-391 is specifically involved in G(q) proteins activation.
Asunto(s)
Asparagina , Proteínas de Unión al GTP/metabolismo , Receptores de Colecistoquinina/química , Receptores de Colecistoquinina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Ácido Aspártico , Sitios de Unión , Células COS , Colecistoquinina/farmacología , Secuencia Conservada , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Fosfatos de Inositol/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/farmacología , Proteína Quinasa C/metabolismo , Ratas , Receptor de Colecistoquinina B , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfección , Fosfolipasas de Tipo C/metabolismoRESUMEN
We have analyzed in Chinese hamster ovary cells the upstream mediators by which the G protein-coupled receptor, gastrin/CCKB, activates the extracellular-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc completely blocked gastrin-stimulated Shc.Grb2 complex formation but partially inhibited ERK-1 activation by this peptide. Expression of Csk, which inactivates Src-family kinases, totally inhibited gastrin-induced Src-like activity detected in anti-Src and anti-Shc precipitates but diminished by 50% Shc phosphorylation and ERK-1 activation. We observed a rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src. IRS-1 complex formation and IRS-1 phosphorylation in Csk-transfected cells. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation was abolished by Csk. Our results demonstrate the existence of two mechanisms in gastrin-mediated ERKs activation. One requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Colecistoquinina/metabolismo , Familia-src Quinasas/metabolismo , Animales , Células CHO , Cricetinae , Replicación del ADN , Activación Enzimática , Proteína Adaptadora GRB2 , Gastrinas/metabolismo , Humanos , Proteínas Sustrato del Receptor de Insulina , Proteína Quinasa 3 Activada por Mitógenos , Fosfoproteínas/metabolismo , Fosforilación , Proteínas/metabolismo , Receptor de Colecistoquinina B , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Tirosina/metabolismoRESUMEN
The molecular events whereby gastrin occupancy of G/CCK(B) receptors leads to phosphatidylinositol (PI) 3-kinase activation have been examined. We report here that this peptide promotes the association between two non-receptor tyrosine kinases, p60Src and p125FAK, and elicits a parallel increase in tyrosine phosphorylation and activity of both kinases. Gastrin-induced PI 3-kinase activity was coprecipitated with p60Src and p125FAK and was inhibited by herbimycin A, the selective Src inhibitor PP-2 or cytochalasin D, which disrupts the actin cytoskeleton and prevents p125FAK activity. These results indicate, for the first time, that a p60Src/p125FAK complex acts upstream of the gastrin-stimulated PI 3-kinase pathway.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Gastrinas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Transducción de Señal , Animales , Activación Enzimática , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Gastrinas/farmacología , Fosforilación , Pruebas de Precipitina , Ratas , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
Gastrin via its G-protein coupled specific receptor induces transcription of c-fos and c-jun genes through a ras-MAPK pathway. Ornithine Decarboxylase (ODC), a growth regulated proto-oncogene, was chosen to investigate gastrin effects on translation initiation of mRNAs exhibiting a 5'UnTranslated Region (5'UTR) responsible for translation repression in quiescent cells. In AR4-2J tumoral cells, we first demonstrated that gastrin increases ODC mRNA translation. Transient transfections with various CAT chimeric constructs suggested a direct involvement of the 5'UTR in this observation. Translation of this group of mRNAs is enhanced by the availability of the cap-binding protein (eIF4E) that is increased after phosphorylation of its specific binding protein eIF4E-BP1. We found that AR4-2J cells over-expressed eIF4E protein which was not modulated by gastrin treatment. Rapamycin which inhibits 4E-BP1 phosphorylation, completely prevents gastrin-mediated increase of ODC translation indicating that 4E-BP1 could be involved in regulating ODC translation. Implication of 4E-BP1 in mediating gastrin effects is corroborated by the capacity of the ligand to affect 4E-BP1 phosphorylation. These results indicate that gastrin enhances ornithine decarboxylase mRNA translation through a rapamycin sensitive pathway and provide the first evidence in the control of 4E-BP1 phosphorylation after occupancy of a G protein-coupled receptor.
Asunto(s)
Proteínas Portadoras , Gastrinas/farmacología , Ornitina Descarboxilasa/genética , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/metabolismo , ARN Mensajero/genética , Animales , Células COS , Inducción Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular , Ornitina Descarboxilasa/biosíntesis , Inhibidores de la Ornitina Descarboxilasa , Fosforilación/efectos de los fármacos , Polienos/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Ratas , Proteínas Represoras/farmacología , Sirolimus , Células Tumorales CultivadasRESUMEN
Gastrin/CCK(B) G protein-coupled receptors have been shown to mediate proliferation stimulated by their endogenous ligands. The present study demonstrates the proliferative effect of arachidonic acid on AR4-2J cells. Gastrin induces an [3H]arachidonic-acid release in a dose-dependent manner. The use of a specific inhibitor of cPLA2, AACOCF3 established the involvement of a cPLA2 in the proliferative effect of gastrin. The results also demonstrate that a cytosolic high-molecular-weight PLA2 is activated by gastrin in AR4-2J cells.
Asunto(s)
Ácido Araquidónico/fisiología , Gastrinas/farmacología , Fosfolipasas A/fisiología , Animales , Calcio/metabolismo , División Celular , Línea Celular , Citosol/enzimología , Activación Enzimática , Fosfolipasas A2 , RatasRESUMEN
The gastrin/CCKB (G/CCKB) G protein-coupled receptor has been shown to mediate the proliferative effects of gastrin on normal and neoplastic gastro-intestinal tissues. In the present study, we examined the signal transduction mechanisms coupled to this receptor. We report here that phosphorylation and activity of the p70S6K, whose major substrate is the ribosomal S6 protein, are enhanced in response to gastrin. These effects were completely reversed by a commonly used PI-3-kinase inhibitor, wortmannin, suggesting that p70S6K may be a downstream target of PI-3-kinase in a signaling cascade induced by gastrin. In addition, blocking PI-3-kinase activity by wortmannin partially decreased gastrin-induced MAPK activation (42% +/- 3) as well as the tyrosine phosphorylation of She (50% +/- 6), an upstream regulator of the Ras-dependent MAPK pathway. These results indicate that at least two signaling pathways lead to MAPK activation by gastrin, only one of which is sensitive to PI-3-kinase inhibitors.
Asunto(s)
Androstadienos/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión al GTP/metabolismo , Gastrinas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Colecistoquinina/fisiología , Proteínas Ribosómicas/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Gastrinas/metabolismo , Neoplasias Pancreáticas , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Polienos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ratas , Receptor de Colecistoquinina B , Proteínas Quinasas S6 Ribosómicas , Sirolimus , Células Tumorales Cultivadas , Wortmanina , Dominios Homologos src/efectos de los fármacosRESUMEN
Glycine-extended gastrin precursors (G-Gly) were considered as processing intermediates devoid of biological activity. However, we have recently identified selective receptors for G-Gly which mediate the proliferative effects of this precursor. Little is known about the signaling pathways activated by G-Gly. In the present study, we demonstrate that PI-3-kinase is rapidly and transiently activated by G-Gly. We also observed a rapid increase in the tyrosine phosphorylation of IRS-1 and an activation of the PI-3-kinase in anti-IRS-1 immunoprecipitates, suggesting that PI-3-kinase may be activated by association with tyrosine phosphorylated IRS-1. We also demonstrated that gastrin precursors activate the serine/threonine kinase, p70 kDa S6 kinase (p70S6K), through a wortmannin sensitive pathway.
Asunto(s)
Gastrinas/farmacología , Glicina/farmacología , Fosfoproteínas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotirosina/metabolismo , Precursores de Proteínas/farmacología , Androstadienos/farmacología , Animales , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Proteínas Sustrato del Receptor de Insulina , Cinética , Neoplasias Pancreáticas , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Células Tumorales Cultivadas , WortmaninaRESUMEN
The proliferative effects of gastrin on normal and neoplastic gastro-intestinal tissues have been shown to be mediated by the gastrin/CCKB (G/CCKB) G-protein-coupled receptors. We have recently reported that gastrin stimulates the tyrosine phosphorylation of Shc proteins and their subsequent association with the Grb2/Sos complex, leading to mitogen-activated protein kinase (MAPK) activation, a pathway known to play an important role in cell proliferation. We undertook the present study to characterize the signalling pathways used by this receptor to mediate the activation of the Shc/Grb2 complex. Since G/CCKB receptor occupancy leads to the activation of the phospholipase C (PLC)/protein kinase C (PKC) pathway, we examined whether PKC stimulation and Ca2+ mobilization contribute to the phosphorylation of Shc proteins and their association with Grb2 in response to gastrin. Our results indicate that Shc proteins are tyrosine phosphorylated and associate with Grb2 in response to phorbol esters, suggesting that activation of PKC is a potential signalling pathway leading to activation of the Shc/Grb2 complex. Inhibition of PKC by GF109203X completely blocked the effect of PMA on Shc tyrosine phosphorylation and its subsequent association with Grb2, but had a partial inhibitory effect on the response to gastrin. Depletion of the intracellular Ca2+ pools by treatment with thapsigargin blocked the increase in intracellular free calcium concentration induced by gastrin and diminished the ability of the peptide to stimulate Shc phosphorylation and recruitment of Grb2. In addition, removal of extracellular Ca2+ partially inhibited the effect of gastrin on Shc phosphorylation as well as its association with Grb2, indicating that the effects of gastrin are also mediated by Ca2+-dependent mechanisms. Furthermore, we show that blockage of the two major early signals generated by activation of PLC, which induced the activation of the Shc/Grb2 complex, also blocked gastrin-induced MAPK activation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/metabolismo , Gastrinas/farmacología , Proteínas Quinasas Activadas por Mitógenos , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Animales , Western Blotting , Activación Enzimática , Proteína Adaptadora GRB2 , Humanos , Proteína Quinasa 3 Activada por Mitógenos , Neoplasias Experimentales , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal/fisiología , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Acetato de Tetradecanoilforbol/farmacología , Tapsigargina/farmacología , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
Caerulein-induced pancreatitis (CIP) in rats is characterized by oedema and cell necrosis followed by spontaneous regeneration. The ras protein as well as ornithine decarboxylase (ODC) play a central role in the transmission of signals induced by growth factors. Therefore, we analyzed these gene products during the course of CIP and during the regeneration of the gland. Growth and biochemical parameters (pancreatic weight, total DNA, RNA and proteins) were determined along with ODC activity and quantitative reverse-transcriptase polymerase chain reaction measurements of mRNA levels. During CIP, the significant increases in pancreatic weight were the result of oedema. During that period, maximal increases in ODC activity were observed at 3 h, in ODC mRNA expression at 2, 3, and 4 h, and in Ki-ras mRNA expression at 1 h. During the 3-day resting period within which no treatment was given, pancreatic weight exhibited its maximal reduction after 2 days in the CIP group. In that same group, the ODC activity reached its maximal level above control after 3 days and ODC and Ki-ras mRNA expression after 1 and 2 days. During the regeneration period of 5 days, the pancreata of the untreated pancreatitis rats did not totally recover, whereas those of the animals receiving the small dose of caerulein (1 microgram) showed full recovery and even a significant increase above control after 5 days. During that period, maximal increases in ODC activity and Ki-ras mRNA expression occurred after 1 day of caerulein treatment; ODC mRNA expression was also significantly increased after 3 and 5 days in the pancreatitis animals with no effect of caerulein treatment. The positive effect of caerulein on Ki-ras mRNA suggests that the cholecystokinin analogue can induce the expression of essential growth-promoting genes.
Asunto(s)
Expresión Génica , Genes ras/genética , Ornitina Descarboxilasa/genética , Pancreatitis/genética , Enfermedad Aguda , Animales , Peso Corporal , Ceruletida , ADN/metabolismo , Masculino , Ornitina Descarboxilasa/metabolismo , Páncreas/enzimología , Páncreas/patología , Pancreatitis/inducido químicamente , Pancreatitis/enzimología , Pancreatitis/patología , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
The growth-promoting effects of gastrin on normal and neoplastic gastrointestinal tissues have been shown to be mediated by the gastrin/CCKB receptor, which belongs to the family of G protein-coupled receptors. However, the downstream signaling pathways activated by gastrin are not well characterized. In the present study, we demonstrate that gastrin stimulates tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), the major cytoplasmic substrate of the insulin receptor. The gastrin-induced phosphorylation of IRS-1 was rapid and transient, occurring within 30 s of treatment and diminishing thereafter. IRS-1 binds several proteins containing Src homology 2 domains through its multiple tyrosine phosphorylation sites. Following gastrin stimulation, we observed a time- and dose-dependent association of IRS-1 with the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase). In addition, activation of PI 3-kinase was detected in anti-IRS-1 immunoprecipitates from gastrin-treated cells, suggesting that tyrosine phosphorylation of IRS-1, which leads to the rapid recruitment of p85, might be one mechanism used by gastrin to activate PI 3-kinase. We have previously reported that tyrosine phosphorylation of Shc and its association with the Grb2-Sos complex may contribute to the activation of the mitogen-activated protein kinase pathway by gastrin. We report here that Grb2 also interacts with tyrosine-phosphorylated IRS-1 in response to gastrin. Taken together, our results suggest that IRS-1 may serve as a converging target in the signaling pathways stimulated by receptors that belong to different families, such as the gastrin/CCKB G protein-coupled receptor and the insulin receptor.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Receptores ErbB/metabolismo , Gastrinas/farmacología , Fosfoproteínas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas/metabolismo , Tirosina/metabolismo , Animales , Activación Enzimática , Proteína Adaptadora GRB2 , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Páncreas/efectos de los fármacos , Páncreas/enzimología , Fosfatidilinositol 3-Quinasas , Fosforilación , Ratas , Células Tumorales CultivadasRESUMEN
For the first time, we have demonstrated in AR4-2J cells, an experimental model of azaserine-induced carcinoma in the rat exocrine pancreas, the co-expression of alpha 1 subunit of dihydropyridine-sensitive Ca2+ channel and the alpha 1 sub-unit of omega-conotoxin-sensitive Ca2+ channel RNA messengers which share homologous sequences with, respectively, rbC II and rbB I sub-types described in the rat brain. These two types of voltage-dependent Ca2+ channels which are functionally expressed, emphasize the acquisition during carcinogenesis of neuroendocrine features of AR4-2J cells. Additionally, using antisense phosphorothioate oligodeoxynucleotide, we demonstrated clearly the involvement of dihydropyridine-sensitive Ca2+ channels in the control of AR4-2J cell proliferation.
Asunto(s)
Canales de Calcio/genética , Canales de Calcio/metabolismo , Calcio/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Animales , Elementos sin Sentido (Genética)/genética , Secuencia de Bases , Agonistas de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Carcinoma/inducido químicamente , Carcinoma/genética , Carcinoma/patología , División Celular/genética , División Celular/fisiología , Cartilla de ADN/química , Dihidropiridinas/farmacología , Relación Dosis-Respuesta a Droga , Venenos de Moluscos/farmacología , Páncreas/citología , Neoplasias Pancreáticas/inducido químicamente , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Reacción en Cadena de la Polimerasa , Ratas , Células Tumorales CultivadasRESUMEN
Glycine-extended gastrin (gastrin-Gly) stimulates proliferation of AR4-2J pancreatic tumor cell line through a specific receptor, different from the gastrin-cholecystokinin B receptor. Our purpose was to determine whether AR4-2J cells produced gastrin-Gly and then whether the peptide was involved in an autocrine loop. First, proliferation of AR4-2J cells in serum-free medium was inhibited by a gastrin anti-sense oligodeoxynucleotide phosphorothioate and by antibodies specific for gastrin-Gly. In contrast, antibodies specific for alpha-amidated gastrin were without effect. By using RT-PCR, we have shown that AR4-2J cells expressed gastrin mRNA. The presence of gastrin-Gly, but not alpha-amidated gastrin, in serum-free media was detected by radioimmunoanalysis. Gel chromatography revealed that the predominant molecular forms secreted were glycine-extended gastrin-34 and gastrin- 17. Furthermore, epidermal growth factor (EGF), a stimulator of gastrin gene transcription, modulates gastrin-Gly secretion by AR4-2J. These data together suggest that gastrin-Gly is an autocrine growth factor for AR4-2J cells and that it participates with EGF in the regulation of AR4-2J-cell proliferation.