RESUMO
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.
Assuntos
Apoptose/fisiologia , Fatores de Transcrição Kruppel-Like/fisiologia , Apoptose/genética , Sequência de Bases , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Dados de Sequência Molecular , Mutação , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína do Retinoblastoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido NucleicoRESUMO
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.
Assuntos
Apoptose , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Receptores de Somatostatina/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Somatostatina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Camundongos , Modelos Biológicos , Mimetismo Molecular/efeitos dos fármacos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
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.
Assuntos
Vírus Defeituosos/fisiologia , Técnicas de Transferência de Genes , Neoplasias Pancreáticas/patologia , Vírus 40 dos Símios/fisiologia , Replicação Viral , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Humanos , Reação em Cadeia da Polimerase , Transdução GenéticaRESUMO
Many reports emphasized the role of gastrin as growth factor on normal gastrointestinal mucosa and pancreas. In the present study, we analyzed the proliferative effects of cholecystokinin (CCK) and gastrin peptides on a rat tumoral pancreatic cell line, AR42J, which possesses both CCKA and CCKB receptor subtypes. The results showed a good correlation between the binding of gastrin to CCKB receptor [Kd 1.125 +/- 0.3 (SD) nM] and its ability to either induce ornithine decarboxylase activity [50% effective concentration, 0.6 +/- 0.3 nM] and [3H]-thymidine incorporation [50% effective concentration, 2 +/- 0.4 nM]. Furthermore, the ability of different cholecystokinin and gastrin antagonists such as proglumide and asperlicin derivatives (respectively, CR1409, CR1505, and L364,718) were tested. We found that all antagonists displaced 125I-labeled gastrin binding, with the following order of potencies: L364,718 greater than CR1409 greater than CR1505 greater than proglumide. Furthermore, the 50% inhibitory concentration of CR1409 and CR1505 to inhibit gastrin stimulated ornithine decarboxylase activity (an early event involved in cell proliferation) and [3H]thymidine incorporation were in agreement with their constants of inhibition (Ki) on gastrin binding. The L364,718 compound, at a concentration which fully occupied the CCKA without affecting the CCKB, had no effect on gastrin stimulated ornithine decarboxylase activity and [3H]thymidine incorporation. In addition, this compound appeared to be a full agonist on CCKB receptor. These results confirm the implication of the CCKB receptor in the proliferative response of AR42J cells to gastrin.
Assuntos
DNA de Neoplasias/biossíntese , Gastrinas/farmacologia , Glutamina/análogos & derivados , Neoplasias Pancreáticas/metabolismo , Proglumida/análogos & derivados , Animais , Ornitina Descarboxilase/análise , Neoplasias Pancreáticas/patologia , Proglumida/farmacologia , Ratos , Receptores da Colecistocinina/análise , Receptores da Colecistocinina/fisiologia , Timidina/metabolismo , Células Tumorais CultivadasRESUMO
The ornithine decarboxylase enzyme (ODC) is the key regulator of polyamine synthesis and is a member of the cellular proto-oncogene family. Its expression becomes constitutively activated by carcinogens, viruses, and oncogenes. ODC mRNA has a long 5' untranslated region that could be important in the regulation of enzyme levels by affecting translation. To test this hypothesis, we have determined the role of this region on the constitutive ODC hyperexpression measured in AR4-2J cells, an azaserine-induced, tumor-derived pancreatic acinar cell line. Construction of expression vectors in which ODC 5' leader sequence was placed flanking the chloramphenicol acetyltransferase reporter gene allowed us to identify three AR4-2J specific, different alternatively spliced ODC 5' leaders. The 5' ends of exons 2 and 3 were lengthened by 17 and 13 bases, respectively. Translation performed in a cell-free system as well as in COS7 transient transfection experiments demonstrated that AR4-2J isoforms induce a strong increase in the rate of translation. These results provide evidence that alternative splicing observed in tumoral cells, coupled with translation regulation, relieves the translation repression mediated by the long and structured 5' untranslated region of the ODC proto-oncogene.
Assuntos
Processamento Alternativo , Ornitina Descarboxilase/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Cloranfenicol O-Acetiltransferase/análise , Cloranfenicol O-Acetiltransferase/biossíntese , Chlorocebus aethiops , Primers do DNA , Repressão Enzimática , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Dados de Sequência Molecular , Pâncreas , Neoplasias Pancreáticas , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Transfecção , Células Tumorais CultivadasRESUMO
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.
Assuntos
Proteínas de Transporte , Gastrinas/farmacologia , Ornitina Descarboxilase/genética , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/metabolismo , RNA Mensageiro/genética , Animais , Células COS , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Ornitina Descarboxilase/biossíntese , Inibidores da Ornitina Descarboxilase , Fosforilação/efeitos dos fármacos , Polienos/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Ratos , Proteínas Repressoras/farmacologia , Sirolimo , Células Tumorais CultivadasRESUMO
A somatostatin-14-degrading activity has been purified to homogeneity from rat pure pancreatic juice. This proteinase was concentrated more than 350-fold in a four-step procedure including ion-exchange and gel filtration. The final preparation contained a single protein with a molecular weight (M(r)) of approx. 29,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The determination of its NH2-terminal sequence led us to conclude that the purified proteinase corresponds to the rat pancreatic elastase II predicted from the cDNA clone isolated by MacDonald in 1982. This anionic proteinase exhibits an isoelectric point of 5.6 and does not contain any carbohydrate moieties in its structure. The proteinase is sensitive to the trypsin inhibitors soybean trypsin inhibitor and N alpha-tosyl-L-lysine-chloromethyl ketone and also to 3,4-dichloroisocoumarin, a general elastase inhibitor. The cleavage products obtained after hydrolysis of somatostatin-14 by the purified elastase, were separated by reversed phase high performance liquid chromatography and identified by amino-acid analysis. The primary hydrolysis was trypsin-like and consisted in an opening of the cyclic structure of somatostatin-14 after the Lys-9 residue leading to the formation of a Y-shaped peptide with the same amino-acid composition as the native peptide. The initial 'trypsin-like specificity' was not observed during the secondary hydrolysis of the Y-shaped peptide; indeed the proteinase seemed more specific for a certain motif in the native peptide rather than for a specific class of amino acid, this last kind of selectivity is commonly observed with trypsin and chymotrypsin. In order to establish that the proteinase possesses an extended recognition site on the substrate rather than a specificity for a class of amino acid, the substrate specificity of the rat pancreatic elastase II was investigated with a series of para-nitroanilide peptides. The proteinase exhibits a large specificity involving peptide chain of at least four amino acids with a preference for bulky residue in P1 or P2. The Km values of 89 microM and 1567 microM obtained for somatostatin-14 and Suc-Ala-Ala-Pro-Met-pNA, respectively, indicate that elastase II has a greater affinity for the natural substrate than for synthetics. This last observation along with the substrate specificity of the proteinase leads us to propose that elastase II could be specifically involved in the regulation of biological functions of somatostatin-14 in the gastrointestinal tract.
Assuntos
Pâncreas/enzimologia , Elastase Pancreática/isolamento & purificação , Suco Pancreático/enzimologia , Sequência de Aminoácidos , Animais , Cinética , Masculino , Dados de Sequência Molecular , Elastase Pancreática/química , Elastase Pancreática/metabolismo , Ratos , Ratos Wistar , Somatostatina/metabolismo , Especificidade por SubstratoRESUMO
We studied the effects of oxyntomodulin (OXM), of its C-terminal (19-37) fragment (OXM (19-37)) and of glucagon (GLU) on somatostatin release, cyclic AMP accumulation and inositol phosphate turnover in somatostatin-secreting RIN T3 cells in culture. Rapid changes in cellular free Ca2+ were also measured using fura-2. Carbachol was used as a control test agent for the parameters involving the inositol phosphate/Ca2+ cascade. OXM, GLU and OXM (19-37) were all able to stimulate somatostatin release with relative ED50 of approx. 1, 22 and 45, respectively. OXM and GLU stimulated cyclic AMP levels with relative ED50 of approx. 1 and 30, respectively, whereas OXM (19-37) was totally ineffective on this parameter. In contrast to carbachol, none of the peptides significantly modified the inositol phosphate turnover or induced rapid changes in cellular free Ca2+. We conclude that the RIN T3 cells contain a receptor-cyclic AMP system similar to that found in gastric mucosa and that this system is linked to somatostatin release. Another receptor-second messenger mechanism linked to somatostatin release is triggered by the (19-37) fragment. This mechanism is not the inositol phosphate/Ca2+ cascade triggered in the same cells by cholinergic agents.
Assuntos
Peptídeos Semelhantes ao Glucagon/farmacologia , Insulinoma/metabolismo , Somatostatina/metabolismo , Animais , Cálcio/metabolismo , Carbacol/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Glucagon/farmacologia , Peptídeos Semelhantes ao Glucagon/química , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Oxintomodulina , Fragmentos de Peptídeos/farmacologia , Ratos , Taxa Secretória/efeitos dos fármacos , Somatostatina/biossíntese , Fatores de Tempo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Elementos Antissenso (Genética)/genética , Sequência de Bases , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Carcinoma/induzido quimicamente , Carcinoma/genética , Carcinoma/patologia , Divisão Celular/genética , Divisão Celular/fisiologia , Primers do DNA/química , Di-Hidropiridinas/farmacologia , Relação Dose-Resposta a Droga , Venenos de Moluscos/farmacologia , Pâncreas/citologia , Neoplasias Pancreáticas/induzido quimicamente , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase , Ratos , Células Tumorais CultivadasRESUMO
Somatostatin has been demonstrated to negatively regulate pancreatic growth in vivo. In this study we used the AR4-2J rat pancreatic acinar tumor cell line to investigate the effect of a stable somatostatin analog, SMS 201-995 (SMS) on cell proliferation. SMS induced an antiproliferative effect on both serum or epidermal growth factor (EGF)-induced cell proliferation; exposure of the cells for 48 h to SMS caused a slight inhibition of serum-induced proliferation (maximal inhibition, 26%) and abolished the growth-promoting effect of EGF. Maximal effect was observed with 10 nM SMS, and half-maximal (IC50) effect with 0.06-0.1 nM SMS. Binding studies with an iodinated derivative of SMS, [125I-Tyr3]SMS, revealed the presence of a single class of high affinity binding sites on AR4-2J plasma membranes with an equilibrium dissociation constant of 0.2 +/- 0.03 nM and a binding site number of 1.1 +/- 0.07 pmol/mg protein. Addition of the nonhydrolyzable GTP analog, guanosine 5-[gamma-thio] triphosphate (GTP gamma S), increased the rate of dissociation of the specifically bound peptide in agreement with the coupling of somatostatin receptors with a GTP-binding regulatory protein. The good agreement between the IC50 for SMS inhibition of cell proliferation and the apparent Kd for binding indicates that the characterized binding sites are the somatostatin receptors that mediate the antiproliferative effect of SMS. When cells were grown in serum-free medium EGF stimulated AR4-2J cell proliferation with half-maximal (ED50) and maximal effects at 0.6 and 10 nM EGF, respectively. This stimulatory effect of EGF was mediated by specific receptors, since binding studies with [125I]EGF indicated that AR4-2J cells contained a single class of EGF receptors (13,000 sites/cell), with an affinity constant for [125I]EGF (Kd = 0.9 +/- 0.09 nM) close to the ED50 for EGF stimulation of cell growth. To examine if SMS-induced growth inhibition involved a cAMP-dependent mechanism we first studied the effect of SMS on cAMP production. SMS had no effect on basal cAMP, but completely inhibited VIP-stimulated cAMP production with an IC50 of 0.2 nM. Pertussis toxin, which is known to abolish the inhibitory effect of somatostatin on adenylate cyclase activity in AR4-2J cells, did not reverse the ability of SMS to inhibit cell proliferation as well as EGF-induced cell proliferation. These data indicate that the antiproliferative effect of SMS does not involve the GTP-binding protein-mediated negative coupling of somatostatin receptors to adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Toxina Adenilato Ciclase , Divisão Celular/efeitos dos fármacos , Proteínas de Ligação ao GTP/fisiologia , Octreotida/farmacologia , Toxina Pertussis , Somatostatina/antagonistas & inibidores , Fatores de Virulência de Bordetella/farmacologia , Animais , Linhagem Celular , AMP Cíclico/biossíntese , Replicação do DNA/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Cinética , Neoplasias Pancreáticas , Receptores de Neurotransmissores/metabolismo , Receptores de Somatostatina , Somatostatina/metabolismo , Somatostatina/farmacologia , Timidina/metabolismoRESUMO
The effects of somatostatin-14 (S14) and somatostatin-28 (S28), a novel intestinal peptide containing somatostatin tetradecapeptide in its C-terminal position, on the bombesin-stimulated release of gastrin, insulin, and glucagon were tested. On iv infusion of bombesin, the increase in the level of glucagon was seen to be twice that of gastrin, and the insulin increase was 8 times that of gastrin. Plasma concentrations of somatostatin were not modified. S14 and S28 inhibited the release of gastrin, insulin, and glucagon; insulin release was inhibited most effectively, with glucagon release next, and gastrin release least inhibited. Based on the exogenous dose needed to produce equal effects, S28 was more potent than S14 on a molar basis, but based on the plasma concentrations needed to produce equal effects, S14 and S28 were equipotent.
Assuntos
Bombesina/farmacologia , Gastrinas/sangue , Glucagon/sangue , Insulina/sangue , Peptídeos/farmacologia , Precursores de Proteínas/farmacologia , Somatostatina/farmacologia , Animais , Cães , Cinética , Radioimunoensaio , Somatostatina/sangue , Somatostatina-28RESUMO
Somatostatin analogs are an alternative medical treatment in patients with TSH-secreting pituitary adenoma. A 31-yr-old infertile woman with a TSH-secreting macroadenoma was treated with continuous sc infusion of 300 micrograms octreotide/day. After 3 months, euthyroid status was restored, and pituitary magnetic resonance imaging showed a reduction of the macroadenoma. Subsequently, the patient was found to be pregnant, and octreotide was stopped after 1 month of gestation. Serum TSH and free thyroid hormone concentrations returned to pretreatment values. At 6 months of pregnancy, a visual field examination was abnormal, and a magnetic resonance imaging scan showed an enlargement of the pituitary adenoma. Reinstitution of octreotide treatment was associated with normalization of TSH and free thyroid hormone concentrations, a rapid improvement of visual fields, and a new reduction in the size of pituitary macroadenoma. Octreotide treatment was continued until an elective cesarean section was performed at 8 months gestation. Despite the presence of immunoreactive octreotide in the umbilical cord, neonatal thyroid parameters were normal, and a physiological rise in TSH with the increase in thyroid hormone concentrations occurred in the neonate. In conclusion, 1) octreotide treatment is effective in controlling TSH-secreting macroadenoma during pregnancy; 2) despite the transplacental passage of immunoreactive octreotide, physiological changes in thyroid parameters occur in the neonate, and 3) exposure of the fetus to octreotide during the first month as well as the last trimester of gestation did not induce any malformation or affect fetal development.
Assuntos
Adenoma/complicações , Adenoma/tratamento farmacológico , Infertilidade Feminina/complicações , Octreotida/uso terapêutico , Neoplasias Hipofisárias/complicações , Neoplasias Hipofisárias/tratamento farmacológico , Complicações Neoplásicas na Gravidez/tratamento farmacológico , Tireotropina/metabolismo , Adenoma/metabolismo , Adulto , Feminino , Humanos , Imageamento por Ressonância Magnética , Neoplasias Hipofisárias/metabolismo , GravidezRESUMO
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.
Assuntos
Moléculas de Adesão Celular/metabolismo , Gastrinas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais , Animais , Ativação Enzimática , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Gastrinas/farmacologia , Fosforilação , Testes de Precipitina , Ratos , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
(Thr28,Nle31)CCK(23-33) (CCK-9) and gastrin(1-17)I (gastrin) inhibited adenylate cyclase activity in membranes from the tumoral rat pancreatic acinar cell line AR 4-2J through a Bordetella pertussis toxin-sensitive mechanism. This contrasted with the stimulatory effect exerted by CCK-9 on adenylate cyclase activity in membranes from normal rat pancreas. The relative potency of CCK-9, gastrin, and related peptides in inhibiting adenylate cyclase, when confronted with previous evidence, suggests that 'non-selective CCK-gastrin CCK-B receptors' predominating over 'selective CCK-A receptors' in the AR 4-2J cell line, favored the coupling of the first receptors to adenylate cyclase through Gi, while CCK-A receptors capable of stimulating the enzyme through Gs were detected only after Bordetella pertussis toxin pretreatment.
Assuntos
Toxina Adenilato Ciclase , Inibidores de Adenilil Ciclases , Colecistocinina/farmacologia , Gastrinas/farmacologia , Pâncreas/enzimologia , Neoplasias Pancreáticas/enzimologia , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologia , Animais , Membrana Celular/enzimologia , Guanosina Trifosfato/farmacologia , Pentagastrina/farmacologia , Fragmentos de Peptídeos/farmacologia , Ratos , Secretina/farmacologia , Tetragastrina/farmacologia , Células Tumorais Cultivadas , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
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.
Assuntos
DNA/biossíntese , Gastrinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Quinases da Família src/metabolismo , Androstadienos/farmacologia , Animais , Células CHO , Cálcio/metabolismo , Cromonas/farmacologia , Cricetinae , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Gastrinas/farmacologia , Humanos , Líquido Intracelular/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Receptor de Colecistocinina B , Receptores da Colecistocinina/genética , Receptores da Colecistocinina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tapsigargina , Transfecção , Wortmanina , Proteínas Quinases p38 Ativadas por Mitógeno , Quinases da Família src/antagonistas & inibidoresRESUMO
Gastrin/CCKB G protein-coupled receptors have been shown to mediate proliferative effects of their endogenous ligands. In the present study, we examined the signal transduction mechanisms linked to the G/CCKB receptor occupancy. We report here that gastrin stimulates MAP kinase activation in a dose- and time-dependent manner, a pathway known to play a key role in cell proliferation. We also characterized the molecular events, upstream of p21-Ras, that may link the MAP kinase pathway to G/CCKB receptors. Gastrin induced a rapid and transient increase in tyrosine phosphorylation of several proteins including the 2 isoforms (46 and 52 kDa) of the adaptor protein Shc. Phosphorylated Shc subsequently associated with a complex that includes Grb2 and the p21-Ras activator, Sos. Our results also indicate that Sos becomes phosphorylated in response to gastrin as shown by a reduction in electrophoretic mobility of the protein. Tyrosine phosphorylation of Shc and subsequent complex formation with Grb2 and Sos appear to be a common mechanism by which tyrosine kinase receptors and the G/CCKB G protein-coupled receptor stimulate the Ras-dependent MAP kinase pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Gastrinas/farmacologia , Proteínas de Membrana/metabolismo , Fosfotirosina/metabolismo , Proteínas/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteína Adaptadora GRB2 , Cinética , Neoplasias Pancreáticas , Fosforilação , Ratos , Receptores da Colecistocinina/fisiologia , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteínas Son Of Sevenless , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Células Tumorais CultivadasRESUMO
Biological active forms of somatostatin are produced by cleavage of large precursors. If the sequence of the pre-proform of somatostatin has been deduced from cDNA structure in several species, little is known about the processing of these large precursors. For this purpose, the analysis of immunoreactive components secreted by the R.I.N. cell line was investigated. After selection of a cell population and culture conditions providing the optimal production of these peptides, analysis of their molecular forms was done by molecular gel filtration. The results show that mainly pro-forms accumulate in the culture medium while besides the pre-proform the smaller immunoreactive species behaving like S-28 and S-14 were found in cell extracts. Incorporation studies in serum free medium showed rapid formation of an intermediate compound eluted at 1.87 V0.
Assuntos
Ilhotas Pancreáticas/metabolismo , Precursores de Proteínas/biossíntese , Somatostatina/biossíntese , Animais , Linhagem Celular , Insulina/metabolismo , Secreção de Insulina , Ratos , Somatostatina-28RESUMO
In rat pancreatic acinar cells, amylase release and Ca2+ mobilization are related to the occupancy of CCKA receptor. The rat pancreatic acinar cell line (AR4-2J) possesses both CCKA (CCKA R) and CCKB (CCKB R) sub-type receptors. Using this cell line we attempted to determine the relative involvement of each sub-type in both amylase release and Ca2+ mobilization. For this purpose we used L 364718 a selective antagonist for CCKA R and PD 135158 a selective antagonist for CCKB R. We showed on AR4-2J cells that: a minority of CCKA R (Kd = 0.7 nM), a classical CCKB R (Kd = 0.93 nM) and a new high affinity gastrin binding site (Kd = 2.1 pM) coexisted; CCK through CCKA R and CCKB R, was more potent to stimulate amylase secretion (EC50 = 34 pM) and Ca2+ mobilization (EC50 = 30 pM) than to occupy its receptor. Gastrin induced a biphasic stimulation of amylase release. Gastrin through CCKB R was equally potent to stimulate amylase release (EC50 = 1.72 nM) and Ca2+ mobilization (EC50 = 3.1 nM), whereas through the high affinity gastrin binding site, gastrin-induced amylase release (EC50 = 0.73 pM) did not correlate with the Ca2+ mobilization (EC50 = 3.1 nM). These results demonstrated for the first time the existence, on AR4-2J cells, of a high affinity gastrin receptor whose occupation by gastrin induces amylase release.
Assuntos
Amilases/metabolismo , Gastrinas/farmacologia , Pâncreas/enzimologia , Suco Pancreático/metabolismo , Receptores da Colecistocinina/fisiologia , Animais , Benzodiazepinonas/farmacologia , Cálcio/metabolismo , Células Cultivadas , Colecistocinina/metabolismo , Devazepida , Gastrinas/metabolismo , Técnicas In Vitro , Indóis/farmacologia , Meglumina/análogos & derivados , Meglumina/farmacologia , Ratos , Receptores da Colecistocinina/antagonistas & inibidoresRESUMO
Gastrin and cholecystokinin (CCK) have proven trophic effects on the gut. We have previously demonstrated that these peptides stimulate an early event in cellular proliferation, namely ornithine decarboxylase activity (ODC), in a rat exocrine pancreatic cell line AR4-2J. Furthermore, this effect is mediated through a G/CCKB receptor. Thus, in the present study we sought to examine the signal transduction mechanisms linked to the G/CCKB receptor occupancy. Both gastrin and CCK induced a rapid (maximum at 40 s) increase in inositol triphosphates (InsP3) and diacylglycerol (DAG) formation in a dose-dependent manner (EC50 = 5.6 nM) that quickly returned to baseline. Although InsP3 levels remained at baseline, DAG levels demonstrated a second gradual increase that was maximal at 15 min. CCK/gastrin efficiency to stimulate DAG and InsP3 formation (EC50 = 5.6 nM) could be correlated to the G/CCKB receptor occupancy, suggesting a coupling of this receptor to phospholipase C. To examine the involvement of protein kinase C (PKC) activation in the increase in ODC activity, we stimulated the AR4-2J cells with the phorbol ester TPA and observed an increase in ODC activity with a maximal effect at 100 nM. TPA stimulation of ODC activity was completely abolished by the PKC inhibitor staurosporine (50 nM). However, 50 nM staurosporine inhibited only 65% of the gastrin and CCK induced increase in ODC activity suggesting that a portion of the G/CCKB receptor-mediated increase in ODC activity is PKC independent.
Assuntos
Proteína Quinase C/metabolismo , Receptores da Colecistocinina/metabolismo , Fosfolipases Tipo C/metabolismo , Alcaloides/farmacologia , Animais , Sítios de Ligação , Colecistocinina/farmacologia , Diglicerídeos/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Gastrinas/farmacologia , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Ornitina Descarboxilase/metabolismo , Pâncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/metabolismo , Proteína Quinase C/antagonistas & inibidores , Ratos , Transdução de Sinais/fisiologia , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
The binding of bombesin to its receptors on normal human pancreatic membranes was investigated using high specific activity, radioiodinated bombesin ([125I]-Tyr4-bombesin), prepared by an oxidative method with chloramine-T. Binding was specific, temperature-dependent, saturable, reversible and linearly related to membranes protein concentration. After a 30 min period of incubation with membranes the degradation of the tracer has never been found superior to 20%. Scatchard analysis of binding data was compatible with a single class of binding sites with a high affinity (0.96 nM) and a Bmax of 753 fmol/mg protein. [125I]-Tyr4-bombesin binding to human pancreatic membranes was competitively inhibited by (1-Tyr4-)bombesin, GRP, the nonapeptide of bombesin and litorin but not by unrelated hormones such as somatostatin, CCK, human gastrin, etc. These results describe for the first time the presence of specific receptors for bombesin on human pancreatic membranes. The binding characteristics obtained are comparable with those found in other species.