RESUMEN
Factors that stimulate gut mucosal proliferation may be beneficial during periods of gut disuse or atrophy. Recently glucagon-like peptide 2 (GLP-2) has been shown to stimulate small bowel growth. The purpose of our study was to compare the trophic effects of GLP-2 with those of neurotensin (NT), a potent gut trophic factor. Mice were randomized to receive either GLP-2, NT, or saline solution (control) for 10 days. The mice were killed on day 11, at which time the jejunum, ileum, and colon were removed, weighed, and DNA and protein content measured. Mice treated with GLP-2 showed a significant increase in the weight of the jejunum, ileum, and colon compared to both control and NT-treated mice. DNA content, a marker of cellular hyperplasia, was significantly increased in the small bowel and colon by treatment with GLP-2 and NT compared to control tissues. Small intestinal protein content, an indicator of cellular hypertrophy, was significantly increased by GLP-2 compared to both NT and control; protein content of the colon was greater in each of the treatment groups compared with control mice. We have demonstrated, for the first time, that GLP-2 stimulates colonic growth. In addition, GLP-2 is a potent trophic factor of normal small intestine with proliferative effects that are equal to or greater than those of NT. Administration of GLP-2 may be useful clinically to enhance small intestinal regeneration and adaptation during periods of disease and in the early phases of the short bowel syndrome.
Asunto(s)
Colon/efectos de los fármacos , Hormonas Gastrointestinales/farmacología , Glucagón/inmunología , Sustancias de Crecimiento/farmacología , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Péptidos/farmacología , Precursores de Proteínas/farmacología , Adaptación Fisiológica , Animales , División Celular/efectos de los fármacos , Colon/citología , ADN/análisis , Femenino , Péptido 2 Similar al Glucagón , Péptidos Similares al Glucagón , Íleon/citología , Íleon/efectos de los fármacos , Enfermedades Intestinales/tratamiento farmacológico , Mucosa Intestinal/citología , Intestino Delgado/citología , Yeyuno/citología , Yeyuno/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neurotensina/farmacología , Tamaño de los Órganos , Proteínas/análisis , Distribución Aleatoria , Regeneración , Síndrome del Intestino Corto/tratamiento farmacológicoRESUMEN
Gastrin-secreting tumors have been identified in ectopic locations including the ovary; the mechanisms regulating gastrin gene expression, its distribution, and signaling pathways in these ectopic tissues are not known. The purpose of our present study was to determine: (1) whether the gastrin gene and peptide could be detected in ovarian cancer cell lines, (2) if functional gastrin releasing peptide receptors (GRP-R) are present, and (3) whether gastrin gene expression is altered by GRP. Five ovarian cancer cell lines (SW626, OVCA 420, OVCA 429, OVCA 432, and OVCA 433) were analyzed. We identified gastrin gene and peptide expression in the SW626 cell line but not in the OVCA lines. SW626 cells express a functional GRP-R that is correctly coupled to the Ca2+ signaling pathway. Treatment of SW626 cells with bombesin, the amphibian equivalent of GRP, inhibited expression of the gastrin gene in a time- and dose-dependent fashion. The SW626 ovarian cancer cell line will provide a useful model to further define regulation and expression of both the gastrin gene and peptide in ectopic (nongastrointestinal) tissues.
Asunto(s)
Cistadenocarcinoma/genética , Gastrinas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas/genética , Northern Blotting , Southern Blotting , Bombesina/farmacología , Cistadenocarcinoma/patología , Femenino , Gastrinas/biosíntesis , Humanos , Neoplasias Ováricas/patología , Reacción en Cadena de la Polimerasa , Radioinmunoensayo , Receptores de Bombesina/análisis , Receptores de Bombesina/efectos de los fármacos , Receptores de Bombesina/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND: Expression of the gene encoding the neurotensin/neuromedin N (NT/N) is developmentally regulated in the gut in a distinctive temporal and spatial fashion. Src kinase, a nonreceptor tyrosine kinase, has been implicated in the growth and differentiation of various tissues; its role in gut differentiation is not known. The purpose of this study was to determine whether the Src signaling pathway plays a role in the activation of the human NT/N promoter. METHODS: Caco-2 cells, a human colon cancer cell line that can differentiate to a small bowel phenotype, were transiently transfected with human NT/N promoter fragments linked to luciferase and various amounts of Src expression plasmids or dominant negative Raf; luciferase and beta-galactosidase activities were measured after 48 hours. RESULTS: Cotransfection of Src resulted in an approximate eightfold increase of NT/N promoter activity; mutation of a proximal activating protein-1/cyclic adenosine monophosphate responsive element site resulted in a dramatic decrease of Src-mediated NT/N induction. Cotransfection with a dominant negative Raf plasmid partially blocked Src-mediated NT/N activation. CONCLUSIONS: Src increases NT/N promoter activity in Caco-2 cells acting, in part, through a proximal AP-1/CRE promoter element. In addition, Src regulation of the NT/N promoter appears to be mediated through a Raf-dependent pathway. We propose that Src may play a role in tissue-specific gene expression in the gut.
Asunto(s)
Neurotensina/biosíntesis , Neurotensina/genética , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/metabolismo , Proteína Tirosina Quinasa CSK , Neoplasias del Colon , Regulación de la Expresión Génica , Humanos , Luciferasas/biosíntesis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Proteínas Recombinantes de Fusión/biosíntesis , Transfección , Células Tumorales Cultivadas , beta-Galactosidasa/biosíntesis , Proteínas ras/metabolismo , Familia-src QuinasasRESUMEN
Gastrin (G-17) is a trophic hormone with a high affinity for the cholecystokinin-B receptor (CCK-BR); the mechanisms linking receptor binding and activation of downstream events to cell growth are not known, and these studies have been hampered by the lack of a cell model. We have established a pancreatic carcinoid cell line, BON, which produces a number of gut hormones; however, these cells lack native CCK-BR. The purpose of our study was to develop a model cell line containing the CCK-BR and to characterize the cellular mechanisms involved in gastrin regulation of human cell growth. BON cells were transfected with an expression plasmid containing the human CCK-BR, and stable clones were selected using G418. Functional CCK-BR was confirmed by reverse transcriptase-polymerase chain reaction, 125I-gastrin binding, and mobilization of intracellular calcium ([Ca2+]i) in response to G-17. Stable transfectants were treated with G-17 (+/-) the CCK-BR antagonist, L365,260 (L-60); growth was assessed using a Coulter counter. G-17 stimulated the growth of the stable clones, whereas the selective CCK-BR antagonist, L-60, abolished this G-17-mediated trophic effect. We have shown that G-17, acting through the CCK-BR, mobilizes [Ca2+]i as a second messenger and stimulates cell growth. Our unique BON cell line, stably transfected with the human CCK-BR, provides a novel paradigm to further delineate signaling mechanisms in gastrin regulation of human cell growth.
Asunto(s)
División Celular/fisiología , Gastrinas/fisiología , Línea Celular , Células Cultivadas , Humanos , Receptor de Colecistoquinina B/fisiología , Transducción de SeñalRESUMEN
BACKGROUND: Bombesin, a gut tetradecapeptide homologous to the mammalian gastrin-releasing peptide (GRP), stimulates the growth of the human gastric cancer line SIIA through specific GRP receptors (GRP-Rs); the cellular mechanisms are not known. The purpose of our study was to (1) confirm functional GRP-R in SIIA and (2) determine whether bombesin alters the expression and binding activity of the AP-1 transcription factors, c-jun and jun-B. METHODS: SIIA cells were treated with bombesin, and intracellular calcium mobilization was measured by means of fura-2 spectrofluorometry. To assess changes in c-jun and jun-B, RNA and protein were extracted for Northern and Western blots, respectively; nuclear protein was extracted for gel mobility shifts to determine AP-1 binding activity. RESULTS: SIIA cells mobilized intracellular calcium in response to bombesin, exhibiting a functional cell-surface GRP-R. Bombesin treatment increased expression of both c-jun and jun-B mRNA by 0.5 hours, with maximal expression at 1 hour; concomitant increases in steady-state levels of c-Jun and JunB protein were identified. Moreover, bombesin increased binding of the AP-1 proteins as shown by gel shifts. CONCLUSIONS: The SIIA human gastric cancer possesses functional GRP-R coupled to the calcium second messenger pathway. Further, bombesin stimulates expression of c-jun and jun-B mRNA and protein and increases binding activity of AP-1 proteins. Delineating the cellular pathways involved in bombesin-mediated gene activation will provide important insights into the mechanisms responsible for normal and neoplastic gut growth.