RESUMEN
BACKGROUND: Colonoscopic screening and surveillance for colorectal cancer could be made safer and more efficient if endoscopists could predict histology without the need to biopsy and perform histopathology on every polyp. Elastic-scattering spectroscopy (ESS), using fiberoptic probes integrated into standard biopsy tools, can assess, both in vivo and in real time, the scattering and absorption properties of tissue related to its underlying pathology. AIMS: The objective of this study was to evaluate prospectively the potential of ESS to predict polyp pathology accurately. METHODS: We obtained ESS measurements from patients undergoing screening/surveillance colonoscopy using an ESS fiberoptic probe integrated into biopsy forceps. The integrated forceps were used for tissue acquisition, following current standards of care, and optical measurement. All measurements were correlated to the index pathology. A machine learning model was then applied to measurements from 367 polyps in 169 patients to prospectively evaluate its predictive performance. RESULTS: The model achieved sensitivity of 0.92, specificity of 0.87, negative predictive value (NPV) of 0.87, and high-confidence rate (HCR) of 0.84 for distinguishing 220 neoplastic polyps from 147 non-neoplastic polyps of all sizes. Among 138 neoplastic and 131 non-neoplastic polyps ≤ 5 mm, the model achieved sensitivity of 0.91, specificity of 0.88, NPV of 0.89, and HCR of 0.83. CONCLUSIONS: Results show that ESS is a viable endoscopic platform for real-time polyp histology, particularly for polyps ≤ 5 mm. ESS is a simple, low-cost, clinically friendly, optical biopsy modality that, when interfaced with minimally obtrusive endoscopic tools, offers an attractive platform for in situ polyp assessment.
Asunto(s)
Adenocarcinoma/diagnóstico , Pólipos Adenomatosos/diagnóstico , Pólipos del Colon/diagnóstico , Colonoscopía/métodos , Neoplasias Colorrectales/diagnóstico , Diagnóstico por Computador/métodos , Análisis Espectral/métodos , Adenocarcinoma/patología , Pólipos Adenomatosos/patología , Inteligencia Artificial , Pólipos del Colon/patología , Neoplasias Colorrectales/patología , Humanos , Sensibilidad y Especificidad , Análisis Espectral/instrumentaciónRESUMEN
BACKGROUND: In 10% to 15% of individuals, inflammatory bowel disease (IBD) is difficult to classify as ulcerative colitis (UC) or Crohn's disease (CD). Previous work has demonstrated that probe-based elastic scattering spectroscopy (ESS) can produce spectra, informed by parameters like tissue ultrastructure and hemoglobin content, capable of differentiating pathologies. This study investigates whether ESS is an in vivo optical biomarker for the presence, activity, and type of IBD in the colon. METHODS: Pilot study, a retrospective data analysis. ESS spectra of endoscopically normal and inflamed colon were obtained from 48 patients with IBD and 46 non-IBD controls. Measurements from patients with IBD were categorized as CD or UC based on clinical diagnosis. Spectra were analyzed using high-dimensional methods. Leave-one-patient-out cross-validation was used to obtain diagnostic performance estimates. RESULTS: Patients with IBD were distinguishable from non-IBD controls with a sensitivity of 0.93 and specificity of 0.91 based on readings from endoscopically normal mucosa, and 0.94 and 0.93 from inflamed mucosa. In patients with IBD, histologically normal and inflamed colon were distinguishable with per-class accuracies of 0.83 and 0.89, respectively; histologically normal from inactive inflammation with accuracies of 0.73 and 0.89, respectively; and inactive from active colitis with accuracies of 0.87 and 0.84, respectively. The diagnosis of CD versus UC was made with per-class accuracies of 0.92 and 0.87 in normal and 0.87 and 0.85 in inflamed mucosa, respectively. CONCLUSIONS: ESS, a simple, low-cost clinically friendly optical biopsy modality, has the potential to enhance the endoscopic assessment of IBD and its activity in real time and may help to distinguish CD from UC.
Asunto(s)
Colitis Ulcerosa/diagnóstico , Enfermedad de Crohn/diagnóstico , Imagen Óptica/métodos , Adulto , Anciano , Colitis Ulcerosa/patología , Colon/patología , Enfermedad de Crohn/patología , Diagnóstico Diferencial , Femenino , Humanos , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Imagen Óptica/normas , Proyectos Piloto , Reproducibilidad de los Resultados , Estudios Retrospectivos , Dispersión de Radiación , Sensibilidad y Especificidad , Índice de Severidad de la Enfermedad , Análisis EspectralRESUMEN
Glucose-dependent insulinotropic polypeptide (GIP) is a mammalian incretin hormone released into the circulation following nutrient ingestion. We examined the functional evolution of GIP and its relationship with insulin to delineate their respective roles in promoting nutrient efficiency. Expression patterns were examined in the sea lamprey (Petromyzon marinus), a basal vertebrate lacking a distinct pancreas, and in the zebrafish, Xenopus laevis, chicken, and mouse, organisms possessing extraintestinal pancreata. Although sea lamprey genomic analysis predicted a potential GIP-like gene, transcripts were not detected, and insulin expression was confined to the caudal pancreatic bud. GIP was detected in both the intestine and pancreas of the zebrafish and X. laevis. In contrast, GIP and insulin expression were limited to the intestine and pancreas, respectively, in chicken and mouse. Phylogenetic analysis of the glucagon-like ligands suggested proglucagon as the common ancestor, supporting the theory that GIP arose as a gene duplication of proglucagon. Insulin-secreting cells in the sea lamprey intestine may have obviated the need for an enteroinsular axis, and zebrafish may represent an evolutionary transition where GIP does not yet function as an incretin hormone. These observations are consistent with the hypothesis that GIP and insulin influence survival advantage by enhancing the efficiency of nutrient absorption and energy storage.
Asunto(s)
Evolución Molecular , Polipéptido Inhibidor Gástrico/genética , Insulina/genética , Petromyzon/genética , Proglucagón/genética , Pez Cebra/genética , Animales , Pollos/genética , Polipéptido Inhibidor Gástrico/metabolismo , Expresión Génica , Insulina/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Páncreas/metabolismo , Petromyzon/metabolismo , Filogenia , Xenopus laevis/genética , Xenopus laevis/metabolismo , Pez Cebra/metabolismoRESUMEN
Glucose-dependent insulinotropic polypeptide (GIP), an important component of the enteroinsular axis, is a potent stimulator of insulin secretion, functioning to maintain nutrient efficiency. Although well-characterized in mammals, little is known regarding GIP transcriptional regulation in Danio rerio (Dr). We previously demonstrated that DrGIP is expressed in the intestine and the pancreas, and we therefore cloned the Dr promoter to compare GIP transcriptional regulation in Dr and mammals. Although no significant homology was indentified between the highly conserved mammalian promoter and the DrGIP promoter, 1072-bp of the DrGIP promoter conferred tissue-specific expression in mammalian cell lines. Deletional analysis of the DrGIP promoter identified two regions that, when deleted, reduced transcription by 75% and 95%, respectively. Mutational analysis of the upstream region suggested involvement of an Nkx binding site, although we were unable to identify the factor binding to this site. The cis element in the downstream region was found to be a GATA binding site. Lastly, overexpression and shRNA experiments identified PAX4 as a potential repressor of DrGIP expression. These findings provide evidence that despite the identification of species-specific transcriptional regulators and differences in GIP expression patterns between D. rerio and mammals, a moderate degree of regulatory conservation appears to exist.
Asunto(s)
Evolución Molecular , Polipéptido Inhibidor Gástrico/genética , Animales , Línea Celular , Línea Celular Tumoral , Ratones , Células 3T3 NIH , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/genética , Ratas , Pez CebraRESUMEN
Although numerous epidemiological studies have provided convincing evidence for an increase in the prevalence of colorectal cancer (CRC) in obese individuals, the precise mechanisms involved have not been elucidated. Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal regulatory peptide whose primary physiologic role is to stimulate postprandial pancreatic insulin secretion. Like insulin, GIP has been linked to enhanced nutrient efficiency, which occurred during the course of evolution. Its expression is increased in obesity, and we thus initiated studies to examine whether GIP might contribute to the pathogenesis of obesity-related CRC. RT-PCR and Western analysis demonstrated the presence of the GIP receptor (GIPR) in several human CRC cell lines. GIP stimulated the proliferation of MC-26 cells, a mouse CRC cell line, in a concentration-dependent manner. Western analysis showed that GIP induced the activity of several downstream signaling molecules known to be involved in cellular proliferation in a concentration- and time-dependent manner. These studies indicate that the presence of GIP receptors in CRC may enable ligand binding and, in so doing, stimulate CRC cell proliferation. The overexpression of GIP, which occurs in obesity, might thereby be contributing to the enhanced rate of carcinogenesis observed in obesity.
Asunto(s)
Neoplasias Colorrectales/patología , Polipéptido Inhibidor Gástrico/farmacología , Animales , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Polipéptido Inhibidor Gástrico/genética , Humanos , Ligandos , Ratones , Obesidad/genética , Obesidad/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , PorcinosRESUMEN
In mammals, glucose-dependent insulinotropic polypeptide (GIP) is synthesized predominately in the small intestine and functions in conjunction with insulin to promote nutrient deposition. However, little is known regarding GIP expression and function in early vertebrates like the zebrafish, a model organism representing an early stage in the evolutionary development of the compound vertebrate pancreas. Analysis of GIP and insulin (insa) expression in zebrafish larvae by RT-PCR demonstrated that although insa was detected as early as 24 h postfertilization (hpf), GIP expression was not demonstrated until 72 hpf, shortly after the completion of endocrine pancreatic development but prior to the commencement of independent feeding. Furthermore, whole mount in situ hybridization of zebrafish larvae showed expression of GIP and insa in the same tissues, and in adult zebrafish, RT-PCR and immunohistochemistry demonstrated GIP expression in both the intestine and the pancreas. Receptor activation studies showed that zebrafish GIP was capable of activating the rat GIP receptor. Although previous studies have identified four receptors with glucagon receptor-like sequences in the zebrafish, one of which possesses the capacity to bind GIP, a functional analysis of these receptors has not been performed. This study demonstrates interactions between the latter receptor and zebrafish GIP, identifying it as a potential in vivo target for the ligand. Finally, food deprivation studies in larvae demonstrated an increase in GIP and proglucagon II mRNA levels in response to fasting. In conclusion, the results of these studies suggest that although the zebrafish appears to be a model of an early stage of evolutionary development of GIP expression, the peptide may not possess incretin properties in this species.
Asunto(s)
Polipéptido Inhibidor Gástrico/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Factores de Edad , Envejecimiento/genética , Envejecimiento/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Privación de Alimentos , Polipéptido Inhibidor Gástrico/genética , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Insulina/genética , Insulina/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/embriología , Larva/genética , Larva/metabolismo , Ligandos , Masculino , Datos de Secuencia Molecular , Páncreas/embriología , Páncreas/metabolismo , Proglucagón/genética , ARN Mensajero/metabolismo , Ratas , Receptores de la Hormona Gastrointestinal/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
A thorough examination of glucose-dependent insulinotropic polypeptide (GIP) expression has been hampered by difficulty in isolating widely dispersed, GIP-producing enteroendocrine K-cells. To elucidate the molecular mechanisms governing the regulation of GIP expression, 14 intestinal and pancreatic cell lines were assessed for their suitability for studies examining GIP expression. Both STC-1 cells and the pancreatic cell line betaTC-3 were found to express GIP mRNA and secrete biologically active GIP. However, levels of GIP mRNA and bioactive peptide and the activity of transfected GIP reporter constructs were significantly lower in betaTC-3 than STC-1 cells. When betaTC-3 cells were analyzed for transcription factors known to be important for GIP expression, PDX-1 and ISL-1, but not GATA-4, were detected. Double staining for GIP-1 and GATA-4 in mouse duodenum demonstrated GATA-4 expression in intestinal K-cells. Exogenous expression of GATA-4 in betaTC-3 cells led to marked increases in both GIP transcription and secretion. Lastly suppression of GATA-4 via RNA interference, in GTC-1 cells, a subpopulation of STC-1 cells with high endogenous GIP expression resulted in a marked an attenuation of GIP promoter activity. Our data support the hypothesis that GATA-4 may function to augment or enhance GIP expression rather than act as an initiator of GIP transcription.
Asunto(s)
Linaje de la Célula , Factor de Transcripción GATA4/metabolismo , Polipéptido Inhibidor Gástrico/genética , Intestinos/citología , Páncreas/citología , Regulación hacia Arriba/genética , Animales , Bioensayo , Línea Celular , Factor de Transcripción GATA4/antagonistas & inhibidores , Polipéptido Inhibidor Gástrico/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Mucosa Intestinal/metabolismo , Proteínas con Homeodominio LIM , Luciferasas/metabolismo , Ratones , Páncreas/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Ratas , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The physiological effects of glucose-dependent insulinotropic polypeptide (GIP) are mediated through specific receptors expressed on target cells. Because aberrant GIP receptor (GIPR) expression has been implicated in abnormal GIP responses associated with type 2 diabetes mellitus and food-induced Cushing's syndrome, we sought to identify factors that regulate the GIPR. We previously demonstrated that sequences between -1 and -100 of the GIPR gene were sufficient to direct transcription in a rat insulinoma cell line (RIN38). In the present study, we compared the 5'-flanking regions of the rat and human GIPR gene and demonstrated 88% identity within the first 92 bp. Subsequent serial deletion analyses showed that the region between -85 and -40 is essential for maximal promoter activity. Within this region, we identified three putative Sp1 binding motifs, located at positions -77, -60, and -50, that can specifically bind both Sp1 and Sp3. Whereas mutation of the Sp1 sites at -50 and -60 led to 36 and 40% reduction in promoter activity, respectively, mutation of the Sp1 motif at -70 did not affect promoter activity. Cotransfection of S2 Schneider cells with GIPR-luciferase chimeric constructs and either Sp1 or Sp3 expression vectors indicated that both Sp1 and the long form of Sp3 activate transcription through binding to the Sp1 sites located between -100 and -40. Lastly, chromatin immunoprecipitation analyses revealed that both Sp1 and Sp3 bind to the GIPR promoter region in RIN38 cells. These results indicate that cell-specific expression of GIPR is associated with the binding of the transcription factors Sp1 and Sp3 to the GIPR promoter.
Asunto(s)
Polipéptido Inhibidor Gástrico/genética , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/genética , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Polipéptido Inhibidor Gástrico/metabolismo , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Unión Proteica , Ratas , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp3/metabolismoRESUMEN
Glucose-dependent insulinotropic polypeptide (GIP) is a potent stimulator of insulin secretion and comprises an important component of the enteroinsular axis. GIP is synthesized in enteroendocrine K-cells located principally in the upper small intestine. The homeobox-containing gene PDX-1 is also expressed in the small intestine and plays a critical role in pancreatic development and in the expression of pancreatic-specific genes. Previous studies determined that the transcription factors GATA-4 and ISL-1 are important for GIP expression. In this study, we demonstrate that PDX-1 is also involved in regulating GIP expression in K-cells. Using immunohistochemistry, we verified the expression of PDX-1 protein in the nucleus of GIP-expressing mouse K-cells and evaluated the expression of PDX-1, serotonin, and GIP in wild-type and PDX-1(-/-) mice at 18.5 d after conception. Although we demonstrated a 97.8% reduction in the number of GIP-expressing cells in PDX-1(-/-) mice; there was no statistical difference in the number of serotonin-positive cells. Additionally, PDX-1 transcripts and protein were detected in a GIP-expressing neuroendocrine cell line, STC-1. Electromobility shift assays using STC-1 nuclear extracts demonstrated the specific binding of PDX-1 protein to a specific regulatory region in the GIP promoter. Using chromatin immunoprecipitation analysis, we demonstrated binding of PDX-1 to this same region of the GIP promoter in intact cells. Lastly, overexpression of PDX-1 in transient transfection assays led to a specific increase in the activity of GIP/Luc reporter constructs. The results of these studies indicate that the transcription factor PDX-1 plays a critical role in the cell-specific expression of the GIP gene.
Asunto(s)
Polipéptido Inhibidor Gástrico/metabolismo , Proteínas de Homeodominio/fisiología , Transactivadores/fisiología , Animales , Línea Celular , Polipéptido Inhibidor Gástrico/genética , Inmunohistoquímica , Intestino Delgado/citología , Intestino Delgado/metabolismo , Ratones , Ratones Noqueados , Sistemas Neurosecretores/citología , Sistemas Neurosecretores/metabolismo , Regiones Promotoras Genéticas/fisiología , Estructura Terciaria de Proteína/fisiología , Ratas , Transactivadores/deficienciaRESUMEN
The prevalence of esophageal adenocarcinoma in the setting of Barrett's metaplasia continues to increase in Western nations at a rate greater than any other cancer. The trophic properties of gastrin have been documented in gastric, pancreatic and colon cancer cell lines, suggesting a potential role for this regulatory peptide in the growth of these malignancies. The aims of these studies were to identify and characterize the presence of functional cholecystokinin type-2 (gastrin) receptors on the membranes of human esophageal adenocarcinoma cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated the presence of cholecystokinin type-2 receptor transcripts in human esophageal adenocarcinoma cell lines. Competitive binding assays revealed specific binding of gastrin in SEG-1 cells (IC50 of 2.4 x 10(-8) M). This finding was confirmed by laser scanning confocal microscopy through internalization of rhodamine green labeled gastrin heptapeptide in SEG-1 cells. Gastrin caused a dose-dependent increase in proliferation of SEG-1 cells when compared to controls. This effect was abolished by co-incubation with L365,260, a CCK-2-specific receptor antagonist. Gastrin-induced phosphorylation of the p44 and p42 mitogen-activated protein kinases was demonstrated by Western blot analysis. In conclusion, the studied human esophageal adenocarcinoma cell lines possess cholecystokinin type-2 (gastrin) receptors. Receptors bind gastrin, resulting in increased proliferation in SEG-1 cells.
Asunto(s)
Adenocarcinoma/patología , Proliferación Celular/efectos de los fármacos , Neoplasias Esofágicas/patología , Gastrinas/farmacología , Receptor de Colecistoquinina B/genética , Adenocarcinoma/metabolismo , Benzodiazepinonas/farmacología , Unión Competitiva , Western Blotting , Neoplasias Esofágicas/metabolismo , Humanos , Fragmentos de Péptidos/farmacología , Compuestos de Fenilurea/farmacología , Fosforilación/efectos de los fármacos , Receptor de Colecistoquinina B/antagonistas & inhibidores , Receptor de Colecistoquinina B/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
BACKGROUND/AIMS: Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that, in the presence of glucose, stimulates insulin secretion from beta-cells. GIP is expressed in gastrointestinal K-cells. Prior analysis of the GIP promoter demonstrated that 193 bases of the promoter are required to direct cell specific expression. Here we sought to identify and characterize the transcription factors involved. RESULTS: By mutational analysis of the GIP promoter in a neuroendocrine cell line (STC-1), we identified two regions located between bases -193 and -182 and bases -156 and -151 that, when independently altered, were responsible for a 90% and 85% reduction in transcription, respectively. When we compared these two regions with known motifs from transcription factor databases, we identified the cis elements as potential GATA and ISL-1 binding sites. With subsequent electrophoretic mobility shift analysis (EMSA) using STC-1 nuclear extracts, we demonstrated the ability of these regions to form specific DNA protein complexes. Furthermore, we utilized antisera to confirm the specific binding of GATA-4 to the upstream site and ISL-1 to the downstream element. CONCLUSION: These findings provide evidence for the involvement of the transcription factors GATA-4 and ISL-1 in the cell-specific expression of the GIP gene.