RESUMEN
This study was conducted to visualize the lobular distribution of enhanced mRNA expression levels of heat shock proteins (HSPs) in liver samples from carbon tetra chloride (CCl4)-treated mice using in-situ hybridization (ISH). Male BALB/c mice given a single oral administration of CCl4 were euthanized 6 hours or 1 day after the administration (6 h or 1 day). Paraffin-embedded liver samples were obtained, ISH for HSPs was conducted, as well as hematoxylin-eosin staining and immunohistochemistry (IHC). At 6 h, centrilobular hepatocellular vacuolization was observed, and increased signals for Hspa1a, Hspa1b, and Grp78, which are HSPs, were noted in the centrilobular area using ISH. At 1 day, zonal hepatocellular necrosis was observed in the centrilobular area, but mRNA signal increases for HSPs were no longer observed there. Some discrepancies between ISH and IHC for HSPs were observed, and they might be partly caused by post-transcriptional gene regulation, including the ribosome quality control mechanisms. It is known that CCl4 damages centrilobular hepatocytes through metabolization by cytochrome P450, mainly located in the centrilobular region, and HSPs are induced under cellular stress. Therefore, our ISH results visualized increased mRNA expression levels of HSPs in the centrilobular hepatocytes of mice 6 hours after a single administration of CCl4 as a response to cellular stress, and it disappeared 1 day after the treatment when remarkable necrosis was observed there.
RESUMEN
N-glycanase 1 (NGLY1) deficiency, an autosomal recessive disease caused by mutations in the NGLY1 gene, is characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, movement disorders and other neurological phenotypes. Because of few animal models that recapitulate these clinical signatures, the mechanisms of the onset of the disease and its progression are poorly understood, and the development of therapies is hindered. In this study, we generated the systemic Ngly1-deficient rodent model, Ngly1-/- rats, which showed developmental delay, movement disorder, somatosensory impairment and scoliosis. These phenotypes in Ngly1-/- rats are consistent with symptoms in human patients. In accordance with the pivotal role played by NGLY1 in endoplasmic reticulum-associated degradation processes, cleaving N-glycans from misfolded glycoproteins in the cytosol before they can be degraded by the proteasome, loss of Ngly1 led to accumulation of cytoplasmic ubiquitinated proteins, a marker of misfolded proteins in the neurons of the central nervous system of Ngly1-/- rats. Histological analysis identified prominent pathological abnormalities, including necrotic lesions, mineralization, intra- and extracellular eosinophilic bodies, astrogliosis, microgliosis and significant loss of mature neurons in the thalamic lateral and the medial parts of the ventral posterior nucleus and ventral lateral nucleus of Ngly1-/- rats. Axonal degradation in the sciatic nerves was also observed, as in human subjects. Ngly1-/- rats, which mimic the symptoms of human patients, will be a useful animal model for preclinical testing of therapeutic options and understanding the detailed mechanisms of NGLY1 deficiency.
Asunto(s)
Trastornos Congénitos de Glicosilación/genética , Trastornos del Movimiento/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/deficiencia , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Animales , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Trastornos Congénitos de Glicosilación/metabolismo , Trastornos Congénitos de Glicosilación/patología , Modelos Animales de Enfermedad , Degradación Asociada con el Retículo Endoplásmico/genética , Enfermedades Hereditarias del Ojo , Técnicas de Inactivación de Genes , Glicosilación , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Enfermedades del Aparato Lagrimal , Trastornos del Movimiento/patología , Mutación/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Sistema Nervioso Periférico/metabolismo , Sistema Nervioso Periférico/patología , Fenotipo , Complejo de la Endopetidasa Proteasomal/genética , RatasRESUMEN
BACKGROUND AND PURPOSE: Neuromedin U (NmU) may be a novel target for obesity treatment owing to its anorectic and energy expenditure enhancing effects. Although two receptors, NMU1 and NMU2, are both responsible for the NmU-mediated anti-obesity effects, the receptor agonist with the most appropriate profiles for treating obesity and diabetes in terms of efficacy and safety is as yet unknown. Thus, we developed and evaluated novel NMU1/2 receptor-selective agonists. EXPERIMENTAL APPROACH: Efficacy and safety were assessed in mice with diet-induced obesity (DIO) and those with leptin-deficient diabetes (ob/ob) through repeated peripheral administration of selective agonists to NMU1 (NMU-6102) and NMU2 (NMU-2084), along with non-selective NMU1/2 agonists (NMU-0002 and NMU-6014). We also performed immunohistochemistry for c-Fos protein expression in the brain to probe their mechanisms of action. KEY RESULTS: Although both non-selective NMU1/2 agonists and the NMU2-selective agonist had high efficacy compared with the NMU1-selective agonist, only the NMU2-selective agonist led to relatively low adverse effects, such as diarrhoea, in DIO mice. However, the non-selective NMU1/2 agonist and the NMU1-selective agonist, but not the NMU2-selective agonist, were effective in diabetic ob/ob mice. Mechanistically, NMU2-selective agonists preferentially activate pro-opiomelanocortin neurons in the hypothalamic arcuate nucleus but not in the paraventricular nucleus. CONCLUSIONS AND IMPLICATIONS: These results suggest that an NMU2 receptor-selective agonist may be a well-balanced drug for the treatment of obesity and that an NMU1 receptor-selective agonist may also be beneficial for treating obesity and diabetes once its side effects are minimized.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Obesidad/tratamiento farmacológico , Oligopéptidos/efectos adversos , Oligopéptidos/uso terapéutico , Receptores de Neurotransmisores/agonistas , Animales , Núcleo Arqueado del Hipotálamo/fisiología , Encéfalo/metabolismo , Masculino , Ratones , Núcleo Hipotalámico Paraventricular/fisiología , Proteínas Proto-Oncogénicas c-fos/metabolismoRESUMEN
Continuous administration of a 14-amino acid peptide YY (PYY) analog, Ac-[d-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys30]PYY(23-36) (4), which has a high binding affinity and agonist activity for the neuropeptide Y2 receptor (Y2R), has previously shown an antiobesity effect in a 2-week diet-induced obesity (DIO) study in mice. However, there remained a possibility to obtain more potent analogs by further improving its pharmacokinetic profile. A combination of the N-terminal 4-imidazolecarbonyl moiety and three amino acid substitutions, trans-4-hydroxy-d-proline (d-Hyp)24, isovaline (Iva)25, and γ-methylleucine (γMeLeu)28, not only improved the binding affinity of the peptide for Y2R but also increased its anorectic activity in lean mice. In a 2-week DIO study in mice, continuous administration of 4-imidazolecarbonyl-[d-Hyp24,Iva25,Pya(4)26,Cha27,36,γMeLeu28,Lys30,Aib31]PYY(23-36) (31, PYY-1119) at a dose of 0.03mg/kg/day showed a highly potent antiobesity effect, with more than 10% body weight reduction.
Asunto(s)
Peso Corporal/efectos de los fármacos , Péptido YY/química , Péptido YY/farmacología , Secuencia de Aminoácidos , Animales , Fármacos Antiobesidad/química , Fármacos Antiobesidad/farmacología , Dieta , Concentración 50 Inhibidora , Masculino , Ratones , Ratones Noqueados , Estructura Molecular , Péptido YY/agonistasRESUMEN
The gastrointestinal peptide, peptide YY3-36 (PYY3-36) and its shorter peptide analogues have been reported to reduce appetite by activating the neuropeptide Y2 receptor (Y2R), which is associated with obesity and other metabolic diseases. A 14-amino acid PYY analogue, Ac-[d-Pro24,Cha27,28,36,Aib31]PYY(23-36) (3), showed high binding affinity and agonist activity for the Y2R, similar to that of PYY3-36, but had weak anorectic activity upon continuous administration in lean mice. Three amino acid substitutions [Pya(4)26, Aib28, Lys30], which contributed to the decreased hydrophobicity of 3, efficiently increased its anorectic activity. The compound containing these three amino acids, Ac-[d-Pro24,Pya(4)26,Cha27,36,Aib28,31,Lys30]PYY(23-36) (22), exerted more potent and durable food intake suppression than that by PYY3-36 in lean mice, as well as excellent Y2R agonist activity (EC50: 0.20nM) and good subcutaneous bioavailability (66.6%). The 11-day continuous administration of 22 at 1mg/kg/day successfully produced antiobese and antidiabetic effects, with more than 20% body weight loss in obese and Type 2 diabetes ob/ob model mice.
Asunto(s)
Fármacos Antiobesidad/farmacología , Ingestión de Alimentos/efectos de los fármacos , Obesidad/tratamiento farmacológico , Péptido YY/farmacología , Animales , Fármacos Antiobesidad/administración & dosificación , Fármacos Antiobesidad/química , Relación Dosis-Respuesta a Droga , Inyecciones Intravenosas , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Estructura Molecular , Péptido YY/administración & dosificación , Péptido YY/química , Receptores de Neuropéptido Y/agonistas , Relación Estructura-ActividadRESUMEN
Gastrointestinal peptides such as peptide YY (PYY) can regulate appetite, which is relevant to the study of obesity. The intraperitoneal bolus administration of PYY3-36 and a 12-amino acid PYY analogue, benzoyl-[Cha27,28,36,Aib31]PYY25-36 (1), showed similar anorectic activity by activating the Y2 receptor (Y2R). However, food intake inhibition and body weight loss were not observed upon continuous subcutaneous administration of 1 with osmotic pumps in diet-induced obese (DIO) mice. N-Terminal elongation of 1, together with amino acid substitution at position 24, led to a hydrophilic 14-amino acid peptide, Ac-[d-Hyp24,Cha27,28,36,Aib31]PYY23-36 (18), that showed higher affinity and more potent agonist activity for Y2R and a robust anorectic activity with potency similar to that of PYY3-36. In addition, the continuous subcutaneous administration of 18 at 0.3 mg/(kg·day) induced significant body weight loss in DIO mice. These results suggest that a short-length PYY analogue can be a lead compound for antiobesity therapy in a sustained-release formulation.
RESUMEN
Neuromedin U (NMU) is a neuropeptide known to regulate food intake and energy homeostasis that is widely distributed in the gastrointestinal tract, hypothalamus, and pituitary. A short form of NMU, porcine NMU-8 has potent agonist activity for the receptors NMUR1 and NMUR2; however, its short half-life precludes its effective use in vivo. To address this limitation, we designed and synthesized NMU-8 analogs modified by polyethylene glycol (PEG) with a molecular weight of 30kDa (PEG30k) via a variety of linkers (i.e., ω-amino- and ω-imino-carboxylic acid linker). Integrated evaluation of NMUR1 and NMUR2 binding affinities in vitro and anorectic activity in mice revealed that the introduction of a linker with a rigid ring group, e.g., 2-(piperazin-1-yl)acetic acid (PipAc), yielded a highly potent anorectic peptide, PEG30k-PipAc-NMU-8 (14), possessing improved receptor binding affinity. Subsequent optimization of the molecular weight of the PEG moiety led to the discovery of a PEG20k conjugate (15), which exhibited significant anti-obesity effect upon once-daily subcutaneous administration in diet-induced obese mice with 10% and 22% body weight loss at doses of 10 and 30nmol/kg, respectively. In addition, 15 reduced the weights of the liver and adipose tissue in a dose-dependent manner and improved the plasma biochemical parameters, e.g., insulin, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and total cholesterol. Thus, our results suggest that 15 (NMU-0002), which showed potent and long-lasting biological profiles in vivo, represents a candidate peptide for investigating the central and peripheral actions of NMU and its potential for clinical use.
Asunto(s)
Fármacos Antiobesidad/farmacología , Neuropéptidos/farmacología , Polietilenglicoles/química , Animales , Fármacos Antiobesidad/farmacocinética , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos C57BL , Neuropéptidos/química , Neuropéptidos/farmacocinética , Porcinos , Pérdida de Peso/efectos de los fármacosRESUMEN
Peripheral administration of PYY3-36, a fragment of peptide YY (PYY), has been reported to reduce food intake by activating the neuropeptide Y2 receptor (Y2R). An N-terminally truncated PYY analogue, benzoyl-[Ala26,Ile28,31]PYY(25-36) (1), showed a relatively potent agonist activity for Y2R but a weak anorectic activity by intraperitoneal administration (2000 nmol/kg) in lean mice because of its markedly poor biological stability in the mouse serum. Notably, two cyclohexylalanine (Cha) substitutions for Tyr residues at positions 27 and 36 (4) improved the stability in the mouse serum concomitant with enhanced anorectic activity. Further optimization at positions 27, 28, 30, and 31 revealed that 21, containing Cha28 and Aib31 residues, showed a more potent anorectic activity than PYY3-36 at a low dose of 300 nmol/kg. The minimum effective dose by intraperitoneal administration of 21 was 30 nmol/kg (ca. 52 µg/kg) in mice, suggesting the biologic potential of short-length PYY3-36 analogues with a potent anorectic effect.
RESUMEN
Metastin/kisspeptin, a hypothalamic peptide, plays a pivotal role in controlling GnRH neurons. Here we studied the effect of chronic sc administration of two kisspeptin analogs, KISS1-305 and TAK-448, on hypothalamic-pituitary-gonadal function in male rats in comparison with a GnRH analogue leuprolide or bilateral orchiectomy (ORX). The prototype polypeptide, KISS1-305 (1-4 nmol/h), caused substantial elevations of plasma LH and testosterone, followed by abrupt reductions of both hormone levels. Notably, testosterone levels were reduced to castrate levels within 3 d and remained depleted throughout the 4-wk dosing period, an effect that was faster and more pronounced than leuprolide (1 nmol/h) dosing. KISS1-305 also reduced genital organ weight more profoundly than leuprolide. In mechanistic studies, chronic KISS1-305 administration only transiently induced c-Fos expression in GnRH neurons, suggesting that GnRH-neural response was attenuated over time. Hypothalamic GnRH content was reduced to 10-20% of control at 3 wk without any changes in Gnrh mRNA expression. Dosing with the investigational peptide TAK-448 was also studied to extend our understanding of hypothalamic-pituitary functions. Similar to ORX, TAK-448 (0.1 nmol/h) depleted testosterone and decreased GnRH content by 4 wk. However, in contrast to ORX, TAK-448 decreased gonadotropin levels in pituitary and plasma samples, implying the suppression of GnRH pulses. These results suggest that chronic administration of kisspeptin analogs disrupts endogenous kisspeptin signals to suppress intrinsic GnRH pulses, perhaps by attenuating GnRH-neural response and inducing continuous GnRH leakage from the hypothalamus. The potential utility of kisspeptin analogs as novel agents to treat hormone-related diseases, including prostate cancer, is discussed.
Asunto(s)
Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Kisspeptinas/farmacología , Neuronas/efectos de los fármacos , Testículo/efectos de los fármacos , Testosterona/sangre , Animales , Hormona Liberadora de Gonadotropina/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Leuprolida/farmacología , Hormona Luteinizante/sangre , Masculino , Neuronas/metabolismo , Orquiectomía , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Ratas , Ratas Sprague-Dawley , Testículo/metabolismoRESUMEN
Prolactin-releasing peptide (PrRP) was isolated as an endogenous ligand of the orphan G-protein coupled receptor hGR3. PrRP has been shown to be involved in the regulation of food intake, stress responses, prolactin secretion and release, blood pressure, and the opioid system. Here we report that PrRP and its receptor, GPR10, were found in the mouse substantia nigra pars compacta (SNpc), the main location of dopaminergic (DA) neurons of the nigrostriatal system. We generated PrRP knockout (KO) mice, and then treated PrRP KO mice and their wild type (WT) littermates with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neuron toxin that selectively damages DA neurons in the SNpc. We found that PrRP KO mice were resistant to MPTP-induced lesions of the nigrostriatal system. These effects were further confirmed by the intracerebroventricular injection of P2L-1C, a monoclonal antibody against PrRP into mice. Taken together, our data established a critical role of PrRP in MPTP intoxication in mice.
Asunto(s)
Encéfalo/metabolismo , Hormonas Hipotalámicas/metabolismo , Intoxicación por MPTP/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacología , Western Blotting , Encéfalo/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Dopamina/metabolismo , Hormonas Hipotalámicas/antagonistas & inhibidores , Hormonas Hipotalámicas/genética , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Noqueados , Neurotoxinas/farmacología , Hormona Liberadora de Prolactina , Receptores Acoplados a Proteínas G/metabolismo , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismoRESUMEN
Glucocorticoids are known to stimulate growth hormone (GH) production but to suppress prolactin (PRL) production. However, previous data were obtained with rather high doses of corticosterone. In this study we examined the effects of various doses (10 (-12) -10 (-7) M) of corticosterone on GH and PRL production in a rat pituitary somatomammotropic cell line, MtT/SM cells, and found that GH mRNA expression was facilitated by high doses (10 (-7) and 10 (-8) M). In contrast, a biphasic effect of corticosterone on PRL mRNA expression and secretion was observed, i.e., high doses (10 (-7) and 10 (-8) M) suppressed and low doses (10 (-12) -10 (-10) M) facilitated them. In an immunofluorescent staining study, the number of PRL immunopositive cells increased with low doses of corticosterone while it decreased with high doses of it, which corresponded to PRL mRNA expression and hormone secretion, respectively. These effects of corticosterone on PRL production were abolished by a glucocorticoid receptor (GR) antagonist, mifepristone. In addition, co-treatment with low doses of corticosterone (10 (-12) -10 (-10) M) and 17beta-estradiol (E(2), 10 nM) additively increased the number of PRL immunopositive cells. Moreover, a 24 h BrdU incorporation experiment suggested that the increase in the number of PRL immunopositive cells treated with low dose corticosterone was caused by novel synthesis of PRL while, on the other hand, that of those treated with E(2) resulted from PRL cell proliferation. Thus, we concluded that corticosterone biphasically regulates PRL production and the sensitivity of E(2) to different degrees.
Asunto(s)
Glucocorticoides/farmacología , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Prolactina/metabolismo , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Corticosterona/farmacología , Relación Dosis-Respuesta a Droga , Estradiol/farmacología , Hormona del Crecimiento/metabolismo , Antagonistas de Hormonas/farmacología , Mifepristona/farmacología , Hipófisis/citología , ARN Mensajero/metabolismo , RatasRESUMEN
A pituitary tumor is considered to be composed of a heterogeneous population of hormone-producing endocrine cells, folliculo-stellate (FS) cells, and potential hormone-inactive progenitor cells to maintain a microenvironment such as that in angiogenesis for tumor development cooperatively. However, the system that maintains such a heterogeneous cell population has not been clarified yet. In the present study, we examined the mechanism for maintaining a heterogeneous cell population using two rat cell lines, MtT/S and MtT/E cells, which are known growth hormone (GH)-producing cells, and their progenitor cells, respectively. We found that conditioned medium of MtT/S cells could stimulate the growth of MtT/E cells. In addition, GH and insulin-like growth factor I (IGF-I) stimulated the growth of MtT/E cells. The messenger RNAs (mRNAs) of receptors for IGF-I and GH were expressed in the MtT/E cells. Moreover, IGF-I receptor inhibitor AG1024 could abolish the growth stimulatory activity in the conditioned medium of MtT/S cells. Therefore, we concluded that somatotropes (MtT/S) maintain their progenitor cells (MtT/E) through the GH-IGF-I signaling and IGF-I directly, which might be involved in the maintenance of progenitors of GH-producing cells and might contribute to pituitary tumor development.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/fisiología , Neoplasias Hipofisarias/patología , Somatotrofos/patología , Células Madre/patología , Animales , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Hormona de Crecimiento Humana/farmacología , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , ARN Mensajero/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Somatotrofos/metabolismo , Células Madre/metabolismo , Células Tumorales CultivadasRESUMEN
Folliculo-stellate (FS) cells are known to act as sustentacular cells or scavenger cells in the anterior lobe. However, the precise function and origin of FS cells are still under discussion. Like brain astrocytes, FS cells contain S-100beta protein, and FS cells can be detected immunocytochemically using antibodies for S-100beta protein after fixation; however, living FS cells can not be detected. The generation of transgenic rats expressing green fluorescent protein (GFP) under the control of S-100beta protein gene promoter may allow the detection of living FS cells, which may be an excellent tool for the study of FS cells. With the aim of generation of transgenic rats, we analyzed the promoter activity of the S-100beta gene and found that intron 1 is important for cell-specific expression of the S-100beta gene. Therefore, we obtained a DNA construct containing GFP gene under a part of the S-100 promoter with intron 1. We transfected the construct into rat embryos and succeeded in generating transgenic rats. The transgenic rats expressed GFP in FS cells specifically in the anterior lobe. GFP is also expressed in other known S-100beta-expressing cells, i.e. brain astrocytes, adipocytes, and chondrocytes. We believe that the newly generated transgenic rats will provide a new approach for the study of FS cells and other S-100beta protein-producing cells.
Asunto(s)
Animales Modificados Genéticamente , Astrocitos/metabolismo , Encéfalo/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Hipófisis/metabolismo , Proteínas S100/metabolismo , Animales , Encéfalo/citología , Separación Celular , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Factores de Crecimiento Nervioso/genética , Hipófisis/citología , Regiones Promotoras Genéticas , Ratas/genética , Ratas Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/genéticaRESUMEN
We previously reported the ectopic differentiation of skeletal muscle cells in a pituitary gland transplanted beneath a kidney capsule. Morphological observation suggested that the skeletal muscle cells may have differentiated from folliculo-stellate (FS) cells in the anterior pituitary gland. However, at that time, we did not confirm this directly with an in vitro system. To obtain direct evidence, we used the Tpit/F1 cell line. The Tpit/F1 cell line was recently established from the pituitary gland of a temperature-sensitive T antigen transgenic mouse and has the characters of pituitary FS cells. Using Tpit/F1 cells, we have found that FS cells of the pituitary are able to differentiate into muscle cells in vitro. Additionally, we showed that the cells have some characteristics of pituitary FS cells and also express pituitary endocrine cell-specific transcription factor (pit-1) and prolactin genes, and can differentiate into striated muscle cells. The anterior pituitary gland is known to be of ectodermal origin, so the differentiation of its cells into striated muscle is completely unexpected. This is the first report of direct evidence of ectopic differentiation of skeletal muscle cells from pituitary cells.
Asunto(s)
Diferenciación Celular/fisiología , Coristoma/metabolismo , Ectodermo/metabolismo , Músculo Esquelético/embriología , Adenohipófisis/embriología , Células Madre/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Animales , Antimetabolitos Antineoplásicos/farmacología , Azacitidina/farmacología , Biomarcadores , Línea Celular , Línea Celular Tumoral/metabolismo , Línea Celular Tumoral/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ectodermo/ultraestructura , Inducción Embrionaria/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Ratones , Microscopía Electrónica , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestructura , Proteína MioD/genética , Proteína MioD/metabolismo , Miogenina/genética , Miogenina/metabolismo , Mioglobina/genética , Mioglobina/metabolismo , Adenohipófisis/metabolismo , Adenohipófisis/ultraestructura , Prolactina/genética , Prolactina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Células Madre/ultraestructura , Factor de Transcripción Pit-1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
To study pituitary tumor formation, we used a rat pituitary tumor cell line, MtT/E, which was derived from an estrogen-induced rat prolactinoma. MtT/E cells are known not to produce any pituitary hormone; however, they do produce the Pit-1 protein, which is known to be a common transcription factor in thyrotropes, somatotropes, and mammotropes. Although MtT/E is a clonal cell line, it exhibits two distinct phenotypes, fibroblastic (F-) and epithelial (E-) cells. We obtained subclonal cell lines from MtT/E cells with characters similar to those of F- and E-cells and called them MtT/E-G1 and MtT/E-B3, respectively. To examine tumor formation by these cells, we implanted them into female Fischer rats. One month later, typical pituitary tumors had appeared in MtT/E-B3-implanted rats; however, tumor formation by MtT/E-G1 was delayed. Interestingly, the tumors formed by MtT/E-B3 cells were intensely vascularized. To examine changes in tumor cell morphology, we performed primary culture and found that spindle-shaped cells appeared. These spindle-shaped cells were immunopositive for the Pit-1 protein, which suggests that they originated from MtT/E-B3 cells. Interestingly, reverse transcriptase polymerase chain reaction showed that both tumors and the cells obtained in primary culture expressed basic fibroblast growth factor (bFGF). By contrast, the original MtT/E-B3 cells did not express bFGF. These results suggested that MtT/E-B3 cells show a change in phenotype during tumor formation; that is, epithelial-type cells change into bFGFexpressing fibroblastic cells. These phenomena, especially the appearance of bFGFexpressing cells in tumor tissue, may explain the extensive angiogenesis in the tumors formed by MtT/E-B3 cells.