RESUMEN
Previously our lab has created a mouse ovarian xenograft model of copy number variation (CNV)-mediated G protein-coupled receptor (GPCR) MAS-driven tumorigenesis, and RNA profiling identified a putative chemokine tumor-induced factor (Tif). Sequence analysis and chemotactic study suggested that Tif was likely to be a hamster homolog of human GROγ (CXCL3) [IJC 125 (2009): 1316-1327]. In the present study, we report the molecular and functional characterization of the Tif gene. Genomic study of CHO-K1 cells indicated that Tif gene consisted of 4 exons, characterized with an antisense B1 element which is embedded in the fourth exon. Two Tif transcripts were identified which shared identical sequences except that a string of 71-nt derived from the antisense B1 element was deficient in the shorter transcript. Of interests, B1-like RNA ladder was detected in xenografts. Functional studies showed that TIF induced chemotaxis and neovessel formation. Pharmacological studies suggested that TIF activated Gi-coupled CXCR2 and induced both calcium mobilization and ERK1/2 phosphorylation, and suppressed forskolin-stimulated cAMP accumulation. In addition, secreted matured TIF functioned as an autocrine factor and promoted anchorage-independent growth. Unexpectedly, TIF delayed the onset of tumor formation, possibly via suppressing proliferation of stromal fibroblasts. However, TIF did not exert any inhibitory effect on tumor growth. Potentially, TIF could be used for preventing cancer relapse.
Asunto(s)
Quimiocinas CXC/genética , Quimiocinas/genética , Animales , Células CHO , Señalización del Calcio/efectos de los fármacos , Quimiocinas/metabolismo , Quimiocinas/farmacología , Quimiocinas CXC/metabolismo , Quimiotaxis , Cricetulus , Humanos , Ratones , Ratones Desnudos , Neovascularización Fisiológica/efectos de los fármacos , Fosforilación , Ratas , Receptores de Interleucina-8B/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Linfoma/metabolismo , Linfoma/terapia , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacocinética , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/aislamiento & purificación , Especificidad de Anticuerpos , Línea Celular Tumoral , Femenino , Linfoma/inmunología , Linfoma/patología , Ratones , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/uso terapéutico , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/aislamiento & purificaciónRESUMEN
Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2) receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.
Asunto(s)
Epítopos/metabolismo , Receptor de Angiotensina Tipo 2/genética , Receptor de Angiotensina Tipo 2/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Expresión Génica , Células HEK293 , Humanos , Peso Molecular , Células PC12 , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Ratas , Receptor de Angiotensina Tipo 2/química , Transfección , UbiquitinaciónRESUMEN
Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig) variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR) step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3). The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx). Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N) protein of SARS-associated coronavirus (SCoV) were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.
Asunto(s)
Región Variable de Inmunoglobulina/genética , Anticuerpos de Cadena Única/genética , Animales , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Biblioteca de Péptidos , Reacción en Cadena de la PolimerasaRESUMEN
DHFR-deficient Chinese hamster ovary (CHO DHFR(-)) cells are the most popular mammalian expression system for inducible amplification of transgene. In order to obtain more stable transfected CHO DHFR(-) cell clones, transfection efficiency of electroporation under different conditions were systemically investigated using plasmid pSV-beta-Gal as reporter gene. Transfection efficiency was proportionally increased with pulse duration and number of pulse applied. In addition, higher transfection efficiency was found in high salt extracellular solution (Berg's and Hank's buffers) than in intracellular solution (cytomix buffer) under the same electroporation condition. The highest transfection efficiency in examined conditions was about 1 in 350 cells (or 0.289%) when cells were electroporated with twice pulses at 400V, 375microF. The present study offers an optimized guideline for introducing exogenous DNA into CHO DHFR(-) cells by electroporation.
Asunto(s)
Células CHO , Electroporación , Expresión Génica , Tetrahidrofolato Deshidrogenasa/genética , Transfección/métodos , Animales , Tampones (Química) , Cricetinae , Cricetulus , Eficiencia , Electroporación/métodos , Genes , Genes Reporteros , Vectores Genéticos , Tetrahidrofolato Deshidrogenasa/deficienciaRESUMEN
Overexpressions of G protein-coupled receptor (GPCR) with elevated downstream signaling events have been reported in various tumors. However, the cellular mechanism that GPCR overexpression leads to tumor formation is largely unknown. The orphan GPCR mas was originally isolated from a human epidermoid carcinoma. In vivo studies of mas-overexpressing cells suggested that xenograft tumor formation was positively correlated with the levels of mas expression. Histochemical analysis indicated that xenograft tumor consisted of mas-transfected and stromal cells. Biochemical analyses revealed that cells overexpressing mas exhibited significantly increased anchorage-independent growth, whereas there was no significant difference in cell proliferation in comparison with empty vector-transfected control cells. Expression profiling using mRNA differential display and Northern analysis indicated an elevated expression of GRO and a novel CXC chemokines, tumor-induced factor (TIF), in mas-transfected cells and xenograft tumor. Bacterially expressed recombinant TIF was found to act as a neutrophil chemoattractant in a chemotactic assay. These results suggest that mas overexpression enables anchorage-independent growth of transformed cells, and interplays of secreted chemokines with stromal cells modulate xenograft tumor formation. Importantly, a novel CXC chemokine, TIF, was identified in the xenograft tumor tissues.
Asunto(s)
Quimiocinas CXC/metabolismo , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Proteínas Proto-Oncogénicas/genética , Receptores Acoplados a Proteínas G/genética , Animales , Northern Blotting , Células CHO , Ciclo Celular , Proliferación Celular , Transformación Celular Neoplásica , Quimiocinas CXC/genética , Quimiotaxis , Cricetinae , Cricetulus , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Neoplasias Experimentales/patología , Proto-Oncogenes Mas , ARN Mensajero/metabolismo , Transfección , Trasplante HeterólogoRESUMEN
Angiotensin and endothelin are vasoactive peptides with neuromodulatory effect, however their interactions in facilitating neurotransmission are largely unknown. In the present study, effort was made to examine how endothelin 1 modulates angiotensin II-potentiated purinergic neurotransmission in prostatic rat vas deferens. Both peptides facilitated field-stimulated muscle contraction in a concentration-dependent manner with Kd values of 16.97+/-6.47 and 2.46+/-0.83 nM for angiotensin II and endothelin 1, respectively. Hill plot analysis gave Hill constants of 0.91+/-0.15 and 0.97+/-0.26 for angiotensin II and endothelin 1, respectively. Correlation analysis indicated that the extent of potentiation by angiotensin II, but not endothelin 1, was proportional to the basal field-stimulated muscle contraction. In the presence of low concentrations of endothelin 1 (< or = 3 nM), angiotensin II-potentiated field-stimulated contraction was further enhanced by endothelin. However, in the presence of high concentrations of endothelin 1 (> or = 10 nM), a much increased basal field-stimulated contraction was observed, and the addition of angiotensin II did not elicit any further enhancement in the contractile response. Intriguingly, after prolonged exposure of prostatic rat vas deferens to a high concentration of endothelin 1, the addition of angiotensin II induced a refractory response to field-stimulation. Taken together, our result indicated that endothelin 1 augmented angiotensin II-facilitated purinergic neurotransmission in prostatic rat vas deferens at low concentrations, but inhibited gradually at high concentrations.
Asunto(s)
Angiotensina II/farmacología , Endotelina-1/farmacología , Próstata/fisiología , Receptores Purinérgicos P2/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Conducto Deferente/efectos de los fármacos , Conducto Deferente/inervación , Angiotensina II/antagonistas & inhibidores , Animales , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Cinética , Masculino , Ratas , Ratas Sprague-Dawley , Conducto Deferente/fisiologíaRESUMEN
In order to explore the role of cytochrome P-450 (CYP) 2E1 in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effects of Sch B treatment on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression were compared between wild-type and cyp2e1 knock-out C57B/6N mice. Cyp2e1 knock-out mice exhibited a significantly smaller degree of Sch B-induced enhancement in hepatic mtGAS when compared with the wild-type counterpart. But Hsp25/70 expression induced by Sch B was not affected. Sch B-induced enhancement of mtGAS was corroborated by the increase in hepatic mitochondrial antioxidant capacity, as assessed by in vitro measurement of oxidant production, with the enhancing effect being slightly reduced in the knock-out mice. Using liver microsomes prepared from wild-type and knock-out mice as a source of CYP, Sch B was found to be a good co-substrate for the CYP-catalyzed reaction, with the rate of NADPH oxidation observable in microsomes prepared from knock-out mice being slower. The CYP-catalyzed reaction with Sch B was associated with a concomitant production of oxidant species, with the extent of oxidant production being reduced in cyp2e1 knock-out mouse microsomes. Taken together, the results indicate that CYP2E1 is partly responsible for the hepatic metabolism of Sch B that may trigger the antioxidant response in vivo.
Asunto(s)
Antioxidantes/farmacología , Citocromo P-450 CYP2E1/metabolismo , Lignanos/farmacología , Mitocondrias Hepáticas/enzimología , Compuestos Policíclicos/farmacología , Animales , Antioxidantes/metabolismo , Catálisis/efectos de los fármacos , Ciclooctanos/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Glutatión/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Chaperonas Moleculares , NADP/metabolismo , Proteínas de Neoplasias/metabolismoRESUMEN
Infection of SARS-associated coronavirus (SARS-CoV) induced a strong anti-nucleocapsid (anti-N) antibody response. However, the pathophysiological significance of the anti-N antibodies in SARS pathogenesis is largely unknown. To profile the anti-N antibodies, a phage-displayed scFv library was prepared from mice immunized with heat-inactivated SARS-CoV-infected Vero E6 cell lysate. Specific anti-N scFvs were isolated by panning against a recombinant nucleocapsid protein and reactivity was confirmed with phage-ELISA. Sequence analysis indicated that two of the isolated anti-N scFv clones were identical and displayed a high homology with an scFv specific for interleukin 11 (IL-11), an anti-inflammatory cytokine derived from bone marrow stroma cells. In a neutralization assay, IL-11-induced STAT 3 phosphorylation in rat intestinal epithelial IEC-18 cells was completely suppressed by the anti-N scFv clone L9N01.
Asunto(s)
Anticuerpos Antivirales/inmunología , Interleucina-11/inmunología , Proteínas de la Nucleocápside/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Reacciones Cruzadas/inmunología , Ratones , Datos de Secuencia Molecular , Proteínas de la Nucleocápside/química , Ratas , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
The peroxisome proliferator-activated receptor alpha (PPARalpha) has been implicated as a key control of fatty acid catabolism during the cellular fasting. However, little is known regarding changes of individual fatty acids in hepatic triacylglycerol (TG) and phospholipid (PL) as a result of starvation. In the present work, the effects of 72 h fasting on hepatic TG and PL fatty acid profiles in PPARalpha-null (KO) mice and their wild-type (WT) counterparts were investigated. Our results indicated that mice deficient in PPARalpha displayed hepatomegaly and hypoketonemia following 72 h starvation. Histochemical analyses revealed that severe fatty infiltration was observed in the livers of KO mice under fasted conditions. Furthermore, 72 h fasting resulted in a 2.8-fold higher accumulation of hepatic TG in KO mice than in WT mice fasted for the same length of time. Surprisingly, the total hepatic PL contents in fasted KO mice decreased by 45%, but no significant change in hepatic PL content was observed in WT mice following starvation. Gas chromatographic analysis indicated that KO mice were deprived of arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids during fasting. Taken together, these results show that PPARalpha plays an important role in regulation of fatty acid metabolism as well as phospholipid homeostasis during energy deprivation.
Asunto(s)
Privación de Alimentos , PPAR alfa/fisiología , Fosfolípidos/metabolismo , Triglicéridos/metabolismo , Ácido 3-Hidroxibutírico/sangre , Animales , Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/metabolismo , Compuestos Azo/farmacología , Northern Blotting , Carbohidratos/química , Colesterol/sangre , Cromatografía de Gases , Colorantes/farmacología , Ácidos Docosahexaenoicos/metabolismo , Ayuno , Ácidos Grasos/metabolismo , Glucógeno/química , Homeostasis , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Naftalenos , PPAR alfa/metabolismo , Ácido Peryódico/farmacología , Fosfolípidos/química , ARN/metabolismo , Factores de Tiempo , Triglicéridos/sangreRESUMEN
Earlier studies indicate that binding sites of type II angiotensin (AT2) receptors are detected all over the pancreas, as well as in the pancreatic exocrine cell line AR4-2J. However, lack of corresponding functional AT2 receptor responses can be detected in the exocrine pancreas. The aim of present study is to determine the protein expression of AT2 receptors in the pancreas by probing with an AT2 receptor-specific antibody, and to examine the role of AT2 receptors in the regulation of pancreatic endocrine hormone release. In Western protein analysis of adult rat tissues, expression of AT2 receptor-immunoreactive bands of 56, 68, and 78 kDa was detected in the adrenal, kidney, liver, salivary glands, and pancreas. In adult rat pancreas, strong immunoreactivity was detected on cells that were located at the outer region of Langerhans islets. Immunohistochemical studies indicated that AT2 receptors colocalized with somatostatin-producing cells in the endocrine pancreas. Consistent with the findings in adult pancreas, abundant expression of AT2 receptors was also detected in immortalized rat pancreatic endocrinal cells lines RIN-m and RIN-14B. To examine the role of AT2 receptors on somatostatin secretion in the pancreas, angiotensin-stimulated somatostatin release from pancreatic RIN-14B cells was studied by an enzyme immunoassay in the absence or presence of various subtype-selective angiotensin analogues. There was a basal release of somatostatin from RIN-14B cells at a rate of 8.72 +/- 4.21 ng/10(6) cells (n = 7). Angiotensin II (1 nM-10 microM) stimulated a biphasic somatostatin release in a dose-dependent manner with an apparent EC50 value of 49.3 +/- 25.9 nM (n = 5), and reached maximal release at 1 microM angiotensin II (982 +/- 147.34% over basal secretion; n = 5). Moreover, the AT2 receptor-selective angiotensin analogue, CGP42112, was 1000 times more potent than the AT1 receptor-selective angiotensin analogue, losartan, in inhibiting angiotensin II-stimulated somatostatin release. These results suggest that angiotensin may modulate pancreatic hormone release via regulation of somatostatin secretion.