ABSTRACT
This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.
Subject(s)
Drug Evaluation, Preclinical/methods , Genes, Reporter/genetics , Receptors, Corticotropin/metabolism , Animals , CHO Cells , Calcitonin/pharmacology , Colforsin/pharmacology , Cricetinae , Cyclic AMP/metabolism , Dimethyl Sulfoxide/pharmacology , Drug Evaluation, Preclinical/economics , Enzyme Induction/drug effects , Gene Expression Regulation/drug effects , Heterotrimeric GTP-Binding Proteins/metabolism , Humans , Luciferases/genetics , Luciferases/metabolism , Protein Binding , Receptors, Corticotropin/antagonists & inhibitors , Receptors, Corticotropin/genetics , Receptors, Melanocortin , Reproducibility of Results , Response Elements/genetics , Sensitivity and Specificity , Time Factors , Transfection , alpha-MSH/analogs & derivatives , alpha-MSH/pharmacologyABSTRACT
We have developed an assay in which modulation of two or more signaling pathways can be assessed concurrently by combining reporter gene systems with fluorescent probe technology. The validation of this method was achieved by indirect analysis of adenylyl cyclase activation with the use of a cyclic AMP response element (CRE)-luciferase reporter system in combination with the measurement of calcium mobilization by Calcium Green-1 AM fluorescence on a fluorescent imaging plate reader. To demonstrate the utility of the method in studying the pharmacology of receptors that couple to more than one G protein, Chinese hamster ovary (CHO) cells, which stably expressed both the CRE-luciferase reporter gene and the human pituitary adenylyl cyclase-activating peptide (PACAP) receptor, were treated with PACAP 1-27 and 1-38. Calcium mobilization and the induction of adenylyl cyclase activity in response to each concentration of peptide were assessed in individuals wells. This assay may also be used to screen for ligands of two or more unrelated receptors simultaneously without compromising the assessment of either signaling pathway. To illustrate this point, Rat-1 fibroblasts, which expressed human alpha1A receptors, were cocultured with CRE-luciferase CHO cells, which expressed human GLP-1 receptors. Calcium mobilization elicited by phenylephrine agonism of the alpha1A receptor was assessed in the same assay as GLP-1-induced activation of adenylyl cyclase. The pEC(50) for each agonist was similar to that observed when the cell lines were not cocultured. The number of different receptors that can be screened per well is limited only by the ability to distinguish different reporter gene signals and fluorescent indicators.
Subject(s)
Calcium/metabolism , GTP-Binding Proteins/metabolism , Genes, Reporter/physiology , Signal Transduction/physiology , Adenylyl Cyclases/drug effects , Adenylyl Cyclases/metabolism , Animals , CHO Cells , Calcitonin/pharmacology , Colforsin/pharmacology , Cricetinae , Cyclic AMP/metabolism , Drug Evaluation, Preclinical/methods , Enzyme Induction , Fluorescent Dyes/pharmacology , GTP-Binding Proteins/drug effects , Humans , Luciferases/pharmacology , Neuropeptides/pharmacology , Pituitary Adenylate Cyclase-Activating Polypeptide , Rats , Receptors, Drug , Signal Transduction/drug effectsABSTRACT
Tumor establishment and metastasis are dependent on extracellular matrix proteolysis, tumor cell migration, and angiogenesis. Urokinase plasminogen activator (uPA) and its receptor are essential mediators of these processes. The purpose of this study was to investigate the effect of a recombinant human uPAR antagonist on growth, establishment, and metastasis of tumors derived from human cancer cell lines. A noncatalytic recombinant protein, consisting of amino acids 1-137 of human uPA and the CH2 and CH3 regions of mouse IgG1 (uPA-IgG), was expressed, purified, and shown to bind specifically to human uPAR and to saturate the surface of human tumor cells which express uPAR. Daily i.p. administration of uPA-IgG to nude mice extended latencies of unstaged tumors derived from Lox melanoma and SW48 colon carcinoma cells by 7.7 and 5.5 days, respectively. uPA-IgG treatment did not affect the growth of Lox or KB tumors staged to 200 mg before antagonist treatment commenced. The effect of uPA-IgG on the establishment of micrometastases was assessed in SCID mice. KB head/neck tumor cells were injected in the tail vein and allowed to seed for 48 h before initiation of daily i.p. injections of uPA-IgG for 24 days. The number of lung colonies ranged between 5 and 30% of vehicle-treated mice in two separate experiments. Furthermore, a single 800 microg dose of uPA-IgG administered 1 h prior to tail vein injection of KB cells reduced lung colony formation to just 3.5% of vehicle-treated SCID mice. These data demonstrate that antagonism of uPAR arrested metastasis and inhibited the establishment of primary tumors and micrometastases. Thus, small molecule uPAR antagonists may serve as useful adjuvant agents in combination with existing cancer chemotherapy.
Subject(s)
Immunoglobulin G/therapeutic use , Neoplasm Metastasis/prevention & control , Neoplasm Proteins/antagonists & inhibitors , Receptors, Cell Surface/antagonists & inhibitors , Recombinant Fusion Proteins/therapeutic use , Animals , Disease Progression , Humans , Immunoglobulin G/pharmacology , Mice , Mice, Nude , Mice, SCID , Neoplasm Proteins/drug effects , Neoplasm Transplantation , Receptors, Cell Surface/drug effects , Receptors, Urokinase Plasminogen Activator , Recombinant Fusion Proteins/pharmacology , Tumor Cells, Cultured/drug effectsABSTRACT
Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPA-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 microg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 microg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA.
Subject(s)
Baculoviridae/genetics , Immunoglobulin G/genetics , Plasminogen Activators/genetics , Recombinant Fusion Proteins/genetics , Urokinase-Type Plasminogen Activator/genetics , Animals , Cell Line , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Humans , Recombinant Fusion Proteins/isolation & purification , SpodopteraABSTRACT
The present study investigated the effect of chronic administration of a kappa opioid receptor agonist on the function of kappa and mu opioid, serotonergic and cholinergic regulation of secretion from the hypothalamo-pituitary-adrenal axis in neonatal rats. After chronic treatment with saline or U50,488H (trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]- benzeneacetamide methane sulfonate), a kappa opioid receptor agonist and subsequent pharmacological challenge, corticosterone (CS) in serum was determined. Kappa tolerance did not develop in pups treated on postnatal days 5-9 with increasing doses of U50,488H (0.5-2.5 mg/kg). When the rats were treated with the same chronic regimen of U50,488H at different stages of development from birth through weaning, only weanling rats became tolerant to U50,488H. In the absence of measurable kappa tolerance, the responses of corticosterone in serum to morphine, quipazine, a serotonin receptor agonist and physostigmine, an inhibitor of acetylcholinesterase, were attenuated in neonatal rats, treated with U50,488H. These studies suggest that kappa tolerance is more difficult to induce in developing rats than in adults and that regulation of the function of the hypothalamo-pituitary-adrenal axis by other neurotransmitter systems is altered by treatment with kappa opioid receptor agonists, even in the apparent absence of tolerance.
Subject(s)
Analgesics/pharmacology , Hypothalamo-Hypophyseal System/physiology , Pituitary-Adrenal System/physiology , Pyrrolidines/pharmacology , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer , Aging , Animals , Animals, Newborn , Corticosterone/blood , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Tolerance , Hypothalamo-Hypophyseal System/drug effects , Hypothalamo-Hypophyseal System/growth & development , Morphine/pharmacology , Physostigmine/pharmacology , Pituitary-Adrenal System/drug effects , Pituitary-Adrenal System/growth & development , Pyrrolidines/administration & dosage , Quipazine/pharmacology , Rats , Rats, Inbred StrainsABSTRACT
Chronic administration of opiates to rats results in HPA axis tolerance and abstinence-induced hypersecretion. The effects of specific mu and kappa tolerance and withdrawal on the functional secretion of the HPA axis were evaluated in this study. Adult male rats were injected s.c. twice daily with saline, morphine or U50,488 for 5 days. Serum adrenocorticotrophic hormone (ACTH) or corticosterone (CS) were determined by radioimmunoassay as measures of HPA axis function. Tolerance to morphine (10 mg/kg) and U50,488 (1 mg/kg), but no cross-tolerance, was observed suggesting the development of mu- or kappa-specific tolerance, respectively. Tolerance does not occur at the pituitary or adrenal levels after these paradigms because ACTH and CS responses to exogenous corticotropin-releasing factor and ACTH, respectively, were not attenuated. CS secretion in response to novelty stress was not affected by either chronic opiate treatment, but the circadian variation of CS levels was slightly blunted after chronic morphine. In contrast, the elevation of CS secretion by quipazine (0.5 mg/kg) and physostigmine (0.1 mg/kg) was attenuated after chronic U50,488, but not morphine administration. Both spontaneous and antagonist-precipitated withdrawal from morphine, but not U50,488, resulted in elevation of CS levels. Low doses of morphine suppressed morphine abstinence-induced CS hypersecretion, whereas, U50,488 and clonidine had no effect. In conclusion, alterations of HPA axis function occur during chronic mu or kappa opiate administration that are receptor-specific and involve multiple neural controls of the HPA axis.
Subject(s)
Hypothalamo-Hypophyseal System/metabolism , Narcotics/pharmacology , Pituitary-Adrenal System/metabolism , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer , Adrenocorticotropic Hormone/metabolism , Analgesics/pharmacology , Animals , Circadian Rhythm , Corticosterone/metabolism , Dose-Response Relationship, Drug , Drug Tolerance , Hypothalamo-Hypophyseal System/physiology , Male , Morphine/pharmacology , Parasympathomimetics/pharmacology , Physostigmine/pharmacology , Pituitary-Adrenal System/physiology , Pyrrolidines/pharmacology , Rats , Rats, Inbred Strains , Receptors, Opioid/physiology , Receptors, Opioid, kappa , Receptors, Opioid, mu , Serotonin/physiology , Stress, Physiological/physiopathology , Substance Withdrawal SyndromeABSTRACT
In order to study the ontogeny of TSH regulation in the rat, we have compared TSH secretion in neonatal and adult rats after treatment with morphine, TRH and apomorphine, clonidine, and exposure to cold. Apomorphine attenuated exogenous TRH-induced TSH release in both neonatal and adult rats. Morphine suppressed TSH levels at every age tested. Although clonidine and cold exposure elicited TSH secretion in adults, neonatal rats did not respond to either treatment. These findings suggest that opioid and dopaminergic controls of TSH release mature before central noradrenergic regulation in developing rats. This lack of noradrenergic control may account for the absence of the response to cold exposure in the neonate.