RESUMEN
Bipotential cells in human trabecular bone explant cultures that express osteoblast characteristics are able to undergo adipogenesis in the presence of 3-isobutyl-1-methylxanthine plus dexamethasone (Nuttall et al. [1998] J Bone Miner Res 13:371-382). The initial studies of these bipotential cells in explant cultures have been extended to examine differential gene expression during osteoblast/adipocyte transdifferentiation. Using differential display, we have identified a gene expressed in trabecular bone explant cultures that is downregulated as these cells differentiate from an osteoblast to an adipocyte phenotype. Homology searching identified this gene as the human urea transporter HUT11. The expression and downregulation of HUT11 have been observed in multiple patient bone explant cultures. The size of the bone explant-derived HUT11 mRNA is approximately 4.4 kb, which is identical to the largest splice variant reported. In this article, we report the cloning and sequencing of this gene from primary human osteoblasts. In addition, we report tissue distribution for the bone explant-derived form of HUT11 mRNA and show a reciprocal relationship between the expression of HUT11 and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma 2, which is a marker of adipocyte differentiation. Because the control of osteoblast/adipocyte transdifferentiation is unknown, selective downregulation of HUT11 during adipogenesis suggests that HUT11 expression may be a marker of the switch from an osteoblast to an adipocyte phenotype. Understanding the role of HUT11 in osteoblasts may provide insights into the mechanism controlling osteoblast and adipocyte differentiation.
Asunto(s)
Adipocitos/metabolismo , Proteínas Portadoras/genética , Diferenciación Celular/genética , Regulación de la Expresión Génica/genética , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Osteoblastos/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Secuencia de Bases , Biomarcadores , Proteínas Portadoras/metabolismo , Células Cultivadas , Clonación Molecular , Dexametasona/farmacología , Regulación hacia Abajo/genética , Histocitoquímica , Humanos , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Transportadores de UreaRESUMEN
A solution-based microscale approach for determination of high-affinity noncovalent complexes from mixtures of compounds is presented, based on capillary isoelectric focusing coupled on-line with electrospray ionization ion trap mass spectrometry. The studies are performed using the src homology 2 domain and tyrosine-phosphorylated peptide ligands as a model system. Tight complexes are formed in solution, preconcentrated up to 2 orders of magnitude and separated on the basis of their isoelectric points. The complexes are then dissociated in the mass spectrometer and the freed ligands identified. Picomole or less amounts of protein reagent are consumed per experiment. Structural information for the ligands involved in tight complex formation may be obtained using the MSn capabilities of the ion trap. The methodology can potentially be used to screen rapidly combinatorial mixtures of compounds for high-affinity ligands.
Asunto(s)
Péptidos/análisis , Fosfotirosina/análisis , Electroforesis Capilar , Focalización Isoeléctrica , Espectrometría de Masas , Espectrofotometría UltravioletaRESUMEN
Expression of tumor necrosis factor alpha (TNF) by lipopolysaccharide-treated human monocytic cells is inhibited by bicyclic imidazoles. We studied the mechanism of action of a representative inhibitor, SK&F 86002, on synthesis of TNF by THP-1 cells. Levels of TNF protein were lowered by SK&F 86002 under conditions where TNF mRNA accumulation was unaffected, suggesting a post-transcriptional action. No effect of SK&F 86002 was detected on the rate of induction of TNF mRNA or steady state levels over a 5 hr period. The kinetics of SK&F 86002 inhibition of TNF protein synthesis coincided with those of anisomycin, not with actinomycin, suggesting an effect of SK&F 86002 on TNF mRNA translation. By using sucrose gradient sedimentation, we showed that quiescent THP-1 cells contained a substantial amount of TNF mRNA which was primarily associated with 43S pre-ribosomal complexes. Activation of the cells with lipopolysaccharide caused an elevation of the TNF mRNA level and increased the proportion associated with polyribosomes. Treatment with lipopolysaccharide plus SK&F 86002 led to a marked accumulation of TNF mRNA in the 43S complex-containing fractions and a concomitant reduction of polysome-associated TNF message. Neither lipopolysaccharide nor SK&F 86002 affected the amount or distribution of cyclophilin mRNA in the same fractions. The results suggest that lipopolysaccharide activates TNF translation at the initiation step and that SK&F 86002 inhibits this activation.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Imidazoles/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Tiazoles/farmacología , Factor de Necrosis Tumoral alfa/genética , Secuencia de Bases , Línea Celular , Centrifugación por Gradiente de Densidad , Medios de Cultivo Condicionados , Humanos , Cinética , Datos de Secuencia Molecular , Monocitos/metabolismo , ARN Mensajero , Ribosomas , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
A synthetic peptide corresponding to a novel protein sequence isolated from bovine kidney was used to immunize rabbits. When applied to Western blots of bovine kidney extracts, antiserum to this peptide recognizes proteins with molecular weights of 23 and 18 KD. Immunohistochemical examination of a variety of bovine and rat tissues with this antiserum revealed a unique distribution of immunoreactivity with the intermediate layers of a variety of stratified epithelia, in addition to renal glomeruli. The pattern of reactivity differed from previously described epithelial markers such as cytokeratins. These results indicate that this antiserum may be useful as a tool for the identification of cells of the intermediate layer of stratified epithelia and, as such, may aid in the study of this differentiating/proliferating tissue compartment.
Asunto(s)
Sueros Inmunes/inmunología , Riñón/inmunología , Péptidos/inmunología , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Western Blotting , Bovinos , Células Epiteliales , Epitelio/inmunología , Inmunohistoquímica , Riñón/citología , Datos de Secuencia Molecular , Peso Molecular , Péptidos/análisis , RatasRESUMEN
We have purified a novel form of epithelial transforming growth factor (TGFe) from bovine kidney. Acid ethanol extracts of kidney were fractionated by size exclusion, reverse phase and cation exchange chromatography and activity was monitored by measuring growth of SW13 adrenocortical carcinoma cells in soft agar. The purified material was highly cationic, bound weakly to heparin and gave a band at 13-15000 Mr by SDS-PAGE following Bolton-Hunter iodination. This band correspond to the migration of biological activity extractable from gel slices. The results suggest that we have isolated a truncated form of TGFe which nonetheless retains biological activity.
Asunto(s)
Riñón/química , Factores de Crecimiento Transformadores/aislamiento & purificación , Animales , Bovinos , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Epitelio/química , Factores de Crecimiento de Fibroblastos/metabolismo , Heparina , Peso Molecular , Factores de Crecimiento Transformadores/química , Factores de Crecimiento Transformadores/metabolismoRESUMEN
Proliferation of SW13 human adrenocortical carcinoma cells under anchorage independent conditions was stimulated in a dose-dependent manner by treatment with the cholesterol biosynthesis inhibitor mevinolin. Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was observed in mevinolin treated cultures. The growth stimulatory effect of mevinolin, but not that of epithelial transforming growth factor, a polypeptide growth factor for SW13 cells, was reversed by exogenous mevalonic acid. However, neither dolichol nor low density lipoprotein supplementation affected the response of SW13 cells to mevinolin. The results suggest that mevalonic acid metabolites may participate in the regulation of anchorage independent growth of SW13 cells.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/patología , Colesterol/biosíntesis , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Lovastatina/farmacología , Ácido Mevalónico/farmacología , Factores de Crecimiento Transformadores/farmacología , Células Tumorales Cultivadas/citología , Adhesión Celular , División Celular , Línea Celular , Humanos , Cinética , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimologíaRESUMEN
Conditioned media collected under serum-free conditions over 24 to 48 h from 18 human colon adenocarcinoma cell lines were analyzed for transforming growth factor, types alpha and beta (TGF-alpha and -beta), and platelet-derived growth factor in assays for anchorage-independent growth and radioreceptor competition. Detectable levels of TGF-alpha, TGF-beta, and platelet-derived growth factor were produced by 17, 16, and 6 cell lines, respectively. Three liters of conditioned medium from highly tumorigenic (HT-29, DLD-1, and SW620) and nontumorigenic (SKCO-1) colon cell lines and from nonneoplastic rat kidney (NRK-52E) and small intestinal (IEC-6) epithelial cells were purified by high-performance liquid chromatography and assayed for TGF-alpha- and TGF-beta-like activity. The highly tumorigenic colon cell lines produced 10- to 45-fold (soft agar), 19- to 90-fold (radioreceptor), and 4- to 35-fold (radioimmunoassay) more TGF-alpha activity compared to the nonneoplastic rat intestinal (IEC) epithelial cells. NRK-52E did not produce detectable TGF-alpha activity. Radioimmunoassay analysis of peak fractions revealed only TGF-alpha immunoreactivity; epidermal growth factor was not detected. Levels of TGF-beta-like material in the colon carcinoma populations were comparable (HT-29) or elevated (DLD-1, SW620) only 3- to 4-fold (soft agar) or 1- to 3-fold (radioreceptor binding) compared to IEC cells or NRK-52E. Growth factor production is an ubiquitous property of colon carcinoma cell lines maintained in vitro and is consistent with this class of molecule, playing a contributory role in regulating cell growth.
Asunto(s)
Neoplasias del Colon/metabolismo , Neoplasias Colorrectales/metabolismo , Receptores ErbB/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Receptores de Superficie Celular/biosíntesis , Factores de Crecimiento Transformadores/biosíntesis , Animales , Cromatografía Líquida de Alta Presión , Medios de Cultivo/análisis , Relación Dosis-Respuesta a Droga , Humanos , Ratas , Receptores de Factores de Crecimiento Transformadores beta , Células Tumorales Cultivadas/metabolismoRESUMEN
Using a monoclonal antibody (P6C3) derived from mice immunized with partially delipidated rat myelin, we detected a single 57.5 kDa protein species (BT57.5) positioned between alpha- and beta-tubulin on nitrocellulose containing electrophoretically separated rat brain myelin proteins. The kinetics of incorporation of 14C-labeled BT57.5 and myelin basic protein into myelin was similar in rats injected intracranially with [14C]amino acids to radioactively label these proteins. Immunoblotting of the separated proteins of the myelinlike fraction of rat brain with the anti-BT57.5 monoclonal antibody occasionally revealed a faint band corresponding to BT57.5 but consistently showed a more prominent protein band that migrated slightly ahead of BT57.5. When the myelin and myelinlike fraction were isolated from rats injected with [14C] amino acids, labeling of the prominent anti-BT57.5 binding protein in the myelinlike fraction preceded that of BT57.5 in myelin, reaching a peak of labeling and then decreasing before BT57.5 reached its peak level. Thus, the prominent anti-BT57.5 protein in the myelinlike fraction might be a metabolic precursor of BT57.5 in myelin or the myelinlike fraction is simply turning over more rapidly and is distinct from the myelin fraction. BT57.5 was detectable in rat, mouse, guinea pig, bovine, and human CNS myelin but not rabbit and was detectable in the peripheral nervous system only in myelin of rats and humans. Finally, in extensively purified myelin, BT57.5 appeared to copurify with myelin basic protein, suggesting that BT57.5 is a constituent of myelin rather than an artifact arising during brain homogenization procedures.
Asunto(s)
Anticuerpos Monoclonales , Proteínas de la Mielina/aislamiento & purificación , Animales , Ratones , Peso Molecular , RatasRESUMEN
We have developed a method for automated data collection from anchorage-independent growth assays by direct interfacing of an Omnicon image analysis system with a VAX mainframe computer network. By use of this interface, data generated with the Omnicon can be acquired and manipulated by the VAX, providing several advantages including high throughput, elimination of operator error, flexibility and speed, and capacity of mainframe data processing. We have applied these techniques to aid in the purification of a novel growth factor for human epithelial cells. Both column elution profiles and dose-response data were processed to graphic formats, and ED50 values for the individual purification steps were obtained by Hill transformation of the dose-response curves. The assay for anchorage-independent growth is widely used for purification of growth factors and testing of chemotherapeutic agents against human tumor cells. The present technique should be useful in facilitating these labor-intensive studies.
Asunto(s)
Ensayo de Unidades Formadoras de Colonias , Interpretación Estadística de Datos , Factores de Crecimiento Transformadores/aislamiento & purificación , Ensayo de Tumor de Célula Madre , Animales , Bovinos , Adhesión Celular , División Celular , Cromatografía Líquida de Alta Presión , Humanos , Riñón/análisisRESUMEN
The skin tumor-initiating activities of several bay-region metabolites of 3-methylcholanthrene (3-MCA) were determined in SENCAR mice. 3-MCA-anti-9,10-diol-7,8-epoxide possessed weak tumor-initiating activity when tested at 100 and 200 nmol/mouse doses (0.27 and 0.67 papillomas per mouse after 18 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA)). 3-MCA-trans-9,10-diol at initiating doses of 50 and 100 nmol/mouse was as active as 3-MCA. 3-MCA-trans-9,10-diol was also tested for mutagenic activity toward V79 cells in cell-mediated assays and found to be approximately 2-times more potent than 3-MCA. The data suggest that 3-MCA-trans-9,10-diol is a proximate carcinogen for mouse skin.
Asunto(s)
Metilcolantreno/análogos & derivados , Neoplasias Cutáneas/inducido químicamente , Animales , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Metilcolantreno/metabolismo , Metilcolantreno/toxicidad , RatonesRESUMEN
The skin tumor-promoting ability of 1,8-dihydroxy-3-methyl-9-anthrone (chrysarobin) was compared with that of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1,8-dihydroxy-9-anthrone (anthralin) in SENCAR mice. Although dose-response comparisons indicated that chrysarobin was several orders of magnitude less potent than TPA for promoting papilloma formation, this anthrone was 1.5 to 2 times more potent than anthralin. Maximal papilloma responses were achieved by 15 weeks of promotion with TPA whereas at least 25 weeks of promotion were necessary to achieve maximal papilloma responses with chrysarobin or anthralin indicating marked differences in tumor latency between the two classes of compounds. Interestingly, at optimal promoting doses, chrysarobin gave a carcinoma response (22% with 0.3 carcinomas per mouse at 45 weeks) similar to that of TPA suggesting that this compound may be more efficient at promoting carcinomas than papillomas. In two-stage promotion experiments, chrysarobin was incapable of functioning independently as a Stage I or II promoter despite its complete promoting activity. Chrysarobin and TPA were compared at optimal promoting doses for their ability to induce: (a) skin edema, (b) epidermal hyperplasia, and (c) epidermal ornithine decarboxylase. In each case, distinct differences were noted between the two compounds. When taken together, the data support the hypothesis that anthracene-derived skin tumor promoters work at least in part by a mechanism different from the phorbol esters.
Asunto(s)
Antracenos/toxicidad , Carcinógenos , Neoplasias Cutáneas/inducido químicamente , Animales , Antralina/toxicidad , Relación Dosis-Respuesta a Droga , Edema/inducido químicamente , Femenino , Hiperplasia , Ratones , Ornitina Descarboxilasa/análisis , Papiloma/inducido químicamente , Piel/patología , Acetato de TetradecanoilforbolRESUMEN
The rates of formation and disappearance of benzo[a]pyrene (B[a]P) DNA-adducts were analyzed in the epidermis of SENCAR mice over a 21-day time course. Mice were treated topically with 200 nmol/mouse of [3H]B[a]P at various times prior to sacrifice. The formation and disappearance of total adducts as well as individual adducts was determined and in addition, the rate of DNA turnover was monitored concurrently so that adduct disappearance could be expressed as a function of epidermal cell turnover. Under these experimental conditions, covalent binding of B[a]P to epidermal DNA reached a peak 24 h after treatment. Interestingly, between 24-48 h after application of the hydrocarbon there was a very rapid drop in the level of bound B[a]P to value approximately 50% of the maximum level at 24 h. Thereafter, the level of bound B[a]P disappeared at a much slower rate. In dual-label experiments, where the epidermal DNA was pre-labeled with [14C]thymidine, [3H]B[a]P DNA-adduct disappearance between 24-48 h was clearly more rapid than could be explained on the basis of epidermal DNA turnover. By 72 h and beyond, however, [3H]B[a]P DNA-adduct disappearance approximately paralleled DNA turnover. Examination of the rate of formation and disappearance of individual B[a]P DNA-adducts (nine individual adducts) suggested that some deoxyadenosine adducts were removed more rapidly than deoxyguanosine adducts. The results indicate that at least some epidermal cells have the capacity to repair B[a]P DNA-adducts. The data are discussed in relation to the process of tumor initiation in mouse skin.
Asunto(s)
Benzo(a)pireno/metabolismo , ADN/metabolismo , Piel/metabolismo , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido , Animales , Benzopirenos/metabolismo , ADN/biosíntesis , Femenino , Ratones , Ratones Endogámicos , Neoplasias Cutáneas/inducido químicamente , TritioRESUMEN
10-Fluoro-7,12-dimethylbenz(a)anthracene (10-F-DMBA) is a more potent skin tumor initiator in SENCAR mice when compared with the parent hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA). To elucidate the mechanism for this difference, the covalent binding of these two hydrocarbons to the DNA of mouse epidermal cells in vivo and in vitro was compared. The quantity of 10-F-DMBA covalently bound to mouse epidermal DNA in vivo was greater than that of DMBA at all doses tested over the range of 4 to 200 nmol/mouse. The magnitude of this binding difference between 10-F-DMBA and DMBA was greater at the higher doses (e.g., 1.5-fold at 4 nmol/mouse versus 3.4-fold at 200 nmol/mouse). These results correlated closely with the dose-response relationships for tumor initiation by the two hydrocarbons. Analysis of the isolated DNA samples by Servacel DHB chromatography revealed the relative proportion of syn-diol-epoxide:DNA adducts derived from DMBA increased dramatically as a function of dose (approximately 30% at 4 nmol/mouse versus approximately 55% at 200 nmol/mouse). Conversely, the relative proportion of syn-diol-epoxide adducts derived from 10-F-DMBA was low and remained essentially constant over the same dose range. High-pressure liquid chromatographic analyses of the DNA adducts derived from DMBA- and 10-F-DMBA-treated mice revealed qualitatively similar profiles. However, as expected, there was a marked reduction in the relative proportion of syn-diol-epoxide:DNA adducts in the profiles of epidermal samples from 10-F-DMBA-treated mice. The major syn-diol-epoxide:deoxy-adenosine adduct was present at a level only 30% that found in high-pressure liquid chromatographic profiles of DMBA samples. Similar results were obtained when primary cultures of mouse epidermal cells were treated with the hydrocarbons. The results suggest that the increased total binding and possibly the decreased proportion of syn-diol-epoxide:DNA adducts confer greater tumor-initiating potency on 10-F-DMBA.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno/análogos & derivados , 9,10-Dimetil-1,2-benzantraceno/metabolismo , ADN/metabolismo , Piel/metabolismo , Animales , Transformación Celular Neoplásica/efectos de los fármacos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Epidermis/metabolismo , Ratones , Relación Estructura-ActividadRESUMEN
Mice of the inbred strain DBA/2 responded to a two-stage, initiation-promotion tumorigenesis protocol when high initiating doses (400 nmol/mouse) of 7,12-dimethylbenz[a]anthracene were utilized. They also responded when N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was used as the initiating agent. The tumor response in both cases was characterized by a rapid rate of tumor development with the maximal tumor responses reached on or before the 15th week of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). When DBA/2 mice were compared with SENCAR mice for promotion sensitivity following initiation with MNNG, the two mouse stocks responded with a nearly identical tumor response. C57BL/6 mice were essentially resistant to TPA promotion regardless of the initiator or the dose of initiator used. A preliminary study was conducted to determine how susceptibility to tumor promotion by TPA was inherited in F1 mice derived from DBA/2 (sensitive) and C57BL/6 (resistant) parents. The B6D2F1 mice were as sensitive as the DBA/2 parent, suggesting that susceptibility in these two inbred mouse strains is inherited as an autosomal dominant trait. The results show that these two inbred mouse strains may provide a model system for studying genetic factors controlling susceptibility to phorbol ester skin tumor promotion.