RESUMEN
Endometrial glandular epithelial cells were subcultured on matrix-coated filters in bicameral chambers in a serum-free chemically defined medium. The cells were untreated or treated with 50 nmol progesterone l-1 or 10 nmol oestradiol l-1 or 10 nmol oestradiol l-1 plus 50 nmol progesterone l-1 and the proteins secreted into the basal or apical compartment were analysed after [35S]methionine labelling. Compared with the untreated cells, oestradiol treatment did not affect the electrophoretic profiles of proteins secreted by the glandular epithelial cells in either compartment. Progesterone treatment induced a decrease in the labelling of 88 and 53 kDa proteins secreted in the apical and basal compartments and an increase in the labelling of a 28 kDa protein. Moreover, progesterone specifically induced the apical secretion of a 137 kDa protein. Interaction between the epithelial and stromal cells was also investigated. When stromal cells were cultured in the basal compartment under the epithelial monolayer, the progesterone effect on the apical secretion of the 137 kDa protein and basal secretion of the 88 and 28 kDa proteins were altered, whereas this progesterone effect was not altered when the epithelial cells were cultured alone in media conditioned with stromal cells. Interactions between epithelial and stromal cells modified the effect of progesterone on protein secretion by the epithelial cells.
Asunto(s)
Endometrio/metabolismo , Progesterona/farmacología , Proteínas/metabolismo , Animales , Comunicación Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electroforesis , Endometrio/citología , Endometrio/efectos de los fármacos , Endotelio/citología , Endotelio/efectos de los fármacos , Estradiol/farmacología , Femenino , Cobayas , Proteínas/análisisRESUMEN
Glandular epithelial cells (GEC) and stromal cells (SC) were isolated from guinea-pig endometrium and cultured separately. After the cells were made quiescent by serum depletion, cell proliferation and c-fos gene expression were investigated. Estradiol-17 beta (E2) alone had no effect on cell proliferation or c-fos gene expression. Cell proliferation was observed in SC and GEC after stimulation with insulin plus EGF and a supplementary effect was obtained when E2 was associated with these factors only in GEC. In GEC, insulin plus EGF had no effect on c-fos expression but an effect was observed when E2 was associated with these factors or with a protein synthesis inhibitor, cycloheximide (Chx) or puromycine. An E2 effect in the presence of Chx was also observed in SC. These data suggest the presence of a labile protein preventing the in vitro estrogenic action in endometrial cells.
Asunto(s)
Endometrio/citología , Factor de Crecimiento Epidérmico/farmacología , Estradiol/farmacología , Expresión Génica/efectos de los fármacos , Genes fos/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Femenino , Cobayas , Técnicas In Vitro , Células del Estroma/citologíaRESUMEN
Guinea pig endometrial stromal cells were cultured in serum-free medium to assess the effects of growth factors and ovarian steroids on cell proliferation. When the cells were made quiescent by serum depletion, [3H]thymidine incorporation was increased by the addition of insulin plus epidermal growth factor (EGF), reaching a peak after 24 h of stimulation. This effect was dose-dependent. Both factors acted synergistically. Estradiol-17 beta (E2), either alone or with various concentrations of growth factors, had no mitogenic effect. Thus, cell proliferation appeared to be estrogen-insensitive, despite a high level of estrogen receptors (19,000 sites per cell). The integrity of these receptors was checked by transfecting cells with a plasmid containing an estrogen-responsive element linked to a CAT gene: E2-induced CAT activity was reduced by the antiestrogen ICI 164,384. Despite the presence of progesterone receptors, the cells, either primed with E2 or not, were not growth-stimulated by progesterone. E2 had no effect on cells cultured in the presence of dextran-coated charcoal-stripped serum. Thus, whatever the culture conditions, stromal cells with functional estrogen receptors were insensitive to the putative mitogenic effects of E2 and progesterone. However, they were highly responsive to the mitogenic effects of insulin and EGF.
Asunto(s)
Endometrio/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Insulina/farmacología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Endometrio/citología , Endometrio/metabolismo , Factor de Crecimiento Epidérmico/administración & dosificación , Receptores ErbB/metabolismo , Estradiol/farmacología , Femenino , Cobayas , Insulina/administración & dosificación , Factor I del Crecimiento Similar a la Insulina/farmacología , Progesterona/farmacología , Receptores de Estrógenos/metabolismo , Timidina/metabolismoRESUMEN
Flavonoids are known to reduce reactive oxygen species released by polymorphonuclear neutrophils (PMNs) in vitro. We have studied the effects of S5682 (Daflon 500 mg), a purified flavonoid fraction composed of 90% diosmin and 10% hesperidin. S5682 produced a dose-dependent inhibition of the luminol chemiluminescence (CL) induced by phorbol myristate acetate on PMNs (IC50 = 5 x 10(-5) M), with no effect on superoxide anion (O2.-) formation and on cellular superoxide dismutase activity as determined by lucigenin-amplified CL. The CL results were confirmed by the hydrogen peroxide (H2O2) determination showing that S5682 reduced H2O2 formed through either PMN stimulation (IC50 = 1.6 x 10(-6) M) or an in vitro enzymatic mechanism (IC50 = 2 x 10(-6) M). S5682 inhibited luminol-dependent CL induced by H2O2 (IC50 = 5 x 10(-6) M). However, O2 was not formed from H2O2 in contact with S5682 and the UV spectrum of this compound was not modified. In contrast, S5682 inhibited luminol-dependent CL induced by H2O2 in the presence of horseradish peroxidase (IC50 = 3 x 10(-6) M), and the UV spectrum of S5682 was modified. Luminol-dependent CL induced by hypochlorite (OCl- 10(-5) M) was also inhibited by S5682 (IC50 = 7 x 10(-5) M). This inhibitory effect was similar to that of sodium azide on myeloperoxidase activity. Moreover, OCl- 5 x 10(-4) M also altered the UV spectrum of S5682 10(-4) M. These results indicate that S5682 could be active on the H2O2-OCl(-)-myeloperoxidase system.
Asunto(s)
Antioxidantes/farmacología , Diosmina/farmacología , Hesperidina , Neutrófilos/efectos de los fármacos , Acridinas , Combinación de Medicamentos , Radicales Libres , Humanos , Mediciones Luminiscentes , Luminol , Neutrófilos/metabolismo , Peroxidasa , Especies Reactivas de Oxígeno , Acetato de TetradecanoilforbolRESUMEN
The purification and characterization of a low-molecular-mass binding protein from female guinea-pig liver cytosol is reported. Its molecular mass (14.4 kDa), amino acid composition, abundance and biological properties identify it as belonging to the Z class of liver cytosolic proteins [Levi, A.J., Gatmaitan, Z. & Arias, I.M. (1969) J. Clin. Invest. 48, 2956-2167]. Among the most important members of this class of proteins are the fatty-acid-binding proteins (FABPs) and the sterol carrier protein2 (SCP2). The guinea-pig Z protein (G-ZP) has some similarities in its amino acid composition and NH2-terminal sequence with those of the rat liver FABP, but its isoelectric point is basic (pI 8.85), like that of SCP2. We also examined its binding affinities for a number of ligands bound by these two proteins. The results show that the purified G-ZP binds dehydroepiandrosterone sulfate, estrone sulfate, oleic acid and cholesterol, but shows no affinity for free steroids such as estrone and DHEA. Thus it can be said that G-ZP has some characteristics of FABPs and some of SCP2 but seems, however, to be different from both these proteins. The purified G-ZP inhibits microsomal DHEA sulfate sulfatase activity in a mixed noncompetitive way. This protein could be involved in the transport and/or metabolism of sulfated steroids.
Asunto(s)
Proteínas Portadoras/metabolismo , Citoplasma/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Proteínas de Plantas , Secuencia de Aminoácidos , Animales , Arilsulfatasas/antagonistas & inhibidores , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Femenino , Cobayas , Focalización Isoeléctrica , Hígado/metabolismo , Microsomas/enzimología , Datos de Secuencia Molecular , Embarazo , Homología de Secuencia de Ácido Nucleico , Esteroles/metabolismo , Esteril-SulfatasaRESUMEN
Certain 99mTc-labeled derivatives of ethylene diamine substituted in NN', when labeled with 99mTc, have been proposed for studying renal excretion, and others for brain imaging. Ethambutol, a well known antituberculosis drug, possesses a structure very similar to these derivatives. We prepared a complex of [99mTc]ethambutol using hypophosphorus acid as the reducing agent. The complex was then analyzed by paper and cellulose F chromatography and by FAB mass spectrometry. A pharmacokinetic study in the mouse and rat indicated that the complex is eliminated by the kidney faster than DTPA, slower than [131I]hippuran and without significant renal retention. The [99mTc]ethambutol complex is easy to prepare and shows good stability in water at physiological pH, making it potentially useful in renal excretion studies.
Asunto(s)
Etambutol/farmacocinética , Compuestos de Organotecnecio/farmacocinética , Animales , Marcaje Isotópico/métodos , Riñón/diagnóstico por imagen , Riñón/metabolismo , Ratones , Cintigrafía , Ratas , Distribución TisularRESUMEN
The effects of estradiol and/or antiestrogens on cholesterol biosynthesis were studied in two breast cancer cell lines. Cholesterogenic activity was evaluated after labeling cells with sodium [14C]acetate for increasing periods of time (up to 24 h) and measuring the incorporation of the radioactivity into nonsaponifiable lipids and into cholesterol, after separation from other labeled metabolites. We compared the effects of estradiol on cholesterogenesis with the well-known effects of this hormone on cell proliferation: estradiol stimulated both cholesterol synthesis and cell growth in MCF-7 cells, but stimulated neither in BT20 cells. The stimulation affected both the 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase step and the post-HMGCoA steps. Only the key enzyme step appeared to be mediated by the estrogen receptor. The hydroxytamoxifen and LY 117018 antiestrogens strongly inhibited cellular cholesterol production in both cell lines. Under the same conditions, cell growth is affected in MCF-7 cells, but not in BT20 (as shown by groups from other laboratories). This demonstrates that de novo synthesis of cholesterol is not essential for cell growth when cells are cultured in the presence of whole serum. The inhibition of cholesterol synthesis by antiestrogens mainly affected the lanosterol demethylation step and the C-27 sterol to cholesterol conversion. This inhibiting effect of antiestrogens was not mediated by the estrogen receptor.
Asunto(s)
Neoplasias de la Mama/metabolismo , Colesterol/biosíntesis , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Acetatos/metabolismo , Ácido Acético , Línea Celular , Femenino , Humanos , Hidroximetilglutaril-CoA Reductasas/metabolismoRESUMEN
The effects of estradiol (E2), 4-hydroxy-tamoxifen (OH-Tam), and LY117018 on cholesterogenesis were investigated in two human breast cancer cell lines (MCF-7 and BT20), and in rat hepatoma (HTC) and fibroblastic (NRK-49F) cell lines. It was found that 10(-10) M E2 stimulated and 10(-8) M OH-Tam inhibited cholesterol synthesis in the estrogen-sensitive MCF-7 cell line. The OH-Tam effect occurred in less than 15 min whereas E2 only stimulated after 8 h. The inhibition of cholesterol synthesis was not reversed by E2. E2 was without effect in the HTC and estrogen-resistant BT20 cell lines whereas OH-Tam was as effective as in the MCF-7 cells. LY117018 had nearly as much effect on cholesterol synthesis as OH-Tam, in both MCF-7 and BT20 cells. Neither E2 nor OH-Tam had any effect on the NRK-49F cell line, even at micromolar concentrations. The three lines (MCF-7, BT20, HTC), whose cholesterol synthesis has been shown to be OH-Tam sensitive, appeared to contain high-affinity antiestrogen binding sites (AEBS); since the OH-Tam-resistant line (NRK) only contained low-affinity AEBS, there appears to be some relationship between OH-Tam sensitivity and high-affinity AEBS content. This suggests that the cholesterogenesis inhibition induced by antiestrogens is ER-independent and may involve AEBS. The cholesterogenesis stimulation induced by E2 occurred via a different pathway that appears to be related to the presence of ER in the cells.