RESUMEN
We conducted 2 experiments to determine lysine loss from 2 lipid-coated lysine products after mixing with silage. In our first experiment, we mixed 2 lipid-coated lysine products, crystalline lysine or crystalline lysine and amounts of lipid identical to amounts included in lipid-coated lysine products, with alfalfa or corn silage that had 2 different amounts of acidity. Lysine appeared to disassociate from lipid-coated lysine products in a nonlinear manner after mixing with either alfalfa or corn silage at different amounts of acidity. Additionally, silage source and acidity affected amounts of lysine released from lipid-coated lysine products after mixing. In a corresponding experiment, in vitro estimates of lysine available to ruminal microbiota after mixing with alfalfa or corn silage at different amounts of acidity were measured by ammonia release. In vitro measures were conducted with or without monensin to allow estimates of effects of monensin on amounts of lysine released from the 2 lipid-coated lysine products. It is unclear whether in vitro estimates of lysine fermentation from lipid-coated lysine are truly reflective of ruminal degradation of lysine from lipid-coated lysine because amounts of time needed to measure differences between different lysine sources were greater than typical estimates of mean ruminal particulate retention time. Nonetheless, monensin apparently reduced ammonia release from lysine, but ammonia release from lipid-coated lysine did not differ from crystalline lysine. Clearly, methods of manufacture together with physical and chemical characteristics of diet can affect amounts of lysine provided from lipid-coated lysine products to ruminants.
Asunto(s)
Digestión/fisiología , Portadores de Fármacos , Fermentación , Lisina/metabolismo , Animales , Dieta , Femenino , Lactancia , Lípidos , Lisina/administración & dosificación , Medicago sativa , Rumen , Ensilaje , Zea maysRESUMEN
BACKGROUND: The use of pulse oximetry in horses is limited due to inadequate readings with conventional transmission sensor probes. OBJECTIVES: The objectives of this study were to 1) develop an improved sensor design for horses to be used at an appropriate anatomical site, and 2) evaluate this design in an experimental study. STUDY DESIGN: In vivo experiment. METHODS: A new sensor design for reflectance pulse oximetry at the buccal mucosa was developed. A conventional Nonin 2000SL sensor for transmission pulse oximetry was included into this design. Three different prototypes (N1, N2a, N2b) were constructed and used with the Nonin 2500A Vet pulse oximetry monitor. Thirteen anaesthetised warmblood horses were included into a desaturation protocol (100-70% SaO2 ). SpO2 and pulse frequency values were recorded, using SaO2 calculated from blood gas analysis and invasive pulse frequency measurements as reference methods. Bias and precision were evaluated by calculations of the root mean square deviation (Arms ). The agreement of the methods was tested with Bland-Altman analysis. RESULTS: The quality of the pulse frequency readings determined the quality of the SpO2 -readings. Good pulse signal strength resulted in a SpO2 -accuracy comparable to that of the original sensor (Nonin 2000SL: Arms = 3%; N1: Arms = 3.60%; N2b: Arms = 3.46%). Especially at heart rates ≤30 bpm, pulse rate readings that were about twice as high as the reference value occurred. Their exclusion from the dataset resulted in a pulse rate accuracy similar to that of the original sensor. Bland-Altman plots showed limits of agreement typical of pulse oximeters. MAIN LIMITATIONS: The pulse frequency accuracy requires further improvement. The usability in clinical cases needs to be tested. CONCLUSIONS: The new sensor design has been shown to be suitable for buccal pulse oximetry in horses.
Asunto(s)
Caballos/fisiología , Monitoreo Fisiológico/veterinaria , Oximetría/veterinaria , Animales , Monitoreo Fisiológico/instrumentación , Oximetría/instrumentación , Oxígeno/sangreRESUMEN
We conducted 2 experiments to determine lysine bioavailability from 2 lipid-coated lysine products. In an in vitro experiment we mixed each lipid-coated lysine product with either alfalfa- or corn-silage at different amounts of acidity. Scanning electron micrographs indicated that surface structure of each lipid-coated lysine particle was eroded after mixing with silage. Additionally, visual evaluation of scanning electron micrographs suggested that peripheral surface abrasion of lipid-coated lysine may be greater when lipid-coated lysine was mixed with alfalfa silage in comparison to corn silage. In a corresponding experiment, in vivo measures of lysine bioavailability to sheep from 2 lipid-coated lysine products and lysine-HCl were determined after mixing in corn silage. Plasma lysine concentrations increased linearly (P < 0.01) in response to abomasal lysine infusion indicating that our model was sensitive to increases in metabolizable lysine flow. Bioavailability of each lipid-coated lysine source and dietary lysine-HCl were calculated to be 23, 15, and 18%, respectively. Even though each dietary source of lysine increased plasma lysine, rates of increases in plasma lysine from one lipid-coated lysine source (linear; P = 0.20) and lysine-HCl (linear; P = 0.11) were not different from plasma lysine levels supported by diet alone. However, the rate of plasma lysine increase in response to lysine from the other lipid-coated lysine source was greater (P = 0.04) than plasma lysine from feed alone. Nonetheless, the rate of plasma lysine increase in response to lipid-coated lysine did not differ (P ≥ 0.70) from the rate of plasma lysine increase from lysine-HCl. Clearly, methods of manufacture, together with physical and chemical characteristics of diet, can impact amounts of metabolizable lysine provided from lipid-coated lysine products. Direct measures of lysine bioavailability from lipid-coated lysine products after mixing with diets should be based on measurements with the products treated similarly to the method of feeding.
RESUMEN
We present a detailed study of the superconducting properties of the weakly pinned, quasi-two-dimensional superconductor 2H-NbSe2, and its intercalated variant NbSe2{CoCp2}0.26. The intercalation of 2H-NbSe2 with the organometallic donor molecule cobaltocene (CoCp2) hardly affects the superconducting properties within the layers. However, the properties perpendicular to the layers change significantly due to the large expansion of the layer spacings of the host lattice in the c-direction by a factor of about two. In particular, the superconducting anisotropy factor Γ increases from 3.3 in the parent compound 2H-NbSe2 up to 4.4 in the intercalated species. Therefore, NbSe2{CoCp2}0.26 is an excellent candidate to analyze how the anisotropy effects the superconducting mechanism in layered dichalcogenides, and to evaluate the various models proposed in the literature to account for the anisotropy in 2H-NbSe2. While a two-gap model and an anisotropic single-gap model are competing concepts to describe the almost linear T(2)-dependence of ΔC/T in low-dimensional dichalcogenides, our comparative study suggests that a single-gap model with an anisotropic Fermi-surface is sufficient to capture the ΔC/T(T) behavior in our samples qualitatively.
RESUMEN
Traumatic limb amputations and transections of the trunk generally indicate high impact velocities. Complete traumatic severances of the trunk in road accidents however are rarely reported in literature. We describe a case where the trunk of a motorcyclist was completely severed as he hit the post of a traffic sign. Based on the documented end position and damage of the motorcycle, an impact velocity range of 52-80 km/h could be determined for the motorcyclist. To our knowledge, this is the first report on the type and mechanism of injury at this comparatively low collision speed range.
Asunto(s)
Traumatismos Abdominales/patología , Accidentes de Tránsito , Motocicletas , Vértebras Torácicas/lesiones , Heridas Penetrantes/patología , Aceleración , Adulto , Patologia Forense , Humanos , Masculino , Vértebras Torácicas/patologíaAsunto(s)
Proteína de Unión a CREB/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Polimorfismo Genético , Trastorno Bipolar/genética , Proteína de Unión a CREB/genética , Proteínas de Ciclo Celular/genética , Línea Celular Transformada/efectos de los fármacos , Colforsina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Nucleares/genética , Receptores Acoplados a Proteínas G , Serina/genética , Transfección/métodosRESUMEN
The photosensitizer 9-capronyloxytetrakis (methoxyethyl) porphycene localizes predominantly in the endoplasmic reticulum (ER) and, to a lesser extent, in mitochondria of murine leukemia L1210 cells. Subsequent irradiation results in the loss of ER > mitochondrial Bcl-2 and an apoptotic response. Although an increase in cytosolic Ca(2+) was observed after irradiation, apoptosis was not inhibited by either the presence of the calcium chelator BAPTA or by the mitochondrial uniporter inhibitor ruthenium amino binuclear complex (Ru360). Moreover, neither reagent prevented the loss of Bcl-2. Ruthenium red (RR) devoid of Ru360 prevented Bcl-2 loss, release of Ca(2+) from the ER and the initiation of apoptosis. Since RR was significantly more sensitive than Ru360 to oxidation by singlet oxygen, we attribute the protective effect of RR to the quenching of reactive oxygen species. Although cytosolic and (to a lesser extent) mitochondrial Ca(2+) levels were elevated after photodynamic therapy, these changes were apparently insufficient to contribute to the development of apoptosis.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Fármacos Fotosensibilizantes/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rojo de Rutenio/farmacología , Animales , Apoptosis , Western Blotting , Relación Dosis-Respuesta a Droga , Leucemia L1210/radioterapia , Potenciales de la Membrana , Ratones , Mitocondrias/fisiología , Fotoquimioterapia , Células Tumorales CultivadasRESUMEN
Ursodeoxycholic acid (UDCA), a relatively nontoxic bile acid, enhanced the apoptotic response of tumor cells to both photosensitizers that cause photodamage to Bcl-2 and to the nonpeptidic Bcl-2/Bcl-x(L) antagonist HA14-1. The latter agent binds to the surface pocket formed by the BH1, BH2 and BH3 domains of Bcl-2 and Bcl-x(L). Fluorescence polarization studies indicated that affinity of HA14-1 for Bcl-2 was enhanced in the presence of UDCA. Moreover, Bcl-2 photodamage was promoted by UDCA using a photosensitizing agent with affinity for the endoplasmic reticulum, a site of Bcl-2 localization. Fluorescence resonance energy transfer (FRET) studies revealed that the proximity of Bcl-2 to a hydrophobic photosensitizing agent embedded in liposomes was enhanced by UDCA. Since photodamage will occur only if a protein is in close contact with a photosensitizing agent, we propose that these findings support the hypothesis that UDCA causes a conformational change in Bcl-2, promoting HA14-1 binding and enhancing affinity for certain membrane-bound photosensitizers.
Asunto(s)
Apoptosis/fisiología , Benzopiranos/farmacología , Retículo Endoplásmico/metabolismo , Nitrilos/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Ácido Ursodesoxicólico/farmacología , Animales , Apoptosis/efectos de los fármacos , Ácidos y Sales Biliares/farmacología , Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Polarización de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Liposomas/química , Ratones , Conformación Molecular , Fármacos Fotosensibilizantes/farmacología , Proteínas Proto-Oncogénicas c-bcl-2 , Células Tumorales CultivadasAsunto(s)
Quimiocinas CXC/genética , Tasa de Filtración Glomerular/fisiología , Péptidos y Proteínas de Señalización Intercelular , Trasplante de Riñón/patología , Trasplante de Riñón/fisiología , Ácido Micofenólico/uso terapéutico , Receptores de Quimiocina/genética , Adolescente , Quimiocina CXCL10 , Quimiocina CXCL9 , Niño , Preescolar , Quimioterapia Combinada , Tasa de Filtración Glomerular/efectos de los fármacos , Humanos , Inmunosupresores/uso terapéutico , Lactante , Interferón gamma/genética , Trasplante de Riñón/inmunología , Ácido Micofenólico/análogos & derivados , Valor Predictivo de las Pruebas , Receptores CXCR3RESUMEN
Photodynamic therapy (PDT) protocols employing lysosomal sensitizers induce apoptosis via a mechanism that causes cytochrome c release prior to loss of mitochondrial membrane potential (DeltaPsi(m)). The current study was designed to determine how lysosomal photodamage initiates mitochondrial-mediated apoptosis in murine hepatoma 1c1c7 cells. Fluorescence microscopy demonstrated that the photosensitizer N-aspartyl chlorin e6 (NPe6) localized to the lysosomes. Irradiation of cultures preloaded with NPe6 induced the rapid destruction of lysosomes, and subsequent cleavage/activation of Bid, pro-caspases-9 and -3. Pro-caspase-8 was not activated. Release of cytochrome c occurred at about the time of Bid cleavage and preceded the loss of DeltaPsi(m). Extracts of purified lysosomes catalyzed the in vitro cleavage of cytosolic Bid, but not pro-caspase-3 activation. Pharmacological inhibition of cathepsin B, L and D activities did not suppress Bid cleavage or pro-caspases-9 and -3 activation. These studies demonstrate that photodamaged lysosomes trigger the mitochondrial apoptotic pathway by releasing proteases that activate Bid.
Asunto(s)
Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Grupo Citocromo c/metabolismo , Precursores Enzimáticos/metabolismo , Lisosomas/enzimología , Mitocondrias/enzimología , Neoplasias/terapia , Fotoquimioterapia , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/efectos de los fármacos , Caspasa 8 , Caspasa 9 , Caspasas/efectos de los fármacos , Catepsina D/efectos de los fármacos , Catepsina D/metabolismo , Extractos Celulares/farmacología , Grupo Citocromo c/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/efectos de los fármacos , Lisosomas/efectos de los fármacos , Lisosomas/ultraestructura , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Neoplasias/enzimología , Neoplasias/fisiopatología , Porfirinas/farmacología , Porfirinas/efectos de la radiación , Células Tumorales CultivadasRESUMEN
T cells are believed to be critical functional targets of Pb immunotoxicity. In this study, low concentrations of lead (i.e., as low as 0.1 microM approximately 2 microg/dl) were found to markedly enhance the allogeneic mixed lymphocyte reaction-an assay of CD4(+) T cell responsiveness. Cell cycle analysis of cells recovered from allogeneic mixed lymphocyte cultures revealed that Pb stimulated a substantial increase in the proportion of cycling alloreactive CD4(+) T cells. The enhanced alloproliferative response was characterized by an increased population of lymphoblasts expressing heightened cell surface expression of CD4 (i.e., CD4(high) cells). Successive rounds of cell division were monitored using the cell division dye 5- (and 6)-carboxyfluorecein diacetate succinimyl ester and it was determined that the CD4(high) subpopulation comprised the expanding alloreactive T cells, which ultimately took on the phenotype of memory/effector T cells (i.e., CD44(high), CD45RB(low), CD69(high), and CD162(high)). Enhancement of T cell proliferation by lead was selective for responsiveness to alloantigen, as lead had no effect on T cell proliferation induced by mitogens or superantigen, processes that unlike alloreactivity are not dependent on antigen presentation. Collectively, these data suggest that Pb enhances alloantigen-specific T cell proliferation through an indirect mechanism involving altered antigen processing/presentation, resulting in marked clonal expansion or repertoire expansion of alloreactive T cell clones. Consistent with this suggestion was the finding that a single exposure to Pb during alloantigen priming elicited a population of CD4(+) T cells that was hyperresponsive to further alloantigen stimulation and neither lead dependent nor lead responsive.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Isoantígenos/inmunología , Plomo/toxicidad , Animales , Antígenos CD/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Recuento de Células , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Femenino , Citometría de Flujo , Memoria Inmunológica/efectos de los fármacos , Memoria Inmunológica/inmunología , Inmunofenotipificación , Plomo/inmunología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos , Mitógenos/inmunología , Mitógenos/farmacología , Superantígenos/inmunología , Superantígenos/farmacologíaRESUMEN
The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances or suppresses the transcriptional activation of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a cell/tissue-specific manner. The basis for these effects is not known. Exposure of the immortalized human breast epithelial cell line MCF10A-Neo to TPA at the time of, or up to 12 hr prior to, the addition of TCDD strongly suppressed the transcriptional activation of CYP1A1 and CYP1B1 (IC(50) approximately 0.5 nM). A recent study (Carcinogenesis 2000;21:1303-12) demonstrated that TPA-treated MCF10A-Neo cells rapidly activate the latent transforming growth factor beta (TGFbeta) in the serum used to supplement the culture medium. The suppressive effects of TPA on CYP1A1 induction by TCDD in MCF10A-Neo cultures could be partially suppressed by: (a) co-incubation of TCDD + TPA-treated cultures with a neutralizing TGFbeta pan antibody; (b) prior removal of latent TGFbeta from the culture medium; or (c) switching cultures to serum- and growth factor-free medium immediately before the addition of TPA and TCDD. Exposure of cultures to TPA 24-48 hr prior to subsequent TPA + TCDD treatment not only inhibited the suppressive effects of TPA, but markedly enhanced CYP1A1 mRNA accumulation. TPA caused a rapid and protracted activation of extracellular signal-regulated kinases (ERKs). Pretreatment of cultures with the mitogen-activated protein kinase kinase (MEK) inhibitor PD184352 [2-(2-chloro-4-iodo-phenylamino)-N-cyclopropyl-methoxy-3,4-difluoro-benzamide] completely inhibited ERK activation by TPA. However, PD184352 did not prevent the suppressive effects of TPA on CYP1A1 activation by TCDD. These studies demonstrate that TPA initiates protein kinase C-dependent, ERK-independent processes that suppress CYP1A1 activation by TCDD in MCF10A-Neo cells. Furthermore, TGFbeta mediates a small portion of this suppressive activity.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Citocromo P-450 CYP1A1/biosíntesis , Sistema Enzimático del Citocromo P-450/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/fisiología , Dibenzodioxinas Policloradas/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factor de Crecimiento Transformador beta/fisiología , Benzamidas/farmacología , Carcinógenos/farmacología , Células Cultivadas , Citocromo P-450 CYP1A1/efectos de los fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1 , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/genética , Interacciones Farmacológicas , Activación Enzimática , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Activación Transcripcional/efectos de los fármacosRESUMEN
Analyses of CYP1A1 mRNA were used to monitor the responsiveness of murine hepatoma 1c1c7 and human monocytic U937 cells in different phases of the cell cycle to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Concentrations of TCDD capable of inducing CYP1A1 were not cytostatic to either cell line. Steady-state CYP1A1 mRNA contents were reduced (45-90%) in TCDD-treated cultures arrested in G2/M as a consequence of exposure to microtubule disrupters (Colcemid, estramustine, vinblastine) or the microtubule stabilizer Taxol, relative to TCDD-treated asynchronous 1c1c7 cultures. The accumulation of mRNAs corresponding to Nmo1, another TCDD-inducible gene of the Ah battery, was also reduced in TCDD-treated G2/M cultures. Quantitative reverse transcriptase-polymerase chain reaction analyses of CYP1A1 heterogeneous nuclear RNA (hnRNA) revealed that Cyp1a1 transcription was suppressed in G2/M cells. This suppression reflected neither changes in the relative content of the proteins comprising the aryl hydrocarbon receptor (AHR) complex nor a suppression of AHR activation and translocation to the nucleus. Release of 1c1c7 cultures arrested in G2/M restored TCDD responsiveness. Centrifugal elutriation of TCDD-treated asynchronously growing U937 cells was used to prepare populations of cells in specific phases of the cell cycle. Within 3 h of TCDD exposure late G1/early S phase cells had CYP1A1 mRNA contents approximately 1.4- and 3-fold higher than the contents of asynchronous/early G1 and G2/M cultures, respectively. These studies suggest that the transcriptional activation of members of the Ah battery by TCDD is cell cycle-dependent, and markedly suppressed in G2/M cells.
Asunto(s)
Citocromo P-450 CYP1A1/biosíntesis , Dioxinas/farmacología , Western Blotting , Proteína Quinasa CDC2/metabolismo , Ciclo Celular/efectos de los fármacos , Centrifugación , Cromosomas/efectos de los fármacos , Cromosomas/ultraestructura , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Ácido Orótico/metabolismo , Dibenzodioxinas Policloradas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Nuclear/biosíntesis , Receptores de Hidrocarburo de Aril/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
While some epidemiological risk factors for breast cancer have been identified, the environmental factors responsible for transformation of mammary epithelial cells are not clear. We have exposed the spontaneously immortalized human mammary epithelial cell line MCF-10A to benzo[a]pyrene and selected transformed clones based on a loss of contact inhibition and anchorage-dependent growth. Cytogenetic studies showed that each of the transformed sublines possess an isochromosome 8q aberration. The c-Myc proto-oncogene, which is positioned at 8q24, was analyzed for changes in expression. Both c-Myc mRNA and protein levels were increased in the transformed clones relative to the parental cells. The transformed clones were not able to grow as tumors in vivo when injected into nude or SCID mice. To determine whether the involvement of chromosome 8 in BP-induced mutagenesis was a reproducible event, transformed clones were selected from three additional independently treated sets of BP-exposed MCF-10A cultures and analyzed by spectral karyotyping (SKY). These transformed sublines also harbored the isochromosome 8q abnormality. Data from this model show that benzo[a]pyrene, a ubiquitous procarcinogen, can induce selectable morphologic changes in a human mammary epithelial cell line, and that these transformed cells possess chromosomal aberrations frequently found in human breast tumors.
Asunto(s)
Benzo(a)pireno/efectos adversos , Mama/patología , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 8/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Animales , Mama/efectos de los fármacos , Pruebas de Carcinogenicidad , Línea Celular , Línea Celular Transformada/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Aberraciones Cromosómicas/genética , Cromosomas Humanos Par 8/genética , Células Clonales , Análisis Citogenético , Análisis Mutacional de ADN , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Genes myc/genética , Humanos , Ratones , Proto-Oncogenes Mas , ARN Mensajero/metabolismoRESUMEN
Arsenite treatment has been found to induce clinical remission in patients with acute promyelocytic leukemia. Although the potential therapeutic value of arsenite may lie in triggering apoptosis, it has not been established that cytotoxicity is the sole mechanism of action. We have used a myelomonocytic leukemia cell line (U937) to characterize the concentration-dependent effects of arsenite on cell growth, viability, apoptosis, and differentiation. Arsenite has multiple effects on U937 cells. Low concentrations of arsenite (i.e., < or = 1 microM) potentiate vitamin-D(3)-induced differentiation. Two markers of monocyte differentiation, Mac-1 expression and nitroblue tetrazolium reduction, are increased in arsenite-exposed, D(3)-costimulated cells. Concentrations of arsenite >10 microM rapidly induce the death of cells irrespective of cell cycle phase. Intermediate concentrations of arsenite (i.e., 5 to 10 microM) are cytostatic initially. Cell cycle analysis using elutriated, synchronous cell populations revealed that intermediate concentrations of arsenite delay both G(1) and G(2) transit. G(2) cells appear to be most sensitive to arsenite, in that transit through G(2)/M is more delayed than transit through G(1), and apoptosis is induced in these cells as they emerge from an aberrant G(2)/M. Arsenite-induced apoptosis was caspase-3 dependent. Arsenite-mediated cytotoxicity was reduced in the presence of the broad caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone; however, caspase inhibition did not reverse arsenite-induced cytostasis. Thus, arsenite has multiple effects on U937 cells that are dependent on concentration and cell cycle phase. Specifically, cell cycle transit and differentiation are more sensitive to arsenite than is the induction of apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Arsenitos/farmacología , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Apoptosis/fisiología , Calcitriol/farmacología , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Ciclo Celular/fisiología , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Factores de Tiempo , Células U937RESUMEN
Numerous flavonoids are ligands of the aryl hydrocarbon receptor (AHR) and function as AHR antagonists and/or agonists. LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] is a widely used inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), and is structurally related to members of the flavonoid family. Concentrations of LY294002 >/= 10 microM were cytostatic, but not cytotoxic, to cultures of the immortalized human breast epithelial cell line MCF10A-Neo. Treatment of MCF10A-Neo cultures with the AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) stimulated the transcriptional activation of CYP1A1, as monitored by measurements of steady-state CYP1A1 mRNA. Pretreatment of cultures with >/= 10 microM LY294002 suppressed the TCDD activation of CYP1A1 (IC(50) approximately 10 microM). Electrophoretic mobility shift assays employing rat liver cytosol demonstrated that concentrations of LY294002 = 400 microM did not transform the AHR into a DNA-binding species. However, the addition of LY294002 to cytosol just prior to TCDD addition completely suppressed AHR transformation by TCDD (IC(50) approximately 35 microM). The PI 3-kinase inhibitor Wortmannin was weakly cytostatic, but not cytotoxic to MCF10A-Neo cultures at concentrations = 500 nM. Exposure of cultures to Wortmannin (10-500 nM) did not suppress TCDD activation of CYP1A1. Analyses of the phosphorylation status of Akt-1, an in vivo substrate of PI 3-kinase, demonstrated that concentrations of LY294002 >/= 50 microM and Wortmannin >/= 10 nM completely suppressed PI 3-kinase activity. Hence, the ability of LY294002 to suppress TCDD-dependent activation of CYP1A1 is unrelated to PI 3-kinase inhibition. Instead, this activity reflects LY294002 functioning as an AHR antagonist. Furthermore, most of the cytostatic activity of LY294002 towards MCF10A-Neo cells is unrelated to the inhibition of PI 3-kinase.
Asunto(s)
Cromonas/farmacología , Citocromo P-450 CYP1A1/biosíntesis , Inhibidores Enzimáticos/farmacología , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Dibenzodioxinas Policloradas/farmacología , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Androstadienos/farmacología , División Celular/efectos de los fármacos , Línea Celular Transformada , Citocromo P-450 CYP1A1/genética , ADN/efectos de los fármacos , ADN/metabolismo , Interacciones Farmacológicas , Inducción Enzimática , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Hidrocarburo de Aril/fisiología , Activación Transcripcional/efectos de los fármacos , WortmaninaRESUMEN
The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle progression were examined in the human breast cell line MCF10A-Neo and a derivative line which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G(1) arrest that lasted approximately 16-24 h (IC(50) approximately 0.5 nM). TPA-treated cultures produced a cytostatic conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3-7 h later and disappeared between 16 and 24 h post-treatment. However, cytostatic conditioned medium could be quickly regenerated by re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor beta (TGF beta) from the culture medium, supplementing the culture medium with anti-TGFbeta or soluble TGF beta(II) receptor, or pre-absorption of conditioned medium with anti-TGF beta all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo proliferation by approximately 50%. Co-treatment with the serine protease inhibitors aprotinin or plasminogen activator inhibitor-1 also suppressed the cytostatic activity of TPA approximately 50%. Conditioned medium isolated from TPA-treated MCF10A-Neo cultures was transiently cytostatic to MCF10A-NeoT cells. The proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, was suppressed at least 72 h following TPA exposure. Conditioned medium isolated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT proliferation for approximately 72 h, but suppressed MCF10A-Neo proliferation for <24 h. These studies suggest that TPA quickly activates proteolytic processes in MCF10A-Neo cells leading to the activation of latent TGF beta supplied by the serum in the culture medium. TPA also stimulates the production of an additional cytostatic factor(s) which signals via a mechanism not involving the TGF beta(II) receptor. Lastly, expression of an activated Ha-ras oncogene alters both the types of cytostatic factors produced following TPA treatment and responsiveness to these factors.
Asunto(s)
Antineoplásicos/farmacología , Mama/efectos de los fármacos , Mama/metabolismo , Genes ras/fisiología , Proteínas Proto-Oncogénicas c-mos/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Antineoplásicos/antagonistas & inhibidores , Aprotinina/farmacología , Mama/citología , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Transformada , Medios de Cultivo Condicionados , Interacciones Farmacológicas , Humanos , Proteína Oncogénica p21(ras)/biosíntesis , Proteína Oncogénica p21(ras)/fisiología , Inhibidor 1 de Activador Plasminogénico/farmacología , Receptores de Estrógenos/fisiología , Inhibidores de Serina Proteinasa/farmacología , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Transformación Genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Cultured cells are commonly exposed to trypsin-containing solutions in order to prepare cell suspensions suitable for subculture. Conditions used to release and disperse monolayers of cultured murine hepatoma 1c1c7 and human breast epithelial MCF10A cells caused the loss (40-95%) of cellular glutathione (GSH), but did not affect viability. Glutathione contents returned to pretrypsinization values within 24 h of replating. In contrast, the GSH contents of cultured rat hepatoma 5L cells were not affected by trypsinization. Exposure of 1c1c7 cultures to H(2)O(2) or etoposide 1 or 24 h after replating resulted in concentration-dependent cytostatic and cytotoxic effects. The concentration-response curves defining the cytostatic and cytotoxic effects of etoposide, and the cytostatic effects of H(2)O(2) were not influenced by the timing of toxicant addition. However, 1c1c7 cultures treated with H(2)O(2) 1 h after replating were more susceptible to the cytotoxic actions of the peroxide than cultures treated 24 h after plating. These studies show that conditions commonly used for the passaging of cultured cells can lead to a transient, but profound loss of GSH in some cell lines. Furthermore, the outcome of cytotoxicity analyses can be influenced by the time elapsed between the plating of cultures and the addition of toxicant.
Asunto(s)
Glutatión/metabolismo , Células Tumorales Cultivadas/patología , Animales , Antineoplásicos/toxicidad , Neoplasias de la Mama/patología , Carcinoma Hepatocelular/patología , Comunicación Celular , Técnicas de Cultivo de Célula/métodos , Muerte Celular , Etopósido/toxicidad , Femenino , Glutatión/farmacocinética , Humanos , Peróxido de Hidrógeno/toxicidad , Neoplasias Hepáticas/patología , Oxidantes/toxicidad , Ratas , Pruebas de ToxicidadRESUMEN
Studies with mouse leukemia L1210 cells revealed that selective lysosomal photodamage caused by any of three photosensitizing agents was followed by a gradual loss of the mitochondrial membrane potential (delta psi m), release of cytochrome c into the cytosol, increased DEVDase activity (a measure of levels of caspase-3) and a limited apoptotic response. Similar effects were observed in the murine hepatoma 1c1c7 cell line. Immunofluorescence techniques employing 1c1c7 cells demonstrated the immediate release of the lysosomal enzyme cathepsin B following lysosomal photodamage. These studies suggest that the cytotoxic effects of lysosomal photodamage are initiated by released lysosomal proteases that either directly and/or indirectly activate caspases as a consequence of the induction of mitochondrial damage.
Asunto(s)
Apoptosis/efectos de los fármacos , Lisosomas/efectos de los fármacos , Fármacos Fotosensibilizantes/toxicidad , Animales , Apoptosis/efectos de la radiación , Catepsina B/análisis , Membranas Intracelulares/efectos de los fármacos , Leucemia L1210 , Luz , Neoplasias Hepáticas Experimentales , Lisosomas/enzimología , Lisosomas/efectos de la radiación , Potenciales de la Membrana/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Células Tumorales CultivadasRESUMEN
Ursodeoxycholic acid (UDCA) protects cells from the apoptotic effects of hydrophobic bile acids and some other cytotoxic agents. We observed the opposite result when assessing the effects of UDCA on the apoptotic response to mitochondrial photodamage induced by photodynamic therapy (PDT). Two photosensitizers with predominantly mitochondrial specificity were used: a porphycene we have designated CPO; and the tin etiopurpurin SnET2. UDCA potentiated the loss of mitochondrial potential, release of cytochrome c into the cytosol, activation of caspase-3, and apoptotic cell death after irradiation of photosensitized murine leukemia L1210 or hepatoma 1c1c7 cells. These effects were not observed when UDCA was added after irradiation. Glyco-UDCA and tauro-UDCA, conjugated forms of UDCA that are formed in vivo, were as effective as UDCA in promoting PDT phototoxicity. Because UDCA does not act by enhancing intracellular accumulation of the photosensitizing agents used in this study, we propose that the mode of action of UDCA involves the sensitization of mitochondrial membranes to photodamage. UDCA is used currently in gastroenterology for several indications. The drug may offer a means for promoting the efficacy of PDT with minimal adverse effects.