RESUMEN
BACKGROUND: Cardamom (Elettaria cardamomum) is a spice and exhibits potent antioxidant and biological activities through distinct molecular mechanisms. However, the anticancer effect of cardamom was not explored yet in Ehrlich solid tumor (EST)-bearing mice. OBJECTIVES: This investigation was aimed to evaluate the anti-cancer effects of green cardamom (GCar) alone or combined with the anti-cancer drug cyclophosphamide in an in vivo model to explore its mechanistic role in tumor cell death in EST-bearing mice. METHODS: Ehrlich ascites tumor cells were injected in the mice and 5 days later the animals treated with GCar and/or cyclophosphamide for 10 days. Twenty-four hours from the last treatment, animals were sacrificed for the different measurements. RESULTS: Data recorded for tumor size, percentage of tumor growth inhibition, tumor growth delay and mean survival time of EST-bearing mice demonstrated the effective role of GCar alone or combined with CPO as a promising anti-cancer agent because it reduced tumor size. GCar elevated the mean survival time of EST-bearing mice compared to that of untreated EST and EST + CPO groups. Analysis of qPCR mRNA gene and protein expression revealed that GCar alone or combined with CPO were promising anticancer agents. After the treatment of EST with GCar, the apoptotic-related genes and proteins were significantly modulated. GCar induced markedly significant decreases in oxidative stress biomarkers and a significant increment in glutathione levels and that of antioxidant enzymes. With a marked diminish in liver and kidney function biomarkers. CONCLUSION: The results revealed that GCar could serve as an apoptotic stimulator agent, presenting a novel and potentially curative approach for cancer treatment, inducing fewer side effects than those of the commercially used anti-cancer drugs, such as CPO.
Asunto(s)
Antineoplásicos , Carcinoma de Ehrlich , Ciclofosfamida , Elettaria , Extractos Vegetales , Animales , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Peso Corporal/efectos de los fármacos , Carcinoma de Ehrlich/química , Carcinoma de Ehrlich/patología , Ciclofosfamida/farmacología , Ciclofosfamida/toxicidad , Masculino , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/química , Neoplasias Experimentales/patología , Extractos Vegetales/farmacología , Extractos Vegetales/toxicidad , Semillas/químicaRESUMEN
Such biological parameters as tumor volume, Ki-67 and p53, which characterize the development of ascites and solid tumor of Ehrlich were evaluated. The kinetic curve of growth of ascites tumor was S-shaped (Gomperts) while that of the solid one--cubic (Speer-Retsky). Ki-67 expression level was found to be in cyclic correlation with duration (3 and 6 day intervals) which might be worth considering when working out therapeutic procedure. Moreover, no increase in cell death was observed when tumor growth slowed down.
Asunto(s)
Ascitis/patología , Biomarcadores de Tumor/análisis , Carcinoma de Ehrlich/patología , Animales , Ascitis/metabolismo , Ascitis/fisiopatología , Carcinoma de Ehrlich/química , Carcinoma de Ehrlich/fisiopatología , Femenino , Antígeno Ki-67/análisis , Cinética , Ratones , Factores de Tiempo , Proteína p53 Supresora de Tumor/análisisRESUMEN
Multidrug resistance (MDR) is a major obstacle to successful application of cancer chemotherapy and also a basic problem in cancer biology. Studies on the molecular basis of MDR have revealed that a number of proteins over express in multidrug resistant cells viz., multidrug resistant MDR1 gene product P-glycoprotein, the multidrug resistance-associated protein (MRP) and enzymes associated with the glutathione (GSH) metabolism. Decreased expression or altered activity of topoisomerase II has also been implicated in MDR. In the present investigation a number of changes in phase II detoxification parameters have been noticed in drug resistant cells but the novel aspect of the present report is the observation that the metal copper is involved in drug resistance. Although copper plays important roles in many human and other biological systems and even in the treatment of cancer but the relation of Cu and drug resistance has not so far been studied in detailed. The present report describes the novel findings that the level of copper increases with the development of drug resistance in Ehrlich ascites carcinoma and in Lewis lung carcinoma cells and also in serum of mice bearing drug resistant cancer cells compared to mice bearing drug sensitive cells; the work indicates the important aspect of treating drug resistant cancer patients by lowering Cu level in the cancerous cells and serum prior to treatment.
Asunto(s)
Carcinoma de Ehrlich/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Cobre/sangre , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Animales , Carcinoma de Ehrlich/sangre , Carcinoma de Ehrlich/química , Carcinoma Pulmonar de Lewis/sangre , Carcinoma Pulmonar de Lewis/química , Catalasa/análisis , Catalasa/metabolismo , Línea Celular Tumoral , Cobre/química , Progresión de la Enfermedad , Riñón/química , Riñón/enzimología , Hígado/química , Hígado/metabolismo , Masculino , Ratones , Relación Estructura-Actividad , Superóxido Dismutasa/análisis , Superóxido Dismutasa/metabolismoRESUMEN
The action of 12-O-tetradecanoyl-13-acetate (TPA) in vitro in a wide range of concentration from 10(-3) mol/l down to ultra-low doses 10(-23) mol/l and dilution 10(-24) mol/l on the microsome membranes isolated from tumor--Ehrlich ascite carcinoma (EAC) has been studied by ESR-method using two spin probes: 5- and 16-doxyl stearates (5- and 16-DS) localized in the different regions of lipid bilayer. From the ESR spectra obtained it was calculated the following parameters: an order of the long axis 5-DS (S) related to order of the fatty acids chains in the lipid bilayer; two rotation correlation times (Tc1 and Tc2) of 16-DC to estimate a microviscosity value and structural-sensitive ones. It was found the stage changes of all these parameters (increase and decrease) as compared with control level (the membranes untreated by TPA) depending on TPA concentration into the range of 10(-3)-10(-24) mol/l; in particular, the most significant shape changes of structural-sensitive parameters have been observed at TPA doses below 10(-16) mol/l. It is concluded that tumor membranes are very sensitive to TPA action in vitro in a wide range of concentration included ultra-low doses.
Asunto(s)
Membrana Celular/efectos de los fármacos , Metabolismo de los Lípidos , Microsomas/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Animales , Carcinoma de Ehrlich/química , Membrana Celular/metabolismo , Óxidos N-Cíclicos/química , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Lípidos/química , Ratones , Ratones Endogámicos , Microsomas/química , Microsomas/metabolismo , Marcadores de Spin , Células Tumorales CultivadasRESUMEN
The paper will reflect on how Ussing has affected my own scientific work and how he created much of the framework within which I have been working. I have used five examples: (i) The first description of a 1:1 exchange diffusion was introduced by Ussing in 1947 and has been found to be of great physiological significance in most cells. We found that Cl- transport in Ehrlich ascites tumor cells (EATC) was completely dominated by an exchange diffusion process, as defined by Ussing, and, thus, the Cl- conductance was much lower than previously estimated from measurements of unidirectional tracer fluxes. This had a major influence on my later description of a swelling-activated Cl- conductance. (ii) The pump-leak steady-state concept for cell volume control was introduced by Krogh in 1946, but it was developed in detail by Leaf and Ussing in 1959. This concept was the basis for me and others, when we later found that the passive ion leaks play an active role in cell volume control. (iii) The use of isotopes and Ussing's famous flux ratio equation provided an ingenious instrument for distinguishing the various transport routes. We used this to identify the Na,K,2Cl cotransport system as accounting for maintaining a [Cl-]i in the EATC far above thermodynamic equilibrium, as well as accounting for the ion uptake during a regulatory volume increase (RVI) in EATC, similar to what Ussing had found in frog skin. (iv) Short-circuit current setup in the Ussing chamber is still used in laboratories around the world to study ion transport across epithelia. A few results on Cl- transport across the operculum epithelium of the small eurohaline fish Fundulus heteroclitus mounted in small Ussing chambers are presented. (v) Shrinkage-activated Na+ conductance and its possible role in isotonic secretion in frog skin glands is finally discussed.
Asunto(s)
Canales de Cloruro/farmacocinética , Canales de Potasio/metabolismo , Canales de Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Transporte Biológico , Carcinoma de Ehrlich/química , Carcinoma de Ehrlich/metabolismo , Membrana Celular/fisiología , Difusión , Conductividad Eléctrica , Células Epiteliales/metabolismo , Retroalimentación , Transporte Iónico/fisiología , Ratones , Modelos Biológicos , Técnicas de Placa-Clamp/métodos , Agua/metabolismoRESUMEN
Lipopolysaccharide fraction isolated from Ehrlich ascites carcinoma (E-LPS) was investigated as an antitumor agent against human leukemia cell ML-2. Marked cell growth inhibition was observed with ML-2 cell accompanied by inhibition of DNA synthesis and perturbation of cell cycle. Induction of differentiation in treated ML-2 cells was observed as indicated by morphological maturation, NBT reducing activity and indirect immunofluorescence.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Ehrlich/metabolismo , Leucemia Mieloide/patología , Lipopolisacáridos/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Animales , Antineoplásicos/aislamiento & purificación , Carcinoma de Ehrlich/química , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , ADN de Neoplasias/biosíntesis , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente , Inhibidores de Crecimiento/farmacología , Humanos , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/metabolismo , Lipopolisacáridos/aislamiento & purificación , Ratones , Trasplante de NeoplasiasRESUMEN
The K+ and Cl- currents activated by hypotonic cell swelling were studied in Ehrlich ascites tumour cells using the whole-cell recording mode of the patch-clamp technique. Currents were measured in the absence of added intracellular Ca2+ and with strong buffering of Ca2+. K+ current activated by cell swelling was measured as outward current at the Cl- equilibrium potential (ECl) under quasi-physiological gradients. It could be abolished by replacing extracellular Na+ with K+, thereby cancelling the driving force. Replacement with other cations suggested a selectivity sequence of K+ > Rb+ > NH4 approximately Na+ approximately Li+; Cs+ appeared to be inhibitory. The current-voltage relationship of the volume-sensitive K+ current was well fitted with the Goldman-Hodgkin-Katz current equation between -130 and +20 mV with a permeability coefficient of around 10(-6) cm s(-1) with both physiological and high-K+ extracellular solutions. The class III antiarrhythmic drug clofilium blocked the volume-sensitive K+ current in a voltage-independent manner with an IC50 of 32 microM. Clofilium was also found to be a strong inhibitor of the regulatory volume decrease response of Ehrlich cells. Cell swelling-activated K+ currents of Ehrlich cells are voltage and calcium insensitive and are resistant to a range of K+ channel inhibitors. These characteristics are similar to those of the so-called background K+ channels. Noise analysis of whole-cell current was consistent with a unitary conductance of 5.5 pS for the single channels underlying the K+ current evoked by cell swelling, measured at 0 mV under a quasi-physiological K+ gradient.
Asunto(s)
Carcinoma de Ehrlich/química , Carcinoma de Ehrlich/metabolismo , Activación del Canal Iónico/fisiología , Canales de Potasio/fisiología , Potasio/metabolismo , Animales , Antiarrítmicos/farmacología , Artefactos , Carcinoma de Ehrlich/patología , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Conductividad Eléctrica , Estimulación Eléctrica , Electrofisiología , Soluciones Hipotónicas/farmacología , Activación del Canal Iónico/efectos de los fármacos , Ratones , Ratones Endogámicos , Compuestos de Amonio Cuaternario/farmacologíaRESUMEN
Our earlier studies have shown that removal of various blocking factors from the sera of tumor-bearing animals and humans by adsorption over heat-attenuated and formalin-fixed-Staphylococcus aureus Cowan I (SAC) containing Protein A (PA) causes antitumor immune response. It was also shown that this procedure caused regression of a wide variety of established animal and human tumors. In the present investigation, the therapeutic potential of inoculation of ascites fluid adsorbed in vitro over non-viable SAC containing PA has been demonstrated in Ehrlich' s ascites tumor (EAT) in mouse. The antitumor effect was evident by a significant decrease in body weight (p<0.001) as well as significant reduction in viability of ascites tumor cells (p<0.001) in peritoneal cavity. However, some of the responding animals died earlier than controls, this may be due to the toxicity associated with therapy. The toxic effects were evident in decreased contents of glutathione, and increased activity of glutathione-S-transferase, decreased activity of microsomal enzymes and also in an early death of some of tumor regressed animals. The probable causes of toxicity of the therapy and prospects of reversing these toxic effects are discussed.
Asunto(s)
Carcinoma de Ehrlich/química , Carcinoma de Ehrlich/metabolismo , Proteína Estafilocócica A/metabolismo , Staphylococcus aureus/química , Albinismo , Animales , Carcinoma de Ehrlich/tratamiento farmacológico , Carcinoma de Ehrlich/mortalidad , Supervivencia Celular/efectos de los fármacos , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Ratones , Microsomas/efectos de los fármacos , Microsomas/enzimología , Oxigenasas de Función Mixta/metabolismo , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/uso terapéutico , Proteínas de Neoplasias/toxicidad , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
Numerous clinical studies with the pentavalent technetium complex of dimercaptosuccinic acid [Tc(V)-DMS] seem to indicate its new role in nuclear oncology. Thus, we questioned what properties of the Tc(V)-DMS molecule associate with its tumoral tissue accumulation. Because studies have reported tumor tissue to be more acidic than normal tissue, acidification might be related to the Tc(V)-DMS localization in tumor tissue. Thus, in the present study, a working hypothesis drew to test the acidification as a plausible factor, and various analytical methods and an in vitro cellular system using Ehrlich ascites tumor cells (EATC) implemented. Analytical methodologies demonstrated the decrease of the overall negative charge of the Tc(V)-DMS molecule, promoted by the acidification of the analytical medium and the sample dilution. In the in vitro cellular experiment, acidification alone showed no effect on the radioactivity accumulation in EATC; nevertheless, if accompanied by a pre-dilution of the Tc(V)-DMS sample added into the cell incubation media, cellular radioactivity accumulation was observed. Thus, acidification as a mediator for the Tc(V)-DMS accumulation in tumoral cells, concurrently with dilution as the promoter of the process, constituted the foundation for discerning the working hypothesis.
Asunto(s)
Ácido Dimercaptosuccínico de Tecnecio Tc 99m/química , Ácido Dimercaptosuccínico de Tecnecio Tc 99m/metabolismo , Ácido Acético/química , Ácidos/química , Ácidos/metabolismo , Animales , Transporte Biológico , Carcinoma de Ehrlich/química , Carcinoma de Ehrlich/metabolismo , Carcinoma de Ehrlich/patología , Cromatografía en Gel , Cromatografía en Capa Delgada , Electroforesis en Acetato de Celulosa/métodos , Concentración de Iones de Hidrógeno , Ligandos , Masculino , Ratones , Ratones Endogámicos , Modelos Químicos , Neoplasias Experimentales , Factores de TiempoRESUMEN
Occasionally new and intriguing roles arise for proteins with well established functions. The alpha-1 serum protease inhibitor (alpha-1 PI) represents another example. Sequence identities exist in the alpha-1 PI and in a nuclear 52-kDa glycoprotein which is involved in resistant DNA-polypeptide complexes. The results of Western blots support the identity of the two proteins and immunocytochemical studies indicate the nuclear location of the alpha-1 PI. Consistently, e.g. Ehrlich ascites tumor cells express the alpha-1 PI, and the fusion protein between the alpha-1 PI and the green fluorescent protein from Aequorea victoria shows intracellular accumulation and partly nuclear location.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , alfa 1-Antitripsina/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Carcinoma de Ehrlich/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/ultraestructura , Endopeptidasa K/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Dodecil Sulfato de Sodio/farmacología , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/aislamiento & purificación , alfa 1-Antitripsina/ultraestructuraRESUMEN
The effect of annexins II, III and V, purified from different species, on the calcium-activated chloride current across the stage-V to stage-VI Xenopus laevis oocyte membrane was tested either directly, using calcium entry mediated by depolarization, by A23187 permeabilization of oocytes or indirectly by quisqualate stimulation of a metabotropic glutamate receptor in the membrane expressed by the oocyte after injection of mRNA. The annexins isolated from the Ehrlich ascites cell, which is a mouse tumor cell, were found to be potent inhibitors of the chloride current, showing half-maximal inhibition at 50 nM, whereas no block was found using bovine or porcine annexins isolated from lung tissue. Of the annexins tested, we found annexin III to be naturally occurring in the oocyte, while only trace amounts of annexins II and V could be demonstrated. The inhibition pattern varied somewhat according to the stimulus method, the inhibition being more complete when an indirect stimulus via the metabotropic receptor was applied compared to a direct calcium stimulus.
Asunto(s)
Anexinas/farmacología , Canales de Cloruro/antagonistas & inhibidores , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Animales , Anexina A2/aislamiento & purificación , Anexina A2/farmacología , Anexina A3/aislamiento & purificación , Anexina A3/farmacología , Anexina A5/aislamiento & purificación , Anexina A5/farmacología , Anexinas/aislamiento & purificación , Anexinas/metabolismo , Calcimicina/farmacología , Calcio/metabolismo , Calcio/farmacología , Carcinoma de Ehrlich/química , Bovinos , Femenino , Técnicas In Vitro , Ionóforos/farmacología , Pulmón/química , Potenciales de la Membrana , Ratones , Especificidad de la Especie , Porcinos , Xenopus laevisRESUMEN
Phosphofructokinase (PFK) subunits and isozymes have been examined in ascites tumor cells and murine mammary gland, the tissue from where this tumor originated. Ascites tumor was found to contain predominantly the C-type subunit, whereas the L-type subunit was more abundant in mammary gland. An altered M-type subunit of lower electrophoretic mobility was found in both cell types and no M4 homotetramers were detected in either of them. Characteristic regulatory properties of ascites tumor PFK, i.e. insensitivity to fructose-1,6-P2 activation and inhibition by P-enolpyruvate, were also observed in the mammary gland isozyme. The nature of these properties and the contribution of the distinct subunit types to fructose-1,6-P2 activation are discussed.
Asunto(s)
Carcinoma de Ehrlich/enzimología , Isoenzimas/química , Glándulas Mamarias Animales/enzimología , Fosfofructoquinasa-1/química , Animales , Carcinoma de Ehrlich/química , Activación Enzimática/efectos de los fármacos , Fructosadifosfatos/farmacología , Isoenzimas/efectos de los fármacos , Masculino , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de los fármacos , Ratones , Fosfoenolpiruvato/farmacología , Fosfofructoquinasa-1/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
We have described a simple and rapid chromatographic method for the analytical and preparative separation of major types of ribosomal ribomononucleotides with Dowex 1-X10 (HCOO-, 37-74 microns) and Dowex 2-X10 (HCOO-, 37-74 microns) columns, by desorption with formiate solutions in 1-2 h. The separation has been achieved for Cp, Ap, Up and Gp, while a mixture of 2'-, and 3'-nucleoside phosphates desorbs as a single peak; with both resins, a successful separation was achieved with a load from 25 micrograms to 1 mg of ribomononucleotide mixture per ml of packed resin. A complete separation was achieved with Dowex 1, while the separation with Dowex 2 resin was even better. The resins cannot separate unusual nucleosides; therefore, our method is suitable for studies of ribonucleic acids with a low content of unusual nucleosides. Our method has been applied for the quantitative determination of the ribomononucleotide composition of 18S and 28S rRNAs, isolated from mammalian tissues: rat liver, mouse kidney and Ehrlich ascites cells. Dowex 1 and Dowex 2 resins afforded similar or identical ribomononucleotide compositions in all cases; analytical data were in agreement with the literature data. Our method is competitive, in several respects, with modern HPLC techniques for the separation of ribomononucleotides.
Asunto(s)
ARN Ribosómico/química , Ribonucleótidos/aislamiento & purificación , Ribosomas/química , Animales , Resinas de Intercambio Aniónico/metabolismo , Carcinoma de Ehrlich/química , Cromatografía por Intercambio Iónico , Hidrólisis , Riñón/química , Hígado/química , Ratones , ARN Ribosómico/análisis , ARN Ribosómico 18S/química , ARN Ribosómico 28S/química , Ratas , Resinas Sintéticas , Ribonucleótidos/análisisRESUMEN
We studied the effects of theanine, a component of green tea leaves, on the antitumor activity of adriamycin (ADR) from the biochemical modulation view point. In vitro, theanine inhibited the ADR efflux from Ehrlich ascites carcinoma cells and maintained the ADR concentration in tumor cells. Theanine enhanced the inhibitory effect of ADR on tumor growth by 2.1-fold in vivo, and increased 2.9-fold the ADR concentration in the tumor, compared to the ADR alone group. An increase in ADR concentration was not observed in normal tissues, such as the heart and liver. Theanine did not enhance, rather tended to normalize the increase of lipid peroxide level and reduction of glutathione peroxidase activity as indicators of the ADR-induced side toxicity.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Doxorrubicina/farmacología , Glutamatos/farmacología , Animales , Antibióticos Antineoplásicos/análisis , Cafeína/farmacología , Carcinoma de Ehrlich/química , Carcinoma de Ehrlich/tratamiento farmacológico , Doxorrubicina/análisis , Glutatión Peroxidasa/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Técnicas In Vitro , Riñón/efectos de los fármacos , Riñón/metabolismo , Peróxidos Lipídicos/metabolismo , Hígado/química , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Miocardio/química , Miocardio/enzimologíaRESUMEN
Monitoring cell activation processes and cell cycle progression in cells of various ontogenetic and phylogenetic origins is one of the base challenges in cellular biology. Flow cytometric analysis by DNA histograms show the cell cycle distribution and indicate the S-phase fractions of cell populations, as well as those cells in Go/G1 and G2/M phases. Human and murine normal diploid cells, K562 tumor cells (human origin) and Ehrlich cells (murine origin) were analysed using a propidium iodide staining procedure for flow cytometry. Our preliminary results suggested that this method may be a useful tool to detect cell populations with quantitative changes in DNA content, and to estimate cell fractions.
Asunto(s)
Carcinoma de Ehrlich/patología , Ciclo Celular , ADN de Neoplasias/análisis , Citometría de Flujo/métodos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Aneuploidia , Animales , Carcinoma de Ehrlich/química , División Celular , Colorantes Fluorescentes , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucocitos Mononucleares/química , Leucocitos Mononucleares/citología , Ratones , Propidio , Células Tumorales Cultivadas/químicaRESUMEN
Differences in the nature of the gangliosides present in two types of Ehrlich ascites tumour (EAT) cells, the adherent and non-adherent EAT cells, were studied. Gangliosides were isolated by DEAE Sephadex column chromatography and analysed by high-performance thin-layer chromatography (HPTLC). The non-adherent EAT (na-EAT) cells which grow in the peritoneal cavity of mice were selected for growth on basement membrane and tissue culture plastic to give the adherent EAT (a-EAT) cells. na-EAT cells contained 1.57 nmol lipid-bound sialic acid per mg protein and at least 12 different gangliosides, including major gangliosides such as GM3, GM2, GM1, GD3, GD1a and GT1b. On the other hand, the ganglioside pattern of a-EAT cells differed significantly from that of na-EAT cells, both quantitatively and qualitatively. The content of lipid-bound sialic acid in a-EAT cells was only 0.24 nmol per mg of protein. The gangliosides in a-EAT cells were characterized as GD1a and trisialogangliosides and, significantly, a-EAT cells did not contain monosialogangliosides. Neutral glycolipids were isolated from both cell lines and their patterns were compared. In contrast to the gangliosides pattern, their neutral glycolipid patterns were similar. Glucosylceramide and lactosylceramide were the major components in both types of cells. In addition to na- and a-EAT cells, a-EAT cells were passaged in mice by intraperitoneal injection, giving rise to a third variant (c/m EAT cells). We analysed the gangliosides in c/m EAT cells to determine whether there was a change in the ganglioside pattern found in na-EAT cells. After repeated passage of c/m EAT cells in mice, the pattern of gangliosides shifted to that of na-EAT cells. Alterations of ganglioside composition may be associated with the growth environment of the murine peritoneal cavity; alternatively, a selection process may have occurred.
Asunto(s)
Carcinoma de Ehrlich/química , Gangliósidos/química , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Cromatografía en Capa Delgada , Femenino , Gangliósidos/aislamiento & purificación , Variación Genética , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Células Tumorales CultivadasRESUMEN
Lactate transport is mediated in most tissues by H+-monocarboxylate-- cotransporters (MCTs). We have cloned and sequenced the lactate transporter from Ehrlich Lettré tumour cells by using the polymerase chain reaction (PCR) to amplify MCT1-related sequence from cDNA. The sequence is 93% and 87% identical to MCT1 from Chinese hamster and human respectively and so represents mouse MCT1. Most differences between MCT1 from Chinese hamster and mouse are conservative substitutions, located in hydrophilic parts of the molecule. Specific antipeptide antibodies confirm the presence of MCT1 protein in membranes from Ehrlich Lettré tumour cells. One difference between the mouse and Chinese hamster MCT1 is the absence of a predicted external consensus sequence for N-linked glycosylation in the mouse sequence. Using N-glycanase-F treatment and an in vitro translation system, we provide evidence that this glycosylation site is not actually utilised in Chinese hamster MCT1. These results are discussed in relation to current understanding of the roles of glycosylation of membrane proteins.
Asunto(s)
Carcinoma de Ehrlich/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Membrana Celular/química , Clonación Molecular , Cricetinae , Cricetulus , Glicosilación , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Ratones , Microsomas/metabolismo , Datos de Secuencia Molecular , Transportadores de Ácidos Monocarboxílicos , Alineación de Secuencia , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
We have previously reported [McCormick and Johnstone (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881] the partial purification of the Na(+)-dependent A-system amino acid transporter from Ehrlich cell plasma membranes and have suggested that a 120-130 kDa peptide, a major component of the purified fraction [octyl glucoside (OG) extract], is involved in Na(+)-dependent amino acid transport. In the present study, N-terminal sequence analysis of the 120-130 kDa peptide revealed a sequence similar to that of the alpha 3 subunit of the integrin alpha 3 beta 1. The presence of alpha 3 beta 1 was confirmed by Western blots of the OG extract probed with anti-alpha 3 or -beta 1 antibodies. Western blots also showed that an antibody originally raised against the 120-130 kDa peptide crossreacts with both the alpha 3 and beta 1 integrin subunits. Co-purification of alpha 3 beta 1 and Na(+)-dependent transport activity suggested that the two activities might be associated. Evidence that alpha 3 plays a role in transport is shown by the fact that an antibody against human alpha 3, but not beta 1, removed transport activity (approximately 25% loss) from cholate-solubilized Ehrlich membranes. Further purification of OG extracts using concanavalin A and wheat-germ lectin columns resulted in the separation of transport activity from the bulk (but not all) of alpha 3 beta 1 integrin without loss of the transport activity. These results indicate that the integrin itself is not essential for amino acid transport. Reconstitution of a purified alpha 3 beta 1-depleted protein fraction showed high levels of Na(+)-dependent, alpha-methylaminoisobutyric-acid-inhibitable amino acid transport in proteoliposomes, whereas reconstituted integrin alone showed little transport activity. However, in the integrin-depleted fractions, high amino acid uptake occurred in K+ which compromised the accurate measurement of the Na(+)-dependent component of uptake. The data suggest that alpha 3 may be associated with the A-system transporter and may modulate the activity of this carrier. Moreover, transfection of K562 and RD cells with human alpha 3 and alpha 2 cDNA showed that the former but not the latter increased A-system transport, thus providing more direct evidence that alpha 3 may modulate A-system transport activity.
Asunto(s)
Carcinoma de Ehrlich/química , Proteínas Portadoras/análisis , Integrinas/análisis , Proteínas de la Membrana/análisis , Sistemas de Transporte de Aminoácidos , Animales , Carcinoma de Ehrlich/ultraestructura , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Membrana Celular/química , Humanos , Integrina alfa3beta1 , Proteínas de la Membrana/química , Peso Molecular , Pruebas de Precipitina , Células Tumorales CultivadasRESUMEN
We have studied by high resolution in situ light and electron microscopic lectin-gold techniques the subcellular distribution of alpha-D-Gal residues using the Griffonia simplicifolia I-B4 isolectin and compared it with that of beta-D-Gal residues as detected with the Datura stramonium lectin in Ehrlich tumour cells grown as ascites or monolayer. The microvillar but not the smooth plasma membrane regions were labelled with the Griffonia simplicifolia I-B4 isolectin whereas both plasma membrane regions were equally well labelled with the Datura stramonium lectin. Elements of the endocytotic/lysosomal system such as coated membrane invaginations and vesicles, early and late endosomes and secondary lysosomes were positive for both alpha-D-Gal and beta-D-Gal residues. A particular feature of Ehrlich tumour cells is an elaborate tubular membrane system located in the pericentriolar region which is labelled throughout by both lectins and represents part of the endosomal system. In the Golgi apparatus labelling with both lectins was observed to commence in trans cisternae which is indirect evidence for a joint distribution of the sequentially acting beta 1,4 and alpha 1,3-galactosyl-transferases.
Asunto(s)
Carcinoma de Ehrlich/química , Galactosa/análisis , Glicoconjugados/análisis , Lectinas/metabolismo , Lectinas de Plantas , Animales , Carcinoma de Ehrlich/ultraestructura , Oro Coloide , Histocitoquímica , Microscopía ElectrónicaRESUMEN
Ehrlich carcinoma (EC) cells isolated from mice at different phases of ascites growth were exposed to hyperthermia (44 degrees C), or oxidative stress (hydrogen peroxide or vikasol), or ATP depletion induced by rotenone. These exposures caused protein aggregation and rapid necrotic death in exponentially growing EC cells. On the contrary, the same cell culture at stationary phase of growth became considerably more resistant to all the above cytotoxic treatments, and the level of aggregated protein was significantly lower in stressed stationary EC cells than that in exponential ones. Comparative immunoblotting has revealed the unexpected expression of inducible 70 kDa heat-shock protein form (HSP68), as well as accumulation of HSP27 and HSP90 in the thermo- and drug-resistant stationary EC cells. It is suggested that the in vivo occurring HSP overexpression in stationary EC cells is an adaptive modulation of the tumor cell phenotype to maintain the viability of ascites EC cells under chronic deficiency of oxygen and nutrients.