RESUMEN
We have shown that antioxidant N-acetylcysteine (NAC, 2-10 mM) quickly (for 2 hours) and completely inactivates the activity of matrix metalloproteinases (gelatinases MMP-2 and MMP-9, and collagenases MMP-1 and MMP-8) secreted by transformed mouse fibroblasts 3T3-SV40 into the medium. The same MMP inhibition took place in the cell-free conditioned medium of HT-1080 fibroblasts, which suggests a direct chemical interaction between NAC and MMP resulting in the loss of MMP activity. Besides inhibitory effect, NAC decreased MMP-1 and MMP-9 (but not MMP-2) production in the cell medium. However, the level of MMP-1 and MMP-9 inhibitor (TIMP-1) remained normal, indicating a shift in the balance between the enzyme and inhibitor. The correlation between MMP-2 level and tissue enzyme inhibitor TIMP-2 was similar in control and NAC treated cells. At the same time, reorganization of type I collagen at the cell surface occurred. All together permits the conclusion that NAC action results in the extracellular matrix remodeling and changing in cellular functions.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Matriz Extracelular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Animales , Línea Celular Transformada , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Matriz Extracelular/enzimología , Matriz Extracelular/ultraestructura , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 8 de la Matriz/genética , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Células 3T3 NIH , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
We studied the changes in the level of reactive oxygen species (ROS) in normal (3T3) and transformed (3T3-SV40) murine fibroblasts under the antioxidant action for 15 min using fluorescent probe carbo- xy-H2DCFDA. We have shown that N-acetylcysteine (NAC) decreased ROS level in both cellular types. Another antioxidant, alpha-lipoic acid (ALA), and its reduced form, dihydrolipoic acid (DHLA), caused the prooxidant effects. Both ALA and DHLA in the concentration range of 0.1-1.25 mM increased ROS level in dose dependent manner in both cellular types. The ability of ALA and DHLA to activate hydrogen peroxide production is discussed.
Asunto(s)
Antioxidantes/farmacología , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Animales , Línea Celular Transformada , Fluoresceínas , Colorantes Fluorescentes , Peróxido de Hidrógeno/farmacología , Ratones , Células 3T3 NIH , Especies Reactivas de Oxígeno/agonistas , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Ácido Tióctico/análogos & derivados , Ácido Tióctico/farmacologíaRESUMEN
In vitro, we studied the effect of melatonin (1mkM) on the sensitivity of murine transformed fibroblasts 3T3-SV40 and murine hepatoma cells MN22a to the lysis by natural killer cells. In vivo, we examined changes in tumorigenic properties of MN22a cells treated with melatonin. It was shown that the sensitivities of both cell types to lysis by killer cells fell sharply fourfold after 24-hour treatment with melatonin; their cytotoxic index value approximated to that of intact cells, insensitive to natural killer cells. After MN22a cells were pre-treated with melatonin for 24 h before injection into syngenic mice, tumorigenesis and development slowed down and mortality rates decreased. Both effects were reversible. All cellular parameters were similar to control ones 24 hrs after evacuation of melatonin. Thus melatonin failed to change cell cycle progression which pointed to an unaltered rate of cell proliferation. Different hypotheses are discussed on the mechanisms of melatonin action which result in transient reversion of transformed phenotype.
Asunto(s)
Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Células Asesinas Naturales/metabolismo , Melatonina/metabolismo , Células 3T3 , Animales , División Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Citotoxicidad Inmunológica/efectos de los fármacos , Fibroblastos/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Endogámicos C3H , Virus 40 de los Simios , Factores de TiempoRESUMEN
We studied the effect of antioxidants such as N-acetylcysteine (NAC, 10 mM) and alpha-lipoic acid (ALA, 1.25 mM) and of the hormone melatonin (1 microM) on the ability of murine hepatoma cells MH22a to develop tumors in syngenic mice (C3HA) after subsutaneous injection. Tumor formation and development slowed down and mouse mortality decreased when the injected cells were pretreated by NAC, ALA or melatonin during 20 h. Melatonin had the most marked effect. Tumors appeared in 100 % cases after 10 days in control mice when untreated cells had been injected; injection of cells pretreated by NAC or ALA resulted in tumor formation only in 40 and 53 % of mice, respectively. When cells were pretreated with melatonin the tumors appeared only in 18-20 days after injection. Until the end of the observation (36 days) 67 % of control mice died, but when the cells were pretreated by NAC or ALA mouse death-rate was 20 and 53 %, respectively. In the case of melatonin we did not observed any dead mice at all. We showed that treatment by antioxidants delayed (NAC) or completely inhibited (ALA) cell cycle of hepatoma cells. Cell cycle was restored after removal of the antioxidants. Melatonin did not change cell cycle phase distribution. We conclude that there is no direct correlation between loss of tumorigenic properties and changing of proliferative activity of hepatoma cells. Different mechanisms of antioxidants and melatonin action resulting in transient tumor phenotype normalization are discussed.
Asunto(s)
Antioxidantes/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Melatonina/farmacología , Acetilcisteína/farmacología , Acetilcisteína/uso terapéutico , Animales , Antioxidantes/uso terapéutico , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/prevención & control , Ciclo Celular/efectos de los fármacos , Citometría de Flujo , Humanos , Inyecciones Subcutáneas , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/prevención & control , Melatonina/uso terapéutico , Ratones , Ratones Endogámicos C3H , Ácido Tióctico/farmacología , Ácido Tióctico/uso terapéutico , Trasplante Isogénico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Embryonic cells regulate the expression of matrix metalloproteinases (MMP) providing remodulation of extracellular matrix, which in turn provides the changes in cell adhesion and migration during the cell development and differentiation. In present work we studied the changes of gelatinases (MMP-2 and MMP-9) and collagenases (MMP-1 and MMP-8) activities in the process of cultivating the primary murine embryonic fibroblasts (MEF). Cultivation was continued for 6 passages, after that the culture died in time. According to gelatin and collagen zymography results, drastic changes of all MMPs activities occurred during the third passage of cell cultivation. The MMP-1 and MMP-9 activity appears in the middle of cultivation and then disappeared at the end. The most important event MEF cultivation is appearance and subsequent reservation of collagenase MMP-8 and active form of gelatinase MMP-2.
Asunto(s)
Embrión de Mamíferos/enzimología , Matriz Extracelular/enzimología , Fibroblastos/enzimología , Animales , Caseínas/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular , Colágeno/metabolismo , Cruzamientos Genéticos , Electroforesis en Gel de Poliacrilamida , Embrión de Mamíferos/citología , Femenino , Fibroblastos/citología , Gelatina/metabolismo , Masculino , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBARESUMEN
Adduced proofs of the telomere shortening are the main or even the sole mechanism of the natural and radiation aging. All apparent contradictions, primary, the absence of exact inverse correlation between residual telomere length and the donor age are explained within the frames of the telomere theory. We try to explain in what way telomere shortening might be the cause of aging and longevity restriction. We also show the inability of the oxidative theory to explain a number of indisputable facts easily explained by the telomere theory, such as unlimited growth of tumor sells or why a new-born child starts to age from zero level rather than the level reached by the cells of his parents at the moment of conception. We postulate that if oxidative damage was entirely absent, telomeres would, nevertheless, shorten with each mitotic cycle because such is the mechanism of DNA replication, and aging would be in progress, which we invariantly observe in the presence of any antioxidants. But if telomeres do not shorten, as happens in transformed cells because telomerase works there, aging does cease and the transformed cells show no senescence. We also observe it in spite of the damaging effect of reactive oxygen species which may be even more than in normal cells.
Asunto(s)
Envejecimiento/fisiología , Envejecimiento/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Telómero/metabolismo , Animales , Senescencia Celular/fisiología , Humanos , Radiación IonizanteRESUMEN
We studied N-acetylcysteine (NAC) ability to change the phenotype properties of several transformed and embryonic cells. We examined human epidermoid carcinoma A431 cells, murine hepatoma MH22a cells, and murine embryonic fibroblasts (MEFs) in terms of the sensitivity to natural killer (NK) recognition and abolishment. We have demonstrated that treatment with NAC (10 mM) results in a loss of susceptibility to NK cell activity by transformed A431 and MH22a cells similar to 3T3-SV40 transformed cells whose partial reversion caused by NAC was revealed by us before. We have shown that MEFs are also sensitive to NK activity and abolished by NK cells as well as transformed cells. MEFs pretreated with 10 mM NAC as well as transformed cells lose their susceptibility to NK cell activity. The loss of cell sensitivity to NK cytolytic activity was accompanied by a reorganization of the actin cytoskeleton and the appearance of well-pronounced stress-fibers.
Asunto(s)
Acetilcisteína/farmacología , Citotoxicidad Inmunológica , Embrión de Mamíferos/efectos de los fármacos , Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Células 3T3 , Actinas/metabolismo , Animales , Línea Celular Transformada , Línea Celular Tumoral , Embrión de Mamíferos/inmunología , Embrión de Mamíferos/ultraestructura , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Humanos , Masculino , Ratones , Fibras de Estrés/metabolismoRESUMEN
The purpose of this study was to compare the effects of two antioxidants, alpha-lipoic acid (ALA) and N-acetylcysteine (NAC) on the sensitivity of 3T3-SV40 fibroblasts to lytic activity of natural killer (NK) cells. ALA (1.25 mM) reduced significantly the fibroblast sensitivity in several hours, whereas NAC (10 mM) did not change it. Subsequent removal of the antioxidants from the cultivation medium resulted in gradual recovery of the sensitivity in the case of ALA and in complete loss of it in the case of NAC. Inactivation of gelatinase MMP-2 (matrix metalloproteinase) using pretreatment of the cells with the inhibitor of MMP, G6001, or specific antibodies to MMP-2 or MMP-9 resulted in decrease of 3T3-SV40 sensitivity to NK cells activity. This effect was similar to that of ALA, not to the NAC one. Pretreatment of NK cells with G6001 did not influence their lytic activity. The results obtained demonstrate that the direct antioxidant, NAC (having reduced thiol groups), and the indirect one, ALA (reducing thiol groups and acting as a direct antioxidant only inside the cell) activate principally different intracellular signal pathways. However, both NAC and ALA pathway includes inactivation of MMP-2.
Asunto(s)
Antioxidantes/farmacología , Transformación Celular Viral/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Células Asesinas Naturales/inmunología , Ácido Tióctico/farmacología , Acetilcisteína/farmacología , Animales , Células 3T3 BALB , Línea Celular Transformada , Transformación Celular Viral/inmunología , Citotoxicidad Inmunológica , Fibroblastos/inmunología , Depuradores de Radicales Libres/farmacología , RatonesRESUMEN
In this study we investigated the effect of alpha-lipoic acid (ALA) in concentration range 0.7-5.0 mM on the intracellular level of reduced glutathione, the cell cycle phase distribution, the structure of microfilaments and microtubules of normal (3T3) and transformed (3T3-SV40) fibroblasts. We obtained that ALA increased the glutathione content in transformed cells, but did not change its level in normal cells, induced cell cycle arrest of 3T3 cells (but not 3T3-SV40 cells), and disrupted actin microfilaments in cells of both lines. The effect of ALA was compared with N-acetylcysteine (NAC) action. The whole complex of findings allows us to affirm that each of these antioxidants acts on its own target molecules in normal and transformed cells and activates different signal and metabolic pathways in these cells. But at the same time the intermediate steps of ALA and NAC action can be common (alteration of the intracellular level of glutathione, reorganization of actin cytoskeleton, etc.).
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Fibroblastos/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Ácido Tióctico/farmacología , Células 3T3 , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular Transformada , Transformación Celular Viral , Fibroblastos/ultraestructura , Glutatión/metabolismo , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Virus 40 de los SimiosRESUMEN
The present work was aimed to examine whether the actin reorganization of 3T3-SV40 cells influences their sensitivity to natural killer (NK) cells activity. The effects of N-acetylcystein (NAC) and latrunculin B, actin depolimerizator, on both cellular parameters were studied. Experiments with NAC demonstrated that 3T3-SV40 sensitivity to NK cells activity remained unchanged under the disordered microfilaments but decreased upon the appearance of structured stress-fibres. The data on latrunculin B action resulted in the opposite conclusion: the more microfilaments disorganization in the presence of latrunculin B the lesser 3T3-SV40 sensitivity to lysis by NK cells. These facts suggest that relations between microfilament integrity in 3T3-SV40 cells and their sensitivity to NK cells are rather independent. The latter confirms our previous conclusion (Gamaley et al., 2006). Decrease in 3T3-SV40 sensitivity to NK cells activity accompanied by actin reorganization resulted from both latrunculin B and NAC action suggests changes in cellular surface, which ultimately lead to inactivation (or loss) of the molecules being activating signals to NK cells.
Asunto(s)
Actinas/fisiología , Línea Celular Transformada/inmunología , Línea Celular Transformada/metabolismo , Células Asesinas Naturales/inmunología , Células 3T3 , Animales , Citotoxicidad Inmunológica , Ratones , Virus 40 de los SimiosRESUMEN
We have shown that 18-20 h cultivation of transformed mouse fibroblasts 3T3-SV40 in the presence of antioxidant, N-acetylcysteine (NAC, 10 mM), did not change their sensitivity to lysis by natural killer (NK) mouse splenocytes. However, in 18-20 h after NAC removal 3T3-SV40 cells demonstrated resistance to NK cell activity. The cytotoxicity index (CI) was reduced up to 4.6 +/- 2.4 % (in comparison with the control value 31.8 +/- 2.4 %) approximating to the value in non-transformed 3T3 mouse fibroblasts. Normal 3T3 cells were resistant to NK action in all experimental conditions (CI varied within 0.7-5.3 %). These results show that NAC can induce partial reversion of transformed phenotype. We suggest that this effect may be due to the NAC-induced modifications of the cell surface and extracellular matrix proteins.
Asunto(s)
Acetilcisteína/farmacología , Citotoxicidad Inmunológica/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Células Asesinas Naturales/inmunología , Células 3T3 , Animales , Línea Celular Transformada , Transformación Celular Viral , Humanos , Células K562 , Ratones , Virus 40 de los Simios , Bazo/inmunologíaRESUMEN
Long-term treatment of transformed 3T3-SV40 mouse fibroblasts with antioxidant N-acetylcysteine decreased cell level of ROS and increased the concentration of reduced glutathione. Removal of N-acetylcysteine from the medium led to the appearance of well-expressed stress fibrils, virtually absent in control cells. In contrast to control cells, these cells were not invaded by apathogenic Escherichia coli A2 strain producing ECP32 protease specifically cleaving actin. Antioxidant N-acetylcysteine can cause partial reversion of transformed phenotype at the expense of a shift of cell redox balance in favor of reduced glutathione.
Asunto(s)
Acetilcisteína/farmacología , Actinas/metabolismo , Transformación Celular Viral/efectos de los fármacos , Endopeptidasas/metabolismo , Escherichia coli/metabolismo , Glutatión/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células 3T3 BALB , Línea Celular Transformada , Transformación Celular Viral/fisiología , Escherichia coli/efectos de los fármacos , Ratones , Microscopía Electrónica , Microscopía Fluorescente , Virus 40 de los SimiosRESUMEN
Effect of antioxidants on actin cytoskeleton in 3T3 fibroblasts and 3T3 fibroblasts transformed with SV40 virus (3T3-SV40 cells) was studied. Antioxidants used were as follows: N-acetyl-L-cysteine (NAC), (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and glutathione in the reduced form (GSH). Both NAC and OTZ are precursors of GSH in the cell, but, in contrast to NAC, OTZ reduces inside the cell forming L-cysteine. The presence of NAC (5-20 mM) in the culture medium of both cell types resulted in loosening of monolayer, fragmentation of stress fibers, and the appearance of amorphous actin structures. As 3T3-SV40 cells contain less actin stress fibers than 3T3 cells, the NAC-induced rearrangements of actin cytoskeleton were stronger in these cells than in 3T3 cells. In contrast to NAC, OTZ (10-20 mM) did not destroy monolayer and did not induce any visible disappearance of stress fibers either in 3T3 or 3T3-SV40 cells. However, in the presence of OTZ, amorphous actin-containing structures were observed in 3T3-SV40 cells. The effect of glutathione on both cell types was similar to that of NAC. The time required for GSH-induced alterations of actin cytoskeleton (about 5 h) was consistent with the increase in the intracellular level of reactive oxygen species (4 h after addition of GSH to the culture medium). Upon removal of the antioxidants from the medium, actin filament structures were reconstructed. However, within 24 h after withdrawal of NAC or GSH, only a partial reconstruction of stress fibers was observed in 3T3 cells. On the contrary, 3T3-SV40 cells demonstrated formation of well-structured actin fibers similar to normal fibroblasts. These results suggest that GSH can act as a pro-oxidant in the absence of oxidative stress.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Antioxidantes/farmacología , Células 3T3 , Acetilcisteína/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Actinas/metabolismo , Animales , Línea Celular Transformada , Glutatión/farmacología , Ratones , Ácido Pirrolidona Carboxílico , Especies Reactivas de Oxígeno/metabolismo , Virus 40 de los Simios , Fibras de Estrés/metabolismo , Tiazoles/farmacología , Tiazolidinas , Factores de TiempoRESUMEN
Reactive oxygen species (ROS) were established to play an important role in cellular signaling as second messengers by integrating different pathways. Recently, we showed that EGF initiated a rapid tyrosine phosphorylation of both EGF-receptor and STAT factors with simultaneous increase in the intracellular ROS level. Now, we have investigated the effect of intracellular red-ox state on EGF- and H2O2-induced activation of EGF receptor, STAT1 and STAT3. We demonstrated that the pretreatment of A431 cells with antioxidant N-acetyl-L-cysteine (NAC) partly reduced the level of EGF-induced phosphorylation of proteins under investigation. Besides, H2O2-induced activation of EGF receptor, and STAT factors was fully prevented by NAC pretreatment. The inhibition of ROS generation by DPI declined EGF-dependent activation of EGF receptor and STAT factors to basal level. Our results demonstrate the essential role of cellular red-ox status in the modulation of EGF-mediated activation of receptor and STAT factors. We have postulated that EGF-induced ROS generation is a very important initial event promoting physiological activation of EGF receptor and subsequent STAT factor activation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Transactivadores/metabolismo , Acetilcisteína , Antioxidantes , Línea Celular Tumoral , Receptores ErbB/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/antagonistas & inhibidores , Compuestos Onio/farmacología , Oxidación-Reducción , Fosforilación , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Factores de TiempoRESUMEN
A comparison of cell cycle phase distribution of 3T3 cells and their transformants 3T3SV40 treated with different substances changing the intracellular level of reactive oxygen species (ROS) has been made. In this study the following glutathione synthesis modulating agents were tested: two precursors of intracellular glutathione, antioxidant N-acetyl-L-cysteine (NAC) (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and inhibitor of glutathione synthesis, DL-buthionine-S, R-sulfoximine (BSO). It has been shown that both NAC (10-20 mM) and OTZ (20 mM) decreased the intracellular level of ROS in both cell lines. OTZ was more potent than NAC. However, only NAC caused changes in cell cycle progression of both cell types in dose-dependent manner. These changes differed in 3T3 and 3T3SV40 cells. Flow cytometric analysis of cell cycle phase distribution indicated that NAC (20 mM) blocked cell cycle in the G1 phase. The G1--arrest was completely reversible after removal of NAC from the medium. NAC (10-20 mM) caused a decrease in S and G2/M phases of transformants 3T3SV40. Moreover, a part of the population died apoptoticaly. Different mechanisms of NAC effect on normal and transformed cells are discussed. It is suggested that there is no strong correlation between cell cycle progression and intracellular level of ROS.
Asunto(s)
Antioxidantes/farmacología , Ciclo Celular/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Células 3T3 , Acetilcisteína/farmacología , Animales , Apoptosis/efectos de los fármacos , Butionina Sulfoximina/farmacología , Línea Celular Transformada , Relación Dosis-Respuesta a Droga , Glutatión/biosíntesis , Ratones , Ácido Pirrolidona Carboxílico , Tiazoles/farmacología , TiazolidinasRESUMEN
Cryptosporidiosis in an opportunistic infection which poses a significant threat to the immunodeficient patients (including people with AIDS). The aim of the present work was to study whether oxidative burst, known as a nonspecific immune response of macrophages, may be modulated in vitro by persisting oocysts of a coccidian pathogen Cryptosporidium parvum. Live oocysts of C. parvum engulfed by murine perithoneal macrophages may persist within the phagosomes and retain their initial morphology for about 7 days following oocyst injection into macrophage monolayer. A short-term interaction of adherent macrophages with C. parvum oocyst suspension for 5-15 min caused oxidative burst in these macrophages. After a while, the intensity of this oxidative burst smoothly decreased to vanish within the following 2-6 h oocyst-macrophage cultivation. Dead oocysts caused no oxidative burst in macrophages. Co-cultivation of macrophages with oocysts for 1.0-1.5 days led to the appearance of macrophages, which had oocysts both contacting the cell surface and existing inside the phagosomes. In these macrophages the oxidative burst in response to the addition of a chemotactic peptide (fMLP) was considerably higher than in uninfected control cells. During a 1-4 day co-cultivation, the degree of oxidative burst caused by fMLP in macrophages containing only phagocytosed oocysts did not differ from that in the non-infected (oocyst-free) control. The data obtained enable us to propose that the products of oxidative burst in macrophages, formed in response to the interaction with C. parvum oocysts, do not kill the oocysts. These can survive for a long time in the phagosomes of macrophages. Such a long persistence of oocysts in phagosomes does not affect the capability of macrophages for oxidative burst in response to the action of fMLP.
Asunto(s)
Criptosporidiosis/metabolismo , Criptosporidiosis/parasitología , Cryptosporidium parvum/fisiología , Macrófagos Peritoneales/parasitología , Estallido Respiratorio , Animales , Células Cultivadas , Macrófagos Peritoneales/fisiología , Ratones , Ratones Endogámicos C57BL , Infecciones Oportunistas/metabolismo , Infecciones Oportunistas/parasitología , Fagocitosis , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Changes in the levels of mRNAs encoding ion transporters (ATP1B1, NHE1, NKCC1), beta-actin, GAPDH, regulators of proliferation and apoptosis (p53, Bcl-2) and kinase hSGK, involved in cell water regulation, were studied using RT PCR in the peripheral human lymphocytes activated with phytohemagglutinin for 4-24 h. The common, "grouped", effect that was found was an increase in the levels of the studied mRNAs after an 8 h activation, sometimes preceded by a delay or slight decrease at the initial stage of 0-4 h. Apart from the common features, some differences were observed in the time courses and amplitudes of the responses of individual mRNAs. The arrangement of the individual mRNA responses in lymphocytes from different donors could differ significantly, thus indicating differential regulation of the studied mRNAs apart from the "grouped" effect. The data obtained confirmed our suggestion that regulation of ion transport at the level of mRNA could be involved in the changes of ion balance at the late stage of lymphocyte activation.
Asunto(s)
Ciclo Celular , Fibroblastos/citología , Especies Reactivas de Oxígeno , Proteínas E1A de Adenovirus/fisiología , Proteínas E1B de Adenovirus/fisiología , Animales , División Celular , Línea Celular Transformada , Medio de Cultivo Libre de Suero , Fibroblastos/metabolismo , RatasRESUMEN
Intracellular level of reactive oxygen species (ROS) and distribution of primary rat embryo fibroblast throughout the cell cycle have been studied. Serum-starved cells were activated by the addition of 10% serum to the culture medium in the presence on N-acetyl-cystein (NAC) and ROS-inhibitors, diphenileniodonium (DPI) and rothenone. It has been shown that serum starvation could block the cells at the G1/S boundary. Activation of serum-starved cells by the addition of serum reactivated the cell cycle and caused cell progression into S phase, which was paralleled with the increase in the intracellular level of ROS. Effects of NAC, PAI and rothenone, similar to that of serum starvation, blocked cell progression into S phase and decreased ROS formation due to the action of serum growth factors. The antiproliferative effect of NAC is discussed.
Asunto(s)
Acetilcisteína/farmacología , Ciclo Celular , Embrión de Mamíferos/citología , Compuestos Onio/farmacología , Especies Reactivas de Oxígeno , Rotenona/farmacología , Células 3T3 , Animales , Células Cultivadas , Medios de Cultivo , Citometría de Flujo , Ratones , Ratas , Receptores de Factores de Crecimiento/metabolismo , Transducción de SeñalRESUMEN
Generation of reactive oxygen species (ROS) in A431 cells, NIH3T3 fibroblasts expressing normal epidermal growth factor (EGF) receptor, L929 fibroblasts, and in mouse peritoneal macrophages (professionally phagocytic cells) upon the effect of different activators has been studied. It has been shown that ROS formation in A431 and NIH3T3 cells upon the effect of EGF is time- and dose-dependent process. A variety of stimuli were used to stimulate macrophage ROS production. However, the effect of only phorbol ester, opsonized zymozan, peptide fMLP, and platelet activating factor led to ROS generation, whereas tumor necrosis factor alpha, interferon gamma, and lipopolysaccharide did not stimulate macrophage oxidative burst. The literature data on ROS generation in a variety of cell types are presented. ROS formed in cells acted upon certain agents are considered as the molecules participating in intracellular signaling.
Asunto(s)
Especies Reactivas de Oxígeno , Animales , Línea Celular , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Ratones , N-Formilmetionina Leucil-Fenilalanina/farmacología , Fagocitos/efectos de los fármacos , Fagocitos/metabolismo , Factor de Activación Plaquetaria/farmacología , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
It has been shown that astrocytes (human glioblastoma U118 cell line) release reactive oxygen species (ROS), including superoxide O2.- and hydrogen peroxide H2O2 following the action of C5a complement component C5a (but not C3a). The effect of C5a (1 nM) is accompanied with hyperpolarization of the astrocyte plasma membrane. Component C3a (100 nM), which is not an inducer of ROS, caused a prolonged depolarization of astrocytes. However, both the agents induced a transient increase in intracellular Ca2+ concentration. The data obtained permit a conclusion that O2.- participates in the intracellular signal transduction, and is involved in the mechanism of hyperpolarization response of astrocytes to the effect of the inducer of ROS, complement component C5a.