RESUMEN
The aim of this study was to evaluate platelet activation in gastric cancer patients with regard to histopathological classification and the presence of distant metastases, by using platelet morphological parameters: MPV, L-PLT, MPC, as well as quantitative evaluation of surface receptor expression: CD41a, CD61, CD42b, CD62P, by flow cytometry at the resting state and after TRAP activation. In gastric cancer patients higher values of MPV and LP, as well as decreased MPC values were determined. Quantitative evaluation of surface antigen expression also revealed higher number of CD41a, CD61 and CD62P molecules, as compared with the platelets in the control group. Significant decrease of CD42b molecules' number after TRAP incubation, and the increased CD41a, CD61 and CD62P expression also point to the retained reactivation capacity of platelets. Good correlation between morphological parameters and the number of CD62P molecules indicates the usefulness of routine tests in evaluation of platelet activation.
Asunto(s)
Adenocarcinoma/metabolismo , Plaquetas/metabolismo , Activación Plaquetaria/fisiología , Neoplasias Gástricas/metabolismo , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Plaquetas/citología , Estudios de Casos y Controles , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Estadificación de Neoplasias , Recuento de Plaquetas , Pronóstico , Neoplasias Gástricas/patologíaRESUMEN
UNLABELLED: Mitogen-activated protein kinase (MAPK) family members, namely MAPK, c-Jun NH2-terminal protein kinase (JNK), and p38MAPK, have been recently reported to have opposing effects on apoptosis. AIM: To determine the activity of MAPKs and the level of Bax, Bcl-2 and p53--proteins known to be involved in the regulation of apoptosis--in pancreatic acini subjected to stressful stimuli leading to cell death. METHODS AND RESULTS: Isolated pancreatic acini were irradiated for 30 min with ultraviolet B (UV-B) or stimulated with supraphysiological concentrations of cholecystokinin (CCK). As it was assessed by means of acridine orange/ethidium bromide staining, irradiation with UV-B induced predominantly apoptosis while necrosis predominated in CCK-stimulated acini. The activity of MAPK, JNK and p38MAPK was determined by means of Western-blotting, with the use of antibodies which recognize active, dually phosphorylated enzymes. Irradiation with UV-B induced a rapid, 3-fold increase in MAPK activity. It had a maximum at 30 min and then gradually declined to reach the normal level at 120 min. Concomitantly, early activation of p38-MAPK was found at 30 min. However, unlike MAPK, p38-MAPK activity was then gradually rising to reach a maximum (5-fold increase) at 180 min. UV-B-induced activation of both kinases was not affected by the pretreatment with antioxidant--N-acetylo-L-cysteine or protein kinase C inhibitor--GF-109203X. In UV-B-irradiated cells, we did not detect any significant JNK activation as well as any significant changes in Bax, Bcl-2 and p53 levels assessed by means of Western-blotting. CONCLUSION: It seems likely that a specific interaction between MAPK and p38MAPK signaling pathway may be involved in the determination of the cell death mechanism in pancreatic acini subjected to stressful stimuli.
Asunto(s)
Apoptosis/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Páncreas/citología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Muerte Celular/efectos de la radiación , Colecistoquinina/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/efectos de la radiación , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de la radiación , Masculino , Páncreas/efectos de los fármacos , Páncreas/efectos de la radiación , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Ratas , Ratas Wistar , Proteína p53 Supresora de Tumor/fisiología , Rayos Ultravioleta , Proteína X Asociada a bcl-2RESUMEN
Although endothelial cells and keratinocytes appear to be the primary cellular targets of sulfur mustard (SM), the role of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) in SM-induced vesication has not been clearly defined. PARP is thought to play a crucial role in DNA repair mechanisms following exposure to alkylating agents like SM. Using a combination of fluorescence microscopy and biochemical assays, we tested the hypothesis that SM causes activation of PARP in endothelial cells and keratinocytes with subsequent loss of nicotinamide adenine dinucleotide (NAD) and depletion of adenosine triphosphate (ATP) levels. To determine if PARP activation accounts for SM-induced vesication, keratinocyte adherence and permeability of endothelial monolayers were measured as in vitro correlates of vesication. As early as 2 to 3 h after exposure to SM concentrations as low as 250 microM, dramatic changes were induced in keratinocyte morphology and microfilament architecture. Exposure to 500 microM SM induced a fourfold increase in PARP activity in endothelial cells, and a two- to threefold increase in keratinocytes. SM induced a dose-related loss of NAD+ in both endothelial cells and keratinocytes. ATP levels fell to approximately 50% of control levels in response to SM concentrations >/=500 microM. SM concentrations >/=250 microM significantly reduced keratinocyte adherence as early as 3 h after exposure. Endothelial monolayer permeability increased substantially with concentrations of SM >250 microM. These observations support the hypothesis that the pathogenic events necessary for SM-induced vesication (i.e., capillary leak and loss of keratinocyte adherence) at higher vesicating doses of SM (>/=500 microM) may depend on NAD loss with PARP activation and subsequent ATP-dependent effects on microfilament architecture. Vesication developing as a result of exposure to lower concentrations of SM presumably occurs by mechanisms that do not depend on loss of cellular ATP (e.g., apoptosis and direct SM-mediated damage to integrins and the basement membrane).
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Gas Mostaza/toxicidad , Poli(ADP-Ribosa) Polimerasas/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Adenosina Trifosfato/análisis , Animales , Bovinos , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/enzimología , Activación Enzimática/efectos de los fármacos , Queratinocitos/enzimología , NAD/análisisRESUMEN
Cell death due to necrosis results in acute inflammation, while death by apoptosis generally does not. The effect of adenosine triphosphate (ATP) on the pattern of cell death induced by oxidants was examined in bovine endothelial cells. ATP levels were altered by hydrogen peroxide (H2O2), glutamine (Gln), and metabolic inhibition (MI), to determine if necrosis can be shifted to apoptosis during oxidant injury. The form of cell death was determined by fluorescence microscopic techniques and the pattern of DNA degradation on agarose gels. ATP levels were measured using the luciferase-luciferin assay. Apoptosis occurred with 100 microM H2O2 without an alteration in ATP levels. ATP was significantly lowered with 5 mM H2O2, and necrosis occurred. MI, in combination with 100 microM H2O2, decreased ATP and resulted in necrosis. MI alone, however, did not cause cell death. Gln partially restored ATP levels in cells injured with 5 mM H2O2 and resulted in a significant increase in apoptosis. DNA laddering on agarose gels confirmed the apoptotic changes seen by fluorescence microscopy. In summary, a threshold level of ATP 25% of basal levels is required for apoptosis to proceed after oxidant stress, otherwise necrosis occurs. Agents like glutamine that enhance ATP levels in oxidant-stressed cells may be potent means of shifting cell death during inflammation to the noninflammatory form of death--apoptosis.
Asunto(s)
Adenosina Trifosfato/farmacología , Apoptosis/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Necrosis , Adenosina Trifosfato/metabolismo , Animales , Bovinos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Glutamina/farmacología , Peróxido de Hidrógeno/farmacología , Microscopía Fluorescente , Oligomicinas/farmacología , Estrés Oxidativo/efectos de los fármacos , Arteria Pulmonar/patología , Factores de TiempoRESUMEN
Little is known about the biochemical "machinery" responsible for the morphological features of apoptosis, although the cytoskeleton is presumed to be involved. Using flow cytometry, polyacrylamide gel electrophoresis, and fluorescence microscopy, we show that apoptosis induced by ultraviolet (UV) irradiation or 80 micrograms/ml etoposide correlates with early transient polymerization and later depolymerization of filamentous (F)-actin and dramatic changes in visible microfilament organization. Depolymerization of F-actin began before the formation of apoptotic bodies and was ultimately composed of decreases in both the detergent-insoluble (40%) and detergent-soluble (50%) pools of F-actin. Dihydrocytochalasin B (H2CB), which blocked apoptotic body formation, depolymerized F-actin in the detergent-insoluble pool only. Visually, H2CB treatment disrupted microfilament organization, resulting in short, brightly stained microfilaments dispersed throughout the cytoplasm. In contrast, apoptotic cells contained a network of fine microfilaments with bright staining concentrated at the site of apoptotic body formation. Together, these results suggest that reorganization of the microfilament network is necessary for the formation of apoptotic bodies and that depolymerization of F-actin may also be a necessary component of the process of apoptosis.
Asunto(s)
Actinas , Apoptosis , Citoesqueleto/patología , Apoptosis/efectos de la radiación , Citoesqueleto/efectos de la radiación , Dimerización , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Células HL-60 , Humanos , Microscopía Fluorescente , Rayos UltravioletaRESUMEN
Sulfur Mustard (SM) is a vesicant or blistering chemical warfare agent, for which there still is no effective therapy. Endothelial cells are one of the major cellular targets for SM. The mechanism of endothelial cell death during SM injury is poorly understood. We studied the effect of exposure of endothelial cells to 0-1000 microM SM over the time course of 2-24 hr to determine the role of apoptotic and necrotic patterns of cell death in endothelial injury induced by SM. SM concentrations < or = 250 microM induced exclusively apoptosis which was observed after 5 hr in 30% of endothelial cells. Exposure to SM concentrations > or = 500 microM caused apoptosis and necrosis to the same extent in 60-85% of all cells after 5 to 6 hr. Necrosis was accompanied by a significant (approximately 50%) depletion of intracellular ATP, while in apoptotic cells ATP remained at the level similar to healthy cells. Interestingly, disruption of the long actin filament stress fibers and rounding of cells preceded other features of apoptosis--DNA fragmentation, membrane budding, and apoptotic body formation. In apoptotic cells, microfilaments formed constricted perinuclear bands, which were not observed in necrotic cells. Pretreatment with 50 mM N-acetyl-L-cysteine (NAC), a sulfhydryl donor and antioxidant, nearly eliminated the apoptotic features of cell death but did not prevent necrosis in response to SM. NAC pretreatment alone induced reorganization of actin filaments into an enhanced network of long stress fibers instead of a dominant cortical band of actin. NAC pretreatment prevented loss of cell adherence and cell rounding following exposure to 250 microM SM. The effect of NAC on cytoskeletal organization and its ability to eliminate SM-induced apoptosis suggests that actin filament organization may be an important element in cellular susceptibility to apoptotic stimuli.