RESUMEN

Disruptions of cell death signalling occur in pathological processes, such as cancer and degenerative disease. Increased knowledge of cell death signalling has opened new areas of therapeutic research, and identifying key mediators of cell death has become increasingly important. Early triggering events in cell death may provide potential therapeutic targets, whereas agents affecting later signals may be more palliative in nature. A group of primary mediators are derivatives of the highly unsaturated fatty acids (HUFAs), particularly oxygenated metabolites such as prostaglandins. HUFAs, esterified in cell membranes, act as critical signalling molecules in many pathological processes. Currently, agents affecting HUFA metabolism are widely prescribed in diseases involving disordered cell death signalling. However, partly due to rapid metabolism, their role in cell death signalling pathways is poorly characterized. Recently, HUFA-derived mediators, the resolvins/protectins and endocannabinoids, have added opportunities to target selective signals and pathways. This review will focus on the control of cell death by HUFA, eicosanoid (C20 fatty acid metabolites) and docosanoid (C22 metabolites), HUFA-derived lipid mediators, signalling elements in the micro-environment and their potential therapeutic applications. Further therapeutic approaches will involve cell and molecular biology, the multiple hit theory of disease progression and analysis of system plasticity. Advances in the cell biology of eicosanoid and docosanoid metabolism, together with structure/function analysis of HUFA-derived mediators, will be useful in developing therapeutic agents in pathologies characterized by alterations in cell death signalling.

Asunto(s)

Muerte Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Eicosanoides/metabolismo , Ácidos Grasos/metabolismo , Microdominios de Membrana/efectos de los fármacos , Terapia Molecular Dirigida , Transducción de Señal/efectos de los fármacos , Animales , Agonistas de Receptores de Cannabinoides , Antagonistas de Receptores de Cannabinoides , Moduladores de Receptores de Cannabinoides/agonistas , Moduladores de Receptores de Cannabinoides/antagonistas & inhibidores , Moduladores de Receptores de Cannabinoides/metabolismo , Membrana Celular/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Eicosanoides/agonistas , Eicosanoides/antagonistas & inhibidores , Ácidos Grasos/agonistas , Ácidos Grasos/antagonistas & inhibidores , Humanos , Microdominios de Membrana/metabolismo , Metabolómica/métodos

RESUMEN

The mechanisms involved in storage-induced damage in platelets are not well understood, but membrane signalling via Ca2+ ion flux may affect mitochondrial H+ gradients and metabolism and the intrinsic pathways of cell death, platelet survival and function. In this study, the effects of blood bank storage conditions, including reduced plasma concentration and interrupted agitation, were evaluated in platelets from 136 healthy donors. Mitochondrial membrane potential (DeltaPsim), an indicator of intrinsic cell death, and its sensitivity to Ca2+ ionophore A23187, were monitored using JC-1 by flow cytometry and fluorescence microscopy. Platelet survival was examined using lactate dehydrogenase release, annexin V binding and caspase-3/7 activity. Decreased plasma concentration and interrupted agitation affected DeltaPsim and caspase-3/7. Over 7 days in 30% plasma DeltaPsim showed a significant reduction (86.3 +/- 1.1% platelets with polarised mitochondria day 1; 79.9 +/- 2.1% day 5; 75.1 +/- 3.8% day 7, P = 0.01 day 1 vs. day 7). Whilst DeltaPsim in agitated platelets in 100% plasma was unchanged up to day 7, interruption of agitation was associated with a 44% reduction in the proportion of platelets with polarised mitochondria after 5 days (56 +/- 11%). The Ca2+ sensitivity of DeltaPsim changed earlier: 5 microM A23187 caused a 20-30% change in the fraction of platelets with polarised mitochondria by day 5. Ca2+ sensitivity also increased during interrupted agitation and reduced plasma concentration. DeltaPsim also correlated with indicators of platelet death, caspase-3 activity and annexin V binding (correlation coefficients of 0.8). In conclusion, changes in Ca2+-sensitive DeltaPsim are involved in the initiation of storage-induced cell death signals that influence platelet count and function in vivo.

Asunto(s)

Plaquetas/fisiología , Conservación de la Sangre , Calcio/fisiología , Muerte Celular/fisiología , Potenciales de la Membrana/fisiología , Membranas Mitocondriales/fisiología , Eliminación de Componentes Sanguíneos/métodos , Calcimicina/farmacología , Caspasa 3/metabolismo , Senescencia Celular/fisiología , Humanos , Ionóforos/farmacología , Recuento de Plaquetas , Factores de Tiempo

Asunto(s)

Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ácidos Araquidónicos/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos/metabolismo , Ácidos Araquidónicos/metabolismo , Ciclooxigenasa 2 , Humanos , Isoenzimas/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Proteínas de la Membrana , Prostaglandina-Endoperóxido Sintasas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo

RESUMEN

The highly unsaturated fatty acids (HUFA) of the n-6 and n-3 series are involved in cell signalling in normal and transformed cells and have recently been associated with pathways leading to tumour cell death. The antitumour activity of three HUFA (arachidonic acid, gamma linolenic acid and eicosapentaenoic acid) were studied in glioma cells and tissue. Using five glioma models, including primary cell suspensions prepared from 46 human glioma samples and an in vivo rat C6 glioma model, we obtained evidence that, following exposure to HUFA, either administered into the medium surrounding human glioma cells or in 16 preparations of multicellular spheroids derived from human and rodent glioma cell lines (C6, MOG, U87, U373) or administered intra-tumourally by infusion using osmotic mini-pumps in 48 rats, glioma regression and apoptosis were detected. Additionally, synergy between gamma irradiation and HUFA administration was observed in 13 experiments analyzing C6 glioma cell apoptosis in vitro. These pro-apoptotic and antiproliferative activities were observed using both C18 and C20 fatty acids of the n-6 and n-3 series, but not when saturated and monounsaturated C18 and C20 fatty acid preparations were used. In the glioma infusion model, in addition to the apoptosis detected in glioma tissue infused with HUFA for 3-7 days, preservation of normal neural tissue and vasculature in adjacent brain was observed. Also, there was little evidence of acute inflammatory infiltration in regressing tumours. Our findings suggest that intraparenchymal infusion of HUFA may be effective in stimulating glioma regression.

Asunto(s)

Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/patología , Ácidos Grasos Insaturados/farmacología , Glioma/patología , Animales , Apoptosis/efectos de la radiación , Ácido Araquidónico/farmacología , Neoplasias Encefálicas/radioterapia , División Celular/efectos de los fármacos , Glioma/radioterapia , Humanos , Etiquetado Corte-Fin in Situ , Ratas , Especies Reactivas de Oxígeno/metabolismo , Células Tumorales Cultivadas , Rayos X , Ácido gammalinolénico/farmacología

RESUMEN

Secreted phospholipase A(2) (sPLA(2)) regulates a variety of cellular functions. The present investigation was undertaken to elucidate the potential role of sPLA(2) in endothelial cell (EC) migration. Bovine aortic endothelial cells (BAECs) exposed to sPLA(2) placed in the lower compartment of a modified Boyden chamber displayed increased migration compared to cells exposed to vehicle. The effect of sPLA(2) on EC migration was time and dose dependent. Migration of BAECs was observed at 30 minutes, increased over 1 to 2 hours, and declined thereafter. At 2 hours of stimulation, sPLA(2) (0.01-2 micromol/L) induced 1.2- to 3-fold increased cell migration compared with media alone. Among the different sPLA(2)s tested, bee venom, Naja naja, and porcine and human pancreatic PLA(2)s all evoked a migratory response in ECs. Moreover, human synovial fluid, obtained from patients with arthritis and containing sPLA(2) activity, induced EC migration. Migration of ECs was significantly reduced after exposure to a catalytic site mutant of pancreatic sPLA(2) with decreased lipolytic activity as compared to wild-type sPLA(2). Similarly, pretreatment of human synovial fluid with p-bromophenacyl bromide, an irreversible inhibitor of sPLA(2), markedly decreased the ability of human synovial fluid to stimulate EC migration. Moreover, migration of ECs was stimulated on exposure to hydrolytic products of sPLA(2) activity including arachidonic acid, lysophosphatidic acid, and lysophosphatidylcholine. These findings suggest that sPLA(2) plays a physiologic role in induction of EC migration. Moreover, the effects of sPLA(2) on EC migration are mediated, at least in part, by its catalytic activity. (Blood. 2000;96:3809-3815)

Asunto(s)

Endotelio Vascular/citología , Fosfolipasas A/farmacología , Sustitución de Aminoácidos , Animales , Aorta , Ácido Araquidónico/farmacología , Artritis/metabolismo , Dominio Catalítico/genética , Dominio Catalítico/fisiología , Bovinos , Técnicas de Cultivo de Célula , Movimiento Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Lisofosfolípidos/farmacología , Páncreas/enzimología , Fosfolipasas A/metabolismo , Solubilidad , Líquido Sinovial/química , Distribución Tisular

RESUMEN

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide synthesized and secreted by vascular endothelial cells. Regulation of ET-1 production occurs at the level of gene transcription. We previously demonstrated a role for arachidonic acid as an intracellular mediator in the regulation of gene expression. This study investigated the role of arachidonic acid in induction of ET-1 production in endothelial cells. Challenge of bovine aortic endothelial cells (BAECs) with arachidonic acid induced a dose- and time-dependent increase in the amount of immunoreactive ET-1 in the supernatant. The maximum effect was observed at concentrations of 20 microM. Release of ET-1 by arachidonic acid was preceded by induction of ET-1 gene expression. Arachidonic acid increased ET-1 gene expression by increasing transcription of the ET-1 gene. The effect of arachidonic acid was mimicked by other polyunsaturated fatty acids, whereas saturated fatty acids had no effect. Moreover, inhibitors of the lipoxygenase pathway blocked arachidonic acid-induced release of ET-1. These results suggest that arachidonic acid stimulated the production of ET-1 in BAECs by inducing ET-1 gene transcription. Arachidonic acid-induced production of ET-1 is dependent on lipoxygenase products of arachidonate metabolism.

Asunto(s)

Ácido Araquidónico/farmacología , Endotelina-1/genética , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Expresión Génica/efectos de los fármacos , Animales , Ácido Araquidónico/antagonistas & inhibidores , Bovinos , Línea Celular , Medios de Cultivo Condicionados/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Relación Dosis-Respuesta a Droga , Endotelina-1/biosíntesis , Ácidos Grasos/farmacología , L-Lactato Deshidrogenasa/metabolismo , Inhibidores de la Lipooxigenasa/farmacología , ARN Mensajero/biosíntesis , Sensibilidad y Especificidad , Factores de Tiempo

RESUMEN

The hallmark of chronic myeloid leukemia (CML) is the presence of the bcr-abl oncogene, which is associated with transforming ability and an intrinsic resistance to induction of apoptosis by genotoxic agents. Arachidonic acid (AA), a biologically active fatty acid, plays a crucial role as a mediator of signaling pathways involved in cell proliferation and survival. In this study, we investigated the potential role of AA as a proapoptotic agent in CML. Pretreatment of human CML isolated progenitor cells with AA (100 microM for 18 h) induced 71-75% inhibition of in vitro colony formation of granulocyte-macrophage colony-forming units, multilineage colony-forming units, and erythroid burst-forming units. This inhibition was significantly greater than the effect on normal progenitor cells (19-39% growth inhibition of erythroid burst-forming units, multilineage colony-forming units, and granulocyte-macrophage colony-forming units). AA also inhibited growth of the bcr-abl-transformed cell line H7.bcr-abl A54. In contrast, a minimal effect of AA on inhibition of cell growth was observed in the parental nontransformed NSF/N1.H7 cell line. The antiproliferative effect of AA was associated with apoptosis. Gamma-linolenic acid, a precursor of AA, also inhibited cell growth, whereas other unsaturated and saturated fatty acids had no effect. Pharmacological inhibition of cyclooxygenase, lipooxygenase, and cytochrome P450 monooxygenase enzymes prior to exposure to AA did not rescue cells from the inhibitory effect of AA. Moreover, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable arachidonate analogue, also inhibited cell growth, suggesting that the effect of AA did not require further metabolism. Treatment with antioxidants prior to stimulation with AA was also ineffective in preventing its antiproliferative effect. Thus, AA inhibited proliferation of CML cells by inducing apoptotic cell death. The signaling mechanisms of AA-induced inhibition of cell growth appeared to be independent of its conversion into eicosanoids or free radical generation.

Asunto(s)

Apoptosis/efectos de los fármacos , Ácido Araquidónico/farmacología , Células Madre Hematopoyéticas/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ácido 5,8,11,14-Eicosatetrainoico/farmacología , Adulto , Animales , Antioxidantes/farmacología , Células de la Médula Ósea/patología , Ácidos Cafeicos/farmacología , División Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/patología , Células Precursoras Eritroides/ultraestructura , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/ultraestructura , Humanos , Indometacina/farmacología , Cetoconazol/farmacología , Masculino , Masoprocol/farmacología , Ratones , Persona de Mediana Edad , Células Tumorales Cultivadas

RESUMEN

Arachidonic acid (AA) plays a signaling role in the induction of several genes. We previously demonstrated that AA induces c-jun gene expression in the stromal cell line +/+.1 LDA 11 by a signaling pathway involving activation of the c-jun amino-terminal kinase (JNK). This study investigated the role of calcium in AA signaling of c-jun activation in +/+.1 LDA 11 cells. AA (10-50 microM) caused a rapid dose-dependent rise in cytosolic calcium. AA-induced calcium mobilization involved both influx of extracellular calcium and the release of intracellular calcium. The importance of calcium was investigated by variation of the extracellular calcium concentration, chelation of intracellular calcium and by calcium ionophore-induced influx of extracellular calcium. AA-induced c-jun gene expression and increased luciferase activity of a construct containing the high affinity AP-1 binding site was decreased in cells preincubated with the intracellular calcium chelator 1,2-bis(o-aminophenoxy)-eThane-N,N,N',N',-tetraacetic acid tetra(aceToxymethyl-esTer) (BAPTA-AM, 10 microM) prior to stimulation with AA. Similarly, chelation of intracellular calcium decreased AA-induced JNK activation. On the contrary, changes in the extracellular calcium concentration had no effect. Also, ionophore A23187 failed to induce c-jun and JNK activation either alone than in combination with AA. These results suggested that calcium was required for AA-dependent activation of c-jun, but that calcium alone was insufficient to induce activation of c-jun. Thus, release of calcium from intracellular stores is implicated in the signaling pathway of AA-induced c-jun activation in stromal cells.

Asunto(s)

Ácido Araquidónico/farmacología , Calcio/metabolismo , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Sitios de Unión , Transporte Biológico , Calcimicina/farmacología , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Retículo Endoplásmico , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/genética , Transducción de Señal , Factor de Transcripción AP-1/metabolismo

RESUMEN

We used four-color fluorescence analysis to compare lineage antigen expression in relationship to CD38 and HLA-DR on CD34+ progenitor cells in adult human bone marrow and mobilized peripheral blood. Each of four progenitor cell subpopulations defined by HLA-DR and CD38 intensity (CD38-/HLA-DR-, CD38-/HLA-DR+, CD38+/HLA-DR+, and CD38+/HLA-DR-) were present in both progenitor cell sources in similar ratios. The most prevalent subpopulation consisted of cells that expressed both CD38 and HLA-DR. Virtually all progenitor cells that lacked CD38 also lacked lineage antigens regardless of their HLA-DR expression. In contrast, the majority of the cells within both CD38+ progenitor cell subpopulations possessed either lineage antigens or the proliferation-associated antigen, CD71. Furthermore, CD71 was expressed on three times the number of CD38+/HLA-DR- cells when compared with the CD38-/HLA-DR- subpopulation. Within CD34+ progenitor cell subpopulations defined by the expression of CD38 and HLA-DR, the CD38+/HLA-DR- component appears to be the most mature, based on the expression of CD71 and various lineage-associated antigens, including representative markers characterizing early lymphoid, myeloid, and erythroid precursors. Thus, selection of the most immature CD34+ progenitor cells based solely on the lack of HLA-DR expression results in isolation of two distinct cell populations with markedly different maturation status and resultant growth characteristics.

Asunto(s)

Antígenos CD , Antígenos de Diferenciación/análisis , Linaje de la Célula , Antígenos HLA-DR/análisis , Células Madre Hematopoyéticas/citología , NAD+ Nucleosidasa/análisis , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Adulto , Antígenos CD34/análisis , Células de la Médula Ósea , División Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células Madre Hematopoyéticas/inmunología , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Glicoproteínas de Membrana

RESUMEN

We have previously shown that arachidonic acid mediates interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha)-induced transcription of c-jun. The signaling pathway of arachidonic acid-induced c-jun transcription was independent of protein kinase C activation and involved a tyrosine kinase-dependent process. The present study was undertaken to further elucidate the signal transduction pathway of arachidonate-induced c-jun transcription. We used a glutathione-S-transferase-c-jun fusion protein containing the aminoterminal domain of c-jun (residues 5 to 89) to explore the hypothesis that arachidonic acid stimulates c-jun amino-terminal kinase (JNK) activity in the murine stromal cell line +/+.1 LDA 11. Extracts from arachidonic acid-treated cells catalyzed phosphorylation of the c-jun fusion protein, indicating stimulation of JNK activity. Similar results were obtained when cells were challenged with IL-1 and TNF-alpha. The effect of arachidonic acid was specific, because extracts from stimulated cells failed to phosphorylate a mutated fusion protein in which serine 63 and 73 of c-jun were each substituted with leucine. Arachidonic acid induced JNK activation in a time- and dose-dependent manner that was not mimicked by saturated fatty acids such as palmitic acid or other unsaturated fatty acids from the n-3, n-6, or n-9 series. Furthermore, other lipids, such as diacylglycerol, phosphatidic acid, and C2-ceramide, failed to induce a significant increase in JNK activity. Treatment of stromal cells with propyl gallate, a dual inhibitor of lipoxygenase and cyclooxygenase enzymes, did not affect the ability of arachidonic acid to induce JNK activation. Moreover, ETYA (5,8,11,14-eicosate-traynoic acid), a nonmetabolizable arachidonate analogue, also induced JNK activation. These results are consistent with the hypothesis that the signal transduction pathway by which arachidonate stimulates c-jun transcription involves activation of the JNK cascade. Furthermore, arachidonic acid itself and not its cyclooxygenase or lipoxygenase metabolites is involved in stimulating JNK activity. Thus, arachidonic acid may act as a second messenger in mediating the effects of IL-1 and TNF-alpha in the activation of c-jun.

Asunto(s)

Ácido Araquidónico/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células del Tejido Conectivo , Interleucina-1/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Sistemas de Mensajero Secundario/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 4 , Fosforilación , Proteínas Recombinantes/farmacología

RESUMEN

Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by a chimeric BCR/ABL gene giving rise to a 210-kD fusion protein with dysregulated tyrosine kinase activity. We investigated the effect of genistein, a protein tyrosine kinase inhibitor, on the in vitro growth of CML and normal marrow-derived multi-potent (colony-forming unit-mix [CFU-Mix]), erythroid (burst-forming unit-erythroid [BFU-E]), and granulocyte-macrophage (colony-forming unit-granulocyte-macrophage [CFU-GM]) hematopoietic progenitors. Continuous exposure of CML and normal marrow to genistein induced a statistically significant and dose-dependent suppression of colony formation. Genistein doses causing 50% inhibition of CML and normal progenitors were not significantly different for CFU-Mix (27 mumol/L v 23 mumol/L), BFU-E (31 mumol/L v 29 mumol/L), and CFU-GM (40 mumol/L v 32 mumol/L v 32 mumol/L). Preincubation of CML and normal marrow with genistein (200 mumol/ L for 1 to 18 hours) induced a time-dependent suppression of progenitor cell growth, while sparing a substantial proportion of long-term culture-initiating cells (LTC-IC) from CML (range, 91% +/- 9% to 32% +/- 3%) and normal marrow (range, 85% +/- 8% to 38% +/- 9%). Analysis of individual CML colonies for the presence of the hybrid BCR/ABL mRNA by reverse transcription-polymerase chain reaction (RT-PCR) showed that genistein treatment significantly reduced the mean +/- SD percentage of marrow BCR/ABL+ progenitors both by continuous exposure (76% +/- 18% v 24% +/- 12%, P < or = .004) or preincubation (75% +/- 16% v 21% +/- 10%, P < or = .002) experiments. Preincubation with genistein reduced the percentage of leukemic LTC-IC from 87% +/- 12% to 37% +/- 12% (P < or = .003). Analysis of individual colonies by cytogenetics and RT-PCR confirmed that genistein-induced increase in the percentage of nonleukemic progenitors was not due to suppression of BCR/ABL transcription. Analysis of nuclear DNA fragmentation by DNA gel electrophoresis and terminal deoxynucleotidyl transferase assay showed that preincubation of CML mononuclear and CD34+ cells with genistein induced significant evidence of apoptosis. These observations show that genistein is capable of (1) exerting a strong antiproliferative effect on CFU-Mix, BFU-E, and CFU-GM while sparing the more primitive LTC-IC and (2) selecting benign hematopoietic progenitors from CML marrow, probably through an apoptotic mechanism.

Asunto(s)

Apoptosis/efectos de los fármacos , Purgación de la Médula Ósea/métodos , Inhibidores Enzimáticos/farmacología , Proteínas de Fusión bcr-abl/genética , Células Madre Hematopoyéticas/efectos de los fármacos , Isoflavonas/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/efectos de los fármacos , ARN Mensajero/análisis , ARN Neoplásico/análisis , Adulto , Anciano , Anciano de 80 o más Años , División Celular/efectos de los fármacos , Fragmentación del ADN , ADN de Neoplasias/análisis , Femenino , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Genisteína , Células Madre Hematopoyéticas/enzimología , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/análisis , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Ensayo de Tumor de Célula Madre

RESUMEN

Receptor and nonreceptor protein tyrosine kinases (PTKs) play a key role in the control of normal and neoplastic cell growth. The availability of PTK inhibitors prompted us to evaluate the effects of genistein, a natural inhibitor of PTKs, on in vitro colony formation by normal multilineage colony-forming units (CFU-Mix), erythroid bursts (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM), long-term culture-initiating cells (LTC-IC) and acute myelogenous leukaemia colony-forming units (CFU-AML). Continuous exposure of normal marrow and blood mononuclear non-adherent cells, blood CD34+CD45RA- cells, and leukaemic blasts to increasing doses of genistein (1-100 microM) resulted in a statistically significant (P < or = 0.05) dose-dependent suppression of CFU-Mix, BFU-E, CFU-GM and CFU-AML growth. Regression analysis showed that growth inhibition was linearly related to genistein concentration. Genistein dose causing 50% inhibition (ID50) of CFU-AML was significantly lower compared to CFU-GM ID50 for marrow (19 v 32 microM, P < or = 0.017), unseparated blood (19 v 44 microM, P < or = 0.028) or CD34+CD45RA- blood (19 v 36, P < or = 0.04). Preincubation of leukaemic blasts with genistein (200 microM) for 1-2h confirmed that CFU-AML were significantly more sensitive than normal marrow and blood CFU-GM to genistein. Preincubation conditions which maximally suppressed leukaemic and normal colony growth spared a substantial percentage of marrow (29 +/- 4%) and blood (40 +/- 3%) LTC-IC. In conclusion, our data demonstrate that: (a) genistein strongly inhibits the growth of normal and leukaemic haemopoietic progenitors; (b) growth inhibition is dose- and time-dependent; (c) leukaemic progenitors are more sensitive than normal progenitors to genistein-induced growth inhibition; (d) genistein exerts a direct toxic effect on haemopoietic cells while sparing a substantial proportion of LTC-IC. The potent CFU-AML growth inhibition associated with the relative resistance of normal LTC-IC strongly supports the use of genistein for marrow purging.

Asunto(s)

Células Madre Hematopoyéticas/efectos de los fármacos , Isoflavonas/farmacología , Leucemia Mieloide Aguda/patología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Genisteína , Células Madre Hematopoyéticas/patología , Humanos , Células Tumorales Cultivadas

RESUMEN

We have previously shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression induced by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) in the murine stromal cell line +/+.1-LDA 11 involves activation of phospholipase A2 (PLA2). Furthermore, induction of GM-CSF gene expression due to release of arachidonic acid as a result of PLA2 activation was mediated by the transcriptional factor c-jun. In the present study, we have investigated the potential mechanism involved in the induction of c-jun gene expression by arachidonic acid. Arachidonic acid induced transcription of c-jun mRNA. Downregulation of protein kinase C (PKC) by chronic exposure of stromal cells to the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 400 nmol/L) did not effect c-jun expression induced by arachidonate. Moreover, pretreatment of cells with the PKC inhibitor, calphostin C (1 mumol/L), caused a marked decrease of c-jun expression induced by TPA, but had no influence on c-jun expression induced by arachidonate. To explore the hypothesis that a tyrosine kinase signalling pathway, independent of PKC activation, was involved in arachidonate-induced c-jun expression, stromal cells were pretreated with the protein tyrosine kinase inhibitor, genistein, before challenge with arachidonic acid. Arachidonate 50 mumol/L)-induced c-jun expression was inhibited, in a dose- and time-dependent manner, by genistein. Genistein similarly inhibited c-jun expression in stromal cells exposed to IL-1 (500 U/mL) plus TNF-alpha (500 U/mL). The potential role of a tyrosine kinase pathway in arachidonate-mediated c-jun expression was further investigated by assaying the tyrosine kinase activity of cells challenged with arachidonic acid, IL-1, and TNF-alpha. Exposure of stromal cells to arachidonic acid induced a 2.1-fold increase in intracellular tyrosine kinase activity determined by phosphorylation of the synthetic peptide, raytide, in the presence of [gamma-32P]-ATP. Similarly, IL-1 and TNF-alpha induced 1.7- and 2.4-fold increases in tyrosine protein kinase activity, respectively. The effect of arachidonic acid on tyrosine kinase activity was inhibited by genistein and was enhanced by sodium vanadate. The increase of protein tyrosine kinase activity detected in arachidonate-stimulated cells was associated, in a dose- and time-dependent fashion, with tyrosine phosphorylation of 240-, 40-, and 29-kD substrates. These results are consistent with the hypothesis that a tyrosine phosphorylation process is triggered by arachidonate as an early event in the signalling pathway that leads to increased expression of c-jun.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

Ácido Araquidónico/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes jun/efectos de los fármacos , Interleucina-1/farmacología , Proteínas Tirosina Quinasas/fisiología , Sistemas de Mensajero Secundario/fisiología , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Genisteína , Humanos , Isoflavonas/farmacología , Ratones , Naftalenos/farmacología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes/farmacología , Transcripción Genética/efectos de los fármacos

RESUMEN

The activity of 1-phosphatidylinositol 4-kinase (EC 2.7.1.67), the first committed ATP-utilizing enzyme of inositol 1,4,5-trisphosphate and diacylglycerol biosynthesis, was determined in a spectrum of rat hepatomas of different growth rates, in sarcoma, and in normal tissues of high cell renewal rates which include differentiating and regenerating liver. A standard isotopic method was developed to measure the enzymic activity in crude particulate extracts. In this assay, the enzyme activity was linear with time for 2 min and proportional with protein concentrations over a range of 0.1 to 1.0 mg per 0.1 ml reaction mixture. The optimum pH for both liver and hepatoma enzyme was 7.4. The apparent Km values of the kinase for ATP, Mg2+, and the substrate phosphatidylinositol in normal liver were 0.03, 10, and 0.2 mM, respectively, and in rapidly growing hepatoma 3924A 0.01, 0.1, and 5.3 mM. The kinase activity in adult rat livers was 0.3 to 0.5 +/- 0.01 nmol/h/mg protein. In hepatomas of slow and intermediate growth rates, kinase activity increased 5.3- to 7.6-fold, and in rapidly proliferating hepatoma 3924A, it was elevated 28.5-fold over that of normal liver. In rat sarcoma, kinase activity was 13.2-fold higher than in normal muscle. To clarify further the linkage between kinase activity and proliferation, enzymic activity was determined in rapidly growing rat tissues. The kinase activity in rat thymus, bone marrow, spleen, and testis increased 8.4-, 7.6-, 5.6- and 5.6-fold, respectively, over the values of normal rat liver; by contrast, in skeletal muscle, liver, heart, and renal cortex, the activities were low. In the rapidly growing neonatal rat liver and in 24-h regenerating liver, activities were 3.4- and 3.0-fold higher than in the adult resting liver. From this study, the relationship of 1-phosphatidylinositol 4-kinase activity with transformation and cell proliferation is clearly apparent in the markedly increased activity in transplantable hepatomas of different growth rates and in sarcoma and is further emphasized by the high activity observed in newborn and regenerating liver and in thymus, bone marrow, spleen, and testis. Since the kinase activity is linked with proliferation and malignancy, it may well be a sensitive target for chemotherapy.

Asunto(s)

Carcinoma Hepatocelular/enzimología , Neoplasias Hepáticas/enzimología , Regeneración Hepática , Hígado/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/análisis , 1-Fosfatidilinositol 4-Quinasa , Animales , Diferenciación Celular , Femenino , Hígado/patología , Hígado/fisiología , Masculino , Ratas , Ratas Endogámicas ACI , Ratas Endogámicas BUF , Ratas Sprague-Dawley , Ratas Wistar , Sarcoma/enzimología , Células Tumorales Cultivadas

RESUMEN

Signal transduction pathways evoked by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) to stimulate expression of other cytokines in mesenchymal cells are not clearly understood. Stimulation of the murine bone marrow stromal cell line +/(+)-1.LDA 11 with IL-1 (500 U/ml) in combination with TNF-alpha (500 U/ml) (IL-1 plus TNF-alpha) induced expression of c-jun mRNA as well as granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA. We investigated the possibility that arachidonic acid metabolites, acting through protein kinase C (PKC) and perhaps also through the PKC-responsive transcription factor c-jun/AP-1, may be responsible for regulating GM-CSF transcription in these stromal cells. Expression of GM-CSF mRNA was preceded by IL-1 plus TNF-alpha induced arachidonate release (assayed using the 3H-derivative). Pretreatment of cells with the phospholipase A2 inhibitor quinacrine (20 microM) inhibited accumulation of both c-jun and GM-CSF mRNA but had no influence on expression of other genes induced by IL-1 and TNF-alpha, including leukemia inhibitory factor (LIF). In addition, quinacrine partially blocked IL-1 plus TNF-alpha induced 3H-arachidonic acid release from prelabeled stromal cells. Furthermore, exogenous arachidonate (10 to 50 microM) induced expression of c-jun. To investigate the role of arachidonate in GM-CSF transcription, we used a reporter vector consisting of the murine GM-CSF promoter linked to firefly luciferase. Transfection efficiency was monitored by assessing expression of a constitutively active gene, RSV-beta galactosidase. In this system, quinacrine significantly inhibited IL-1 plus TNF-alpha induced GM-CSF transcription assayed with the reporter construct. Exogenous arachidonic acid alone (10 microM) increased activity of GM-CSF reporter vector 1.5-fold over control. These results are consistent with the hypothesis that arachidonate metabolites are involved in the signaling pathway that leads to IL-1 plus TNF-alpha induced GM-CSF gene expression. Thus, transcriptional activation of GM-CSF gene is mediated, in part, by the arachidonate cascade.

Asunto(s)

Ácidos Araquidónicos/metabolismo , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Interleucina-1/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Ácidos Araquidónicos/fisiología , Northern Blotting , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Células de la Médula Ósea , Línea Celular , Relación Dosis-Respuesta a Droga , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Ratones , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-jun/análisis , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Quinacrina/farmacología , ARN Mensajero/análisis , ARN Mensajero/genética , Transducción de Señal/fisiología , Transcripción Genética , Transfección , Tritio

RESUMEN

Previous studies have demonstrated that although both mammalian and avian erythrocytes express an inducible inositol bisphosphate-specific phospholipase C, only the latter possess the guanine nucleotide-binding protein (Gp) that regulates this activity. In confirmation of previous reports, turkey erythrocyte plasma membranes responded to guanosine 5'-0-(3-thio)triphosphate (GTP-gamma-S) and fluoroaluminates with hydrolysis of phosphoinositides, release of inositol phosphates, and generation of diacylglycerol, whereas human erythrocyte plasma membranes exhibited no such changes when incubated with known activators of guanine nucleotide regulatory proteins. We next contrasted responses of intact turkey and human erythrocytes to fluoroaluminates to develop a model to investigate the cellular effects of Gp activation. When turkey erythrocytes were exposed to fluoroaluminates, cellular levels of diacylglycerol and phosphatidic acid rapidly increased as phosphoinositides were hydrolyzed. The alterations in the lipid composition of turkey erythrocytes effected by fluoroaluminates were remarkable; phosphatidic acid levels increased over 30-fold, whereas levels of polyphosphoinositides were decreased to less than 10% of those present before stimulation. In contrast, fluoroaluminates caused only minor alterations in the diacylglycerol and phospholipid content of intact human erythrocytes. To define the role of inositol-specific phospholipase C activation in the transmembrane conveyance of extracellular Ca++, we compared the influx of extracellular Ca++ in human and turkey erythrocytes exposed to fluoroaluminates. Fluoroaluminates initiated a sustained influx of extracellular 45Ca++ into turkey, but not human, erythrocytes. These results provide strong support for the hypothesis that Gp activation results in an influx of calcium into stimulated cells. Moreover, the data demonstrate that comparison of responses of human and turkey erythrocytes to fluoroaluminates provides a well-defined method for investigating the mechanisms and consequences of Gp activation in intact cells.

Asunto(s)

Calcio/metabolismo , Eritrocitos/metabolismo , Fluoruros/farmacología , Proteínas de Unión al GTP/fisiología , Fosfatidilinositoles/metabolismo , Animales , Diglicéridos/biosíntesis , Membrana Eritrocítica/metabolismo , Espacio Extracelular/metabolismo , Humanos , Hidrólisis , Fosfatos de Inositol/biosíntesis , Iones , Fosfolípidos/sangre , Pavos

RESUMEN

A case of refractory anemia with sideroblastosis and a number of bone-marrow blasts slightly over the limit which separates the I/II and III FAB-subtypes of myelodysplastic syndromes is described. The leukemic-like type of in vitro growth and the multiple karyotypic changes observed in the bone-marrow cells at presentation were both indicators of the malignant nature of the disorder and underlined the importance of these studies in assessing diagnosis and prognosis in patients with preleukemic disorders. The role that the chromosome aberrations, del(11)(q14) and del(18)(q21), both found in 100% of the bone-marrow metaphases examined, may play in the pathogenesis of the disease is also discussed.

Asunto(s)

Anemia Refractaria con Exceso de Blastos/genética , Deleción Cromosómica , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 18 , Síndromes Mielodisplásicos/genética , Anciano , Anemia Refractaria con Exceso de Blastos/patología , Médula Ósea/patología , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Femenino , Humanos , Cariotipificación , Síndromes Mielodisplásicos/patología , Cromosoma X

RESUMEN

We previously showed that the proliferative response of a serum- and interleukin-3 (IL-3)-dependent murine myeloid cell line, NFS/N1-H7, was partially inhibited by pertussis toxin as a result of toxin-induced increased adenylate cyclase activity. In the present studies, we examined the role of the phosphoinositide cycle in the proliferative response of these cells and demonstrated that there was no change in PIP (phosphatidylinositol bisphosphate)-specific phospholipase C activity in response to IL-3 alone. However, serum caused a pertussis toxin-insensitive increase in PIP2-specific phospholipase C activity as reflected by decreased cellular levels of 32P-labelled PIP2. Proliferation of a subline selected from val-12-mutant H-ras-transfected NFS-H7 cells, clone E5, was insensitive to pertussis toxin, occurred in the absence of serum but remained serum-stimulatable and absolutely dependent on IL-3. This val-12 mutant ras-expressing cell line showed an increase in 32P-labelled PIP (phosphatidylinositol phosphate) in response to serum whereas the parent cell line did not. Membrane fractions from 32P-labelled ras-transfected cells displayed higher GTP gamma S-, GTP-, or F(-)-stimulated PIP2-specific phospholipase C activity compared to membranes from the parent cell line. Thus serum-dependence and adenylate cyclase-mediated pertussis toxin-sensitivity of the parent cell line was bypassed by val-12 mutant ras p21, possibly as a result of increased PIP2-specific phospholipase C activity.

Asunto(s)

Proteínas Proto-Oncogénicas p21(ras)/fisiología , Fosfolipasas de Tipo C/metabolismo , Toxina de Adenilato Ciclasa , Animales , Sangre , División Celular/fisiología , Línea Celular , Membrana Celular/metabolismo , Medios de Cultivo , Interleucina-3/fisiología , Cinética , Ratones , Mutación , Toxina del Pertussis , Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositoles/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Timidina/metabolismo , Factores de Virulencia de Bordetella/farmacología

RESUMEN

Taken together, the above reports indicate that the IMP dehydrogenase inhibitors are valuable probes for investigation of the biological functions of guanine nucleotides in intact cells. While these agents have minor effects on levels of other nucleotides and enzymes, non-specific effects can be monitored by addition of guanine or guanosine to provide substrates for the salvage pathway of guanine nucleotide synthesis. The most important question yet to be resolved in employing these agents is why incomplete depletion of intracellular guanine nucleotides results in such dramatic effects on G-protein function. Since the level of GTP in resting cells is approximately 0.5 mM, even a 90% reduction in GTP levels should leave enough nucleotide to adequately activate most known G-proteins, as the latter display high binding affinities for guanine nucleotides in cell free systems. Several explanations have been proposed to account for this disparity. Much of the intracellular guanine nucleotide may be bound or compartmentalized and therefore unable to interact with certain G-proteins. Possibly, G-proteins in the intracellular environment possess a much lower affinity for GTP that they do in cell free system. It may be to the cells' advantage that relatively minor fluctuations in levels of GTP result in pronounced alterations in the biological function of G-proteins as this effect may provide a physiologically important mechanism for the regulation of G-proteins in vivo. Further studies are necessary to clarify the mechanisms involved in the regulation of the biological function of G-proteins and oncogene products by guanine nucleotides in intact cells.

Asunto(s)

Proteínas de Unión al GTP/fisiología , IMP Deshidrogenasa/antagonistas & inhibidores , Animales , Diferenciación Celular/fisiología , ADN/biosíntesis , Inhibidores Enzimáticos , Nucleótidos de Guanina/metabolismo , Oncogenes/fisiología , Transducción de Señal/fisiología

Asunto(s)

Separación Celular/métodos , Leucocitos/citología , Adulto , Femenino , Filtración , Humanos , Masculino