RESUMEN
In order to investigate whether and how a modification of mitochondrial metabolism can affect yeast sensitivity to programmed cell death (PCD) induced by acetic acid (AA-PCD), yeast cells were grown on raffinose, as a sole carbon source, which, differently from glucose, favours mitochondrial respiration. We found that, differently from glucose-grown cells, raffinose-grown cells were mostly resistant to AA-PCD and that this was due to the activation of mitochondrial retrograde (RTG) response, which increased with time, as revealed by the up-regulation of the peroxisomal isoform of citrate synthase and isocitrate dehydrogenase isoform 1, RTG pathway target genes. Accordingly, the deletion of RTG2 and RTG3, a positive regulator and a transcription factor of the RTG pathway, resulted in AA-PCD, as shown by TUNEL assay. Neither deletion in raffinose-grown cells of HAP4, encoding the positive regulatory subunit of the Hap2,3,4,5 complex nor constitutive activation of the RTG pathway in glucose-grown cells due to deletion of MKS1, a negative regulator of RTG pathway, had effect on yeast AA-PCD. The RTG pathway was found to be activated in yeast cells containing mitochondria, in which membrane potential was measured, capable to consume oxygen in a manner stimulated by the uncoupler CCCP and inhibited by the respiratory chain inhibitor antimycin A. AA-PCD resistance in raffinose-grown cells occurs with a decrease in both ROS production and cytochrome c release as compared to glucose-grown cells en route to AA-PCD.
Asunto(s)
Ácido Acético/farmacología , Apoptosis/efectos de los fármacos , Mitocondrias/metabolismo , Rafinosa/farmacología , Saccharomyces cerevisiae/crecimiento & desarrollo , Transducción de Señal/efectos de los fármacos , Citocromos c/metabolismo , Eliminación de Gen , Glucosa/farmacología , Concentración de Iones de Hidrógeno/efectos de los fármacos , Immunoblotting , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Fosforilación/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Adhesion of normal epithelial cells to the extracellular matrix (ECM) is essential for survival. Cell detachment from ECM induces a specific form of programmed cell death (PCD) termed anoikis. BRCA2, a tumor suppressor gene whose mutations confer predisposition to cancer, has been implicated in the regulation of DNA repair, transcription, cell proliferation, and apoptosis. However, the potential role of BRCA2 in the regulation of anoikis has not been investigated. Here, we found that suppression of BRCA2 expression by short hairpin RNA promoted resistance to anoikis in prostate, breast and thyroid normal epithelial cells, which was accompanied by reduced caspases 3/7 levels and activity. Using yeast as a model, we assessed that expression of human BRCA2 does not induce cell death by itself but it can promote acetic acid-induced PCD (AA-PCD). Induction of BRCA2 expression decreased cell survival and increased the number of cells positive to different apoptotic markers, including DNA fragmentation and phosphatidylserine externalization en route to AA-PCD. A higher increase in ROS levels occurred in the early phase of AA-PCD in BRCA2-expressing yeast cells compared with non-expressing cells. Accordingly, a delay in the initial burst of ROS levels was observed in BRCA2-knockdown anoikis-resistant human cells. Treatment with the antioxidants N-acetylcysteine or ascorbic acid reduced sensitivity to anoikis in human cells and inhibited AA-PCD in yeast cells expressing BRCA2. Taken together, these results show a new function of BRCA2 protein as modulator of anoikis sensitivity through an evolutionarily-conserved molecular mechanism involving regulation of ROS production and/or detoxification by BRCA2 during PCD processes.
Asunto(s)
Ácido Acético/farmacología , Anoicis , Proteína BRCA2/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Acetilcisteína/farmacología , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Proteína BRCA2/genética , Mama/citología , Caspasas/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Silenciador del Gen , Humanos , Masculino , Próstata/citología , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Glándula Tiroides/citologíaRESUMEN
BRCA2 (breast cancer 2, early onset) is a tumor suppressor gene that confers increased susceptibility for prostate cancer (PCa). Previous in vitro experiments demonstrated that Skp2, an E3 ubiquitin ligase aberrantly overexpressed in PCa, is involved in the proteolytic degradation of BRCA2 in PCa cells, suggesting that the BRCA2-Skp2 interaction may play a role in prostate tumorigenesis. Herein, we investigated BRCA2 and Skp2 expression during PCa development using a prostate TMA. Although luminal and basal benign prostate epithelium exhibited moderate to strong nuclear BRCA2 immunostaining, the intensity and number of positive nuclei decreased significantly in high-grade prostatic intraepithelial neoplasia and PCa. Decreased frequency and intensity of nuclear BRCA2 labeling were inversely correlated with Skp2 expression in high-grade prostatic intraepithelial neoplasia and PCa. To functionally assess the effects of BRCA2 and Skp2 expression on prostate malignant transformation, we overexpressed Skp2 in normal immortalized prostate cells. Skp2 overexpression reduced BRCA2 protein and promoted cell growth and migration. A similar phenotype was observed after reduction of BRCA2 protein levels using specific BRCA2 small-interfering RNA. Forced BRCA2 expression in Skp2-overexpressing stable transfectants inhibited the migratory and growth properties by >60%. These results show that loss of BRCA2 expression during prostate tumor development is strongly correlated with both migratory behavior and cancer growth and include Skp2 as a BRCA2 proteolytic partner in vivo.
Asunto(s)
Proteína BRCA2/biosíntesis , Transformación Celular Neoplásica/genética , Neoplasias de la Próstata/metabolismo , Proteínas Quinasas Asociadas a Fase-S/biosíntesis , Proteína BRCA2/genética , Western Blotting , Movimiento Celular , Proliferación Celular , Humanos , Inmunohistoquímica , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Quinasas Asociadas a Fase-S/genética , Análisis de Matrices Tisulares , Transfección , Regulación hacia ArribaRESUMEN
DS (Down's syndrome) is the most common human aneuploidy associated with mental retardation and early neurodegeneration. Mitochondrial dysfunction has emerged as a crucial factor in the pathogenesis of numerous neurological disorders including DS, but the cause of mitochondrial damage remains elusive. In the present study, we identified new molecular events involved in mitochondrial dysfunction which could play a role in DS pathogenesis. We analysed mitochondrial respiratory chain function in DS-HSFs (Down's syndrome human foetal skin fibroblasts; human foetal skin fibroblasts with chromosome 21 trisomy) and found a selective deficit in the catalytic efficiency of mitochondrial complex I. The complex I deficit was associated with a decrease in cAMP-dependent phosphorylation of the 18 kDa subunit of the complex, due to a decrease in PKA (protein kinase A) activity related to reduced basal levels of cAMP. Consistently, exposure of DS-HSFs to db-cAMP (dibutyryl-cAMP), a membrane-permeable cAMP analogue, stimulated PKA activity and consequently rescued the deficit of both the cAMP-dependent phosphorylation and the catalytic activity of complex I; conversely H89, a specific PKA inhibitor, suppressed these cAMP-dependent activations. Furthermore, in the present paper we report a 3-fold increase in cellular levels of ROS (reactive oxygen species), in particular superoxide anion, mainly produced by DS-HSF mitochondria. ROS accumulation was prevented by db-cAMP-dependent activation of complex I, suggesting its involvement in ROS production. Taken together, the results of the present study suggest that the drastic decrease in basal cAMP levels observed in DS-HSFs participates in the complex I deficit and overproduction of ROS by DS-HSF mitochondria.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Fibroblastos/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Síndrome de Down/genética , Síndrome de Down/metabolismo , Humanos , Transducción de SeñalRESUMEN
Cell homeostasis results from the balance between cell capability to adapt or succumb to environmental stress. Mitochondria, in addition to supplying cellular energy, are involved in a range of processes deciding about cellular life or death. The crucial role of mitochondria in cell death is well recognized. Mitochondrial dysfunction has been associated with the death process and the onset of numerous diseases. Yet, mitochondrial involvement in cellular adaptation to stress is still largely unexplored. Strong interest exists in pharmacological manipulation of mitochondrial metabolism and signaling. The yeast Saccharomyces cerevisiae has proven a valuable model organism in which several intracellular processes have been characterized in great detail, including the retrograde response to mitochondrial dysfunction and, more recently, programmed cell death. In this paper we review experimental evidences of mitochondrial involvement in cytoprotection and propose yeast as a model system to investigate the role of mitochondria in the cross-talk between prosurvival and prodeath pathways.
Asunto(s)
Citoprotección , Mitocondrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Muerte Celular , Supervivencia Celular , Homeostasis , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/fisiología , Estrés FisiológicoRESUMEN
The use of non-mammalian model organisms, including yeast Saccharomyces cerevisiae, can provide new insights into eukaryotic PCD (programmed cell death) pathways. In the present paper, we report recent achievements in the elucidation of the events leading to PCD that occur as a response to yeast treatment with AA (acetic acid). In particular, ROS (reactive oxygen species) generation, cyt c (cytochrome c) release and mitochondrial function and proteolytic activity will be dealt with as they vary along the AA-PCD time course by using both wild-type and mutant yeast cells. Two AA-PCD pathways are described sharing common features, but distinct from one another with respect to the role of ROS and mitochondria, the former in which YCA1 acts upstream of cyt c release and caspase-like activation in a ROS-dependent manner and the latter in which cyt c release does not occur, but caspase-like activity increases, in a ROS-independent manner.
Asunto(s)
Ácido Acético/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/fisiología , Caspasas/metabolismo , Citocromos c/metabolismo , Peróxido de Hidrógeno/metabolismo , Mitocondrias/metabolismo , Oxidantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
A central role for mitochondrial dysfunction has been proposed in the pathogenesis of DS (Down's syndrome), a multifactorial disorder caused by trisomy of human chromosome 21. To explore whether and how abnormalities in mitochondrial energy metabolism are involved in DS pathogenesis, we investigated the catalytic properties, gene expression and protein levels of certain proteins involved in mitochondrial ATP synthesis, such as F1F0-ATPase, ANT (adenine nucleotide translocator) and AK (adenylate kinase), in DS-HSF (human skin fibroblasts with trisomic karyotype), comparing them with euploid fibroblasts. In DS-HSF, we found a strong impairment of mitochondrial ATP synthesis due to a reduction in the catalytic efficiency of each of the investigated proteins. This impairment occurred in spite of unchanged gene expression and an increase in ANT and AK protein content, whereas the amount of ATPase subunits was selectively reduced. Interestingly, exposure of DS-HSF to dibutyryl-cAMP, a permanent derivative of cAMP, stimulated ANT, AK and ATPase activities, whereas H89, a specific PKA (protein kinase A) inhibitor, suppressed this cAMPdependent activation, indicating an involvement of the cAMP/PKA-mediated signalling pathway in the ATPase, ANT and AK deficit. Consistently, DS-HSF showed decreased basal levels of cAMP and reduced PKA activity. Despite the impairment of mitochondrial energy apparatus, no changes in cellular energy status, but increased basal levels of L-lactate, were found in DS-HSF, which partially offset for the mitochondrial energy deficit by increasing glycolysis and mitochondrial mass.These results provide new insight into the molecular basis for mitochondrial dysfunction in DS and might provide a molecular explanation for some clinical features of the syndrome.
Asunto(s)
Adenilato Quinasa/metabolismo , Metabolismo Energético/genética , Fibroblastos/enzimología , Mitocondrias/enzimología , Translocasas Mitocondriales de ADP y ATP/metabolismo , ATPasas de Translocación de Protón/metabolismo , Trisomía/genética , Adenosina Trifosfato/biosíntesis , Adenilato Quinasa/genética , Línea Celular , Respiración de la Célula/genética , Cromosomas Humanos Par 21/genética , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN Mitocondrial/metabolismo , Fibroblastos/patología , Regulación Enzimológica de la Expresión Génica , Humanos , Cinética , Ácido Láctico/metabolismo , Translocasas Mitocondriales de ADP y ATP/genética , Fosforilación Oxidativa , ATPasas de Translocación de Protón/genética , Piel/patologíaRESUMEN
Having confirmed that externally added L-lactate can enter cerebellar granule cells, we investigated whether and how L-lactate is metabolized by mitochondria from these cells under normal or apoptotic conditions. (1) L-lactate enters mitochondria, perhaps via an L-lactate/H+ symporter, and is oxidized in a manner stimulated by ADP. The existence of an L-lactate dehydrogenase, located in the inner mitochondrial compartment, was shown by immunological analysis. Neither the protein level nor the Km and Vmax values changed en route to apoptosis. (2) In both normal and apoptotic cell homogenates, externally added L-lactate caused reduction of the intramitochondrial pyridine cofactors, inhibited by phenylsuccinate. This process mirrored L-lactate uptake by mitochondria and occurred with a hyperbolic dependence on L-lactate concentrations. Pyruvate appeared outside mitochondria as a result of external addition of L-lactate. The rate of the process depended on L-lactate concentration and showed saturation characteristics. This shows the occurrence of an intracellular L-lactate/pyruvate shuttle, whose activity was limited by the putative L-lactate/pyruvate antiporter. Both the carriers were different from the monocarboxylate carrier. (3) L-lactate transport changed en route to apoptosis. Uptake increased in the early phase of apoptosis, but decreased in the late phase with characteristics of a non-competitive like inhibition. In contrast, the putative L-lactate/pyruvate antiport decreased en route to apoptosis with characteristics of a competitive like inhibition in early apoptosis, and a mixed non-competitive like inhibition in late apoptosis.
Asunto(s)
Apoptosis/fisiología , Cerebelo/metabolismo , Ácido Láctico/metabolismo , Mitocondrias/metabolismo , Potasio/farmacología , Animales , Apoptosis/efectos de los fármacos , Transporte Biológico , Western Blotting , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glucosa/farmacología , Cinética , L-Lactato Deshidrogenasa/metabolismo , Ácido Láctico/farmacocinética , Ácido Láctico/farmacología , Mitocondrias/efectos de los fármacos , Modelos Biológicos , NAD/metabolismo , Oxidación-Reducción/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
To investigate whether and how mitochondria can change in plant programmed cell death (PCD), we used the non-photosynthetic Tobacco Bright Yellow 2 (TBY-2) cells. These can be synchronized to high levels, stand out in terms of growth rate and homogeneity and undergo PCD as a result of heat shock. Using these cells we investigated the activity of certain mitochondrial proteins that have a role in providing ATP and/or other nucleoside triphosphates (NTPs). We show that, already after 2 h from the heat shock, when cell viability remains unaffected, the rate of ADP/ATP exchange due to adenine nucleotide translocator (ANT) activity, and the rate of the reactions catalysed by adenylate kinase (ADK; EC 2.7.4.3) and nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) are inhibited in a non-competitive-like manner. In all cases, externally added ascorbate partially prevented the inhibition. These effects occurred in spite of minor (for ANT) or no changes in the mitochondrial protein levels as immunologically investigated. Interestingly, a decrease of both the steady state level of the ascorbate pool and of the activity of l-galactono-gamma-lactone dehydrogenase (GLDH) (EC 1.3.2.3), the mitochondrial enzyme catalysing the last step of ascorbate biosynthesis, were also found.
Asunto(s)
Adenilato Quinasa/metabolismo , Apoptosis/fisiología , Mitocondrias/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Nicotiana/fisiología , Nucleósido-Difosfato Quinasa/metabolismo , Línea Celular , Fumarato Hidratasa/metabolismo , Calor , Immunoblotting , Cinética , Octoxinol , Espectrometría de Fluorescencia , Factores de Tiempo , Nicotiana/metabolismoRESUMEN
BRCA2 is a multifunctional tumor suppressor protein which plays critical roles in DNA repair, transcription, and cell proliferation, and the loss of which has been linked to the biology of several types of cancers. Here, on prostate adenocarcinoma specimens from 80 patients, we demonstrate that BRCA2 protein is lost in carcinoma cells compared to normal and hyperplastic prostate epithelium. Using highly metastatic prostate cancer PC-3 cells, we show that while BRCA2 depletion by small-interfering RNA promoted migration onto the extracellular matrix proteins fibronectin, laminin, and collagens, as well as invasion through the reconstituted basement membrane matrix Matrigel by more than 140%, recombinant BRCA2 overexpression decreased both phenomena by 57-80% and changed cell morphology from angular and spindle to round and compact. The BRCA2 inhibitory effect on cancer cell migration and invasion resulted from down-regulation of matrix metalloproteinase (MMP)-9 protein levels due to increased MMP-9 proteolysis, and was signaled through inhibition of PI3-kinase/AKT and activation of MAPK/ERK pathway. In BRCA2-overexpressing PC-3 cells, transient transfection with a constitutively active PI3-kinase mutant or treatment with the MAPK/ERK inhibitor PD98059 rescued MMP-9 levels and restored the migratory and invasive capabilities. Consistently, PI3-kinase inhibition with a dominant-negative mutant or MAPK/ERK activation with a gain-of-function mutant reduced MMP-9 levels and prevented migration and invasion in wild-type PC-3 cells. These results provide novel evidence showing that a functional BRCA2 protein may limit the metastatic potential of neoplastic cells by down-regulating MMP-9 production through inhibition of PI3-kinase/AKT and activation of MAPK/ERK, effectively hindering cancer cell migration and invasion.
Asunto(s)
Proteína BRCA2/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias de la Próstata/enzimología , Regulación hacia Arriba , Proteína BRCA2/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
To investigate the role of catalase and superoxide dismutase (SOD) in the acetic acid (AA) induced yeast programmed cell death (AA-PCD), we compared Saccharomyces cerevisiae cells (C-Y) and cells individually over-expressing catalase T (CTT1-Y) and Cu,Zn-SOD (SOD1-Y) with respect to cell survival, hydrogen peroxide (H2O2) levels and enzyme activity as measured up to 200 min after AA treatment. AA-PCD does not occur in CTT1-Y, where H2O2 levels were lower than in C-Y and the over-expressed catalase activity decreased with time. In SOD1-Y, AA-PCD was exacerbated; high H2O2 levels were found, SOD activity increased early, remaining constant en route to AA-PCD, but catalase activity was strongly reduced.
Asunto(s)
Ácido Acético/farmacología , Apoptosis/efectos de los fármacos , Catalasa/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Microscopía Fluorescente , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimologíaRESUMEN
To gain insight into the processes by which acetic acid-induced programmed cell death (AA-PCD) takes place in yeast, we investigated both cytochrome c release from yeast mitochondria and mitochondrial coupling over the time course of AA-PCD. We show that the majority of cytochrome c release occurs early in AA-PCD from intact coupled mitochondria which undergo only gradual impairment. The released cytochrome c can be reduced both by ascorbate and by superoxide anion and in turn be oxidized via cytochrome c oxidase, thus working both as a ROS scavenger and a respiratory substrate. Late in AA-PCD, the released cytochrome c is degraded.
Asunto(s)
Ácido Acético/metabolismo , Apoptosis , Citocromos c/metabolismo , Depuradores de Radicales Libres/metabolismo , Mitocondrias/metabolismo , Saccharomyces cerevisiae/enzimología , Ácido Acético/farmacología , Ácido Ascórbico/farmacología , Electrones , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
BACKGROUND: The pyridoxal-5'-phosphate (PLP)-dependent or vitamin B6-dependent enzymes that catalyze manifold reactions in the metabolism of amino acids belong to no fewer than four evolutionarily independent protein families. The multiple evolutionary origin and the essential mechanistic role of PLP in these enzymes argue for the cofactor having arrived on the evolutionary scene before the emergence of the respective apoenzymes and having played a dominant role in the molecular evolution of the B6 enzyme families. Here we report on an attempt to re-enact the emergence of a PLP-dependent protoenzyme. The starting protein was pancreatic ribonuclease A (RNase), in which active-site Lys41 or Lys7 readily form a covalent adduct with PLP. RESULTS: We screened the PLP adduct of wild-type RNase and two variant RNases (K7R and K41R) for catalytic effects toward L- and D-amino acids. RNase(K41R)-PLP, in which the cofactor is bound through an imine linkage to Lys7, qualifies for a model proto-B6 enzyme by the following criteria: (1) covalent linkage of PLP (internal aldimine); (2) catalytic activity toward amino acids that depends on formation of an imine linkage with the substrate (external aldimine); (3) adjoining binding sites for the cofactor and amino acid moiety that facilitate the transimination reaction of the internal to the external aldimine and stabilize the resulting noncovalent complex of the coenzyme-substrate adduct with the protein; (4) reaction specificity, the only detectable reactions being racemization of diverse amino acids and beta-decarboxylation of L-aspartate; (5) acceleration factors for racemization and beta-decarboxylation of >103 over and above that of PLP alone; (6) ribonuclease activity that is 103-fold lower than that of wild-type RNase, attenuation of a pre-existing biological activity being indispensable for the further evolution as a PLP-dependent protoenzyme. CONCLUSION: A single amino acid substitution (Lys41Arg) and covalent binding of PLP to active-site Lys7 suffice to turn pancreatic ribonuclease A into a protein catalyst that complies with all plausible criteria for a proto-B6 enzyme. The study thus retraces in a model system what may be considered the committed step in the molecular evolution of a potential ancestor of a B6 enzyme family.
Asunto(s)
Arginina/química , Evolución Molecular , Lisina/química , Fosfato de Piridoxal/metabolismo , Ribonucleasa Pancreática/química , Vitamina B 6/química , Sustitución de Aminoácidos , Sitios de Unión , Catálisis , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ribonucleasa Pancreática/genética , Vitamina B 6/metabolismoRESUMEN
Mitochondrial dysfunction resulting from mitochondrial DNA (mtDNA) mutations and/or depletion has been correlated with cancer progression and drug resistance. To investigate the role of mtDNA in prostate cancer progression, we used LNCaP and PC-3 prostate carcinoma cells as experimental model. Compared to minimally invasive androgen-dependent LNCaP cells, highly invasive androgen-independent PC-3 cells, as well as androgen-independent DU145 and C4-2 cells, exhibited significantly reduced mtDNA content. In PC-3 cells, reduction of mtDNA was accompanied by decreased mitochondrial membrane potential (DeltaPsi(m)), increased migration onto the basement membrane protein laminin-1, reduced chemosensitivity to paclitaxel (IC(50)=110 nM vs. 22 nM) and decreased expression of poly(ADP-ribose) polymerase (PARP)-1. To investigate the relationship between mtDNA depletion and these phenotypic characteristics, we established mtDNA-depleted LNCaP cells [Rho(-)] by long-term exposure to ethidium bromide or treated wild-type LNCaP cells with a mitochondrial ionophore, carbonyl cyanide m-chlorophenylhydrazone. Both manipulations resulted in DeltaPsi(m) loss, acquisition of invasive cytology, increased motility onto laminin-1, reduced sensitivity to paclitaxel (IC(50)= approximately 100 nM) and approximately 75% reduction in PARP-1 protein levels, resembling PC-3 cells. Overall, these results provide novel evidence demonstrating that mtDNA depletion in early prostate carcinoma may contribute to the acquisition of a more invasive phenotype that is less sensitive to paclitaxel-induced apoptosis.
Asunto(s)
Andrógenos/metabolismo , ADN Mitocondrial/genética , Neoplasias Hormono-Dependientes/genética , Poli(ADP-Ribosa) Polimerasas/genética , Neoplasias de la Próstata/genética , Apoptosis/efectos de los fármacos , Carcinógenos/farmacología , Línea Celular Tumoral , Ensayos de Migración Celular , Movimiento Celular/genética , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , ADN Mitocondrial/antagonistas & inhibidores , ADN Mitocondrial/biosíntesis , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Etidio/farmacología , Humanos , Masculino , Potencial de la Membrana Mitocondrial , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Neoplasias Hormono-Dependientes/patología , Paclitaxel/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/biosíntesis , Neoplasias de la Próstata/patologíaRESUMEN
An early increase in ROS production is characteristic of cerebellar granule cells undergoing apoptosis in the presence of 5 mM KCl. However, the sources of this increase have not been investigated in detail. In particular whether there is a single enzymatic source or the increase in ROS production is the consequence of the involvement of different enzymes has not been studied in depth. Different enzymatic pathways may indeed contribute to the up-regulation of intracellular ROS production either directly or via side-chain reactions and a number of candidate enzymes are known to be involved in the apoptotic process in various cell types. The aim of this study was to identify the cellular sources of the ROS generated by CGCs undergoing apoptosis by low K+. A panel of specific inhibitors against phospholipase, cytochromes P450, cyclooxygenase, lipoxygenase, xanthine oxidase, ribonucleotide reductase and NADPH oxidase were used. We provide evidence that no single source of ROS can be identified in apoptotic CGCs, but the ROS generated through the arachidonic acid (AA) pathways, mainly via lipoxygenase activities, seems to be the most prominent.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Potasio/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Animales , Ácido Araquidónico/metabolismo , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Lipooxigenasa/metabolismo , Inhibidores de la Lipooxigenasa/farmacología , Modelos Biológicos , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfolipasas/antagonistas & inhibidores , Fosfolipasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Ratas , Ratas Wistar , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/metabolismo , Superóxidos/análisis , Xantina Oxidasa/antagonistas & inhibidores , Xantina Oxidasa/metabolismoRESUMEN
The interactions between cancer cells and the extracellular matrix (ECM) regulate cancer progression. The beta1C and beta1A integrins, two cytoplasmic variants of the beta1 integrin subfamily, are differentially expressed in prostate cancer. Using gene expression analysis, we show here that the beta1C variant, an inhibitor of cell proliferation, which is down-regulated in prostate cancer, up-regulates insulin-like growth factor-II (IGF-II) mRNA and protein levels. In contrast, beta1A does not affect IGF-II levels. We provide evidence that beta1C-mediated up-regulation of IGF-II levels increases adhesion to Laminin-1, a basement membrane protein down-regulated in prostate cancer, and that the beta1C cytoplasmic domain contains the structural motif sufficient to increase cell adhesion to Laminin-1. This autocrine mechanism that locally supports cell adhesion to Laminin-1 via IGF-II is selectively regulated by the beta1 cytoplasmic domain via activation of the growth factor receptor binding protein 2-associated binder-1/SH2-containing protein-tyrosine phosphatase 2/phosphatidylinositol 3-kinase pathway. Thus, the concurrent local loss of beta1C integrin, of its ligand Laminin-1, and of IGF-II in the tumor microenvironment may promote prostate cancer cell invasion and metastasis by reducing cancer cell adhesive properties. It is, therefore, conceivable that reexpression of beta1C will be sufficient to revert a neoplastic phenotype to a nonproliferative and highly adherent normal phenotype.
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/biosíntesis , Integrina beta1/fisiología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células CHO , Adhesión Celular/fisiología , Cricetinae , Citoplasma/metabolismo , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Integrina beta1/biosíntesis , Integrina beta1/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Laminina/metabolismo , Masculino , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatasa 2 , Estructura Terciaria de Proteína , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transfección , Regulación hacia ArribaRESUMEN
To find out whether and how proteasome is involved in plant programmed cell death (PCD) we measured proteasome function in tobacco cells undergoing PCD as a result of heat shock (HS-PCD). Reactive oxygen species (ROS) production, cytochrome c levels and caspase-3-like protease activation were also measured in the absence or presence of MG132, a proteasome inhibitor. We show that proteasome activation occurs in early phase of HS-PCD upstream of the caspase-like proteases activation; moreover inhibition of proteasome function by MG132 results in prevention of PCD perhaps due to the prevention of ROS production, cytochrome c release and caspase-3-like protease activation.
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Apoptosis/fisiología , Nicotiana/citología , Nicotiana/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular , Citocromos c/metabolismo , Citosol/metabolismo , Respuesta al Choque Térmico , Calor , Leupeptinas/farmacología , Mitocondrias/metabolismo , Inhibidores de Proteasas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Nicotiana/efectos de los fármacosRESUMEN
BRCA2 is central to an utterly diverse biological behavior elicited after integrin-mediated normal and prostate cancer cell adhesion to basement membrane (BM) and extracellular matrix (ECM) proteins. Unlike normal cells, adhesive stimuli in cancer cells activate PI 3-kinase/AKT signaling resulting in BRCA2 degradation and unchecked cancer cell proliferation and metastasis. However, the precise mechanisms involved in normal BRCA2 homeostasis are unknown. We investigated ERK and AKT phosphorylation in normal (PNT1A) and cancer (PC-3) prostate cells after adhesion to ECM and the effects upon BRCA2 and cell proliferation. PNT1A cell adhesion to ECM triggered MAPK/ERK signaling resulting in upregulation of BRCA2 mRNA and protein, with negligible effects upon cell proliferation. Disruption of MAPK/ERK with PD98059 prevented any BRCA2 upregulation inhibiting DNA synthesis below basal levels. PC-3 cells exhibited a defective MAPK/ERK pathway that was unresponsive to adhesion to the ECM, which instead triggered PI 3-kinase/AKT signaling leading to BRCA2 protein depletion and cell proliferation. Reconstitution of MAPK/ERK by recombinant expression of a constitutively active form of MAPK kinase 1 (MEK1) effectively reversed the neoplastic phenotype by increasing BRCA2 expression and preventing any aberrant cell proliferation at rest and upon interaction with ECM proteins. Our results suggest that aberrant loss of MAPK/ERK activity in prostate cancer may play a pivotal role in the malignant phenotype, and provide evidence that interventions aimed at bypassing the signaling block are able to effectively reverse neoplastic unchecked cell proliferation.
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Proteína BRCA2/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasias de la Próstata/patología , Adhesión Celular , División Celular , Línea Celular Tumoral , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/genética , Humanos , Immunoblotting , Masculino , Proteínas Quinasas Activadas por Mitógenos/genética , ARN Interferente Pequeño/genética , Timidina/metabolismo , TransfecciónRESUMEN
Although it is recognized that ATP plays a part in apoptosis, whether and how its level changes en route to apoptosis as well as how ATP is synthesized has not been fully investigated. We have addressed these questions using cultured cerebellar granule cells. In particular, we measured the content of ATP, ADP, AMP, IMP, inosine, adenosine and L-lactate in cells undergoing apoptosis during the commitment phase (0-8 h) in the absence or presence of oligomycin or/and of citrate, which can inhibit totally the mitochondrial oxidative phosphorylation and largely the substrate-level phosphorylation in glycolysis, respectively. In the absence of inhibitors, apoptosis was accompanied by an increase in ATP and a decrease in ADP with 1:1 stoichiometry, with maximum ATP level found at 3 h apoptosis, but with no change in levels of AMP and its breakdown products and with a relatively low level of L-lactate production. Consistently, there was an increase in the cell energy charge and in the ratio ([ATP][AMP])/[ADP](2). When the oxidative phosphorylation was completely blocked by oligomycin, a decrease of the ATP content was found both in control cells and in cells undergoing apoptosis, but nonetheless cells still died by apoptosis, as shown by checking DNA laddering and by death prevention due to actinomycin D. In this case, ATP was provided by anaerobic glycolysis, as suggested by the large increase of L-lactate production. On the other hand, citrate itself caused a small decrease in ATP level together with a huge decrease in L-lactate production, but it had no effect on cell survival. When ATP level was further decreased due to the presence of both oligomycin and citrate, death occurred via necrosis at 8 h, as shown by the lack of DNA laddering and by death prevention found due to the NMDA receptor antagonist MK801. However, at a longer time, when ATP level was further decreased, cells died neither via apoptosis nor via glutamate-dependent necrosis, in a manner similar to something like to energy catastrophe. Our results shows that cellular ATP content increases in cerebellar granule cell apoptosis, that the role of oxidative phosphorylation is facultative, i.e. ATP can also derive from anaerobic glycolysis, and that the type of cell death depends on the ATP availability.
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Adenosina Trifosfato/metabolismo , Apoptosis/fisiología , Cerebelo/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenilato Quinasa/metabolismo , Anaerobiosis , Animales , Cerebelo/citología , Cerebelo/efectos de los fármacos , Ácido Cítrico/farmacología , Fragmentación del ADN , Glucólisis , Ácido Láctico/biosíntesis , Oligomicinas/farmacología , Fosforilación Oxidativa , Ratas , Ratas WistarRESUMEN
Yeast cells lacking the metacaspase-encoding gene YCA1 (Deltayca1) were compared with wild-type (WT) cells with respect to the occurrence, nature and time course of acetic-acid triggered death. We show that Deltayca1 cells undergo programmed cell death (PCD) with a rate lower than that of the WT and that PCD in WT cells is caused at least in part by the caspase activity of Yca1p. Since in Deltayca1 cells this effect is lost, but z-VAD-fmk does not prevent both WT and Deltayca1 cell death, PCD in WT cells occurs via a Yca1p caspase and a non-caspase route with similar characteristics.