RESUMEN
RAS signalling is involved in the control of several metabolic pathways including glycolysis, mitochondrial respiration and glutamine metabolism. Importantly, we have found here that loss of PDHK4, a key regulator of the pyruvate dehydrogenase complex, caused a profound cell growth inhibition in tumour cells harbouring KRAS mutations. Using isogenic cells and a panel of colorectal and lung cell lines we demonstrated that KRAS mutant cells showed a dependency on PDHK4 whereas KRAS wild-type cells were significantly resistant to PDHK4 knockdown. We have found that PDHK4 plays a role in the post-translational regulation of mutant KRAS activity. Depletion of PDHK4 causes disruption of KRAS cellular localization, a reduction in KRAS activity which, in turn, results in reduced MAPK signalling. Interestingly, PDHK4 and KRAS depletion resulted in a similar metabolic phenotype consisting of a reduction of glucose and fatty acid oxidation. Moreover, stable expression of PDHK4 increased localization of activated KRAS at the plasma membrane and induced tumour cell growth in vitro and in vivo. Taken together these data support a model where PDHK4 regulates KRAS signalling and its tumorigenic properties and suggest that inhibition of PDHK4 could represent a novel therapeutic strategy to target KRAS mutant colorectal and lung cancers.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Pulmonares/genética , Proteínas Quinasas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/patología , Mutación , Transducción de Señal/genéticaRESUMEN
Peroxisome proliferators (PPs) are a diverse group of chemicals that cause hepatic proliferation, suppression of apoptosis, peroxisome proliferation and liver tumours in rodents. The biochemical response to PPs involves changes in the expression of peroxisomal beta-oxidation enzymes and fatty acid transport proteins such as acyl-CoA oxidase and liver fatty acid binding protein. The response to PPs is mediated by the peroxisome proliferator-activated receptor alpha (PPARalpha) and the livers of PPARalpha-null transgenic mice do not develop tumours in response to PPs. In order to identify the molecular pathways underlying the adverse effects of PPs in rodent liver, we carried out two-dimensional differential gel electrophoresis to provide quantitative proteomic analyses of diethylhexylphthalate (DEHP)-treated wild-type or PPARalpha-null mouse livers. Since tumourigenesis is both PP- and PPARalpha-dependent, analyses were focused on these changes. Fifty-nine proteins were identified where altered expression was both PPARalpha- and PP-dependent. In addition, six proteins regulated by the deletion of PPARalpha were identified, possibly indicating an adaptive change in response to the loss of this receptor. The proteins that we identified as being regulated by PPARalpha are known to be involved in lipid metabolism pathways, but also in amino acid and carbohydrate metabolism, mitochondrial bioenergetics and in stress responses including several genes not previously reported to be regulated by PPARalpha. These data provide novel insights into the pathways utilised by PPs and may assist in the identification of early markers rodent nongenotoxic hepatocarcinogenesis.
Asunto(s)
Dietilhexil Ftalato/toxicidad , Hígado/metabolismo , Proliferadores de Peroxisomas/toxicidad , Peroxisomas/metabolismo , Proteoma/análisis , Animales , Western Blotting , Electroforesis en Gel Bidimensional , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Peroxisomas/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de TranscripciónRESUMEN
Fluorescence two-dimensional differential gel electrophoresis (2-D DIGE*) is a new development in protein detection for two-dimensional gels. Using mouse liver homogenates (control and paracetamol (N-acetyl-p-aminophenol, APAP)-treated), we have determined the quantitative variation in the 2-D DIGE process and established statistically valid thresholds for assigning quantitative changes between samples. Thresholds were dependent on normalised spot volume, ranged from approximately 1.2 fold for large volume spots to 3.5 fold for small volume spots and were not markedly affected by the particular cyanine dye combination or by multiple operators carrying out the dye labelling reaction. To minimise the thresholds, substantial user editing was required when using ImageMaster 2D-Elite software. The difference thresholds were applied to the test system and quantitative protein differences were determined using replicate gels of pool samples and single gels from multiple individual animals (control vs treated in each gel). Throughout, the differences revealed with a particular cyanine dye combination were mirrored almost without exception when the dye combination was reversed. Both pool and individual sample analyses provided unique data to the study. The inter-animal response variability in inbred mice was approximately nine times that contributed by the 2-D DIGE process. A number of the most frequently observed protein changes resulting from APAP-treatment were identified by mass spectrometry. Several of these can be rationalised based on available data on the mechanism of APAP hepatotoxicity but others cannot, indicating that proteomics can provide further insights into the biochemical basis of APAP toxicity.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Proteoma/aislamiento & purificación , Acetaminofén/toxicidad , Animales , Biotecnología , Fluorescencia , Colorantes Fluorescentes , Hígado/química , Hígado/efectos de los fármacos , Masculino , Espectrometría de Masas/métodos , Ratones , Proteínas/aislamiento & purificaciónRESUMEN
Peroxisome proliferators are nongenotoxic rodent-liver carcinogens that have been shown to cause both an induction of hepatocyte proliferation and a suppression of apoptosis. Both epidermal growth factor (EGF) and the peroxisome proliferator nafenopin induce DNA replication in primary rat hepatocyte cultures, but apparently through different signalling pathways. However, both EGF and nafenopin require tumour necrosis factor alpha (TNFalpha) signalling to induce DNA replication. By examining proteins isolated from rat primary hepatocyte cultures using two-dimensional gel electrophoresis and mass spectrometry, we found that proteins showing an altered expression pattern in response to nafenopin differed from those showing altered expression in response to EGF. However, many proteins showing altered expression upon stimulation with TNFalpha were common to both the EGF and nafenopin responses. These proteome profiling experiments contribute to a better understanding of the molecular mechanisms involved in the response to peroxisome proliferators. We found 32 proteins with altered expression upon stimulation with nafenopin, including muscarinic acetylcholine receptor 3, intermediate filament vimentin and the beta subunit of the ATP synthase. These nonperoxisomal protein targets offer insights into the mechanisms of peroxisome proliferator-induced carcinogenesis in rodents and provide opportunities to identify toxicological markers to facilitate early identification of nongenotoxic carcinogens.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Hígado/citología , Nafenopina/farmacología , Proliferadores de Peroxisomas/farmacología , Proteoma/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Actinas/biosíntesis , Animales , Células Cultivadas , Replicación del ADN/efectos de los fármacos , Bases de Datos Factuales , Dimetilformamida/farmacología , Electroforesis en Gel Bidimensional , Expresión Génica/efectos de los fármacos , Procesamiento de Imagen Asistido por Computador , Focalización Isoeléctrica , Hígado/metabolismo , Espectrometría de Masas , Estrés Oxidativo , Ratas , Receptores Muscarínicos/metabolismo , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Regulación hacia Arriba , Vimentina/biosíntesisRESUMEN
Microparticle generation during cardiopulmonary bypass was monitored continuously in 60 adult patients who underwent open-heart operations. Echo-ultrasound transducers of 5 mHz frequency were interposed in a bubble oxygenator arterial line proximal and distal to a commercially available micropore filter. Ordinary perfusion events correlated with an increase in embolic counts and were recorded graphically. Calculation of filter efficiency revealed that all filters decreased measurable embolic counts. Platelet and leukocyte determinations and plasma hemoglobin values were not altered beyond limits ordinarily encountered during perfusion without filters. No patient in any filter group experienced postoperative respiratory distress, diffuse pulmonary infiltrate, or low PaO2. The 20 mu woven nylon mesh filter and the Dacron-wool filter showed greater than 90% effectiveness in removing recorded particles. Insertion of a cardiotomy filter did not appreciably alter recorded embolic counts distal to the arterial line filter.