RESUMEN
Most tumours have an aberrantly activated lipid metabolism1,2 that enables them to synthesize, elongate and desaturate fatty acids to support proliferation. However, only particular subsets of cancer cells are sensitive to approaches that target fatty acid metabolism and, in particular, fatty acid desaturation3. This suggests that many cancer cells contain an unexplored plasticity in their fatty acid metabolism. Here we show that some cancer cells can exploit an alternative fatty acid desaturation pathway. We identify various cancer cell lines, mouse hepatocellular carcinomas, and primary human liver and lung carcinomas that desaturate palmitate to the unusual fatty acid sapienate to support membrane biosynthesis during proliferation. Accordingly, we found that sapienate biosynthesis enables cancer cells to bypass the known fatty acid desaturation pathway that is dependent on stearoyl-CoA desaturase. Thus, only by targeting both desaturation pathways is the in vitro and in vivo proliferation of cancer cells that synthesize sapienate impaired. Our discovery explains metabolic plasticity in fatty acid desaturation and constitutes an unexplored metabolic rewiring in cancers.
Asunto(s)
Ácidos Grasos/química , Ácidos Grasos/metabolismo , Redes y Vías Metabólicas , Neoplasias/metabolismo , Neoplasias/patología , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Proliferación Celular , Ácido Graso Desaturasas/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ácidos Oléicos/metabolismo , Palmitatos/metabolismo , Ácidos Palmíticos/metabolismo , Estearoil-CoA Desaturasa/metabolismoRESUMEN
Aberrant activation in fibroblast growth factor signaling has been implicated in the development of various cancers, including squamous cell lung cancer, squamous cell head and neck carcinoma, colorectal and bladder cancer. Thus, fibroblast growth factor receptors (FGFRs) present promising targets for novel cancer therapeutics. Here, we evaluated the activity of a novel pan-FGFR inhibitor, rogaratinib, in biochemical, cellular and in vivo efficacy studies in a variety of preclinical cancer models. In vitro kinase activity assays demonstrate that rogaratinib potently and selectively inhibits the activity of FGFRs 1, 2, 3 and 4. In line with this, rogaratinib reduced proliferation in FGFR-addicted cancer cell lines of various cancer types including lung, breast, colon and bladder cancer. FGFR and ERK phosphorylation interruption by rogaratinib treatment in several FGFR-amplified cell lines suggests that the anti-proliferative effects are mediated by FGFR/ERK pathway inhibition. Furthermore, rogaratinib exhibited strong in vivo efficacy in several cell line- and patient-derived xenograft models characterized by FGFR overexpression. The observed efficacy of rogaratinib strongly correlated with FGFR mRNA expression levels. These promising results warrant further development of rogaratinib and clinical trials are currently ongoing (ClinicalTrials.gov Identifiers: NCT01976741, NCT03410693, NCT03473756).
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Piperazinas/farmacología , Pirroles/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Tiofenos/farmacología , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Distribución Aleatoria , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeting the vascular endothelial growth factor signaling axis in glioblastoma inevitably leads to tumor recurrence and a more aggressive phenotype. Therefore, other angiogenic pathways, like the angiopoietin/tunica interna endothelial cell kinase (TIE) signaling axis, have become additional targets for therapeutic intervention. Here, we explored whether targeting the receptor tyrosine kinase TIE-2 using a novel, highly potent, orally available small molecule TIE-2 inhibitor (BAY-826) improves tumor control in syngeneic mouse glioma models. BAY-826 inhibits TIE-2 phosphorylation in vitro and in vivo as demonstrated by suppression of Angiopoietin-1- or Na3 VO4 -induced TIE-2 phosphorylation in glioma cells or extracts of lungs from BAY-826-treated mice. There was a trend toward prolonged survival upon single-agent treatment in two of four models (SMA-497 and SMA-540) and there was a significant survival benefit in one model (SMA-560). Co-treatment with BAY-826 and irradiation was ineffective in one model (SMA-497), but provided synergistic prolongation of survival in another (SMA-560). Decreased vessel densities and increased leukocyte infiltration were observed, but might be independent processes as the effect was also observed in single treatment modalities. These data demonstrate that TIE-2 inhibition may improve tumor response to treatment in highly vascularized tumors such as glioblastoma.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/enzimología , Modelos Animales de Enfermedad , Glioma/enzimología , Receptor TIE-2/antagonistas & inhibidores , Receptor TIE-2/metabolismo , Animales , Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Glioma/tratamiento farmacológico , Isoinjertos , Ratones , Ratones Endogámicos C57BL , Resultado del Tratamiento , Carga TumoralRESUMEN
Prostate cancer is a frequent malignancy in older men and has a very high 5-year survival rate if diagnosed early. The prognosis is much less promising if the tumor has already spread outside the prostate gland. Targeted treatments mainly aim at blocking androgen receptor (AR) signaling and initially show good efficacy. However, tumor progression due to AR-dependent and AR-independent mechanisms is often observed after some time, and novel treatment strategies are urgently needed. Dysregulation of the PI3K/AKT/mTOR pathway in advanced prostate cancer and its implication in treatment resistance has been reported. We compared the impact of PI3K/AKT/mTOR pathway inhibitors with different selectivity profiles on in vitro cell proliferation and on caspase 3/7 activation as a marker for apoptosis induction, and observed the strongest effects in the androgen-sensitive prostate cancer cell lines VCaP and LNCaP. Combination treatment with the AR inhibitor darolutamide led to enhanced apoptosis in these cell lines, the effects being most pronounced upon cotreatment with the pan-PI3K inhibitor copanlisib. A subsequent transcriptomic analysis performed in VCaP cells revealed that combining darolutamide with copanlisib impacted gene expression much more than individual treatment. A comprehensive reversal of the androgen response and the mTORC1 transcriptional programs as well as a marked induction of DNA damage was observed. Next, an in vivo efficacy study was performed using the androgen-sensitive patient-derived prostate cancer (PDX) model LuCaP 35 and a superior efficacy was observed after the combined treatment with copanlisib and darolutamide. Importantly, immunohistochemistry analysis of these treated tumors showed increased apoptosis, as revealed by elevated levels of cleaved caspase 3 and Bcl-2-binding component 3 (BBC3). In conclusion, these data demonstrate that concurrent blockade of the PI3K/AKT/mTOR and AR pathways has superior antitumor efficacy and induces apoptosis in androgen-sensitive prostate cancer cell lines and PDX models.
Asunto(s)
Neoplasias de la Próstata , Proteínas Proto-Oncogénicas c-akt , Masculino , Humanos , Anciano , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Androgénicos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Caspasa 3 , Andrógenos , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Próstata/genética , Proliferación Celular , Apoptosis , Línea Celular TumoralRESUMEN
BACKGROUND: Regorafenib was previously shown to reduce tumor-associated macrophages and potently inhibit colony-stimulating factor 1 receptor (CSF1R), also known as CD115, in biochemical assays. The CSF1R signaling pathway is essential in the biology of the mononuclear/phagocyte system, which can promote the development of cancer. METHODS: A deeper investigation of regorafenib's effects on CSF1R signaling was performed using preclinical in vitro and in vivo studies with syngeneic CT26 and MC38 mouse models of colorectal cancer. Peripheral blood and tumor tissue were analyzed mechanistically by flow cytometry using antibodies against CD115/CSF1R and F4/80 and by ELISA for chemokine (C-C motif) ligand 2 (CCL2) levels. These read-outs were correlated with drug levels for the detection of pharmacokinetic/pharmacodynamic relationships. RESULTS: Potent inhibition of CSF1R by regorafenib and its metabolites M-2, M-4, and M-5 was confirmed in vitro in RAW264.7 macrophages. The dose-dependent growth inhibition of subcutaneous CT26 tumors by regorafenib was associated with a significant reduction in both the number of CD115hi monocytes in peripheral blood and the number of selective subpopulations of intratumoral F4/80hi tumor-associated macrophages. CCL2 levels were not affected by regorafenib in blood but increased in tumor tissue, which may contribute to drug resistance and prevent complete tumor remission. An inverse relationship between regorafenib concentration and the number of CD115hi monocytes and CCL2 levels was observed in peripheral blood, supporting the mechanistic involvement of regorafenib. CONCLUSIONS: These findings may be clinically useful in optimizing drug dosing using blood-based pharmacodynamic markers and in identifying resistance mechanisms and ways to overcome them by appropriate drug combinations.
Asunto(s)
Neoplasias Colorrectales , Macrófagos , Ratones , Animales , Monocitos , Piridinas/farmacología , Piridinas/uso terapéutico , Piridinas/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismoRESUMEN
Rogaratinib (BAY 1163877) is a highly potent and selective small-molecule pan-fibroblast growth factor receptor (FGFR) inhibitor (FGFR1-4) for oral application currently being investigated in phaseâ 1 clinical trials for the treatment of cancer. In this publication, we report its discovery by deâ novo structure-based design and medicinal chemistry optimization together with its pharmacokinetic profile.
Asunto(s)
Descubrimiento de Drogas , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Tiofenos/farmacología , Humanos , Modelos Moleculares , Estructura Molecular , Piperazinas/química , Inhibidores de Proteínas Quinasas/química , Pirroles/química , Bibliotecas de Moléculas Pequeñas/química , Tiofenos/químicaRESUMEN
The lactate transporter SLC16A1/monocarboxylate transporter 1 (MCT1) plays a central role in tumor cell energy homeostasis. In a cell-based screen, we identified a novel class of MCT1 inhibitors, including BAY-8002, which potently suppress bidirectional lactate transport. We investigated the antiproliferative activity of BAY-8002 in a panel of 246 cancer cell lines and show that hematopoietic tumor cells, in particular diffuse large B-cell lymphoma cell lines, and subsets of solid tumor models are particularly sensitive to MCT1 inhibition. Associated markers of sensitivity were, among others, lack of MCT4 expression, low pleckstrin homology like domain family A member 2, and high pellino E3 ubiquitin protein ligase 1 expression. The antitumor effect of MCT1 inhibition was less pronounced on tumor xenografts, with tumor stasis being the maximal response. BAY-8002 significantly increased intratumor lactate levels and transiently modulated pyruvate levels. In order to address potential acquired resistance mechanisms to MCT1 inhibition, we generated MCT1 inhibitor-resistant cell lines and show that resistance can occur by upregulation of MCT4 even in the presence of sufficient oxygen, as well as by shifting energy generation toward oxidative phosphorylation. These findings provide insight into novel aspects of tumor response to MCT1 modulation and offer further rationale for patient selection in the clinical development of MCT1 inhibitors. Mol Cancer Ther; 17(11); 2285-96. ©2018 AACR.
Asunto(s)
Aminobenzoatos/farmacología , Benzoatos/farmacología , Biomarcadores de Tumor/metabolismo , Resistencia a Antineoplásicos , Transportadores de Ácidos Monocarboxílicos/antagonistas & inhibidores , Sulfonas/farmacología , Simportadores/antagonistas & inhibidores , Aminobenzoatos/química , Animales , Benzoatos/química , Transporte Biológico/efectos de los fármacos , Radioisótopos de Carbono , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Fluorescencia , Humanos , Concentración de Iones de Hidrógeno , Ácido Láctico/metabolismo , Ratones SCID , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Pirimidinonas/farmacología , Ácido Pirúvico/metabolismo , Sulfonas/química , Simportadores/metabolismo , Tiofenos/farmacología , Resultado del Tratamiento , Xenopus laevisRESUMEN
Histone deacetylases (HDACs) catalyze the deacetylation of epsilon-acetyl-lysine residues within the N-terminal tail of core histones and thereby mediate changes in the chromatin structure and regulate gene expression in eukaryotic cells. So far, surprisingly little is known about the substrate specificities of different HDACs. Here, we prepared a library of fluorogenic tripeptidic substrates of the general format Ac-P(-2)-P(-1)-Lys(Ac)-MCA (P(-1), P(-2)=all amino acids except cysteine) and measured their HDAC-dependent conversion in a standard fluorogenic HDAC assay. Different HDAC subtypes can be ranked according to their substrate selectivity: HDAH > HDAC8 > HDAC1 > HDAC3 > HDAC6. HDAC1, HDAC3, and HDAC6 exhibit a similar specificity profile, whereas both HDAC8 and HDAH have rather distinct profiles. Furthermore, it was shown that second-site modification (e.g., phosphorylation) of substrate sequences as well as corepressor binding can modulate the selectivity of enzymatic substrate conversion.
Asunto(s)
Histona Desacetilasas/química , Histona Desacetilasas/clasificación , Especificidad por Sustrato , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Activación Enzimática , Datos de Secuencia Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
Signaling cascades integrate extracellular stimuli primarily through regulated protein-protein interactions (PPIs). Intracellular signal transduction strictly depends on PPIs occurring at the membrane and in the cytosol. To monitor constitutive and regulated protein interactions within living mammalian cells, we have developed a biological assay termed split TEV. We engineered inactive fragments of the NIa protease from the tobacco etch virus (TEV protease) that regain activity only when coexpressed as fusion constructs with interacting proteins. Functional reconstitution of TEV protease fragments can be monitored with 'proteolysis-only' reporters, which can be previously silent fluorescent and luminescent reporter proteins. Additionally, proteolytically cleavable inactive transcription factors can be combined with any downstream reporter gene of choice to yield 'transcription-coupled' reporter systems. Thus, split TEV combines the advantages of split enzyme- and reporter gene-mediated assays, and provides full flexibility with regard to the final readout. In a first biological application, we monitored neuregulin-induced ErbB2/ErbB4 receptor tyrosine kinase heterodimerization.
Asunto(s)
Fenómenos Fisiológicos Celulares , Fragmentos de Péptidos/metabolismo , Huella de Proteína/métodos , Huella de Proteína/tendencias , Mapeo de Interacción de Proteínas/métodos , Mapeo de Interacción de Proteínas/tendencias , Proteoma/metabolismo , Fragmentos de Péptidos/química , Proteoma/químicaRESUMEN
In order to evaluate the methylotrophic yeast Pichia pastoris as means for high-yield production of homogenous D(2S) receptor protein, we have expressed the unmodified D(2S) receptor and various D(2S) receptor fusion constructs under the transcriptional control of the highly inducible promotor of the P. pastoris alcoholoxidase 1 gene in strain SMD1163. Fusion of the D(2S) receptor gene to the alpha-factor preprosequence proved to be essential for receptor production. For the receptor fusion constructs a gene dosage of more than two copies per cell increased production levels three- to sixfold. Adding various dopaminergic ligands to the induction medium increased yields up to tenfold, reaching 51,500 +/- 5700 receptors/cell. Immunoblot analysis of the effect of tunicamycin on D(2S) receptor fusion proteins and immunoprecipitation of metabolically labeled wild-type and glycosylation-deficient D(2S) receptor fusion proteins revealed that the high-mannose-type glycosylation of the D(2S) receptor prevents cleavage of the alpha-factor prosequence by the Kex2 endopeptidase. Abolishing glycosylation restored correct processing. Immunogold electron microscopy showed that recombinant yeast cells overproducing the D(2S) receptor developed membrane stacks harboring the receptor protein. The pharmacological profile of the recombinant D(2S) receptor was similar to that reported for neuronal D(2) receptors independent of glycosylation and processing. In conclusion, the D(2S) receptor can readily be produced in P. pastoris with high yield suitable for receptor purification and future structural studies.
Asunto(s)
Clonación Molecular , Pichia/genética , Receptores de Dopamina D2/genética , Dosificación de Gen , Vectores Genéticos , Humanos , Ligandos , Pichia/metabolismo , Receptores de Dopamina D2/metabolismoRESUMEN
The human dopamine D2S receptor was expressed in the methylotrophic yeast Pichia pastoris, where the receptor with a molecular mass of approximately 40kDa exhibited specific and saturable binding properties. The dopamine antagonist [3H]spiperone showed an average dissociation constant K(d) of 0.6+/-0.17 nM for the dopamine D2S receptor. The receptor was solubilized using the non-ionic detergent dodecylmaltoside and purified by affinity chromatography using a Ni(2+) chelate (His-Trap) column or by batch extraction with an anti-FLAG M1 affinity resin. The receptor maintained its biological activity after solubilization and purification from the membrane protein fraction. A 244- or 185-fold enrichment, as judged by an increase in specific binding, was obtained after adsorption to the His-Trap or anti-FLAG materials, respectively.
Asunto(s)
Pichia/genética , Receptores de Dopamina D2/aislamiento & purificación , Membrana Celular/metabolismo , Ácidos Cólicos/química , Medios de Cultivo , Humanos , Peso Molecular , Pichia/metabolismo , Unión Proteica , Receptores de Dopamina D2/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Sensibilidad y Especificidad , SolubilidadRESUMEN
Functional gamma-aminobutyric acid(B) (GABA(B)) receptors assemble from two subunits, GABA(B(1)) and GABA(B(2).) This heteromerization, which involves a C-terminal coiled-coil interaction, ensures efficient surface trafficking and agonist-dependent G-protein activation. In the present study, we took a closer look at the implications of the intracellular C termini of GABA(B(1)) and GABA(B(2)) for G-protein coupling. We generated a series of C-terminal mutants of GABA(B(1)) and GABA(B(2)) and tested them for physical interaction, surface trafficking, coupling to adenylyl cyclase, and G-protein-gated inwardly rectifying potassium channels in human embryonic kidney (HEK) 293 cells as well as on endogenous calcium channels in sympathetic neurons of the superior cervical ganglion (SCG). We found that the C-terminal interaction contributes only partly to the heterodimeric assembly of the subunits, indicating the presence of an additional interaction site. The described endoplasmic reticulum retention signal within the C terminus of GABA(B(1)) functioned only in the context of specific amino acids, which constitute part of the GABA(B(1)) coiled-coil sequence. This finding may provide a link between the retention signal and its shielding by the coiled coil of GABA(B(2).) In HEK293 cells, we observed that the two well-known GABA(B) receptor antagonists [S-(R*,R*)]-[3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxypropyl](cyclohexylmethyl) phosphinic acid (CGP54626) and (+)-(2S)-5,5-dimethyl-2-morpholineacetic acid (SCH50911) CGP54626 and SCH50911 function as inverse agonists. The C termini of GABA(B(1)) and GABA(B(2)) strongly influenced agonist-independent G-protein coupling, although they were not necessary for agonist-dependent G-protein coupling. The C-terminal GABA(B) receptor mutants described here demonstrate that the active receptor conformation is stabilized by the coiled-coil interaction. Thus, the C-terminal conformation of the GABA(B) receptor may determine its constitutive activity, which could be a therapeutic target for inverse agonists.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores de GABA-B/metabolismo , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Células Cultivadas , AMP Cíclico/metabolismo , Electrofisiología , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Eliminación de Gen , Humanos , Datos de Secuencia Molecular , Mutación , Canales de Potasio/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de GABA-B/química , Receptores de GABA-B/genética , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
G-protein-coupled receptors (GPCRs) are the largest group of cell surface molecules involved in signal transduction and are receptors for a wide variety of stimuli ranging from light, calcium and odourants to biogenic amines and peptides. It is assumed that systematic genomic data-mining has identified the overwhelming majority of all remaining GPCRs in the genome. Here we report the cloning of a novel orphan GPCR which was identified in a search for erythropoietin-induced genes in the brain as a strongly up-regulated gene. This unknown gene coded for a protein which had a seven-transmembrane topology and key features typical of GPCRs of the A family but a low overall identity to all known GPCRs. The protein, coded ee3, has an unusually high evolutionary conservation and is expressed in neurons in diverse areas of the CNS with relation to integrative functions or motor tasks. A yeast two-hybrid screen for interacting proteins revealed binding to the microtubule-associated protein (MAP) 1b. Coupling to MAP1a has been described for another cognate GPCR, the 5-hydroxytryptamine (5HT) 2a receptor. Surprisingly, we found complete colocalization of ee3 and the 5HT2a receptor. The interaction with MAP1b proved to be critical for the stability or folding of ee3 as in mice lacking MAP1b the ee3 protein was undetectable by immunohistochemistry, although messenger RNA levels remained unchanged. We propose that ee3 is a highly interesting new orphan GPCR with potential connections to erythropoietin and 5HT2a receptor signalling.
Asunto(s)
Eritropoyetina/farmacología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Receptor de Serotonina 5-HT2A/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Clonación Molecular , Humanos , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Especificidad de Órganos , Ratas , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
It has been suggested that the behavior and function of Paneth cells in metaplasia are different from those found in normal intestinal mucosa. In this study, we investigated whether calnexin, a protein involved in secretory pathways, might be associated with differentiation and function of Paneth cells in normal small intestine, in complete intestinal metaplasia of the stomach, and in Paneth cell-rich adenomas. Differentiation and function of Paneth cells was monitored by Ki67, lysozyme, and morphologic features. Using a newly established monoclonal antibody, we found that calnexin is regularly synthesized by Paneth cells of normal small intestine. In these cells, the staining intensity of calnexin was inversely correlated with their content of secretory granules (lysozyme). In contrast, Paneth cells of intestinal metaplasia and Paneth cell-rich adenomas showed a reduced immunostaining of both calnexin and lysozyme. Moreover, these Paneth cells synthesized the proliferation marker Ki67, a phenomenon that was never observed in Paneth cells of normal small intestine. In vitro experiments using CaCo2 cells showed that the expression of calnexin is not directly affected by the induction of mitosis. In conclusion, calnexin probably reflects the status of Paneth cell differentiation and function. The results do not necessarily indicate that calnexin has a function in Paneth cell proliferation.
Asunto(s)
Calnexina/biosíntesis , Células de Paneth/citología , Adenoma/metabolismo , Adenoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Células CACO-2/metabolismo , Células CACO-2/patología , Calnexina/análisis , Diferenciación Celular/fisiología , Femenino , Mucosa Gástrica/metabolismo , Humanos , Inmunohistoquímica , Intestino Delgado/citología , Intestino Delgado/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Metaplasia/metabolismo , Metaplasia/patología , Persona de Mediana Edad , Muramidasa/metabolismo , Células de Paneth/metabolismo , Células de Paneth/patología , Vesículas Secretoras/enzimología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estómago/patología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologíaRESUMEN
Cerebral ischaemia induces transcriptional changes in a number of pathophysiologically important genes. Here we have systematically studied gene expression changes after 90 min and 24 h of permanent focal ischaemia in the mouse by an advanced fragment display technique (restriction-mediated differential display). We identified 56 transcriptionally altered genes, many of which provide novel hints to ischaemic pathophysiology. Particularly interesting were two pro-apoptotic genes (Grim19 and Tdag51), whose role in cerebral ischaemia and neuronal cell death has not been recognized so far. Among the unknown sequences, we identified a gene that was rapidly and transiently up-regulated. The encoded protein displayed high homology to the MARK family of serine-threonine protein kinases and has recently been described as MARKL1/MARK4. Here we demonstrate that this protein is a functional protein kinase with the ability to specifically phosphorylate a cognate peptide substrate for the AMP-kinase family. Upon overexpression in heterologous cells, the functional wild-type protein, but not its kinase-dead mutant, led to decreased cell viability. We conclude that the up-regulation of this kinase during focal ischaemia may represent an interesting new target for pharmacological intervention.