RESUMEN
CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1(K/K)) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1(K/K) mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.
Asunto(s)
Neuronas Motoras/metabolismo , Neuronas Motoras/patología , ARN de Transferencia de Tirosina/metabolismo , Factores de Transcripción/metabolismo , Esclerosis Amiotrófica Lateral , Animales , Animales Recién Nacidos , Axones/metabolismo , Axones/patología , Muerte Celular , Diafragma/inervación , Pérdida del Embrión , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Exones/genética , Femenino , Fibroblastos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Atrofia Muscular Espinal , Enfermedades Neuromusculares/metabolismo , Enfermedades Neuromusculares/patología , Estrés Oxidativo , Procesamiento Postranscripcional del ARN , ARN de Transferencia de Tirosina/genética , Proteínas de Unión al ARN , Respiración , Nervios Espinales/citología , Factores de Transcripción/deficiencia , Proteína p53 Supresora de Tumor/metabolismo , Tirosina/genética , Tirosina/metabolismoRESUMEN
PAX5-JAK2 has recently been identified as a novel recurrent fusion gene in B-cell precursor acute lymphoblastic leukemia, but the function of the encoded chimeric protein has not yet been characterized in detail. Herein we show that the PAX5-JAK2 chimera, which consists of the DNA-binding paired domain of PAX5 and the active kinase domain of JAK2, is a nuclear protein that has the ability to bind to wild-type PAX5 target loci. Moreover, our data provide compelling evidence that PAX5-JAK2 functions as a nuclear catalytically active kinase that autophosphorylates and in turn phosphorylates and activates downstream signal transducers and activators of transcription (STATs) in an apparently noncanonical mode. The chimeric protein also enables cytokine-independent growth of Ba/F3 cells and therefore possesses transforming potential. Importantly, the kinase activity of PAX5-JAK2 can be efficiently blocked by JAK2 inhibitors, rendering it a potential target for therapeutic intervention. Together, our data show that PAX5-JAK2 simultaneously deregulates the PAX5 downstream transcriptional program and activates the Janus kinase-STAT signaling cascade and thus, by interfering with these two important pathways, may promote leukemogenesis.
Asunto(s)
Janus Quinasa 2/genética , Proteínas de Fusión Oncogénica/genética , Factor de Transcripción PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Regulación Leucémica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/metabolismo , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inhibidores de Proteínas Quinasas/farmacología , Factores de Transcripción STAT/metabolismo , Transcriptoma , Células Tumorales CultivadasRESUMEN
The ß subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain ß subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of ß isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual ß variants in specific neuronal functions. In the present study, an alternatively spliced ß4 subunit lacking the variable N terminus (ß4e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons. Compared with the other two known full-length ß4 variants (ß4a and ß4b), ß4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of ß4 subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (ß4-null) mice individually reconstituted with ß4a, ß4b, and ß4e. Notably, the number of genes regulated by each ß4 splice variant correlated with the rank order of their nuclear-targeting properties (ß4b > ß4a > ß4e). Together, these findings support isoform-specific functions of ß4 splice variants in neurons, with ß4b playing a dual role in channel modulation and gene regulation, whereas the newly detected ß4e variant serves exclusively in calcium-channel-dependent functions.
Asunto(s)
Canales de Calcio/genética , Expresión Génica/genética , Neuronas/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Canales de Calcio/metabolismo , Femenino , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Glucocorticoid (GC) resistance is a continuing clinical problem in childhood acute lymphoblastic leukaemia (ALL) but the underlying mechanisms remain unclear. A proteomic approach was used to compare profiles of the B-lineage ALL GC-sensitive cell line, PreB 697, and its GC-resistant sub-line, R3F9, pre- and post-dexamethasone exposure. PAX5, a transcription factor critical to B-cell development was differentially regulated in the PreB 697 compared to the R3F9 cell line in response to GC. PAX5 basal protein expression was less in R3F9 compared to its GC-sensitive parent and confirmed to be lower in other GC-resistant sub-lines of Pre B 697 and was associated with a decreased expression of the PAX5 transcriptional target, CD19. Gene set enrichment analysis showed that increasing GC-resistance was associated with differentiation from preB-II to an immature B-lymphocyte stage. GC-resistant sub-lines were shown to have higher levels of phosphorylated JNK compared to the parent line and JNK inhibition caused re-sensitization to GC. Exploiting this maturation may be key to overcoming GC resistance and targeting signalling pathways linked to the maturation state, such as JNK, may be a novel approach.
Asunto(s)
Antineoplásicos/farmacología , Linfocitos B/efectos de los fármacos , Dexametasona/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de Neoplasias/biosíntesis , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteómica/métodos , Apoptosis/efectos de los fármacos , Linfocitos B/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/fisiología , Exones/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Reacción en Cadena de la Polimerasa Multiplex , Mutación , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/fisiología , Fosforilación/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Glucocorticoids (GCs) cause apoptosis in malignant cells of lymphoid lineage by transcriptionally regulating a plethora of genes. As a result, GCs are included in almost all treatment protocols for lymphoid malignancies, particularly childhood acute lymphoblastic leukemia (chALL). The most commonly used synthetic GCs in the clinical setting are prednisolone and dexamethasone. While the latter has a higher activity and more effectively reduces the tumor load in patients, it is also accompanied by more serious adverse effects than the former. Whether this difference might be explained by regulation of different genes by the two GCs has never been addressed. RESULTS: Using a recently developed GC bioassay based on a GC-responsive reporter construct in human Jurkat T-ALL cells, we found ~7-fold higher biological activity with dexamethasone than prednisolone. Similarly, 1.0e-7 M dexamethasone and 7.0e-7 M prednisolone triggered similar cell death rates in CCRF-CEM-C7H2 T-chALL cells after 72 hours of treatment. Using microarray-based whole genome expression profiling and a variety of statistical and other approaches, we compared the transcriptional response of chALL cells to 6 hour exposure to both synthetic GCs at the above concentrations. Our experiments did not detect any gene whose regulation by dexamethasone differed significantly from that by prednisolone. CONCLUSIONS: Our findings suggest that the reported differences in treatment efficacy and cytotoxicity of dexamethasone and prednisolone are not caused by inherent differences of the 2 drugs to regulate the expression of certain genes, but rather result either from applying them in biologically in-equivalent concentrations and/or from differences in their pharmacokinetics and - dynamics resulting in different bioactivities in tumor cells and normal tissues.
Asunto(s)
Dexametasona/farmacología , Genes Relacionados con las Neoplasias/efectos de los fármacos , Glucocorticoides/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Prednisolona/farmacología , Apoptosis/efectos de los fármacos , Niño , Humanos , Células Jurkat , Transcripción Genética/efectos de los fármacosRESUMEN
BACKGROUND: FOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells. METHODS: The effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor. RESULTS: We report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells. CONCLUSION: The combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.
Asunto(s)
Neoplasias Encefálicas/genética , Factores de Transcripción Forkhead/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas/genética , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Sitios de Unión/genética , Neoplasias Encefálicas/patología , Catalasa/metabolismo , Etopósido/farmacología , Proteína Forkhead Box O3 , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Peróxido de Hidrógeno/toxicidad , Péptidos y Proteínas de Señalización Intracelular , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neuroblastoma/patología , Peroxisomas/efectos de los fármacos , Peroxisomas/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , beta Catenina/metabolismoRESUMEN
BACKGROUND: Glucocorticoids (GCs) are natural stress induced steroid hormones causing cell cycle arrest and cell death in lymphoid tissues. Therefore they are the central component in the treatment of lymphoid malignancies, in particular childhood acute lymphoblastic leukemia (chALL). GCs act mainly via regulating gene transcription, which has been intensively studied by us and others. GC control of mRNA translation has also been reported but has never been assessed systematically. In this study we investigate the effect of GCs on mRNA translation on a genome-wide scale. RESULTS: Childhood T- (CCRF-CEM) and precursor B-ALL (NALM6) cells were exposed to GCs and subjected to "translational profiling", a technique combining sucrose-gradient fractionation followed by Affymetrix Exon microarray analysis of mRNA from different fractions, to assess the translational efficiency of the expressed genes. Analysis of GC regulation in ribosome-bound fractions versus transcriptional regulation revealed no significant differences, i.e., GC did not entail a significant shift between ribosomal bound and unbound mRNAs. CONCLUSIONS: In the present study we analyzed for the first time possible effects of GC on the translational efficiency of expressed genes in two chALL model systems employing whole genome polysome profiling. Our results did not reveal significant differences in translational efficiency of expressed genes thereby arguing against a potential widespread regulatory effect of GCs on translation at least in the investigated in vitro systems.
Asunto(s)
Glucocorticoides/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Transcriptoma , Línea Celular Tumoral , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , ARN Mensajero/metabolismoRESUMEN
Approximately 25% of childhood acute lymphoblastic leukemias carry the ETV6/RUNX1 fusion gene. Despite their excellent initial treatment response, up to 20% of patients relapse. To gain insight into the relapse mechanisms, we analyzed single nucleotide polymorphism arrays for DNA copy number aberrations (CNAs) in 18 matched diagnosis and relapse leukemias. CNAs were more abundant at relapse than at diagnosis (mean 12.5 vs 7.5 per case; P=.01) with 5.3 shared on average. Their patterns revealed a direct clonal relationship with exclusively new aberrations at relapse in only 21.4%, whereas 78.6% shared a common ancestor and subsequently acquired distinct CNA. Moreover, we identified recurrent, mainly nonoverlapping deletions associated with glucocorticoid-mediated apoptosis targeting the Bcl2 modifying factor (BMF) (n=3), glucocorticoid receptor NR3C1 (n=4), and components of the mismatch repair pathways (n=3). Fluorescence in situ hybridization screening of additional 24 relapsed and 72 nonrelapsed ETV6/RUNX1-positive cases demonstrated that BMF deletions were significantly more common in relapse cases (16.6% vs 2.8%; P=.02). Unlike BMF deletions, which were always already present at diagnosis, NR3C1 and mismatch repair aberrations prevailed at relapse. They were all associated with leukemias, which poorly responded to treatment. These findings implicate glucocorticoid-associated drug resistance in ETV6/RUNX1-positive relapse pathogenesis and therefore might help to guide future therapies.
Asunto(s)
Eliminación de Gen , Glucocorticoides/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Transducción de Señal/genética , Disparidad de Par Base/genética , Niño , Preescolar , Células Clonales , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Variaciones en el Número de Copia de ADN/genética , Resistencia a Antineoplásicos/genética , Femenino , Reordenamiento Génico de Linfocito T/genética , Humanos , Lactante , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Glucocorticoides/metabolismo , RecurrenciaRESUMEN
Mitochondria are signal-integrating organelles involved in cell death induction. Mitochondrial alterations and reduction in energy metabolism have been previously reported in the context of glucocorticoid (GC)-triggered apoptosis, although the mechanism is not yet clarified. We analyzed mitochondrial function in a GC-sensitive precursor B-cell acute lymphoblastic leukemia (ALL) model as well as in GC-sensitive and GC-resistant T-ALL model systems. Respiratory activity was preserved in intact GC-sensitive cells up to 24h under treatment with 100 nM dexamethasone before depression of mitochondrial respiration occurred. Severe repression of mitochondrial respiratory function was observed after permeabilization of the cell membrane and provision of exogenous substrates. Several mitochondrial metabolite and protein transporters and two subunits of the ATP synthase were downregulated in the T-ALL and in the precursor B-ALL model at the gene expression level under dexamethasone treatment. These data could partly be confirmed in ALL lymphoblasts from patients, dependent on the molecular abnormality in the ALL cells. GC-resistant cell lines did not show any of these defects after dexamethasone treatment. In conclusion, in GC-sensitive ALL cells, dexamethasone induces changes in membrane properties that together with the reduced expression of mitochondrial transporters of substrates and proteins may lead to repressed mitochondrial respiratory activity and lower ATP levels that contribute to GC-induced apoptosis.
Asunto(s)
Glucocorticoides/efectos adversos , Membranas Mitocondriales/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Antineoplásicos Hormonales/efectos adversos , Antineoplásicos Hormonales/farmacología , Línea Celular Tumoral , Respiración de la Célula/efectos de los fármacos , Respiración de la Célula/genética , Dexametasona/farmacología , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Humanos , Análisis por Micromatrices , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/fisiología , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/patología , Membranas Mitocondriales/fisiología , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMEN
Glucocorticoids (GC) actuate apoptosis as well as cell cycle arrest in lymphocytes, and included as core element in the lymphoid malignancy treatment. Despite clinical significance of GC and considerable efforts to understand it, the molecular basis of GC regulated cell death and the resistance phenomenon remains, however, poorly understood. Using Affymetrix-based whole genome expression profiling our group has previously identified a number of prominent glucocorticoid-response genes (Blood 107: 2061, 2006). Promyelocytic leukemia zinc finger (PLZF) was one of the best candidate genes. This study was proposed to investigate the possible role of PLZF in GC regulated cell death in leukemic model cell line NALM6. To this end, we generated NALM6 cell line (bulk) transduced with a retroviral expression vectors, pHR-SFFV-PLZF-IRES-Puro (U426) and pHR-SFFV-Venus-IRES-Puro (U417), as control, for constitutive gene-expression. HEK293T cells were transfected transiently to generate viral particles. These cell lines were characterized by Western blotting and used to assay the effect of constitutive PLZF expression. In conclusion, we report that bona fide transcription repressor PLZF, which turned out as prominent GC-regulated gene both in vivo and in vitro situations was found to enhance the GC-induced cell death (basal) in leukemic model cell line NALM6 after 48 and 72h time points.
Asunto(s)
Apoptosis/efectos de los fármacos , Glucocorticoides/farmacología , Factores de Transcripción de Tipo Kruppel/fisiología , Leucemia/tratamiento farmacológico , Línea Celular Tumoral , Células HEK293 , Humanos , Leucemia/patología , Proteína de la Leucemia Promielocítica con Dedos de ZincRESUMEN
Scaffold proteins regulate intracellular MAP kinase signaling by providing critical spatial and temporal specificities. We have shown previously that the scaffold protein MEK1 partner (MP1) is localized to late endosomes by the adaptor protein p14. Using conditional gene disruption of p14 in livers of mice (p14(Δhep) ) we analyzed protein and transcript signatures in tissue samples. Further biological network analysis predicted that the differentially expressed transcripts and proteins are involved in cell cycle progression and regulation of cellular proliferation. Although some of the here identified signatures were previously linked to phospho-ERK activity, most of them were novel targets of the late endosomal p14/MP1/MEK/ERK signaling module. Finally, the proliferation defect was confirmed in a chemically induced liver regeneration model in p14(Δhep) knockout mice.
Asunto(s)
Biomarcadores/metabolismo , Perfilación de la Expresión Génica , Proteínas/fisiología , Proteoma/metabolismo , Animales , Biomarcadores/análisis , Electroforesis en Gel Bidimensional , Femenino , Immunoblotting , Integrasas/metabolismo , Regeneración Hepática , Masculino , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteoma/análisis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Loss of CDKN2A/p16(INK4A) in hematopoietic stem cells is associated with enhanced self-renewal capacity and might facilitate progression of damaged stem cells into pre-cancerous cells that give rise to leukemia. This is also reflected by the frequent loss of the INK4A locus in acute lymphoblastic T-cell leukemia. T-cell acute lymphoblastic leukemia cells designed to conditionally express p16(INK4A) arrest in the G(0)/G(1) phase of the cell cycle and show increased sensitivity to glucocorticoid- and tumor necrosis factor receptor superfamily 6-induced apoptosis. To investigate the underlying molecular mechanism for increased death sensitivity, we interfered with specific steps of apoptosis signaling by expression of anti-apoptotic proteins. We found that alterations in cell death susceptibility resulted from changes in the composition of pro- and anti-apoptotic BCL2 proteins, i.e. repression of MCL1, BCL2, and PMAIP1/Noxa and the induction of pro-apoptotic BBC3/Puma. Interference with Puma induction by short hairpin RNA technology or retroviral expression of MCL1 or BCL2 significantly reduced both glucocorticoid- and FAS-induced cell death in p16(INK4A)-reconstituted leukemia cells. These results suggest that Puma, in concert with MCL1 and BCL2 repression, critically mediates p16(INK4A)-induced death sensitization and that in human T-cell leukemia the deletion of p16(INK4A) confers apoptosis resistance by shifting the balance of pro- and anti-apoptotic BCL2 proteins toward apoptosis protection.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Regulación hacia Abajo , Proteína Ligando Fas/metabolismo , Leucemia/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Regulación Leucémica de la Expresión Génica , Glucocorticoides/metabolismo , Humanos , Leucemia/genética , Leucemia/fisiopatología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
BACKGROUND: Glucocorticoids (GCs) cause apoptosis and cell cycle arrest in lymphoid cells and constitute a central component in the therapy of lymphoid malignancies, most notably childhood acute lymphoblastic leukemia (ALL). PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-2), a kinase controlling glucose metabolism, was identified by us previously as a GC response gene in expression profiling analyses performed in children with ALL during initial systemic GC mono-therapy. Since deregulation of glucose metabolism has been implicated in apoptosis induction, this gene and its relatives, PFKFB1, 3, and 4, were further analyzed. METHODS: Gene expression analyses of isolated lymphoblasts were performed on Affymetrix HGU133 Plus 2.0 microarrays. GCRMA normalized microarray data were analyzed using R-Bioconductor packages version 2.5. Functional gene analyses of PFKFB2-15A and -15B isoforms were performed by conditional gene over-expression experiments in the GC-sensitive T-ALL model CCRF-CEM. RESULTS: Expression analyses in additional ALL children, non-leukemic individuals and leukemic cell lines confirmed frequent PFKFB2 induction by GC in most systems sensitive to GC-induced apoptosis, particularly T-ALL cells. The 3 other family members, in contrast, were either absent or only weakly expressed (PFKFB1 and 4) or not induced by GC (PFKFB3). Conditional PFKFB2 over-expression in the CCRF-CEM T-ALL in vitro model revealed that its 2 splice variants (PFKFB2-15A and PFKFB2-15B) had no detectable effect on cell survival. Moreover, neither PFKFB2 splice variant significantly affected sensitivity to, or kinetics of, GC-induced apoptosis. CONCLUSIONS: Our data suggest that, at least in the model system investigated, PFKFB2 is not an essential upstream regulator of the anti-leukemic effects of GC.
Asunto(s)
Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Dexametasona/uso terapéutico , Glucocorticoides/uso terapéutico , Linfocitos/efectos de los fármacos , Fosfofructoquinasa-2/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Animales , Apoptosis/genética , Supervivencia Celular , Niño , Doxiciclina/farmacología , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Células Jurkat , Linfocitos/enzimología , Linfocitos/patología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfofructoquinasa-2/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Ratas , Factores de Tiempo , TransfecciónRESUMEN
Late endosomal/lysosomal adaptor and MAPK and mTOR activator (LAMTOR/Ragulator) is a scaffold protein complex that anchors and regulates multiprotein signaling units on late endosomes/lysosomes. To identify LAMTOR-modulated endolysosomal proteins, primary macrophages were derived from bone marrow of conditional knockout mice carrying a specific deletion of LAMTOR2 in the monocyte/macrophage cell lineage. Affymetrix-based transcriptomic analysis and quantitative iTRAQ-based organelle proteomic analysis of endosomes derived from macrophages were performed. Further analyses showed that LAMTOR could be a novel regulator of foam cell differentiation. The lipid droplet formation phenotype observed in macrophages was additionally confirmed in MEFs, where lipidomic analysis identified cholesterol esters as specifically downregulated in LAMTOR2 knockout cells. The data obtained indicate a function of LAMTOR2 in lipid metabolism.
Asunto(s)
Diferenciación Celular , Células Espumosas/metabolismo , Metabolismo de los Lípidos , Macrófagos/metabolismo , Proteínas/metabolismo , Animales , Células Cultivadas , Ésteres del Colesterol/metabolismo , Células Espumosas/citología , Gotas Lipídicas/metabolismo , Macrófagos/citología , Ratones , Proteínas/genética , TranscriptomaRESUMEN
Glucocorticoid (GC)-induced apoptosis plays a major role in the treatment of acute lymphoblastic leukemia (ALL) and related malignancies. Members of the BCL2 family of pro- and anti-apoptotic proteins are regulated by GC, but to what extent these regulations contribute to GC-induced cell death and resistance development is poorly understood. Using primary lymphoblasts from ALL children during systemic GC monotherapy and related cell lines, we have previously shown that the response of the BCL2 rheostat to GC was dominated by induction of the pro-apoptotic BH3-only molecules BMF and BCL2L11/Bim, but we also observed an unexpected significant repression of the pro-apoptotic BCL2 protein PMAIP1/Noxa. Here, we report that GC represses Noxa mRNA levels and also interferes with its protein stability in a proteasome-dependent manner. Prevention of GC-mediated Noxa repression by conditional expression of transgenic Noxa changed the kinetics of GC-induced apoptosis to resemble cell death induced by BimEL alone. Hence, GC appear to activate functionally relevant pro- as well as anti-apoptotic pathways in ALL cells. Interfering with the anti-apoptotic component of the GC response might contribute to improved therapeutic approaches and circumvention of resistance to this therapy.
Asunto(s)
Glucocorticoides/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Cinética , Proteínas de la Membrana/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Transcripts for the human glucocorticoid receptor (NR3C1) are known to contain alternative first exons 1A1, 1A2, and 1A3 from the distal promoter or 1D, 1E, 1B, 1F, 1C, or 1H from the proximal promoter. Here, we report two additional alternative first exons identified by Rapid amplification of cDNA ends (RACE)-PCR. The first, exon 1I, starts approximately 700 bp downstream of the splice donor site of the longest form of exon 1A, 1A3, considerably extending the known distal promoter region with a region containing conserved transcription factor-binding sites as well as a potential glucocorticoid response element (GRE) that differs from the consensus GRE in only two positions. The second, exon 1J, is part of the proximal promoter region and resides between exons 1D and 1E. Since this has been determined by quantitative real-time reverse transcriptase (RT)-PCR, exon 1I is used foremost in cells of the T-lymphocyte lineage. In the T-ALL cell line CEM-C7H2, which is sensitive to glucocorticoid-induced apoptosis, transcripts containing alternative first exons from the distal as well as the proximal promoter regions were markedly autoinduced by glucocorticoid treatment, with more pronounced relative induction in the distal promoter. Neither transcript was autoinduced in the related, resistant cell lines CEM-C1, and CEM-C7R5. In contrast, the glucocorticoid-sensitive PreB697 cell line strongly autoinduced transcripts from the proximal promoter, but not transcripts from the distal promoter, to relevant levels. Therefore, the autoinductive feedback loop implicated in glucocorticoid-induced apoptosis cannot universally rely on the distal promoter of the glucocorticoid receptor.
Asunto(s)
Empalme Alternativo/genética , Exones , Receptores de Glucocorticoides/genética , Secuencia de Bases , Línea Celular , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos , Regiones Promotoras Genéticas , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Receptores de Glucocorticoides/biosíntesis , Análisis de Secuencia de ADNRESUMEN
Glucocorticoids (GCs) specifically induce apoptosis in malignant lymphoblasts and are thus pivotal in the treatment of acute lymphoblastic leukemia (ALL). However, GC-resistance is a therapeutic problem with an unclear molecular mechanism. We generated approximately 70 GC-resistant sublines from a GC-sensitive B- and a T-ALL cell line and investigated their mechanisms of resistance. In response to GCs, all GC-resistant subclones analyzed by real-time polymerase chain reaction (PCR) showed a deficient up-regulation of the GC-receptor (GR) and its downstream target, GC-induced leucine zipper. This deficiency in GR up-regulation was confirmed by Western blotting and on retroviral overexpression of GR in resistant subclones GC-sensitivity was restored. All GC-resistant subclones were screened for GR mutations using denaturing high-pressure liquid chromatography (DHPLC), DNA-fingerprinting, and fluorescence in situ hybridization (FISH). Among the identified mutations were some previously not associated with GC resistance: A484D, P515H, L756N, Y663H, L680P, and R714W. This approach revealed three genotypes, complete loss of functional GR in the mismatch repair deficient T-ALL model, apparently normal GR genes in B-ALLs, and heterozygosity in both. In the first genotype, deficiency in GR up-regulation was fully explained by mutational events, in the second by a putative regulatory defect, and in the third by a combination thereof. In all instances, GC-resistance occurred at the level of the GR in both models.
Asunto(s)
Resistencia a Antineoplásicos , Glucocorticoides/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Receptores de Glucocorticoides/metabolismo , Línea Celular Tumoral , Reparación de la Incompatibilidad de ADN , Resistencia a Antineoplásicos/genética , Glucocorticoides/metabolismo , Humanos , Mutación , Receptores de Glucocorticoides/genética , Factores de Transcripción/metabolismoRESUMEN
Establishment of stably transfected mammalian cells with conditional expression of antiproliferative or proapoptotic proteins is often hampered by varying expression within bulk-selected cells and high background in the absence of the inducing drug. To overcome such limitations, we designed a gene expression system that transcribes the tetracycline-dependent rtTA2-M2-activator, TRSID-silencer, and selection marker as a tricistronic mRNA from a single retroviral vector. More than 92% of bulk-selected cells expressed enhanced green fluorescent protein or luciferase over more than three orders of magnitude in an almost linear, dose-dependent manner. To functionally test this system, we studied how dose-dependent expression of p27(Kip1) affects proliferation and viability of SH-EP neuroblastoma cells. Low to moderate p27(Kip1) expression caused transient G(0)-G(1) accumulation without reduced viability, whereas high p27(Kip1) levels induced significant apoptosis after 72 hours. This proves that this expression system allows concentration-dependent analysis of gene function and implicates p27(Kip1) as a critical regulator of both proliferation and apoptosis in SH-EP neuroblastoma cells.
Asunto(s)
Apoptosis/genética , Perfilación de la Expresión Génica/métodos , Vectores Genéticos/genética , Proteínas Represoras/genética , Retroviridae/genética , Tetraciclina/farmacología , Transactivadores/genética , Ciclo Celular/genética , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Doxiciclina/farmacología , Proteínas Fluorescentes Verdes/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Neuroblastoma/genética , Neuroblastoma/patología , Proteínas Represoras/efectos de los fármacos , Transactivadores/efectos de los fármacos , Transfección , Tristetraprolina/efectos de los fármacos , Tristetraprolina/genética , Células Tumorales CultivadasRESUMEN
This work evaluated gene expression differences between a hanging-drop 3D NSCLC model and 2D cell cultures and their in-vivo relevance by comparison to patient-derived data from The Cancer Genome Atlas. Gene expression of 2D and 3D cultures for Colo699 and A549 were assessed using Affymetrix HuGene 1.0 ST gene chips. Biostatistical analyses tested for reproducibility, comparability and significant differences in gene expression profiles between cell lines, experiments and culture methods. The analyses revealed a high interassay correlation within specific culture systems proving a high validity. 979 genes were altered in A549 and 1106 in Colo699 cells due to 3D cultivation. The overlap of changed genes between the cell lines was small (149), but the involved pathways in the reactome and GO- analyses showed a high overlap with DNA methylation, cell cycle, SIRT1, PKN1 pathway, DNA repair and oxidative stress as well known cancer-associated representatives. Additional specific GSEA-analyses revealed changes in immunologic and endothelial cell proliferation pathways, whereas hypoxic, EMT and angiogenic pathways were downregulated. Gene enrichment analyses showed 3D-induced gene up-regulations in the cell lines 38 to be represented in in-vivo samples of NSCLC patients using data of The Cancer Genome Atlas. Thus, our 3D NSCLC model might provide a tool for early drug development and investigation of microenvironment-associated mechanisms. However, this work also highlights the need for further individualization and model adaption to address remaining challenges.
RESUMEN
Cancer cell lines are used worldwide in biological research, and data interpretation depends on unambiguous attribution of the respective cell line to its original source. Short-tandem-repeat (STR) profiling (DNA fingerprinting) is the method of choice for this purpose; however, the genetic stability of cell lines under various experimental conditions is not well defined. We tested the effect of long-term culture, subcloning, and generation of drug-resistant subclones on fingerprinting profiles in four widely used leukemia cell lines. The DNA fingerprinting profile remained unaltered in two of them (U937 and K562) throughout 12 months in culture, and the vast majority of subclones derived therefrom by limiting dilution after long-term culture revealed the same profile, indicating a high degree of stability and clonotypic homogeneity. In contrast, two other cell lines (CCRF-CEM and Jurkat) showed marked alterations in DNA fingerprinting profiles during long-term culture. Limiting dilution subcloning revealed extensive clonotypic heterogeneity with subclones differing in up to eight STR loci from the parental culture. Similar heterogeneity was observed in subclones generated by selection culture for drug resistance where DNA fingerprinting proved useful in identifying possible resistance mechanisms. Thus, common tissue culture procedures may dramatically affect the fingerprinting profile of certain cell lines and thus render definition of their origin difficult.