RESUMEN
The genetic architecture of adaptation in natural populations has not yet been resolved: it is not clear to what extent the spread of beneficial mutations (selective sweeps) or the response of many quantitative trait loci drive adaptation to environmental changes. Although much attention has been given to the genomic footprint of selective sweeps, the importance of selection on quantitative traits is still not well studied, as the associated genomic signature is extremely difficult to detect. We propose 'Evolve and Resequence' as a promising tool, to study polygenic adaptation of quantitative traits in evolving populations. Simulating replicated time series data we show that adaptation to a new intermediate trait optimum has three characteristic phases that are reflected on the genomic level: (1) directional frequency changes towards the new trait optimum, (2) plateauing of allele frequencies when the new trait optimum has been reached and (3) subsequent divergence between replicated trajectories ultimately leading to the loss or fixation of alleles while the trait value does not change. We explore these 3 phase characteristics for relevant population genetic parameters to provide expectations for various experimental evolution designs. Remarkably, over a broad range of parameters the trajectories of selected alleles display a pattern across replicates, which differs both from neutrality and directional selection. We conclude that replicated time series data from experimental evolution studies provide a promising framework to study polygenic adaptation from whole-genome population genetics data.
Asunto(s)
Adaptación Fisiológica/genética , Evolución Molecular , Genética de Población , Modelos Genéticos , Sitios de Carácter Cuantitativo , Alelos , Frecuencia de los Genes , Aptitud Genética , Densidad de Población , Selección GenéticaRESUMEN
Evolve and resequence (E&R) is a new approach to investigate the genomic responses to selection during experimental evolution. By using whole genome sequencing of pools of individuals (Pool-Seq), this method can identify selected variants in controlled and replicable experimental settings. Reviewing the current state of the field, we show that E&R can be powerful enough to identify causative genes and possibly even single-nucleotide polymorphisms. We also discuss how the experimental design and the complexity of the trait could result in a large number of false positive candidates. We suggest experimental and analytical strategies to maximize the power of E&R to uncover the genotype-phenotype link and serve as an important research tool for a broad range of evolutionary questions.
Asunto(s)
Adaptación Fisiológica/genética , Evolución Biológica , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Animales , Drosophila melanogaster/genética , Frecuencia de los Genes , Estudios de Asociación Genética , Genotipo , Fenotipo , Polimorfismo de Nucleótido Simple , Proyectos de InvestigaciónRESUMEN
The Ewing sarcoma (ES) EWS-FLI1 chimeric oncoprotein is a prototypic aberrant ETS transcription factor with activating and repressive regulatory functions. We report that EWS-FLI1-repressed promoters are enriched in forkhead box (FOX) recognition motifs, and identify FOXO1 as a EWS-FLI1-suppressed regulator orchestrating a major subset of EWS-FLI1-repressed genes. In addition to FOXO1 regulation by direct promoter binding of EWS-FLI1, its subcellular localization and activity is regulated by cyclin-dependent kinase 2- and AKT-mediated phosphorylation downstream of EWS-FLI1. Restoration of nuclear FOXO1 expression in ES cells impaired proliferation and significantly reduced clonogenicity. Gene-expression profiling revealed a significant overlap between EWS-FLI1-repressed and FOXO1-activated genes. As a proof of principle for a potential therapeutic application of our findings, the treatment of ES cell lines with methylseleninic acid (MSA) reactivated endogenous FOXO1 in the presence of EWS-FLI1 in a dose- and time-dependent manner and induced massive cell death dependent on FOXO1. In an orthotopic xenograft mouse model, MSA increased FOXO1 expression in the tumor paralleled by a significant decrease in ES tumor growth. FOXO1 reactivation by small molecules may therefore serve as a promising strategy for a future ES-specific therapy.
Asunto(s)
Neoplasias Óseas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Sarcoma de Ewing/metabolismo , Animales , Antineoplásicos/farmacología , Secuencia de Bases , Sitios de Unión , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Línea Celular Tumoral , Proliferación Celular , Secuencia de Consenso , Quinasa 2 Dependiente de la Ciclina/metabolismo , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Silenciador del Gen , Humanos , Ratones , Proteínas de Fusión Oncogénica/genética , Compuestos de Organoselenio/farmacología , Fosforilación , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteína Proto-Oncogénica c-fli-1/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/genética , Transcripción Genética , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Though p53 mutations are rare in ES, there is a strong indication that p53 mutant tumours form a particularly bad prognostic group. As such, novel treatment strategies are warranted that would specifically target and eradicate tumour cells containing mutant p53 in this subset of ES patients. METHODS: PRIMA-1(Met), also known as APR-246, is a small organic molecule that has been shown to restore tumour-suppressor function primarily to mutant p53 and also to induce cell death in various cancer types. In this study, we interrogated the ability of APR-246 to induce apoptosis and inhibit tumour growth in ES cells with different p53 mutations. RESULTS: APR-246 variably induced apoptosis, associated with Noxa, Puma or p21(WAF1) upregulation, in both mutant and wild-type p53 harbouring cells. The apoptosis-inducing capability of APR-246 was markedly reduced in ES cell lines transfected with p53 siRNA. Three ES cell lines established from the same patient at different stages of the disease and two cell lines of different patients with identical p53 mutations all exhibited different sensitivities to APR-246, indicating cellular context dependency. Comparative transcriptome analysis on the three cell lines established from the same patient identified differential expression levels of several TP53 and apoptosis-associated genes such as APOL6, PENK, PCDH7 and MST4 in the APR-246-sensitive cell line relative to the less APR-246-sensitive cell lines. CONCLUSION: This is the first study reporting the biological response of Ewing sarcoma cells to APR-246 exposure and shows gross variability in responses. Our study also proposes candidate genes whose expression might be associated with ES cells' sensitivity to APR-246. With APR-246 currently in early-phase clinical trials, our findings call for caution in considering it as a potential adjuvant to conventional ES-specific chemotherapeutics.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Óseas/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Quinuclidinas/farmacología , Sarcoma de Ewing/genética , Proteína p53 Supresora de Tumor/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias Óseas/patología , Técnicas de Silenciamiento del Gen , Humanos , Análisis por Micromatrices , Mutación/fisiología , ARN Interferente Pequeño/farmacología , Sarcoma de Ewing/patología , Transcriptoma , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/antagonistas & inhibidoresRESUMEN
Despite dramatic reduction in sequencing costs with the advent of next generation sequencing technologies, obtaining a complete mammalian genome sequence at sufficient depth is still costly. An alternative is partial sequencing. Here, we have sequenced a reduced representation library of an Iberian sow from the Guadyerbas strain, a highly inbred strain that has been used in numerous QTL studies because of its extreme phenotypic characteristics. Using the Illumina Genome Analyzer II (San Diego, CA, USA), we resequenced â¼ 1% of the genome with average 4 × depth, identifying 68,778 polymorphisms. Of these, 55,457 were putative fixed differences with respect to the assembly, based on the genome of a Duroc pig, and 13,321 were heterozygous positions within Guadyerbas. Despite being highly inbred, the estimate of heterozygosity within Guadyerbas was â¼ 0.78 kb(-1) in autosomes, after correcting for low depth. Nucleotide variability was consistently higher at the telomeric regions than on the rest of the chromosome, likely a result of increased recombination rates. Further, variability was 50% lower in the X-chromosome than in autosomes, which may be explained by a recent bottleneck or by selection. We divided the whole genome in 500 kb windows and we analyzed overrepresented gene ontology terms in regions of low and high variability. Multi organism process, pigmentation and cell killing were overrepresented in high variability regions and metabolic process ontology, within low variability regions. Further, a genome wide Hudson-Kreitman-Aguadé test was carried out per window; overall, variability was in agreement with neutral expectations.
Asunto(s)
Mapeo Cromosómico/métodos , Análisis de Secuencia de ADN/métodos , Porcinos/genética , Animales , Secuencia de Bases , Femenino , Variación Genética , Genoma , Genómica/métodos , Endogamia , Polimorfismo de Nucleótido Simple , Alineación de SecuenciaAsunto(s)
Apoptosis/efectos de los fármacos , Desoxiglucosa/farmacología , Glucocorticoides/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Animales , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , HumanosRESUMEN
Glucocorticoids (GCs) induce apoptosis in lymphoid lineage cells and are therefore used in the therapy of acute lymphoblastic leukemia (ALL) and related malignancies. MicroRNAs (miRNAs) and the related mirtrons are ~22 nucleotide RNAs derived from polymerase-II transcripts and implicated in the control of essential biological functions, including apoptosis. Whether GCs regulate miRNA-encoding transcription units is unknown. We investigated miRNA/mirtron expression and GC regulation in 8 leukemia/lymphoma in vitro models and 13 ALL children undergoing systemic GC monotherapy using a combination of expression profiling techniques, real time reverse transcription (RT)-PCR and northern blotting to detect mature miRNAs and/or their precursors. We found that mature miRNA regulations can be inferred from expression data of their host genes. Although a simple miRNA-initiated canonical pathway to GC-induced apoptosis or cell cycle arrest did not emerge, we identified several miRNAs/mirtrons that were regulated by GC in patients and cell lines, including the myeloid-specific miR-223 and the apoptosis and cell cycle arrest-inducing miR15 ~ 16 clusters. In an in vitro model, overexpression of miR15b ~ 16 mimics increased and silencing by miR15b ~ 16 inhibitors decreased GC sensitivity. Thus, the observed complex changes in miRNA/mirtron expression during GC treatment might contribute to the anti-leukemic GC effects in a cell context-dependent manner.
Asunto(s)
Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , MicroARNs/análisis , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Apoptosis , Línea Celular Tumoral , ADN Polimerasa II/genética , Perfilación de la Expresión Génica , Glucocorticoides/uso terapéutico , Humanos , MicroARNs/genética , ARN MensajeroRESUMEN
Until recently, only a few microsatellites have been available for Cucurbita, thus their development is highly desirable. The Austrian oil-pumpkin variety Gleisdorfer Olkürbis (C. pepo subsp. pepo) and the C. moschata cultivar Soler (Puerto Rico) were used for SSR development. SSR-enriched partial genomic libraries were established and 2,400 clones were sequenced. Of these 1,058 (44%) contained an SSR at least four repeats long. Primers were designed for 532 SSRs; 500 primer pairs produced fragments of expected size. Of these, 405 (81%) amplified polymorphic fragments in a set of 12 genotypes: three C. moschata, one C. ecuadorensis, and eight C. pepo representing all eight cultivar groups. On an average, C. pepo and C. moschata produced 3.3 alleles per primer pair, showing high inter-species transferability. There were 187 SSR markers detecting polymorphism between the USA oil-pumpkin variety "Lady Godiva" (O5) and the Italian crookneck variety "Bianco Friulano" (CN), which are the parents of our previous F(2) mapping population. It has been used to construct the first published C. pepo map, containing mainly RAPD and AFLP markers. Now the updated map comprises 178 SSRs, 244 AFLPs, 230 RAPDs, five SCARs, and two morphological traits (h and B). It contains 20 linkage groups with a map density of 2.9 cM. The observed genome coverage (Co) is 86.8%.
Asunto(s)
Mapeo Cromosómico , Cucurbita/genética , ADN de Plantas/genética , Ligamiento Genético , Genoma de Planta , Repeticiones de Microsatélite , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Cromosomas de las Plantas/genética , Cartilla de ADN , Marcadores Genéticos , Biblioteca Genómica , Genotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADNRESUMEN
Glucocorticoid (GC)-induced apoptosis is essential in the treatment of acute lymphoblastic leukemia (ALL) and related malignancies. Pro- and anti-apoptotic members of the BCL2 family control many forms of apoptotic cell death, but the extent to which this survival 'rheostat' is involved in the beneficial effects of GC therapy is not understood. We performed systematic analyses of expression, GC regulation and function of BCL2 molecules in primary ALL lymphoblasts and a corresponding in vitro model. Affymetrix-based expression profiling revealed that the response included regulations of pro-apoptotic and, surprisingly, anti-apoptotic BCL2 family members, and varied among patients, but was dominated by induction of the BH3-only molecules BMF and BCL2L11/Bim and repression of PMAIP1/Noxa. Conditional lentiviral gene overexpression and knock-down by RNA interference in the CCRF-CEM model revealed that induction of Bim, and to a lesser extent that of BMF, was required and sufficient for apoptosis. Although anti-apoptotic BCL2 members were not regulated consistently by GC in the various systems, their overexpression delayed, whereas their knock-down accelerated, GC-induced cell death. Thus, the combined clinical and experimental data suggest that GCs induce both pro- and anti-apoptotic BCL2 family member-dependent pathways, with the outcome depending on cellular context and additional signals feeding into the BCL2 rheostat.
Asunto(s)
Apoptosis/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Niño , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Glucocorticoides/uso terapéutico , Humanos , Proteínas de la Membrana/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Among all Bcl2 homology domain 3 (BH3)-only proteins known to date, APR/PMAIP1/Noxa, albeit showing weak proapoptotic potential on its own, appears to be crucial in fine-tuning cell death decisions by targeting the prosurvival molecule Mcl1 for proteasomal degradation. This event appears critical for cell death induction along the mitochondrial Bcl2-regulated apoptosis pathway in response to factor deprivation or DNA damage, presumably by sensitizing the cell toward the action of additional BH3-only protein family members. This review aims to summarize the function of Noxa in normal physiology, stress-induced cell death and tumorigenesis.
Asunto(s)
Apoptosis/fisiología , Daño del ADN , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Secuencia de Aminoácidos , Animales , Niño , Regulación de la Expresión Génica , Humanos , Invertebrados/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/fisiología , Neoplasias/etiología , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Acetato de Tetradecanoilforbol/farmacología , Proteína p53 Supresora de Tumor/fisiología , Vertebrados/metabolismoRESUMEN
High CD99 expression levels and rearrangements of the EWS gene with ETS transcription factor genes characterize the Ewing's sarcoma family of tumors (ESFT). CD99 is a cell surface glycoprotein whose engagement has been implicated in cell proliferation as well as upregulation and transport of several transmembrane proteins in hematopoietic cells. In ESFT, antibody ligation of CD99 induces fast homotypic cell aggregation and cell death although its functional role in these processes remains largely unknown. Here, using an RNAi approach, we studied for the first time the consequences of modulated CD99 expression in six different ESFT cell lines, representing the most frequent variant forms of EWS gene rearrangement. CD99 suppression resulted in growth inhibition and reduced migration of ESFT cells. Among genes whose expression changes in response to CD99 modulation, the potassium-channel modulatory factor KCMF1 was consistently upregulated. In a series of 22 primary ESFT, KCMF1 expression levels inversely correlated with CD99 abundancy. Cells forced to express ectopic KCMF1 showed a similar reduction in migratory ability as CD99 silenced ESFT cells. Our results suggest that in ESFT, high CD99 expression levels contribute to the malignant properties of ESFT by promoting growth and migration of tumor cells and identify KCMF1 as a potential metastasis suppressor gene downregulated by high constitutive CD99 expression in ESFT.
Asunto(s)
Antígenos CD/fisiología , Neoplasias Óseas/patología , Moléculas de Adhesión Celular/fisiología , Sarcoma de Ewing/patología , Ubiquitina-Proteína Ligasas/metabolismo , Antígeno 12E7 , Neoplasias Óseas/metabolismo , Movimiento Celular , Proliferación Celular , Ensayo de Unidades Formadoras de Colonias , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/farmacología , Sarcoma de Ewing/metabolismo , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genética , Regulación hacia ArribaRESUMEN
The cell cycle inhibitor p16(INK4A) is frequently inactivated in acute lymphoblastic T-cell leukemia (T-ALL). We analyzed mechanisms and consequences of p16(INK4A) reconstitution in T-ALL cells lacking this tumor suppressor. CCRF-CEM cells with tetracycline-regulated p16(INK4A) expression underwent stable G1-phase cell cycle arrest for 72 h followed by massive apoptosis. p16(INK4A) expression caused pRB hypophosphorylation and repression of certain E2F target genes. Interestingly, cyclin E and c-Myc were not affected, suggesting pRB/E2F-independent expression of these E2F targets. Cyclin E/CDK2, however, was inactive due to stabilization and redistribution of p27(Kip1) from CDK4/CDK6 to CDK2. Analyses of c-Myc target genes suggested that c-Myc was transcriptionally inactive, which correlated with hypophosphorylation of the c-Myc inhibitor p107. Thus, p16(INK4A), although unable to repress the expression of deregulated cyclin E and c-Myc, functionally inactivated these potential oncogenes. p16(INK4A)-arrested cells showed morphologic changes, induction of T-cell-specific surface markers and repression of telomerase activity, suggesting differentiation. Moreover, p16(INK4A) reconstitution was associated with increased cellular volume, normal protein synthesis rates and elevated ATP levels. Taken together, p16(INK4A) reconstitution in p16(INK4A)-deficient T-ALL cells induced cell cycle arrest in the presence of cyclin E and c-Myc expression, uncoupled growth from cell cycle progression and caused a sequential process of growth, differentiation and apoptosis.
Asunto(s)
Ciclina E/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Genes myc/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatología , Apoptosis/fisiología , Biomarcadores de Tumor , Diferenciación Celular/fisiología , División Celular/fisiología , Niño , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Fase G1/fisiología , Regulación Leucémica de la Expresión Génica , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteína p107 Similar a la del Retinoblastoma , Linfocitos T/patología , Linfocitos T/fisiología , Telomerasa/metabolismo , Activación Transcripcional/fisiologíaRESUMEN
The ability of glucocorticoids (GC) to efficiently kill lymphoid cells has led to their inclusion in essentially all chemotherapy protocols for lymphoid malignancies. This review summarizes recent findings related to the molecular basis of GC-induced apoptosis and GC resistance, and discusses their potential clinical implications. Accumulating evidence suggests that GC may induce cell death via different pathways resulting in apoptotic or necrotic morphologies, depending on the availability/responsiveness of the apoptotic machinery. The former might result from regulation of typical apoptosis genes such as members of the Bcl-2 family, the latter from detrimental GC effects on essential cellular functions possibly perpetuated by GC receptor (GR) autoinduction. Although other possibilities exist, GC resistance might frequently result from defective GR expression, perhaps the most efficient means to target multiple antileukemic GC effects. Numerous novel drug combinations are currently being tested to prevent resistance and improve GC efficacy in the therapy of lymphoid malignancies.
Asunto(s)
Apoptosis/fisiología , Glucocorticoides/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Muerte Celular/genética , Muerte Celular/fisiología , Resistencia a Antineoplásicos/genética , Expresión Génica , Glucocorticoides/metabolismo , Glucocorticoides/uso terapéutico , Humanos , Leucemia Linfoide/tratamiento farmacológico , Leucemia Linfoide/genética , Ratones , Modelos Biológicos , Mutación , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/fisiología , Activación Transcripcional/genética , Activación Transcripcional/fisiologíaRESUMEN
The genus Flavivirus includes a number of important human pathogens that impose major health problems in large regions of the world. The emergence of flaviviruses in new geographic regions (e.g., West Nile virus in North America) and rapid socioeconomic changed in many developing countries where flaviviruses such as dengue virus and Japanese encephalitis virus and endemic demand the development of new vaccines against these diseases. Using tick-borne encephalitis virus as a model we have established a new method to generate attenuated flavivirus strains that may be useful for generating cost-effective and safe live vaccines. This method relies on the specific introduction of deletions into one of the structural proteins, the capsid protein C. These deletions remove parts or all of an internal stretch of hydrophobic amino acid residues that probably is involved in virion assembly. We observed that remarkably long deletions were tolerated, yielding viable viral mutants that were highly attenuated in the mouse model but efficiently induced protective immunity. Biochemical analyses suggested that attenuation was caused by an assembly defect of infectious virions but the mutants produced ample amounts of non-infections subviral particles. The generation of viable mutants with deletions longer that 16 amino acid residues depended on additional, spontaneously emerging mutations within protein C that increased the hydrophobicity of the mutant protein. Although the second-site mutations increased infectivity, they did not restore neuroinvasiveness. Mouse experiments demonstrated excellent safety and immunogenicity profiles for these mutants.
Asunto(s)
Infecciones por Flavivirus/prevención & control , Flavivirus/genética , Flavivirus/patogenicidad , Animales , Proteínas de la Cápside/genética , Flavivirus/fisiología , Infecciones por Flavivirus/transmisión , Humanos , Mutagénesis , Eliminación de SecuenciaRESUMEN
Glucocorticoids (GC) induce apoptosis in malignant lymphoblasts, but the mechanism of this process as well as that of the clinically important GC resistance is unknown. We investigated GC resistance in Jurkat T-ALL cells in which ectopic GC receptor (GR) restores GC sensitivity, suggesting deficient GR expression. Jurkat cells expressed one wild-type and one mutated (R477H) GR allele. GR(R477H) ligand-binding-dependent nuclear import, as revealed by live-cell microscopy of YFP-tagged GR, was unaffected. Transactivation and transrepression were markedly impaired; however, GR(R477H) did not act in a dominant-negative manner, that is, did not prevent cell death, when introduced into a GC-sensitive cell line by retroviral gene transfer. Contrary to another GR heterozygous, but GC-sensitive, T-ALL model (CCRF-CEM), Jurkats expressed lower basal GR levels and did not auto-induce their GR, as revealed by 'real-time' RT-PCR and immunoblotting. Absent GR auto-induction could not be restored by transgenic GR and, hence, was not caused by reduced basal GR levels. Thus, inactivation of one GR gene results in haploinsufficiency if associated with lack of GR auto-induction.
Asunto(s)
Resistencia a Antineoplásicos/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Receptores de Glucocorticoides/genética , Animales , Apoptosis/genética , Apoptosis/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Western Blotting , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Dexametasona/farmacología , Genes Dominantes/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Heterocigoto , Humanos , Células Jurkat , Cinética , Luciferasas de Renilla/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Virus del Tumor Mamario del Ratón/genética , Microscopía Confocal , Mutación Puntual , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Ensayo de Unión Radioligante , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Transfección , Triamcinolona/metabolismoRESUMEN
Glucocorticoids (GC) induce cell cycle arrest and apoptosis in lymphoblastic leukemia cells. To investigate cell cycle effects of GC in the absence of obscuring apoptotic events, we used human CCRF-CEM leukemia cells protected from cell death by transgenic bcl-2. GC treatment arrested these cells in the G1 phase of the cell cycle due to repression of cyclin D3 and c-myc. Cyclin E and Cdk2 protein levels remained high, but the kinase complex was inactive due to increased levels of bound p27(Kip1). Conditional expression of cyclin D3 and/or c-myc was sufficient to prevent GC-induced G1 arrest and p27(Kip1) accumulation but, importantly, did not interfere with the induction of apoptosis. The combined data suggest that repression of both, c-myc and cyclin D3, is necessary to arrest human leukemia cells in the G1 phase of the cell division cycle, but that neither one is required for GC-induced apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ciclinas/metabolismo , Glucocorticoides/farmacología , Leucemia Linfoide/metabolismo , Leucemia Linfoide/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Ciclina D3 , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Fase G1/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Glucocorticoids (GC) control cell cycle progression and induce apoptosis in cells of the lymphoid lineage. Physiologically, these phenomena have been implicated in regulating immune functions and repertoire generation. Clinically, they form the basis of inclusion of GC in essentially all chemotherapy protocols for lymphoid malignancies. In spite of their significance, the molecular mechanisms underlying the anti-leukemic GC effects and the clinically important phenomenon of GC resistance are still unknown. This review summarizes recent findings related to GC-induced apoptosis, cell cycle arrest, and GC resistance with particular emphasis on acute lymphoblastic leukemia (ALL). We hypothesize that under conditions of physiological Bcl-2 expression, GC might induce classical programmed cell death by directly perturbing the Bcl-2 rheostat. In the presence of anti-apoptotic Bcl-2 proteins, cell death might result from accumulating catabolic and/or other detrimental GC effects driven by, and critically dependent on, GC receptor (GR) autoinduction. Although still controversial, there is increasing evidence for release of apoptogenic factors through pores in the outer mitochondrial membrane, rather than deltapsiloss-dependent membrane rupture, with maintenance of mitochondrial function at least in the early phase of the death response. GC-induced cell cycle arrest in ALL cells appears to be independent of apoptosis induction and vice versa, and critically depends on repression of both cyclin-D3 and c-myc followed by increased expression of the cyclin-dependent kinase inhibitor, p27Kip1. Since development of GC-resistant clones requires both cell cycle progression and survival, GC resistance might frequently result from structural or regulatory defects in GR expression, perhaps the most efficient means to target both pathways concurrently.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Glucocorticoides/uso terapéutico , Leucemia Linfoide/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Humanos , Leucemia Linfoide/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Glucocorticoid (GC) resistance is a phenomenon of major significance in a number of clinical situations, including the therapy of lymphoid malignancies. Resistance may concern all, or just selected, GC effects, it may be absolute or just reflect a state of reduced sensitivity and, clinically relevant, be reversible or irreversible. Numerous molecular mechanisms can be envisaged acting either 'upstream' in the GC-triggered signaling pathway, i.e. at the level of the GC receptor (GR), or 'downstream' at the level of the GC-regulated genes responsible for individual GC effects. In lymphoid malignancies, GCs have anti-leukemic effects through the induction of apoptosis and/or cell cycle arrest. In this condition evidence for only a small number of mechanisms for GC resistance has been provided, mostly at the level of the GR. Herein, we review reports and hypotheses regarding 'upstream' and 'downstream' mechanisms for GC resistance in lymphoblastic leukemia and present an in vitro GC resistance model that might allow identification of resistance mechanisms.