RESUMEN
Cross-disease genome-wide association studies (GWASs) unveil pleiotropic loci, mostly situated within the non-coding genome, each of which exerts pleiotropic effects across multiple diseases. However, the challenge "W-H-W" (namely, whether, how, and in which specific diseases pleiotropy can inform clinical therapeutics) calls for effective and integrative approaches and tools. We here introduce a pleiotropy-driven approach specifically designed for therapeutic target prioritization and evaluation from cross-disease GWAS summary data, with its validity demonstrated through applications to two systems of disorders (neuropsychiatric and inflammatory). We illustrate its improved performance in recovering clinical proof-of-concept therapeutic targets. Importantly, it identifies specific diseases where pleiotropy informs clinical therapeutics. Furthermore, we illustrate its versatility in accomplishing advanced tasks, including pathway crosstalk identification and downstream crosstalk-based analyses. To conclude, our integrated solution helps bridge the gap between pleiotropy studies and therapeutics discovery.
Asunto(s)
Pleiotropía Genética , Estudio de Asociación del Genoma Completo , Humanos , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido SimpleAsunto(s)
Proteínas de Fusión bcr-abl , Trasplante de Células Madre Hematopoyéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Adulto , Proteínas de Fusión bcr-abl/genética , Masculino , Femenino , Persona de Mediana Edad , Trasplante de Células Madre Hematopoyéticas/métodos , Medición de Riesgo , Anciano , Adulto Joven , AdolescenteRESUMEN
ETV6::ACSL6 represents a rare genetic aberration in hematopoietic neoplasms and is often associated with severe eosinophilia, which confers an unfavorable prognosis requiring additional anti-inflammatory treatment. However, since the translocation is unlikely to produce a fusion protein, the mechanism of ETV6::ACSL6 action remains unclear. Here, we performed multi-omics analyses of primary leukemia cells and patient-derived xenografts from an acute lymphoblastic leukemia (ALL) patient with ETV6::ACSL6 translocation. We identified a super-enhancer located within the ETV6 gene locus, and revealed translocation and activation of the super-enhancer associated with the ETV6::ACSL6 fusion. The translocated super-enhancer exhibited intense interactions with genomic regions adjacent to and distal from the breakpoint at chromosomes 5 and 12, including genes coding inflammatory factors such as IL-3. This led to modulations in DNA methylation, histone modifications, and chromatin structures, triggering transcription of inflammatory factors leading to eosinophilia. Furthermore, the bromodomain and extraterminal domain (BET) inhibitor synergized with standard-of-care drugs for ALL, effectively reducing IL-3 expression and inhibiting ETV6::ACSL6 ALL growth in vitro and in vivo. Overall, our study revealed for the first time a cis-regulatory mechanism of super-enhancer translocation in ETV6::ACSL6ALL, leading to an ALL-accompanying clinical syndrome. These findings may stimulate novel treatment approaches for this challenging ALL subtype.
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Proteína ETS de Variante de Translocación 6 , Elementos de Facilitación Genéticos , Eosinofilia , Interleucina-3 , Proteínas de Fusión Oncogénica , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteínas Proto-Oncogénicas c-ets , Proteínas Represoras , Translocación Genética , Animales , Humanos , Ratones , Eosinofilia/genética , Eosinofilia/metabolismo , Eosinofilia/patología , Regulación Leucémica de la Expresión Génica , Interleucina-3/genética , Interleucina-3/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Mutations in epigenetic regulators are common in relapsed pediatric acute lymphoblastic leukemia (ALL). Here, we uncovered the mechanism underlying the relapse of ALL driven by an activating mutation of the NSD2 histone methyltransferase (p.E1099K). Using high-throughput drug screening, we found that NSD2-mutant cells were specifically resistant to glucocorticoids. Correction of this mutation restored glucocorticoid sensitivity. The transcriptional response to glucocorticoids was blocked in NSD2-mutant cells due to depressed glucocorticoid receptor (GR) levels and the failure of glucocorticoids to autoactivate GR expression. Although H3K27me3 was globally decreased by NSD2 p.E1099K, H3K27me3 accumulated at the NR3C1 (GR) promoter. Pretreatment of NSD2 p.E1099K cell lines and patient-derived xenograft samples with PRC2 inhibitors reversed glucocorticoid resistance in vitro and in vivo. PRC2 inhibitors restored NR3C1 autoactivation by glucocorticoids, increasing GR levels and allowing GR binding and activation of proapoptotic genes. These findings suggest a new therapeutic approach to relapsed ALL associated with NSD2 mutation. SIGNIFICANCE: NSD2 histone methyltransferase mutations observed in relapsed pediatric ALL drove glucocorticoid resistance by repression of the GR and abrogation of GR gene autoactivation due to accumulation of K3K27me3 at its promoter. Pretreatment with PRC2 inhibitors reversed resistance, suggesting a new therapeutic approach to these patients with ALL.This article is highlighted in the In This Issue feature, p. 1.
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Inhibidores Enzimáticos/uso terapéutico , Glucocorticoides/uso terapéutico , Histona Metiltransferasas/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Proteínas Represoras/genética , Línea Celular Tumoral/efectos de los fármacos , Supervivencia Celular , Niño , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/farmacología , Femenino , Glucocorticoides/farmacología , Humanos , Masculino , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologíaRESUMEN
BACKGROUND: Acute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy. Glucocorticoids form a critical component of chemotherapy regimens and resistance to glucocorticoid therapy is predictive of poor outcome. We have previously shown that glucocorticoid resistance is associated with upregulation of the oncogene C-MYC and failure to induce the proapoptotic gene BIM. METHODS: A high-throughput screening (HTS) campaign was carried out to identify glucocorticoid sensitisers against an ALL xenograft derived from a glucocorticoid-resistant paediatric patient. Gene expression analysis was carried out using Illumina microarrays. Efficacy, messenger RNA and protein analysis were carried out by Resazurin assay, reverse transcription-PCR and immunoblotting, respectively. RESULTS: A novel glucocorticoid sensitiser, 2-((4,5-dihydro-1H-imidazol-2-yl)thio)-N-isopropyl-N-phenylacetamide (GCS-3), was identified from the HTS campaign. The sensitising effect was specific to glucocorticoids and synergy was observed in a range of dexamethasone-resistant and dexamethasone-sensitive xenografts representative of B-ALL, T-ALL and Philadelphia chromosome-positive ALL. GCS-3 in combination with dexamethasone downregulated C-MYC and significantly upregulated BIM expression in a glucocorticoid-resistant ALL xenograft. The GCS-3/dexamethasone combination significantly increased binding of the glucocorticoid receptor to a novel BIM enhancer, which is associated with glucocorticoid sensitivity. CONCLUSIONS: This study describes the potential of the novel glucocorticoid sensitiser, GCS-3, as a biological tool to interrogate glucocorticoid action and resistance.
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Antineoplásicos/farmacología , Proteína 11 Similar a Bcl2/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animales , Dexametasona/farmacología , Descubrimiento de Drogas/métodos , Glucocorticoides/farmacología , Humanos , Receptores de Glucocorticoides/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inhibition of the Menin (MEN1) and MLL (MLL1, KMT2A) interaction is a potential therapeutic strategy for MLL-rearranged (MLL-r) leukemia. Structure-based design yielded the potent, highly selective, and orally bioavailable small-molecule inhibitor VTP50469. Cell lines carrying MLL rearrangements were selectively responsive to VTP50469. VTP50469 displaced Menin from protein complexes and inhibited chromatin occupancy of MLL at select genes. Loss of MLL binding led to changes in gene expression, differentiation, and apoptosis. Patient-derived xenograft (PDX) models derived from patients with either MLL-r acute myeloid leukemia or MLL-r acute lymphoblastic leukemia (ALL) showed dramatic reductions of leukemia burden when treated with VTP50469. Multiple mice engrafted with MLL-r ALL remained disease free for more than 1 year after treatment. These data support rapid translation of this approach to clinical trials.
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Cromatina/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas Proto-Oncogénicas/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Cromatina/genética , Regulación Leucémica de la Expresión Génica/genética , Reordenamiento Génico/efectos de los fármacos , Reordenamiento Génico/genética , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genéticaRESUMEN
Glucocorticoids play a critical role in the treatment of lymphoid malignancies. While glucocorticoid efficacy can be largely attributed to lymphocyte-specific apoptosis, its molecular basis remains elusive. Here, we studied genome-wide lymphocyte-specific open chromatin domains (LSOs), and integrated LSOs with glucocorticoid-induced RNA transcription and chromatin modulation using an in vivo patient-derived xenograft model of acute lymphoblastic leukemia (ALL). This led to the identification of LSOs critical for glucocorticoid-induced apoptosis. Glucocorticoid receptor cooperated with CTCF at these LSOs to mediate DNA looping, which was inhibited by increased DNA methylation in glucocorticoid-resistant ALL and non-lymphoid cell types. Our study demonstrates that lymphocyte-specific epigenetic modifications pre-determine glucocorticoid resistance in ALL and may account for the lack of glucocorticoid sensitivity in other cell types.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Cromatina/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Glucocorticoides/farmacología , Linfocitos/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Apoptosis/efectos de los fármacos , Azacitidina/administración & dosificación , Azacitidina/farmacología , Cromatina/genética , Cromatina/metabolismo , Dexametasona/administración & dosificación , Dexametasona/farmacología , Resistencia a Antineoplásicos/genética , Glucocorticoides/administración & dosificación , Humanos , Linfocitos/metabolismo , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismoRESUMEN
Myc oncoproteins exert tumorigenic effects by regulating expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisite for Myc-induced transcriptional activation, and DOT1L is the only known histone methyltransferase that catalyzes H3K79 methylation. Here, we show that N-Myc upregulates DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. shRNA-mediated depletion of DOT1L reduced mRNA and protein expression of N-Myc target genes ODC1 and E2F2 DOT1L bound to the Myc Box II domain of N-Myc protein, and knockdown of DOT1L reduced histone H3K79 methylation and N-Myc protein binding at the ODC1 and E2F2 gene promoters and reduced neuroblastoma cell proliferation. Treatment with the small-molecule DOT1L inhibitor SGC0946 reduced H3K79 methylation and proliferation of MYCN gene-amplified neuroblastoma cells. In mice xenografts of neuroblastoma cells stably expressing doxycycline-inducible DOT1L shRNA, ablating DOT1L expression with doxycycline significantly reduced ODC1 and E2F2 expression, reduced tumor progression, and improved overall survival. In addition, high levels of DOT1L gene expression in human neuroblastoma tissues correlated with high levels of MYCN, ODC1, and E2F2 gene expression and independently correlated with poor patient survival. Taken together, our results identify DOT1L as a novel cofactor in N-Myc-mediated transcriptional activation of target genes and neuroblastoma oncogenesis. Furthermore, they characterize DOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Cancer Res; 77(9); 2522-33. ©2017 AACR.
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Metiltransferasas/genética , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Transcripción Genética , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Metiltransferasas/antagonistas & inhibidores , Ratones , Proteína Proto-Oncogénica N-Myc/biosíntesis , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Regiones Promotoras GenéticasRESUMEN
Purpose: Robust preclinical models of pediatric acute lymphoblastic leukemia (ALL) are essential in prioritizing promising therapies for clinical assessment in high-risk patients. Patient-derived xenograft (PDX) models of ALL provide a clinically relevant platform for assessing novel drugs, with efficacy generally assessed by enumerating circulating human lymphoblasts in mouse peripheral blood (PB) as an indicator of disease burden. While allowing indirect measurement of disease burden in real time, this technique cannot assess treatment effects on internal reservoirs of disease. We explore benefits of bioluminescence imaging (BLI) to evaluate drug responses in ALL PDXs, compared with PB monitoring. BLI-based thresholds of drug response are also explored.Experimental Design: ALL PDXs were lentivirally transduced to stably express luciferase and green fluorescent protein. In vivo PDX responses to an induction-type regimen of vincristine, dexamethasone, and L-asparaginase were assessed by BLI and PB. Residual disease at day 28 after treatment initiation was assessed by flow cytometric analysis of major organs. BLI and PB were subsequently used to evaluate efficacy of the Bcl-2 inhibitor venetoclax.Results: BLI considerably accelerated and enhanced detection of leukemia burden compared with PB and identified sites of residual disease during treatment in a quantitative manner, highlighting limitations in current PB-based scoring criteria. Using BLI alongside enumeration of human lymphoblasts in PB and bone marrow, we were able to redefine response criteria analogous to the clinical setting.Conclusions: BLI substantially improves the stringency of preclinical drug testing in pediatric ALL PDXs, which will likely be important in prioritizing effective agents for clinical assessment. Clin Cancer Res; 23(14); 3744-55. ©2017 AACR.
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Diagnóstico por Imagen/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico por imagen , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Humanos , Lentivirus/genética , Luciferasas/genética , Mediciones Luminiscentes , Ratones , Células Neoplásicas Circulantes/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangre , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Patient derived xenografts (PDXs) have become a vital, frequently used, component of anti-cancer drug development. PDXs can be serially passaged in vivo for years, and shared across laboratories. As a consequence, the potential for mis-identification and cross-contamination is possible, yet authentication of PDXs appears limited. We present a PDX Authentication System (PAS), by combining a commercially available OpenArray assay of single nucleotide polymorphisms (SNPs) with in-house R studio programs, to validate PDXs established in individual mice from acute lymphoblastic leukemia biopsies. The PAS is sufficiently robust to identify contamination at levels as low as 3%, similar to the gold standard of short tandem repeat (STR) profiling. We have surveyed a panel of PDXs established from 73 individual leukemia patients, and found that the PAS provided sufficient discriminatory power to identify each xenograft. The identified SNP-discrepant PDXs demonstrated distinct gene expression profiles, indicating a risk of contamination for PDXs at high passage number. The PAS also allows for the authentication of tumor cells with complex karyotypes from solid tumors including prostate cancer and Ewing's sarcoma. This study highlights the demands of authenticating PDXs for cancer research, and evaluates a reliable authentication platform that utilizes a commercially available and cost-effective system.
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Genotipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Neoplasias de la Próstata/genética , Sarcoma de Ewing/genética , Animales , Línea Celular Tumoral , Quimerismo , Aberraciones Cromosómicas , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Ratones , Ratones SCID , Polimorfismo de Nucleótido Simple , Análisis de Matrices Tisulares , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glucocorticoids are critical components of combination chemotherapy regimens in pediatric acute lymphoblastic leukemia (ALL). The proapoptotic BIM protein is an important mediator of glucocorticoid-induced apoptosis in normal and malignant lymphocytes, whereas the antiapoptotic BCL2 confers resistance. The signaling pathways regulating BIM and BCL2 expression in glucocorticoid-treated lymphoid cells remain unclear. In this study, pediatric ALL patient-derived xenografts (PDXs) inherently sensitive or resistant to glucocorticoids were exposed to dexamethasone in vivo. Microarray analysis showed that KLF13 and MYB gene expression changes were significantly greater in dexamethasone-sensitive than -resistant PDXs. Chromatin immunoprecipitation (ChIP) analysis detected glucocorticoid receptor (GR) binding at the KLF13 promoter to trigger KLF13 expression only in sensitive PDXs. Next, KLF13 bound to the MYB promoter, deactivating MYB expression only in sensitive PDXs. Sustained MYB expression in resistant PDXs resulted in maintenance of BCL2 expression and inhibition of apoptosis. ChIP sequencing analysis revealed a novel GR binding site in a BIM intronic region (IGR) that was engaged only in dexamethasone-sensitive PDXs. The absence of GR binding at the BIM IGR was associated with BIM silencing and dexamethasone resistance. This study has identified novel mechanisms of opposing BCL2 and BIM gene regulation that control glucocorticoid-induced apoptosis in pediatric ALL cells in vivo.
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Proteínas Reguladoras de la Apoptosis/genética , Apoptosis/genética , Proteínas de la Membrana/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas/genética , Receptores de Glucocorticoides/genética , Animales , Antineoplásicos Hormonales/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteína 11 Similar a Bcl2 , Western Blotting , Inmunoprecipitación de Cromatina , Dexametasona/farmacología , Resistencia a Antineoplásicos/genética , Ensayo de Inmunoadsorción Enzimática , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Endogámicos NOD , Ratones SCID , Análisis de Secuencia por Matrices de Oligonucleótidos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Glucocorticoides/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Aldo-keto reductase 1C3 (AKR1C3, EC 1.1.1.188) metabolises steroid hormones, prostaglandins and xenobiotics, and activates the dinitrobenzamide mustard prodrug PR-104A by reducing it to hydroxylamine PR-104H. Here, we describe a functional assay for AKR1C3 in cells using the fluorogenic probe coumberone (a substrate for all AKR1C isoforms) in conjunction with a specific inhibitor of AKR1C3, the morpholylurea SN34037. We use this assay to evaluate AKR1C3 activity and PR-104A sensitivity in human leukaemia cells. SN34037-sensitive reduction of coumberone to fluorescent coumberol correlated with AKR1C3 protein expression by immunoblotting in a panel of seven diverse human leukaemia cell lines, and with SN34037-sensitive reduction of PR-104A to PR-104H. SN34037 inhibited aerobic cytotoxicity of PR-104A in high-AKR1C3 TF1 erythroleukaemia cells, but not in low-AKR1C3 Nalm6 pre-B cell acute lymphocytic leukaemia (B-ALL) cells, although variation in PR-104H sensitivity confounded the relationship between AKR1C3 activity and PR-104A sensitivity across the cell line panel. AKR1C3 mRNA expression showed wide variation between leukaemia patients, with consistently higher levels in T-ALL than B-ALL. In short term cultures from patient-derived paediatric ALL xenografts, PR-104A was more potent in T-ALL than B-ALL lines, and PR-104A cytotoxicity was significantly inhibited by SN34037 in T-ALL but not B-ALL. Overall, the results demonstrate that SN34037-sensitive coumberone reduction provides a rapid and specific assay for AKR1C3 activity in cells, with potential utility for identifying PR-104A-responsive leukaemias. However, variations in PR-104H sensitivity indicate the need for additional biomarkers for patient stratification.
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3-Hidroxiesteroide Deshidrogenasas/metabolismo , Antineoplásicos/metabolismo , Fluorometría/métodos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Compuestos de Mostaza Nitrogenada/metabolismo , Profármacos/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/genética , Aerobiosis , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Médula Ósea/enzimología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Células HCT116 , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Humanos , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Hidroxiprostaglandina Deshidrogenasas/genética , Leucocitos/enzimología , Morfolinas/química , Morfolinas/metabolismo , Compuestos de Mostaza Nitrogenada/farmacocinética , Compuestos de Mostaza Nitrogenada/farmacología , Oxidación-Reducción , Profármacos/farmacocinética , Profármacos/farmacología , Especificidad por Sustrato , Factores de Tiempo , Urea/análogos & derivados , Urea/química , Urea/metabolismoRESUMEN
BACKGROUND: In the bone marrow mesenchymal stromal cells and osteoblasts form functional niches for hematopoietic stem and progenitor cells. This microenvironment can be partially mimicked using in vitro co-culture systems. In this study, we examined the oxygen tension in three distinct compartments in a co-culture system of purified CD34(+) cells and mesenchymal stromal cells with regard to different spatial localizations. DESIGN AND METHODS: Hypoxic cells in the co-culture were visualized by pimonidazole staining. Hematopoietic cell distribution, and functional and phenotypic characteristics were analyzed by flow cytometry. The secretion of vascular endothelial growth factor and stromal-derived factor-1 by mesenchymal stromal cells in low oxygen co-cultures was determined by an enzyme-linked immunosorbent assay. The effect of co-culture medium on the hematopoietic cell migration potential was tested in a transwell assay. RESULTS: In co-cultures under atmospheric oxygen tension, regions of low oxygen tension could be detected beneath the feeder layer in which a reservoir of phenotypically more primitive hematopoietic cells is located in vitro. In low oxygen co-culture, the adhesion of hematopoietic cells to the feeder layer was decreased, whereas hematopoietic cell transmigration beneath mesenchymal stromal cells was favored. Increased vascular endothelial growth factor-A secretion by mesenchymal stromal cells under low oxygen conditions, which increased the permeability of the monolayer, was responsible for this effect. Furthermore, vascular endothelial growth factor-A expression in low oxygen mesenchymal stromal cells was induced via hypoxia-inducible factor signaling. However, stromal cell-derived factor-1 secretion by mesenchymal stromal cells was down-regulated under low oxygen conditions in a hypoxia-inducible factor-independent manner. CONCLUSIONS: We demonstrate for the first time that differences in oxygen tension cause selective modification of hematopoietic cell and mesenchymal stromal cell interactions in a co-culture system, thus confirming that oxygen tension plays a critical role in the interaction between hematopoietic cells and the niche environment.
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Células Madre Hematopoyéticas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Oxígeno/metabolismo , Nicho de Células Madre/fisiología , Antígenos CD34/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Adhesión Celular/genética , Movimiento Celular/genética , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Técnicas de Cocultivo , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Humanos , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inmunofenotipificación , Integrinas/genética , Integrinas/metabolismo , Unión Proteica , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Hematopoietic stem cells located in the bone marrow interact with a specific microenvironment referred to as the stem cell niche. Data derived from ex vivo co-culture systems using mesenchymal stromal cells as a feeder cell layer suggest that cell-to-cell contact has a significant impact on the expansion, migratory potential and 'stemness' of hematopoietic stem cells. Here we investigated in detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex vivo expansion. DESIGN AND METHODS: In the co-culture system, we defined three distinct localizations of hematopoietic stem cells relative to the mesenchymal stromal cell layer: (i) those in supernatant (non-adherent cells); (ii) those adhering to the surface of mesenchymal stromal cells (phase-bright cells) and (iii) those beneath the mesenchymal stromal cells (phase-dim cells). Cell cycle, proliferation, cell division and immunophenotype of these three cell fractions were evaluated from day 1 to 7. RESULTS: Phase-bright cells contained the highest proportion of cycling progenitors during co-culture. In contrast, phase-dim cells divided much more slowly and retained a more immature phenotype compared to the other cell fractions. The phase-dim compartment was soon enriched for CD34(+)/CD38(-) cells. Migration beneath the mesenchymal stromal cell layer could be hampered by inhibiting integrin beta1 or CXCR4. CONCLUSIONS: Our data suggest that the mesenchymal stromal cell surface is the predominant site of proliferation of hematopoietic stem cells, whereas the compartment beneath the mesenchymal stromal cell layer seems to mimic the stem cell niche for more immature cells. The SDF-1/CXCR4 interaction and integrin-mediated cell adhesion play important roles in the distribution of hematopoietic stem cells in the co-culture system.
Asunto(s)
Células Madre Hematopoyéticas/citología , Mesodermo/citología , Células del Estroma/citología , Células de la Médula Ósea/metabolismo , Adhesión Celular , Técnicas de Cultivo de Célula , Ciclo Celular , Movimiento Celular , Proliferación Celular , Técnicas de Cocultivo , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunofenotipificación , Integrina beta1/metabolismo , Mesodermo/metabolismo , Receptores CXCR4/metabolismo , Células del Estroma/metabolismoRESUMEN
Primary human multipotent mesenchymal stromal cells (MSCs) are capable of self renewal or differentiation into several different lineages, including osteoblasts, chondrocytes and adipocytes. However, upon prolonged in vitro culture, MSCs tend to undergo spontaneous osteogenic differentiation. Here, we address the possible role of endogenous osteogenic bone morphogenetic proteins (BMPs) in in situ osteoblastic differentiation of human MSCs. Human MSCs consistently express biologically active BMP-2, BMP-4 and BMP-6 in addition to all BMP-activated receptors, which are functional as shown by the induction of alkaline phosphatase (ALP) activity and up-regulation of osteogenic genes (ALP, BSP1, collagen I and Runx2) following BMP-2 exposure. Since glycosaminoglycans (GAGs) have been implicated in the modulation of the osteogenic bioactivity of BMPs, we reduced sulphated cell surface GAGs by NaClO(3) treatment and found significantly reduced osteogenic gene expression and ALP activity, suggesting that this was partly due to the reduced biological activity of endogenous BMPs. Antagonising osteogenic BMP activity led to a significant reduction in the ALP activity and down-regulation of the transcription factor Runx2 associated with osteogenic development. Blocking BMP receptor type I kinase function with dorsomorphin demonstrated that endogenous osteogenesis was independent of Smad activation but was dependent on phosphatidylinositol 3-kinase (PI-3K). Inclusion of the PI-3K kinase inhibitor Ly294002 significantly reduced osteogenic gene expression and ALP activity. Spontaneous mineralisation was also abrogated following PI-3K inhibition. Thus, endogenous BMPs could contribute to spontaneous osteogenesis through Smad-independent PI-3K-dependent signalling.
Asunto(s)
Células de la Médula Ósea/metabolismo , Proteínas Morfogenéticas Óseas/biosíntesis , Células Madre Mesenquimatosas/metabolismo , Fosfatasa Alcalina/biosíntesis , Células de la Médula Ósea/citología , Receptores de Proteínas Morfogenéticas Óseas/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas/genética , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Osteogénesis , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transducción de Señal , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
OBJECTIVE: To investigate the impact of direct contact between mesenchymal stromal cells (MSCs) and CD133(+) hematopoietic stem cells in terms of expansion potential, differentiation, migratory capacity, and gene expression profile. MATERIALS AND METHODS: CD133(+)-purified hematopoietic progenitor cells were cultured for 7 days on subconfluent MSCs supplemented with growth-factor-containing medium. After ex vivo expansion, nonadherent and adherent cells were collected and analyzed separately. RESULTS: The adherent cell population was less differentiated than the nonadherent fraction. CXCR4 was upregulated in the adherent fraction, which was associated with a higher migration capacity toward a stromal cell-derived factor-1 gradient. Colony-forming unit granulocyte-macrophage and long-term culture-initiation cell assays demonstrated a higher clonogenicity and repopulating capacity of the adherent fraction. Genes involved in adhesion, cell-cycle control, motility, and self-renewal were more highly expressed in the adherent fraction. CONCLUSION: Adhesion and direct cell-to-cell contact with an MSC feeder layer supports ex vivo expansion, migratory potential, and stemness of CD133(+) hematopoietic progenitor cells.
Asunto(s)
Antígenos CD/metabolismo , Movimiento Celular/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glicoproteínas/metabolismo , Células Madre Hematopoyéticas , Células Madre Mesenquimatosas/metabolismo , Péptidos/metabolismo , Células del Estroma/metabolismo , Antígeno AC133 , Western Blotting , Adhesión Celular , División Celular/fisiología , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células del Estroma/citología , Propiedades de SuperficieRESUMEN
In order to develop a novel biomaterial, films of chitosan blended with poly(3-hydroxybutyrate) (PHB) were prepared by an emulsion blending technique and their properties were characterized. Scanning electron microscopy (SEM) showed that PHB microspheres were formed and were entrapped in chitosan matrices, which made the film surface rough. With increasing PHB content, the roughness of the film surface increased, while the swelling capability of the films decreased. In a wet state, the blended films exhibited a lower elastic modulus, a higher elongation-at-break and a higher tensile strength compared with chitosan films. Cell-culture experiments revealed that the blended films had better cytocompatibility than chitosan films. To explore the potential application of the blended material in tissue engineering, the porous blended scaffolds were fabricated and their pore morphology was observed by SEM. The results revealed that not only pore structure but also pore wall morphology of the blended scaffolds could be controlled by selecting the parameters of the fabrication process. These advantageous properties indicate that the blended chitosan/PHB material is promising for tissue engineering applications.