RESUMEN
Master regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation.
Asunto(s)
Anemia , Factor de Transcripción GATA1 , Diferenciación Celular/genética , Cromatina/genética , Inmunoprecipitación de Cromatina , Eritropoyesis/genética , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , HumanosRESUMEN
Although preoperative autologous blood donation (PABD) has many advantages, there has been a decrease in the performance due to a decrease in the residual risk of allogeneic blood transfusion. In allogeneic blood transfusion, anti HLA antibodies and donor-specific antibodies mediate antibody-mediated rejection, which results in graft failure. PABD for anemic patients such as those with end-stage renal disease (ESRD) and a kidney transplant is relatively contraindicated. In this study, we aimed to investigate the characteristics of patients who underwent PABD and elucidate the safety and feasibility of PABD. We performed PABD safely in ten ESRD patients and nine kidney transplant patients and retrospectively analyzed medical records of the hospital. All kidney transplant patients avoided allogeneic blood transfusion, but 4 out of 10 ESRD patients had allogeneic blood transfusion, even if their blood donation volume was larger than those of the kidney transplant patients. It depends on the type of operation; cardiovascular surgery was more common in ESRD patients, and orthopedic surgery was more common in kidney transplant patients. There was profuse bleeding in cardiovascular surgery compared to orthopedic surgery because of longer operation time of the former. Completely avoiding allogeneic blood transfusion in major surgery was rather difficult even if PABD was performed. To prevent the formation of anti- HLA antibodies, PABD would be considered for ESRD patients undergoing kidney transplantation and kidney transplant patients that are potential candidates for secondary kidney transplantation.
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Pérdida de Sangre Quirúrgica/prevención & control , Transfusión de Sangre Autóloga , Fallo Renal Crónico/cirugía , Trasplante de Riñón , Cuidados Preoperatorios , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Cell-free and concentrated ascites reinfusion therapy (CART) has been suggested to be able to treat malignant ascites more safely and effectively with chemotherapy because of its ability to retain serum protein and albumin. Although the characteristics of cancer types and CART and the clinical implications of combination therapy with antitumor agents are becoming widespread, there are limited reports on its efficacy and complications. METHODS: In this prospective observational national post-marketing study, 128 patients with malignancies received 300 CART sessions at 22 centers. After excluding other malignancies, the patients were divided into four groups: gynecological malignancies with chemotherapy (GYC+; 18 cases and 36 times) and without chemotherapy (GYC-; 35 cases and 52 times), and gastrointestinal malignancies with chemotherapy (GIC+; 8 cases and 16 times) and without chemotherapy (20 cases and 58 times). RESULTS: There were significant reductions in the body weight in all groups and significant reductions in abdominal circumference and significant improvements in the diet and Eastern Cooperative Oncology Group performance status only in the GYC+ group. The total serum protein and albumin increased significantly in all groups, except for the GIC+ group, before and after CART. There was no significant difference in the presence or absence of antitumor medication. CONCLUSION: With CART, there were differences in the improvement of the clinical symptoms between malignancy groups. The combination of CART and antineoplastic agents may be as safe as CART alone in cases of exudative malignant ascites.
RESUMEN
Diamond-Blackfan anemia is a congenital bone marrow failure syndrome characterized by red blood cell (RBC) aplasia with varied malformations in infants. Elevated activity of adenosine deaminase (ADA) has been considered as a useful biomarker of Diamond-Blackfan anemia, and ADA assay has been shown to be more sensitive than genetic diagnosis. Approximately, 80% of the examined patients showed elevated ADA activity, whereas genetic tests of ribosome subunit genes identified mutations in approximately 60% of the patients. We previously reported that reduced glutathione (GSH) levels in RBCs may serve as a biomarker of Diamond-Blackfan anemia. In this study, to confirm the universality of our data, we extended the analysis to seven RBC enzymes and GSH of 14 patients with Diamond-Blackfan anemia and performed a cross-analysis study using enzyme activity assay and recently reported proteome data. Statistical analysis revealed that both data exhibited high similarity, upregulation in the hexokinase and pentose-phosphate pathway, and downregulation in glycolytic enzymes such as phosphofructokinase and pyruvate kinase, in the RBCs obtained from the subjects with Diamond-Blackfan anemia. The only discrepancy between enzyme activity and proteome data was observed in glucose-6-phosphate dehydrogenase (G6PD), as increased G6PD activity showed no relation with the significant elevation in protein levels. These results suggest that our enzymatic activity data of Diamond-Blackfan anemia are universal and that the enzymatic activation of G6PD via a hitherto-unveiled mechanism is another metabolic feature of RBCs of Diamond-Blackfan anemia.
Asunto(s)
Anemia de Diamond-Blackfan/sangre , Anemia de Diamond-Blackfan/enzimología , Eritrocitos/enzimología , Adolescente , Aminohidrolasas/sangre , Biomarcadores/sangre , Estudios de Casos y Controles , Niño , Preescolar , Regulación hacia Abajo , Glucosafosfato Deshidrogenasa/sangre , Glutatión/sangre , Glucólisis , Humanos , Lactante , Japón , Vía de Pentosa Fosfato , Regulación hacia ArribaRESUMEN
Hereditary hemolytic anemia (HHA) is a group of monogenic diseases arising from the increased destruction of circulating erythrocytes. HHA is caused by germline mutations in genes encoding components of the red blood cell membrane, hemoglobin, and enzymes. Comprehensive gene analyses have identified various HHA-associated germline defects. However, early HHA diagnosis can be difficult in newborns because they present with hydrops and severe jaundice, which require urgent blood transfusions. Considering neonatal physiological hemolysis and pediatric infection, we select efficient diagnostic procedures following the exclusion of "syndromic hemolytic diseases". Clinical sequencing is performed for atypical cases, although phenotypic and laboratory tests remain essential for the verification of pathogenicity when certain variants are identified. The diagnostic gene panel can also be useful for predicting prognosis and determining treatment options. Although transfusion-dependent adult patients with HHA are rare in Japan, their management remains challenging. Clinical trials of new drugs and genetic controls are ongoing in other countries. However, the long-term management of a small group of patients with severe HHA must still be addressed in Japan. Here, we review the strategy and clinical significance of using genetic diagnostic methods for HHA in newborns.
Asunto(s)
Anemia Hemolítica Congénita , Membrana Eritrocítica , Eritrocitos , Hemólisis , Humanos , Recién Nacido , JapónRESUMEN
Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia with mutations in ribosomal protein (RP) genes. Elevated activity of erythrocyte adenosine deaminase (eADA) has been utilized as a biomarker of DBA. We examined erythrocyte reduced glutathione (GSH) as well as eADA in 22 patients in 18 DBA families, in whom RP gene mutations had been identified. Simultaneous evaluation of both eADA and GSH demonstrated that all examined DBA patients showed elevated values of either eADA or GSH, whereas presence of both eADA and GSH elevation was able to distinguish DBA patients from 34 normal controls and 14 unaffected members of the DBA families. Furthermore, a support vector machines analysis using both eADA and GSH levels yielded a formula to differentiate DBA from both normal controls and non-DBA family members. To confirm the usefulness of the formula, we analyzed additional 7 patients diagnosed by the clinical criteria. Although eADA showed within normal values in 3 patients, all of these patients were diagnosed as 'DBA' by use of the formula. Because extensive analysis of the RP genes failed to detect no causative mutation in approximately 40% of clinically diagnosed DBA patients, GSH may be useful an additional biomarker for diagnosis of DBA.
Asunto(s)
Anemia de Diamond-Blackfan/diagnóstico , Eritrocitos/química , Glutatión/sangre , Adenosina Desaminasa , Biomarcadores/sangre , Estudios de Casos y Controles , Familia , Femenino , Humanos , Masculino , Mutación , Proteínas Ribosómicas/genéticaRESUMEN
Phosphatidylserine is localized exclusively to the inner leaflet of the membrane lipid bilayer of most cells, including erythrocytes. This asymmetric distribution is critical for the survival of erythrocytes in circulation since externalized phosphatidylserine is a phagocytic signal for splenic macrophages. Flippases are P-IV ATPase family proteins that actively transport phosphatidylserine from the outer to inner leaflet. It has not yet been determined which of the 14 members of this family of proteins is the flippase in human erythrocytes. Herein, we report that ATP11C encodes a major flippase in human erythrocytes, and a genetic mutation identified in a male patient caused congenital hemolytic anemia inherited as an X-linked recessive trait. Phosphatidylserine internalization in erythrocytes with the mutant ATP11C was decreased 10-fold compared to that of the control, functionally establishing that ATP11C is a major flippase in human erythrocytes. Contrary to our expectations phosphatidylserine was retained in the inner leaflet of the majority of mature erythrocytes from both controls and the patient, suggesting that phosphatidylserine cannot be externalized as long as scramblase is inactive. Phosphatidylserine-exposing cells were found only in the densest senescent cells (0.1% of total) in which scramblase was activated by increased Ca(2+) concentration: the percentage of these phosphatidylserine-exposing cells was increased in the patient's senescent cells accounting for his mild anemia. Furthermore, the finding of similar extents of phosphatidylserine exposure by exogenous Ca(2+)-activated scrambling in both control erythrocytes and the patient's erythrocytes implies that suppressed scramblase activity rather than flippase activity contributes to the maintenance of phosphatidylserine in the inner leaflet of human erythrocytes.
Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Anemia Hemolítica Congénita/genética , Anemia Hemolítica Congénita/metabolismo , Eritrocitos/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Adenosina Trifosfatasas/deficiencia , Adolescente , Anemia Hemolítica Congénita/diagnóstico , Transporte Biológico Activo , Biomarcadores , Calcio/metabolismo , Índices de Eritrocitos , Eritrocitos/efectos de los fármacos , Humanos , Masculino , Proteínas de Transporte de Membrana/deficiencia , Mutación , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacologíaRESUMEN
Research and development of personalized cancer vaccines as precision medicine are ongoing. We predicted human leukocyte antigen (HLA)-compatible cancer antigen candidate peptides based on patient-specific cancer genomic profiles and performed a Phase I clinical trial for the safety and tolerability of cancer vaccines with human platelet lysate-induced antigen-presenting cells (HPL-APCs) from peripheral monocytes. Among the five enrolled patients, two patients completed six doses per course (2-3 × 107 cells per dose), and an interim analysis was performed based on the immune response. An immune response was detected by enzyme-linked immunosorbent spot (ELISpot) assays to HLA-A*33:03-matched KRASWT, HLA-DRB1*09:01-compliant KRASWT or G12D, or HLA-A*31:01-matched SMAD4WT, and HLA-DRB1*04:01-matched SMAD4G365D peptides in two completed cases, respectively. Moreover, SMAD4WT-specific CD8+ effector memory T cells were amplified. However, an attenuation of the acquired immune response was observed 6 months after one course of cancer vaccination as the disease progressed. This study confirmed the safety and tolerability of HPL-APCs in advanced and recurrent cancers refractory to standard therapy and is the first clinical report to demonstrate the immunoinducibility of personalized cancer vaccines using HPL-APCs. Phase II clinical trials to determine immune responses with optimized adjuvant drugs and continued administration are expected to demonstrate efficacy.
RESUMEN
Hereditary stomatocytosis (HSt) is a type of congenital hemolytic anemia caused by abnormally increased cation permeability of erythrocyte membranes. Dehydrated HSt (DHSt) is the most common subtype of HSt and is diagnosed based on clinical and laboratory findings related to erythrocytes. PIEZO1 and KCNN4 have been recognized as causative genes, and many related variants have been reported. We analyzed the genomic background of 23 patients from 20 Japanese families suspected of having DHSt using a target capture sequence and identified pathogenic/likely pathogenic variants of PIEZO1 or KCNN4 in 12 families.
RESUMEN
Diamond-Blackfan anaemia (DBA) shares clinical features with two recently reported sporadic cases of dyserythropoietic anaemia with a cryptic GATA1 splicing mutation (c.871-24 C>T). We hypothesized that some patients clinically diagnosed with DBA but whose causative genes were unknown may carry the intronic GATA1 mutation. Here, we examined 79 patients in our DBA cohort, who had no detectable causative genes. The intronic GATA1 mutation was identified in two male patients sharing the same pedigree that included multiple cases with anaemia. Cosegregation of this mutation and disease in multiple family members provide evidence to support the pathogenicity of the intronic GATA1 mutation.
RESUMEN
Genetic causes of undiagnosed hemolytic anemia in nineteen patients were analyzed by whole-exome sequencing, and novel COL4A1 variants were identified in four patients (21%). All patients were complicated with congenital malformations of the brain, such as porencephaly or schizencephaly. In these patients, hemolysis became less severe within 2 months after birth, and red cell transfusion was no longer required after 50 days, whereas chronic hemolysis continued.
RESUMEN
Diamond-Blackfan anemia (DBA) is a congenital red-cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. It is not known how the RPS19 deficiency impairs erythropoiesis and proliferation of hematopoietic progenitors. To elucidate molecular mechanisms in RPS19-deficient DBA, we analyzed the effects of RPS19 deficiency on erythropoietin (EPO)-induced signal transduction, cell cycle, and apoptosis in RPS19-deficient TF-1 cells. We did not find any abnormality in EPO-induced signal transduction. However, RPS19-deficient TF-1 cells showed G0/G1 arrest (82% vs. 58%; p < .05) together with accumulation of p21 and p27. The fraction of apoptotic cells detected by Annexin V analysis also increased compared with control cells (13% vs. 3.1%; p < .05). Western blot analysis of apoptosis-related proteins showed that the level of bcl-2 and Bad was decreased and Bax was increased in RPS19-deficient TF-1 cells. Moreover, primary CD34-positive cells from DBA patients detected by Annexin V analysis also generated a higher number of apoptotic cells compared with normal CD34-positive cells during in vitro culture (38% vs. 8.9%; n = 5; p < .001). Finally, we show that although RPS19 silencing reduces EPO-induced development of erythroid progenitors expressing glycophorin A (GPA), RPS19 silencing in cells already expressing GPA does not affect GPA expression. These findings indicate that RPS19 deficiency causes apoptosis and accelerated loss of erythroid progenitors in RPS19-deficient DBA.
Asunto(s)
Anemia de Diamond-Blackfan/sangre , Anemia de Diamond-Blackfan/patología , Proteínas Ribosómicas/deficiencia , Anemia de Diamond-Blackfan/genética , Anexina A5/metabolismo , Apoptosis , Línea Celular , Proliferación Celular , Eritropoyesis/efectos de los fármacos , Eritropoyesis/fisiología , Eritropoyetina/farmacología , Glicoforinas/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Modelos Biológicos , Mutación , ARN Interferente Pequeño/genética , Receptores de Eritropoyetina/metabolismo , Proteínas Recombinantes , Proteínas Ribosómicas/antagonistas & inhibidores , Proteínas Ribosómicas/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Krüppel-like factor 1 (KLF1), a transcription factor controlling definitive erythropoiesis, is involved in sequential control of terminal cell division and enucleation via fine regulation of key cell cycle regulator gene expression in erythroid lineage cells. Type IV congenital dyserythropoietic anemia (CDA) is caused by a monoallelic mutation at the second zinc finger of KLF1 (c.973G>A, p.E325K). We recently diagnosed a female patient with type IV CDA with the identical missense mutation. To understand the mechanism underlying the dyserythropoiesis caused by the mutation, we generated induced pluripotent stem cells (iPSCs) from the CDA patient (CDA-iPSCs). The erythroid cells that differentiated from CDA-iPSCs (CDA-erythroid cells) displayed multinucleated morphology, absence of CD44, and dysregulation of the KLF1 target gene expression. In addition, uptake of bromodeoxyuridine by CDA-erythroid cells was significantly decreased at the CD235a+/CD71+ stage, and microarray analysis revealed that cell cycle regulator genes were dysregulated, with increased expression of negative regulators such as CDKN2C and CDKN2A. Furthermore, inducible expression of the KLF1 E325K, but not the wild-type KLF1, caused a cell cycle arrest at the G1 phase in CDA-erythroid cells. Microarray analysis of CDA-erythroid cells and real-time polymerase chain reaction analysis of the KLF1 E325K inducible expression system also revealed altered expression of several KLF1 target genes including erythrocyte membrane protein band 4.1 (EPB41), EPB42, glutathione disulfide reductase (GSR), glucose phosphate isomerase (GPI), and ATPase phospholipid transporting 8A1 (ATP8A1). Our data indicate that the E325K mutation in KLF1 is associated with disruption of transcriptional control of cell cycle regulators in association with erythroid membrane or enzyme abnormalities, leading to dyserythropoiesis.
Asunto(s)
Anemia Diseritropoyética Congénita , Diferenciación Celular/genética , Células Eritroides , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Células Madre Pluripotentes Inducidas , Factores de Transcripción de Tipo Kruppel , Mutación Missense , Adulto , Sustitución de Aminoácidos , Anemia Diseritropoyética Congénita/genética , Anemia Diseritropoyética Congénita/metabolismo , Anemia Diseritropoyética Congénita/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Eritroides/metabolismo , Células Eritroides/patología , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: Red blood cell pyruvate kinase (R-PK) deficiency is the most common glycolytic enzyme defect associated with hereditary non-spherocytic hemolytic anemia. Cases with the most severe deficiency die in the peri- or neonatal period and no specific therapy exists at present. To test whether the targeted overexpression of the normal R-PK gene in erythroid cells could reduce hemolysis in R-PK mutant mice, we performed a genetic rescue study using human R-PK transgenic mice. DESIGN AND METHODS: Human R-PK promoter driven with human mLCR of the human b-globin locus was used for the erythroid-specific expression of human R-PK in murine erythrocytes. The transgenic lines were mated with homozygous R-PK mutant mice and subsequently backcrossed. Mutant homozygotes with the mLCR-R-PK transgene were examined for any therapeutic effects of transgene expression. RESULTS: Two PK transgenic lines, hRPK_lo and hRPK_hi, were obtained. R-PK activity of the transgenic mice reached as high as three times that of the animals with the endogenous PK gene. Overexpression of human R-PK in the homozygous mutant mice successfully reduced hemolytic anemia. Improvements of hemolysis were evaluated by hemoglobin concentration, reticulocyte count, and spleen weight, which showed significant correlations with the levels of expression of the transgene. Recovery from metabolic disturbance in mutant red blood cells was shown as normalized concentrations of the glycolytic intermediates upstream of PK. In addition, there was a remarkable negative correlation between R-PK activity and the number of TUNEL-positive erythroid progenitors in the spleen. INTERPRETATION AND CONCLUSIONS: These results indicate that overexpression of the wild-type PK gene in mutant erythroid cells ameliorates both erythroid apoptosis and the shortened red blood cell lifespan observed in PK mutant mice. It is likely that the level of transgene expression required to achieve evident therapeutic effects should be equivalent to or more than that of the endogenous PK gene. This gene-addition strategy may be suitable for clinical application if there is a high level of transgene expression of R-PK in erythroid progenitors/red blood cells.
Asunto(s)
Anemia Hemolítica/terapia , Terapia Genética/métodos , Piruvato Quinasa/deficiencia , Anemia Hemolítica/etiología , Anemia Hemolítica Congénita no Esferocítica/etiología , Anemia Hemolítica Congénita no Esferocítica/terapia , Animales , Apoptosis , Envejecimiento Eritrocítico , Eritrocitos/enzimología , Eritrocitos/patología , Humanos , Ratones , Ratones Transgénicos , Piruvato Quinasa/genéticaRESUMEN
We performed post-marketing surveillance to evaluate the safety and efficacy of cell-free and concentrated ascites reinfusion therapy (CART). In total, 356 CART sessions in 147 patients at 22 centers were performed. The most common primary disease was cancer (128 cases, 300 sessions). Mean amount of ascites collected was 3.7 L, and mean concentration ratio was 9.2. Mean amount of reinfused protein was 67.8 g (recovery rate, 72.0%). Performance status, dietary intake, urine volume, body weight and abdominal circumference were significantly improved after CART. Body temperature increased significantly, by 0.3°C on average. Concomitant steroids and/or NSAIDs use before reinfusion was significantly and negatively associated with increases in body temperature. Most adverse events were fever and chills. This study examined a large number of patients compared with previous studies, and showed that CART is an effective and relatively safe treatment for refractory ascites, such as malignant ascites.
Asunto(s)
Ascitis/patología , Ascitis/terapia , Líquido Ascítico/patología , Fluidoterapia/métodos , Adulto , Anciano , Anciano de 80 o más Años , Ascitis/etiología , Presión Sanguínea , Temperatura Corporal , Femenino , Fluidoterapia/efectos adversos , Humanos , Infusiones Parenterales , Masculino , Persona de Mediana Edad , Vigilancia de Productos Comercializados , Resultado del Tratamiento , Adulto JovenRESUMEN
To investigate the role of protein kinase Cdelta (PKCdelta) in angiotensin II-induced facilitation mechanisms of hypoxic neuronal damage and whether candesartan, an AT1 receptor antagonist, can suppress these mechanisms, we performed in vitro experiments which were free from vascular components using PC12 cells under hypoxic (12 h)/reoxygenation (0-48 h) conditions. Angiotensin II apparently increased the basal expression level of PKCdelta phosphorylated at Ser(643) before hypoxia, promoted the cleavage of PKCdelta to its catalytic fragment, and fostered the progression of DNA fragmentation after hypoxia. Candesartan inhibited both phosphorylation and cleavage of PKCdelta and suppressed the angiotensin II-induced facilitation of DNA fragmentation under hypoxic/reoxygenation conditions. However, PD123319, an AT2 receptor antagonist, influenced neither PKCdelta nor the angiotensin II-induced facilitation of DNA fragmentation. Furthermore, in PC12 cells expressing the ATP-binding mutant of PKCdelta (PKCdelta(K376R)) acting as a dominant-negative protein, both phosphorylation and cleavage of PKCdelta were attenuated and DNA fragmentation was markedly suppressed regardless of the presence of angiotensin II. These findings suggest that angiotensin II-induced facilitation of DNA fragmentation under hypoxic conditions is mediated by PKCdelta, and the mechanisms can be suppressed by the candesartan mediated blockade of the AT1 receptor.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Angiotensina II/antagonistas & inhibidores , Bencimidazoles/farmacología , Muerte Celular/efectos de los fármacos , Proteína Quinasa C/fisiología , Tetrazoles/farmacología , Animales , Compuestos de Bifenilo , Western Blotting/métodos , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Regulación de la Expresión Génica/efectos de los fármacos , Imidazoles/farmacología , Etiquetado Corte-Fin in Situ/métodos , Ratones , Células PC12 , Fosforilación , Proteína Quinasa C-delta , Piridinas/farmacología , Ratas , Serina/metabolismo , Factores de Tiempo , Transfección/métodosRESUMEN
A real-time quantitative-polymerase chain reaction (RQ-PCR) targeting the immunoglobulin heavy chain (IgH) gene has been used for the quantification of minimal residual disease (MRD) in B-cell hematological malignancies. In non-Hodgkin lymphoma (NHL), experimental costs are increased, as a large number of primer-probe sets are required because of diversity, due to somatic and ongoing mutations of the IgH gene. We developed an allele-specific oligonucleotide (ASO) combined with a germline consensus probe-based RQ-PCR assay and examined MRD in peripheral blood stem cells (PBSC). The IgH consensus probes were adapted in seven (50%) of 14 amplifiable cases. Patients with heavily contaminating tumor cells in PBSC relapsed after PBSC transplantation. Our strategy will contribute to the development of a cost-efficient, precisely quantitative and systemic detection assay for MRD in NHL.
Asunto(s)
Genes de Inmunoglobulinas/genética , Linfoma de Células B/terapia , Neoplasia Residual/diagnóstico , Células Neoplásicas Circulantes/patología , Trasplante de Células Madre de Sangre Periférica/métodos , Reacción en Cadena de la Polimerasa/métodos , Adolescente , Adulto , Anciano , Alelos , Secuencia de Bases , Secuencia de Consenso , Cartilla de ADN/economía , Cartilla de ADN/normas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Oligonucleótidos , Trasplante de Células Madre de Sangre Periférica/normas , Reacción en Cadena de la Polimerasa/economía , Reacción en Cadena de la Polimerasa/normas , Sensibilidad y Especificidad , Trasplante AutólogoRESUMEN
The transforming growth factor-beta (TGF-beta) superfamily encompasses the ligands and receptors for TGF-beta, bone morphogenic proteins (BMPs), and Activins. Cellular response to ligand is context-dependent and may be controlled by specificity and/or redundancy of expression of these superfamily members. Several pathways within this family have been implicated in the proliferation, differentiation, and renewal of hematopoietic stem cells (HSCs); however, their roles and redundancies at the molecular level are poorly understood in the rare HSC. Here we have characterized the expression of TGF-beta superfamily ligands, receptors, and Smads in murine HSCs and in the Lhx2-hematopoietic progenitor cell (Lhx2-HPC) line. We demonstrate a remarkable likeness between these two cell types with regard to expression of the majority of receptors and Smads necessary for the transduction of signals from TGF-beta, BMP, and Activin. We have also evaluated the response of these two cell types to various ligands in proliferation assays. In this regard, primary cells and the Lhx2-HPC line behave similarly, revealing a suppressive effect of Activin-A that is similar to that of TGF-beta in bulk cultures and no effect of BMP-4 on proliferation. Signaling studies that verify the phosphorylation of Smad2 (Activin and TGF-beta) and Smad1/5 (BMP) confirm cytosolic responses to these ligands. In addition to providing a thorough characterization of TGF-beta superfamily expression in HSCs, our results define the Lhx2-HPC line as an appropriate model for molecular characterization of Smad signaling.
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Células Madre Hematopoyéticas/metabolismo , Proteínas Smad/metabolismo , Activinas/metabolismo , Activinas/farmacología , Animales , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Subunidades beta de Inhibinas/metabolismo , Subunidades beta de Inhibinas/farmacología , Ligandos , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Smad/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1RESUMEN
The Smad-signaling pathway downstream of the transforming growth factor-beta superfamily of ligands is an evolutionarily conserved signaling circuitry with critical functions in a wide variety of biologic processes. To investigate the role of this pathway in the regulation of hematopoietic stem cells (HSCs), we have blocked Smad signaling by retroviral gene transfer of the inhibitory Smad7 to murine HSCs. We report here that the self-renewal capacity of HSCs is promoted in vivo upon blocking of the entire Smad pathway, as shown by both primary and secondary bone marrow (BM) transplantations. Importantly, HSCs overexpressing Smad7 have an unperturbed differentiation capacity as evidenced by normal contribution to both lymphoid and myeloid cell lineages, suggesting that the Smad pathway regulates self-renewal independently of differentiation. Moreover, phosphorylation of Smads was inhibited in response to ligand stimulation in BM cells, thus verifying impairment of the Smad-signaling cascade in Smad7-overexpressing cells. Taken together, these data reveal an important and previously unappreciated role for the Smad-signaling pathway in the regulation of self-renewal of HSCs in vivo.
Asunto(s)
Diferenciación Celular/fisiología , Proliferación Celular , Células Progenitoras Mieloides/fisiología , Transducción de Señal/fisiología , Proteína smad7/metabolismo , Animales , Células Cultivadas , Expresión Génica , Ligandos , Ratones , Células Progenitoras Mieloides/citología , Fosforilación , Procesamiento Proteico-Postraduccional/fisiología , Proteína smad7/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. No models exist for RPS19-deficient DBA and the molecular pathogenesis is unknown. To establish an in vitro inducible model for DBA, human erythroid leukemic cell lines, TF-1 and UT-7 cells, were cotransduced with a lentiviral vector expressing the green fluorescent protein (GFP) gene and small interfering RNA (siRNA) against RPS19 controlled by a tet operator regulatory element and another transactivator vector containing the red fluorescent protein (RFP) gene and the cDNA encoding a tetracycline-controllable transcriptional repressor. Following transduction, the RFP-positive and GFP-negative cell population was sorted by flow cytometry. Upon incubation with doxycycline (0.5 mug/ml), more than 98% of cells expressed GFP and the siRNA. Significant suppression of erythroid differentiation, cell growth, and colony formation was observed in cells treated with siRNA against RPS19 but not in cells treated with a control vector. These findings show that RPS19 plays an important role in the regulation of hematopoietic cell proliferation and erythroid differentiation. These novel cell lines represent models for RPS19-deficient DBA and can be used to identify the molecular mechanisms in RPS19-deficient DBA.