RESUMEN
BACKGROUND: Continuous infusion (CI) of recombinant FVIII (rFVIII) concentrates has been reported as an effective and safe method to achieve haemostasis during major surgeries or severe bleeding events. For more effective and safer CI, better understanding of in vivo recovery (IVR) and clearance (CL) issues is imperative. OBJECTIVE: We investigated the following factors affecting IVR and CL using univariate and multivariate regression analyses during 47 CIs in 34 patients: rFVIII concentrate type, haemophilia severity, blood type, the presence of hepatitis C virus (HCV) or human immunodeficiency virus (HIV), age and body mass index (BMI). RESULTS: The mean IVR was 1.64 ± 0.49 IU dL-1 per IU kg-1 , and the mean CL during CI was 3.56 ± 1.57 mL h-1 kg-1 . The univariate and multivariate regression analyses showed that the CL of octocog alfa was significantly lower than that of rurioctocog alfa (P = 0.043 and 0.0034, respectively). There was a significant difference in BMI in the univariate and multivariate regression analyses (P = 0.0403 and 0.0376, respectively). CONCLUSIONS: This study indicated that CL during CI was potentially affected by the type of rFVIII concentrate used and BMI.
Asunto(s)
Factor VIII/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Factor VIII/farmacocinética , Humanos , Bombas de Infusión , Persona de Mediana Edad , Proteínas Recombinantes/farmacocinética , Estudios Retrospectivos , Adulto JovenRESUMEN
Fms-like tyrosine kinase 3 (FLT3) is expressed in hematopoietic progenitor cells. An internal tandem duplication (ITD) of FLT3 (FLT3/ITD) is the most frequent mutation in human adult acute myeloid leukemia (AML). FLT3/ITD contributes to the constitutive activation of FLT3 itself and its downstream signal components, mitogen-activated protein kinase and signal transducers and activators of transcription 5 (STAT5), and enables interleukin (IL)-3-dependent cell lines to grow autonomously. In the present study, we showed the specific association of FLT3/ITD with Lyn, which led to the phosphorylation of Lyn in vivo. We also demonstrated that FLT3/ITD receptors displayed a higher affinity to bind to Lyn than wild-type FLT3 receptors in vitro and that this affinity was relative to the intensity of tyrosil phosphorylation of the receptor. Both treatment with small interfering RNA (siRNA) targeting Lyn and the Src family kinase inhibitor PP2 suppressed the IL-3-independent growth of FLT3/ITD-expressing 32D cells (FLT3/ITD-32D), reducing the constitutive phosphorylation of Lyn and STAT5. PP2 treatment of mice transplanted with FLT3/ITD-32D cells blocked the onset of tumors and decreased the size of established tumors. These results demonstrate that Lyn is an important component of the signal transduction pathway specific to FLT3/ITD and can be a therapeutic target in the treatment of AML with FLT3/ITD.
Asunto(s)
Leucemia Mieloide/enzimología , Proteínas Mutantes/metabolismo , Proteínas de Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/metabolismo , Familia-src Quinasas/metabolismo , Enfermedad Aguda , Secuencia de Aminoácidos , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular/efectos de los fármacos , Línea Celular/trasplante , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/genética , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/genética , Proteínas de Neoplasias/genética , Fosforilación , Fosfotirosina/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión/fisiología , Factor de Transcripción STAT5/metabolismo , Secuencias Repetidas en Tándem , Transfección , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/genética , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
The cellular components of the hematopoietic stem cell niche have been gradually identified. However, the niche for malignant hematopoiesis remains to be elucidated. Here, using human leukemia cells, which could be transplanted to immunodeficient mice, we studied the in vivo homing, proliferation and survival sites by immunohistopathology, compared with the corresponding sites for cord blood CD34(+) (CBCD34(+)) cells. The human leukemia cells initially localized on the surface of osteoblasts in the epiphysial region, and expanded to the inner vascular and diaphysial regions within 4 weeks. The percentage of CD34(+) leukemia cells in the bone marrow was transiently increased up to 50%. In vivo 5-bromo-2'-deoxyuridine labeling revealed that the epiphysis was the most active site for leukemia cell proliferation. CBCD34(+) cells showed the similar pattern of homing and proliferation to leukemia cells. After high-dose administration of cytosine-1-beta-D-arabinofuranoside, residual leukemia cells were localized in the perivascular endothelium as well as in contact with the trabecular endosteum. These findings suggest that xenotransplantation into immunodeficient mice provides a useful model to study the leukemia niche.
Asunto(s)
Leucemia/patología , Animales , Antígenos CD34 , Arabinonucleósidos/farmacología , Bromodesoxiuridina , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Trasplante de Células Madre de Sangre del Cordón Umbilical , Células Madre Hematopoyéticas/patología , Humanos , Antígenos Comunes de Leucocito , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Células Tumorales CultivadasRESUMEN
High-dose methotrexate (Hd-MTX) therapy has recently been applied to the treatment of adult acute lymphoblastic leukemia (ALL) based on pediatric protocols; however, its effectiveness for adult ALL has not yet been confirmed in a rigorous manner. We herein conducted a randomized phase III trial comparing Hd-MTX therapy with intermediate-dose (Id)-MTX therapy. This study was registered at UMIN-CTR (ID: C000000063). Philadelphia chromosome (Ph)-negative ALL patients aged between 25 and 64 years of age were enrolled. Patients who achieved complete remission (CR) were randomly assigned to receive therapy containing Hd-MTX (3 g/m2) or Id-MTX (0.5 g/m2). A total of 360 patients were enrolled. The CR rate was 86%. A total of 115 and 114 patients were assigned to the Hd-MTX and Id-MTX groups, respectively. The estimated 5-year disease-free survival rate of the Hd-MTX group was 58%, which was significantly better than that of the Id-MTX group at 32% (P=0.0218). The frequencies of severe adverse events were not significantly different. We herein demonstrated the effectiveness and safety of Hd-MTX therapy for adult Ph-negative ALL. Our results provide a strong rationale for protocols containing Hd-MTX therapy being applied to the treatment of adult ALL.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Biomarcadores de Tumor , Esquema de Medicación , Femenino , Humanos , Masculino , Metotrexato/administración & dosificación , Persona de Mediana Edad , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inducción de Remisión , Análisis de Supervivencia , Resultado del Tratamiento , Adulto JovenRESUMEN
Sequence analysis of the immunoglobulin heavy chain complementarity determining region (CDR)-III of B-lineage cells at various stages has provided important insights concerning B cell maturation and selection. Knowledge of human CDR-III sequences has been relatively limited compared with that of the murine system. We analyzed the CDR-III sequences of B cell precursor acute lymphoblastic leukemia (pre-B ALL) cells in 23 newly diagnosed and 10 relapsed patients, in order to elucidate the organization of CDR-III in B cell precursors. We found a very low frequency of somatic mutations in D and JH regions, preferential use of DLR, DXP, DHQ52, and DN elements, and of 3' side JH segments, and no predominant usage of D coding frames. Unusual joinings such as VH-D-D-JH and VH-JH were observed in three, and one sequences, respectively. We compared the CDR-III sequences derived from 10 patients between diagnosis and relapse. Two of them had three spots of mutated nucleotides at relapse, all of which were found in the N region near the D segments. Our data showed the possibility of somatic mutation at relapse, in addition to developmentally regulated rearrangement of the immunoglobulin gene at the stage of B cell precursors.
Asunto(s)
Linfoma de Burkitt/inmunología , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Células Madre Neoplásicas/inmunología , Adolescente , Adulto , Secuencia de Bases , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , RecurrenciaRESUMEN
Secondary failure of platelet recovery (SFPR), which is a delayed decline in platelet count after primary recovery following myeloablative hematopoietic SCT, is a significant problem in allogeneic SCT. However, its clinical characteristics have not been well described in autologous SCT for acute myeloid leukemia. We reviewed 11 consecutive patients who had received autologous or syngeneic SCT for acute promyelocytic leukemia. Seven of 11 patients (64%) had SFPR, which is defined as a decline in the platelet count to less than 30,000/microl for more than 7 days. The median onset of SFPR was day 36 (range, 25-51 days) and the median duration of thrombocytopenia was 13 days (range, 4-25 days). Of nine patients who received busulfan-containing preparative regimens, seven (78%) had SFPR and one had delayed primary platelet count recovery. Neither patient who received cyclophosphamide and total body irradiation as preparative regimens had SFPR. The clinical courses of SFPR were transient and self-limited. SFPR was not associated with relapse of underlying diseases, graft failure or other fatal morbidities. The unexpectedly high prevalence and the characteristics of SFPR may provide additional information on management following autologous SCT for acute myeloid leukemia.
Asunto(s)
Leucemia Promielocítica Aguda/cirugía , Trasplante de Células Madre de Sangre Periférica/efectos adversos , Trombocitopenia/etiología , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Recuento de Plaquetas , Trasplante Autólogo , Trasplante IsogénicoRESUMEN
Although tyrosine kinase inhibitors (TKIs) have significantly improved the prognosis of chronic myeloid leukemia (CML), the ability of TKIs to eradicate CML remains uncertain and patients must continue TKI therapy for indefinite periods. In this study, we performed whole-exome sequencing to identify somatic mutations in 24 patients with newly diagnosed chronic phase CML who were registered in the JALSG CML212 study. We identified 191 somatic mutations other than the BCR-ABL1 fusion gene (median 8, range 1-17). Age, hemoglobin concentration and white blood cell counts were correlated with the number of mutations. Patients with mutations ⩾6 showed higher rate of achieving major molecular response than those<6 (P=0.0381). Mutations in epigenetic regulator, ASXL1, TET2, TET3, KDM1A and MSH6 were found in 25% of patients. TET2 or TET3, AKT1 and RUNX1 were mutated in one patient each. ASXL1 was mutated within exon 12 in three cases. Mutated genes were significantly enriched with cell signaling and cell division pathways. Furthermore, DNA copy number analysis showed that 2 of 24 patients had uniparental disomy of chromosome 1p or 3q, which disappeared major molecular response was achieved. These mutations may play significant roles in CML pathogenesis in addition to the strong driver mutation BCR-ABL1.
Asunto(s)
Proteínas de Unión al ADN/genética , Dioxigenasas/genética , Histona Demetilasas/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Factores de Edad , Variaciones en el Número de Copia de ADN/genética , Resistencia a Antineoplásicos/genética , Epigénesis Genética/genética , Femenino , Proteínas de Fusión bcr-abl/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Recuento de Leucocitos , Masculino , Mutación , Inhibidores de Proteínas Quinasas/administración & dosificación , Transducción de Señal , Secuenciación del ExomaRESUMEN
Two distinct forms of fms-like tyrosine kinase (FLT3) gene aberrations, internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations, have been recognized in a substantial proportion of patients with acute myeloid leukemia (AML). To investigate their prognostic significance, we performed a meta-analysis of the four published studies that provided survival information according to the FLT3 status: ITD, TKD mutation, and wild type. The summary hazard ratios for disease-free survival (DFS) were 1.88 (95% confidence interval (CI) 1.58-2.23; P<0.001) for FLT3 mutations, 1.86 (95% CI: 1.52-2.29; P<0.001) for ITD, and 1.90 (95% CI: 1.40-2.60; P<0.001) for TKD mutation. The corresponding ratios for overall survival were 1.61 (95% CI: 1.37-1.89; P<0.001), 1.68 (95% CI: 1.39-2.03; P<0.001), and 1.37 (95% CI: 0.94-2.01; P=0.104). Neither white blood cell count at diagnosis nor cytogenetic risk category was a significant source of heterogeneity. These findings indicate that FLT3 mutations have an adverse effect on the outcome for AML, and that the negative impact of TKD mutation seems comparable to that of ITD with regard to DFS. Although it should be borne in mind that this meta-analysis was based on data abstracted from observational studies, these results may justify the risk-adapted therapeutic strategies for AML according to the FLT3 status.
Asunto(s)
Leucemia Mieloide/genética , Mutación , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Secuencias Repetidas en Tándem , Enfermedad Aguda , Supervivencia sin Enfermedad , Humanos , Leucemia Mieloide/mortalidad , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Proto-Oncogénicas/química , Proteínas Tirosina Quinasas Receptoras/química , Tasa de Supervivencia , Tirosina Quinasa 3 Similar a fmsRESUMEN
To clarify the role of fragile histidine triad (FHIT) in hematological malignancies, we examined the methylation status and the expression level of the FHIT gene in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cells in comparison with the methylation of the p15(INK4B) gene. The FHIT methylation was found in 13 of 94 (13.8%) AML and 22 of 40 (55.0%) MDS cases, but not in normal mononuclear cells (MNCs). Both the frequency and density of methylation increased in the advanced-stages MDS and the relapsed AML cases. Although FHIT and p15(INK4B) methylations were not correlated in MDS and AML, increased FHIT methylation at the relapse in AML was associated with p15(INK4B) methylation. The median expression level in AML was significantly higher than in normal MNCs, although the median expression level in those with methylation was significantly lower than in those without methylation. Furthermore, the methylation level at relapse was significantly higher than at diagnosis in AML. These results suggested that FHIT methylation was accumulated through the disease progression of MDS and AML, and the role of the FHIT gene as a tumor suppressor seemed different in AML and MDS.
Asunto(s)
Ácido Anhídrido Hidrolasas/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Leucemia Mieloide/genética , Síndromes Mielodisplásicos/genética , Proteínas de Neoplasias/genética , Enfermedad Aguda , Azacitidina/análogos & derivados , Azacitidina/farmacología , Médula Ósea/patología , Proteínas de Ciclo Celular/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Decitabina , Genes Supresores de Tumor , Humanos , Leucemia Mieloide/etiología , Leucemia Mieloide/patología , Síndromes Mielodisplásicos/etiología , Síndromes Mielodisplásicos/patología , ARN Mensajero/análisis , Recurrencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
Intravascular large B-cell lymphoma (IVLBCL) is a distinct disease entity with the peculiar characteristic that tumor cells proliferate within vessels. Despite recent advances in understanding the disease from clinical aspects, the underlying pathogenesis remains unknown. Here we demonstrate analyses of IVLBCL biology using four xenograft mouse models established from primary IVLBCL samples. In all four models, the main characteristic of IVLBCL tumor cell proliferation within vessels was retained. Time-lapse engraftment analyses revealed that the tumor cells initially engrafted and proliferated in the sinusoids and vessels in the liver and then engrafted and proliferated in multiple organs. Intriguingly, serial passage of tumor cells from the adrenal gland of a transplanted mouse developed from primary patient bone marrow cells into a second mouse showed that the tumor cells mainly distributed into the adrenal gland in the second mouse, implying the existence of clonal selection and/or evolution at engraftment of a specific organ. Gene expression profiling analyses demonstrated that the gene set associated with cell migration was enriched for normal peripheral blood B cells, indicating that inhibition of cell migration might be involved in IVLBCL pathogenesis. In conclusion, the mouse xenograft models described here are essential tools for uncovering IVLBCL biology.
Asunto(s)
Xenoinjertos/patología , Linfoma de Células B Grandes Difuso/patología , Neoplasias Vasculares/patología , Anciano , Animales , Movimiento Celular , Proliferación Celular , Femenino , Supervivencia de Injerto , Humanos , Cadenas Pesadas de Inmunoglobulina/análisis , Hígado/irrigación sanguínea , Masculino , Ratones , Persona de Mediana Edad , Especificidad de ÓrganosRESUMEN
We have recently identified an internal tandem duplication of the human Flt3 gene in approximately 20% of acute myeloid leukemia (AML) cases. In the present study, the wild-type and the mutant Flt3 genes were transfected into two IL-3-dependent cell lines, 32D and BA/F3 cells. Mutant Flt3-transfected cells exhibited autonomous growth while wild-type Flt3-transfected cells with the continuous stimulation of Flt3 ligand exhibited a minimal proliferation. Cells expressing mutant Flt3 showed constitutive activation of STAT5 and MAP kinase. In contrast, Flt3 ligand stimulation caused rapid activation of MAP kinase but not STAT5 in cells expressing wild-type Flt3. Finally, we found constitutive activation of MAP kinase and STAT5 in all clinical samples of AML patients with mutant Flt3. Our study shows the significance of internal tandem duplication of Flt3 receptors for leukemia cell expansion.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Duplicación de Gen , Interleucina-3/fisiología , Proteínas de la Leche , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transactivadores/metabolismo , Secuencia de Aminoácidos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Muerte Celular/efectos de los fármacos , División Celular/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Inhibidores de Crecimiento/farmacología , Humanos , Leucemia Eritroblástica Aguda/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Datos de Secuencia Molecular , Mutación/genética , Fosforilación , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Secuencias Repetitivas de Aminoácido , Factor de Transcripción STAT5 , Células Tumorales Cultivadas , Tirosina Quinasa 3 Similar a fmsRESUMEN
To characterize multiple myeloma (MM) from the viewpoint of the immunoglobulin (Ig) gene structure, we compared the transcripts of the Ig heavy chain variable region from 23 MM samples with 221 clones of the gamma, alpha and mu chain transcripts amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) from normal bone marrow (BM) cells. The usage of D and JH gene segments and the length of the N regions were the same between MM and the normal gamma, alpha and mu transcripts. Compared with the known germline VH genes, the frame work regions (FWRs) and complementarity determining regions (CDRs) of the VH segments mutated at rates of 8.3 +/- 4.7% and 15.9 +/- 7.7%, respectively, which were the same as the normal gamma and alpha (gamma/alpha) transcripts and higher than the normal mu transcripts. The replacement/silent (R/S) ratios of the mutations in FWRs and CDRs were 1.9 +/- 1.3 and 2.7 +/- 1.8, respectively, which were the same as the gamma/alpha and mu transcripts. On the other hand, we detected the clone-specific mu transcripts by RT-PCR using the primers corresponding to the each respective CDR-III and the constant region of the mu chain in three of the studied six MM samples, suggesting the involvement of a pre-switched B cell in some cases of MM. These findings suggested that the cellular origin of MM is heterogeneous, but that the Ig structure in MM reflects normal B cell maturation to plasma cell through mutation and selection.
Asunto(s)
Médula Ósea/inmunología , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Linfocitos/inmunología , Mieloma Múltiple/genética , Análisis de Secuencia de ADN , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Secuencia de Bases , Células de la Médula Ósea , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mieloma Múltiple/inmunología , Alineación de SecuenciaRESUMEN
Immature B lineage acute lymphoblastic leukemia (ALL) is divided into two subtypes, 'pre-B' and 'early pre-B' ALL, by the presence or absence of cytoplasmic immunoglobulin (cIg). To study their clonal origin, we compared mu-chain transcripts in six cIg+ and eight cIg- ALL samples (CD10+/- CD19+ surface Ig-) with those in the normal phenotypic counterparts (CD10+ CD19+ surface Ig-) sorted from the bone marrow (BM). Northern blot analysis showed that the cIg+ ALL samples expressed greater amounts of mu-chain transcripts than the cIg- ALL samples. In the ALL samples and their counterparts, sequence analysis of mu-chain transcripts revealed infrequent somatic mutations of the V(H) genes and the similar usage of D and J(H) gene segments, but the length of complementarity determining region (CDR)-3 in the ALL samples was longer than that in the counterparts (50.0 +/- 15.5 vs 40.8 +/- 12.7 bp, P = 0.01). The mu-chain transcripts in the six cIg+ ALL samples and the counterparts (119/120 clones) had productive sequences, whereas those in the eight cIg- ALL samples had nonsense codons and/or frame shifts in their CDR-3. Our data suggest that a phenotype of ALL, 'pre-B' or 'early pre-B', is associated with V(H)-D-J(H) gene recombinatorial events, and that the CD10+ CD19+ surface Ig- population in the BM is not simply the cellular origin of ALL.
Asunto(s)
Linfocitos B/fisiología , Médula Ósea/fisiología , Linfoma de Burkitt/genética , Genes de Inmunoglobulinas , Hematopoyesis , Antígenos CD19/análisis , Secuencia de Bases , Separación Celular , Niño , Preescolar , Femenino , Citometría de Flujo , Reordenamiento Génico de Cadena Pesada de Linfocito B , Humanos , Región Variable de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/genética , Masculino , Datos de Secuencia Molecular , Neprilisina/análisis , ARN Mensajero/genética , ARN Neoplásico/genética , Transcripción GenéticaRESUMEN
We studied the organization, diversification and clinical significance of the immunoglobulin light chain (IgL) variable region genes expressed in 17 kappa-chain and 16 lambda-chain producing multiple myeloma (MM) samples. The V genes from 31 MM samples had over 84.9% homology to the known germline Vkappa/lambda genes, whereas one Vkappa and one Vlambda gene had only 75.5% and 65.9% homology, respectively. While all five Jkappa segments were equally used, only Jlambda-1 or Jlambda-2/3 was used among seven Jlambda segments. N nucleotide addition was found at two Vkappa-Jkappa and five Vlambda-Jlambda junctions. The lambda-chain complementarity determining region (CDR)-3 was longer and more variable than the kappa-chain CDR-3 mainly due to junctional flexibility of Vlambda and Jlambda segments. Somatic mutations were more frequent in the Jlambda than the Jkappa segments, and were distributed in the CDR-3 as well as the frame work region (FWR)-4. Those of the Jkappa segments, however, were limited to FWR-4. In FWR-4, replacement mutations were clustered at codon 106 of kappa-chain and 103 of lambda-chain. Thus nucleotide mutation or conservation was dependent on position, indicating a structural necessity of IgL for the development of myeloma cells in addition to a non-random distribution of mutations. There was no characteristic IgL sequence according to the isotype of M-protein, clinical stage or renal complication.
Asunto(s)
Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Secuencia de Bases , Femenino , Expresión Génica , Reordenamiento Génico de Cadena Ligera de Linfocito B , Genes de Inmunoglobulinas/genética , Humanos , Región de Unión de la Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/biosíntesis , Cadenas kappa de Inmunoglobulina/biosíntesis , Cadenas kappa de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/biosíntesis , Cadenas lambda de Inmunoglobulina/genética , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Transcripción GenéticaRESUMEN
All-trans retinoic acid (ATRA) induces complete remission in patients with acute promyelocytic leukemia (APL). However, ATRA sometimes causes retinoic acid syndrome (RAS) characterized by respiratory distress, pleural effusions, fever and weight gain. To investigate the pathophysiology of RAS, we generated an animal model by injecting an APL cell line, NB4, into immunodeficient mice. When NOD/scid mice were injected intravenously with fully differentiated NB4 cells (1 x 10(7)) and then given a daily administration of ATRA, three of 12 mice died of pulmonary edema within 14 days. Pathologically, dilated lung capillary vessels and alveolar effusions were observed. After the injection, NB4 cells were detected in the lung within 2 days and in the pleural effusion later on. The gene expression levels of CXC chemokines (MIP-2 and KC) and ICAM-1 were increased in the lung and heart by the ATRA administration. In immunohistochemical analyses, MIP-2 was clearly detected in alveolar macrophages of the lung in mice with RAS. Dexamethasone treatment prevented the development of RAS and decreased the CXC chemokine mRNA expression in the lung. These findings suggested that the activation of adhesion molecules for leukocytes and expression of CXC chemokines in the lung are closely involved in triggering RAS.
Asunto(s)
Antineoplásicos/efectos adversos , Leucemia Promielocítica Aguda/patología , Tretinoina/efectos adversos , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Doxorrubicina/uso terapéutico , Corazón/efectos de los fármacos , Corazón/fisiología , Humanos , Inyecciones Intravenosas , Leucemia Promielocítica Aguda/tratamiento farmacológico , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/efectos de los fármacos , Edema Pulmonar/etiología , Inducción de Remisión , Síndrome , Tretinoina/administración & dosificación , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
Arsenic trioxide (As2O3)-treatment is effective in acute promyelocytic leukemia (APL) patients with t(15;17). Clinically achievable concentrations of As2O3 induce apoptosis in NB4, an APL cell line, in vitro. Here, to study the mechanism of As2O3-induced apoptosis, we established an As2O3-resistant subline, NB4/As. Growth of NB4/As was inhibited by 50% after 2 day-treatment (IC50) at 1.6 microM As2O3, whereas IC50 of NB4 was 0.3 microM. Degradation of PML-RARalpha and change of the PML-subcellular localization were similarly induced by As2O3 in NB4 and NB4/As, suggesting that their contribution to apoptosis is small. Treatment with 1 microM As2O3 induced the activation of caspase 3 as well as a loss of mitochondrial transmembrane potential (deltapsim) in NB4 but not in NB4/As. Caspase 8 and Bid were also activated by As2O3 in NB4 but not in NB4/As. In NB4, an inhibitor of caspase 8 blocked not only the activation of caspase 3 but also the loss of deltapsim. Neither cell line expressed CD95/Fas, and agonistic anti-Fas antibody (CH-11) failed to cause apoptosis. Neither antagonistic anti-CD95/Fas antibody nor anti-Fas ligand antibodies influenced the As2O3-induced apoptosis. NB4/As had a higher concentration of intracellular glutathione (GSH) than NB4 (96 vs 32 nmol/mg). Reduction of the GSH level by buthionine sulfoxide (BSO) completely restored the sensitivity to As2O3 in NB4/As. Furthermore, caspase activation and the loss of deltapsim were recovered by combination treatment with BSO. These findings suggest that the As2O3 treatment activates caspase 8 in a CD95-independent but GSH concentration-dependent manner. In combination with BSO, As2O3 might be applied to therapy of leukemia/cancers which are insensitive to the clinically achievable concentrations of As2O3.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Caspasas/metabolismo , Leucemia Promielocítica Aguda/patología , Óxidos/farmacología , Antineoplásicos/uso terapéutico , Trióxido de Arsénico , Arsenicales/uso terapéutico , Caspasa 8 , Caspasa 9 , Activación Enzimática , Glutatión/metabolismo , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Óxidos/uso terapéutico , Células Tumorales Cultivadas , Receptor fas/inmunologíaRESUMEN
An internal tandem duplication of the juxtamembrane (JM) domain of FLT3, a family of ligand-activated receptor tyrosine kinases, has been found in 20% of cases of acute myeloid leukemia (AML), and this mutation is correlated with leukocytosis and a poor prognosis. As a therapeutic approach, we previously reported that herbimycin A (HA) inhibited the growth of tandemly duplicated FLT3 (TDFLT3)-transformed cells (Leukemia 2000; 14: 374). Here, we have investigated the mechanism behind the cytotoxicity of HA, an ansamycin derivative which is now known to target Hsp90. The treatment with HA or another Hsp90 inhibitor, radicicol, induced selective apoptosis in TDFLT3-transformed 32D cells (TDFLT3/32D). The tyrosine-phosphorylation of TDFLT3 was inhibited by HA, whereas FLT3 ligand-induced phosphorylation of wild-type FLT3 (WtFLT3) was not. The downstream signal molecules MAPK, Akt and STAT5a were also dephosphorylated by HA in TDFLT3/32D. Immunoprecipitation analysis showed that TDFLT3 but not WtFLT3 formed a complex with Hsp90, and that the HA treatment dissociated TDFLT3 from the Hsp90 chaperone complex. These findings imply that targeting of Hsp90 will facilitate the development of anti-TDFLT3 therapy, and that Hsp90 is closely involved in the oncogenic activation of FLT3.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactonas/farmacología , Proteínas de la Leche , Proteínas de Neoplasias/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/fisiología , Quinonas/farmacología , Proteínas Tirosina Quinasas Receptoras/fisiología , Animales , Benzoquinonas , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/metabolismo , Proteínas HSP90 de Choque Térmico/química , Interleucina-3/farmacología , Lactamas Macrocíclicas , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrólidos , Sustancias Macromoleculares , Ratones , Células Mieloides/efectos de los fármacos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fosforilación , Pliegue de Proteína , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/genética , Rifabutina/análogos & derivados , Factor de Transcripción STAT5 , Secuencias Repetidas en Tándem , Transactivadores/metabolismo , Tirosina Quinasa 3 Similar a fmsRESUMEN
To study the molecular mechanism of the differentiation induced by retinoic acid (RA) in acute promyelocytic leukemia (APL), we established a new RA-resistant NB4 subline, NB4/RA. The NB4/RA cells were neither differentiated by a single or a combination of RA isoforms, nor by the addition of clotrimazole (P450-inhibitor) or interferon gamma. However, the combination of RA and 8-(4-chlorophenylthio) adenosine cyclic 3',5'-monophosphate (a cAMP analog, 8-CPT-cAMP) induced differentiation. Immunostaining of NB4/RA cells using anti-PML antibody showed a microgranular pattern which was not restored even by the combination of RA and 8-CPT-cAMP, whereas the microgranular pattern in NB4 cells was rapidly restored to the normal speckled pattern by RA. Western blot analysis revealed that RA alone or the combination with 8-CPT-cAMP did not down-regulate PML-RARalpha in NB4/RA cells, which was in contrast to NB4 cells. The PML-RARalpha fusion gene and transcript in NB4/RA cells were conserved as well as the RARalpha gene and transcripts. Sequence analysis of the PML-RARalpha transcript in NB4/RA cells indicated a Pro (CCG) to Leu (CTG) mutation at codon 900 (type L) in AF-2 domain, while the RARalpha transcript had a normal sequence. These data suggest that differentiation of APL by RA is triggered directly through PML-RARalpha, and is associated with its degradation. Furthermore, there might be another mechanism of differentiation which does not require the down-regulation of PML-RARalpha and the restoration of the PML-staining pattern.
Asunto(s)
Antígenos CD/genética , Diferenciación Celular/efectos de los fármacos , Leucemia Promielocítica Aguda/genética , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Tretinoina/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Técnicas de Cultivo de Célula , Regulación hacia Abajo , Resistencia a Antineoplásicos , Humanos , Immunoblotting , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Datos de Secuencia Molecular , Mutación Puntual , Receptores de Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Análisis de Secuencia de ADN , Tretinoina/metabolismo , Células Tumorales CultivadasRESUMEN
The development of therapy-related acute myeloid leukemia (t-AML) has become a growing concern over the past decade, because of the increase in the percentage of long-term survivors of primary malignancy. We reviewed 17 cases with etoposide-related acute promyelocytic leukemia (APL) reported in the literature. The close association between treatment with etoposide for Langerhans cell histiocytosis (LCH) and the development of etoposide-related APL was demonstrated among Japanese and Italians. Our data on the breakpoints (b/ps) of the PML and RARalpha genes are presented. It is suggested that chromatin structure might be more important than specific consensus sequence in the distribution of b/ps in etoposide-related APL.
Asunto(s)
Antineoplásicos Fitogénicos/efectos adversos , Etopósido/efectos adversos , Leucemia Promielocítica Aguda/etiología , Proteínas de Neoplasias/genética , Neoplasias Primarias Secundarias , Proteínas de Fusión Oncogénica/genética , Adulto , Anciano , Secuencia de Bases , Niño , Preescolar , Cromosomas Humanos Par 15 , Cromosomas Humanos Par 17 , Femenino , Humanos , Lactante , Leucemia Promielocítica Aguda/genética , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Neoplasias Primarias Secundarias/genética , Inhibidores de Topoisomerasa IIRESUMEN
We sequentially analyzed the immunoglobulin heavy chain variable (IgH V) region gene of leukemia cells obtained from a chronic myeloid leukemia (CML) patient who had three episodes of B-lymphoid crisis after bone marrow transplantation. Southern blot analysis using the JH probe showed different rearranged bands at each crisis, although the same rearranged bands of the BCR gene were observed. We amplified and sequenced the IgH V region gene of the leukemia cells by reverse transcriptase polymerase chain reaction (RT-PCR) using the primers corresponding to the consensus 5'VH and mu constant regions. The dominant leukemia clone at each crisis had a unique VH-D-JH rearrangement; VH4A (V79)-DLR2-J5 (clone-1), VH4B (DP70)-DK4-J6 (clone-2) and VH4A (V79)-DN4-J6 (clone-3) at the first, second and third crises, respectively. Further analysis by PCR amplification using the consensus 5'VH and clone-specific primers revealed that clone-1 underwent VH4-->VH3 replacement at the second crisis, and that clone-3 was already in existence at the first crisis. Moreover, the DN4-J6 joining clone, in which the sequence was the same as that of clone-3, was identified at the first and third crises by PCR amplification using primers corresponding to the region upstream of the DN4 segment and DN4-J6 boundary of clone-3. These observations suggest that multiple clones were generated from the progenitor cells of blast crisis, which were transformed at a very early stage of B-lymphocyte ontogeny, by continuing rearrangement mechanisms of the IgH genes, and that the dominant clone at each crisis was undergoing change.