RESUMEN
Infant acute lymphoblastic leukemia (ALL) is frequently characterized by the t(4;11)(q21;q23) cytogenetic abnormality encoding the MLL/AF4 oncogene, increased HOX gene expression and a pro-B/monocytoid phenotype. We have previously established a novel MLL/AF4-positive cell line, B-lineage 3 (BLIN-3), which retains several features of normal B-lineage development (functional Ig gene rearrangement and apoptotic sensitivity to stromal cell withdrawal) not generally observed in infant ALL. We now use microarray analysis to identify patterns of gene expression in BLIN-3 that may modulate MLL/AF4 oncogenesis and contribute to the retention of normal B-lineage developmental characteristics. Comparison of 6815 expressed genes in BLIN-3 with published microarray data on leukemic blasts from t(4;11) patients indicated that BLIN-3 was unique in lacking the expression of certain HOX-A cluster genes. These results were validated by RT-PCR showing no expression of HOX A7 or HOX A9 in BLIN-3. A HOX C8 promoter reporter was active in BLIN-3, indicating that lack of HOX gene expression in BLIN-3 was not due to a nonfunctional MLL/AF4. Our results suggest that B-lineage development can proceed in t(4;11) leukemic blasts in the absence of HOX-A gene expression.
Asunto(s)
Linfocitos B/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Homeodominio/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proto-Oncogenes , Factores de Transcripción , Linfocitos B/patología , Línea Celular Tumoral , Linaje de la Célula , Regulación Leucémica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina , Humanos , Lactante , Proteína de la Leucemia Mieloide-Linfoide , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatología , Regiones Promotoras Genéticas , Factores de Elongación TranscripcionalRESUMEN
The most common chromosomal abnormality of infant acute lymphoblastic leukemia (ALL) is the t(4;11)(q21;q23) that gives rise to the MLL/AF4 fusion gene. Leukemic blasts expressing MLL/AF4 are arrested at an early progenitor stage with lymphoid or monocytoid characteristics. A novel B-lineage ALL cell line termed B-lineage-3 (BLIN-3) requiring human bone marrow (BM) stromal cell contact and interleukin-7 (IL-7) for optimal proliferation has been established. BLIN-3 cells have a CD19(+)/CD10(-) phenotype typical of infant ALL, and they harbor the t(4;11)(q21;q23) chromosomal translocation. Reverse transcription-polymerase chain reaction and Western blot analysis confirmed the presence of the MLL/AF4 fusion mRNA and protein in BLIN-3. Initial BLIN-3 cultures had a pro-B cell phenotype and did not express cytoplasmic or surface mu heavy chain. After approximately 5 months in culture on BM stromal cells plus IL-7, BLIN-3 sublines emerged expressing mu heavy chain and VpreB on the cell surfaces (ie, pre-B-cell receptor [BCR](+)). BLIN-3 cells expressing pre-BCR had the t(4;11)(q21;q23) translocation and expressed the MLL/AF4 fusion protein. Cross-linking the BLIN-3 pre-BCR led to enhanced cell proliferation, demonstrating that BLIN-3 expressed a functional pre-BCR. Increased acquisition of surface pre-BCR in BLIN-3 sublines was associated with loss of DJ rearrangements and the appearance of VDJ rearrangements. These results indicate that expression of the MLL/AF4 fusion protein is compatible with BM stromal cell and cytokine dependency, functional immunoglobulin gene segment rearrangement, and subsequent expression of a potentially diverse antigen receptor repertoire. Thus, the expression of MLL/AF4 is compatible with the normal developmental program of human B-lineage cells.
Asunto(s)
Linfocitos B/patología , Diferenciación Celular , Células Madre Hematopoyéticas/patología , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Anticuerpos Monoclonales/farmacología , Células de la Médula Ósea/fisiología , División Celular , Supervivencia Celular , Reactivos de Enlaces Cruzados , Femenino , Citometría de Flujo , Expresión Génica , Humanos , Cadenas Ligeras de Inmunoglobulina/análisis , Lactante , Interleucina-7/farmacología , Proteína de la Leucemia Mieloide-Linfoide , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/fisiología , Células Tumorales CultivadasRESUMEN
The acquisition of genetic abnormalities in human B-lineage acute lymphoblastic leukemia (ALL) culminates in the clonal expansion of bone marrow (BM)-derived leukemic blasts. However, the response of leukemic cells to signals transduced by the BM microenvironment is not completely understood. The present study describes a new human B-lineage ALL cell line designated BLIN-4 (B LINeage-4). BLIN-4 cells respond to multiple cytokines/human BM stromal cell-derived molecules. One subline (BLIN-4E) undergoes cell death in the absence of BM stromal cells or cytokines and slowly proliferates on human BM stromal cells supplemented with interleukin (IL)-7 + FLT3-ligand. Another subline (BLIN-4L) slowly proliferates in the absence of cytokines and BM stromal cells and shows robust proliferation on BM stromal cells supplemented with IL-7 + FLT3-ligand. Although human BM stromal cells are comparable with IL-7 + FLT3-ligand in supporting proliferation of BLIN-4L cells, neutralizing antibody experiments demonstrate that BLIN-4L expansion on BM stromal cells is IL-7/FLT3-ligand independent. BLIN-4L could also respond to human thymic stromal lymphopoietin. BLIN-4E and BLIN-4L have the identical immunoglobulin heavy chain rearrangement and a CD10(+)/CD19(+)/CD20(-)/CD22(+)/CD40(+)/mu heavy chain(-) phenotype. The original BM leukemic blasts harbored a ring chromosome 4 with a low percentage of cells also having either trisomy 8 or trisomy 18. The BLIN-4 sublines maintained the ring chromosome 4, but the trisomy 8 and trisomy 18 segregated into BLIN-4E and BLIN-4L, respectively. Thus, the BLIN-4 sublines exhibit biological characteristics consistent with a potential evolution in B-lineage ALL involving subclones with decreasing requirements on the BM microenvironment.
Asunto(s)
Células de la Médula Ósea/citología , Linfoma de Burkitt/patología , Citocinas/farmacología , Células Tumorales Cultivadas , Adolescente , Células de la Médula Ósea/metabolismo , Linfoma de Burkitt/genética , División Celular/efectos de los fármacos , Linaje de la Célula , Supervivencia Celular/efectos de los fármacos , Células Clonales , Medios de Cultivo Condicionados , Femenino , Humanos , Interleucina-7/farmacología , Proteínas de la Membrana/farmacología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptores de Interleucina-7/biosíntesis , Células del Estroma/metabolismo , Tirosina Quinasa 3 Similar a fms , Linfopoyetina del Estroma TímicoRESUMEN
Eukaryotic cell division is regulated by cyclins, cyclin-dependent kinases (CDK), and cyclin-dependent kinase inhibitors (CKI). Genes encoding these proteins are mutated or deleted in many types of cancer. For example, 20%-30% of B-lineage acute lymphoblastic leukemias (ALL) have deletions in the CKI known as INK4a. The contribution of INK4a deletions to the progression of B-lineage ALL is uncertain, partially due to a paucity of data on expression in normal B-cell precursors. We therefore conducted a comparative analysis of normal and leukemic human B-cell development for the expression of cyclins, CDK, and CKI. Specific stages of human B-cell development from normal bone marrow were purified by fluorescence-activated cell sorting. The sorted populations and B-lineage ALL cell lines (BLIN-1, 2, 3, 4) were examined for expression of cyclins, CDK, and CKI by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting.RT-PCR analysis showed that cyclin D2, cyclin D3, CDK4, and CDK6 were ubiquitously expressed in normal B-cell development and in the BLIN ALL cell lines. The p19(INK4d) CKI was the most commonly expressed member of the INK4 family, whereas p16(INK4a) was more weakly and variably expressed. Expression of the p57(KIP2) CKI varied as a function of the stage of B-cell development. Analysis of normal B-cell precursors by Western blotting indicated that CDK4, CDK6, p19(INK4d), and p57(KIP2) were expressed, whereas p16(INK4a) was not detected. Cyclin D/CDK expression in normal and leukemic human B-cell precursors is similar to expression of these proteins in human and murine mature B cells. In contrast, the ubiquitous expression of p19(INK4d) has not been previously described in human or murine B-lineage cells. Our results suggest that loss of INK4a may only minimally contribute to tumor cell progression in B-lineage ALL, since expression of INK4d could provide a compensatory function as a cyclin-dependent kinase inhibitor.
Asunto(s)
Linfocitos B/metabolismo , Células de la Médula Ósea/metabolismo , Proteínas de Ciclo Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos , Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Proteínas Proto-Oncogénicas , Proteínas de Saccharomyces cerevisiae , Antígenos CD19/análisis , Antígenos CD34/análisis , Proteínas Portadoras/genética , Células Cultivadas , Ciclina D2 , Ciclina D3 , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Inhibidor p19 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Embrión de Mamíferos , Proteínas Fúngicas/genética , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Motoras Moleculares , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Serina-Treonina Quinasas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Mammalian B-cell development can be viewed as a developmental performance with several acts. The acts are represented by checkpoints centered around commitment to the B-lineage and functional Ig gene rearrangement--culminating in expression of the pre-B-cell receptor (pre-BCR) and the BCR. Progression of cells through these checkpoints is profoundly influenced by the fetal liver and adult bone marrow (BM) stromal cell microenvironments. Our laboratory has developed a model of human B-cell development that utilizes freshly isolated/non-transformed human BM stromal cells as an in vitro microenvironment. Human CD34+ hematopoietic stem cells plated in this human BM stromal cell microenvironment commit to the B lineage and progress through the pre-BCR and BCR checkpoints. This human BM stromal cell microenvironment also provides survival signals that prevent apoptosis in human B-lineage cells. Human B-lineage cells exhibit differential expression of Notch receptors and human BM stromal cells express the Notch ligand Jagged-1. These results suggest a potential role for Notch in regulating B-lineage commitment and/or progression through the pre-BCR and BCR checkpoints.
Asunto(s)
Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Células del Estroma/inmunología , Apoptosis , Proteínas de Unión al Calcio , Diferenciación Celular , División Celular , Linaje de la Célula , Citocinas/fisiología , Desarrollo Embrionario y Fetal , Células Madre Hematopoyéticas/inmunología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Interleucina-7/fisiología , Proteína Jagged-1 , Proteínas de la Membrana/fisiología , Modelos Biológicos , Receptores Notch , Proteínas Serrate-Jagged , Transducción de Señal , Linfopoyetina del Estroma TímicoRESUMEN
Development of mammalian B-lineage cells is characterized by progression through a series of checkpoints defined primarily by rearrangement and expression of immunoglobulin genes. Progression through these checkpoints is also influenced by stromal cells in the microenvironment of the primary tissues wherein B-cell development occurs, ie, fetal liver and bone marrow and adult bone marrow. This review focuses on the developmental biology of human bone marrow B-lineage cells, including perturbations that contribute to the origin and evolution of B-lineage acute lymphoblastic leukemia and primary immunodeficiency diseases characterized by agammaglobulinemia. Recently described in vitro and in vivo models that support development and expansion of human B-lineage cells through multiple checkpoints provide new tools for identifying the bone marrow stromal cell-derived molecules necessary for survival and proliferation. Mutations in genes encoding subunits of the pre-B cell receptor and molecules involved in pre-B cell receptor signaling culminate in X-linked and non-X-linked agammaglobulinemia. A cardinal feature of these immunodeficiencies is an apparent apoptotic sensitivity of B-lineage cells at the pro-B to pre-B transition. On the other end of the spectrum is the apoptotic resistance that accompanies the development of B-lineage acute lymphoblastic leukemia, potentially a reflection of genetic abnormalities that subvert normal apoptotic programs. The triad of laboratory models that mimic the bone marrow microenvironment, immunodeficiency diseases with specific defects in B-cell development, and B-lineage acute lymphoblastic leukemia can now be integrated to deepen our understanding of human B-cell development.
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Adulto , Agammaglobulinemia/inmunología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/patología , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/patología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Síndromes de Inmunodeficiencia/inmunología , MamíferosRESUMEN
The Notch genes encode a conserved family of receptors that influence developmental fate in many species. Prior studies have indicated that Notch-1 and Notch-2 signaling influence the development of hematopoietic stems cells and thymocytes, but little is known regarding Notch expression and function in B-lineage cells. We analyzed the expression of Notch receptors and Notch ligands in human B-lineage cells and bone marrow (BM) stromal cells. Notch-1 mRNA and protein is expressed throughout normal B cell development and in leukemic B-lineage cells. In contrast, Notch-2 expression is limited to pre-B cells expressing low levels of surface mu. The Notch ligand Delta is expressed in BM B-lineage cells. The Notch ligand Jagged-1 is not expressed in B-lineage cells, but is expressed in BM stromal cells. These results suggest a model wherein lateral signaling between Notch and Delta on B-lineage cells and/or Notch/Jagged-1 interactions between B-lineage cells and BM stromal cells may regulate human B cell development.
Asunto(s)
Linfocitos B/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Factores de Transcripción , Animales , Secuencia de Bases , Linaje de la Célula , Cartilla de ADN , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos C57BL , Receptor Notch1 , Receptor Notch2 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Certain VH genes are predominantly expressed in mature B cells. We hypothesized that several, mutually nonexclusive VH-dependent mechanisms operating at distinct stages during B cell development may be responsible for overrepresentation of these VH genes. In the present study, we have assessed whether one of the mechanisms involves preferential rearrangement at the pro-B cell stage. The frequency of individual VH4 and VH3 genes in rearrangement libraries from FACS-purified human CD34+/CD19+ pro-B and CD34-/CD19+ pre-B cells was assessed. The in-frame and out-of-frame rearrangements from both cell populations were analyzed using a high resolution PAGE system. The frequencies of individual VH gene segments among out-of-frame rearrangements from pro-B cells were determined, because these frequencies should reflect only processes before the translation of the mu-heavy chain and should not be biased by selection mechanisms. Our results demonstrate that, at the pro-B cell stage, the V4-34, V4-39, and V4-59 gene segments are the most frequently rearranged VH4 family genes, and the V3-23 and V3-30 gene segments are the most frequently rearranged VH3 family genes. This finding suggests that the predominant expression of these VH genes in peripheral mature B cells is determined to a significant degree by their preferential rearrangement during V-DJ recombination.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Adulto , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Feto , Frecuencia de los Genes/inmunología , Humanos , Hígado/inmunología , Hígado/metabolismo , Familia de Multigenes/inmunología , Sistemas de Lectura/genética , Sistemas de Lectura/inmunología , Células Madre/inmunología , Células Madre/metabolismoRESUMEN
This study compared human murine stromal cells for their capacity to support human hematopoietic stem cell (HSC) development into the B lineage. FACS sorted human fetal bone marrow (BM) HSC (CD34+CD19- or CD34+/CD10-/CD19-/CD45RA) were cultured on human fetal BM stromal cells, human skin fibroblasts, or murine S17 stromal cells and analyzed by flow cytometry or reverse transcriptase polymerase chain reaction. CD34+CD19- HSC on human BM stromal cells or fibroblasts differentiated into B-lineage cells with a continuum in density of surface CD19 expression, and some cells expressing micro/kappa or micro/lambda B-cell receptors. In contrast, CD19+ cells from S17 cultures had two- to fourfold higher levels of CD19, but no cells expressing B-cell receptors. The number and percentage of CD19+ cells was high, intermediate, or low in the human BM, human fibroblast, or murine S17 stromal cell cultures, respectively. Reverse transcriptase polymerase chain reaction analysis showed that TdT, CD19, and DHQ52-J(H) rearrangements were expressed at comparable levels when CD34+/CD19- HSC were plated on human or murine stromal cells. In contrast, CD34+/CD10-/CD19-/CD45RA HSC plated on human or murine stromal cells expressed CD19 in both cultures, but TdT was only expressed in human stromal cell cultures. We conclude that human BM stromal cell, human skin fibroblasts, and murine S17 stromal cell cultures can provide complementary and comparative tools for identification of stromal cell ligands with potentially unique functions in regulating human B-cell development.
Asunto(s)
Linfocitos B/citología , Células de la Médula Ósea/fisiología , Fibroblastos/fisiología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/fisiología , Células del Estroma/fisiología , Animales , Antígenos CD19/inmunología , Células de la Médula Ósea/efectos de los fármacos , Moléculas de Adhesión Celular/fisiología , Linaje de la Célula , Células Cultivadas , Técnicas de Cocultivo , Medio de Cultivo Libre de Suero , ADN Nucleotidilexotransferasa/biosíntesis , Citometría de Flujo , Expresión Génica , Reordenamiento Génico de Linfocito B , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Interleucina-3/farmacología , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/citologíaRESUMEN
Clonal expansion of B-cell precursor acute lymphoblastic leukemia (ALL) is potentially regulated by survival, growth, and death signals transduced by the bone marrow (BM) microenvironment. Using a human BM stromal cell culture that supports the growth of normal human B-cell precursors, we established a pre-B ALL cell line designated BLIN-2. BLIN-2 has a clonal rearrangement of the Ig heavy chain locus, a dic(9;20) chromosomal abnormality, and a bi-allelic deletion of the p16(INK4a) and p19(ARF) genes. The most interesting feature of BLIN-2 is an absolute dependence on adherent human BM stromal cells for sustained survival and growth. BLIN-2 cultured in the absence of BM stromal cells undergo apoptosis, and direct contact with viable BM stromal cells is essential for optimal growth. BLIN-2 cells also grow on vascular cell adhesion molecule-1 (VCAM-1)-negative human skin fibroblasts, making it unlikely that a very late antigen-4 (VLA-4)/VCAM-1 interaction is required for BLIN-2 growth. Western blot analysis of BLIN-2 cells cultured in the presence or absence of BM stromal cells demonstrates that contact of BLIN-2 with BM stromal cells induces hyperphosphorylation of Rb. In contrast, the pre-B ALL cell line BLIN-1, which has a bi-allelic deletion of p16(INK4a) p19(ARF) but does not require BM stromal cells for growth, does not undergo Rb phosphorylation after BM stromal cell contact. The BLIN-2 cell line will facilitate identification of ligand/receptor interactions at the B-cell precursor/BM stromal cell interface and may provide new insight into microenvironmental regulation of leukemic cell survival and growth.
Asunto(s)
Células de la Médula Ósea/fisiología , Comunicación Celular , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Apoptosis , Ciclo Celular , Células Cultivadas , Cromosomas Humanos Par 9/genética , Técnicas de Cocultivo , Femenino , Fibroblastos/fisiología , Genes p16 , Humanos , Hibridación Fluorescente in Situ , Lactante , Masculino , Fosforilación , Reacción en Cadena de la Polimerasa , Procesamiento Proteico-Postraduccional , Proteína de Retinoblastoma/fisiología , Células del Estroma/fisiología , Células Tumorales Cultivadas , Molécula 1 de Adhesión Celular Vascular/fisiologíaRESUMEN
Functional immunoglobulin gene rearrangement is a sine qua non for successful B cell development in mammalian bone marrow, but other factors are also important. Studies reported during the past year have contributed new insight into the surface receptor complexes and signaling outcomes that influence the fate of B cell precursors. Identification and characterization of secreted and membrane-associated stromal cell products, and their actions on B-cell precursors, was a parallel area of ongoing investigation.
Asunto(s)
Linfocitos B/citología , Animales , Médula Ósea/fisiología , Diferenciación Celular/fisiología , Expresión Génica/fisiología , Hematopoyesis/genética , Humanos , Ratones , Receptores de Antígenos de Linfocitos B/fisiología , Transducción de Señal/fisiologíaRESUMEN
Contact with bone marrow stromal cells is crucial for the normal growth and development of B-cell precursors. We have previously shown that human bone marrow stromal cell tyrosine kinase activity can be activated by direct contact with B-lymphoid cells (J Immunol 155:2359, 1995). In the present study, we show that increased tyrosine phosphorylation of focal adhesion kinase, paxillin, and extracellular-related kinase 2 (or p42 MAP kinase) accounted for the major changes occurring in stromal cell tyrosine phosphorylation after 5 to 10 minutes of contact with the RAMOS B-lymphoma cell line. Although adhesion of B-cell precursors to stromal cells is primarily mediated by very late antigen-4 (VLA-4) and vascular cell adhesion molecule-1 (VCAM-1), VLA-4-deficient and adhesion-deficient RAMOS cells were equally capable of stimulating stromal cell tyrosine phosphorylation. Similar changes in the tyrosine phosphorylation pattern of stromal cells were induced by contact with normal human B-cell precursors and several other B-lineage cell lines. After 5 to 30 minutes of contact with stromal cells, no change in protein tyrosine phosphorylation was detected in RAMOS or normal human B-cell precursors removed from stromal cells. Pretreatment of stromal cells with cytochalasin D abrogated contact-mediated enhancement of stromal cell tyrosine phosphorylation, suggesting that an intact cytoskeleton was essential. These results suggest that B-cell contact activates stromal cell signaling cascades that regulate cytoskeletal organization and transcription, independent of the interaction mediated by VLA-4 and VCAM-1.
Asunto(s)
Linfocitos B/citología , Células de la Médula Ósea , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Moléculas de Adhesión Celular/metabolismo , Comunicación Celular , Proteínas del Citoesqueleto/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Tirosina/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Adhesión Celular , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Integrina alfa4beta1 , Integrinas/metabolismo , Linfoma de Células B/enzimología , Linfoma de Células B/patología , Proteína Quinasa 1 Activada por Mitógenos , Paxillin , Fosforilación , Receptores Mensajeros de Linfocitos/metabolismo , Células del Estroma/citología , Células del Estroma/enzimología , Células Tumorales CultivadasRESUMEN
Although the underlying genetic defect in multiple myeloma is unknown, activating mutations in the N- and K-ras oncogenes are common. Recent studies have suggested that ras mutations are associated with disease progression. We have introduced an activated N-ras12, N-ras61, or K-ras12 cDNA into the interleukin 6 (IL-6)-dependent multiple myeloma cell line ANBL6 to determine the effect of N- and K-ras on the growth/death properties of ANBL6. All three transduced cell populations demonstrate a growth advantage over the parent ANBL6 when propagated on normal human bone marrow stromal cells. In the absence of bone marrow stromal cells, augmentation of growth was observed in all three mutant ras-expressing populations at optimal and suboptimal concentrations of IL-6. Furthermore, in the absence of IL-6, all mutant ras populations demonstrated an augmentation in DNA synthesis when compared to the parent ANBL6. However, growth of the K-ras12 population in the absence of IL-6 was significantly inhibited when compared to the mutant N-ras populations. This could be explained by the observation that in the absence of IL-6, N-ras12 and N-ras61 suppress apoptosis, whereas K-ras12 does not. We also found that mutant ras expression could result in early protection from glucocorticoid-induced apoptosis similar to that observed by the addition of IL-6. However, the combination of mutant ras and IL-6 could completely block the glucocorticoid induction of apoptosis in long-term cultures. These data suggest that mutations in different ras family members may have similar or distinct effects on myeloma tumor growth and death and may alter the response to glucocorticoid treatment.
Asunto(s)
Genes ras , Interleucina-6/farmacología , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Mutación , Apoptosis/efectos de los fármacos , Apoptosis/genética , Células de la Médula Ósea , Ciclo Celular/genética , División Celular/genética , Células Cultivadas , ADN/biosíntesis , Glucocorticoides/farmacología , Humanos , Recombinación Genética , Células del Estroma , Transducción Genética , Células Tumorales CultivadasRESUMEN
The B cell-specific cell surface molecule CD19 is expressed at all stages of B cell development, including normal plasma cells, and mediates signal transduction via interaction with cytoplasmic effector proteins. Cross-linking CD19 on early human B lineage cells induces the formation of a CD19/Vav/phosphatidylinositol-3 kinase complex, tyrosine phosphorylation of CD19 and Vav, and activation of the Ras pathway. To further explore the ramifications of CD19 signaling, the current study examined whether phosphorylation of Elk-1, activation of activator protein-1 (AP-1), or activation of nuclear factor-kappaB (NF-kappaB) transcription factors occurred following CD19 cross-linking. The cells used were the BLIN-1 pre-B cell line expressing low levels of cell surface mu heavy chain associated with surrogate light chain and the 1E8 immature B cell line expressing cell surface mu/kappa. Lysates from CD19 cross-linked 1E8 cells induced robust phosphorylation of an Elk-1 fusion protein in vitro, whereas no phosphorylation of Elk-1 fusion protein occurred using lysates from CD19 cross-linked BLIN-1 cells. An electrophoretic mobility shift assay employing AP-1 and NF-kappaB consensus oligonucleotides was used to demonstrate that AP-1 -binding activity increased, while constitutive NF-kappaB-binding activity was not enhanced, following 2 h of CD19 cross-linking in 1E8 cells. Supershift experiments revealed that JunD and c-Fos proteins mediated anti-CD19 induced AP-1-binding activity in 1E8 cells. In contrast, CD19 cross-linking in BLIN-1 cells resulted in the induction of NF-kappaB, but had no apparent effect on AP-1-binding activity. These data suggest that CD19-mediated signal transduction activates different transcription factors at juxtaposed stages of B cell development that may culminate in the activation or suppression of distinct sets of genes.
Asunto(s)
Antígenos CD19/metabolismo , Linfocitos B/metabolismo , Proteínas de Unión al ADN , Factores de Transcripción , Humanos , FN-kappa B/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción AP-1/metabolismo , Células Tumorales Cultivadas , Proteína Elk-1 con Dominio etsRESUMEN
Mammalian hematopoietic stem cell (HSC) commitment and differentiation into lymphoid lineage cells proceed through a series of developmentally restricted progenitor compartments. A complete understanding of this process, and how it differs from HSC commitment and differentiation into cells of the myeloid/erythroid lineages, requires the development of model systems that support HSC commitment to the lymphoid lineages. We now describe a human bone marrow stromal cell culture that preferentially supports commitment and differentiation of human HSC to CD19+ B-lineage cells. Fluorescence activated cell sorterpurified CD34++/lineage-cells were isolated from fetal bone marrow and cultured on human fetal bone marrow stromal cells in serum-free conditions containing no exogenous cytokines. Over a period of 3 weeks, CD34++/lineage- cells underwent commitment, differentiation, and expansion into the B lineage. Progressive changes included: loss of CD34, acquisition of and graded increases in the level of cell surface CD19, and appearance of immature B cells expressing mu/kappa or mu/lambda cell surface Ig receptors. The tempo and phenotype of B-cell development was not influenced by the addition of IL-7 (10 ng/ml), or by the addition of goat anti-IL-7 neutralizing antibody. These results indicate a profound difference between mouse and human in the requirement for IL-7 in normal B-cell development, and provide an experimental system to identify and characterize human bone marrow stromal cell-derived molecules crucial for human B lymphopoiesis.
Asunto(s)
Linfocitos B/inmunología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Interleucina-7/farmacología , Animales , Anticuerpos Monoclonales , Antígenos CD/análisis , Antígenos CD19/análisis , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Médula Ósea/embriología , Médula Ósea/inmunología , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Feto , Citometría de Flujo , Cabras , Hematopoyesis/inmunología , Humanos , Cadenas kappa de Inmunoglobulina/inmunología , Cadenas lambda de Inmunoglobulina/inmunología , Cadenas mu de Inmunoglobulina/inmunología , Inmunofenotipificación , Cinética , Mamíferos , Ratones , Receptores Inmunológicos/biosíntesis , Especificidad de la Especie , Células del Estroma , Factores de TiempoRESUMEN
A common sequela to allogeneic or autologous bone marrow transplantation (BMT) is a delay in the reconstitution of a functional B-cell immune response. Therefore, we examined whether the posttransplant BM microenvironment is deficient in supporting the proliferation and/or differentiation of B-cell precursors. BM stromal cell cultures were established from patients who received allogeneic or autologous BMT for acute lymphoblastic leukemia, Hodgkin's disease, or non-Hodgkin's lymphoma. These cultures were then compared with normal donor BM stromal cell cultures for expression of adhesion molecules and the capacity to support the adhesion and interleukin-7 (IL-7)-dependent growth of normal B-cell precursors. Analysis of BM stromal cell cultures established from 28 BMT recipients showed a significantly reduced expression of cell surface vascular cell adhesion molecule-1 (VCAM-1/CD106), compared with normal donor BM stromal cells. Transplant BM stromal cell CD106 expression was responsive to regulatory cytokines in a manner qualitatively comparable with normal donor BM stromal cells. The level of B-cell precursor adhesion to transplant BM stromal cells correlated with the level of CD106 expression. Of 19 evaluable transplant BM stromal cell cultures, eight exhibited a reduced capacity to support the growth of CD19+/light chain- normal B-cell precursors. The capacity of transplant BM stromal cells to support B-cell precursor growth correlated with the level of CD106 expression, and the level of B-cell precursor adhesion. Our collective results may provide new mechanistic insight into why B-cell recovery is delayed post-BMT and underscore the importance of VCAM-1/CD106 in regulating B lymphopoiesis.
Asunto(s)
Linfocitos B/citología , Células de la Médula Ósea , Moléculas de Adhesión Celular/análisis , Hematopoyesis/fisiología , Células del Estroma/química , Células del Estroma/fisiología , Trasplante de Médula Ósea , Adhesión Celular , Diferenciación Celular , División Celular , Células Cultivadas , Citometría de Flujo , Enfermedad de Hodgkin/terapia , Humanos , Interleucina-1/farmacología , Interleucina-4/farmacología , Interleucina-7/farmacología , Linfoma no Hodgkin/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Molécula 1 de Adhesión Celular VascularAsunto(s)
Linfocitos B/citología , Células de la Médula Ósea , Hematopoyesis , Células Madre Hematopoyéticas/citología , Médula Ósea/embriología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Cultivadas , Medio de Cultivo Libre de Suero , Regulación del Desarrollo de la Expresión Génica , Reordenamiento Génico de Linfocito B , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Interleucina-7/farmacología , Hígado/citología , Hígado/embriologíaRESUMEN
The interaction of normal human B cell precursors with the bone marrow (BM) stromal cells that support their growth and maturation is mediated by adhesion of B cell CD49d/CD29 (alpha 4 beta 1 integrin or VLA-4) to stromal cell signal transduction, we generated CD49d-deficient variants of the RAMOS early b Burkitt's lymphoma cell line. RAMOS was chosen due to its lack of expression of CD49e (alpha 5 integrin) and CD44, which could potentially interact with stromal cells or their extracellular matrix. In contrast to the parental RAMOS cell line, CD49d-deficient RAMOS cell lines did not adhere to primary human BM stromal cells. Tyrosine phosphorylation of several stromal cell proteins was stimulated within 5 to 10 min of contact with either CD49d-expressing or CD49d-deficient RAMOS. These proteins included a 110-kDa substrate tentatively identified as pp125FAK. In contrast, mAb cross-linking of stromal cell CD106 had no significant effect on protein tyrosine phosphorylation. Il-6 production by stromal cells was enhanced up to fourfold over 3 days when stromal cells were cultured in contact with RAMOS cells, regardless of CD49d expression. Stromal cell IL-6 production was not significantly enhanced by RAMOS cells cultured above the stromal cells in Transwell inserts. Thus, we have demonstrated through the use of paired CD49d-expressing and CD49d-deficient RAMOS cell lines that protein tyrosine kinase activity and IL-6 production by primary human BM stromal cells can be enhanced in a manner that is dependent on B cell contact but independent of CD49d-mediated adhesion. These results indicate the potential for B cells to interact with cells in their microenvironment and activate feedback mechanisms mediated by stromal cells, which may regulate lymphopoiesis.
Asunto(s)
Linfocitos B/metabolismo , Médula Ósea/enzimología , Interleucina-6/biosíntesis , Proteínas Tirosina Quinasas/biosíntesis , Células del Estroma/enzimología , Anticuerpos Monoclonales/inmunología , Antígenos CD/fisiología , Médula Ósea/metabolismo , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/inmunología , Reacciones Cruzadas/inmunología , Inducción Enzimática , Humanos , Integrina alfa4 , Células del Estroma/metabolismo , Células Tumorales Cultivadas , Molécula 1 de Adhesión Celular VascularRESUMEN
Multiple myeloma (MM) is a late-stage B-cell cancer with an unknown etiology. Activating mutations of the N-ras and K-ras oncogenes occur with a high frequency in myeloma and, therefore, may play a role in the pathogenesis of the disease. To study the role of N-ras-activating mutations in the regulation of myeloma tumor growth, we introduced a constitutively active N-ras cDNA containing a glutamine to arginine (CAA-CGA) amino acid substitution at codon 61 into the interleukin 6 (IL-6)-dependent myeloma cell line ANBL6. Expression of the mutant N-ras cDNA resulted in significant IL-6-independent growth, as well as augmentation of growth at suboptimal concentrations of IL-6. The IL-6-independent growth pattern was not the result of activation of autocrine IL-6 production in the mutant N-ras-expressing population because neutralizing antibodies to the IL-6 receptor and to IL-6 had no effect on the rate of DNA synthesis in the absence of IL-6. Furthermore, mutant N-ras expression decreased the percentage of cells undergoing apoptosis in the absence of IL-6. These data suggest that activating mutations of the ras oncogenes may result in growth factor independence accompanied by a suppression of apoptosis in MM. Therefore, the use of therapies designed to block IL-6 action in MM may have less of an impact on tumors bearing activated ras mutations.