RESUMEN

Flow cytometry is a powerful tool for quantitative biology because it can perform single-cell analysis of large cell populations using multiple parameters. Results are often visualized as a two-dimensional scatter or contour plot. Because these plots can be relatively diffuse, it is not always straightforward to discern a relationship between measured parameters. We have demonstrated that quantitative trends can be fit to the single-cell data generated from a heterogeneous population. We engineered Abelson virus-transformed pre-B cells to express a broad range of oncogenic Ras levels. Instead of individual cultures with individual expression levels, a continuous range of levels was expressed by different cells in one heterogeneous culture. We then stained cells for downstream Erk phosphorylation to monitor MAPK signaling or employed an E2F-responsive genetic reporter to monitor cell-cycle activity. Subsequent analysis by flow cytometry and locally weighted scatterplot smoothing (LOWESS) revealed that increasing Ras oncogene expression led to increasing MAPK signaling. In contrast, E2F activity peaked at an optimal, intermediate level of Ras. To make this analytical method widely available to others, we have provided a software application that performs LOWESS on any two-parameter population data collected by flow cytometry.

Asunto(s)

Citometría de Flujo/métodos , Proteínas Oncogénicas v-abl/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Proteínas ras/metabolismo , Virus de la Leucemia Murina de Abelson/genética , Animales , Ciclo Celular/genética , Línea Celular , Línea Celular Transformada , Transformación Celular Neoplásica/genética , Factores de Transcripción E2F/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Sistema de Señalización de MAP Quinasas/genética , Ratones , Proteínas Oncogénicas v-abl/genética , Fosforilación , Reproducibilidad de los Resultados , Análisis de la Célula Individual/métodos , Programas Informáticos , Proteínas ras/genética

RESUMEN

Class I PI3K-dependent signaling regulates cell proliferation, differentiation, and survival. Analysis of gene-deficient mice revealed specific roles for the hematopoietically expressed PI3K catalytic subunits, p110gamma and p110delta, in development and function of T and B lymphocytes. However, the functional redundancy between these two PI3K isoforms in the B cell lineage remains unclear. Here, we demonstrate that p110delta and p110gamma are expressed in B cells at early developmental stages. Normal B cell differentiation requires both isoforms, as p110gamma/p110delta double deficiency causes an increased percentage of CD43(hi)/B220(+)/CD19(-) cells as compared with single deficiency. Interestingly, initial transformation efficiency of B cell precursors was strongly reduced in double-deficient cells following transformation by p185 bcr-abl or v-abl oncogenes as compared with single-deficient cells. The requirement of p110gamma and p110delta in B cell development is underlined by reduced splenic B cell numbers of p110gamma/p110delta double-deficient mice and of lethally irradiated wild-type mice reconstituted with double-deficient BM. Moreover, the peripheral maintenance of p110gamma/p110delta double-deficient T and B cells was highly impaired following adoptive transfer of double-deficient splenocytes into wild-type mice. Functionally, LPS stimulation of splenocytes revealed proliferation defects resulting in decreased survival of p110gamma/p110delta double-deficient B cells, which correlated with impaired induction of D-type cyclins and Bcl-X(L). Surprisingly, this was not observed when purified B cells were analyzed, indicating a contribution of likely cell-extrinsic factor(s) to the impaired proliferation of double-deficient B cells. Thus, we provide novel evidence that p110gamma and p110delta have overlapping and cell-extrinsic roles in the development, peripheral maintenance, and function of B cells.

Asunto(s)

Linfocitos B/citología , Proliferación Celular , Transformación Celular Neoplásica , Fosfatidilinositol 3-Quinasas/fisiología , Virus de la Leucemia Murina de Abelson/genética , Traslado Adoptivo , Animales , Linfocitos B/metabolismo , Western Blotting , Médula Ósea/metabolismo , Diferenciación Celular , Fosfatidilinositol 3-Quinasa Clase I , Fosfatidilinositol 3-Quinasa Clase Ib , Femenino , Citometría de Flujo , Genes abl/fisiología , Isoenzimas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal

RESUMEN

Specific inhibitors of PI3K isoforms are currently evaluated for their therapeutic potential in leukemia. We found that BCR/ABL(+) human leukemic cells express PI3Kdelta and therefore explored its impact on leukemia development. Using PI3Kdelta-deficient mice, we define a dual role of PI3Kdelta in leukemia. We observed a growth-promoting effect in tumor cells and an essential function in natural killer (NK) cell-mediated tumor surveillance: Abelson-transformed PI3Kdelta-deficient cells induced leukemia in RAG2-deficient mice with an increased latency, indicating that PI3Kdelta accelerated leukemia progression in vivo. However, the absence of PI3Kdelta also affected NK cell-mediated tumor surveillance. PI3Kdelta-deficient NK cells failed to lyse a large variety of target cells because of defective degranulation, as also documented by capacitance recordings. Accordingly, transplanted leukemic cells killed PI3Kdelta-deficient animals more rapidly. As a net effect, no difference in disease latency in vivo was detected if both leukemic cells and NK cells lack PI3Kdelta. Other tumor models confirmed that PI3Kdelta-deficient mice succumbed more rapidly when challenged with T- or B-lymphoid leukemic or B16 melanoma cells. Thus, the action of PI3Kdelta in the NK compartment is as relevant to survival of the mice as the delayed tumor progression. This dual function must be taken into account when using PI3Kdelta inhibitors as antileukemic agents in clinical trials.

Asunto(s)

Vigilancia Inmunológica/genética , Células Asesinas Naturales/inmunología , Leucemia/inmunología , Fosfatidilinositol 3-Quinasas/genética , Virus de la Leucemia Murina de Abelson/genética , Animales , Muerte Celular/genética , Muerte Celular/inmunología , Línea Celular Transformada , Fosfatidilinositol 3-Quinasa Clase I , Progresión de la Enfermedad , Regulación Leucémica de la Expresión Génica , Humanos , Células Jurkat , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Leucemia/genética , Leucemia/metabolismo , Leucemia/mortalidad , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/mortalidad , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Células Tumorales Cultivadas

RESUMEN

Transformation by Abelson murine leukemia virus (Ab-MLV) is a multistep process in which growth-stimulatory signals from the v-Abl oncoprotein and growth-suppressive signals from the p19(Arf)-p53 tumor suppressor pathway oppose each other and influence the outcome of infection. The process involves a proliferative phase during which highly viable primary transformants expand, followed by a period of marked apoptosis (called "crisis") that is dependent on the presence of p19(Arf) and p53; rare cells that survive this phase emerge as fully transformed and malignant. To understand the way in which v-Abl expression affects p19(Arf) expression, we examined changes in expression of Arf during all stages of Ab-MLV transformation process. As is consistent with the ability of v-Abl to stimulate Myc, a transcription factor known to induce p19(Arf), Myc and Arf are induced soon after infection and p19(Arf) is expressed. At these early time points, the infected cells remain highly viable. The onset of crisis is marked by an increase in p19(Arf) expression and a change in localization of the protein from the nucleoplasm to the nucleolus. These data together suggest that the localization and expression levels of p19(Arf) modulate the effects of the protein during oncogenesis and reveal that the p19(Arf)-mediated response is subject to multiple layers of regulation that influence its function during Ab-MLV-mediated transformation.

Asunto(s)

Virus de la Leucemia Murina de Abelson/genética , Apoptosis , Linfocitos B/virología , Transformación Celular Viral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Animales , Linfocitos B/patología , Línea Celular Transformada , Cruzamientos Genéticos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Citometría de Flujo , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Heterocigoto , Indoles/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH

RESUMEN

Abelson murine leukemia virus (Ab-MLV) arose from a recombination between gag sequences in Moloney MLV (Mo-MLV) and the c-abl proto-oncogene. The v-Abl oncoprotein encoded by Ab-MLV contains MA, p12, and a portion of CA sequences derived from the gag gene of Mo-MLV. Previous studies indicated that alteration of MA sequences affects the biology of Mo-MLV and Ab-MLV. To understand the role of these sequences in Ab-MLV transformation more fully, alanine substitution mutants that affect Mo-MLV replication were examined in the context of Ab-MLV. Mutations affecting Mo-MLV replication decreased transformation, while alanine mutations in residues dispensable for Mo-MLV replication did not. The altered v-Abl proteins displayed aberrant subcellular localization that correlated to transformation defects. Immunofluorescent analyses suggested that aberrant trafficking of the altered proteins and improper interaction with components of the cytoskeleton were involved in the phenotype. Similar defects in localization were observed when the Gag moiety containing these mutations was expressed in the absence of abl-derived sequences. These results indicate that MA sequences within the Gag moiety of the v-Abl protein contribute to proper localization by playing a dominant role in trafficking of the v-Abl molecule.

Asunto(s)

Virus de la Leucemia Murina de Abelson/metabolismo , Productos del Gen gag/química , Productos del Gen gag/metabolismo , Virus de la Leucemia Murina de Moloney/metabolismo , Proteínas Oncogénicas v-abl/química , Proteínas Oncogénicas v-abl/metabolismo , Virus de la Leucemia Murina de Abelson/química , Virus de la Leucemia Murina de Abelson/genética , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Productos del Gen gag/genética , Modelos Moleculares , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/química , Virus de la Leucemia Murina de Moloney/genética , Mutación/genética , Oligosacáridos/química , Oligosacáridos/metabolismo , Proteínas Oncogénicas v-abl/genética , Péptidos/química , Péptidos/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

The Abelson murine leukemia virus (Ab-MLV), like other retroviruses that contain v-onc genes, arose following a recombination event between a replicating retrovirus and a cellular oncogene. Although experimentally validated models have been presented to address the mechanism by which oncogene capture occurs, very little is known about the events that influence emerging viruses following the recombination event that incorporates the cellular sequences. One feature that may play a role is the genetic makeup of the host in which the virus arises; a number of host genes, including oncogenes and tumor suppressor genes, have been shown to affect the pathogenesis of many murine leukemia viruses. To examine how a host gene might affect an emerging v-onc gene-containing retrovirus, we studied the weakly oncogenic Ab-MLV-P90A strain, a mutant that generates highly oncogenic variants in vivo, and compared the viral populations in normal mice and mice lacking the p53 tumor suppressor gene. While variants arose in both p53+/+ and p53-/- tumors, the samples from the wild-type animals contained a more diverse virus population. Differences in virus population diversity were not observed when wild-type and null animals were infected with a highly oncogenic wild-type strain of Ab-MLV. These results indicate that p53, and presumably other host genes, affects the selective forces that operate on virus populations in vivo and likely influences the evolution of oncogenic retroviruses such as Ab-MLV.

Asunto(s)

Virus de la Leucemia Murina de Abelson/genética , Virus de la Leucemia Murina de Abelson/patogenicidad , Genes p53 , Leucemia Experimental/genética , Leucemia Experimental/virología , Infecciones por Retroviridae/genética , Infecciones por Retroviridae/virología , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/virología , Virus de la Leucemia Murina de Abelson/aislamiento & purificación , Animales , Secuencia de Bases , ADN Viral/genética , Femenino , Variación Genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Mutación , Virulencia/genética , Integración Viral/genética

RESUMEN

The transcription factor BKLF (basic Krüppel-like factor, KLF3) is a member of the Krüppel-like factors (KLF) family. KLF members harbor a characteristic C-terminal zinc-finger DNA-binding domain and bind preferentially to CACCC-motifs. BKLF is highly expressed in haematopoietic and erythoid cells and works either as repressor or activator of transcription in various genes. BKLF-deficient mice display myeloproliferative disorders and abnormalities in haematopoiesis. Other members of the KLF-family such as GKLF and BCL11A have been implicated in tumorigenesis, however, for BKLF such association has not yet been demonstrated. We report here that a single Abelson-murine leukemia virus (A-MuLV) provirus is present in the genome of the hypermutating murine pre-B cell line 18-81. The provirus has integrated into the locus of the transcription factor BKLF. In contrast to other A-MuLV transformed pre-B cell lines, BKLF is highly transcribed in cell line 18-81. BKLF transcripts originate from the retroviral long terminal repeats (LTRs) and BKLF protein can be detected by gel shift retardation assay. We hypothesize on a potential role of BKLF deregulation in tumorigenesis and/or in the induction of somatic hypermutation in cell line 18-81.

Asunto(s)

Virus de la Leucemia Murina de Abelson/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Provirus/genética , Factores de Transcripción/metabolismo , Animales , Línea Celular Transformada , Línea Celular Tumoral , Transformación Celular Viral , Ensayo de Cambio de Movilidad Electroforética , Etiquetas de Secuencia Expresada , Reordenamiento Génico de Linfocito B , Cambio de Clase de Inmunoglobulina , Factor 4 Similar a Kruppel , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Hipermutación Somática de Inmunoglobulina , Secuencias Repetidas Terminales , Transcripción Genética , Dedos de Zinc

RESUMEN

The v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) induces pre-B-cell transformation. Signals emanating from the SH2 domain of the protein are required for transformation, and several proteins bind this region of v-Abl. One such protein is the adaptor molecule Shc, a protein that complexes with Grb2/Sos and facilitates Ras activation, an event associated with Ab-MLV transformation. To test the role this interaction plays in growth and survival of infected pre-B cells, dominant-negative (DN) Shc proteins were coexpressed with v-Abl and transformation was examined. Expression of DN Shc reduced Ab-MLV pre-B-cell transformation and decreased the ability of v-Abl to stimulate Ras activation and Erk phosphorylation in a Raf-dependent but Rac-independent fashion. Further analysis revealed that Shc is required for v-Abl-mediated Raf tyrosine 340 and 341 phosphorylation, an event associated with Erk phosphorylation. In contrast to effects on proliferation, survival of the cells and activation of Akt were not affected by expression of DN Shc. Together, these data reveal that v-Abl-Shc interactions are a critical part of the growth stimulatory signals delivered during transformation but that they do not affect antiapoptotic pathways. Furthermore, these data highlight a novel role for Shc in signaling from v-Abl to Raf.

Asunto(s)

Virus de la Leucemia Murina de Abelson/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis , Transformación Celular Viral/fisiología , Virus de la Leucemia Murina de Abelson/química , Virus de la Leucemia Murina de Abelson/genética , Animales , Línea Celular , Proliferación Celular , Proteína Adaptadora GRB2 , Regulación Viral de la Expresión Génica , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Proteínas ras/metabolismo

RESUMEN

Aberrant activation of the JAK-STAT pathway has been implicated in tumor formation; for example, constitutive activation of JAK2 kinase or the enforced expression of STAT5 induces leukemia in mice. We show here that the Janus kinase TYK2 serves an opposite function. Mice deficient in TYK2 developed Abelson-induced B lymphoid leukemia/lymphoma as well as TEL-JAK2-induced T lymphoid leukemia with a higher incidence and shortened latency compared with WT controls. The cell-autonomous properties of Abelson murine leukemia virus-transformed (A-MuLV-transformed) TYK2(-/-) cells were unaltered, but the high susceptibility of TYK2(-/-) mice resulted from an impaired tumor surveillance, and accordingly, TYK2(-/-) A-MuLV-induced lymphomas were easily rejected after transplantation into WT hosts. The increased rate of leukemia/lymphoma formation was linked to a decreased in vitro cytotoxic capacity of TYK2(-/-) NK and NKT cells toward tumor-derived cells. RAG2/TYK2 double-knockout mice succumbed to A-MuLV-induced leukemia/lymphoma faster than RAG2(-/-)TYK2(+/-) mice. This defines NK cells as key players in tumor surveillance in Abelson-induced malignancies. Our observations provide compelling evidence that TYK2 is an important regulator of lymphoid tumor surveillance.

Asunto(s)

Leucemia de Células B/inmunología , Leucemia Experimental/inmunología , Proteínas Tirosina Quinasas/metabolismo , Virus de la Leucemia Murina de Abelson/genética , Virus de la Leucemia Murina de Abelson/metabolismo , Animales , Animales Recién Nacidos , Transformación Celular Neoplásica , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Leucemia de Células B/patología , Leucemia Experimental/patología , Leucemia de Células T/inmunología , Leucemia de Células T/patología , Hígado/citología , Hígado/patología , Ratones , Ratones Noqueados , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Nucleares , Proteínas Tirosina Quinasas/genética , Bazo/citología , Bazo/patología , Tasa de Supervivencia , TYK2 Quinasa

RESUMEN

Suppression of apoptosis is an important feature of the Abelson murine leukemia virus (Ab-MLV) transformation process. During multistep transformation, Ab-MLV-infected pre-B cells undergo p53-dependent apoptosis during the crisis phase of transformation. Even once cells are fully transformed, an active v-Abl protein tyrosine kinase is required to suppress apoptosis because cells transformed by temperature-sensitive (ts) kinase mutants undergo rapid apoptosis after a shift to the nonpermissive temperature. However, inactivation of the v-Abl protein by a temperature shift interrupts signals transmitted via multiple pathways, making it difficult to identify those that are critically important for the suppression of apoptosis. To begin to dissect these pathways, we tested the ability of an SH2 domain Ab-MLV mutant, P120/R273K, to rescue aspects of the ts phenotype of pre-B cells transformed by the conditional kinase domain mutant. The P120/R273K mutant suppressed apoptosis at the nonpermissive temperature, a phenotype correlated with its ability to activate Akt. Apoptosis also was suppressed at the nonpermissive temperature by constitutively active Akt and in p53-null pre-B cells transformed with the ts kinase domain mutant. These data indicate that an intact Src homology 2 (SH2) domain is not critical for apoptosis suppression and suggest that signals transmitted through Akt and p53 play an important role in the response.

Asunto(s)

Virus de la Leucemia Murina de Abelson/fisiología , Apoptosis , Linfocitos B/virología , Células de la Médula Ósea/virología , Transformación Celular Viral , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Virus de la Leucemia Murina de Abelson/genética , Animales , Línea Celular Transformada , Humanos , Ratones , Proteínas Oncogénicas v-abl/genética , Proteínas Oncogénicas v-abl/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Proteína p53 Supresora de Tumor/genética , Dominios Homologos src

RESUMEN

The v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) induces transformation of pre-B cells in vivo and in vitro and can transform immortalized fibroblast cell lines in vitro. Although the kinase activity of the protein is required for these events, most previously studied mutants encoding truncated v-Abl proteins that lack the extreme carboxyl terminus retain high transforming capacity in NIH 3T3 cells but transform lymphocytes poorly. To understand the mechanisms responsible for poor lymphoid transformation, mutants expressing a v-Abl protein lacking portions of the COOH terminus were compared for their ability to transform pre-B cells. Although all mutants lacking sequences within the COOH terminus were compromised for lymphoid transformation, loss of amino acids in the central region of the COOH terminus, including those implicated in JAK interaction and DNA binding, decreased transformation twofold or less. In contrast, loss of the extreme COOH terminus rendered the protein unstable and led to rapid proteosome-mediated degradation, a feature that was more prominent when the protein was expressed in Ab-MLV-transformed lymphoid cells. These data indicate that the central portion of the COOH terminus is not essential for lymphoid transformation and reveal that one important function of the COOH terminus is to stabilize the v-Abl protein in lymphoid cells.

Asunto(s)

Virus de la Leucemia Murina de Abelson/fisiología , Transformación Celular Viral , Regulación Viral de la Expresión Génica , Linfocitos/virología , Proteínas Oncogénicas v-abl/química , Células 3T3 , Virus de la Leucemia Murina de Abelson/química , Virus de la Leucemia Murina de Abelson/genética , Animales , Secuencia de Bases , Línea Celular , Línea Celular Transformada , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Eliminación de Gen , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Oncogénicas v-abl/genética , Proteínas Oncogénicas v-abl/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo

RESUMEN

Abelson murine leukemia virus-transformed cell lines have provided a critical model system for studying the regulation of B cell development. However, transformation by v-Abl blocks B cell development, resulting in the arrest of these transformants in an early pre-B cell-like state. We report here that treatment of Abelson virus-transformed pre-B cell lines with the small molecule Abl kinase inhibitor (STI571) results in their differentiation to a late pre-B cell-like state characterized by induction of immunoglobulin (Ig) light chain gene rearrangement. DNA microarray analyses enabled us to identify two genes inhibited by v-Abl that encode the Igk 3' enhancer-binding transcription factors Spi-B and IRF-4. We show that enforced expression of these two factors is sufficient to induce germline Igk transcription in Abelson-transformed pro-B cell lines. This suggests a key role for these factors, and perhaps for c-Abl itself, in the regulated activation of Ig light chain gene rearrangement.

Asunto(s)

Virus de la Leucemia Murina de Abelson , Linfocitos B/citología , Linfocitos B/virología , Proteínas Oncogénicas v-abl/antagonistas & inhibidores , Virus de la Leucemia Murina de Abelson/genética , Virus de la Leucemia Murina de Abelson/patogenicidad , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Secuencia de Bases , Benzamidas , Diferenciación Celular/efectos de los fármacos , Línea Celular Transformada , ADN/genética , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Reordenamiento Génico de Cadena Ligera de Linfocito B/efectos de los fármacos , Mesilato de Imatinib , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Piperazinas , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Factores de Transcripción/genética

RESUMEN

Abelson murine leukemia virus (Ab-MLV) encodes the v-Abl protein tyrosine kinase and induces transformation of immortalized fibroblast lines and pre-B cells. Temperature-sensitive mutations affecting the kinase domain of the protein have demonstrated that the kinase activity is absolutely required for transformation. Despite this requirement, mutations affecting other regions of v-Abl modulate transformation activity. The SH2 domain and the highly conserved FLVRES motif within it form a phosphotyrosine-binding pocket that is required for interactions between the kinase and cellular substrates. To understand the impact of SH2 alterations on Ab-MLV-mediated transformation, we studied the Ab-MLV mutant P120/R273K. This mutant encodes a v-Abl protein in which the beta B5 arginine at the base of the phosphotyrosine-binding pocket has been replaced by a lysine. Unexpectedly, infection of NIH 3T3 or pre-B cells with P120/R273K revealed a temperature-dependent transformation phenotype. At 34 degrees C, P120/R273K transformed about 10-fold fewer cells than wild-type virus of equivalent titer; at 39.5 degrees C, 300-fold fewer NIH 3T3 cells were transformed and pre-B cells were refractory to transformation. Temperature-dependent transformation was accompanied by decreased phosphorylation of Shc, a protein that interacts with the v-Abl SH2 and links the protein to Ras, and decreased induction of c-Myc expression. These data suggest that alteration of the FLVRES pocket affects the ability of v-Abl to interact with at least some of its substrates in a temperature-dependent fashion and identify a novel type of temperature-sensitive Abelson virus.

Asunto(s)

Virus de la Leucemia Murina de Abelson/patogenicidad , Transformación Celular Viral , Mutación , Dominios Homologos src/genética , Células 3T3 , Virus de la Leucemia Murina de Abelson/genética , Virus de la Leucemia Murina de Abelson/fisiología , Animales , Linfocitos B/virología , Línea Celular Transformada , Genes Virales , Ratones , Proteínas Oncogénicas v-abl/química , Proteínas Oncogénicas v-abl/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Premenopausia , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Temperatura , Proteínas ras/genética , Proteínas ras/metabolismo

RESUMEN

V(D)J recombination is the mechanism by which antigen receptor genes are assembled. The site-specific cleavage mediated by RAG1 and RAG2 proteins generates two types of double-strand DNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although these DNA breaks are mainly resolved into coding joints and signal joints, they can participate in a nonstandard joining process, forming hybrid and open/shut joints that link coding ends to signal ends. In addition, the broken DNA molecules excised from different receptor gene loci could potentially be joined to generate interlocus joints. The interlocus recombination process may contribute to the translocation between antigen receptor genes and oncogenes, leading to malignant transformation of lymphocytes. To investigate the underlying mechanisms of these nonstandard recombination events, we took advantage of recombination-inducible cell lines derived from scid homozygous (s/s) and scid heterozygous (s/+) mice by transforming B-cell precursors with a temperature-sensitive Abelson murine leukemia virus mutant (ts-Ab-MLV). We can manipulate the level of recombination cleavage and end resolution by altering the cell culture temperature. By analyzing various recombination products in scid and s/+ ts-Ab-MLV transformants, we report in this study that scid cells make higher levels of interlocus and hybrid joints than their normal counterparts. These joints arise concurrently with the formation of intralocus joints, as well as with the appearance of opened coding ends. The junctions of these joining products exhibit excessive nucleotide deletions, a characteristic of scid coding joints. These data suggest that an inability of scid cells to promptly resolve their recombination ends exposes the ends to a random joining process, which can conceivably lead to chromosomal translocations.

Asunto(s)

Transformación Celular Neoplásica/genética , ADN Nucleotidiltransferasas/metabolismo , ADN de Neoplasias/genética , Proteínas de Unión al ADN , Reordenamiento Génico de Cadena Ligera de Linfocito B , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Inmunodeficiencia Combinada Grave/genética , Virus de la Leucemia Murina de Abelson/genética , Virus de la Leucemia Murina de Abelson/fisiología , Animales , Apoptosis/genética , Transformación Celular Viral , Proteína Quinasa Activada por ADN , Activación Enzimática , Regulación Leucémica de la Expresión Génica , Genes de Inmunoglobulinas , Genes bcl-2 , Genotipo , Cadenas J de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Ratones SCID , Proteínas de Neoplasias/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Inmunodeficiencia Combinada Grave/complicaciones , Células Tumorales Cultivadas , VDJ Recombinasas

RESUMEN

In many tumor systems, analysis of cells for loss of heterozygosity (LOH) has helped to clarify the role of tumor suppressor genes in oncogenesis. Two important tumor suppressor genes, p53 and the Ink4a/Arf locus, play central roles in the multistep process of Abelson murine leukemia virus (Ab-MLV) transformation. p53 and the p53 regulatory protein, p19Arf, are required for the apoptotic crisis that characterizes the progression of primary transformed pre-B cells to fully malignant cell lines. To search for other tumor suppressor genes which may be involved in the Ab-MLV transformation process, we used endogenous proviral markers and simple-sequence length polymorphism analysis to screen Abelson virus-transformed pre-B cells for evidence of LOH. Our survey reinforces the role of the p53-p19 regulatory pathway in transformation; 6 of 58 cell lines tested had lost sequences on mouse chromosome 4, including the Ink4a/Arf locus. Consistent with this pattern, a high frequency of primary pre-B-cell transformants derived from Ink4a/Arf +/- mice became established cell lines. In addition, half of them retained the single copy of the locus when the transformation process was complete. These data demonstrate that a single copy of the Ink4a/Arf locus is not sufficient to fully mediate the effects of these genes on transformation.

Asunto(s)

Virus de la Leucemia Murina de Abelson/genética , Linfocitos B/virología , Proteínas Portadoras/genética , Transformación Celular Viral , Genes Supresores de Tumor , Células Madre Hematopoyéticas/virología , Pérdida de Heterocigocidad , Animales , Mapeo Cromosómico , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Genes p53/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA

RESUMEN

The human lambda 5 (hu lambda 5) gene is the structural homologue of the murine lambda 5 (m lambda 5) gene and is transcriptionally active in pro-B and pre-B lymphocytes. The lambda 5 and VpreB polypeptides together with the Ig mu H chain and the signal-transducing subunits, Ig alpha and Ig beta, comprise the pre-B cell receptor. To further investigate the pro-B/pre-B-specific transcription regulation of hu lambda 5 in an in vivo model, we generated mouse lines that contain a 28-kb genomic fragment encompassing the entire hu lambda 5 gene. High levels of expression of the transgenic hu lambda 5 gene were detected in bone marrow pro-B and pre-B cells at the mRNA and protein levels, suggesting that the 28-kb transgene fragment contains all the transcriptional elements necessary for the stage-specific B progenitor expression of hu lambda 5. Flow cytometric and immunoprecipitation analyses of bone marrow cells and Abelson murine leukemia virus-transformed pre-B cell lines revealed the hu lambda 5 polypeptide on the cell surface and in association with mouse Ig mu and mouse VpreB. Finally, we found that the hu lambda 5 transgene is able to rescue the pre-B lymphocyte block when bred onto the m lambda 5-/- background. Therefore, we conclude that the hu lambda 5 polypeptide can biochemically and functionally substitute for m lambda 5 in vivo in pre-B lymphocyte differentiation and proliferation. These studies on the mouse and human pre-B cell receptor provide a model system to investigate some of the molecular requirements necessary for B cell development.

Asunto(s)

Subgrupos de Linfocitos B/inmunología , Cadenas lambda de Inmunoglobulina/genética , Glicoproteínas de Membrana/genética , Transgenes/inmunología , Virus de la Leucemia Murina de Abelson/genética , Animales , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Sitios de Unión de Anticuerpos/genética , 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 , Cruzamientos Genéticos , Femenino , Regulación de la Expresión Génica/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina , Inmunoglobulina de Cadenas Ligeras Subrogadas , Cadenas lambda de Inmunoglobulina/biosíntesis , Cadenas lambda de Inmunoglobulina/metabolismo , Cadenas mu de Inmunoglobulina/metabolismo , Inmunofenotipificación , Masculino , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos B/biosíntesis , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Células Madre/citología , Células Madre/inmunología , Células Madre/metabolismo , Testículo/inmunología , Testículo/metabolismo , Timo/inmunología , Timo/metabolismo

RESUMEN

Abelson murine leukemia virus (Ab-MLV) transforms NIH 3T3 and pre-B cells via expression of the v-Abl tyrosine kinase. Although the enzymatic activity of this molecule is absolutely required for transformation, other regions of the protein are also important for this response. Among these are the SH2 domain, involved in phosphotyrosine-dependent protein-protein interactions, and the long carboxyl terminus, which plays an important role in transformation of hematopoietic cells. Important signals are sent from each of these regions, and transformation is most likely orchestrated by the concerted action of these different parts of the protein. To explore this idea, we compared the ability of the v-Src SH2 domain to substitute for that of v-Abl in the full-length P120 v-Abl protein and in P70 v-Abl, a protein that lacks the carboxyl terminus characteristic of Abl family members. Ab-MLV strains expressing P70/S2 failed to transform NIH 3T3 cells and demonstrated a greatly reduced capacity to mediate signaling events associated with the Ras-dependent mitogen-activated protein (MAP) kinase pathway. In contrast, Ab-MLV strains expressing P120/S2 were indistinguishable from P120 with respect to these features. Analyses of additional mutants demonstrated that the last 162 amino acids of the carboxyl terminus were sufficient to restore transformation. These data demonstrate that an SH2 domain with v-Abl substrate specificity is required for NIH 3T3 transformation in the absence of the carboxyl terminus and suggest that cooperativity between the extreme carboxyl terminus and the SH2 domain facilitates the transmission of transforming signals via the MAP kinase pathway.

Asunto(s)

Virus de la Leucemia Murina de Abelson/fisiología , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Transformación Celular Viral , Proteínas Fúngicas , Proteínas Oncogénicas v-abl/química , Proteínas Oncogénicas v-abl/metabolismo , Dominios Homologos src , Células 3T3 , Virus de la Leucemia Murina de Abelson/química , Virus de la Leucemia Murina de Abelson/genética , Animales , Línea Celular , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Factores de Transcripción GATA , Proteína Adaptadora GRB2 , Regulación Viral de la Expresión Génica , Genes fos , Prueba de Complementación Genética , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Oncogénicas v-abl/genética , Fosforilación , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Proteínas ras/metabolismo , Dominios Homologos src/genética

RESUMEN

Tumorigenesis is a multistep process that involves the activation of oncogenes and the inactivation of tumor suppressor genes. The transforming activity of the v-Abl oncogene of Abelson murine leukemia virus (A-MuLV) in immortal cell lines has been well studied, while the effects of v-Abl in primary fibroblasts are less clear. Here we show that v-Abl causes cell cycle arrest in primary mouse embryonic fibroblasts (MEFs) and elevated levels of both p53 and the cyclin-dependent kinase inhibitor p21Cip. p53-/- or p19ARF-/- MEFs were resistant to v-Abl-induced cell cycle arrest. Although wild-type MEFs were resistant to v-Abl transforming activity, p53-/- or p19ARF-/- MEFs were susceptible. The results indicate that loss of p19ARF and p53 function plays an important role during the transformation of primary cells by v-Abl. We suggest that although v-Abl is a potent oncogene, its full potential transforming activity cannot be realized until the ARF-, and p53-dependent growth inhibitory pathway is disabled. We also show that p53 is not the mediator of v-Abl toxicity in immortal fibroblasts and does not determine the susceptibility of immortal fibroblasts to v-Abl transformation.

Asunto(s)

Transformación Celular Neoplásica , Genes abl , Genes p53 , Proteínas Oncogénicas v-abl/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Virus de la Leucemia Murina de Abelson/genética , Animales , Células Cultivadas , Embrión de Mamíferos , Fibroblastos/citología , Fibroblastos/patología , Regulación Neoplásica de la Expresión Génica , Ratones , Ratones Noqueados , Transcripción Genética , Proteína p14ARF Supresora de Tumor , Proteína p53 Supresora de Tumor/deficiencia

RESUMEN

The Ink4a/Arf locus encodes p16(Ink4a) and p19(Arf) and is among the most frequently mutated tumor suppressor loci in human cancer. In mice, many of these effects appear to be mediated by interactions between p19(Arf) and the p53 tumor-suppressor protein. Because Tp53 mutations are a common feature of the multistep pre-B cell transformation process mediated by Abelson murine leukemia virus (Ab-MLV), we examined the possibility that proteins encoded by the Ink4a/Arf locus also play a role in Abelson virus transformation. Analyses of primary transformants revealed that both p16(Ink4a) and p19(Arf) are expressed in many of the cells as they emerge from the apoptotic crisis that characterizes the transformation process. Analyses of primary transformants from Ink4a/Arf null mice revealed that these cells bypassed crisis. Because expression of p19(Arf) but not p16 (Ink4a) induced apoptosis in Ab-MLV-transformed pre-B cells, p19(Arf) appears to be responsible for these events. Consistent with the link between p19(Arf) and p53, Ink4a/Arf expression correlates with or precedes the emergence of cells expressing mutant p53. These data demonstrate that p19(Arf) is an important part of the cellular defense mounted against transforming signals from the Abl oncoprotein and provide direct evidence that the p19(Arf)-p53 regulatory loop plays an important role in lymphoma induction.

Asunto(s)

Virus de la Leucemia Murina de Abelson/genética , Apoptosis , Transformación Celular Neoplásica , Genes p53 , Proteínas/fisiología , Proteína p53 Supresora de Tumor/fisiología , Células 3T3 , Animales , Linfocitos B , Línea Celular Transformada , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Genes p16 , Humanos , Ratones , Proteínas/genética , Proteínas Recombinantes/metabolismo , Transfección , Proteína p14ARF Supresora de Tumor

RESUMEN

The mechanism by which early lymphoid cells are selectively transformed by v-Abl is currently unknown. Previous studies have shown constitutive activation of IL-4 and IL-7 signaling pathways, as measured by activation of Janus protein kinase (JAK)1, JAK3, STAT5, and STAT6, in pre-B cells transformed by v-Abl. To determine whether activation of these cytokine signaling pathways by v-Abl is important in the cellular events induced by the Abelson murine leukemia virus, the effects of IL-4 and IL-7 on pre-B cells transformed with a temperature-sensitive v-Abl mutant were examined. Whereas IL-4 had little or no effect, IL-7 delayed both the apoptosis and cell cycle arrest that occur upon v-Abl kinase inactivation. IL-7 also delayed the decreases in the levels of c-Myc, Bcl-2, and Bcl-xL that occur upon loss of v-Abl kinase activity. IL-7 did not maintain v-Abl-mediated differentiation arrest of the pre-B cells, as activation of NF-kappaB and RAG gene transcription was unaffected by IL-7. These results identify a potential role for IL-7 signaling pathways in transformation by v-Abl while demonstrating that a combination of IL-4 and IL-7 signaling cannot substitute for an active v-Abl kinase in transformed pre-B cells.

Asunto(s)

Virus de la Leucemia Murina de Abelson/fisiología , Linfocitos B/virología , Transformación Celular Viral/fisiología , Citocinas/fisiología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Interleucina-7/fisiología , Proteínas de la Leche , Proteínas Oncogénicas v-abl/fisiología , Proteínas Tirosina Quinasas/fisiología , Transducción de Señal/efectos de los fármacos , Virus de la Leucemia Murina de Abelson/genética , Animales , Apoptosis/efectos de los fármacos , Linfocitos B/citología , Ciclo Celular/efectos de los fármacos , Proteínas de Unión al ADN/fisiología , Genes abl , Genes bcl-2 , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Humanos , Interleucina-4/fisiología , Interleucina-7/farmacología , Janus Quinasa 1 , Janus Quinasa 3 , Ratones , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Factor de Transcripción STAT5 , Factor de Transcripción STAT6 , Transactivadores/fisiología , Factores de Transcripción/fisiología , Proteína bcl-X