RESUMEN
The cooperation of oncogenes in the transformation of primary rat Schwann cells is a strikingly synergistic process. We have explored the molecular mechanisms involved. Activation of an inducible Raf kinase results in morphologically transformed cells that are arrested in G1 via the induction of p21(CiP1) and subsequent inhibition of cyclin/cdk activity. In contrast, coexpression of SV40 large T (LT) or a dominant-negative mutant of p53 abolishes p21(CiP1) induction and alleviates the growth arrest. Moreover in this scenario, Raf activation results in an increase in the specific activity of cyclin/cdk complexes with Raf and LT cooperating to superinduce cyclin A/cdk2 activity and stimulate proliferation in the absence of mitogens. Thus, signaling by Raf and its cooperating partners converges at the regulation of cyclin/cdk complexes, with the cellular responses to Raf modulated by p53.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Oncogénicas de Retroviridae/metabolismo , Animales , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Ciclo Celular/genética , División Celular/efectos de los fármacos , Transformación Celular Neoplásica , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , ADN/biosíntesis , Activación Enzimática , Fase G1/genética , Proteínas Oncogénicas v-raf , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Wistar , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Oncogénicas de Retroviridae/genética , Células de Schwann/patología , Células de Schwann/fisiología , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
E7 is the major transforming protein of human papillomavirus type 16 (HPV16). It has been found to associate with the retinoblastoma protein Rb1. We investigated whether HPV16 E7 protein was associated with other cellular proteins, in particular with those involved in cell cycle control. Immunoprecipitates from CaSki cell extracts with an anti E7 monoclonal antibody contained a histone H1 kinase. Recombinant E7, synthesized in yeast, when mixed with protein extracts from epithelial cells bound histone H1 kinase activity in vitro. The in vivo and the in vitro-formed E7-kinase complex had the same periodicity of activity during the cell cycle, being most active in S and G2/M. Immunoblotting of E7 immunoprecipitates with an antibody raised against the p33CDK2, revealed a 33 kDa protein band not detected by an anti-p34cdc2 antibody, suggesting that the E7-associated kinase activity is due to the p33CDK2. The interaction appears to be via cyclin A, since probing of similar immunoblots showed a 50 kDa band corresponding to cyclin A. The association of E7 with cyclin A appeared to be direct, not involving Rb 1 or other proteins.
Asunto(s)
Ciclinas/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Fase G2 , Humanos , Proteínas E7 de Papillomavirus , Pruebas de Precipitina , Protamina Quinasa/metabolismo , Proteína de Retinoblastoma/metabolismo , Fase SRESUMEN
Infection of 1-day-old chicks with reticuloendotheliosis virus strain T induces a neoplastic disease that kills the chicks 7 to 14 days postinfection. In association with reticuloendotheliosis-associated virus (REV-A), reticuloendotheliosis virus T (REV-T) induces tumors that are predominantly immunoglobulin M (IgM) negative. We examined a variety of REV-T(REV-A)-induced tumors and tumor-derived cell lines and concluded that the principal IgM-negative tumors that develop in REV-T(REV-A)-infected chicks are neither pre-B or pre-B-pre-T but rather mature T lymphoid and myeloid. Without exception, the immunoglobulin heavy- and light-chain loci were in germ line configuration. Furthermore, the cell lines expressed neither sterile transcripts of the heavy- or light-chain immunoglobulin genes nor elevated levels of c-myb, two characteristics associated with murine pre-B lymphomas. Cell lines were also examined by using monoclonal antibodies for expression of a variety of cell surface markers expressed on B lymphocytes and/or T lymphocytes and/or myeloid cells. These reagents defined two types of IgM-negative tumor cell lines, one CIa+ CT-3+ (T lymphoid) and the other CIa+ CT-3-. By using the same approaches, tumor development was examined following REV-T(REV-A) infection at 1 and 3 weeks post-hatching of cyclophosphamide-treated chicks shown to be devoid of B-lymphoid cells. Again, the tumors that developed were either CIa+ CT-3+ (T lymphoid) or CIa+ CT-3-. Furthermore, the frequency and rate with which IgM-negative tumors developed in cyclophosphamide-treated chicks were not different from those observed in normal chicks. In 3-week-old cyclophosphamide-treated chicks, the presence of CIa+ CT-3- tumors bearing hematopoietic lineage markers, such as CLA-3 and 5M19, are most likely to have been derived from cells within the myeloid lineage.
Asunto(s)
Células de la Médula Ósea , Transformación Celular Neoplásica , Enfermedades Linfáticas/microbiología , Oncogenes , Proteínas Oncogénicas de Retroviridae/genética , Retroviridae/genética , Linfocitos T/citología , Animales , Anticuerpos Monoclonales , Antígenos de Superficie/análisis , Biomarcadores , Línea Celular , Células Cultivadas , Embrión de Pollo , Pollos , Ciclofosfamida/uso terapéutico , ADN/aislamiento & purificación , Sondas de ADN , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina , Cadenas Ligeras de Inmunoglobulina/genética , Enfermedades Linfáticas/tratamiento farmacológico , Proteínas Oncogénicas v-rel , Proteínas Tirosina Quinasas/genética , ARN/aislamiento & purificación , Linfocitos T/inmunologíaRESUMEN
The vast majority of immunoglobulin-expressing mature chicken B lymphocytes contain one functionally rearranged and one unrearranged allele of the immunoglobulin light chain (IgL) gene. Therefore, nearly all IgL V-J rearrangements present in mature chickens are in-frame. In contrast, the Ig genes of mature mammalian B cells contain a high proportion of out-of-frame V-J joints. To investigate the basis for this difference, gene rearrangement at the chicken IgL locus was characterized during embryonic development and in mature B-cell lines. Joining of the single functional variable (VL) segment with the single joining (JL) segment occurs in cells in multiple tissues during a transient period of chicken embryogenesis. Only one-third of the V-J joints cloned from days 10-12 of development are in-frame. An increasing proportion of in-frame V-J joints is observed within the bursa of Fabricius at successively later stages of development. Our data suggest that the bursa of Fabricius serves during embryonic development as a site of selective amplification of cells that have undergone productive V-J joining, such that nearly all V-J joints present in postembryonic B cells are in-frame. The high frequency of rearranged alleles joined in-frame that is found in posthatching bursal cells and mature B-cell lines appears to result from a low frequency with which cells undergo IgL rearrangement at both alleles, rather than from an increase in the precision of V-J joining in avian species.
Asunto(s)
Linfocitos B/citología , Bolsa de Fabricio/embriología , Pollos/inmunología , Reordenamiento Génico de Cadena Ligera de Linfocito B , Genes de Inmunoglobulinas , Cadenas Ligeras de Inmunoglobulina/genética , Alelos , Animales , Secuencia de Bases , Bolsa de Fabricio/citología , Embrión de Pollo , Pollos/genética , Conversión Génica , Cadenas J de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Datos de Secuencia Molecular , Selección GenéticaRESUMEN
Chicken immunoglobulin light chain (IgL) gene rearrangement has been characterized. Rearrangement of the single variable (VL) segment with the single joining (JL) segment within the chicken IgL locus results in the deletion of the DNA between VL and JL from the genome. This deletion is accomplished by a molecular mechanism in which a precise joining of the IgL recombination signal sequences leads to the formation of a circular episomal element. The circular episome is an unstable genetic element that fails to be propagated during B cell development. Evidence was obtained that the formation of the circular episome is accompanied by the addition of a single nonrandom base to both the VL and JL coding segments. The subsequent joining of the VL and JL segments appears to occur at random, as we observed at least 25 unique V-J junction sequences, 11 of which are out-of-frame. A novel recombination mechanism that accounts for the observed features of chicken IgL gene rearrangement is discussed.
Asunto(s)
Reordenamiento Génico de Cadena Ligera de Linfocito B , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Deleción Cromosómica , Datos de Secuencia Molecular , Plásmidos , Relación Estructura-ActividadRESUMEN
The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain locus demonstrated that 20 of 22 lines contained a single rearranged allele. With respect to specific restriction enzyme sites within the V lambda 1 gene, the active allele in any given clone was either diversified or nondiversified. In contrast, examination of the heavy-chain loci within these lines demonstrated that 16 of the 22 had both alleles rearranged. Further diversification of the V lambda 1 locus did not occur after prolonged in vitro passage of the cell lines. We propose that v-rel expression arrests diversification of the light-chain locus in these lymphoid cells, allowing the production of stable, clonal B-cell populations. The development of these and similar cell lines will make it possible to identify specific stages of avian lymphoid ontogeny and to study the mechanism of rearrangement and diversification in the avian B lymphocyte.
Asunto(s)
Reordenamiento Génico de Cadena Pesada de Linfocito B , Reordenamiento Génico de Cadena Ligera de Linfocito B , Genes Virales , Virus de la Reticuloendoteliosis/genética , Retroviridae/genética , Transcripción Genética , Animales , Antígenos de Superficie/genética , Linfocitos B/inmunología , Línea Celular , Pollos , Genes de Inmunoglobulinas , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/genética , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/inmunologíaRESUMEN
We have documented the effect of two nondefective helper viruses, reticuloendotheliosis virus A (REV-A) and chick syncytial virus (CSV) infection on bursal tissue. REV-A infection results in bursal atrophy, destroying both its structural and functional integrity. In contrast, the bursae in CSV-infected chicks, while reduced slightly in size, appear both structurally and functionally normal. REV-A-induced bursal atrophy is not a result of viral replication in the B-lymphocyte as (a) both viruses are capable of inducing, with equal efficiency, the formation of preneoplastic lesions containing proliferating B lymphocytes and (b) it appears that equivalent amounts of viral antigen are expressed in the bursae of chicks infected with either virus. We have examined the phenotype of tumors induced by the replication-defective virus REV-T when replicated by the two different helper viruses, REV-A and CSV. In REV-T(REV-A)-infected chicks, the majority of tumors that develop are negative for IgM expression. In contrast, the majority of tumors induced by REV-T(CSV) infection are IgM+. This finding is confirmed by recovery of IgM- cell lines from REV-T(REV-A)-infected chicks and IgM+ cell lines from REV-T(CSV)-infected chicks. In addition, repopulation studies show that bursal-derived cells that are IgM+ serve as target cells for REV-T(CSV)-induced lymphomas. This study demonstrates, therefore, that REV-T can induce IgM+, B cell lymphomas with high efficiency. We conclude that infections by the helper viruses, REV-A and CSV, differ dramatically in their effects on the composition of the population of cells that serve as targets for REV-T-induced neoplasia.