RESUMEN
An acidic domain (AD) of gp130 was previously found to interact with the Src family kinase (SFK) Hck. Here, the influence of myristoylated peptides derived from this AD was assessed in the mouse myeloma cell line, 7TD1. The IL-6-dependent growth of 7TD1 cells was reduced by approximately 75%, if 100 microM of myristoylated 18mer peptide (18AD) was included in the growth medium, but was unaffected by a control peptide with scrambled sequence (18sc). A similar differential inhibition by peptides 18AD and 18sc was observed for the erythropoietin-dependent growth of BaF-EH cells expressing chimeric erythropoietin receptor-gp130 and human Hck and for the human myeloma cell line INA-6. While the peptide 18AD concentration inhibiting 50% was approximately 30 microM in 7TD1 and BaF-EH cells, peptide 18AD did not significantly inhibit growth of IL-6-independent MM1.S myeloma and OKT1 hybridoma cells or of BaF-EH cells supplied with IL-3. Treatment with 100 microM peptide 18AD caused the same degree or 60% of apoptosis induction as IL-6 deprivation in 7TD1 or INA-6 cells, respectively. Co-immunoprecipitation experiments revealed that peptide 18AD interfered with the association of Hck and gp130 in 7TD1 lysates in a concentration-dependent manner. IL-6-treatment of INA-6 cells induced the kinase activities of Fyn, Lyn and Hck, but not Src, and the IL-6-induced SFK activities were inhibited by peptide 18AD. Expression in 7TD1 cells of a kinase-inactive Hck mutant (K269R) elicited a dominant-negative effect on cell number increases providing further evidence that SFKs are required for gp130 signalling in myeloma cells.
Asunto(s)
Receptor gp130 de Citocinas/antagonistas & inhibidores , Interleucina-6/antagonistas & inhibidores , Mieloma Múltiple/enzimología , Fragmentos de Péptidos/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Transporte Biológico , Ciclo Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Receptor gp130 de Citocinas/química , Receptor gp130 de Citocinas/metabolismo , Receptor gp130 de Citocinas/farmacología , Humanos , Ratones , Datos de Secuencia Molecular , Mieloma Múltiple/patología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-hck/metabolismo , Factor de Transcripción STAT3/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
CX+/CX- and Colo+/Colo- tumor sublines with stable heat shock protein 70 (Hsp70) high and low membrane expression were generated by fluorescence activated cell sorting of the parental human colon (CX2) and pancreas (Colo357) carcinoma cell lines, using an Hsp70-specific antibody. Two-parameter flow cytometry revealed that Hsp70 colocalizes with Bag-4, also termed silencer of death domain, not only in the cytosol but also on the plasma membrane. After nonlethal gamma-irradiation, the percentage of membrane-positive cells and the protein density of Hsp70 and Bag-4 were found to be strongly upregulated in carcinoma sublines with initially low expression levels (CX-, Colo-). Membrane expression of Hsp70 was also elevated in Bag-4 overexpressing HeLa cervix carcinoma cells when compared to neo-transfected cells. In response to gamma-irradiation, neo-transfected HeLa cells behaved like Hsp70/Bag-4 low-expressing CX- and Colo-, and Bag-4-transfected HeLa cells like Hsp70/Bag-4 high-expressing carcinoma sublines CX+ and Colo+. Immunoprecipitation studies further confirmed colocalization of Hsp70 and Bag-4 but also point to an association of Hsp70 and Hsp40 on the plasma membrane of CX+ and Colo+ cells; on CX- and Colo- tumor sublines, Hsp40 was detectable in the absence of Hsp70 and Bag-4. Other co-chaperones including Hsp60 and Hsp90 were neither found on the cell surface of CX+/CX-, Colo+/Colo- nor on HeLa neo-/HeLa Bag-4-transfected tumor cells. Functionally, Hsp70/Bag-4 and Hsp70/Hsp40 membrane-positive tumor cells appeared to be better protected against radiation-induced effects, including G2/M arrest and growth inhibition, on the one hand. On the other hand, membrane-bound Hsp70, but neither Bag-4 nor Hsp40, served as a recognition site for the cytolytic attack mediated by natural killer cells.
Asunto(s)
Células Asesinas Naturales/fisiología , Proteínas de la Membrana/fisiología , Tolerancia a Radiación/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Apoptosis/efectos de la radiación , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Neoplasias del Colon/patología , Citotoxicidad Inmunológica/inmunología , Citotoxicidad Inmunológica/efectos de la radiación , Rayos gamma , Expresión Génica/efectos de la radiación , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/fisiología , Células HeLa , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/fisiología , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/efectos de la radiación , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Transfección , Regulación hacia ArribaRESUMEN
Homing behavior and function of autoimmune CD4+ T cells in vivo was analyzed before and during EAE, using MBP-specific T cells retrovirally engineered to express the gene of green fluorescent protein. The cells migrate from parathymic lymph nodes to blood and to the spleen. Preceding disease onset, large numbers of effector cells invade the CNS, with only negligible numbers left in the periphery. In early EAE, most (>90%) infiltrating CD4+ cells were effector cells. Migratory effector cells downregulate activation markers (CD25, OX-40) but upregulate several chemokine receptors and adsorb MHC class II on their membranes. Within the CNS, the effector cells are reactivated, with upregulated proinflammatory cytokines and downmodulated T cell receptor-associated structures, presumably reflecting autoantigen recognition in situ.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Movimiento Celular , Encefalomielitis Autoinmune Experimental/inmunología , Animales , Sistema Nervioso Central/inmunología , Proteínas Fluorescentes Verdes , Antígenos de Histocompatibilidad Clase II/inmunología , Inyecciones Intraperitoneales , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Activación de Linfocitos/inmunología , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Ratas , Ratas Endogámicas LewRESUMEN
Human plasmacytoid precursor dendritic cells (ppDC) are a major source of type I IFN upon exposure to virus and bacteria, yet the stimulus causing their maturation into DCs is unknown. After PBMC activation with immunostimulatory bacterial DNA sequences (CpG-DNA) we found that ppDC are the primary source of IFN-alpha. In fact, either CpG-DNA or dsRNA (poly(I:C)) induced IFN-alpha from purified ppDC. Surprisingly, only CpG-DNA triggered purified ppDC survival, maturation, and production of TNF, GM-CSF, IL-6, and IL-8, but not IL-10 or IL-12. Known DC activators such as CD40 ligation triggered ppDC maturation, but only IL-8 production, while bacterial LPS was negative for all activation criteria. An additional finding was that only CpG-DNA could counteract IL-4-induced apoptosis in ppDC. Therefore, CpG-DNA represents a pathogen-associated molecular pattern for ppDC. In contrast to these finding, CpG-DNA, like LPS, caused TNF, IL-6, and IL-12 release from PBMC and purified monocytes; however, differentiation of monocytes into DCs with GM-CSF and IL-4 unexpectedly resulted in refractoriness to CpG-DNA, but not LPS. Taken together, these results suggest that within a DC subset a multiplicity of responses can be generated by distinct environmental stimuli and that responses to a given stimulus may be dissimilar between DC subsets.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Islas de CpG/inmunología , ADN Bacteriano/farmacología , Células Dendríticas/citología , Células Dendríticas/inmunología , Integrina alfaXbeta2 , Oligodesoxirribonucleótidos/farmacología , Receptores de Interleucina-3/biosíntesis , Células 3T3 , Animales , Diferenciación Celular/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células Cultivadas , Citocinas/biosíntesis , Células Dendríticas/metabolismo , Células Dendríticas/microbiología , Escherichia coli/inmunología , Humanos , Integrina alfaXbeta2/biosíntesis , Interferón Tipo I/biosíntesis , Subunidad alfa del Receptor de Interleucina-3 , Isoantígenos/fisiología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/farmacología , Ratones , Monocitos/citología , Monocitos/inmunología , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Células Plasmáticas/microbiología , Poli I-C/farmacología , Células Madre/citología , Células Madre/inmunología , Células Madre/metabolismo , Células Madre/microbiología , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Chemokines and their receptors have been identified as major regulators controlling the functional organization of secondary lymphoid organs. Here we show that expression of CXC chemokine receptor 5 (CXCR5), a chemokine receptor required for B cell homing to B cell follicles, defines a novel subpopulation of B helper T cells localizing to follicles. In peripheral blood these cells coexpress CD45RO and the T cell homing CC chemokine receptor 7 (CCR7). In secondary lymphoid organs, CD4(+)CXCR5(+) cells lose expression of CCR7, which allows them to localize to B cell follicles and germinal centers where they express high levels of CD40 ligand (CD40L), a costimulatory molecule required for B cell activation and inducible costimulator (ICOS), a recently identified costimulatory molecule of the CD28 family. Thus, when compared with CD4(+)CD45RO(+)CXCR5(-) cells, CD4(+)CD45RO(+)CXCR5(+) tonsillar T cells efficiently support the production of immunoglobulin (Ig)A and IgG. In contrast, analysis of the memory response revealed that long-lasting memory cells are found within the CD4(+)CD45RO(+)CXCR5(-) population, suggesting that CXCR5(+)CD4 cells represent recently activated effector cells. Based on the characteristic localization within secondary lymphoid organs, we suggest to term these cells "follicular B helper T cells" (T(FH)).
Asunto(s)
Formación de Anticuerpos , Linfocitos B/inmunología , Tejido Linfoide/inmunología , Receptores de Quimiocina/biosíntesis , Receptores de Citocinas/biosíntesis , Receptores Mensajeros de Linfocitos/biosíntesis , Linfocitos T Colaboradores-Inductores/metabolismo , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/genética , Linfocitos T CD4-Positivos/clasificación , Linfocitos T CD4-Positivos/inmunología , Ligando de CD40/biosíntesis , Fraccionamiento Celular , Citocinas/biosíntesis , Centro Germinal/citología , Centro Germinal/inmunología , Humanos , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Memoria Inmunológica/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles , Leucocitos Mononucleares/clasificación , Leucocitos Mononucleares/inmunología , Tejido Linfoide/citología , Receptores CCR7 , Receptores CXCR5 , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunología , Receptores de Citocinas/genética , Receptores de Citocinas/inmunología , Receptores Mensajeros de Linfocitos/genética , Receptores Mensajeros de Linfocitos/inmunología , Linfocitos T Colaboradores-Inductores/clasificación , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
The product of the proto-oncogene c-myc (myc) is a potent activator of cell proliferation. In Burkitt lymphoma (BL), a human B-cell tumor, myc is consistently found to be transcriptionally activated by chromosomal translocation. The mechanisms by which myc promotes cell cycle progression in B-cells is not known. As a model for myc activation in BL cells, we have established a human EBV-EBNA1 positive B-cell line, P493-6, in which myc is expressed under the control of a tetracycline regulated promoter. If the expression of myc is switched off, P493-6 cells arrest in G0/G1 in the presence of serum. Re-expression of myc activates the cell cycle without inducing apoptosis. myc triggers the expression of cyclin D2, cyclin E and Cdk4, followed by the activation of cyclin E-associated kinase and hyper-phosphorylation of Rb. The transcription factor E2F-1 is expressed in proliferating and arrested cells at constant levels. The Cdk inhibitors p16, p21, p27 and p57 are expressed at low or not detectable levels in proliferating cells and are not induced after repression of myc. Ectopic expression of p16 inhibits cell cycle progression. These data suggest that myc triggers proliferation of P493-6 cells by promoting the expression of a set of cell cycle activators but not by inactivating cell cycle inhibitors.
Asunto(s)
Linfoma de Burkitt/fisiopatología , Proteínas de Ciclo Celular/fisiología , Ciclo Celular/fisiología , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Linfoma de Burkitt/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Fosforilación , Inhibidores de la Síntesis de la Proteína/farmacología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteína de Retinoblastoma/metabolismo , Proteína p107 Similar a la del Retinoblastoma , Tetraciclina/farmacología , Células Tumorales CultivadasRESUMEN
Human CMV has evolved multiple strategies to interfere with immune recognition of the host. A variety of mechanisms target Ag presentation by MHC class I molecules resulting in a reduced class I cell-surface expression. This down-regulation of class I molecules is expected to trigger NK cytotoxicity, which would have to be counteracted by the virus to establish long-term infection. Here we describe that the human CMV open reading frame UL40 encodes a canonical ligand for HLA-E, identical with the HLA-Cw03 signal sequence-derived peptide. Expression of UL40 in HLA-E-positive target cells conferred resistance to NK cell lysis via the CD94/NKG2A receptor. Generation of the UL40-derived HLA-E ligand was also observed in TAP-deficient cells. The presence of a functional TAP-independent HLA-E ligand in the UL40 signal sequence implicates this viral gene as an important negative regulator of NK activity.
Asunto(s)
Citomegalovirus/inmunología , Citotoxicidad Inmunológica/inmunología , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunosupresores/farmacología , Células Asesinas Naturales/inmunología , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Células Cultivadas , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibroblastos/virología , Humanos , Células K562 , Ligandos , Datos de Secuencia Molecular , Oligopéptidos , Sistemas de Lectura Abierta/inmunología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/aislamiento & purificación , Péptidos/inmunología , Procesamiento Proteico-Postraduccional/inmunología , Transfección , Proteínas Virales/genética , Antígenos HLA-ERESUMEN
We previously reported that cell surface expression of hsp70, the major stress inducible member of the 70-kDa heat shock protein family, is inducible by nonlethal heat as well as by treatment with the membrane-interactive compound alkyl-lysophospholipid 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) selectively on human tumor cell lines. Plasma membrane expression of hsp70 increases selectively the sensitivity of tumor cells to lysis and, therefore, might play an important role in the antitumor immune response. Here, we demonstrate that a combined treatment consisting of sublethal heat (41.8 degrees C) and a noncytotoxic concentration of ET-18-OCH3 (25 micrograms/mL) results in a synergistic increase in the amount of cell membrane-bound hsp70 on leukemic K562 cells and on freshly isolated bone marrow of a chronic myelogeneous leukemia (CML) patient, but not on peripheral blood lymphocytes or CD34+ hematopoietic progenitor cells of healthy human individuals. Under these conditions the repopulating capacity of progenitor cells was not influenced. The increased hsp70 membrane expression on leukemic K562 cells results in a significantly increased sensitivity to lysis mediated by natural killer cells. In contrast to leukemic cells, the lysis of peripheral blood lymphocytes and CD34+ progenitor cells that lack expression of hsp70 on their plasma membrane was not negatively influenced by this treatment. A nonspecific disruption of the plasma membrane could be excluded, because treatment with a nontoxic concentration of the detergent Tween20 did not have an influence on hsp70 cell surface expression or on the sensitivity to lysis. Our findings might have further clinical implications with respect to purging of bone marrow from patients suffering from leukemia at sublethal conditions to induce a tumor-selective immune response.
Asunto(s)
Antineoplásicos/farmacología , Purgación de la Médula Ósea/métodos , Membrana Celular/metabolismo , Proteínas HSP70 de Choque Térmico/biosíntesis , Células Madre Hematopoyéticas/efectos de los fármacos , Calor , Células K562/efectos de los fármacos , Éteres Fosfolípidos/farmacología , Adulto , Células Sanguíneas , Células de la Médula Ósea , Ensayo de Unidades Formadoras de Colonias , Sangre Fetal/citología , Regulación Leucémica de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Células Madre Hematopoyéticas/citología , Humanos , Células K562/citología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Células Tumorales Cultivadas/efectos de los fármacos , Ensayo de Tumor de Célula MadreRESUMEN
Bacterial DNA and immunostimulatory (i.s.) synthetic CpG-oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as "nature's adjuvant" since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG-ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigen-presenting cell (APC) function. Unfractionated murine bone marrow-derived DC and FACS-fractionated MHC class IIlow (termed immature DC) or MHC class IIhigh populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG-ODN. Similar to lipopolysaccharide, i.s. CpG-ODN caused up-regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL-12, IL-6 and TNF-alpha. CpG-ODN-activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B-driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG-ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.
Asunto(s)
Islas de CpG/inmunología , ADN Bacteriano/inmunología , Células Dendríticas/inmunología , Oligodesoxirribonucleótidos/inmunología , Adyuvantes Inmunológicos , Animales , Células de la Médula Ósea , Antígenos CD40/metabolismo , Células Cultivadas , Inducción Enzimática , Femenino , Antígeno HLA-B7/metabolismo , Hematopoyesis , Antígenos de Histocompatibilidad Clase II/metabolismo , Interleucina-12/biosíntesis , Interleucina-6/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Citotóxicos/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Regulación hacia Arriba , beta-Galactosidasa/metabolismoRESUMEN
The immune phenotype of canine hematopoietic progenitor cells was studied by immunoseparation and culturing of separated cells. Two separation methods were used, the magnetic cell sorting system (MACS) and the fluorescence activated cell sorter (FACS). For separation rat anti dog antibodies Dog 13 and Dog 14 directed against Thy-1, and Dog 26 as well as cross-reactive mouse anti human antibodies IOT2a and 7.2 directed against MHC class II were used. Separated cell populations were cultured in semisolid agar before and after long-term culture on a pre-established irradiated stromal cell layer. After 28 days, adherent and nonadherent cells were harvested from long-term culture. The MACS system allowed separation of cells into positive and negative fractions. Long-term culture-initiating cells (LTC-IC) were found in both the Thy-1+ and the Thy-1- fraction, but the content of LTC-IC was higher in the Thy-1+ fraction. The MACS system did not allow separation of progenitor cells according to the expression of MHC class II antigen detected by Dog 26 and the cross-reactive antibodies IOT2a and 7.2. In contrast to the MACS system the FACS allowed separation of negative, low-positive and high-positive cell populations. Low-positive fractions were well defined for Thy-1 and less well defined for MHC class II. CFU before and after long-term culture were exclusively observed in the low positive fraction (Thy-1(lo+)). Using MHC class II antibody Dog 26 LTC-IC were found mainly in the negative and low positive fraction, and CFU were observed mainly in the low and high positive fraction. In conclusion pluripotent canine hematopoietic precursor cells are low positive for Thy-1 and for MHC class II. In this respect canine hematopoietic progenitor cells are comparable to those of mouse and man.
Asunto(s)
Antígenos de Superficie/inmunología , Células Madre Hematopoyéticas/inmunología , Inmunofenotipificación , Animales , Células Cultivadas , Perros , Citometría de Flujo , Humanos , Ratones , RatasRESUMEN
Evidence is accumulating that members of the heat shock protein 70 (HSP70) family are found on the cell surface of certain tumor cells where they elicit a strong antitumor immune response. We demonstrated that HSP72, the major heat-inducible form of the HSP70 group, is located on the cell surface of approximately 60% of the human colon carcinoma cells CX2 with two different mAbs by indirect immunofluorescence, by electron microscopy, and by selective cell surface biotinylation. In an effort to analyze the role of HSP72 cell surface expression as a tumor-specific recognition structure within an "autologous" tumor system, the CX2 cells were separated into a stably HSP72 high expressing (CX+: >90%) and a stably HSP72 low expressing (CX-: <20%) subline. The expression "autologous" was written in parentheses to indicate that the colon carcinoma sublines CX+ and CX- derived from the original CX2 tumor cell line differ with respect to the cell surface expression pattern of HSP72, whereas they exhibit an identical cell surface expression pattern of MHC and cellular adhesion molecules (e.g., intercellular cellular adhesion molecule, neural cellular adhesion molecule, vascular cellular adhesion molecule). Within this "autologous" tumor cell system, we demonstrate that the sensitivity to lysis mediated by adherent non-MHC-restricted effector cells correlates (p < 0.05) with the amount of HSP72 that is expressed on the cell surface. Blocking studies using an HSP72-specific mAb revealed that HSP72 might act in an MHC-unrestricted manner as a tumor-specific recognition structure for a distinct NK cell population.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Choque Térmico/metabolismo , Células Asesinas Naturales/inmunología , Antígenos de Superficie/metabolismo , Carcinoma/metabolismo , Carcinoma/patología , Ciclo Celular , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Citotoxicidad Inmunológica , Citometría de Flujo , Proteínas del Choque Térmico HSP72 , Humanos , Inmunidad Celular , Inmunohistoquímica , Células Tumorales CultivadasRESUMEN
Treatment of cells with DNA damaging agents leads to induction of a variety of genes involved in different cellular processes. We have applied a lacZ-based gene trap strategy to search for new mammalian genes induced by genotoxic stress. A population of 32 x 10(3) neo r clones stably transfected with a gene trap vector was obtained, stained with fluorescein di-beta-d-galactopyranoside and analyzed by flow activated cell sorting and replica plating. This strategy allowed isolation of 30 neo r 'putative inducible' cell lines expressing lacZ only after a DNA damaging treatment. For three clones the site of integration and the degree of inducibility after UV treatment were determined by Southern blot and beta-galactosidase measurement respectively. One cell line (clone VI) showed a single integration site and a reproducible 3-fold induction of beta-galactosidase activity following UV irradiation. Fused transcripts were isolated from induced cells and a portion of the trapped gene was amplified by rapid amplification of cDNA ends. Sequence analysis and comparison with available gene and protein databanks revealed that the gene was novel.
Asunto(s)
Daño del ADN , Técnicas Genéticas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Células CHO , Clonación Molecular , Cricetinae , ADN/genética , ADN/aislamiento & purificación , Vectores Genéticos , Hibridación Fluorescente in Situ , Operón Lac , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Transfección , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
The tumor-suppressor p53 inhibits cell cycle progression by direct transactivation of the p21WAF1/CIP1/SDI1 gene, which encodes a universal inhibitor of cyclin dependent kinases (cdk). The proto-oncogene product c-Myc induces cell cycle progression and, in the absence of survival factors, apoptosis. However, a direct link between the cell cycle machinery and c-Myc has not yet been established. We show that c-Myc has not yet been established. We show that c-Myc abrogates a p53-induced G1-arrest without elevating the expression of cdks or cyclins involved in the G1/S-transition. Instead, the results suggest that c-Myc interferes with the inhibitory action of p21 on cdk/cyclin-complexes by inducing a heat-labile inhibitor of p21. The inactivation of p21 and related cdk-inhibitors may explain several of the oncogenic actions of c-Myc, including the induction of proliferation, immortalisation and the inhibition of differentiation. Modulation of cdk activity by the induction of an inhibitor of cdk-inhibitors represents a novel mechanism of cell cycle regulation in mammalian cells.
Asunto(s)
Ciclinas/antagonistas & inhibidores , Fase G1/genética , Genes p53 , Proteínas Proto-Oncogénicas c-myc/fisiología , Células 3T3 , Animales , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Activación Enzimática , Genes myc , Ratones , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
Infection of primary B-lymphocytes by Epstein-Barr virus (EBV) leads to growth transformation of these B-cells in vitro. EBV nuclear antigen 2 (EBNA2), one of the first genes expressed after EBV infection of B-cells, is a transcriptional activator of viral and cellular genes and is essential for the transforming potential of the virus. We generated conditional EBV mutants by expressing EBNA2 as chimeric fusion protein with the hormone binding domain of the estrogen receptor on the genetic background of the virus. Growth transformation of primary normal B-cells by mutant virus resulted in estrogen-dependent lymphoblastoid cell lines expressing the chimeric EBNA2 protein. In the absence of estrogen about half of the cells enter a quiescent non-proliferative state whereas the others die by apoptosis. EBNA2 is thus required not only for initiation but also for maintenance of transformation. Growth arrest occurred at G1 and G2 stages of the cell cycle, indicating that functional EBNA2 is required at different restriction points of the cell cycle. Growth arrest is reversible for G1/G0 cells as indicated by the sequential accumulation and modification of cell cycle regulating proteins. EBV induces the same cell cycle regulating proteins as polyclonal stimuli in primary B-cells. These data suggest that EBV is using a common pathway for B-cell activation bypassing the requirement for antigen, T-cell signals and growth factors.
Asunto(s)
Antígenos Virales/genética , Linfocitos B/virología , Ciclo Celular , Transformación Celular Viral , Proteínas de Unión al ADN/genética , Herpesvirus Humano 4/genética , Antígenos Virales/metabolismo , Ciclo Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr , Estrógenos/farmacología , Citometría de Flujo , Fase G1 , Fase G2 , Herpesvirus Humano 4/crecimiento & desarrollo , Humanos , Hibridación Fluorescente in Situ , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión , Transducción de SeñalRESUMEN
Permeabilization of L1210 cells by lithotripter shock waves in vitro was monitored by evaluating the accumulation of fluorescein-labeled dextrans with a relative molecular mass ranging from 3,900-2,000,000. Incubation with labeled dextran alone caused a dose- and time-dependent increase in cellular fluorescence as determined by flow cytometry, with a vesicular distribution pattern in the cells consistent with endocytotic uptake. Shock wave exposure prior to incubation with labeled dextran revealed similar fluorescence intensities compared to incubation with labeled dextran alone. When cells were exposed to shock waves in the presence of labeled dextran, mean cellular fluorescence was further increased, indicating additional internalization of the probe. Confocal laser scanning microscopy confirmed intracellular fluorescence of labeled dextran with a diffuse distribution pattern. Fluorescence-activated cell sorting with subsequent determination of proliferation revealed that permeabilized cells were viable and able to proliferate. Permeabilization of the membrane of L1210 cells by shock waves in vitro allowed loading of dextrans with a relative molecular mass up to 2,000,000. Permeabilization of tumor cells by shock waves provides a useful tool for introducing molecules into cells which might be of interest for drug targeting in tumor therapy in vivo.
Asunto(s)
Permeabilidad de la Membrana Celular , Membrana Celular/fisiología , Leucemia L1210/fisiopatología , Litotricia , Animales , Dextranos , Citometría de Flujo , Fluoresceína-5-Isotiocianato/análogos & derivados , Cinética , Ratones , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Human cord blood or bone marrow cells expressing the CD34 surface antigen include a population of pluripotent progenitors. We identified and isolated a subpopulation of cells coexpressing CD34 and c-kit, a transmembrane receptor with tyrosine kinase activity. Novel monoclonal antibodies (16A6, 14A3, 3D6) directed against the extracellular domain of c-kit were used for immunofluorescence labeling and sorting of low-density mononuclear cells (MNCs) from umbilical cord blood and bone marrow. The frequency of c-kit-labeled MNCs from cord blood (mean 5.0% +/- 2.1%, n = 16) was similar to that from adult bone marrow (mean 3.7% +/- 1.3%, n = 4). On average, 1.4% of CD34-positive cells were recorded in cord blood and 2.1% in bone marrow MNCs. Roughly 60% of CD34-positive cells coexpressed c-kit. The ability of CD34+/c-kit+ cells to form multilineage colonies (CFU-GEMM) was assayed after sorting with an antibody that did not show any significant effect on c-kit ligand (RL) or granulocyte/macrophage colony-stimulating factor (GM-CSF)-induced colony formation. For CD34+/c-kit+ cells, we found a 20- to 50-fold enrichment as against total MNCs, and a 2-fold enrichment if compared with the CD34+/c-kit-population. To study expression of c-kit in lymphocytic precursors, monoclonal anti-CD7 or anti-CD10 antibodies were used simultaneously. In contrast to CD34-expressing cells, however, no consistent double-labeled subpopulation of lymphocytic cells was detected. Furthermore, coexpression of CD38 (73% +/- 14%, n = 4) or CD33 (29% +/- 12%, n = 5) on a majority of c-kit-positive cells showed their lineage commitment to erythropoiesis and granulocytopoiesis.
Asunto(s)
Antígenos CD/análisis , Sangre Fetal/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas Proto-Oncogénicas/análisis , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Anticuerpos Monoclonales , Antígenos CD34 , Antígenos de Diferenciación/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Linfocitos B/metabolismo , Células de la Médula Ósea , Separación Celular , Células Cultivadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Granulocitos/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Macrófagos/citología , Glicoproteínas de Membrana , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas c-kit , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Linfocitos T/metabolismoRESUMEN
We investigated the ultrastructural effects of recombinant tumor necrosis factor alpha (TNF) on primary leukemia cells of 8 patients (4 cases of acute myelogenous leukemia and 4 cases of chronic myelogenous leukemia) as well as on bone marrow cells of 1 normal control. The cells were kept in liquid culture for up to 92 h in the presence of up to 10,000 U/ml of recombinant human TNF without adding colony-stimulating factors. Under these conditions the concentration of viable leukemic cells decreased by 14 to 53%, compared to control cultures. In acute myelogenous leukemia, all cases to some degree developed an enlargement of mitochondria; in 2 cases prominent cytoplasmic processes, and in 2 cases cytoplasmic vacuoles were observed. In chronic myelogenous leukemia, an enlargement and deformation of all cell types was observed to varying degrees. In the normal bone marrow sample only minor cytoplasmic changes occurred. In all cultures apoptotic changes were rarely observed and--if present--were observed also in cultures without TNF. When the DNA of leukemic cells treated with TNF was separated on agarose, no fragments characteristic of apoptosis were visible. Our results demonstrate that TNF does not induce direct cytotoxicity or apoptosis in acute or chronic myeloid leukemias and are compatible with the notion that some leukemic cells may be activated or stimulated by TNF. The mitochondrion appears to be one of the primary targets of TNF. Electron microscopy is useful for monitoring the changes induced by TNF.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucemia Mieloide Aguda/patología , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis , División Celular , Supervivencia Celular , Citoplasma/ultraestructura , ADN de Neoplasias/aislamiento & purificación , Electroforesis en Gel de Agar , Humanos , Microscopía Electrónica , Mitocondrias/ultraestructura , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/administración & dosificación , Vacuolas/ultraestructuraRESUMEN
We assume the existence of a specific G1 protein which is an initiator of DNA replication. This initiator is supposed to be synthesized according to Michaelis-Menten kinetics. In order to start DNA replication, it is assumed that this G1 specific protein must be produced in a required amount. Intra-cellular growth inhibitors and extra-cellular growth factors control the production of the initiator. This model allows to calculate the average G1 phase time as a function of the various chemical concentrations of nutrients, enzymes, growth inhibitors and growth factors. This model is compared to cell kinetics experiments on a leukemic cell line responding to Interleukin 3 deprivation. The curves giving the experimental average G1 phase times with respect to Interleukin-3 concentrations are fitted by the mathematical model with a quite good agreement.
Asunto(s)
Fase G1/fisiología , Interleucina-3/metabolismo , Leucemia Experimental/fisiopatología , Modelos Biológicos , Células Cultivadas , ADN/biosíntesis , CinéticaRESUMEN
In order to test a mathematical model of G1/S-phase transition, the proliferative response of the murine myeloid interleukin 3 (IL-3) dependent cell line NFS-78 to graded reduction of IL-3 levels was measured. Exponentially growing cells were exposed to bromodeoxyuridine (BUdR), which replaces thymidine (TdR) in the DNA double strands during DNA synthesis. After incubation periods ranging from 3 to 36 h the cells were fixed and stained with a fluorescence dye mixture of Hoechst 33258 and ethidium bromide (EB) and subsequently analyzed in a two-parametrical flow cytometer. The BUdR-quenched TdR-specific Hoechst 33258 fluorescence of each cell provides information on the cell cycle location at the start of incubation and on whether or not a cell has divided. The DNA-specific EB fluorescence provides information on the actual cell cycle location at the end of the incubation period. From the 2-dimensional fluorescence distributions the efflux from G1-phase was calculated. Upon IL-3 reduction the cells showed accumulation in the G1-phase along with a reduction in the progression rate through the other phases of the cell cycle. By staining with the vital dye Hoechst 33342 as well as with propidium iodide (PI) it was further possible to show that cell death after IL-3 withdrawal occurred in all phases of the cell cycle.
Asunto(s)
Fase G1/fisiología , Interleucina-3/metabolismo , Modelos Biológicos , Animales , Ciclo Celular/fisiología , Línea Celular , ADN/biosíntesis , Citometría de Flujo , RatonesRESUMEN
APO-1 is a cell surface molecule that induces apoptosis when ligated with the monoclonal antibody anti-APO-1. Expression of APO-1 and response to anti-APO-1 was investigated in a number of Epstein-Barr virus (EBV)-positive and -negative Burkitt lymphoma (BL) cell lines, in EBV-immortalized lymphoblastoid cell lines, and in cells from fresh BL biopsies. APO-1 was not expressed in EBV-negative cell lines and in EBV-positive BL cell lines with a phenotype corresponding to BL tumor biopsy cells (CD10+, CD21-, CD23-, CD30-, CD39-, CDw70-, CD77+). Accordingly, fresh BL cells obtained from three BL biopsies were APO-1 negative. EBV-positive BL cell lines that had acquired a lymphoblastoid phenotype (CD10-, CD21+, CD23+, CD30+, CD39+, CDw70+, CD77-) upon prolonged in vitro cultivation, as well as normal B-lymphoblastoid cell lines, expressed a high density of APO-1. APO-1 may, therefore, be regarded as a B-cell activation marker. APO-1 expression is not the only prerequisite for anti-APO-1-induced apoptosis because 6 of 7 APO-1-expressing EBV-positive BL cell lines were not sensitive to anti-APO-1, whereas all lymphoblastoid cell lines were killed by anti-APO-1. The sensitivity of lymphoblastoid cell lines to anti-APO-1-mediated apoptosis may open a new therapeutic approach for the treatment of EBV-induced lymphoproliferative lesions in immunocompromised individuals, because these are composed of cells with a lymphoblastoid phenotype.