RESUMO
During the past few years studies from several laboratories have utilized gene disruption approaches to define the function of members of the Stat family of transcription factors. The results have demonstrated that each family member has unique, critical, non-redundant functions in signal transduction through members of the cytokine receptor superfamily. Many of the family members mediate functions associated with innate or acquired immunity. With the availability of mice deficient in one or more of the Stats, critical experiments are possible to evaluate the roles of Stat signal transduction pathways in cellular transformation as well as evaluating their specific roles in a range of cellular responses to cytokines.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas do Leite , Receptores de Citocinas/fisiologia , Transdução de Sinais , Transativadores/fisiologia , Animais , Proteínas de Ligação a DNA/genética , Humanos , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , Fator de Transcrição STAT3 , Fator de Transcrição STAT4 , Fator de Transcrição STAT5 , Fator de Transcrição STAT6 , Transativadores/genéticaRESUMO
Janus kinase-3 (JAK3) deficiency has recently been identified as a cause of severe combined immunodeficiency (SCID) in humans. We used a mouse model of Jak3-deficient SCID to test a gene therapy approach for treatment of this disease. Transfer of a Jak3 retroviral vector to repopulating hematopoietic stem cells resulted in increased numbers of T and B lymphocytes, reversal of hypogammaglobulinemia, restoration of T-cell activation upon stimulation with mitogens, and development of an antigen-specific immune response after immunization. Analysis for vector copy number in lymphoid and myeloid populations showed a large in vivo selective advantage for Jak3-expressing lymphoid cells. These results show that gene replacement is a feasible treatment strategy for this disease and that naturally occurring in vivo selection of corrected cells is an important advantage of this approach.
Assuntos
Linfócitos B/imunologia , Terapia Genética/métodos , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/terapia , Linfócitos T/imunologia , Agamaglobulinemia/etiologia , Agamaglobulinemia/terapia , Animais , Formação de Anticorpos , Células da Medula Óssea/citologia , Transplante de Medula Óssea/métodos , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Janus Quinase 3 , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Reação em Cadeia da Polimerase , Proteínas Tirosina Quinases/biossíntese , Retroviridae , Imunodeficiência Combinada Severa/enzimologia , Baço/imunologiaRESUMO
The humoral immune response against endogenous ecotropic murine leukemia viruses (MuLV) was examined in irradiated and control C57BL/6 mice. Control mice developed antibodies against MuLV slowly throughout life. In contrast, within 2-3 mo after irradiation 90% of irradiated C57BL/6 mice had developed detectable antibodies against MuLV. The characteristics of this immune response, however, were identical in control and irradiated mice in terms of peak titers, specificity for endogenous ecotropic MuLV, and reactivity against the ecotropic viruses' glycoprotein (gp71). Moreover, the rate of appearance of antibodies against MuLV in irradiated mice and the peak titers were generally not affected by age at irradiation, dose of irradiation (two, three, or four treatments of 175 R), or bone marrow reconstitution. Although the ability of irradiation to accelerate the appearance of antibody in a population of C57BL/6 mice suggested activation of endogenous ecotropic MuLV, there was no apparent correlation between the appearance of this immune response or its persistence and the development of lymphoma. Thus, the incidence of lymphoma was comparable in mice that: (a) developed no immune response; (b) developed an immune response only transiently after irradiation; or (c) developed an immune response which persisted until death from lymphoma. Moreover, experimental conditions that alter the ability of irradiation to induce leukemia, such as age, dose, or bone marrow reconstitution did so without significantly altering either the rate of appearance of a humoral immune response to MuLV or its peak titers. The results, therefore, fail to demonstrate any seroepidemological relationship between endogenous ecotropic MuLV and radiation-induced leukemia.
Assuntos
Anticorpos Antivirais/biossíntese , Formação de Anticorpos/efeitos da radiação , Vírus da Leucemia Murina , Leucemia Induzida por Radiação , Linfoma/etiologia , Camundongos Endogâmicos C57BL/microbiologia , Vírus AKR da Leucemia Murina/imunologia , Fatores Etários , Animais , Epitopos , Vírus da Leucemia Murina de Friend/imunologia , Vírus da Leucemia Murina/imunologia , Leucemia Induzida por Radiação/etiologia , Leucemia Induzida por Radiação/imunologia , Camundongos , Camundongos Endogâmicos C57BL/imunologia , Timoma/etiologia , Neoplasias do Timo/etiologiaRESUMO
The radioimmune precipitation assay using (3)H-labeled AKR leukemia virus was applied to the detection and quantitation of natural serum antibodies directed against endogenous murine leukemia virus (MuLV) envelope antigens B6C3F(1) and BALB/c mice, which have low natural incidences of leukemia and lymphoma, and AKR mice, which have a high incidence, were used in this study. Sera from mice of various age groups were assayed. A marked difference in age-associated levels of the autogenous immune response to endogenous murine leukemia virus was detected, and the quantitative differences among these strains were inversely related to the incidence of lymphoma. The radioimmune precipitation test as applied was 500 times more sensitive than virus neutralization. That the reactions we have observed are specific is suggested by several lines of evidence, including the nonreactivity of normal hamster and absorbed rat serum, the positive reaction of absorbed rat anti-AKR serum, the inhibition of precipitation of labeled virus by purified unlabeled virus, and isopycnic gradient analysis of the reactive products.
Assuntos
Anticorpos Antivirais/análise , Vírus da Leucemia Murina/imunologia , Radioimunoensaio , Vírus AKR da Leucemia Murina/imunologia , Animais , Antígenos Virais , Células Cultivadas , Centrifugação com Gradiente de Concentração , Precipitação Química , Embrião de Mamíferos , Fibroblastos , Vida Livre de Germes , Cobaias/imunologia , Leucina/metabolismo , Linfoma/imunologia , Masculino , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Ratos/imunologia , Trítio , Cultura de VírusRESUMO
The expression of terminal deoxynucleotidyl transferase (TdT) in the thymus and bone marrow of irradiated mice has been examined. Mice given the leukemogenic regimen of irradiation of four weekly doses of 175 rads starting at 1 mo of age show a long-term elimination of TdT activity in the bone marrow and a reduction of TdT activity in thymocytes. In such mice, the reappearance of normal levels of TdT in the thymus appears to only be associated with the onset of overt leukemia. This effect on TdT expression was shown to be uniquely associated with the leukemogenic regimen of irradiation in that nonleukemogenic irradiation or variations such as bone marrow reconstitution or age which reduce leukemias did not show the same phenotypic effects on TdT expression. The basis for the loss of TdT-positive cells was shown not to be due to the lack of the requisite factors involved in differentiation, but rather to the ability of leukemogenic doses of irradiation to reduce or eliminate an inducible bone marrow stem cell. These results are discussed with respect to the possible mechanisms involved in radiation-induced leukemias in mice.
Assuntos
DNA Nucleotidilexotransferase/metabolismo , DNA Nucleotidiltransferases/metabolismo , Leucemia Experimental/enzimologia , Leucemia Induzida por Radiação/enzimologia , Animais , Medula Óssea/enzimologia , Relação Dose-Resposta à Radiação , Linfonodos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/enzimologia , Timectomia , Timo/enzimologiaRESUMO
The expression of endogenous ecotropic viruses in radiation-induced thymomas of C57BL/6 mice was examined. Competition radioimmunoassays for AKR MuLV gp71, p30, and p12 were used for viral antigen expression. 3 of 40 lymphomas had readily detectable ecotropic gp71 at levels of 95-689 ng/mg protein; the remainder of the tumors had no detectable gp71 (less than 1.0 ng/mg protein). 30 thymomas were characterized by the presence of MuLV p30 at levels of 1-10 ng/mg protein, levels that were comparable to those found in thymus extracts from age-matched, nonirradiated control. 10 tumors were characterized by having p30 levels of 10-30 ng/mg protein. In one tumor significant levels of AKR MuLV p12 were detectable. Since B-tropic and N-tropic viruses from C57BL/6 mice have glycoproteins (gp71) indistinguishable from AKR MuLV gp71 and the N-tropic virus had a p12 serologically identical to AKR MuLV p12, these results demonstrate that overt endogenous B-tropic virus was detectable in 2 of 40 thymomas and endogenous N-tropic virus was detectable in 1 of 40 thymomas. The lack of overt expression of gp71 or p12 was also confirmed by cytotoxicity assays using monospecific antisera to these viral proteins. Radiation-induced lymphomas were also examined for the presence of reverse transcriptase after chromatography of tissue extracts on poly G-Sepharose. One tumor, which was characterized by the lack of gp71, also had no detectable reverse transcriptase; whereas one tumor with gp71 was characterized by readily detectable levels of reverse transcriptase in cellular extracts. The presence of viral RNA was examined using AKR cDNA. Low levels of RNA capable of hybridizing with AKR cDNA were found in age-matched, nonirradiated mice; these hybrids had Tm's of 72 degrees C, while hybrids with AKR MuLV 70S RNA had Tm's of 80 degrees C. In 1 of 12 thymomas the concentration of hybridizable RNA and the Tm of the hybrids were identical to control values. In 9 of 12 thymomas the concentration of hybridizable sequences increased approximately three-to fivefold and the Tm of these hybrids varied from 73 to 75 degrees C. In 1 of 12 thymomas the concentration of hybridizable sequences increased over 100-fold, hybridized completely with AKR MuLV cDNA, and the hybrids had Tm's of 79 degrees C. This thymoma was also characterized by the presence of the AKR MuLV type of gp71 and p12. One tumor was characterized by a 10-to 100-fold increase in hybridizable sequences, which only partially hybridized with AKR MuLV cDNA, and hybrids had a Tm of 73 degrees C. This tumor was characterized by the presence of AKR MuLV gp71 but not AKR MuLV p12. The results taken together demonstrate that overt endogenous ecotropic virus expression is only rarely detectable in radiation-induced thymomas of C57BL/6 mice.
Assuntos
Antígenos Virais/análise , Vírus da Leucemia Murina , Leucemia Induzida por Radiação , Camundongos Endogâmicos C57BL/microbiologia , RNA Viral/análise , Timoma/microbiologia , Neoplasias do Timo/microbiologia , Vírus AKR da Leucemia Murina , Animais , Reações Antígeno-Anticorpo , Testes Imunológicos de Citotoxicidade , Vírus da Leucemia Murina/imunologia , Leucemia Induzida por Radiação/microbiologia , Camundongos , DNA Polimerase Dirigida por RNA/análise , Timoma/etiologia , Neoplasias do Timo/etiologiaRESUMO
The hematopoietic cell kinase (hck) is a member of the src family of tyrosine kinases, and is primarily expressed in myeloid cells. Hck expression increases with terminal differentiation in both monocyte/macrophages and granulocytes and is further augmented during macrophage activation. Recent evidence has implicated src-related tyrosine kinases in critical signaling pathways in other hematopoietic lineages. Herein we demonstrate that manipulation of the level of hck expression in the murine macrophage cell line BAC1.2F5 alters the responsiveness of these cells to activation by bacterial lipopolysaccharide (LPS) but does not affect survival or proliferation. Overexpression of an activated mutant of hck in BAC1.2F5 cells augments tumor necrosis factor (TNF) production in response to LPS, whereas inhibition of endogenous hck expression, by antisense oligonucleotides, interferes with LPS-mediated TNF synthesis. Together, these observations suggest that hck is an important component of the signal transduction pathways in activated macrophages.
Assuntos
Macrófagos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Sequência de Bases , Benzoquinonas , Linhagem Celular , Expressão Gênica , Lactamas Macrocíclicas , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Oligonucleotídeos Antissenso/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-hck , Quinonas/farmacologia , RNA Mensageiro/genética , Rifabutina/análogos & derivadosRESUMO
The expression of the murine TCR-gamma genes was examined in a series of IL-3-dependent and growth factor-independent cell lines. All of the IL-3-dependent cell lines, but none of the IL-3-independent lines, expressed high levels of one or more of the gamma genes but did not express the TCR-beta genes. None of the cell lines expressing the gamma loci contained detectable genomic gamma gene rearrangements. Sequencing of cDNA clones from two of the cell lines demonstrated that transcription was from nonrearranged gamma loci based on the presence of sequences in the cDNAs that are found immediately 5' of the J gamma 4 and J gamma 2 genes. The expression of gamma transcripts was dependent upon IL-3 and no transcripts were detectable within 6-8 h after the removal of IL-3. Readdition of IL-3, but not granulocyte CSF, resulted in the reappearance of gamma transcripts within 30 min. The results demonstrate that IL-3 regulates the expression of nonrearranged gamma loci. Since expression is required for rearrangement, it can be hypothesized that IL-3 may influence the ability of lymphoid/myeloid progenitors to commit to the T cell lineage.
Assuntos
Rearranjo Gênico do Linfócito T , Interleucina-3/fisiologia , Receptores de Antígenos de Linfócitos T/genética , Transcrição Gênica , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Linhagem Celular , Clonagem Molecular , DNA/isolamento & purificação , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T gama-delta , Infecções por Retroviridae/genéticaRESUMO
By competition radioimmune assays with antisera against AKR murine leukemia virus (MuLV) gp 71 or antisera against xenotropic virus, and iodinated AKR MuLV gp71 or BALB(X) gp71, antigens serologically indistinguishable from the viral antigens can be detected in tissues of normal mice in the absence of overt virus expression. An antigen serologically indistinguishable from AKR MuLV gp71 can be readily detected in normal bone marrow cells of the common strains of mice including NIH Swiss, 129/J, and SWR/J, as well as in Mus cervicolor and Mus musculus casteneus. In contrast, this antigen is not detected in normal spleen, thymus, lymph nodes, or serum. Similarly, an antigen serologically indistinguishable from BALB(X) gp71 was found in all normal mouse sera examined. This antigen was not present in fetal liver, perfused adult liver, thymus, spleen, lymph nodes, or bone marrow of the mice examined. An equivalent antigen was detected in sera from Mus musculus casteneus but not in sera from Mus cervicolor.
Assuntos
Vírus AKR da Leucemia Murina/imunologia , Antígenos Virais , Vírus da Leucemia Murina/imunologia , Animais , Antígenos Virais/análise , Medula Óssea/análise , DNA Viral/análise , Camundongos , Camundongos Endogâmicos , Hibridização de Ácido Nucleico , Especificidade de Órgãos , RNA Viral/análise , Radioimunoensaio , Ratos , Ratos Endogâmicos F344 , Proteínas Virais/análiseRESUMO
Terminal deoxynucleotidyl transferase (TdT) expression in bovine serum albumin (BSA) gradient-fractionated bone marrow cells was examined in NIH Swiss nu/nu and thymectomized C57BL/6 mice. In nude mice, TdT levels were approximately 10% of those of thymus-bearing littermates. In C57BL/6 mice, thymectomy caused a time-dependent loss of TdT activity in bone marrow cells. To determine whether or not not the apparent thymic requirement for TdT expression in bone marrow was mediated by thymic hormones, we examined the effects of thymosin fraction 5. Treatment of either NIH Swiss nu/nu or thymectomized C57BL/6 mice with thymosin fraction 5 restored the levels of TdT activity in BSA gradient-fractionated bone marrow cells to normal. Moreover, treatment of BSA gradient-fractionated bone marrow cells from NIH Swiss nu/nu or thymectomized C57BL/6 mice in tissue culture with thymosin fraction 5 induced TdT expression. In tissue culture, TdT induction was optimal with 25 ng/ml of thymosin fraction 5, it occurred within 6 h, and it was completely inhibited by actinomycin D. The effect was specific in that neither control nor spleen fraction 5-treated cells were induced to express TdT. These data demonstrate that TdT expression in bone marrow cells is under the direct control of thymic polypeptide hormones.
Assuntos
Medula Óssea/enzimologia , DNA Nucleotidiltransferases/biossíntese , Timosina/farmacologia , Hormônios do Timo/farmacologia , Animais , Células Cultivadas , Indução Enzimática/efeitos dos fármacos , Linfonodos/enzimologia , Camundongos , Camundongos Nus/metabolismo , Baço/enzimologia , TimectomiaRESUMO
The viral antigenic determinants recognized in an autogenous immune response in mice against their endogenous C-type virus have been identified by SDS-polyacrylamide gel electrophoresis of immune precipitates between various sera and H(3)-labeled intact or disrupted AKR leukemia virus. Normal B6C3F(1) [(C57BL/6 x C3H/Anf)F(1)] serum reacts with viral envelope antigens having mol wt of approximately 68,000, 43,000, and 17,000. In addition, minor reactions with viral antigens having mol wts of approximately 19,000 and 15,000 are demonstrable. The 68,000 and 43,000 mol wt antigens can be labeled with [(3)H]glucosamine and may correspond to the major viral envelope antigens M(2) and M(1), respectively. The antigens recognized by autogenous immune sera do not differ with respect to age of the animal, nor are they significantly different in sera from various strains of mice (BALB/c, C57BL/6, and C3H/Anf). These results suggest that the age-asociated and strain variations in the autogenous immune response, as determined by radioimmune precipitation assays against intact virus, are due to quantitative and qualitative alterations of antibody levels against common antigens.
Assuntos
Anticorpos Antivirais , Isoanticorpos , Vírus da Leucemia Murina/imunologia , Fatores Etários , Animais , Antígenos Virais/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Feminino , Glucosamina/metabolismo , Soros Imunes , Marcação por Isótopo , Leucina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Testes de Precipitina , Vírus Rauscher/efeitos dos fármacos , Vírus Rauscher/imunologia , Tensoativos/farmacologia , TrítioRESUMO
The genetic linkage of the endogenous C3H/HeJ C-type ecotropic virus to phosphoglucomutase-1 (0.28, recombinant fraction) on chromosome 5 was established by means of serological assays of backcrossed mice. With a combination of serological techniques and DNA-DNA hybridization the BALB/c endogenous ecotropic virus was shown to be either closely linked or allelic with the C3H/HeJ locus.
Assuntos
Genes Virais , Camundongos Endogâmicos BALB C/microbiologia , Camundongos Endogâmicos C3H/microbiologia , Fosfoglucomutase/genética , Retroviridae/genética , Alelos , Animais , Genes , Ligação Genética , Vírus da Leucemia Murina/genética , Camundongos , Camundongos Endogâmicos/genética , FenótipoRESUMO
The interleukin-2 receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta, and IL-2R gamma chains, the last of which is also used in the receptors for IL-4, IL-7, and IL-9. Stimulation with IL-2 induces the tyrosine phosphorylation and activation of the Janus kinases Jak1 and Jak3. Jak1 and Jak3 were found to be selectively associated with the "serine-rich" region of IL-2R beta and the carboxyl-terminal region of IL-2R gamma, respectively. Both regions were necessary for IL-2 signaling. Furthermore, Jak3-negative fibroblasts expressing reconstituted IL-2R became responsive to IL-2 after the additional expression of Jak3 complementary DNA. Thus, activation of Jak1 and Jak3 may be a key event in IL-2 signaling.
Assuntos
Interleucina-2/farmacologia , Proteínas Tirosina Quinases/metabolismo , Receptores de Interleucina-2/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Humanos , Janus Quinase 1 , Janus Quinase 3 , Receptores de Interleucina-2/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tirosina/metabolismoRESUMO
The Janus tyrosine kinases (Jaks) play a central role in signaling through cytokine receptors. Although Jak1, Jak2, and Tyk2 are widely expressed, Jak3 is predominantly expressed in hematopoietic cells and is known to associate only with the common gamma (gamma c) chain of the interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 receptors. Homozygous mutant mice in which the Jak3 gene had been disrupted were generated by gene targeting. Jak3-deficient mice had profound reductions in thymocytes and severe B cell and T cell lymphopenia similar to severe combined immunodeficiency disease (SCID), and the residual T cells and B cells were functionally deficient. Thus, Jak3 plays a critical role in gamma c signaling and lymphoid development.
Assuntos
Linfócitos B/imunologia , Proteínas Tirosina Quinases/fisiologia , Linfócitos T/imunologia , Envelhecimento/imunologia , Animais , Antígenos CD/metabolismo , Quimera , Feminino , Marcação de Genes , Interleucina-7/metabolismo , Interleucina-7/farmacologia , Janus Quinase 3 , Ativação Linfocitária , Contagem de Linfócitos , Subpopulações de Linfócitos/imunologia , Tecido Linfoide/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Proteínas Tirosina Quinases/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-7 , Transdução de SinaisRESUMO
A variety of cytokines, lymphokines and growth factors function by interacting with receptors that are members of the cytokine receptor superfamily. These receptors share extracellular motifs and have limited similarity in their cytoplasmic domains. Although lacking catalytic domains, this family of receptors couples ligand binding with the induction of tyrosine phosphorylation. Recent studies have shown that this is mediated by members of the Janus kinase (JAK) family of cytoplasmic protein tyrosine kinases. JAKs physically associate with the membrane-proximal region of the ligand-bound receptor, leading to their tyrosine phosphorylation and activation. The activated JAKs phosphorylate the receptors as well as cytoplasmic proteins belonging to a family of transcription factors called the signal transducers and activators of transcription (STATs), providing a novel signaling pathway that is shared by all members of the cytokine receptor superfamily.
Assuntos
Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas , Receptores de Citocinas/fisiologia , Transdução de Sinais , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Citocinas/fisiologia , Janus Quinase 1 , Janus Quinase 2 , Dados de Sequência Molecular , Proteínas Tirosina Quinases/química , Homologia de SequênciaRESUMO
Many cytokines mediate their biological effects through interaction with a distinct family of receptors termed the cytokine receptor superfamily. Although members of this family lack catalytic domains, they couple ligand binding to tyrosine phosphorylation. Recent studies have shown that a novel family of cytoplasmic protein tyrosine kinases, termed the Janus kinases (Jaks), associate with the cytokine receptors and are catalytically activated after ligand binding. The activated Jaks phosphorylate and activate members of a novel family of transcription factors termed signal transducers and activators of transcription (Stats). In addition, many cytokines induce the phosphorylation of SHC, Vav and the p85 subunit of PI-3 kinase. The region of the receptors proximal to the cytoplasmic membrane is required for Jak association, mitogenesis, Stat activation and Vav phosphorylation. The membrane-distal region, which contains the major sites of tyrosine phosphorylation, is required for phosphorylation of SHC and p85, not for mitogenesis, thus allowing functional dissection of the signaling pathways activated by cytokines.
Assuntos
Citocinas/fisiologia , Proteínas de Ligação a DNA/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/fisiologia , Proteínas/fisiologia , Proteínas Proto-Oncogênicas , Receptores de Citocinas/fisiologia , Transdução de Sinais , Transativadores/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Consenso , Proteínas de Drosophila , Ativação Enzimática , Janus Quinase 1 , Janus Quinase 2 , Janus Quinase 3 , Janus Quinases , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Fosforilação , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , TYK2 Quinase , Fatores de TranscriçãoRESUMO
When spleen cells of 5-fluorouracil (5-FU)-treated mice were cultured in the presence of interleukin 3 (IL-3), most colonies revealed IL-3 concentration-dependent colony formation except for mast cell colonies and blast cell colonies. While most colonies were smaller in lower concentrations of IL-3, the size of the blast cell colonies were similar between high and low IL-3 groups. These data suggested that blast cell colony development requires less IL-3 than the development of multilineage colonies from blast cell colonies. This notion was supported by experiments in which IL-3 was added twice to cultures of spleen cells of 5-FU-treated mice. When low concentrations of IL-3 were added on day 7, there was a reduction in the number of multilineage colonies formed without an effect on the number of blast cell colonies. Using this information, we developed a culture system that favors blast cell colony formation by cells of normal mice. When low (20 U/ml) concentrations of IL-3 were added to cultures of spleen cells of normal mice on day 7 of incubation in media containing 2-5% fetal calf serum, blast cell colonies were the predominant colony type. The blast cell colonies revealed high but variable secondary replating efficiencies. These data suggest that multipotential progenitors may become less sensitive to IL-3 as they differentiate in culture.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Linfocinas/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Meios de Cultura , Feminino , Interleucina-3 , Camundongos , Baço/citologiaRESUMO
Medium conditioned by activated T lymphocytes stimulates the in vitro proliferation of pluripotent hematopoietic stem cells (spleen colony-forming units [CFU-S]) but the factors involved have not been identified. Because the lymphokine interleukin 3 (IL-3) enhances in vitro colony formation by committed hematopoietic progenitor cells, we examined the effect of IL-3 on the in vitro proliferation of CFU-S using an 11-d spleen colony assay. When mouse marrow cells were placed in liquid culture, CFU-S content declined progressively and by 96 h only 13% of the CFU-S remained. By contrast, after 96 h in the presence of 20 U/ml of IL-3, the number of CFU-S were the same as that in the initial inoculum. Although the number of CFU-S eventually declined, they could still be recovered after 264 h of culture. In the absence of IL-3, the number of CFU-S synthesizing DNA was negligible; in its presence, greater than 20% of the CFU-S were in cycle. IL-3 stimulated CFU-S proliferation at a concentration of 0.2 U/ml. The dose-response curve was similar to that observed for other biologic effects of the lymphokine, and as little as 1 h of exposure to IL-3 enhanced the survival of CFU-S in vitro. Treatment of marrow cells with anti-Thy 1.2 antibody and complement before exposure to IL-3 did not inhibit spleen colony formation, but treatment of the cells with anti-Thy 1.2 antibody and complement after exposure to IL-3 reduced CFU-S recovery after 96 h of culture by 45%. The cell composition of day 11 spleen colonies formed by IL-3-treated marrow cells was similar to that of colonies formed by untreated marrow cells. Finally, day 11 CFU-S persisting in the marrow of mice treated with 5-fluorouracil required IL-3 for proliferation in vitro. Taken together, these data indicate that IL-3 promotes the proliferation of CFU-S in vitro, increases the number of CFU-S synthesizing DNA, but does not alter their commitment program, and the target cell population includes CFU-S with self-renewal and marrow-repopulating ability.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Linfocinas/farmacologia , Animais , Células da Medula Óssea , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Meios de Cultura/farmacologia , Relação Dose-Resposta a Droga , Interleucina-3 , Camundongos , Baço , Linfócitos T/metabolismoRESUMO
Protein tyrosine phosphorylation and dephosphorylation have been implicated in the growth and functional responses of hematopoietic cells. Recent studies have identified a novel protein tyrosine phosphatase, termed hematopoietic cell phosphatase (HCP) or PTP1C, that is predominantly expressed in hematopoietic cells. HCP encodes a cytoplasmic phosphatase that contains two src homology 2 (SH2) domains. Since SH2 domains have been shown to target the association of signal-transducing molecules with activated growth factor receptors containing intrinsic protein kinase activity, we assessed the association of HCP with two hematopoietic growth factor receptors, c-Kit and c-Fms. The results demonstrate that HCP transiently associates with ligand-activated c-Kit but not c-Fms and that this association occurs through the SH2 domains. In both colony-stimulating factor 1- and stem cell factor-stimulated cells, there is a marginal increase in tyrosine phosphorylation of HCP. Lastly, HCP can dephosphorylate autophosphorylated c-Kit and c-Fms in in vitro reactions. The potential role of HCP in stem cell factor signal transduction is discussed.
Assuntos
Fatores de Crescimento de Células Hematopoéticas/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Western Blotting , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Fatores de Crescimento de Células Hematopoéticas/isolamento & purificação , Humanos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Megacariócitos , Dados de Sequência Molecular , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosfotirosina , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas c-kit , Receptor de Fator Estimulador de Colônias de Macrófagos/isolamento & purificação , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptores de Fator Estimulador de Colônias/isolamento & purificação , Receptores de Fator Estimulador de Colônias/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Fator de Células-Tronco , Células Tumorais Cultivadas , Tirosina/análogos & derivados , Tirosina/análiseRESUMO
Activation of the Evi-1 zinc finger gene is a common event associated with transformation of murine myeloid leukemias. To characterize the gene product, we developed antisera against various protein domains. These antisera primarily detected a 145-kilodalton nuclear protein that bound double-stranded DNA. Binding was inhibited by chelating agents and partially restored by zinc ions.