RESUMO
The incidence of prostate cancer is increasing in western countries because of population aging. Prostate cancer begins as an androgen-dependent disease, but it can become androgen independent at a later stage or in tumors recurring after an antihormonal treatment. Although many genetic events have been described to be involved in androgen-dependent and/or -independent prostate cancer growth, little is known about the contribution of epigenetic events. Here we have examined the possibility that the methyl-CpG-binding protein MECP2 might play a role in controlling the growth of prostate cancer cells. Inhibition of MECP2 expression by stable short hairpin RNA stopped the growth of both normal and cancer human prostate cells. In addition, ectopic expression of the MECP2 conferred a growth advantage to human prostate cancer cells. More importantly, this expression allowed androgen-dependent cells to grow independently of androgen stimulation and to retain tumorigenic properties in androgen-depleted conditions. Analysis of signaling pathways showed that this effect is independent of androgen receptor signaling. Instead, MECP2 appears to act by maintaining a constant c-myc level during antihormonal treatment. We further show that MECP2-expressing cells possess a functional p53 pathway and are still responsive to chemotherapeutic drugs.
Assuntos
Proliferação de Células , Proteína 2 de Ligação a Metil-CpG/fisiologia , Neoplasias da Próstata/patologia , Androgênios/fisiologia , Antineoplásicos Hormonais/farmacologia , Sobrevivência Celular , Humanos , Masculino , Próstata/citologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores Androgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Two distinct c-mil-related cDNA clones have been isolated from a chicken embryo cDNA library. Results presented here show that the single chicken c-mil gene is coding for two c-mil mRNA species, different by at least 60 base pairs and generated by an alternative splicing mechanism. These mRNA molecules can be translated into two distinct proteins of 73 and 71 kilodaltons.
Assuntos
Proto-Oncogenes , Splicing de RNA , RNA Mensageiro/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Clonagem Molecular , Enzimas de Restrição do DNA , Éxons , Dados de Sequência MolecularRESUMO
After differential screening of a cDNA library constructed from quail neuroretina cells (QNR) infected with the v-myc-containing avian retrovirus MC29, we have isolated a cDNA clone, Pax-QNR, homologous to the murine Pax-6, which is mutated in the autosomal dominant mutation small eye of mice and in the disorder aniridia in humans. Here we report the characterization of the Pax-QNR proteins expressed in the avian neuroretina. From bacterially expressed Pax-QNR peptides, we obtained rabbit antisera directed against different domains of the protein: paired domain (serum 11), domain between the paired domain and homeodomain (serum 12), homeodomain (serum 13), and carboxyl-terminal part (serum 14). Sera 12, 13, and 14 were able to specifically recognize five proteins (48, 46, 43, 33, and 32 kDa) in the neuroretina. In contrast to proteins of 48, 46, and 43 kDa, proteins of 33 and 32 kDa were not recognized by the paired antiserum (serum 11). Paired-less and paired-containing proteins exhibited the same half-life (6 h) and were phosphorylated mostly on serine residues. Immunoprecipitations performed with subcellular fractions of neuroretinas showed that the paired-containing proteins were located in the nucleus, whereas the 33- and 32-kDa proteins were found essentially in the cytoplasmic compartment. However, immunofluorescence experiments performed after transient transfections showed that p46 and p33/32 were also located in vivo into the nucleus. Thus, the Pax-QNR/Pax-6 gene can produce proteins with two DNA-binding domains as well as proteins containing only the DNA-binding homeodomain.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Codorniz/metabolismo , Retina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Anticorpos , Sequência de Bases , Embrião de Galinha , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Fosforilação , Codorniz/genética , Coelhos , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
Expression of the P100gag-mil protein of avian retrovirus MH2 in cultured chicken embryo neuroretina cells was previously shown to result in the proliferation of normally quiescent cell populations. We show here that long terminal repeat activation of the carboxy terminus of the c-mil gene is sufficient to induce neuroretina cell proliferation.
Assuntos
Divisão Celular , Proteínas Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Retina/citologia , Animais , Embrião de Galinha , Mapeamento Cromossômico , Coturnix , Regulação da Expressão Gênica , Produtos do Gene gag , Sequências Repetitivas de Ácido Nucleico , Proteínas dos Retroviridae/genética , TransfecçãoRESUMO
Rel/nuclear factor kappaB transcription factors were shown to have either pro- or antiapoptotic as well as pro- or antiproliferative functions, and it is often assumed that the outcome of their activation depends on the cell type or cellular context. Inconsistent with this assumption, we show here that cRel is able in one cell type to inhibit proliferation, protect against apoptosis induced by tumor necrosis factor alpha (TNF-alpha) + cycloheximide (CHX), and increase the basal rate of apoptosis. Both the effects of proliferation inhibition and protection against TNF-alpha + CHX-induced apoptosis are massive and occur in the same cells. Using reverse transcription-PCR, Western blot and immunofluorescence, and transactivation assays, we found that the manganese superoxide dismutase (MnSOD), an enzyme that converts O2*- in H2O2, is up-regulated by cRel through a kappaB site in intron 2. Inhibition of MnSOD induction by antisense oligonucleotides and overexpression of MnSOD respectively reverts and mimics both the antiproliferative and antiapoptotic effects of cRel, suggesting that they both occur via the induction of this gene. On one hand, MnSOD could improve the efficiency of cRel-overexpressing cells in eliminating toxic O2*- produced on TNF-alpha treatment, explaining why they escape TNF-alpha-induced apoptosis. On the other hand, cRel-overexpressing cells should accumulate H2O2. We present evidence linking this H2O2 accumulation to the proliferation arrest induced by cRel. Therefore, different effects on proliferation and apoptosis could arise from the induction of MnSOD and thus coexist in cRel-overexpressing cells.
Assuntos
Apoptose/fisiologia , Proteínas Proto-Oncogênicas c-rel/fisiologia , Superóxido Dismutase/biossíntese , Apoptose/efeitos dos fármacos , Sequência de Bases , Ciclo Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Cicloeximida/toxicidade , Regulação Enzimológica da Expressão Gênica , Vetores Genéticos , Células HeLa , Humanos , Peróxido de Hidrogênio/metabolismo , Íntrons/genética , Mitocôndrias/enzimologia , NF-kappa B/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Oxirredução , Inibidores da Síntese de Proteínas/toxicidade , Proteínas Proto-Oncogênicas c-rel/biossíntese , Proteínas Proto-Oncogênicas c-rel/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/fisiologiaRESUMO
We have previously shown that introduction of the v-myc oncogene in chick or quail embryos at E3 induces rapidly growing heart rhabdomyomas. We now report that a retrovirus containing one or two other oncogenes induces additional pathologies specified by the v-myc-associated oncogene. The v-mil/myc combination introduced at E3 induces, in addition to heart rhabdomyomas, tumors of proliferating cells aggregated onto the luminal aspect of vessels in both chick and quail embryos. In the quail these cells react positively with the quail-specific mAb QH1, which recognizes endothelial and most hemopoietic cells, while chick intravascular cells do not react with the chick-specific mAb VIA2 that recognizes hemopoietic cells. Thus the v-mil/myc tumors appear to be of endothelial origin. The v-myb-ets/myc combination injected at E3 induces cardiorhabdomyomas and aggressive VIA2-positive hemopoietic tumors in chick embryos, but only the v-myc-induced cardiorhabdomyomas in quail embryos. When injected into hatched animals, v-myc alone transforms hemopoietic and perhaps endothelial cells, but not cardiac cells. Thus the developmental stage at which a cell type can be transformed by v-myc and another associated oncogene depends on as yet undefined species-specific factors. More importantly, several examples of oncogene cooperation in vivo are adduced by these experiments. The type of cell transformed is specified by the viral oncogene combination.
Assuntos
Transformação Celular Neoplásica/genética , Genes myc/genética , Neoplasias Cardíacas/genética , Oncogenes , Rabdomioma/genética , Neoplasias Cutâneas/genética , Animais , Embrião de Galinha , Embrião não Mamífero , Imunofluorescência , Proteínas Oncogênicas v-raf , Oncogenes/genética , Proteínas Tirosina Quinases/genética , Codorniz , Proteínas Oncogênicas de Retroviridae/genéticaRESUMO
We show that a construct designated as MAHEVA, which encodes oncogenes v-myc from MH2 virus and v-erbA from AEV under the control of the LTR of MH2, induces rapidly growing heart rhabdomyosarcomas, when it is injected in E3 but not E5 chick embryos. A similar pathology has previously been observed with MC29, within the same limited time frame. The tumors, which expressed P61-63myc, P75gag-erbA and Pr76gag proteins were detectable from E14 onwards. Compared with MC29, MAHEVA induced a secondary anomaly, not detectable prior to E17. This is the appearance of cartilage nodules within the heart rhabdomyosarcomas. The constant location of these nodules inside the rhabdomyosarcomas and their delayed appearance suggests that the chondrocytes originate from myoblasts prevented from differentiating by the expression of the v-myc product. This interpretation is supported by the appearance of chondrocytes in E3 heart muscle cells infected in vitro with MAHEVA.
Assuntos
Embrião de Mamíferos/efeitos dos fármacos , Embrião não Mamífero , Desenvolvimento Embrionário e Fetal/genética , Proteína Oncogênica p55(v-myc)/farmacologia , Proteínas Oncogênicas de Retroviridae/farmacologia , Animais , Cartilagem/patologia , Linhagem Celular , Embrião de Galinha , Sinergismo Farmacológico , Expressão Gênica/genética , Neoplasias Cardíacas/induzido quimicamente , Neoplasias Cardíacas/metabolismo , Neoplasias Cardíacas/patologia , Proteína Oncogênica p55(v-myc)/genética , Proteína Oncogênica p55(v-myc)/metabolismo , Proteínas Oncogênicas v-erbA , Testes de Precipitina , Proteínas Oncogênicas de Retroviridae/genética , Proteínas Oncogênicas de Retroviridae/metabolismo , Rabdomiossarcoma/induzido quimicamente , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Transcrição Gênica/genéticaRESUMO
The retina is an integral part of the central nervous system, and consists of two layers, the outer pigmented layer and the inner sensory layer or neuroretina (NR). The NR layer contains several strata of cells (glial and neuronal) derived from proliferating neuroectodermal precursors that differentiate after terminal mitosis. In vitro, NR cells can differentiate not only into neuronal and glial types, but also into pigment and lens cells. Quail (Coturnix coturnix japonica) NR cells (QNR) infected with MC29 transforming retrovirus become pigmented after several passages in vitro. In order to characterize the genes expressed in these pigmented MC29 QNR, a cDNA library was prepared from these cells. After differential screening we have isolated a cDNA clone which identifies an RNA expressed in NR but not in the pigmented layer of the retina. This cDNA encodes a protein related to that of Drosophila, mouse and zebrafish paired box- and homeobox-containing segmentation genes and is called Pax-QNR. The expression of Pax-QNR in the NR is confined to the ganglionic cell layer and to the lower part of the inner nuclear layer containing the amacrine or correlation neurones.
Assuntos
Coturnix/genética , Expressão Gênica , Genes Homeobox , Retina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/isolamento & purificação , Biblioteca Gênica , Dados de Sequência Molecular , Mutação , Hibridização de Ácido Nucleico , Especificidade de ÓrgãosRESUMO
Human mature erythrocytes have been considered as unable to undergo programmed cell death (PCD), due to their lack of mitochondria, nucleus and other organelles, and to the finding that they survive two conditions that induce PCD in vitro in all human nucleated cells, treatment with staurosporine and serum deprivation. Here we report that mature erythrocytes can undergo a rapid self-destruction process sharing several features with apoptosis, including cell shrinkage, plasma membrane microvesiculation, phosphatidylserine externalization, and leading to erythrocyte disintegration, or, in the presence of macrophages, to macrophage ingestion of dying erythrocytes. This regulated form of PCD was induced by Ca(2+) influx, and prevented by cysteine protease inhibitors that allowed erythrocyte survival in vitro and in vivo. The cysteine proteinases involved seem not to be caspases, since (i) proforms of caspase 3, while present in erythrocytes, were not activated during erythrocyte death; (ii) cytochrome c, a critical component of the apoptosome, was lacking; and (iii) cell-free assays did not detect activated effectors of nuclear apoptosis in dying erythrocytes. Our findings provide the first identification that a death program can operate in the absence of mitochondria. They indicate that mature erythrocytes share with all other mammalian cell types the capacity to self-destruct in response to environmental signals, and imply that erythrocyte survival may be modulated by therapeutic intervention.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Eritrócitos/fisiologia , Mitocôndrias/fisiologia , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Caspase 3 , Caspases/metabolismo , Caspases/farmacologia , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Eritrócitos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leupeptinas/metabolismo , Leupeptinas/farmacologia , Ativação de Macrófagos/imunologia , Camundongos , Modelos Biológicos , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologiaRESUMO
In the ets gene family of transcription factors, elk1 belongs to the subfamily of Ternary Complex Factors (TCFs) which bind to the Serum Response Element (SRE) in conjunction with a dimer of Serum Response Factors (SRFs). In this communication we report the isolation of cDNAs from the mouse elk1 gene, containing the full coding sequence homologous (87% identical) to the human gene, and the structure and organization of 22 kb of the mouse elk1 locus. The coding sequence is spread through 5 exons (numbered 1 to 5): exons 1 to 4 range from 102 bp to 447 bp and exon 5 is at least 620 bp. Exon 0 was not found in the 8.5 kb sequence upstream of exon 1. The intron between exons 1 and 2 is 4 kb long and the 3 other introns are less than 500 bp long. This information will be useful to engineer targeted mutations of this gene in mice and to determine the genomic structure of the other TCF genes.
Assuntos
Proteínas de Ligação a DNA , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Genoma , Humanos , Camundongos , Dados de Sequência Molecular , Análise de Sequência de DNA , Proteínas Elk-1 do Domínio etsRESUMO
After in vitro EBV infection of peripheral blood lymphocytes (PBL), we previously obtained IL-2-independent T-cell lines expressing EBNA1 and LMP1 viral latent genes. One tumorigenic clone, NC5, was further characterized for chromosomal abnormalities, rearrangement and expression of oncogenes, and constitutive or induced activation of cellular transduction pathways. NC5 as well as TC cells derived from an NC5-induced tumor exhibited the same few chromosomal abnormalities absent in normal PBL and B-cell lines (LCLs) from the same donor. No rearrangement or altered expression of C-MYC, BCL-2 and NF-KB2 oncogenes could be detected. In contrast, we found high levels of BCL-X and thioredoxin (TRX), as markers of EBV infection or T-cell activation/transformation status. No constitutive activation of NF-kappa B or STAT transcriptional complexes was observed in these cells. For NF-kappa B, this was in apparent contradiction with its reported inducibility mediated by LMP1, taking into account that NF-kappa B was still inducible by TNF alpha or PMA and ionomycin. Our results highlight independence of EBV protein-mediated transformation towards classical cellular pathways in T-lymphocytes.
RESUMO
Rel/nuclear factor (NF)-kappaB transcription factors play a major role in the regulation of programmed cell death. A few anti-apoptotic Rel/NF-kappaB target genes have been characterized; they act either downstream in the apoptotic pathway or upstream, for example at the tumor necrosis factor (TNF) receptor level. We found using DNA arrays, reverse transcription-polymerase chain reaction, and immunofluorescence that Rel/NF-kappaB factors up-regulate DcR1, a receptor for TNF-related apoptosis-inducing ligand (TRAIL), a cytokine of the TNF family that induces apoptosis in tumor cells. Four related receptors bind TRAIL, two death receptors (DR4 and DR5) that signal apoptosis and two decoy receptors (DcR1 and DcR2) that act as dominant negative inhibitors of TRAIL-mediated apoptosis. DcR1 is devoid of an intracellular domain and is anchored at the cell surface membrane by a glycophospholipid. Our results indicate that overexpression of cRel or activation of endogenous Rel/NF-kappaB factors by TNFalpha in HeLa cells up-regulates DcR1 without changing the expression of DcR2, DR4, and DR5 and makes cells resistant against TRAIL-induced apoptosis. This resistance is a consequence of DcR1 up-regulation, because it was abolished when DcR1 was removed from the cell surface by a phosphatidylinositol phospholipase C. Therefore, Rel/NF-kappaB transcription factors could regulate the sensitivity of cells to TRAIL, by controlling the ratio of TRAIL-decoy to -death receptors.
Assuntos
Apoptose/fisiologia , Glicoproteínas de Membrana/fisiologia , NF-kappa B/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Regulação para Cima/fisiologia , Proteínas Reguladoras de Apoptose , Sequência de Bases , Primers do DNA , Imunofluorescência , Proteínas Ligadas por GPI , Células HeLa , Humanos , Membro 10c de Receptores do Fator de Necrose Tumoral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Transcrição RelA , Receptores Chamariz do Fator de Necrose TumoralRESUMO
The v-mil oncogene of the avian retrovirus MH2 is expressed as a fusion protein with viral gag determinants in infected cells. This P100gag-mil protein accounts for the proliferation of chicken embryo neuroretina cells (CNR) induced by MH2 in vitro. We constructed a series of mutants by in-frame deletions in different parts of the gag and mil domains and tested their ability to induce CNR growth. We show that gag sequences, as well as 200-base-pair 5' mil sequences, were not required to induce such a proliferation. However, gag sequences seem to contribute to a full proliferation of growing CNR. In contrast, deletions in the kinase domain abolish this induction. In particular, by deleting only 9 nucleotides localized around the unique SphI site of v-mil, we produced a totally inactive mutant (BalSp). This mutant directs the synthesis of a v-mil protein lacking the dipeptide Tyr-Leu, which is conserved in almost all the members of the large protein kinase family, and a histidine residue highly conserved in Ser-Thr protein kinase members.
Assuntos
Ciclo Celular , Proteínas Oncogênicas Virais/genética , Oncogenes , Retina/citologia , Proteínas dos Retroviridae/genética , Animais , Células Cultivadas , Deleção Cromossômica , Mapeamento Cromossômico , Coturnix , Análise Mutacional de DNA , Regulação da Expressão Gênica , Produtos do Gene gag , Retina/microbiologia , Relação Estrutura-Atividade , Transfecção , Replicação ViralRESUMO
We have previously shown that schistosome-derived inhibitory factors (SDIF) inhibited lymphocyte proliferation and induced immunosuppression. Crude SDIF was purified by successive gel filtration and reverse-phase high-performance liquid chromatography. Purified SDIF preparations strongly inhibited the proliferation of different T cell line cells, while other cell lines (B cells, macrophages and fibroblasts) were almost not affected by SDIF. The inhibition of T cell proliferation by SDIF was not mediated through an Interleukin-2-dependent mechanism since both Interleukin-2-dependent and -independent T cells were inhibited. SDIF-activity was absorbed by cells in a time- and cell-number-dependent fashion at 4 degrees C, suggesting the existence of a possible receptor for SDIF. However, the difference in sensitivity to SDIF proliferation inhibition could not be attributed to the presence or absence of this receptor since cells from SDIF-sensitive and SDIF-resistant cell lines absorbed SDIF activity in the same way.
Assuntos
Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Schistosoma/imunologia , Absorção , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Humanos , Imunossupressores/isolamento & purificação , Imunossupressores/metabolismo , Camundongos , Linfócitos T/imunologiaRESUMO
After differential screening of a complementary DNA library constructed from quail neuroretina cells (QNR) infected with the v-myc-containing avian retrovirus MC29, we have isolated a complementary DNA clone which identifies a mRNA essentially expressed in the neuronal layer of the retina. This complementary DNA encodes a protein containing paired box and homeobox domains. This gene, called Pax-QNR, is homologous to the murine Pax-6 and human AN genes, which are mutated in the autosomal dominant mutation small eye (Sey) of the mouse and aniridia in humans. Here, we report the genomic exon-intron organization, as well as the existence of alternative splicing events taking place at both the 5' end and the middle part of the gene. A Pax-QNR clone translated in reticulocyte lysate directed the synthesis of a 46 kilodalton protein able to bind specifically to the e5 sequence present upstream of the Drosophila even-skipped gene and target of the Drosophila paired protein. The Pax-QNR paired and homeobox domains individually expressed in bacteria are both able to recognize the e5 sequence.
Assuntos
Genes Homeobox , Codorniz/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Mapeamento Cromossômico , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Éxons , Genoma , Dados de Sequência Molecular , Codorniz/embriologia , Transcrição GênicaRESUMO
Primary cultures of human monocyte-derived macrophages (n = 50) were characterized in order to use this cellular model to establish a proteomic map of macrophages. Peripheral blood mononuclear cells were isolated from healthy donors' blood using density gradient centrifugation. The cell culture quality was checked in respect of several morphological and molecular aspects. The homogeneity and purity of cells was assessed after 12 days' primary culture with phase microscopy, immunocytochemistry and flow cytometry. Monocytes were completely differentiated into macrophages within 12 days as shown by phase microscopy. On day 12, all cells expressed CD68 antigen and were negative for CD3. Flow cytometry experiments showed a purity of the primary culture on day 12, in a range between 76% and 98% of CD14+ cells. The functionality of cells was characterized for the presence of ECE-1 as an intracellular marker and for the presence of MMP-9 as a marker secreted into the culture medium. This study allowed to determine criteria of quality and functionality for the primary culture of monocyte-derived macrophages. Cultures meeting these criteria will be used for the proteomic analysis and the establishment of the reference map.
Assuntos
Macrófagos/fisiologia , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Receptores de Lipopolissacarídeos/fisiologia , Macrófagos/citologia , Macrófagos/enzimologia , Metaloproteinase 3 da Matriz , Metaloproteinase 9 da Matriz , Microscopia de Contraste de Fase , Monócitos/fisiologiaRESUMO
Besides its crucial role in type I hypersensitivity reactions, IgE is involved in anti-parasite immunity. This role has been clearly demonstrated in both human and rat schistosomiasis, but remains controversial in the mouse. Since the cellular distribution of the high affinity IgE receptor, Fc epsilon RI, differs in humans and mice, it might explain the differences in effector function of IgE between the two species. In humans, eosinophils and macrophages induce IgE-dependent cytotoxicity toward Schistosoma mansoni larvae, which involves Fc epsilon RI in the case of eosinophils. In the present study, we have investigated the expression and function of Fc epsilon RI in rat eosinophils and macrophages. We demonstrate, by flow cytometry, fluorescence microscopy, and western blot analysis, that in rats, as in humans, a functional alpha gamma 2 trimeric Fc epsilon RI is expressed on eosinophils and macrophages. We also show that these two cell types can induce IgE-mediated, Fc epsilon RI-dependent cellular cytotoxicity toward schistosomula. These results thus provide a molecular basis for the differences observed between rat and mouse regarding IgE-mediated anti-parasite immunity.
Assuntos
Eosinófilos/metabolismo , Macrófagos Peritoneais/metabolismo , Receptores de IgE/biossíntese , Animais , Citotoxicidade Celular Dependente de Anticorpos , Testes Imunológicos de Citotoxicidade , Eosinófilos/imunologia , Citometria de Fluxo , Imunoglobulina E/fisiologia , Larva/imunologia , Macrófagos Peritoneais/imunologia , Camundongos , Ratos , Ratos Endogâmicos , Receptores de IgE/química , Receptores de IgE/fisiologia , Schistosoma mansoni/imunologia , Células Tumorais Cultivadas , Regulação para Cima/imunologiaRESUMO
Epstein-Barr virus (EBV) is a human lymphotropic virus whose main targets have traditionally been described as B lymphocytes and epithelial cells. Here we report the isolation and characterization of largely monoclonal transformed human T-cell lines infected by EBV. The transformed T cells expressed CD2, CD3, and either CD4 or CD8 surface molecules and more generally displayed the phenotype of naive T cells with a complete and clonal rearrangement of the T-cell receptor. None of the cell lines expressed B cells, natural killer, or myeloid antigens or had immunoglobulins genes rearrangement. They grew in the absence of growth factor; however, they all secreted interleukin-2 after mitogenic activation. Polymerase chain reaction (PCR) analysis showed the presence of EBV DNA in all these cell lines. Moreover, Southern blot analysis of one of these cell lines shows the presence of circular episomic EBV DNA, and by Northern blot or reverse transcriptase-PCR analysis, only the expression of Epstein-Barr nuclear antigen-1 (EBNA-1) and latent membrane protein-1 (LMP-1) genes was detected. Finally, the complete transformed phenotype of this T-cell line was shown by its injection into nude or recombination activating gene 2 (RAG2)-deficient mice that led to the formation of solid tumors.
Assuntos
Linhagem Celular Transformada , Transformação Celular Viral , Herpesvirus Humano 4/fisiologia , Linfócitos T/virologia , Animais , Antígenos CD/análise , Células Clonais , DNA Viral/análise , Proteínas de Ligação a DNA , Regulação Viral da Expressão Gênica , Rearranjo Gênico do Linfócito T , Herpesvirus Humano 4/isolamento & purificação , Humanos , Imunofenotipagem , Interleucina-2/metabolismo , Linfoma de Células T/patologia , Linfoma de Células T/virologia , Camundongos , Camundongos Knockout , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Proteínas Nucleares , RNA Viral/biossíntese , RNA Viral/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/metabolismo , Linfócitos T/patologia , Transplante Heterólogo , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
The lactic acid bacteria (LAB) are safe microorganisms which are mainly used for the preparation of fermented foods and for probiotic applications. The potential of LAB as live vehicles for the production and delivery of therapeutic molecules such as antigens is also being actively investigated today. However, very little is known about the fate of live LAB when administered in vivo and about the interaction of these microorganisms with the nasal or gastrointestinal ecosystem. For future applications, it is essential to be able to discriminate the biotherapeutic strain from the endogenous microflora and to unravel the mechanisms underlying the postulated health-beneficial effect. We therefore started to investigate both aspects in a mouse model with two LAB species presently under development as live vaccine vectors, i.e., Lactococcus lactis and Lactobacillus plantarum. We have constructed different expression vectors carrying the gfp (green fluorescent protein [GFP]) gene from the jellyfish Aequoria victoria, and we found that this visible marker was best expressed when placed under the control of the inducible strong nisA promoter from L. lactis. Notably, a threshold amount of GFP was necessary to obtain a bright fluorescent phenotype. We further demonstrated that fluorescent L. plantarum NCIMB8826 can be enumerated and sorted by flow cytometry. Moreover, tagging of this strain with GFP allowed us to visualize its phagocytosis by macrophages in vitro and ex vivo and to trace it in the gastrointestinal tract of mice upon oral administration.
Assuntos
Vacinas Bacterianas/genética , Lactobacillus/genética , Lactobacillus/fisiologia , Proteínas Luminescentes/genética , Animais , Clonagem Molecular , Contagem de Colônia Microbiana , Citometria de Fluxo , Marcadores Genéticos , Vetores Genéticos , Proteínas de Fluorescência Verde , Immunoblotting , Lactobacillus/imunologia , Lactobacillus/metabolismo , Proteínas Luminescentes/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , FagocitoseRESUMO
Several studies have shown that full transformation of primary rodent fibroblasts can be achieved in vitro through the cooperation of two oncogenes (usually one nuclear and one cytoplasmic) classified on the basis of different complementation groups. We have shown previously that cooperation between v-mil (cytoplasmic, serine-threonine kinase product), and v-myc (nuclear, DNA-binding product) is required to transform 7-day-old chicken neuroretina cells, which in usual culture medium do not rapidly proliferate. v-mil induces sustained growth of chicken neuroretina cells without transformation; v-myc fails to stimulate the proliferation of chicken neuroretina cells but is required to achieve transformation of the proliferating cells. Here, we present results indicating that the P135gag-myb-ets nuclear protein of avian erythroblastosis virus E26 is able to induce proliferation but not transformation of chicken neuroretina cells. v-myc is required in addition to P135gag-myb-ets to achieve chicken neuroretina cell transformation. In contrast, we found that the P135gag-myb-ets and P100gag-mil proteins are not able to cooperate in this system.