RESUMO
Recent advances in the diagnostic of myeloproliferative neoplasms (MPNs) discovered CALRETICULIN (CALR) mutations as a major driver in these disorders. In contrast to JAK2 mutations being mainly associated with polycythaemia vera, CALR mutations are only associated with primary myelofibrosis (PMF) and essential thrombocythaemia (ET). CALR mutations are present in the majority of PMF and ET patients lacking JAK2 and MPL mutations. As these CALR mutations are absent from reactive bone marrow (BM) lesions their presence indicates ET or PMF. So far these mutations are detectable only by molecular assays. Their molecular detection is cumbersome because of the great CALR mutation heterogeneity. Therefore, the availability of a simple assay would be of great help. All CALR mutations reported lead to a frameshift generating a new 36 amino-acid C-terminus. We generated a monoclonal antibody (CAL2) to this C-neoterminus by immunizing mice with a representative peptide and compared its performance with Sanger sequencing data in 173 MPNs and other BM diseases. There was a 100% correlation between the molecular and the CAL2 immunohistochemical (IHC) assays. Thus, the detection of CALR mutations by the CAL2 IHC is a specific, sensitive, rapid, simple and low-cost method.
Assuntos
Anticorpos Monoclonais/imunologia , Medula Óssea/química , Calreticulina/genética , Mutação , Sequência de Aminoácidos , Animais , Biópsia , Calreticulina/análise , Formaldeído , Células HEK293 , Humanos , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Inclusão em ParafinaRESUMO
BACKGROUND AND PURPOSE: NF-κB-driven inflammation is negatively regulated by the zinc finger protein A20. Gibberellic acid (GA3 ) is a plant-derived diterpenoid with documented anti-inflammatory activity, which is reported to induce A20-like zinc finger proteins in plants. Here, we sought to investigate the anti-inflammatory effect of GA3 in airway epithelial cells and determine if the anti-inflammatory action relates to A20 induction. EXPERIMENTAL APPROACH: Primary nasal epithelial cells and a human bronchial epithelial cell line (16HBE14o-) were used. Cells were pre-incubated with GA3 , stimulated with Pseudomonas aeruginosaâ LPS; IL-6 and IL-8 release, A20, NF-κB and IκBα expression were then evaluated. To determine if any observed anti-inflammatory effect occurred via an A20-dependent mechanism, A20 was silenced using siRNA. KEY RESULTS: Cells pre-incubated with GA3 had significantly increased levels of A20 mRNA (4 h) and protein (24 h), resulting in a significant reduction in IL-6 and IL-8 release. This effect was mediated via reduced IκBα degradation and reduced NF-κB (p65) expression. Furthermore, the anti-inflammatory action of GA3 was abolished in A20-silenced cells. CONCLUSIONS AND IMPLICATIONS: We showed that A20 induction by GA3 attenuates inflammation in airway epithelial cells, at least in part through its effect on NF-κB and IκBα. GA3 or gibberellin-derived derivatives could potentially be developed into anti-inflammatory drugs for the treatment of chronic inflammatory diseases associated with A20 dysfunction.
Assuntos
Anti-Inflamatórios/farmacologia , Células Epiteliais/efeitos dos fármacos , Giberelinas/farmacologia , Inflamação/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Pseudomonas aeruginosa/química , RNA Mensageiro/metabolismo , Mucosa Respiratória/metabolismo , Relação Estrutura-AtividadeRESUMO
Stimulation of the TNF receptors CD30 and CD95 exerts opposite effects. Crosstalk of both receptors is unknown. We aimed to reveal regulatory mechanisms of CD30-induced effects on CD95 signaling of cALCL cell lines. "CD30/CD95 crosstalk analysis" was performed in cALCL cell lines by comparison of CD30 or CD95 stimulation and CD30/CD95 costimulation. Receptor expression and induction of apoptosis was investigated by flow cytometry. mRNA expression of CD30-inducible genes (cFLIP, TRAF1, cIAP2, and A20) was compared by semiquantitative reverse transcription (RT-RQ-) PCR in stimulated and unstimulated cells. Protein expression of IκBα, p100/p52, caspase-8, caspase-3, and cFLIP was analyzed by immunoblotting. A lentiviral-based shRNA-mediated approach was used to inhibit cFLIP expression. CD30/CD95 crosstalk experiments revealed that CD30 ligation leads to NFκB-mediated cFLIP upregulation in cALCL cells, which in turn enhanced resistance to CD95-mediated apoptosis. This effect is based on the CD30-induced upregulation of cFLIP. Knockdown of cFLIP restores sensitivity to CD95-mediated apoptosis. We conclude that the anti-apoptotic function of CD30 antibodies should be kept in mind if CD30 antibody-based therapeutic concepts for ALCL lymphoma are considered.
Assuntos
Apoptose/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Antígeno Ki-1/genética , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/patologia , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/patologia , Receptor Cross-Talk , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Receptor fas/genética , Linhagem Celular Tumoral , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/patologia , Regulação para Cima/genéticaRESUMO
Although therapy of CD30-positive lymphomas such as classical Hodgkin lymphoma and anaplastic large cell lymphoma has been improved considerably during the last decades, patients suffer from high toxicity of current therapeutic regimens. Since CD30 expression is very restricted, CD30-positive tumors are well suited for immunotherapeutic approaches. Several distinct immunotherapeutic approaches with chimeric, humanized, and bispecific antibodies as well as immunotoxins are already described. In this report, we give a short overview of CD30-targeting approaches in humans. Furthermore, we introduce two novel anti-CD30 fusion proteins consisting of the single chain variable fragment of the CD30 monoclonal antibody Ber-H2 and human interleukin-2, evaluate their biological activity in a human CD30-positive syngeneic murine model, and demonstrate the immunological mechanisms leading to tumor rejection by these reagents. The data indicate that there are several promising approaches in CD30-targeted immunotherapy. The findings of the anti-CD30 IL-2 constructs suggest that these fusion proteins are particularly useful to remove small, residual tumors.
Assuntos
Anticorpos Monoclonais/imunologia , Sobrevivência Celular/efeitos dos fármacos , Interleucina-2/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Sistemas de Liberação de Medicamentos , Humanos , Interleucina-2/imunologia , Antígeno Ki-1/imunologia , Proteínas Recombinantes de Fusão/imunologiaRESUMO
AIMS: To investigate T-bet expression profiles in various lymphoid tissue diseases caused by intracellular pathogens and to compare them in disorders without an infective aetiology. Murine and in vitro experiments have shown that the expression/induction of T-bet, the master regulator of Th1 differentiation, can be achieved by obligate intracellular pathogens and high interferon (IFN)-gamma levels. METHODS: Lymph node biopsies were analysed immunohistochemically employing single and double labelling for T-bet and CD20, CD4, CD8 and CD30 detection. RESULTS: In disorders associated with high IFN-gamma levels and intracellular pathogens (infectious mononucleosis, HIV-associated lymphadenopathy, cat-scratch disease, and toxoplasmic lymphadenitis), T-bet-expressing CD4 cells were accompanied by significant numbers of T-bet-positive CD8 and B cells. A similar profile was also found in histiocytic necrotizing (Kikuchi) lymphadenitis, a disease of unknown cause. In contrast, T-bet expression in disorders without an infective aetiology was observed in only a small portion of lymphocytes. CONCLUSIONS: Increased T-bet expression does not only identify intracellular infections in lymphoid tissue associated with high IFN-gamma levels, but also implies that, under these conditions, it becomes induced in B cells, which apparently support the Th1 response. T-bet expression in Kikuchi lymphadenitis underscores the hypothesis that it is caused by an intracellular microorganism.
Assuntos
Tecido Linfoide/patologia , Fatores de Transcrição/biossíntese , Antígenos CD20/análise , Complexo CD3/análise , Antígenos CD4/análise , Antígenos CD8/análise , Hiperplasia do Linfonodo Gigante/metabolismo , Hiperplasia do Linfonodo Gigante/patologia , Doença da Arranhadura de Gato/metabolismo , Doença da Arranhadura de Gato/patologia , Infecções por HIV/complicações , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Linfadenite Histiocítica Necrosante/metabolismo , Linfadenite Histiocítica Necrosante/patologia , Histiocitose de Células de Langerhans/metabolismo , Histiocitose de Células de Langerhans/patologia , Histiocitose Sinusal/metabolismo , Histiocitose Sinusal/patologia , Humanos , Hiperplasia , Imuno-Histoquímica , Mononucleose Infecciosa/metabolismo , Mononucleose Infecciosa/patologia , Antígeno Ki-1/análise , Linfonodos/química , Linfonodos/patologia , Doenças Linfáticas/complicações , Doenças Linfáticas/metabolismo , Doenças Linfáticas/patologia , Tecido Linfoide/metabolismo , Tonsila Palatina/química , Tonsila Palatina/patologia , Dermatopatias/complicações , Dermatopatias/metabolismo , Dermatopatias/patologia , Baço/química , Baço/patologia , Proteínas com Domínio T , Toxoplasmose/complicações , Toxoplasmose/metabolismo , Toxoplasmose/patologia , Tuberculose/complicações , Tuberculose/metabolismo , Tuberculose/patologiaRESUMO
One of the main functions of the tumor necrosis factor receptor (TNFR) family is induction of apoptosis. CD30, a member of the TNFR superfamily is overexpressed in highly proliferating tumors such as anaplastic large cell lymphoma (ALCL) and Hodgkin's lymphoma (HL). CD30 stimulation leads to apoptosis and growth arrest in cultured ALCL, but not in Hodgkin-Reed-Sternberg cells. To identify changes in the transcriptional program responsible for these opposing effects, we performed gene expression analysis in CD30-stimulated ALCL (Karpas 299) and HL (KM-H2) cell lines using cDNA microarrays. Selected genes were validated by real-time PCR. Hierarchical clustering was applied to the whole dataset and separated the cell lines clearly with respect to their origin. In HL, there were only minor CD30-specific alterations, whereas ALCL unequivocally showed a pronounced CD30-specific transcriptional response. Ninety-three genes (6.6% of total) were deregulated by more than a factor of two after CD30 stimulation in ALCL cells. The majority of genes identified are involved in cell cycle regulation and apoptosis. mRNA expression patterns further indicate that in contrast to HL, CD30 stimulation in ALCL induces cell death via the CD95-CD95 ligand (CD95L) pathway and the TNF-R1/TNF-R2 crosstalk. These data provide a detailed view on the transcriptional changes upon CD30 stimulation and may explain the observed functional differences of HL and ALCL.
Assuntos
Apoptose/genética , Ciclo Celular/genética , Regulação Leucêmica da Expressão Gênica , Antígeno Ki-1 , Linfoma Difuso de Grandes Células B/genética , Transdução de Sinais , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Doença de Hodgkin/genética , Doença de Hodgkin/metabolismo , Humanos , Antígeno Ki-1/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Análise de Sequência com Séries de OligonucleotídeosRESUMO
BACKGROUND: Since 2003, reports have been published on necrosis of the jaw bones possibly being associated with the administration of bisphosphonates. Bisphosphonates are highly active inhibitors of osteoclasts which have been used prophylactically or symptomatically in the treatment of plasmocytoma, bone metastasis of malignant disease, tumor-associated hypercalcaemia and in the treatment of osteoporosis. Due to the importance of this side effect of bisphosphonates, we report six cases. CASE REPORTS: Six patients (two women and four men) with a median age of 69 years (range 55-37) were diagnosed with osteonecrosis of the maxilla and/or mandible. These osteonecroses did not react adequately to local treatment and systemic therapy with antibiotics. Four patients suffered from plasmocytoma and two patients had a history of metastasising breast cancer. Besides cytostatic chemotherapies, all patients received bisphosphonates over an extended period. DISCUSSION: Bisphosphonates are considered an important standard in the treatment of plasmocytoma and bone metastasis due to malignancies. Since 2003, several reports have been published describing patients in whom therapy resistant osteonecrosis of jaw bones occurred either after dental extractions or spontaneously. Until then, unknown side effects of bisphosphonate therapy had been suspected. Since patients with malignant diseases receive cytostatic therapy and a range of other drugs, including bisphosphonates, enhancement of the side effects may be presumed. CONCLUSIONS: The probable association of the therapeutic use of bisphosphonates and the occurence of jaw bone necrosis has to be studied in further investigations. Patients receiving bisphosphonates should be followed-up regularly to avoid the occurrence of extended osteonecrotic lesions, which should be diagnosed early and treated adequately.
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Neoplasias da Mama/tratamento farmacológico , Difosfonatos/efeitos adversos , Doenças Maxilomandibulares/induzido quimicamente , Osteonecrose/induzido quimicamente , Plasmocitoma/tratamento farmacológico , Adulto , Idoso , Neoplasias Ósseas/patologia , Neoplasias da Mama/patologia , Difosfonatos/uso terapêutico , Feminino , Humanos , Arcada Osseodentária/patologia , Doenças Maxilomandibulares/patologia , Masculino , Pessoa de Meia-Idade , Osteonecrose/patologia , Plasmocitoma/patologia , Radiografia PanorâmicaRESUMO
The alpha-chain of the IL-15R (IL-15Ralpha) serves as the specific, high-affinity receptor for IL-15. It is expressed by lymphoid and nonlymphoid cells, including B cell lymphoma lines. In this study, we have further explored IL-15Ralpha-mediated signaling in activated primary B cells and in Raji cells, a human B-lymphoblastoid cell line which expresses the IL-15Ralpha and IL-2Rgamma chains, but lacks the IL-2Rbeta chain. Stimulation of Raji cells with IL-15 induces their proliferation and rescues them from C2-ceramide-induced apoptosis. By immunoprecipitation and Western blotting, we show that treatment of Raji cells and activated primary B cells with IL-15 induces coprecipitation of Syk kinase with the IL-15Ralpha chain. Upon association, the activated Syk kinase phosphorylates the IL-15Ralpha chain as well as phospholipase Cgamma, which coprecipitates with Syk. Furthermore, transfection of Raji cells with stem-loop Syk antisense oligonucleotides prevents IL-15Ralpha and phospholipase Cgamma phosphorylation as well as the inhibition of apoptosis by IL-15. Mutation of a defined region of the intracellular signaling portion of IL-15Ralpha (Tyr227) abrogates both the IL-15Ralpha/Syk association and IL-15Ralpha phosphorylation. Taken together, this suggests that Syk kinase physically and functionally associates with the IL-15Ralpha chain in B cells and that Syk plays a key role in mediating IL-15-induced signal transduction, thus accounting for the distinct functional consequences of IL-15 vs IL-2 binding to B cells.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Precursores Enzimáticos/metabolismo , Interleucina-15/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Interleucina-2/fisiologia , Transdução de Sinais/imunologia , Esfingosina/análogos & derivados , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/imunologia , Subpopulações de Linfócitos B/efeitos dos fármacos , Subpopulações de Linfócitos B/enzimologia , Cálcio/antagonistas & inibidores , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Ativação Enzimática/imunologia , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/genética , Precursores Enzimáticos/fisiologia , Humanos , Interleucina-15/antagonistas & inibidores , Interleucina-15/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Células Jurkat , Células K562 , Ativação Linfocitária , Mutagênese Sítio-Dirigida , Oligonucleotídeos Antissenso/farmacologia , Fosfolipase C gama , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , Receptores de Interleucina-15 , Receptores de Interleucina-2/antagonistas & inibidores , Receptores de Interleucina-2/biossíntese , Receptores de Interleucina-2/metabolismo , Transdução de Sinais/genética , Esfingosina/farmacologia , Quinase Syk , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Tumorais Cultivadas , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo , Domínios de Homologia de src/imunologiaRESUMO
The CD30 antigen is a member of the tumor necrosis factor receptor (TNFR) family which is overexpressed on the surface of the tumor cells of Hodgkin's lymphoma, anaplastic large cell lymphoma (ALCL), and embryonal carcinoma of the testis. In this study the entire cd30 gene which is more than 24000 bp long and organized in eight exons was characterized by analyzing cosmid and phage lambda clones from human placental libraries with long-range polymerase chain reaction (PCR) and sequencing. Differences to other genes of the TNFR family were detected in the region encoding the extracellular domain of the cd30 gene. In nearly all other TNFR genes, the coding region of each cysteine-rich repeat is interrupted by one intron, i.e., the 3-4 cysteine-rich repeats of these receptors are encoded by at least 4-5 exons, whereas the six cysteine-rich repeats of the cd30 gene are encoded by two exons, i.e., each of these exons encode three cysteine-rich repeats. In addition, we also found a genetic polymorphism of tetranucleotide ATCC-repeats in the 5' part of the CD30 promoter. This region was amplified by PCR from seven CD30 overexpressing human lymphoid cell lines and five human tissues with an absent or very low CD30 expression. The amplification products showed length differences of more than 550 bp. The number of the ATCC-repeats was higher in CD30(+) cell lines than in normal tissues. Comparison of the individual PCR products in reporter gene assays revealed that the CD30 promoter activity increased with the length of this polymorphic region up to eightfold. The data suggest that the number of ATCC-repeats in the 5' region of the CD30 promoter modulates the regulation of CD30 expression.
Assuntos
Doença de Hodgkin/genética , Antígeno Ki-1/genética , Repetições Minissatélites/genética , Sequência de Bases , Clonagem Molecular , Primers do DNA , Doença de Hodgkin/imunologia , Doença de Hodgkin/patologia , Humanos , Antígeno Ki-1/imunologia , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Células Tumorais CultivadasRESUMO
Anaplastic large cell lymphoma (ALCL) represents a generally recognized group of large cell lymphomas. Defining features consist of a proliferation of predominantly large lymphoid cells with strong expression of the cytokine receptor CD30 and a characteristic growth pattern. With the use of molecular and clinical criteria, 3 entities of ALCL have been identified: primary systemic anaplastic lymphoma kinase (ALK)(+) ALCL, primary systemic ALK(-) ALCL, and primary cutaneous ALCL. ALK expression is caused by chromosomal translocations, most commonly t(2;5). ALK(+) ALCL predominantly affects young male patients and, if treated with chemotherapy, has a favorable prognosis. It shows a broad morphologic spectrum, with the "common type," the small cell variant, and the lymphohistiocytic variant being most commonly observed. The knowledge of the existence of these variants is essential in establishing a correct diagnosis. ALK(-) ALCL occurs in older patients, affecting both genders equally and having an unfavorable prognosis. The morphology and the immunophenotype of primary cutaneous ALCL show an overlap with that of lymphomatoid papulosis. Both diseases have an excellent prognosis, and secondary systemic dissemination is only rarely observed. The described ALCL entities usually derive from cytotoxic T cells. In contrast, large B-cell lymphomas with anaplastic morphology are believed to represent not a separate entity but a morphologic variant of diffuse large B-cell lymphoma. Malignant lymphomas with morphologic features of both Hodgkin disease and ALCL have formerly been classified as Hodgkin-like ALCL. Recent immunohistologic studies, however, suggest that ALCLs Hodgkin-like represent either cases of tumor cell-rich classic Hodgkin disease or (less commonly) ALK(+) ALCL or ALK(-) ALCL. (Blood. 2000;96:3681-3695)
Assuntos
Linfoma Anaplásico de Células Grandes/genética , Quinase do Linfoma Anaplásico , Rearranjo Gênico , Doença de Hodgkin , Humanos , Imunofenotipagem , Linfoma Anaplásico de Células Grandes/imunologia , Linfoma Anaplásico de Células Grandes/patologia , Proteínas Nucleares/genética , Nucleofosmina , Proteínas Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases , Translocação GenéticaRESUMO
One of the most peculiar immunohistological characteristics of the tumour cells of Hodgkin's lymphoma, anaplastic large cell lymphoma (ALCL), and embryonal carcinoma of the testis is the expression of the CD30 antigen. Physiologically, CD30 expression is restricted to a few activated lymphocytes in normal lymphoid tissue and a small population of decidual cells. To clarify the reasons behind this highly restricted expression pattern and to learn about the combination of transcription factors involved in this regulation in Hodgkin's lymphoma and other CD30(+) malignancies, the 5'-flanking regulatory region of the cd30 gene was analysed. The major transcription start site was determined to be 270 bases upstream of the translational start codon in the Hodgkin's lymphoma-derived cell lines L591 and L428. Reporter gene assays revealed that the CD30 promoter (-413 to 84) induces a 50- to 1000-fold higher luciferase expression in CD30(+) human lymphoid cell lines (Co, Jurkat, and the Hodgkin's lymphoma-derived cell line L540) than in CD30(-) human lymphoid cell lines (DG75, SUP-T1, and U698M), CD30(-) human carcinoma cell lines (HeLa and MCF-7), or COS1 cells. Deletion analysis defined a TATA-less, minimal promoter sequence from -164 to 84. The transcription factor Sp1 and members of the Ets family induce CD30 expression, whereas the transcription factor Sp3 diminishes its induction. These data suggest that a high Sp1/Sp3 expression ratio and a peculiar expression pattern of the Ets transcription factors are involved in the overexpression of CD30 and might contribute to the transformation of CD30(+) tumour cells.
Assuntos
Antígeno Ki-1/genética , Linfoma Anaplásico de Células Grandes/genética , Regiões Promotoras Genéticas/genética , Sequência de Bases , Transformação Celular Neoplásica/genética , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Antígeno Ki-1/metabolismo , Linfoma Anaplásico de Células Grandes/metabolismo , Dados de Sequência Molecular , Mutação , Fatores de Transcrição , Células Tumorais CultivadasRESUMO
OX40 is a member of the TNF receptor family, expressed on activated T cells. It is the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukemia virus type-I (HTLV-I)-producing cells. In a T cell line, OX40 surface expression was shown to be induced by HTLV-I Tax alone. To understand molecular mechanisms of OX40 gene regulation and modulation by HTLV-I Tax, we have cloned the human OX40 gene and analyzed its 5'-flanking region. By reporter gene analysis with progressive 5' deletions from nucleotides -1259 to -64, we have defined a 157-bp DNA fragment as a minimal promoter for constitutive expression. In addition, we show that in the OX40+ cell line, Co, Tax is able to further increase OX40 surface expression. Up-regulation of OX40 promoter activity by Tax requires two upstream NF-kappaB sites, which are not active in the constitutive OX40 expression. Their deletion abrogates Tax responsiveness in reporter gene analysis. The site-directed mutagenesis of each NF-kappaB site demonstrates that cooperative NF-kappaB binding is a prerequisite for Tax-directed activity as neither site alone is sufficient for a full Tax responsiveness of the OX40 promoter. Upon Tax expression, both sites bind p65 and c-Rel. These data provide new insight into the direct regulation of OX40 by Tax and add to our understanding of the possible role of the OX40/OX40 ligand system in the proliferation of HTLV-I+ T cells.
Assuntos
Produtos do Gene tax/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Receptores do Fator de Necrose Tumoral , Transcrição Gênica/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/imunologia , Sequência de Bases , Clonagem Molecular , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/imunologia , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptores OX40 , Ativação Transcricional/imunologia , Células Tumorais Cultivadas , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Originally, expression of the CD30 antigen was shown to be typical of the tumour cells of Hodgkin's disease (HD) and of anaplastic large cell lymphomas (ALCLs). In reactive lymphoid tissue, CD30 is expressed only in a small population of activated lymphoid blasts. Since then, several reports have been published describing CD30 expression in non-lymphoid tissues and malignancies, such as embryonal carcinomas (ECs), seminomas, cultivated macrophages, two histiocytic neoplasms, decidual cells, and mesotheliomas. As CD30 detection is important in the differential diagnosis of HD and ALCL, the expression of CD30 in different non-lymphoid tissues was re-evaluated by immunohistology and in situ hybridization. Extra-lymphoid CD30 expression was found in 48/51 cases of EC or EC components of germ cell tumours, in decidual cells of 1/10 cases, in activated mesothelium in 16/28 pleural and peritoneal effusions, and in small foci of tumour cells in 2/8 mesotheliomas. CD30 expression was not confirmed in 27 germ cell tumours of the testis without an EC component nor in cultivated macrophages and 17 histiocytic malignancies. The knowledge of these CD30 expression patterns is important for the immunohistological differential diagnosis of anaplastic tumours. The absence of CD30 expression in reactive and neoplastic macrophages does not favour the concept that HD and ALCL are derived from these cells.
Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Antígeno Ki-1/metabolismo , Mesotelioma/metabolismo , Neoplasias Embrionárias de Células Germinativas/metabolismo , Adolescente , Adulto , Líquido Ascítico/metabolismo , Técnicas de Cultura de Células , Decídua/metabolismo , Epitélio/metabolismo , Feminino , Humanos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Derrame Pleural/metabolismoRESUMO
Interleukin-15 (IL-15) is a potent inhibitor of several apoptosis pathways. One prominent path toward apoptosis is the ligand-induced association of TNF receptor 1 (TNFR1) with death domain adaptor proteins. Studying if and how IL-15 blocks TNFR1-mediated apoptosis in a murine fibroblast cell line (L929), we show here that IL-15 blocks TNFR1-induced apoptosis via IL-15Ralpha chain signaling. The intracellular tail of IL-15Ralpha shows sequence homologies to the TRAF2 binding motifs of CD30 and CD40. Most important, binding of IL-15 to IL-15Ralpha successfully competes with the TNFR1 complex for TRAF2 binding, which may impede assembly of key adaptor proteins to the TNFR1 complex, and induces IkappaBalpha phosphorylation. Thus, IL-15Ralpha chain stimulation is a powerful deflector of cell death very early in the apoptosis signaling cascade, while TNF-alpha and IL-15 surface as major opponents in apoptosis control.
Assuntos
Apoptose/efeitos dos fármacos , Interleucina-15/farmacologia , Proteínas/fisiologia , Receptores de Interleucina-2/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Sítios de Ligação , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Citometria de Fluxo , Células L , Substâncias Macromoleculares , Camundongos , Proteínas/química , Receptores de Interleucina-15 , Receptores de Interleucina-2/química , Receptores de Interleucina-2/genética , Receptores do Fator de Necrose Tumoral/fisiologia , Proteínas Recombinantes/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fator 2 Associado a Receptor de TNF , Transfecção , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Apaf-1, the human C. elegans Ced-4 homologue, plays a crucial role in mitochondrial mediated apoptosis. To determine whether a mutant Apaf-1 is involved in the pathogenesis of malignant lymphomas, we performed sequence analysis of its transcriptional gene product. Therefore, cDNAs coding for Apaf-1 were examined in six lymphoma derived cell lines, in three non-lymphoid tumor cell lines, as well as in peripheral blood lymphocytes and in tissue from heart, kidney, and liver by RT-PCR. These studies did not disclose the expected tumor related alterations but led instead to the identification of three novel forms of Apaf-1. These forms vary in length, but contain all functional domains formerly characterized in the Apaf-1 protein. The results indicate that the previously published sequence is not the Apaf-1 form most widely expressed.
Assuntos
Proteínas/genética , Sequência de Aminoácidos , Apoptose , Fator Apoptótico 1 Ativador de Proteases , Doença de Hodgkin/genética , Humanos , Linfócitos/química , Linfoma/genética , Dados de Sequência Molecular , Mutação , Proteínas/análise , Proteínas/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Células Tumorais CultivadasRESUMO
Several members of the tumour necrosis factor receptor (TNF-R) superfamily can induce cell death. For TNF-R1, Fas/APO-1, DR3, DR6, TRAIL-R1 and TRAIL-R2, a conserved 'death domain' in the intracellular region couples these receptors to activation of caspases. However, it is not yet known how TNF receptor family members lacking a death domain, such as TNF-R2, CD40, LT-betaR, CD27 or CD30, execute their death-inducing capability. Here we demonstrate in different cellular systems that cytotoxic effects induced by TNF-R2, CD40 and CD30 are mediated by endogenous production of TNF and autotropic or paratropic activation of TNF-R1. In addition, stimulation of TNF-R2 and CD40 synergistically enhances TNF-R1-induced cytotoxicity. These findings describe a novel pro-apoptotic mechanism induced by some members of the TNF-R family.
Assuntos
Antígenos CD/fisiologia , Apoptose , Antígenos CD40/fisiologia , Antígeno Ki-1/fisiologia , Proteínas de Membrana/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos/farmacologia , Antígenos CD/química , Antígenos CD/genética , Apoptose/efeitos dos fármacos , Antígenos CD40/genética , Inibidores de Caspase , Caspases/metabolismo , Proteína Ligante Fas , Humanos , Interferon gama/antagonistas & inibidores , Interferon gama/farmacologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Mutação , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interferon/genética , Receptores de Interferon/fisiologia , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Regulação para Cima , Proteína bcl-X , Receptor de Interferon gamaRESUMO
The tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1) is a member of the recently defined TRAF family. It takes part in the signal transduction of the TNF receptor 2 (TNFR2), the lymphotoxin-beta receptor (LT-betaR), CD40, CD30, and LMP1; is induced by LMP1 in vitro; and protects lymphoid cells from apoptosis. To identify the cells in which TRAF1 is active in vivo, we studied TRAF1 transcripts in normal lymphoid tissue, in Epstein-Barr virus (EBV)-induced lymphoproliferations, and in malignant lymphomas with special reference to those that overexpress the cytokine receptor CD30 and CD40 of the TNF receptor family at the single-cell level using a radioactive in situ hybridization. In normal lymphoid tissue, TRAF1 message proved to be absent from all resting B and T cells as well as from macrophages and accessory cells (follicular dendritic cells and interdigitating cells) and present in few perifollicular and intrafollicular lymphoid blasts. In contrast, there was a high and consistent TRAF1 overexpression in EBV-induced lymphoproliferations and Hodgkin's disease. Nearly all non-Hodgkin's lymphoma show low or no TRAF1 expression. Only some cases of diffuse large B-cell lymphoma showed a moderate to high TRAF1 signal. Several of the latter cases were EBV+. These data confirm that TRAF1 is an inducible molecule and indicates its deregulation in the mentioned disorders with the potential of a blockage of the apoptotic pathway.
Assuntos
Infecções por Vírus Epstein-Barr/metabolismo , Expressão Gênica , Doença de Hodgkin/metabolismo , Transtornos Linfoproliferativos/virologia , Proteínas/genética , Células de Reed-Sternberg/metabolismo , Humanos , Mononucleose Infecciosa/metabolismo , Tecido Linfoide/metabolismo , Transtornos Linfoproliferativos/metabolismo , Receptores do Fator de Necrose Tumoral , Fator 1 Associado a Receptor de TNFRESUMO
Costimulation of TNFR80 can strongly enhance TNFR60-induced cell death. In this study, we show that this enhancement is TNFR60 selective, as neither TNF-related apoptosis-inducing ligand/Apo2 ligand-, Apo1/Fas-, ceramide-, nor daunorubicin-mediated cell death was affected by costimulation of TNFR80. We further demonstrate that TNFR-associated factor 2 (TRAF2) is critically involved in both negative and positive regulation of TNF-induced cell death. Overexpression of TRAF2 and of a TRAF2 mutant, deficient in nuclear factor-kappaB activation, selectively desensitized and enhanced, respectively, TNFR60-induced cell death in HeLa cells. However, upon costimulation of TNFR80, which mediates activation of nuclear factor-kappaB and the c-Jun amino-terminal kinase via TRAF2, TNF-induced cell death is drastically enhanced in parental and TRAF2-transfected, but not in TRAF2 (87-501)-transfected cells. These data point to a critical role of TRAF2 in the apoptotic TNFR cross talk, whereby the TNFR80-dependent enhancement of TNFR60-induced cell death is due to TNFR80-mediated negative regulation of TRAF2 function(s). An interference with TRAF2 function was confirmed independently by analysis of c-Jun amino-terminal kinase activation via TNFR60 upon prestimulation of TNFR80. We propose that the apoptotic TNFR cross talk is based on TNFR80-mediated abrogation of antiapoptotic TRAF2-dependent signaling pathways initiated by TNFR60, but not Apo1/Fas or the apoptotic TNF-related apoptosis-inducing ligand receptors.
Assuntos
Adjuvantes Imunológicos/fisiologia , Antígenos CD/fisiologia , Proteínas de Transporte/fisiologia , Células HeLa/imunologia , Proteínas Quinases Ativadas por Mitógeno , Receptores do Fator de Necrose Tumoral/fisiologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral , Antígenos CD/química , Antígenos CD/metabolismo , Proteínas Reguladoras de Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Ceramidas/farmacologia , Daunorrubicina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Células HeLa/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Ligantes , Glicoproteínas de Membrana/fisiologia , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Fator 2 Associado a Receptor de TNF , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/fisiologia , Receptor fas/fisiologiaRESUMO
Although the expression patterns of the members of the tumour necrosis factor receptor and ligand families have extensively been studied by flow-cytometry on stimulated peripheral blood mononuclear cells (PBMNC), little or no flow-cytometric or immunohistological data exist about their expression in lymphoid tissue. According to the data obtained from stimulated PBMNC, several members of these molecule families (e.g. CD40 ligand [CD40L], CD30, CD27, hOX40) have been considered to be either T-cell restricted or strongly T-cell associated. The present study on samples from palatine tonsils revealed that most of these molecules are also expressed by tonsillar B cells. The additional analysis of the co-expression of these molecules also disclosed the existence of CD40+/CD40L+ and CD27+/CD70+ (CD27L+) lymphoid cells in tonsillar tissue.
Assuntos
Linfócitos B/metabolismo , Ligantes , Receptores do Fator de Necrose Tumoral/metabolismo , Antígenos CD/metabolismo , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Tonsila Palatina/imunologia , Linfócitos T/metabolismoRESUMO
Interleukin 15 (IL-15) shares many functional properties with interleukin 2 (IL-2), although both cytokines probably also exert distinct functions. In order to screen for functional differences between IL-2 and IL-15 with respect to the control of T cell functions, we have stimulated human T lymphoblasts (hTBl) with IL-2 and/or IL-15 and have assessed the resulting changes in the following parameters: T cell proliferation; expression of various relevant surface markers; cytokine and receptor (alpha-chain) transcription; and IL-2 and IL-15 activity. Both cytokines equally upregulate standard activation markers such as CD25 and CD95 and downregulate CD27. However, IL-2 upregulates CD30, TNF receptor type II and CD40L expression significantly stronger than IL-15. IL-15 potentiates Con A-induced IL-2 secretion. Even though hTBl transcribe the IL-15 gene, they do not secrete IL-15 activity. These observations suggest that both cytokines can differentially regulate T cells, e.g. T cell functions relevant to the control of cell cycle progression and apoptosis, and/or that they can stimulate different T cell subsets. Moreover, IL-15 may potentiate IL-2-driven T cell responses.