RESUMO
Hodgkin lymphoma (HL) in older patients appears to be a different disease compared with younger patients with historically lower survival rates. This is related to a variety of factors, including increased treatment-related toxicity, the presence of comorbidities, and biologic differences. In order to better assess the clinical characteristics, treatment strategies, and outcome of this particular population, we conducted a population-based, retrospective analysis including 269 patients with HL older than 60 years (median age 71 years, range 60-94), treated between 2000 and 2017 in 15 referral centers across Switzerland. Primary endpoints were overall survival (OS), progression-free survival (PFS), and cause-specific survival (CSS). The vast majority of patients were treated with curative intent, either with a combined modality approach (chemotherapy followed by radiation therapy) or with systemic therapy. At a median follow-up of 6.6 years (95% confidence interval [CI], 6.0-7.6), 5-year PFS was 52.2% (95% CI, 46.0-59.2), 5-year OS was 62.5% (95% CI, 56.4-69.2), and 5-year CSS was 85.1.8% (95% CI, 80.3-90.1) for the entire cohort. A significant difference in terms of CSS was observed for patients older than 71 years in comparison to patients aged 60-70 years (hazard ratio 2.6, 1.3-5.0, p = 0.005). Bleomycin-induced lung toxicity (BLT) was documented in 26 patients (17.7%) out of the 147 patients exposed to this compound and was more frequent in patients older than 71 years (15/60, 25%). Outcome of HL pts older than 71 years appeared to decrease substantially in comparison to the younger counterpart. Treatment-related toxicities appeared to be relevant, in particular, BLT. New, potentially less toxic strategies need to be investigated in prospective clinical trials in this particular frail population.
Assuntos
Doença de Hodgkin/epidemiologia , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , SuíçaRESUMO
The receptor subunit gp130 transduces multiple cell type-specific activities of the leukemia inhibitory factor (LIF)/interleukin (IL)-6 family of cytokines through the signal transducer and activator of transcription (STAT) and src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-2/ras/Erk pathways. To define STAT-dependent physiological responses, we generated mice with a COOH-terminal gp130(DeltaSTAT) "knock-in" mutation which deleted all STAT-binding sites. gp130(DeltaSTAT) mice phenocopyed mice deficient for IL-6 (impaired humoral and mucosal immune and hepatic acute phase responses) and LIF (failure of blastocyst implantation). However, unlike mice with null mutations in any of the components in the gp130 signaling pathway, gp130(DeltaSTAT) mice also displayed gastrointestinal ulceration and a severe joint disease with features of chronic synovitis, cartilaginous metaplasia, and degradation of the articular cartilage. Mitogenic hyperresponsiveness of synovial cells to the LIF/IL-6 family of cyto-kines was caused by sustained gp130-mediated SHP-2/ras/Erk activation due to impaired STAT-mediated induction of suppressor of cytokine signaling (SOCS) proteins which normally limits gp130 signaling. Therefore, the joint pathology in gp130(DeltaSTAT) mice is likely to arise from the disturbance of the otherwise balanced activation of the SHP-2/ras/Erk and STAT signaling cascades emanating from gp130.
Assuntos
Antígenos CD/genética , Antígenos CD/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Proteínas Repressoras , Transativadores/genética , Transativadores/fisiologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Receptor gp130 de Citocina , Primers do DNA/genética , Implantação do Embrião/genética , Implantação do Embrião/fisiologia , Feminino , Artropatias/etiologia , Artropatias/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Mutantes , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Úlcera Péptica/etiologia , Úlcera Péptica/patologia , Gravidez , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de CitocinaAssuntos
Angioedema/diagnóstico , Edema/etiologia , Neoplasias de Cabeça e Pescoço/diagnóstico , Linfoma de Células T/diagnóstico , Paniculite/diagnóstico , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biópsia , Ciclofosfamida/administração & dosagem , Diagnóstico Diferencial , Doxorrubicina/administração & dosagem , Edema/diagnóstico , Face , Neoplasias de Cabeça e Pescoço/complicações , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Linfoma de Células T/complicações , Linfoma de Células T/tratamento farmacológico , Linfoma de Células T/patologia , Paniculite/complicações , Paniculite/tratamento farmacológico , Paniculite/patologia , Valor Preditivo dos Testes , Prednisolona/administração & dosagem , Fatores de Risco , Resultado do Tratamento , Vincristina/administração & dosagemRESUMO
The antiretroviral agent nelfinavir has antimyeloma activity and can overcome resistance to bortezomib. Our phase I/II trial investigated whether adding nelfinavir to lenalidomide-dexamethasone can overcome lenalidomide resistance in lenalidomide-refractory multiple myeloma (MM). Twenty-nine patients were included (high-risk cytogenetic aberrations 31%; ≥2 prior therapy lines 93%; lenalidomide-bortezomib double-refractory 34%). Twenty-four patients (83%) had prior bortezomib and 10 (34%) were lenalidomide-bortezomib double-refractory. They received four cycles of nelfinavir 2500 mg/day with standard-dose lenalidomide (25 mg days 1-21) and dexamethasone (40/20 mg days 1, 8, 15, 22). Minor response or better was achieved in 16 patients (55%; 95% CI 36-74%), including 40% of those who were lenalidomide-bortezomib double-refractory, and partial response or better in nine patients (31%; 95% CI 15-51%). Median progression-free survival was 3.4 (95% CI 2.0-4.9) months and median overall survival 21.6 (13.0-50.1) months. Lenalidomide-related pneumonitis, pneumonia, and neutropenic fever occurred, but there were no unexpected adverse events. Peripheral blood mononuclear cells showed a 45% (95% CI 40-51%) reduction in total proteasome activity from baseline and significant induction of unfolded protein response and autophagy. Thus, nelfinavir-lenalidomide-dexamethasone is an active oral combination in lenalidomide-refractory MM.
Assuntos
Anti-Inflamatórios/uso terapêutico , Dexametasona/uso terapêutico , Inibidores da Protease de HIV/uso terapêutico , Lenalidomida/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Nelfinavir/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anti-Inflamatórios/farmacologia , Dexametasona/farmacologia , Feminino , Inibidores da Protease de HIV/farmacologia , Humanos , Lenalidomida/farmacologia , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Nelfinavir/farmacologiaRESUMO
Chemotherapy with G-CSF is used to mobilize peripheral stem cells in multiple myeloma (MM) patients, with plerixafor as a rescue strategy for poorly mobilizing patients. Preclinical studies suggested that the nonsteroidal anti-inflammatory drug meloxicam enhances the mobilization of CD34+ cells. In this single-center study, we evaluated whether adding meloxicam to chemotherapy/G-CSF mobilization increases peripheral hematopoietic CD34+ cell levels and reduces the need of using plerixafor. We prospectively compared two consecutive cohorts of MM patients in first remission mobilized with G-CSF and non-myelosuppressive chemotherapy with vinorelbine or gemcitabine. The second cohort additionally received oral meloxicam. The cohorts comprised 84 patients without meloxicam (-M) and 66 patients with meloxicam (+M). Meloxicam was well tolerated and associated with similar hematologic engraftment after transplantation and equal survival rates. However, the meloxicam group had higher CD34+ cell levels on day 8 of the mobilization procedure (53 200 versus 35 600 CD34+ cells/mL; P=0.007), and fewer patients needed >1 collection day (+M: 6 (9%) patients versus -M: 16 (19%) patients; P=0.04). This resulted in reduced plerixafor administrations (+M: 7 (11%) patients versus -M: 18 (21%) patients; P=0.03) and less costs. Our data suggest that meloxicam enhances the mobilization of hematopoietic CD34+ blood cells in MM patients.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Mobilização de Células-Tronco Hematopoéticas/métodos , Meloxicam/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/cirurgia , Transplante de Células-Tronco de Sangue Periférico/métodos , Adulto , Idoso , Anti-Inflamatórios não Esteroides/farmacologia , Feminino , Humanos , Masculino , Meloxicam/farmacologia , Pessoa de Meia-Idade , Mieloma Múltiplo/patologiaRESUMO
ADAM22 is one of three catalytically inactive ADAM family members highly expressed in the brain. Preliminary functional studies suggest possible roles in epilepsy and myelination. We report an additional eight new splice variants of human ADAM22. Analysis of the altered splicing patterns of ADAM22 mRNAs in glioma allows us to suggest alternate splicing patterns in normal brain compared to glioma may represent differential use of exon 32. We also report diversity in the 5' leader sequences of ADAM22 mRNAs as a consequence of alternate transcriptional initiation sites. ADAM22 has an additional transcriptional initiation element producing transcripts lacking the exon 1 sequence including the signal peptide. Variable transcriptional initiation in exon 1 produces a range of ADAM22 5' leader sequence lengths, all of which are significantly longer than those described in NCBI reference sequences. Longer 5' leader sequences contain a second upstream AUG codon which acts to inhibit ADAM22 translation.
Assuntos
Proteínas ADAM/genética , Glioma/patologia , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , Proteínas ADAM/química , Proteínas ADAM/metabolismo , Processamento Alternativo , Encéfalo/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Códon , Códon de Terminação , Primers do DNA , Éxons , Etiquetas de Sequências Expressas , Genes Reporter , Glioma/metabolismo , Humanos , Rim/citologia , Luciferases/metabolismo , Estadiamento de Neoplasias , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Isoformas de Proteínas , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Sítio de Iniciação de TranscriçãoRESUMO
The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.
Assuntos
DNA Viral/genética , Genes Virais , Polyomavirus/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Regulação da Expressão GênicaRESUMO
Primary brain tumors of glial origin often overexpress epidermal growth factor receptors (EGF-Rs). This may be associated with amplification of the EGF-R gene. We have examined tissue from 23 glioblastoma multiforme tumors and found amplification and rearrangement of the EGF-R gene in four of these. The cytoplasmic domain of the EGF-R gene was invariably amplified in these four tumors, while the epidermal growth factor binding domain was not uniformly amplified in three of these tumors. Western blot analysis of the EGF-R protein revealed high levels of a truncated EGF-R protein in two of the four tumors with EGF-R gene amplification.
Assuntos
Receptores ErbB/genética , Amplificação de Genes , Glioblastoma/genética , Citoplasma/análise , Receptores ErbB/análise , Humanos , Peso Molecular , RNA Mensageiro/análiseRESUMO
Ten cell lines were established from different biopsies from nine patients with small cell lung cancer (SCLC). These were established from metastases in the marrow (7), breast (1), pleural fluid (1), and spinal cord (1) and had been in culture for periods varying from 1 to 11 months. All cell lines exhibited typical cytological features of SCLC, and produced neuron specific enolase. All lines examined (5) contained dense core granules and were tumorigenic when injected intracranially into nude mice. None of the six cell lines tested had an amplified oncogene (c-myc or n-myc) previously reported to be amplified in SCLC. Detailed chromosome analyses were undertaken and showed that a structural abnormality of chromosome 3(p11p23) was a frequent (6 of 10) but not invariable finding. Our study and one other indicate that there is not a unique chromosome abnormality present in all cases of SCLC, although loss of chromosome 13 and structural abnormalities of chromosome 3 were frequently found. A feature of these SCLC cell lines established from metastatic deposits was the complexity of the karyotypes due to numerical and structural chromosomal abnormalities.
Assuntos
Carcinoma de Células Pequenas/genética , Neoplasias Pulmonares/genética , Biópsia , Linhagem Celular , Deleção Cromossômica , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 3 , Feminino , Amplificação de Genes , Humanos , Cariotipagem , Masculino , Microscopia Eletrônica , OncogenesRESUMO
Gliomas are highly invasive, invariably fatal intracerebral tumors. It seems that receptors for hyaluronan are required for the invasive process. Hyaluronan is a major component of the extracellular matrix in the brain, and all of the gliomas express CD44, the principal receptor for hyaluronan. To investigate the role of lysosomal hyaluronidases on tumor invasion we overexpressed hyaluronidase-2 (HYAL2) in murine astrocytoma cells. We found that high expression of HYAL2 accelerated intracerebral tumor growth dramatically, whereas the same cells formed s.c. tumors within the same time as the parental cells. The brain tumors were highly vascularized and more invasive than the control tumors. It seems that the interactions of the HYAL2-expressing tumor cells with the hyaluronan-containing extracellular matrix in the brain mediate these effects, whereas the same cells in a s.c. environment, which lacks the high hyaluronan level, behave like the parental cells.
Assuntos
Astrocitoma/enzimologia , Neoplasias Encefálicas/enzimologia , Hialuronoglucosaminidase/fisiologia , Neoplasias Cutâneas/enzimologia , Animais , Divisão Celular , Glioma/enzimologia , Receptores de Hialuronatos/biossíntese , Hialuronoglucosaminidase/biossíntese , Hialuronoglucosaminidase/genética , Camundongos , Invasividade Neoplásica , Transplante de Neoplasias , RNA Mensageiro/biossínteseRESUMO
Using FDC-P1 derived cell lines which ectopically express either the wild type or mutant forms of the murine CSF-1 receptor in which individual tyrosine residues have been replaced with phenylalanine, we analysed the requirement for tyrosine residues of the receptor for the activation of STAT proteins in response to CSF-1. We found Y706 to be required for efficient activation of STAT1. The activation of STAT3 was not affected by the mutation of Y706 to phenylalanine. The addition of phosphopeptides spanning Y708 of the human CSF-1 receptor (identical with the sequence surrounding Y706 of the murine receptor) to electrophoretic mobility shift assays led to competition of the formation of STAT1 containing complexes, SIF-B and SIF-C with the DNA probe. These phosphopeptides did, however, not affect the formation of the STAT3 containing complex, SIF-A, with the probe. Replacement of Y807 with phenylalanine led to a complete block of activation of all STAT proteins in response to CSF-1, however, this phosphotyrosine does not appear to represent a STAT binding site of the receptor as a phosphopeptide spanning Y809 of the human CSF-1 receptor could not compete any STAT/DNA complex formation in electrophoretic mobility shift assays.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Proteínas Tirosina Quinases , Receptor de Fator Estimulador de Colônias de Macrófagos/efeitos dos fármacos , Transativadores/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Camundongos , Fosforilação , Proteínas/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Transdução de Sinais , TYK2 QuinaseRESUMO
There is currently much interest in the mechanisms of action of antiproliferative agents and their effects on cell cycle machinery. In the present study we examined the mechanisms of action of four unrelated agents known to inhibit proliferation of CSF-1-stimulated bone marrow-derived macrophages (BMM). We report that 8-bromo-cAMP (8Br-cAMP) and lipopolysaccharide (LPS) potently reduced CSF-1-stimulated cyclin D1 protein, and cyclin-dependent kinase (cdk) 4 mRNA and protein levels, while the inhibitory effects of the Na+/ H+ antiport inhibitor 5-(N',N'-dimethyl) amiloride (DMA) and interferon gamma (IFN gamma ) were only weak. All agents repressed CSF-1-stimulated retinoblastoma protein phosphorylation. Furthermore, 8Br-cAMP and to a lesser extent IFN gamma, also reduced CSF-1-stimulated levels of E2F DNA binding activity in a macrophage cell line, BAC1.2F5. An explanation for the different effects of the agents is that 8Br-cAMP and LPS were found to arrest BMM in early/mid-G1, while IFN gamma and DMA arrested cells in late G1 or early S phase. These data indicate that (1) different antiproliferative agents can arrest the same cell type at distinct checkpoints in G1 and (2) effects of antiproliferative agents on cell cycle machinery is linked to the position at which they arrest cells in G1.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Fase G1/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Proteínas Proto-Oncogênicas , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Sequência de Bases , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Ciclina D1 , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/farmacologia , Ciclinas/metabolismo , DNA/metabolismo , Fatores de Transcrição E2F , Fase G1/fisiologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Oncogênicas/metabolismo , Fosforilação/efeitos dos fármacos , Proteína do Retinoblastoma/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/metabolismoRESUMO
Actinomycin D, known for its suppression of cellular RNA synthesis and for the reduction of the rate of synthesis of double-stranded DNA by the RNA tumor virus RNA-dependent DNA polymerase, was found to interact with single-stranded DNA in such a way as to inhibit DNA . DNA and DNA . RNA hybridizations. This finding is discussed in the light of the observation that DNA elongation during DNA synthesis of RNA tumor viruses is blocked in vitro in the presence of actinomycin D. It thus supports the model that hybridization is a necessary step during RNA tumor virus DNA synthesis.
Assuntos
Vírus da Leucose Aviária/metabolismo , Vírus da Mieloblastose Aviária/metabolismo , DNA Viral/síntese química , Dactinomicina/farmacologia , Hibridização de Ácido Nucleico/efeitos dos fármacos , DNA Viral/biossíntese , Modelos Biológicos , Replicação ViralRESUMO
Recombinant human cytokines were examined for their effects on plasminogen activator inhibitor-1 (PAI-1) production by human articular cartilage and chondrocyte monolayer cultures. Cartilage and chondrocytes were cultured with and without added cytokines and the conditioned media assayed for PAI-1 by a specific enzyme-linked immunosorbent assay, and mRNA levels determined by Northern blot analysis. Tumor necrosis factor alpha (TNF alpha) reduced, and transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (bFGF) increased, the levels of PAI-1 antigen and mRNA in the culture fluids and cell extracts, respectively. The effects of TNF alpha and TGF-beta on PAI-1 antigen levels were both time- and concentration-dependent; optimum doses being 10-100 pM TNF alpha and 0.4-0.8 nM TGF-beta, with each cytokine exerting its effect on PAI-1 antigen levels within 8 h of addition to culture. TNF alpha (and interleukin-1 alpha) also countered the effects of TGF-beta and bFGF. The anti-inflammatory drugs, indomethacin and dexamethasone, did not appear to modulate PAI-1 levels in cultures of cartilage tissue. The inhibition of PAI-1 levels by cytokines and reagents which stimulate cartilage resorption (i.e., TNF alpha, interleukin-1 alpha, retinoic acid) and enhancement by cytokines which counter it (i.e., TGF-beta, bFGF) further implicate plasminogen activator in the mechanism(s) of cartilage degradation in diseases such as arthritis.
Assuntos
Cartilagem Articular/metabolismo , Citocinas/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Artropatias/metabolismo , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Idoso , Anti-Inflamatórios/farmacologia , Cartilagem Articular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Inibidor 1 de Ativador de Plasminogênio/genética , Ativadores de Plasminogênio/metabolismo , RNA Mensageiro/análise , Regulação para CimaRESUMO
Inactivation of the tumour suppressor p53 is the most common defect in cancer cells. p53 is a sequence specific transcription factor that is activated in response to various forms of genotoxic stress to induce cell cycle arrest and apoptosis. Induction of p53 is subjected to complex and strict control through several pathways, as it will often determine cellular fate. The p73 protein shares strong structural and functional similarities with p53 such as the potential to activate p53 responsive genes and the ability to induce apoptosis. In addition to alternative splicing at the carboxyl terminus which yields several p73 isoforms, a p73 variant lacking the N-terminal transactivation domain (Delta Np73) was described in mice. In this study, we report the cloning and characterisation of the human Delta Np73 isoforms, their regulation by p53 and their possible role in carcinogenesis. As in mice, human Delta Np73 lacks the transactivation domain and starts with an alternative exon (exon 3'). Its expression is driven by a second promoter located in a genomic region upstream of this exon, supporting the idea of two independently regulated proteins, derived from the same gene. As anticipated, Delta Np73 is capable of regulating TAp73 and p53 function since it is able to block their transactivation activity and their ability to induce apoptosis. Interestingly, expression of the Delta Np73 is strongly up-regulated by the TA isoforms and by p53, thus creating a feedback loop that tightly regulates the function of TAp73 and more importantly of p53. The regulation of Delta Np73 is exerted through a p53 responsive element located on the Delta N promoter. Expression of Delta Np73 not only regulates the function of p53 and TAp73 but also shuts off its own expression, once again finely regulating the whole system. Our data also suggest that increased expression of Delta Np73, functionally inactivating p53, could be involved in tumorogenesis. An extensive analysis of the expression pattern of Delta Np73 in primary tumours would clarify this issue.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Genes p53 , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem de Organismos , Proteínas de Ligação a DNA/genética , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases , Regiões Promotoras Genéticas , Ativação Transcricional/genética , Ativação Transcricional/fisiologia , Transfecção , Células Tumorais Cultivadas , Proteína Tumoral p73 , Proteínas Supressoras de TumorRESUMO
Lipopolysaccharide (LPS) is a powerful macrophage-activating agent and antimitogen. We recently showed that LPS unexpectedly induces cyclin D2 in macrophages. Since LPS stimulates macrophages to produce autocrine-acting cytokines, we examined whether LPS induction of cyclin D2 was mediated by one such type of cytokine, type I interferons (IFN). We report that bone marrow-derived macrophages (BMM) lacking a component of the type I interferon receptor (IFNAR-1) do not express cyclin D2 mRNA or protein in response to LPS stimulation (0.01-1 microg/ml for 7-30 h). Consistent with this result, addition of anti-IFN-alpha/beta neutralizing antibodies reduced levels of LPS-stimulated cyclin D2 in normal BMM. Furthermore, IFN-alpha alone induced cyclin D2 mRNA and protein in normal BMM. Thus, we have identified a new role for type I IFN in macrophages, namely, as essential mediators of LPS-stimulated cyclin D2 expression.
Assuntos
Ciclinas/biossíntese , Interferon Tipo I/fisiologia , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Ciclina D2 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos KnockoutRESUMO
The promoter of the human urokinase plasminogen activator (uPA) gene contains a sequence identical with the retinoblastoma control element (RCE) of the murine c-fos gene, as well as several Sp1 binding sites. In a number of cell lines, the uPA promoter is activated during enforced expression of the retinoblastoma protein, pRB. Electrophoretic mobility-shift assays revealed that the RCE sequence of the uPA gene forms only one specific DNA-protein complex that does not contain pRB. The formation of the RCE-protein complex can be inhibited by 20 molar excess of the unlabeled RCE sequences and by 5 molar excess of the unlabeled E2F binding site. The RCE of the human uPA gene interacts specifically with a protein, which appears to be distinct from members of the E2F family of proteins, Sp1, ATF2, and Elf-1, which are all transcription factors shown to be regulated by pRB.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regiões Promotoras Genéticas , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Sequência de Bases , Sítios de Ligação , Neoplasias da Mama , Linhagem Celular , Cloranfenicol O-Acetiltransferase/biossíntese , Sequência Consenso , Genes fos , Células HeLa , Humanos , Pulmão , Camundongos , Leite , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , TransfecçãoRESUMO
To study the feasibility of a therapy for thalassemia based on addition of a correctly functioning globin gene to bone marrow stem cells, we have developed retroviral vectors that can transfer the human beta-globin gene into pluripotent hematopoietic stem cells of the mouse. Mice reconstituted with virus-infected bone marrow cells showed long-term tissue-specific expression of human beta-globin RNA and protein. Recently, we have redesigned the retroviral vector to improve the efficiency of stem cell infection and to raise the level of globin expression obtained from the virally transduced gene. Removal of a portion of the second intron of the beta-globin gene resulted in the accumulation of a higher level of full-length viral RNA in retrovirus packaging cell lines, and these cell lines produced beta-globin virus particles at substantially higher titers. Addition of fragments from the locus activation region (LAR) of the beta-like globin gene cluster to the retroviral vectors increased beta-globin expression in infected murine erythroleukemia (MEL) cells. Fragments from the -18 and -10.9 kbp DNase I-hypersensitive sites of the LAR increased human beta-globin RNA levels to 35% and 132% of the endogenous mouse beta maj-globin RNA level, respectively. Increased expression was also found for neomycin phosphotransferase RNA, which was transcribed from the retroviral long terminal repeat (LTR), showing that the LAR fragments also activated expression from a nearby heterologous promoter. These results are discussed in the context of the efficacy and safety of gene therapy for chronic anemia in humans.