RESUMO
Gain of 1q is one of the most common alterations in cancer and has been associated with adverse clinical behaviour in ependymoma. The aim of this study was to investigate this region to gain insight into the role of 1q genes in intracranial paediatric ependymoma. To address this issue we generated profiles of eleven ependymoma, including two relapse pairs and seven primary tumours, using comparative genome hybridisation and serial analysis of gene expression. Analysis of 656 SAGE tags mapping to 1q identified CHI3L1 and S100A10 as the most upregulated genes in the relapse pair with de novo 1q gain upon recurrence. Moreover, three more members of the S100 family had distinct gene expression profiles in ependymoma. Candidates (CHI3L1, S100A10, S100A4, S100A6 and S100A2) were validated using immunohistochemistry on a tissue microarray of 74 paediatric ependymoma. In necrotic cases, CHI3L1 demonstrated a distinct staining pattern in tumour cells adjacent to the areas of necrosis. S100A6 significantly correlated with supratentorial tumours (P<0.001) and S100A4 with patients under the age of 3 years at diagnosis (P=0.038). In conclusion, this study provides evidence that S100A6 and S100A4 are differentially expressed in clinically relevant subgroups, and also demonstrates a link between CHI3L1 protein expression and necrosis in intracranial paediatric ependymoma.
Assuntos
Neoplasias do Sistema Nervoso Central/genética , Cromossomos Humanos Par 1 , Ependimoma/genética , Proteínas S100/genética , Neoplasias do Sistema Nervoso Central/patologia , Criança , Pré-Escolar , Ependimoma/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , RNA Mensageiro/genética , RecidivaRESUMO
Mutations in the retinoblastoma (pRb) tumor suppressor pathway including its cyclin-cdk regulatory kinases, or cdk inhibitors, are a hallmark of most cancers and allow unrestrained E2F-1 transcription factor activity, which leads to unregulated G1-to-S-phase cell cycle progression. Moderate levels of E2F-1 overexpression are tolerated in interleukin 3 (IL-3)-dependent 32D.3 myeloid progenitor cells, yet this induces apoptosis when these cells are deprived of IL-3. However, when E2F activity is augmented by coexpression of its heterodimeric partner, DP-1, the effects of survival factors are abrogated. To determine whether enforced E2F-1 expression selectively sensitizes cells to cytotoxic agents, we examined the effects of chemotherapeutic agents and radiation used in cancer therapy. E2F-1 overexpression in the myeloid cells preferentially sensitized cells to apoptosis when they were treated with the topoisomerase II inhibitor etoposide. Although E2F-1 alone induces moderate levels of p53 and treatment with drugs markedly increased p53, the deleterious effects of etoposide in E2F-1-overexpressing cells were independent of p53 accumulation. Coexpression of Bcl-2 and E2F-1 in 32D.3 cells protected them from etoposide-mediated apoptosis. However, Bcl-2 also prevented apoptosis of these cells upon exposure to 5-fluorouracil and doxorubicin, which were also cytotoxic for control cells. Pretreating E2F-1-expressing cells with ICRF-193, a second topoisomerase II inhibitor that does not damage DNA, protected the cells from etoposide-induced apoptosis. However, ICRF-193 cooperated with DNA-damaging agents to induce apoptosis. Therefore, topoisomerase II inhibition and DNA damage can cooperate to selectively induce p53-independent apoptosis in cells that have unregulated E2F-1 activity resulting from mutations in the pRb pathway.
Assuntos
Antineoplásicos/farmacologia , Apoptose/fisiologia , Proteínas de Transporte , Proteínas de Ciclo Celular , Dano ao DNA , Proteínas de Ligação a DNA , Inibidores da Topoisomerase II , Fatores de Transcrição/fisiologia , Células da Medula Óssea , Ciclo Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dicetopiperazinas , Doxorrubicina/farmacologia , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Fluoruracila/farmacologia , Raios gama , Expressão Gênica , Humanos , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Malignant transformation occurs in cells that overexpress c-Myc or that inappropriately activate E2F-1. Transformation occurs after the selection of cells that have acquired resistance to apoptosis that is triggered by these oncogenes, and a key mediator of this cell death process is the p53 tumor suppressor. In IL-3-dependent immortal 32D.3 myeloid cells the ARF/p53 apoptotic pathway is inactivated, as these cells fail to express ARF. Nonetheless, both c-Myc and E2F-1 overexpression accelerated apoptosis when these cells were deprived of IL-3. Here we report that c-Myc or E2F-1 overexpression suppresses Bcl-2 protein and RNA levels, and that restoration of Bcl-2 protein effectively blocks the accelerated apoptosis that occurs when c-Myc- or E2F-1-overexpressing cells are deprived of IL-3. Blocking p53 activity with mutant p53 did not abrogate E2F-1-induced suppression of Bcl-2. Analysis of immortal myeloid cells engineered to overexpress c-Myc and E2F-1 DNA binding mutants revealed that DNA binding activity of these oncoproteins is required to suppress Bcl-2 expression. These results suggest that the targeting of Bcl-2 family members is an important mechanism of oncogene-induced apoptosis, and that this occurs independent of the ARF/p53 pathway.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Genes bcl-2 , Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Fatores de Transcrição/fisiologia , Animais , Apoptose/genética , Linhagem Celular Transformada , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , DNA/genética , DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Genes myc , Genes p53 , Interleucina-3/farmacologia , Camundongos , Mutação , Células Mieloides/efeitos dos fármacos , Células Mieloides/patologia , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes de Fusão/fisiologia , Temperatura , Fatores de Transcrição/genética , Transfecção , Proteína Supressora de Tumor p14ARF/deficiência , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p14ARF/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Proteína bcl-XRESUMO
The p55 tumor necrosis factor (TNF) receptor and the Fas (CD95/APO-1) receptor share an intracellular domain necessary to induce apoptosis, suggesting they utilize common signaling pathways. To define pathways triggered by Fas and TNF-alpha we utilized human CEM-C7 T-cells. As expected, stimulation of either receptor induced apoptosis and TNF-alpha-induced signaling included the activation of NF-kappaB. Surprisingly, Fas-induced signaling also triggered the activation of NF-kappaB in T cells, yet the kinetics of NF-kappaB induction by Fas was markedly delayed. NF-kappaB activation by both pathways was persistent and due to the sequential degradation of IkappaB-alpha and IkappaB-beta. However, the kinetics of IkappaB degradation were different and there were differential effects of protease inhibitors and antioxidants on NF-kappaB activation. Signaling pathways leading to activation of apoptosis were similarly separable and were also independent of NF-kappaB activation. Thus, the Fas and TNF receptors utilize distinct signal transduction pathways in T-cells to induce NF-kappaB and apoptosis.
RESUMO
Integration host factor (IHF) binds to two sites near the origin of transfer of the conjugative antibiotic resistance plasmid, R100. DNase I footprinting shows that one site is immediately adjacent to oriT and the gene X promoter, and another is adjacent to the traM promoter. A third site, known only from retardation gels, is near the traJ promoter. The relative promoter activities of genes X, traJ and traM are reduced in himA mutants (IHF-), as measured by chloramphenicol-resistance assays. Transcript analyses by Northern blots showed a reduction in size of the principal gene X and traJ transcripts in the absence of IHF.
Assuntos
Proteínas de Bactérias/genética , Conjugação Genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Regiões Promotoras Genéticas , Sequência de Bases , Resistência ao Cloranfenicol , Mapeamento Cromossômico , Desoxirribonuclease I , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Fatores Hospedeiros de Integração , Dados de Sequência Molecular , Mutação , Plasmídeos , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The fertility control gene finP, the transfer gene traM, and the transfer origin, oriT, of plasmid R100 were isolated on a single 1.2-kilobase EcoRV fragment and were then subcloned as HaeIII fragments. The sequence of the 754-base-pair finP-containing fragment is reported here. In addition to the finP gene, the sequence includes all but two bases of the R100 traM open reading frame and apparently all of the leader mRNA sequence and amino end of the traJ gene of R100. The sequence contains two open reading frames which encode small proteins on the opposite strand from the traM and traJ genes. It also shows two sets of inverted repeats that have the characteristics of transcription terminators. One set is positioned as if it was the traM terminator, and the other set, which is downstream from the first, sits in the middle of the leader mRNA sequence for traJ. On the bottom strand, this inverted repeat has the structure of a rho-independent terminator. Other less-stable inverted repeats overlap this second terminator in the same way as is seen in attenuation sequences, and the two separate small open reading frames on the bottom strand also totally overlap the stem of the rho-independent terminator, suggesting that their translation would cause shifting of termination to the bottom strand homolog of the putative traM terminator. The finP gene product was not identified, but the gene was mapped to the sequence which contains the traJ gene. It either overlaps traJ or is antisense to it.
Assuntos
Conjugação Genética , Genes Bacterianos , Fatores R , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , Escherichia coli/genética , Sequências Repetitivas de Ácido Nucleico , Regiões Terminadoras Genéticas , Transcrição GênicaRESUMO
Transcription from the promoter of mouse mammary tumor virus is subject to both positive and negative control by cellular factors, and proviral promoter elements that mediate a basal level of transcription must in some way respond to these cellular regulatory signals. Several such elements, including a TATA box, a region containing three octamer-related sequences, and a binding site for nuclear factor 1, have been previously defined. Additional promoter mutations have allowed a fourth basal promoter element to be identified near the transcription initiation site between +2 and +10. Sequence alterations within this element affect transcription both in vivo and in vitro. Gel electrophoresis mobility shift and DNase I footprinting assays define a nuclear protein, termed initiation site-binding protein, that specifically recognizes this region of the promoter. Optimal levels of transcription from the mouse mammary tumor virus promoter require initiation site-binding protein, as demonstrated by a correlation between protein affinity and transcriptional activity and by specific inhibition of transcription in vitro by an oligonucleotide capable of titrating the protein from transcriptionally active fractions.