RESUMO
Four isoforms of the human fibroblast growth factor 2 (FGF-2), with different intracellular localizations and distinct effects on cell phenotype, result from alternative initiations of translation at three CUG and one AUG start codons. We showed here by Western immunoblotting and immunoprecipitation that the CUG-initiated forms of FGF-2 were synthesized in transformed cells, whereas "normal" cells almost exclusively produced the AUG-initiated form. CUG-initiated FGF-2 was induced in primary skin fibroblasts in response to heat shock and oxidative stress. In transformed cells and in stressed fibroblasts, CUG expression was dependent on cis-elements within the 5' region of FGF-2 mRNA and was not correlated to mRNA level, indicating a translational regulation. UV cross-linking experiments revealed that CUG expression was linked to the binding of several cellular proteins to FGF-2 mRNA 5' region. Since translation of FGF-2 mRNA was previously shown to occur by internal ribosome entry, a nonclassical mechanism already described for picornaviruses, the cross-linking patterns of FGF-2 and picornavirus mRNAs were compared. Comigration of several proteins, including a p60, was observed. However, this p60 was shown to be different from the p57/PTB internal entry factor, suggesting a specificity towards FGF-2 mRNA. We report here a process of translational activation of the FGF-2 CUG-initiated forms in direct relation with trans-acting factors specific to transformed and stressed cells. These data favor a critical role of CUG-initiated FGF-2 in cell transformation and in the stress response.
Assuntos
Transformação Celular Viral , Códon de Iniciação , Fator 2 de Crescimento de Fibroblastos/genética , Estresse Oxidativo , Biossíntese de Proteínas , Animais , Western Blotting , Células COS , Linhagem Celular Transformada , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Fator 2 de Crescimento de Fibroblastos/biossíntese , Células HeLa , Temperatura Alta , Humanos , Proteínas de Neoplasias/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Células Tumorais CultivadasRESUMO
Four isoforms of human fibroblast growth factor 2 (FGF-2) result from alternative initiations of translation at three CUG start codons and one AUG start codon. Here we characterize a new 34-kDa FGF-2 isoform whose expression is initiated at a fifth initiation codon. This 34-kDa FGF-2 was identified in HeLa cells by using an N-terminal directed antibody. Its initiation codon was identified by site-directed mutagenesis as being a CUG codon located at 86 nucleotides (nt) from the FGF-2 mRNA 5' end. Both in vitro translation and COS-7 cell transfection using bicistronic RNAs demonstrated that the 34-kDa FGF-2 was exclusively expressed in a cap-dependent manner. This contrasted with the expression of the other FGF-2 isoforms of 18, 22, 22.5, and 24 kDa, which is controlled by an internal ribosome entry site (IRES). Strikingly, expression of the other FGF-2 isoforms became partly cap dependent in vitro in the presence of the 5,823-nt-long 3' untranslated region of FGF-2 mRNA. Thus, the FGF-2 mRNA can be translated both by cap-dependent and IRES-driven mechanisms, the balance between these two mechanisms modulating the ratio of the different FGF-2 isoforms. The function of the new FGF-2 was also investigated. We found that the 34-kDa FGF-2, in contrast to the other isoforms, permitted NIH 3T3 cell survival in low-serum conditions. A new arginine-rich nuclear localization sequence (NLS) in the N-terminal region of the 34-kDa FGF-2 was characterized and found to be similar to the NLS of human immunodeficiency virus type 1 Rev protein. These data suggest that the function of the 34-kDa FGF-2 is mediated by nuclear targets.
Assuntos
Códon de Iniciação , Fator 2 de Crescimento de Fibroblastos/biossíntese , Capuzes de RNA , Células 3T3 , Animais , Células COS , Sobrevivência Celular , Fator 2 de Crescimento de Fibroblastos/genética , Produtos do Gene rev/genética , Células HeLa , Humanos , Camundongos , Sinais de Localização Nuclear , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , RNA MensageiroRESUMO
Anaplastic large cell lymphomas (ALCLs) are frequently associated with the t(2;5)(p23;q35) translocation, leading to the expression of NPM-ALK, a fusion protein linking nucleophosmin and anaplastic lymphoma kinase, a receptor tyrosine kinase. In ALCLs, dimerization of NPM-ALK leads to constitutive autophosphorylation and activation of the kinase, necessary for NPM-ALK oncogenicity. To investigate whether NPM-ALK, like other oncogenic tyrosine kinases, can inhibit drug-induced apoptosis, we permanently transfected NPM-ALK into Jurkat T-cells. As in ALCLs, NPM-ALK was expressed as a constitutively kinase-active 80 kDa protein, and could be detected by immunocytochemistry in nucleoli, nuclei and cytoplasm. Doxorubicin-induced apoptosis (assessed by cell morphology and annexin V-FITC binding) was significantly inhibited in two independent NPM-ALK-expressing clones (5.2+/-1.8 and 7.5+/-0.8% apoptosis), compared to control vector-transduced cells (36+/-6.7%). Similar results were observed with etoposide. In contrast, Fas-induced apoptosis was not inhibited. Cytochrome c release into the cytosol was delayed in doxorubicin-, but not anti-Fas-treated transfectant cells, indicating that apoptosis inhibition occurred upstream of mitochondrial events. Using NPM-ALK mutants, we demonstrated that inhibition of drug-induced apoptosis: (1) requires functional kinase activity, (2) does not involve phospholipase C-gamma, essential for NPM-ALK-mediated mitogenicity and (3) appears to be phosphoinositide 3-kinase independent, despite a strong Akt/PKB activation observed in wild type NPM-ALK-expressing cells. These results suggest that the NPM-ALK antiapoptotic and mitogenic pathways are distinct.
Assuntos
Apoptose/efeitos dos fármacos , Glicoproteínas de Membrana/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases/fisiologia , Linfócitos T/metabolismo , Receptor fas/fisiologia , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Apoptose/genética , Apoptose/fisiologia , Sítios de Ligação , Cromonas/farmacologia , Grupo dos Citocromos c/metabolismo , Doxorrubicina/farmacologia , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Proteína Ligante Fas , Humanos , Isoenzimas/metabolismo , Células Jurkat/efeitos dos fármacos , Células Jurkat/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Morfolinas/farmacologia , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfolipase C gama , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes de Fusão/fisiologia , Linfócitos T/efeitos dos fármacos , Transfecção , Fosfolipases Tipo C/metabolismoRESUMO
Under certain conditions, T4 gene 32 protein is known to increase the efficiency of different enzymes, such as Taq DNA polymerase, reverse transcriptase, and telomerase. In this study, we compared the efficiency of the SMART PCR cDNA synthesis kit with and without the T4 gene 32 protein. The use of this cDNA synthesis procedure, in combination with T4 gene 32 protein, increases the yield of RT-PCR products from approximately 90% to 150%. This effect is even observed for long mRNA templates and low concentrations of total RNA (25 ng). Therefore, we suggest the addition of T4 gene 32 protein in the RT-PCR mixture to increase the efficiency of cDNA synthesis, particularly in cases when low amounts of tissue are used.
Assuntos
Proteínas de Ligação a DNA , Reação em Cadeia da Polimerase/métodos , Taq Polimerase , Proteínas Virais , DNA Complementar , RNA MensageiroRESUMO
This report describes a case of anaplastic large cell lymphoma with the canonical t(2;5)(p23;q35) translocation in association with duplication of the short arm of the non-translocated chromosome 2, as demonstrated by two colour fluorescence in situ hybridisation. Because the tumour cells were tetraploid, these abnormalities were in duplicate, with four copies of the full length ALK gene and two copies of the t(2;5)(p23;q35) translocation. Despite multiple copies of the normal ALK gene, immunohistochemical, reverse transcriptase polymerase chain reaction, and western blot analysis demonstrated that only the fusion gene NPM/ALK was expressed and that normal ALK genes remained silent. Although based on a single case, these data indicate that structural rather than numerical abnormalities of the ALK gene are implicated in the pathogenesis of anaplastic large cell lymphomas.
Assuntos
Cromossomos Humanos Par 2 , Cromossomos Humanos Par 5 , Linfoma Difuso de Grandes Células B/genética , Proteínas Tirosina Quinases/genética , Translocação Genética , Quinase do Linfoma Anaplásico , Criança , Duplicação Gênica , Humanos , Hibridização in Situ Fluorescente , Masculino , Receptores Proteína Tirosina QuinasesRESUMO
Dysregulated expression of factors that control protein synthesis is associated with poor prognosis of many cancers, but the underlying mechanisms are not well defined. Analysis of the diffuse large B-cell lymphoma (DLBCL) translatome revealed selective upregulation of mRNAs encoding anti-apoptotic and DNA repair proteins. We show that enhanced synthesis of these proteins in DLBCL is mediated by the relief of repression that is normally imposed by structure in the 5'-untranslated regions of their corresponding mRNAs. This process is driven by signaling through mammalian target of rapamycin, resulting in increased synthesis of eukaryotic initiation factor (eIF) 4B complex (eIF4B), a known activator of the RNA helicase eIF4A. Reducing eIF4B expression alone is sufficient to decrease synthesis of proteins associated with enhanced tumor cell survival, namely DAXX, BCL2 and ERCC5. Importantly, eIF4B-driven expression of these key survival proteins is directly correlated with patient outcome, and eIF4B, DAXX and ERCC5 are identified as novel prognostic markers for poor survival in DLBCL. Our work provides new insights into the mechanisms by which the cancer-promoting translational machinery drives lymphomagenesis.
Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Regiões 5' não Traduzidas , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Humanos , Linfoma Difuso de Grandes Células B/patologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
Division inhibition caused by the minCD gene products of Escherichia coli is suppressed specifically at mid-cell by MinE protein expressed at physiological levels. Excess MinE allows division to take place also at the poles, leading to a minicell-forming (Min-) phenotype. In order to investigate the basis of this topological specificity, we have analysed the ability of truncated derivatives of MinE to suppress either minCD-dependent division inhibition in a chromosomal delta(minB) background, or the division inhibition exerted by MinCD at the cell poles in a minB+ strain. Our results indicate that these two effects are not mediated by identical interactions of MinE protein. In addition, gel filtration and the yeast two-hybrid system indicated that MinE interacts with itself by means of its central segment. Taken together, our results favour a model in which wild-type MinE dimer molecules direct the division inhibitor molecules to the cell poles, thus preventing polar divisions and allowing non-polar sites to divide. This model explains how excess MinE, or an excess of certain MinE derivatives which prevent the accumulation of the division inhibitor at the poles, can confer a Min- phenotype in a minB+ strain.
Assuntos
Proteínas de Bactérias/genética , Divisão Celular/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Supressão Genética , Sequência de Aminoácidos , Proteínas de Ciclo Celular , Dados de Sequência Molecular , Fenótipo , Plasmídeos/genética , Análise de Sequência , Deleção de SequênciaRESUMO
Fibroblast growth factor 2 (FGF-2) belongs to a family of 18 genes coding for either mitogenic differentiating factors or oncogenic proteins, the expression of which must be tightly controlled. We looked for regulatory elements in the 5823-nucleotide-long 3'-untranslated region of the FGF-2 mRNA that contains eight potential alternative polyadenylation sites. Quantitative reverse transcription-polymerase chain reaction revealed that poly(A) site utilization was cell type-dependent, with the eighth poly(A) site being used (95%) in primary human skin fibroblasts, whereas proximal sites were used in the transformed cell lines studied here. We used a cell transfection approach with synthetic reporter mRNAs to localize a destabilizing element between the first and second poly(A) sites. Although AU-rich, the FGF-2-destabilizing element had unique features: it involved a 122-nucleotide direct repeat, with both elements of the repeat being required for the destabilizing activity. These data show that short stable FGF-2 mRNAs are present in transformed cells, whereas skin fibroblasts contain mostly long unstable mRNAs, suggesting that FGF-2 mRNA stability cannot be regulated in transformed cells. The results also provide evidence of a multilevel post-transcriptional control of FGF-2 expression; such a stringent control prevents FGF-2 overexpression and permits its expression to be enhanced only in relevant physiological situations.
Assuntos
Fator 2 de Crescimento de Fibroblastos/biossíntese , RNA Mensageiro/biossíntese , Regiões 3' não Traduzidas/genética , Monofosfato de Adenosina , Animais , Sequência de Bases , Células COS , Fator 2 de Crescimento de Fibroblastos/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Plasmídeos , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , TransfecçãoRESUMO
Five fibroblast growth factor 2 (FGF-2) isoforms are synthesized from human FGF-2 mRNA by a process of alternative initiation of translation. The regulation of FGF-2 isoform expression by the mRNA 5823-nucleotide-long 3'-untranslated region containing eight alternative polyadenylation sites was examined. Because previous studies had shown that FGF-2 expression was regulated in primary cells but not in transformed cells, primary human skin fibroblasts were used in this study. Using an approach of cell transfection with synthetic reporter mRNAs, a novel translational enhancer (3'-TE) was identified in the 1370-nucleotide mRNA segment located upstream from the eighth poly(A) site. Deletion mutagenesis showed that the 3'-TE was composed of two domains with additive effects. The 3'-TE exhibited the unique feature of modulating the use of FGF-2 alternative initiation codons, which favored the relative expression of CUG-initiated isoforms. Interestingly, the use of an alternative polydenylation site removing the 3'-TE was detected in skin fibroblasts in response to heat shock and cell density variations. At high cell densities, 3'-TE removal was correlated with a loss of CUG-initiated FGF-2 expression. These data show that the FGF-2 mRNA 3'-untranslated region is able to modulate FGF-2 isoform expression by the coupled processes of translation activation and alternative polyadenylation.
Assuntos
Regiões 3' não Traduzidas , Elementos Facilitadores Genéticos , Fator 2 de Crescimento de Fibroblastos/genética , Poli A/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Regulação da Expressão Gênica/genética , Humanos , MutagêneseRESUMO
Anaplastic lymphoma kinase (ALK)-positive lymphomas are characterized by expression of a hybrid protein, comprising the cytoplasmic portion of the ALK tyrosine kinase fused to a partner protein. This hybrid kinase is often encoded by the nucleophosmin (NPM) NPM-ALK fusion gene resulting from the (2;5)(p23;q35) chromosomal translocation. However, the ALK gene at 2p23 may also be involved in 2 variant translocations, namely t(1;2)(q25;p23) and t(2;3)(p23;q21), which create the TPM3-ALK and TFG-ALK fusion genes, respectively. We report here 2 lymphomas with an unusual finely granular cytoplasmic ALK staining pattern, clearly different from the pattern observed in ALK-positive lymphomas carrying NPM-ALK or its variants. A cloned complementary DNA sequence from 1 of these 2 lymphomas contained the ALK gene fused to the second clathrin heavy chain gene (also referred to as clathrin heavy polypeptide-like gene) (CLTCL). The distinctive granular cytoplasmic staining pattern for ALK was likely to be due to binding of the fusion protein to clathrin-coated vesicles. The CLTCL gene is constitutively expressed in lymphoid cells and therefore presumably contributes an active promoter for the CLTCL-ALK gene. The fusion protein had a molecular weight (250 kd) that differs from all known ALK products, and it was autophosphorylated in an in vitro kinase assay, confirming that it is constitutively active and hence capable of contributing to malignant transformation. These 2 cases, therefore, represent a hitherto undescribed mechanism of ALK activation in lymphoma and further illustrate the diversity of fusion partners for the ALK gene.
Assuntos
Cromossomos Humanos Par 1 , Cromossomos Humanos Par 2 , Cromossomos Humanos Par 3 , Clatrina/genética , Linfoma Difuso de Grandes Células B/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Translocação Genética , Sequência de Aminoácidos , Quinase do Linfoma Anaplásico , Sequência de Bases , Pré-Escolar , Feminino , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Tirosina Quinases/biossíntese , Receptores Proteína Tirosina QuinasesRESUMO
The majority of anaplastic large cell lymphomas (ALCL) are associated with chromosomal abnormalities affecting the anaplastic lymphoma kinase (ALK) gene which result in the expression of hybrid ALK fusion proteins in the tumor cells. In most of these tumors, the hybrid gene comprises the 5' region of nucleophosmin (NPM) fused in frame to the 3' portion of ALK, resulting in the expression of the chimeric oncogenic tyrosine kinase NPM-ALK. However, other variant rearrangements have been described in which ALK fuses to a partner other than NPM. Here we have identified the moesin (MSN) gene at Xq11-12 as a new partner of ALK in a case of ALCL which exhibited a distinctive membrane-restricted pattern of ALK labeling. The hybrid MSN-ALK protein had a molecular weight of 125 kd and contained an active tyrosine kinase domain. The unique membrane staining pattern of ALK is presumed to reflect association of moesin with cell membrane proteins. In contrast to other translocations involving the ALK gene, the ALK breakpoint in this case occurred within the exonic sequence coding for the juxtamembrane portion of ALK. Identification of the genomic breakpoint confirmed the in-frame fusion of the whole MSN intron 10 to a 17 bp shorter juxtamembrane exon of ALK. The breakpoint in der(2) chromosome showed a deletion, including 30 bp of ALK and 36 bp of MSN genes. These findings indicate that MSN may act as an alternative fusion partner for activation of ALK in ALCL and provide further evidence that oncogenic activation of ALK may occur at different intracellular locations.