RESUMO
OBJECTIVES: Mutational signatures (MS) are gaining traction for deriving therapeutic insights for immune checkpoint inhibition (ICI). We asked if MS attributions from comprehensive targeted sequencing assays are reliable enough for predicting ICI efficacy in non-small cell lung cancer (NSCLC). METHODS: Somatic mutations of m = 126 patients were assayed using panel-based sequencing of 523 cancer-related genes. In silico simulations of MS attributions for various panels were performed on a separate dataset of m = 101 whole genome sequenced patients. Non-synonymous mutations were deconvoluted using COSMIC v3.3 signatures and used to test a previously published machine learning classifier. RESULTS: The ICI efficacy predictor performed poorly with an accuracy of 0.51-0.09+0.09, average precision of 0.52-0.11+0.11, and an area under the receiver operating characteristic curve of 0.50-0.09+0.10. Theoretical arguments, experimental data, and in silico simulations pointed to false negative rates (FNR) related to panel size. A secondary effect was observed, where deconvolution of small ensembles of point mutations lead to reconstruction errors and misattributions. CONCLUSION: MS attributions from current targeted panel sequencing are not reliable enough to predict ICI efficacy. We suggest that, for downstream classification tasks in NSCLC, signature attributions be based on whole exome or genome sequencing instead.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Análise Mutacional de DNA , Inibidores de Checkpoint Imunológico , Neoplasias Pulmonares , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Conjuntos de Dados como Assunto , Análise Mutacional de DNA/métodos , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Resultado do Tratamento , Simulação por Computador , Aprendizado de Máquina , Mutação PuntualRESUMO
INTRODUCTION: Detection of mutations in patients with myeloid neoplasms (MNs) has shown great potential for diagnostic and prognostic purposes. Next-generation sequencing (NGS) is currently implemented for the diagnostic profiling of the four major MN subgroups. METHODS: First, we validated the targeted NGS approach using the TruSight Myeloid panel. Next, we screened 287 patients with a clinical suspicion of MN and 61 follow-up patients with documented MN. RESULTS: Validation of the NGS workflow resulted in maximal precision, accuracy, sensitivity, and specificity for gene variants with an allele frequency of at least 5% and a minimal read depth of 300. In our diagnostic screen, we identified at least one somatic mutation in 89% of patients with proven MN. Of the 155 newly diagnosed MN cases, 126 (81%) showed at least one mutation, confirming clonality. Moreover, the co-occurrence of mutated genes in the different MN subentities facilitates their classification and justifies the diagnostic use of a pan-myeloid panel. Furthermore, several of these mutations provide additional prognostic information independently of traditional prognostic scoring systems. CONCLUSION: Pan-myeloid targeted NGS fits elegantly in the routine diagnostic approach of MNs allowing for an improved diagnosis, subclassification, and prognosis.
Assuntos
Neoplasias Hematológicas , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Transtornos Mieloproliferativos , Análise Mutacional de DNA/instrumentação , Análise Mutacional de DNA/métodos , Feminino , Seguimentos , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Humanos , Masculino , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/genéticaRESUMO
X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4(-/-) mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.
Assuntos
Variação Genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Adulto , Animais , Células Cultivadas , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Estudos de Coortes , Quinases Ciclina-Dependentes/genética , Sequenciamento de Nucleotídeos em Larga Escala , Histona Acetiltransferases/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Neurônios/metabolismo , Neurônios/patologia , Proteínas Nucleares/genética , RNA Mensageiro/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Submicroscopic duplications along the long arm of the X-chromosome with known phenotypic consequences are relatively rare events. The clinical features resulting from such duplications are various, though they often include intellectual disability, microcephaly, short stature, hypotonia, hypogonadism and feeding difficulties. Female carriers are often phenotypically normal or show a similar but milder phenotype, as in most cases the X-chromosome harbouring the duplication is subject to inactivation. Xq28, which includes MECP2 is the major locus for submicroscopic X-chromosome duplications, whereas duplications in Xq25 and Xq26 have been reported in only a few cases. Using genome-wide array platforms we identified overlapping interstitial Xq25q26 duplications ranging from 0.2 to 4.76 Mb in eight unrelated families with in total five affected males and seven affected females. All affected males shared a common phenotype with intrauterine- and postnatal growth retardation and feeding difficulties in childhood. Three had microcephaly and two out of five suffered from epilepsy. In addition, three males had a distinct facial appearance with congenital bilateral ptosis and large protruding ears and two of them showed a cleft palate. The affected females had various clinical symptoms similar to that of the males with congenital bilateral ptosis in three families as most remarkable feature. Comparison of the gene content of the individual duplications with the respective phenotypes suggested three critical regions with candidate genes (AIFM1, RAB33A, GPC3 and IGSF1) for the common phenotypes, including candidate loci for congenital bilateral ptosis, small head circumference, short stature, genital and digital defects.
Assuntos
Anormalidades Múltiplas/genética , Blefaroptose/congênito , Duplicação Cromossômica , Doenças Genéticas Ligadas ao Cromossomo X/genética , Adulto , Animais , Blefaroptose/genética , Estatura/genética , Criança , Fissura Palatina/genética , Feminino , Dedos/anormalidades , Humanos , Deficiência Intelectual/genética , Cariotipagem , Masculino , Camundongos , Camundongos Transgênicos , Microcefalia/genética , SíndromeRESUMO
Genome-wide array comparative genome hybridization has become the first in line diagnostic tool in the clinical work-up of patients presenting with intellectual disability. As a result, chromosome X-copy number variations are frequently being detected in routine diagnostics. We retrospectively reviewed genome wide array-CGH data in order to determine the frequency and nature of chromosome X-copy number variations (X-CNV) in a cohort of 2222 sporadic male patients with intellectual disability (ID) referred to us for diagnosis. In this cohort, 68 males were found to have at least one X-CNV (3.1%). However, correct interpretation of causality remains a challenging task, and is essential for proper counseling, especially when the CNV is inherited. On the basis of these data, earlier experience and literature data we designed and propose an algorithm that can be used to evaluate the clinical relevance of X-CNVs detected in sporadic male ID patients. Applied to our cohort, 19 male ID patients (0.85%) were found to carry a (likely) pathogenic X-CNV.
Assuntos
Cromossomos Humanos X/genética , Variações do Número de Cópias de DNA , Deficiência Intelectual/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Hibridização Genômica Comparativa , Aconselhamento Genético , Loci Gênicos , Genoma Humano , Humanos , Lactente , Deficiência Intelectual/diagnóstico , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Focal dermal hypoplasia (FDH), Goltz or Goltz-Gorlin syndrome, is an X-linked dominant multisystem disorder characterized primarily by involvement of the skin, skeletal system and eyes. We screened for mutations in the PORCN gene in eight patients of Belgian and Finnish origin with firm clinical suspicion of FDH. First, we performed quantitative PCR (qPCR) analysis to define the copy number at this locus. Next, we sequenced the coding regions and flanking intronic sequences of the PORCN gene. Three de novo mutations were identified in our patients with FDH: a 150-kb deletion removing six genes including PORCN, as defined by qPCR and X-array-CGH, and two heterozygous missense mutations; c.992T>G (p.L331R) in exon 11 and c.1094G>A (p.R365Q) in exon 13 of the gene. Both point mutations changed highly conserved amino acids and were not found in 300 control X chromosomes. The three patients in whom mutations were identified all present with characteristic dermal findings together with limb manifestations, which were not seen in our mutation-negative patients. The clinical characteristics of our patients with PORCN mutations were compared with the previously reported mutation-positive cases. In this report, we summarize the literature on PORCN mutations and associated phenotypes.
Assuntos
Hipoplasia Dérmica Focal/genética , Proteínas de Membrana/genética , Mutação/genética , Aciltransferases , Adolescente , Sequência de Aminoácidos , Sequência de Bases , Criança , Análise Mutacional de DNA , Éxons/genética , Feminino , Humanos , Recém-Nascido , Masculino , Proteínas de Membrana/química , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genéticaRESUMO
We present the first comprehensive study, to our knowledge, on genomic chromosomal analysis in syndromic craniosynostosis. In total, 45 patients with craniosynostotic disorders were screened with a variety of methods including conventional karyotype, microsatellite segregation analysis, subtelomeric multiplex ligation-dependent probe amplification) and whole-genome array-based comparative genome hybridisation. Causative abnormalities were present in 42.2% (19/45) of the samples, and 27.8% (10/36) of the patients with normal conventional karyotype carried submicroscopic imbalances. Our results include a wide variety of imbalances and point to novel chromosomal regions associated with craniosynostosis. The high incidence of pure duplications or trisomies suggests that these are important mechanisms in craniosynostosis, particularly in cases involving the metopic suture.
Assuntos
Aberrações Cromossômicas , Segregação de Cromossomos , Craniossinostoses/genética , Repetições de Microssatélites , Humanos , Cariotipagem , Hibridização de Ácido Nucleico/métodos , Polimorfismo GenéticoRESUMO
Studies to identify the genetic defects associated with X-linked mental retardation (XLMR) in males have revealed tens of genes important for normal brain development and cognitive functioning in men. Despite extensive efforts in breakpoint cloning of chromosomal rearrangements and mutation screening of candidate genes on the X chromosome, still many XLMR families and sporadic cases remain unsolved. It is now clear that submicroscopic copy number changes on the X chromosome can explain about 5% of these idiopathic cases. Interestingly, beside gene deletions, an increase in gene dosage due to genomic duplications seems to contribute to causality more often than expected. Since larger duplications on the X chromosome are tolerated compared to deletions, they often harbour more than one gene hampering the identification of the causal gene. In contrast to copy number variations (CNVs) on autosomes, most disease-associated CNVs on the X chromosome in males are inherited from their mothers who normally do not present with any clinical symptoms due to non-random X inactivation. Here, we review the different methods applied to study copy number alterations on the X chromosome in patients with cognitive impairment, discuss those CNVs that are associated with disease and elaborate on the genes and mechanisms involved. At the end, we will resume in vivo assays to study the relation of CNVs on the X chromosome and mental disability.
Assuntos
Dosagem de Genes/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Animais , Deleção Cromossômica , Humanos , Modelos Genéticos , Seleção Genética , Cromossomo X/genéticaAssuntos
Deleção Cromossômica , Cromossomos Humanos Par 4/genética , Análise de Sequência com Séries de Oligonucleotídeos , Mapeamento Físico do Cromossomo , Criança , Pré-Escolar , Quebra Cromossômica/genética , Feminino , Marcadores Genéticos/genética , Genótipo , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Masculino , Hibridização de Ácido Nucleico , Fenótipo , Polimorfismo Genético/genética , SíndromeRESUMO
BACKGROUND: The gene encoding fatty acid CoA ligase 4 (FACL4) is mutated in families with non-specific X linked mental retardation (MRX) and is responsible for cognitive impairment in the contiguous gene syndrome ATS-MR (Alport syndrome and mental retardation), mapped to Xq22.3. This finding makes this gene a good candidate for other mental retardation disorders mapping in this region. METHODS: We have screened the FACL4 gene in eight families, two MRX and six syndromic X linked mental retardation (MRXS), mapping in a large interval encompassing Xq22.3. RESULTS: We have found a missense mutation in MRX68. The mutation (c.1001C>T in the brain isoform) cosegregates with the disease and changes a highly conserved proline into a leucine (p.P375L) in the first luciferase domain, which markedly reduces the enzymatic activity. Furthermore, all heterozygous females showed completely skewed X inactivation in blood leucocytes, as happens in all reported females with other FACL4 point mutations or deletions. CONCLUSIONS: Since the FACL4 gene is highly expressed in brain, where it encodes a brain specific isoform, and is located in hippocampal and cerebellar neurones, a role for this gene in cognitive processes can be expected. Here we report the third MRX family with a FACL4 mutation and describe the development of a rapid enzymatic assay on peripheral blood that we propose as a sensitive, robust, and efficient diagnostic tool in mentally retarded males.
Assuntos
Coenzima A Ligases/genética , Testes Genéticos/métodos , Deficiência Intelectual Ligada ao Cromossomo X/enzimologia , Deficiência Intelectual Ligada ao Cromossomo X/genética , Mutação de Sentido Incorreto/genética , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Adolescente , Adulto , Substituição de Aminoácidos/genética , Extratos Celulares/química , Linhagem Celular , Criança , Cromossomos Humanos X/genética , Coenzima A Ligases/sangue , Feminino , Triagem de Portadores Genéticos/métodos , Humanos , Lactente , Leucina/genética , Linfócitos/química , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/sangue , Deficiência Intelectual Ligada ao Cromossomo X/etiologia , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Linhagem , Prolina/genética , Aberrações dos Cromossomos SexuaisRESUMO
This review covers the history and nosology of X-linked mental retardation (XLMR) in which the following, largely clinically based, subclassification was used: fragile X syndrome (FRAXA), syndromic forms (MRXS) and non-specific forms (MRX). After the discovery of the FMR2 gene at the FRAXE site, 10 MRX genes have been identified in the last 6 years. A short description is given of the strategies used to identify the genes that cause mental retardation (MR). Furthermore, their potential functions and the association with MR will be discussed. It is emphasized that mutations in several of these MR genes can result in non-specific, as well as in syndromic forms of XLMR. Present findings stress the importance of accurate clinical evaluation. Most considerably, genotype-phenotype correlation studies of affected individuals in XLMR families with MRX gene mutations are necessary to define the criteria of MRX vs MRXS subclassification.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X/classificação , Deficiência Intelectual Ligada ao Cromossomo X/genética , Animais , Saúde da Família , Humanos , Deficiência Intelectual Ligada ao Cromossomo X/etiologia , Mutação , SíndromeRESUMO
BACKGROUND: Although X-linked mental retardation (XLMR) affects 2%-3% of the human population, little is known about the underlying molecular mechanisms. Recent interest in this topic led to the identification of several genes for which mutations result in the disturbance of cognitive development. RESULTS: We identified a novel gene that is interrupted by an inv(X)(p21.1;q22) in a male patient with a syndromic form of mental retardation. Molecular analysis of both breakpoint regions did not reveal an interrupted gene on Xp, but identified a novel nuclear RNA export factor (NXF) gene cluster, Xcen-NXF5-NXF2-NXF4-NXF3-Xqter, in which NXF5 is split by the breakpoint, leading to its functional nullisomy. The predicted NXF5 protein shows high similarity with the central part of the presumed mRNA nuclear export factor TAP/NXF1. Functional analysis of NXF5 demonstrates binding to RNA as well as to the RNA nuclear export-associated protein p15/NXT. In contrast to TAP/NXF1, overexpression studies localized NXF5 in the form of granules in the cell body and neurites of mature hippocampal neurons, suggesting a role in mRNA transport. The two newly identified mouse nxf homologs, nxf-a and nxf-b, which also map on X, show highest mRNA levels in the brain. CONCLUSIONS: A novel member of the nuclear RNA export factor family is absent in a male patient with a syndromic form of mental retardation. Although we did not find direct evidence for the involvement of NXF5 in MR, the gene could be involved in development, possibly through a process in mRNA metabolism in neurons.
Assuntos
Deleção de Genes , Deficiência Intelectual/genética , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático , Proteínas de Ligação a RNA/genética , Cromossomo X , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Inversão Cromossômica , Clonagem Molecular , Citoplasma/metabolismo , Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Deficiência Intelectual/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Família Multigênica , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Homologia de Sequência de Aminoácidos , SíndromeRESUMO
Chemokines contribute to the inflammatory response by selective attraction of various leukocytic cell types. Human GCP-2 was originally identified by amino acid sequence analysis as a CXC chemokine co-produced with IL-8 by osteosarcoma cells. Furthermore, the complete coding domain of human GCP-2 was disclosed by means of RT-PCR. Similarly, mouse GCP-2 was isolated from fibroblastoid and epithelial cells and completely identified by sequence analysis. Human and mouse GCP-2 share 61% identical amino acids. Both chemokines occur as multiple NH2-terminally truncated forms. The shorter forms of mouse, but not those of human, GCP-2 showed a higher neutrophil chemotactic potency and gelatinase B releasing capacity. Mouse GCP-2 was a more potent neutrophil activator than human GCP-2, natural mouse KC, and MIP-2. Human GCP-2 was not chemotactic for monocytes, lymphocytes, or eosinophils. Quantitative studies of mRNA expression in diploid fibroblasts revealed GCP-2 induction by IL-1beta. Human GCP-2 induced [Ca2+]i increase in neutrophils, which was reciprocally desensitized by IL-8, GROalpha, and ENA-78. Human GCP-2 induced [Ca2+]i increases and chemotactic responses in both CXCR1- and CXCR2-transfected cells. Finally, GCP-2 provoked neutrophil accumulation and plasma extravasation in rabbit skin. In humans, GCP-2 complements the activity of IL-8 as neutrophil chemoattractant and activator but it constitutes a major neutrophil chemokine in the mouse. GCP-2 induces neutrophil chemotaxis and activation but it might also contribute to detrimental tissue damage in sepsis, acute respiratory distress syndrome, acute hypersensitivity reactions, and autoimmune diseases. It might also influence the invasive capacity of GCP-2-secreting tumor cells.
Assuntos
Quimiocinas CXC/fisiologia , Quimiocinas/fisiologia , Regulação da Expressão Gênica/fisiologia , Receptores de Quimiocinas/metabolismo , Sequência de Aminoácidos , Animais , Quimiocina CXCL6 , Quimiocinas/genética , Quimiocinas/metabolismo , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
The murine monoclonal antibody REGA-3G12 selectively and specifically inhibits the activity of human gelatinase B. The cDNA fragments which encode the variable regions of the light and heavy chains were isolated by PCR-mediated cloning and sequenced. Single-chain Fv expression constructs for Escherichia coli were generated in which c-myc tag sequences were encoded. Inducible expression of the scFv and secretion to the periplasm were obtained with higher yields when the c-myc tag sequence was positioned at the amino-terminal side. The inhibitory activity of purified scFv on neutrophil gelatinase B was tested in a gelatin degradation assay and it was found to possess a similar specific activity as that of the intact monoclonal antibody and of the pepsin-clipped F(ab')2 derivative. This shows for the first time that inhibition of soluble enzymes with scFv is possible and opens new perspectives for the treatment of diseases with excessive and detrimental enzyme production in the host.
Assuntos
Escherichia coli/genética , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/farmacologia , Região Variável de Imunoglobulina/genética , Inibidores de Metaloproteinases de Matriz , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Humanos , Fragmentos de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/farmacologia , Metaloproteinase 9 da Matriz , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de SequênciaRESUMO
The authors describe the design and validation of a competitive RT-PCR method for the efficient and reproducible quantitation of mRNA molecules of IFN-gamma, TNF-alpha, IL-4 and IL-10 in mouse spleen RNA extracts. Before being subjected to RT-PCR, the RNA extracts were supplemented with internal control RNAs (IC-RNAs), which were constructed by inserting DNA fragments in the cDNA of the respective cytokines. The efficiency of amplification of the target and the IC-RNA was shown to remain equal over a wide range of cycle numbers. Reproducibility was such that differences in mRNA contents that were greater than 17% could be detected between two RNA samples run in parallel. Normal mouse spleen tissue was found to contain 10(7)-10(8) molecules of TNF-alpha, IFN-gamma, IL-4 and IL-10 mRNA per micrograms total RNA extracted. Injection of animals with anti-CD3 antibody, a well-known cytokine inducer, resulted in a moderate increase in TNF-alpha and IL-10 mRNA levels (14- and 24-fold, respectively), and in a substantially greater increase in the levels of mRNA for IL-4 and IFN-gamma (199- and 851-fold, respectively). These results demonstrate an accurate and reliable quantitation of cytokine mRNA levels in animal tissues.
Assuntos
Citocinas/análise , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/análise , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Complexo CD3/imunologia , Citocinas/genética , Interferon gama/genética , Interleucina-10/genética , Interleucina-4/genética , Lasers , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Valores de Referência , Reprodutibilidade dos Testes , Espectrometria de Fluorescência , Baço/efeitos dos fármacos , Baço/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
Human osteosarcoma cells secrete a novel C-X-C chemokine called granulocyte chemotactic protein-2 (GCP-2), which was previously identified by amino acid sequencing of the purified natural protein. In order to understand the role of this new protein in inflammatory reactions, we cloned GCP-2 DNA sequences to generate recombinant protein and specific DNA probes and primers. By means of PCR on cloned cDNA of osteosarcoma cells induced by interleukin-1 beta and fibroblasts induced by lipopolysaccharide plus dsRNA, the complete coding domain of GCP-2 was isolated. This sequence was cloned into the bacterial expression vector pHEN1 and, after induction, GCP-2 was secreted into the periplasm of Escherichia coli. Recombinant GCP-2 (rGCP-2) was purified and characterized by SDS/PAGE as a monomeric 6.5-kDa protein and by amino-terminal sequencing. The chemoattractive potency of GCP-2 for neutrophilic granulocytes was about 10-times less than that of interleukin-8 and the minimal effective dose was 10 ng/ml. However, at optimal dose (100 ng/ml) the maximal chemotactic response was comparable with that of interleukin-8. Both characteristics correspond with those of natural GCP-2. In addition, intracellular calcium release in neutrophils by recombinant GCP-2 was achieved with as little as 10 ng/ml. Quantitation studies using reverse transcriptase and the polymerase chain reaction revealed higher GCP-2 mRNA production in normal fibroblasts than in tumor cells. When compared with epithelial-cell-derived neutrophil-activating peptide-78 (ENA-78) mRNA, the GCP-2 mRNA levels were higher in all cell lines tested. In addition, GCP-2 and ENA-78 expression seem to be differentially regulated in that phorbol ester and lipopolysaccharide have opposing effects on their mRNA induction in diploid fibroblasts and epithelial cells, respectively. Interleukin-1 was demonstrated to be a general inducer for both chemokines, while interferon-gamma down-regulates their mRNA expression. The availability of recombinant GCP-2 together with the quantitation studies on mRNA expression will help to further elucidate the biological role of GCP-2 during the inflammatory response.
Assuntos
Quimiocinas CXC , Quimiocinas/biossíntese , Quimiocinas/genética , Vetores Genéticos , Interleucina-8/genética , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Sequência de Aminoácidos , Sequência de Bases , Quimiocina CXCL5 , Quimiocina CXCL6 , Quimiocinas/química , Quimiocinas/isolamento & purificação , Quimiotaxia de Leucócito , Clonagem Molecular , Códon/isolamento & purificação , Células Epiteliais , Epitélio/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Granulócitos/fisiologia , Humanos , Interleucina-8/análogos & derivados , Dados de Sequência Molecular , Ativação de NeutrófiloAssuntos
Anticorpos/farmacologia , Ciclosporina/farmacologia , Enterotoxinas/farmacologia , Terapia de Imunossupressão/métodos , Interferon gama/imunologia , Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Proteína Ligante Fas , Tolerância Imunológica , Interferon gama/biossíntese , Interleucina-2/biossíntese , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/biossíntese , Superantígenos/farmacologia , Linfócitos T/efeitos dos fármacos , Transcrição GênicaRESUMO
Stimulated MG-63 osteosarcoma cells have been used as a source to purify and identify the monocyte chemokines MCP-1, MCP-2 and MCP-3. In comparison with MCP-1, the production yields of MCP-2 and MCP-3 in these cells are rather low and variable. Although the protein sequence of human MCP-2 is identified, its DNA sequence remains elusive. A degenerate primer set was used to isolate an MCP-2 gene fragment from the chemokine YAC contig on human chromosome 17. Based on the gene sequence of MCP-2, a unique primer set was synthesized and used to screen cDNA libraries for the presence of MCP-2 transcripts by PCR. The complete MCP-2 cDNA was cloned from a human bone marrow cDNA library and sequenced. The cDNA-derived protein sequence was identical to that of purified natural MCP-2, except for Gln46 which replaced Lys46. There seem thus to exist two MCP-2 allelic variants because at position 46 the codons of two residues (Lys46 and Gln46) were detected in individual genomes. As shown by Northern hybridization, the MCP-2 steady-state mRNA levels in normal diploid fibroblasts were increased by IL-1 beta, IFN-gamma and the double-stranded RNA poly rI:rC. RT-PCR analysis showed induction of MCP-2 mRNA in MG-63 cells by IFN-gamma and IL-1 beta. The regulated production of MCP-2 by tumor cells and normal mesenchymal cells is indicative of a role in neoplasia and inflammatory host responses.