RESUMO
Rabies virus is a neurovirulent RNA virus, which causes about 59,000 human deaths each year. Treatment for rabies does not exist due to incomplete understanding of the pathogenesis. MALT1 mediates activation of several immune cell types and is involved in the proliferation and survival of cancer cells. MALT1 acts as a scaffold protein for NF-κB signaling and a cysteine protease that cleaves substrates, leading to the expression of immunoregulatory genes. Here, we examined the impact of genetic or pharmacological MALT1 inhibition in mice on disease development after infection with the virulent rabies virus strain CVS-11. Morbidity and mortality were significantly delayed in Malt1-/- compared to Malt1+/+ mice, and this effect was associated with lower viral load, proinflammatory gene expression, and infiltration and activation of immune cells in the brain. Specific deletion of Malt1 in T cells also delayed disease development, while deletion in myeloid cells, neuronal cells, or NK cells had no effect. Disease development was also delayed in mice treated with the MALT1 protease inhibitor mepazine and in knock-in mice expressing a catalytically inactive MALT1 mutant protein, showing an important role of MALT1 proteolytic activity. The described protective effect of MALT1 inhibition against infection with a virulent rabies virus is the precise opposite of the sensitizing effect of MALT1 inhibition that we previously observed in the case of infection with an attenuated rabies virus strain. Together, these data demonstrate that the role of immunoregulatory responses in rabies pathogenicity is dependent on virus virulence and reveal the potential of MALT1 inhibition for therapeutic intervention.IMPORTANCE Rabies virus is a neurotropic RNA virus that causes encephalitis and still poses an enormous challenge to animal and public health. Efforts to establish reliable therapeutic strategies have been unsuccessful and are hampered by gaps in the understanding of virus pathogenicity. MALT1 is an intracellular protease that mediates the activation of several innate and adaptive immune cells in response to multiple receptors, and therapeutic MALT1 targeting is believed to be a valid approach for autoimmunity and MALT1-addicted cancers. Here, we study the impact of MALT1 deficiency on brain inflammation and disease development in response to infection of mice with the highly virulent CVS-11 rabies virus. We demonstrate that pharmacological or genetic MALT1 inhibition decreases neuroinflammation and extends the survival of CVS-11-infected mice, providing new insights in the biology of MALT1 and rabies virus infection.
Assuntos
Encéfalo/imunologia , Inflamação/prevenção & controle , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/fisiologia , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Linfócitos T/imunologia , Animais , Encéfalo/metabolismo , Encéfalo/virologia , Células Cultivadas , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Raiva/imunologia , Raiva/metabolismo , Linfócitos T/patologia , Linfócitos T/virologiaRESUMO
BACKGROUND: The mitogen-activated protein kinase (MAPK) phosphatases or dual specificity phosphatases (DUSPs) are a family of proteins that catalyse the inactivation of MAPK in eukaryotic cells. Little is known of the expression, regulation or function of the DUSPs in human neoplasia. METHODS: We used RT-PCR and quantitative PCR (qPCR) to examine the expression of DUSP16 mRNA. The methylation in the DUSP16 CpG island was analysed using bisulphite sequencing and methylation-specific PCR. The activation of MAPK was determined using western blotting with phospho-specific antibodies for extra-cellular signal-related kinase (ERK), p38 and c-Jun N-terminal kinase (JNK). The proliferation of cell lines was assessed using the CellTiter 96 Aqueous One assay. RESULTS: The expression of DUSP16, which inactivates MAPK, is subject to methylation-dependent transcriptional silencing in Burkitt's Lymphoma (BL) cell lines and in primary BL. The silencing is associated with aberrant methylation in the CpG island in the 5' regulatory sequences of the gene blocking its constitutive expression. In contrast to BL, the CpG island of DUSP16 is unmethylated in other non-Hodgkin's lymphomas (NHLs) and epithelial malignancies. In BL cell lines, neither constitutive nor inducible ERK or p38 activity varied significantly with DUSP16 status. However, activation of JNK was increased in lines with DUSP16 methylation. Furthermore, methylation in the DUSP16 CpG island blocked transcriptional induction of DUSP16, thereby abrogating a normal physiological negative feedback loop that limits JNK activity, and conferred increased cellular sensitivity to agents, such as sorbitol and anthracycline chemotherapeutic agents that activate JNK. CONCLUSION: DUSP16 is a new epigenetically regulated determinant of JNK activation in BL.
Assuntos
Linfoma de Burkitt/genética , Fosfatases de Especificidade Dupla/genética , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Linhagem Celular Tumoral , Metilação de DNA , Fosfatases de Especificidade Dupla/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Transdução de SinaisRESUMO
Mucosa-associated lymphoid tissue (MALT) lymphoma is a heterogeneous form of a B-cell non-Hodgkin's lymphoma with extranodal location. The gastrointestinal tract is the most common site of disease, but involvement of multiple other organ systems has been documented. Four translocations, t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21) and t(3;14)(p13;q32), are specifically associated with MALT lymphoma. Remarkably, the genes targeted by at least three of these translocations are involved in one and the same pathway, leading to the activation of nuclear factor-kappaB (NF-kappaB). This review presents MALT lymphoma as a model of how sustained inflammation increases the risk of genotoxic insults and how these genetic events initiate oncogenesis.
Assuntos
Linfoma de Zona Marginal Tipo Células B/etiologia , Animais , Antígenos de Bactérias/imunologia , Autoantígenos/imunologia , Subpopulações de Linfócitos B/imunologia , Caspases/genética , Caspases/fisiologia , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 1/ultraestrutura , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 11/ultraestrutura , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 14/ultraestrutura , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 18/ultraestrutura , Doença Crônica , Gastrite/complicações , Gastrite/tratamento farmacológico , Gastrite/imunologia , Gastrite/microbiologia , Neoplasias Gastrointestinais/etiologia , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/microbiologia , Regulação Neoplásica da Expressão Gênica , Genes de Imunoglobulinas , Infecções por Helicobacter/complicações , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Inflamação/complicações , Linfoma de Zona Marginal Tipo Células B/tratamento farmacológico , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma de Zona Marginal Tipo Células B/imunologia , Linfoma de Zona Marginal Tipo Células B/microbiologia , Camundongos , Camundongos Transgênicos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B/fisiologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/fisiologia , Translocação GenéticaRESUMO
Mutations in the ras gene are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in malignant melanoma (MM). We searched for point mutations in the N-ras gene in a large series of primary and metastatic MM from 81 different retrospectively selected patients using the very sensitive denaturing gradient gel electrophoresis technique, followed by sequencing. The classical codon 12 and codon 61 mutations were found in 21 and 17% of the cases, respectively. No codon 13 mutation was found. A novel mutation at codon 18 of exon 1, consisting of a substitution of alanine (GCA) by threonine (ACA), was found in 15% of the primary MMs but in none of the metastatic MMs. All of the other cases were free of mutations. Using microdissected cells from distinctive MM growth phases as source of DNA for mutation analysis, this particular N-ras exon 1 mutation at codon 18 was already present in the radial growth phase and preserved throughout the successive growth phases; it was also found in a dysplastic nevi in continuity with a MM, indicating a clonal relationship between both lesions. Our findings also illustrate the clonal relationship between the distinctive growth phases in MM and suggest the codon 18 mutation to occur early in MM development. The MM in patients with this mutation were significantly thinner than those without a codon 18 mutation (P = 0.0257). Statistical analysis, comparing the group of codon 18 patients with the group of patients with the classical mutations and without mutations, revealed a highly significant difference in overall outcome. The cumulative probability of developing metastasis was significantly lower for the group patients with a codon 18 mutation (P = 0.0130). We can thus conclude that this codon 18 mutation identifies a group of patients with better prognosis than patients with melanoma that harbor wild-type sequence or classical activating point mutations in codon 12 or 61. Preliminary nucleotide binding measurements could not detect a difference between wild-type Ras protein and the mutant Ras(A18T) protein. However, for a precise elucidation of the role of the N-Ras(A18T) mutant in melanoma, additional studies aimed to measure the affinity to guanine nucleotide exchange factors and GTPase-activating proteins are needed.
Assuntos
Genes ras/genética , Melanoma/genética , Mutação Puntual , Códon , DNA de Neoplasias/genética , Éxons , Feminino , Guanilil Imidodifosfato/metabolismo , Humanos , Masculino , Melanoma/patologia , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Prognóstico , Estudos Retrospectivos , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
We identified a fusion between ETV6 on 12p13 and MDS1/EVI1 on 3q26 in a t(3;12)(q26;p13) found in two cases of myeloproliferative disorder. The resulting chimeric transcript consists of the first two exons of ETV6 fused to MDS1 sequences, which in turn is fused to the second exon of the EVI1 gene. It has recently been reported that MDS1 can be expressed in normal tissues both as a single gene and fused to EVI1. ETV6 does not contribute any known functional domain to the predicted fusion protein. Association with blast crisis and myelodysplastic syndrome-derived leukemia, bad prognosis, and relative complex karyotype are in agreement with observations made in other cases of t(3;12)(q26;p13). Furthermore, a comparison can be made with the formation of an AML1/MDS1/EVI1 fusion gene in translocations (3;21)(q26;q22).
Assuntos
Anemia Refratária com Excesso de Blastos/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 3 , Proteínas de Ligação a DNA/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Translocação Genética , Adulto , Sequência de Aminoácidos , Feminino , Quinase 3 da Glicogênio Sintase , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-ets , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Recently, a new recurrent t(12;21)(pl3;q22) has been identified in a B-cell lineage childhood acute lymphoblastic leukemia (ALL). The translocation results in a fusion of two known genes, ETV6/TEL (12p13) and AML1 (21q22), previously shown to be involved in the pathogenesis of myeloid disorders. We report results of cytogenetic fluorescence in situ hybridization and molecular studies of a B-cell childhood common ALL with a cryptic 12;21 translocation. Aberrations identified in this case involve both chromosomes 12 and include not only the ETV6-AML1 gene fusion and two different microdeletions of ETV6 but also the hemizygous loss of CDKN1B, D12S119, and KRAS2 loci and a putative rearrangement of the second CDKN1B allele as a result of an inv(12)(p13q24). Moreover, it was shown that the AML1-ETV6 reciprocal chimeric transcript was not present in the malignant cells, and hence may not play a major role in leukemogenesis. In addition, the putative loss of wild-type function of CDKN1B and ETV6 could indicate a synergistic effect of both genes in the pathogenesis of this leukemia case.
Assuntos
Aberrações Cromossômicas/genética , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 21 , Ciclinas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Fatores de Transcrição/genética , Adolescente , Alelos , Sequência de Bases , Bandeamento Cromossômico , Deleção Cromossômica , Transtornos Cromossômicos , Inibidor de Quinase Dependente de Ciclina p21 , Primers do DNA/química , Feminino , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Neoplásico/genética , Translocação GenéticaRESUMO
A patient is described with de novo acute non-lymphocytic leukemia of megakaryoblastic lineage with tri-lineage myelodysplasia. This patient was studied cytogenetically and using molecular genetic techniques throughout her clinical course. She had an N-ras mutation at diagnosis which persisted despite a bone marrow transplant, and acquired a Philadelphia chromosome associated with a P190 BCR-ABL transcript at clinical relapse 3 months post-transplantation.
Assuntos
Genes ras , Leucemia Megacarioblástica Aguda/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adulto , Sequência de Bases , Bandeamento Cromossômico , Feminino , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Megacarioblástica Aguda/patologia , Dados de Sequência Molecular , Oligonucleotídeos/química , Reação em Cadeia da Polimerase , Fatores de TempoRESUMO
INTRODUCTION: T-cell prolymphocytic leukemia is a rare form of mature leukemia which occurs in adults and in younger patients suffering ataxia telangiectasia. Among others, complex chromosome aberrations of chromosome 12 have been described in this disease. We searched for deletions of the 12p13 region as the result of these chromosome rearrangements. MATERIAL AND METHODS: Paired leukemic and non-leukemic cells were obtained from a series of 21 patients suffering T-cell prolymphocytic leukemia. Loss of heterozygosity was searched for by microsatellite typing using a fluorescent automated laser DNA sequencer to analyze the amplification products. Proteins were analyzed by Western blot. Southern blot analysis of one patient was conducted. RESULTS AND CONCLUSION: Loss of heterozygosity of the 12p13 region, including the ETV6 and CDKN1B genes, was detected in nine of these 21 cases (43%). Western and Southern blot analyses of one case demonstrated a biallelic deletion which did not include ETV6. Taken together, our results defined a minimal region of deletion of less than one Mb flanked by the markers b312C2T7 and D12S320, excluding ETV6 as a candidate gene. Deletion of the 12p13 region is thus a highly recurrent genetic event in T-cell prolymphocytic leukemia.
Assuntos
Centrômero/genética , Deleção Cromossômica , Cromossomos Humanos Par 12 , Proteínas de Ligação a DNA/genética , Leucemia Prolinfocítica/genética , Leucemia de Células T/genética , Perda de Heterozigosidade , Proteínas Repressoras/genética , Mapeamento Cromossômico , DNA de Neoplasias/sangue , DNA de Neoplasias/isolamento & purificação , Marcadores Genéticos , Humanos , Repetições de Microssatélites , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas c-ets , Mapeamento por Restrição , Transcrição Gênica , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
We found a new type of rare HRAS1 alleles arising de novo in the lymph node of lymphoma patients. Rare alleles of 6.2 kb (a0.1) and 7.3 kb (a2.3) are displayed in the presence of common alleles of 6.6 kb (a1) and 7.6 kb (a3), respectively. These rare alleles lack nucleotide sequences, present in the common alleles in the 5' part between the BamHI and XmaIII site. In addition, the rare alleles may have the classic A-G mutation in the intron at position 2719. In contrast to what is observed for rare constitutional alleles, the VTR region of these rare alleles has the same size as the common alleles. At present the significance of this new type of alleles is not clear.
Assuntos
Alelos , Linfonodos/metabolismo , Linfoma/genética , Proteína Oncogênica p21(ras)/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Bases , DNA de Cadeia Simples , Genótipo , Humanos , Dados de Sequência Molecular , Polimorfismo de Fragmento de RestriçãoRESUMO
Chromosomal abnormalities involving the short arm of chromosome 12 have been frequently observed in a broad spectrum of hematological malignancies. Recently, a gene located in this chromosomal region and implicated in leukemogenesis was identified. The gene, called ETV6 (previously known as TEL) is a new member of the ETS family, a group of genes thought to act as transcriptional activators. The gene spans 240 kb and consists of eight exons coding for a helix-loop-helix (HLH) and a DNA-binding domain. ETV6 was originally identified in a t(5;12)(q33;p13) occurring in a chronic myelomonocytic leukemia (CMML). Recent reports, however, show its involvement in a growing number of translocations associated with myeloid as well as lymphoid leukemias. At the molecular level fusions of ETV6 with PDGFRB (5q33), ABL (9q34), MNI(22q11) and AML1(21q22) have already been identified. Analysis of these chimeric proteins indicates that distinct domains of ETV6 can be involved in different fusion products, thus ETV6 can provide transcriptional and dimerization properties for partner genes, or the gene itself can act as an altered transcriptional factor. At least two clinico-pathological entities associated with ETV6 rearrangements have emerged as distinct disorders. The first one is a chronic myeloid malignancy characterized by t(5;12)(q33;p13), monocytosis and/or eosinophilia. The second entity is a type of childhood acute lymphoblastic leukemia (ALL) hallmarked by t(12;21)(p13;q22), and is shown to be the most frequent but cytogenetically largely undetectable chromosomal anomaly in childhood ALL.
Assuntos
Proteínas de Ligação a DNA/genética , Rearranjo Gênico , Neoplasias Hematológicas/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 12 , Humanos , Proteínas Proto-Oncogênicas c-ets , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
The API2-MALT1 fusion oncoprotein is created by the recurrent t(11;18)(q21;q21) chromosomal translocation in mucosa-associated lymphoid tissue (MALT) lymphoma. We identified receptor interacting protein-1 (RIP1) as a novel API2-MALT1-associated protein, and demonstrate that RIP1 is required for API2-MALT1 to stimulate canonical nuclear factor kappa B (NF-κB). API2-MALT1 promotes ubiquitination of RIP1 at lysine (K) 377, which is necessary for full NF-κB activation. Furthermore, we found that TNF receptor-associated factor 2 (TRAF2) recruitment is required for API2-MALT1 to induce RIP1 ubiquitination, NF-κB activation and cellular transformation. Although both TRAF2 and RIP1 interact with the API2 moiety of API2-MALT1, this moiety alone is insufficient to induce RIP1 ubiquitination or activate NF-κB, indicating that API2-MALT1-dependent RIP1 ubiquitination represents a gain of function requiring the concerted actions of both the API2 and MALT1 moieties of the fusion. Intriguingly, constitutive RIP1 ubiquitination was recently demonstrated in several solid tumors, and now our study implicates RIP1 ubiquitination as a critical component of API2-MALT1-dependent lymphomagenesis.
Assuntos
Linfoma de Zona Marginal Tipo Células B/genética , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/fisiologia , Fator 2 Associado a Receptor de TNF/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células HEK293 , Humanos , Imunoprecipitação , Linfoma de Zona Marginal Tipo Células B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Oncogenes , Proteínas de Ligação a RNA/genética , Fator 2 Associado a Receptor de TNF/genética , Transfecção , UbiquitinaçãoAssuntos
Cromossomos Humanos Par 16 , Cromossomos Humanos Par 5 , Fusão Gênica , Leucemia Mielomonocítica Crônica/genética , Síndrome de Noonan/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Transcrição Gênica , Adulto , Sequência de Bases , Feminino , Humanos , Hibridização in Situ Fluorescente , Translocação GenéticaAssuntos
Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 18/genética , Neoplasias Pulmonares/genética , Linfoma de Zona Marginal Tipo Células B/genética , Translocação Genética , Primers do DNA/química , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We previously isolated a cDNA fragment homologous to the Drosophila Bicaudal-D gene (Bic-D) using a hybridization selection procedure with cosmids derived from the short arm of human chromosome 12. A PCR-mediated cDNA cloning strategy was applied to obtain the coding sequence of the human homologue (BICD1) and to generate a partial mouse (Bicdh1) cDNA. The Drosophila Bicaudal-D gene encodes a coiled coil protein, characterized by five alpha-helix domains and a leucine zipper motif, that forms part of the cytoskeleton and mediates the correct sorting of mRNAs for oocyte- and axis-determining factors during oogenesis. Analysis of the predicted amino acid sequence of the BICD1 cDNA clones indicates that the sequence similarity is essentially limited to the amphipatic helices and the leucine zipper, but the conserved order of these domains suggests a similar function of the protein in mammalians. A database search further indicates the existence of a second human homologue on chromosome arm 9q and a Caenorhabditis elegans homologue. Northern blot analysis indicates that both the human and the murine homologues produce an mRNA species of congruent with9.5 kb expressed in brain, heart, and skeletal muscle and during mouse embryonic development. The conserved structural characteristics of the BICD1 protein and its expression in muscle and especially brain suggest that BICD1 is a component of a cytoskeleton-based mRNA sorting mechanism conserved during evolution.
Assuntos
Proteínas do Citoesqueleto , Proteínas de Drosophila , Proteínas de Insetos/genética , Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas de Insetos/metabolismo , Zíper de Leucina , Camundongos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Distribuição TecidualRESUMO
An assay is described in which 11 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be screened simultaneously. Six different exons of the CFTR gene are amplified in a single multiplex amplification. Biotinylated dUTP is incorporated into the different fragments during the amplification process. A sample of this mixture is then hybridized to 21 different poly-dT tailed oligonucleotide probes which are bound to a nylon membrane. In order to screen the different mutations in a single step hybridization, the length of the different oligonucleotides and the amount used in the assay were optimized. The detection is performed by binding avidin-alkaline phosphatase to the biotin, followed by a chemiluminescent reaction. By means of this fast and sensitive assay, about 85% of all the cystic fibrosis mutations in the Belgian population can be detected.
Assuntos
Proteínas de Membrana/genética , Mutação/genética , Sequência de Bases , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística , Humanos , Immunoblotting/métodos , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico , Sondas de Oligonucleotídeos/genéticaRESUMO
To map human chromosome 12p aberrations by fluorescence in situ hybridization (FISH), cosmids were isolated or obtained for 14 known 12p loci (D12S119, D12S158, D12S178, D12S370, D12S380E, A2M, CACNL1A1, FGF6, GAPD, KRAS2, PRB1, PZP, TPI1, and VWF). Using two-color FISH with three labeled probes to interphase nuclei, and to prometaphase chromosomes where possible, the order of these loci was sequentially determined to be pter-D12S158-D12S380E-CACNL1A1-FGF6- D12S370-VWF-GAPD-TPI1-A2M-PZP-PRB1-D12S 178-D12S119-KRAS2-cen. Two cell lines were analyzed with this set of cosmids. The EBV-transformed cell line TA carries a der(12) with a deletion of bands 12p13.1-->p11.2.D12S178, D12S119, and KRAS2 were absent in the der(12), whereas the other loci were present. The second cell line, GM01203A, exhibits a balanced t(4;12)(4q25; 12p13.3) with a breakpoint between FGF6 and D12S370.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 12 , Cosmídeos , Hominidae/genética , Animais , Núcleo Celular/ultraestrutura , Células Cultivadas , Mapeamento Cromossômico , Humanos , Hibridização in Situ Fluorescente/métodos , Interfase , Linfócitos/citologia , MasculinoRESUMO
BACKGROUND: Extranodal marginal zone cell lymphoma (MZCL) of MALT-type share similar features with nodal and splenic MZCL regarding morphology and immunophenotype. At the genetic level, recent cytogenetic studies have shown that t(11;18) is a recurring abnormality in extranodal MALT-type MZCL but has hitherto never been reported in nodal or splenic MZCL. The aim of the present study was to determine the prevalence of t(11;18) in a large series of nodal, splenic and extranodal MALT-type MZCL, using a sensitive real-time RT-PCR method. MATERIALS AND METHODS: Ninety-three MZCL cases were divided on clinical grounds into 61 extranodal MALT-type, 19 splenic and 12 nodal MZCL. One case that presented with a massive splenomegaly but for which also gastro-intestinal localisations were found, was left unclassified. A real-time RT-PCR method for the detection of the API2-MLT fusion resulting from t(11;18) was performed on RNA extracted from frozen tissue sections. RESULTS: The API2-MLT fusion was detected in 12 cases, which were all extranodal MALT-type lymphomas of the stomach, except for one case. The remaining positive case was the unclassified case, for which the translocation was detected in the spleen and in hilar lymph node tissue. CONCLUSIONS: While similarities between MZCL from different anatomic sites have lend us to propose that all MZCL have a common normal counterpart, the almost exclusive detection of t(11;18) in gastric MALT-type lymphoma favours its recognition as a separate lymphoma entity. The absence of the translocation in nodal and splenic MZCL challenges the idea of these lymphomas being secondary to MALT-type lymphomas of the gut. The unclassified case illustrates the inadequate approaches available at present to identify and define the various MZCL.
Assuntos
Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 18/genética , Metástase Linfática/genética , Linfoma de Zona Marginal Tipo Células B/genética , Neoplasias Esplênicas/genética , Translocação Genética , Apoptose/genética , Fusão Gênica Artificial , Primers do DNA , Rearranjo Gênico , Humanos , Imunoglobulina G/genética , Imuno-Histoquímica , Metástase Linfática/patologia , Linfoma de Zona Marginal Tipo Células B/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Esplênicas/patologiaRESUMO
The t(11;18)(q21;q21) between the inhibitor of apoptosis API2 and the MLT gene is a distinct feature of marginal zone B-cell lymphomas of MALT-type. Hitherto the chimeric API2-MLT transcripts are all "in-frame" and predominantly fuse exon 7 of API2 to different MLT exons. Recurrent chromosomal translocations are common in lymphoid neoplasms and might represent by-products of the rearrangement processes generating antigen receptor diversity. The genomic structure of the MLT gene was determined to facilitate amplification of the genomic breakpoint junctions from 5 MALT-type lymphomas with t(11;18). Their sequence analysis showed scattering of the chromosome 11 breakpoints in intron 7 of API2 whereas rearrangements in MLT occurred in intron 2, 4, 7, or 8, respectively. Sequences around the junctions did not display recognition signal sequences mediating lymphocytic V(D)J recombination or other sequence motifs associated with recombination. The breakpoints occurred in a copy of an AluSx repeat in three cases, but interchromosomal Alu-mediated homologous recombination could be ruled out as the repeat resided only on one of the participating chromosomes. The t(11;18) was associated with a deletion in 4 out of 5 cases, ranging in size from 53 bp up to more than 200 kb. These deletions were observed on one or sometimes both derivative chromosomes that might indicate the susceptibility of these regions for breakage. Our data suggest that the API2-MLT fusion might result from a non-homologous end joining event after multiple double-strand breaks. The clustering of breaks in intron 7 of API2 and the consistent "in frame" API2-MLT fusions could therefore reflect certain functional constraints crucial for clonal outgrowth.
Assuntos
Quebra Cromossômica/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 18/genética , Linfoma de Zona Marginal Tipo Células B/genética , Proteínas de Neoplasias , Proteínas/química , Proteínas/genética , Translocação Genética/genética , Apoptose/genética , Sequência de Bases , Caspases , Cromossomos Humanos Par 11/química , Cromossomos Humanos Par 18/química , Humanos , Proteínas Inibidoras de Apoptose , Dados de Sequência Molecular , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Análise de Sequência de DNA , Neoplasias Gástricas/química , Neoplasias Gástricas/genéticaRESUMO
We have characterized 117 cDNAs isolated by direct cDNA selection using pools of human chromosome 12p cosmids. Sequencing revealed that 41 clones did overlap with other cDNAs. Of the remaining 76 cDNA sequences, 11 matched previously identified human chromosome 12p genes and 3 matched previously determined cDNA sequences, including the retinoblastoma binding protein 2 (RBBP2), the cyclin-dependent kinase inhibitor KIP1, and an expressed sequence tag. For each of the 76 cDNAs specific selection by a genomic cosmid clone was confirmed. STSs were developed for all cosmids, among them 3 polymorphic simple sequence repeats associated with, respectively, the TNFR related protein, CD27, and SCNN1. Regional assignment of the STSs by PCR analysis with somatic cell hybrids and fluorescence in situ hybridization showed that the majority of the loci map to chromosome 12p13, similar to the distribution of the known 12p genes. Evidence was found for the duplication on 12p of a region containing a polymorphic simple sequence repeat and sequences of two different cDNAs.