RESUMO
MicroRNAs (miRNAs) regulate cell proliferation and differentiation by controlling the expression of proteins involved in many signaling pathways. Recent studies have shown that dysregulation of miRNA expression is associated with increased tumorigenicity and a poor prognosis in several types of cancers. The miRNA let-7b is one of the severely downregulated miRNAs in mixed-lineage leukemia (MLL)-rearranged acute lymphoblastic leukemia (ALL) patients. In vitro transfection of leukemogenic MLL fusion genes into human embryonic kidney-293 cells suppressed let-7b expression. In leukemic cells with an MLL fusion gene, the regulatory region for let-7b expression was hypermethylated, and its expression was partially recovered after culturing the cells with the demethylating agent 5-azacitidine. These results suggest that loss of let-7b expression may be one of the consequences of oncogenic MLL fusion proteins, and contributes to leukemogenesis possibly through the upregulation of let-7b-regulated target genes with leukemogenic potential in hematopoietic cells. The enforced expression of let-7b in ALL cell lines with an MLL fusion gene inhibited their growth, indicating the possible use of let-7b as a new therapeutic tool for refractory infant ALL with an MLL fusion gene.
Assuntos
Metilação de DNA , Regulação Leucêmica da Expressão Gênica , Rearranjo Gênico , MicroRNAs/genética , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Sequência de Bases , Proliferação de Células , Histona-Lisina N-Metiltransferase , Humanos , Lactente , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasAssuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/genética , Piperazinas/uso terapêutico , Mielofibrose Primária/genética , Pirimidinas/uso terapêutico , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzamidas , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 5 , Doença Crônica , Evolução Fatal , Humanos , Mesilato de Imatinib , Leucemia Mieloide Aguda/etiologia , Masculino , Proteínas de Fusão Oncogênica/biossíntese , Mielofibrose Primária/complicações , Mielofibrose Primária/tratamento farmacológico , Transcrição Gênica , Translocação GenéticaRESUMO
We report a novel MLL-associated chromosome translocation t(11;14)(q23;q24) in a child who showed signs of acute undifferentiated leukemia 3 years after intensive chemotherapy that included the topoisomerase-II inhibitor VP 16. Screening of a cDNA library of the patient's leukemic cells showed a novel fusion transcript between MLL and the Gephyrin (GPHN) gene on 14q24. The resulting MLL-GPHN fusion gene encodes MLL AT hook motifs and a DNA methyltransferase homology domain fused to the C-terminal half of Gephyrin, including a presumed tubulin binding site and a domain homologous to the Escherichia coli molybdenum cofactor biosynthesis protein MoeA. Genomic breakpoint analysis showed potential in vitro topoisomerase-II DNA-binding sites spanning the breakpoints in both MLL and GPHN but no flanking sequences that might mediate homologous recombination. This suggests that MLL-GPHN may have been generated by VP 16/topoisomerase-II-induced DNA double-strand breaks, followed by error-prone DNA repair via non-homologous end joining. Gephyrin was originally identified as a submembraneous scaffold protein that anchors and immobilizes postsynaptic membrane neurotransmitter receptors to underlying cytoskeletal elements. It also is reported to bind to phosphatidylinositol 3,4,5-triphosphate binding proteins involved in actin dynamics and downstream signaling and interacts with ATM-related family member RAFT1. Gephyrin domains in the chimeric protein therefore could contribute novel signal sequences or might modify MLL activity by oligomerization or intracellular redistribution.
Assuntos
Proteínas de Transporte/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 14/genética , Proteínas de Ligação a DNA/genética , Leucemia Monocítica Aguda/genética , Proteínas de Membrana/genética , Proteínas de Fusão Oncogênica/genética , Proto-Oncogenes , Fatores de Transcrição , Translocação Genética , Sequência de Aminoácidos , Sequência de Bases , Pré-Escolar , Quebra Cromossômica/genética , Clonagem Molecular , Feminino , Histona-Lisina N-Metiltransferase , Humanos , Dados de Sequência Molecular , Proteína de Leucina Linfoide-MieloideAssuntos
Cromossomos Humanos Par 12 , Cromossomos Humanos Par 15 , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptor trkC/genética , Proteínas Repressoras/genética , Criança , Pré-Escolar , Aberrações Cromossômicas , Feminino , Expressão Gênica , Humanos , Masculino , Proteínas Proto-Oncogênicas c-ets , Sensibilidade e Especificidade , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
TEL-AML1 fusion resulting from the t(12;21)(p13;q22) is one of the most common genetic abnormalities in childhood acute lymphoblastic leukemia. Recent findings that site-specific cleavage of the MLL gene can be induced by chemotherapeutic agents such as topoisomerase-II inhibitors suggest that apoptogenic agents can cause chromosomal translocations in hematopoietic cells. This study demonstrates a possible relationship between exposure to apoptogenic stimuli, TEL breaks, and the formation of TEL-AML1 fusion in immature B lymphocytes. Short-term culture of immature B cell lines in the presence of apoptogenic stimuli such as serum starvation, etoposide, or salicylic acid induced double-strand breaks (DSBs) in intron 5 of the TEL gene and intron 1 of the AML1 gene. TEL-AML1 fusion transcripts were also identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in cell lines treated by serum starvation or aminophylline. DSBs within the TEL gene were also associated with fusion to other unknown genes, presumably as a result of chromosomal translocation. We also examined 67 cord blood and 147 normal peripheral blood samples for the existence of in-frame TEL-AML1 fusion transcripts. One cord blood sample (1.5%) and 13 normal peripheral blood samples (8.8%) were positive as detected by nested RT-PCR. These data suggest that breakage and fusion of TEL and AML1 may be relatively common events and that sublethal apoptotic signals could play a role in initiating leukemogenesis via the promotion of DNA damage.
Assuntos
Apoptose , Linfócitos B/ultraestrutura , Quebra Cromossômica , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Aminofilina/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Sequência de Bases , Mapeamento Cromossômico , Subunidade alfa 2 de Fator de Ligação ao Core , Meios de Cultura Livres de Soro/farmacologia , Etoposídeo/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Recém-Nascido , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , RNA Mensageiro/biossíntese , Ácido Salicílico/farmacologia , Transcrição Gênica , Translocação Genética , Células Tumorais CultivadasRESUMO
We analyzed 32 patients with various hematological malignancies including acute myelocytic leukemia and non-Hodgkin lymphoma with a breakpoint at 11q22-q25 of chromosome 11, but who did not have rearrangements of the MLL/ALL-1 gene. The breakpoint in each patient was identified by fluorescence in situ hybridization using 21 cosmid probes and 2 YAC probes. Breakpoints for each "rearrangement" involving translocations such as t(1;11), t(2;11), inv(11), t(11;15), and t(10;11) found in 5 of the 11 patients had breakpoints in a small region from Ccl11-430 to Ccl11-526 at 11q22-q23.1. Furthermore, breakpoints for chromosome deletions at 11q21-q23 in 10 patients were located in the same region as that of translocations. A commonly deleted region among 8 patients was identified from Ccl11-526 to Ccl11-555 at 11q23.1. Fluorescence in situ hybridization analysis revealed that breakpoints for additive chromosome [add(11)] aberrations, which had additional material of unknown origin at 11q23 to 11q25 in 11 patients, were not located at 11q23 but rather at the more telomeric site of Ccl11-503 to VIJ(2)2072 at 11q25. These results indicated that the patients had several restricted breakpoint sites, which means that these chromosomal regions have recurrent oncogenes and tumor suppressor genes for pathogenesis for leukemia and lymphoma.
Assuntos
Cromossomos Humanos Par 11/genética , Leucemia Mieloide/genética , Linfoma de Células B/genética , Linfoma de Células T/genética , Translocação Genética/genética , Doença Aguda , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 10/genética , Feminino , Rearranjo Gênico do Linfócito B/genética , Rearranjo Gênico do Linfócito T/genética , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-IdadeRESUMO
The ETV6/TEL gene has been reported to fuse to PDGFRbetab MDS1/EVI1, BTL, ACS2, STL, JAK2, ABL, CDX2, TRKC, AML1, and MN1. Among them, PDGFRbeta, ABL, JAK2, and TRKC are tyrosine kinases (TK). We identified a novel ETV6 partner gene, ARG (ABL-related gene or ABL2), another TK gene in a cell line established from a patient with acute myelogenous leukemia (AML-M3) with a t(15;17)(q22;q11.2) and a t(1;12)(q25;p13), which has the remarkable feature to differentiate to mature eosinophils in culture with all-trans retinoic acid and cytokines. The ETV6/ARG transcripts consisted of exon 1 to 5 of ETV6 and the 3' portion of ARG starting from exon 1B or exon 2, resulting in an open reading frame for a fusion protein consisting of the entire PNT oligomerization domain of ETV6 and all of the functional domains of ARG including the TK domain. This is the same protein structure as identified in the other ETV6 TK fusion proteins. The reciprocal ARG/ETV6 transcript was not expressed, and the normal ETV6 allele was not deleted or rearranged. Although the ABL is known to be involved in various human malignancies, ARG has not been involved in human malignancies despite its high homology to ABL. Thus, this is the first report showing involvement of ARG in human leukemia. The ETV6/ARG protein may be involved in the unique differentiation capacity of this cell line. (Blood. 2000;95:2126-2131)
Assuntos
Cromossomos Humanos Par 12 , Cromossomos Humanos Par 1 , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Leucemia Promielocítica Aguda/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Translocação Genética , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Northern Blotting , Southern Blotting , Humanos , Hibridização in Situ Fluorescente , Masculino , Modelos Genéticos , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-ets , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Chromosome translocations involving band 12p13 are known to be involved in a variety of hematologic malignancies, some of them resulting in rearrangement of the ETV6/TEL gene. Applying the fluorescence in situ hybridization (FISH) method, we found a cryptic translocation t(12;15)(p13;q25) in an adult acute myeloid leukemia (AML) patient. Hybridization with cosmid probes showed that the ETV6 gene was rearranged in this translocation. A patient-specific cDNA library was screened with ETV6 cDNA, and a novel fusion transcript was identified between the ETV6 and TRKC/NTRK3 gene located on 15q25. TRKC is a receptor tyrosine kinase that is activated by neurotrophin-3 (NT-3). It is known to be expressed broadly in neural tissues but not in hematologic cells, so far. ETV6-TRKC chimeric transcript encoded the pointed (PNT) domain of the ETV6 gene that fused to the protein-tyrosine kinase (PTK) domain of the TRKC gene. Two types of fusion transcript were determined, one that included the entire PTK domain of TRKC and the other in which the 3'-terminal 462 bp of TRKC was truncated within the PTK domain. Western blot analysis showed the expression of both chimeric proteins of 52 and 38 kD in size. Our results suggest that chimeric PTK expressed in the leukemic cells may contribute to cellular transformation by abnormally activating TRK signaling pathways. Moreover, this is the first report on truncated neurotrophin receptors associated in leukemia.
Assuntos
Cromossomos Humanos Par 12 , Cromossomos Humanos Par 15 , Proteínas de Ligação a DNA/genética , Leucemia Mieloide/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator de Crescimento Neural/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Translocação Genética , Doença Aguda , Adulto , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Rearranjo Gênico , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-ets , Receptor trkC , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Fluorescence in situ hybridization (FISH) analysis was applied to detect t(12;21) using two yeast artificial chromosome probes and cosmid probes covering the TEL(ETV6) and the AML1 gene to clarify the incidence of abnormality of t(12;21) in Japanese childhood acute lymphoblastic leukemia (ALL). We detected seven TEL/AML1 fusion positive patients (9.5%), all of whom were diagnosed as B-lineage ALL, among 74 childhood ALL. On the other hand, no TEL/AML1 fusion positive patients were found among 37 adult ALL. The incidence among Japanese seemed to be lower than that among other nations. Of the seven patients with the TEL/AML1 fusion, five exhibited normal karyotype, one was t(8;12)(q11;p13), i(21q) and the remaining one exhibited a near-triploid karyotype in conventional G-banding. The FISH method clearly demonstrated that all patients with the TEL/AML1 fusion had subpopulations of leukemic cells with deletion of the normal TEL allele, which is significant for understanding the progression of leukemia with t(12;21).
Assuntos
Cromossomos Humanos Par 12 , Cromossomos Humanos Par 21 , Hibridização in Situ Fluorescente , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Repressoras , Translocação Genética , Southern Blotting , Criança , Pré-Escolar , Proteínas de Ligação a DNA/genética , Feminino , Deleção de Genes , Humanos , Masculino , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-ets , Fatores de Transcrição/genética , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
A 42-year-old woman had been given a diagnosis of malignant lymphoma, follicular, small cleaved cell. She had undergone chemotherapy including etoposide (1,500 mg/total) and was in her second complete remission. Four years and 4 months later, refractory anemia with excess of blasts (RAEB) with dup(11) (q21q23) x 2 developed in the patient and progressed to acute myeloid leukemia (AML-M5b). Despite regression of the AML to RAEB, a clone with the additional abnormality of del(20) (q11q13.1) appeared and transformed the RAEB into AML-M6. Rearrangement of the MLL gene was observed, and FISH analysis demonstrated that the signal sequences from the gene's 5' and 3'-terminal regions had detached. RT-PCR techniques detected a tandem duplication of MLL gene exons 2 through 8. This was considered to be one of the mechanisms behind the formation of the 11q23 abnormality observed in this patient.
