RESUMO
Diverse protocadherin-alpha genes (Pcdha, also called cadherin-related neuronal receptor or CNR) are expressed in the vertebrate brain. Their genomic organization involves multiple variable exons and a set of constant exons, similar to the immunoglobulin (Ig) and T-cell receptor (TCR) genes. This diversity can be used to distinguish neurons. Using polymorphisms that distinguish the C57BL/6 and MSM mouse strains, we analyzed the allelic expression of the Pcdha gene cluster in individual neurons. Single-cell analysis of Purkinje cells using multiple RT-PCR reactions showed the monoallelic and combinatorial expression of each variable exon in the Pcdha genes. This report is the first description to our knowledge of the allelic expression of a diversified receptor family in the central nervous system. The allelic and combinatorial expression of distinct variable exons of the Pcdha genes is a potential mechanism for specifying neuron identity in the brain.
Assuntos
Caderinas/genética , Variação Genética , Neurônios/metabolismo , Animais , Éxons , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Modelos Genéticos , Dados de Sequência Molecular , Família Multigênica , Células de Purkinje/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Genome stability is regulated by the balance between efficiencies of the repair machinery and genetic alterations such as mutations and chromosomal rearrangements. It has been postulated that deregulation of class switch recombination (CSR) and somatic hypermutation (SHM), which modify the immunoglobulin (Ig) genes in activated B cells, may be responsible for aberrant chromosomal translocations and mutations of non-Ig genes that lead to lymphocyte malignancy. However, the molecular basis for these genetic instabilities is not clearly understood. Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce both CSR and SHM in artificial substrates in fibroblasts as well as B cells. Here we show that constitutive and ubiquitous expression of AID in transgenic mice caused both T cell lymphomas and dysgenetic lesions of epithelium of respiratory bronchioles (micro-adenomas) in all individual mice. Point mutations, but not translocations, were massively introduced in expressed T cell receptor (TCR) and c-myc genes in T lymphoma cells. The results indicate that AID can mutate non-Ig genes including oncogenes, implying that aberrant AID expression could be a cause of human malignancy.
Assuntos
Transformação Celular Neoplásica , Citidina Desaminase/metabolismo , Animais , Sequência de Bases , Southern Blotting , Primers do DNA , Citometria de Fluxo , Tecido Linfoide/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Mutação Puntual , Receptores de Antígenos de Linfócitos T/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A 17-year-old boy developed therapy-related acute myeloid leukemia (t-AML) 3 years after the cessation of chemo- and radiotherapy for undifferentiated sarcoma of the liver. At the onset of the t-AML, his white blood cell count was 900/microL with a 46,XY,t(2;3)(p21;q26),del(5)(q?) karyotype. Despite intensive chemotherapy and two hematopoietic stem cell transplants, he died of the leukemia. At the terminal phase, his white blood cell count surpassed 30,000/microL and the Philadelphia (Ph) chromosome appeared. Expression of EVI1 in bone marrow cells was remarkably high at the onset of t-AML, although it was not detected at the end of therapy for the sarcoma. Polymerase chain reaction analysis of bone marrow cells revealed that mRNA for the bcr-abl chimera was negative at the onset of t-AML and positive at the terminal phase. These results suggest that EVI1 overexpression was the major factor contributing to leukemogenesis, and the late appearance of the Ph chromosome is closely associated with the progression to an aggressive form of leukemia.
Assuntos
Proteínas de Ligação a DNA/genética , Segunda Neoplasia Primária/genética , Cromossomo Filadélfia , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Adolescente , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação Leucêmica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/genética , Neoplasias Hepáticas/terapia , Proteína do Locus do Complexo MDS1 e EVI1 , Masculino , RNA Mensageiro/metabolismo , Sarcoma/terapia , Regulação para CimaRESUMO
A new cell line, STR-428 was established from ascites tumor cells of a malignant effusion lymphoma patient without human herpes virus-8 (HHV-8) infection. STR-428 cells showed an immunophenotype of mature B-cells and produced few cytokines related to lymphomatous effusion. Karyotypic and genetic analysis revealed complex translocations including t(14;18)(q32;q21) effecting IgH/BCL2 and der(8)t(3;8)(q27;q24) involving c-MYC. STR-428 represents a unique, B-cell lymphoma cell line carrying concurrent rearrangement of BCL2 and c-MYC genes with features distinct from those of HHV-8-related primary effusion lymphoma. This cell line may be a valuable tool, other than HHV-8, to investigate the pathogenesis of primary lymphomatous effusion.
Assuntos
Herpesvirus Humano 8/fisiologia , Linfoma/genética , Derrame Pleural Maligno/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Translocação Genética , Southern Blotting , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 18/genética , Citocinas , Humanos , Técnicas Imunoenzimáticas , Imunofenotipagem , Hibridização in Situ Fluorescente , Linfoma/metabolismo , Linfoma/virologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
We conducted a retrospective analysis of the clinical features of 20 patients with severe eosinophilia at our institution, including 10 cases of hypereosinophilic syndrome (HES) (5 definite and 5 probable cases) and 10 cases of other eosinophilic disorders. Of the 20 patients, 14 initially received prednisolone treatment, which resulted in rapid improvement and normalization of eosinophilia within 8 weeks; however, 2 patients with splenomegaly showed poor control of eosinophilia in response to corticosteroid treatment. In addition, the FIP1L1-PDGFRA fusion gene was detected only in these 2 cases. One of the FIP1L1-PDGFRA - positive HES cases featured bone marrow fibrosis. Treatment of this patient with imatinib mesylate resulted in a dramatic improvement of eosinophilia, organomegaly, and the bone marrow fibrosis. Taken together, our data and previous reports suggest that FIP1L1-PDGFRA - positive HES is a distinct clinical entity with myeloproliferative features and showing a poor response to corticosteroid treatment.
Assuntos
Corticosteroides/uso terapêutico , Síndrome Hipereosinofílica/genética , Síndrome Hipereosinofílica/patologia , Transtornos Mieloproliferativos/genética , Proteínas de Fusão Oncogênica/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Adulto , Idoso , Medula Óssea/patologia , Eosinofilia/tratamento farmacológico , Eosinofilia/genética , Eosinofilia/patologia , Feminino , Fibrose/etiologia , Humanos , Síndrome Hipereosinofílica/tratamento farmacológico , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Prednisolona/uso terapêutico , Prognóstico , Estudos Retrospectivos , EsplenomegaliaRESUMO
A new cell line, designated UCH1, was established from a patient with splenic marginal zone lymphoma (SMZL). UCH1 cells feature a mature B-cell phenotype, characterized by surface IgM +, kappa+, CD5-, CD10-, CD19+ and CD20+. The BCL2 and BCL6 genes retained their germ-line configurations and overexpression of cyclin D1 was not detected. UCH1 cells carry numerical and structural aberrations in chromosome 3, but these were too complex to be analyzed with the conventional G-banding method. Spectral karyotyping (SKY) and fluorescence in situ hybridization analysis clearly demonstrated the presence of a balanced translocation between chromosomes 8 and 14 [t(8;14)(q24;q32)] in the complex aberrations involving chromosome 3. The results of Southern blot analysis supported this finding by showing rearrangement of the c-myc gene in UCH1 cells. SKY analysis also identified a translocation involving chromosome band 18q21, to which BCL2 and MALT1 genes were assigned, suggesting their implication in the development or progression of SMZL.
Assuntos
Linhagem Celular Tumoral , Cromossomos Humanos Par 3 , Linfoma/genética , Linfoma/patologia , Translocação Genética , Idoso , Linfócitos B/metabolismo , Rearranjo Gênico , Humanos , Imunoglobulina M/metabolismo , Imunofenotipagem , Hibridização in Situ Fluorescente , Cariotipagem , Leucócitos Mononucleares/metabolismo , Masculino , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Nuclei isolated from green fluorescent protein-marked neurons in the cerebral cortex of juvenile mice (14-21 d after birth) were injected into enucleated oocytes that were allowed to develop into blastocysts. Embryonic stem (ES) cell lines were established from the inner cell mass of 76 cloned blastocysts after injecting 2026 neuronal nuclei. Some ES cells were injected individually into enucleated oocytes (nuclear transfer). Other ES cells were transferred into the blastocoeles of tetraploid blastocysts (tetraploid complementation). Two-cell embryos after nuclear transfer were transferred to the oviducts of surrogate mothers. Four (1.5%) of 272 nuclear-transferred two-cell embryos developed to term, and two (0.7%) developed into fertile adults. Nineteen (1.9%) of 992 tetraploid blastocysts receiving ES cells reached term, and 10 (1.0%) developed into adults. These findings demonstrate that some of the nuclei of differentiated neurons in the cerebral cortex of juvenile mice maintain developmental pluripotency.
Assuntos
Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Córtex Cerebral/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Animais , Blastocisto/fisiologia , Córtex Cerebral/embriologia , Clonagem de Organismos , Embrião de Mamíferos , Marcadores Genéticos , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Camundongos , Plasmídeos , Poliploidia , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
A 26-year-old man with idiopathic hypereosinophilic syndrome (HES) was treated with imatinib mesylate following a 5-year history of prednisolone therapy. The patient had hypereosinophilia (absolute eosinophil counts >1500/microL) occurring in cyclic oscillations as well as histologically diagnosed eosinophilic vasculitis, bursitis, and periodic soft-tissue swellings. Laboratory data revealed high levels of serum tryptase and increased numbers of mast cells in the bone marrow, but serum interleukin 5 levels were within the normal range. The disease initially responded well to 100 mg/day of imatinib mesylate but recurred 8 weeks later. Thereafter, a daily 200-mg dose was temporarily effective. Despite the response to imatinib, the FIP1L1-PDGFRA fusion gene was not detected by fluorescence in situ hybridization analysis. Additional molecular and cytogenetic studies showed neither translocations of platelet-derived growth factor receptor (PDGFR) genes nor mutations in the c-KIT or the PDGFR genes. Although imatinib mesylate is a choice of treatment for patients with HES, its precise molecular mechanism in individual cases remains to be clarified.
Assuntos
Antineoplásicos/farmacologia , Síndrome Hipereosinofílica/tratamento farmacológico , Piperazinas/farmacologia , Pirimidinas/farmacologia , Adulto , Benzamidas , Análise Mutacional de DNA , Humanos , Mesilato de Imatinib , Masculino , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Recidiva , Resultado do TratamentoRESUMO
A 2-year-old girl with Down syndrome (DS) developed acute megakaryoblastic leukemia (AMKL) following a transient myeloproliferative disorder (TMD). The blast cells showed an altered karyotype of 47,XX,r(7),+21c. Serial cytogenetic studies during the course of the illness showed rapid stepwise clonal chromosome changes, including a ring chromosome 7, associated with treatment refractoriness. We reviewed 10 published cases of Down syndrome-related AMKL (DS-AMKL) showing chromosome 7 abnormalities and found that these changes do not carry the same prognostic weight as for non-DS children. For DS-AMKL, therefore, other prognostic factors besides clonal cytogenetic changes need to be identified for planning optimal therapy.
Assuntos
Aberrações Cromossômicas , Transtornos Cromossômicos , Cromossomos Humanos Par 7 , Síndrome de Down/genética , Leucemia Megacarioblástica Aguda/genética , Pré-Escolar , Progressão da Doença , Síndrome de Down/complicações , Feminino , Humanos , Leucemia Megacarioblástica Aguda/complicações , Cromossomos em Anel , Cariotipagem Espectral , TrissomiaRESUMO
Primary effusion lymphoma (PEL) is recognized as a unique lymphoma entity, which occurs exclusively in body cavities as a serous lymphomatous effusion without tumor formation or organ infiltration. We established a cell line of B-cell origin from a pericardial effusion of a 63-year-old Japanese PEL patient who did not have human immunodeficiency virus infection. This PEL cell line had human herpesvirus-8 (HHV-8) and Epstein-Barr virus (EBV) infection. We named this cell line RM-P1. This cell line showed complex chromosomal abnormalities that could not be identified by G-banding. However, spectral karyotyping analysis determined the origin and organization of all unidentified chromosomal abnormalities. When inoculated into the peritoneal cavity of 8 severe combined immunodeficiency (SCID) mice depleted of natural killer cells, RM-P1 cells induced solid tumor with ascites in all animals tested. These tumor and ascitic cells had the same immunogenotypic features as those of the original RM-P1. These 2 types of cells were positive for both HHV-8 and EBV as demonstrated using polymerase chain reaction. Fluorescence-activated cell sorting analyses showed that neither tumors nor ascitic cells grown in SCID mice expressed leukocyte function-associated antigen (LFA)-1alpha (CD11a), LFA-1lbeta (CD18), LFA-2 (CD2), LFA-3 (CD58), intercellular adhesion molecule (ICAM)-1 (CD54), ICAM-2 (CD102), ICAM-3 (CD50), or leukocyte endothelial adhesion molecule (LECAM)-1 (CD62L), suggesting that these cytoadhesion molecules are not involved in tumor formation of RM-P1 cells in vivo. The establishment of the RM-P1 cell line and the animal model of PEL may provide insights for understanding the relationship between these viruses and PEL and for understand the mechanism for PEL.
Assuntos
Linfoma/patologia , Derrame Pleural Maligno/patologia , Células Tumorais Cultivadas/citologia , Animais , Moléculas de Adesão Celular/análise , Divisão Celular , Herpesvirus Humano 4 , Herpesvirus Humano 8 , Humanos , Cariotipagem , Linfoma/virologia , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Transplante de Neoplasias , Derrame Pleural Maligno/virologia , Células Tumorais Cultivadas/transplante , Células Tumorais Cultivadas/virologiaRESUMO
We report a case of acute myeloid leukemia (AML), M2 subtype according to the French-American-British (FAB) classification, with extramedullary myeloblastoma of the uterus and a masked type of variant translocation of t(8;21)(q22;q22). A 45-year-old Japanese woman presented with metrorrhagia, and AML (M2) with uterine invasion was diagnosed. The patient received an allogeneic peripheral blood stem cell transplantation after remission, and her pelvis was irradiated locally. Cytogenetic study at first showed t(8;17)(q22;p13) by G-banding. Spectral karyotyping (SKY) analysis modified this interpretation to a 3-way translocation involving chromosomes 8,17, and 21 and identified a masked type of variant t(8;21)(q22;q22) translocation. Results of fluorescence in situ hybridization using the AML1/ETO probe, and of detection of the AML1/ETO fusion transcript by reverse transcriptase-polymerase chain reaction were consistent with the karyotyping result. SKY analysis is useful to compensate for the limitations of cytogenetic studies.
Assuntos
Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Leucemia Mieloide Aguda/genética , Cariotipagem Espectral , Translocação Genética , Subunidade alfa 2 de Fator de Ligação ao Core , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Proteína 1 Parceira de Translocação de RUNX1 , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Neoplasias Uterinas/complicaçõesRESUMO
Acute myeloblastic leukemia cases carrying the translocation t(8;16) (p11;p13) are characterized by the M4 and M5 subtypes, erythrophagocytosis by the blast cells and a poor prognosis, suggesting a new clinical entity. The t(8;16) fuses the MOZ gene which encodes a histone acetyltransferase, located on 8p11 with the CBP gene which also encodes a histone acetyltransferase, located on 16p13, and recent reports suggested that the chimeric transcription MOZ-CBP is essential for leukemogenesis. A 68-year-old woman who had been treated mainly with paclitaxel and carboplatin for preceding ovarian cancer was admitted to our hospital, complaining of right breast mass. She was diagnosed as having breast cancer and acute monocytic leukemia (M5b). Cytogenetic study with spectral karyotyping analysis revealed the development of 47 XX, + 8, t(8;16)(p11;p13). Eleven cases of therapy-related t(8;16) leukemia including the present case have been reported, but prior treatment with paclitaxel and carboplatin-based chemotherapy has never been reported. The relation of histone acetylase and therapy-related leukemia is discussed.
Assuntos
Cromossomos Humanos Par 16 , Cromossomos Humanos Par 8 , Leucemia Monocítica Aguda/genética , Segunda Neoplasia Primária/genética , Translocação Genética , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carboplatina/efeitos adversos , Transformação Celular Neoplásica/genética , Feminino , Humanos , Leucemia Monocítica Aguda/induzido quimicamente , Segunda Neoplasia Primária/induzido quimicamente , Neoplasias Ovarianas/complicações , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/efeitos adversosRESUMO
Thrombocytosis is a rare finding in acute myeloblastic leukemia (AML). Here, we describe a patient with AML who relapsed with marked thrombocytosis. The patient was initially diagnosed as having AML (M4) with a low platelet count. The patient was started on combination chemotherapy including high-dose etoposide and achieved complete remission. However, the patient relapsed six months later with an extremely high platelet count (72.5 x 10(4)/microl). Cytogenetic analysis at relapse revealed the development of t(2;14)(p13;q32). Despite the repeated combination chemotherapy, the patient died with progressive disease. This case suggests that the additional chromosomal aberration t(2;14)(p13;q32) may be related to abnormal thrombocytosis in AML.
Assuntos
Cromossomos Humanos Par 14 , Cromossomos Humanos Par 2 , Leucemia Mielomonocítica Aguda/complicações , Trombocitose/genética , Translocação Genética , Adulto , Análise Citogenética , Progressão da Doença , Evolução Fatal , Humanos , Leucemia Mielomonocítica Aguda/diagnóstico , Leucemia Mielomonocítica Aguda/genética , Masculino , Contagem de Plaquetas , Recidiva , Trombocitose/etiologiaRESUMO
Myeloproliferative neoplasms (MPN) are multiple disease entities characterized by clonal expansion of one or more of the myeloid lineages (i.e. granulocytic, erythroid, megakaryocytic and mast cell). JAK2 mutations, such as the common V617F substitution and the less common exon 12 mutations, are frequently detected in such tumor cells and have been incorporated into the diagnostic criteria published by the World Health Organization since 2008. However, the mechanism by which these mutations contribute to MPN development is poorly understood. We examined gene expression profiles of MPN patients focusing on genes in the JAK-STAT signaling pathway using low-density real-time PCR arrays. We identified the following 2 upregulated genes in MPN patients: a known target of the JAK-STAT axis, SOCS3, and a potentially novel target, SPI1, encoding PU.1. Induction of PU.1 expression by JAK2 V617F in JAK2-wildtype K562 cells and its downregulation by JAK2 siRNA transfection in JAK2 V617F-positive HEL cells supported this possibility. We also found that the ABL1 kinase inhibitor imatinib was very effective in suppressing PU.1 expression in BCR-ABL1-positive K562 cells but not in HEL cells. This suggests that PU.1 expression is regulated by both JAK2 and ABL1. The contribution of the two kinases in driving PU.1 expression was dominant for JAK2 and ABL1 in HEL and K562 cells, respectively. Therefore, PU.1 may be a common transcription factor upregulated in MPN. PU.1 is a transcription factor required for myeloid differentiation and is implicated in erythroid leukemia. Therefore, expression of PU.1 downstream of activated JAK2 may explain why JAK2 mutations are frequently observed in MPN patients.
Assuntos
Substituição de Aminoácidos/genética , Neoplasias da Medula Óssea/sangue , Neoplasias da Medula Óssea/genética , Janus Quinase 2/genética , Transtornos Mieloproliferativos/sangue , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Regulação para Cima/genética , Idoso , Neoplasias da Medula Óssea/enzimologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Janus Quinase 2/metabolismo , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/genética , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
Feasibility of chromosomal manipulation in mammalian cells was first reported 15 years ago. Although this technique is useful for precise understanding of gene regulation in the chromosomal context, a limited number of laboratories have used it in actual practice because of associated technical difficulties. To overcome the practical hurdles, we developed a Cre-mediated chromosomal recombination system using fluorescent proteins and various site-specific recombinases. These techniques enabled quick construction of targeting vectors, easy identification of chromosome-rearranged cells, and rearrangement leaving minimum artificial elements at junctions. Applying this system to a human cell line, we successfully recapitulated two types of pathogenic chromosomal translocations in human diseases: MYC/IgH and BCR/ABL1. By inducing recombination between two loxP sites targeted into the same chromosome, we could mark cells harboring deletion or duplication of the inter-loxP segments with different colors of fluorescence. In addition, we demonstrated that the intrachromosomal recombination frequency is inversely proportional to the distance between two recombination sites, implicating a future application of this frequency as a proximity sensor. Our method of chromosomal manipulation can be employed for particular cell types in which gene targeting is possible (e.g. embryonic stem cells). Experimental use of this system would open up new horizons in genome biology, including the establishment of cellular and animal models of diseases caused by translocations and copy-number variations.
Assuntos
Cromossomos/ultraestrutura , DNA Nucleotidiltransferases/genética , Corantes Fluorescentes/metabolismo , Técnicas Genéticas , Vetores Genéticos , Animais , Linhagem Celular , Drosophila , Dosagem de Genes , Marcação de Genes , Humanos , Hibridização in Situ Fluorescente , Integrases/genética , Recombinação Genética , Translocação GenéticaRESUMO
Acute lymphoblastic leukemia (ALL) with chromosome aberration t(8;14)(q11.2;q32) mostly affects patients younger than 20 years old. One third of patients with this translocation have been reported to have Down syndrome. This translocation has been reported rarely in patients over the age of 50. Here we report a 71-year-old male ALL patient who carried t(8;14)(q11.2;q32). Fluorescence in situ hybridization (FISH) analysis revealed the involvement of CCAAT enhancer-binding protein delta (CEBPD) gene on chromosome 8, and IgH gene on chromosome 14. This case provides a new aspect for considering this clinical entity.
Assuntos
Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 8/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteína delta de Ligação ao Facilitador CCAAT/genética , Aberrações Cromossômicas , Evolução Fatal , Genes de Cadeia Pesada de Imunoglobulina , Humanos , Hibridização in Situ Fluorescente , Masculino , Recidiva Local de Neoplasia/patologiaRESUMO
PURPOSE: Seventy to eighty percent of rhabdomyosarcoma (RMS) tumors retain wild-type p53. The tumor suppressor p53 plays a central role in inducing cell cycle arrest or apoptosis in response to various stresses. p53 protein levels are regulated by MDM2 through ubiquitin-dependent degradation. In this study, we evaluated whether nutlin-3, a recently developed small-molecule antagonist of MDM2, has an effect on p53-dependent cell cycle arrest and apoptosis in cultured human RMS cell lines. EXPERIMENTAL DESIGN: Five RMS cell lines with different p53 statuses and MDM2 expression levels were treated with nutlin-3. Gene expression patterns, cell viability, cell cycle, and apoptosis after nutlin-3 treatment, and antitumor activity of combination treatment with vincristine or actinomycin D were assessed. RESULTS: Significant p53 activation was observed in wild-type p53 cell lines after nutlin-3 treatment. p53 activation led to cell cycle arrest in parallel with increased p21 expression. Furthermore, these cell lines underwent p53-dependent apoptosis, concomitant with elevation of proapoptotic genes and activation of caspase-3. The effect of nutlin-3 was almost the same in terms of half maximal inhibitory concentration and apoptosis whether or not MDM2 was overexpressed. Nutlin-3 did not induce either cell cycle arrest or apoptosis in p53 mutant cell lines. A combination of vincristine or actinomycin D with nutlin-3 enhanced the antitumor activity in RMS cell lines with wild-type p53. CONCLUSIONS: Nutlin-3 effectively restored p53 function in both normal MDM2 expression and MDM2 overexpression RMS cell lines with wild-type p53. p53 restoration therapy is a potential therapeutic strategy for refractory RMS with wild-type p53.