RESUMO
Neuroblastoma is the most common extra-cranial solid tumour in children. Natural killer (NK) cells are innate lymphocytes that are known to mediate the direct cytotoxicity of neuroblastoma tumour cells. Natural variation in the highly polymorphic killer immunoglobulin-like receptors (KIR) and their cognate human leukocyte antigen (HLA) class I ligands results in considerable diversity in NK cell function. As the early onset of neuroblastoma suggests the contribution of genetic factors, we investigated if individual KIR genes, combined KIR gene haplotypes or compound KIR-HLA ligand genotypes could influence susceptibility to neuroblastoma. Genotype analysis of the KIR genes as well as their three major HLA class I ligand groups, HLA-C1, HLA-C2 and HLA-Bw4, was carried out in a cohort of 201 neuroblastoma patients compared with 240 healthy control subjects using polymerase chain reaction with sequence-specific primers. We found a significant increase in the frequency of KIR2DL2 (P = 0.019) as well as KIR2DS2 (P = 0.008) in patients with neuroblastoma compared with the healthy control group. While the incidence of the least inhibitory compound KIR-HLA-C genotype, KIR2DL3 in the presence of HLA-C1 was slightly reduced in neuroblastoma patients, this did not reach statistical significance (P = 0.069). In summary, while KIR-HLA compound genotypes have previously been implicated in predicting treatment outcomes in neuroblastoma, here we show that the presence of the individual KIR genes, KIR2DL2 and KIR2DS2, irrespective of HLA-C genotype is associated with the onset of this embryonal malignancy.
Assuntos
Predisposição Genética para Doença , Neuroblastoma/genética , Receptores KIR2DL2/genética , Receptores KIR/genética , Alelos , Estudos de Casos e Controles , Centrômero/genética , Estudos de Coortes , Sequência Conservada/genética , Antígenos HLA-C/genética , Haplótipos , Humanos , Ligantes , Telômero/genéticaRESUMO
AIMS/HYPOTHESIS: HNF1A-MODY is a monogenic form of diabetes caused by mutations in the HNF1A gene. Here we identify, for the first time, HNF1A-MODY-associated microRNAs (miRNAs) that can be detected in the serum of HNF1A-MODY carriers. METHODS: An miRNA array was carried out in rat INS-1 insulinoma cells inducibly expressing the common human Pro291fsinsC-HNF1A frame shift mutation. Differentially expressed miRNAs were validated by quantitative real-time PCR. Expression of miRNAs in the serum of HNF1A-MODY carriers (n = 31), MODY-negative family members (n = 10) and individuals with type 2 diabetes mellitus (n = 17) was quantified by absolute real-time PCR analysis. RESULTS: Inducible expression of Pro291fsinsC-HNF1A in INS-1 cells caused a significant upregulation of three miRNAs (miR-103, miR-224, miR-292-3p). The differential expression of two miRNAs (miR-103 and miR-224) was validated in vitro. Strongly elevated levels of miR-103 and miR-224 could be detected in the serum of HNF1A-MODY carriers compared with MODY-negative family controls. Serum levels of miR-103 distinguished HNF1A-MODY carriers from HbA1c-matched individuals with type 2 diabetes mellitus. CONCLUSIONS/INTERPRETATION: Our study demonstrates that the pathophysiology of HNF1A-MODY is associated with the overexpression of miR-103 and miR-224. Furthermore, our study demonstrates that these miRNAs can be readily detected in the serum of HNF1A-MODY carriers.
Assuntos
Diabetes Mellitus Tipo 2/genética , Fator 1-alfa Nuclear de Hepatócito/genética , MicroRNAs/genética , Animais , Mutação da Fase de Leitura/genética , Insulinoma/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Fator 1 de Transcrição de Linfócitos T/genéticaRESUMO
Developing targeted therapies for high grade gliomas (HGG), the most common primary brain tumor in adults, relies largely on glioma cultures. However, it is unclear if HGG tumorigenic signaling pathways are retained under in-vitro conditions. Using array comparative genomic hybridization and immunohistochemical profiling, we contrasted the epidermal and platelet-derived growth factor receptor (EGFR/PDGFR) in-vitro pathway status of twenty-six primary HGG cultures with the pathway status of their original HGG biopsies. Genomic gains or amplifications were lost during culturing while genomic losses were more likely to be retained. Loss of EGFR amplification was further verified immunohistochemically when EGFR over expression was decreased in the majority of cultures. Conversely, PDGFRα and PDGFRß were more abundantly expressed in primary cultures than in the original tumor (p<0.05). Despite these genomic and proteomic differences, primary HGG cultures retained key aspects of dysregulated tumorigenic signaling. Both in-vivo and in-vitro the presence of EGFR resulted in downstream activation of P70s6K while reduced downstream activation was associated with the presence of PDGFR and the tumor suppressor, PTEN. The preserved pathway dysregulation make this glioma model suitable for further studies of glioma tumorigenesis, however individual culture related differences must be taken into consideration when testing responsiveness to chemotherapeutic agents.
Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Hibridização Genômica Comparativa , Genômica , Glioma/genética , Glioma/metabolismo , Proteômica , Adulto , Idoso , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Glioma/patologia , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Adulto JovemRESUMO
BACKGROUND: Neuroblastoma remains a major cause of cancer-linked mortality in children. miR-204 has been used in microRNA expression signatures predictive of neuroblastoma patient survival. The aim of this study was to explore the independent association of miR-204 with survival in a neuroblastoma cohort, and to investigate the phenotypic effects mediated by miR-204 expression in neuroblastoma. METHODS: Neuroblastoma cell lines were transiently transfected with miR-204 mimics and assessed for cell viability using MTS assays. Apoptosis levels in cell lines were evaluated by FACS analysis of Annexin V-/propidium iodide-stained cells transfected with miR-204 mimics and treated with chemotherapy drug or vehicle control. Potential targets of miR-204 were validated using luciferase reporter assays. RESULTS: miR-204 expression in primary neuroblastoma tumours was predictive of patient event-free and overall survival, independent of established known risk factors. Ectopic miR-204 expression significantly increased sensitivity to cisplatin and etoposide in vitro. miR-204 direct targeting of the 3' UTR of BCL2 and NTRK2 (TrkB) was confirmed. CONCLUSION: miR-204 is a novel predictor of outcome in neuroblastoma, functioning, at least in part, through increasing sensitivity to cisplatin by direct targeting and downregulation of anti-apoptotic BCL2. miR-204 also targets full-length NTRK2, a potent oncogene involved with chemotherapy drug resistance in neuroblastoma.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , MicroRNAs/farmacologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Receptor trkB/efeitos dos fármacos , Análise de Variância , Animais , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Modelos Animais de Doenças , Intervalo Livre de Doença , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Etoposídeo/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos SCID , Neuroblastoma/mortalidade , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Reação em Cadeia da Polimerase em Tempo Real , Receptor trkB/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
Neuroblastoma (NB) is one of the most common forms of cancer in children, accounting for 15% of pediatric cancer deaths. The clinical course of these tumors is highly variable and is dependent on such factors as age at presentation, stage, ploidy and genomic abnormalities. Hemizygous deletion of chromosome 1p occurs in approximately 30% of advanced stage tumors, is associated with a poor prognosis, and likely leads to the loss of one or more tumor suppressor genes. We show here that microRNA (miRNA)-34a (1p36.23) is generally expressed at lower levels in unfavorable primary NB tumors and cell lines relative to normal adrenal tissue and that reintroduction of this miRNA into three different NB cell lines causes a dramatic reduction in cell proliferation through the induction of a caspase-dependent apoptotic pathway. As a potential mechanistic explanation for this observation, we demonstrate that miR-34a directly targets the messenger ribonucleic acid (mRNA) encoding E2F3 and significantly reduces the levels of E2F3 protein, a potent transcriptional inducer of cell-cycle progression. Furthermore, miR-34a expression increases during retinoic acid-induced differentiation of the SK-N-BE cell line, whereas E2F3 protein levels decrease. Thus, adding to the increasing role of miRNAs in cancer, miR-34a may act as a suppressor of NB tumorgenesis.
Assuntos
Apoptose , Genes Supressores de Tumor , MicroRNAs/fisiologia , Neuroblastoma/genética , Linhagem Celular Tumoral , Criança , Deleção Cromossômica , Cromossomos Humanos Par 1 , Fator de Transcrição E2F3/genética , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologiaRESUMO
3D scaffold-based in vitro cell culturing is a recent technological advancement in cancer research bridging the gap between conventional 2D culture and in vivo tumours. The main challenge in treating neuroblastoma, a paediatric cancer of the sympathetic nervous system, is to combat tumour metastasis and resistance to multiple chemotherapeutic drugs. The aim of this study was to establish a physiologically relevant 3D neuroblastoma tissue-engineered system and explore its therapeutic relevance. Two neuroblastoma cell lines, chemotherapeutic sensitive Kelly and chemotherapeutic resistant KellyCis83 were cultured in a 3D in vitro model on two collagen-based scaffolds containing either glycosaminoglycan (Coll-GAG) or nanohydroxyapatite (Coll-nHA) and compared to 2D cell culture and an orthotopic murine model. Both neuroblastoma cell lines actively infiltrated the scaffolds and proliferated displaying >100-fold increased resistance to cisplatin treatment when compared to 2D cultures, exhibiting chemosensitivity similar to orthotopic xenograft in vivo models. This model demonstrated its applicability to validate miRNA-based gene delivery. The efficacy of liposomes bearing miRNA mimics uptake and gene knockdown was similar in both 2D and 3D in vitro culturing models highlighting the proof-of-principle for the applicability of 3D collagen-based scaffolds cell system for validation of miRNA function. Collectively, this data shows the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. While neuroblastoma is the specific disease being focused upon, the platform may have multi-functionality beyond this tumour type. STATEMENT OF SIGNIFICANCE: Traditional 2D cell cultures do not completely capture the 3D architecture of cells and extracellular matrix contributing to a gap in our understanding of mammalian biology at the tissue level and may explain some of the discrepancies between in vitro and in vivo results. Here, we demonstrated the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. The ability to test drugs in this reproducible and controllable tissue-engineered model system will help reduce the attrition rate of the drug development process and lead to more effective and tailored therapies. Importantly, such 3D cell models help to reduce and replace animals for pre-clinical research addressing the principles of the 3Rs.
Assuntos
Colágeno/química , Técnicas de Transferência de Genes , Neuroblastoma , Alicerces Teciduais/química , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neuroblastoma/terapiaRESUMO
Neuroblastoma is characterized by numerous recurrent large-scale chromosomal imbalances and gene amplifications which are associated with poor clinical outcome. The most common include MYCN amplification, loss of 1p, 3p and 11q, and gain of 17q genomic regions. Two of these abnormalities, MYCN amplification and loss of 11q, define different genetic subtypes of the disease with vastly different global gene expression profiles. The progress towards the identification of the genes and genetic pathways that have been affected by these abnormalities is reviewed and high resolution mapping of the chromosomal breakpoint regions using oligonucleotide array CGH (oaCGH) is discussed. oaCGH analysis is proving useful for both defining minimal regions of overlap of imbalances, as well as providing information on the molecular mechanisms that generate the chromosomal imbalances. These high resolution analyses have also permitted the detection of micro-deletions in the tumors that further assist in identifying genes important for neuroblastoma pathogenesis.
Assuntos
Aberrações Cromossômicas , Neuroblastoma/genética , Sítios Frágeis do Cromossomo , Humanos , Neuroblastoma/patologia , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , PrognósticoRESUMO
Polyethylene glycol-mediated fusion of Chinese hamster ovary (CHO) cells with mouse Cl1D cells produced interspecific somatic cell hybrids which slowly segregated CHO chromosomes. Cytogenetic and isozyme analysis of HAT- and bromodeoxyuridine-selected hybrid subclones and of members of a hybrid clone panel retaining different combinations of CHO chromosomes enabled provisional assignments of the following enzyme loci to CHO chromosomes: TK, GALK, and ACP1 to chromosome 7; TK and GALK to chromosome Z13; ACP1, ADA, and ITPA to chromosome Z8; and ADA and ITPA to chromosome Z9. These genetic markers reflect the origin of each of these Z group chromosomes and indicate the functional activity of alleles located on rearranged chromosomes. Identification of diploid electrophoretic shift mutations for ADA and ITPA was consistent with those observations. Assignment of the functional TK locus in TK+/- CHO-AT3-2 cells indicated that gene deletion may be responsible for TK hemizygosity in this subline.
Assuntos
Cromossomos , Cricetinae/genética , Cricetulus/genética , Enzimas/genética , Genes , Alelos , Animais , Linhagem Celular , Mapeamento Cromossômico , Feminino , Células Híbridas/enzimologia , Camundongos , Ovário , Translocação GenéticaRESUMO
Gene amplification has been associated with multidrug resistance (MDR) in several drug-resistant Chinese hamster ovary (CHO) cell lines which exhibit cross-resistance to other unrelated, cytotoxic drugs. In situ hybridization studies (Teeter et al., J. Cell Biol., in press) suggested the presence of an amplified gene associated with the MDR phenotype on the long arm of either of the largest CHO chromosomes (1 or Z1) in vincristine-resistant cells. In this study, somatic cell hybrids were constructed between these vincristine-resistant CHO cells and drug-sensitive murine cells to determine the functional relationship between the chromosome bearing the amplified sequences and the MDR phenotype. Hybrids exhibited primary drug resistance and MDR in an incomplete dominant fashion. Hybrid clones and subclones segregated CHO chromosomes. Concordant segregation between vincristine resistance, the MDR phenotype, the presence of the MDR-associated amplified sequences, overexpression of the gene located in those sequences, and CHO chromosome Z1 was consistent with the hypothesis that there is an amplified gene on chromosome Z1 of the vincristine-resistant CHO cells which is responsible for the MDR in these cells. A low level of discordance between CHO chromosomes Z8 and 2 and the drug resistance phenotype suggests that these chromosomes may contain genes involved with the MDR phenotype.
Assuntos
Resistência a Medicamentos , Amplificação de Genes , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Demecolcina/toxicidade , Doxorrubicina/toxicidade , Feminino , Células Híbridas/citologia , Células Híbridas/efeitos dos fármacos , Camundongos , Hibridização de Ácido Nucleico , Ovário , Fenótipo , Vincristina/toxicidadeRESUMO
Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines.
Assuntos
Mapeamento Cromossômico , Amplificação de Genes , Metalotioneína/genética , Animais , Bandeamento Cromossômico , Cricetinae , Cricetulus , Feminino , Hibridização de Ácido NucleicoRESUMO
We describe here the derivation, characterization, and use of clonal cadmium-resistant (Cdr) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylamide gel electrophoresis, we showed that the stable Cdr phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 microM, coordinate amplification of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which we have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cdr variants expressing abundant MT and their corresponding Cds parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expression of the isometallothioneins.
Assuntos
Cádmio/farmacologia , Amplificação de Genes , Regulação da Expressão Gênica , Metalotioneína/genética , Animais , Cricetinae , Cricetulus , DNA/análise , Enzimas de Restrição do DNA/metabolismo , Resistência a Medicamentos , Eletroforese em Gel de Poliacrilamida , Hibridização de Ácido NucleicoRESUMO
The genes for the M2 subunit of ribonucleotide reductase (RRM2), ornithine decarboxylase (ODC1), and 55,000-Daltons protein (P5), are amplified in hydroxyurea-resistant hamster and human cell lines. These genomic sequences have been mapped to hamster chromosome 7 and to human chromosome 2p24-25 near the cytogenetic location of the N-myc gene. We now report that genomic sequences homologous to N-myc are amplified in hydroxyurea-resistant hamster lung cell line, 600H, and the N-myc gene segregates with hamster chromosome 7 in mouse-hamster somatic cell hybrids. The conserved linkage group consisting of the RRM2, ODC1, P5, and N-myc in the hamster and human genomes prompted our investigation of human neuroblastomas. We report here that genomic DNA from 1 of 6 primary neuroblastoma tumors containing amplified N-myc also contains amplified sequences homologous to a hamster ODC cDNA.
Assuntos
Amplificação de Genes , Hidroxiureia/farmacologia , Neuroblastoma/genética , Ornitina Descarboxilase/genética , Proto-Oncogenes , Adolescente , Animais , Linhagem Celular , Criança , Pré-Escolar , Cricetinae , DNA/análise , Resistência a Medicamentos , Humanos , Lactente , Células Tumorais CultivadasRESUMO
Neuroblastoma, one of the most common tumors of childhood, presents at diagnosis with a vast number of recurrent chromosomal imbalances that include hyperdiploidy for whole chromosomes, partial loss of 1p, 3p, 4p, 11q, 14q, partial gain of 1q, 7q, 17q and amplification of MYCN. These abnormalities are nonrandomly distributed in neuroblastoma as loss of 3p and 11q rarely occur in MYCN amplified neuroblastomas. Here, we report on a patient who had a non-MYCN amplified 3p-/11q- neuroblastoma at diagnosis who subsequently developed a high level of MYCN amplification in bone marrow metastases 41 months after induction of complete remission. The tumor at diagnosis had low level unbalanced gain of distal 2p. In order to assess the frequency of low level gain of distal 2p in neuroblastoma, we examined the comparative genomic hybridization results from 60 neuroblastomas. Among non-MYCN amplified neuroblastomas, 8/45 (18%) had low level gain of distal 2p. Low level gain for a segment of 2p (i.e. a region larger than the 2p23-->p24 undergoing amplification) was also detected in five of the 15 tumors that had high level MYCN amplification. The possibility that low level gain of distal 2p is a risk factor for high level MYCN amplification is discussed.
Assuntos
Aneuploidia , Cromossomos Humanos Par 2 , Amplificação de Genes , Genes myc , Neuroblastoma/genética , Neoplasias Abdominais/genética , Neoplasias das Glândulas Suprarrenais/genética , Criança , Pré-Escolar , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Estadiamento de Neoplasias , Neuroblastoma/patologia , Hibridização de Ácido Nucleico/métodos , TrissomiaRESUMO
SCORE, a program for computer-assisted scoring of Southern blots of clone DNA, retains the use of expert human judgment while taking over much of the drudgery of the scoring task. The primary functions of the program are to help make an aligned overlay of the fluorescence gel image and the autoradiogram blot image, to keep track of band and lane locations and to store the resulting data directly into a database. Use of SCORE has resulted in greatly increased efficiency and accuracy.
Assuntos
Southern Blotting , Software , Autorradiografia , Mapeamento Cromossômico/métodos , Impressões Digitais de DNA/métodos , Eletroforese em Gel de Ágar , Humanos , Processamento de Imagem Assistida por Computador/métodosRESUMO
Fluorescence in situ hybridization analysis (FISH) was used to map the constitutional chromosome 16 pericentric inversion breakpoints inv(16)(p13q22) detected in one individual (II-2) from a large kindred [Bianchi et al., 1992: Am J Med Genet 43:791-795]. The breakpoints found in individual II-2 mapped to distinctly different locations than the chromosome 16 pericentric inversion breakpoints commonly acquired in acute nonlymphocytic leukemia. The constitutional pericentric inversion breakpoints also do not map to regions where low abundance repetitive DNA sequences found in bands 16p13 and q22 are located. The results indicate that low abundance, chromosome 16-specific repetitive DNA sequences in bands p13 and q22 are probably not causally related to the inversion that is found in many members of a large kindred [Bianchi et al., 1992].
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 16/ultraestrutura , Adulto , Mapeamento Cromossômico , Cosmídeos , Feminino , Corantes Fluorescentes , Humanos , Hibridização in Situ Fluorescente , Indóis , Leucemia Mieloide Aguda/genéticaRESUMO
Neuroblastoma, the most common extracranial solid tumor of childhood, is associated with a number of genetic abnormalities that are prognostically significant. The most common abnormalities are associated with aggressive clinical behavior and include deletion of distal chromosome 1p, NMYC amplification, and unbalanced gain of the long arm of chromosome 17. There are also other recurrent, but less frequent abnormalities, the clinical significance of which is uncertain. These less common abnormalities include deletion 3p, 11q, and 14q. To gain further clinical insight into some of the less commonly observed abnormalities in neuroblastoma, we performed comparative genomic hybridization (CGH) analysis on 24 primary and metastatic neuroblastomas (6 stage 2, 5 stage 3, 11 stage 4, and 2 stage 4). Nineteen of these tumors were prechemotherapy. A total of 190 abnormalities were detected from these tumors. Four of the 24 tumors studied showed loss of 11q material, with 3 of these tumors also possessing distal chromosome 3p deletions. Our results provide confirmation that deletion of chromosome 3p is nonrandomly associated with deletion of chromosome 11q in neuroblastoma. However, analysis of our results, along with other results reported in the literature, indicate that there is no statistically significant association between 3p and 11q loss and more clinically aggressive tumors.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 3 , Neuroblastoma/genética , Neoplasias Abdominais/genética , Biópsia , Aberrações Cromossômicas , Humanos , Metástase Neoplásica , Hibridização de Ácido Nucleico , Neoplasias Pélvicas/genética , Neoplasias Torácicas/genéticaRESUMO
Neuroblastoma exhibiting deletion of a segment of the long arm of chromosome 11 represents a genetic subtype of tumor that is distinct from those exhibiting MYCN amplification or 1p deletion. The 11q- genetic subtype is further characterized by gain of 17q and loss of distal 3p material. Gain of 11p material has also been reported in neuroblastoma with 11q loss, but at a considerably lower frequency than gain of 17q or loss of the distal 3p region. Our results, however, indicate that gain of 11p may occur more frequently in 11q- neuroblastoma than what was previously realized. Comparative genomic hybridization analyses of neuroblastoma tissue from eleven patients indicated that six of 11 tumors (55%) with loss of 11q also possessed gain of 11p. The shortest region of 11p gain was 11p11.2-->p14. G-banding and fluorescence in situ hybridization analysis performed on tumor cells from primary and metastatic sites from one patient allowed us to infer that gain of 11p arose secondarily to the abnormality that led to the loss of 11q material. Gain of an entire chromosome 7 was detected in 17 of 43 (40%) tumors, whereas gain of 7q was detected in 5 of 43 (12%) tumors. Unlike gain of 11p, gain of an entire chromosome 7 appears to be prevalent in all tumor stages and is not limited to the 11q- tumor subtype. Gain of 7q, however, is more prevalent in higher stage tumors. G-band cytogenetic analysis indicated that an unbalanced t(3;7) was responsible for the gain of 7q and loss of 3p material in one case. We discuss the possibility that gain of 7/7q, and 11p material may contribute to either tumorigenesis or progression.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 11/ultraestrutura , Cromossomos Humanos Par 7/ultraestrutura , Amplificação de Genes , Neuroblastoma/genética , Neoplasias Abdominais/genética , Neoplasias Abdominais/patologia , Transformação Celular Neoplásica/genética , Bandeamento Cromossômico , Deleção Cromossômica , Coloração Cromossômica , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 7/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metástase Linfática , Neuroblastoma/patologia , Hibridização de Ácido Nucleico , Deleção de Sequência , Neoplasias Torácicas/genética , Neoplasias Torácicas/patologiaRESUMO
Loss of 11q material occurs in approximately 30% of advanced stage neuroblastoma and defines a distinct genetic subtype of this disease. These tumors almost always possess unbalanced gain of the 17q, along with many additional recurrent chromosomal imbalances. Loss of 11q and gain of 17q is often the consequence of an unbalanced translocation between the long arms of both chromosomes, but because of the involvement of other chromosomal mechanisms, the actual frequency of t(11;17) is unknown. In addition, chromosomal breakpoint positions for the t(11;17) are variable in different tumors, with breakpoints on neither the 11q nor 17q being well defined. We have used interphase fluorescence in situ hybridization analysis to detect a der(11)t(11;17) in a series of neuroblastomas with 11q loss/17q gain using a statistical approach which could be applicable to the detection of translocations in other solid tumors. The frequency of der(11)t(11;17) was approximately 90% in our neuroblastoma series. A balanced t(11;17) was also detected in a MYCN amplified tumor, which is a distinctly different genetic subtype from the 11q- tumors. Breakpoint positions on 11q were determined to be variable, whereas all breakpoints on 17q appeared to cluster proximal to position 43.1 Mb on the DNA sequence map. The majority of tumors had large numbers of nuclei with 2 or more copies of der(11)t(11;17), which led to unbalanced gain of 11p, and further increases in 17q imbalance. The prevalence of t(11;17) in neuroblastoma warrants additional studies to further define the range in variation in breakpoint positions on both chromosomes and to elucidate the molecular mechanisms that lead to this important and interesting recurrent genetic abnormality.
Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 17 , Neuroblastoma/genética , Translocação Genética , Cromossomos Artificiais Bacterianos , Humanos , Hibridização in Situ Fluorescente , Hibridização de Ácido Nucleico/métodosRESUMO
Neuroblastoma (NBL) is the most common solid tumor in infants and accounts for 15% of all pediatric cancer deaths. Several risk factors predict NBL outcome: age at the time of diagnosis, stage, chromosome alterations and MYCN (V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma-Derived Homolog) amplification, which characterizes the subset of the most aggressive NBLs with an overall survival below 30%. MYCN-amplified tumors develop exceptional chemoresistance and metastatic capacity. These properties have been linked to defects in the apoptotic machinery, either by silencing components of the extrinsic apoptotic pathway (e.g. caspase-8) or by overexpression of antiapoptotic regulators (e.g. Bcl-2, Mcl-1 or FLIP). Very little is known on the implication of death receptors and their antagonists in NBL. In this work, the expression levels of several death receptor antagonists were analyzed in multiple human NBL data sets. We report that Lifeguard (LFG/FAIM2 (Fas apoptosis inhibitory molecule 2)/NMP35) is downregulated in the most aggressive and undifferentiated tumors. Intringuingly, although LFG has been initially characterized as an antiapoptotic protein, we have found a new association with NBL differentiation. Moreover, LFG repression resulted in reduced cell adhesion, increased sphere growth and enhanced migration, thus conferring a higher metastatic capacity to NBL cells. Furthermore, LFG expression was found to be directly repressed by MYCN at the transcriptional level. Our data, which support a new functional role for a hitherto undiscovered MYCN target, provide a new link between MYCN overexpression and increased NBL metastatic properties.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Membrana/metabolismo , Neuroblastoma/patologia , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Antibacterianos/toxicidade , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Adesão Celular , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Doxiciclina/toxicidade , Feminino , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Proteína Proto-Oncogênica N-Myc , Metástase Neoplásica , Estadiamento de Neoplasias , Neuroblastoma/metabolismo , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores de Morte Celular/antagonistas & inibidores , Receptores de Morte Celular/metabolismo , Transplante Heterólogo , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
MicroRNAs (miRNAs) contribute to the pathogenesis of many forms of cancer, including the pediatric cancer neuroblastoma, but the underlying mechanisms leading to altered miRNA expression are often unknown. Here, a novel integrated approach for analyzing DNA methylation coupled with miRNA and mRNA expression data sets identified 67 epigenetically regulated miRNA in neuroblastoma. A large proportion (42%) of these miRNAs was associated with poor patient survival when underexpressed in tumors. Moreover, we demonstrate that this panel of epigenetically silenced miRNAs targets a large set of genes that are overexpressed in tumors from patients with poor survival in a highly redundant manner. The genes targeted by the epigenetically regulated miRNAs are enriched for a number of biological processes, including regulation of cell differentiation. Functional studies involving ectopic overexpression of several of the epigenetically silenced miRNAs had a negative impact on neuroblastoma cell viability, providing further support to the concept that inactivation of these miRNAs is important for neuroblastoma disease pathogenesis. One locus, miR-340, induced either differentiation or apoptosis in a cell context dependent manner, indicating a tumor suppressive function for this miRNA. Intriguingly, it was determined that miR-340 is upregulated by demethylation of an upstream genomic region that occurs during the process of neuroblastoma cell differentiation induced by all-trans retinoic acid (ATRA). Further biological studies of miR-340 revealed that it directly represses the SOX2 transcription factor by targeting of its 3'-untranslated region, explaining the mechanism by which SOX2 is downregulated by ATRA. Although SOX2 contributes to the maintenance of stem cells in an undifferentiated state, we demonstrate that miR-340-mediated downregulation of SOX2 is not required for ATRA induced differentiation to occur. In summary, our results exemplify the dynamic nature of the miRNA epigenome and identify a remarkable network of miRNA/mRNA interactions that significantly contribute to neuroblastoma disease pathogenesis.