RESUMO
Assessing the impact of variants of unknown significance (VUS) on splicing is a key issue in molecular diagnosis. This impact can be predicted by in silico tools, but proper evaluation and user guidelines are lacking. To fill this gap, we embarked upon the largest BRCA1 and BRCA2 splice study to date by testing 272 VUSs (327 analyses) within the BRCA splice network of Unicancer. All these VUSs were analyzed by using six tools (splice site prediction by neural network, splice site finder (SSF), MaxEntScan (MES), ESE finder, relative enhancer and silencer classification by unanimous enrichment, and human splicing finder) and the predictions obtained were compared with transcript analysis results. Combining MES and SSF gave 96% sensitivity and 83% specificity for VUSs occurring in the vicinity of consensus splice sites, that is, the surrounding 11 and 14 bases for the 5' and 3' sites, respectively. This study was also an opportunity to define guidelines for transcript analysis along with a tentative classification of splice variants. The guidelines drawn from this large series should be useful for the whole community, particularly in the context of growing sequencing capacities that require robust pipelines for variant interpretation.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Patologia Molecular/métodos , Patologia Molecular/normas , Splicing de RNA/genética , Éxons/genética , Feminino , HumanosRESUMO
BACKGROUND: Congenital secondary erythrocytoses are due to deregulation of hypoxia inducible factor resulting in overproduction of erythropoietin. The most common germline mutation identified in the hypoxia signaling pathway is the Arginine 200-Tryptophan mutant of the von Hippel-Lindau tumor suppressor gene, resulting in Chuvash polycythemia. This mutant displays a weak deficiency in hypoxia inducible factor α regulation and does not promote tumorigenesis. Other von Hippel-Lindau mutants with more deleterious effects are responsible for von Hippel-Lindau disease, which is characterized by the development of multiple tumors. Recently, a few mutations in gene for the prolyl hydroxylase domain 2 protein (PHD2) have been reported in cases of congenital erythrocytosis not associated with tumor formation with the exception of one patient with a recurrent extra-adrenal paraganglioma. DESIGN AND METHODS: Five PHD2 variants, four of which were novel, were identified in patients with erythrocytosis. These PHD2 variants were functionally analyzed and compared with the PHD2 mutant previously identified in a patient with polycythemia and paraganglioma. The capacity of PHD2 to regulate the activity, stability and hydroxylation of hypoxia inducible factor α was assessed using hypoxia-inducible reporter gene, one-hybrid and in vitro hydroxylation assays, respectively. RESULTS: This functional comparative study showed that two categories of PHD2 mutants could be distinguished: one category with a weak deficiency in hypoxia inducible factor α regulation and a second one with a deleterious effect; the mutant implicated in tumor occurrence belongs to the second category. CONCLUSIONS: As observed with germline von Hippel-Lindau mutations, there are functional differences between the PHD2 mutants with regards to hypoxia inducible factor regulation. PHD2 mutation carriers do, therefore, need careful medical follow-up, since some mutations must be considered as potential candidates for tumor predisposition.
Assuntos
Mutação em Linhagem Germinativa , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Mutantes/metabolismo , Policitemia/genética , Policitemia/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Adolescente , Adulto , Sequência de Bases , Células Cultivadas , Feminino , Células HEK293 , Humanos , Hidrólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia , Masculino , Pessoa de Meia-Idade , Pró-Colágeno-Prolina Dioxigenase/genética , Adulto JovemRESUMO
BACKGROUND: Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an autosomal dominant disorder predisposing humans to cutaneous and uterine leiomyomas; in 20% of affected families, type 2 papillary renal cell cancers (PRCCII) also occur with aggressive course and poor prognosis. HLRCC results from heterozygous germline mutations in the tumour suppressor fumarate hydratase (FH) gene. METHODS: As part of the French National Cancer Institute (INCa) 'Inherited predispositions to kidney cancer' network, sequence analysis and a functional study of FH were preformed in 56 families with clinically proven or suspected HLRCC and in 23 patients with isolated PRCCII (5 familial and 18 sporadic). RESULTS: The study identified 32 different germline FH mutations (15 missense, 6 frameshifts, 4 nonsense, 1 deletion/insertion, 5 splice site, and 1 complete deletion) in 40/56 (71.4%) families with proven or suspected HLRCC and in 4/23 (17.4%) probands with PRCCII alone, including 2 sporadic cases. 21 of these were novel and all were demonstrated as deleterious by significant reduction of FH enzymatic activity. In addition, 5 asymptomatic parents in 3 families were confirmed as carrying disease-causing mutations. CONCLUSIONS: This study identified and characterised 21 novel FH mutations and demonstrated that PRCCII can be the only one manifestation of HLRCC. Due to the incomplete penetrance of HLRCC, the authors propose to extend the FH mutation analysis to every patient with PRCCII occurring before 40 years of age or when renal tumour harbours characteristic histologic features, in order to discover previously ignored HLRCC affected families.
Assuntos
Carcinoma de Células Renais/genética , Fumarato Hidratase/genética , Neoplasias Renais/genética , Mutação , Adulto , Idoso , Linhagem Celular Tumoral , Códon sem Sentido , Feminino , Mutação da Fase de Leitura , Deleção de Genes , Rearranjo Gênico , Genótipo , Mutação em Linhagem Germinativa , Humanos , Mutação INDEL , Leiomiomatose/congênito , Leiomiomatose/genética , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Síndromes Neoplásicas Hereditárias , Linhagem , Neoplasias Cutâneas , Neoplasias UterinasRESUMO
PURPOSE: The aim was to investigate whether germline polymorphisms within candidate genes known or suspected to be involved in fluorouracil (FU), oxaliplatin, and irinotecan pathways were associated with toxicity and clinical outcome in patients with metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: Blood samples from 349 patients included in the Fédération Francophone de Cancérologie Digestive 2000-05 randomized trial, which compared FU plus leucovorin (LV5FU2) followed by FU, leucovorin, and oxaliplatin (FOLFOX) followed by FU, leucovorin, and irinotecan (FOLFIRI; sequential arm) with FOLFOX followed by FOLFIRI (combination arm) in terms of progression-free survival (PFS) and overall survival, were collected. Twenty polymorphisms within the DPD, TS, MTHFR, ERCC1, ERCC2, GSTP1, GSTM1, GSTT1, and UGT1A1 genes were genotyped. RESULTS: The ERCC2-K751QC allele was independently associated with an increased risk of FOLFOX-induced grade 3 or 4 hematologic toxicity (P = .01). In the sequential arm, TS-5'UTR3RG and GSTT1 alleles were independently associated with response to LV5FU2 (P = .009) and FOLFOX (P = .01), respectively. The effect of oxaliplatin on tumor response increased with the number of MTHFR-1298C alleles (test for trend, P = .008). The PFS benefit from first-line FOLFOX was restricted to patients with 2R/2R (hazard ratio [HR] = 0.39; 95% CI, 0.23 to 0.68) or 2R/3R (HR = 0.59; 95% CI, 0.42 to 0.82) TS-5'UTR genotypes, respectively. Conversely, patients with the TS-5'UTR 3R/3R genotype did not seem to benefit from the adjunction of oxaliplatin (HR = 0.96; 95% CI, 0.66 to 1.40; trend between the three HRs, P = .006). CONCLUSION: A pharmacogenetic approach may be a useful strategy for personalizing and optimizing chemotherapy in mCRC patients and deserves confirmation in additional prospective studies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Camptotecina/análogos & derivados , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Genótipo , Humanos , Irinotecano , Leucovorina/administração & dosagem , Leucovorina/efeitos adversos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Compostos Organoplatínicos/administração & dosagem , Compostos Organoplatínicos/efeitos adversos , Oxaliplatina , Farmacogenética , Polimorfismo Genético , Estudos Prospectivos , Resultado do TratamentoRESUMO
Germline mutations of the CDKN2A gene are found in melanoma-prone families and individuals with multiple sporadic melanomas. The encoded protein, p16(INK4A), comprises four ankyrin-type repeats, and the mutations, most of which are missense and occur throughout the entire coding region, can disrupt the conformation of these structural motifs as well as the association of p16(INK4a) with its physiological targets, the cyclin-dependent kinases (CDKs) CDK4 and CDK6. Assessing pathogenicity of nonsynonymous mutations is critical to evaluate melanoma risk in carriers. In the current study, we investigate 20 CDKN2A germline mutations whose effects on p16(INK4A) structure and function have not been previously documented (Thr18_Ala19dup, Gly23Asp, Arg24Gln, Gly35Ala, Gly35Val, Ala57Val, Ala60Val, Ala60Arg, Leu65dup, Gly67Arg, Gly67_Asn71del, Glu69Gly, Asp74Tyr, Thr77Pro, Arg80Pro, Pro81Thr, Arg87Trp, Leu97Arg, Arg99Pro, and [Leu113Leu;Pro114Ser]). By considering genetic information, the predicted impact of each variant on the protein structure, its ability to interact with CDK4 and impede cell proliferation in experimental settings, we conclude that 18 of the 20 CDKN2A variants can be classed as loss of function mutations, whereas the results for two remain ambiguous. Discriminating between mutant and neutral variants of p16(INK4A) not only adds to our understanding of the functionally critical residues in the protein but provides information that can be used for melanoma risk prediction.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Mutação em Linhagem Germinativa , Melanoma/genética , Linhagem Celular , Proliferação de Células , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/química , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Saúde da Família , Testes Genéticos , Humanos , Melanoma/diagnóstico , Modelos Moleculares , Mutação de Sentido Incorreto , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
PURPOSE: The aim of this study was to explore the effect of several demographic, biological, and pharmacogenetic covariates on the disposition of imatinib and its main metabolite (CGP74588) in both adults and children. EXPERIMENTAL DESIGN: Thirty-three children with solid malignancies included in a phase II exploratory study and 34 adults with gastrointestinal stromal tumors received 340 mg/m(2) and 400 mg imatinib, respectively. Plasma imatinib and CGP74588 concentrations observed on day 1 and at steady-state were analyzed by a population pharmacokinetic method (NONMEM) to evaluate the effect of age, body weight, age, sex, albuminemia, plasma alpha1-acid glycoprotein (AGP), and eight polymorphisms corresponding to ABCB1, ABCG2, CYP3A4, CYP3A5, and AGP (pharmacogenetic data available for 46 of 67 patients). RESULTS: Analysis of the whole data set in 67 patients showed that apparent clearance (CL/F) of imatinib was positively correlated with body weight and albuminemia and negatively with AGP. By considering these three covariates, the interindividual variability on CL/F decreased from 47% to 19%. The apparent clearance of CGP74588 was similarly dependent on both body weight and AGP and significantly lower (30% reduction) at steady-state. By adding genotype status to the final covariate imatinib model, a 22% reduction in CL/F was observed in heterozygous compared with wild-type patients corresponding to ABCG2 c.421C>A (P<0.05). CONCLUSIONS: By considering morphologic and biological covariates, a unique covariate model could be used to accurately describe imatinib pharmacokinetics in patients ages 2 to 84 years. Morphologic and biological characteristics have a stronger influence than pharmacogenetics on imatinib pharmacokinetics.
Assuntos
Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/metabolismo , Piperazinas/farmacologia , Piperazinas/farmacocinética , Pirimidinas/farmacologia , Pirimidinas/farmacocinética , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Benzamidas , Proteínas Sanguíneas/metabolismo , Peso Corporal , Criança , Pré-Escolar , Glicoproteínas/sangue , Humanos , Mesilato de Imatinib , Taxa de Depuração Metabólica/genética , Pessoa de Meia-Idade , Farmacogenética , Piperazinas/metabolismo , Polimorfismo Genético , Grupos Populacionais , Pirimidinas/metabolismo , Albumina Sérica , Fatores Sexuais , Adulto JovemRESUMO
p73, a p53-related gene, encodes two classes of isoforms with opposing functions: (1) a full-length transactivation-competent p73 protein (TAp73) with tumour suppressor activity; and (2) a group of NH2-terminally truncated, transactivation-deficient p73 proteins, deltaEx2p73, deltaEx2-3p73, deltaNp73 and deltaN'p73 (collectively named deltaTAp73) with oncogenic activity. In this study, for the first time, we analyse the deregulations of TAp73 and deltaTAp73 in head and neck squamous cell cancer (HNSCC) and compare them to p53 status. We found that all the p73 isoforms in HNSCC tissue were upregulated with respect to those in normal adjacent tissue. Concomitant upregulations of p73 transcripts were often found in cancer tissue but not in normal tissue. p53 mutations and p73 transcript alterations are not mutually exclusive. All the HNSCC specimens studied had at least one p53 mutation and/or one deltaTAp73 transcript alteration. Although both the deltaNp73 and the TAp73 transcripts were found to be upregulated in head and neck cancers, the predominant protein in the cancers analysed was deltaNp73. TAp73, in contrast, was only weakly expressed. This finding is highly relevant and sheds light on the puzzling question of the biological significance of TAp73 upregulation in cancers. deltaNp73 protein levels were significantly overexpressed in HNSCC tissue compared to matched normal tissue (p = 0.003). Furthermore, a trend was found for better overall survival in patients with a low expression of deltaNp73. Our results show that the deregulation of both the p53 and the p73 pathways plays an important role in inducing head and neck cancers.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Células Escamosas/genética , Proteínas Nucleares/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Western Blotting , Primers do DNA/genética , Componentes do Gene , Genótipo , Humanos , Mutação/genética , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Tumoral p73RESUMO
PURPOSE: From epidemiological studies it appears that breast cancer (BC) and cutaneous melanoma (CMM) in the same individual occur at a higher frequency than expected by chance. Genetic factors common to both cancers can be suspected. Our goal was to estimate the involvement of "high risk" genes in patients presenting these two neoplasia, selected irrespectively from family history and age at diagnosis. EXPERIMENTAL DESIGN: Eighty two patients with BC and CMM were screened for BRCA1, BRCA2, TP53, CDKN2A and CDK4 (exon 2) germline mutations. RESULTS: Deleterious mutations were identified in 6 patients: two carriers of a BRCA1 germline mutation, two carriers of TP53 germline mutations (one of which also harbored a BRCA2 deleterious mutation, the other one a BRCA2 unclassified variant), and two carriers of a CDKN2A germline mutation. In addition, 6 variants of unknown signification were identified in BRCA1 or BRCA2 genes. Regarding family history, 3/13 (23%) patients with a positive family history of BC or CMM were carriers of a germline mutation, whereas only 3/69 (4%) patients without family history were carriers of a germline mutation. CONCLUSION: Our findings show that few patients with BC and CMM who lacked family histories of these cancers are carriers of deleterious germline mutations in four of the five genes we examined. We describe for the first time, two simultaneous BRCA2 and TP53 mutations, suggesting that analysis in more than one gene could be performed if a patient's personal or familial history does not match a single syndrome.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Mutação em Linhagem Germinativa/genética , Melanoma/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Quinase 4 Dependente de Ciclina/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Neoplasias Cutâneas/genéticaRESUMO
BACKGROUND: To identify new molecular markers of bone marrow dissemination in human neuroblastoma (NB), we studied the transcriptome profiles of malignant neuroblasts established from the human MYCN-amplified IGR-N-91 model. METHODS: This experimental model includes human neuroblastoma cells derived from a subcutaneous stage 4 disease, myocardium (Myoc) and bone marrow (BM) metastatic cells. RESULTS: Gene expression profiles obtained with Agilent oligo microarrays revealed a set of 107 differentially expressed genes in the metastatic neuroblasts. This set included up-regulated genes involved in chemoresistance, cell motility, neuronal structure/signaling, and the recently characterized GALNT13 gene encoding a glycosyltransferase that initiates mucin-type O-glycosylation. Because the glycosylation process is involved in the progression of primary tumor to metastatic disease, we investigated whether the most strongly up-regulated gene, GALNT13, might be a marker of bone marrow involvement in stage 4 NB patients. Importantly, in the BM of healthy adults no GALNT13 transcript was detected with analysis by quantitative (n = 3) and nested reverse transcription-PCR (n = 4) assays. In contrast, GALNT13 transcripts were detected in 23/23 cytologically involved BM samples obtained at diagnosis of stage 4 NB patients and in 5/27 cytologically noninvolved BM samples obtained from patients with stage 1-4 and 4S and treated stage 4 NB. The quantitative measurements of tyrosine hydroxylase (TH), ganglioside D2 synthase, dopa decarboxylase, and GALNT13 transcript values were compared in the same NB patients, and the results showed that GALNT13 expression was most highly correlated to poor clinical outcome at diagnosis. CONCLUSION: We propose ppGalNAc-T13 as a new informative marker for the molecular diagnosis of BM involvement and the follow-up of minimal residual disease in NB patients.
Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias da Medula Óssea/diagnóstico , Perfilação da Expressão Gênica , N-Acetilgalactosaminiltransferases/biossíntese , Neuroblastoma/diagnóstico , Adolescente , Adulto , Animais , Neoplasias da Medula Óssea/metabolismo , Neoplasias da Medula Óssea/secundário , Linhagem Celular Tumoral , Criança , Pré-Escolar , Neoplasias Cardíacas/diagnóstico , Neoplasias Cardíacas/metabolismo , Neoplasias Cardíacas/secundário , Humanos , Lactente , Recém-Nascido , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neuroblastoma/metabolismo , Neuroblastoma/secundário , RNA Mensageiro/biossíntese , Análise de Sobrevida , Transplante HeterólogoRESUMO
BACKGROUND: Advanced stage and relapsing neuroblastoma (NB) has a poor prognosis with frequent treatment failures, warranting new treatment options and enhanced local tumor control. Treatment with conditionally replicative adenoviruses (CRAds) has shown effectiveness in various preclinical cancer models, but has not yet been evaluated for local control of NB. Here, we tested the efficacy of the CRAd AdDelta24 and of two AdDelta24 derivatives against NB. Derivative AdDelta24-425S11 infects cells deficient in coxsackie/adenovirus receptor (CAR) via the epidermal growth factor receptor (EGFR). Derivative AdDelta24-p53 expresses the tumor suppressor protein p53 to promote oncolysis. METHODS: Expression of CAR and EGFR, and p53 pathway and DNA damage responses were analyzed in six NB cell lines and two xenografts derived from primary NB using immunohistochemistry, reporter gene transactivation, Western blot and fluorescence-activated cell sorting (FACS) analysis. Efficacy of AdDelta24, AdDelta24-425S11 and AdDelta24-p53 against NB was evaluated in vitro by cell viability analysis and in vivo by monitoring subcutaneous xenograft tumor growth in mice and by histological analysis of treated tumors. RESULTS: Neuroblastoma cell lines were sensitive to oncolysis by AdDelta24, with a higher susceptibility of those with functional p53 and intact DNA damage responses. Compared to AdDelta24, AdDelta24-p53 exhibited enhanced oncolytic potency on all NB cell lines independent of their p53 status and AdDelta24-425S11 was more effective against CAR-low IGR-NB8 cells. Moreover, five daily intratumoral injections of 10(8) plaque-forming units (pfu) of AdDelta24-p53 or AdDelta24-425S11 into subcutaneous IGR-NB8 and IGR-N91 xenografts at an advanced tumor stage yielded significant tumor growth delays (TGD). In contrast, at this dose, AdDelta24 did not cause significant TGD of neuroblastoma xenografts. Injection of AdDelta24-p53 was associated with extensive cell lysis, apoptotic cell death, and fibrous fascicles in the tumors. CONCLUSION: CRAds expressing p53 and targeted towards EGFR appear promising new agents for local control in the treatment of neuroblastoma.
Assuntos
Adenoviridae/fisiologia , Receptores ErbB/metabolismo , Terapia Genética , Neuroblastoma/terapia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/virologia , Efeito Citopatogênico Viral/fisiologia , Dano ao DNA , Feminino , Marcação de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/uso terapêutico , Humanos , Camundongos , Camundongos Nus , Neuroblastoma/virologia , Receptores Virais/metabolismo , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although screening for large deletions or duplications of the BRCA1 gene is becoming a routine component of the molecular diagnosis of familial breast cancer, little is known about the occurrence of such rearrangements in the BRCA2 gene. Because of the high frequency of BRCA2 mutations in breast cancer families with at least one case of male breast cancer, we selected a cohort of 39 such families, tested negative for mutations in the coding regions of BRCA1 and BRCA2, and developed an assay for BRCA2 rearrangements, based on quantitative multiplex PCR of short fluorescent fragments (QMPSF). We found three rearrangements: (1) a deletion of exons 12 and 13; (2) a duplication of exons 1 and 2; and (3) a complete deletion of BRCA2. We determined the boundaries of the deletion of exons 12 and 13, showing that it resulted from an unequal recombination between Alu sequences. We mapped the complete BRCA2 deletion, which extends over at least 298 kb and showed that it does not affect APRIN/AS3, previously characterized as a tumor suppressor gene, but it comprises several loci corresponding to proven or putative transcripts of unknown functional significance. These data suggest that screening for BRCA2 rearrangements should be done, especially in male breast cancer families tested negative for BRCA1 and BRCA2 mutations.
Assuntos
Neoplasias da Mama Masculina/genética , Rearranjo Gênico , Genes BRCA2 , Humanos , Masculino , MutaçãoRESUMO
Familial gastrointestinal stromal tumor (GIST) is a rare autosomal dominant genetic disorder associated with KIT germline mutations. In sporadic forms of the disease, somatic mutations target either KIT or PDGFRA genes. In a kindred in which 5 individuals had GIST, no germline mutation in KIT coding sequence has been detected. We hypothesized that the PDGFRA gene could be a predisposing gene in familial GIST. We sequenced PDGFRA exons 12 and 18 because several somatic mutations were identified within this region. We detected a germline PDGFRA missense mutation, 2675G > T, resulting in a tyrosine substitution for the highly conserved aspartic acid at codon 846. This mutation showed perfect cosegregation with the GIST phenotype among the 7 family members tested. Interestingly, PDGFRA Asp846 is homologous to codon 820, which is located in the KIT tyrosine kinase II domain. In a previous study, a KIT germline Asp820Tyr mutation was detected in a Japanese kindred in which 6 individuals had GIST. Transfection of a KIT820Tyr complementary DNA in nude mice was found to be tumorigenic confirming the oncogenic potential of this mutation. The present study shows that PDGFRA is a second familial GIST predisposing gene. These results indicate a further example of involvement of structurally related genes in familial cancer syndromes.
Assuntos
Neoplasias Gastrointestinais/genética , Mutação em Linhagem Germinativa/genética , Mutação de Sentido Incorreto/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Adulto , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , LinhagemRESUMO
p73, the first p53 gene homologue, encodes an array of p73 proteins including p73 alpha full-length (TAp73 alpha) and amino-truncated isoforms (Delta Np73 alpha), two proteins with opposite biological functions. TAp73 alpha can induce tumor suppressive properties, while Delta Np73 alpha antagonizes p53 as well as TAp73 in a dominant-negative manner. In human malignant neuroblasts, p53 protein is wild-type but known to be excluded from the nucleus, therefore disabling its function as a tumor suppressor. The present study investigates whether there is a functional link between p73 isoforms and p53 in neuroblastoma. Experiments were performed on two neuroblastoma cell lines differing in their p53 status, e.g. wild-type p53 SH-5Y5Y cells and mutated p53 IGR-N-91 cells. Data indicate that (i) both TA- and Delta N-p73 alpha enhance p53 protein level in SH-SY5Y cells, whereas level remains unchanged in IGR-N-91 cells; (ii) only in SH-SY5Y cells does forced TAp73 alpha overexpression markedly induce nuclear accumulation of p53 protein; (iii) p21 protein expression is increased in both cell lines infected with TAp73, suggesting that, in IGR-N-91 cells, p21 is induced by p73 through a p53-independent pathway; (iv) in the SHSY5Y cell line, Btg2 expression is strongly enhanced in cells overexpressing TA, and to a lesser extent in cells overexpressing Delta N. Taken together our results suggest that TAp73 may restore p53 function in NB with wild-type nonfunctional p53, but not in NB with mutated p53.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Ativação Transcricional/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Genes Supressores de Tumor , Humanos , Proteínas Imediatamente Precoces/metabolismo , Mutação , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Nucleares/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células Tumorais Cultivadas , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor , Quinases Ativadas por p21RESUMO
Medullary carcinoma is a poorly differentiated breast cancer with a high histologic grade and a paradoxically good prognosis. It accounts for only 3 percent of all breast cancers except in BRCA-1 families, in which it can account for as many as 13 percent of cancers. To date, only histologic criteria have been used to define this tumor type. In an attempt to more clearly define the genetic pathway leading to this subtype of cancer, we recently demonstrated that nearly 100 percent of these carcinomas display p53 mutations. In the present study, we extended our analysis to include HIN-1, a candidate tumor suppressor that has been shown to be silenced by methylation in the majority of breast tumors. In striking contrast to unselected sporadic invasive ductal carcinoma, we show that medullary carcinomas do not display a high frequency of HIN-1 methylation (p less than 0.001). This feature is also found in BRCA-1 associated tumors that shared several histologic characteristics with medullary carcinomas of the breast. Medullary carcinoma of the breast should therefore be considered to be a unique entity defined by specific histologic and molecular traits.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal/genética , Carcinoma Medular/genética , Citocinas/genética , Metilação de DNA , Genes BRCA1/fisiologia , Proteínas Supressoras de Tumor/genética , Neoplasias da Mama/patologia , Carcinoma Ductal/patologia , Carcinoma Medular/patologia , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Genes p53/fisiologia , Predisposição Genética para Doença , Humanos , Mutação/genética , Regiões Promotoras Genéticas , RNA Neoplásico/genética , RNA Neoplásico/metabolismoRESUMO
Despite intensive high-dose chemotherapy and autologous hematopoietic stem cell transplantation, disseminated neuroblastoma (NB) frequently proves to be chemosensitive but not chemocurable, and more often so in NB-presenting MYCN amplification. To assess the direct relationship between the MYCN oncogene and chemoresistance acquisition during NB metastatic dissemination, we have studied MYCN and MDR1 genes using the human IGR-N-91 ectopic xenograft metastatic model. This characterized experimental in vitro model includes human neuroblasts derived from a subcutaneous primary tumor xenograft, disseminated blood cells, myocardium, and bone marrow (BM) metastatic cells. All IGR-N-91-derived neuroblasts harbor a consistent MYCN genomic content but, unlike primary tumor xenograft, BM, and myocardium, human neuroblasts elicit a concomitant increase in MYCN and MDR1 transcripts levels, consistent with chemoresistance phenotype and active P-gp. In contrast, no variation of MRP1 transcript level was associated with the metastatic process in this model. Using an MDR1 promoter-CAT construct, we have shown that the MycN protein activates MDR1 transcription both in exogenous transient MYCN-transfected SK-N-SH cells and in endogenous BM metastatic neuroblasts with an increase in the MYCN transcript level. Band-shift experiments indicate that IGR-N-91 cells enriched with the MycN transcription factor do bind to two E-box motifs localized within the MDR1 promoter. Overall, our data indicate that MYCN overexpression increment contributes to the acquired drug resistance that occurs throughout the NB metastatic process.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Regulação Neoplásica da Expressão Gênica , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Criança , Resistência a Múltiplos Medicamentos/fisiologia , Humanos , Masculino , Camundongos , Camundongos Nus , Proteína Proto-Oncogênica N-Myc , Metástase Neoplásica , Transplante de Neoplasias , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Regiões Promotoras Genéticas , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
A large germline deletion removing exons 1 to 22 of the BRCA1 gene has been previously detected using quantitative PCR based methods (QMPSF and real time PCR gene dosage assay) in a woman affected with breast and ovarian cancer. Here, we report its characterisation by using colour bar code on combed DNA of the BRCA1 region. The 5' boundary is located in a Alu Y sequence in NBR1 intron 18 whereas the 3' boundary is located in a Alu Sc sequence in BRCA1 intron 22. This 161 kb deletion encompassing the NBR1, PsiBRCA1, NBR2 and BRCA1 genes is the largest BRCA1 deletion reported so far. No specific phenotype was associated with the hemizygosity of these four genes.
Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Deleção de Genes , Neoplasias Ovarianas/genética , Proteínas/genética , Sequência de Bases , Cromossomos Humanos Par 17/genética , Análise Mutacional de DNA/métodos , Saúde da Família , Feminino , França , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência MolecularRESUMO
Neuroblastoma is the most frequent solid extracranial neoplasm of childhood, with a median age of presentation of under 2 years. This tumour is highly malignant in patients older than 12 months of age with metastatic disease. Clinical studies have confirmed that amplification of the MYCN proto-oncogene is one of the best prognostic indicators of poor outcome. Approximately 30% of neuroblastoma tumours present MYCN amplification at diagnosis. Far less is known about the incidence and consequences of overrepresentation of the gene due to duplication or rearrangement of the chromosome arm in which the gene is situated. This study has analysed 110 neuroblastomas by FISH and has detected a gain of 1-3 copies per cell of MYCN in 8% of MYCN-non-amplified tumours. In these primary tumours, cells gained small numbers of additional MYCN genes by two mechanisms: formation of an isochromosome 2p, or an unbalanced translocation involving the short arm of chromosome 2 (with MYCN) and various partner chromosomes. Quantitative RT-PCR showed three- to seven-fold elevated MYCN expression in three tumours. Although the follow-up time to date is still short, clinical outcome suggests that low-level overexpression of the MYCN gene does not enhance tumour aggressiveness and rapidity of disease progression, as is often seen in neuroblastoma with MYCN amplification. It is hypothesized that the small elevation in MYCN expression could alter the regulation of apoptosis, as has been shown in experimental models.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Cromossomos Humanos Par 2/genética , Humanos , Hibridização in Situ Fluorescente , Lactente , Proteína Proto-Oncogênica N-Myc , Ploidias , Reação em Cadeia da Polimerase/métodos , Proto-Oncogene Mas , RNA Mensageiro/genética , RNA Neoplásico/genética , Regulação para CimaRESUMO
Treatment of malignant gliomas remains a major challenge in adults and children because of high treatment failure. The E1B 55 kDa-gene deleted adenovirus, ONYX-015 (ONYX Pharmaceuticals), was demonstrated to replicate selectively in and lyse tumor cells. Currently ongoing clinical trials of ONYX-015 in head and neck tumors are promising. Here, we demonstrate ONYX-015-mediated cell lysis and antitumor activity in three of four s.c. human malignant glioma xenografts deriving from primary tumors. Intratumoral injections of ONYX-015, 1 x 10(8) plaque-forming units daily for 5 consecutive days, yielded significant tumor growth delay in the p53 mutant xenografts IGRG88 and the p53 wild-type IGRG93 and IGRG121 treated at an advanced tumor stage. The p53 wild-type tumors IGRG93 and IGRG121 experienced 45% and 82% complete tumor regressions. Four and 8 of 11 animals, respectively, survived tumor free 4 months after treatment. Widespread intratumoral adenoviral replication was observed in tumor cells of these two xenografts compared with only scattered replication in the p53-mutant tumors. In addition to a fast tumor growth rate, wild-type p53 status was associated with increased antitumor activity of the E1B-attenuated virus, and induction of functional p53 may therefore determine adenoviral cytolysis in tumor cells. In conclusion, ONYX-015 displayed a major antitumor activity in human xenografts derived from primary malignant glioma supporting its development in the treatment of these highly malignant tumors.
Assuntos
Adenoviridae/fisiologia , Proteínas E1B de Adenovirus/genética , Glioblastoma/terapia , Glioblastoma/virologia , Proteína Supressora de Tumor p53/fisiologia , Adenoviridae/genética , Animais , Efeito Citopatogênico Viral , Feminino , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Receptores Virais/biossíntese , Ativação Transcricional , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastic tumors (NTs), occurring in early childhood, display a wide spectrum of differentiation. Recurrent deletions involving the p73 locus are frequently observed in undifferentiated NTs. To address the question of the possible implication of p73 in neuroblastic differentiation, we investigated the status of the expression of this gene in a panel of differentiated and undifferentiated tumors. Although mutations were not found, p73 transcript profiles differed between undifferentiated and differentiated tumors. The frequency of the transcripts lacking exon 2 (species 1-3) appeared to be higher in undifferentiated than in differentiating and differentiated NTs. In contrast, products from using an alternate promoter (DeltaN-p73) were present in all NTs. In addition, only DeltaN-p73, but not full-length proteins, were detected by immunoblotting, suggesting a greater stability of N-truncated isoforms. Importantly, as in the adrenal medulla, most NTs showed p73-positive immunohistological staining with a cellular distribution and intensity varying according to the neuronal differentiation. Surprisingly, we observed redistribution of p73 from the nucleus to the cytoplasm during neuroblastic differentiation. Our data suggest that, in undifferentiated NTs, a link may exist between the accumulation of DeltaN-p73alpha variants and the "nuclear exclusion" of p53.