RESUMO
Benzene is extensively used in industry despite its leukemogenic activity, representing a significant occupational hazard. We investigated if long-term treatment with low-doses hydroquinone (HQ), a benzene metabolite, might be sufficient to alter in vitro the epigenetic signature underlining LINE-1 sequences, a poorly explored step in health risks associated with benzene exposure. In HL-60 cell line, exploring the epigenetic events occurring in chromatin, we found the transient instauration of the distinctive signature combining the repressive H3Lys27 tri-methylation mark and the activating H3Lys4 tri-methylation mark (H3K27me3/H3K4me3), indicating a tendency toward a poised chromatin conformation. These alterations are lost in time after short-term treatments, while the long-term setting, performed using a concentration within the levels of total HQ in peripheral blood of benzene-exposed workers, showed a gradual increase in H3K4me3. We observed the absence of statistically significant variations in DNA methylation and expression levels of LINE-1, despite a decrease in protein levels of UHRF1, DNA methyl-transferases and histone methyl-transferases. In conclusion, in vitro treatment with low-dose HQ determined the instauration of a reversible poised state of chromatin in LINE-1 sequences, suggesting that prolonged exposure could cause persistent epigenetic alterations.
Assuntos
Hidroquinonas/toxicidade , Elementos Nucleotídeos Longos e Dispersos/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cromatina/metabolismo , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Células HL-60 , Histonas/metabolismo , Humanos , Metiltransferases/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Biobanking or collection and storage of specimens for future research purposes have become an essential tool in many fields of biomedical research and aims to provide a better understanding of disease mechanisms as well as the identification of disease-specific biomarkers that can navigate in complex diseases. In this study, we assessed the use of Flinders Technology Associates (FTA) cards as a long-term storage device for cervical specimens with suspected human papillomavirus (HPV) infections. HPV detection and genotyping results in liquid-based transport media were compared to HPV results from FTA cards. The overall agreement for the presence of any HPV infection between liquid-based medium and FTA cards stored for 1 year at ambient temperature was 100%. Reproducibility analysis of HPV detection and genotyping from FTA cards demonstrated that FTA cards are a reliable medium to store and preserve viral nucleic acids. Biobanking of cervical cells on FTA cards may provide a key resource for epidemiological and retrospective HPV studies.
Assuntos
Técnicas Citológicas/métodos , DNA Viral/isolamento & purificação , Papillomaviridae/isolamento & purificação , Preservação Biológica/métodos , Manejo de Espécimes/métodos , Virologia/métodos , Adulto , Idoso , Colo do Útero/virologia , DNA Viral/genética , Feminino , Técnicas de Genotipagem/métodos , Humanos , Pessoa de Meia-Idade , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Reprodutibilidade dos Testes , Estudos Retrospectivos , Adulto JovemRESUMO
The authors, all in charge of the administration of one of the 7 French Cancéropôles, reply to the article authored by Audrey Vézian, and -provide an alternative and more supportive view of the initiatives -sponsored by these regional cancer research networks.
Assuntos
Pesquisa Biomédica/organização & administração , Neoplasias , Política Pública , HumanosRESUMO
Previous studies have shown that DNA can be transferred from dying engineered cells to neighboring cells through the phagocytosis of apoptotic bodies, which leads to cellular transformation. Here, we provide evidence of an uptake of apoptotic-derived cervical cancer cells by human mesenchymal cells. Interestingly, HeLa (HPV 18+) or Ca Ski (HPV16+) cells, harboring integrated high-risk HPV DNA but not C-33 A cells (HPV-), were able to transform the recipient cells. Human primary fibroblasts engulfed the apoptotic bodies effectively within 30 minutes after co-cultivation. This mechanism is active and involves the actin cytoskeleton. In situ hybridization of transformed fibroblasts revealed the presence of HPV DNA in the nucleus of a subset of phagocytosing cells. These cells expressed the HPV16/18 E6 gene, which contributes to the disruption of the p53/p21 pathway, and the cells exhibited a tumorigenic phenotype, including an increased proliferation rate, polyploidy and anchorage independence growth. Such horizontal transfer of viral oncogenes to surrounding cells that lack receptors for HPV could facilitate the persistence of the virus, the main risk factor for cervical cancer development. This process might contribute to HPV-associated disease progression in vivo.
Assuntos
Apoptose , Transformação Celular Viral , Papillomaviridae/fisiologia , Neoplasias do Colo do Útero/patologia , Proliferação de Células , Transformação Celular Viral/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Fibroblastos/citologia , Genes Virais/genética , Células HeLa , Humanos , Mesoderma/citologia , Oncogenes/genética , Papillomaviridae/genética , Poliploidia , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismoRESUMO
Topoisomerase I is a privileged target for widely used anticancer agents such as irinotecan. Although these drugs are classically considered to be DNA-damaging agents, increasing evidence suggests that they might also influence the tumor environment. This study evaluates in vivo cellular and molecular modifications induced by irinotecan, a topoisomerase I-directed agent, in patient-derived colon tumors subcutaneously implanted in athymic nude mice. Irinotecan was given intraperitoneally at 40 mg/kg five times every 5 d, and expression profiles were evaluated at d 25 in tumors from treated and untreated animals. Unexpectedly, the in vivo antitumor activity of irinotecan was closely linked to a downregulation of hypoxia-inducible factor-1α (HIF1A) target genes along with an inhibition of HIF1A protein accumulation. The consequence was a decrease in tumor angiogenesis leading to tumor size stabilization. These results highlight the molecular basis for the antitumor activity of a widely used anticancer agent, and the method used opens the way for mechanistic studies of the in vivo activity of other anticancer therapies.
Assuntos
Antineoplásicos/uso terapêutico , Camptotecina/análogos & derivados , DNA Topoisomerases Tipo I/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Patológica/tratamento farmacológico , Inibidores da Topoisomerase I/uso terapêutico , Animais , Camptotecina/uso terapêutico , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Irinotecano , Masculino , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Colon carcinogenesis encompasses the stepwise accumulation of genomic aberrations correlated with the transition of aberrant crypt-adenoma-carcinoma. Recent data have revealed that, in addition to the microsatellite-instable phenotype, the chromosome instability pathway, representing four fifth of the colon carcinoma, could be involved in heterogeneous molecular alterations. Our project was aimed at determining the existence of distinct molecular subtypes in 159 non-microsatellite-instable colon polyps and their correlation with histology and dysplasia, using allelotyping, MGMT promoter gene methylation status, and K-RAS mutation analyses. Allelic imbalance, MGMT methylation, and K-RAS mutations arise in 62%, 39%, and 32% of polyps, respectively. Only 14% of polyps had no alterations. A 2-way hierarchical clustering analysis of the allelic imbalances identified subgroups of polyps according to their allelic imbalance frequency and distribution. Not only tubulovillous adenoma but also high-grade adenomas were correlated with high global allelic imbalance frequency (P = .005 and P = .003), with allelic imbalance at microsatellites targeting chromosomes 1, 6, and 9. In conclusion, the data presented in this study show that a large heterogeneity exists in the molecular patterns of alterations in precancerous colon lesions, favoring different modes of tumor initiation. Therefore, molecular alterations correlated with tubulovillous-type and high-grade dysplasia could represent targets identifying predictive factors of progression.
Assuntos
Adenoma/genética , Neoplasias do Colo/genética , Pólipos do Colo/genética , Instabilidade de Microssatélites , Adenoma/patologia , Idoso , Desequilíbrio Alélico , Neoplasias do Colo/patologia , Pólipos do Colo/patologia , Ilhas de CpG/genética , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , DNA de Neoplasias/análise , Progressão da Doença , Feminino , Humanos , Masculino , Proteínas Supressoras de Tumor/genéticaRESUMO
INTRODUCTION: The purpose of this study was to investigate the gene expression of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) in circulating mononuclear cells harvested from septic shock patients on drotrecogin-α activated (DAA) in order to determine whether this treatment has any effect on the inflammation phase. METHODS: We conducted a prospective cohort study in two intensive care departments. Blood samples were collected at inclusion (T1) and 36 hours later (T2) to measure plasma cytokines and the changes in intracellular TNF-α, IL-10 and IFN-γ mRNA expressions using the real-time quantitative polymerase chain reaction (RT-qPCR). Thirty-two septic shock patients were included: 16 with DAA at 24 µg/kg/h for 96 hours (DAA+) and 16 control (DAA-) eligible but contraindicated for DAA because of low platelet count. RESULTS: The basal characteristics were similar in both groups: mortality (50%), plasma cytokine concentrations, and baseline IFN-γ, TNF-α and IL-10 mRNA expressions (DAA+ vs. DAA-). At T2, there was a significant IFN-γ gene down-regulation in DAA+ but not in DAA- patients (-0.34 (-0.62; +1.54) vs. +1.41 (+0.35; +5.87), P = 0.008). In survivors, DAA administration was associated with a down-expression of both IFN-γ (-0.65 (-0.93; 0.48) vs. +0.7 (-0.04; +1.26), P = 0.01) and IL-10 (-0.78 (-0.92; -0.6) vs. -0.18 (-0.68; +0.46), P = 0.038). In the non-survivors, DAA infusion was associated with IL-10 over-expression when compared with survivors (+0.54 (-0.35; +11.52) vs. -0.78 (-0.92; -0.6), P < 0.001). CONCLUSIONS: In this study, lack of IL-10 gene down-expression despite a 36-hour infusion of DAA is an ominous sign in septic shock patients suggesting that DAA is not able to reverse the outcome. Our results suggest that DAA can decrease the expression of anti-inflammatory cytokines in septic shock patients. IL-10 or IFN-γ gene down-expression could represent markers of DAA response.
Assuntos
Regulação para Baixo/fisiologia , Interleucina-10/antagonistas & inibidores , Interleucina-10/genética , Leucócitos Mononucleares/metabolismo , Proteína C/administração & dosagem , Choque Séptico/sangue , Idoso , Estudos de Coortes , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Infusões Intravenosas , Interleucina-10/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Pessoa de Meia-Idade , Projetos Piloto , Estudos Prospectivos , Proteínas Recombinantes/administração & dosagem , Choque Séptico/tratamento farmacológico , Choque Séptico/patologiaAssuntos
Corantes Fluorescentes/química , Novobiocina/química , Proteômica/métodos , DNA Girase/química , DNA Girase/genética , DNA Girase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodaminas/química , Coloração e RotulagemRESUMO
PURPOSE: Despite recent progress, colon cancer is often resistant to combination chemotherapy, highlighting the need for development of novel therapeutic approaches. An attractive target is hypoxia-inducible factor-1alpha (HIF-1alpha), a key transcription factor with a pivotal role in tumor cell metabolism. One potential class of therapeutic agents targeting HIF-1alpha are mammalian target of rapamycin inhibitors such as rapamycin. A second class are topoisomerase I inhibitors, such as irinotecan, which are able to inhibit the accumulation of HIF-1alpha. We here investigated whether combination of rapamycin and irinotecan was active in human colon cancer models. EXPERIMENTAL DESIGN: Human metastatic tumors were xenografted in nude mice and treated with low doses of irinotecan alone, rapamycin alone, or combination of both drugs. The cellular effects of irinotecan and rapamycin were further characterized for HT-29 and HCT-116 colon cancer cells in vitro. RESULTS: In contrast to single-agent therapy, xenografted tumors treated with combination of irinotecan and rapamycin showed potent inhibition of the mammalian target of rapamycin/HIF-1alpha axis, which was accompanied by a dramatic reduction in tumor volume. In vitro experiments showed that exposure to low concentrations of the two drugs resulted in massive HT-29 cell death under hypoxic, but not normoxic, conditions, in full agreement with a cytotoxic effect mediated through HIF-1alpha rather than through induction of genotoxic lesions. HCT-116 cells were less sensitive to the combined treatment due to constitutive activation of phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways. CONCLUSION: These results identify HIF-1alpha as a promising target and provide a rationale for clinical trials of low-dose irinotecan and rapamycin combination toward metastatic colon cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Camptotecina/análogos & derivados , Neoplasias do Colo/tratamento farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Proteínas Quinases/fisiologia , Sirolimo/administração & dosagem , Animais , Camptotecina/administração & dosagem , Camptotecina/farmacologia , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Glicólise/efeitos dos fármacos , Humanos , Irinotecano , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Proteína Supressora de Tumor p53/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Crohn's disease is considered to be caused either by an excess of T-cell effector functions and/or by a defective regulatory T-cell compartment. The aim of this study was to assess in Crohn's disease the frequency of circulating CD4(+)CD25(high) T cells that possess regulatory T-cell functions and CD4(+)CD25(low) T cells that contain activated T cells. Flow cytometry of peripheral blood was used to assess CD4(+)CD25(high) and CD4(+)CD25(low) T-cell frequencies in a cohort of 66 patients with Crohn's disease in comparison to 19 patients with ulcerative colitis and 31 healthy individuals enrolled as controls. The CD4(+)CD25(high) T-cell frequency was significantly lowered in naïve Crohn's disease (P = 0.013) and in ulcerative colitis (P = 0.001). CD4(+)CD25(low) T-cell frequency was increased in Crohn's disease (P = 0.0001) and in ulcerative colitis (P = 0.0002). Both CD4(+)CD25(high) and CD4(+)CD25(low) T-cell frequencies are altered in naïve Crohn's disease resulting in an imbalance between both populations and a relative contraction of the CD4(+)CD25(high) T-cell population.
Assuntos
Antígenos CD4/sangue , Doença de Crohn/imunologia , Subunidade alfa de Receptor de Interleucina-2/sangue , Linfócitos T Reguladores , Adulto , Idoso , Contagem de Células Sanguíneas , Colite Ulcerativa/sangue , Colite Ulcerativa/imunologia , Doença de Crohn/sangue , Feminino , Citometria de Fluxo , Humanos , Subpopulações de Linfócitos/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
Human UHRF1 belongs to the unique mammalian family of proteins which contain a SET- and RING finger-associated (SRA) domain. This 180-residue domain has been reported to play key roles in the functions of the protein. It allows UHRF1 to bind methylated DNA, histone deacetylase 1 and DNA methyltransferase 1, suggesting a bridge between DNA methylation and the histone code. No structural data is available for any SRA domain. Native and SeMet-labelled SRA domains of human UHRF1 were overexpressed in Escherichia coli cells, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. A complete MAD data set was collected to 2.2 A resolution at 100 K. Crystals of the SeMet-labelled protein belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 53.78, c = 162.05 A.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cristalização , Cristalografia/métodos , DNA/metabolismo , Metilação de DNA , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Ligação Proteica , Ubiquitina-Proteína LigasesRESUMO
Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m- and h-UHRF1) is a histone-binding protein expressed only in proliferating cells. During pericentromeric heterochromatin (PH) replication, Np95 specifically relocalizes to chromocenters where it highly concentrates in the replication factories that correspond to less compacted DNA. Np95 recruits HDAC and DNMT1 to PH and depletion of Np95 impairs PH replication. Here we show that Np95 causes large-scale modifications of chromocenters independently from the H3:K9 and H4:K20 trimethylation pathways, from the expression levels of HP1, from DNA methylation and from the cell cycle. The PHD domain is essential to induce this effect. The PHD domain is also required in vitro to increase access of a restriction enzyme to DNA packaged into nucleosomal arrays. We propose that the PHD domain of Np95-ICBP90 contributes to the opening and/or stabilization of dense chromocenter structures to support the recruitment of modifying enzymes, like HDAC and DNMT1, required for the replication and formation of PH.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Centrômero/ultraestrutura , Heterocromatina/fisiologia , Acetilação , Animais , Ciclo Celular , Cromatina/química , Metilação de DNA , Heterocromatina/química , Histonas/química , Camundongos , Modelos Biológicos , Células NIH 3T3 , Nucleossomos/metabolismo , Estrutura Terciária de Proteína , Interferência de RNA , Ubiquitina-Proteína LigasesRESUMO
INTRODUCTION: Recent clinical success of epidermal growth factor (EGFR)-tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) have raised hopes that targeting other deregulated growth factor signaling, such as the hepatocyte growth factor/MET pathway, will lead to new therapeutic options for NSCLC. Furthermore, NSCLC present secondary EGFR-TKIs resistance related to exons 20 and 19 EGFR mutations or more recently to MET amplification. The aim of this study was to determine MET copy number related to EGFR copy number and K-Ras mutations in a targeted TKI naive NSCLC cohort. METHODS: We investigated 106 frozen tumors from surgically resected NSCLC patients. Genes copy number of MET and EGFR were assessed by quantitative relative real-time polymerase chain reaction and K-Ras mutations by sequencing. RESULTS: MET is amplified in 22 cases (21%) and deleted in nine cases (8.5%). EGFR is amplified in 31 cases (29%). K-Ras is mutated in 11 cases (10.5%). As observed for EGFR amplification, MET amplification is never associated with K-Ras mutation. MET amplification could be associated with EGFR amplification. MET amplification is not related to clinical and pathologic features. MET amplification and EGFR amplification showed a trend toward poor prognosis in adenocarcinomas. CONCLUSION: In EGFR-TKIs naive NSCLC patients, MET amplification is a frequent event, which could be associated with EGFR amplification, but not with K-Ras mutation. MET amplification may identify a subset of NSCLC for new targeted therapy. It will also be important to evaluate MET copy number to properly interpret future clinical trials.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Dosagem de Genes , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Fatores de Crescimento/genética , Adenocarcinoma/genética , Adenocarcinoma/secundário , Adenocarcinoma/cirurgia , Adenocarcinoma Bronquioloalveolar/genética , Adenocarcinoma Bronquioloalveolar/secundário , Adenocarcinoma Bronquioloalveolar/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/secundário , Carcinoma de Células Escamosas/cirurgia , Estudos de Coortes , Análise Mutacional de DNA , Receptores ErbB/genética , Feminino , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas c-met , Proteínas Proto-Oncogênicas p21(ras) , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Proteínas ras/genéticaRESUMO
Dysregulated cell growth or differentiation due to misexpression of developmental critical factors seems to be a decisive event in oncogenesis. As osteosarcomas are histologically defined by malignant osteoblasts producing an osteoid component, we prospected in pediatric osteosarcomas treated with OS94 protocol the genomic status of several genes implied in ossification processes. In 91 osteosarcoma cases, we focused on the analysis of the fibroblast growth factor receptors (FGFRs) TWIST, APC, and MET by allelotyping, real-time quantitative polymerase chain reaction, gene sequencing, and protein polymorphism study. Our study supports the frequent role of TWIST, APC, and MET as osteosarcoma markers (50%, 62%, and 50%, respectively). TWIST and MET were mainly found to be deleted, and no additional APC mutation was identified. Surprisingly, FGFRs are abnormal in only < 30%. Most of these factors and their abnormalities seem to be linked more or less to one clinical subgroup, but the most significant correlation is the link of MET, TWIST, and APC abnormalities to a worse outcome and their combination within abnormal tumors. A wider cohort is mandatory to define more robust molecular conclusions, but these results are to be considered as the beginning of a more accurate basis for diagnosis, in search of targeted therapies, and to further characterize prognostic markers.
Assuntos
Fatores de Coagulação Sanguínea/fisiologia , Neoplasias Ósseas/etiologia , Neoplasias Ósseas/metabolismo , Proteínas Nucleares/fisiologia , Osteogênese/fisiologia , Osteossarcoma/etiologia , Osteossarcoma/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Proteína 1 Relacionada a Twist/fisiologia , Criança , Humanos , Proteínas Proto-Oncogênicas c-metRESUMO
Virtually all cancer biological attributes are heterogeneous. Because of this, it is currently difficult to reconcile results of cancer transcriptome and proteome experiments. It is also established that cancer somatic mutations arise at rates higher than suspected, but yet are insufficient to explain all cancer cell heterogeneity. We have analyzed sequence variations of 17 abundantly expressed genes in a large set of human ESTs originating from either normal or cancer samples. We show that cancer ESTs have greater variations than normal ESTs for >70% of the tested genes. These variations cannot be explained by known and putative SNPs. Furthermore, cancer EST variations were not random, but were determined by the composition of the substituted base (b0) as well as that of the bases located upstream (up to b - 4) and downstream (up to b + 3) of the substitution event. The replacement base was also not randomly selected but corresponded in most cases (73%) to a repetition of b - 1 or of b + 1. Base substitutions follow a specific pattern of affected bases: A and T substitutions were preferentially observed in cancer ESTs. In contrast, cancer somatic mutations [Sjoblom T, et al. (2006) Science 314:268-274] and SNPs identified in the genes of the current study occurred preferentially with C and G. On the basis of these observations, we developed a working hypothesis that cancer EST heterogeneity results primarily from increased transcription infidelity.
Assuntos
Transformação Celular Neoplásica/genética , Etiquetas de Sequências Expressas , Variação Genética/genética , Neoplasias/genética , Sequência de Bases , Humanos , RNA Mensageiro/genética , Vimentina/genéticaRESUMO
In our previous study, a frequent rearrangement at 4q12 has been identified by allelotyping in our large and homogeneous population of pediatric osteosarcomas and it was significantly linked to c-kit protein overexpression. To confirm and understand the involvement of KIT in this tumor, the next step of the study was designed to detect the potential mutations of KIT gene by sequencing the frequently mutated exons 6, 8, 10, 11, 13, 17 and 21 and, in case of unmutated samples, to confirm the genomic amplifications of the wild-type receptor by real-time quantitative PCR (QPCR). A new microsatellite and QPCR targeting PDGFRA was also added to check the accuracy of the 4q11-12 locus. These techniques were performed in 74 pediatric high-grade osteosarcomas treated with the OS94 protocol. Surprisingly, no mutations were found, but, only DNA amplification of KIT gene in the entire population. PDGFRA gene QPCR revealed an unexpected result of predominant deletions in the rearranged tumors. All these results confirm the major role of the 4q11-12 locus and specifically the involvement of c-kit wild-type receptor overexpression in pediatric osteosarcomas and leads us to believe that inhibitors targeting this receptor could have a therapeutic effect in a selected group of patients.
Assuntos
Neoplasias Ósseas/genética , Osteossarcoma/genética , Proteínas Proto-Oncogênicas c-kit/genética , Adolescente , Adulto , Sequência de Bases , Neoplasias Ósseas/tratamento farmacológico , Criança , Pré-Escolar , Cromossomos Humanos Par 4 , Primers do DNA , Feminino , Humanos , Masculino , Osteossarcoma/tratamento farmacológico , Reação em Cadeia da Polimerase , Receptores de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: Drotrecogin alpha (activated) (DAA), or recombinant human activated protein C, is a new treatment in sepsis-induced multiple organ failure, leading to significant reduction in the mortality rate, thanks to its anticoagulant properties. It has been suggested that DAA has anti-inflammatory and antiapoptotic effects in sepsis animal models. This study investigates the potential actions of DAA on circulating mononuclear cells apoptosis in human septic shock. DESIGN: Prospective, cohort study. SETTING: Two intensive care wards and two research laboratories in a university hospital. PATIENTS: Twenty-two septic shock patients with DAA treatment (DAA+), 19 septic shock patients without DAA treatment (DAA-), and 14 healthy controls were successively enrolled, but only 20 DAA+ and 16 DAA- patients fulfilled criteria for statistical analysis. INTERVENTIONS: Blood samples were collected at inclusion and 24 hrs later. MEASUREMENTS AND MAIN RESULTS: Circulating mononuclear cell apoptosis levels were assessed by flow cytometry with annexin V, and variations of the apoptotic rheostats (Bax/Bcl-2 and Bax/Bcl-xl ratios) were analyzed by real-time reverse transcription-polymerase chain reaction. Apoptosis was significantly increased in septic shock patients (DAA+, 12 +/- 6.4%; DAA-, 10.4 +/- 5%) vs. healthy patients (3.4 +/- 2.1%, p < .001). Twenty-four hours after DAA infusion, apoptosis was significantly lower in the DAA+ group compared with DAA- ones (respectively, 11.7 +/- 5.3% and 16.2 +/- 7.6%, p < .001). At inclusion, DAA+ and DAA- groups showed comparable Bax/Bcl-2 ratio (DAA+, 0.92 +/- 0.9; DAA-, 1.32 +/- 0.87) and Bax/Bcl-xl ratio (DAA+, 2 +/- 1.04; DAA-, 1.31 +/- 0.93). In contrast, 24 hrs later we observed a significant decrease in these ratios, indicating an antiapoptotic effect in the DAA+ group (Bax/Bcl-2, 0.39 +/- 0.27; Bax/Bcl-xl, 0.68 +/- 0.35) compared with the DAA- group (Bax/Bcl-2, 1.81 +/- 1.1; Bax/Bcl-xl, 1.22 +/- 0.92, p = .001 and p = .039, respectively). CONCLUSIONS: In vivo, in human septic shock, DAA has antiapoptotic effects on circulating mononuclear cells, assessed by a significant decrease of both the Bax/Bcl-2 and Bax/Bcl-xl ratios.
Assuntos
Anti-Infecciosos/uso terapêutico , Leucócitos Mononucleares/química , Proteína C/uso terapêutico , Choque Séptico , Proteína X Associada a bcl-2/efeitos dos fármacos , Proteína bcl-X/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Anti-Infecciosos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Feminino , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Humanos , Infusões Intravenosas , Contagem de Leucócitos , Escore Lod , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Proteína C/farmacologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Choque Séptico/sangue , Choque Séptico/tratamento farmacológico , Proteína X Associada a bcl-2/análise , Proteína X Associada a bcl-2/genética , Proteína bcl-X/análise , Proteína bcl-X/genéticaRESUMO
In colorectal cancer, the molecular alterations that lead to metastasis are not clearly established, probably because of their high genetic complexity. To identify combinations of genetic changes involved in tumor progression and metastasis, we focused on chromosome instable (CIN) colon cancers. We compared by allelotyping of 33 microsatellites, the genomic alterations of 38 primary colon tumors with the synchronously resected matched liver metastases (CLM). We observed that (i) the number of patients with alterations at certain loci did not differ significantly between the whole primary tumor and the paired CLM, (ii) a group of patients had fewer alterations in the metastasis when compared with the matched primary tumor. A 2-way hierarchical unsupervised clustering of the allelotyping data revealed 2 tumor subtypes that have different levels of CIN (CIN-High, CIN-Low). Both subtypes have a minimal common set of alterations at chromosomes 8p, 17p and 18q, but does not include alteration at 5q or mutation at K-Ras. These 2 subtypes were also observed using a collection of 104 independent primary CIN colon tumors. In addition, we found a third subtype, consisting of tumors with a very low number of alterations not associated with specific loci (CIN-Very Low). We found that colon carcinogenesis may require a minimal set of alterations and that, in contrast to the current hypothesis, the level of CIN does not correlate with tumor progression. Therefore, our results suggest that metastasis potential could be present at very early stages of tumor development.
Assuntos
Aberrações Cromossômicas , Neoplasias do Colo/genética , Instabilidade Genômica/genética , Neoplasias Hepáticas/genética , Alelos , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 8/genética , Análise por Conglomerados , Neoplasias do Colo/classificação , Neoplasias do Colo/patologia , Feminino , Genótipo , Humanos , Neoplasias Hepáticas/secundário , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To detect the presence of tumour DNA in urine of patients with renal cancer based on microsatellite analysis. MATERIAL AND METHOD: Blinded, comparative, experimental study conducted between July 1996 and December 2003. Preoperative urine and blood samples were collected from 69 patients with renal cancer (pT1 to pT4). The control population comprised 35 patients with a benign urological disease. Loss of heterozygosity (LOH) and allele amplification were investigated by analysing allele imbalances between urinary DNA and blood lymphocyte DNA. Twenty-six loci were analysed, including 23 microsatellites. We studied the sensitivity and specificity of this analysis as a function of stage, grade and invasion of renal cavities by the tumour. RESULTS: The sensitivity of the test was 61% for pT1a and the global sensitivity was 62.3%. A significant difference was observed in favour of low nuclear grades (p < 0.05). More than 80% of tumours detected were identified by 4 microsatellites. The specificity was 83%. Renal cavities were invaded by the tumour in 38% of cases. No correlation was observed between invasion of renal cavities and the result of the test. CONCLUSION: The results are encouraging, particularly for small tumours and justify development of clinical applications.
Assuntos
DNA de Neoplasias/urina , Neoplasias Renais/diagnóstico , Neoplasias Renais/urina , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
The identification of genes as markers for chromosome aberrations in specific tumors might facilitate oncogenesis mechanism comprehension, cancer detection, prediction of clinical outcomes, and response to therapy. Previous physiologic and oncologic data identified the TWIST gene as a marker for mesodermal derivative and bone tissue differentiation, but its contribution to bone malignancies has not been investigated. In the present study, search for genomic alterations in high-grade pediatric osteosarcomas was focused on the 7p21 region, and more specifically on the TWIST gene. In a cohort of 74 patients, we observed by allelotyping that 31 of 68 informative tumors were rearranged at the TWIST locus. Among them, analysis by quantitative PCR (QPCR) revealed that, surprisingly, mostly deletions (22/68), but also amplifications (9/68), of the TWIST gene were detected. Furthermore, deletions at TWIST were statistically correlated to other molecular abnormalities, like alterations at the APC or c-kit loci, as well as to clinical features such as a poor outcome. This work shows that the TWIST gene seemed to be involved in high-grade pediatric osteosarcomas and is a new marker with a possible initial predictive value.