RESUMO
We previously described a selective bile duct ligation model to elucidate the process of hepatic fibrogenesis in children with biliary atresia or intrahepatic biliary stenosis. Using this model, we identified changes in the expression of alpha smooth muscle actin (α-SMA) both in the obstructed parenchyma and in the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin and TGF-β1, molecules known to be involved in hepatic fibrogenesis, were unchanged when analyzed by semiquantitative polymerase chain reaction (RT-PCR). Thus, the molecular mechanisms involved in the modulation of liver fibrosis in this experimental model are not fully understood. This study aimed to evaluate the molecular changes in an experimental model of selective bile duct ligation and to compare the gene expression changes observed in RT-PCR and in real-time quantitative PCR (qRT‐PCR). Twenty-eight Wistar rats of both sexes and weaning age (21-23 days old) were used. The rats were separated into groups that were assessed 7 or 60 days after selective biliary duct ligation. The expression of desmin, α-SMA and TGF-β1 was examined in tissue from hepatic parenchyma with biliary obstruction (BO) and in hepatic parenchyma without biliary obstruction (WBO), using RT-PCR and qRT‐PCR. The results obtained in this study using these two methods were significantly different. The BO parenchyma had a more severe fibrogenic reaction, with increased α-SMA and TGF-β1 expression after 7 days. The WBO parenchyma presented a later, fibrotic response, with increased desmin expression 7 days after surgery and increased α-SMA 60 days after surgery. The qRT‐PCR technique was more sensitive to expression changes than the semiquantitative method.
Assuntos
Animais , Feminino , Masculino , Actinas/metabolismo , Colestase/complicações , Desmina/metabolismo , Cirrose Hepática/etiologia , Fígado/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fator de Crescimento Transformador beta1/metabolismo , Análise de Variância , Actinas/genética , Atresia Biliar , Ductos Biliares/cirurgia , Colágeno Tipo I/biossíntese , Modelos Animais de Doenças , Desmina/genética , Expressão Gênica , Ligadura , Cirrose Hepática/metabolismo , Fígado/cirurgia , Ratos Wistar , Fator de Crescimento Transformador beta1/genéticaRESUMO
We previously described a selective bile duct ligation model to elucidate the process of hepatic fibrogenesis in children with biliary atresia or intrahepatic biliary stenosis. Using this model, we identified changes in the expression of alpha smooth muscle actin (α-SMA) both in the obstructed parenchyma and in the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin and TGF-ß1, molecules known to be involved in hepatic fibrogenesis, were unchanged when analyzed by semiquantitative polymerase chain reaction (RT-PCR). Thus, the molecular mechanisms involved in the modulation of liver fibrosis in this experimental model are not fully understood. This study aimed to evaluate the molecular changes in an experimental model of selective bile duct ligation and to compare the gene expression changes observed in RT-PCR and in real-time quantitative PCR (qRT-PCR). Twenty-eight Wistar rats of both sexes and weaning age (21-23 days old) were used. The rats were separated into groups that were assessed 7 or 60 days after selective biliary duct ligation. The expression of desmin, α-SMA and TGF-ß1 was examined in tissue from hepatic parenchyma with biliary obstruction (BO) and in hepatic parenchyma without biliary obstruction (WBO), using RT-PCR and qRT-PCR. The results obtained in this study using these two methods were significantly different. The BO parenchyma had a more severe fibrogenic reaction, with increased α-SMA and TGF-ß1 expression after 7 days. The WBO parenchyma presented a later, fibrotic response, with increased desmin expression 7 days after surgery and increased α-SMA 60 days after surgery. The qRT-PCR technique was more sensitive to expression changes than the semiquantitative method.
Assuntos
Actinas/metabolismo , Colestase/complicações , Desmina/metabolismo , Cirrose Hepática/etiologia , Fígado/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fator de Crescimento Transformador beta1/metabolismo , Actinas/genética , Análise de Variância , Animais , Ductos Biliares/cirurgia , Atresia Biliar , Colágeno Tipo I/biossíntese , Desmina/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Ligadura , Fígado/cirurgia , Cirrose Hepática/metabolismo , Masculino , Ratos Wistar , Fator de Crescimento Transformador beta1/genéticaRESUMO
Amplification of the MYCN gene in neuroblastomas is a potent biological marker of highly aggressive tumors, which are invariably fatal unless sound clinical management is applied. To determine the usefulness of semi-quantitative differential PCR (SQ-PCR) for accurate quantification of MYCN gene copy number, we evaluated the analytical performance of this method by comparing the results obtained with it for 101 tumor samples of neuroblastoma to that obtained by absolute and relative real-time PCR. Similar results were obtained for 100 (99%) samples, no significant difference was detected between the median log10 MYCN copy number (1.53 by SQ-PCR versus 1.55 by absolute real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). In the comparison of SQ-PCR and relative real-time PCR, SQ-PCR versus relative real-time PCR concordant results were found in 100 (99%) samples, no significant difference was found in median log10 MYCN copy number (1.53 by SQ-PCR versus 1.27 by relative real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). These findings indicate that the performance of SQ-PCR was comparable to that of real-time PCR for the amplification and quantification of MYCN copy number. Thus, SQ-PCR can be reliably used as an alternative assay in laboratories without facilities for real-time PCR.
Assuntos
Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Reação em Cadeia da Polimerase/métodos , Feminino , Amplificação de Genes , Humanos , Masculino , Proteína Proto-Oncogênica N-MycRESUMO
Amplification of the MYCN gene in neuroblastomas is a potent biological marker of highly aggressive tumors, which are invariably fatal unless sound clinical management is applied. To determine the usefulness of semi-quantitative differential PCR (SQ-PCR) for accurate quantification of MYCN gene copy number, we evaluated the analytical performance of this method by comparing the results obtained with it for 101 tumor samples of neuroblastoma to that obtained by absolute and relative real-time PCR. Similar results were obtained for 100 (99 percent) samples, no significant difference was detected between the median log10 MYCN copy number (1.53 by SQ-PCR versus 1.55 by absolute real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). In the comparison of SQ-PCR and relative real-time PCR, SQ-PCR versus relative real-time PCR concordant results were found in 100 (99 percent) samples, no significant difference was found in median log10 MYCN copy number (1.53 by SQ-PCR versus 1.27 by relative real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). These findings indicate that the performance of SQ-PCR was comparable to that of real-time PCR for the amplification and quantification of MYCN copy number. Thus, SQ-PCR can be reliably used as an alternative assay in laboratories without facilities for real-time PCR.
Assuntos
Feminino , Humanos , Masculino , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Reação em Cadeia da Polimerase/métodos , Amplificação de GenesRESUMO
BACKGROUND: The purpose of this study was to examine the relationships between growth in children with sickle cell anemia and the different beta-globin haplotypes, as well as components of the insulin-like growth factor (IGF)/insulin-like growth factor binding protein (IGFBP) axis. PATIENTS AND METHODS: Growth parameters and plasma concentrations of growth hormone (GH), IGF-I, and IGFBP-3 were studied in 41 children with sickle cell anemia whose haplotypes were defined. RESULTS: Plasma concentrations of IGF-I (total, free, and free/total fraction) and IGFBP-3 were significantly reduced in all patients with sickle cell anemia compared with the healthy children. Patients with the CAR/CAR haplotype had significantly lower mean growth velocity compared with those with Ben/Ben. When the GH/IGF axis elements were compared in relation with the different haplotypes, total IGF-I levels in CAR/CAR patients were significantly lower compared with levels in patients with Ben/Ben. A positive correlation was found between hematocrit and total IGF-I and between fetal hemoglobin percentages and the z-scores for total IGF-I and IGFBP-3. There was a positive correlation between age, weight, height, bone age, and the various elements of the GH/IGF-I axis when all groups were considered, although the correlation was lost when the auxologic data were expressed as standard deviation score for age. Growth velocity and the z-score for growth velocity were not correlated with any element of the axis. CONCLUSIONS: The positive relationship between hematocrit and fetal hemoglobin percentages with total IGF-I, free/total IGF-I, and IGFBP-3 in patients with sickle cell anemia could show that the delayed growth of these patients may be linked to intrinsic factors of the disease, which also determine the low circulating concentrations of the various elements of the GH/IGF-I axis. It is reasonable to assume that decrease of total IGF-I concentrations in patients with CAR/CAR haplotype is secondary to the severity of the disease.
Assuntos
Anemia Falciforme/fisiopatologia , Globinas/genética , Hormônio do Crescimento Humano/sangue , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Fator de Crescimento Insulin-Like I/metabolismo , Adolescente , Anemia Falciforme/sangue , Anemia Falciforme/genética , Criança , Pré-Escolar , Feminino , Deleção de Genes , Transtornos do Crescimento/sangue , Transtornos do Crescimento/etiologia , Transtornos do Crescimento/genética , Haplótipos , Humanos , Masculino , Família MultigênicaRESUMO
BACKGROUND: The recent introduction of sensitive RT-PCR-based techniques for the detection of epithelial antigen expression, such as CK-19, in the peripheral blood and bone marrow of breast cancer patients may provide an opportunity to evaluate tumor response at the molecular level, even in the absence of measurable disease while patients are still receiving chemotherapy. METHODS: We studied serially collected blood samples of 53 patients with breast cancer before, during, and after adjuvant, neoadjuvant, and palliative chemotherapy to evaluate its effects on the expression of CK-19 measured by RT-PCR. RESULTS: The percentage of CK-19 RT-PCR positivity decreased consistently from 43% (23/53) before chemotherapy to 14.3% (7/49), and to 18.9% (7/37) after 3 and 6 cycles, respectively (chi-square for linear trend = 7.948; p = 0.0048). Furthermore, there was a significant correlation between a negative CK-19 at three months and the response to chemotherapy (p = 0.024). CONCLUSION: We conclude that RT-PCR negativity for CK-19 expression at 3 months after the beginning of chemotherapy correlates with tumor response and, as treatment progresses, there is a significant trend for the occurrence of more negative RT-PCR results. Further studies are needed to confirm if this technique can be useful to assess response to chemotherapy in patients without measurable disease and if negativation of CK-19 expression while on chemotherapy is of prognostic significance.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/análise , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Regulação Neoplásica da Expressão Gênica , Queratinas/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto , Idoso , Progressão da Doença , Feminino , Humanos , Pessoa de Meia-Idade , Cuidados Paliativos , Valor Preditivo dos Testes , Resultado do TratamentoRESUMO
Aiming to verify if insulin-like growth factor type I and its receptor (IGF-IR) are implicated on pathophysiology of chronic myelogenous leukemia (CML), we studied 35 patients with CML in chronic phase at diagnosis or during interferon-alpha (IFN-A) or hydroxyurea treatments. Cytometry flow analysis and reverse transcription PCR (RT-PCR) molecular assay for IGF-IR expression on peripheral blood cells from CML patients diagnosed didn't show statistical differences from the control group. Hydroxyurea treated patients had lower expression of IGF-IR in granulocytes, lymphocytes and monocytes (P<0.01). We found statistical higher percentage of T and B lymphocytes positive for IGF-IR on IFN-A treated patients (P<0.001). Also an increase of IGF-IR mRNA expression could be detected in this group when compared with patients in hydroxyurea therapy (P<0.05). Our study suggest that IGF-IR is not directly implicated on CML installation and that the increased expression of IGF-IR on lymphoid cells of IFN-A treated patients could contribute to the immune recognition of malignant cell clone by enhancing immunocompetent cell proliferation and action.
Assuntos
Antineoplásicos/uso terapêutico , Interferon-alfa/uso terapêutico , Leucemia Mieloide de Fase Crônica/sangue , Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Receptor IGF Tipo 1/biossíntese , Adolescente , Adulto , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Criança , Feminino , Citometria de Fluxo , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Humanos , Hidroxiureia/uso terapêutico , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/fisiologia , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
A balanced de novo translocation t(X;1) is described in a girl with severe haemophilia B. The translocated X was shown cytologically to be preferentially active, and methylation analysis of the DXS255 locus confirmed the skewed X-inactivation with the paternal allele being the active one. Cytogenetic and molecular analysis showed that this chromosomal rearrangement led to the deletion of at least part of the factor IX gene. Therefore, the girl was heterozygous for factor IX deficiency and expression of her clinical phenotype was the result of the inactivation of the normal maternal X chromosome. The localization of one of the X chromosome translocation breakpoints in YAC clone 957F9, that was demonstrated to map distally to the factor IX gene, revealed the complexity of this chromosomal rearrangement.
Assuntos
Cromossomos Humanos Par 1 , Fator IX/genética , Deleção de Genes , Hemofilia B/genética , Translocação Genética , Cromossomo X , Southern Blotting , Criança , Bandeamento Cromossômico , Mecanismo Genético de Compensação de Dose , Feminino , Heterozigoto , Humanos , Hibridização in Situ FluorescenteRESUMO
RAS mutations can be detected in a variable number of patients with myeloproliferative disorders such as myelodysplastic syndromes and acute myeloid leukemia, but are rare events in chronic myelogenous leukemia in chronic phase. However, there is good evidence supporting the involvement of RAS signalling pathway in CML and this could be due to alterations in RAS activity regulatory proteins. The neurofibromatosis (NF1) gene down-regulates the RAS signal transduction pathway through the inhibitory function of its GAP-related domain (GRD) on RAS protein. The loss or alteration of neurofibromin (the NF1 protein) may produce a disfunction similar to point mutations in the RAS gene resulting in the permanent stimulation of the RAS signal transduction pathway. Mutations involving the GRD region of the NF1 gene (GRD-NF1) have been described in a variety of tumors such as colon carcinoma and astrocytoma. Germline mutations and deletions in the NF1 gene, as seen in neurofibromatosis type 1, are also associated with certain myeloid disorders. In the present work, we sought to identify mutations in the codons 12/13 and 61 of RAS gene and in the Lys-1423 codon of GRD-NF1, which are well known hot spots in these genes, in a group of 36 adults and ten children with chronic myelogenous leukemia in chronic phase and blast crisis. Using the PCR-SSCP and the allele-specific restriction assay (ASRA) techniques, we were not able to observe any RAS or NF1 detectable mutation. These findings suggest that RAS and GRD-NF1 mutations are not involved either in chronic phase or in the progression to blast crisis in chronic myelogenous leukemia in adults and children.
Assuntos
Genes da Neurofibromatose 1 , Genes ras , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas/genética , Adulto , Criança , Pré-Escolar , Análise Mutacional de DNA , Proteínas Ativadoras de GTPase , Humanos , Mutação , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Proteínas Ativadoras de ras GTPaseRESUMO
The MYCN oncogene is amplified in 20% of childhood neuroblastoma and is associated independently with poor prognosis. Alteration of the p53 tumor supressor gene, in contrast, occurs infrequently in these tumors. In this report, we described a 3-year-old girl with stage IV neuroblastoma. Molecular analysis revealed, both MYCN gene amplification and a point mutation of the p53 tumor supressor gene. To our knowledge, this is the first reported case of neuroblastoma with genetic alterations of both these genes.
Assuntos
Amplificação de Genes , Genes myc/genética , Genes p53/genética , Neuroblastoma/genética , Mutação Puntual/genética , Southern Blotting , Pré-Escolar , DNA de Neoplasias/análise , Éxons/genética , Evolução Fatal , Feminino , Humanos , Estadiamento de Neoplasias , Neuroblastoma/patologia , Prognóstico , Análise de Sequência de DNARESUMO
BACKGROUND: Early detection of haematogenous dissemination of epithelial tumours afforded by the analysis of epithelial antigen expression in the peripheral blood mononuclear fraction (PBMN) and bone marrow may confer a worse prognosis to patients with carcinoma. Cytokeratin 19 is a protein normally expressed by epithelial cells including normal and malignant mammary cells. Previous studies have demonstrated that analysis of cytokeratin 19 expression by the reverse transcriptase-polymerase chain reaction (RT-PCR) can detect one epithelial cell in as many as 10(5)-10(7) haematopoetic cells. Despite its sensitivity concern has been voiced recently about the specificity of this technique owing to the detection of cytokeratin 19 expression in the PBMN of normal volunteers and the bone marrow of patients with haematological malignancies. AIMS: To assess the sensitivity and specificity of RT-PCR detection of cytokeratin 19 in PBMN of normal female blood donors. METHODS: Blood was taken from 52 normal female blood donors and PBMN separated through Fycol gradient centrifugation. Cytokeratin 19 was measured using a two step nested RT-PCR assay. RESULTS: No amplification was found in the first step for any of the samples studied, whereas in the second step amplification was observed in 10 of the 52 samples. Both steps could detect one MCF-7 cell (the cytokeratin 19 positive control) in 10(6) CEM (cytokeratin 19 negative control) cells. CONCLUSIONS: As both PCR steps are sensitive to the 10(-6) level, performing only the first amplification step may decrease the non-specificity of this method. Further studies are needed to define the specificity and sensitivity of this technique in blood and bone marrow specimens of women with breast cancer.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Queratinas/sangue , Reação em Cadeia da Polimerase/métodos , Biomarcadores Tumorais/genética , Doadores de Sangue , Feminino , Expressão Gênica , Humanos , Queratinas/genética , RNA Mensageiro/genética , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
We searched for mutations of the p53 gene in 25 phaeochromocytomas using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of the entire conserved region of the gene, encompassing exons 4-8; expression of the p53 protein was assessed by immunohistochemistry. No mutations were found, while a polymorphism in codon 72 was observed. Immunohistochemistry revealed nuclear p53 overexpression in one tumour sample. We conclude that mutations of the 'hotspot' region of the p53 gene do not seem to play a role in the pathogenesis of phaeochromocytoma.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Genes p53 , Feocromocitoma/genética , Adolescente , Adulto , Sequência de Bases , Criança , Análise Mutacional de DNA , DNA de Neoplasias/genética , Éxons/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Síndromes Neoplásicas Hereditárias/genética , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita SimplesRESUMO
The actual significance of the type of BCR-ABL rearrangement in chronic myeloid leukemia (CML) prognosis remains controversial. Also, the molecular events that lead to CML progression are largely unknown. We analyzed the M-BCR breakpoint position in 64 CML patients by Southern blot and correlated the molecular findings with the cytogenetic, hematologic, and clinical data. No statistically significant differences were found with respect to the clinical and hematologic data presented at diagnosis or in the median duration of chronic phase (CP) and survival between the groups of patients with 5' and 3' breakpoints. We also studied by PCR-SSCP and direct sequencing the p53 gene in patients with specimens available in both chronic phase and blast crisis. We identified p53 mutations in 17% of the blast crisis samples analyzed, whereas no abnormalities were found in CP. This finding suggests that only in a minor fraction of cases are lesions in the p53 gene involved in transformation. Given the present findings, along with previous reports, we believe that a novel mechanism to explain the heterogeneity of CML should be postulated and actively pursued, as should the identification of secondary molecular events more consistently involved in progression.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas Oncogênicas/genética , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas , Transformação Genética , Proteína Supressora de Tumor p53/genética , Adolescente , Adulto , Idoso , Sequência de Bases , Criança , Sondas de DNA , Feminino , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Prognóstico , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-bcr , Análise de SobrevidaRESUMO
In this preliminary report the results of PCR for detection of DNA sequences (65 KDa antigen) of Mycobacterium tuberculosis in CSF samples from 20 patients are registered. In 10 patients there were clinical and laboratory findings suggesting the diagnosis of tuberculous meningitis (test group). In the other 10 patients, clinical and laboratory findings suggested meningitis or meningo-encephalitis from other etiologies (control group). In 7 patients from the test group antigenic DNA sequences of Mycobacterium tuberculosis were found in CSF by PCR; positive results were not registered in the control group.
Assuntos
Reação em Cadeia da Polimerase , Tuberculose Meníngea/diagnóstico , HumanosRESUMO
Nesse relato preliminar säo registrados os resultados da pesquisa de PCR para detecçäo de sequências de DNA (antígeno 65 KDa) do Mycobacteruium tuberculosis no LCR. Foram estudadas amostras de LCR de 20 pacientes: em 10 havia suspeita clínica e laboratorial de neurotuberculose (grupo de teste); nos outros 10 havia suspeita diagnóstica de meningite ou menigoencefalite de outras etiologias ( grupo controle). Em 7 dos 10 pacientes do primeiro grupo a pesquisa de sequências antigênicas de DNA do Mycobacterium tuberculosis por PCR foi positiva; em nenhum dos pacientes do grupo controle a pesquisa foi positiva