RESUMEN
OBJECTIVE: Fine needle cytology (FNC) is the first-line diagnostic method to determine the benign or malignant nature of thyroid nodules. The gray zone of cytological classifications remains, however, a crucial and challenging area for cytopathologists. DESIGN, PATIENTS AND MEASUREMENTS: In the present study, 141 thyroid cytological samples, with ultrasonographic suspicious features, have been prospectively analysed. Molecular analyses were performed by an innovative technology using two multiplex PCRs for the amplification of BRAF, N-H-K-RAS and RET exon genes. RNA samples were studied for RET/PTC1 and RET/PTC3 rearrangements by PCR amplification, and the conditions were set-up to study, with a single experiment, both wild-type PAX8 and PAX8/PPARÉ£ rearrangements. In total, 111 samples were examined for BRAF, N-H-KRAS and RET genes. An ultrasonographic, cytological and molecular correlation was also carried out in an attempt to suggest a possible way to manage the patients with thyroid nodules. Cyto-histological correlation was available in 115 cases, and it was used to verify the global diagnostic accuracy of this combined approach. RESULTS: According to the histopathological diagnosis, FNC accuracy was 100% for TIR5 and metastases; 89% for TIR4; 84% for TIR3A and 58% for TIR3B. About 11% of the studied samples showed either RET-PTC1 or RET/PTC3 chromosomal rearrangements, and only one sample simultaneously presented RET/PTC1 and RET/PTC3 rearrangements. PAX8/PPARÉ£ rearrangement was found in 6% of the samples. CONCLUSIONS: A multidisciplinary approach to the thyroid is therefore necessary to develop innovative methods suitable for an improved diagnostic and prognostic definition of thyroid cancer.
Asunto(s)
Neoplasias de la Tiroides/diagnóstico por imagen , Neoplasias de la Tiroides/diagnóstico , Nódulo Tiroideo/diagnóstico por imagen , Nódulo Tiroideo/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia con Aguja Fina , Análisis Mutacional de ADN , Exones/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factor de Transcripción PAX8/genética , Estudios Prospectivos , Proteínas Proto-Oncogénicas B-raf/genética , Cáncer Papilar Tiroideo/diagnóstico , Cáncer Papilar Tiroideo/diagnóstico por imagen , Cáncer Papilar Tiroideo/genética , Neoplasias de la Tiroides/genética , Nódulo Tiroideo/genética , Adulto JovenRESUMEN
Thyroid cancer is the most common malignancy of the endocrine system and includes well-differentiated forms, namely papillary and follicular carcinomas, and the poorly differentiated and undifferentiated forms that result from the transformation of thyroid follicular cells (anaplastic carcinomas). Notably, 5-10% of all thyroid cancers are medullary thyroid cancers that arise from parafollicular cells also known as C cells. The most common genetic mutations in papillary and follicular thyroid cancers are point mutations of the BRAF or RAS genes, while the most common chromosomal alterations are RET/PTC and PAX8/PPARγ rearrangements. The most frequent initial manifestation of thyroid cancer is the appearance of a nodule most of which are benign; indeed, less than 5% are malignant. However, some cases are misdiagnosed, and many patients undergo unnecessary surgery. Therefore, an accurate pre-surgery evaluation is crucial. The most reliable diagnostic test for thyroid nodules is fine needle aspiration (FNA) cytology, which accurately distinguishes between a benign and malignant lesion in most cases. However, cytological discrimination between malignant and benign follicular cancer is often difficult because of poor quality samples. Here we describe rapid methods to create a positive control and identify the PAX8/PPARγ rearrangement in FNA thyroid samples by molecular biology.
RESUMEN
POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1) is an emerging cancer-related gene that is downregulated in different human malignancies, including thyroid cancer, where its levels gradually decrease going from papillary thyroid carcinomas (PTC) to poorly differentiated and undifferentiated highly aggressive anaplastic carcinomas (ATC). The restoration of PATZ1 expression in thyroid cancer cells reverted their malignant phenotype by inducing mesenchymal-to-epithelial transition, thus validating a tumor suppressor role for PATZ1 and suggesting its involvement in thyroid cancer progression. Here, we investigated the consequences of the homozygous and heterozygous loss of PATZ1 in the context of a mouse modeling of PTC, represented by mice carrying the RET/PTC1 oncogene under the thyroid specific control of the thyroglobulin promoter RET/PTC1 (RET/PTC1TG). The phenotypic analysis of RET/PTC1TG mice intercrossed with Patz1-knockout mice revealed that deficiency of both Patz1 alleles enhanced thyroid cancer incidence in RET/PTC1TG mice, but not the heterozygous knockout of the Patz1 gene. However, both RET/PTC1TG;Patz1+/- and RET/PTC1TG;Patz1-/- mice developed a more aggressive thyroid cancer phenotype-characterized by higher Ki-67 expression, presence of ATCs, and increased incidence of solid variants of PTC-than that shown by RET/PTC1TG; Patz1+/+ compound mice. These results confirm that PATZ1 downregulation has a critical role in thyroid carcinogenesis, showing that it cooperates with RET/PTC1 in thyroid cancer progression.
RESUMEN
BAG3 protein is an apoptosis inhibitor and is highly expressed in Anaplastic Thyroid Cancer. We investigated the entire set of proteins modulated by BAG3 silencing in the human anaplastic thyroid 8505C cancer cells by using the Stable-Isotope Labeling by Amino acids in Cell culture strategy combined with mass spectrometry analysis. By this approach we identified 37 up-regulated and 54 down-regulated proteins in BAG3-silenced cells. Many of these proteins are reportedly involved in tumor progression, invasiveness and resistance to therapies. We focused our attention on an oncogenic protein, CAV1, and a tumor suppressor protein, SERPINB2, that had not previously been reported to be modulated by BAG3. Their expression levels in BAG3-silenced cells were confirmed by qRT-PCR and western blot analyses, disclosing two novel targets of BAG3 pro-tumor activity. We also examined the dataset of proteins obtained by the quantitative proteomics analysis using two tools, Downstream Effect Analysis and Upstream Regulator Analysis of the Ingenuity Pathways Analysis software. Our analyses confirm the association of the proteome profile observed in BAG3-silenced cells with an increase in cell survival and a decrease in cell proliferation and invasion, and highlight the possible involvement of four tumor suppressor miRNAs and TP53/63 proteins in BAG3 activity.
RESUMEN
The most frequent initial manifestation of thyroid cancer is the appearance of a nodule. More than 20% of the general population has a palpable thyroid nodule and the percentage rises to 70% based on ultrasound identification. In 95% of cases the nodule is simply a hyperplastic or benign lesion. The most reliable diagnostic test for thyroid nodules is fine needle aspiration (FNA), but cytological discrimination between malignant and benign follicular neoplasms remains difficult. Cytological analysis is now, almost routinely, being combined with molecular genetics to enable the pathologist to make a more objective diagnosis. In this study, we performed the molecular analysis using a new simplified procedure that involves a panel of BRAF, RAS, RET and RET/PTC gene mutations in easily obtainable FNA samples, in the attempt to improve the efficacy of the FNA diagnosis of thyroid nodules and thus patient management. In this new procedure, PCR and sequencing analysis are used to detect point mutations, and, in parallel, RT-PCR is used to detect the chimeric RET/PTC1 and RET/PTC3 transcripts in RNA extracted from FNA.
Asunto(s)
Reacción en Cadena de la Polimerasa Multiplex , Mutación , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/genética , Biomarcadores de Tumor , Biopsia con Aguja Fina , Análisis Mutacional de ADN/métodos , Exones , Reordenamiento Génico , Genes Esenciales , Humanos , Técnicas de Diagnóstico Molecular , Reacción en Cadena de la Polimerasa Multiplex/métodos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas ras/genéticaRESUMEN
A neoadjuvant clinical trial was previously conducted in patients with resectable colorectal cancer liver metastases (CRLM). At a median follow up of 28 months, 20/33 patients were dead of disease, 8 were alive with disease and 5 were alive with no evidence of disease. To shed further insight into biological features accounting for different outcomes, the expression of CXCR4-CXCL12-CXCR7, TLR2-TLR4, and the programmed death receptor-1 (PD-1)/programmed death-1 ligand (PD-L1) was evaluated in excised liver metastases. Expression profiles were assessed through qPCR in metastatic and unaffected liver tissue of 33 CRLM neoadjuvant-treated patients. CXCR4 and CXCR7, TLR2/TLR4, and PD-1/PD-L1 mRNA were significantly overexpressed in metastatic compared to unaffected liver tissues. CXCR4 protein was negative/low in 10/31, and high in 21/31, CXCR7 was negative/low in 16/31 and high in 15/31, CXCL12 was negative/low in 14/31 and high in 17/31 CRLM. PD-1 was negative in 19/30 and positive in 11/30, PD-L1 was negative/low in 24/30 and high in 6/30 CRLM. Stromal PD-L1 expression, affected the progression-free survival (PFS) in the CRLM population. Patients overexpressing CXCR4 experienced a worse PFS and cancer specific survival (CSS) (p = 0.001 and p = 0.0008); in these patients, KRAS mutation identified a subgroup with a significantly worse CSS (p < 0.01). Thus, CXCR4 and PD-L1 expression discriminate patients with the worse PFS within the CRLM evaluated patients. Within the CXCR4 high expressing patients carrying Mut-KRAS in CRLM identifies the worst prognostic group. Thus, CXCR4 targeting plus anti-PD-1 therapy should be explored to improve the prognosis of Mut-KRAS-high CXCR4-CRLMs.
RESUMEN
Ovarian cancer is the most lethal gynecological malignancy and the high mortality rate is associated with advanced-stage disease at the time of the diagnosis. In order to find new tools to make diagnosis of Epithelial Ovarian Cancer (EOC) at early stages we have analyzed the presence of specific HMGA2 mRNA in the plasma of patients affected by this neoplasm. HMGA2 overexpression represents a feature of several malignances including ovarian carcinomas. Notably, we detected HMGA2 specific mRNA in the plasma of 40 out 47 patients with EOC, but not in the plasma of healthy donors. All cases found positive for HMGA2 mRNA in the plasma showed HMGA2 protein expression in EOC tissues. Therefore, on the basis of these results, the analysis of circulating HMGA2 specific mRNA might be considered a very promising tool for the early diagnosis of EOC.
Asunto(s)
Proteína HMGA2/genética , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , ARN Mensajero/sangre , ARN Mensajero/genética , Adulto , Anciano , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Femenino , Proteína HMGA2/metabolismo , Humanos , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/sangre , Neoplasias Ováricas/sangreRESUMEN
Fine-needle cytology (FNC) is frequently used to diagnose thyroid nodules discovered by palpation or imaging studies. Molecular tests on FNC material may increase its diagnostic accuracy. We report a case of a classic papillary thyroid carcinoma combined with a mucoepidermoid carcinoma correctly identified on FNC. The papillary component had a classic immunophenotype (CK19+, TTF1+), while the mucoepidermoid one was only focally CK19+. Point mutations (BRAF and RAS) and rearrangements (RET/PTC) of the papillary component have been also investigated on FNC samples, with resulting concurrent rearrangements of RET/PTC1 and RET/PTC3, but no point mutations. The histogenesis of combined papillary and mucoepidermoid carcinoma of the thyroid still remains partly unsettled, and further genomic studies are needed to shed some more light on this peculiar neoplasm.
Asunto(s)
Carcinoma Mucoepidermoide/diagnóstico , Carcinoma/diagnóstico , Tumor Mixto Maligno/diagnóstico , Neoplasias de la Tiroides/diagnóstico , Adulto , Biopsia con Aguja Fina , Carcinoma/metabolismo , Carcinoma/patología , Carcinoma Mucoepidermoide/metabolismo , Carcinoma Mucoepidermoide/patología , Carcinoma Papilar , Diagnóstico Diferencial , Femenino , Humanos , Inmunohistoquímica , Tumor Mixto Maligno/metabolismo , Tumor Mixto Maligno/patología , Técnicas de Diagnóstico Molecular , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patologíaRESUMEN
Hereditary cancers account for approximately 10 % of breast and ovarian cancers. Mutations of the BRCA1 and BRCA2 genes, encoding two proteins involved in DNA repair, underlie most cases of such hereditary cancers. Women with BRCA mutations develop breast cancer in 50-80 % of cases and ovarian cancer in 10-40 % of cases. Assessing BRCA mutational status is needed to direct the clinical management of women with predisposition to these hereditary cancers. However, BRCA screening constitutes a bottleneck in terms of costs and time to deliver results. We developed a PCR-based assay using 73 primer pairs covering the entire coding regions of BRCA1 and BRCA2. PCR primers, containing at the 5' end the universal M13 primer sequences, were pre-spotted in 96-well plates. Following PCR, direct sequencing was performed using M13 primers, allowing to standardize the conditions. PCR amplification and sequencing were successful for each amplicon. We tested and validated the assay on 10 known gDNAs from patients with Hereditary breast and ovarian cancer (HBOC). Our strategy is a promising time and cost-effective method to detect BRCA mutations in the clinical setting, which is essential to formulate a personalized therapy for patients with HBOC.
Asunto(s)
Análisis Mutacional de ADN/métodos , Genes BRCA1 , Genes BRCA2 , Mutación , Reacción en Cadena de la Polimerasa/métodos , Neoplasias de la Mama/genética , Estudios de Casos y Controles , Cartilla de ADN , Femenino , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Ováricas/genéticaRESUMEN
BAG3, a member of the BAG family of heat shock protein (HSP) 70 cochaperones, is expressed in response to stressful stimuli in a number of normal cell types and constitutively in a variety of tumors, including pancreas carcinomas, lymphocytic and myeloblastic leukemias, and thyroid carcinomas. Down-regulation of BAG3 results in cell death, but the underlying molecular mechanisms are still elusive. Here, we investigated the molecular mechanism of BAG3-dependent survival in human osteosarcoma (SAOS-2) and melanoma (M14) cells. We show that bag3 overexpression in tumors promotes survival through the NF-kappaB pathway. Indeed, we demonstrate that BAG3 alters the interaction between HSP70 and IKKgamma, increasing availability of IKKgamma and protecting it from proteasome-dependent degradation; this, in turn, results in increased NF-kappaB activity and survival. These results identify bag3 as a potential target for anticancer therapies in those tumors in which this gene is constitutively expressed. As a proof of principle, we show that treatment of a mouse xenograft tumor model with bag3siRNA-adenovirus that down-regulates bag3 results in reduced tumor growth and increased animal survival.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Regulación Neoplásica de la Expresión Génica , Quinasa I-kappa B/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Femenino , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , FN-kappa B/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
HMGA protein overexpression is associated with a highly malignant phenotype and it is also causally related to neoplastic cell transformation. Our previous results have shown that HMGA1 was not expressed in normal breast tissue whereas HMGA1 staining was intense in 25% of hyperplastic lesions with cellular atypia and in 60% of sporadic ductal carcinomas. Moreover, HMGA1 protein levels were significantly correlated with c-Erb-B2 expression. These results suggested HMGA1 expression as a novel prognostic factor in breast ductal carcinomas. In order to investigate whether the HMGA1 detection might have a prognostic role also for inherited breast carcinomas we have analysed the expression of the HMGA1 proteins in 116 breast familial carcinomas associated with BRCA1 or BRCA2 or negative for mutations in both genes (BRCAX). HMGA1 expression was weakly positive in 23 (20%), moderately positive in 34 (29%) and strongly positive in 20 (17%) breast carcinomas, and was not detected in 39 of them (34%). Statistical analysis of the immunostaining data showed that HMGA1 was significantly overexpressed, with a more intense staining, in BRCA2 (p=0.0009) and BRCAX (p=0.0134) patients compared to BRCA1 ones. Furthermore, in BRCA2 positive patients, the expression of HMGA1 seems to correlate with a favourable prognosis with a median overall survival of 65 months and a 5-year survival rate of 80% for HMGA1-negative patients, while median overall survival in the HMGA1-positive subsets was not reached with 5-year survival rates ranging from 84% to 100% of patients (p=0.0198). Conversely, no correlation was found between HMGA1 expression and overall survival in patients carrying inherited mutations in the BRCA1 and in BRCAX patients.
Asunto(s)
Neoplasias de la Mama/genética , Proteína HMGA1a/metabolismo , Proteínas de Neoplasias/metabolismo , Adulto , Anciano , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Femenino , Genes BRCA1 , Genes BRCA2 , Mutación de Línea Germinal , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Persona de Mediana Edad , Linaje , Adulto JovenRESUMEN
NF-kappaB is constitutively activated in primary human thyroid tumors, particularly in those of anaplastic type. The inhibition of NF-kappaB activity in the human anaplastic thyroid carcinoma cell line, FRO, leads to an increased susceptibility to chemotherapeutic drug-induced apoptosis and to the blockage of their ability to form tumors in nude mice. To identify NF-kappaB target genes involved in thyroid cancer, we analyzed the secretome of conditioned media from parental and NF-kappaB-null FRO cells. Proteomic analysis revealed that the neutrophil gelatinase-associated lipocalin (NGAL), a protein involved in inflammatory and immune responses, is secreted by FRO cells whereas its expression is strongly reduced in the NF-kappaB-null FRO cells. NGAL is highly expressed in human thyroid carcinomas, and knocking down its expression blocks the ability of FRO cells to grow in soft agar and form tumors in nude mice. These effects are reverted by the addition of either recombinant NGAL or FRO conditioned medium. In addition, we show that the prosurvival activity of NGAL is mediated by its ability to bind and transport iron inside the cells. Our data suggest that NF-kappaB contributes to thyroid tumor cell survival by controlling iron uptake via NGAL.
Asunto(s)
Proteínas de Fase Aguda/metabolismo , Regulación Neoplásica de la Expresión Génica , Lipocalinas/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Neoplasias de la Tiroides/enzimología , Neoplasias de la Tiroides/patología , Proteínas de Fase Aguda/genética , Línea Celular Tumoral , Supervivencia Celular , Salud , Humanos , Quinasa I-kappa B/metabolismo , Inmunohistoquímica , Lipocalina 2 , Lipocalinas/genética , Proteómica , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , Neoplasias de la Tiroides/genéticaRESUMEN
We have analysed the expression of the HMGA2 gene in a panel of normal and neoplastic thyroid tissues by immunohistochemistry and quantitative RT-PCR. HMGA2 protein was detectable in four out of 21 follicular carcinomas, 30 out of 45 papillary carcinomas, and 11 out of 12 undifferentiated carcinomas. As far as follicular thyroid adenomas are concerned, only three cases of the 31 analysed showed HMGA2 protein expression, whereas it was absent in seven normal thyroid tissues and in 12 hyperplastic nodules. Quantitative RT-PCR showed that almost all the papillary thyroid carcinomas and 13 out of 16 follicular thyroid carcinomas express much higher HMGA2 specific mRNA levels in comparison to normal thyroids and adenomas. Therefore, our data support the quantitative RT-PCR analysis of HMGA2 expression, rather than immunohistochemistry, as a powerful tool for the diagnosis of thyroid neoplasias.
Asunto(s)
Adenocarcinoma Folicular/genética , Carcinoma Papilar/genética , Proteína HMGA2/metabolismo , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Neoplasias de la Tiroides/genética , Proteína HMGA2/genética , Humanos , Inmunohistoquímica , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A3 adenosine receptor (A3AR) agonists have been reported to modulate cellular proliferation. This work was aimed to investigate the expression and the possible implication of A3AR in the human thyroid carcinomas. Normal thyroid tissue samples did not express A3 adenosine receptor, while primary thyroid cancer tissues expressed high level of A3AR, as determined by immunohistochemistry analysis. In human papillary thyroid carcinoma cell line, NPA, at concentrations > or =10 microM, the A3AR-selective agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (Cl-IB-MECA) produced inhibition of cell growth, by blocking the G(1) cell cycle phase in a concentration- and time-dependent manner. This effect was well correlated with a reduction of protein expression of cyclins D1 and E2 after 24 hours of Cl-IB-MECA treatment. Moreover Cl-IB-MECA induced dephosphorylation of ERK1/2 in a time- and concentration-dependent manner, which in turn inhibits cell proliferation. The effect of Cl-IB-MECA was not prevented by A3AR antagonists, MRS1191 or MRS1523 or FA385. Furthermore, neither nucleoside transporter inhibitors, Dypiridamole and NBTI, nor the A1, A2A and A2B receptors antagonists were able to block the response to Cl-IB-MECA. Although Cl-IB-MECA has been shown to influence cell death and survival in other systems through an A3AR-mediated mechanism, in NPA cells the growth inhibition induced by micromolar concentrations of Cl-IB-MECA is not related to A3AR activation and hence that its effects on human papillary carcinoma cell line seem to be independent of the presence of this receptor subtype.
Asunto(s)
Agonistas del Receptor de Adenosina A3 , Adenosina/análogos & derivados , Proliferación Celular/efectos de los fármacos , Neoplasias de la Tiroides/patología , Adenosina/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ciclinas/metabolismo , Relación Dosis-Respuesta a Droga , Fase G1/efectos de los fármacos , Humanos , Inmunohistoquímica , Factores de TiempoRESUMEN
OBJECTIVE: To evaluate an in vivo pro-oxidant state in patients with ataxia telangiectasia (AT). METHODS: A set of oxidative stress endpoints were measured in 9 AT homozygotes, 16 AT heterozygotes (parents) and 83 controls (grouped in age ranges as for patients and parents, respectively). The following analytes were measured: (a) leukocyte 8-hydroxy-2'-deoxyguanosine (8-OHdG); (b) blood glutathione (GSSG and GSH); and (c) plasma levels of glyoxal (Glx) and methylglyoxal (MGlx). RESULTS: AT patients displayed a significant decrease in blood GSSG (p=0.012) and in MGlx plasma concentrations (p=0.012). A non-significant decrease in the GSSG:GSH ratio (p=0.1) and a non-significant increase in 8-OHdG and Glx levels were observed in AT patients vs. young controls (age range 4-35 years). AT heterozygotes failed to display any significant changes vs. adult controls (age range 36-68 years). CONCLUSION: No significant increase in oxidative stress biomarkers was detected in blood from AT patients. The decrease in GSSG and MGlx levels in AT patients may suggest an adaptive response to a pro-oxidant state in AT-related target organs.
Asunto(s)
Ataxia Telangiectasia/sangre , Glutatión/sangre , Estrés Oxidativo/fisiología , 8-Hidroxi-2'-Desoxicoguanosina , Adaptación Fisiológica , Adolescente , Adulto , Niño , Preescolar , Daño del ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/sangre , Femenino , Glioxal/sangre , Humanos , Masculino , Piruvaldehído/sangreRESUMEN
BACKGROUND: Fos-related antigen 1 (FRA-1) is an immediate early gene encoding a member of AP-1 family of transcription factors involved in cell proliferation, differentiation, apoptosis, and other biological processes. fra-1 gene overexpression has an important role in the process of cellular transformation, and our previous studies suggest FRA-1 protein detection as a useful tool for the diagnosis of thyroid neoplasias. Here we investigate the expression of the FRA-1 protein in benign and malignant breast tissues by immunohistochemistry, Western blot, RT-PCR and qPCR analysis, to evaluate its possible help in the diagnosis and prognosis of breast neoplastic diseases. METHODS: We investigate the expression of the FRA-1 protein in 70 breast carcinomas and 30 benign breast diseases by immunohistochemistry, Western blot, RT-PCR and qPCR analysis. RESULTS: FRA-1 protein was present in all of the carcinoma samples with an intense staining in the nucleus. Positive staining was also found in most of fibroadenomas, but in this case the staining was present both in the nucleus and cytoplasm, and the number of positive cells was lower than in carcinomas. Similar results were obtained from the analysis of breast hyperplasias, with no differences in FRA-1 expression level between typical and atypical breast lesions; however the FRA-1 protein localization is mainly nuclear in the atypical hyperplasias. In situ breast carcinomas showed a pattern of FRA-1 protein expression very similar to that observed in atypical hyperplasias. Conversely, no FRA-1 protein was detectable in 6 normal breast tissue samples used as controls. RT-PCR and qPCR analysis confirmed these results. Similar results were obtained analysing FRA-1 expression in fine needle aspiration biopsy (FNAB) samples. CONCLUSION: The data shown here suggest that FRA-1 expression, including its intracellular localization, may be considered a useful marker for hyperplastic and neoplastic proliferative breast disorders.
Asunto(s)
Enfermedades de la Mama/metabolismo , Enfermedades de la Mama/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Biopsia con Aguja Fina , Enfermedades de la Mama/genética , Humanos , Hiperplasia/genética , Hiperplasia/metabolismo , Hiperplasia/patología , Inmunohistoquímica , Neoplasias/genética , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/genéticaRESUMEN
CONTEXT: We previously showed that BAG3 protein, a member of the BAG (Bcl-2-associated athanogene) co-chaperone family, modulates apoptosis in human leukemias. The expression of BAG3 in other tumor types has not been extensively investigated so far. OBJECTIVE: The objective of this study was to analyze BAG3 expression in thyroid neoplastic cells and investigate its influence in cell apoptotic response to TNF-related apoptosis-inducing ligand (TRAIL). DESIGN, SETTING, AND PATIENTS: We investigated BAG3 expression in human thyroid carcinoma cell lines, including NPA, and the effect of BAG3-specific small interfering RNA on TRAIL-induced apoptosis in NPA cells. Subsequently, we analyzed BAG3 expression in 30 benign lesions and 56 carcinomas from patients of the Naples Tumor Institute Fondazione Senatore Pascale. MAIN OUTCOME MEASURES: The main outcome measures were: analysis of BAG3 protein in NPA cells by Western blot and immunocytochemistry; analysis of apoptosis in TRAIL-stimulated NPA cells by flow cytometry; and evaluation of BAG3 expression in specimens from thyroid lesions by immunohistochemistry. RESULTS: BAG3 was expressed in human thyroid carcinoma cell lines; small interfering RNA-mediated downmodulation of its levels significantly (P < 0.0195) enhanced NPA cell apoptotic response to TRAIL. The protein was not detectable in 19 of 20 specimens of normal thyroid or goiters, whereas 54 of 56 analyzed carcinomas (15 follicular, 28 papillary, and 13 anaplastic) were clearly positive for BAG3 expression. CONCLUSIONS: BAG3 downmodulates the apoptotic response to TRAIL in human neoplastic thyroid cells. The protein is specifically expressed in thyroid carcinomas and not in normal thyroid tissue or goiter.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Apoptosis/efectos de los fármacos , Carcinoma/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Neoplasias de la Tiroides/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/metabolismo , Carcinoma/patología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Inmunohistoquímica , ARN Interferente Pequeño/farmacología , Neoplasias de la Tiroides/patologíaRESUMEN
Oxidative stress has been associated with Down syndrome (DS) and with its major phenotypic features, such as early ageing. In order to evaluate an in vivo pro-oxidant state, the following analytes were measured in a group of DS patients aged 2 months to 57 years: (a) leukocyte 8-hydroxy-2'-deoxyguanosine (8-OHdG); (b) blood glutathione; (c) plasma levels of: glyoxal (Glx) and methylglyoxal (MGlx); some antioxidants (uric acid, UA, ascorbic acid, AA and Vitamin E), and xanthine oxidase (XO) activity. A significant 1.5-fold increase in 8-OHdG levels was observed in 28 DS patients vs. 63 controls, with a sharper increase in DS patients aged up to 30 years. The GSSG:GSH x 100 ratio was significantly higher in young DS patients (< 15 years), in contrast to DS patients aged >or=15 years that showed a significant decrease in the GSSG:GSH x 100 ratio ratio vs. controls of the respective age groups. Plasma Glx levels were significantly higher in young DS patients, whereas no significant difference was detected in DS patients aged >or=15 years. Unlike Glx, the plasma levels of MGlx were found to be significantly lower in DS patients vs. controls. A significant increase was observed in plasma levels of UA in DS patients that could be related to an increased plasma XO activity in DS patients. The plasma concentrations of AA were also increased in young (< 15 years) DS patients, but not in older patients vs. controls in the same age range. The levels of Vitamin E in DS patients did not differ from the values determined in control donors. The evidence for a multiple pro-oxidant state in young DS patients supports the role of oxidative stress in DS phenotype, with relevant distinctions according to patients' ages.
Asunto(s)
Envejecimiento/metabolismo , Síndrome de Down/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Adolescente , Adulto , Factores de Edad , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The hypothesis was tested that Werner syndrome (WS) phenotype might be associated with an in vivo prooxidant state. A set of redox-related endpoints were measured in three WS patients, two of their parents, and 99 controls within a study of some cancer-prone and/or ageing-related genetic disorders. The following analytes were measured: (a) leukocyte 8-hydroxy-2'-deoxyguanosine; (b) glutathione from whole blood, and (c) plasma levels of glyoxal, methylglyoxal, 8-isoprostane, and some plasma antioxidants (uric acid, ascorbic acid, alpha- and gamma-tocopherol). Leukocyte 8-hydroxy-2'-deoxyguanosine levels showed a significant increase in the 3 WS patients vs. 85 controls (p<10(-7)). The disulfide glutathione:glutahione ratio was significantly altered in WS patients (p=0.005). Glyoxal and methylglyoxal levels were significantly increased (p=0.018 and p=0.007, respectively). The plasma levels of uric acid (p=0.002) and ascorbic acid (p=0.003) were also increased significantly in WS patients and in their parents. No significant alterations were found in the plasma levels of alpha- and gamma-tocopherol, nor of 8-isoprostane. This is the first report of in vivo alterations of oxidative stress parameters in WS patients. Further investigations on more extensive study populations are warranted to verify the relevance of an in vivo prooxidant state in WS patients.
Asunto(s)
Oxidación-Reducción , Síndrome de Werner/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Antioxidantes/análisis , Cromatografía Líquida de Alta Presión , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análisis , Dinoprost/análogos & derivados , Dinoprost/sangre , Femenino , Glutatión/sangre , Glioxal/sangre , Heterocigoto , Humanos , Leucocitos/metabolismo , Masculino , Persona de Mediana Edad , Piruvaldehído/sangre , Síndrome de Werner/genéticaRESUMEN
Some selected oxidative stress parameters were measured in 56 Fanconi anaemia (FA) patients (42 untransplanted and 14 transplanted), 54 FA heterozygotes (parents) and 173 controls. Untransplanted FA patients showed a highly significant increase in leukocyte 8-hydroxy-2'-deoxyguanosine (8-OHdG) (P = 0.00003) and a borderline increase (P = 0.076) in urinary levels of 8-OHdG versus child controls. These increases were more pronounced in female FA patients (P = 0.00005 for leukocyte 8-OHdG and P = 0.021 for urinary 8-OHdG). Female FA patients also displayed a highly significant excess of spontaneous chromosomal breaks versus male patients (P = 0.00026), in the same female:male ratio ( approximately 1.4) as detected for both leukocyte and urine 8-OHdG levels. Plasma methylglyoxal (MGlx) levels were increased in untransplanted FA patients versus child controls (P = 0.032). The increases in leukocyte and urinary 8-OHdG and in MGlx levels were detected in young FA patients (< or =15 years), whereas patients aged 16-29 years failed to display any differences versus controls in the same age group. A significant increase in oxidized:reduced glutathione (GSSG:GSH) ratio was observed (P = 0.046) in the FA patients aged < or =15 years, whereas those aged 16-29 years, both untransplanted and transplanted, displayed a decrease (P = 0.06) in the GSSG:GSH ratio versus the controls of the respective age groups. No significant changes were detected in plasma levels of vitamin C, vitamin E or uric acid. Transplanted FA patients showed lesser alterations in leukocyte 8-OHdG and in GSSG:GSH ratio versus untransplanted patients. The parents of FA patients displayed a significant increase in plasma MGlx levels (P = 0.0014) versus adult controls. The results suggest a gender- and age-related modulation of oxidative stress in FA patients. The observed increase in urinary 8-OHdG in untransplanted FA patients suggests a proficient removal of oxidized DNA bases.