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[This retracts the article DOI: 10.1128/JVI.56.1.284-292.1985.].
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AIMS: The UbcH10 ubiquitin-conjugating enzyme plays a key role in regulating mitosis completion. We have previously reported that UbcH10 overexpression is associated with aggressive thyroid, ovarian and breast carcinomas. The aim of this study was to investigate UbcH10 expression in human lymphomas. METHODS AND RESULTS: Cell lines and tissue samples of Hodgkin's lymphoma (HL) and of non-Hodgkin's lymphoma (NHL) were screened for UbcH10 expression at transcriptional and translational levels. UbcH10 expression was related to the grade of malignancy. In fact, it was low in indolent tumours and high in a variety of HL and NHL cell lines and in aggressive lymphomas. It was highest in Burkitt's lymphoma, as shown by quantitative real-time polymerase chain reaction and by tissue microarray immunohistochemistry. Flow cytometry of cell lines confirmed that UbcH10 expression is cell-cycle dependent, steadily increasing in S phase, peaking in G(2)/M phase and dramatically decreasing in G(0)/G(1) phases. We also showed that UbcH10 plays a relevant role in lymphoid cell proliferation, since blocking of its synthesis by RNA interference inhibited cell growth. CONCLUSIONS: Taken together, these results indicate that UbcH10 is a novel lymphoid proliferation marker encompassing the cell cycle window associated with exit from mitosis. Its overexpression in aggressive lymphomas suggests that UbcH10 could be a therapeutic target in this setting.
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Enfermedad de Hodgkin/enzimología , Linfoma no Hodgkin/enzimología , Enzimas Ubiquitina-Conjugadoras/genética , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Enfermedad de Hodgkin/patología , Humanos , Linfoma no Hodgkin/patología , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Our group has recently demonstrated the overexpression of the UbcH10 gene in undifferentiated thyroid carcinomas. Subsequently, a clear correlation between UbcH10 overexpression and a reduced survival in ovarian carcinoma patients has been described indicating UbcH10 as a valid prognostic marker in this neoplastic disease. Here we have extended the analysis of the UbcH10 expression to neoplastic breast diseases. We demonstrated, by tissue micro-arrays immunohistochemical studies, a significant difference (p=0.0001) in the mean percentage of UbcH10 stained cells between benign (0.22%) and malignant (11.01%) neoplastic lesions. High UbcH10 expression was associated with intense Ki-67 staining (p=0.015) and ErbB2 positivity (p=0.092). The suppression of the ErbB2 expression in breast carcinoma cell lines induces a reduction of UbcH10 level. Consistently, the inhibition of breast carcinoma cell growth was achieved following the block of UbcH10 protein synthesis by RNA interference. Therefore, these results suggest the perspective of a therapy of aggressive breast carcinomas based on the suppression of the UbcH10 function.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/enzimología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , División Celular , Línea Celular Tumoral , Genes erbB-2/genética , Humanos , Inmunohistoquímica , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Elevated expression of HMGA1 and HMGA2 proteins is correlated with a highly malignant phenotype in several human tumors. We previously demonstrated that the block of HMGA2 protein synthesis prevented rat thyroid cell transformation by murine retroviruses. Suppression of HMGA2 synthesis was associated with lack of induction of HMGA1 proteins suggesting that both HMGA1 and HMGA2 play a role in the process of neoplastic transformation. To determine the role of the HMGA1 gene in thyroid cell transformation, we blocked HMGA1 protein synthesis by an antisense methodology. Here we report that transfection of an HMGA1 cDNA antisense construct into a normal rat thyroid cell line (FRTL-5 Cl2), followed by infection with Kirsten murine sarcoma virus (KiMSV), generated a transformed cell line that expresses high levels of the v-ras-Ki oncogene and that does not require thyroid-stimulating hormones for growth. However, this cell line does not show the malignant phenotype, i.e., it neither grows in soft agar nor induces tumors after injection in athymic mice. Moreover, the lack of the neoplastic phenotype in the virus-infected thyroid cells carrying the HMGA1 antisense construct correlates with the absence of induction of AP-1 transcriptional activity.
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Transformación Celular Viral/fisiología , Proteína HMGA1a/fisiología , Virus del Sarcoma Murino de Kirsten/fisiología , Proteína Oncogénica p21(ras)/fisiología , Glándula Tiroides/citología , Animales , Línea Celular Transformada/trasplante , Transformación Celular Viral/genética , Células Cultivadas , ADN sin Sentido/genética , ADN Complementario/genética , Genes ras , Proteína HMGA1a/deficiencia , Proteína HMGA1a/genética , Proteína HMGA2/fisiología , Virus del Sarcoma Murino de Kirsten/genética , Ratones , Ratones Desnudos , Fenotipo , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes de Fusión/fisiología , Especificidad de la Especie , Factor de Transcripción AP-1/metabolismo , Transfección , Ensayo de Tumor de Célula MadreRESUMEN
INTRODUCTION: UbcH10 codes for the cancer related E2 Ubiquitin Conjugating Enzyme, an enzymatic molecule with a key role in the ubiquitin-proteasome pathway. Current studies have suggested a critical role of UbcH10 in a variety of malignancies, including human thyroid, breast, ovarian and colorectal carcinomas. The aim of this study has been to extend the analysis of UbcH10 expression to lung cancer. This neoplasia represents one of the leading cause of cancer mortality worldwide, and new tools for an accurate diagnosis/prognosis are needed. METHODS: The expression levels of UbcH10 were analysed in human non-small cell lung carcinoma (NSCLC) by quantitative RT-PCR and tissue microarray immunohistochemistry, and these values were correlated with the clinicopathological features of the patients affected by NSCLC. RESULTS: Our results demonstrate that UbcH10 is overexpressed in NSCLC compared to the normal lung tissue. Moreover, UbcH10 expression is significantly higher in squamous cell and large cell carcinomas than in adenocarcinomas, and directly and inversely correlated with the mutational status of p53 and EGFR, respectively. The suppression of UbcH10 expression by RNAi resulted in a drastic reduction of proliferation and migration abilities of lung carcinoma cell lines. CONCLUSION: These results, taken together, indicate that UbcH10 overexpression has a critical role in lung carcinogenesis, and the evaluation of UbcH10 expression levels may be a new tool for the characterisation of NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Genes p53 , Neoplasias Pulmonares/genética , Mutación , Enzimas Ubiquitina-Conjugadoras/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Análisis Mutacional de ADN , Epistasis Genética/genética , Epistasis Genética/fisiología , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Genes p53/fisiología , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Mutación/fisiología , Análisis de Matrices Tisulares , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/fisiologíaRESUMEN
CONTEXT: Thyroid carcinoma is one of the most common malignancies of the endocrine system, and, despite the high frequency of oncogene activation in thyroid neoplastic lesions, the tumor suppressor genes involved in thyroid carcinogenesis remain unidentified. Our previous data implicated a link between the CL2/CCDC80 gene and thyroid cancer. OBJECTIVE: The objective of the study was to examine the expression of the CL2/CCDC80 gene in human thyroid carcinomas in the attempt to determine whether it plays a role in thyroid carcinogenesis. DESIGN: We evaluated the expression of CL2/CCDC80 in a large number of thyroid neoplastic tissue samples differing in degree of malignancy. We also investigated the effects of its restoration in 2 human thyroid carcinoma cell lines characterized by very low levels of CL2/CCDC80 expression. RESULTS: CL2/CCDC80 expression was much lower in almost all the thyroid carcinomas analyzed than in normal thyroid tissues and was lowest in follicular variants of papillary carcinomas. Loss of heterozygosity partially accounted for CL2/CCDC80 down-regulation in thyroid carcinoma samples. Restoration of CL2/CCDC80 expression in the 2 human thyroid anaplastic carcinoma cell lines resulted in a higher susceptibility to apoptosis and suppression of the malignant phenotype. CL2/CCDC80 expression positively regulated the expression of E-cadherin, thereby halting cancer progression. CONCLUSIONS: These results indicate that CL2/CCDC80 is a putative tumor suppressor gene in thyroid carcinogenesis.
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Carcinoma/metabolismo , Regulación hacia Abajo , Glicoproteínas/metabolismo , Pérdida de Heterocigocidad , Proteínas de Neoplasias/metabolismo , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Apoptosis , Carcinoma/genética , Carcinoma/patología , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar Folicular/genética , Carcinoma Papilar Folicular/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/patología , Citoplasma/metabolismo , Citoplasma/patología , Proteínas de la Matriz Extracelular , Estudios de Asociación Genética , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular , Italia , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Transporte de Proteínas , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Glándula Tiroides/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética , Regulación hacia ArribaRESUMEN
BACKGROUND: Thyroid fine-needle aspiration (FNA) samples belonging to the follicular neoplasm/suspicious for malignancy classes are controversial. The authors identified UbcH10 as a marker useful in the diagnosis of several neoplasms, including thyroid cancer. Here, analysis of UbcH10 expression by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry was applied to FNAs. METHODS: A series of 84 follicular neoplasm/suspicious for malignancy FNAs with histological follow-up (30 malignant) was prospectively collected. UbcH10 immunostaining was performed on cell blocks and compared with that of the proliferation marker Ki-67. At the mRNA level, UbcH10 was compared with CCND2 and PCSK2 expression, these latter being the best performing components of the previously reported 3-gene assay; to determine the diagnostic accuracy, the area under the curve (AUC) of the receiver operating characteristic (ROC) curve for each gene individually and in combination was evaluated. RESULTS: UbcH10 and Ki-67 shared a similar pattern; although UbcH10 expression was higher in malignant than in benign lesions (P < .001), staining was sporadic, and the cutoff value derived by the ROC analysis was too low (1.25%) for routine application. Conversely, UbcH10 expression assessment by quantitative RT-PCR was effective. UbcH10 mRNA levels associated with malignant histology were significantly higher than those associated with benign histology (P = .02). The AUC was 0.74 for UbcH10, 0.81 for CCDN2, 0.62 for PCSK2, and 0.84 for UbcH10 and CCND2 combination. CONCLUSIONS: UbcH10 quantitative RT-PCR analysis, rather than immunohistochemistry, is useful to increase the detection of malignancy in thyroid FNAs. UbcH10 may be added as a panel component in quantitative RT-PCR-based assays.
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Biomarcadores de Tumor/análisis , Biopsia con Aguja Fina , Neoplasias de la Tiroides/diagnóstico , Enzimas Ubiquitina-Conjugadoras/análisis , Enzimas Ubiquitina-Conjugadoras/genética , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/genética , Femenino , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Masculino , Persona de Mediana Edad , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/genéticaRESUMEN
Using gene expression profiling, we found that the CBX7 gene was drastically down-regulated in six thyroid carcinoma cell lines versus control cells. The aims of this study were to determine whether CBX7 is related to the thyroid cancer phenotype and to try to identify new tools for the diagnosis and prognosis of thyroid cancer. We thus evaluated CBX7 expression in various snap-frozen and paraffin-embedded thyroid carcinoma tissues of different degrees of malignancy by quantitative reverse transcription-PCR and immunohistochemistry, respectively. CBX7 expression progressively decreased with malignancy grade and neoplasia stage. Indeed, it decreased in an increasing percentage of cases going from benign adenomas to papillary (PTC), follicular, and anaplastic (ATC) thyroid carcinomas. This finding coincides with results obtained in rat and mouse models of thyroid carcinogenesis. CBX7 loss of heterozygosity occurred in 36.8% of PTC and in 68.7% of ATC. Restoration of CBX7 expression in thyroid cancer cells reduced growth rate, with a retention in the G(1) phase of the cell cycle, suggesting that CBX7 can contribute to the proliferation of the transformed thyroid cells. In conclusion, loss of CBX7 expression correlates with a highly malignant phenotype in thyroid cancer patients.
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Adenocarcinoma Folicular/genética , Carcinoma Papilar/genética , Carcinoma/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Neoplasias de la Tiroides/genética , Adenocarcinoma Folicular/metabolismo , Adenocarcinoma Folicular/patología , Adenoviridae/genética , Animales , Western Blotting , Carcinoma/metabolismo , Carcinoma/patología , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patología , Línea Celular Tumoral , Proliferación Celular , Cromosomas Humanos Par 22/genética , Ensayo de Unidades Formadoras de Colonias , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Pérdida de Heterocigocidad , Ratones , Ratones Desnudos , Complejo Represivo Polycomb 1 , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándula Tiroides/metabolismo , Glándula Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patologíaRESUMEN
In the last years, an increasing number of experiments has provided compelling evidence for a casual role of Ras protein mutations, resulting in their constitutive activation, in thyroid carcinogenesis. However, despite the clear involvement of Ras proteins in thyroid carcinogenesis, the nature of most of the target genes, whose expression is modulated by the Ras-induced signaling pathways and that are ultimately responsible for Ras-induced cellular transformation, remains largely unknown. To analyze Ras-dependent modulation of gene expression in thyroid cells we took advantage of a differentiated rat thyroid cell line, FRTL-5. As a model for Ras-dependent thyroid transformation, we used FRTL-5 cells infected with the Kirsten murine sarcoma virus, carrying the v-Ki-Ras oncogene. The infected cells (FRTL-5 v-Ki-Ras) have lost expression of the thyroid differentiation markers and also are completely transformed. We hybridized two different Affimetrix chips containing probe sets interrogating both known rat genes and ESTs for a total of more than 17,000 sequences using mRNA extracted from FRTL-5 and FRTL-5 v-Ki-Ras cell lines. We identified about 50 genes whose expression was induced and about 40 genes whose expression was downregulated more than 10-fold by Ras. We confirmed the differential expression of many of these genes in FRTL-5 v-Ki-Ras as compared to parental cells by using alternative techniques. Remarkably, we investigated the expression of some of the Ras-regulated genes in human thyroid carcinoma cell lines and tumor samples, our results, therefore, providing a new molecular profile of the genes involved in thyroid neoplastic transformation.
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Transformación Celular Neoplásica , Perfilación de la Expresión Génica , Genes ras , Neoplasias de la Tiroides/genética , Animales , Antígeno CD24/análisis , Línea Celular Tumoral , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , RatasRESUMEN
High mobility group A 1 (HMGA1) proteins are chromatinic factors, which are absent or expressed at very low levels in normal adult tissues, while they are over-expressed in several human malignant tumors. In this study, HMGA1 protein expression was investigated by immunohistochemistry in a series of 44 epithelial ovarian specimens, which included four normal ovarian tissues, 29 primary invasive carcinomas, one metastatic ovarian tumor and 10 low malignant potential (LMP) tumors. HMGA1 staining was not detected in normal ovarian surface epithelium, which is the area from which ovarian adenocarcinoma frequently arises. HMGA1 proteins were expressed at low levels in some LMP tumors, whereas they were present in abundance in most of the primary ovarian adenocarcinomas. RT-PCR and western blot analysis correlated with immunohistochemical data. We demonstrated that the suppression of HMGA1 protein synthesis by an adenovirus carrying the HMGA1 gene in antisense orientation (Ad-Yas-GFP) inhibited the growth of two human ovarian carcinoma cell lines (OVCAR-5 and OVCAR-8). These results confirm HMGA1 over-expression as a general feature of human malignant neoplasias, including ovarian cancer and suggest that suppression of HMGA1 protein synthesis by an antisense adenoviral vector may represent a new and promising gene therapy for the treatment of ovarian cancer.