RESUMEN

Previously, we reported that adenovirus E1a protein behaves as a tumor suppressor in human cells. It apparently functions by transcriptionally inducing an array of epithelial cell adhesion genes, while repressing other cell-type specific genes, thus producing an epithelial phenotype. Concomitantly, the cells become sensitive to anoikis (apoptosis of epithelial cells detached from extracellular matrix), potentially causing tumor suppression. E1a protein interacts with the nuclear acetylases p300, CBP and P/CAF, and also with the co-repressor protein CtBP. In this study, we have determined the role of these interactions in E1a's phenotypic effects on human tumor cells. The results indicate that E1a's interaction with CtBP activates at least three epithelial cell adhesion gene promoters. The E-cadherin repressor appeared to be the CtBP-interacting protein delta EF1/ZEB, which bound the ras-repressible E-boxes of the E-cadherin promoter. The E1a-CtBP interaction also contributed to anoikis-sensitization. E1a's interactions with the nuclear acetylases conferred epithelial morphologies but did not activate epithelial genes. These latter interactions did not sensitize tumor cells to anoikis but nevertheless conferred tumor suppression. These results implicate CtBP as an antagonist of the epithelial phenotype and anoikis. They also indicate a new but undefined role for nuclear acetylases in maintaining the transformed phenotype.

Asunto(s)

Proteínas E1A de Adenovirus/metabolismo , Apoptosis , Cadherinas/genética , Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Fosfoproteínas/fisiología , Factores de Transcripción , Proteínas E1A de Adenovirus/genética , Oxidorreductasas de Alcohol , Línea Celular Transformada , Proteínas de Unión al ADN/metabolismo , Desmoplaquinas , Epitelio/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Células Tumorales Cultivadas , Homeobox 1 de Unión a la E-Box con Dedos de Zinc , Dedos de Zinc

RESUMEN

The human IGF2 gene belongs to a group of imprinted genes clustered on the short arm of chromosome 11, band p15.5. It contains 9 exons and spans over 30 kb. IGF2 mRNA overexpression has been reported in human tumours and in some inherited growth disorders. It was recently demonstrated that IGF2 mRNA overexpression contributes to tumour progression and that loss of parental imprinting as well as altered transcription factors are contributing to this overexpression. We have reported structural alterations in the 3' region of the IGF2 gene in two colorectal tumours that overexpressed the IGF2 transcript by 200- and 800-fold. We cloned by the vectorette-PCR strategy, genomic DNA fragments containing the breakpoints from these tumours. The sequencing of these fragments positioned the breakpoint 2 kb downstream the IGF2 gene in one tumour, and in exon 9 in the second. Both breakpoints occurred in regions containing repetitive elements: a TGGA repeat we have identified downstream the gene, and the (CA)n repetition in exon 9. We hypothesize that a negative regulatory element, located downstream the IGF2 gene, has been deleted following these structural alterations and leads to IGF2 gene overexpression.

Asunto(s)

Aberraciones Cromosómicas , Trastornos de los Cromosomas , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Factor II del Crecimiento Similar a la Insulina/genética , ARN Neoplásico/biosíntesis , Clonación Molecular , Reordenamiento Génico , Impresión Genómica , Humanos , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , Análisis de Secuencia de ADN

RESUMEN

The HER2 proto-oncogene product is overexpressed in 30% of breast cancers, and this correlates with poor prognosis. Increased levels of HER2 mRNA in breast cancer cell lines result from increased gene transcription. We report the identification of a new 17-bp-long cis sequence located between positions -506 and -489 from the transcription start site. This sequence is recognized by a trans-activating factor that we tentatively named HER2 transcription factor (HTF). This factor, involved in the increased transcription of the HER2 gene in the BT-474 mammary tumor cells, has a molecular weight of about Mr 50,000. HTF can also bind, but with a lower affinity, to a related cis sequence present in the epidermal growth factor receptor promoter. Interestingly, the HTF binding activity is high in nuclear extracts from several mammary tumor cells overexpressing the HER2 gene.

Asunto(s)

Neoplasias de la Mama/genética , Proteínas de Neoplasias/genética , Receptor ErbB-2/genética , Transactivadores/genética , Activación Transcripcional , Secuencia de Bases , Unión Competitiva , Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Electroforesis , Receptores ErbB/metabolismo , Femenino , Genes erbB-2 , Humanos , Datos de Secuencia Molecular , Peso Molecular , Proteínas de Neoplasias/metabolismo , Oligonucleótidos/metabolismo , Proto-Oncogenes Mas , ARN Mensajero/metabolismo , Receptor ErbB-2/metabolismo , Transactivadores/metabolismo , Factor de Transcripción AP-2 , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas

RESUMEN

Hyperplasia without and with atypia is considered to be a precursor lesion for certain breast carcinomas. The cytogenetic events and the molecular pathology involved in the multistep process from normal to invasive carcinoma are unknown. To characterise the sequence of early genetic abnormalities of chromosome 17q and their biological consequences in the pathogenesis of breast cancer, we performed immunohistochemistry on 451 breast tissues including 180 normal breast specimens, 28 hyperplastic lesions without atypia and 44 with atypia, 100 cases of ductal carcinoma in situ (DCIS) and 99 cases of invasive ductal carcinoma. We correlated the overexpression of the c-ErbB-2 protein, the histological and the recently proposed differentiation classification of DCIS with the extent of DCIS. For fluorescence in situ hybridisation (FISH) analysis, different probes spanning the 17q region including the c-erbB-2 gene locus and those which are found adjacent, were used. Reverse painting and comparative genomic hybridisation (CGH) were performed on several breast cancer cell lines. c-ErbB-2 overexpression was observed in only 29% of DCIS and 23% of invasive carcinomas, but not in hyperplastic and normal tissue. c-ErbB-2 overexpression is correlated with poor differentiation in DCIS but not in invasive carcinoma. In DCIS, there was no correlation with the histological subtype classification. The average extent of DCIS is significantly increased from 13.81 mm in c-ErbB-2 negative cases to 29.37 mm in c-ErbB-2 positive cases. The increase was considered to be a possible consequence of the overexpression and is probably due to the previously described motility enhancing effect of the c-ErbB-2 protein. The histological and differentiation classification of DCIS did not correlate with the extent of disease. Using FISH, amplified genes at 17q12, always including the c-erbB-2 gene, were detected in all cases of DCIS and invasive carcinoma with c-ErbB-2 overexpression. The centromeric region and the NF1 locus, which is located between the centromere and c-erbB-2, were not amplified in any of the DCIS and invasive breast carcinomas, but co-amplification of the myeloperoxidase gene was detected in 3/5 DCIS and 1/5 invasive carcinomas with c-ErbB-2 overexpression. In contrast to c-erbB-2, immunohistochemical overexpression of their respective gene products was not observed. FISH, reverse painting and CGH show similar amplified genes with amplified c-erbB-2 in c-ErbB-2 overexpressing SK-BR-3 and BT474 human breast cancer cells. The amplified genes are part of two different amplicons. Extensive modifications of the 17q chromosomal region, caused by translocation, were also observed in these cell lines. It is concluded that the modifications of chromosome 17q, inducing overexpression of c-ErbB-2 protein, occur at the level of transition from hyperplasia to DCIS. They are preserved in invasive carcinoma with overexpression of c-ErbB-2 protein. This had led to the hypothesis that these modifications at 17q may lead to a larger extent of DCIS.

Asunto(s)

Neoplasias de la Mama/etiología , Carcinoma Ductal de Mama/etiología , Cromosomas Humanos Par 17 , Amplificación de Genes , Receptor ErbB-2/genética , Translocación Genética , Mama/química , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/química , Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/química , Carcinoma Ductal de Mama/genética , Transformación Celular Neoplásica/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Genes erbA/genética , Genes erbB-2/genética , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Cariotipificación , Proteínas Oncogénicas v-erbA/análisis , Proteínas Oncogénicas v-erbA/genética , Proteínas Oncogénicas v-erbA/metabolismo , Peroxidasa/análisis , Peroxidasa/genética , Peroxidasa/metabolismo , Receptor ErbB-2/análisis , Receptor ErbB-2/metabolismo

RESUMEN

The expression of members of the IGF system in a mesothelioma from a patient suffering from hypoglycemia, in term placenta and HT29 colon adenocarcinoma cells were compared. Very high levels of IGF-II mRNA and protein were detected in the mesothelioma. Moreover, half of the IGF-II protein took the high-molecular-weight form. We also analyzed the parental imprinting status and the promoter usage of the IGF-II gene. Our results showed loss of imprinting (LOI) in the mesothelioma while the imprinting was maintained in HT29 cells, expressing moderate levels of the transcript. Promoter P4 was active in the three tissues we analyzed, whereas IGF-II mRNA transcription from promoter P3 was only detected in the mesothelioma and the placenta, expressing comparably high levels of the transcript. IGF-II gene structure was identical in the analyzed tissues and cells. The type-I receptor mRNA expression was very low in the tumor. IGFBP-2, -4 and -5 mRNAs were detected in the mesothelioma, while IGFBP-2, -3 and -5 transcripts were detected in the placenta. IGFBP-1 and -6 transcripts were not detected.

Asunto(s)

Factor II del Crecimiento Similar a la Insulina/genética , Mesotelioma/metabolismo , Neoplasias Pleurales/metabolismo , Somatomedinas/metabolismo , Anciano , Anciano de 80 o más Años , Alelos , Northern Blotting , Femenino , Expresión Génica/genética , Impresión Genómica , Células HT29 , Humanos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Masculino , Mesotelioma/química , Placenta/química , Placenta/metabolismo , Neoplasias Pleurales/química , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Ribosómico 18S/análisis , ARN Ribosómico 18S/genética , Radioinmunoensayo , Receptor IGF Tipo 1/análisis , Receptor IGF Tipo 1/genética , Transcripción Genética/genética

RESUMEN

A 6-kilobase fragment extending at the 5'-end of the c-erbB2 protooncogene was isolated from a normal human lymphocyte genomic DNA library. The full-length fragment and five subfragments with identical 3'-ends were obtained by progressive unidirectional deletion from the 5'-end and were cloned in front of the luciferase reporter gene. The hybrid genes were analyzed for transcriptional activity in human mammary cell lines synthesizing low (HBL-100 and T-47D), moderate (MDA-MB-453), or high (BT-474) amounts of the c-erbB2 mRNA and were also analyzed in HeLa cells. Gene-specific expression was observed, indicating the presence of multiple cis-acting sequences in the c-erbB2 promoter. A major negative element is located in the -2- to -4-kilobase region. It is flanked on both sides by positive elements that display enhanced transcriptional activity in the BT-474 tumor cells only. While predominant in the low-expressing cells, the effect of the repressor appears to be overcome by the distal transactivator in the high-expressing BT-474 cells, resulting in a 15 to 50 times increase in luciferase activity relative to the HBL-100 and T-47D cells, respectively. Cell-specific expression relies on the trans-acting factors present in the different cell lines. The formation of cell-specific protein-DNA complexes was demonstrated by gel retardation assay.

Asunto(s)

Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica/genética , Genes Reguladores/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Genes Reguladores/fisiología , Genes Reporteros/genética , Células HeLa , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptor ErbB-2 , Mapeo Restrictivo , Transfección , Células Tumorales Cultivadas

RESUMEN

BT474 and SK-BR-3 mammary adenocarcinoma cells contain eight copies of the c-erbB2 gene but overexpress the mRNA 80 times over the levels measured in normal breast or in the HBL-100 cell line. Using Northern blot analysis and molecular titration based on RNAase protection assay, the decrease in the c-erbB2 mRNA level was monitored in BT474 cells treated with actinomycin D from 1 up to 24 h. The c-erbB2 degradation rate during the first 12 h corresponds to a calculated c-erbB2 mRNA half-life of approximately 7 h. Forty percent of the mRNA present in the cells before treatment remains undegraded after transcription has been blocked for 24 h. Pretreatment with cycloheximide results in complete mRNA degradation in 24 h, suggesting that labile proteins stabilize part of the c-erbB2 mRNA population. Comparison with the c-erbB2 mRNA turnover in HBL-100 'normal' cells indicated that the accumulation of the c-erbB2 gene product in the tumor cells is not the result of stabilization of the messenger. Rather, it is correlated with an increased rate of c-erbB2 mRNA transcription as indicated by run-on transcription assays. Both BT474 and SK-BR-3 tumor cell lines were found to synthesize 20-40 times more c-erbB2 mRNA than HBL-100 cells.

Asunto(s)

Adenocarcinoma/genética , Neoplasias de la Mama/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/metabolismo , Cicloheximida/farmacología , Dactinomicina/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas In Vitro , Proto-Oncogenes , ARN Neoplásico/metabolismo , Receptor ErbB-2 , Células Tumorales Cultivadas