RESUMO
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.
Assuntos
Neoplasias da Mama/genética , Proteínas de Neoplasias/genética , Receptor ErbB-2/genética , Transativadores/genética , Ativação Transcricional , Sequência de Bases , Ligação Competitiva , Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eletroforese , Receptores ErbB/metabolismo , Feminino , Genes erbB-2 , Humanos , Dados de Sequência Molecular , Peso Molecular , Proteínas de Neoplasias/metabolismo , Oligonucleotídeos/metabolismo , Proto-Oncogene Mas , RNA Mensageiro/metabolismo , Receptor ErbB-2/metabolismo , Transativadores/metabolismo , Fator de Transcrição AP-2 , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Reguladores/genética , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes Reguladores/fisiologia , Genes Reporter/genética , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor ErbB-2 , Mapeamento por Restrição , Transfecção , Células Tumorais CultivadasRESUMO
The presence of c-erbB2, TGF-beta 1 and pS2 mRNAs was examined in primary breast tumours. The c-erbB2 mRNA was overexpressed in 34% of the tumours. There was a positive, statistically significant correlation between c-erbB2 gene overexpression and nodal status. TGF-beta 1 mRNA was detected in 84% of the tumours, regardless of their clinical status. When possible, the c-erbB2 and TGF-beta 1 proteins were identified immunohistochemically on frozen sections from the same tumours. For TGF-beta 1, the mRNA and immunohistochemical results were divergent in 6 cases, 5 of which did contain clearly detectable mRNA but did not stain with the antibody. The pS2 mRNA was detected in 22% of the tumours and in the BT474 cell line. There was a significant correlation between the presence of pS2 mRNA and of oestrogen receptors. No statistically significant correlation was observed between pS2 and TGF-beta 1 genes expression and the clinical parameters of the tumours.
