Characterization of an intramolecular protein-protein interaction in c-Ets1 and its viral homologue v-Ets.

The proto-oncoprotein c-Ets1 is a well-known transcription factor that belongs to the Ets family and plays a role in haematopoietic differentiation, angiogenesis, and carcinogenesis. Ets family members share a unique DNA binding domain, the Ets domain, and are known to control DNA binding activity and transcriptional activation by autoinhibition. In c-Ets1 the DNA binding domain as well as N-terminal and C-terminal inhibitory domains are involved in autoinhibitory regulation. It has been proposed that intramolecular interactions are part of the autoinhibitory mechanism. We applied a GST pull-down assay as well as a two-hybrid system in yeast to show an interaction between the DNA binding domain of c-Ets1 and a domain N-terminal thereof. We have mapped the interaction sites within both of the c-Ets1 domains. Comparison of the analogous intramolecular interaction in c-Ets1 and in v-Ets revealed that the interaction we detected is stronger in v-Ets than in c-Ets1. In view of previous findings from DNA binding studies, and kinetic experiments as well as structural data, our results suggest a new model as to how intramolecular interactions might participate in the regulation of DNA binding. Binding of c-Ets1 to DNA temporarily changes the intramolecular pattern of interaction in c-Ets1, leading to an increase in affinity of c-Ets1 to DNA. In full-length c-Ets1 the intramolecular interactions re-form spontaneously and the protein-DNA complex dissociates. The interaction we characterize herein might increase the DNA binding affinity temporarily in DNA-bound c-Ets1. In v-Ets in which the C-terminal domain is mutated this interaction appears to lead to strong DNA binding affinity. Therefore, changes in v-Ets might contribute to the tumorigenic process by altering intramolecular interactions.

A novel breast cancer cell line initially established from pleural effusion: evolution towards a more aggressive phenotype.

Many human breast cancer cell lines have been in culture for several years, serving as model systems for studying aspects of breast cancer biology. Molecular alterations might occur in these cells during cultivation, and it remains unknown to which extent findings in these cell lines can be related to human disease. Hereby, we describe the establishment of a breast cancer cell line, MW1, from malignant pleural effusion. We compare expression patterns of several molecular markers in breast biopsy tissue, in cultivated tumor cells derived from pleural effusion reflecting the metastatic state, and in late passages of a lineage derived from the pleural culture. Our data show that expression of estrogen and progesterone receptors was lost in the cultivated tumor cells derived from pleural effusion as shown by immunohistochemical staining. Cytokeratin expression patterns remained luminal. During cultivation, the growth rate of MW1 cells increased dramatically and the morphology underwent alterations. As shown by Western blotting, E-cadherin expression remained unchanged whereas P-cadherin expression had increased after 4 years of cultivation of the cell line. Integrin beta4 expression was low in early passages of the pleural effusion whereas the cell line exhibited high expression levels of beta4. HGF receptor (c-Met), EGF receptor, VEGF and VEGF receptor-2 (KDR) expression was detectable by semiquantitative RT-PCR and remained unchanged during cultivation. In contrast, VEGF receptor-1 (flt-1) expression showed lower expression after 4 years of cultivation. The cell line migrated towards HGF, but not towards VEGF. This study provides exemplary insight into the molecular metamorphosis tumor cells undergo in vivo or in vitro on their way from the primary tumor via an equivalent of the metastatic state and during the development of a clonal cell line.