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Epithelial-to-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is increasingly considered as an important event during malignant tumor progression and metastasis. To identify molecular players involved in EMT and metastasis, we performed expression profiling of a set of combined in vitro/in vivo cellular models, based on clonal, fully polarized mammary epithelial cells. Seven closely related cell pairs were used, which were modified by defined oncogenes and/or external factors and showed specific aspects of epithelial plasticity relevant to cell migration, local invasion and metastasis. Since mRNA levels do not necessarily reflect protein levels in cells, we used an improved expression profiling method based on polysome-bound RNA, suitable to analyse global gene expression on Affymetrix chips. A substantial fraction of all regulated genes was found to be exclusively controlled at the translational level. Furthermore, profiling of the above multiple cell pairs allowed one to identify small numbers of genes by cluster analysis, specifically correlating gene expression with EMT, metastasis, scattering and/or oncogene function. A small set of genes specifically regulated during EMT was identified, including key regulators and signaling pathways involved in cell proliferation, epithelial polarity, survival and transdifferentiation to mesenchymal-like cells with invasive behavior.

Understanding the mechanism and specificity of substrate binding in the cytochrome P450 (P450) superfamily is an important step toward explaining its key role in drug metabolism, toxicity, xenobiotic degradation, and several biosynthetic pathways. Here we investigate the ligand exit pathways and mechanisms of P450cam (CYP101), P450BM-3 (CYP102), and P450eryF (CYP107A1) by using random expulsion molecular dynamics and classical molecular dynamics simulations. Although several different pathways are found for each protein, one pathway is common to all three. The mechanism of ligand exit along this pathway is, however, quite different in the three different proteins. For P450cam, small backbone conformational changes, in combination with aromatic side chain rotation, allow for the passage of the rather rigid, compact, and hydrophobic substrate, camphor. In P450BM-3, larger transient backbone changes are observed on ligand exit. R47, situated at the entrance to the channel, appears important in guiding negatively charged fatty acid substrates in and out of the active site. In P450eryF, an isolated buried arginine, R185, stabilized by four hydrogen bonds to backbone carbonyl oxygen atoms, is located in the exit channel and is identified as having a particularly unusual functionality, dynamically gating channel opening. The results for these three P450s suggest that the channel opening mechanisms are adjusted to the physico-chemical properties of the substrate and can kinetically modulate protein-substrate specificity.

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