Publications

The balance between detoxification and bioactivation of a compound in a particular species or organ is highly dependent on the relative amounts and activities of the different forms of cytochrome P450 (P450) that are expressed. Therefore, knowledge of the catalytic specificities and regulation of individual P450 forms is of paramount importance in predicting and/or rationalizing species, strain, and individual differences in xenobiotic metabolism as well as metabolic interactions between compounds, both endogenous and exogenous. The emergence in recent years of a battery of isoform-selective chemical inhibitors that can be used in vitro and in vivo in experimental animals and humans has greatly facilitated the identification of individual cytochromes P450 responsible for specific bioactivation and detoxification reactions. Many of these inhibitors are mechanism-based and owe their selectivity to metabolism by the target enzyme. Such compounds have also proven valuable as probes of the catalytic mechanism of cytochromes P450, for identifying amino acid residues of importance for the various functions of the enzyme, for assessing the physiological roles of P450-derived oxidation products of endogenous compounds, in chemical-induced models of acute hepatic porphyria, and for studying protein turnover. The identification of isoform-selective, nontoxic inhibitors of individual human cytochromes P450 raises the real possibility of modulation of human drug metabolism for therapeutic purposes.

The integrin alpha v beta 6 has been shown to be a fibronectin-binding protein. To determine whether the cytoplasmic and transmembrane domains of alpha v beta 6 are necessary for binding to fibronectin, a truncated, secreted form of the integrin lacking these domains was engineered and expressed in Chinese hamster ovary cells. Fibronectin affinity chromatography demonstrated that the secreted integrin, like its full-length counterpart, was capable of binding fibronectin. Monoclonal antibodies were made to secreted alpha v beta 6 and to beta 6-transfected NIH 3T3 cells. In experiments designed to determine whether alpha v beta 6 can mediate cell attachment to fibronectin, full-length human beta 6 was expressed in Chinese hamster ovary cells and in the human colon carcinoma cell line SW480. beta 6-expressing cells were identified by alpha v beta 6-specific antibodies, and the beta 6-transfectants were used in cell-adhesion assays. In Chinese hamster ovary cells, human beta 6 associated with hamster alpha v but was incapable of mediating cell attachment to fibronectin. However, expression of beta 6 in these cells had the dominant negative effect of decreasing alpha v beta 5-dependent adhesion to vitronectin. In SW480 cells, beta 6 expression conferred the ability to bind to fibronectin even in the presence of inhibitory antibodies against beta 1 integrins. In such cells, fibronectin binding ability could be blocked by an antibody to alpha v integrins. These results constitute the first direct evidence that alpha v beta 6 mediates cell attachment to fibronectin.