ROTTIERS

Oxidative metabolism and gene expression: Gene discovery array analysis

Pieter Rottiers, Vera Goossens and Johan Grooten

Laboratory of Molecular Biology, Flanders Interuniversity Institute for Biotechnology (VIB) and University of Ghent, Ghent, Belgium

Treatment of the mouse fibrosarcoma cell line L929 with tumor necrosis factor (TNF) induces necrotic cell death by a mechanism that depends on production of reactive oxygen intermediates (ROI) by mitochondria (Goossens et al., 1995). Besides oxygen, bioenergetic pathways characteristic of tumor cells (usage of glutamine instead of glucose as oxidative substrate) markedly enchanced ROI production and thus influenced the sensitivity of the cell to TNF-induced necrosis (Goossens et al., 1996). Besides resistance to TNF cytotoxicity L929 cells that have been adapted to use glucose (normal metabolism) instead of glutamine (tumor-specific metabolism) as oxidative substrate exhibit, a differentiated morphology and decreased rate of cell division (Goossens et al., 1996). Apparently, the oxidative metabolism of the cell affect also features characteristic of transformed cells namely uncontrolled growth and dedifferentiation. To verify whether altered gene expression underlies this differential behaviour, we performed PCR-based suppression subtraction hybridization to identify genes, which are differentially expressed between L929 cells dependent on glutamine (TNF-sensitive; dedifferentiated morphology) and L929 cells dependent on glucose (TNF-resistant; differentiated morphology). The subtracted PCR products were hybridized on GDA filters (Genome Systems), spotted with 18,000 non-redundant mouse cDNA clones. Subsequent analysis identified genes known to be involved in the oxidative metabolism of the cell or to contribute to signal transduction pathways, and resistance/sensitivity to apoptosis. In addition a large number of unknown genes were revealed. This result establishes a firm link between oxidative metabolism and gene expression.

Goossens, V., Grooten, J., De Vos, K. & Fiers, W. (1995) Direct evidence for Tumor Necrosis Factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc. Natl. Acad. Sci. U.S.A. 92: 8115-8119.

Goossens, V., Grooten, J. & Fiers, W. (1996) The oxidative metabolism of glutamine - A modulator of reactive oxygen intermediate-mediated cytotoxicity of tumor necrosis factor in L929 fibrosarcoma cells. J. Biol. Chem. 271: 192-196.