Serial induction of mutations by ethylnitrosourea in PC12 cells: a new model for a phenotypical characterization of the neurotoxic response to 6-hydroxydopamine

Here we show that the serial generation of allelic mutations by treatment with the mutagen ethylnitrosourea (ENU) in PC 12 cells provides a new model for the phenotypical dissection of biological properties. We tested this approach in the neurotoxic 6-OHDA model of Parkinson's disease in PC12 cells which has been widely used as an in vitro model for the investigation of the molecular pathogenesis of neuronal cell death and for novel treatment approaches. ENU treatment at doses of 0.2 and 0.3 mg/ml for I h resulted in 35 and 25% surviving PC12 cells, respectively, which showed mutation frequencies of approximately 10 mutations per genome. Clones derived from single ENU treated PC 12 cells showed marked differences in their resistance against 6-OHDA. The phenotypical analysis of resistant and sensitive clones showed a differential transcriptional regulation of multiple genes. The applicability of this approach could be demonstrated by the identification of the rat TM9SF1 gene coding for a transmembrane protein of the nonaspanin superfamily as a regulated gene in PC 12 clones resistant against 6-OHDA. Our data demonstrate the suitability of this model for the investigation of the molecular pathogenesis of neurodegeneration and for high-throughput analysis, e.g. for drug discovery. (C) 2004 Elsevier B.V. All rights reserved.