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In Vitro Transformation of Syrian Hamster Epidermal Cells by N-Methyl-N'-nitro-N-nitrosoguanidine¹

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 [N-C. S., C. R. Y. S., R. W. T., A. W. H.]; Department of Biochemistry, Medical University of South Carolina, Charleston, South Carolina 29403 [L. C.]; and FMC Corporation, Princeton, New Jersey 08540 [W-P. F.]

The selection of Syrian hamster epidermal cells which do not terminally differentiate has provided a quantitative focus assay for in vitro chemical transformation. One-day-old Syrian hamster epidermal cells plated at 5 x 10⁶/100-mm dish were treated for 5 hr with various concentrations of N-methyl-N'-nitro-N-nitrosoguanidine. After 4 weeks, the normal epidermal cells began to terminally differentiate to keratinized squamous cells and died, but transformed epidermal colonies grew to higher cell densities and appeared as darker areas against a lightly stained normal cell background. Transformed epidermal foci were isolated and subcultured for at least 15 passages, whereas normal epidermal cells could not be subcultured under the same conditions. The transformed cells assumed the typical cobblestone-like morphology of epithelial cells, retained desmosomes and tonofilaments, and were able to use citrulline in place of arginine. Argininosuccinate synthetase (EC 6.3.4.5) activity was significantly higher in the epidermal cells than in fibroblasts. The injection of 5 x 10⁶ cells of two transformed epidermal cell lines into athymic nude mice resulted in the formation of tumors which were identified as keratinizing squamous carcinomas.

¹ Research was sponsored by the Environmental Protection Agency, Interagency Agreement 40-516-75, and the Office of Health and Environmental Research, United States Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation.

² To whom requests for reprints should be addressed, at Biology Division, Oak Ridge National Laboratory, P. O. Box Y, Oak Ridge, Tenn. 37830.

Received 6/19/80. Accepted 1/16/81.