Selection of glucocorticoid-resistant mutations from an AtT-20 cell line containing a glucocorticoid-regulated...

AtT-20/IDG8 cells contain the stably transfected, selectable gene, neomycin phosphotransferase, under negative glucocorticoid regulation. Thus, when cultured in the simultaneous presence of the neomycin analogue, G418, and dexamethasone, AtT-20/IDG8 cells fail to grow. Our hypothesis was that mutated AtT-20/IDG8 cells capable of growth in such medium would have a defect in the glucocorticoid-mediated regulation of the neor gene. AtT-20/IDG8 cells were chemically mutagenized using ethyl-methane sulfonate and cloned in the presence of G418 and dexamethasone. Fourteen clones were obtained and loss of glucocorticoid control of neor expression was confirmed in them all. The naturally occurring gene, pro-opiomelanocortin, which is down-regulated by glucocorticoids in parent AtT-20/IDG8 cells, was down-regulated by dexamethasone in ten of the mutant lines, indicating that in those cells the receptor was functional in spite of aberrant regulation of neor. In the other four lines, pro-opiomelanocortin regulation was lost, also suggesting that a general transcription factor, such as the receptor, had been altered. These results indicate that multiple factors are involved in glucocorticoid-mediated gene regulation and that new, informative mutations can be produced after insertion of a regulated, selectable gene into a previously non-selectable cell line.