Adrenocorticotropin‐resistant mutants of the Y1 adrenal cell line fail to express the adrenocorticotropin receptor

Abstract

This report examines the basis for adrenocorticotropin (ACTH) resistance in two mutant clones (Y6 and OS3) derived from the ACTH-responsive Y1 mouse adrenocortical tumor cell line. These two mutants were originally characterized by their failure to respond to ACTH with increased adenylyl cyclase activity and as a consequence were resistant to the steroidogenic effects of the hormone. We now demonstrate that ACTH resistance in the Y6 and OS3 mutants results from the failure to express the gene encoding the ACTH receptor. Whereas parental Y1 cells express ACTH receptor transcripts at low levels and are stimulated by ACTH or 8-bromo-cAMP to increase the accumulation of ACTH receptor transcripts approximately twofold, the Y6 and OS3 mutants do not express receptor transcripts either in the presence or absence of 8-bromo-cAMP. The gene encoding the ACTH receptor appears to be present in the Y6 and OS3 mutants, as determined by Southern blot hybridization analysis. Moreover, in the Y6 mutant the ACTH receptor gene appears to be silenced by a modification that is reversed following the growth of the cells as tumors in mice. Clonal isolates of Y6 cells grown as tumors recover the ability to express ACTH receptor transcripts at low but detectable levels and acquire the ability to respond to ACTH with increased adenylyl cyclase activity. Finally, Y6 and OS3 cells transformed with a gene encoding the mouse β₂-adrenergic receptor respond to the β-adrenergic agonist, isoproterenol, in a manner that is indistinguishable from the similarly transformed parent Y1 cell line. These latter results demonstrate the functional integrity of the adenylyl cyclase system in the ACTH-resistant mutants and indicate that the failure to express ACTH receptor transcripts limits the responsiveness of these clones.