ACTH and MSH Production by a Single Cloned Mouse Pituitary Tumor Cell Line

Abstract

The production of ACTH and MSH was studied in the cloned, functional, murine pituitary tumor cell line, AtT-20. Extracts of normal whole LAFj mouse pituitaries, tumors induced by injecting AtT-20 cells into recipient LAF X mice, cultured AtT-20 cells, and the medium in which the cells were cultured were subjected to molecular sieve chromatography on Sephadex G-SO fine resin. The original extracts and the Sephadex fractions were assayed for bioactive ACTH and MSH. Each of the extracts contained both ACTH and MSH. The ACTH existed as two distinct moieties, one of which eluted earlier than the ACTH standard. The MSH eluted in later fractions and thus was a separate molecular entity from the ACTH's. The extracts and fractions were subjected to radioimmunoassays for ACTH, a-MSH, and P-MSH. Both ACTH moieties were immunologically indistinguishable from highly purified human ACTH standard. The MSH in normal mouse pituitary and AtT-20 tumor was immunologically indistinguishable from a-MSH standard, but less than one-fourth of the MSH biologic activity could be accounted for by immunoreactive a-MSH. The MSH in the cultured AtT-20 cells and culture medium, which eluted earlier from the Sephadex column, failed to react in the a-MSH radioimmunoassay. The MSH in each extract reacted with antibodies in the P-MSH radioimmunoassay, generating competition curves parallel to each other but less steep than P-MSH standard. Thus, the MSH was immunologically similar, but not identical, to any known P-MSH. The normal mouse pituitary and the AtT-20 tumor cell synthesize two ACTH's, the larger of which may be a precursor molecule, and a smaller MSH molecule which has immunochemical similarities to both a-MSH and P- MSH. {Endocrinology 92: 385, 1973) T HE KNOWN ACTH's and MSH's 5 from several species have important similarities: they are all produced in the adenohypophysis, they are all relatively short polypeptides that share a common sequence of amino acids, and they all have both intrinsic adrenocorticotropic and melanocyte stimulating activities. Previous studies from our laboratory have shown that the concentrations of ACTH and P-MSH, the principal

Common precursor to corticotropins and endorphins

“…From this information it is possible to calculate that the culture medium contains 80-90% as many moles of endorphin-related peptides as moles of ACTH-related peptides (Fig. 1) (3,(25)(26)(27).…”

Synthesis of opiate peptides by a clonal pituitary tumor cell line.

“…The tumor cells later were adapted to culture conditions (10) and cloned (22). The clonal cells synthesize and secrete ACTH as well as biologically active melanotropin not identical to either a-or f3-melanotropin (11). The presence of melanotropins and endorphins in AtT-20 cells strongly suggests that the cells synthesize f3-lipotropin and contain specific proteases that catalyze the conversion of fl-lipotropin to endorphin and melanotropin peptides.…”

Characterization of a common precursor to corticotropin and beta-lipotropin: cell-free synthesis of the precursor and identification of corticotropin peptides in the molecule.

“…High molecular weight forms of corticotropin (ACTH) have been observed in extracts of pituitary glands and pituitary tumor cells (1)(2)(3)(4). Studies of the biosynthesis of ACTH in a mouse pituitary tumor cell line (AtT-20/D-16v line) that contains four molecular weight (Mr) classes of ACTH suggest that the Mr 31,000 ACTH in these cells is a precursor of Mr 23,000, 13,000, and 4500 classes of ACTH (5).…”