Constitutive and basal secretion from the endocrine cell line, AtT-20

The ACTH nonexpressor was subcloned two additional times to ensure that no cells in the culture were expressing ACTH (HYA.15.6), then further characterized. Sibling clones of cells that expressed ACTH were also isolated during the subcloning of HY.15.6 for use as ATCH-expressing control cells (HYA.15.10). AtT-20 cells normally extend processes when plated on glass or plastic substrates. This process can be enhanced by incubating cells in growth medium containing 5 mM 8-Br-cAMP (Burgess and Kelly, 1985) . When HYA.15.6 or HYA.15.10 cells were plated on glass coverslips, however, they both extended long, thin processes without stimulation with the secretagogue, 8-Br-cAMP. ACTH could be detected by immunofluorescence in HYA.15.10 cells but not in HYA.15.6 cells (Fig. 1) . A sensitive radioimmunoassay was used to detect ACTH in cell extracts. Wild type AtT-20 cells contained 29.9 ng ACTH/#g cell protein, HYA.15.10 cells contained 1.4 ng ACTH/#g cell protein, and HYA.15.6 ceils contained less than 0.10 ng ACTH/#g cell protein (the limit of detection of this assay), at least 300-fold less than wild type cells.