Processing of high-molecular-weight form adrenocorticotropin in human adrenocorticotropin-secreting tumor cell line (DMS-79) after transfection of prohormone convertase 1/3 gene

Abstract

Ectopic ACTH-producing tumors preferentially secrete biologically inactive ACTH precursors and ACTH-related fragments. DMS-79 is known to secrete unprocessed high-molecular-weight (HMW) form ACTH. To determine whether prohormone convertase (PC) 1/3 is involved in the abnormal processing of proopiomelanocortin (POMC), we studied whether PC1/3 and 2 genes are expressed in DMS-79, and whether overexpression of PC1/3 gene affects POMC processing pattern. Steady-state mRNA levels of PC1/3 and 2 were determined by real-time RT-PCR. Molecular weights of ACTH-related peptides were determined by chromatographical analyses coupled with ACTH and beta-endorphin (beta-END) radioimmunoassays. PC1/3 gene was transfected into DMS-79 by retrovirus transduction using pMX-IP vector encoding PC1/3 cDNA. The steady-state mRNA levels of PC1/3 and 2 in DMS-79 were lower than those in ACTH-secreting and nonfunctioning pituitary tumors. DMS-79 predominantly secreted HMW form with both ACTH and beta-END immunoreactivities by size-exclusion chromatography. After purification by immunoaffinity chromatography with anti-ACTH antibody, the apparent molecular weight of HMW form ACTH was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining. After retroviral transfection of PC1/3 cDNA into DMS-79 and puromycin selection, PC1/3 stably-expressing cell line (DMS-79T) secreted two immunoreactive ACTH components, a major one coeluting with ACTH(1-39) and a minor one as a HMW form as well as two beta- END immunoreactive components coeluting with beta-lipotropic hormone and beta-END, respectively. Thus, we have established PC1/3 stably-expressing cell line (DMS-79T) capable of proteolytically processing ACTH precursor molecule(s) into mature ACTH and beta-END.