Processing of high-molecular-weight form adrenocorticotropin in human adrenocorticotropin-secreting tumor cell line (DMS-79) after transfection of prohormone convertase 1/3 gene.
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.
Tateno
T
T
Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyo-ku,Tokyo, Japan. ttateno.cme@tmd.ac.jp
Kato
M
M
Tani
Y
Y
Yoshimoto
T
T
Oki
Y
Y
Hirata
Y
Y
D000236:Adenoma / Q000378:metabolism; D000324:Adrenocorticotropic Hormone / Q000096:biosynthesis* / Q000378:metabolism*; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D008175:Lung Neoplasms; D008970:Molecular Weight; D010911:Pituitary Neoplasms / Q000378:metabolism; D011333:Pro-Opiomelanocortin / Q000378:metabolism*; D045664:Proprotein Convertase 1 / Q000235:genetics* / Q000378:metabolism; D045706:Proprotein Convertase 2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D012190:Retroviridae / Q000235:genetics; D055752:Small Cell Lung Carcinoma; D014162:Transfection; D001615:beta-Endorphin / Q000378:metabolism
D012333:RNA, Messenger; D001615:beta-Endorphin; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2