Journal of Endocrinological Investigation

, Volume 33, Issue 2, pp 113–117 | Cite as

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

  • T. Tateno
  • M. Kato
  • Y. Tani
  • T. Yoshimoto
  • Y. Oki
  • Y. Hirata
Original Articles


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 β-endorphin (β-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 β-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 β-END immunoreactive components coeluting with β-lipotropic hormone and β-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 β-END.


DMS-79 ectopic ACTH syndrome high-molecular-weight form ACTH prohormone convertase 1/3 


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  1. 1.
    Steiner DF. The proprotein convertases. Curr Opin Chem Biol 1998, 2: 31–9.PubMedCrossRefGoogle Scholar
  2. 2.
    Seidah NG, Benjannet S, Hamelin J, et al. The subtilisin/kexin family of precursor convertases. Emphasis on PC1, PC2/7B2, POMC and the novel enzyme SKI-1. Ann N Y Acad Sci 1999, 885: 57–74.PubMedCrossRefGoogle Scholar
  3. 3.
    Seidah NG, Gaspar L, Mion P, Marcinkiewicz M, Mbikay M, Chrétien M. cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases. DNA Cell Biol 1990, 9: 789.PubMedCrossRefGoogle Scholar
  4. 4.
    Day R, Schafer MK, Watson SJ, Chrétien M, Seidah NG. Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary. Mol Endocrinol 1992, 6: 485–97.PubMedGoogle Scholar
  5. 5.
    Benjannet S, Rondeau N, Day R, Chrétien M, Seidah NG. PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci U S A 1991, 88: 3564–8.PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Raffin-Sanson ML, de Keyzer Y, Bertagna X. Proopiomelanocortin, a polypeptide precursor with multiple functions: from physiology to pathological conditions. Eur J Endocrinol 2003, 149: 79–90.PubMedCrossRefGoogle Scholar
  7. 7.
    Pritchard LE, White A: Neuropeptide processing and its impact on melanocortin pathways. Endocrinology 2007, 148: 4201–7.PubMedCrossRefGoogle Scholar
  8. 8.
    Tateno T, Izumiyama H, Doi M, et al. Differential gene expression in ACTH -secreting and non-functioning pituitary tumors. Eur J Endocrinol 2007, 157: 717–24.PubMedCrossRefGoogle Scholar
  9. 9.
    Tateno T, Izumiyama H, Doi M, Akashi T, Ohno K, Hirata Y. Defective expression of prohormone convertase 1/3 in silent corticotroph adenoma. Endocr J 2007, 54: 777–82.PubMedCrossRefGoogle Scholar
  10. 10.
    Hirata Y, Yamamoto H, Matsukura S, Imura H. In vitro release and biosynthesis of tumor ACTH in ectopic ACTH producing tumors. J Clin Endocrinol Metab 1975, 41: 106–14.PubMedCrossRefGoogle Scholar
  11. 11.
    Vieau D, Seidah NG, Mbikay M, Chretien M, Bertagna X. Expression of the prohormone convertase PC2 correlates with the presence of corticotropin-like intermediate lobe peptide in human adrenocorticotropin-secreting tumors. J Clin Endocrinol Metab 1994, 79: 1503–6.PubMedGoogle Scholar
  12. 12.
    Hale AC, Besser GM, Rees LH. Characterization of pro-opiomelanocortin-derived peptides in pituitary and ectopic adrenocorti-cotrophin-secreting tumours. J Endocrinol 1986, 108: 49–56.PubMedCrossRefGoogle Scholar
  13. 13.
    Tsuchiya K, Minami I, Tateno T, et al. Malignant gastric carcinoid causing ectopic ACTH syndrome: discrepancy of plasma ACTH levels measured by different immunoradiometric assays. Endocr J 2005, 52: 743–50.PubMedCrossRefGoogle Scholar
  14. 14.
    White A, Gibson S: ACTH precursors: biological significance and clinical relevance. Clin Endocrinol (Oxf) 1998, 48: 251–5.CrossRefGoogle Scholar
  15. 15.
    Bertagna XY, Nicholson WE, Sorenson GD, Pettengill OS, Mount CD, Orth DN: Corticotropin, lipotropin, and beta-endorphin production by a human nonpituitary tumor in culture: evidence for a common precursor. Proc Natl Acad Sci U S A 1978, 75: 5160–4.PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Shichiri M, Kato H, Doi M, Marumo F, Hirata Y. Induction of max by adrenomedullin and calcitonin gene-related peptide antagonizes endothelial apoptosis. Mol Endocrinol 1999, 13: 1353–63.PubMedCrossRefGoogle Scholar
  17. 17.
    Tateno T, Nakamura N, Hirata Y, Hirose S. Role of C-terminus of Kir7.1 potassium channel in cell-surface expression. Cell Biol Int 2006, 30: 270–7.PubMedCrossRefGoogle Scholar
  18. 18.
    Matsuno A, Okazaki R, Oki Y, Nagashima T. Secretion of high-molecular-weight adrenocorticotropic hormone from a pituitary adenoma in a patient without Cushing stigmata. Case report. J Neurosurg 2004, 101: 874–7.Google Scholar
  19. 19.
    Yamakita N, Murai T, Oki Y, et al. Adrenal insufficiency after incomplete resection of pituitary macrocorticotropinoma of Cushing’s disease: role of high molecular weight ACTH. Endocr J 2001, 48: 43–51.PubMedCrossRefGoogle Scholar
  20. 20.
    Orth DN, Nicholson WE. High molecular weight forms of human ACTH are glycoproteins. J Clin Endocrinol Metab 1977, 44: 214–7.PubMedCrossRefGoogle Scholar
  21. 21.
    Donald RA. ACTH and related peptides. Clin Endocrinol (Oxf) 1980, 12: 491–524.CrossRefGoogle Scholar
  22. 22.
    Eipper BA, Mains RE. Peptide analysis of a glycoprotein form of adrenocorticotropic hormone. J Biol Chem 1977, 252: 8821–32.PubMedGoogle Scholar
  23. 23.
    Goodman LJ, Gorman CM. Autoproteolytic activation of the mouse prohormone convertase mPC1. Biochem Biophys Res Commun 1994, 201: 795–804.PubMedCrossRefGoogle Scholar
  24. 24.
    Lindberg I, Ahn SC, Breslin MB. Cellular distributions of the prohormone processing enzymes PC1 and PC2. Mol Cell Neurosci 1994, 5: 614–22.PubMedCrossRefGoogle Scholar
  25. 25.
    Milgram SL, Mains RE: Differential effects of temperature blockade on the proteolytic processing of three secretory granule-associated proteins. J Cell Sci 1994, 107: 737–45.PubMedGoogle Scholar
  26. 26.
    Sorenson GD, Pettengill OS, Brinck-Johnsen T, Cate CC, Maurer LH. Hormone production by cultures of small-cell carcinoma of the lung. Cancer 1981, 47: 1289–96.PubMedCrossRefGoogle Scholar

Copyright information

© Italian Society of Endocrinology (SIE) 2010

Authors and Affiliations

  • T. Tateno
    • 1
  • M. Kato
    • 1
  • Y. Tani
    • 1
  • T. Yoshimoto
    • 1
  • Y. Oki
    • 2
  • Y. Hirata
    • 1
  1. 1.Department of Clinical and Molecular EndocrinologyTokyo Medical and Dental University Graduate SchoolHamamatsuJapan
  2. 2.The Second Department of Internal MedicineHamamatsu University School of MedicineHamamatsuJapan

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