title: Loss of ACTH expression in cultured human corticotroph macroadenoma cells is consistent with loss of the POMC gene signal sequence.
abstract: The proopiomelanocortin (POMC) gene is highly expressed in the pituitary gland where the resulting mRNA of 1200 base pairs (bp) gives rise to a full-length protein sequence. In peripheral tissues however both shorter and longer POMC variants have been described, these include for example placental tissue which contain 800 (truncated at the 5' end) and 1500 as well as the 1200 bp transcripts. The importance of the 800 bp transcript is unclear as the lack of a signal sequence renders the molecule to be non-functional. This transcript has not been previously demonstrated in the pituitary gland. In this report we show evidence of a 5' truncated POMC gene in human pituitary corticotroph macroadenoma cells (JE) maintained in primary culture for >1 year. The original tumour tissue and the derived cells during early passage (up to passage 4-5) immunostained for ACTH and in situ hybridisation confirmed the presence of the POMC gene in the cultured cells. These cells also secreted 15-40 pg/10(5) cells/24 h ACTH. In addition, as expected RT-PCR demonstrated the presence of all three POMC gene exons and is thus indicative of a full-length POMC gene. In late culture passages (passages 8-15) JE cells ceased to express ACTH and cell growth became very slow due presumably to cells reaching their Hayflick limit. ACTH immunostaining in these cells was undetectable and ACTH secretion was also at the detection limits of the assay and no greater than 10 pg/10(5) cells/24 h. ACTH precursor molecules were also undetectable. RT-PCR for the POMC gene in these late passage cells showed that only exon 3 was detectable, in contrast to early passage cells where all three exons were present. In summary we isolated in culture, human pituitary cells that possessed initially all three exons of the POMC gene and immunostained for ACTH. On further passaging these cells showed a loss of exons 1 and 2 in the POMC gene and a loss of ACTH immunostaining and secretion. We would like to suggest that the loss of ACTH peptide expression in these late passage cells is in part due to the loss of the POMC signal sequence. An alternative explanation for our findings is that there were originally two populations of corticotrophs in the cultures, one of which possessed the full-length POMC gene and the other only the 5' truncated POMC transcript and it is these latter cells which survived in culture. In either scenario this is the first report of the 5' truncated POMC gene occurring in pituitary cells.
authors.affiliation: Department of Medicine, University of Wales College of Medicine, Heath Park, CF14 4XN, Cardiff, UK.
authors.forename: D A
authors.initials: DA
authors.lastname: Rees
authors.forename: P J
authors.initials: PJ
authors.lastname: Hepburn
authors.forename: A M
authors.initials: AM
authors.lastname: McNicol
authors.forename: K
authors.initials: K
authors.lastname: Francis
authors.forename: B
authors.initials: B
authors.lastname: Jasani
authors.forename: M D
authors.initials: MD
authors.lastname: Lewis
authors.forename: W E
authors.initials: WE
authors.lastname: Farrell
authors.forename: B M
authors.initials: BM
authors.lastname: Lewis
authors.forename: M F
authors.initials: MF
authors.lastname: Scanlon
authors.forename: J
authors.initials: J
authors.lastname: Ham
mesh_terms: D000236:Adenoma; D000324:Adrenocorticotropic Hormone / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005091:Exons / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization; D010902:Pituitary Gland / Q000166:cytology*; D010911:Pituitary Neoplasms; D011333:Pro-Opiomelanocortin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone