Loss of ACTH expression in cultured human corticotroph macroadenoma cells is consistent with loss of the POMC gene signal sequence

D. A. Rees, P. J. Hepburn, A. M. McNicol, K. Francis, B. Jasani, M. D. Lewis, W. E. Farrell, B. M. Lewis, M. F. Scanlon, J. Ham

Research output: Contribution to journalArticle

9 Citations (Scopus)

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/105 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/105 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.

Original languageEnglish
Pages (from-to)51-57
Number of pages7
JournalMolecular and Cellular Endocrinology
Volume189
Issue number1-2
DOIs
Publication statusPublished - Mar 28 2002
Externally publishedYes

Fingerprint

Corticotrophs
Pro-Opiomelanocortin
Protein Sorting Signals
Adrenocorticotropic Hormone
Genes
Exons
Base Pairing
Tissue
Pituitary Gland
Molecules
Cell growth
Polymerase Chain Reaction
Cell culture

Keywords

  • ACTH
  • Culture
  • Exon
  • Human pituitary
  • POMC
  • Signal

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Loss of ACTH expression in cultured human corticotroph macroadenoma cells is consistent with loss of the POMC gene signal sequence. / Rees, D. A.; Hepburn, P. J.; McNicol, A. M.; Francis, K.; Jasani, B.; Lewis, M. D.; Farrell, W. E.; Lewis, B. M.; Scanlon, M. F.; Ham, J.

In: Molecular and Cellular Endocrinology, Vol. 189, No. 1-2, 28.03.2002, p. 51-57.

Research output: Contribution to journalArticle

Rees, DA, Hepburn, PJ, McNicol, AM, Francis, K, Jasani, B, Lewis, MD, Farrell, WE, Lewis, BM, Scanlon, MF & Ham, J 2002, 'Loss of ACTH expression in cultured human corticotroph macroadenoma cells is consistent with loss of the POMC gene signal sequence', Molecular and Cellular Endocrinology, vol. 189, no. 1-2, pp. 51-57. https://doi.org/10.1016/S0303-7207(01)00755-9
Rees, D. A. ; Hepburn, P. J. ; McNicol, A. M. ; Francis, K. ; Jasani, B. ; Lewis, M. D. ; Farrell, W. E. ; Lewis, B. M. ; Scanlon, M. F. ; Ham, J. / Loss of ACTH expression in cultured human corticotroph macroadenoma cells is consistent with loss of the POMC gene signal sequence. In: Molecular and Cellular Endocrinology. 2002 ; Vol. 189, No. 1-2. pp. 51-57.
@article{1de271dfac8d4aa89fb651f8fa2840ab,
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/105 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/105 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.",
keywords = "ACTH, Culture, Exon, Human pituitary, POMC, Signal",
author = "Rees, {D. A.} and Hepburn, {P. J.} and McNicol, {A. M.} and K. Francis and B. Jasani and Lewis, {M. D.} and Farrell, {W. E.} and Lewis, {B. M.} and Scanlon, {M. F.} and J. Ham",
year = "2002",
month = "3",
day = "28",
doi = "10.1016/S0303-7207(01)00755-9",
language = "English",
volume = "189",
pages = "51--57",
journal = "Molecular and Cellular Endocrinology",
issn = "0303-7207",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - Loss of ACTH expression in cultured human corticotroph macroadenoma cells is consistent with loss of the POMC gene signal sequence

AU - Rees, D. A.

AU - Hepburn, P. J.

AU - McNicol, A. M.

AU - Francis, K.

AU - Jasani, B.

AU - Lewis, M. D.

AU - Farrell, W. E.

AU - Lewis, B. M.

AU - Scanlon, M. F.

AU - Ham, J.

PY - 2002/3/28

Y1 - 2002/3/28

N2 - 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/105 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/105 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.

AB - 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/105 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/105 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.

KW - ACTH

KW - Culture

KW - Exon

KW - Human pituitary

KW - POMC

KW - Signal

UR - http://www.scopus.com/inward/record.url?scp=0037187333&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037187333&partnerID=8YFLogxK

U2 - 10.1016/S0303-7207(01)00755-9

DO - 10.1016/S0303-7207(01)00755-9

M3 - Article

VL - 189

SP - 51

EP - 57

JO - Molecular and Cellular Endocrinology

JF - Molecular and Cellular Endocrinology

SN - 0303-7207

IS - 1-2

ER -