An amphotropic retroviral vector expressing a mutant gsp oncogene: Effects on human thyroid cells in vitro

Mircea Ivan, M. Ludgate, V. Gire, J. A. Bond, D. Wynford-Thomas

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Point mutations of the gsp protooncogene (encoding the α-subunit of the G(s) protein) that constitutively activate the cAMP signaling pathway are a common feature of and a plausible causative mechanism for thyroid hyperfunctioning adenomas (hot nodules). To investigate the extent to which mutant gsp acting alone can induce proliferation of thyroid follicular cells, we generated an amphotropic retroviral vector (based on the pBABE-neo plasmid and psi-CRIP packaging line) to permit stable introduction of a hemagglutinin tagged Gln227→Leu mutant gsp gene into normal human thyrocytes in vitro. The biological activity of the vector was confirmed by detection of HA- tagged Gsp protein expression and induction of cAMP synthesis in selected target cells. Normal human thyroid follicular cells in primary monolayer culture were infected with the gsp retroviral vector or with corresponding vectors expressing mutant H-ras or neo only as positive and negative controls, respectively. Although, as before, mutant ras generated 10-20 well differentiated epithelial colonies/dish of 105 infected cells, with an average lifespan of 15-20 population doublings, only small groups of no more than 15-50 differentiated thyrocytes were observed with the gsp vector. In addition to standard conditions (10% FCS), infections were performed in reduced serum (1% FCS, TSH, and insulin), in the presence of isobutylylmethylxanthine, or in the presence of agents capable of closing gap junctions, with no significant difference in outcome. Although little or no proliferative response was observed regardless of the conditions, there was clear evidence of morphological response (rearrangement of the actin cytoskeleton and increased cell size). The results suggest that gsp mutation may not be a sufficient proliferogenic stimulus by itself to account for hot nodule formation.

Original languageEnglish (US)
Pages (from-to)2702-2709
Number of pages8
JournalJournal of Clinical Endocrinology and Metabolism
Volume82
Issue number8
DOIs
StatePublished - 1997
Externally publishedYes

Fingerprint

Oncogenes
Thyroid Gland
Gap Junctions
Hemagglutinins
Product Packaging
Actin Cytoskeleton
Cell Size
Point Mutation
Bioactivity
Proteins
Plasmids
Insulin
Actins
Monolayers
Packaging
Mutation
Genes
In Vitro Techniques
Thyroid Epithelial Cells
Infection

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology, Diabetes and Metabolism

Cite this

An amphotropic retroviral vector expressing a mutant gsp oncogene : Effects on human thyroid cells in vitro. / Ivan, Mircea; Ludgate, M.; Gire, V.; Bond, J. A.; Wynford-Thomas, D.

In: Journal of Clinical Endocrinology and Metabolism, Vol. 82, No. 8, 1997, p. 2702-2709.

Research output: Contribution to journalArticle

@article{30fdad86a51a424fafe70def094d0793,
title = "An amphotropic retroviral vector expressing a mutant gsp oncogene: Effects on human thyroid cells in vitro",
abstract = "Point mutations of the gsp protooncogene (encoding the α-subunit of the G(s) protein) that constitutively activate the cAMP signaling pathway are a common feature of and a plausible causative mechanism for thyroid hyperfunctioning adenomas (hot nodules). To investigate the extent to which mutant gsp acting alone can induce proliferation of thyroid follicular cells, we generated an amphotropic retroviral vector (based on the pBABE-neo plasmid and psi-CRIP packaging line) to permit stable introduction of a hemagglutinin tagged Gln227→Leu mutant gsp gene into normal human thyrocytes in vitro. The biological activity of the vector was confirmed by detection of HA- tagged Gsp protein expression and induction of cAMP synthesis in selected target cells. Normal human thyroid follicular cells in primary monolayer culture were infected with the gsp retroviral vector or with corresponding vectors expressing mutant H-ras or neo only as positive and negative controls, respectively. Although, as before, mutant ras generated 10-20 well differentiated epithelial colonies/dish of 105 infected cells, with an average lifespan of 15-20 population doublings, only small groups of no more than 15-50 differentiated thyrocytes were observed with the gsp vector. In addition to standard conditions (10{\%} FCS), infections were performed in reduced serum (1{\%} FCS, TSH, and insulin), in the presence of isobutylylmethylxanthine, or in the presence of agents capable of closing gap junctions, with no significant difference in outcome. Although little or no proliferative response was observed regardless of the conditions, there was clear evidence of morphological response (rearrangement of the actin cytoskeleton and increased cell size). The results suggest that gsp mutation may not be a sufficient proliferogenic stimulus by itself to account for hot nodule formation.",
author = "Mircea Ivan and M. Ludgate and V. Gire and Bond, {J. A.} and D. Wynford-Thomas",
year = "1997",
doi = "10.1210/jc.82.8.2702",
language = "English (US)",
volume = "82",
pages = "2702--2709",
journal = "Journal of Clinical Endocrinology and Metabolism",
issn = "0021-972X",
publisher = "The Endocrine Society",
number = "8",

}

TY - JOUR

T1 - An amphotropic retroviral vector expressing a mutant gsp oncogene

T2 - Effects on human thyroid cells in vitro

AU - Ivan, Mircea

AU - Ludgate, M.

AU - Gire, V.

AU - Bond, J. A.

AU - Wynford-Thomas, D.

PY - 1997

Y1 - 1997

N2 - Point mutations of the gsp protooncogene (encoding the α-subunit of the G(s) protein) that constitutively activate the cAMP signaling pathway are a common feature of and a plausible causative mechanism for thyroid hyperfunctioning adenomas (hot nodules). To investigate the extent to which mutant gsp acting alone can induce proliferation of thyroid follicular cells, we generated an amphotropic retroviral vector (based on the pBABE-neo plasmid and psi-CRIP packaging line) to permit stable introduction of a hemagglutinin tagged Gln227→Leu mutant gsp gene into normal human thyrocytes in vitro. The biological activity of the vector was confirmed by detection of HA- tagged Gsp protein expression and induction of cAMP synthesis in selected target cells. Normal human thyroid follicular cells in primary monolayer culture were infected with the gsp retroviral vector or with corresponding vectors expressing mutant H-ras or neo only as positive and negative controls, respectively. Although, as before, mutant ras generated 10-20 well differentiated epithelial colonies/dish of 105 infected cells, with an average lifespan of 15-20 population doublings, only small groups of no more than 15-50 differentiated thyrocytes were observed with the gsp vector. In addition to standard conditions (10% FCS), infections were performed in reduced serum (1% FCS, TSH, and insulin), in the presence of isobutylylmethylxanthine, or in the presence of agents capable of closing gap junctions, with no significant difference in outcome. Although little or no proliferative response was observed regardless of the conditions, there was clear evidence of morphological response (rearrangement of the actin cytoskeleton and increased cell size). The results suggest that gsp mutation may not be a sufficient proliferogenic stimulus by itself to account for hot nodule formation.

AB - Point mutations of the gsp protooncogene (encoding the α-subunit of the G(s) protein) that constitutively activate the cAMP signaling pathway are a common feature of and a plausible causative mechanism for thyroid hyperfunctioning adenomas (hot nodules). To investigate the extent to which mutant gsp acting alone can induce proliferation of thyroid follicular cells, we generated an amphotropic retroviral vector (based on the pBABE-neo plasmid and psi-CRIP packaging line) to permit stable introduction of a hemagglutinin tagged Gln227→Leu mutant gsp gene into normal human thyrocytes in vitro. The biological activity of the vector was confirmed by detection of HA- tagged Gsp protein expression and induction of cAMP synthesis in selected target cells. Normal human thyroid follicular cells in primary monolayer culture were infected with the gsp retroviral vector or with corresponding vectors expressing mutant H-ras or neo only as positive and negative controls, respectively. Although, as before, mutant ras generated 10-20 well differentiated epithelial colonies/dish of 105 infected cells, with an average lifespan of 15-20 population doublings, only small groups of no more than 15-50 differentiated thyrocytes were observed with the gsp vector. In addition to standard conditions (10% FCS), infections were performed in reduced serum (1% FCS, TSH, and insulin), in the presence of isobutylylmethylxanthine, or in the presence of agents capable of closing gap junctions, with no significant difference in outcome. Although little or no proliferative response was observed regardless of the conditions, there was clear evidence of morphological response (rearrangement of the actin cytoskeleton and increased cell size). The results suggest that gsp mutation may not be a sufficient proliferogenic stimulus by itself to account for hot nodule formation.

UR - http://www.scopus.com/inward/record.url?scp=0030754654&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030754654&partnerID=8YFLogxK

U2 - 10.1210/jc.82.8.2702

DO - 10.1210/jc.82.8.2702

M3 - Article

C2 - 9253357

AN - SCOPUS:0030754654

VL - 82

SP - 2702

EP - 2709

JO - Journal of Clinical Endocrinology and Metabolism

JF - Journal of Clinical Endocrinology and Metabolism

SN - 0021-972X

IS - 8

ER -