title: Human IM-9 lymphoblasts as a model of the growth hormone-insulin-like growth factor axis: gene expression, and interactions of ligands with receptors and binding proteins.
abstract: Human IM-9 lymphoblasts bind growth hormone (hGH) and insulin-like growth factors (IGFs). We have systematically examined the IM-9 cells as a valuable model of the interaction of hGH and the IGFs at the cellular level. Cells were cultured in medium with 10% serum and for a subset of experiments cultured in serum-free medium. Binding of [125I]hGH and [125I]IGF-I and -II to intact IM-9 cells was measured: unlabeled hGH inhibited binding of [125I]hGH (half max. 20 ng/ml). Binding of [125I]IGF-I was inhibited by IGF-I (half max. 7.5 ng/ml), IGF-II (half max. 60 ng/ml), and insulin and anti IGF-I receptor antibody (alpha IR3). [125I]IGF-II was inhibited by IGF-II (half max. 15 ng/ml), IGF-I (half max. 500 ng/ml), insulin (half max. 250 ng/ml) but not by alpha IR3. Crosslinking experiments with [125I]IGF-II and DSS as the crosslinking agent and analysis of radioligand-receptor complexes by SDS-PAGE under reducing conditions revealed that [125I]IGF-II bound to a 250 kDa and a 135 kDa receptor species. The latter possibly represents an insulin-type receptor whereas the 250 kDa species had the characteristics of the IGF-II/M6P receptor. When IM-9 cell conditioned medium was analyzed in ligand blotting experiments with either [125I]IGF-I or -II a 30 kDa IGFBP species was detected on the autoradiographs. Also, IGF-II immunoreactivity (approx. 1 ng/ml medium) was measured in the cell conditioned medium using an IGF-BP blocked RIA employing [125I]IGF-II. In a subset of experiments IM-9 cells were homogenized in 4 M guanidinium-thiocyanate and RNA extracted in 5.7 M CsCl. Denatured RNA was electrophoresed on 0.8% agarose gels and transferred to a nylon membrane, fixed and the blots hybridized with cDNA probes. Probes were labeled with [32P]dCTP using a random prime labeling procedure: a Pst I 700 bp fragment of the human IGF-I cDNA, a 554 bp Pst I-Sal I fragment of the IGF-II cDNA, a 614 bp Pst I fragment of the IGF-I receptor cDNA and a 663 bp Pst I fragment of the IGF-II/M6P receptor. Autoradiographs of Northern blots showed specific hybridization with the IGF-I probe at 3.7 kb and with the IGF-II probe at 5.3 kb. No signal was detected with the IGF-I receptor cDNA probe. Hybridization with the IGF-II/M6P receptor probe yielded a 9 kb RNA species.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Children's Hospital, Department of Paediatric Endocrinology, University of Munich, Germany.
authors.forename: Y
authors.initials: Y
authors.lastname: Yang
authors.forename: A
authors.initials: A
authors.lastname: Hoeflich
authors.forename: U
authors.initials: U
authors.lastname: Kessler
authors.forename: B
authors.initials: B
authors.lastname: Barenton
authors.forename: W
authors.initials: W
authors.lastname: Blum
authors.forename: H P
authors.initials: HP
authors.lastname: Schwarz
authors.forename: W
authors.initials: W
authors.lastname: Kiess
mesh_terms: D001667:Binding, Competitive; D015152:Blotting, Northern; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D013006:Growth Hormone / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000378:metabolism* / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008214:Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D011869:Radioligand Assay; D017526:Receptor, IGF Type 1 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017527:Receptor, IGF Type 2 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D007328:Insulin; D017527:Receptor, IGF Type 2; D011986:Receptors, Somatotropin; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D013006:Growth Hormone; D017526:Receptor, IGF Type 1