Affordable Access

Publisher Website

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

Regulatory Peptides
Publication Date
DOI: 10.1016/0167-0115(93)90334-5
  • Insulin-Like Growth Factor
  • Insulin-Like Growth Factor Receptor
  • Im-9 Lymphoblast
  • Growth Hormone
  • Growth Hormone Receptor
  • Insulin-Like Growth Factor Binding Protein
  • Biology


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 (αIR3). [ 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 αIR3. 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. Specific signals on Northern blots were also detected with a labeled 650 bp Hind III- Xba I fragment of the hGH cDNA and a 670 bp Eco RI- Hind III fragment of the human GH receptor cDNA. In conclusion, IM-9 cells express many constituents of the complex hGH-IGF axis including ligands, receptors and binding proteins. We propose that the study of the IM-9 cells can provide further insight into the relationship between these classes of hormones and binding proteins.

There are no comments yet on this publication. Be the first to share your thoughts.