HUMAN IM-9 LYMPHOBLASTS AS A MODEL OF THE GROWTH HORMONE-INSULIN-LIKEGROWTH-FACTOR AXIS - GENE-EXPRESSION, AND INTERACTIONS OF LIGANDS WITH RECEPTORS AND BINDING-PROTEINS

Human IM-9 lymphoblasts bind growth hormone (hGH) and insulin-like gro wth factors (IGFs). We have systematically examined the IM-9 cells as a valuable model of the interaction of hGH and the IGFs at the cellula r level. Cells were cultured in medium with 10% serum and for a subset of experiments cultured in serum-free medium. Binding of [I-125]hGH a nd [I-125]IGF-I and -II to intact IM-9 cells was measured: unlabeled h GH inhibited binding of [I-125]hGH (half max. 20 ng/ml). Binding of [I -125]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 (alphaIR3 ). [I-125]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 alphaIR 3. Crosslinking experiments with [I-125]IGF-II and DSS as the crosslin king agent and analysis of radioligand-receptor complexes by SDS-PAGE under reducing conditions revealed that [I-125]IGF-II bound to a 250 k Da and a 135 kDa receptor species. The latter possibly represents an i nsulin-type receptor whereas the 250 kDa species had the characteristi cs of the IGF-II/M6P receptor. When IM-9 cell conditioned medium was a nalyzed in ligand blotting experiments with either [I-125]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 [I-125]IGF-I I. In a subset of experiments IM-9 cells were homogenized in 4 M guani dinium-thiocyanate and RNA extracted in 5.7 M CsCl. Denatured RNA was electrophoresed on 0.8% agarose gels and transferred to a nylon membra ne, fixed and the blots hybridized with cDNA probes. Probes were label ed with [P-32]dCTP using a random prime labeling procedure: a Pst I 70 0 bp fragment of the human IGF-I cDNA, a 554 bp Pst I-Sal I fragment o f 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. Autoradiograph s 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 Northe rn blots were also detected with a labeled 650 bp Hind III-Xba I fragm ent 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 furt her insight into the relationship between these classes of hormones an d binding proteins.