Inability of a mouse cell line transformed to produce biologically active recombinant human insulin-like growth factor I (IGF-I) to respond to exogenously added IGF-I |
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Authors: | M A Cascieri N S Hayes B Kelder J J Kopchick G G Chicchi E E Slater M L Bayne |
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Institution: | Merck, Sharp, and Dohme Research Laboratories, Rahway, New Jersey 07065. |
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Abstract: | A plasmid expression vector encoding human insulin-like growth factor I (hIGF-I) in the form of a 97-amino acid precursor protein containing the first 27 amino acids of prebovine GH and the 70 amino acids of hIGF-I has been used to transform mouse L cells. A stably transformed mouse L cell clone has been isolated which expresses and secretes hIGF-I. The secreted peptide comprises 3% of the protein in conditioned medium. IGF-I can be purified to homogeneity in 2 chromatographic steps. One liter of conditioned medium yields approximately 200 micrograms purified peptide. Amino-terminal sequence analysis confirms that the signal peptide has been proteolytically hydrolyzed from the precursor protein before secretion to form Ala0]hIGF-I. The recombinant peptide and serum-derived hIGF-I are equipotent as inhibitors of the binding of 125I]IGF-I to the type 1 receptor of human placenta and to a crude preparation of acid-stable human serum binding proteins. The peptides are equipotent in 2 in vitro assays, the stimulation of the rate of 2-1,2-N-3H]deoxyglucose transport in BC3H1 cells and the stimulation of methyl-3-3H]thymidine incorporation into DNA in A10 cells. In contrast to a control mouse L cell line, DNA synthesis in the Ala0]IGF-I-secreting line is completely unresponsive to Thr59]IGF-I, while it responds normally to calf serum (10%). Thus, the Ala0]IGF-I-secreting line is selectively desensitized to IGF-I. The binding of 125I]IGF-I to both lines is identical, indicating that the loss of responsiveness to IGF-I is not due to a loss of cell surface receptor. The ability to render mouse L cells unresponsive to IGF-I is transferred in the conditioned medium of the Ala0]IGF-I-secreting cell line. In addition, pretreatment of control cells with Thr59]IGF-I (10 nM) results in attenuation of the response to a subsequent dose of IGF-I. These data indicate that prolonged exposure to high levels of IGF-I may cause a postreceptor-mediated desensitization to IGF-I. Alternatively, IGF-I may promote secretion of an inhibitor of IGF-mediated DNA synthesis. |
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