Marjorie E. Svoboda

Expression of a biologically active analogue of somatomedin-C/insulin-like growth factor I

A synthetic gene coding for an analogue of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) was synthesized by solid support phosphoramidite chemistry and subsequently cloned and expressed in Escherichia coli as a fusion protein. The gene, designed with a threonine codon substituted for a methionine codon at position 59 was expressed fused to an eight-amino acid leader peptide under the direction of the E. coli tryptophan promoter. The fusion protein, termed L0-[Thr59]-Sm-C/IGF-I was purified extensively (greater than 97%) and found to be 60% as active as native Sm-C/IGF-I in a radioimmunoassay and 50% as potent as native Sm-C/IGF-I in a radioreceptor assay. Like native Sm-C/IGF-I it was also mitogenic for Balb/c 3T3 cells. After removal of the eight amino acid leader peptide by cyanogen bromide treatment, the resulting threonine analogue, termed [Thr59]-Sm-C/IGF-I was 80% as potent as native Sm-C/IGF-I in both the RIA and the radioreceptor assays. It was also mitogenic in Balb/c 3T3 cells. These two analogues, therefore, display biological activities similar to human-derived Sm-C/IGF-I.

Efficient pubification of somatomedin-C/insulin-like growth factor I using immunoaffinity chromatography

Somatomedin-C/insulin-like growth factor I was purified from human plasma using a monoclonal antibody affinity column. Combining immunoaffinity chromatography with standard protein purification methods resulted in an overall recovery of 18%. The 35 micrograms of somatomedin-C/insulin-like growth factor I purified from 500 ml of plasma appeared as a single band when analyzed by polyacrylamide gel electrophoresis and could be used in radioimmunoassay and receptor binding studies.

Rodent Studies on the Potential Relevance of Insulin-Like Growth Factor (IGF-I) to Ovarian Physiology

The recurring process of ovarian follicular growth is an exponential rather than a linear process characterized by substantial dramatic proliferation and differentiation of the granulosa cell. Although the pivotal role(s) of gonadotropins and of gonadal steroids in this explosive agenda is well recognized, the variable fate of follicles subject to comparable gonadotropic support suggests the existence of additional intraovarian regulatory mechanism(s). Among potential novel intraovarian regulators, insulin-like growth factor I (IGF-I) has been receiving increasingly intense scrutiny (1–2). Taken together these studies strongly suggest the existence of an intraovarian autocrine control mechanism, wherein IGF-I may serve as the central signal, and the granulosa cell its site of production, reception, and action. Viewed in this light, IGF-I may promote the growth and/or differentiation of the granulosa cell, acting for the most part as an amplifier of gonadotropin action. Granulosa cell-derived IGF-I may also provide paracrine input to the nearby theca-interstitial cell compartment in the interest of coordinated follicular function.

[19] Derivation of monoclonal antibodies to human somatomedin C/insulin-like growth factor I

Somatomedin C, also called insulin-like growth factor I (Sm-C/IGF-I), is a highly conserved polypeptide required for the proliferation of many cell types. Since several attempts in our laboratory to recover monoclonal antibody-secreting hybrids to this peptide by the direct fusion of hyperimmunized splenocytes with myeloma cells had been unsuccessful, we modified our approach by coculturing hyperimmunized BALB/c splenocytes and a small amount of the antigen for 5 days prior to fusion with the P3X63Ag.8.653 myeloma cell line. Of 88 microcultures at risk, specific antibody was detected in 24. Two clones were expanded in ascites fluid and characterized as to isotype, affinity, and specificity. Both were IgG1,kappa and bound human Sm-C/IGF-I with affinity constants of 1.09 and 1.01 X 10(10) liter/mol, respectively. Both clones were quite specific for Sm-C/IGF-I with inconsequential binding to insulin-like growth factor II, multiplication-stimulating activity, any of the chymotryptic fragments of Sm-C/IGF-I, insulin preparations, hGH, hTSH, mEGF, or mouse albumin. In vitro boosting after primary in vivo immunization appears to provide monoclones of an IgG isotype in contrast to primary in vitro immunization, which reportedly favors an IgM isotype. The antibodies produced in this study have proved to be extraordinarily useful in defining the physiologic role of Sm-I/IGF-I with immunoneutralization techniques and in the purification of human Sm-C/IGF-I by affinity chromatography.

Action of Somatomedins on Cell Growth

Research on growth factors wsa initiated in our laboratory with the erroneous expectation that the growth hormone-dependent factor that stimulated DNA synthesis in cartilage would prove different from the growth hormone-dependent “sulfation” factor that stimulated proteoglycan synthesis. The dual-isotope method we established to monitor the separation of these putative factors, however, quickly established that the same humoral signal that stimulated DNA synthesis also stimulated the differentiated functions characteristic of chondrocytes.(1) Since increased synthesis of proteins and proteoglycans could be demonstrated before cell division had occurred, expression of differentiated cell functions was clearly a primary event and not the result of more highly differentiated cells arising as a consequence of cellular proliferation.

Ovarian Insulin-Like Growth Factor I: Basic Concepts Leading to Potential Clinical Outlets

Amongst potential novel intraovarian regulators, insulin-like growth factor I (IGF-I) has been the subject of increasingly intense investigation1. Taken together, these studies strongly suggest the existence of an intraovarian autocrine control mechanism, wherein IGF-I may serve as the central signal, and the granulosa cell its site of production, reception, and action. In doing so, IGF-I may promote the replication and/or cytodifferentiation of the developing granulosa cell, acting largely (but not exclusively) as an amplifier of gonadotropin action. Furthermore, IGF-I (presumptively of granulosa cell origin) may also provide paracrine input to the adjacent theca-interstitial cell compartment in the interest of coordinated follicular development.

100 RECEPTOR BINDING OF SOMATOMEDIN|[sol]|INSULIN-LIKE GROWTH FACTORS (Sm|[sol]|IGFs) ON CULTURED RAT ASTROGLIAL CELLS

In previous studies, we identified binding sites for Sm/IGFs on cultured astroglial cells purified from neonatal rat cerebral cortices by high resolution autoradiography. To further characterize these binding sites, we performed traditional binding studies. Binding for each Sm/IGF is specific, saturable and reversible, and 90% of the binding occurs within 6 hours at 4°C. Competitive binding with Sm-C/IGF-I yields curvilinear Scatchard plots, while studies with IGF-II and MSA yields linear plots, suggesting that two receptor species bind Sm-C/IGF-I and one binds IGF-II and MSA. These findings are supported by affinity crosslinking studies with labeled Sm/IGFs, using disuccinimidyl suberate as a crosslinking agent, followed by solubilization, reduction and analysis on SDS PAGE. 125I-Sm-C/IGF-I binds to moieties with Mr of 260K (the type II receptor) and 130K (α subunit of the type I receptor), while 125I-IGF-II and 125I-MSA binds primarily to the Mr 260K moiety. Concurrent studies of Sm/IGF stimulation of 3H-thymidine incorporation into astroglial cells reveal that both IGF-II and MSA are more potent than Sm-C/IGF-I. These data suggest that astroglial cells possess both type I and type II Sm/IGF receptors and that proliferation in response to Sm/IGFs can be mediated through type II receptors.

53 EFFECT OF A MONOCLONAL ANTIBODY AGAINST THE SOMATO MEDIN|[sol]|INSULIN-LIKE GROWTH FACTOR TYPE I RECEPTOR ON IGF-II BINDING TO HUMAN PLACENTAL MEMBRANES

Human placental membranes (HPM) have been employed in radio-receptor assays (RRA) for somatomedins/insulin-like growth factors (Sm/IGF). Although HPM contain both Sm/IGF Type I and II receptors, previous studies suggest that RRAs using HPM and either 125I-Sm-C/IGF-I or 125I-IGF-II primarily measure interaction with the Type I receptor (Nissley SP, Rechler MM, Clin Endo Metab, 13:43, 1984). We have found that a monoclonal antibody (αIR-3) to the Type I receptor which blocks 125I-Sm-C binding does not block 125I-IGF-II binding to HPM in the RRA. This suggests that the interaction of IGF-II with the Type I receptor is less than predicted by earlier competition studies. To further investigate the nature of IGF-II binding, 125I-IGF-II was covalently crosslinked to HPM, solubilized, reduced and analyzed on SDS-PAGE. Auto radiography revealed a heavily labeled band at Mr 260K (Type II receptor), and a less intense band at Mr 135K. The Mr 135K band migrates nearly identically with the α subunits of both the Type I and insulin receptors; its intensity, however, was not attenuated by α IR-3 or by the presence of 100 ng/ml insulin. Parallel studies with radiolabeled Sm-C showed a marked inhibition of binding to the α subunit in the presence of α IR-3. These results suggest that IGF-II may bind to a 135K moiety that is discrete from previously described receptors. The nature of this moiety has not yet been defined.