Joyce A. Romanus

Autonomous growth of a human neuroblastoma cell line is mediated by insulin-like growth factor II.

Insulin-like growth factor II (IGF-II) mRNA was increased in two of eight neuroblastomas and in eight of eight pheochromocytomas, tumors of the adrenal medulla that occur in childhood and adulthood, respectively. RNA encoding the type I IGF receptor, the receptor thought to mediate the mitogenic effects of IGF-I and IGF-II, also was uniformly expressed in these cells. To assess the role of IGF-II in the growth of these tumor cells, we have used the SK-N-AS cultured neuroblastoma cell line, which can be continuously propagated in mitogen-free medium, as a model system. Our results strongly suggest that IGF-II, synthesized by SK-N-AS cells and acting through type I IGF receptors, contributes to the autonomous growth of this tumor cell line. (a) SK-N-AS cells synthesized large amounts of IGF-II RNA and secreted greater than 50 ng/ml of IGF-II (as determined by specific radioimmuno- and radioreceptor assays). Little, if any, IGF-I RNA or immunoreactive IGF-I were detected. (b) SK-N-AS cells possess type I IGF receptors. (c) Exogenous IGF-I and IGF-II stimulated DNA synthesis in SK-N-AS cells, and this stimulation was abolished by a blocking antibody to the type I IGF receptor. (d) This anti-receptor antibody also abolished the multiplication of SK-N-AS cells in the absence of added mitogens. We conclude that IGF-II is an autocrine growth factor for SK-N-AS cells and suggest that this mechanism may contribute to the growth of some adrenal medullary tumors.

Receptors for insulinlike growth factor I are defective in fibroblasts cultured from a patient with leprechaunism.

We previously have demonstrated that fibroblasts from a patient with leprechaunism exhibited markedly decreased insulin binding to insulin receptors and that the ability of insulin to stimulate glucose incorporation in the patients cells was greatly impaired. In addition, the insulinlike growth factor, multiplication-stimulating activity (MSA), also exhibited an impaired ability to stimulate glucose incorporation in the patients fibroblasts, although in normal fibroblasts this response appears to be mediated by an insulinlike growth factor receptor. The present study examines 125I-labeled insulinlike growth factor I (IGF-I) binding to patients and control fibroblasts. 125I-labeled IGF-I binds to a specific IGF-I receptor in normal fibroblasts. At steady state, binding was inhibited by unlabeled IGF-I, IGF-II, MSA III-2, MSA II, insulin, and proinsulin, in order of potency, but not by high concentrations of epidermal growth factor and human growth hormone, chemically unrelated polypeptides 125I-labeled IGF-I binding to patients cells was decreased by approximately 75%, whereas binding of epidermal growth factor to its cell surface receptors was unaffected. Computer curve-fitting of untransformed equilibrium binding data suggests that the decreased binding resulted from a decreased Ka for IGF-I. The ability of the patients IGF-I receptor to recognize insulin also appears to be altered. Impaired IGF-I binding by the leprechaun patients fibroblasts may contribute to the abnormal biological response to insulinlike growth factors observed in vitro and to the in utero growth retardation.

Neonatal Rat Islet Cell Cultures Synthesize Insulin-like Growth Factor I

Monolayer cultures of islet B-cells were established from neonatal rat pancreas. Serum-free media conditioned by these cultures for 72 h were concentrated and fractionated on Sephadex G-50 at acid pH into a high-molecular-weight pool containing binding protein for insulin-like growth factors (IGFs) and a low-molecular-weight pool containing IGFs. IGF activity in the IGF pool was demonstrated by a specific radioreceptor assay using rat liver plasma membranes and 125I-labeled rat IGF-II. The IGF in islet cell media was characterized further by radioimmunoassays specific for human IGF-I and for rat IGF-II. Islet cell IGF was identified as predominantly IGF-I or a closely related species and not IGF-II. Levels of ∼15–50 ng IGF-I (based on human IGF-I standard)/106 islet cells accumulated in media after 72 h, and presumably represented synthesis by the islet cells. Concentrations of IGF-I attained in culture media, ∼0.1 ng/mf, were sufficient to stimulate [3H]thymidine incorporation into B-cells. Growth hormone did not consistently increase IGF-I synthesis, suggesting that the previously described effects of growth hormone on islet cell replication do not result from stimulation of IGF-I synthesis by islet cells. Thus, although the IGF-I synthesized by islet cells may be a physiologically relevant growth factor for these cells, the mitogenic effects of growth hormone in islet cells appear to be independent and not mediated by IGF-I.

The 34 kilodalton insulin-like growth factor binding proteins in human cerebrospinal fluid and the A673 rhabdomyosarcoma cell line are human homologues of the rat BRL-3A binding protein

Three members of a family of insulin-like growth factor binding proteins have been identified by nucleotide sequencing of cDNA clones: the binding subunit of the 150 kDa IGF-binding protein complex in human serum, the 30 kDa IGF binding protein in human amniotic fluid, and a 30 kDa binding protein (BP-3A) isolated from the rat BRL-3A cell line. The present study demonstrates by molecular hybridization and immunoreactivity that the human counterpart of rat BP-3A is a 34 kDa IGF binding protein that is present in human cerebrospinal fluid and is synthesized and secreted by the A673 human rhabdomyosarcoma cell line.

[25] Purification of rat insulin-like growth factor II

Publisher Summary Dulak and Temin first described the purification of multiplication-stimulating activity (MSA) or rat IGF-II (rIGF-II) from serum-free medium conditioned by the BRL-3A rat liver cell line. Results of biosynthetic labeling experiments suggest that the various size rIGF-II species are derived from a common pro-rIGF-II (∼20 kDa). The smallest rIGF-II species (MSA-III-2) has the highest specific activity in radioreceptor assays and bioassays, and it is most easily purified to homogeneity. However, greater quantities of the 8700-Da species (MSA-II) are produced by the BRL-3A cells, and a mixture of MSA-II species can be obtained by following a two-step purification procedure. This chapter discusses the culture of the BRL-3A rat liver cell line and the measurement of rIGF-II during purification. The chapter also discusses rat IGF-II purification and evaluates rIGF-II preparations.

Characterization and Cloning of a Rat Insulin-Like Growth Factor Binding Protein

The insulin-like growth factors, IGF-I and IGF-II, occur complexed to specific binding proteins in blood and other extracellular fluids.1 IGF-binding protein complexes of 150 kDa predominate in adult human and rat serum2,3, and also have been observed in human and porcine milk 4,5, and human fibroblast conditioned media6,7. Acid pH irreversibly dissociates the 150 kDa binding protein complex into an ~40 kDa acid-stable binding subunit.1 Recently, Baxter8 provided evidence for the existence of a second subunit of ~100 kDa that is unstable at acid pH and does not bind IGFs.

Insulin-Like Growth Factors in Fetal Growth

The insulin-like growth factors, IGF-I and IGF-II, are single-chain polypeptides chemically related to insulin that are synthesized in multiple fetal and adult tissues, and stimulate DMA synthesis and cell differentiation (1,2). IGF-I is regulated by growth hormone and nutritional factors, and promotes bone elongation in childhood (2). Based on results initially obtained in rats, IGF-II was proposed to play a role in fetal growth and development. This paper will review some of the evidence in support of this hypothesis, and discuss its applicability to human fetal development.

Biosynthesis of Rat Insulinlike Growth Factor II in Intact Cells and Cell-Free Translation

Dulak and Temin(1) first reported that the BRL-3A cell line established from normal rat liver secreted a family of polypeptides, which they termed MSA, that had multiplication-stimulating activity for chick embryo fibroblasts. MSA was purified by Moses and colleagues from conditioned media using Dowex chromatography, Sephadex G-75 gel filtration in 1 M acetic acid, and preparative gel electrophoresis, and shown to appear in multiple forms of Mr 16.3K, 8.7K, and 7.1K.(2) Marquardt et al. (3) purified MSA by a different purification scheme (i.e., Bio-Gel P-10 in 1 M acetic acid and high-performance liquid chromatography), and determined the amino acid sequence of a Mr 7484-dalton form. Mr 7484 MSA appears to correspond to our Mr 7.1K species. It is identical to human IGF-II at 62 of 67 amino acid loci, establishing that BRL-MSA represents the rat homologue of IGF-II.(3)