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J F Perdue

Publications and source records attributed to J F Perdue.

At least 19 recordsLinked to original sources
PubMed1996

Comparative effects of insulin-like growth factor II (IGF-II) and IGF-II mutants specific for IGF-II/CIM6-P or IGF-I receptors on in vitro hematopoiesis.

This report presents the results of studies investigating the effect of insulin-like growth factor II (IGF-II) on the proliferation and differentiation of CD34+ bone marrow cells in serum-substituted liquid cultures. Bone marrow cells were enriched for CD34+ cells and then placed in liquid cultures supplemented with either interleukin 3 (IL-3) or IL-3 and c-kit ligand with and without the addition of IGF-II. When CD34+ cells were incubated with IL-3, cellularity increased throughout four weeks of culture. Cellularity was twofold greater when cultures also contained IGF-II. IGF-II also promoted an increase in cellularity in cultures with IL-3 and c-kit ligand. In combination with IL-3 or IL-3 and c-kit ligand, IGF-II promoted an earlier differentiation of granulocytes, as well as an increase in the number of megakaryocyte lineage cells. There were approximately two-fold more colony-forming units for granulocytes and macrophages (CFU-GM) and burst-forming units for erythroid cells (BFU-E) in cultures containing both IL-3 and IGF-II than in cultures with IL-3 alone. These results demonstrate that in cytokine-supplemented media, physiological concentrations of IGF-II augmented both the proliferation and differentiation of CD34+ bone marrow cells while maintaining a greater number of progenitor cells. To identify the receptors through which IGF-II enhances in vitro hematopoiesis, IGF-II was substituted with one of the mutant forms of IGF-II that selectively interacts with either IGF-II/CIM6-P receptors or with IGF-I and insulin receptors. The results with the mutant forms of IGF-II demonstrate that IGF-II augments in vitro hematopoiesis primarily through its interaction with IGF-I and possibly insulin receptors, rather than IGF-II/CIM6-P receptors.

Antigens, CD34↗
PubMed1996

The expression and characterization of human recombinant proinsulin-like growth factor II and a mutant that is defective in the O-glycosylation of its E domain.

In humans, newly synthesized proinsulin-like growth factor II (pro-IGF-II), i.e. IGF-II with an E domain extension of 89 amino acids, is 0-glycosylated on Thr75. As an approach to define the role that glycosylation of the E domain serves in the processing, secretion, and biological activities of IGF-II and to identify the sites of endoproteolytic processing, we constructed a mutant that encodes carbohydrate-free prepro-IGF-II. The mutant and wild-type prepro-IGF-II were expressed in NIH-3T3 cells, and the protein products were analyzed by SDS-PAGE followed by immunoblots with antipeptide antibodies to human and homologous rat E domain sequences. Transfectants that express glycosylated pro-IGF-II, i.e. xz97 and G11 cells, have intracellular forms of the growth factor with apparent Mr (appMr) of 21, 23, and 27K. NIH-3T3 xz95 cells, i.e. transfected with DNA that is missing the 0-glycosylation sequence, could also synthesize pro-IGF-II with an appMr of 21K. However, they did not accumulate the 23K and 27K forms of presumably glycosylated growth factor. None of the transfected NIH 3T3 cells processed much pro-IGF-II intracellularly, as the appMr 21K, 23K, and 27K forms had terminal E domain amino acid sequences that were recognized by antibodies to the homologous rat peptide sequence Met117 to Gln156. Subsequent to their secretion, the IGF-II in xz97 and G11 cells accumulated in the conditioned medium mostly as two partially processed species with appMr, of 17K and 14K, respectively. The IGF-II that accumulated in the conditioned medium of the xz95 cells had an appMr of 11K. As evidenced by a decrease in mass after treatment with neuraminidase and 0-glycosidase, the 17-kDa form of pro-IGF-II secreted by the NIH-3T3 xz97 cells was 0-glycosylated, whereas that secreted by the xz95 cells was oligosaccharide free. All of the pro-IGF-II forms have E domain amino acid sequences that reacted with antipeptide Ab to the Asp69 to Lys88 sequence. However, appMr 17K IGF-II, but not 14K IGF-II, also contained a larger E domain that was recognized by Ab to the sequence Phe89 to Arg101. The final step in the processing of 11- to 17-kDa IGF-II at Arg68 and the generation of mature IGF-II did not occur in the NIH-3T3 transfectants and is similar to what has been observed in human embryonic cells and mesenchymal tumors. The failure to remove the glycosylated E domain peptide from appMr, 14K and 17K IGF-II did not affect their binding to IGF-II/cation-independent mannose-6 phosphate receptors or presumably to IGF-I receptors, because in in vitro mitogenic assays they were equipotent with mature IGF-II. Unglycosylated pro-IGF-II from the NIH-3T3 xz95 cells also bound to these receptors. However, it was about 10 times more potent than IGF-II in stimulating thymidine incorporation into NIH-3T3 i24 IGF-IR cells, possibly because of the absence of negatively charged sialic acid and/or steric occlusion.

3T3 Cells↗
PubMed1995

N-terminal deletion mutants of insulin-like growth factor-II (IGF-II) show Thr7 and Leu8 important for binding to insulin and IGF-I receptors and Leu8 critical for all IGF-II functions.

To define the role of the N-terminal region of insulin-like growth factor-II (IGF-II) in its binding to insulin and IGF receptors, deletion mutants des-(1-5)-, des-(1-7)-, and des-(1-8)-recombinant (r) IGF-II, and the Gly8 for Leu substitution mutant of rIGF-II were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The binding affinity and mitogenic activity of these rIGF-II mutants as well as commercially available des-(1-6)-rIGF-II were analyzed. While the relative affinity of des-(1-5)- and des-(1-6)-rIGF-II for purified human insulin and IGF-I receptors remained at > or = 50% levels of that of rIGF-II, the affinity of des-(1-7)-rIGF-II decreased to approximately 10% and approximately 3%, respectively, of that of rIGF-II. When the octapeptide including Leu8 was removed prior to the Cys9-Cys47 intrachain bond, the relative affinity of this deletion mutant, des-(1-8)-rIGF-II, for these receptors dramatically decreased to < 1% of that of rIGF-II. Substituting Gly8 for Leu in rIGF-II decreased the affinity of this mutant for the IGF-I and insulin receptors to about the same extent. These results suggest that the side chains of Thr7 and Leu8 may play an important role in retaining all of the IGF-II functions. Decreases in the relative affinity for binding of the mutants to these receptors paralleled the decreases in their mitogenic potency for cultured Balb/c 3T3 cells. Although the relative affinity of des-(1-8)- or [Gly8]rIGF-II for rat IGF-II/CIM6-P (cation-independent mannose 6-phosphate) receptors was also < 1% of that of rIGF-II, the relative affinities of des-(1-5)-, des-(1-6)-, and des-(1-7)-rIGF-II for these receptors was significantly greater than that of rIGF-II. These results clearly demonstrate that Thr7 and Leu8 are important for binding to insulin and IGF-I receptors and Leu8 is critical for expression of all IGF-II functions.

3T3 Cells↗
PubMed1995

Hypoglycemia in pregnancy secondary to a non-islet cell tumor of the pleura and ectopic insulin-like growth factor II hormone production.

BACKGROUND: In nondiabetic women, pregnancy alone rarely causes clinical hypoglycemia. Non-islet cell tumors have recently been shown to be associated with the production of insulin-like growth factor II (IGF-II) and a paraneoplastic syndrome resulting in hypoglycemia. A case report and review of pathophysiologic mechanisms involved is presented. CASE: A 38-year-old multigravida presented suffering from clinical and biochemical hypoglycemia, which was found to be secondary to a mesothelioma of the pleura and ectopic IGF-II production. Tumor resection was performed during the 13th gestational week. The mother became euglycemic immediately after the surgery and remained asymptomatic. Clinical indicators of pregnancy and an ultrasound scan after the surgery were consistent with a normal viable fetus. CONCLUSION: Symptomatic hypoglycemia and other medical conditions occurring during pregnancy require immediate diagnosis and treatment. In addition to the more common causes, documented cases of medical conditions due to paraneoplastic syndromes of ectopic hormone production during pregnancy have been described. This case establishes the non-islet cell tumor with IGF-II-induced hypoglycemia as another such syndrome to be considered when evaluating hypoglycemia in pregnancy.

Adult↗
PubMed1993

Binding of mutants of human insulin-like growth factor II to insulin-like growth factor binding proteins 1-6.

A family of six specific insulin-like growth factor binding proteins (IGFBPs) modulates the biological actions of the insulin-like growth factors, IGF-I and IGF-II. In the present study, we determined the binding affinity of purified human IGFBPs 1-6 for recombinant human IGF-II mutants whose binding to IGF-I, IGF-II/mannose 6-phosphate, and insulin receptors was previously reported (Sakano, K., Enjoh, T., Numata, F., Fujiwara, H., Marumoto, Y., Higashihashi, N., Sato, Y., Perdue, J. F., and Fujita-Yamaguchi, Y. (1991) J. Biol. Chem. 266, 20626-20635). Of the regions studied, the most important determinants of IGF-II binding to the IGFBPs were A-domain residues 48-50 and B-domain residue 26. Substitution of residues 48-50 with the analogous residues from human insulin (Thr-Ser-Ile) reduced binding to IGFBP-1, -5, and -6 more than 50-fold and to IGFBP-4 by 15-50-fold; binding to IGFBP-2 and -3 was reduced 6-12-fold. The same substitution markedly reduced binding to the IGF-II/mannose 6-phosphate receptor but not to IGF-I or insulin receptors. Although substitution of residues 54 and 55 with the analogous residues from IGF-I (Arg-Arg) abolished binding to the IGF-II/mannose 6-phosphate receptor, binding to IGFBPs was not substantially affected. Substitution of Phe26 with Ser or Leu, which decreased binding to the IGF-I and insulin receptors, reduced binding to IGFBP-1 and -6 up to 80-fold, but had lesser effects on the other IGFBPs. [Leu27]IGF-II and [Leu43]IGF-II, which had a more markedly reduced affinity for the IGF-I and insulin receptors than did [Ser26]IGF-II, were bound by the IGFBPs with relatively unchanged affinity compared with IGF-II. Thus, the determinants of IGF-II binding to IGFBPs partially overlap those for the IGF-II/mannose 6-phosphate receptor and overlap those for the IGF-I receptor to a lesser extent. IGFBP-1 and IGFBP-6 are most sensitive to changes in IGF-II structure, although IGFBP-1 binds IGF-I and IGF-II with equal affinity, whereas IGFBP-6 has a marked preferential binding affinity for IGF-II. IGF-II mutants with selective impairment in recognition by specific IGFBPs or receptors will provide a useful tool for dissecting the role of the different IGF binding macromolecules in the mediation of IGF-II actions.

Amino Acid Sequence↗
PubMed1993

Characterization of new monoclonal antibodies to human insulin-like growth factor-II and their application in western immunoblot analysis.

Four immunoglobulin G1 class monoclonal antibodies (mAbs; 1D5, 1D9, 2B11, and 2H11) were produced against recombinant human insulin-like growth factor-II (rhIGF-II). Enzyme-linked immunosorbent assay established that these four mAbs specifically recognized rhIGF-II and hIGF-II, but not rhIGF-I. mAbs 1D5, 1D9, and 2H11 did not cross-react with mouse rIGF-II, although there are only six amino acid differences between mouse IGF-II and human IGF-II. The epitope for each mAb was partially defined by enzyme-linked immunosorbent assay using mouse-human chimera IGF-II mutants and other IGF-II mutants that were prepared by site-directed mutagenic procedures. These results indicated that the epitopes of mAbs 1D5, 1D9, and 2H11 are in the C-domain of hIGF-II around Ala32 to Ser36, and that of 2B11 is in the carboxyl-terminal end of the B- and A-domains of hIGF-II. A specific and sensitive RIA was developed using mAb 2H11. In this RIA, IGF-II variant ([RLPG/S29]IGF-II) and rhIGF-II competed equally with [125I]IGF-II for binding to mAb 2H11. Similar results were produced when mAbs 1D5, 1D9, and 2B11 substituted for 2H11. The potential usefulness of mAb 2H11 in an immunoblot procedure to characterize the heterogeneity of IGF-II in the sera and tumor tissues of patients with nonislet cell tumor hypoglycemia was evaluated. A procedure that combined acid-ethanol extraction of serum or tumor tissues and immunoaffinity concentration of the extracted IGF-II with mAb 2H11-immobilized resin was found to be an effective way to prepare the samples. In Western immunoblots, a quantity of rhIGF-II as low as 3 ng could be identified, whereas 200 ng rhIGF-I or rat IGF-II were not recognized. The levels of IGF-II in the sera of 12 patients with nonislet cell tumor hypoglycemia varied from normal to about twice normal. The mol wt (M(r)) of this IGF-II was between 10-17K. There was little of the processed 7.5K M(r) IGF-II in the sera of these patients. Finally, the source of the high M(r) forms of IGF-II was the tumor, because the ratios of high M(r) forms of IGF-II to 7.5K IGF-II changed dramatically from 99:1 and 91:9 to 4:96 and 32:68 in two patients after successful excision of their tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Glycosylated insulin-like growth factor II promoted expansion of granulocyte-macrophage colony-forming cells in serum-deprived liquid cultures of human peripheral blood cells.

This report presents the results of studies investigating the effect of a glycosylated form of insulin-like growth factor II with an apparent molecular weight of 15,000 (appM(r) = 15K IGF-II) and one with a molecular weight of 7500 (M(r) = 7.5K IGF-II) on the expansion of granulocyte-macrophage colony-forming cells (GM-CFC) in human peripheral blood cells. Blood cells were enriched for GM-CFC and other CFCs, and liquid cultures of these cells were established in serum-deprived medium supplemented with either interleukin-3 (IL-3) alone (no insulin or IGF added to the medium) or with IL-3 plus M(r) = 7.5K recombinant (r) IGF-II or appM(r) = 15K IGF-II. After incubation for 3 days or 1 week, the blood cells were subcultured in plasma clots, and the number of colonies detected at 7 days (D7) and at 14 days (D14) was used to calculate the number of D7 GM-CFC and D14 GM-CFC. The number of GM-CFC in liquid cultures of blood cells incubated for 3 days with IL-3 alone was similar to the number added at day 0. By 1 week, the number of D14 GM-CFC and D7 GM-CFC had increased to 3.5 +/- 0.9-fold (p = .03) and two- to 50-fold (p = .04) of the number at day 3, respectively. There were 1.5- to six-fold more D7 GM-CFC in cultures of blood cells incubated for 1 week with IL-3 plus either 100 ng/mL M(r) = 7.5K IGF-II or 200 ng/mL appM(r) = 15K IGF-II than after incubation with IL-3 alone. appM(r) = 15K IGF-II also promoted a two-fold increase in the number of D14 GM-CFC. appM(r) = 15K IGF-II promoted a greater increase in D14 GM-CFC than incubation with IL-3 alone even for blood samples in which M(r) = 7.5K IGF-II did not promote such an increase. The results of these studies demonstrate that physiologic concentrations of appM(r) = 15K IGF-II and M(r) = 7.5K IGF-II increased the number of GM-CFC more than IL-3 alone and suggest that appM(r) = 15K IGF-II was more potent than M(r) = 7.5K IGF-II in augmenting IL-3-induced expansion of GM-CFC in serum-deprived liquid cultures of peripheral blood cells.

Blood Cells↗
PubMed1992

The identification of O-glycosylated precursors of insulin-like growth factor II.

A procedure that combined ion exchange, gel permeation, and insulin-like growth factor-binding protein 3 (IGF-BP-3) affinity chromatography with chromatofocusing and reversed-phase high pressure liquid chromatography was used to isolate high molecular weight precursors of human insulin-like growth factor II (IGF-II) from acetic acid extracts of Cohn fraction IV1. Two precursors had isoelectric points (pI) of 5.1 and 5.4 and apparent Mr values of 15,000 and 11,500, respectively. An apparent Mr = 16,000 RLPG/Ser29 variant of IGF-II was also identified in the acetic acid extracts. Amino-terminal amino acid sequencing of the major E domain-containing peptide that had been isolated from apparent Mr = 15,000 IGF-II (pI 5.1), following its digestion with the endoprotease Lys-C, indicated the carboxyl terminus of this precursor was near or at Lys88. During the sequencing of this peptide, a sharply reduced yield of derivatized amino acid occurred at cycle 10, indicating that Thr75 had been posttranslationally modified, possibly by O-glycosylation. To evaluate this possibility, the 125I-labeled high molecular weight IGF-IIs and their endoprotease-generated peptides were treated with glycosidases, and their effects were determined from the change in relative mobilities of the polypeptide and peptides during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Neuraminidase treatment of apparent Mr = 15,000 and 11,500 IGF-II reduced their Mr values to a common value of 10,500. When the desialylated precursors of IGF-II were treated with O-glycosidase, but not when treated with N-glycosidase, the Mr values were reduced further to about 10,000. This was the Mr value that would be predicted for an unglycosylated form of precursor IGF-II that had a carboxyl-terminal end at or near Lys88. When the Ser66-Lys88 endoprotease-generated E domain peptides from pI5.1 and 5.4 high Mr IGF-II were treated with the glycosidases, they had relative mobility changes during sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were similar to those of the intact precursors. Finally, the association of O-linked oligosaccharide with the E domain peptide of IGF-II was confirmed by demonstrating the specificity of binding of the Ser66-Lys88 asialoglycopeptide to jackfruit lectin.

Amino Acid Sequence↗
PubMed1992

Glucose utilization in a patient with hepatoma and hypoglycemia. Assessment by a positron emission tomography.

Tumor glucose use in patients with non-islet-cell tumors has been difficult to measure, particularly in hepatoma, because of hepatic involvement by neoplasm. We studied a patient with nonhepatic recurrence of hepatoma after successful liver transplantation. Tumor tissue contained messenger RNA for insulin-like growth factor-II (IGF-II), and circulating high molecular weight components and E-peptide of IGF-II were increased. Glucose use measured by isotope dilution with [3-3H]glucose was 7.94 mg/kg fat-free mass per min, and splanchnic glucose production was 0.93 mg/kg fat-free mass per min. Glucose uptake and glucose model parameters were independently measured in tissues by positron emission tomography with 18F-fluoro-2-deoxy-D-glucose. Glucose uptake by heart muscle, liver, skeletal muscle, and neoplasm accounted for 0.8, 14, 44, and 15% of total glucose use, respectively. Model parameters in liver and neoplasm were not significantly different, and glucose transport and phosphorylation were twofold and fourfold greater than in muscle. This suggests that circulating IGF-II-like proteins are partial insulin agonists, and that hypoglycemia in hepatoma with IGF-II production is predominantly due to glucose uptake by skeletal muscle and suppression of glucose production.

Adult↗
PubMed1991

The design, expression, and characterization of human insulin-like growth factor II (IGF-II) mutants specific for either the IGF-II/cation-independent mannose 6-phosphate receptor or IGF-I receptor.

Five mutants of recombinant insulin-like growth factor-II (rIGF-II) that bound with high affinity to either the IGF-II/cation-independent mannose 6-phosphate (IGF-II/CIM6-P) or the IGF-I receptor were prepared by site-directed mutagenic procedures, expressed as fusion proteins in the larva of Bombyx mori or Escherichia coli, purified to homogeneity, renatured, and characterized in terms of their receptor binding affinities and specificities as well as their biological activities. Class I mutants in which Phe26, Tyr27, and Val43 were substituted with Ser, Leu, and Leu, respectively, bound to enriched preparations of rat placental IGF-II/CIM6-P receptors with apparent equilibrium dissociation constants (Kd(app)) that were only slightly greater, i.e. 0.10, 0.05, and 0.06 nM, than that of rIGF-II (0.04 nM) or hIGF-II (0.03 nM). In contrast, replacing Phe26 with Ser resulted in 5- and 20-fold decreases in the affinities of this mutant for highly purified human placental IGF-I and insulin receptors, respectively. The affinities of the two other Class I mutants, [Leu27]- and [Leu43]rIGF-IIs, for these two receptors were reduced 80- to 220-fold. The affinities of Class II mutants, i.e. [Thr48,Ser49,Ile50]- and [Arg54,Arg55] rIGF-IIs, for IGF-I receptors were as potent as rIGF-II; however, they bound very poorly or not at all to the IGF-II/CIM6-P receptor. In the binding study of those mutant rIGF-IIs, IGF-II was observed to have an unexpectedly high affinity for pure human placental insulin receptor preparations. For example, the affinities of hIGF-II, rIGF-II, and two Class II rIGF-II mutants for the insulin receptor were only 3-, 9-, and 5-fold less, respectively, than that of porcine insulin. In two biological assay systems, i.e. the stimulation of DNA synthesis in Balb/c 3T3 cells and glycogen synthesis in HepG2 cells, the Kd(app) of the rIGF-II mutants for the IGF-I receptor but not the IGF-II/CIM6-P receptor correlated with their abilities to produce biological responses.

3T3 Cells↗
PubMed1991

Binding specificities and transducing function of the different molecular weight forms of insulin-like growth factor-II (IGF-II) on IGF-I receptors.

In a study that was reported from this laboratory, the mitogenic potency of an apparent mol wt (appMr) of 15,000 precursor form of human insulin-like growth factor-II (hIGF-II) was shown to be greater than that of completely processed hIGF-II for human fetal-derived fibroblasts, and both were more potent than rIGF-I. Since it is generally acknowledged that the stimulation of cell replication by the IGFs is mediated by IGF-I receptors, we undertook to determine whether differences between the receptors' affinity for the two Mr forms of hIGF-II and recombinant IGF-I (rIGF-I) or between its efficiency to couple specific growth factor occupancy to the activation of protein kinase could explain the greater replicating potential of appMr 15,000 hIGF-II. Equilibrium dissociation, i.e. Kd, and inhibition, i.e. Ki, constants were determined by measuring the ability of rIGF-I, hIGF-II, appMr 15,000 hIGF-II, insulin, and the antireceptor monoclonal antibody alpha IR-3 to compete with 125I-labeled rIGF-I and hIGF-II for binding to purified preparations of IGF-I receptors prepared from an enriched source of fetal membrane, i.e. human term placenta. The results of these experiments established that 1) hIGF-II and appMr 15,000 hIGF-II bind to the IGF-I receptor with the same affinity as rIGF-I, e.g. with Kd and Ki values between 0.03-0.07 nM; 2) the total binding capacity, i.e. Ro, for IGF-I binding was not statistically different from the Ro calculated for IGF-II binding; and 3) the statistical analysis of 12 data sets from the competitive binding experiments for goodness of fit indicated that a 1-site model for IGF-I and -II binding was a better fit of the data than a 2-site model. Measurements of the stimulation of IGF-I receptor autophosphorylation at low ligand concentrations established that appMr 15,000 hIGF-II and hIGF-II were more effective than rIGF-I in coupling receptor occupancy to the activation of its protein kinase. At saturating ligand concentrations, the 3 had similar potencies. The original preparation of appMr 15,000 hIGF-II contains a mixture of forms with acidic isoelectric points (pIs) and was more potent than Mr 7,500 IGF-II in stimulating receptor autophosphorylation. These results are consistent with the relative potencies of this preparation, hIGF-II, and rIGF-I in stimulating the replication of 12-week-old fetal dermal fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1989

Purification and characterization of an insulin-like growth factor II variant from human plasma.

An insulin-like growth factor II variant (IGF-II variant) was purified from Cohn fraction IV1 of human plasma by ion exchange, gel filtration, and reversed-phase high pressure liquid chromatography. The amino-terminal sequence of the first 35 amino acid residues showed a replacement of Ser-29 of IGF-II with the tetrapeptide Arg-Leu-Pro-Gly of IGF-II variant. Peptides isolated and sequenced after digestion with endoproteinase Asp-N and endoproteinase Glu-C disclosed no differences with the sequence predicted from an IGF-II variant cDNA clone isolated by Jansen, M., van Shaik, F. M. A., van Tol, H., Van den Brande, J. L., and Sussenbach, J. S. (1985) FEBS Lett., 179, 243-246. The molecular ion of intact IGF-II variant was 7809.4 mass units, as measured by plasma desorption mass spectrometry. This is in close agreement with the molecular ion of 7812.8 mass units calculated from the determined sequence and indicates the entire amino acid sequence had been accounted for. Binding of IGF-II variant to purified insulin-like growth factor I (IGF-I) receptors demonstrated a 2-3-fold lower affinity for this receptor compared with IGF-I or IGF-II. The dissociation constants for IGF-I, IGF-II, and IGF-II variant are 0.23, 0.38, and 0.80 nM, respectively. In a growth assay, the concentration of IGF-II and IGF-II variant required to stimulate the half-maximal growth of MCF-7 cells was 4 and 13 nM, respectively. Finally, the amount of IGF-II variant that can be purified by this method constitutes approximately 25% of the total IGF-II isolated from Cohn fraction IV1 of human plasma.

Amino Acid Sequence↗
PubMed1989

Expression of insulin-like growth factor-II mRNA in fetal kidney and Wilms' tumor. An in situ hybridization study.

The pattern of insulin-like growth factor II (IGF-II) mRNA expression in developing kidney and Wilms' tumor was examined with in situ hybridization. In developing kidney, IGF-II was primarily expressed in blastemal cells and lost with their differentiation. In triphasic Wilms' tumor, a similar relationship was found. But in a monomorphous Wilms', tumor cells with epithelial differentiation expressed IGF-II mRNA. These data suggest that IGF-II may be involved in fetal nephrogenesis, that its expression is inversely coupled to normal epithelial differentiation, and that this differentiation may be aberrantly regulated in Wilms' tumor.

Child, Preschool↗
PubMed1988

Insulin-like growth factor II mRNA expression in human breast cancer.

Insulin-like growth factor II is a growth factor important in fetal development. Several cancer tissues and cell lines have been reported to express IGF-II and rat IGF-II is mitogenic for breast cancer cell lines. Using Northern analysis and ribonuclease protection assays, IGF-II mRNA was detected in normal fibroblasts and in the established breast cancer cell line, T47D. In this cell line, steady state levels of IGF-II message were increased by treatment with estradiol. 10 nM IGF-II, purified from human serum, was mitogenic for breast cancer cell lines. In vitro, IGF-II may act as an autocrine growth factor for some cell lines. RNA derived from breast cancer, pathologically normal breast tissue, and benign breast disease also contained IGF-II mRNA. When paired samples of normal and cancer tissue were obtained from the breast of the same patient, the level of IGF-II mRNA expression in the normal tissue was at least that found in the cancer. This is consistent with previous observations that show IGF-II is expressed in mesenchyme. These findings suggest that in breast cancer IGF-II is produced by stromal tissue elements and potentially by the malignant epithelial cells. Therefore, IGF-II may function as an autocrine or a paracrine growth factor in different breast tumors.

Blotting, Northern↗
PubMed1987

Presence of somatomedin receptors on primary human breast and colon carcinomas.

Competitive binding techniques were used to study the interaction of insulin-like growth factor I (IGF-I) with a plasma membrane-enriched subcellular fraction purified from primary breast and colon carcinoma specimens obtained at surgery. The presence of specific binding sites for IGF-I was detected in all tumour specimens studied. Scatchard analysis and competition studies with insulin and insulin-like growth factor-II (IGF-II) revealed the presence of specific IGF-I receptors, showing a Kd-value of approximately 2 nM. These results are consistent with the hypothesis that somatomedins play a role in determining the proliferative behaviour of human breast and colon tumors, and suggest that recent laboratory studies showing dependence of neoplastic cells on somatomedins for optimum proliferation may have clinical relevance.

Breast Neoplasms↗
PubMed1987

Purification and characterization of a unique high molecular weight form of insulin-like growth factor II.

A form of insulin-like growth factor II (IGF-II) with a mol wt of 15,000 has been purified to homogeneity from human Cohn fraction IV1-4. This protein has an amino-terminal sequence through the first 28 residues that is identical to 7.5K IGF-II. The amino acid composition of 15K IGF-II, however, indicates that its carboxyl-terminal region may be different from that predicted from the analysis of IGF-II cDNA clones. The affinities of 15K IGF-II for receptors on rat placental membranes and for an IGF-binding protein that was isolated from the medium of cultured buffalo rat liver cells were similar to those of the 7.5K form of the growth factor. A best-fit analysis of data from the binding of the two mol wt forms of IGF-II to receptors on rat placental membranes by the LIGAND program was consistent with a model in which 7.5K and 15K IGF-II bound to one site with Kd values of 0.27 +/- 0.03 and 0.38 +/- 0.04, respectively. There was an indication that 15K IGF-II also bound to a second low affinity site on the membrane. In mitogenesis assays performed on human fibroblasts isolated from the skin of two fetuses of an early gestational age, 15K IGF-II stimulated the incorporation of [3H]thymidine into DNA at a half-maximal concentration, i.e. ED50, of 5.7 and 5.0 nM. In these experiments, the ED50 values for 7.5K IGF-II were 8.7 and 15 nM.

Amino Acid Sequence↗