The distribution, ultrastructure, and chemistry of microfilaments in cultured chick embryo fibroblasts.

The distribution, ultrastructure, and chemistry of microfilaments in cultured chick embryo fibroblasts were studied by thin sectioning of flat-embedded untreated and glycerol-extracted cells, histochemical and immunological electron microscopic procedures, and the negative staining of cells cultured on electron microscopic grids. In these cultured cells, the microfilaments are arranged into thick bundles that are disposed longitudinally and in looser arrangements in the fusiform-shaped cells. In the latter case, they are concentrated along the margins of the flattened cell, on the dorsal surface, and particularly at the ends of the cell and its ventral surface, where contact is made with the plastic dish or with other cells. Extracellular filaments, presumably originating from within the cell, are found at these points of contact. The microfilaments are composed in part of an actin-like protein. These filaments are between 70 and 90 A in diameter, they are stable in 50% glycerol, they have an endogenous ATPase (myosin-like?) associated with them, they bind rabbit muscle heavy meromyosin, and they specifically bind antibody directed against isolated actin-like protein. In the cultured chick embryo fibroblasts, the microfilaments are essential for the establishment and maintenance of form, and they are probably critical elements for adhesion and motility. The microfilaments might also serve as stabilizers of intramembranous particle fluidity.

Ultrastructural distribution of cell surface antigens in avian tumor virus-infected chick embryo fibroblasts.

The distribution of neoantigens in the surface membrane of avian tumor virus-infected chicken embryo fibroblasts was examined on carbon replicas of cell cultures using hemocyanin-labeled antibody. New determinants appearing on the cell surface of virally infected but not transformed cells are thought to be common with components of the viral envelope. These antigens were found to exist in a diffuse, random array on the dorsal cell surface, with a denser accumulation along the cell processes. In living cells, surface antigens are capable of several types of redistribution when activated by reaction with antibody. Leukosis virus-infected (non-transformed) cells showed two apparently independent modes of redistribution: a relocation of some antibody-related sites to the cell margin; or an involvement of essentially all sites in randomly dispersed aggregates. Viral antigenic sites on sarcoma virus-infected (transformed) cells, reacted with antibody, were able to produce weak marginal relocation; but revealed a more striking tendency to migrate to some central location. The centripetal coalescence thus formed resembles the "cap" noted in other systems. Prior aggregation into "patches" may not be a prerequisite for such cap formation. Tumor-specific surface antigen detection and mapping was attempted by this technique, but results were equivocal. An antigen possibly characteristic of rapidly dividing cells occurred in a sparse, diffuse fashion over the surface of morphologically distinct "round" cells.

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.

Failure to confirm evidence for a nonvirion tumor-specific surface antigen in avian retrovirus-transformed cells.

Triton X-100 or Nonidet P40-deoxycholate extracts of [3H]fucose-labeled Rous sarcoma virus-transformed chick embryo fibroblasts were examined by indirect immunoprecipitation for the presence of a tumor-specific neoantigen of 100,000 daltons. Extracts were incubated with immune IgG from Rous tumor-sensitized chickens, and the resultant antigen-antibody complexes were precipitated with rabbit antichicken IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactivity appeared between the migration positions of proteins having molecular weights of 65,000 and 95,000 daltons and at about 30,000 daltons. These antigens were group-specific, and thei precipitation could be inhibited by competition with extracts from cultured fibroblasts that had been infected with a nontransforming avian leukosis virus. They were not precipitated with IgG from unimmunized chickens or chickens immunized with the culture supernatants of uninfected chick embryo fibroblasts. In contrast to results reported recently, the present results could not confirm by immunoprecipitation, the existence of a tumor neoantigen different from that associated with viral components. However, the tumor-specific antigen possibly existed on the cell surface but was not preserved in these detergent extracts.

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.

Stereospecific D-glucose transport in mixed membrane and plasma membrane vesicles derived from cultured chick embryo fibroblasts.

Mixed membrane vesicles prepared from cultured chick embryo fibroblasts possess a stereospecific D-glucose transport system, the properties of which are identical to those of the system in intact cells. Uptake of D-glucose proceeds without chemical alteration. The rate of stereospecific uptake of D-glucose into the mixed vesicles is 70% greater than that of the homogenate and uptake is directly proportional to membrane protein concentration. Stereospecific D-glucose uptake appears linear for 0.3 min, reaches a maximum at 2--5 min, and declines to zero by 5 h as L-glucose enters the vesicles. Uptake is osmotically sensitive and inhibited by cytochalasin B (Ki = 0.13 microM) and the structural analogues of D-glucose : D-mannose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, D-galactose and maltose, but not by sucrose of L-glucose. Uphill counterflow can be demonstrated and the apparent activation energy displays a transition from 47.7 kcal/mol below 11 degrees C to 18.1 kcal/mol above 11 degrees C. Stereospecific uptake rates of mixed vesicles prepared from Rous sarcoma virus-transformed cells are increased 30% over control values, and are increased 66% in vesicles derived from cells incubated for 24 h in glucose-free medium. Plasma membrane vesicles prepared from these cells by a dextran cushion centrifugation procedure display a 9-fold increase in the specific activity of stereospecific D-glucose uptake relative to the homogenate. Extraction of these membranes with dimethylmaleic anhydride (5 mg/mg protein) results in substantial or complete removal of major polypeptides of molecular weight 40 000, 55 000, 75 000, 78 000 and 200 000 with no loss in total uptake activity. Following extraction, major polypeptides of molecular weight 28 000, 33 000 and 68 000 remain in the membrane residue.

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.

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.

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.

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)

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)

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.

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.

An airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors.

A method is described in which high-speed centrifugation of membranes through an oil phase is used to separate membrane-bound and detergent-solubilized polypeptide receptor-iodinated ligand complexes from unbound ligands. Three centrifuges, the Brinkmann Eppendorf (5412), the Beckman Microfuge B and the Beckman Airfuge were evaluated for this capability. Under the conditions described, the Beckman Airfuge surpassed the others in recovering previously 125I- and 32P-labelled cell membranes. The Airfuge method was compared with the more classically employed membrane filtration method to measure specific [125I]insulin and [125I]thrombin binding to human placental membranes and an enriched plasma membrane fraction from mouse embryo fibroblasts, respectively, are found to be 4 to 6 times more sensitive. For example, specific binding of ligand to its receptor was demonstrated with 5 micrograms of protein. With slight modifications, the polyethyleneglycol 6000 method of precipitating 125I-labelled ligand-soluble receptor complexes can be adapted to the Airfuge sedimentation through oil procedure.