Parental imprinting of the genes for IGF-II and its receptor.
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Animals↗
Biomedical subjects
C Polychronakos
Publications and source records attributed to C Polychronakos.
Parental genomic imprinting of the human IGF2 gene.
The mouse igf2 gene, coding for the insulin-like growth factor II (IGF-II) is parentally imprinted, only the gene copy derived from the father is expressed. To know whether IGF2, the human homologue, is also imprinted, we used an ApaI polymorphism at the 3' untranslated region in order to distinguish between mRNA derived from each copy of the gene in placentae from heterozygote human fetuses, studied after careful removal of the decidua. Six term and two pre-term placentae of heterozygotes were studied, and in each case the cDNA contained only one of the two alleles present in the genomic DNA. In three cases the mother was homozygous for the non-expressed allele, allowing assignment of paternal origin to the transcribed gene copy. We conclude that, as in the mouse, human IGF2 is parentally imprinted.
Alleles↗
Mitogenic effects of insulin and insulin-like growth factors on PA-III rat prostate adenocarcinoma cells: characterization of the receptors involved.
Four transplantable cell lines (PA-I, II, III, and IV) derived from four Lobund-Wistar (L-W) rats that manifested spontaneous prostate cancer have demonstrated metastatic capacity in visceral organs. Interestingly, PA-III cells, when deposited over the scapula or calvarium of the Lobund-Wistar rat, could produce lytic and blastic reactions on rat skeleton. Since growth factors and growth factor receptors have been implicated in bone remodeling, cancer biology, and metastatic growth of cancer cells, we have examined 1) the effects of insulin and insulin-like growth factors (IGF-I and IGF-II) on the proliferation of PA-III cells; and 2) the presence of specific receptors for these peptides. IGF-I (0.5 to 100 ng/ml), IGF-II (0.5 to 100 ng/ml), and insulin (0.5 to 10 micrograms/ml) stimulated tritiated thymidine uptake and increased the number of PA-III cells in culture. Receptor studies demonstrated the presence of specific bindings sites for IGF-I and II but not for insulin. The number and affinity of the receptor sites were: IGF-I (nb = 675 fmol/100 g protein, Kd = 0.56 nmol) and IGF-II (nb = 225 fmol/100 g protein, Kd = 0.71 nmol). Molecular characterization of IGF binding sites by polyacrylamide gel electrophoresis under denaturing conditions indicated only the presence for the type I IGF receptor. The presence of the IGF-I receptor and the absence of IGF-II and insulin receptors are discussed in relation to the capacity of PA-III cells to produce bone lesions on the L-W rat.
Adenocarcinoma↗
Effect of tamoxifen on serum insulinlike growth factor I levels in stage I breast cancer patients.
Insulinlike growth factor I (IGF-I) has been shown to be a potent mitogen for breast cancer cells in vitro, and IGF-I receptors have been demonstrated on human primary breast neoplasms. In a randomized, placebo-controlled study, we document that administration of the antiestrogen tamoxifen to patients with breast cancer was associated with a statistically significant (P = .002) reduction in the serum level of IGF-I. The mean IGF-I level was 1.4 U/mL in the placebo-treated group and 0.9 U/mL in the tamoxifen-treated group. Because serum IGF-I level is growth hormone (GH) dependent and because data suggest that the pubertal surge in GH and IGF-I levels is sex steroid dependent, we speculate that the mechanism underlying our observation may involve blockade by tamoxifen of estrogen action in the hypothalamic-pituitary axis. We conclude that tamoxifen treatment reduces IGF-I levels and that this reduction may contribute to the therapeutic effect of the drug.
Aged↗
Insulinlike growth factor I: a potent mitogen for human osteogenic sarcoma.
Insulinlike growth factor I (IGF-I) is among the peptide mitogens that play key roles in the regulation of normal skeletal growth. To investigate the possibility that certain skeletal neoplasms retain a sensitivity to mitogenic stimulation by IGF-I, we studied the effects of this growth factor on human osteosarcoma. Competitive-binding assays and affinity-labeling experiments on membranes prepared from MG-63 immortalized human osteosarcoma cells and primary human osteogenic sarcoma cells demonstrate the presence of specific IGF-I receptors. Furthermore, we show that IGF-I is a potent stimulator of proliferation of MG-63 cells in vitro and is active at concentrations as low as 10(-10) M. A blocking antibody against the IGF-I receptor (alpha-IR3) significantly reduces IGF-I-stimulated proliferation in a dose-dependent manner. These results are consistent with the hypothesis that at least a subset of human osteogenic sarcomas are responsive to IGF-I and indicate that it may be possible to exploit this responsiveness therapeutically.
Affinity Labels↗
Effects of mannose-6-phosphate on receptor-mediated endocytosis of insulin-like growth factor-II.
The insulin-like growth factor-II (IGF-II) and lysosomal enzymes containing a mannose-6-phosphate (M6P) recognition site bind to different sites of the same receptor molecule. We have observed that M6P increases the receptor-mediated uptake of IGF-II into IM-9 cells. We now confirm this phenomenon in a different line, the H-35 rat hepatoma cells, and present additional characterization of the underlying mechanisms. When incubated in the presence of radiolabeled IGF-II, H-35 cells accumulated, in a time-dependent fashion, radioactivity that was resistant to removal by trypsin digestion at 15 C, indicating that it was endocytosed. In the presence of 3 mM M6P, endocytosed counts were approximately 2-fold higher after 5 min of incubation, an enhancement that peaked at 10 min, then declined, but was still evident after 40 min (1.5-fold). The rate of release of cell-associated IGF-II, degraded or intact, as measured in a chase experiment, was not affected by M6P. These observations indicate that M6P increased accumulation of IGF-II by accelerating its rate of endocytosis rather than by interfering with IGF-II degradation or with the recycling of intact hormone-receptor complexes to the cell surface. Electrophoresis after affinity cross-linking of labeled cells demonstrated that the enhancement in radioactivity could be located at a molecular size of approximately 250 kDa, corresponding to IGF-II-receptor complexes. Preincubation with M6P did not significantly alter the specific binding of IGF-II to the cell surface of H-35 cells, as measured by a binding assay at 4 C. Finally, pretreatment with cycloheximide for up to 8 h, to remove all newly synthesized lysosomal enzymes bound to the M6P/IGF-II receptor, did not affect IGF-II endocytosis beyond what could be accounted for by some loss of receptor, suggesting that the observed effect of M6P is due to the binding of M6P itself to the receptor and not to displacement of lysosomal enzymes. We conclude that simultaneous occupancy of the M6P/IGF-II receptor by ligands on both binding sites enhances its rate of endocytosis.
Animals↗
Enhancement of cytosolic tyrosine kinase activity by propylthiouracil-induced hyperplasia in the rat thyroid.
Hyperplasia of the thyroid gland induced by propylthiouracil (PTU) is a well established model of rapid cell proliferation in vivo. Recent evidence indicates that tyrosine kinase activity is associated with growth factor receptors and oncogene protein products and may have an important regulatory action in the control of cell growth. Thus, we examined tyrosine kinase activity in rat thyroid membrane and cytosol preparations at rest and during PTU-induced hyperplasia. Although kinase activity was present in a crude microsomal membrane preparation, no change was observed during thyroid growth. In contrast, tyrosine kinase activity assayed with the artificial substrate poly(Glu,Na:Tyr) 4:1 was present in normal rat thyroid cytosol and increased 2- to 6-fold during the rapid phase of hyperplasia in the first 5-10 days of PTU treatment. It declined to control values by day 15, when the size and DNA content of the thyroid reached a plateau. Preincubation of the cytosolic preparations with several peptides known to bind to and activate growth factor receptor tyrosine kinases failed to enhance the activity, suggesting, along with the cytosolic localization, that the activity was distinct from these receptors. By gel filtration chromatography and polyacrylamide gel electrophoresis, tyrosine kinase activity was associated with a 55 kDa protein. Partial purification over a poly(Glu,Na:Tyr)4:1-Sepharose column, yielded a protein that appeared capable of autophosphorylation. It is suggested that this tyrosine kinase plays a role in mediating the growth-promoting effects of this model of thyroid cell hyperplasia.
Animals↗
Mannose 6-phosphate increases the affinity of its cation-independent receptor for insulin-like growth factor II by displacing inhibitory endogenous ligands.
The insulin-like growth factor II (IGF-II) and glycoprotein lysosomal enzymes containing mannose 6-phosphate (M6P) bind with high affinity to two separate sites on the same receptor molecule (Morgan et al. Nature 329:301). The addition of free M6P significantly increases the affinity of some preparations of the M6P/IGF-II receptor (M6P/IGF-II-R) for IGF-II. We conducted this study to test the hypothesis that this effect is the result of displacement of M6P-related ligands that inhibit IGF-II binding. First we found that although M6P caused a 66% increase in the binding of IGF-II to microsomes prepared from IM9 cells, it had no effect, under identical conditions, on binding to receptor on the surface of intact cells. Secondly, extensive washing of rat liver microsomes in the presence of 3 mM M6P, followed by removal of the M6P by further washing, abolished the effect by raising binding to levels seen in the presence of M6P. M6P, then, had no additional effect. Finally, when IGF-II-affinity purified receptor was repurified by ultracentrifugation on a sucrose gradient, binding to the pure receptor peak was not affected by M6P. We conclude that there is no intrinsic positive cooperativity between free M6P and the IGF-II-binding site of the M6P/IGF-II-R. The reported M6P-induced increase in IGF-II binding appears to be due to the displacement of contaminating inhibitory endogenous ligands.
Animals↗
Characterization of insulin-like growth factor I (IGF-I) receptors of human breast cancer cells.
Studies of binding of IGF-I to a plasma-membrane-enriched subcellular fraction prepared from MCF-7 human breast cancer cells reveal the presence of 0.2 pmols specific binding sites for this mitogen per mg membrane protein, with an equilibrium affinity constant of 1.45 nM-1. Competition studies with insulin, IGF-II, and an anti-IGF-I receptor antibody are consistent with the presence of specific IGF-I receptors, and SDS-PAGE showed binding to a 130 kDa subunit identical to that of receptors from human placenta. In addition, we show that IGF-I is more potent than estradiol and comparable to EGF in stimulating in vitro proliferation of MCF-7 cells, and that IGF-I-stimulated proliferation of these cells is inhibited by a blocking monoclonal antibody against the IGF-I receptor. These results demonstrate that IGF-I is an important mitogen for MCF-7 cells and that the mitogenic effect is mediated by specific IGF-I receptors.
Breast Neoplasms↗
Transient growth deceleration in normal short children. A potential source of bias in growth studies.
We have examined the records of 21 short children evaluated for growth hormone deficiency and found not to be deficient. Their growth velocity was evaluated for at least 6 months, both before and after testing. Without any specific therapeutic intervention, growth velocity was significantly higher after testing, as compared with before. We attribute this apparent "therapeutic" effect of testing to a selection bias, due to the fact that, in normal clinical practice, children are selected for testing immediately following a period of slow growth, and that decelerations of growth are very often transient. Studies of growth-stimulating treatments using children as their own controls should, for this reason, be interpreted with caution.
Adolescent↗
Endocytosis of receptor-bound insulin-like growth factor II is enhanced by mannose-6-phosphate in IM9 cells.
The insulin-like growth factor II (IGF-II), and glycoproteins containing mannose 6-phosphate (M6P), bind to two different sites of the same receptor molecule (Morgan et al, Nature 329:301, 1987). To study the interactions between the two ligands on their common receptor in intact cells, we examined the effect of free M6P on IGF-II binding and endocytosis in the IM9 human lymphoblastoid cell line. M6P, up to a 3 mM concentration, had no effect on the binding of IGF-II to the cell surface receptor of intact IM9 cells at 4 degrees C. By contrast, when IM9 cells were incubated with 125I-IGF-II at 37 degrees C, 1mM M6P increased cell-associated radioactivity by twofold. The increase was resistant to acid wash at 4 degrees C, and therefore assumed to represent endocytosed IGF-II. Acid-washable radioactivity was no different, confirming that, in intact cells, M6P does not affect IGF-II surface binding. In addition, preincubation of cells with M6P at 37 degrees C for up to 3 hours did not change the abundance of receptor on the cell surface, as measured by a subsequent 4 degrees C binding assay. We conclude that M6P causes a shift of IGF-II-occupied receptors form the cell surface to intracellular locations without affecting surface binding of this ligand in IM9 cells. The effect could be produced by the binding of M6P itself, or by the displacement of endogenous phosphomannosylated ligands.
Cell Line↗
Endocytosis of receptor-bound insulin-like growth factor II is enhanced by mannose-6-phosphate in IM9 cells.
The insulin-like growth factor II (IGF-II), and glycoproteins containing mannose 6-phosphate (M6P), bind to two different sites of the same receptor molecule (Morgan et al, Nature 329:301, 1987). To study the interactions between the two ligands on their common receptor in intact cells, we examined the effect of free M6P on IGF-II binding and endocytosis in the IM9 human lymphoblastoid cell line. M6P, up to a 3 mM concentration, had no effect on the binding of IGF-II to the cell surface receptor of intact IM9 cells at 4 degrees C. By contrast, when IM9 cells were incubated with 125I-IGF-II at 37 degrees C, 1 mM M6P increased cell-associated radioactivity by twofold. The increase was resistant to acid wash at 4 degrees C, and therefore assumed to represent endocytosed IGF-II. Acid-washable radioactivity was no different, confirming that, in intact cells, M6P does not affect IGF-II surface binding. In addition, preincubation of cells with M6P at 37 degrees C for up to 3 hours did not change the abundance of receptor on the cell surface, as measured by a subsequent 4 degrees C binding assay. We conclude that M6P causes a shift of IGF-II-occupied receptors form the cell surface to intracellular locations without affecting surface binding of this ligand in IM9 cells. The effect could be produced by the binding of M6P itself, or by the displacement of endogenous phosphomannosylated ligands.
Cell Line↗
Impact of SMBG on control of diabetes as measured by HbA1. 3-yr survey of a juvenile IDDM clinic.
Three hundred twelve diabetic children and adolescents were seen in our diabetic clinic and instructed to test their capillary blood glucose (CBG) twice daily and to use an algorithm to adjust their short-acting insulin. Of this group, 219 youngsters had a full 3-yr period of observation. At each clinic visit, blood was obtained for fasting blood glucose and HbA1 and, once a year, cholesterol and triglycerides were also measured. Patient and parent accuracy in measuring CBG was found to be adequate. The changes over time in HbA1 were nondifferential across age and sex, and there was no difference in the level of HbA1 between age and sex groups, the number of tests reported to have been done by the patients, the number of injections of insulin per day, or the serum cholesterol. There was a significant relationship between the HbA1 and the fasting blood glucose (P less than .001) measured by the laboratory as well as with the serum triglyceride (P less than .01). The failure to improve diabetic control, despite measures that would have been expected to do so, was believed to relate more to a lack of compliance than to a flaw in the therapeutic approach. It was interesting to note that the adolescent patients in the study were in no worse control than the younger children in the group. Although better technical skills are available today to manage diabetes, the psychosocial approach to patient motivation requires improvement.
Adolescent↗
Increase in specific binding of insulin-like growth factor (IGF) II to type 1 IGF receptors on erythrocytes of hypopituitary children receiving growth hormone therapy.
Specific receptor binding for insulin-like growth factors (IGFs) is measurable in young erythrocytes. Cells of similar age, Fraction A, can be reproducibly obtained by dextran gradient centrifugation from 5-10 ml of blood. We now report IGF-II specific binding to Fraction A erythrocytes from normal children and children with growth hormone deficiency. Normal controls (Group 1) were 5 male volunteers (14.7 +/- .6 years, mean +/- SEM) and 10 children with constitutional short stature (11.4 +/- 1.6 years) who had normal 6-hour daytime growth hormone profiles and plasma IGF-I values. Twelve growth hormone deficient children (Group 2), aged 13.7 +/- 1.1 years, had samples taken after 2 months without growth hormone therapy and again following 2 months with growth hormone (0.1 U/kg 3 times per week) therapy. The percent of total erythrocytes in Fraction A did not differ in the two groups of children. Group 1 had IGF-II specific binding of 10.2 +/- 0.6% (per 3 X 10(9) cells). IGF-II specific binding was less in Group 2 at 6.6 +/- 0.8% (p less than 0.002). With growth hormone therapy, IGF-II specific binding increased to 10.4 +/- 1.0% (p less than 0.02), a value not different from that seen in Group 1. Corresponding plasma IGF-II and IGF-I values showed a positive correlation with IGF-II specific binding (r = 0.54 and r = 0.56 respectively, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Specificity of insulin-like growth factor binding to type-II IGF receptors in rabbit mammary gland and hypophysectomized rat liver.
We have reevaluated IGF binding specificity to membrane receptors in rabbit mammary gland (RMG) and hypophysectomized rat liver (HRL) using recombinant DNA-derived and synthetic analogues of human IGF-I and highly purified IGF-II. SDS-PAGE demonstrated that [125I]IGF-I bound to type-I IGF receptors in RMG; this binding was inhibited in a similar fashion by the IGF-I analogues (IC50 = 10 ng/ml) and to a lesser extent by IGF-II (IC50 = 60 ng/ml). [125I]IGF-II bound to type-II IGF receptors in both RMG and HRL. The IC50 for IGF-II was 9 and 3 ng/ml with RMG and HRL, respectively. At a dose as high as 1 microgram/ml, IGF-I analogues inhibited less than 20% of [125I]IGF-II binding. These results suggest that IGF-I has little or no affinity for type-II IGF receptors.
Animals↗
Management of thyroid nodules in children: a 20-year experience.
The medical records and surgical slides of 58 patients with the diagnosis of thyroid nodules (solitary nodule in 50 patients) are reviewed. The most common cause of thyroid nodules in this series is follicular adenoma (27 patients or 46%). A nuclear scan (technetium or radioactive iodine) was performed in 55 patients, of which 40 showed a cold nodule. Twelve of the 40 cold nodules were malignant (30%). However, for solitary nodules the incidence of cancer is 27%. This last figure is significantly greater than the one recently reported by Hung et al (18.5%). Available diagnostic methods are reviewed and the clinical management as derived from our experience is presented.
Adolescent↗
Glycogen storage disease type I.
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Blood Glucose↗
Increase in the number of type II insulin-like growth factor receptors during propylthiouracil-induced hyperplasia in the rat thyroid.
We have observed that membranes isolated from rat thyroids contain receptors for the insulin-like growth factors (IGF). As IGFs are known to be important mediators of tissue growth, we conducted this study to determine whether modulation of thyroid IGF receptors might be involved in TSH-stimulated hyperplasia. A substantial increase in both the weight of the thyroid and its DNA content was observed within 2 days of exposing adult male rats to 0.1% propylthiouracil (PTU) in their drinking water. Serum T4 reached unmeasurable levels and serum TSH rose 3-fold over control by the tenth day of treatment. [125I]Iodo-human(h)IGF-II binding to membranes isolated from hyperplastic glands was significantly higher than control beginning at 2 days. A maximum was reached after 5 days (13.3 +/- 0.8%/25 micrograms protein vs. a control level of 6.7 +/- 0.7%, mean +/- SEM). The increase had disappeared by 15 days of PTU exposure, paralleling the drastic fall in the growth rate of the glands. This increase in binding was specific for the thyroid, as it was not seen in other organs. In both treated and control animals, the receptor involved was shown to be type II by preferential binding to IGF-II, lack of interaction with insulin, and molecular sizing. The observed increase in binding could be accounted for by an increase in receptor site number, the affinity remaining essentially the same. We conclude that the TSH-stimulated hyperplasia of the rat thyroid, induced by PTU, is associated with an increase in the binding sites of the type II IGF receptor. This observation raises the possibility that modulation of this receptor may play a role in the mediation of the mitogenic effect of TSH on the thyroid gland.
Animals↗