Acta Padiatr S c a d [Suppl] 367: 61-13, 1990
Insulin-Like Growth Factor II Effects Mediated through Insulin-Like Growth Factor II Receptors M. TALLY and K. HALL From the Deparrment of Endocrinology, Karolinska Institute, Stockholm, Sweden
ABSTRACT. Tally, M. and HaU, K. (Department of Endocrinology, Karolinska Institute, Stockholm, Sweden). Insulin-like growth factor 11 effects mediated through insulin-like growth factor I1 receptors. Acta Paediatr Scand [Suppl] 367: 67, 1990. Insulin-like growth factor 11 (IGF-II) resembles the homologous peptide insulin-like growth factor I (IGF-I) in that it stimulates cellular growth in vitro. This effect is generally believed to be mediated through IGF type 1 receptors; the role of the IGF type 2 receptor remains, as yet, unknown. IGF-11 has been shown to stimulate clonal expansion in cells from the human erythroleukaemia cell line K562, which displays binding of IGF-II and insulin but not IGF-I. This IGF-11 effect was dose-dependent and correlated to the amount of specific binding; IGF-I did not stimulate growth. A similar effect on clonal growth was observed in the human T-ceU line Jurkat. Furthermore, IGF-11 was found to stimulate the cytotoxic activity of natural killer cells (as does interleukin 2). This effect was not inhibited by addition of IGF binding protein 1. Thus, it can be concluded that IGF-11, besides demonstrating standard IGF properties, exhibits unique biological effects in certain cells. Key wonls: Insulin-like growth faetor II, insulin-like growth factor II receptor.
The polypeptides insulin-like growth factor I (IGF-I) and insulin-like growth factor I1 (IGF-11) are present in the blood and peripheral tissues of humans. Homologous peptides are also present in most other mammals (1-5). IGF-I is regulated by growth hormone, whereas no regulators of IGF-I1 are known in humans. IGF-I and IGF-I1 have separate specific receptors: type 1 and type 2 IGF receptors. The amino acid sequence of the type 1 IGF receptor, deduced from cDNA (6), has been shown to be structurally homologous to the amino acid sequence of the insulin receptor (7). IGF-II and insulin can compete with IGF-I for the type 1 IGF receptor, but the affinity of insulin for the receptor is approximately 1% of that of IGF-I. The type 2 IGF receptor is a monomer with a molecular weight of 250 kDa. When the amino acid sequence of the type 2 IGF receptor was deduced from cDNA (8), it was found to be identical to the cation-independent mannose-6-phosphate (M6-P) receptor (9-1 2). The receptor binds IGF-I1 and M6-P at different sites (10, 13). No autophosphorylation by intrinsic kinase activity has been observed as a response to ligand binding at this receptor, but the molecule can be a substrate for phosphorylation of tyrosine residues (14). Since specific IGF-I1 binding was defined, it has become clear that insulin does not compete with IGF-I1 for the type 2 IGF receptor (15, 16). On the other hand, IGF-I has been reported to have affinity for the IGF-I1 receptor with potencies that vary according to the IGF-I and IGF-I1 preparations used. Growth effects of IGF-I in vitro have been reported in numerous tissues and from several species (17). The growth-stimulating effects of IGF-I1 in vitro have generally been found to be 10-100% of those of IGF-I (18, 19). It has been proposed that the growth-promoting effects of IGF-I1 are mediated through the type 1 IGF receptor. This proposal is supported by the finding that monoclonal antibodies to the type 1 IGF receptor block IGF-11-stimulated 3H-thymidine incorporation into DNA in fibroblasts (20, 21). Several antibodies raised against the IGF-WM6-P receptor have been used to study the effect of IGF-I1 on processes in a variety of cells. These include DNA synthesis in H-35 hepatoma cells (22), and uptake of 3H-deoxyglucose, 14C-methyl-aminoisobutyric acid and 3H-leucine in L6 myoblasts (23). As the IGF-I1 effects observed in these systems were not inhibited by these antibodies, it was concluded that they were not mediated through the IGF-I1 receptor. To provide a model for the evaluation of the specific growth effects of IGF-11, human cell lines were screened for their expression of binding sites for IGF-I and IGF-11, in order to find cells lacking receptors for IGF-I. Two types of cells that exhibit binding of IGF-I1 but not IGF-I and that respond specifically to IGF-I1 were found (both of haematopoietic origin): transformed cells in culture and lymphocytes from healthy donors.
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METHODS Materials. Human IGF-I and IGF-I1 were purified and further separated, as previously described (24, 25). Recombinant IGF-I1 was produced as described elsewhere (26) and recombinant IGF-I was a gift from Kabi, Sweden. Synthetic IGF-I and IGF-I1 were supplied by Professor Choh Hao Li, University of California, San Francisco. Insulin-like growth factor binding protein 1 (IGFBP-I) was purified from human amniotic fluid, as described previously (27). Cell culture. K562 and Jurkat (28) cells were maintained in RPMI 1640 medium supplemented with 5 % fetal calf serum (FCS). Three days after subculture, cells were sedimented by centrifugation (500 g, 1 minute), and thereafter washed three times in saline and Hepes buffer before resuspension in assay buffer. Isolation of lymphocytes. T-lymphocytes were isolated from healthy blood donors by Ficoll-Hypaque (29) centrifugation, washed with phosphate buffered saline and resuspended in RPMI 1640 with 15% newborn calf serum (NCS) and antibiotics. Adherent cells were removed by incubation in plastic Petri dishes for 60 minutes at 37°C. T-lymphocytes were further purified by nylon-wool adherence chromatography. Isolation of natural killer cells. The T-cell population was fractionated by ‘panning’ (30) using solid-phase coupled monoclonal antibodies against the CD3 antigen. Isolated cells were cultured in RPMI 1640 with 5% FCS or 1 % bovine serum albumin (BSA). Cell binding and radioreceptor assay. Cell binding assays were carried out in siliconized or albumin-treated tubes, as described elsewhere (31). Incubations were carried out overnight at 0 4 ° C . Clonal expansion assay. Cells were washed three times in RPMI 1640 without FCS, and diluted to 90,OOO cells/ml in RPMI 1640 with 3 % FCS. A sterile 1 % agar solution was prepared, and kept warm in water. Peptides were lyophilized, dissolved and diluted in RPMI 1640 with 0.25% human serum albumin to 1.5 times more than the desired final concentration. Aliquots (1 ml) were sterile-filtered into siliconized glass tubes and the cell suspension (50 pl) and agar (0.5 ml) were added. Four aliquots (250 pl each) were transferred to multiwell plates and incubated for 5-6 days in 95% 0,and 5 % CO,. Colonies of more than five cells were counted. Cyroroxic assay. Target cells (K562 or Daudi) were incubated with 5’Cr for 1 hour at 37°C. washed three times and resuspended in RPMI 1640 with 1 % BSA. Pretreated natural killer (NK)cells (effector cells) were added to target cells in microtitre plates (total volume 150 p1) and incubated for 4 hours at 37°C. Microplates were centrifuged at 250 xg for 2 minutes and supernatants (75 pl) were aspirated and radioactivity measured to determine the specific lysis of target cells.
RESULTS The binding of 1251-IGF-IIand its competitive inhibition by native and synthetic IGF-I1 and native, synthetic and recombinant IGF-I to K562 cells is shown in Fig. 1. It is clear that none of the three types of IGF-I investigated can compete with IGF-I1 binding in these cells in concentrations of up to 1 pg/ml, whereas both the IGF-I1 preparations used compete to a similar degree with the labelled IGF-11. This pattern was consistent at incubation temperatures of 4 and 37°C (incubation times, 16 hours and 15 minutes, respectively).
0
1 10 100 1000 IGF-I1 (ng/ml)
Fig. 1. Competitive inhibition of the binding of labelled IGF-I1 to K562 cells at 4°C by the following peptides: synthetic IGF-I1 (0-0); native IGF-I (0-0); synthetic IGF-I native IGF-I1 (0-0); ( 0 - 0 ) and recombinant IGF-I (A---A). Reproduced with permission from Tally M, Enberg G, Li CH, Hall K. The specificity of the human IGF-2 receptor. Biochem Biophys Res Commun 1987; 147: 1206-12.
IGF-N and its receptors
Acta Paediatr Scand [Suppl] 367
50
c
OL
T
10% FCS
0
100
10
1
IGF-I1 (ng/ml)
Fig. 2. Growth of K562 cells as measured by formation of colonies in semi-solid agar in the presence of 10% FCS or increasing concentrations of IGF-I1 (A),IGF-I ( 0 )or insulin (0). The number of colonies is measured per 0.5 cm2. Reproduced with permission form Tally M, Li CH, Hall K. IGF-2 stimulated growth mediated by the somatomedin-type 2 receptor. Biochem Biophys Res Commun 1987;148: 811-16.
3.0-
"**
Q
T
a,
Q
Pg
5 = 2.0 C(D
ag .o ' cE o
:g 1.0 $2 a, ;0-
*.
T
L
Insulin
I II Controls
IGF-I
0
IGF-I1
1 10 IGF-I1 (nghnl)
100
Fig. 3. (a) Specific binding of 'Z51-labelledinsulin, IGF-I and IGF-I1 evaluated in the presence of unlabelled insulin (1 p g / m l ) , IGF-I (100nglml), and IGF-II(100 ng/ml), respectively on the T-cell line Jurkat (*p < 0.05, * * p < 0.01,***p < 0.001).(b) Growth stimulation of IGF-I1 on Jurkat cells evaluated as colony formation in semi-solid agar in the presence of (I) 10% FCS, (11) 10 ng/ml IGF-I in 0.1% FCS, and increasing concentrations of FCS (0.1% ( O ) , 1 % (.) and 5% (0))and IGF-11. Standard deviations of quadruple determinations are indicated.
69
I0
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Acta Paediatr Scand [Suppl] 367
D T
IL-2
FCS
IGF-I1 (ng/ml)
Fig. 4. The percentage stimulation of killing activity of NK cells over the 1 % BSA control by 100 IU IL-2, 5% FCS and increasing concentrations of IGF-I1 (2, 20, 200 ng/ml). Error bars indicate SEM for six
experiments.
The growth-promoting effects of insulin, IGF-I and IGF-I1 were evaluated by clonal expansion in semi-solid agar, counting colonies of five cells or more after a 5-day incubation. As shown in Fig. 2, IGF-II, and to some extent insulin, stimulated the formation of colonies, whereas IGF-I had no effect in this assay. As K562 is a cell line containing cells with features resembling pre-erythrocytes, other transformed cells of haematopoietic origin were evaluated for their distribution of receptors for insulin, IGF-I and IGF-11. The T-cell line Jurkat displayed specific binding of IGF-11, but very little or no IGF-I and insulin binding, as shown in Fig. 3a. Fig. 3b shows the growthstimulating effects of IGF-I1 on these cells, evaluated in three different concentrations of FCS (0.1, 1 and 5 %). IGF-I at a concentration of 10 n g / d caused no stimulation of growth. A previous investigation of T-lymphocytes from healthy donors indicated that they possess a very low ISF-I1 binding capacity that is increased after a 48-hour stimulation with a nonspecific antigen, phytohaemagglutinin (PHA) (32). When the T-cells were subfractionated, using CD3 antibodies, into small granular lymphocytes and NK cells, it was observed that the NK fraction had a 2-3 times higher specific binding of IGF-11, compared to the undifferentiated T-cells. Evaluation of the effects of IGF-I1 on the activity of the killer cells revealed that a 24-48-hour stimulation caused a concentration-dependent increase of the killing activity, as is shown in Fig. 4. No stimulation at all could be achieved with the same concentrations of IGF-I, insulin or GH, and IGFBP-1 had no inhibiting effect on the IGF-IIstimulated killing activity (data not shown). DISCUSSION A prerequisite to demonstrate the specific effects of IGF-I1 upon ligand binding has been the use of tissues or cells lacking response to IGF-I. Assay systems where only a minor IGF-I effect can be detected fail to fulfil these demands. The fact that IGF-I1 cross-reacts with the IGF-I receptor has obscured the net result of IGF-I1 stimulation from detection. The data presented here, however, disclose that IGF-I1 can act specifically, under certain conditions, in tissues where IGF-I exhibits no detectable biological response and demonstrates
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no significant amount of biriing. Furthermore, no cross-reactivity of IGF-I for the IGF-I1 receptor was found in these human cells, a finding supported by results with other human tissues (33). This is consistent with the characteristics of the purified human IGF-II/M6-P receptor (34). This would seem to indicate a difference between human and rat receptors, as IGF-I has been shown to have affinity for the rat IGF-I1 receptor (35). Both the K562 and Jurkat cell lines, as well as lymphocytes isolated from healthy subjects, are of haematopoietic origin (36). The few previous reports on specific growth effects of IGFI1 have been consistent with this finding. Daniak and Kreczko (37) showed that rat IGF-I1 stimulated both burst and colony formation in human bone marrow cultures, and Schimpff et al. (38) demonstrated a stimulatory effect on the 3H-thymidine incorporation in PHAstimulated T-cells. That IGF-I1 stimulated the growth of K562 cells was later confirmed by Blanchard et al. (39). They measured the effects of IGF-11, two preparations of IGF-I, crude IGFs, and insulin on the incorporation of 3H-thymidine into DNA. IGF-I1 was the most potent stimulator of DNA synthesis, but recombinant IGF-I also produced a considerable stimulation. This may be explained by the presence of low concentrations of type 1 IGF receptors in the strain of K562 cells used in their experiments. IGF-I1 had a marked effect on the activity of the NK cells isolated from healthy donors, compared to the 5% FCS control. Nevertheless, the effect obtained with 100 IU of interleukin 2 (IL-2) by far exceeds this effect. However, the highest concentration of IGF-I1 used was only 200 nglml. As the circulating concentration of IGF-I1 (bound to IGF binding proteins) in healthy humans ranges from 500 to 800 ng/ml, a concentration of 200 ng/ml may not be sufficient to result in maximal stimulation of DNA synthesis under these conditions. IGF-I-stimulated multiplication has been found to be inhibited by the addition of IGFBP-1, probably due to the resulting decrease in the active concentration of IGF-I. IGFBP-1 did not, however, inhibit the stimulation of NK cell activity by IGF-11. This might indicate that the active concentration of IGF-I1 is normally high and that the V,, for the IGF-I1 effect is not reached with the concentrations used. Affinity labelling, SDS-PAGE and autoradiography of intact, IGF-I1 responding cells (32) resulted in two labelled bands: a large protein with a molecular weight of approximately 250 kDa, probably corresponding to the previously purified and sequenced IGF-II/M6-P receptor (8), and a smaller protein with an approximate molecular weight of 75-80 m a . Surprisingly, no detectable labelling of the 250 kDa protein was found in Jurkat cells, and in the lymphocytes responding to IGF-I1 from healthy subjects, the 250 kDa band was barely detectable. The 75-80 kDa band was, however, always labelled to a large extent. This small molecular weight protein has yet to be purified and fully characterized, but its binding kinetics seem to differ from those of the 250 kDa IGF-II/M6-P receptor. When the 75-80 kDa is affinity labelled on intact cells, displacement of the radioactive affinity ligand can be achieved with 50 pglml unlabelled IGF-11 in the presence of 300 pg/ml insulin in order to inhibit the degradation of IGF-11, as insulin cannot compete for IGF-I1 binding sites. A partly purified fraction, however, displays specificity for IGF-I1 in a soluble competitive binding assay (unpublished data). The biological effects of IGF-I1 on lymphocytes from healthy donors are similar to several effects found when such cells are stimulated with IL-2. When small granular lymphocytes (CD3+, non-killer cells) were stimulated (unpublished data) with IGF-11 at a concentration of 200 ng/ml, an increased expression of the small IL-2 receptor (the Tac-antigen, 55 kDa) was found. This is also an effect obtained after IL-2 stimulation. In conclusion, it has been shown that IGF-I1 is a growth factor, acting through its own specific binding. This is shown in three cell types. However, the possibility that the effect of IGF-I1 may be indirect cannot yet be excluded, considering that in all experiments described the cells were incubated with IGF-I1 for 24-48 hours. It is possible that IGF-II may act by releasing another factor, thus acting in an autocrine fashion, either by inducing a growth promoter or by suppressing a growth inhibitor.
ACKNOWLEDGEMENT This study was supported by a grant from the Swedish Medical Research Council no. 4224.
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REFERENCES 1. Rinderknecht E, Humbel RE. Polypeptides with nonsuppressible insulin-like and cell-growth promoting
activities in human serum. Isolation, chemical characterization, and some biological properties of form I and 11. Proc Natl Acad Sci USA 1976; 73: 2365-9. 2. Rinderknecht E, Humbel RE. Primary structure of insulin-like growth factor 11. FEBS Lett 1978; 89: 2834. 3. Hall K, Sara VR. Growth and somatomedins. In: Munson PL, Diszfalusy E, Glover J, eds. Vitamins and hormones. New York: Academic Press, 1983; 40: 175-232. 4. Nissley SP, Rechler MM. Insulin-like growth factors: biosynthesis, receptors, and carrier proteins in hormonal proteins and peptides. In: Li CH, ed. Growth factors. New York: Academic Press, 1984; 12: 127-203. 5 . Froesch ER, Schmid C, Schwander J. Zapf J. Actions of insulin-like growth factors. Annu Rev Physiol 1985; 47: 443-67. 6. Ullrich A, Gray A, Tam AW er al. Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J 1986; 10: 2503- 12. 7. Ullrich A, Bell JR, Chen EY et 01. Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature 1985; 313: 756-61. 8. Morgan DO, Edman JC, Standring DN er al. Insulin-like growth factor I1 receptor as a multifunctional binding protein. Nature 1987; 329: 301-7. 9. Lobel P, Dahms N, Kornfeld S. Cloning and sequence analysis of the cation-independent mannose 6-phosphate receptor. J Biol Chem 1988; 263: 2563-70. 10. Roth RA, Stover C, Hari J et nl. Interactions of the receptor for insulin-like growth factor I1 with mannose-6-phosphate and antibodies to the mannose-6-phosphate receptor. Biochem Biophys Res Commun 1987; 149: 600-6. 1 1. Roth RA. Structure of the receptor for insulin-like growth factor 11: the puzzle amplified. Science 1988; 239: 1269-7 1. 12. Kiess W, Blickenstaff GD, Sklar MM, Thomas CL, Nissley SP, Sahagian GG. Biochemical evidence that the type I1 insulin-like growth factor receptor is identical to the cation-independent mannose-6-phosphate receptor. J Biol Chem 1988; 263: 9339-44. 13. Waheed A, Braulke T, Junghans U, von Figura K. Mannose-6-phosphate/insulin-likegrowth factor I1 receptor: the two types of ligands bind simultaneously to one receptor at different sites. Biochem Biophys _ _ Res Commun 1988; 152: 1248-54. 14 Corvera S, Whitehead RE, Mottola C, Czech MP. The insulin-like growth factor I1 receptor is phosphorylated by a tyrosine kinase in adipocyte plasma membranes. J Biol Chem 1986; 261: 7675-9. 15. King GL, Kahn CR, Rechler MM, Nissley SP. Direct demonstration of separate receptors for growth and metabolic activities of insulin and multiplication-stimulating activity (an insulin-like growth factor) using antibodies to the insulin receptor. J Clin Invest 1980; 66: 130-40. 16. Kasuga M, Van Obberghen E, Nissley SP, Rechler MM. Demonstration of two subtypes of insulin-like growth factor receptors by affinity cross-linking. J Biol Chem 1981; 256: 5305-8. 17. Van Wyk JJ. The somatomedins: biological actions and physiologic control mechanisms in hormonal proteins and peptides. In: Li CH, ed. Growth factors. New York: Academic Press, 1984; 12: 81-125. 18. Rechler MM, Fryklund L, Nissley SP et al. Purified human somatomedin A and rat multiplication stimulating activity. Mitogens for cultured fibroblasts that crossreact with the same growth peptide receptors. Eur J Biochem 1978; 82: 5-12. 19. Enberg G, Tham A, Sara VR. The influence of purified somatomedins and insulin on foetal rat brain DNA synthesis in vitro. Acta Physiol Scand 1985; 125: 305-8. 20. Conover CA, Misra P, Hintz RL, Rosenfeld RG. Effect of an anti-insulin-like growth factor I receptor antibody on insulin-like growth factor I1 stimulation of DNA synthesis in human fibroblasts. Biochem Biophys Res Commun 1986; 139: 501-8. 21. Furlanetto RW, DiCarlo JN, Wisehart C. The type I1 insulin-like growth factor receptor does not mediate deoxyribonucleic acid synthesis in human fibroblasts. J Clin Endocrinol Metab 1987; 64: 1142-9. 22. Mottola C , Czech MP. The type I1 insulin-like growth factor receptor does not mediate increased DNA synthesis in H-35 hepatoma cell. J Biol Chem 1984; 259: 12705-13. 23. Kiess W, Haskell JF, Lee L er al. An antibody that blocks insulin-like growth factor (IGF) binding to the type I1 IGF receptor is neither an agonist nor an inhibitor of IGF-stimulated biological responses in L6 myoblasts. J Biol Chem 1987; 262: 12745-51. 24. Enberg G, Carlquist M, Jornvall H, Hall K. The characterization of somatomedin A, isolated by microcomputer-controlled chromatography, reveals an apparent identity to insulin-like growth factor 1. Eur J Biochem 1984; 143: 117-24. 25. Tally M, Florell K, Enberg G. An effective method for the separation of insulin-like growth factors 1 and 2 during the purification process. Bioscience Rep 1988; 8: 293-7. 26. Hammarberg B, Moks T, Tally M er al. Different stability of recombinant human insulin-like growth factor I1 in Escherichia coli and Sraphylococcus aureus. J Biotechnol; in press. 27. P6voa G, Enberg G, Jornvall H,Hall K. Isolation and characterization of a somatomedin-binding protein from mid-term human amniotic fluid. Eur J Biochem 1984; 144: 199-204.
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28. Weiss A, Stobo JD. Requirement for the coexpression of T3 and the T cell antigen receptor on a malignant human T cell line. J Exp Med 1984; 160: 1284-99. 29. Boyum A. Separation of leucocytes from blood and bone marrow. Scand J Clin Lab Invest [Suppl] 1968; 21: 97. 30. Wysocki LJ, Sato VL. ‘Panning’ for lymphocytes: a method for cell selection. Proc Natl Acad Sci USA 1978; 75: 2844-8. 31. Tang X-Z,Tally M, Jondal M, Hall K. Characterization of insulin binding to the erythroleukemia cell line K 562. Biochem Biophys Res Commun 1983; 117: 826-33. 32. Tally M. Insulin-like growth factor II. Studies on binding and biological effects in target tissues. Thesis, Karolinska Institutet; 1989. 33. Tally M, Enberg G, Li CH, Hall K. The specificity of the human IGF-2 receptor. Biochem Biophys Res Commun 1987; 147: 1206-12. 34. Roth RA, Steele-Perkins G, Hari J et al. Insulin and insulin-like growth factor receptors and responses. Cold Spring Harbor Symp Quant Biol 1988; 53: 537-43. 35. Rosenfeld RG, Conover CA, Hodges D er al. Heterogeneity of insulin-like growth factor-I affinity for the insulin-like growth factor-I1 receptor. Comparison of natural, synthetic and recombinant DNA-derived insulin-like growth factor-I. Biochem Biophys Res Commun 1987; 143: 199-205. 36. Tally M, Li CH, Hall K. IGF-2 stimulated growth mediated by the somatomedin-type 2 receptor. Biochem Biophys Res Commun 1987; 148: 81 1-16. 37. Daniak N, Kreczko S. Interactions of insulin-like growth factor 11, and platelet derived growth factor in erythropoietic culture. J Clin Invest 1985; 76: 1 2 3 7 4 2 . 38. Schimpff R-M, Repellin A-M, Salvatoni A, Thieriot-Prevost G, Chatelain P. Effect of purified somatomedins on thymidine incorporation into lectin-activated human lymphocytes. Acta Endocrinol (Copenh) 1983; 102: 21-6. 39. Blanchard MM, Barenton B, Sullivan A, Foster B, Guyda HJ, Posner BI. Characterization of the insulinlike growth factor (IGF) receptor in K562 erythroleukemia cells; evidence for a biological function for the type I1 IGF receptor. Mol Cell Endocrinol 1988; 56: 235-44.
(M.T.) Department of Endocrinology Karolinska Institute S-104 01 Stockholm Sweden
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Insulin-Like Growth Factor II Effects Mediated through Insulin-Like Growth Factor II Receptors M. TALLY and K. HALL From the Deparrment of Endocrinology, Karolinska Institute, Stockholm, Sweden
ABSTRACT. Tally, M. and HaU, K. (Department of Endocrinology, Karolinska Institute, Stockholm, Sweden). Insulin-like growth factor 11 effects mediated through insulin-like growth factor I1 receptors. Acta Paediatr Scand [Suppl] 367: 67, 1990. Insulin-like growth factor 11 (IGF-II) resembles the homologous peptide insulin-like growth factor I (IGF-I) in that it stimulates cellular growth in vitro. This effect is generally believed to be mediated through IGF type 1 receptors; the role of the IGF type 2 receptor remains, as yet, unknown. IGF-11 has been shown to stimulate clonal expansion in cells from the human erythroleukaemia cell line K562, which displays binding of IGF-II and insulin but not IGF-I. This IGF-11 effect was dose-dependent and correlated to the amount of specific binding; IGF-I did not stimulate growth. A similar effect on clonal growth was observed in the human T-ceU line Jurkat. Furthermore, IGF-11 was found to stimulate the cytotoxic activity of natural killer cells (as does interleukin 2). This effect was not inhibited by addition of IGF binding protein 1. Thus, it can be concluded that IGF-11, besides demonstrating standard IGF properties, exhibits unique biological effects in certain cells. Key wonls: Insulin-like growth faetor II, insulin-like growth factor II receptor.
The polypeptides insulin-like growth factor I (IGF-I) and insulin-like growth factor I1 (IGF-11) are present in the blood and peripheral tissues of humans. Homologous peptides are also present in most other mammals (1-5). IGF-I is regulated by growth hormone, whereas no regulators of IGF-I1 are known in humans. IGF-I and IGF-I1 have separate specific receptors: type 1 and type 2 IGF receptors. The amino acid sequence of the type 1 IGF receptor, deduced from cDNA (6), has been shown to be structurally homologous to the amino acid sequence of the insulin receptor (7). IGF-II and insulin can compete with IGF-I for the type 1 IGF receptor, but the affinity of insulin for the receptor is approximately 1% of that of IGF-I. The type 2 IGF receptor is a monomer with a molecular weight of 250 kDa. When the amino acid sequence of the type 2 IGF receptor was deduced from cDNA (8), it was found to be identical to the cation-independent mannose-6-phosphate (M6-P) receptor (9-1 2). The receptor binds IGF-I1 and M6-P at different sites (10, 13). No autophosphorylation by intrinsic kinase activity has been observed as a response to ligand binding at this receptor, but the molecule can be a substrate for phosphorylation of tyrosine residues (14). Since specific IGF-I1 binding was defined, it has become clear that insulin does not compete with IGF-I1 for the type 2 IGF receptor (15, 16). On the other hand, IGF-I has been reported to have affinity for the IGF-I1 receptor with potencies that vary according to the IGF-I and IGF-I1 preparations used. Growth effects of IGF-I in vitro have been reported in numerous tissues and from several species (17). The growth-stimulating effects of IGF-I1 in vitro have generally been found to be 10-100% of those of IGF-I (18, 19). It has been proposed that the growth-promoting effects of IGF-I1 are mediated through the type 1 IGF receptor. This proposal is supported by the finding that monoclonal antibodies to the type 1 IGF receptor block IGF-11-stimulated 3H-thymidine incorporation into DNA in fibroblasts (20, 21). Several antibodies raised against the IGF-WM6-P receptor have been used to study the effect of IGF-I1 on processes in a variety of cells. These include DNA synthesis in H-35 hepatoma cells (22), and uptake of 3H-deoxyglucose, 14C-methyl-aminoisobutyric acid and 3H-leucine in L6 myoblasts (23). As the IGF-I1 effects observed in these systems were not inhibited by these antibodies, it was concluded that they were not mediated through the IGF-I1 receptor. To provide a model for the evaluation of the specific growth effects of IGF-11, human cell lines were screened for their expression of binding sites for IGF-I and IGF-11, in order to find cells lacking receptors for IGF-I. Two types of cells that exhibit binding of IGF-I1 but not IGF-I and that respond specifically to IGF-I1 were found (both of haematopoietic origin): transformed cells in culture and lymphocytes from healthy donors.
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METHODS Materials. Human IGF-I and IGF-I1 were purified and further separated, as previously described (24, 25). Recombinant IGF-I1 was produced as described elsewhere (26) and recombinant IGF-I was a gift from Kabi, Sweden. Synthetic IGF-I and IGF-I1 were supplied by Professor Choh Hao Li, University of California, San Francisco. Insulin-like growth factor binding protein 1 (IGFBP-I) was purified from human amniotic fluid, as described previously (27). Cell culture. K562 and Jurkat (28) cells were maintained in RPMI 1640 medium supplemented with 5 % fetal calf serum (FCS). Three days after subculture, cells were sedimented by centrifugation (500 g, 1 minute), and thereafter washed three times in saline and Hepes buffer before resuspension in assay buffer. Isolation of lymphocytes. T-lymphocytes were isolated from healthy blood donors by Ficoll-Hypaque (29) centrifugation, washed with phosphate buffered saline and resuspended in RPMI 1640 with 15% newborn calf serum (NCS) and antibiotics. Adherent cells were removed by incubation in plastic Petri dishes for 60 minutes at 37°C. T-lymphocytes were further purified by nylon-wool adherence chromatography. Isolation of natural killer cells. The T-cell population was fractionated by ‘panning’ (30) using solid-phase coupled monoclonal antibodies against the CD3 antigen. Isolated cells were cultured in RPMI 1640 with 5% FCS or 1 % bovine serum albumin (BSA). Cell binding and radioreceptor assay. Cell binding assays were carried out in siliconized or albumin-treated tubes, as described elsewhere (31). Incubations were carried out overnight at 0 4 ° C . Clonal expansion assay. Cells were washed three times in RPMI 1640 without FCS, and diluted to 90,OOO cells/ml in RPMI 1640 with 3 % FCS. A sterile 1 % agar solution was prepared, and kept warm in water. Peptides were lyophilized, dissolved and diluted in RPMI 1640 with 0.25% human serum albumin to 1.5 times more than the desired final concentration. Aliquots (1 ml) were sterile-filtered into siliconized glass tubes and the cell suspension (50 pl) and agar (0.5 ml) were added. Four aliquots (250 pl each) were transferred to multiwell plates and incubated for 5-6 days in 95% 0,and 5 % CO,. Colonies of more than five cells were counted. Cyroroxic assay. Target cells (K562 or Daudi) were incubated with 5’Cr for 1 hour at 37°C. washed three times and resuspended in RPMI 1640 with 1 % BSA. Pretreated natural killer (NK)cells (effector cells) were added to target cells in microtitre plates (total volume 150 p1) and incubated for 4 hours at 37°C. Microplates were centrifuged at 250 xg for 2 minutes and supernatants (75 pl) were aspirated and radioactivity measured to determine the specific lysis of target cells.
RESULTS The binding of 1251-IGF-IIand its competitive inhibition by native and synthetic IGF-I1 and native, synthetic and recombinant IGF-I to K562 cells is shown in Fig. 1. It is clear that none of the three types of IGF-I investigated can compete with IGF-I1 binding in these cells in concentrations of up to 1 pg/ml, whereas both the IGF-I1 preparations used compete to a similar degree with the labelled IGF-11. This pattern was consistent at incubation temperatures of 4 and 37°C (incubation times, 16 hours and 15 minutes, respectively).
0
1 10 100 1000 IGF-I1 (ng/ml)
Fig. 1. Competitive inhibition of the binding of labelled IGF-I1 to K562 cells at 4°C by the following peptides: synthetic IGF-I1 (0-0); native IGF-I (0-0); synthetic IGF-I native IGF-I1 (0-0); ( 0 - 0 ) and recombinant IGF-I (A---A). Reproduced with permission from Tally M, Enberg G, Li CH, Hall K. The specificity of the human IGF-2 receptor. Biochem Biophys Res Commun 1987; 147: 1206-12.
IGF-N and its receptors
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50
c
OL
T
10% FCS
0
100
10
1
IGF-I1 (ng/ml)
Fig. 2. Growth of K562 cells as measured by formation of colonies in semi-solid agar in the presence of 10% FCS or increasing concentrations of IGF-I1 (A),IGF-I ( 0 )or insulin (0). The number of colonies is measured per 0.5 cm2. Reproduced with permission form Tally M, Li CH, Hall K. IGF-2 stimulated growth mediated by the somatomedin-type 2 receptor. Biochem Biophys Res Commun 1987;148: 811-16.
3.0-
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Q
T
a,
Q
Pg
5 = 2.0 C(D
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:g 1.0 $2 a, ;0-
*.
T
L
Insulin
I II Controls
IGF-I
0
IGF-I1
1 10 IGF-I1 (nghnl)
100
Fig. 3. (a) Specific binding of 'Z51-labelledinsulin, IGF-I and IGF-I1 evaluated in the presence of unlabelled insulin (1 p g / m l ) , IGF-I (100nglml), and IGF-II(100 ng/ml), respectively on the T-cell line Jurkat (*p < 0.05, * * p < 0.01,***p < 0.001).(b) Growth stimulation of IGF-I1 on Jurkat cells evaluated as colony formation in semi-solid agar in the presence of (I) 10% FCS, (11) 10 ng/ml IGF-I in 0.1% FCS, and increasing concentrations of FCS (0.1% ( O ) , 1 % (.) and 5% (0))and IGF-11. Standard deviations of quadruple determinations are indicated.
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Acta Paediatr Scand [Suppl] 367
D T
IL-2
FCS
IGF-I1 (ng/ml)
Fig. 4. The percentage stimulation of killing activity of NK cells over the 1 % BSA control by 100 IU IL-2, 5% FCS and increasing concentrations of IGF-I1 (2, 20, 200 ng/ml). Error bars indicate SEM for six
experiments.
The growth-promoting effects of insulin, IGF-I and IGF-I1 were evaluated by clonal expansion in semi-solid agar, counting colonies of five cells or more after a 5-day incubation. As shown in Fig. 2, IGF-II, and to some extent insulin, stimulated the formation of colonies, whereas IGF-I had no effect in this assay. As K562 is a cell line containing cells with features resembling pre-erythrocytes, other transformed cells of haematopoietic origin were evaluated for their distribution of receptors for insulin, IGF-I and IGF-11. The T-cell line Jurkat displayed specific binding of IGF-11, but very little or no IGF-I and insulin binding, as shown in Fig. 3a. Fig. 3b shows the growthstimulating effects of IGF-I1 on these cells, evaluated in three different concentrations of FCS (0.1, 1 and 5 %). IGF-I at a concentration of 10 n g / d caused no stimulation of growth. A previous investigation of T-lymphocytes from healthy donors indicated that they possess a very low ISF-I1 binding capacity that is increased after a 48-hour stimulation with a nonspecific antigen, phytohaemagglutinin (PHA) (32). When the T-cells were subfractionated, using CD3 antibodies, into small granular lymphocytes and NK cells, it was observed that the NK fraction had a 2-3 times higher specific binding of IGF-11, compared to the undifferentiated T-cells. Evaluation of the effects of IGF-I1 on the activity of the killer cells revealed that a 24-48-hour stimulation caused a concentration-dependent increase of the killing activity, as is shown in Fig. 4. No stimulation at all could be achieved with the same concentrations of IGF-I, insulin or GH, and IGFBP-1 had no inhibiting effect on the IGF-IIstimulated killing activity (data not shown). DISCUSSION A prerequisite to demonstrate the specific effects of IGF-I1 upon ligand binding has been the use of tissues or cells lacking response to IGF-I. Assay systems where only a minor IGF-I effect can be detected fail to fulfil these demands. The fact that IGF-I1 cross-reacts with the IGF-I receptor has obscured the net result of IGF-I1 stimulation from detection. The data presented here, however, disclose that IGF-I1 can act specifically, under certain conditions, in tissues where IGF-I exhibits no detectable biological response and demonstrates
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no significant amount of biriing. Furthermore, no cross-reactivity of IGF-I for the IGF-I1 receptor was found in these human cells, a finding supported by results with other human tissues (33). This is consistent with the characteristics of the purified human IGF-II/M6-P receptor (34). This would seem to indicate a difference between human and rat receptors, as IGF-I has been shown to have affinity for the rat IGF-I1 receptor (35). Both the K562 and Jurkat cell lines, as well as lymphocytes isolated from healthy subjects, are of haematopoietic origin (36). The few previous reports on specific growth effects of IGFI1 have been consistent with this finding. Daniak and Kreczko (37) showed that rat IGF-I1 stimulated both burst and colony formation in human bone marrow cultures, and Schimpff et al. (38) demonstrated a stimulatory effect on the 3H-thymidine incorporation in PHAstimulated T-cells. That IGF-I1 stimulated the growth of K562 cells was later confirmed by Blanchard et al. (39). They measured the effects of IGF-11, two preparations of IGF-I, crude IGFs, and insulin on the incorporation of 3H-thymidine into DNA. IGF-I1 was the most potent stimulator of DNA synthesis, but recombinant IGF-I also produced a considerable stimulation. This may be explained by the presence of low concentrations of type 1 IGF receptors in the strain of K562 cells used in their experiments. IGF-I1 had a marked effect on the activity of the NK cells isolated from healthy donors, compared to the 5% FCS control. Nevertheless, the effect obtained with 100 IU of interleukin 2 (IL-2) by far exceeds this effect. However, the highest concentration of IGF-I1 used was only 200 nglml. As the circulating concentration of IGF-I1 (bound to IGF binding proteins) in healthy humans ranges from 500 to 800 ng/ml, a concentration of 200 ng/ml may not be sufficient to result in maximal stimulation of DNA synthesis under these conditions. IGF-I-stimulated multiplication has been found to be inhibited by the addition of IGFBP-1, probably due to the resulting decrease in the active concentration of IGF-I. IGFBP-1 did not, however, inhibit the stimulation of NK cell activity by IGF-11. This might indicate that the active concentration of IGF-I1 is normally high and that the V,, for the IGF-I1 effect is not reached with the concentrations used. Affinity labelling, SDS-PAGE and autoradiography of intact, IGF-I1 responding cells (32) resulted in two labelled bands: a large protein with a molecular weight of approximately 250 kDa, probably corresponding to the previously purified and sequenced IGF-II/M6-P receptor (8), and a smaller protein with an approximate molecular weight of 75-80 m a . Surprisingly, no detectable labelling of the 250 kDa protein was found in Jurkat cells, and in the lymphocytes responding to IGF-I1 from healthy subjects, the 250 kDa band was barely detectable. The 75-80 kDa band was, however, always labelled to a large extent. This small molecular weight protein has yet to be purified and fully characterized, but its binding kinetics seem to differ from those of the 250 kDa IGF-II/M6-P receptor. When the 75-80 kDa is affinity labelled on intact cells, displacement of the radioactive affinity ligand can be achieved with 50 pglml unlabelled IGF-11 in the presence of 300 pg/ml insulin in order to inhibit the degradation of IGF-11, as insulin cannot compete for IGF-I1 binding sites. A partly purified fraction, however, displays specificity for IGF-I1 in a soluble competitive binding assay (unpublished data). The biological effects of IGF-I1 on lymphocytes from healthy donors are similar to several effects found when such cells are stimulated with IL-2. When small granular lymphocytes (CD3+, non-killer cells) were stimulated (unpublished data) with IGF-11 at a concentration of 200 ng/ml, an increased expression of the small IL-2 receptor (the Tac-antigen, 55 kDa) was found. This is also an effect obtained after IL-2 stimulation. In conclusion, it has been shown that IGF-I1 is a growth factor, acting through its own specific binding. This is shown in three cell types. However, the possibility that the effect of IGF-I1 may be indirect cannot yet be excluded, considering that in all experiments described the cells were incubated with IGF-I1 for 24-48 hours. It is possible that IGF-II may act by releasing another factor, thus acting in an autocrine fashion, either by inducing a growth promoter or by suppressing a growth inhibitor.
ACKNOWLEDGEMENT This study was supported by a grant from the Swedish Medical Research Council no. 4224.
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REFERENCES 1. Rinderknecht E, Humbel RE. Polypeptides with nonsuppressible insulin-like and cell-growth promoting
activities in human serum. Isolation, chemical characterization, and some biological properties of form I and 11. Proc Natl Acad Sci USA 1976; 73: 2365-9. 2. Rinderknecht E, Humbel RE. Primary structure of insulin-like growth factor 11. FEBS Lett 1978; 89: 2834. 3. Hall K, Sara VR. Growth and somatomedins. In: Munson PL, Diszfalusy E, Glover J, eds. Vitamins and hormones. New York: Academic Press, 1983; 40: 175-232. 4. Nissley SP, Rechler MM. Insulin-like growth factors: biosynthesis, receptors, and carrier proteins in hormonal proteins and peptides. In: Li CH, ed. Growth factors. New York: Academic Press, 1984; 12: 127-203. 5 . Froesch ER, Schmid C, Schwander J. Zapf J. Actions of insulin-like growth factors. Annu Rev Physiol 1985; 47: 443-67. 6. Ullrich A, Gray A, Tam AW er al. Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J 1986; 10: 2503- 12. 7. Ullrich A, Bell JR, Chen EY et 01. Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature 1985; 313: 756-61. 8. Morgan DO, Edman JC, Standring DN er al. Insulin-like growth factor I1 receptor as a multifunctional binding protein. Nature 1987; 329: 301-7. 9. Lobel P, Dahms N, Kornfeld S. Cloning and sequence analysis of the cation-independent mannose 6-phosphate receptor. J Biol Chem 1988; 263: 2563-70. 10. Roth RA, Stover C, Hari J et nl. Interactions of the receptor for insulin-like growth factor I1 with mannose-6-phosphate and antibodies to the mannose-6-phosphate receptor. Biochem Biophys Res Commun 1987; 149: 600-6. 1 1. Roth RA. Structure of the receptor for insulin-like growth factor 11: the puzzle amplified. Science 1988; 239: 1269-7 1. 12. Kiess W, Blickenstaff GD, Sklar MM, Thomas CL, Nissley SP, Sahagian GG. Biochemical evidence that the type I1 insulin-like growth factor receptor is identical to the cation-independent mannose-6-phosphate receptor. J Biol Chem 1988; 263: 9339-44. 13. Waheed A, Braulke T, Junghans U, von Figura K. Mannose-6-phosphate/insulin-likegrowth factor I1 receptor: the two types of ligands bind simultaneously to one receptor at different sites. Biochem Biophys _ _ Res Commun 1988; 152: 1248-54. 14 Corvera S, Whitehead RE, Mottola C, Czech MP. The insulin-like growth factor I1 receptor is phosphorylated by a tyrosine kinase in adipocyte plasma membranes. J Biol Chem 1986; 261: 7675-9. 15. King GL, Kahn CR, Rechler MM, Nissley SP. Direct demonstration of separate receptors for growth and metabolic activities of insulin and multiplication-stimulating activity (an insulin-like growth factor) using antibodies to the insulin receptor. J Clin Invest 1980; 66: 130-40. 16. Kasuga M, Van Obberghen E, Nissley SP, Rechler MM. Demonstration of two subtypes of insulin-like growth factor receptors by affinity cross-linking. J Biol Chem 1981; 256: 5305-8. 17. Van Wyk JJ. The somatomedins: biological actions and physiologic control mechanisms in hormonal proteins and peptides. In: Li CH, ed. Growth factors. New York: Academic Press, 1984; 12: 81-125. 18. Rechler MM, Fryklund L, Nissley SP et al. Purified human somatomedin A and rat multiplication stimulating activity. Mitogens for cultured fibroblasts that crossreact with the same growth peptide receptors. Eur J Biochem 1978; 82: 5-12. 19. Enberg G, Tham A, Sara VR. The influence of purified somatomedins and insulin on foetal rat brain DNA synthesis in vitro. Acta Physiol Scand 1985; 125: 305-8. 20. Conover CA, Misra P, Hintz RL, Rosenfeld RG. Effect of an anti-insulin-like growth factor I receptor antibody on insulin-like growth factor I1 stimulation of DNA synthesis in human fibroblasts. Biochem Biophys Res Commun 1986; 139: 501-8. 21. Furlanetto RW, DiCarlo JN, Wisehart C. The type I1 insulin-like growth factor receptor does not mediate deoxyribonucleic acid synthesis in human fibroblasts. J Clin Endocrinol Metab 1987; 64: 1142-9. 22. Mottola C , Czech MP. The type I1 insulin-like growth factor receptor does not mediate increased DNA synthesis in H-35 hepatoma cell. J Biol Chem 1984; 259: 12705-13. 23. Kiess W, Haskell JF, Lee L er al. An antibody that blocks insulin-like growth factor (IGF) binding to the type I1 IGF receptor is neither an agonist nor an inhibitor of IGF-stimulated biological responses in L6 myoblasts. J Biol Chem 1987; 262: 12745-51. 24. Enberg G, Carlquist M, Jornvall H, Hall K. The characterization of somatomedin A, isolated by microcomputer-controlled chromatography, reveals an apparent identity to insulin-like growth factor 1. Eur J Biochem 1984; 143: 117-24. 25. Tally M, Florell K, Enberg G. An effective method for the separation of insulin-like growth factors 1 and 2 during the purification process. Bioscience Rep 1988; 8: 293-7. 26. Hammarberg B, Moks T, Tally M er al. Different stability of recombinant human insulin-like growth factor I1 in Escherichia coli and Sraphylococcus aureus. J Biotechnol; in press. 27. P6voa G, Enberg G, Jornvall H,Hall K. Isolation and characterization of a somatomedin-binding protein from mid-term human amniotic fluid. Eur J Biochem 1984; 144: 199-204.
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28. Weiss A, Stobo JD. Requirement for the coexpression of T3 and the T cell antigen receptor on a malignant human T cell line. J Exp Med 1984; 160: 1284-99. 29. Boyum A. Separation of leucocytes from blood and bone marrow. Scand J Clin Lab Invest [Suppl] 1968; 21: 97. 30. Wysocki LJ, Sato VL. ‘Panning’ for lymphocytes: a method for cell selection. Proc Natl Acad Sci USA 1978; 75: 2844-8. 31. Tang X-Z,Tally M, Jondal M, Hall K. Characterization of insulin binding to the erythroleukemia cell line K 562. Biochem Biophys Res Commun 1983; 117: 826-33. 32. Tally M. Insulin-like growth factor II. Studies on binding and biological effects in target tissues. Thesis, Karolinska Institutet; 1989. 33. Tally M, Enberg G, Li CH, Hall K. The specificity of the human IGF-2 receptor. Biochem Biophys Res Commun 1987; 147: 1206-12. 34. Roth RA, Steele-Perkins G, Hari J et al. Insulin and insulin-like growth factor receptors and responses. Cold Spring Harbor Symp Quant Biol 1988; 53: 537-43. 35. Rosenfeld RG, Conover CA, Hodges D er al. Heterogeneity of insulin-like growth factor-I affinity for the insulin-like growth factor-I1 receptor. Comparison of natural, synthetic and recombinant DNA-derived insulin-like growth factor-I. Biochem Biophys Res Commun 1987; 143: 199-205. 36. Tally M, Li CH, Hall K. IGF-2 stimulated growth mediated by the somatomedin-type 2 receptor. Biochem Biophys Res Commun 1987; 148: 81 1-16. 37. Daniak N, Kreczko S. Interactions of insulin-like growth factor 11, and platelet derived growth factor in erythropoietic culture. J Clin Invest 1985; 76: 1 2 3 7 4 2 . 38. Schimpff R-M, Repellin A-M, Salvatoni A, Thieriot-Prevost G, Chatelain P. Effect of purified somatomedins on thymidine incorporation into lectin-activated human lymphocytes. Acta Endocrinol (Copenh) 1983; 102: 21-6. 39. Blanchard MM, Barenton B, Sullivan A, Foster B, Guyda HJ, Posner BI. Characterization of the insulinlike growth factor (IGF) receptor in K562 erythroleukemia cells; evidence for a biological function for the type I1 IGF receptor. Mol Cell Endocrinol 1988; 56: 235-44.
(M.T.) Department of Endocrinology Karolinska Institute S-104 01 Stockholm Sweden
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