Short Communications Pitfalls in Insulin-Like Growth Factor (IGF) Receptor Binding Studies Using Cells in Monolayer Culture M. J. Duclos1 and C. Goddard Department of Cellular and Molecular Biology, AFRC, Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Roslin, Midlothian, United Kingdom
Introduction
Materials and Methods
Hormone binding to cell monolayers directly in culRecombinant cDNA-derived human IGF-I and ture dishes is a widely used technique in the analysis of receptors. It is IGF-II were obtained from Bachem (Saffron Walden, UK) or from rapid, convenient and theoretically allows study of the receptor in conAmgen (Amersham International, Buckinghamshire, UK). Monoditions similar to those in which biological effects mediated by the ligcomponent porcine insulin was obtained from Novo Laboratories and are measured. Many such studies have been conducted using the (Copenhagen, Denmark). The peptides were iodinated by a modificainsulin-like growth factors (IGF-I and IGF-II) in different cell types tion of the chloramine T method as described earlier (Duclos and Godand these have allowed the characterisation of two distinct receptors dard 1990). (type I and type II). The type I IGF receptor is considered to mediate most if not all of the effects of IGF peptides. However, discrepancies Primary muscle satellite cells were prepared from have been reported between the biological activities and binding breast muscle obtained from 5 day old chicks and maintained in culpotencies of the related peptides insulin, IGF-I and IGF-II for the type ture in multiwell plates as previously described (Duclos, Wilkie and I IGF receptor. For instance, a difference between the concentration Goddard 1991). The procedure employed an obligatory 24 h period in DMEM containing 10% fetal calf serum (DMEM/10% FCS) to of IGF-II required to displace [125I]-labelled IGF-I binding and to inallow cells to seed efficiently on gelatin coated plates. Subsequently, itiate biological activity has been reported in the BC3H1 muscle cell line (De Vroede, Romanus, Standert, Pollet, Nissley and Rechler 1984) cells were growth restricted for 24 h in DMEM/0.02 % FCS. and the L6 muscle cell line {Ballard, Read, Francis, Bagley and Wallace 1986) although not by all investigators (Beguinot, Kahn, Moses Binding studies were performed as described (Duand Smith 1985; Ewton, Fahlen and Florini 1987). This has been inter- clos, Wilkie and Goddard 1991). Briefly, the medium was harvested, preted as evidence that the biological activity was mediated by a type I the cell monolayer was washed twice with PBS and incubated with receptor and that the binding data is a reflection of substantial binding DMEM containing 0.5% Bovine Serum Albumin (BSA, RIA grade; of IGF-I to the type II receptor. Sigma, Poole, UK), [l25I]-labelled IGF-I or IGF-II (about 100,000 cpm/ml equivalent to 0.4 ng/ml) and appropriate amounts of cold peptide at 15 °C for 4 h or 24 h at 4 °C. The monolayer was washed We have conducted binding studies using IGF-I and IGF-II on chicken muscle cells in monolayer culture according to stanthree times with ice-cold PBS and solubilised with 0.2% SDS before dard procedures used for different muscle cell lines (De Vroede et al. counting for radioactivity. Binding in the absence of cells was con1984; Beguinot et al. 1985; Ballard et al. 1986; Ewton, Fahlen and ducted under the same conditions on gelatin coated plates which had Florini 1987). As the type II receptor is absent in chicken tissues {Can- been incubated at 37 °C in an atmosphere of 5% CO2 in DMEM/10% field and Komfeld 1989), IGF peptides were expected to bind only to FCS or DMEM/2% Ultroser (Gibco-BRL, Paisley, UK) for the first the type I receptor. However, unusually high concentrations of insulin 24 h and DMEM/0.02 % FCS or DMEM/0.004% Ultroser for the (40 ug/1) were required to displace [125I]-labelled IGF-I and IGF-II last 20 h before the binding experiment. For some experiments, culture binding and IGF-II was more potent than IGF-I in displacing either plates were pretreated overnight with 10% BSA which was aspirated ligand. Moreover, IGF binding increased rather than decreased when before cell culture and subsequent binding studies to cells or to plates fewer cells were seeded, raising questions about the validity of the syssimilarly treated in the absence of cells. tem. We overcame this difficulty by preincubating the culture plates with bovine serum albumin (BSA) and characterised IGF binding in Results chicken muscle satellite cells under these conditions (Duclos, Wilkie and Goddard 1991). In the absence of cells, no specific binding of either ligand was observed on culture plates which had not been treated with We demonstrate in this paper that IGF peptides are FCS. In contrast, binding of [125l]-labelled hIGF-I and IGF-II was obable to bind to culture plates treated with serum supplemented culture served using serum treated culture plates. This binding could be inmedium, in the absence of cells, with characteristics usually observed hibited by the addition of either unlabelled IGF peptide and was for specific binding to receptors. This IGF binding activity is probably higher in the absence than in the presence of cells (Fig. la). Under due to the adhesion of specific IGF binding proteins to the culture these conditions no specific binding of [ I]-labelled insulin to the plates which interfere with receptor binding at the cell surface. plates was observed (data not shown). Similar binding was observed on plates (in the absence of cells) whether they were coated with gelatin, collagen, or not coated (data not shown). When a serum substiPresent address: tute was used (DMEM/2% Ultroser), binding to empty plates was Unite d'Endocrinologie de la Croissance et du Metabolisme markedly reduced compared to that seen with FCS (Fig. lb), although Station de Recherches Avicoles, INRA, Nouzilly 125TI to a lesser extent in the case of IGF-II. The binding of [125I]-labelled F-37380 Monnaie (France) Horm.metab.Res.23(1991)562-564 © Georg Thieme Verlag Stuttgart • New York
Received: 8 March 1991
Accepted: 12 Aug. 1991 after revision
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
562
Pitfalls in IGF Receptor Binding Studies
Horm. metab. Res. 23 (1991)
563
IGF-I or IGF-II to the FCS treated plates was displaced in a dose dependent manner by increasing concentrations of the corresponding unlabelled ligand (Fig. Id). The affinity constants calculated from this data were in the range of 109 1/mole. Pretreatment of the plates with BSA resulted in a complete inhibition of subsequent IGF-I and IGF-II binding to the culture plates in the absence of cells (Fig. lc). Under these conditions specific binding of IGF-I was linearly and significantly correlated (r = 0.96; p < 0.01; df = 11) with the number of cells seeded at the beginning of the culture (Table 1) as would be expected for association of ligand with cell membrane receptor.
Fig. 1 Binding of [125l]-labelled IGF-I and IGF-II to multiwell plates in the presence or in the absence of satellite cells. All plates were coated with gelatin prior to culture or any other treatment. Satellite cells (+cells, Fig. 1a) were plated at a density of 100,000 cells/cm2 and incubated in DMEM/10% FCS for 24 h followed by DMEM/0.02% FCS for the next 20 h. Empty plates receiving no cells (-cells, Fig. 1a) were treated similarly. Binding was carried out, after two washes with PBS, for 24 h at 4 °C (Fig. 1a) or for 4 h at 15 °C (Fig. 1b to 1d). In Fig. 1b, empty plates were treated prior to binding experiments with DMEM containing FCS (10% then 0.02%) as for cell culture (FCS), or Ultroser (2% then 0.004%) (Ultroser). In Fig. 1c, prior to treatment with DMEM containing FCS (10% then 0.02%) and binding, half of the empty plates (BSA) were pretreated with BSA (10%) while the other half (FCS) were not. In Fig. 1 d, binding of [1251 ]-labelled IGF-I or IGF-II to empty plates, treated with FCS (without BSA pretreatment) was inhibited by the corresponding unlabelled peptide. Bo = binding of iodinated ligand alone; B = binding of the iodinated ligand in the presence of the indicated concentration of the corresponding unlabelled ligand; NSB = non specific binding measured in the presence of unlabelled IGF-I or IGF-II (100 ng/ml); SB = specific binding (Bo-NSB). All values are expressed as mean±standard error of the mean of two to five replicates and as % of the total counts.
Our results indicate that despite the omission of serum from late stages of culture and with extensive washing, components originating from fetal calf serum used in medium allowing seeding of cells interfere with subsequent IGF binding studies on cell monolayers. This can lead to erroneous results in term of specificity and apparent receptor number. The specificity and high affinity for IGF-I and IGF-II strongly argue that these components are in fact specific IGF binding proteins (IGF-BP), which have been shown to inhibit IGF binding to its receptor when present in solution (De Vroede, Tseng, Katsoyannis, Nissley and Redder 1986). The effect of pretreatment with BSA suggests that these IGF-BP adhere to the plastic or gelatin surface in a non-specific manner, permitting subsequent specific binding of the IGF peptides. The results are different from those obtained with IGF-BP in solution (De Vroede et al. 1986) which showed that [125I]-labelled IGF binding was paradoxically increased by the addition of low concentrations of unlabelled IGF. In the case of contamination with adherent IGF-BP the modifications are much less evident but result in unusual specificity and possibly a high number of binding sites. There are a number of possibilities for avoiding this problem. The development of a synthetic culture medium suitable for the entire length of the culture is likely to be the most preferable. It is not presently available and even under these conditions it would be difficult to rule out the possibility that IGF-BP secreted by the cells would not adhere to the plates. Alternatively, binding studies on cells suspensions or on membrane preparations would be acceptable but other constraints such as low yields and therefore the requirement for very large cell preparations apply. Analogs of IGF-I have recently been characterised, which bind to the type I receptor but do not bind to IGF-BP (Szabo, Mottershead, Ballard and Wallace 1988; McCusker, Camacho-Hubner, Bayne, Cascieri and Clemmons 1990) and could be used to specifically assess these receptors even in the presence of IGFBP. However, the IGF-BP would still interfere with unlabelled IGF-I and IGF-II if they were used to inhibit the binding of the iodinated analog and it would therefore be difficult to establish the specificity of the receptor for the native peptides. We have shown that this pitfall can be overcome by pretreatment of the culture plates with BSA to inhibit IGF-BP adhesion. We presume that the BSA occupies non-specific
Table 1 Effect of cell density on [125l]-labelled IGF-I binding to monolayers. All plates were coated with gelatin (0.1 %) and subsequently pretreated with BSA (10%), before muscle satellite cells were plated at the indicated densities. For the first 24 h the cells were incubated in DMEM/10% FCS, which was subsequently replaced by DMEM/0.02% (20 h). Binding was carried out, after two washes with PBS, for 4 h at 15 °C. Bo = binding of iodinated ligand alone; NSB = non specific binding measured in the presence of unlabelled IGF-I (100 ng/ml); SB = specific binding (Bo-NSB). All values are expressed as mean±standard error of the mean, as % of the total counts; (n)=the number of replicates. Experiment BSA pretreated no cells 100,000 cells/cm2 200,000 cells/cm2 300,000 cells/cm2
Bo (%) 0.30 ±0.03 0.97 ±0.20 1.70 ±0.23 2.97 ±0.24
NSB (%) (4) (2) (2) (2)
0.13 ±0.02 0.23 ±0.01 0.29 ±0.03 0.30 ±0.04
SB (%) (2) (2) (2) (2)
0.17 ±0.03 0.73 ±0.20 1.44±0.19 2.67 ±0.24
(4) (2) (2) (4)
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Discussion
564 Horm. metab. Res. 23 (1991)
These observations indicate that binding studies involving IGF peptides conducted on cells in monolayer culture must be carefully interpreted especially if serum has been used at any time of the culture. Interference with IGF-BP whether originating from serum or produced by the cells can lead to erroneous results. This is especially true if the receptor number is relatively low. We suggest that until this is carefully ruled out it is difficult to interpret experiments in which discrepancies are observed between binding properties and biological properties of IGF peptides and insulin.
References Ballard, F. J., L. C. Read, G. L. Francis, C. J. Bagley, J. C. Wallace: Biochem. J. 233:223-230 (1986) Beguinot, F., R. Kahn, A. C. Moses, R. J. Smith: J. Biol. Chem. 260: 15892-15898(1985) Canfield, W.M.,S.Kornfeld.J.Biol.Chem.264:7100-7103(1989) De Vroede, M. A., J. A. Romanus, M. L. Standert, R. J. Pollet, S. P. Nissley, M. M. Rechler: Endocrinology 114: 1917-1929 (1984) De Vroede, M. A., L. H. Y. Tseng, G. Katsoyannis, S. P. Nissley, M. M. Rechler.]. Clin. Invest. 77:602-613 (1986) Duclos,M. J., C. Goddard.].Endocr. 125:199-206(1990) Duclos, M. J., R. S. Wilkie, C. Goddard:]. Endocr. 126:35-42 (1991) Ewton, D. Z., S. L. Fahlen, J. R. Florini: Endocrinology 120: 115—123 (1987) McCusker, R. H., C. Camacho-Hubner, M. L. Bayne, M. A. Cascieri, D.R. Clemmons:]. Cell. Physiol. 144:244-253 (1990) Szabo, L., D. G. Mottershead, F. J. Ballard, J. C. Wallace: Biochem. Biophys. Res. Comm. 151:207-214(1988)
Requests for reprints should be addressed to: Acknowledgements We would like to thank Dr. J. Simon for helpful discussion and advice in the preparation of this manuscript and Ross Breeders (Newbridge, Midlothian, UK) for a generous supply of animals. The cost of this research has been funded in part by a grant from Institut National de la Recherche Agronomique (AIP-94714): 'Mecanismes cellulaires et endocriniens de la Croissance' to M. J. D.
M. J. Duclos Unite d'Endocrinologie de la Croissance et du Metabolisme Station de Recherches Avicoles, INRA F-37380 Nouzilly (France)
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
binding sites on the plastic or gelatin which otherwise bind the IGFBP. BSA pretreatment of the culture plates allows the measurement of IGF-I binding which increases linearly with cell number and unequivocally represents cell surface binding (Table 1). Under these conditions, the presence of type I IGF receptors has been characterised in satellite cells prepared from 5 day-old chicks and as reported in other chicken tissues, the absence of type II receptors in these cells has been observed {Duclos, Wilkie and Goddard 1991). In addition, a good agreement was observed between the potency of IGF-I, IGF-II and insulin to inhibit iodinated IGF-I binding to these cells and their potency to stimulate DNA synthesis {Duclos, Wilkie and Goddard 1991).
M. J. Duclos and C. Goddard
Introduction
Materials and Methods
Hormone binding to cell monolayers directly in culRecombinant cDNA-derived human IGF-I and ture dishes is a widely used technique in the analysis of receptors. It is IGF-II were obtained from Bachem (Saffron Walden, UK) or from rapid, convenient and theoretically allows study of the receptor in conAmgen (Amersham International, Buckinghamshire, UK). Monoditions similar to those in which biological effects mediated by the ligcomponent porcine insulin was obtained from Novo Laboratories and are measured. Many such studies have been conducted using the (Copenhagen, Denmark). The peptides were iodinated by a modificainsulin-like growth factors (IGF-I and IGF-II) in different cell types tion of the chloramine T method as described earlier (Duclos and Godand these have allowed the characterisation of two distinct receptors dard 1990). (type I and type II). The type I IGF receptor is considered to mediate most if not all of the effects of IGF peptides. However, discrepancies Primary muscle satellite cells were prepared from have been reported between the biological activities and binding breast muscle obtained from 5 day old chicks and maintained in culpotencies of the related peptides insulin, IGF-I and IGF-II for the type ture in multiwell plates as previously described (Duclos, Wilkie and I IGF receptor. For instance, a difference between the concentration Goddard 1991). The procedure employed an obligatory 24 h period in DMEM containing 10% fetal calf serum (DMEM/10% FCS) to of IGF-II required to displace [125I]-labelled IGF-I binding and to inallow cells to seed efficiently on gelatin coated plates. Subsequently, itiate biological activity has been reported in the BC3H1 muscle cell line (De Vroede, Romanus, Standert, Pollet, Nissley and Rechler 1984) cells were growth restricted for 24 h in DMEM/0.02 % FCS. and the L6 muscle cell line {Ballard, Read, Francis, Bagley and Wallace 1986) although not by all investigators (Beguinot, Kahn, Moses Binding studies were performed as described (Duand Smith 1985; Ewton, Fahlen and Florini 1987). This has been inter- clos, Wilkie and Goddard 1991). Briefly, the medium was harvested, preted as evidence that the biological activity was mediated by a type I the cell monolayer was washed twice with PBS and incubated with receptor and that the binding data is a reflection of substantial binding DMEM containing 0.5% Bovine Serum Albumin (BSA, RIA grade; of IGF-I to the type II receptor. Sigma, Poole, UK), [l25I]-labelled IGF-I or IGF-II (about 100,000 cpm/ml equivalent to 0.4 ng/ml) and appropriate amounts of cold peptide at 15 °C for 4 h or 24 h at 4 °C. The monolayer was washed We have conducted binding studies using IGF-I and IGF-II on chicken muscle cells in monolayer culture according to stanthree times with ice-cold PBS and solubilised with 0.2% SDS before dard procedures used for different muscle cell lines (De Vroede et al. counting for radioactivity. Binding in the absence of cells was con1984; Beguinot et al. 1985; Ballard et al. 1986; Ewton, Fahlen and ducted under the same conditions on gelatin coated plates which had Florini 1987). As the type II receptor is absent in chicken tissues {Can- been incubated at 37 °C in an atmosphere of 5% CO2 in DMEM/10% field and Komfeld 1989), IGF peptides were expected to bind only to FCS or DMEM/2% Ultroser (Gibco-BRL, Paisley, UK) for the first the type I receptor. However, unusually high concentrations of insulin 24 h and DMEM/0.02 % FCS or DMEM/0.004% Ultroser for the (40 ug/1) were required to displace [125I]-labelled IGF-I and IGF-II last 20 h before the binding experiment. For some experiments, culture binding and IGF-II was more potent than IGF-I in displacing either plates were pretreated overnight with 10% BSA which was aspirated ligand. Moreover, IGF binding increased rather than decreased when before cell culture and subsequent binding studies to cells or to plates fewer cells were seeded, raising questions about the validity of the syssimilarly treated in the absence of cells. tem. We overcame this difficulty by preincubating the culture plates with bovine serum albumin (BSA) and characterised IGF binding in Results chicken muscle satellite cells under these conditions (Duclos, Wilkie and Goddard 1991). In the absence of cells, no specific binding of either ligand was observed on culture plates which had not been treated with We demonstrate in this paper that IGF peptides are FCS. In contrast, binding of [125l]-labelled hIGF-I and IGF-II was obable to bind to culture plates treated with serum supplemented culture served using serum treated culture plates. This binding could be inmedium, in the absence of cells, with characteristics usually observed hibited by the addition of either unlabelled IGF peptide and was for specific binding to receptors. This IGF binding activity is probably higher in the absence than in the presence of cells (Fig. la). Under due to the adhesion of specific IGF binding proteins to the culture these conditions no specific binding of [ I]-labelled insulin to the plates which interfere with receptor binding at the cell surface. plates was observed (data not shown). Similar binding was observed on plates (in the absence of cells) whether they were coated with gelatin, collagen, or not coated (data not shown). When a serum substiPresent address: tute was used (DMEM/2% Ultroser), binding to empty plates was Unite d'Endocrinologie de la Croissance et du Metabolisme markedly reduced compared to that seen with FCS (Fig. lb), although Station de Recherches Avicoles, INRA, Nouzilly 125TI to a lesser extent in the case of IGF-II. The binding of [125I]-labelled F-37380 Monnaie (France) Horm.metab.Res.23(1991)562-564 © Georg Thieme Verlag Stuttgart • New York
Received: 8 March 1991
Accepted: 12 Aug. 1991 after revision
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
562
Pitfalls in IGF Receptor Binding Studies
Horm. metab. Res. 23 (1991)
563
IGF-I or IGF-II to the FCS treated plates was displaced in a dose dependent manner by increasing concentrations of the corresponding unlabelled ligand (Fig. Id). The affinity constants calculated from this data were in the range of 109 1/mole. Pretreatment of the plates with BSA resulted in a complete inhibition of subsequent IGF-I and IGF-II binding to the culture plates in the absence of cells (Fig. lc). Under these conditions specific binding of IGF-I was linearly and significantly correlated (r = 0.96; p < 0.01; df = 11) with the number of cells seeded at the beginning of the culture (Table 1) as would be expected for association of ligand with cell membrane receptor.
Fig. 1 Binding of [125l]-labelled IGF-I and IGF-II to multiwell plates in the presence or in the absence of satellite cells. All plates were coated with gelatin prior to culture or any other treatment. Satellite cells (+cells, Fig. 1a) were plated at a density of 100,000 cells/cm2 and incubated in DMEM/10% FCS for 24 h followed by DMEM/0.02% FCS for the next 20 h. Empty plates receiving no cells (-cells, Fig. 1a) were treated similarly. Binding was carried out, after two washes with PBS, for 24 h at 4 °C (Fig. 1a) or for 4 h at 15 °C (Fig. 1b to 1d). In Fig. 1b, empty plates were treated prior to binding experiments with DMEM containing FCS (10% then 0.02%) as for cell culture (FCS), or Ultroser (2% then 0.004%) (Ultroser). In Fig. 1c, prior to treatment with DMEM containing FCS (10% then 0.02%) and binding, half of the empty plates (BSA) were pretreated with BSA (10%) while the other half (FCS) were not. In Fig. 1 d, binding of [1251 ]-labelled IGF-I or IGF-II to empty plates, treated with FCS (without BSA pretreatment) was inhibited by the corresponding unlabelled peptide. Bo = binding of iodinated ligand alone; B = binding of the iodinated ligand in the presence of the indicated concentration of the corresponding unlabelled ligand; NSB = non specific binding measured in the presence of unlabelled IGF-I or IGF-II (100 ng/ml); SB = specific binding (Bo-NSB). All values are expressed as mean±standard error of the mean of two to five replicates and as % of the total counts.
Our results indicate that despite the omission of serum from late stages of culture and with extensive washing, components originating from fetal calf serum used in medium allowing seeding of cells interfere with subsequent IGF binding studies on cell monolayers. This can lead to erroneous results in term of specificity and apparent receptor number. The specificity and high affinity for IGF-I and IGF-II strongly argue that these components are in fact specific IGF binding proteins (IGF-BP), which have been shown to inhibit IGF binding to its receptor when present in solution (De Vroede, Tseng, Katsoyannis, Nissley and Redder 1986). The effect of pretreatment with BSA suggests that these IGF-BP adhere to the plastic or gelatin surface in a non-specific manner, permitting subsequent specific binding of the IGF peptides. The results are different from those obtained with IGF-BP in solution (De Vroede et al. 1986) which showed that [125I]-labelled IGF binding was paradoxically increased by the addition of low concentrations of unlabelled IGF. In the case of contamination with adherent IGF-BP the modifications are much less evident but result in unusual specificity and possibly a high number of binding sites. There are a number of possibilities for avoiding this problem. The development of a synthetic culture medium suitable for the entire length of the culture is likely to be the most preferable. It is not presently available and even under these conditions it would be difficult to rule out the possibility that IGF-BP secreted by the cells would not adhere to the plates. Alternatively, binding studies on cells suspensions or on membrane preparations would be acceptable but other constraints such as low yields and therefore the requirement for very large cell preparations apply. Analogs of IGF-I have recently been characterised, which bind to the type I receptor but do not bind to IGF-BP (Szabo, Mottershead, Ballard and Wallace 1988; McCusker, Camacho-Hubner, Bayne, Cascieri and Clemmons 1990) and could be used to specifically assess these receptors even in the presence of IGFBP. However, the IGF-BP would still interfere with unlabelled IGF-I and IGF-II if they were used to inhibit the binding of the iodinated analog and it would therefore be difficult to establish the specificity of the receptor for the native peptides. We have shown that this pitfall can be overcome by pretreatment of the culture plates with BSA to inhibit IGF-BP adhesion. We presume that the BSA occupies non-specific
Table 1 Effect of cell density on [125l]-labelled IGF-I binding to monolayers. All plates were coated with gelatin (0.1 %) and subsequently pretreated with BSA (10%), before muscle satellite cells were plated at the indicated densities. For the first 24 h the cells were incubated in DMEM/10% FCS, which was subsequently replaced by DMEM/0.02% (20 h). Binding was carried out, after two washes with PBS, for 4 h at 15 °C. Bo = binding of iodinated ligand alone; NSB = non specific binding measured in the presence of unlabelled IGF-I (100 ng/ml); SB = specific binding (Bo-NSB). All values are expressed as mean±standard error of the mean, as % of the total counts; (n)=the number of replicates. Experiment BSA pretreated no cells 100,000 cells/cm2 200,000 cells/cm2 300,000 cells/cm2
Bo (%) 0.30 ±0.03 0.97 ±0.20 1.70 ±0.23 2.97 ±0.24
NSB (%) (4) (2) (2) (2)
0.13 ±0.02 0.23 ±0.01 0.29 ±0.03 0.30 ±0.04
SB (%) (2) (2) (2) (2)
0.17 ±0.03 0.73 ±0.20 1.44±0.19 2.67 ±0.24
(4) (2) (2) (4)
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Discussion
564 Horm. metab. Res. 23 (1991)
These observations indicate that binding studies involving IGF peptides conducted on cells in monolayer culture must be carefully interpreted especially if serum has been used at any time of the culture. Interference with IGF-BP whether originating from serum or produced by the cells can lead to erroneous results. This is especially true if the receptor number is relatively low. We suggest that until this is carefully ruled out it is difficult to interpret experiments in which discrepancies are observed between binding properties and biological properties of IGF peptides and insulin.
References Ballard, F. J., L. C. Read, G. L. Francis, C. J. Bagley, J. C. Wallace: Biochem. J. 233:223-230 (1986) Beguinot, F., R. Kahn, A. C. Moses, R. J. Smith: J. Biol. Chem. 260: 15892-15898(1985) Canfield, W.M.,S.Kornfeld.J.Biol.Chem.264:7100-7103(1989) De Vroede, M. A., J. A. Romanus, M. L. Standert, R. J. Pollet, S. P. Nissley, M. M. Rechler: Endocrinology 114: 1917-1929 (1984) De Vroede, M. A., L. H. Y. Tseng, G. Katsoyannis, S. P. Nissley, M. M. Rechler.]. Clin. Invest. 77:602-613 (1986) Duclos,M. J., C. Goddard.].Endocr. 125:199-206(1990) Duclos, M. J., R. S. Wilkie, C. Goddard:]. Endocr. 126:35-42 (1991) Ewton, D. Z., S. L. Fahlen, J. R. Florini: Endocrinology 120: 115—123 (1987) McCusker, R. H., C. Camacho-Hubner, M. L. Bayne, M. A. Cascieri, D.R. Clemmons:]. Cell. Physiol. 144:244-253 (1990) Szabo, L., D. G. Mottershead, F. J. Ballard, J. C. Wallace: Biochem. Biophys. Res. Comm. 151:207-214(1988)
Requests for reprints should be addressed to: Acknowledgements We would like to thank Dr. J. Simon for helpful discussion and advice in the preparation of this manuscript and Ross Breeders (Newbridge, Midlothian, UK) for a generous supply of animals. The cost of this research has been funded in part by a grant from Institut National de la Recherche Agronomique (AIP-94714): 'Mecanismes cellulaires et endocriniens de la Croissance' to M. J. D.
M. J. Duclos Unite d'Endocrinologie de la Croissance et du Metabolisme Station de Recherches Avicoles, INRA F-37380 Nouzilly (France)
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
binding sites on the plastic or gelatin which otherwise bind the IGFBP. BSA pretreatment of the culture plates allows the measurement of IGF-I binding which increases linearly with cell number and unequivocally represents cell surface binding (Table 1). Under these conditions, the presence of type I IGF receptors has been characterised in satellite cells prepared from 5 day-old chicks and as reported in other chicken tissues, the absence of type II receptors in these cells has been observed {Duclos, Wilkie and Goddard 1991). In addition, a good agreement was observed between the potency of IGF-I, IGF-II and insulin to inhibit iodinated IGF-I binding to these cells and their potency to stimulate DNA synthesis {Duclos, Wilkie and Goddard 1991).
M. J. Duclos and C. Goddard
