Insulin-like growth factor receptors in normal and tumorous adult human adrenocortical glands
Matthias M Weber, Christoph J Auernhammer, Wieland Kiess1 and Dieter Engelhardt
Medical Department II, Laboratory of Endocrine Research, Klinikum Großhadern, University of Munich, Germany and 1 Department of Pediatrics, University of Giessen, Germany
(Correspondence should be addressed to M M Weber, Medizinische Klinik II, Klinikum Großhadern, Marchioninistr. 15, 81377 München, Germany)
Abstract
We have identified and characterized insulin-like growth factor (IGF)-I and IGF-II/mannose-63 phosphate (IGF-II/M6P) receptors in normal adult human adrenocortical tissue. Furthermore, web investigated the IGF-I receptor concentration and binding characteristics in benign and carcinomatous adrenocortical tumors. Membrane preparations of 14 normal adrenocortical glands showed a mean specific 125I-IGF-I binding (SB) of 5.0 ±0.5% and a competition by unlabeled ligands which is characteristic of the IGF-I receptor. The Scatchard analysis revealed a single class of high affinity binding sites with a dissociation constant (Ka) of 0.16 + 0.03 nmol/l, and a receptor concentration (RC) of 19. 2± 2.5 nmol/kg protein. Affinity cross-linking experiments with normal and tumorous adrenocortical tissue displayed a band at an apparent molecular mass of 135 kDa, corresponding to the size of the normal a-subunit of the IGF-I receptor. In agreement, 125I-IGF-II binding to normal adult human adrenocortical membranes was characteristic for the IGF-II/M6P receptor, and the Scatchard analysis revealed the presence of a single class of high affinity binding sites (SB 7.5 + 0.5% RC 1137 ± 265 nmol/kg protein, Ka 2.20 ± 0.46 nmol/l, n = 6). The identity of the IGF-II/M6P6 receptor in adrenocortical tissue was further confirmed by Western blotting showing a specific band” at 220 kDa. When 125I-IGF-I binding in adrenocortical hyperplasias (SB 4. 1± 0.4%, RC 19.6 9 2.0 nmol/kg protein, Ka 0.19 ± 0.04 nmol/l, n=4) and adenomas (SB 4.0 ± 1.1%, RC 17.5 + 3.1 nmol/kg protein, Ka 0.21 + 0.04 nmol/l, n=4) was compared with the 125I-IGF-I binding in normal adrenocortical tissue, similar IGF-I receptor concentration and binding kinetics were found. Inb contrast, three out of four hormonally active adrenocortical carcinomas showed a strongly elevated” specific 1251-IGF-I binding with a 3- to 4-fold increase in IGF-I receptor concentration, as compared with normal adrenocortical tissue. This resulted in a significantly higher mean specific binding and receptor concentration in adrenocortical carcinomas, while the binding kinetics and the size of the a-subunit of the IGF-I receptor remained unaltered (n = 4, SB 13.8 ± 4.2%, RC 72.2 ± 21 .3 nmol/kg protein, Ka 0.17 ± 0.02 nmol/l). In summary, we show that intact IGF-I and IGF-II receptors are present in normal adult human adrenocortical tissue. While the abundance of the IGF-I receptor in adrenocortical hyperplasias and adenomas was similar to normal tissue, a strong overexpression of the intact IGF-I receptor was found in three out of four adrenocortical carcinomas.
European Journal of Endocrinology 136 296-303
Introduction
The insulin-like growth factors (IGF)-I and IGF-II are involved in the regulation of cell growth and differ- entiation. IGFs are synthesized by a variety of tissues where they act in an autocrine/paracrine manner. The IGF-I receptor (IGF-IR) is a transmembrane tyrosine kinase receptor which has high affinity for IGF-I (dissociation constant (Ka) 0.2-1.5 nmol/l), a 2- to 15-fold lower affinity for IGF-II, and a 100- to 1000-fold lower affinity for insulin. The structurally distinct IGF-II/ mannose-6-phosphate receptor (IGF-II/M6P-R) has no tyrosine kinase activity, binds IGF-II with high affinity
(Ka 0.02-1 nmol/l), IGF-I with more than 500-fold lower affinity, and has no affinity for insulin. While mose effects of IGF-I and IGF-II are mediated through interaction with the IGF-IR, the function of the IGF-IIg M6P-R remains unclear. Furthermore, IGFs bind to a variety of IGF-binding proteins which are present in most tissues and take part in the regulation of IGF action (1-4).
Recent findings indicate that the IGF-IR plays a central role in the mechanism of transformation and tumorigenesis (5-7). IGFs inhibit apoptosis, promote tumor growth, and induce transformation and meta- stasis in many types of malignancies. Elevated levels of
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IGF-II and, to a lesser extent, of IGF-I have been found in Wilm’s tumors, glioblastomas, sarcomas, pheochromo- cytomas, hepatomas, and colon, breast, lung and adrenal carcinomas. Most, if not all, mitogenic effects of IGF-I and IGF-II are mediated through the IGF-IR, and the majority of tumors express intact IGF-IR. Increased levels of the IGF-IR have been described in lung tumors, breast carcinomas and Wilm’s tumors (8, 9). In breast cancer, IGF-IR expression directly corre- lates with estrogen receptor expression (6). However, the expression of the IGF-IR has not been correlated so far with any prognostic factor for breast cancer or any other malignancy (10). Overexpression of the human IGF-IR promotes ligand-dependent neoplastic transfor- mation and IGF-induced mitogenesis can be inhibited by IGF-IR antibody (aIR3), IGF-IR antisense oligonucleo- tides or a dominant negative IGF-IR mutant (5-7, 10-16).
IGFs play an important role in the regulation of growth and differentiation of the adrenal gland. IGF peptides are synthesized by the adrenal gland of various species including the human, and both IGFs have been found to induce steroidogenesis and mitogenesis in ovine, bovine, rat and human adrenocortical cells (17-20). In the adult bovine and human adrenal gland IGF-II enhances the steroidogenic effect of adreno- corticotropic hormone (ACTH) more potently than IGF-I. This effect is mediated through interaction with the IGF-IR, and modulated by locally produced IGF- binding proteins (21, 22). In the actively growing fetal human adrenal gland, high levels of IGF-II are expressed, whereas in adult adrenal tissue only low IGF-II levels are found. It is assumed that IGF-II mediates the ACTH-induced fetal adrenal growth since ACTH induces IGF-II gene expression in human fetal adrenocortical cells, and IGF-II is mitogenic in these cells (23-26). Overexpression of IGF-II, which might contribute to the neoplastic cell proliferation, has been found in functional human adrenocortical carcinomas and in pheochromocytomas (27-35). Although the presence of both types of IGF receptors has been identified in normal and tumorous human adrenal tissue by PCR and immunohistochemistry, no data exist on the binding characteristics and concentrations of the IGF-IR and IGF-II/M6P-R in adult human adrenal glands and adrenal tumors (36, 37). In order to clarify further the significance of the IGF system in the tumorigenesis of the human adrenal gland, we exam- ined the binding characteristics and concentrations of both IGF receptors in normal adult human adreno- cortical glands, and compared them to the IGF-IR binding in adrenocortical tumors of various origins.
Methods
Tissue samples
Tissue from normal human adrenal glands was obtained from 14 patients who underwent total unilateral
nephrectomy due to renal carcinoma. The removed adrenal glands were found to be normal after morpho- logical and histopathological examination. Adrenal tumor specimens were obtained from patients who underwent surgery due to adrenocortical hyperplasia (with hypercortisolism due to pituitary Cushing’s disease (n = 2), ectopic ACTH syndrome (n = 1), bilateral macronodular adrenal hyperplasia (n= 1)), adreno- cortical adenoma (Cushing’s adenoma (n= 2), Conn’s adenoma (n= 2)), and adrenocortical carcinoma (hor- monally active carcinomas with hypercortisolism (n=2), hyperandrogenism (n=1), cortisol-secreting intra-abdominal metastasis (n= 1)). The tumors were classified clinically and the histological diagnosis was performed according to the histopathological criteria defined by Weiss (38). Immediately after surgical removal, the adrenal tissue was dissected by the pathologist, and a sample of fresh non-necrotic adrenal tissue was provided. The adrenocortical and medullary tissue was separated as described, and the samples were stored in liquid nitrogen until further use (39).
Materials
Recombinant human (rh) IGF-I and IGF-II were kindly provided by Kabi/Pharmacia (Stockholm, Sweden), 125I- iodotyrosyl-rhIGF-I (125I-IGF-I) and 125I-IGF-II with a specific activity of 2 × 10° Ci/mol were purchased from Amersham Buchler (Braunschweig, Germany). The polyclonal rabbit antiserum 3637 against the human IGF-II/M6P receptor was kindly provided by Dr S P Nissley (NIH, Bethesda, USA).
Membrane preparations
Membrane preparations were performed as described previously (40). Briefly, the frozen tissue samples were thawed on ice in homogenization buffer (0-25 mol/l sucrose, 0.25 mg/l antipain and 100 mg/l phenyl- methyl sulfonyl fluoride (PMSF)). After mechanical homogenization, the homogenate was centrifuged at 600 g for 10 min. The supernatant was saved and centrifuged at 10 000 g for 30 min, adjusted to a final concentration of 0.1 mol/l NaCl and 0.2 x 10-3 mol/l Mg2SO4, and centrifuged at 100 000 g for 90 min. The membrane pellet was resuspended in membrane buffer (50 mmol/l Tris-HCI, pH 7.4, 0.25 mg/l antipain, 100 mg/l PMSF) and centrifuged twice at 100 000 g for 90 min. The membrane pellet was finally resuspen- ded in membrane buffer and the protein concentration was determined.
Binding studies
Binding studies of 1251-IGF-I and 1251-IGF-II to mem- brane preparations were performed as described (40, 41). Briefly, aliquots of membranes accounting for 160 µg protein for the IGF-I radioreceptor assay (RRA)
(a)
100
I-IGF-I binding (%)
75
IGF-11
50
Insulin
IGF-I
25
125
0
0
0.25
1
4
16
1024
(b)
I-IGF-II binding (%)
100
IGF-1
Insulin
75
IGF41
50
25
125
0
0
0.25
1
4
16
1024
Ligand (mmol/l)
and 80 µg protein for the IGF-II RRA were incubated with a total of 20 000 c.p.m. 125I-IGF-I or 125I-IGF-II. Increasing concentrations of unlabeled IGF-I or IGF-II were added (0-16 nmol/l), and the final volume was adjusted to 400 ul with binding buffer (Medium 199 containing 0.2% bovine serum albumin, 50 mM HEPES buffer (pH 8), 150mm NaCl and 1.2 mm MgSO4). Incubation was performed for 2.5 h at room tempera- ture. Afterwards, membranes were centrifuged at 600 g for 10 min, the supernatant was discarded and 1 ml PBS was added. Washing was repeated twice. Membrane- bound radioactivity was measured in a gamma-counter. The binding capacity and receptor kinetics were calculated by Scatchard analysis (40-42) using a computer-based analysis device (Ligand). When
repeated IGF-IR binding studies were performed with the same normal or tumorous tissue samples, the variation of specific binding and receptor concentration was found to be less than 10%.
Affinity cross-linking
Affinity cross-linking experiments of 1251-IGF-I with membrane preparations from normal adrenal glands and adrenocortical carcinomas were performed as described previously (40). Adrenocortical membranes were incubated with 125I-IGF-I (200 000 c.p.m.) and unlabeled ligands as indicated, washed, and incubated in Dubnoff minimum essential medium with 0.2% BSA and 0.1 mol/l disuccinimidylsuberate for 15 min. The reaction was quenched with 10 mmol/l EDTA, the cells lysed and the lysates subjected to SDS-PAGE gel electrophoresis (6% acrylamide bis) under reducing conditions. Gels were dried and analyzed by autoradio- graphy.
Western blotting
Western blotting of the IGF-II/M6P receptor was performed as described (40, 43). Briefly, SDS-PAGE gel electrophoresis (6% acrylamide) under non-reducing conditions was performed on cell lysates (200 µg/lane) and followed by a protein transfer to nitrocellulose. After incubation with polyclonal rabbit antiserum against the IGF-II/M6P receptor, samples were analyzed using the horseradish peroxidase method.
Results
IGF-I and IGF-II binding to normal adrenocortical tissue
Binding of 1251-IGF-I to human adrenocortical mem- branes and competition by unlabeled IGF-I and IGF-II are shown in Fig. la. The mean specific binding of 1251-IGF-I to membranes of 14 normal human adrenal glands was 5.02 + 0.49% and was effectively displaced by unlabeled IGF-I with a 50% displacement (ED50) at 0.17 ± 0.02 nmol/l (Table 1). In contrast, significantly higher concentrations of IGF-II were necessary for a 50% displacement, demonstrating an 8-fold lower affin- ity of IGF-II to the adrenocortical IGF-IR. Insulin only at umol/l concentrations displaced specific 125I-IGF-I
| n | Specific binding (%) | ED50 (nmol/l) | Ka (nmol/I) | Receptor concentration (nmol/kg protein) | |
|---|---|---|---|---|---|
| Normal adrenal gland | 14 | 5.0 ± 0.5 | 0·17 ± 0·02 | 0·16 ± 0.03 | 19-2 + 2.5 |
| Adrenocortical hyperplasia | 4 | 4·1 ± 0.4 | 0.23 ± 0.08 | 0.19 ± 0.04 | 19.6 ± 2.0 |
| Adrenocortical adenoma | 4 | 4.0 ± 1.1 | 0-28 ± 0.15 | 0·21 ± 0-04 | 17.5 + 3.1 |
| Adrenocortical carcinoma | 4 | 13.8 ± 4.2 | 0·23 ±0.04 | 0·17 ± 0.02 | 72.2 + 21-3 |
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KDa
200
100
50
binding from the IGF-IR (Fig. 1a). Scatchard analysis revealed a single class of high affinity binding sites with a Ka of 0.16 + 0.03 nmol/l and a receptor concentra- tion of 19.24 ± 2.51 nmol/kg protein (Table 1). In agreement, the mean specific binding of 125I-IGF-II to 6 normal adrenocortical glands was 7.49 ± 0-46% and could be displaced by unlabeled IGF-II with an ED50 of 3.74 + 0.47 nmol/l, while IGF-I and insulin at similar doses did not compete for 125I-IGF-II binding. Data of a representative experiment showing the binding of 25I-IGF-II to human adrenocortical membranes and 125
IGF-I receptor concentration (nmol/kg)
100
80
60
40
20
I
*
0
normal
adenoma
hyperplasia
carcinoma
competition by unlabeled IGF-II, IGF-I and insulin are shown in Fig. 1b. When the binding data of 125I-IGF-II were subjected to Scatchard analysis, a single class of high affinity binding sites was found with a Ka of 2.20 ± 0.46 nmol/l and a receptor concentration of 1137 ± 264 nmol/kg protein.
Western blotting of the IGF-II/M6P receptor
Western blotting of human adult adrenocortical membranes with an anti-human IGF-II/M6P-R anti- body revealed the presence of a specific band at the expected molecular size of 220 kDa (Fig. 2), thus con- firming the presence of IGF-II/M6P receptors on normal adult human adrenocortical cells by immuno- logical criteria.
IGF-I binding to adrenocortical hyperplasias and adenomas
The binding kinetics of 1251-IGF-I to four adrenocortical hyperplasias and four adrenocortical adenomas were investigated. All examined hyperplasias and adenomas showed 125I-IGF-I binding which was comparable to those obtained in normal adrenal glands (Table 1). The mean specific 125I-IGF-I binding was 4.06 ± 0.4% in adrenocortical hyperplasias and 4.03 ± 1.11% in mem- branes of adrenocortical adenomas. No difference was observed between 1251-IGF-I binding of adenomas caus- ing Cushing’s syndrome or Conn’s syndrome. In accord- ance with normal adrenocortical tissue, Scatchard analysis showed a single class of high affinity binding sites with similar binding kinetics, indicating the presence of similar concentrations of intact IGF-IR on normal, hyperplastic and adenomatous adrenocortical tissue (Fig. 3).
IGF-I binding to adrenocortical carcinomas
In comparison to normal, hyperplastic or adenomatous adrenocortical tissue, the specific binding of 1251-IGF-I to adrenocortical carcinomas was 3-fold higher in two hormonally active adrenocortical carcinomas (14.7 and 15.4%), and 4.5-fold higher in a cortisol-secreting adrenocortical metastasis (22.7%). The fourth cortisol- secreting adrenocortical carcinoma showed a specific binding of 125I-IGF-I which was within the normal range. Although the mean specific binding of 125I-IGF-I to membranes of adrenocortical carcinomas was sig- nificantly higher than in normal adrenocortical glands (P<0.01 by non-paired t-testing), IGF-I was equally potent in displacing the labeled ligand from carcinoma- tous (ED50) 0.23 + 0.04 nmol/l) as well as normal adrenocortical membranes. Scatchard analysis showed a single class of high affinity binding sites with normal binding kinetics in all examined carcinomas (Ka 0.17 ± 0.02 nmol/l) but 4- to 5-fold higher concentrations of
15
0.20
specific binding (%)
12
0.15
10
bound / free
7.5
0.10
5
0.05
2.5
0
0
0
0.06
0.25
1
4
16
0
0.01 0.02 0.03 0.04
unlabeled IGF-I
bound IGF-I (nM)
normal
carcinoma
the IGF-IR in three out of four carcinomas as compared with the mean receptor concentration of normal adreno- cortical tissue (Fig. 3, Table 1). A representative com- parison of the competitive binding data and Scatchard analysis between a normal and a carcinomatous
KDa
normal carcinoma
1 2 3 4
200
100
50
- + - +
unlabeled IGF-I
adrenocortical gland with elevated IGF-IR concentrations is shown in Fig. 4.
Affinity cross-linking of the IGF-I receptor
Affinity cross-linking of normal human adrenocortical membranes with 125 I-IGF-I and subsequent gel electro- phoresis revealed an autoradiographic band at 135 kDa, corresponding to the size of the intact a-subunit of the IGF-IR (Fig. 5, lane 1). In addition, a less intense band with a molecular mass of over 200 kDa was observed which most likely represents a cross-linked a-subunit dimer (37). The binding of 125I-IGF-I could be inhibited by competition with excess unlabeled IGF-I (Fig. 5, lane 2). Affinity cross-linking of membranes of the cortisol- secreting adrenocortical carcinoma showed a similar band at 135 kDa with the same apparent molecular weight as the intact a-subunit of the IGF-IR in normal adrenal membranes. In accordance with the increased specific binding and calculated IGF-IR concentrations, however, the band of the carcinoma was more intense than the band obtained with equivalent amounts of normal adrenocortical membranes (Fig. 5, lane 3). Similarly, the formation of this band was inhibited by competition with an excess of unlabeled IGF-I (Fig. 5, lane 4). No specific bands at a low molecular mass (less than 50 kDa) were detected after cross-linking adreno- cortical membranes with 125I-IGF-I or 125I-IGF-II, making a contamination of the membrane preparations with IGF-binding proteins unlikely.
Discussion
In the present study we have demonstrated the presence of intact IGF-IR and IGF-II/M6P-R on normal adult adrenocortical cells. The competition experiments with 125 I-IGF-I binding to membrane preparations of 14 normal adrenocortical glands and subsequent Scatchard analysis confirmed the presence of a specific, single class of high-affinity binding sites for IGF-I with a Ka of 0.16 ±0.03 nmol/l and a receptor concentration of 19.24 ± 2.51 nmol/kg membrane protein. In compar- ison to IGF-I, unlabeled IGF-II exhibited 8-fold and insulin over 100-fold reduced affinity for the IGF-IR. These binding characteristics are typical for the IGF-IR and similar to those reported for other tissues including bovine and rat adrenal glands (1, 40, 41, 44). Addi- tional proof for the presence of the IGF-IR in adreno- cortical cells was obtained by cross-linking of 1251-IGF-I to a receptor complex with an apparent molecular mass of 135 kDa, which is equivalent to the size of the IGF-IR «-subunit reported in other studies (45). Our data confirm and extend the findings of previous studies which demonstrate the presence of IGF-IR in normal human adrenal glands by immunohistochemistry, in situ hybridization, autoradiography or reverse transcription (RT)-PCR (36, 37, 46-48). Additionally, we show for the first time the binding characteristics and concentrations
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of the IGF-II/M6P-R in normal adult human adreno- cortical tissue. The competition experiments with 125I- IGF-II binding to normal adrenocortical membrane preparations exhibited the typical binding character- istics of the IGF-II/M6P-R, with only IGF-II, but not IGF-I and insulin, being able to compete for labeled IGF-II binding. In comparison to the bovine adrenal gland (40), the Scatchard analysis revealed abundant high-affinity binding sites for IGF-II with a Ka of 2.2 + 0.46 nmol/l and a receptor concentration of 1137 + 264 nmol/kg membrane protein. The presence of the intact IGF-II/ M6P-R was further confirmed immunologically by Western blotting and is in accordance with the demonstration of IGF-II/M6P-R mRNA by RT-PCR (27). Due to the limited amount of tissue available, the IGF-II/ M6P-R studies could not be extended to adrenocortical glands of tumorigenic origin.
The role of IGFs and their receptors in the adult human adrenal gland has not been investigated extensively so far. In agreement with the bovine adrenal gland, we have recently been able to show that IGF-II is more potent than IGF-I in stimulating androgen and cortisol secretion from adult human adrenocortical cells. This effect is mediated by the IGF-IR and modulated by locally produced IGF-binding proteins (22, 39). Therefore, the IGF-IR seems to play an important role in the maintanance of the differentiated function of the normal adult human adrenal gland by mediating the steroidogenic effects of IGF-I and IGF-II. In the present study we further investigated the IGF-IR concentration and binding characteristics in adreno- cortical tumors of various origins. Although each histological group of non-neoplastic tumors includes different clinical manifestations, both benign adreno- cortical adenomas as well as bilateral adrenal hyper- plasias express the typical IGF-IR at the same concentration and with the same binding character- istics as normal adrenocortical tissue. This finding is in accordance with other studies which found IGF-IR mRNA or immunoreactivity in most of the investigated adrenal adenomas and hyperplasias (27, 37). In parallel to the IGF-IR, the expression of IGF-I and IGF-II is reported to be the same in normal, adenomatous and hyperplastic adrenal glands (24).
The most prominent finding of this study is the strong overexpression of the IGF-IR in two hormonally active sporadic adrenocortical carcinomas and in one cortisol- secreting adrenocortical metastasis. Although the carcinomatous membranes showed a significantly elevated mean specific 1251-IGF-I binding, the displace- ment of the tracer by unlabeled ligands was comparable to normal membranes, and the Scatchard analysis revealed similar dissociation constants. In addition, the cross-linking studies showed no difference in the apparent molecular weight of the a-subunit between normal and tumorous tissue, indicating the overexpres- sion of the intact IGF-IR in three out of four adreno- cortical carcinomas. Our results are consistent with an
immunohistochemical study on 94 human adrenal tumors obtained after autopsy (37). Although the IGF-IR concentration, due to the immunohistochemical approach, could not be quantified exactly, Kamio et al. (37) report an increased intensity of IGF-IR staining in adrenocortical carcinomas (n=64) as opposed to adenomas. In another study, the presence of IGF-IR mRNA in adrenocortical carcinomas was demonstrated by RT-PCR (27). Recently, 10- to 100-fold increased levels of IGF-II mRNA and protein have been reported in adrenocortical carcinomas (27-30). The mechanism and functional significance of the overexpression of IGF-II and the IGF-IR in a subgroup of adrenocortical carcino- mas remains unknown at present. Reincke et al. (49) observed mutations of the tumor suppressor gene p53 in 3 out of 11 human adrenocortical carcinomas. Since the intact tumor suppressor protein p53 causes a repression of the IGF-II and IGF-IR expression (50) the loss of intact p53 by mutation could contribute to the observed overexpression of IGF-II and the IGF-IR in some adrenocortical carcinomas. The mitogenic effect of IGF-II is dependent on the presence of the IGF-IR (1, 5), and we have recently shown that in the adult bovine and human adrenal gland the steroidogenic effect of IGF-II is mediated through interaction with the IGF-IR (21, 22). It is evident therefore, that high local levels of IGF-II in combination with elevated IGF-IR concentra- tions would represent a significant growth advantage of the adrenocortical carcinoma cell and could contribute to the highly malignant phenotype, at least in a subgroup of this rare type of cancer.
Overexpression of the human insulin-like growth factor-I receptor promotes ligand-dependent neoplastic transformation (14, 15) and the absence of IGF-IR prevents malignant growth and transformation in vitro and in vivo (10). Although an increasing body of experi- mental evidence indicates that the IGF-IR plays a pivotal role in tumorigenesis, and in several human malig- nancies an overexpression of the IGF-IR has been reported, the clinical implications of IGF-IR expression in human tumors remains to be elucidated. In the present study we show that the intact IGF-IR is present in normal human adult adrenocortical glands as well as in all adrenocortical tumors investigated. While the abundance of the IGF-IR in benign adrenocortical hyper- plasias and adenomas is similar to normal glands, we found a strong increase in the IGF-IR number in three out of four adrenocortical carcinomas. Further studies are needed to elucidate whether the increase of IGF-IR and IGF-II peptide concentrations in part of the malig- nant adrenocortical tumors is merely an epiphenomenon or represents a specific step in tumorigenesis.
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Received 11 September 1996
Accepted 19 December 1996