Overexpression of the insulin-like growth factor I receptor in human pheochromocytomas

in Journal of Molecular Endocrinology

In order to determine the role of the IGF-I receptor (IGF-IR) in human pheochromocytomas we have compared the expression of the IGF-IR in normal tissues and in pheochromocytomas with regard to the IGF-IR mRNA levels and ligand binding. By semiquantitative reverse transcription polymerase chain reaction (RT-PCR), the mRNA of the IGF-IR could be detected in all samples of normal adrenomedullary cells (n=13) and pheochromocytomas (n=16). However, pheochromocytomas exhibited 2.8-fold higher mean IGF-IR mRNA levels than normal adrenomedullary cells (2.8±0.5×105 molecules/μg RNA vs 7.8±1.2×105 molecules/μg RNA; P < 0.001). This overexpression of the IGF-IR in pheochromocytomas could be confirmed at the protein level by binding studies. Radioligand assays and Scatchard analysis revealed a single class of high affinity IGF-IR binding sites with a similar dissociation constant (K d: 0.32±0.1 nmol/l vs 0.22±0.08 nmol/l) for both normal adrenomedullary cells and pheochromocytomas. However, specific 125I-labeled IGF-I binding and the calculated receptor concentration were significantly elevated in pheochromocytomas as compared with normal adrenomedullary cells (58.3±5 vs 24.3±12 nmol/kg protein; P < 0.05). In summary, our results demonstrate significant overexpression of the IGF-IR in human pheochromocytomas. This suggests a possible role of the IGF system in the pathogenesis of adrenal neoplasia and thus IGF-IR may be a target for future therapeutic approaches.

Abstract

In order to determine the role of the IGF-I receptor (IGF-IR) in human pheochromocytomas we have compared the expression of the IGF-IR in normal tissues and in pheochromocytomas with regard to the IGF-IR mRNA levels and ligand binding. By semiquantitative reverse transcription polymerase chain reaction (RT-PCR), the mRNA of the IGF-IR could be detected in all samples of normal adrenomedullary cells (n=13) and pheochromocytomas (n=16). However, pheochromocytomas exhibited 2.8-fold higher mean IGF-IR mRNA levels than normal adrenomedullary cells (2.8±0.5×105 molecules/μg RNA vs 7.8±1.2×105 molecules/μg RNA; P < 0.001). This overexpression of the IGF-IR in pheochromocytomas could be confirmed at the protein level by binding studies. Radioligand assays and Scatchard analysis revealed a single class of high affinity IGF-IR binding sites with a similar dissociation constant (Kd: 0.32±0.1 nmol/l vs 0.22±0.08 nmol/l) for both normal adrenomedullary cells and pheochromocytomas. However, specific 125I-labeled IGF-I binding and the calculated receptor concentration were significantly elevated in pheochromocytomas as compared with normal adrenomedullary cells (58.3±5 vs 24.3±12 nmol/kg protein; P < 0.05). In summary, our results demonstrate significant overexpression of the IGF-IR in human pheochromocytomas. This suggests a possible role of the IGF system in the pathogenesis of adrenal neoplasia and thus IGF-IR may be a target for future therapeutic approaches.

Keywords:

Introduction

Previous studies by our own group and others have shown a critical role for the insulin-like growth factor (IGF) system in either normal adrenocortical cells or adrenocortical tumors (Weber et al. 1997, Boulle et al. 1998, Fottner et al. 1998, 2001). In malignant adrenocortical carcinomas, overexpression of IGF peptides (mainly IGF-II), receptors (IGF-IR) and binding proteins (IGFBP-2) has been observed. In contrast, expression of IGF-I peptides and receptors appears to be unaltered in adrenocortical hyperplasia and adenomas.

The IGF system has also been shown to play a potent regulatory role in cell proliferation and maintenance of sympathetic ganglia and adrenal medulla. In adult adrenal medulla and sympathetic ganglia, gene expression of IGF-I and IGF-II and their receptors was proven at the mRNA level (Haselbacher et al. 1987, Gelato & Vassalotti 1990, Ilvesmäki et al. 1993). IGFs have been shown to promote chromaffin cell survival and proliferation in vivo (Frödin & Gammeltoft 1994); moreover IGF-I enhances catecholamine synthesis (Hwang & Choi 1996) and secretion (Dahmer et al. 1990) in these cells.

Interestingly, alterations in the IGF system also seem to play an important role in tumors originating from the adrenal medulla or sympathetic ganglia, like pheochromocytomas or neuroblastomas. Overexpresion of the IGF-I receptor in neuroblastoma cells results in resistance to apoptosis leading to unregulated growth (Singleton et al. 1996). IGF-II (Sullivan et al. 1995) and IGFBP-2 (Menouny et al. 1997) are widely expressed in human neuroblastomas possibly enhancing and/or modulating IGF-I receptor activation. Overall, alterations in the IGF system have been shown to modulate neuroblastoma growth in vitro, thus underlining its importance in the growth-maintenance of these highly malignant tumors (reviewed in Zumkeller & Schwab 1999).

Overexpression of IGF-II mRNA and peptide has been described in human pheochromocytomas (Haselbacher et al. 1987, Gelato & Vassalotti 1990). However, no information about the abundance of the IGF-I receptor in human pheochromocytomas compared with normal adrenomedullary tissue is available to date. In order to evaluate whether the IGF-IR is overexpressed in human pheochromocytomas, we therefore investigated the expression of the IGF-IR in normal adult adrenomedullary tissue and in pheochromocytomas with regard to mRNA levels and ligand binding in vivo.

Materials and methods

Materials and sample collection

Recombinant human IGF-I was purchased from Boehringer (Mannheim, Germany), (3-[125I]iodotyrosyl) IGF-I (human recombinant, specific activity 2000 Ci/mmol) was purchased from Amersham Buchler GmbH & CoKG (Braunschweig, Germany). Molecular biology reagents for reverse transcription (RT)-PCR were obtained from Promega (San Diego, CA, USA) and Gibco BRL (Eggenheim, Germany).

According to the guidelines of the ethics committee of the Ludwig-Maximilians-University of Munich, normal adrenal tissue was obtained from patients undergoing surgical treatment for renal neoplasia with concomitant ipsilateral adrenalectomy. Immediately after surgical removal, the tissue was dissected by the pathologist and a sample of fresh, non-necrotic adrenal tissue was provided. All adrenal glands were found to be normal after morphological and histopathological examination. Due to the large amount of protein necessary for the binding studies, tissue from only five adrenal glands provided enough material for membrane preparation and consecutive binding assays. If only small parts of the intact adrenal gland were available after pathological analysis, the tissue was used for mRNA isolation. Pheochromocytomas were obtained from patients undergoing surgical treatment of adrenal neoplasia. All patients gave written informed consent. Immediately after surgical removal, samples of normal adrenal tissue and of pheochromocytomas were dissected by the pathologist and a sample of fresh non-necrotic representative adrenal tissue was provided. All adrenal glands were found to be normal after morphological and histopathological examination. For the tumor samples, necrotic and ulcerative portions were removed when necessary and the presence of at least 90% tumor cells was verified histologically. For the preparation of adrenomedullary tissue samples, adrenal glands were freed from the periadrenal fat tissue, incised longitudinally and the adrenal medulla was separated from the cortex under a dissecting microscope. For RNA extraction and membrane preparation, tissue samples were snap frozen in liquid nitrogen and stored at −70°C until further analysis.

The mean age of the pheochromocytoma patients at the time of surgery was 43.5 years and the female/male ratio was 1.2:1. One of the pheochromocytomas was classified as malignant due to the presence of distant metastases (lung and local intestinal lymph-node metastases). The mean tumor size was 3.4 ± 1.2 cm. Two of the 17 histologically verified pheochromocytomas occurred as part of multiple endocrine neoplasia type 2 (MEN 2) and three occurred as part of von Hippel-Lindau disease. The mean age of the patients undergoing adrenalectomy due to renal neoplasia was 64.3 years and the female/male ratio was 1:1.4.

RT-PCR

For measurement of IGF-IR levels in 13 normal adrenal glands and 16 pheochromocytomas, a commercially available competitive quantitative RT-PCR method (Clontech Laboratories, Palo Alto, CA, USA) was used, as previously described in detail (Weber et al. 2002). The MIMIC PCR technique utilizes an exogenous internal standard (MIMIC), which competes for the same primers as the target IGF-I receptor DNA. With knowledge of the amount of MIMIC DNA added in serial dilutions to the amplification reactions, the amount of the target template could be determined, and thus the amount of initial IGF-IR. The competitive internal standard, which contains the identical primer binding sites used to amplify the IGF-IR DNA was generated by amplifying a BamH1/EcoR1 fragment of v-erB with two composite primers. In these composite primers (40-mer), the first 20 nucleotides are complementary to IGF-II or the IGF-IR and the following 20 nucleotides are complementary to v-erB. The internal standard was synthesized, purified and quantified by spectophotometry as described by the manufacturer (Clontech Laboratories). Amplification of the competitive internal standard generated a 288 bp fragment. The primers used to amplify the human IGF-IR and the internal standard respectively were: sense 5′ACAGAG AACCCCAAGACTGAGG3′, antisense: 5′TGATGTT GTAGGTGTCTGCGGC3′, corresponding to nucleotides 2095–2116 (exon 10) and 2341–2320 (exon 11) of the human IGF-IR cDNA sequence (Ullrich et al. 1986). Amplification of the target DNA with these intron-overlapping primers yielded one specific 247 bp fragment of the IGF-IR, thereby excluding amplification of contaminating DNA. The PCR products obtained were confirmed by sequencing. In pilot experiments, the exponential phase of the amplification was determined for the target DNA and the internal standard. Subsequently, a cycle number that was in the middle of the linear amplification range (21–27 cycles) was chosen. In the system used, the efficiencies of amplification of target cDNA and competitive internal standard DNA were equal (Gilliland et al. 1990, Alms et al. 1996, Becker-Andre et al. 1989, Kutoh et al. 1998, Zhang et al. 1998).

For RNA extraction, 50 mg tissue specimens were incubated with 1 ml cell lysis buffer (Trizol, Gibco, Grand Island, NY, USA) for 5 min, then total cellular RNA was isolated using the acid-guanidinium isothio-cyanate phenol-chloroform extraction method as described by Chomczynski & Sacchi (1987). The assessment of RNA integrity was evaluated by inspection of the 28S and 18S ribosomal RNA bands using gel electrophoresis, and the concentration and purity of the RNA were further determined by ultraviolet spectrophotometry; RNA was stored at −70 °C until analyzed. For reverse transcription of extracted RNA to cDNA, 1.0 μg total RNA template was incubated for 60 min at 37 °C in 20 μl reaction volume containing 1 × 1st strand buffer (50 mM Tris/HCl, 75 mM KCl, 3 mM MgCl2), 0.5 mM of each deoxynucleotide, 1.8 μg random primer, 10 mM dithiothreitol (DTT), 20 U ribonuclease inhibitor RNasin (1.0 U/μl), and 240 U Moloney murine leukemia virus reverse transcriptase (12 U/μl). The reaction was stopped by incubating at 95 °C for 5 min, and samples were placed on ice or stored at −20 °C for further analysis. Subsequently, PCRs were performed in a thermal cycler (Gene Amp PCR System 2400, Perkin-Elmer, Weiterstadt, Germany and icycler, BioRad, Munich, Germany): 2 μl RT product and internal MIMIC standard in serial dilution were amplified in a volume of 50 μl, containing 1 × PCR buffer (10 mM Tris–HCl, pH 9.0, 50 mM KCl), 1.0 mM MgCl2, 200 μM each deoxynucleotide, 0.6 pmol/μl each primer, and 0.06 U/μl Taq DNA polymerase. The first denaturation step (95 °C for 6 min) was followed by 29 cycles with a 1 min denaturing step at 95 °C, a 1 min annealing step, starting at 70 °C and decreasing by 0.5 °C with each cycle to a minimum of 65 °C, and a 1.5 min elongation step at 72 °C. As a final extension step, the reaction was heated to 72 °C for 6 min and then cooled. PCR products were electrophoresed on a 1.5 agarose-gel with a 1 Kb DNA ladder followed by ethidium-bromide staining. The stained gel was analyzed with a computerized scanner and image analyzing software (NIH Image, version 1.61, National Institute of Health, Bethesda, MD, USA). Routinely, negative controls without input RNA or with omitted RT-step were included. For quantification of the target mRNA levels, equal amounts of target cDNA were amplified with different dilutions of known amounts of MIMIC DNA. After RT-PCR, the ratios of MIMIC to target band intensity were determined, and the concentration of a 1:1 MIMIC/target ratio was calculated as described (Kutoh et al. 1998). For each sample, an initial estimate of IGF-IR mRNA was performed with a single dose of internal standard DNA, followed by a narrow titration of internal standards around this estimated value, according to the method of Alms et al.(1996). Results were expressed as number of molecules per μg total RNA. The RNA of each sample was reverse transcribed and analyzed by RT-PCR in duplicate in two separate experiments. Using this method, the intra-assay coefficient of variation for IGF-IR mRNA quantification was 3%, and the interassay coefficient of variation was 11%.

IGF-I binding studies

125I-IGF-I binding studies were performed with membrane preparations of 5 normal adrenomedullary tissue samples and 17 pheochromocytomas as previously described (Weber et al. 1997). Briefly, tissue samples were homogenized mechanically in homogenization buffer (0.25 M sucrose, 0.25 mg/l antipain and 100 mg/l phenylmethyl sulfonyl fluoride (PMSF) and centrifuged at 600 g for 10 min. The supernatant was centrifuged at 10 000 g for 30 min, adjusted to a final concentration of 0.1 mol/l NaCl and 0.2 × 10−3 mol/l Mg2SO4, centrifuged twice at 100 000 g for 90 min and resuspended in membrane buffer (50 mM Tris–HCl, pH 7.4; 0.25 mg/l antipain and 100 mg/l PMSF). Aliquots of 80 μg membrane protein were incubated for 3 h at room temperature together with 125I-IGF-I (20 000 c.p.m.) and increasing concentrations of unlabeled IGF-I in 400 μl binding buffer (Medium 199 containing 0.2% bovine serum albumin, 150 mM NaCl and 1.2 mM MgSO4). Membrane bound radioactivity was measured and receptor kinetics were calculated by Scatchard analysis (Scatchard 1949) using a standard software program (Ligand, NICHHD, NIH, Bethesda, MD, USA).

Statistical analysis

All data are expressed as means ± s.e.m. Comparative data were analyzed by multivariate analysis and paired t-test with significance defined as P < 0.05, unless otherwise stated.

Results

IGF-I receptor mRNA expression in human pheochromocytomas

The expression of IGF-IR mRNA in human pheochromocytomas was compared with normal adrenal medulla by quantitative RT-PCR of tissue samples from 16 patients with pheochromocytomas and from 13 normal adrenal glands. Amplification of cDNA with primers located in exons 10 and 11 yielded one specific PCR product of 247 bp (Fig. 1). Sequence analysis showed that these products were identical with the cDNA sequence of the human IGF-IR (data not shown). No products were detected when the RT reaction or the input RNA were omitted. Furthermore, the same size and intensity of the amplification product was observed when the RNA was treated with DNase prior to RT-PCR, excluding possible amplification of contaminating DNA. Expression of the human IGF-IR gene was detected in the tissues of all patients. In the normal adrenal medulla, IGF-IR was expressed at 2751 ± 541 × 103 molecules/μg RNA, while significantly higher levels were observed in tumor samples, with a mean expression of 7772 ± 1203 × 103 molecules/μg RNA (P < 0.001). Figure 1 shows a representative result of the quantitative RT-PCR analysis from two samples of normal adrenomedullary and pheochromocytoma tissue. When the IGF-IR mRNA levels of pheochromocytoma tissue samples were analyzed (Fig. 2), in contrast to normal adrenal glands, a wide heterogeneity in IGF-IR mRNA levels was observed (2303 − 20 520 × 103 molecules/μg RNA) (Table 1). However, IGF-IR mRNA levels of pheochromocytoma tissue samples showed 2.83-fold higher IGF-IR levels than normal adrenomedullary tissue (P < 0.01). Seventy-five percent of human pheochromocytomas showed a more than 2-fold overexpression of the IGF-IR as compared with normal adrenal medulla. IGF-IR expression was unrelated to tumor size, age, sex and catecholamine secretion. However, the highest IGF-IR mRNA level of all investigated tissue specimens was seen in the only malignant pheochromocytoma investigated in this series.

IGF binding to normal adrenomedullary and pheochromocytoma tissue

Binding kinetics of 125I-IGF-I to membranes from five normal adrenal medullas and 17 pheochromocytomas was investigated. The mean specific binding of 125I-IGF-I to membranes from normal human adrenal medulla was 3.6 ± 0.9%. 125I-IGF-I binding could be effectively displaced by unlabeled IGF-I with a 50% displacement (ED50) at 6.1 ± 2.0 ng/ml. In contrast, significantly higher concentrations of IGF-II were necessary for a 50% displacement, and insulin was effective only at micromolar concentrations (data not shown). Scatchard analysis revealed a single-class of high-affinity binding sites with a Kd of 0.32 ± 0.1 nmol/l and a receptor concentration of 2.43 ± 1.2 nmol/kg protein (Fig. 3). In comparison with normal adrenomedullary tissue, membrane preparations from the pheochromocytoma tissues showed a significantly higher specific 125I-IGF-I binding of 7.0 ± 1.1%, as well as an elevated mean IGF-IR concentration of 5.8 ± 0.5 nmol/kg protein (P < 0.05). The mean IGF-IR concentration was about 2.4-fold higher in pheochromocytomas as compared with normal adrenomedullary tissue. However, IGF-I was equally potent in displacing the labeled ligand from pheochromocytoma membranes (ED50 5.6 ± 1.0 ng/ml), and the Scatchard analysis showed a single class of high affinity binding sites with normal binding kinetics in all examined pheochromocytomas (Kd 0.20 ± 0.09 nmol/l), indicating overexpression of normal intact human IGF-IR in human pheochromocytomas. A representative comparison of the Scatchard plots for a pair of normal adrenomedullary and pheochromocytoma tissue samples is shown in Fig. 3.

Discussion

The presence of IGF-I receptors in pheochromocytoma cells has already been described in samples from human adrenal tumors (Kamino et al. 1991) as well as in PC12 cells, a cell line derived from rat pheochromocytoma (Dahmer et al. 1989). However, information about the IGF-I receptor expression in human pheochromocytoma cells in comparison with normal adrenomedullary tissue has not been available to date.

The present study is the first to report a significant overexpression of the IGF-I receptor in human pheochromocytomas. The analysis of tissue samples from human pheochromocytomas via RT-PCR showed markedly elevated levels of IGF-IR mRNA in pheochromocytoma cells compared with normal adult adrenal medulla. In up to 85% of all pheochromocytomas examined, a more than twofold higher expression of IGF-IR mRNA was observed, with a mean 2.5-fold overexpression. A comparable IGF-IR overexpression was also present at the protein level as confirmed by Scatchard analysis. The binding kinetics of the IGF-I receptors in pheochromocytoma cells were similar to those observed in normal adrenomedullary tissue, suggesting that the abundant IGF-I receptors in pheochromocytomas are functionally intact.

In PC12 cells, IGF-I receptors have been shown to be important for the stimulation of cell replication (Dahmer et al. 1989) and the IGFs are potent mitogens stimulating cell proliferation three times over basal. IGF-I was 10 times more potent in stimulating DNA synthesis than IGF-II, suggesting that these effects are mediated by the IGF-IR (Dahmer & Perlman 1988, Nielsen & Gammeltoft 1988). Moreover, binding affinities to the IGF-IR correlate directly with the ability of IGF-I and IGF-II to completely prevent apoptosis in PC12 cells (Forbes et al. 2002). In these cells, promotion of cell growth and proliferation by IGF-I is exerted by the ERK pathway (Foncea et al. 1997), whereas for prevention of apoptosis the phosphatidylinositol 3-kinase pathway is involved (Kulik et al. 1997).

Overexpression of the IGF-I receptor is not a phenomenon confined to adrenal tumors. In common malignant tumors such as colorectal and gastric cancer as well as in prostate and breast cancer, a strong overexpression of the IGF-IR has been observed. It promotes ligand-dependent neoplastic transformation and there is a quantitative relationship between tumorigenesis and IGF-IR levels, while the absence of IGF-IR prevents malignant growth and transformation (Moschos & Mantzoros 2002). Additionally, in these tumors a positive correlation between IGF-IR over-expression and malignant phenotype has been observed. The mechanisms responsible for enhanced IGF-IR expression in pheochromocytomas and other malignancies are still unclear. However, expression of the IGF-IR is regulated by a variety of factors, including tumor suppressor genes, transcription factors and other growth factors. In several different IGF-IR overexpressing malignant cell systems such as colorectal, gastric, and adrenocortical cancer as well as in osteosarcoma and hematopoetic cells, alterations of tumor suppressor genes and transcription factors important for IGF-IR regulation, such as p53 and Sp1, have been demonstrated (Werner et al. 2000, Baserga et al. 2003). In normal cells, expression of wild-type p53 was shown to inhibit IGF-IR gene expression, whereas mutant p53 upregulates IGF-IR gene expression in several different tumors. In adrenocortical carcinomas, mutations within the conserved regions of p53 have been found in approximately 30% of malignant adrenocortical tumors, whereas mutations are rarely found in benign adrenocortical adenomas (Fottner et al. 2004). Similar results have been found in colorectal-cancer and osteosarcoma cells, suggesting a role for p53 in upregulating IGF-IR expression (Ohlsen et al. 1998, Durai et al. 2005). Additionally, the expression of the IGF-IR has been shown to be regulated by the transcription factor, Sp1 via specific binding sites within the IGF-IR promoter region. In human gastric cancer, overexpression of the IGF-IR strongly correlated with Sp1 expression and with an advanced tumor stage, increased lymph node metastasis and predicted a poor survival, whereas enforced down-regulation of Sp1 and IGF-IR expression suppressed growth and metastasis of gastric cancer in animal models (Wang et al. 2003, Jiang et al. 2004). Therefore, altered expression of the IGF-IR, one of the down-stream effectors of Sp1, may play an important role in cancer growth and metastasis. Furthermore, the frequently observed elevated IGF-II concentration in malignancies overexpressing the IGF-IR might additionally contribute to the overexpression of IGF-I receptors in these tumors. In CaCo-2 human colon carcinoma cells, it has been shown that stable overexpression of IGF-II resulted in increased IGF-IR expression with increased proliferation and anchorage-independent growth (Hoeflich et al. 1996), and in colorectal carcinomas a positive correlation between the expression of IGF-II and IGF-I receptors has been reported (Weber et al. 2002).

The molecular mechanisms by which overexpression of the IGF-IR is induced in pheochromocytoma-associated hereditary syndromes like MEN or von Hippel-Lindau disease are still unclear. However, in renal carcinoma cells (RCC) wild-type von Hippel-Lindau gene (VHL) has been shown to block protein kinase C-delta (PKC-delta), an important downstream signaling molecule of IGF-IR-mediated cell proliferation and transformation. In mutated VHL, this tumor suppressor function gets potentially lost. VHL has also been shown to regulate the protein expression levels of IGF-IR (Li et al. 1998, Datta et al. 2000). It is therefore tempting to speculate if alterations in PKC-delta-mediated pathways are involved in the increased expression of IGF-IR in human pheochromocytoma cells.

Previous studies by our own group and by others have shown a critical role of the IGF system in either normal adrenocortical cells or in adrenocortical tumors (Weber et al. 1997, Boulle et al. 1998, Fottner et al. 1998, 2001). Several authors report the effects of high amounts of IGF-II in human adrenal pheochromocytomas on protein and mRNA levels (Hasselbacher et al. 1987, Gelato & Vassalotti 1990) despite unaltered levels of IGF-I. Compared with normal adrenomedullary tissue, 20 times more immunoreactive IGF-II per gram of tissue was measured in samples from human pheochromocytomas. IGF-II seems to be secreted by pheochromocytoma cells in an autocrine or paracrine manner, supporting tumor growth locally, while the IGF-II serum levels remain unaltered (Gelato & Vassalotti 1990). We speculate that the marked overexpression of the IGF receptor type I and IGF-II in human pheochromocytoma cells results in a state of constitutive growth stimulation in vivo. In malignant adrenocortical carcinomas, overexpression of IGF peptides (mainly IGF-II), receptors (IGF-IR) and binding proteins (IGFBP-2) has been observed. In contrast, expression of IGF-I peptides and receptors appears to be unaltered in adrenocortical hyperplasia and adenomas. Adrenocortical carcinoma, a rare, highly malignant subtype of cancer, showed a 3- to 4-fold increase in IGF-IR expression and a 10- to 100-fold increase in IGF-II expression (Liu 1995, Weber et al. 1997). Functionally, an autocrine stimulatory loop contributing to adrenocortical tumorigenesis may underlie this specific expression pattern. A similar pattern of high IGF-II and concomitant IGF-IR overexpression has previously been reported for neuroblastoma cells (Leventhal et al. 1990) and more recently by our group in human colon carcinomas (Weber et al. 2002). The exact role of the frequently observed overexpression of IGF-binding proteins in parallel with the overexpression of the IGF-I receptor and IGF ligands is still unclear and although high concentrations, especially of IGFBP-2, are a frequent finding in a variety of malignant tumors such as adrenocortical, prostate, breast and colonic cancer, the functional significance remains unclear. Since IGFBPs modulate cellular bioavailability of IGFs and, in addition, have been shown to directly regulate tumor growth and invasion (Hoeflich et al. 2001), it is likely that overxpression of IGFBPs in cancer is not merely an epiphenomenon. Similar to these results, one study also reports a higher expression of IGFBP-2 in human pheochromocytomas in comparison with normal adrenal glands (Ilvesmäki et al. 1998) and recently published data show that IGFBP-2 plays a critical role in neuroblastoma cell proliferation, migration and invasion, thus pointing to an important role of IGFBP-2 in chromaffin cell tumors (Russo et al. 2005). However, additional studies are necessary to further characterize the role of IGFBPs in human pheochromocytomas.

Since overexpression of IGF-IR promotes neoplastic growth (Kaleko et al. 1990) and absence of the IGF-IR has been shown to prevent malignant transformation (Rubin & Baserga 1995), it is tempting to speculate about a possible role of IGF-IR in malignant transformation of human pheochromocytoma cells. It would be interesting to elucidate if the degree of IGF-IR expression in pheochromocytomas correlates with the tumor size and a more malignant phenotype, as has previously been reported for other malignant tumors such as colorectal, gastric and mammary cancers and in adrenocortical carcinomas (Fottner et al. 2004, Foulstone et al. 2005). In the present study, no correlation between clinical characteristics, such as catecholamine secretion or tumor size could be found (Table 1). In contrast, in the subsequent studies, one malignant pheochromocytoma (characterized by the presence of distant metastases) has been examined, and this showed the strongest overexpression of IGF-IR of all investigated pheochromocytomas. This could support the hypothesis mentioned above. However, due to the small number of tumors examined in this study, at this point there is no clear evidence for a correlation between the degree of IGF-IR overexpression and other clinical characteristics and a more malignant phenotype.

Further investigation is needed to clarify if the observed overexpression of IGF-IR is part of a functionally relevant mechanism promoting tumor growth in human pheochromocytoma and possibly promoting malignant transformation of these cells. If so, the IGF system might be an interesting focus for new therapeutic approaches.

Table 1

IGF-I-receptor mRNA levels and clinical characteristics of evaluated normal adrenomedullary tissue and pheochromocytomas

Catecholamine secretion (μg/24 h)
DiagnosisAdrenaline (Norm: 4–20 μg/d)Noradrenaline (Norm: 20–105 μg/d)IGF-IR-mRNA ×103 molecules/μg RNA
MEN IIa, multiple endocrine neoplasia IIa; VHL, von Hippel-Lindau.
Patient sex/age
♂/58Normal adrenal medulla513
♀/72Normal adrenal medulla625
♂/72Normal adrenal medulla798
♀/61Normal adrenal medulla2622
♀/70Normal adrenal medulla2623
♀/55Normal adrenal medulla2620
♂/58Normal adrenal medulla2554
♂/51Normal adrenal medulla2953
♂/68Normal adrenal medulla2964
♂/66Normal adrenal medulla3648
♀/64Normal adrenal medulla3283
♀/71Normal adrenal medulla3762
♀/69Normal adrenal medulla7200
♂/49PheochromocytomaSporadic/benign246.2688.12303
♂/51PheochromocytomaSporadic/benign187.2543.62850
♂/33PheochromocytomaMEN IIa/benign155.52557.44446
♂/29PheochromocytomaVHL/benign187.61455.65130
♀/34PheochromocytomaMEN IIa/benign88.31866.85244
♂/41PheochromocytomaSporadic/benign46.8546.75700
♂/38PheochromocytomaSporadic/benign286.41433.16042
♀/48PheochromocytomaSporadic/benign345.9685.96384
♂/52PheochromocytomaSporadic/benign172.5571.66384
♀/44PheochromocytomaSporadic/benign285.61479.16452
♀/56PheochromocytomaSporadic/benign455.61885.37068
♂/36PheochromocytomaSporadic/benign155.4894.39120
♀/30PheochromocytomaVHL/benign187.23655.69918
♀/35PheochromocytomaVHL/benign245.61465.712540
♀/62PheochromocytomaSporadic/benign99.41006.414250
♀/58PheochromocytomaSporadic/malignant286.53148.620520
Figure 1
Figure 1

Representative result of the RT-PCR analysis of normal adrenomedullary tissue and a human pheochromocytoma. (A) Ethidium bromide staining of amplification products separated on an agarose gel. One microgram total RNA was reverse transcribed and amplified together with serial dilutions of internal control DNA using the same IGF-IR primers. The figure shows a typical titration experiment performed using 10−2- to 10−6-fold dilutions of the internal standard (100 amol/l) and fixed amounts of the target cDNA. M, marker; C, control. (B) Representative linear regression analysis of the internal standard titration curve obtained with the DNA from a pheochromocytoma (▪) and normal adrenomedullary tissue (•). The values on the ordinate are the log of the internal standard/target cDNA ratio. The values on the abscissa are dilutions of the internal standard on a log scale. The titration point corresponds to the intersection with the 0 axis.

Citation: Journal of Molecular Endocrinology 36, 2; 10.1677/jme.1.01975

Figure 2
Figure 2

Mean IGF-IR expression 1000 molecules/μg RNA in human pheochromocytomas (shaded bar; n=16) and normal adrenomedullary tissue (open bar; n=13).

Citation: Journal of Molecular Endocrinology 36, 2; 10.1677/jme.1.01975

Figure 3
Figure 3

(A) Scatchard analysis of the competitive 125I-IGF-I binding data to human pheochromocytomas (solid symbols) and normal adrenomedullary tissue (shaded symbols). Each point represents the mean of duplicate determinations of a single representative experiment. (B) Binding characteristics of the IGF-IR in human pheochromocytomas and normal human adrenomedullary tissue. Results are means ±s.e.m. of 5 (normal) and 17 (pheochromocytomas) independently performed binding experiments. *=P < 0.05.

Citation: Journal of Molecular Endocrinology 36, 2; 10.1677/jme.1.01975

This work is part of the doctoral thesis of S K.

Funding

This work was supported by a DFG Grant WE 1356/4–2 to M M W. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

  • AlmsWJ Braun-Elwert L James SP Yurovsky VV & White B 1996 Simultaneous quantitation of cytokine mRNAs by reverse transcription-polymerase chain reaction using multiple internal standard cRNAs. Diagnostic and Molecular Pathology588–97.

    • Search Google Scholar
    • Export Citation
  • BasergaR Peruzzi F & Reiss R 2003 The IGF-I receptor in cancer biology. International Journal of Cancer107873–877.

  • Becker-AndreM & Hahlbrook K 1989 Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Research179437–9446.

    • Search Google Scholar
    • Export Citation
  • BoulleN Logie A Gicquel C Perin L & Le Bouc Y 1998 Increased levels of insulin-like growth factor II (IGF-II) and IGF-binding protein-2 are associated with malignancy in sporadic adrenocortical tumors. Journal of Clinical Endocrinology and Metabolism831713–1720.

    • Search Google Scholar
    • Export Citation
  • ChomczynskiP & Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Annals of Biochemistry162156–159.

    • Search Google Scholar
    • Export Citation
  • DahmerMK & Perlman RL 1988 Insulin and insulin-like growth factors stimulate desoxyribonucleic acid synthesis in PC12 pheochromocytoma cells. Endocrinology1222109–2113.

    • Search Google Scholar
    • Export Citation
  • DahmerMK Ji L & Perlman RL 1989 Characterisation of insulin-like growth factors-I receptors in PC12 pheochromocytoma cells and bovine adrenal medulla. Journal of Neurochemistry531036–1042.

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  • DahmerMK Hart PM & Perlman RL 1990 Studies on the effect of insulin-like growth factor I on catecholamine secretion from chromaffin cells. Journal of Neurochemistry54931–936.

    • Search Google Scholar
    • Export Citation
  • DattaK Nambudripad R Pal S Zhou M Cohen HT & Mukhopadhyay D 2000 Inhibition of insulin-like growth factorI-mediated cell signaling by the von Hippel-Lindau gene product in renal cancer. Journal of Biological Chemistry27520700–20706.

    • Search Google Scholar
    • Export Citation
  • DuraiR Yang W Gupta S Seifalian AM & Winslet MC 2005 The role of the insulin-like growth factor system in colorectal cancer: review of current knowledge. International Journal of Colorectal Disease20203–220.

    • Search Google Scholar
    • Export Citation
  • FonceaR Andersson M Ketterman A Blakesley V Sapag-Hagar M Sugden PH Le Roith D & Lavandero S 1997 Insulin-like growth factor-I rapidly activates multiple signal transduction pathways in cultured rat cardiac myocytes. Journal of Biological Chemistry27219115–19124.

    • Search Google Scholar
    • Export Citation
  • ForbesBE Hartfield PJ McNeil KA Surinya KH Milner SJ Cosgrove LJ & Wallace JC 2002 Characteristics of binding of insulin-like growth factor (IGF)-I and IGF-II analogues to the type 1 IGF receptor determined by BIAcore analysis. European Journal of Biochemistry269961–968.

    • Search Google Scholar
    • Export Citation
  • FottnerC Engelhardt D & Weber MM 1998 Regulation of steroidogenesis by insulin-like growth factors (IGFs) in adult human adrenocortical cells: IGF-I and more potently IGF-II preferentially enhance androgen biosynthesis through interaction with the IGF-I receptor and IGF-binding proteins. Journal of Endocrinology158409–417.

    • Search Google Scholar
    • Export Citation
  • FottnerC Elmlinger M Engelhardt D & Weber MM 2001 Identification and characterization of insulin-like growth factor (IGF) binding protein expression and secretion by adult human adrenocortical cells: differential regulation by IGFs and adrenocorticotrophin. Journal of Endocrinology168465–474.

    • Search Google Scholar
    • Export Citation
  • FottnerC Höflich A Wolf E & Weber MM 2004 Role of the insulin-like growth factor system in adrenocortical growth control and carcinogenesis. Hormone and Metabolic Research36397–405.

    • Search Google Scholar
    • Export Citation
  • FoulstoneE Prince S Zaccheo O Burns JL Harper J Jacobs C Church D & Hassan AB 2005 Insulin-like growth factor ligands receptors and binding proteins in cancer. Journal of Pathology205145–153.

    • Search Google Scholar
    • Export Citation
  • FrödinM & Gammeltoft S 1994 Insulin-like growth factors act synergistically with basic fibroblast growth and nerve growth factor to promote chromaffin cell proliferation. PNAS911771–1775.

    • Search Google Scholar
    • Export Citation
  • GelatoMC & Vassalotti J 1990 Insulin-like growth factor II: possible local growth factor in pheochromocytoma. Journal of Clinical Endocrinology and Metabolism711168–1174.

    • Search Google Scholar
    • Export Citation
  • GillilandG Perrin S Blanchard K & Bunn HF 1990 Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. PNAS872725–2729.

    • Search Google Scholar
    • Export Citation
  • HaselbacherGK Irminger JC Zapf J Ziegler WH & Humbel RE 1987 Insulin-like growth factor II in human adrenal pheochromocytomas and Wilms tumors: expression at the mRNA and protein level. PNAS841104–1106.

    • Search Google Scholar
    • Export Citation
  • HoeflichA Yang Y & Rascher W 1996 Coordinate expression of insulin-like growth factor II (IGF-II) and IGF-II/mannose-6-phosphate receptor mRNA during differentiation of human colon carcinoma cells (caco-2). European Journal of Endocrinology13549–59.

    • Search Google Scholar
    • Export Citation
  • HoeflichA Reisinger E Lahm H Kiess W Blum WF Kolb HJ Weber MM & Wolf E 2001 Insulin-like growth factor binding protein 2 in tumorigenesis: protector or promoter? Cancer Research618601–8610.

    • Search Google Scholar
    • Export Citation
  • HwangO & Choi HJ 1996 Induction of gene expression of the catecholamine-synthesizing enzymes by insulin-like growth factor I. Journal of Neurochemistry651988–1996.

    • Search Google Scholar
    • Export Citation
  • IlvesmäkiV Kahri AI Miettinen PJ & Voutilainen R 1993 Insulin-like growth factors (IGFs) and their receptors in adrenal tumors: high IGF-II expression in functional adrenocortical carcinomas. Journal of Clinical Endocrinology and Metabolism77852–858.

    • Search Google Scholar
    • Export Citation
  • IlvesmäkiV Liu J Heikkilä A Kahri AI & Voutilainen R 1998 Expression of insulin-like growth factor binding protein 1–6 genes in adrenocortical tumors and pheochromocytomas. Hormone and Metabolic Research30619–623.

    • Search Google Scholar
    • Export Citation
  • JiangX Wang L Gong W Wei D Le X Yao J Ajani J Abbruzzese JL Huang S & Xie K 2004 A high expression of insulin-like growth factor I receptor is associated with increased expression of transcription factor Sp1 and regional lymph node metastasis of human gastric cancer. Clinical and Experimental Metastasis21755–764.

    • Search Google Scholar
    • Export Citation
  • KalekoM Rutter WJ & Miller AD 1990 Overexpression of the human insulin-like growth factor I promotes ligand-dependent neoplastic transformation. Molecular Cell Biology10464–473.

    • Search Google Scholar
    • Export Citation
  • KaminoT Shigematsu K Kawai K & Tsuchiyama H 1991 Immunoreactivity and receptor expression of insulin-like growth factor I and insulin in human adrenal tumors. American Journal of Pathology13883–91.

    • Search Google Scholar
    • Export Citation
  • KulikG Klippel A & Weber MJ 1997 Antiapoptotic signaling by the insulin-like growth factor I receptor phosphatidylinositol 3-kinase. Molecular and Cellular Biology171595–1606.

    • Search Google Scholar
    • Export Citation
  • KutohE Boss O Levasseur M & Giacobino JP 1998 Quantification of the full length leptin receptor (OB-Rb) in human brown and white adipose tissue. Life Sciences62445–451.

    • Search Google Scholar
    • Export Citation
  • LeventhalPS Randolph AE Vesbit TE Schenone A Windebank AJ & Feldmann EL 1990 Insulin-like growth factor as a paracrine growth factor in human neuroblastoma cells. Experimental Cell Research221179–186.

    • Search Google Scholar
    • Export Citation
  • LiW Jiang YX Zhang J Soon L Flechner L Kapoor V Pierce JH & Wang LH 1998 Protein kinase C-delta is an important signaling molecule in insulin-like growth factor I receptor-mediated cell transformation. Molecular and Cellular Biology185888–5898.

    • Search Google Scholar
    • Export Citation
  • MenounyM Binoux M & Babajko S 1997 Role of insulin.like growth factor binding protein-2 and its limited proteolysis in neuroblastoma cell proliferation: modulation by transforming growth factor-beta and retinoic acid. Endocrinology138683–690.

    • Search Google Scholar
    • Export Citation
  • MoschosSJ & Mantzoros CS 2002 The role of the IGF system in cancer: from basic to clinical studies and clinical applications. Oncology63317–332.

    • Search Google Scholar
    • Export Citation
  • NielsenFC & Gammeltoft S 1988 Insulin-like growth factors are mitogens for rat pheochromocytoma PC 12 cells. Biochemical and Biophysical Research Communications1541018–1023.

    • Search Google Scholar
    • Export Citation
  • OhlssonC Kley N Werner H & LeRoith D 1998 p53 regulates insulin-like growth factor-I (IGF-I) receptor expression and IGF-I induced tyrosine phosphorylation in an osteosarcoma cell line: interaction with p53 and Sp1. Endocrinology1391101–1107.

    • Search Google Scholar
    • Export Citation
  • RubinR & Baserga R 1995 Insulin-like growth factor I receptor. Its role in cell proliferation apoptosis and tumorigenicity. Laboratory Investigations73311–331.

    • Search Google Scholar
    • Export Citation
  • RussoVC Schutt BS Andaloro E Ymer SI Hoeflich A Ranke MB Bach LA & Werther GA 2005 Insulin-like growth factor binding protein-2 binding to extracellular matrix plays a critical role in neuroblastoma cell proliferation migration and invasion. Endocrinology1464445–4455.

    • Search Google Scholar
    • Export Citation
  • ScatchardG1949 The attraction of proteins for small molecules and ions. Annals of the New York Academy of Sciences51660–672.

  • SingletonJR Randolph AE & Feldman EL 1996 Insulin-like growth factor I receptor prevents apoptosis and enhances neuroblastoma tumorigenesis. Cancer Research564522–4529.

    • Search Google Scholar
    • Export Citation
  • SullivanKA Castle VP Hanash SM & Feldmann EL 1995 Insulin-like growth factor II in the pathogenesis of human neuroblastoma. American Journal of Pathology1471790–1798.

    • Search Google Scholar
    • Export Citation
  • UllrichA Gray A Tam AW Yang-Feng T Tsubokawa M Collins C Henzel W Le Bon T Kathuria S Chen E et al. 1986 Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specifity. EMBO Journal52503–2512.

    • Search Google Scholar
    • Export Citation
  • WangL Wei D & Huang S 2003 Transcription factor Sp1 expression is a significant predictor of survival in human gastric cancer. Clinical Cancer Research96371–6380.

    • Search Google Scholar
    • Export Citation
  • WeberMM Auernhammer C Kiese W & Engelhardt D 1997 Insulin-like growth factor receptors in normal and tumorous adult human adrenocortical glands. European Journal of Endocrinology139296–303.

    • Search Google Scholar
    • Export Citation
  • WeberMM Fottner C Liu SB Jung MC Engelhardt D & Baretton GB 2002 Overexpression of the insulin-like growth factor I receptor in human colon carcinomas. Cancer952086–2095.

    • Search Google Scholar
    • Export Citation
  • WernerH Shalita-Chesner M Abramovitch S Idelman G Shaharabani-Gargir L & Glaser T 2000 Regulation of the insulin-like growth factor-I receptor gene by oncogenes and anti-oncogenes: implications in human cancer. Molecular Genetics and Metabolism71315–320.

    • Search Google Scholar
    • Export Citation
  • ZhangW Thorton WH & MacDonald RS 1998 Insulin-like growth factor-I and -II receptor expression in rat colon mucosa is affected by dietary lipid intake. Journal of Nutrition182158–165.

    • Search Google Scholar
    • Export Citation
  • ZumkellerW & Schwab M 1999 Insulin-like growth factor system in neuroblastoma tumorigenesis and apoptosis: potential diagnostic and therapeutic perspectives. Hormone and Metabolic Research31138–141.

    • Search Google Scholar
    • Export Citation

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  • View in gallery

    Representative result of the RT-PCR analysis of normal adrenomedullary tissue and a human pheochromocytoma. (A) Ethidium bromide staining of amplification products separated on an agarose gel. One microgram total RNA was reverse transcribed and amplified together with serial dilutions of internal control DNA using the same IGF-IR primers. The figure shows a typical titration experiment performed using 10−2- to 10−6-fold dilutions of the internal standard (100 amol/l) and fixed amounts of the target cDNA. M, marker; C, control. (B) Representative linear regression analysis of the internal standard titration curve obtained with the DNA from a pheochromocytoma (▪) and normal adrenomedullary tissue (•). The values on the ordinate are the log of the internal standard/target cDNA ratio. The values on the abscissa are dilutions of the internal standard on a log scale. The titration point corresponds to the intersection with the 0 axis.

  • View in gallery

    Mean IGF-IR expression 1000 molecules/μg RNA in human pheochromocytomas (shaded bar; n=16) and normal adrenomedullary tissue (open bar; n=13).

  • View in gallery

    (A) Scatchard analysis of the competitive 125I-IGF-I binding data to human pheochromocytomas (solid symbols) and normal adrenomedullary tissue (shaded symbols). Each point represents the mean of duplicate determinations of a single representative experiment. (B) Binding characteristics of the IGF-IR in human pheochromocytomas and normal human adrenomedullary tissue. Results are means ±s.e.m. of 5 (normal) and 17 (pheochromocytomas) independently performed binding experiments. *=P < 0.05.

  • AlmsWJ Braun-Elwert L James SP Yurovsky VV & White B 1996 Simultaneous quantitation of cytokine mRNAs by reverse transcription-polymerase chain reaction using multiple internal standard cRNAs. Diagnostic and Molecular Pathology588–97.

    • Search Google Scholar
    • Export Citation
  • BasergaR Peruzzi F & Reiss R 2003 The IGF-I receptor in cancer biology. International Journal of Cancer107873–877.

  • Becker-AndreM & Hahlbrook K 1989 Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Research179437–9446.

    • Search Google Scholar
    • Export Citation
  • BoulleN Logie A Gicquel C Perin L & Le Bouc Y 1998 Increased levels of insulin-like growth factor II (IGF-II) and IGF-binding protein-2 are associated with malignancy in sporadic adrenocortical tumors. Journal of Clinical Endocrinology and Metabolism831713–1720.

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    • Export Citation
  • ChomczynskiP & Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Annals of Biochemistry162156–159.

    • Search Google Scholar
    • Export Citation
  • DahmerMK & Perlman RL 1988 Insulin and insulin-like growth factors stimulate desoxyribonucleic acid synthesis in PC12 pheochromocytoma cells. Endocrinology1222109–2113.

    • Search Google Scholar
    • Export Citation
  • DahmerMK Ji L & Perlman RL 1989 Characterisation of insulin-like growth factors-I receptors in PC12 pheochromocytoma cells and bovine adrenal medulla. Journal of Neurochemistry531036–1042.

    • Search Google Scholar
    • Export Citation
  • DahmerMK Hart PM & Perlman RL 1990 Studies on the effect of insulin-like growth factor I on catecholamine secretion from chromaffin cells. Journal of Neurochemistry54931–936.

    • Search Google Scholar
    • Export Citation
  • DattaK Nambudripad R Pal S Zhou M Cohen HT & Mukhopadhyay D 2000 Inhibition of insulin-like growth factorI-mediated cell signaling by the von Hippel-Lindau gene product in renal cancer. Journal of Biological Chemistry27520700–20706.

    • Search Google Scholar
    • Export Citation
  • DuraiR Yang W Gupta S Seifalian AM & Winslet MC 2005 The role of the insulin-like growth factor system in colorectal cancer: review of current knowledge. International Journal of Colorectal Disease20203–220.

    • Search Google Scholar
    • Export Citation
  • FonceaR Andersson M Ketterman A Blakesley V Sapag-Hagar M Sugden PH Le Roith D & Lavandero S 1997 Insulin-like growth factor-I rapidly activates multiple signal transduction pathways in cultured rat cardiac myocytes. Journal of Biological Chemistry27219115–19124.

    • Search Google Scholar
    • Export Citation
  • ForbesBE Hartfield PJ McNeil KA Surinya KH Milner SJ Cosgrove LJ & Wallace JC 2002 Characteristics of binding of insulin-like growth factor (IGF)-I and IGF-II analogues to the type 1 IGF receptor determined by BIAcore analysis. European Journal of Biochemistry269961–968.

    • Search Google Scholar
    • Export Citation
  • FottnerC Engelhardt D & Weber MM 1998 Regulation of steroidogenesis by insulin-like growth factors (IGFs) in adult human adrenocortical cells: IGF-I and more potently IGF-II preferentially enhance androgen biosynthesis through interaction with the IGF-I receptor and IGF-binding proteins. Journal of Endocrinology158409–417.

    • Search Google Scholar
    • Export Citation
  • FottnerC Elmlinger M Engelhardt D & Weber MM 2001 Identification and characterization of insulin-like growth factor (IGF) binding protein expression and secretion by adult human adrenocortical cells: differential regulation by IGFs and adrenocorticotrophin. Journal of Endocrinology168465–474.

    • Search Google Scholar
    • Export Citation
  • FottnerC Höflich A Wolf E & Weber MM 2004 Role of the insulin-like growth factor system in adrenocortical growth control and carcinogenesis. Hormone and Metabolic Research36397–405.

    • Search Google Scholar
    • Export Citation
  • FoulstoneE Prince S Zaccheo O Burns JL Harper J Jacobs C Church D & Hassan AB 2005 Insulin-like growth factor ligands receptors and binding proteins in cancer. Journal of Pathology205145–153.

    • Search Google Scholar
    • Export Citation
  • FrödinM & Gammeltoft S 1994 Insulin-like growth factors act synergistically with basic fibroblast growth and nerve growth factor to promote chromaffin cell proliferation. PNAS911771–1775.

    • Search Google Scholar
    • Export Citation
  • GelatoMC & Vassalotti J 1990 Insulin-like growth factor II: possible local growth factor in pheochromocytoma. Journal of Clinical Endocrinology and Metabolism711168–1174.

    • Search Google Scholar
    • Export Citation
  • GillilandG Perrin S Blanchard K & Bunn HF 1990 Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. PNAS872725–2729.

    • Search Google Scholar
    • Export Citation
  • HaselbacherGK Irminger JC Zapf J Ziegler WH & Humbel RE 1987 Insulin-like growth factor II in human adrenal pheochromocytomas and Wilms tumors: expression at the mRNA and protein level. PNAS841104–1106.

    • Search Google Scholar
    • Export Citation
  • HoeflichA Yang Y & Rascher W 1996 Coordinate expression of insulin-like growth factor II (IGF-II) and IGF-II/mannose-6-phosphate receptor mRNA during differentiation of human colon carcinoma cells (caco-2). European Journal of Endocrinology13549–59.

    • Search Google Scholar
    • Export Citation
  • HoeflichA Reisinger E Lahm H Kiess W Blum WF Kolb HJ Weber MM & Wolf E 2001 Insulin-like growth factor binding protein 2 in tumorigenesis: protector or promoter? Cancer Research618601–8610.

    • Search Google Scholar
    • Export Citation
  • HwangO & Choi HJ 1996 Induction of gene expression of the catecholamine-synthesizing enzymes by insulin-like growth factor I. Journal of Neurochemistry651988–1996.

    • Search Google Scholar
    • Export Citation
  • IlvesmäkiV Kahri AI Miettinen PJ & Voutilainen R 1993 Insulin-like growth factors (IGFs) and their receptors in adrenal tumors: high IGF-II expression in functional adrenocortical carcinomas. Journal of Clinical Endocrinology and Metabolism77852–858.

    • Search Google Scholar
    • Export Citation
  • IlvesmäkiV Liu J Heikkilä A Kahri AI & Voutilainen R 1998 Expression of insulin-like growth factor binding protein 1–6 genes in adrenocortical tumors and pheochromocytomas. Hormone and Metabolic Research30619–623.

    • Search Google Scholar
    • Export Citation
  • JiangX Wang L Gong W Wei D Le X Yao J Ajani J Abbruzzese JL Huang S & Xie K 2004 A high expression of insulin-like growth factor I receptor is associated with increased expression of transcription factor Sp1 and regional lymph node metastasis of human gastric cancer. Clinical and Experimental Metastasis21755–764.

    • Search Google Scholar
    • Export Citation
  • KalekoM Rutter WJ & Miller AD 1990 Overexpression of the human insulin-like growth factor I promotes ligand-dependent neoplastic transformation. Molecular Cell Biology10464–473.

    • Search Google Scholar
    • Export Citation
  • KaminoT Shigematsu K Kawai K & Tsuchiyama H 1991 Immunoreactivity and receptor expression of insulin-like growth factor I and insulin in human adrenal tumors. American Journal of Pathology13883–91.

    • Search Google Scholar
    • Export Citation
  • KulikG Klippel A & Weber MJ 1997 Antiapoptotic signaling by the insulin-like growth factor I receptor phosphatidylinositol 3-kinase. Molecular and Cellular Biology171595–1606.

    • Search Google Scholar
    • Export Citation
  • KutohE Boss O Levasseur M & Giacobino JP 1998 Quantification of the full length leptin receptor (OB-Rb) in human brown and white adipose tissue. Life Sciences62445–451.

    • Search Google Scholar
    • Export Citation
  • LeventhalPS Randolph AE Vesbit TE Schenone A Windebank AJ & Feldmann EL 1990 Insulin-like growth factor as a paracrine growth factor in human neuroblastoma cells. Experimental Cell Research221179–186.

    • Search Google Scholar
    • Export Citation
  • LiW Jiang YX Zhang J Soon L Flechner L Kapoor V Pierce JH & Wang LH 1998 Protein kinase C-delta is an important signaling molecule in insulin-like growth factor I receptor-mediated cell transformation. Molecular and Cellular Biology185888–5898.

    • Search Google Scholar
    • Export Citation
  • MenounyM Binoux M & Babajko S 1997 Role of insulin.like growth factor binding protein-2 and its limited proteolysis in neuroblastoma cell proliferation: modulation by transforming growth factor-beta and retinoic acid. Endocrinology138683–690.

    • Search Google Scholar
    • Export Citation
  • MoschosSJ & Mantzoros CS 2002 The role of the IGF system in cancer: from basic to clinical studies and clinical applications. Oncology63317–332.

    • Search Google Scholar
    • Export Citation
  • NielsenFC & Gammeltoft S 1988 Insulin-like growth factors are mitogens for rat pheochromocytoma PC 12 cells. Biochemical and Biophysical Research Communications1541018–1023.

    • Search Google Scholar
    • Export Citation
  • OhlssonC Kley N Werner H & LeRoith D 1998 p53 regulates insulin-like growth factor-I (IGF-I) receptor expression and IGF-I induced tyrosine phosphorylation in an osteosarcoma cell line: interaction with p53 and Sp1. Endocrinology1391101–1107.

    • Search Google Scholar
    • Export Citation
  • RubinR & Baserga R 1995 Insulin-like growth factor I receptor. Its role in cell proliferation apoptosis and tumorigenicity. Laboratory Investigations73311–331.

    • Search Google Scholar
    • Export Citation
  • RussoVC Schutt BS Andaloro E Ymer SI Hoeflich A Ranke MB Bach LA & Werther GA 2005 Insulin-like growth factor binding protein-2 binding to extracellular matrix plays a critical role in neuroblastoma cell proliferation migration and invasion. Endocrinology1464445–4455.

    • Search Google Scholar
    • Export Citation
  • ScatchardG1949 The attraction of proteins for small molecules and ions. Annals of the New York Academy of Sciences51660–672.

  • SingletonJR Randolph AE & Feldman EL 1996 Insulin-like growth factor I receptor prevents apoptosis and enhances neuroblastoma tumorigenesis. Cancer Research564522–4529.

    • Search Google Scholar
    • Export Citation
  • SullivanKA Castle VP Hanash SM & Feldmann EL 1995 Insulin-like growth factor II in the pathogenesis of human neuroblastoma. American Journal of Pathology1471790–1798.

    • Search Google Scholar
    • Export Citation
  • UllrichA Gray A Tam AW Yang-Feng T Tsubokawa M Collins C Henzel W Le Bon T Kathuria S Chen E et al. 1986 Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specifity. EMBO Journal52503–2512.

    • Search Google Scholar
    • Export Citation
  • WangL Wei D & Huang S 2003 Transcription factor Sp1 expression is a significant predictor of survival in human gastric cancer. Clinical Cancer Research96371–6380.

    • Search Google Scholar
    • Export Citation
  • WeberMM Auernhammer C Kiese W & Engelhardt D 1997 Insulin-like growth factor receptors in normal and tumorous adult human adrenocortical glands. European Journal of Endocrinology139296–303.

    • Search Google Scholar
    • Export Citation
  • WeberMM Fottner C Liu SB Jung MC Engelhardt D & Baretton GB 2002 Overexpression of the insulin-like growth factor I receptor in human colon carcinomas. Cancer952086–2095.

    • Search Google Scholar
    • Export Citation
  • WernerH Shalita-Chesner M Abramovitch S Idelman G Shaharabani-Gargir L & Glaser T 2000 Regulation of the insulin-like growth factor-I receptor gene by oncogenes and anti-oncogenes: implications in human cancer. Molecular Genetics and Metabolism71315–320.

    • Search Google Scholar
    • Export Citation
  • ZhangW Thorton WH & MacDonald RS 1998 Insulin-like growth factor-I and -II receptor expression in rat colon mucosa is affected by dietary lipid intake. Journal of Nutrition182158–165.

    • Search Google Scholar
    • Export Citation
  • ZumkellerW & Schwab M 1999 Insulin-like growth factor system in neuroblastoma tumorigenesis and apoptosis: potential diagnostic and therapeutic perspectives. Hormone and Metabolic Research31138–141.

    • Search Google Scholar
    • Export Citation