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Schwerpunkt Endokrinologie und Stoffwechselerkrankungen, I Medizinische Klinik und Poliklinik der Johannes Gutenberg Universität Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany
(Requests for offprints should be addressed to M M Weber; Email: MMWeber{at}uni-mainz.de)
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Abstract |
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Introduction |
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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.
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Materials and methods |
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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 x 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 x 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 x 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.
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Results |
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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 x 103 molecules/µg RNA, while significantly higher levels were observed in tumor samples, with a mean expression of 7772 ± 1203 x 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 x 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.
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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.
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Discussion |
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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.
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Acknowledgements |
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Funding |
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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.
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References |
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Received in final form 29 December 2005
Accepted 17 January 2006
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) 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.
