BACKGROUND High concentrations of insulin‐like growth factor (IGF)‐I and IGF‐II have been demonstrated in human colonic adenocarcinomas and exert mitogenic effects through paracrine/autocrine interactions with the IGF‐I receptor (IGF‐IR). However, definitive studies of IGF‐IR expression in these tissues have not been performed. METHODS To study changes in the levels of the IGF‐IR in colorectal carcinoma, we analyzed the expression of IGF‐IR in 40 paired samples of normal and carcinomatous colonic tissue by quantitative reverse‐transcription–polymerase chain reaction (RT‐PCR), immunohistochemistry, and ligand binding. RESULTS As measured by RT‐PCR, the IGF‐IR mRNA ratio in paired tumor and adjacent normal mucosa was higher than 2.0 in 32 of 40 (80%) samples. The overall mean IGF‐IR mRNA level was five‐fold higher in tumor versus adjacent normal mucosa (P < 0.0001). Overexpression of IGF‐IR in colon carcinomas was confirmed at the protein level by immunohistochemistry and receptor‐binding studies. Colon carcinoma cells exhibited a positive staining for IGF‐IR in 91% of all tumors (30 of 33) whereas the adjacent normal colonic epithelial cells showed only a very faint or no significant IGF‐IR immunoreactivity. Radioligand assays and Scatchard analysis in both tissue types revealed a single class of high‐affinity IGF‐IR–binding sites with a similar dissociation constant (Kd; 0.14 ± 0.02 nmol/L, n = 18). However, specific 125IGF‐I–binding and receptor concentrations were elevated in tumor membranes compared with normal mucosa (33.6 ± 5.6 vs. 22.7 ± 3.4 fmol/mg protein, P < 0.05). IGF‐I affinity crosslinking and sodium dodecyl sulfate–polyacrylamide gel electrophoresis displayed specific bands corresponding to the size of the normal α‐subunit of the IGF‐IR that were more intense in carcinomatous samples. IGF‐II mRNA levels were significantly elevated in colorectal carcinomas (P < 0.0001). The IGF‐II mRNA ratio in tumor versus normal tissue was elevated more than twofold in 28 of 40 paired samples and a positive correlation was observed between the overexpression of IGF‐II and IGF‐IR in the tumors. CONCLUSIONS Our results demonstrate that, in addition to IGF‐II, a strong overexpression of IGF‐IR is found in the majority of colorectal carcinomas, supporting the hypothesis of an important role of the IGF system in the pathogenesis of colorectal carcinoma. Cancer 2002;95:2086–95. © 2002 American Cancer Society. DOI 10.1002/cncr.10945

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von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome predisposing to retinal, cerebellar and spinal hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma and pancreatic tumors. Clinically two types of the disease can be distinguished: VHL type 1 (without pheochromocytoma) and VHL type 2 (with pheochromocytoma). We report VHL germline mutations and trends in phenotypic variation in families from central Europe. We identified 28 mutations in 53/65 (81.5%) families with 18 (64%) mutations being unique to this population. Whereas types and distribution of mutations as well as a strong correlation of missense mutations with the VHL 2 phenotype were similar to those identified in other populations, these families have provided new insights into the molecular basis for variability in the VHL 2 phenotype. Seven different missense mutations in exons 1 and 3 varied in their biological consequences from a minimal VHL 2 phenotype with pheochromocytoma only to a full VHL 2 phenotype with RCC and pancreatic lesion. These findings contribute to a better understanding of the fundamental mechanisms of VHL disease and its phenotypic variability. Further, we have provided rapid VHL screening for the families in central Europe, which has resulted in improved diagnosis and clinical management.

We have identified and characterized insulin-like growth factor (IGF)-I and IGF-II/mannose-6-phosphate (IGF-II/M6P) receptors in normal adult human adrenocortical tissue. Furthermore, we 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 (Kd) 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 alpha-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, Kd 2.20 +/- 0.46 nmol/l, n = 6). The identity of the IGF-II/M6P 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 +/- 2.0 nmol/kg protein, Kd 0.19 +/- 0.04 nmol/l, n = 4) and adenomas (SB 4.0 +/- 1.1%, RC 17.5 +/- 3.1 nmol/kg protein, Kd 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. In contrast, three out of four hormonally active adrenocortical carcinomas showed a strongly elevated specific 125I-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 alpha-subunit of the IGF-I receptor remained unaltered (n = 4, SB 13.8 +/- 4.2%, RC 72.2 +/- 21.3 nmol/kg protein, Kd 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.

Although the effect of insulin-like growth factors (IGFs) in fetal adrenocortical cells has been investigated extensively, the role of the IGF system in the adult human adrenal gland remains unclear. In the present study we investigated the effect of recombinant human IGF-I and IGF-II on cortisol, dehydroepiandrosterone sulfate (DHEA-S) and cAMP synthesis in adult human adrenocortical cells in primary culture. Both IGFs stimulate basal as well as adrenocorticotropin (ACTH)-induced steroid secretion in a time-and dose-dependent fashion. While both IGFs (6·5 nM) induced only a moderate 2-fold increase in basal cortisol output after 48 h, the effect on basal DHEA-S secretion was significantly stronger, with a 2·7-and 3·7-fold stimulation by IGF-I and IGF-II respectively. Similarly, IGF-II enhanced ACTH-induced cortisol and DHEA-S secretion more potently than IGF-I. In doseresponse experiments, the maximum stimulation of ACTH-induced DHEA-S secretion was induced by 1·6 nM IGF-I (2-fold increase) or IGF-II (2·9-fold increase), while the maximum response of cortisol secretion was elicited only at 13 nM IGF-I (2-fold increase) or IGF-II (2·5-fold increase). This resulted in a significant shift of the DHEA-S dose-response curves to the left, indicating a relative selective stimulation of androgen biosynthesis by physiologically low concentrations (0·4-3·2 nM) of IGF-II, and less potently by IGF-I. At all doses tested, the steroidogenic effect of IGF-II was significantly stronger than the effect of IGF-I. Although both IGF receptors are present in adult human adrenocortical cells, the steroidogenic effect of IGF-II is mediated through the IGF-I receptor, since [Arg 54,55 ]IGF-II, which only binds to the IGF-I receptor, was equipotent with native IGF-II, whereas [Leu 27 ]IGF-II, which preferentially binds to the type II IGF receptor, did not show any effect. In addition, [des 1-3 ]IGF-I, which exhibits only minimal binding to IGFBPs, was significantly more potent than native IGF-I in stimulating adrenal steroid biosynthesis, and elicited almost the same maximum stimulatory effect as IGF-II and [des [1][2][3][4][5][6] ]IGF-II. By Western ligand blotting of conditioned medium it was shown that adult human adrenocortical cells secrete various IGF-binding proteins (IGFBPs), which are induced differentially by treatment with ACTH. In conclusion, these results demonstrate that: (1) IGF-II stimulates basal as well as ACTH-induced DHEA-S and cortisol secretion from adult human adrenocortical cells more potently than IGF-I; (2) both IGFs predominantly stimulate androgen biosynthesis; (3) the steroidogenic effect of IGF-I and IGF-II is mediated through interaction with the IGF-I receptor; (4) the different steroidogenic potency of IGF-I and IGF-II might be explained by interaction of these ligands with locally produced IGFBPs. These data indicate that the IGF system plays an important role in the regulation of the differentiated function of adult human adrenocortical cells.