opposite situation was found in Wilms tumors, where IGF-II content was in the same range as in nontumor tissues despite increased expression of IGF-ll mRNA.
Proc. Nati. Acad. Sci. USA Vol. 84, pp. 1104-1106, February 1987 Medical Sciences
Insulin-like growth factor II in human adrenal pheochromocytomas and Wilms tumors: Expression at the mRNA and protein level (somatomedins)
G. K. HASELBACHER*, J.-C. IRMINGER*, J. ZAPFt, W. H. ZIEGLERt,
AND
R. E. HUMBEL*
*Department of Biochemistry, University of Zfrich, 8057 Zfrich, and tDepartment of Internal Medicine, University Hospital, 8091
Zurich, Switzerland
Communicated by RolfLuft, October 1, 1986 (received for review June 30, 1986)
in size and in 5'-untranslated sequence, regulation and/or function of plasma IGF-II from liver and brain IGF-II might differ (J.-C.I., unpublished data). That IGF-II is a regulatory peptide for brain development has been postulated (15, 16). To find whether IGF-II occurred in other tissues derived from neural crest, adrenal medulla (the cells of which are modified sympathetic neurons) and adrenal pheochromocytomas were examined. Because of the reported elevated expression of IGF-II transcripts in Wilms tumors (17), the content of this substance was also measured in three Wilms tumors.
ABSTRACT Two forms of insulin-like growth factor (IGF) II with molecular masses of 10 and 7.5 kDa, respectively, were found in tumor tissue from human adrenal pheochromocytomas. The tumors contained 5.3-7.1 jig of immunoreactive IGF-H per g of tissue, which is about 20 times more than in adrenal medulla. The total bioactive IGF in the pheochromocytomas exceeded that in normal liver or kidney, which contained only the 7.5-kDa IGF-II species, by a factor of -'100. By contrast, the amount of IGF-I was just measurable and did not vary significantly between tumor and normal tissue. The high amounts of IGF-ll in the pheochromocytomas were not reflected, however, by a corresponding increase of mRNA. The opposite situation was found in Wilms tumors, where IGF-II content was in the same range as in nontumor tissues despite increased expression of IGF-ll mRNA.
MATERIALS AND METHODS Tissue Extraction. Three normal human adrenal glands were obtained 5-8 hr after death in accordance with Ethical Committee protocol. Chromaffin tissue was excised (700 mg) and extracted in 1.4 ml of 1 M acetic acid/0.1% Triton X; a 100,000-g supernatant was prepared and chromatographed on Bio-Gel P-100 (150 x 1.5 cm) in 0.5 M acetic acid. Tissue from three adrenal pheochromocytomas and two Wilms tumors (0.4 x 1 cm in size) were obtained at surgery and immediately frozen on dry ice. Some normal kidney tissue adhering to the tumor was removed and served as a normal control. Diagnosis of each tumor was confirmed by histological examination and in cases of pheochromocytoma by also determining catecholamine levels. No tumors showed evidence of malignancy. The tissue from the pheochromocytomas was extracted as described above except that the extraction medium contained an additional 100 pg/ml of the following protease inhibitors: leupeptin, pepstatin, antipain, chymostatin (Peptide Institute, Osaka, Japan), and aprotinin (Bayer, Leverkusen, F.R.G.). Tissue from human liver was obtained from an organ donor; the liver had been perfused with 0.9% NaCl before storage in liquid nitrogen. Assays. RIA (18) and determination ofbiological activity by fat cell assay (19) were done as described on lyophilized aliquots after elution from Bio-Gel P-100 or after affinity
The insulin-like growth factors (IGFs) I and II, or somatomedins, are polypeptide hormones related to insulin by structure and function (1). In vitro, the insulin-like and mitogenic activities of IGF-I and -II are similar (2). In vivo, IGF-I mimics the effects of growth hormone. IGF-I is therefore considered to be the major somatomedin in humans, whereas IGF-II is considerably less effective in this respect (3). In fact, the main function of IGF-II in humans is not clear. In rodents, IGF-II may function as a fetal growth factor; the levels of IGF-II in fetal rat plasma are high and decline after birth (4). However, in human fetal plasma the IGF-II levels are low and increase until birth and adulthood (5). In rodents, the liver seems to be a major source of plasma IGF-I and -II (6, 7). Several other tissues produce IGF-I (8) and possibly also IGF-II (cf. ref. 9). In humans the pituitary and several brain regions also contain significant amounts of IGF-II, but not of IGF-I (10). Explants of brain and pituitary produce IGF (11), and human thalamic tissue expresses mRNA for IGF-II (J.-C.I., unpublished data). The predominant form of IGF-II in serum is the 7.5-kDa species (12), although the presence of a 10-kDa variant IGF-II has also been demonstrated (13). Significant amounts of 10-kDa IGF-II have been found previously in cerebrospinal fluid (14), in pituitary, and in brain (10) only. About 15% of the total IGF-II in serum occurs in 10-kDa form (14). Of this, the main component has been identified as a partially processed prohormone containing a C-terminal extension of 21 residues and a substitution of Cys-Gly-Asp for Ser-33 (13). The amino acid sequence of IGF-II derived from a cDNA isolated from thalamic mRNA has recently been found identical to that of serum IGF-II (J.-C.I., unpublished data). The 10-kDa form of IGF-II found in brain and pituitary is therefore likely to be partially processed pro-IGFII. Because IGF-II transcripts from liver and thalamus differ
purification. Affinity Purification. IGF-II from pheochromocytoma was further purified on a Sepharose column to which monoclonal antibodies against IGF (MAB 43) had been coupled. This particular antibody recognizes an epitope common to IGF-I and -II (20). Samples were applied in 0.1 M sodium phosphate buffer at pH 7.4 and eluted with 1 M acetic acid. Dot Blot Procedure. Five micrograms of total RNA was dissolved in 20 ,ul of 50% deionized formamide/6% formaldehyde, incubated at 50°C for 1 hr and chilled on ice. Two-fold serial dilutions were then applied to nylon filters, and the blots were hybridized with 32p IGF cDNA (21).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: IGF, insulin-like growth factor(s).
1104
Proc. Natl. Acad. Sci. USA 84 (1987)
Medical Sciences: Haselbacher et al. I I 1 P-4
a. 0
i,
150 FractiQn, ml
FIG. 1. Elution profile of immunoreactive IGF from an adrenal pheochromocytoma after gel filtration. An extract of a tissue fragment (448 mg) was prepared and chromatographed on Bio-Gel P-100. Fifty microliter-aliquots were assayed for IGF-II (.-) and 4'00-p aliquots for IGF-I (---) in specific RIAs.
RESULTS AND DISCUSSION Fig. 1 illustrates the elution profile from Bio-Gel P-100 of immunoreactive IGF extracted from one of the pheochromocytomas. In normal adrenal tissue and in pheochromocytomas, peaks of 10 kDa and of 7.5 kDa of IGF-II were found, but no significant amounts of IGF-I were identified. Serial dilutions of the affinity-purified IGF-I from the pheochromocytoma gave linear curves in the RIA and fat cell assays using pure IGF-I and -}I as standards. The ratios of biological to radioimmunological activities were the same as that for pure IGF-II isolated from serum. The calculated contents of IGFs in adrenal medulla, in each of the three pheochromocytomas, and in liver, kidney, and two Wilms tumors are summarized in Table 1. The mean ratio of IGF-II to IGF-I was 146 in pheochromocytomas, whereas in the other tissues examined this ratio was six. The pheochromocytomas contained about 20 times more IGF-II than did adrenal medulla and contained about 100 times more IGF-II than did liver and kidney. The amount of 10-kDa IGF-I1 varied little from 3.8 to 4.4 pug/g among pheochromocytomas, whereas the 7.5-kDa IGF-II varied from 1.5 to 3.1 ,g/g. The sum of the 7.5- and 10-kDa IGF-IIs in Wilms tumors was 4-6 times higher than IGF-II levels in normal kidney that contained the 7.5-kDa species only. Table 1. Amounts of immunoreactive IGF in various tissue extracts Ag/g of tissue
1105
Because ofhigh levels of IGF-II in pheochromocytoma, we investigated the expression of IGF-II mRNA in these tumors. Total RNA was extracted from a pheochromocytoma, a Wilms tumor, normal kidney, and liver. The amounts of mRNA were compared by dot spot analysis as shown in Fig. 2. Approximately 4 times more mRNA was present in the pheochromocytoma and 30 times more mRNA was present in Wilms tumor as compared with that in normal kidney and liver. In Wilms tumors the expression of IGF-II mRNA has been reported to be 10 to 100 times higher than in normal kidney (17), but measurements of IGF-II contents have not accompanied these data. Clearly, the roughly 30-fold increase in mRNA in Wilms tumor is not reflected by a corresponding increase in IGF-II. In contrast, in pheochromocytoma we find a 100-fold increase in IGF-II that is not reflected by a corresponding increase in mRNA. We recently observed a 5-kb transcript in pheochromocytoma in addition to the 6-kb transcript present in normal adrenal and kidney and Wilms tumor (J.-C.I., unpublished data). The discrepancy between the levels of nRNA and IGF-II suggests a control of translational efficiency of IGF-II mRNA. We cannot, however, exclude that in Wilms tumors part of the mRNA is nonfunctional and therefore not translated at all, or that IGF-II is degraded and/or secreted more rapidly than in normal tissues. Whether the additional 5-kb transcript is translated more efficiently and is thus responsible for high IGF-II levels in pheochromocytoma remains unclear. The 5'-untranslated sequences of the 6-kb and the 5-kb transcripts in pheochromocytoma differ (J.-C.I., unpublished data) and might explain the different translational efficiencies of these two mRNAs. Our results show that not only brain tissue but also neural crest-derived tissue such as the adrenal medulla contain appreciable amounts of IGF-II; in contrast, IGF-I was scarcely detectable. However, a variant IGF-I has recently been reported in human brain (22), and the antiserum used for the RIA may not have recognized this truncated form. Previous immunochemical studies in pheochromocytomas have documented the presence of several peptide hormones such as enkephalin and somatostatin (23), vasoactive intestinal peptidq% corticotropin, (3endorphin and calcitonin (24), growth hormone-releasing factor (25) and neuropeptide Y (26) in pheochromocytomas. Our demonstration of greatly elevated levels of IGF-II in this tumor is thus significant. Whether high expression of IGF-II in pheochromocytoma results from tumor transformation or is a contributing factor 1
2
3
4
A
B
*
IGF-II
IGF-I 7.5 kDa 10 kDa 7.5 kDa 0.03 1.5 3.8 Pheochromocytoma 1 0.04 3.1 4.0 Pheochromocytoma 2 0.06 2.2 4.4 Pheochromocytoma 3 0.02 0.18 0.11 Adrenal medulla 0.02 0.08 0 Liver 0.03 0.06 0 Kidney ND 0.10 0.12 Wilms tumor 1 0.24 0.10 0.14 Wilms tumor 2 The amounts expressed as ,g equivalents were determined by RIA for IGF-I and -II after chromatography of the tissue extracts on Bio-Gel P-100.
FIG. 2. Dot spot analysis of total RNA from different tissues. Rows correspond to RNA from the following tissues: liver, A; kidney, B; Wilms tumor 1, C; and pheochromocytoma 3, D. RNA amounts were as follows: 2.5 Ag, lane 1; 1.2 ,ug, lane 2; 0.6 ,ug, lane 3; 0.3 ,ug, lane 4, and 0.15 ug, lane 5.
1106
Medical Sciences: Haselbacher et al.
(as has been suggested in the case of Wilms tumor; ref. 17) is at present open to speculation. We are indebted to Dr. E. Schoenle for providing us with the tissue from Wilms tumors, Ms. Jiang Zhi-Ping for her technical assistance, and Ms. A. M. Mosca for secretarial help. This work was supported by Swiss National Science Foundation Grant 3.328.82. 1. Humbel, R. E. (1984) in Hormonal Proteins and Peptides XII, ed. Li, C. H. (Academic, New York), pp. 57-79. 2. Rirderknecht, E. & Humbel, R. E. (1976) Proc. Natl. Acad. Sci. USA 73, 2365-2369. 3. Schoenle, E., Zapf, J., Humbel, R. E. & Froesch, E. R. (1982) Nature (London) 296, 252-253. 4. Adams, S. O., Nissley, S. P., Handwerger, S. & Rechler, M. M. (1983) Nature (London) 302, 150-153. 5. Ashton, I. K., Zapf, J., Einschenk, I. & Mackenzie, I. Z. (1985) Acta Endocrinol. (Copenhagen) 110, 558-563. 6. Acquaviva, A. M., Bruni, C. B., Nissley, S. P. & Rechler, M. M. (1982) Diabetes 31, 656-658, 7. Schwander, J. C., Hauri, C. & Zapf, J. (1983) Endocrinology 113, 297-305. 8. D'Ercole, A. J., Applewhite, G. T. & Underwood, L. E. (1980) Dev. Biol. 75, 315-328. 9. Nissley, P. S. & Rechler, M. M. (1984) in Hormonal Proteins and Peptides XII, ed. Li, C. H. (Academic, New York), pp. 129-147. 10. Haselbacher, G., Schwab, M. E., Pasi, A. & Humbel, R. E. (1985) Proc. Natl. Acad. Sci. USA 82, 2153-2157. 11. Binoux, M., Hossenlopp, P., Lassarre, C. & Hardouin, N. (1981) FEBS Lett. 124, 178-184. 12. Rinderknecht, E. & Humbel, R. E. (1978) J. Biol. Chem. 253, 2769-2776. 13. Zumstein, P. P., Lfithi, C. & Humbel, R. E. (1985) Proc. Notl. Acad. Sci. USA 82, 3169-3172.
Proc. Natl. Acad Sci.
USIA 84 (1987)
14. Haselbacher, G. & Humbel, R. (1982) Endocrinology 110, 1822-1824. 15. Sara, V. R., Hall, K. & Wetterberg, L. (1981) in The Biology of Normal Human Growth, eds. Ritzen, M., Aperia, A., Hall, K., Larsson, A., Zetterberg, A. & Zetterstrom, R. (Raven, New York), pp. 241-252. 16. Schoenle, E., Haselbacher, G., Briner, J., Janzer, R. C., Gammeltoft, S., Humbel, R. & Prader, A. (1986) J. Pediatr. (St. Louis) 108, 737-740. 17. Reeve, A. E., Eccles, M. R., Wilins, R. J., Bell, G. I. & Milow, L. J. (1985) Nature (London) 317, 258-260. 18. Zapf, J., Walter, H. & Froesch, E. R. (1981) J. Clin. Invest. 68, 1321-1330. 19. Zapf, J., Schoenle, E., Jagars, G., Sand, I., Grunwald, J. & Froesch, E. R. (1979) J. Clin. Invest. 63, 1077-1084. 20. Laubli, U. K., Baier, W., Binz, H., Celio, M. R. & Humbel, R. E. (1982) FEBS Lett. 149, 109-112. 21. Blanchard, J.-M., Pechaczyk, M., Dani, C., Chambard, J.-C., Franchi, A., Ponyssegur, J. & Jeanteur, P. (1985) Nature (London) 317, 443-445. 22. Carlsson-Skwirut, C., Jornvall, H., Holmgren, A., Andersson, C., Bergman, T., Lundquist, G., Sjbgren, B. & Sara, V. R. (1986) FEBS Lett. 201, 46-50. 23. Lundberg, J. M., Hamberger, B., Schulzberg, M., Hokfelt, T., Granberg, P.-O., Efendic, S., Terenius, L., Goldstein, M. & Luft, R. (1979) Proc. NatI. Acad. Sci. USA 76, 4079-4083. 24. Hassoun, J., Monges, G. & Giraud, P. (1984) Am. J. Pathol. 114, 56-63. 25. Toshiaki, S., Haruhiko, S., Ryuichi, Y., Kazuhito, K., Mineko, I., Eiji, H., Kazuo, H. & Shiro, S. (1984) N. Engl. J. Med. 311, 1520. 26. Adrien, T. E., Terenghi, G., Brown, M. J., Allen, J. M., Bacarese-Hamilton, A. J. & Polak, J. M. (1983) Lancet li, 540-542.
