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Type 1 IGF receptors in specimens of Wilms' tumor and adjacent nonneoplastic kidneytissue. These recep-. PREVIOUS STUDIES of several types of malignant.
American Journal of Pathology, Vol. 130, No. 3, March 1988 Copyright © American Association of Pathologists

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Detection of Type 1 Insulinlike Growth Factor (IGF) Receptors in Wilms' Tumors From the Departments of Pathology and Medicine, Medical University of South Carolina, Charleston, South Carolina

TED GANSLER, MD, KATHERINE D. ALLEN, BS, CHARLES F. BURANT, MD, PhD, TERESA INABNETT, BS, AMY SCOTT, MD, MARIA G. BUSE, MD, DONALD A. SENS, PhD, and A. JULIAN GARVIN, MD, PhD

Insulinlike growth factors (IGFs) are peptide hormones that regulate proliferation and differentiation of several types of normal and neoplastic cells. Recent studies of Wilms' tumor, a childhood kidney neoplasm, have detected increased expression of IGF-2 mRNA and protein. The present report describes detection of Type 1 IGF receptors in specimens of Wilms' tumor and adjacent nonneoplastic kidney tissue. These recep-

tors recognize both IGF-1 and IGF-2, and binding of either peptide activates endogenous tyrosine kinase activity ofthe Type 1 IGF receptor ,6 subunit. These data indicate that Wilms' tumors contain receptors that recognize and respond to exogenous IGF in vitro, and that autocrine IGF production might contribute to the in-

PREVIOUS STUDIES of several types of malignant neoplasms have detected expression of growth factor genes and production of peptide growth factors (GFs). These factors include transforming growth factors (TGF-a and TGF-f6), platelet-derived growth factor (PDGF), and insulinlike growth factors (IGF- 1 and IGF-2).' Cells containing receptors recognizing these GFs may, under appropriate conditions, respond to GF exposure by proliferation and/or substrate-independent growth. Most GF receptors are transmembrane glycoproteins with endogenous tyrosine kinase activity. ' The mitogenic action of IGF- l and IGF-2 is mediated by the Type 1 IGF receptor, a 320-kd glycoprotein with two 1 35-kd binding a subunits and two 95-kd tyrosine kinase /) subunits.26 Although the Type 2 IGF receptor binds IGF-2 with high affinity, this receptor has no kinase activity, does not stimulate cell proliferation, and its exact function is currently not known.7 Wilms' tumor (WT) is a malignant renal neoplasm most common in 1-4-year-olds. Recent studies of WT have revealed increased expression of IGF-2 mRNA and increased content of IGF-2 protein in comparison with adjacent normal renal tissue.8-'0 In

order to investigate the significance of autocrine IGF-2 production by WT, we have assayed extracts of WT and normal renal tissue for the presence of IGF receptors and associated tyrosine kinase activity.

creased proliferation and abnormal differentiation of these cells in vivo. (AmJ Pathol 1988, 130:431-435)

Materials and Methods Tissue Samples Portions of tumor and adjacent normal kidney tissue (NK) were obtained from 2 cases of Wilms' tumor. Specimens were frozen immediately after surgical removal and stored at -70 C until use. In all cases, the diagnosis of WT was confirmed by histologic examination. Both cases used for this study displayed classic morphology, consisting mostly of blasSupported by NIH Grants CA37887 and DKO2001 and by a Medical University of South Carolina Institutional Grant.

Accepted for publication January 6, 1988. Address reprint requests to Ted Gansler, MD, Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425.

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tema with scattered stromal and tubular elements. Anaplasia was not evident.

with that of cross-linked multimers of rabbit phosphorylase b.

Receptor Purification Tissue was finely ground in a mortar containing liquid nitrogen and then homogenized at 4 C in buffer containing 50 mM N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid (HEPES), 1.5% Triton X- 100,4 mM ethylenediamine tetraacetic acid disodium salt, 1 trypsin-inhibitory unit/ml aprotinin, 1 mg/ml benzamidine, 1 mg/ml benzoyl arginine ethyl ester, 1 mg/ml N-a-p-tosyl-L-arginine methyl ester, 0.03 mg/ml leupeptin, and 2 mM phenylmethylsulfonylfluoride, pH 7.4. After centrifugation (l0,OOOg, 10 minutes, 4 C), the supernate was stirred at 4 C for 30 minutes and centrifuged a second time (1 50,000g, 90 minutes, 4 C). This supernate was applied to a wheat germ agglutinin-agarose (WGA) column, washed with 50 mM HEPES/0. 1% Triton X- 100, and eluted with HEPES/Triton containing 0.3 M N-acetylglucosamine.'

Receptor Autophosphorylation Aliquots of WGA eluate from WT and NK were diluted to obtain equal binding activity (final protein concentration, 0.07 and 0.2 mg/ml for WT and NK, respectively), and incubated as previously described with y-32P-ATP (5,uM, 5-15 mCi/4umol) in 25mM HEPES (pH 7.4), 0.1% Triton X-100, 0.1% bovine serum albumin, 5 mM MnCl2, and 10 mM MgCl2 with varying concentrations of IGF-1 or IGF-2."' IGF-1 and IGF-2 was tested at two concentrations, one in the region ofthe Kd and the other 10-100-fold higher. After a 30-minute incubation at 23 C, the reaction was stopped by addition of Laemmli's sample buffer with 0.7 M 2-mercaptoethanol, and samples were analyzed by 7% SDS-PAGE. After the labeled bands were localized by autoradiography, they were excised from the dried gel and solubilized overnight in H202, and the associated radioactivity was quantitated by liquid scintillation counting.

IGF-1 Binding Aliquots of the WGA eluates (0.2 mg/ml protein) were incubated overnight at 4 C with 1.4 X 10-'0M '25I-IGF- 1 (274,Ci/,ig, 1 Ci = 37 GBq) (Amersham, Arlington Heights, Ill) and varying concentrations of unlabeled recombinant human IGF-1 (Amgen Biologicals, Thousand Oaks, Calif) or rat IGF-2 (rIGF-2, multiplication stimulating activity, MSA) (Collaborative Research, Lexington, Mass) in binding buffer (50 mM HEPES, 0.1 % Triton X- 100, 150 mM NaCl, 0.1% bovine serum albumin, 100 U/ml Bacitracin). Receptor was precipitated by addition of polyethylene glycol (12.5%) and bovine gamma globulin (0.1 mg/ml), and the associated radioactivity counted.' Receptor Cross-linking WGA eluates (0.2 mg/ml protein) were incubated with 9.4 X 10-'°M '251-IGF-l and 0 or 10-7 M unlabeled IGF- 1 in binding buffer, overnight at 4 C. Disuccinimidyl suberate (2 mM), dissolved in dimethyl sulfoxide, was added to cross-link ligand to receptor. After 15 minutes at 4 C, Laemmli's 4X sample buffer was added. The sample was boiled for 3 minutes in the presence or absence of 2-mercaptoethanol (0.7 M). After discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (3-10% gradient), gels were stained with Coomassie blue, dried, and the labeled receptors detected by autoradiography. The position oflabeled bands was compared

Results Affinity cross-linking studies reveal association of 125I-IGF-I with a 320-kd glycoprotein (Figure IA). Upon reduction, the IGF remains associated with a 135-kd subunit (Figure 1B). These observations are identical to previous descriptions of Type 1 IGF receptors isolated from a variety of tissues.'2"3 The 320kd bands in Figure lB represent incompletely reduced receptors. Receptor labeling was prevented by addition of 10- M unlabeled IGF- 1. '25I-IGF-I was displaced from WT and NK receptor preparations by unlabeled IGF- 1 with a Kd = 101 M. Values of Kd using IGF-2 (MSA) were 5 X 10-8 M and 3 X 10-8 M for WT and normal kidney, respectively (Figure 2). Specific binding of '25I-IGF- 1 was greater in WT than in NK. The ratio of IGF bound (per milligram protein of WGA extract) by WT to IGF bound by adjacent NK was 3.01 for Case 1 and 3.08 for Case 2. Incubation of WT and normal kidney extracts with IGF- 1 or IGF-2 stimulated autophosphorylation of Type 1 IGF receptors in a dose-dependent manner. Reduction of disulfide bonds prior to electrophoresis demonstrated phosphorylation of the 95-kd ,B subunit. Both basal and maximally stimulated kinase activity was greater in WT, even after WT extracts were diluted threefold to adjust binding activity to that of NK extracts (Table 1). Although IGF- 1 was much more effective than IGF-2 in displacing binding of

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Figure 3-Autophosphorylation of IGF receptors. Extracts from NK 2 (Lanes 1-5) or WT 2 (Lanes 6-10) were incubated with y-=P-ATP. Lanes 1 and 6 show basal kinase activity. Kinase activity was stimulated by 1 0 M IGF-1 (Lanes 2 and 7), 10-7M IGF-1 (Lanes 3 and 8), 5 X 106 M IGF-2 (Lanes 4 and 9) or 5 X 10-7M IGF-2 (Lanes 5 and 10). Reduced proteins were separated by 5% SDS-PAGE.

Figure 1-Identification of IGF receptors from NK and WT. Extracts of NK 1 (Lanes 1 and 2), NK 2 (Lanes 3 and 4), WT 1 (Lanes 5 and 6), and WT 2 (Lanes 7 and 8) were incubated with '251-lGF-1 in the presence (Lanes 2,4,6, and 8) or absence (Lanes 1, 3, 5, and 7) of 10-7M unlabeled IGF-1. Crosslinking with disuccinimidyl suberate was followed by nonreducing (A) or reducing (B) SDS-PAGE on 3-10% gradient gels.

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Figure 2-Competition of 1251-IGF-1 binding. Extracts of NK 1 (A) or WT 1 (B) were incubated with '251-lGF-1 and various concentrations of unlabeled human IGF-1 (open circles) or rat IGF-2 (closed circles). Binding is expressed as per cent of control (no unlabeled IGF added).

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Table 1 -IGF Receptor Kinase Activity

32p in I subunit band (cpm) IGF-1 NK WT WT/NK

IGF-2

Basal

10- M

10-7 M

5 X 108 M

5 X 10-7 M

1569 2325 1.48

1717 2977 1.73

1943 3368 1.73

1751 2983 1.70

1958 3743 1.91

Basal and IGF-stimulated receptor autophosphorylation reactions were performed as described in Materials and Methods. Extracts of normal kidney (NK) and Wilms' tumor (WT) from Case 2 were diluted so that each assay tube contained equivalent binding activity. After SDS-PAGE and autoradiography, the band corresponding to the 95-kd ,B subunit was excised and its radioactivity counted. The above data represent the mean of duplicate determinations.

I251-IGF- 1, the two peptides had similar activity in stimulatingf, subunit phosphorylation at high concentrations (10-7M).

Discussion Peptide growth factors, including PDGF, EGF, TGFs, IGF- 1, and IGF-2 are mitogenic for many types of neoplastic and nonneoplastic cells."',46 Alterations of GF, GF receptors, or GF-related oncogene products have been described in a wide variety of neoplasms and are believed to be important in expression of the transformed phenotype.' Specimens of WT have been shown to contain abnormally high levels of IGF-2 protein and mRNA.8'-0 IGFs also function in control of fetal growth, postnatal skeletal growth, and in compensatory hypertrophy and hyperplasia of renal tissue after unilateral nephrectomy. 17-19 The IGF-2 gene is located on Chromosome 11 (Ip 1 5) near the region (Ip 1 3) frequently deleted in familial cases of WT.8 This suggests that deletion or mutation of the WT locus might increase production of IGF-2, thereby stimulating cell proliferation and inhibiting terminal differentiation. It is not currently known whether the IGF-2 gene is transcriptionally activated by its proximity to an abnormal WT locus or if alteration of the WT locus induces expression of multiple fetal or embryonic genes at various remote chromosomal loci. Because the mitogenic activity of IGF-2 is mediated by the Type 1 IGF receptor, demonstration of this receptor is essential to evaluating the role of autocrine IGF production by WT.24 In the present study, Type 1 IGF receptors were identified by SDS-PAGE of cross-linked 125I-IGF- 1receptor complexes under reducing and nonreducing conditions. There was no detectable difference in apparent molecular weight between receptors (or their

subunits) from WT and NK. Extracts of WT bound approximately three times as much '25I-IGF- 1 as did NK extracts with equal protein concentration. Because WT contain increased levels of IGF-2, it was necessary to demonstrate binding of IGF-2 to the Type 1 receptors from WTs. Binding of '25I-IGF- 1 was displaced by recombinant human IGF- 1 or by partially purified rat IGF-2 (multiplication stimulating activity or MSA). Although the mechanisms by which IGFs regulate intracellular processes are not yet completely understood, it is currently believed that the first step in signal generation involves phosphorylation of tyrosine residues on the receptorfl subunit." 7 MSA, as well as IGF- 1, significantly increased tyrosine kinase activity of receptor preparations from WT and nonneoplastic kidney tissue. Basal kinase activity was much higher in WT, suggesting that activation may have occurred in vivo. Maximal IGF-stimulated kinase activity was also greater in WT. WT kinase activity exceeded that of NK even when WT extracts were diluted threefold to correct for increased receptor content. These data indicate that WT cells possess specific receptors that recognize IGFs and that these receptors are activated by IGF binding in vitro. It appears likely that these receptors are stimulated in vivo by the high levels of IGF-2 produced by WT cells. Although the IGF-2 content of WT is elevated by a factor of four to six,'0 this difference might be amplified by the threefold increase in IGF binding noted in the present study. Increased IGF binding in WT is probably associated with an increase in receptor-mediated internalization and degradation, suggesting that measurements of IGF content may underestimate the elevation in the rate of IGF synthesis. Thus, abnormal autocrine IGF production may be at least partially responsible for increased proliferation and inhibition of terminal differentiation of this neoplasm.

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5. Flier JS, Usher P, Moses AC: Monoclonal antibody to the type 1 insulin-like growth factor (IGF-1) receptor blocks IGF- 1 receptor-mediated DNA synthesis: Clarification of the mitogenic mechanisms of IGF- 1 and insulin in human skin fibroblasts. Proc Natl Acad Sci

USA 1986, 83:664-668 6. Mottola C, Czech MP: The type II insulin-like growth factor receptor does not mediate increased DNA synthesis in H-35 hepatoma cells. J Biol Chem 1984, 259:12705-12713 7. Morgan DO, Edman JC, Standring DN, Fried VA, Smith MC, Roth RA, Rutter WA: Insulin-like growth factor II receptor as a multifunctional binding protein. Nature 1987, 329:301-307 8. Reeve AE, Eccles MR, Wilkins RJ, Bell GI, Millow UJ: Expression of insulin-like growth factor-II transcripts in Wilms' tumor. Nature 1985, 317:258-260 9. Scott J, Cowell ME, Robertson ME, Priestly LM, Wadey R, Hopkins B, Bell GI, Rall LB, Graham CF, Knott TJ: Insulin-like growth factor II in Wilms' tumors and embryonic tissues. Nature 1985, 317:260262 10. Haselbacher GK, Irminger J-C, Zapf J, Ziegler WH, Humbel RE: Insulin-like growth factor II in human adrenal pheochromocytomas and Wilms' tumors: Expression at the mRNA and protein level. Proc Natl Acad Sci USA 1987, 84:1104-1106 11. Burant CF, Treutelaar MK, Landreth GE, Buse MG: Phosphorylation of insulin receptors solubilized from rat skeletal muscle. Diabetes 1984, 33:704-708 12. Kasuga M, Van Obberghen E, Nissley SP, Rechler MM: Demonstration of two subtypes of insulin-like growth factor receptors by affinity cross-linking. J Biol Chem 1981, 256:5305-5308

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13. Massague J, Czech MP: The subunit structure of two distinct receptors for insulin-like growth factors I and II and their relationship to the insulin receptor. J Biol Chem 1982, 257:5038-5045 14. Nagarajan L, Anderson WB, Nissley SP, Rechler MM, Jetten AM: Production of insulin-like growth factor-II (MSA) by endoderm-like cells derived from embryonal carcinoma cells: Possible mediator of embryonic cell growth. J Cell Physiol 1985, 124:199-206 15. Huff KD, Kaufman D, Gabbay KH, Spencer EM, Lippman ME, Dickson RB: Secretion of an insulin-like growth factor-I-related protein by human breast cancer cells. Cancer Res 1986, 46:4613-4619 16. DeLarco JE, Todaro GJ: A human fibrosarcoma cell line producing multiplication stimulating activity (MSA)-related peptides. Nature 1978, 272:356-358 17. Fruesh ER, Schmid C, Schwander J, ZapfJ: Actions of insulin-like growth factors. Ann Rev Physiol 1985, 47:443-467 18. Stiles AD, Sosenko IRS, D'Ercole J, Smith BT: Rela-

tion of kidney tissue somatomedin-C/insulin-like growth factor I to postnephrectomy renal growth in the rat. Endocrinology 1985, 117:2397-2401 19. Fagin JA, Melmed S: Relative increase in insulin-like growth factor I messenger ribonucleic acid levels in compensatory renal hypertrophy. Endocrinology 1987, 120:718-724

Acknowledgments The authors appreciate the editorial assistance of Ms. Sandy Nelson, as well as the photographic expertise of Ms. Leslie Jensen and Mr. James Nicholson.