(CANCER RESEARCH 53. 2255-2259. May 15. 1993] Insulin-like Growth Factor 1 Receptors Are Increased in Estrogen-induced Kidney Tïimors1 Satya Narayan and Deodutta Roy2 Seatv Center for Molecular Science, University of Texas Medical Branch, Galveston. Texas 77550 ¡S.NJ: and Department of Environmental Health Sciences, University of Alabama. Hirmiiif>harn, Alabama 352V4 [D. R.f ABSTRACT We have previously demonstrated that membrane receptor protein tyrosine kinase(s) activities are higher in estrogen-induced kidney tumors in comparison with such activities in the normal kidney. In the present work we have investigated the growth factor binding sites in estrogen- induced kidney tumor and in normal kidney membranes in an attempt to understand the mechanism of activation of membrane protein tyrosine kinase(s) and their possible relationship to the induction of estrogen- induced tumors. The characteristics of the normal hamster kidney mem brane insulin-like growth factor 1 (IGF-1) receptor are similar to those reported for kidney and extrarenai tissues of other rodents. The binding of 1 "IK.I -1 to the normal kidney or tumor membranes was saturable and dependent on time, protein, pH, and temperature. The binding of ''5I- IGF-1 to the tumor membranes was significantly higher when compared to the binding activity of the membranes obtained from age-matched normal kidney. The Scatchard analysis of the binding data of both tumor and normal kidney revealed a single class binding site for IGF-1 with k,, of 1.7 and 1.8 IIMand maximum binding capacities of 4150 and 2050 fmol/mg protein, respectively. Therefore, the difference observed in I25I- IGF-1 binding between tumor and normal kidney membranes was due to an increase in the number of IGF-1 binding sites with no change in the affinity of receptors for IGF-1. An enhanced level of IGF-1 receptors in tumor membranes also was visualized by autoradiography following af finity labeling of membrane proteins subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis. Under reducing conditions of electro- phoresis, two molecular bands of Mr 240,000 and M, 130,000 were evident. The M, 130,000 band represents the a subunit of IGF-1 receptors, and the M, 240,000 band may represent the aggregates of the receptor subunits which were not reduced completely. IGF-1 stimulated normal kidney or tumor membrane protein tyrosine kinase(s) (wheat germ lectin agarose- purified membrane proteins) in a dose-dependent fashion. Therefore, the alteration of IGF-1 binding activity of the tumor membrane receptors and stimulation of IGF-1-mediated membrane protein tyrosine kinase activity in tumor tissues suggest that events coupled to this membrane receptor may play a role in estrogen stimulation of renal carcinoma. INTRODUCTION Estrogens are carcinogenic to both animals and humans. In addition to their carcinogenic actions, estrogens are known to control the growth of normal and neoplastic cells (1, 2). The mechanisms of estrogen-induced carcinogenesis or estrogen-regulated growth of nor mal and neoplastic cells are not clear. Studies using a variety of estrogen-dependent breast or pituitary normal and cancer cell lines have shown that estrogen controls the cell growth by regulating the expression of growth factors and/or their receptors (3). Among the growth factors known to elicit mitogenic responses in estrogen-de pendent cell lines (IGF-1,3 EOF, and basic fibroblast growth factor). Received 12/4/92; accepted 3/12/93. The costs of publication of this article were defrayed in pan by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This research was supported by a grant from the N1H (CA52584). 2 To whom all requests for reprints should be addressed, at Department of Environ mental Health Sciences. University of Alabama, 720 S. 20th St., Birmingham, AL 35294- 0008. ' The abbreviations used are: IGF, insulin-like growth factorts); SDS, sodium dodecyl sulfate: TGF, transforming growth factor; VIP, vasoactive intestinal peptide: WGA. aga- rose wheat germ lectin; HEPES. 4-(2-hydroxyethyl)-l-pipcrazineethanesulfonic acid; BSA. bovine serum albumin: EGF, epidermal growth factor; PGT, poly(Glu, Na-Tyr), 4:1; the IGF-1 has been shown to elicit strongest growth stimulatory ef fects in vitro and in vivo in a variety of normal and neoplastic cells (4-8). The IGF-1 or somatomedin C is growth hormone-dependent polypeptide growth factor with insulin-like activity. The mitogenic or transforming activity of IGF-1 is mediated through binding to its specific receptor on the cell surface. The IGF-1 receptor is a glyco- protein that consists of a and ß subunits. The extracellular a subunit contains the receptor binding domain. The transmembrane spanning ß subunit contains tyrosine kinase activity which mediates the growth promoting actions of type I IGF receptor. An immediate response of ligand binding to IGF-1 receptors is the autophosphorylation of its own receptor and an increase in the property of tyrosine kinase ac tivity. The phosphorylated site(s) and tyrosine kinase domain of the IGF-1 receptor reside in the same polypeptide chain of the receptors (6). Most studies related to the evaluation of mitogenic properties of IGF-1 in cancer cells have utilized an in vitro system (3-5). Less information is available about the possible role of IGF-1 and -2 or of IGF-1 and -2 receptors in transition from normal to malignant cells and in growth of neoplastic cells in vivo. One of the organs widely used to study the relationship between the level of IGF or their receptors and growth responses after acute treatment with physiological doses of estrogen is uterine tissues (7, 8). However, no in vivo data are available relative to type 1 or 2 IGF receptors after chronic treatment with carcinogenic doses of estrogen or in estrogen-induced tumors in animals. In this study we have identified and characterized the IGF-1 receptors in normal and in neoplastic hamster kidney tissues. We show in this study that en hanced levels of IGF-1 receptors are present in estrogen-induced kidney tumors in comparison with levels found in age-matched nor mal hamster kidney. MATERIALS AND METHODS Chemicals. Diethylstilbestrol and PGT were purchased from Sigma Chem ical Co.. St. Louis, MO. Insulin, gastrin, EGF, TGF-ß,and IGF were purchased from Bachern, Inc.. Torrence. CA. VIP was purchased from Peninsula Labo ratory. Belmont, CA. '-5I-IGF-1 (2000 Ci/mmol) was purchased from Amer- sham. Arlington Heights. IL, and WGA was purchased from Pharmacia. All other chemicals were the highest grade commercially available. Tumor Induction by Estrogen Treatment to Hamsters. Male Syrian hamsters (6-8 weeks old) were purchased from Sasco. Laboratory chow and water were provided ad libitum. Hamsters were treated with one s.c. implant of diethylstilbestrol (25 mg containing 10% cholesterol) for 8 months (9). The hamsters which did not receive any treatment were considered controls and were kept for the same period of time under similar conditions. After 8 months of treatment the hamsters were killed, and the tumors in kidney were scored by gross visual examination. Kidney from treated hamsters were separated into tumor and tumor free tissue (surrounding to the tumor). Some of the kidney tumors were fixed in 10% buffered formalin and histologically evaluated using eosin-hematoxylin stain. Preparation of Crude Plasma Membranes. Plasma membranes were pre pared from age-matched normal kidney, tumor surrounding tissues, and tumors as described previously (10) with some modifications. The tissues were washed and homogenized in buffer containing 20 m.MHEPES (pH 7.5), 2 imi PMSF, 0.1% soybean trypsin inhibitor, 100 units/ml trasylol, and 0.1% bacitrasin. The EG», the concentration of the unlabeled ligand (IGF-1) required to produce 50* com petitive displacement of 12'I-IGF-I binding to membrane. 2256 Research. on December 2, 2021. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from