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Epidermal Growth Factor Suppresses Insulin-Like Growth Factor Binding

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Epidermal Growth Factor Suppresses Insulin-Like Growth Factor Binding (CANCER RESEARCH54, 3160-3166, June 15, 19941 Epidermal Growth Factor Suppresses Insulin-like Growth Factor Binding Protein 3 Levels in Human Papillomavirus Type 16-immortalized Cervical Epithelial Cells and Thereby Potentiates the Effects of Insulin-like Growth Factor 1' Joan R. Hembree,2Chapla Agarwal, and Richard L. Ecke& Departments of Physiology and Biophysics (J. R. H., C. A., R. L E.J, Demwtology [R. L. E.J, Reproductive Biology fR L. E.J, and Biochemistiy [J. R. H., R. L E.], Case Western Reserve University School ofMedicine, CIeveIand@Ohio 44106-4970 ABSTRACT growth. The types and quantities of IGFBPs produced are dependent on cell type and are influenced by a variety of hormones and growth Human ectocervical epithelial cells are a primary target for infection by factors (7—9).The effects of changes in IGFBP levels and/or type oncogemc papillomaviruses, which are strongly implicated as causative on IGF-I action are complex, in some cases potentiating (10—13) agents in the genesis of cervical cancer. Growth factors have been Impli and in others inhibiting (10, 14—16)IGF-I action. In the case of cated as agents that stimulate proliferation and enhance the possibility of malignant transformation. In the present study we utilize several human inhibition, it appears that soluble IGFBPs sequester IGFs and paplllomavirus (HPV) type 16-Immortalized eCtOCerVIcaIepithellal cell prevent their interaction with cell surface-associated IGFBPs lines to Investigate the effects of epidermal growth factor (EGF) and and/or IGF receptors (14—16). Potentiation of IGF-I action by insulin-like growth factor I (IGF-I) on cell proliferation and the produc IGFBP-3 has recently been attributed to association of IGFBP-3 tion of IGF binding proteins (IGFBPs). ECE16-1 cells, an HPV16-immor with the cell surface and subsequent alteration of IGFBP-3 and talized/nontumorigenic cell line, maintaIned in defined medium, produce IGF-I receptor activity (17). The studies indicate that IGF activity and release high levels ofIGFBP-3 (38/42 kDa) as well as smaller amounts is tightly controlled by IGFBPs, suggesting that regulation of ofa 24-kDa IGFBP. Supplementation ofdefined medium with EGF causes IGFBPs by other growth factors could have profound effects on a dose-dependent increase In cell growth and a concomitant decrease in IGF-mediated cellular responses. the levels of IGFBP-3 released Into the culture medium. EGF suppression of IGFBP-3 is maintained even when EGF-stimulated cell growth is The ectocervical epithelium is the target of the oncogenic HPV suppressed 67% due to the simultaneous presence of 3 ag/mI of TGFØ1, forms (HPV16, HPV18, HPV31, and HPV33) which are etiological Indicating that EGF suppression ofIGFBP-3 levels is independent of EGF agents in the development ofcervical carcinoma (18—20).Members of effects on cell growth. EGF suppression of IGFBP-3 production is carve the EGF and IGF families are thought to contribute to the process of lated with a reduction in IGFBP-3 mRNA leveL In the presence of EGF, malignant transformation (21—24).However, the role of such growth the growth response of the cells to ng amounts of IGF-I Is significantly factors in the process of cancer development in cervical epithelial enhanced. Moreover, the simultaneous presence of both EGF and IGF-I cells is poorly understood. Members of the EGF and IGF families are reduces the level of IGFBP-3 more emciently than EGF alone. We also recognized mitogens for epithelia in vitro. At least two members of observe that the IGFBP-3 level is decreased and the 24-kDa IGFBP level the EGF family, TGFa (25) and amphiregulin (26) are produced by is Increased In HPV16-posltlve tumorigenic versus nontumorigenic cell epithelial cells. IGF-I is not produced by epithelial cells (27, 28); lines. This is the first report ofEGF acting as a positive regulator of IGF-I however, IGFs derived from the underlying fibroblasts accumulate in action via the IGFBPs. On the basis of these findings, we propose that EGF stimulates ECE16-1 cell growth via a dual-action mechanism by (a) the epithelial cell layers. IGFBPs have been reported to be produced stimulating growth directly via the EGF mitogenic pathway and (b) by the simple epithelium of the kidney (2), intestine (28), and breast stimulating growth indirectly by reducing the levels of inhibitory IGFBPs epithelium (4, 29, 30). However, the types of IGFBPs produced by and thereby potentiating the effects ofIGF-L In addition, the observation cervical epithelium, a nonkeratinizing, squamous epithelial cell that more highly transformed cell types produce lower levels of IGFBP-3 type, have not been characterized. and higher levels of 24-kDa IGFBP suggests that tumor cells in more In this study we examine the interplay between EGF and IGF-I in advanced cervical cancers may have an altered response to IGF-L the regulation of cervical epithelial cell growth and IGFBP production using an HPV16-immortalized ectocervical epithelial cell line, ECE16-1 (31). ECE16-1 cells were generated by transfection of INTRODUCTION primary cultured ectocervical epithelial cells and are a well-charac The mitogenic activity of the IGFs4 is influenced by the presence of terized model for the study of HPV16 effects on cervical cell function IGFBPs (1). Both IGFBPs and IGFs are produced by a wide variety (31, 32). We present evidence that EGF stimulates ECE16-1 cell of cells (2—6),suggesting that the balance of these factors in the growth via a dual-action mechanism by (a) stimulating growth di cellular microenvironment plays an important role in regulating cell rectly via the EGF mitogenic pathway and (b) stimulating growth indirectly by reducing the levels of inhibitory IGFBPs and thereby Received 7/9/93; accepted 4/15/94. potentiating the effects of IGF-I. IGFBPs represent a previously The costs of publication of this article were defrayed in part by the payment of page unrecognized link between the EGF and the IGF signaling systems. charges. This article must therefore be hereby marked advertisement in accordance with A reduction in IGFBP levels via an EGF-dependent mechanism 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant from the American Institute for Cancer Research may be an important mechanism for the promotion of cervical (R. L. E.) and utilized the facilities of the Skin Diseases Research Center of Northeast tumor development. Ohio (NIH AR49750). 2 Supported as a postdoctoral trainee in the Metabolism Training Program (NIH DK07319). 3 To whom requests for reprints should be addressed, at Department of Physiology and MATERIALS AND METHODS Biophysics, Case Western Reserve University School of Medicine, 2109 Adelbert Road, Cleveland, OH 44106-4970. Chemicals. Dulbecco's modified Eagle's medium, F-12 medium, nones 4 The abbreviations used are: IGF, insulin-like growth factor, IGFBP, insulin-like sential amino acids, L-glutamine, trypsin, and antibiotics were purchased from growth factor binding protein; IGF-I, insulin-like growth factor I; HPV, human papillo mavirus; EGF, epidermal growth factor; TGFa, transforming growth factor a; DM, Gibco (Grand Island, NY). Fetal calf serum, insulin, bovine serum albumin, defined medium; TBS, 10 msi Tris-HC1(pH 7.4), 150 mat Naa, and 0.2% sodium azide; hydrocortisone, T3-transferrin, and adenine were obtained from Sigma (1cm cDNA, complementary DNA. icalCo.(St. Louis,MO).CholeratoxinwaspurchasedfromICNBiomedicals 3160 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1994 American Association for Cancer Research. EGFANDTHE EFFECESOFIGF-I (Costa Mesa, CA). Human recombinant EGF (Upstate Biotechnology Inc., days with intensifying screens. The intensity of the signal was determined by Lake Placid, NY), human recombinant IGF-I (Collaborative Biomedical Prod densitometry. ucts, Bedford, MA), or human TGF@1(R & D Systems, Minneapolis, MN) Nucleic Acid Methods. RNA was isolated as previously described (38) was added to medium as indicated in the text. (3-[1@I]iodotyrosyl)IGF-I and from ECE16-1 cells treatedas described in the text. PolyadenylatedRNA, [32P]dCl'P were purchased from Amersham (Arlington Heights, IL). selected from total RNA by binding to oligodeoxythymidylate cellulose, was Prestained molecular weight standards were purchased from Bio-Rad Labors fractionated on a formaldehyde-containing agarose gel (39) and transferred to tories (Richmond, CA). R7 rabbit anti-human IGFBP-3 antiserum was kindly Biodyne A membranes. The membrane was hybridized with cDNAs encoding provided by Dr. R. L Baxter (Royal Prince Alfred Hospital, Camperdown, human IGFBP-3 or glyceraldehyde-3-phosphate dehydrogenase labeled with Australia). EGF neutralizing antibody AB-236-NA was obtained from R & D [32PJdCTPby random priming. Autoradiograms of Northern blots were quan Systems. Human IGFBP-3 cDNA, spanning nucleotides —50to + 1264 base titated by densitometry. Statistics. Data were analyzed using analysis of variance and Dunnett's pairs relative to the translation start codon (33) was kindly provided by Dr. D. multiple range test for comparison with a single control group. Differences R. Powell (Baylor College of Medicine, Houston, TX). Human glyceralde between two groups were analyzed by Student's t test for two independent hyde-3-phosphate dehydrogenase partial cDNA (34) was kindly provided by samples. Experiments were performed in duplicate or triplicate and repeated at Dr. R. Wu (Cornell University, Ithaca, NY). Proteins were transferred to least twice or as indicated in the figure legends. nitrocellulose membranes (0.45 pm; Schleicher and Schuell, Keene, NH) and RNA was transferredto BiodyneA membranes(ICN Biomedicals,Costa Mesa, CA). Densitometry of autoradiograms was performed using a SciScan RESULTS 5000 optical scanner (United States Biochemicals, Cleveland, OH). EGF and TGF@1Regulation of ECE16-1 Cell Proliferation. To Cell Proliferation Studies. ECE16-1 cells were grown as previouslyde investigate the effects of individual growth factors on ECE16-1 cell scribed (31).
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