Growth Factor Action in Lung Tumors Factor-binding Protein-2 in the Regulation of Insulin-like Role for Membrane and Secreted Insulin-like Growth
نویسندگان
چکیده
The insulin-like growth factors (IGFs) have been implicated in the autocrine and/or paracrine growth of a number of tumor types, including lung tumors. Importantly, insulin-like growth factor-binding proteins (IGFBPs), which both enhance and inhibit the physiological and biological actions of the IGFs, have been shown to be secreted in vitro by a wide range of tumors. In particular, IGFBP-2 is frequently produced by human tumor cells, suggesting that this protein may be an important determinant of IGF action in tumors. In the present study, we investigated IGFBP-2 effects in lung tumor cells by examining the influence of IGFBP-2 on IGF-receptor interaction and the biological actions of IGF-I and IGF-II. Affinity cross-linking studies demonstrated expression of type-I and type-II IGF receptors on small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells and the presence of abundant membraneassociated IGFBP in SCLC cells but not in NSCLC cells. An antiserum specific for IGFBP-2 was used in immunoprecipitation and immunoblotting studies which demonstrated that the membrane-associated IGFBP identified by affinity cross-linking in SCLC cells is IGFBP-2. In NSCLC cells, both IGF-I and IGF-II bound predominantly to IGF.I receptors, whereas in SCLC cells binding was principally to surface-associated IGFBP-2. SCLC cells failed to respond to IGF-I and -II stimulation in a DNA synthesis assay. For NSCLC cells, IGF-II was a more potent stimulator of DNA synthesis than IGF-I. Soluble IGFBP-2 inhibited the binding of radiolabeled IGF-I and -II to both SCLC and NSCLC cells in a concentration-dependent manner and inhibited IGF-stimulated DNA synthesis in NSCLC cells. These observations indicate that both soluble and membrane-associated IGFBP-2 compete with IGF receptors for ligand binding and, thus, are likely to be important determinants of IGF responsiveness. The findings of the present study also indicate that the type-I receptor on NSCLC cells contains a high-affinity binding site for IGF-II which presumably mediates the biological effects of IGF-II in these cells, thereby implicating IGF-II in the autocrine/paracrine growth of NSCLC. I N T R O D U C T I O N Several reports have shown that SCLC 2 cell lines secrete and respond to exogenous IGFs, indicating an autocrine role for these peptides in SCLC cell proliferation (1--6). Al though molecular characterization of SCLC-secreted IGFs has not been formally addressed, recent reverse transcriptase-polymerase chain reaction studies indicate that approximately 50% of SCLC and 30% of NSCLC cell lines express the I G F I gene and that almost all SCLC and NSCLC lines show I G F I I gene expression (7). In addition to IGF secretion, lung tumor cell lines also secrete an array of IGFBPs (3, 7-9). Six distinct classes of these proteins have been identified to date: IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6 (10, 11). T h e s e proteins bind the IGFs, often with different relative affinities, and in so doing are thought to modulate the physiological and cellular Received 3/3/93; accepted 7/28/93. The costs of publication of this article were defrayed in part 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. 1 To whom requests for reprints should be addressed, at MRC Clinical Oncology and Radiotherapeutics Unit, Hills Road, Cambridge, CB2 2QH, England. 2 The abbreviations used are: SCLC, small cell lung cancer; IGFs, insulin-like growth factors; NSCLC, non-small cell lung cancer; IGFBPs, insulin-like growth factor-binding proteins; rh, recombinant human; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; Cys, cysteine. actions of these peptides (12). Our recent studies have shown that coexpression of multiple binding protein genes occurs in several lung tumor cell lines (7). However, whereas IGFBP-1 and IGFBP-3 were variably and often weakly expressed in lung tumor cell lines, all SCLC and NSCLC cell lines showed a high level of I G F B P 2 gene expression. Interestingly, a follow-up study of IGFBP production in a wide variety of tumor types showed IGFBP-2 expression in all cell lines derived from solid tumors (13). These findings raise the possibility that IGFBPs, and particularly IGFBP-2, may influence tumor cell responsiveness to the autocrine and/or paracrine actions of the IGFs. In the present study, we addressed this question in lung tumors by investigating the effect of IGFBP-2 on IGF-receptor interaction and the biological actions of IGF-I and IGF-II. M A T E R I A L S AND M E T H O D S Cells. The SCLC cell line NCI-H69 was donated by Drs. D. Carney and A. Gazdar (National Cancer Institute, Navy Medical Oncology Branch, Bethesda, MD). The derivation and characterization of the SCLC cell lines COR-L88, COR-L32, COR-IA2, COR-L47, and COR-L51 and the large cell NSCLC cell line COR-L23 have been described previously (14). These, together with the squamous cell lung carcinoma cell line BEN and the lung adenocarcinoma cell line MOR, were obtained from Dr. P. R. Twentyman, and the squamous cell lung cancer cell line LUDLU-1 was kindly supplied by Dr. P. H. Rabbitts (both from the Clinical Oncology and Radiotherapeutics Unit, Medical Research Council Centre, Cambridge, United Kingdom). All cell lines were routinely grown in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% fetal calf serum, 10 /zg penicillin/ml, and 10/xg streptomycin/ml (all Gibco BRL, Paisley, Scotland). Peptides and Radiochemicals. Recombinant IGF-I and IGF-II were obtained from Calbiochem (Nottingham, United Kingdom) or from Amersham International (Aylesbury, United Kingdom). lzsI-IGF-I (specific activity, 2000 Ci/mmol), 12sI-IGF-II (specific activity, 2000 Ci/mmol), [6-3H]thymidine (specific activity, 27 Ci/mmol), and [methyl-laC]thymidine (specific activity, 57 mCi/mmol) were purchased from Amersham International. The production and characterization of Cys 281 rh IGFBP-2 have been described elsewhere (15, 16). The rabbit anti-type-I receptor antiserum (aiR3) (17) was obtained from Cambridge Bioscience (Cambridge, United Kingdom), and rabbit anti-IGFBP-2 antiserum was from Upstate Biotechnology Inc. (Lake Placid, NY). Characterization of IGF-binding Sites. Cells in exponential growth were harvested either using a cell scraper for adherent cultures of MOR or by centrifugation for the COR-L88 suspension culture. After three washes in phosphate-buffered saline containing 0.8 mg pepstatin/ml, 2 mg aprotinin/ml, and 2 mg leupeptin/ml, cells were resuspended on ice in lysis buffer consisting of 1 mM Tris base containing 0.8 mg pepstatin/ml, 2 mg aprotinin/ml, and 2 mg leupeptin/ml. Ceils were disrupted by sequential passage through 21and 26-gauge syringe needles. The suspension was then centrifuged at 450 • g at 4~ for 10 min. The resulting supernatant was further centrifuged at 50,000 • g for 1 h. The pellet was resuspended in lysis buffer, and protein determinations were carried out. Cell membranes (50 /~g) were incubated at 25~ with 125I-IGF-I or 125IIGF-II (1 • 106cpm) in 5 mM Tris buffer with 0.5% BSA in the presence or absence of unlabeled 100 nM IGF-I, 100 nM IGF-II, 10 p~g/ml insulin, or 10 /xg/ml MR3. In experiments in which anti-receptor antibody was used, membranes and antibody were preincubated for 1 h at room temperature before the addition of the radiolabeled peptide. The samples were incubated at room temperature in a final volume of 1 ml for 2.5 h. The samples were washed with 1 ml of buffer and centrifuged at 4500 • g for 10 min at 4~ The pellet was resuspended in 1 ml 5 mM Tris buffer without BSA, and the radiolabeled
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The insulin-like growth factors (IGFs) have been implicated in the autocrine and/or paracrine growth of a number of tumor types, including lung tumors. Importantly, insulin-like growth factor-binding proteins (IGFBPs), which both enhance and inhibit the physiological and biological actions of the IGFs, have been shown to be secreted in vitro by a wide range of tumors. In particular, IGFBP-2 is ...
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