Peptides. Vol. 8, pp. 103-107.Copyright© PergamonJournals Ltd., 1987. Printedin the U.S.A. 0196-9781/87$3.00 + .00 High Affinity Binding of Cholecystokinin to Small Cell Lung Cancer Cells DARBY G. YODER AND TERRY W. MOODY 1 Department of Biochemistry, The George Washington University School of Medicine and Health Sciences Washington, DC 20037 Received 22 June 1986 YODER, D. G. AND T. W. MOODY. High affinity binding of cholecystokinin to small cell lung cancer cells. PEPTIDES 8(1) 103-107, 1987.---The binding of '2'~I-BoltonHunter-cholecystokinin octapeptide ('2'~I-BH-CCK-8) to small cell lung cancer cell lines was investigated. ~zSI-BH-CCK-8 bound with high affinity (Kd=2.4 nM) to an apparent single class of sites (1700/cell) using cell line NCI-H209. Binding was time dependent and the ratio of specific/nonspecific binding was 8/1. Pharmacology studies indicated that gastrin, caerulein, CCK-33 and nonsulfated CCK-8 were potent inhibitors of specific ~2~I-BH-CCK-8binding whereas CCK-26-32-NH2 was not. Because CCK receptors are present on small cell lung cancer cells, CCK may function as a regulatory peptide in this disease. CCK CCK receptors Small cell lung cancer Neuroendocrine peptides NEUROPEPTIDES such as cholecystokinin (CCK) are bio- logically active in the CNS and periphery [24]. In the brain, CCK may function as a neuromodulator and is colocalized with dopamine in some CNS neurons [13,14]. In the periphery, CCK is a potent satiety agent [10] and causes contraction of the gall bladder [7] and secretion of enzymes from the pan- creas [11]. Mutiple forms of CCK, in particular CCK-33 and CCK-8, have been identified in central and peripheral nerves [6,18]. In addition, CCK has been identified in endocrine cells of the small intestine mucosa [2] and in lung extracts [25]. In particular, CCK was present in moderate densities (0.4 pmol/g wet tissue) in the rat upper trachea and major bronchi but lower levels were also present in the inner and middle lung [26]. When released from brain neurons, CCK may interact with receptors which have been identified in the CNS [12, 15, 28]. Also, CCK receptors are present in pancreatic acini, the trachea, gall bladder and stomach [16, 23, 29, 31]. We have found that certain tumors such as small cell lung cancer (SCLC) are enriched in their neuroendocrine properties. In this regard, cell lines derived from SCLC biopsy specimens have high levels of bombesin-like peptides [20] and neurotensin [22]. When secreted, these neuropep- tides may interact with the receptors for bombesin-like pep- tides [21] and neurotensin [1] which are present on certain SCLC cells. While the role of neurotensin in SCLC remains unknown, bombesin-like peptides function as autocrine growth factors [5]. Because other neuropeptides may be associated with SCLC, we investigated the role of CCK in this disease. Here we report that high affinity binding sites for CCK are present on SCLC cells. METHOD The human tumor cell lines were cultured in serum free HITES medium (RPMI-1640 containing 10-8 M hydrocortisone, 5 /zg/ml bovine insulin, l0 /~g/ml human transferrin, 10-8 M /3-estradiol and 3 × 10-s M Na2SeO3) or serum supplemented medium (RPMI-1640 containing 10% heat inactivated fetal bovine serum) as described previously [3,9]. The cells were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Two days after a medium change, the cells were har- vested by centrifugation at 1,000×g for 10 min. The cells were resuspended in HITES medium and used in receptor binding studies. Two×106 cells were placed in buffer [HITES medium which contained 0.25% bovine serum al- bumin (BSA) and 100/zg/ml bacitracin]. Routinely, the cells were incubated with 100,000 cpm of Ie5I-BH-CCK-8 (2200 Ci/mmol) in the absence or presence of 1 tzM CCK-8, at 37°C for 60 rain; the total assay volume was 200/xl. The cells were pelleted using a Beckman microfuge B for 1 min and the pellet, which contained bound peptide, rinsed twice with 200 /xl of buffer to remove free peptide. Protein was determined using the method of Lowry et al. [19]. SCLC cells were also assayed for immunoreactive CCK. SCLC ceils (107) were extracted in boiling 2 N acetic acid and homogenized using a Kontes tissue disrupter. An aliquot was saved for protein determination and the remainder of the sample was centrifuged at 10,000xg for 10 min. The super- natant was removed, frozen, lyophilized and resuspended in radioimmunoassay buffer (0.25% BSA in PBS). The SCLC extract was incubated with antisera which recognized the 'Requests for reprints should be addressed to Dr. Terry W. Moody, Department of Biochemistry, The George Washington University Medical Center, 2300 Eye Street, N.W., Washington, DC 20037. 103