ICANCER RESEARCH 46, 1214-1218, March 1986] Secretin/Vasoactive Intestinal Peptide-stimulated Secretion of Bombesin/ Gastrin Releasing Peptide from Human Small Cell Carcinoma of the Lung1 Louis Y. Korman,2 Desmond N. Carney, Marc L. Citron, and Terry W. Moody Medica/ Service (151W), Veterans Administration Medical Center, Washington, DC 20422 [L. Y.K., M.L.C.]; Department of Medicine and Biochemistry George Washington University School of Medicine, Washington, DC 20037 [T. W. M.¡;and National Cancer Institute-Navy Medical Oncology Branch National Cancer Institute and National Naval Medical Center, Bethesda, Maryland [D. N. C.¡ ABSTRACT autocrine factor for SCCL (12) growth. We studied the mecha nism of BLI secretion in several SCCL cell lines by examining Bombesin/gastrin releasing peptide-like immunoreactivity (BLI) the action of agents that increase intracellular cAMP. is found in the majority of small cell carcinoma of the lung (SCCL) Because of the results of these in vitro studies and the fact cell lines examined. Because BLI is present in high concentration that secretin stimulates hormone release in patients with gastrin in SCCL we studied the mechanism of BLI secretion from several producing tumors (Zollinger-Ellison syndrome), we examined the SCCL cell lines and in patients with SCCL. In cell line NCI-H345 action of i.v. secretin infusion on plasma BLI levels in several the structurally related polypeptide hormones secretin, vasoac- patients with SCCL, non-SCCL lung tumors, and patients with tive intestinal peptide, and peptide histidine isoleucine as well as theophylline, a phosphodiesterase inhibitor, N6,O2'-dibutyryl out any cancer. cyclic adenosine 3':5'-monophosphate, a cyclic nucleotide ana logue, increased BLI release by 16-120% and cyclic adenosine MATERIALS AND METHODS 3':5'-monophosphate by 36-350%. Similar results were ob Cell Culture. SCCL cell line NCI-H345 was established using a bone tained in SCCL cell line NCI-H209. i.v. injection of secretin (2 biopsy from a patient with SCCL who had relapsed following intensive units/kg) significantly increased plasma BLI in 2 patients with combination chemotherapy. In culture NCI-H345 is aneuploid, clonogenic extrapulmonary SCCL. These data suggest that SCCL cells in soft agarose, tumorigenic in nude mice, expresses elevated levels of 3,4-dihydroxy-L-phenylalanine decarboxylase (198 units/mg protein), possess receptors for secretin/vasoactive intestinal peptide and that receptor occupation stimulates in vitro and In vivo BLI neuron specific enolase (4075 ng/mg protein), and produces high quan tities of BLI (4.7 pmol/mg protein (2). The cell line was maintained secretion. continuously for 24 mo in a serum free defined HITES medium (13). Cells were incubated in 75-cm2 Falcon tissue culture flasks in a humidified INTRODUCTION atmosphere of 5% CO2/95% air at 37°C. One to 2 wk prior to each experiment stock cultures were passaged and then 3 days prior to use In the United States, small cell carcinoma of the lung accounts the cells were resuspended in fresh HITES medium. Other SCCL cell for 25,000-30,000 new cases of primary lung cancer each year lines were maintained as described previously (2). (1). Unlike the other major forms of lung cancer, in vivo and in BLI Radioimmunoassay. Rabbit antisera to bombesin was obtained vitro studies indicate that SCCL3 expresses several unique cy- as described previously (14). The antiserum recognizes bombesin and the structurally related gastrin releasing peptide equally well, because tochemical and functional characteristics including the presence they have the same COOH-terminal heptapeptide, and does not cross- of high levels of 3,4-dihydroxy-L-phenylalanine decarboxylase, react with secretin, VIP, or PHI (14). For the BLI determination cells or dense core neurosecretory granules, and the secretion of peptide supernatant was extracted with boiling 2 N acetic acid, centrifuged at hormones (2-4). These findings suggest that SCCL may origi 2,000 x g for 10 min to remove paniculate material, and the supernatant nate from endocrine (Kultschitzky) cells in the lung (5). However, was frozen, lyophilized, and stored at -70°C. The assay was performed recent studies indicate that SCCL may be derived from a he- by resuspending samples in 140 mw NaCI/16 mw NaPO4 (pH 7.4) matopoietic stem cell (6). Although several polypeptide hor containing 0.25% bovine serum albumin and incubating with rabbit anti- mones including adrenocorticotropic hormone, calcitonin, phys- bombesin serum at 1:100,000 dilution for 1 h at 4°C,then adding 5000 cpm of 125l-labeled tyrosine4 bombesin for a total vol of 400 pi and alaemin, arginine vasopressin, and neurotensin are produced by incubated for 16 h at 4°C.At the end of the incubation 200 pi of normal SCCL, BLI is frequently present (4, 7-9). Prior investigations rabbit serum at 1:200 dilution was added followed by 100 pi of goat anti- have demonstrated that hormone secretion from cultured endo rabbit serum at 1:20 dilution. After 30 min, 200 pi of 12% polyethylene crine cells including SCCL (10) may be mediated by the intracel- glycol was added to enhance precipitation and the samples were centri lular second messenger cAMP (11) and bombesin may be an fuged at 1000 x g for 20 min. The supernatant was removed and the pellet assayed for antibody-tracer complex in a LKB gamma counter Received 2/18/85; revised 7/30/85, 11/26/8S; accepted 12/2/85. 1This work was supported by funds from the Veterans Administration Research (LKB, Rockville, MD). Service and by National Cancer Institute grant CA-33767. Presented in part at the Venous blood samples were centrifuged in a Sorvall RC-5B refriger Annual Meeting of the American Federation for Clinical Research, Washington DC ated centrifuge at 500 x g for 10 min and the plasma was removed and April 1983. stored at -70°C. Prior to assay the plasma was thawed and processed 2 To whom requests for reprints should be addressed, at Gastroenterology Section (151 W), Veterans Administration Medical Center, 50 Irving St., NW, Wash using a modification of the method of Bohlen ef a/. (15). In brief, 2 ml of ington, DC 20422. plasma were added to 6 ml of 5% formic acid, 15% trifluoracetic acid, 3 The abbreviations used are: SCCL, small cell carcinoma of the lung; BLI, 9% HCI, and 1% NaCI. The precipitated plasma proteins were centrifuged bombesin/gastrin releasing peptide-like immunoreactivity; cAMP, cyclic AMP; VIP, at 2000 x g for 10 min and the supernatant was applied to a Sep-Pak vasoactive intestinal peptide; PHI, peptide histidine isoleucine; HITES (medium), RPMI 1640 medium supplemented with hydrocortisone (10 nM), bovine insulin (5 (Millipore). The peptides were eluted from the Sep-Pak using ethanol/0.2 fig/ml), transferrin (10 jig/ml), 17^-estradiol (10 nw), and sodium selenite (30 nM)- N HCI (1/1), and the eluate was frozen, lyophilized, and assayed for BLI cGMP, cyclic GMP. (14). CANCER RESEARCH VOL. 46 MARCH 1986 1214 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1986 American Association for Cancer Research. BOMBESIN/GASTRIN RELEASING PEPTIDE SECRETION FROM HUMAN SCCL Cyclic AMP Radioimmunoassay. Cyclic AMP was determined using mg protein by 5 min which declined to 39 pmol/mg protein by a minor modification (16) of the procedure described by Brooker ef al. 60 min. Similarly, 1 I¿Msecretin, a gastrointestinal peptide with (17). At the times specified iced (4°C)absolute ethanol was added to an equal vol of either the cell suspension or the cell pellet, kept at 4°Cfor significant sequence homology to VIP (20), increased cAMP from 15 min and then frozen and stored at -70°C. Samples were thawed and 17 to 42 pmol/mg protein by 60 min. Without additions cAMP varied between 10 and 25 pmol/mg protein during the 60-min centrifuged at 10,000 x g for 5 min, and an aliquot of supernatant was incubation. Equivalent results were obtained for cell line NCI- removed and assayed for cAMP. Protein was measured by the method described by Lowry ef at. (18). H209 (data not shown). Secretin Infusion. Secretin (2 units/kg) was injected over a 30-s Fig. 2 shows that 1 »Msecretin increased BLI in the superna period into a large antecubital vein via a freely flowing i.v. At 15 min, just tant from 300 to 480 fmol/mg protein after 5 min. Secretion then prior to and at 2, 5,10, 30, and 60 min after secretin injection 5-ml blood remained constant in the presence of 1 /IM secretin. In contrast, samples were obtained, added to 0.5 ml of 20 HIM ethyleneglycol bis(/3- without additions supernatant BLI remained constant at 250 aminoethyl ether)-A/,A/,/v",A/'-tetraacetic acid and bacitracin (pH 7.4), 1 fmol/mg protein throughout the time course of the experiment. mg/ml and placed on ice. Symptoms, blood pressure, pulse, and respi These data indicate that 1 /.M secretin stimulated a rapid release ratory rate were monitored during and for 2 h after secretin injection and of BLI into the supernatant and this release coincided with the no untoward effects occurred. Informed consent, approved by the Med secretin-stimulated increase in cAMP. Similar results were ob ical Center Human Studies Subcommittee, was obtained from the pa tained using VIP. Because both the cAMP levels and secretion tients. Materials. RPM11640 with glutamine was obtained from Grand Island of BLI was maximal after 5 min this time point was used in all Biological, Grand Island, NY; VIP, PHI, secretin, bombesin, physalaemin, subsequent in vitro experiments. Table 1 shows that at 5 min, neurotensin, and cholecystokinin-octapeptide from Peninsula Laborato when secretagogue-stimulated cAMP was maximal, 1 ^M VIP, ries, Belmont, CA; bovine plasma albumin (Fraction V) from Armour PHI, or secretin significantly (21) increased BLI release by 25, Pharmaceutical, Phoenix, AZ; all other chemicals were from Sigma Chemical, St.