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Human Lung Small-Cell Carcinoma Contains Bombesin (Radioimmunoassay/Immunohistochemistry/Peptide Hormones/High-Performance Liquid Chromatography) MICHAEL D

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Human Lung Small-Cell Carcinoma Contains Bombesin (Radioimmunoassay/Immunohistochemistry/Peptide Hormones/High-Performance Liquid Chromatography) MICHAEL D Proc. Nati Acad. Sci. USA Vol. 79, pp. 2379-2383, April 1982 Medical Sciences Human lung small-cell carcinoma contains bombesin (radioimmunoassay/immunohistochemistry/peptide hormones/high-performance liquid chromatography) MICHAEL D. ERISMAN*t, R. ILONA LINNOILA*t, OSCAR HERNANDEZ§, RICHARD P. DiAUGUSTINE*, AND LAWRENCE H. LAZARUS*¶ *Laboratory of Pulmonary Function and Toxicology, and §Laboratory of Environmental Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Communicated by George H. Hitchings, December 18, 1981 ABSTRACT The presence of immunoreactive bombesin in a tide in the tumor, (ii) its quantitation in extracts by radioim- human lung small-cell carcinoma grown in nude mice was estab- munoassay, and (iii) analysis by gel-filtration chromatography lished by several criteria: (i) Radioimmunoassay of tissue extracts and reverse-phase HPLC. for bombesin revealed approximately 6.5 pmol/g of tissue; (ii) bombesin was found in 12-14% of the tumor cells by immuno- histochemical localization; (iii) gel filtration ofsmall-cell carcinoma MATERIALS AND METHODS extract on Sephadex G-75 and Bio-Gel P-4 gave only a single peak Tumor. A SCC resected from a 51-year-old man with few ofimmunoreactivity, which occurred at the elution volume ofbom- apparent clinical endocrinopathies was propagated subcutane- besin; and (iv) reverse-phase HPLC of acid-solubilized extracts ously in athymic (nude) mice by Reid et al. (22) and generously separated the immunoreactive material into three discrete peaks, supplied by G. Sato (University of California, San Diego). Tu- one of which eluted with a retention time identical to that of syn- mors from two separate passages were analyzed separately. The thetic bombesin. The presence of bombesin may represent the tumors, 8.71 and 6.36 g wet weight, were removed and sec- ectopic expression of this peptide in small-cell carcinoma, because tioned for histology, and the remainders of each were minced immunoreactive bombesin was found in human fetal and neonatal at 10C. They were homogenized in 5 ml of ice-cold 0.01 M so- lung but apparently not in adult lung tissue [Wharton, J., Polak, dium phosphate buffer, pH 6.8, containing 1 mM phenylmeth- J. M., Bloom, S. R., Ghatei, M. A., Solcia, E., Brown, M. R. & ylsulfonyl fluoride. The homogenate was slowly added to 25 ml Pearse, A. G. E. (1978) Nature (London) 273, 769-770]. The im- munoreactive bombesin previously found in mammalian tissues ofboiling water, boiled 30 min, and cooled on ice; then formic is considerably larger than amphibian bombesin; these data sub- acid was added to a final concentration of 1.0 M (23, 24) and the stantiate the presence of a mammalian form of bombesin in a hu- mixture was stirred for 60 min. The extract was clarified by cen- man tumor that may have a structure similar to that of the am- trifugation (37,000 X g for 30 min at 40C), and the supernatant phibian peptide. was freeze-dried. The yield was approximately 33 mg of dry extract per g of tissue. Small oat-cell carcinoma accounts for 10-25% of identifiable Immunohistochemistry. Sections of tumor (0.1 cm3) were human lung cancers (1). This neoplasm is characterized by a frozen to the temperature of liquid nitrogen, lyophilized, and rapid growth rate and a propensity to metastasize (1) and is fre- fixed with either diethylpyrocarbonate or parabenzoquinone quently a source for "ectopic" hormone production (1-7). Many vapors (25, 26); other sections (0.03 cm3) were fixed either in ofthe clinical symptoms manifested by patients with small-cell 10% buffered formalin or Bouin's fluid and embedded in par- carcinoma (SCC) can be attributed to increased levels ofectopic affin. Samples for electron microscopy (0.03 cm3) were fixed in hormones (5-7). For example, the syndrome of inappropriate 2.6% glutaraldehyde/2.0% formaldehyde (wt/vol) and embed- secretion of antidiuretic hormone is apparently due to ded in Epon. Immunohistochemistry used an improved im- [Arg]vasopressin (5, 6, 8), whereas Cushing syndrome is con- munoglobulin-enzyme bridge technique (27) with antisera sidered to be caused by ectopic adrenocorticotropin (ACTH) against the following peptides at 1:1,000 dilution: ,B subunit of formation and secretion, which is nonsuppressible by dexa- human chorionic gonadotropin (hCGB; batch 2, Pituitary Hor- methasone (3, 5, 7). Other symptoms may be due to the pres- mone Distribution Program, National Institutes of Health), ence ofpresently unknown or undiscovered peptide hormones. bombesin (see below), physalaemin (PS-1) (23, 24), ACTH (A7, In this regard, bombesin, a tetradecapeptide from anuran skin, midregion specific) (28), Mr 16,000 region of pro-opiomelano- elicits numerous physiological and pharmacological responses cortin (B. Eipper, University of Colorado) and human /3-en- (9, 10) that resemble some of the clinical symptoms observed dorphin (V. Fang, University of Chicago). Sheep anti-rabbit for SCC.(5, 11). Bombesin-like immunoreactivity was detected gamma globulin was purchased from Antibodies, Inc. (Davis, in numerous mammalian tissues (12-20), including human fetal CA), and rabbit anti-peroxidase serum and sheep anti-rabbit and neonatal lung, but is apparently absent from adult lung (21). antiserum were from Cappel Laboratories (Cochranville, PA). Because many immunological and enzymological changes that Controls for the specificity of the interaction included incuba- appear in tumors resemble those normally associated with fetal tion ofserial sections oftissues with antigen-inactivated antisera tissues, it seemed reasonable to evaluate the presence ofbom- besin in a lung neoplasm noted for ectopic hormone production. Abbreviations: SCC, small-cell carcinoma; ACTH, adrenocorticotropin; Bombesin in a human lung SCC grown in nude mice was dem- RIA, radioimmunoassay; GH, somatotropin (growth hormone), hCG', ,B subunit of human chorionic gonadotropin. onstrated by (i) immunohistochemical localization of this pep- t Present address: Institute for Cancer Research, Fox Chase, Phila- delphia, PA 19111. The publication costs ofthis article were defrayed in part by page charge t Present address: Laboratory ofPathology, NCI, NIH, Bethesda, MD payment. This article must therefore be hereby marked "advertise- 20205. nent" in accordance with 18 U. S. C. §1734 solely to indicate this fact. ¶ To whom reprint requests should be addressed. 2379 Downloaded by guest on September 24, 2021 2380 Medical Sciences: Erisman et al. Proc. Natl. Acad. Sci. USA 79 (1982) FIG. 1. Immunohistochemical localization of bombesin in SCC. (A) Hematoxylin/eosin stain of a diethylpyrocarbonate vapor-fixed (26) sample. (x 120.) (B-F) Immunostaining (black areas denoted by arrows) on tissue fixed with parabenzoquinone vapor (25), which givesthe photomicrographs their grayish tone. (B-D) Consecutive serial sections (6 am) immunostained with hCGB antiserum (B), physalaemin antiserum (C), and ACTH antiserum (D) and counterstained with toluidine blue. (x 290.) (E andF) Serial sections (12 gim) immunostained with bombesin (E) andphysalaemin (F) antisera. (x290.) (100-250 ug of peptide per ml) or normal rabbit serum. Ar- Radioimmunoassays (RIAs). [Tyr8]Bombesin (donated by J. gyrophilia was determined by using the silver impregnation E. Rivier, the Salk Institute, La Jolla, CA) was iodinated and method of Grimelius (29) on Bouin's fixed paraffin sections. purified according to published procedures (30). Antiserum to Downloaded by guest on September 24, 2021 Medical Sciences: Erisman et al. Proc. Natl. Acad. Sci. USA 79 (1982) 2381 bombesin was raised in rabbits by injecting TPCK-trypsin Immunohistochemistry. Previous studies showed that vapor- (Worthington)-digested bombesin (des-<Glu'-Arg3-bombesin) fixed tissues gave the best results for the immunohistochemical coupled to keyhole limpet hemocyanin with carbodiimide (31). localization of bombesin-like (12, 15) and physalaemin-like im- Physalaemin was immunoassayed as published (23, 24). We munoreactivities in mammalian tissues (24). In the present thank C. Cooper for assays of calcitonin and gastrin and J. van study positive immunostaining was observed after incubation Wyk for somatomedin C (both at the University of North Car- with antisera to several peptides and hormones: (i) hCGB was olina, Chapel Hill). The hCG/3 level was determined by En- found in approximately 9-13% of the tumor cells in several docrine Sciences (Tarzana, CA), and human somatotropin fields ofview (Fig. 1B); (ii) physalaemin was observed in 9-11% (hGH, human growth hormone) was measured with RIA kits of the cells (Fig. 1 C and F); and (iii) bombesin occurred in supplied by CIS Radiopharmaceuticals (Bedford, MA). 12-14% of the cells (Fig. 1E). The specific immunoprecipitate The bombesin RIA mixture contained 50 mM sodium phos- formed by these antisera was located in either the same single phate at pH 7.6, 0.025% bovine serum albumin, 0.085% gamma cells or groups ofcells, on the basis oftheir localization in serial globulins, 1% Trasylol, 30 mM 2-mercaptoethanol, 25 mM sections. Groups of 2-15 immunopositive cells were scattered NaCl, 5,000 cpm of "WI-labeled [Tyr8]bombesin, antiserum unevenly throughout the specimen. Cells containing ACTH- (BM-XII-165-4; 1:4,000 final dilution), and either bombesin like immunoreactivity were infrequent and did not occur in the standards (Bachem Fine Chemicals, Torrance, CA) or extract same region as cells immunopositive for bombesin, physalae- in a final volume of 100 ,1. Incubation ofassay mixtures
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