(CANCER RESEARCH 49, 2755-2760, May 15, 1989] Corticotropin-releasing Factor and Vasopressin Production in the Rat Pituitary Tumor 7315a: Biochemical and Immunohistochemical Studies1 MaríaInésMoranoand Fernando E. Estivariz2 Centro de Estudios Endocrinos, Facultad de Ciencias Medicas, Universidad Nacional de La Plata, Casilla de Correo 455, 1900 La Plata, Argentina ABSTRACT (16). We have found it interesting to study the production and secretion of hypothalamic factors by this tumor, principally In order to investigate the production and secretion of hypothalamic those involved in the control of POMC peptides secretion. In factors by the prolactin and proopiomelanocortin (POMC)-derived, pep- tide-producing, transplanta blu rat pituitary tumor 7315a, we determined the present report, we determined the tumor content and in the concentrations of corticotropin-releasing factor (CRF)- and vasopres- vitro secretion of CRF and AVP, compared with the production sin (AVP)-like immunoreactivities (IR) in the tumor extracts [14.0 ±1.6 of rat prolactin and POMC peptides in the 7315a tumor. In (SE) and 4.2 ±0.9 pmol/g, respectively] and incubation media (0.26 ± addition, we have investigated the Chromatographie character 0.01 and 0.07 ±0.01pmol/107 cells/h, respectively). Total peptide content istics of the CRF- and AVP-like immunoreactivities, and fi correlated well with tumor weight. Moreover, there is a very good nally, we have explored the possibility of distinct populations correlation between the CRF and AVP IR, but not as good between CRF of tumor cells producing the different hormones. or AVP IR and POMC peptide IR tumor contents. Tumor extracts were chromatographed on Sephadex G-75 and compared with chromatograms of stalk median eminence (SME) extracts from normal Buffalo rats. CRF MATERIALS AND METHODS IR in tumor chromatograms gave an unusual pattern of peaks. About 31% of the total CRF IR was eluted in the high molecular weight region. Materials. All reagents used were of analytical grade. Collagenase The major portion of CRF IR was located in a wide region of lower Type IV, deoxyribonuclease I type DM-100, BSA (RIA grade), molec molecular weight. The AVP radioimmunoassay revealed a similar pattern ular weight markers for gel filtration chromatography, PMSF, 3,3'- of peaks in tumor and SME chromatograms. A propressophysin-like diaminobenzidine, Polypep, and dextran (M, 70,000) were provided by peak and a smaller peak coeluting with synthetic AVP were detected. Sigma, St. Louis, MO. Sephadex G-75 Superfine and chromatography Immunohistochemical staining of consecutive sections of the tumor indi columns were from Pharmacia, Uppsala, Sweden. PAP was from Dako cated that AVP and CRF are often found in the same cell, but the CRF Corporation, Santa Barbara, CA. Norit-A-activated charcoal and and AVP-producing cells are clearly distinct from the POMC peptide- polyethylene glycol (M, 6,000) were purchased from Pablo Zubizarreta producing cells. Ward, Buenos Aires, Argentina. Vycor glass was from Corning Glass Works, New York, NY. Penicillin and streptomycin were supplied by Lepetit Laboratories, Buenos Aires, Argentina. Eagle I lei.a medium INTRODUCTION was from Difco, Detroit, MI. Radioiodine (125I isotope) was from The presence of POMC3-derived peptides has been demon Amersham International Pic., Bucks. Radioiodinated LH-RH was from New England Nuclear, Boston, MA. Synthetic human ACTH (1-39) strated in human pituitary and ectopie ACTH-secreting tumors which may give rise to Cushing's syndrome (1-5). Moreover, [hACTH (1-39)], ACTH (1-24), ACTH (17-39), and a-MSH were supplied by Drs. P. A. Desaulles and W. Rittel, Ciba-Geigy, Basle, since the first demonstration of CRF-like activity in two types Switzerland. Synthetic JV-terminal POMC (1-48) [N-POMC (1-48)] of ectopie ACTH-producing tumors (pancreatic tumor and lung was a gift from Dr. J. Ramachandran, Genentech Inc., San Francisco, oat cell carcinoma) by Upton and Amatruda (6), considerable CA. Human N-POMC (1-76) [hN-POMC (1-76)] and h/3-LPH were evidence has been accumulating on ectopie production of both purified at St. Bartholomew's Hospital, London and kindly donated by ACTH and CRF in various human tumors (7-11). In the rat, Dr. P. J. Lowry, Department of Physiology and Biochemistry, Univer there is some information about CRF-like activity in the ACTH, sity of Reading, England. 73-MSH was a gift from Dr. P. Owens, growth hormone and prolactin-producing pituitary tumor MtT/ University of Newcastle, New South Wales, Australia, and MT was donated by Dr. M. Manning, Medical College of Ohio, OH. Human ß- F4 (12). On the other hand, it has been suggested that tumors endorphin, (Phe2,Nle4) ACTH (1-24), rat and ovine CRF (rCRF and can produce factors which resemble other hypothalamic hor oCRF), Tyr°-rCRF(rCRF with a tyrosine in position 0), sauvagine, mones and affect pituitary hormone secretion (13-15). (Arg')-AVP, (Arg')-AVT, LH-RH, and human angiotensins II and III The 7315a tumor is a Irimethyl-aniline induced trunspkmta (hA II and hA III) were purchased from Peninsula Laboratories, Bel- ble rat pituitary tumor that secretes prolactin and POMC- mont, CA. OT (Syntocinon) was from Sandoz Laboratories, Buenos derived peptides. In this respect, our previous studies have Aires, Argentina. Desacetyl a-MSH and C-terminal a-MSH antiserum analyzed the entire spectrum of POMC peptides produced by were kindly provided by Dr. A. Eberle, Institute of Molecular Biology the 7315a tumor, as a first step towards explaining the effects and Biophysics, Zurich, Switzerland. Rabbit anti-0-endorphin E-286, exerted by the tumor on the hypothalamo-pituitary-adrenal axis anti-rCRF 11 and anti-A II E-44 sera were donated by Dr. E. Spinedi, CRRIE, La Plata, Argentina. The anti-AVP/AVT sera AVPR and AVP4 Received 8/12/88; revised 1/18/89; accepted 1/23/89. were a gift from Dr. S. Blähser,Justus Liebig Universität, Giesen, The costs of publication of this article were defrayed in part by the payment Germany, and rabbit anti-oCRF serum was donated by Dr. G. Gillies, of page charges. This article must therefore be hereby marked advertisement in St. Bartholomew's Hospital, London, England. The anti-N-POMC (1- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Work supported by Grant PID-3-901102 from the National Research Council 76) sera C4 and D4, anti-ACTH serum AC 1-7 and goat anti-rabbit 7- of Argentina (CONICET). globulin were produced in our laboratory. Rat prolactin RIA materials 2 Present address: Neuroendocrinology Unit, Department of Neurobiology, were supplied by the NIADDK Rat Pituitary Hormone Distribution 1MBICE, Casilla de Correo 403, 1900 La Plata, Argentina. To whom requests for reprints should be addressed. Program. 3The abbreviations used are: POMC, proopiomelanocortin; A, angiotensin; Biological Specimens. The 7315a pituitary tumor was kindly provided ACTH, adrenocorticotropin; AVP, vasopressin; AVT, vasotocin; BSA, bovine by Dr. R. M. MacLeod from the Department of Medicine, University serum albumin; CRF, corticotropin-releasing factor; IR, immunoreactivity; LH- of Virginia, School of Medicine, Charlottesville, VA, in 1981 and RH, luteinizing hormone-releasing hormone; /5-LPH, /3-lipotropin; MSH. mela- notropin; MT, mesotocin; OT, oxitocin; PAP, peroxidase-antiperoxidase com maintained in our laboratory since then by successive transfer in Buffalo plex; PMSF, phenylmethylsulphonyl fluoride; N-POMC, N-terminal POMC; rats originally obtained from Simonsen Labs, Gilroy, CA, and subse RIA, radioimmunoassay; SME, stalk median eminence. quently bred in our laboratory. Transplants were performed as described 2755 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. CRF AND AVP IN 7315a TUMOR previously (16) by s.c. inoculation of tumor pieces or dispersed tumor Ojeda, and McCann (22), using an antiserum provided by Dr. A. cells (approximately IO7) suspended in Eagle's HeLa medium, in the Arimura (Tulane University). The detection limit of the assay was 4 suprascapular region of inbred adult Buffalo rats. Animals were kept pg/tube. The rat prolactin RIA was performed using the NIADDK RIA at 21 ±2°Cin a 12-h light/12-h darkness cycle with water and food kit. The intra- and interassay coefficients were less than 8 and 12%, available ad libitum. Since the discovery of CRF and AVP in our tumor respectively, in all the RIA systems employed. line, we have tested and found these peptides in another sample of the Immunohistochemical Methods. One portion of each tumor was 7315a tumor from Dr. MacLeod, recently provided by him. routinely fixed in Elftman's fluid (23), embedded in paraffin, and Peptide Extraction and Chromatography. Tumor tissue was removed sectioned at 3-^m thickness. The presence of POMC peptides, CRF from donor rats between Days 15 and 50 after inoculation. Segments and AVP were immunohistochemically demonstrated as published of nonnecrotic tumor tissue were rapidly homogenized in either 200 elsewhere (24). Adjacent sections were immunostained by the peroxi- mM HC1 or acetic acid (1 M) with PMSF (l IHM)(l g tumor/2.5 ml dase-antiperoxidase (PAP) unlabeled antibody enzyme technique, using acid mixture) with or without brief boiling to inactivate proteolytic as the oxygen acceptor 3,3'-diaminobenzidine. For comparison, pitui- enzymes. Tumor homogenates were immediately centrifuged at 12,000 taries and SME from normal adult Buffalo rats were fixed and treated x g at 0°Cfor60 min. SME (consisting of the hypothalamic-hypophy- in the same way. As negative controls, the first antisera were replaced sial stalk, with a small portion of basal hypothalamic tissue attached to by rabbit normal serum or absorbed against their corresponding anti it) was dissected from normal adult Buffalo rats according to Linton gens. The antibodies employed were identical to those described in "Radioimmunoassays." and Lowry (17), and processed in conditions similar to those for the tumor tissue.