[CANCER RESEARCH 47, 1087-1092, February 15, 1987] Analysis of Prolactin and Growth Hormone Production in Hyperplastic and Neoplastic Rat Pituitary Tissues by the Hemolytic Plaque Assay1 Ricardo V. Lloyd,2 Kimberlee Coleman, Kristina Fields, and Veena Nath Department of Pathology, University of Michigan Medical Center, Ann Arbor, Michigan 48109 ABSTRACT measure antibody secretion by individual B-cells (26-30) and has also been used with streptococcal protein A to detect The reverse hemolytic plaque assay (RHPA) was used to detect antibody-secreting cells (31). With the application of this tech hormone release from cultured normal, hyperplastic, and neoplastic rat pituitary cells. Hyperplastic pituitary cells were produced by SjC.di- nique to the study of pituitary cells, several investigators have ethylstilbestrol (DES) treatment (10 mg hi Silastic tubes) for 3, 6, and 9 observed MS cells which were secreting both PRL and GH in weeks. Neoplastic pituitary cells from rats with MtT/W 15transplantable the rat anterior pituitary gland (18,19). In this report, we used tumors treated with DES for 3 weeks were also analyzed. Aliquots of the the reverse hemolytic plaque assay to examine the effects of same cells were also analyzed by immunocytochemical staining. DES chronic estrogen treatment on PRL-secreting cells, GH-secret- treatment resulted hi an increase in prolactin (PRI.)-producing cells in ing cells, and MS cells in rats with transplantable pituitary hyperplastic pituitaries compared to untreated pituitaries after 9 weeks tumors and in non-tumor-bearing animals. A combination of of treatment by the RHPA [61.2 ±5.2 (SE) versus 32 ±3.0] and by the reverse hemolytic plaque assay and ICC which confirms the immunocytochemical staining [70.9 ±2.4 versus 36 ±1.4].The percent presence of MS cells in normal and neoplastic pituitary tissue age of mammosomatotropic cells decreased from 113 ±3.8 to 4.2 ± is also described. 2.6% in pituitary cells from these same groups of animals. After 3 weeks of DES treatment hi rats with MtT/W 15 tumor, there was an increase in growth hormone (GH)-producing cells and a decrease in PHI .-producing MATERIALS AND METHODS cells when analyzed by the RHPA (control: percentage of GH, 363 ± 6.2; percentage of PRL, 39.0 ±1.6 versus DES-treated tumors: percent Animals. Female 40-day-old Wistar-Furth rats (Harían Sprague age of GH of 68.2 ±1.9; and percentage of PRL, 3.2 ±1.8%). The Dawley, Madison, WI) were implanted s.c. in the right flank with a 1- percentage of mammosomatotropic cells declined from 12.4 ±23 to 0.77 mm3 portion of tumor as described previously (13). The animals were ±2.4%. A combined procedure of RHPA followed by immunocytochem maintained on 12 h light and 12 h darkness and were allowed to feed ical staining on the same slides also revealed a decline in mammosoma ad libitum. After 20 to 35 days, a palpable tumor was detected in all totropic cells after chronic DES treatment in hyperplastic and neoplastic animals. The animals were divided into 3 groups and some were treated MtT/W 15 rumor cells. These results show that DES has different effects with 10 mg DES (Sigma Chemical Co., St. Louis, MO) or 10 mg on PRL and GH secretion and storage hi hyperplastic pituitary and in progesterone (Sigma) in Silastic tubings for 3 weeks or received empty the MtT/WlS pituitary rumor cells. Silastic tubings. After 3 weeks, the rats were sacrificed and the tumors were used for study. Non-tumor-bearing 60-day-old female Wistar- INTRODUCTION Furth rats were implanted with DES capsules for 3, 6, and 9 weeks after which time they were sacrificed and the pituitaries were used for Chronic administration of estrogens is known to cause PRL3 subsequent studies. Control rats with empty Silastic tubings were sac cell hyperplasia and pituitary tumor development in rats (1- rificed between 80 and 120 days of age. All animals were killed by 11). Recent experiments by several investigators have shown decapitation between 8 and 10 a.m. Cell Culture. Anterior pituitaries and tumors were dispersed in 0.25% that estrogens can also inhibit the growth of transplantable rat trypsin in DMEM (Grand Island Biological Company, Grand Island, pituitary tumors such as the MtT/F4 and MtT/W15 (5, 6, 9, NY). After tissue was sliced into 1.0-mm fragments, they were rinsed 12, 13). Recent studies from our laboratory based on studies with DMEM to remove the excess RBC and then trypsinized for 45 with transplantable pituitary tumor tissues (13) and in vitro min at 37°Cwithgentle stirring. Monodispersed cells were prepared as synthesis of PRL or GH in pituitary tumor cells from DES- reported previously (32) and then centrifuged at 250 x g for 5 min. treated rats (14) suggested that the inhibition of growth of the Approximately 1-1.5 x 10' cells/10 mg of pituitary tumor tissue was M+T/W15 tumor by DES was associated with an increase in usually obtained. Viability of pituitary cells and tumor usually exceeded GH production and a decrease in the PRL-producing cells (5, 95%. DMEM with penicillin (10,000 units/ml), streptomycin (10,000 fig/ml), Fungizone (25 Mg/ml), 15% horse serum, and 2.5% fetal bovine 14). serum were used to culture the cells for 18-24 h at 37'C in an The development of a reverse hemolytic plaque assay for atmosphere of 5% CO2-95% air. pituitary cells has enabled investigators to analyze hormone production by individual cells (15-25). This technique is ex RHPA. The RHPA procedure was done according to the method of Neil and Frawley et al. (15). oRBC were obtained from Colorado Serum tremely useful for studying heterogeneous cell populations in Company (Denver, CO). The oRBC were concentrated by centrifuga- which the cells of interest constitute only a small percentage of tion at 500 x g for 10 min to give 1.0 ml of packed cells. After 3 the total population. This method has been used commonly to washings in saline (0.9 g of NaCl/100 ml of water), 1 ml of protein A (0.5 mg/ml of PBS) (Sigma) and 10 ml of CrCl3 (0.1 mg/ml of saline) Received6/26/86;revised9/19/86,11/7/86;accepted11/12/86. were mixed and then added to the 1-ml packed volume of washed The costs of publication of this article were defrayed in part by the payment oRBC. The cells were incubated for l h at 30*C and then washed once 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. with saline and twice with DMEM containing 0.1% BSA (Sigma) and 1This work was supported by NIH Grant CA37238 and CTR Grant 1850. »suspended in 25 ml of DMEM-0.1% BSA for storage at 4*C. Protein 2To whom requests for reprints should be addressed, at Department of A-complexed oRBCs were used within 5 days. Pathology, The University Hospital, 1500 E. Medical Center Drive, 2G332, Ann Arbor, MI 48109-0054. Cultured pituitary cells were removed from the flask by a brief 3The abbreviations used are: PRL, prolactin; GH, growth hormone; DES, incubation in 0.25% trypsin for 5 min. The assay was conducted in a diethylstilbestrol; MS, mammosomatotropic; DMEM, Dulbecco's modified Ea 30-f/l Cunningham incubation chamber (28) constructed on a poly-L- gle's medium; RPHA, reverse hemolytic plaque assay; oRBC, ovine red blood lysine-coated glass microscopic slide. Tape was placed 20 mm apart cells; BSA, bovine serum albumin; ICC, immunocytochemistry; NIADDK, Na tional Institute of Alcoholism, Diabetes, and Diseases of the Kidney; PBS, across the slide and a 22-mnr glass coverslip was placed over the two phosphate-buffered saline; GRH, growth hormone releasing hormone. pieces of tape. Equal volumes of monodispersed pituitary cells (1.0 x 1087 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1987 American Association for Cancer Research. PRL AND GH PRODUCTION IN PITUITARY TUMORS lO'/ml) and 5 x 10" cell/ml protein A-coated oRBC both in DMEM localization of GH and PRL was done by staining with both antisera with 0.1% BSA containing 1 mg/ml ascorbic acid were mixed and concurrently. infused by capillary action into the Cunningham chamber (IS, 28). The The specificity of the antisera used for ICC was tested by absorbtion cells were allowed to attach to the poly-L-lysine-washed slides for 60 with NIADDK iodination grade antigen. Ten Mg/ml of purified GH min in the CO¡incubator at 37°C.The unattached cells were then and PRL abolished ICC staining of their respective antiserum, while washed from the chamber by placing fresh DMEM-0.1% BSA through PRL or GH staining was not abolished with 100 Mg/ml of GH or PRL, one side of the Cunningham chamber and drawing it through to the respectively. Likewise, omission of the primary antiserum, the bio other side with absorbent paper. The chamber was then filled with a tinylated IgG, or the avidin-biotin complex peroxidase reagent resulted solution of DMEM-0.1% BSA containing rabbit anti-rat PRL or mon in no staining. Substitution of rabbit or monkey serum for primary key anti-rat GH antiserum at a final dilution of 1:100 each. Human antisera did not produce staining. GRH 1-44 [1 x IO"7 M; Peninsula Labs (Belmont, CA)] was added to Combined Reverse Hemolytic Plaque Assay and Immunocytochemis all assays for GH production.