[CANCER RESEARCH 43, 2536-2540, June 1983] 0008-5472/83/0043-0000$02.00 Glucocorticoid Effects on Lipopolysaccharide-stimulated Murine B-Cell Leukemia Line (BCL,) Cells1 Deborah A. Roess, Thomas S. Ruh, Clifford J. Bellone, and Mary F. Ruh2 Departments of Physiology and Microbiology, St. Louis University, School of Medicine, St. Louis, Missouri 63104 ABSTRACT The effects of glucocorticoids were studied in lipopolysac- charide (LPS)-stimulated splenic murine B-cell leukemia line one (BCL,) cells. At 24 hr, LPS caused a 3-fold increase in [3H]- thymidine incorporation compared to similarly cultured unstimu- lated cells. Triamcinolone acetonide (TA) and dexamethasone at a concentration of 1CT8 M reduced [3H]thymidine incorporation 80 and 53%, respectively, while estradici at concentrations of 10~10to 10"5 M had no effect. A 500-fold excess of cortexolone or progesterone blocked the response of 10~8 M TA by 42 and 38%, respectively, indicating that the glucocorticoid response could be inhibited by antiglucocorticoids. The maximum rate of thymidine incorporation in LPS-stimulated cells occurred at 24 hr, a time at which 10~5 M TA present in parallel cultures from the initiation of LPS stimulation showed a 79% reduction in [3H]thymidine incorporation. If TA was added at any time after the initiation of LPS stimulation, the degree of decrease in nucleotide incorporation was not as marked. Therefore, maxi mum TA effect in LPS-stimulated BCL! cells occurred when TA was added to cultures at the onset of mitogen stimulation. We also measured glucocorticoid-specific receptor in whole cells both before LPS stimulation and in BCLi cells incubated 24 hr in the presence of LPS. The equilibrium dissociation constant, the number of sites/cell, and the hormone specificity of the gluco corticoid receptor were similar prior to and at the peak of mitogen stimulation. INTRODUCTION The availability of BCL,3 presents a unique opportunity to study the effects of glucocorticoids in proliferating B-cells. The tumor is monoclonal (25) as evidenced by: (a) the surface im- munoglobulin is IgM, all bearing A light chains; (fa) the immune- globulin receptors appear to have identical V regions as evi denced by highly cross-reactive idiotypic determinants; and (c) the secreted ¡mmunoglobulin is also IgM with A light chains and bears the surface idiotypes. Whereas most lymphoid tumors are actively proliferating but appear to be locked into a fixed differ- entiative stage, this is not the case for the BCL, cell line. BCL, cells can be stimulated in vitro by the B-cell mitogen, LPS to both proliferate and differentiate into immunoglobulin-secreting cells. Although the LPS response is not as pronounced as that 1This work was supported by Grant 24034, awarded by the National Cancer Institute, the BiomédicalResearch Support Grant of the USPHS to the St. Louis University School of Medicine, and the McBhde-Love Foundation. 2To whom requests for reprints should be addressed, at Department of Physi ology, 1402 South Grand Boulevard, St. Louis, Mo. 63104. 3The abbreviations used are: BCL,, B-cell leukemia line one; LPS, lipopolysac- charide; TA, triamcinolone acetonide; dThd, thymidine; RPMI 1640, Roswell Park Memorial Institute Tissue Culture Medium 1640; HBSS, Hanks' balanced salt solution. Received September 21, 1982; accepted March 3, 1983. seen in normal murine spleen cells (7), the spleen cell population passed by BCL, cell inoculation into host animals has an advan tage in being comparatively homogeneous after about 6 weeks. Splenic BCLi cells are available in large numbers which also facilitates correlating glucocorticoid effects with receptor char acteristics in stimulated B-cells. Examination of the literature reveals very few reports which critically study the effects of corticosteroids directly on B-lym- phocytes during different stages of differentiation. Our previous studies have shown that LPS-stimulated murine B-cells were glucocorticoid sensitive and that, during proliferation, this sensi tivity was mediated through the glucocorticoid receptor (16). Because the proliferating B-cell becomes progressively less sen sitive to glucocorticoid treatment with time (17), it was of interest to study BCL, cells prior to and at the peak of LPS-induced mitogenesis. The large number of available cells would also make it possible to study glucocorticoid receptors in the BCL, cells. Thus, these tumor cells offer an ideal tool to study biochemical events in homogeneous population of B-cells during differentia tion. MATERIALS AND METHODS Chemicals. [1,2,4-3H]TA (specific activity, 26 Ci/mmol) was obtained from Amersham/Searle Corp. (Chicago, III.). [mef/)y/-3H]dThd (specific activity, 2 Ci/mmol) was purchased from New England Nuclear (Boston, Mass.). LPS (Escherichia coli serotype 055:B5) and unlabeled steroids were obtained from Sigma Chemical Co. (St. Louis, Mo.). RPMI 1640 with 25 rtiM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.2, and HBSS, pH 7.2, were purchased from Grand Island Biological Co. (Grand Island, N. Y.). Fetal calf serum (Lot 200014) was obtained from K. C. Biological (Lenexa, Kans.). BCL, Cells. BCL, cells were passed by injection of 1 x 106 cells i.p. into male BALB/c mice. After about 6 weeks, spleens were removed aseptically and placed in HBSS at 4°, and all further steps were per formed under sterile conditions. Spleens were gently teased to isolate lymphocytes, the debris was removed by sedimentation, and the cells were washed 3 times at 4°in HBSS by centrifugation at 400 x g for 10 min. On the average, each spleen contained at least 1 x 109 cells. Initial cell viability using trypan blue dye exclusion was 90%. dThd Incorporation. Optimal conditions for dThd incorporation were determined. Cells were used at a concentration of 2 x 106 cells/ml in RPMI 1640 supplemented with 5% fetal bovine serum, 2 x 10~3 M glutamine, penicillin (100 ID/ml), streptomycin (100 ¿ig/ml),and 2-mer- captoethanol at a final concentration of 5 x 10~5 M. LPS (£coli serotype 055:B5) at a concentration of 50 Mg/ml gave a 3- to 4-fold increase in [3H]dThd incorporation from 24 to 48 hr. Cells (0.180 ml) were incubated in Costar 96-well tissue culture clusters (BélicoGlass, Inc., Vineland, N. J.) with various glucocorticoids in RPMI 1640:ethanol added to wells at a 10-fold concentration in a volume of 0.02 ml under sterile conditions to give the final concentrations indicated. The ethanol concentration per well was less than 0.01%. Incubations were for 48 hr in 5% CO2 in air at 37°.[3H]dThd (1 ¿iCi/well)was added to each well at 42 hr, and the cells were collected and washed 6 hr later on glass fiber filters using a MASH II harvester (Microbiological Associates, Bethesda. Md.). Filters 2536 CANCER RESEARCH VOL. 43 Research. on October 25, 2021. © 1983 American Association for Cancer cancerres.aacrjournals.org Downloaded from