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(CANCER RESEARCH 50. 1337-1345. February 15. I990| Immunocytochemical Localization of the Glucocorticoid Receptor in Steroidsensitive and -resistant Human Leukemic Cells1 Tony Antakly,2 Dajan O' Donnell, and E. Brad Thompson Department of Anatomy, McGill L'niversity, 3640 L'nirersily Street, Montreal, Quebec, Canada H3A 2B2 [T. A..D. OJ; and Department of Human Biological Chemistryana Genetics, t 'niversity of Texas Medical Rranch. Galveston, Texas [E. B. T.J glucocorticoid receptor levels assayed by ligand binding was found (15, 17). The variability in absolute number of binding Because of their lympholytic action, glucocorticoids are included in sites per cell from patient-to-patient has precluded, however, most therapeutic regimens for the treatment of lymphomas and leukemuch practical clinical use of such data. mias. I In presence of functional glucocorticoid receptor may be predictive One problem may be the fact that in many instances the cells of the response to hormonal therapy in these diseases. \Ve have developed assayed are a mixture of normal and malignant cells. In addi an ¡imminiii \nn lii'iniial procedure for human glucocorticoid receptor (GR) in order to assess its level and subcellular distribution in a well- tion, recent flow cytometry data using monoclonal antibodies studied system of childhood lymphoblastic leukemia cells (GEM), »here to the human glucocorticoid receptor suggests that even in a sensitive and resistant subclones have been established. Several Fixation clonal line of steroid-sensitive, receptor-containing cells, there may be considerable cell-to-cell variation in receptor content and cell permeabilization protocols were compared. The most sensitive and reproducible one for light microscopy was prefixation in Bouin's (18).1 Thus, it would appear that combined ligand-binding and ABSTRACT solution followed by cytocentrifugation. L'sing various polyclonal and monoclonal antibodies, the GR was consistently localized predominantly in the cell cytoplasm in the absence of steroid. We compared localization of GR following glucocorticoid treatment in the glucocorticoid-sensitive clone CEM C7 with resistant subclones (4R4, 3R43, ICR27). Upon incubation with glucocorticoid, an increase in nuclear staining was clearly observed in the steroid-sensitive C7 cells. Although the resistant cell lines contain immunoreactive GR, they failed to show nuclear transloca tion following glucocorticoid treatment. This outlines the importance of the immunocytochemical procedure to distinguish between sensitive and resistant leukcmic cells. Whether this test could be used prospectively to help select glucocorticoid therapy in human leukemias and lymphomas can now be examined. INTRODUCTION Because of their lympholytic actions, glucocorticoids are included in many therapeutic regimens for the treatment of various forms of leukemias and lymphomas (1,2). These actions may be both direct on the malignant cell and indirect, through the down-regulation of lymphokines upon which some, but not all such malignancies depend (3). The exact cause of the direct lympholytic action of glucocorticoids is unknown; it may in volve induction of a lethal substance or deinduction of a critical gene driving cell replication (4-6). In any case it is clear from studies done in tissue culture that the presence of adequate quantities of properly functioning intracellular glucocorticoid receptors is a necessary though not sufficient component for lymphocytolysis (7-9). These facts, plus the success in predict ing response to endocrine therapy in breast cancer simply by evaluating the level of estrogen receptors (10-14) led to several studies attempting to do so in various leukemias and lympho mas (15, 16). The diversity of these diseases, coupled with their relative rarity, resulted in no simple, overall correlation being found. However, in chronic lymphocytic leukemia, in certain types of childhood acute lymphoblastic leukemia and in malig nant lymphomas, a correlation between response to glucocorticoid-containing or single agent glucocorticoid therapy and Received 6/13/88; revised 9/7/89; accepted 10/26/89. The costs of publication of this article were defrayed in part by the payment 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. ' This work was supported by grants from the National Cancer Institute of Canada (to T. A.) and The National Cancer Institute of the United States (to E. B. T.). This paper is dedicated to the memory of Howard J. Eisen (7/31/42-2/ 7/87). 2To whom requests for reprints should be addressed. immunocytochemical analysis might provide a much better evaluation for patient material in those leukemias/lymphomas potentially able to respond to glucocorticoids than biochemical radioligand assays alone. In addition to the above, a second issue, basic to the action of steroids, is addressed herein. Recent studies on estrogen and progesterone receptors have localized them almost exclusively in nuclei, challenging the classic biochemical model proposing that these receptors are located in the cytoplasm and only associate with nuclei after binding ligand (19, 20). Yet accu mulating immunocytochemical reports on glucocorticoid recep tors in several tissues indicate a cytoplasmic localization of unliganded receptor (21-23). Since specific antibodies to the human GR4 have been prepared (24, 25), we have developed a sensitive immunocytochemical procedure requiring minimal amounts of human cells to assess the level and subcellular distribution of GR in normal and neoplastic human tissues and subsequently evaluate its usefulness as a predictive tool for glucocorticoid response. The present paper describes the eval uations that lead to that immunocytochemical procedure, and show the localization of the human GR in a well-studied cell culture system of human childhood leukemia (8, 26-28, and reviews in refs. 7, 15). We demonstrate cytoplasmic localization of the human GR in these lymphoid cells and show that sensitive, but not resistant cell lines show a degree of nuclear translocation following dexamethasone treatment correspond ing to that determined by biochemical assays. MATERIALS AND METHODS Cell Culture The isolation and characterization of the glucocorticoid-sensitive, receptor-positive (r*) CEM-C7 clonal cell line and two types of glucocorticoid-lysis-resistant cell lines, activation labile (r""") and receptor deficient (r~) derived from it have been already described in detail (8, 15, 26-28). Two r"cl/lclones were employed, 4R4 and 3R43; the r~ clone used was ICR27. Each of these cell lines was grown as stationary suspension cultures in RPMI 1640 medium (GIBCO, NY) supple mented with 5% fetal calf serum (GIBCO). Where indicated, sera were previously treated with dextran-coated charcoal (DCC) (Pharmacia and Fisher Scientific) to remove endogenous steroids; this procedure re moved over 99% of added ['Hjdexamethasone. Cells were maintained 3 D. Marchetti, B. Barlogie. and E. B. Thompson, unpublished data. 4 The abbreviations used are: GR, glucocorticoid receptor; DCC, dextrancoated charcoal; PBS, phosphate buffered saline. 1337 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research. GLUCOCORTICOID RECEPTOR IN HUMAN LEUKEM1C CELLS in a tissue culture incubator at 37°Cwith a humidified atmosphere of 94% air and 6% CO2 at a density of 0.5-2 x IO6cells/ml. Preincubation with Dexamethasone In order to study the effect of incubation with glucocorticoid on the localization of the glucocorticoid receptor, cells were first collected by low speed centrifugation (100 x g. clinical centrifuge) washed twice with serum-free medium and finally incubated with or without DCCtreated serum in the presence of IO"6 M dexamethasone (Sigma, St. Louis, MO) or the vehicle used to dissolve the steroid (ethanol. final concentration 0.1%) for 15, 30, or 60 min at 37°C.Subsequently the cells were collected by centrifugation as above and processed for fixation and immunocytochemistry as described below. Fixation and Cell Preparation for Immunocytochemistry In order to obtain reliable and reproducible immunocytochemical results, cells must be fixed to cross-link the proteins and cellular elements in situ and to allow access to the immunocytochemical re agents. However, results may vary' depending upon the tissue prepara tion and type of fixative used and subsequent procedures followed for sectioning the cells and enhancing their permeability to antibodies. In all these situations, a compromise must be reached for a maximum preservation of the ultrastructure of the cells close to the native state and minimum denaturation of the antigenic sites. With these issues in mind we have previously developed an immunocytochemical procedure for the intracellular and intranuclear localization of a variety of antigens in whole cells in culture (30). This procedure was adapted to the present study as described below. We also evaluated detergent permeabilization of cell membranes by treating the prefixed cells with either Triton X100, Nonidet-P40, or Saponin at a concentration of 0.1, 0.2%, 0.5, and 1% for 2-30 min. Control experiments (see below) were also system atically performed to insure proper penetration of the antibodies to the cellular compartments, the nucleus in particular and to avoid artefacts caused by poor fixation, diffusion, and antigen denaturation. The fixation protocols and types of cell preparation are shown in Table 1. Briefly, four methods for fixation and cell preparation were compared as follows. Preparation of Cell Suspensions. The cells were collected by centrif ugation and resuspended in the indicated fixative as shown in the legend for various periods of time. Following three quick rinses in isotonic PBS, the cells were washed overnight in PBS (18-20 h) at 4°C. Subsequently, the cells were collected by centrifugation, incubated in suspension with the glucocorticoid receptor antibody with or without prior detergent permeabilization as above (see "Fixation"). Preparation of Cytospins. Two methods were tested: cells were cytospun with or without prefixation. In the first method, following fixation of the cells as indicated above (see "Preparation of Cell Suspensions"), the cells were resuspended in 49i bovine serum albumin. Subsequently, cells were adhered onto gelatin-coated glass slides using a ShandonEliott cytocentrifuge at 1100 rpm for 5 min (1 x IO5 cells per slide). Following cytospinning, the cells were air dried. In the second method, the cells were cytospun without prefixation and finally postfixed in one of the fixatives indicated in Table 1 for 30 min. In all cases cytospins were air dried and immediately processed for immunocytochemistry or alternatively, stored at -20'C for up to 3 months. Preparation of Paraffin-embedded Sections. The cells prefixed as indicated in Table 1 were subsequently collected as a compact cell pellet before being embedded in a thin layer of 1% agarose and finally routinely processed for paraffin embedding. These embedded pellets were subsequently cut at 5 urn, affixed onto glass slides, and stored at room temperature until processed for the localization of the glucocor ticoid receptor. Frozen Sections. The cultured cells were collected by centrifugation as a compact cell pellet and immediately frozen in liquid N2 without prefixation. The frozen pellets were covered with OCT embedding compound (Tissue-Tek, Miles Scientific catalog no. 4583) and sec tioned in a cryotome (Reichert, Austria) at —¿30°C. Sections S-j/m each Immunocytochemical Procedure The tissue slides were processed for immunocytochemistry as de scribed earlier (21) with one of the glucocorticoid receptor antibodies listed below. Briefly, the slides were incubated with the primary anti bodies for 18-24 h at 4°Cthen washed three times in PBS (15 min with agitation). The reacted antibodies were revealed using biotinlabeled antimouse or antirabbit IgG and the avidin-biotin complex (Vector Labs, Bringhame, CA). Four washes in PBS were done as above. Alternatively the antibodies were revealed either by the peroxidase-anti-peroxidase method using previously documented second an tibodies (30) or by fluorescein-labeled antibodies (Cappel, PA). Peroxidase activity was demonstrated by the diaminobenzidine (Sigma) cytochemical reaction (31). In this report, "staining" and "stain" refer to the dark brown reaction products resulting from the peroxidative polymerization of diaminobenzidine. The slides were ob served by at least two observers who were not aware of the slide's identity. This double-blind procedure is necessary to insure objective recording of results. The staining of the preparation was scored on a scale of + (low) to 5+ (very high). This is a widely accepted method of evaluating immunocytochemical staining. In order to identify cell mor phology, slides were weakly counterstained with 0.12% méthylène blue. Glucocorticoid Receptor Antibodies In order to demonstrate the validity of the immunocytochemical reaction, two sets of independently developed antibodies were used. First, three polyclonal antibodies (882, 884, and 202) to the human glucocorticoid receptor purified from IM9 cells (24) were used, they contained approximately 20-25 mg protein/ml. Second, a monoclonal antibody to the glucocorticoid receptor purified from human Hela cells (32) used in our earlier studies (33) contained 21 mg protein/ml. Data shown in Tables I and 2 are based on studies using all the above antibodies. Immunocytochemical Controls To confirm the specificity of the immunocytochemical staining, the reaction was shown to be eliminated by absorbing the antibody with purified receptor as indicated in Fig. \B. Additional controls were performed by substituting the receptor antibodies with preimmune serum. For the monoclonal antibody, culture media nonimmune hybridoma cells or nonimmune mouse immunoglobulin (Sigma, MO) were used. The data summarized in this paper represents approximately 3000 tissue slide preparations. Most experiments were repeated 5-10 times. RESULTS Effect of Fixation. Table 1 summarizes the pattern of subcel lular localization of the glucocorticoid receptor using different fixation and preparation procedures. With all fixation proce dures that gave a satisfactory staining and morphological pres ervation, a predominantly cytoplasmic localization was ob served in the absence of dexamethasone or endogenous gluco corticoid. A small percentage of cells showed mixed nuclear and cytoplasmic labeling but no exclusively nuclear labeled cells were seen either in absence (Figs. 1-3) or presence (Fig. 4) of dexamethasone. Immunoreactivity was stronger using Bouin's fixative but quite satisfactory results were obtained using Zamboni and 4% paraformaldehyde; the latter resulted, as expected, in a good morphological preservation. The addition of glutaraldehyde to the paraformaldehyde fixative, even in minute amounts, did not improve the staining intensity and resulted in an artefactual staining of the cell periphery (not shown) presumably reflecting poor penetration of the antibodies. As expected, cells prefixed were collected on warm glass slides and air dried for 15 min before and cytospun resulted in a good morphological resolution at fixation as described in Table 1. 1338 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research. GLUCOCORTICOID RECEPTOR IN HUMAN LEUKEMIC CELLS procedure (Figs. 1-3). However, whereas permeabilization is needed when cells are fixed with paraformaldehyde, this pro cedure is not needed in Bouin-fixed cells (Fig. 4). In order to further demonstrate that our experimental con Cell suspension ditions allow penetration of antibodies to the nucleus, Cos cells 2% Parai? +++ E Mostly cyto. & low nucÃ-. 2% Paraf. + 0.01% Glut. ++ E Mostly cyto. & low transfected with SV40 which express the T-antigen in their nucÃ-.,artefact on cell nucleus, were processed simultaneously with C7 cells for imperiphery 4% Paraf. +++ ¡ munocytochemistry. The C7 cells were stained with a glucocor Mostly cyto. & low nucÃ-. Mostly cyto. & low nucÃ-. Zamboni +++ E ticoid receptor antibody, whereas Cos cells were stained with Bouin ++++ E-G Cyto. & nucÃ-. an antibody to the T-antigen (34). Fig. 5 demonstrates that the T-antigen is solely localized in the cell nuclei. Cytospin With prefixation Effect of pH on the Staining Pattern: Demonstration of an 1% or 0.4% Glut. + E Nonspecific staining on Artefact. Immunocytochemistry is normally performed at pH cell periphery 2% Paraf.+ 0.1% Glut. ++ G Nonspecific staining on 7-7.6. Unless otherwise indicated, the staining reported in this cell periphery paper is performed at pH 7.6. Accidentally, we found that when 2% Paraf. +++ G Mostly cyto. & low nucÃ-. the pH was dropped below 5, even for short periods of time (5 4% Paraf. ++++ G Mostly cyto. & low nucÃ-. Mostly cyto. & low nucÃ-. Bouin +++++ G min), the staining of the glucocorticoid receptor became nu Zamboni ++++ G Mostly cyto. &.low nucÃ-. clear. This staining at low pH was considered artefactual be Carnoy P Without prefixation cause the preimmune control serum also showed significant 100% methanol + G Cyto. periphery only. staining of nuclei in cells stained at pH < 5. This intriguing nucÃ-,negative Ethanol + acetic acid' + G finding is currently under further investigation and will be Cyto. periphery only. nucÃ-,negative reported elsewhere. Fig. 3, A and B, contrasts the staining at pH 7.6 and 4. This observation highlights the need to keep the Paraffin-embedded pellet then pH close to 7.6 during staining to avoid artefactual localization. sectioned 4% Paraf. Mostly cyto., some nu Glucocorticoid Receptor Immunoreactivity using Various An clear staining variable tibodies. All antibodies mentioned above gave a reproducible among cells 4% Paraf. + 0.05% Glut. and specific staining, there were, however, quantitative differ Bouin Cyto. & nucÃ-. ences with respect to staining intensity and titer. Monoclonal antibody 11 gave an optimal signal at 1/50 to 1/100. Polyclonal Unfixed frozen cell pellei, sec thenpostfixedNo tioned at -30°C, antibodies 882, 884, and 201 gave a strong specific staining at dilution range from 1/100 to 1/4000 depending upon the fixative4% Paraf.BouinFormalin P+ weak nucÃ-. fixation procedure. Control experiments in which the glucocor PPPPArtifactsCyto..Cyto., ticoid receptor antibody was preabsorbed with purified gluco vapor100% corticoid receptor resulted in a decrease and in some cases a ethanolAcetoneP+ near complete absence of immunoreactivity (Fig. IE). Other * Morphological preservation: E, excellent; G. good: P. poor. controls mentioned above were devoid of staining (Fig. l, D 'Abbreviations: Glut., glutaraldehyde; Paraf.. paraformaldehyde: cyto., cytoand £;Fig. 2, insert; 3C; Fig. 4£). plasmic staining: nucÃ-.,nuclear staining: Dcx, dexamethasone. Difference between the GR Localization in Steroid-sensitive ' Ethanol-acetic acid = 95% ethanol + 5% acetic acid. and Steroid-resistant Cell Lines: Nuclear Translocation. Table 2 summarizes the results of the relative staining intensity with the light microscope only but not at the electron microscope;5 glucocorticoid receptor antibody in various cell lines. It is clear however the quality of these preparations may vary depending that all cell lines studied contain glucocorticoid receptor im on the cytocentrifugation parameters (speed, time, handling). munoreactivity, although some of them are resistant to gluco Finally, unlike other systems, e.g., cell surface markers, stain corticoid action. In general 4R4 and 3R43 cells showed weaker ing of the glucocorticoid receptor requires prefixing of all cells staining than C7 cells. Although ICR27 cells contain very few before centrifugation in order to avoid loss of antigen and glucocorticoid binding sites, they were clearly immunologically deformation of cell morphology. When unfixed cells were cypositive. Next, we studied whether glucocorticoid has an effect tospun then postfixed, neither the staining nor the morphology on the subcellular distribution of GR. We had previously re was satisfactory (Table 1 and Fig. 3). ported a striking increase in nuclear staining in vivo of rat liver Effect of Cell Permeabilization. Aldehyde fixation is known and pituitary cells following adrenalectomy and treatment with to result in a tight cross-linking of cellular proteins; conse glucocorticoid (21 ). We observed a slight but detectable increase quently, antibody penetration must be facilitated by opening in nuclear staining of glucocorticoid-treated CEM-C7 cells, "holes" into the cellular membranes. We have previously estab particularly in paraformaldehyde-fixed cell suspensions and lished an experimental protocol for performing immunocytoBouin-fixed, paraffin-embedded preparations (not shown). In chemistry on paraformaldehyde-fixed whole monolayer cell preliminary experiments, the striking glucocorticoid effect ob cultures using ethanol as a permeabilizing agent (30). In con served in the rat in vivo was not as dramatic in CEM cells in trast to permeabilization using detergents such as Triton, the vitro. At first, we attributed this difference to the in vitro model ultrastructure and cell surface morphology (scanning electron microscopy) of ethanol-permeabilized cells is remarkably well system. However, after attempting numerous types of cell prep arations, fixatives and permeabilization agents, discussed preserved. In the present study we compared permeabilization above, we were able to detect a significant increase in nuclear by ethanol (50% 2 min, 70% 2 min); 0.1, 0.2, 0.5%, 1% Triton staining of glucocorticoid treated CEM C7 cytospun BouinX-100; 0.5% saponin; or 0.2% Nonidet P-40. The most con fixed cells (Fig. 4). This nuclear "translocation" phenomenon sistent and reproducible staining was obtained with the ethanol seen in C7 (glucocorticoid-sensitive) cells but not in the gluco5T. Antakly et al., unpublished data. corticoid-resistant cells 4R4, 3R43, or ICR27 (Fig. 4). Cl cells 1339 Table I Effect affixation on immunocvtochemical staining of(}R in CEM C7 cells Stain Morpholintensity ogy" Fixation Localization Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research. GLUCOCORTICOID RECEPTOR IN HUMAN LEUKEMIC CELLS Fig. 1. Immunoperoxidase localization of glucocorticoid receptor in CEM-C7 (A and and B) and CEM-C1 (C) cells prefixed with 4% paraformaldehyde and stained in suspen sion with antiserum 882 (1/100) (A and C) or with the monoclonal antibody (0.2 mg protein per ml). D. control preparation where the antiscrum was preabsorbed with pu rified glucocorticoid receptor extracted from C7 cells (24). Preabsorbed conditions were as described previously (21). /.. control in which the monoclonal antibody was replaced by culture medium from nonimmune hybridoma cells (0.2 mg protein/ml). These intact cells were photographed in suspension. In A, B, and C, note strong staining in the cytoplasm and a weaker one in the nucleus. Some hetero geneity in staining intensity among cells is observed, a few cells are weakly or even unstained. No staining is observed in D and E. Bar. lOjim. also showed nuclear translocation (Table 2). In addition, it is interesting to note that in general, at high glucocorticoid recep tor antibody concentrations from 1/100 to 1/200, where the staining intensity is very high it was sometimes difficult to see difference between cells incubated in the presence or absence of dexamethasone, cortisone, or cortisol acetate at either of the concentrations tested. Heterogeneity of Glucocorticoid Receptor Expression. Within any given cell preparation, the intensity of staining was variable among individual cells. Although heterogeneity is a character istic of a cell population and of neoplastic cells in particular (18, 35) we are investigating glucocorticoid receptor gene expression in individual cells using /// situ hybridization to a cloned glucocorticoid receptor cRNA probe.6 Our preliminary results show that the GR mRNA labeling intensity was variable among individual cells. In another set of experiments we subcloned wild type CEM-C7 using soft agar technique described earlier (36). Four subclones, generated from individual cells, were processed for immunocytochemistry or in situ hybridiza tion of the glucocorticoid receptor as described above. Results (not shown) demonstrated a heterogeneity of the labeling com parable to the C7 wild type (Figs. 1-4). DISCUSSION Technical Aspects of GR Localization. In the present study, we developed a reliable method for the immunocytochemical 6 T. Antakly and D. O'Donnell, unpublished data. staining of the glucocorticoid receptor in a well-established model system of human leukemic cells. The difficulty of proc essing such cells for immunocytochemistry lies in the fact that they grow as suspensions. In the present experiments, we dem onstrated that the most suitable and reproducible method for light microscopy is fixation of the cells in Bouin's solution while in suspension; then adhering them to glass slides by cytocentrifugation. Such slides can be easily processed through out the immunocytochemical procedure. This procedure would be readily adaptable to cells obtained from peripheral blood or marrow. As contrasted to formaldehyde fixation, the use of Bouin's solution avoids the need for permeabilization agents. If electron microscopic localization is desired, formaldehyde fixation is necessary to preserve the ultrastructural morphol ogy.5 In the case of formaldehyde fixation our results herein and in monolayer cultures of epithelial cells (30) show that permeabilization with ethanol is sufficient for antibody pene tration to various cell compartments, including the nucleus. Furthermore, this mild ethanol treatment produced minimal artifacts in cell morphology. A number of immunocytochemical studies, particularly on surface antigens, used unfixed cells for cytocentrifugation followed by postfixation in ethanol-acetic acid. When this method was followed for the GR, staining artifacts were observed, presumably due to the extreme flatten ing of the unfixed cells which resulted in a thin rim of cytoplasm pushed to the periphery. We observed a narrow band of cyto plasm which was stained moderately, plus little if any nuclear 1340 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research. GLUCOCORTICOID RECEPTOR IN HUMAN LEUKEMIC CELLS Fig. 2. Immunofiuorcscence localization of the glucocorticoid receptor in C7 cells cultured in the presence of 5*7 fetal bovine serum, prefixed in 2rr paraformaldehyde and stained in suspension with antibody 202. 1:200. The cell cytoplasm of most but not all cells is labeled, some nuclei are also labeled. Insert, a control preparation stained with preimmune serum at an identical dilution. Bar. 10 .in. staining (presumably due to poor penetration of antibody) (Fig. 3D). A general conclusion from this study was that, in lymphoid cells, fixation is necessary to maintain native cell morphology and probably to stabilize antigens. Such a conclusion regarding the need for fixation has been reached in a number of studies on the GR and estrogen receptor in a variety of cell types (13, 14, 21, 22, 33, 37, 38). In previous studies we demonstrated GR staining in histológica! sections of Bouin-fixed paraffin- embedded rat tissues (21 ). This method was successfully applied herein to the CEM cell line. However, we noted two drawbacks; the signal is weaker and the morphology is less well defined, particularly the cytoplasm. The disadvantage of paraffin sec tions as compared to unembedded cells (suspensions or cytospins) is probably due to the physical difficulty in obtaining good quality sections of tissue pellet from dispersed (8-10 /^m) lymphoid cells and to antigen denaturation during paraffin embedding. The main advantage of paraffin sections remains that lymphoid cells previously obtained from leukemia or lymphoma patients and processed for pathology, can be used for the immunocytochemical staining of GR. Thus, the patients can be studied retrospectively. Physiological Relevance of GR Localization. Previous immunochemical studies have shown that in several nonlymphoid cells and tissues, the level of the GR in the nucleus increases following glucocorticoid treatment (22, 25, 39,40). In addition, using a monoclonal antibody, Robertson et al., showed a similar localization of glucocorticoid receptor in CEM-C7 cells (25). The present study used a variety of fixation and cell preparation procedures, as well as monoclonal and polyclonal antibodies, to demonstrate that the glucocorticoid receptor in the human lymphoid CEM-C7 cells is localized largely in the cytoplasm and that following glucocorticoid treatment the GR level is increased in the nucleus. This partial nuclear translocation by immunocytochemistry corresponds well to the biochemical data: CEM-C7 cells show only 40% nuclear "translocation" by the classical method of physically separating nuclei and cyto plasm after administration of radioligand to whole cells (7, 8, 43). Moreover, we obtained identical localization using anti bodies to a synthetic peptide in the amino terminus of the GR (23). Thus, the present results are in full agreement with pre vious studies on rat liver and pituitary paraffin sections (30), rat brain vibratome sections (40), rat monolayer pituitary cells,6 pH Fig. 3. Effect of changing no. of fixation conditions and the changing the pH of the staining media on the immunoperoxidase lo calization of the glucocorticoid receptor in C7 cells. The cells were adhered to glass slides using a cytocentrifuge (cytospun). The cells were either prefixed in 2ri paraformaldehyde (A-C). or unfixed (D) prior to cytospin. Only in £>. the cells were postfixed in 95r¿methanol5% acetic acid following cytospin. Cell prepa rations were stained with antiserum 882 1/200 (A. B, 1)) or preimmune serum 1/200 (C). In B. the cells were stained in diaminobenzidine at pH 4.0 instead of the usual pH 7.6. this resulted in an almost exclusive nuclear stain ing. ßar(forall micrographs), 10 ¿im. pH 4-0 7.6 © © 1341 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research. GLUCOCORTICOID RECEPTOR IN HUMAN LEUKEMIC CELLS C7 dex © © control © Fig. 4. Effect of dexamethasone on the immunoperoxidase staining glucocorticoid receptor in the steroid-sensitive C7 cells (I, B) or in the steroid-resistant 4R4 cells (C, D). The cells were incubated for 30 min at 37'C with: A, C. the vehicle or B. D dexamethasone. The cells were subsequently fixed in Bouin and cytocentrifuged before being processed for immunocytochemistry with antibody 882 1:4000. The immunocytochemical staining is brown. The cells were finally counterstained with méthylène blue in order to distinguish cell morphology and enhance color contrast. Note an increase in the brown staining of the nuclei of cells exposed to dexamethasone in C7 but not 4R4 cells. E. is a control preparation in which the primary antibody was substituted by nonimmune immunoglobulin (0.2 mg/ml). F. is an immunocytochemical control in which preimmune serum 1:4000 was used. Bar, 10 ,<m. 1342 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research. GLUCOCORTICOID RECEPTOR IN HUMAN LEl'KEMIC CELLS glucocorticoid receptor is present in two types of steroid resist ant subclones act1 (4R4 and 3R43) and r~ cells, but show no nuclear translocation. Biochemically, this seems to be due to a defect whereby the receptor loses its steroid-binding capability under activating conditions (8, 9). It is also noteworthy that the act1 clone 4R4, shown in Fig. 5, displays a lower intensity of staining, corresponding to its lower level of binding sites and GR mRNA (8, 45). In the case of the "receptorless" (r~) cells, Fig. 5. Immunocytochemical localization of the T-antigen in Cos cells previ ously infected with SV40 virus. Cells were fixed as in Fig. 1. Immunocytochemistry was done as in the case of GR except that antibody to T-antigen was used. This is a phase-contrast micrograph. Note that the staining is exclusively in nuclei. Bar. 10 pm. Table 2 Subcellular localization and (jR staining intensity in CEM cell lines of [3H1Cell dexamethasom sites"14,0001 lineC7Cl4R43R43ICR27No. localizationintensity steroid++++ (—) Largely cyto-plasmic weaknuclear+++ 2.0005.0003,000±Same++ Same++ Same+++ or< none 1000Subccllular Same(+) steroidCytoplasmic, increasednuclearCytoplasmic, increasednuclearCytoplasmic. increasein no nuclearCytoplasmic, increasein no nuclearCytoplasmic. increasein no nuclear " Values taken from Ref. 7. rat hepatoma and human carcinoma monolayers (22). All these studies converge to the conclusion that in most cell types in the absence of steroid, the glucocorticoid receptor is largely cytoplasmic, and that at least part of it translocates to the nucleus following exposure to glucocorticoid. Our study shows that it is specifically so in leukemic cells. This conclusion is further reinforced by molecular genetic studies obtained from transfected cells which transiently express high levels of GR; in this case, GR translocation was observed following glucocorticoid treatment (41). The localization of the glucocorticoid receptor is therefore different from the reported exclusive nuclear local ization of the estrogen and progesterone receptors even in the absence of steroid (42). The nuclear translocation shown here in CEM-C1 cells which though in full agreement with biochemical data (7, 43) may seem unexpected since CEM-C1 cells are in part resistant to glucocorticoid lysis. However, the CEM-C1 cells show glutamine synthetase induction by glucocorticoid (7). Since glutamine synthetase induction occurs at the transcriptional level, one would expect to find glucocorticoid receptors in the nucleus. The observation that CEM-C1 cells are not lysed at short-term interval (24 h) treatment with dexamethasone strongly sug gested that these cells are altered in one or more nonreceptor steps of the lysis pathway (7). Recent studies6 have shown that Cl are growth arrested after longer periods of incubation with dexamethasone (>4 days). While the presence of glucocorticoid receptor is a prerequisite for glucocorticoid response (44), we find that immunoreactive ICR 27, our finding that they contain immunoreactive receptor [reported here and published as an abstract (46)] was subse quently confirmed in two papers published while this manu script was in revision (45, 47). In addition, ICR 27 cells were shown to contain glucocorticoid receptor mRNA in comparable quantity to wild type C7 cells (47). Thus, the data herein again agree with and support the biochemical findings. Although somatic cell hybrids have shown this is not a dominant phenotype (48) there is still a possibility that the abnormal receptor behavior may be due to abnormal association with Cytoplasmic factors (49-55) causing sequestration there. In this context, there is evidence that, in the cytoplasm, GR can bind to the heat-shock protein (hsp 90) from which it presumably disso ciates before entering the nucleus (49-54). Furthermore, the glucocorticoid antagonist RU 486 was shown to block gluco corticoid action by preventing the hsp 90 from dissociating from the GR (53, 54). Other cystolic factors could also inhibit the activation of GR. In recent studies (55), an inhibitor of GR activation which stabilizes the steroid binding ability of the unoccupied GR has been purified. Interestingly, the lack of expression of immunoglobulin K chain was recently shown to be associated with sequestering of a specific nuclear /raws-acting factor in the cytoplasm of nonexpressing cells (56). It is also possible that loss of steroid binding causes failure of the GR to interact with nuclear pores. Transport of nuclear proteins, such as nucleoplasmin and SV40 large-T involve two steps; binding to nuclear pore elements by specific amino acid signal sequences and translocation through nuclear pores (57, 58). One of us have demonstrated glucocorticoid-dependent presence of GR on nuclear envelope preparations in vivo (59) suggesting that translocation of GR to the nucleus also involves interaction with nuclear envelope elements. Interestingly, the GR and other steroid receptors bear homology to the plasminogen and largeT nuclear location signals (60, 6 1). Mutations in the GR nuclear location signal region were shown to result in an absence of GR nuclear translocation (41). Clone ICR-27 represents a different case. It is grossly defi cient in glucocorticoid receptor binding (9), but contains unex pectedly high levels of GR immunoreactive protein and GR mRNA (45, 47). This is in agreement with the high level of Cytoplasmic immunocytochemical reaction in ICR-27 cells seen in this study, in an earlier communication (46) and on flow cytometry (18). Taken together, the present data strongly sup port the view that bound ligand is involved in the process whereby the GR enters the nucleus. Our data are consistent with the studies of Becker et al. who showed the necessity of ligand in vivo for the effective binding of GR to GR element sites, and consequent gene induction (62). ACKNOWLEDGMENTS We thank Jeff Harmon for helpful discussions and for supplying additional amounts of the rabbit polyclonal antibody. We also thank John Cidlowski for a generous supply of human monoclonal antibody, Mike Gannon for initial technical help, and Donna Keays for typing the manuscript. 1343 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1990 American Association for Cancer Research. 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