Progress in Histo- and Cytochemistry, Vol. 26 W. Graumann / J. Drukker (Eds.), Histochemistry of Receptors © Fischer Verlag· Stuttgart· Jena . New York· 1992
2.5 Multi-assay performance of a monoclonal antibody directed against the androgen receptor TH. H. VAN DER KWAST, N.D.ZEGERS, G.JENSTER, W.BOERSMA, A.O.BRINKMANN,
J. TRAPMAN
Department of Pathology and Department of Endocrinology and Reproduction, Erasmus University Rotterdam (The Netherlands), Department of Immunology, TNO Medical Biological Laboratory, Rijswijk (The Netherlands)
Introduction Demonstration of androgen receptors (AR) has thus far relied on ligand binding techniques performed on tissue homogenates or else autoradiography using the specific radiolabeled ligand RI881 (e.g. GORELIC et al. 1987; PETERS and BARRACK 1987). Disadvantages of ligand binding methods performed on tissue homogenates are their sensitivity to proteolysis, their tendency to bind to other steroid binding proteins, and the impossibility of visualizing cellular heterogeneity for AR expression. In addition, these methods require that the ligand be dissociated from the ~eceptor prior to testing. In parallel with the development of immunocytochemical detection methods for other steroid receptors, several research groups have recently focussed their efforts on generating AR specific poly- and monoclonal antibodies that could be applied in various immunochemical assay systems (LUBAHN et al. 1988; CHANG et al. 1989; VAN LAAR et al. 1989; HUSSMAN et al. 1990). Inability to perform a large scale purification of the AR molecule due to its vulnerability to proteolysis and its low tissue content led us to employ the synthetic peptide (SP) strategy for purposes of antibody generation (ZEGERS et al. 1991). Following an alternative approach some laboratories, including our own, were also able to generate antibodies against fusion proteins where a large part of the molecule had been produced by recombinant DNA techniques (CHANG et al. 1989; VAN LAAR et al. 1989). As a consequence the amino acid sequence of the immunoreactive epitope is not known. Obviously, use of these strategies became feasible as a result of the elucidation of the AR cDNA sequence (LUBAHN et al. 1988; TRAPMAN et al. 1988; FABER et al. 1989). Steroid hormone receptors, including AR, consist of three domains: the N-terminal part which is involved in the regulation of gene transcription, a DNA binding domain and a steroid binding domain at the C-terminus (CARSON-JURICA et al. 1990). Interaction of steroid receptors with the specific ligand may lead to transformation of the AR molecule e. g. due to dimerization of AR molecules, conformational changes of the tertiary protein structure, phosphorylation of some amino acid residues, binding to specific DNA sites
62 . Th. H. van der Kwast et al.
and the dissociation of heat shock protein 90 (hsp90) (VAN LAAR et al. 1990). The effects of ligand binding on immunoreactivity using our epitope specific antibody was investigated with immunochemical methods, including immunocytochemistry.
Methods and results Generation of antibodies to SP61 Selection of SP61 was based on analysis of primary and secondary structure parameters such as the hydrophylic nature and expected flexibility of the hAR molecule (ZEGERS et al. 1991). The selected oligopeptide contained a stretch of 20 aminoacids that did not show homology with sequences of other steroid receptors. Homology of SP61 with sequences of other proteins could further be excluded by matching with protein sequences obtained from the protein sequence database (Protein Identification Resource, National Biomedical Research Foundation, Washington, D.C., USA). Immunization of rabbits and mice was performed with a conjugate of SP61 and keyhole limpet hemocyanin. In a direct ELISA using as testing agent SP61 conjugated to the carrier protein BSA and in an agarose precipitation assay antisera were evaluated for their immunoreactivity. Preimmune rabbit sera served as a negative control. After immunization of mice with a conjugate consisting of (glutaraldehyde activated) keyhole limpet hemocyanin and SP61, a fusion was performed of mouse spleen cells with SP2/0 plasmocytoma cells. Out of 768 hybridomas 107 SP61 binding clones were identified by ELISA. In the agarose precipitation assay 15 positive anti-hAR clones were selected. Only one of these clones, designated F39.4, showed the expected nuclear immunoreactivity with prostatic cells in immunohistochemistry (Fig. 1). Surprisingly, some of the hybrid om as produced antibodies with an unexpected immunohistochemical staining pattern (Fig. 2). Seven of the clones with the highest reactivity in immunoprecipitation assay were subcloned inlcuding the clone F39.4 (ZEGERS et al. 1991 ).
Immunohistochemistry The commonly used procedure for immunohistochemical demonstration of the estrogen receptor yielded optimal results. Particularly, incubation with chilled methanol was essential for obtaining an intense nuclear staining. Thus, frozen sections of 5 /lm thickness were fixed immediately for 10 min in 4% buffered formalin, rinsed in phosphate buffered saline and subsequently dehydrated in chilled methanol (4 min, -20°C) and acetone (2 min, -20°C). Sections were then transferred to PBS for 10 min. To minimize nonspecific binding of reagents in subsequent steps, sections were treated with normal goat serum or normal rabbit serum diluted 1: 10 in PBS for 15 min at room temperature. Incubations of the appropriate dilutions of the primary antibody were performed overnight at 4°C. Immunoreactivity was visualized with an indirect conjugated peroxidase method using diaminobenzidine as a substrate. As it was noted that the peroxidase antiperoxidase complex (PAP) yielded better results the latter method was also frequently employed for the visualization of the AR.
Monoclonal antibody against the androgen receptor . 63
Fig. 1. Immunostaining of prostatic tissue with a selective nuclear staining of stromal and epithelial cells by F39.4.
Fig. 2. Unexpected cytoplasmic staining of the basal epithelial cels of prostatic glands with an antibody reactive in ELISA with SP61.
64 . Th. H. van der Kwast et al.
Polyclonal antibodies directed against SP61 were raised in two rabbits. Without the requirement of absorption with putatively AR negative human tissues, both antisera exhibited a predominantly nuclear staining reaction of the secretory epithelial cells of the prostate and seminal vesicle, whereas virtually no reactivity was seen in lymphatic tissues (RUIZEVELD DE WINTER et al. 1989). Preimmune sera also yielded some nuclear staining if applied in high titers; but at dilutions used for immunohistochemical staining with the immune sera this rather faint but definite nuclear staining was diluted out. Using monoclonal antibody F39A, the immunoreactivity pattern of various human tissues was compared with previously reported biochemical ligand binding results obtained on these tissues. Male and female reproductive organs were tested as well as tissues from the digestive, respiratory and urinary tracts, lymphatic tissue, skeletal and myocardial muscle and endocrine organs. In all male and most female reproductive organs F39A immunostaining was found in both epithelial and stromal cells including smooth muscle cells. This staining was predominantly nuclear. Only in some prostatic glands was a dotted cytoplamic staining surrounding the nucleus occasionally seen (Fig. 3). Substantiation of the specificity of the reactivity of the polyclonal anti-AR antibodies as well as monoclonal antibody F39A with SP61 blocking experiments was performed either with the SP61 conjugate or with another AR eDNA derived synthetic peptide conjugate (SP60) as a control. Accordingly, appropriately fixed frozen sections of hyperplastic prostate tissue were incubated with mixtures of primary antibody and variable concentrations of the peptide conjugates. It was found that at a concentration of 0.25 ng SP-KLH/ml a specific blocking of the nuclear immunostaining reaction could be achieved.
Fig. 3. Prostatic ductular structures with perinuclear cytoplasmic staining reaction by F39.4.
Monoclonal antibody against the androgen receptor . 65
Specificity The hAR has a molecular weight of 110 kD. Western blot analysis showed that F39.4 is reactive with a protein (with an apparent size of 110 kD) in a lysate from the AR positive human prostate carcinoma cell line LNCaP. The specificity of F39.4 as a ligand was tested with goat anti-mouse immunoglobulin (GaM) agarose testing using nuclear extracts from LNCaP (androgen receptor), NHIK (glucocorticoid), MCF7 (estrogen receptor) and T47D (progesterone receptor), and radiolabeled R1881, dexamethasone, oestradiol, and RS020. Slight cross-reactivity with oestrogen receptors was seen only at a very high antibody concentration. No cross-reactivity was found with the ligand-bound native progesterone or glucocorticoid receptor (RUIZEVELD DE WINTER et al. 1991).
Effect of ligand binding on F39.4 immunoreactivity Since ligand binding may result in (conformational) changes of the AR molecule an investigation was made of whether ligand binding of the receptor would affect its immunoreactivity with F39.4. Several approaches were used: (l)The AR positive cell line LNCaP was cultured in the presence of the steroid R1881 or in culture medium containing charcoal stripped fetal calf serum; cytospin preparations of these cultured cells were then immunostained with F39.4. Only a slight decrease in immunoreactivity was seen in the LNCaP cells cultured in steroid deprived serum as compared with the androgen (R1881) stimulated culture. Previous experiments on AR RNA expression in androgen stimulated and androgen deprived LNCaP cells had indicated an increase rather than a decrease in AR mRNA after androgen depletion (TRAPMAN et al. 1990). (2) A series of transurethrally resected prostate cancer specimens obtained at the time of progression during androgen ablation therapy were examined with F39.4. Androgen ablation
ANDROGEN INDUCED NUCLEAR LOCALIZATION OF AR - R1881
AR constructs
N
DNA
N
c
+
R1881
c
Fig.4. Induction of nuclear localization of AR in COS cells transiently transfected with expression plasmid containing intact AR gene or AR gene with a deletion in DNA and hinge region.
66 . Th. H. van der Kwast et al.
therapy consisted of bilateral orchiectomy or endocrine therapy. In both cases, a high AR expression was fond in the majority of the tumors (VAN DER KWAST et al. 1991). (3) COS cells were transfected with expression plasmids containing the intact hAR gene or constructs of the AR gene lacking part of the DNA-binding domain and part of the hinge region. The transiently transfected COS cells were cultured for 2 days in the presence of R1881 or in medium containing steroid depleted fetal calf serum. It was found that, in the presence of R1881, F39.4 selectively stained the nuclei of transfected cells, while androgen depletion led to a cytoplasmic staining reaction in addition to (diminished) nuclear staining. Cells transfected with the DNA-domain deleted hAR expression plasmid showed an exclusively cytoplasmic staining reaction in the absence of the ligand, but a nuclear reactivity could be induced by culturing the cells in the presence of R1881 GENSTER et al. 1991; see Fig. 4).
Discussion In this paper we showed that the SP immunization strategy led to the successful generation of a mouse monoclonal antibody exhibiting multi-assay specificity for the human AR. The immunohistochemical screening on cryostat sections of multiple tissues including the prostate proved necessary to eliminate hybridomas producing antibodies exhibiting unwanted immunohistochemical reactivity though they seemed to be specific in ELISA or GAM agarose assay. The specificity of F39.4 as well as rabbit antisera raised against SP61 in respect of the SP was eventually confirmed by the ability to eliminate reactivity by blocking with the specific SP. Furthermore, immunohistochemical analysis of multiple tissues revealed a staining pattern which was largely consistent with the results obtained previously with ligand binding methods (RUIZEVELD DE WINTER et al. 1991). In contrast to the ubiquitous nuclear localization of AR in some ductal structures in prostatic tissue an exclusively speckled perinuclear staining was observed (Fig. 3). Possibly no nuclear transport of the AR can take place in these cells. Transfection experiments showed that the nuclear localization of AR is dependent in part on the presence of the specific ligand GENSTER et al. 1991). In contrast to the results obtained with transfected COS cells a predominant cytoplasmic localization of AR was, however, not found in prostatic carcinoma tissues of patients under androgen ablation therapy (VAN DER KWAST et al. 1991). Interaction of the specific ligand with AR probably leads to phosphorylation and release of hsp 90 as well as dimerization of AR molecules. This may lead to an alteration in the exposure of epitopes. From our results it is apparent that immunoreactivity with F39.4 was not strongly influenced by interaction or depletion of the ligand. Observed variations in intensity of immunostaining in the presence or absence of androgens may therefore be considered as an effect of up- or downregulation of AR expression by androgen.
References BRINKMANN, A. 0., FABER, P. W., VAN ROOI], H. C. et al.: The human androgen receptor: domain structure, genomic organization and regulation of expression. - J. Steroid Biochem. 34, 307-310 (1989). CARSON-JURICA, M. A., SCHRADER, W. T., O'MALLEY, B. W.: Steroid receptor family: structure and functions. - Endocr. Rev. 11, 201-211 (1990).
Monoclonal antibody against the androgen receptor . 67 CHANG, c., CHODAK, G., SARAC, E., TAKEDA, H., LIAO, S.: Prostate androgen receptor: immunohistochemical localization and mRNA characterization. - J. Steroid Biochem. 34,311-313 (1989). FABER, P. W., KUIPER, G. G. J. M., VAN ROOI], H. C. J., VAN DER KORPUT, J. A. G. M., BRINKMANN, A. 0., TRAPMAN, J.: The N-terminal domain of the human androgen receptor is encoded by one, large exon. Mol. cell. Endocrinol. 61, 257-262 (1989). GORELIC, L. S., LAMM, D. L., RAMzy, I., RADWIN, H. M., SHAIN, S. A.: Androgen receptors in biopsy specimens of prostate adenocarcinoma. Heterogeneity of distribution and relation to prognostic significance of receptor measurements for survival of advanced cancer patients. - Cancer 60,211-219 (1987). JENSTER, G., VAN DER KORPUT, J. A. G. M., VAN VROONHOVEN, C. et al.: Domains of the human androgen receptor involved in steroid-binding, transcriptional activation and subcellular localization. Mol. Endrocrinol. 5, 1395-1404 (1991). LUBAHN, D. B., JOSEPH, D. R., SAR, M., TAN, J., HIGGS, H. N., LARSON, R. E., FRENCH, F. S., WILSON, E. M.: The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate. - Mol. Endocrinol. 2, 2165-1275 (1988). HUSSMAN, D. A., WILSON, C. M., MCPHAUL, M. J., TILLEY, W. D., WILSON, J. D.: Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. - Endocrinol. 126, 2359-2368 (1990). PETERS, C. A., BARRACK, E. R.: Androgen receptor localization in the human prostate: demonstration of heterogeneity using a new method of steroid receptor autoradiography. - J. Steroid Biochem. 27,533-541 (1987). RUIZEVELD DE WINTER, J. A., TRAPMAN, J., VERMEY, M., et al.: Androgen receptor expression in human tissues: An immunohistochemical study. J. Histochem. Cytochem. 39,927-936, 1991. VAN DER KWAST, TH. H., SCHALKEN, J., RUIZEVELD DE WINTER, J. A., et al. Androgen receptors in endocrinetherapy-resistant human prostate cancer. Int.-J. Cancer 48, 189-193 (1991). VAN LAAR, J. H., BERREVOETS, C. A., TRAPMAN, J., ZEGERS, N. D., BRINKMANN, A. 0.: Hormone-dependent androgen receptor phosphorylation is accompanied by receptor transformation in human lymph node carcinoma of the prostate cells. - J. bioI. Chern. 266, 3734-3738 (1991). VAN LAAR, J. H., VOORHORST-OGINK, M. M., ZEGERS, N. D., et al.. Characterization of polyclonal antibodies against the N-terminal domain of the human androgen receptor. - Mol. Cell. Endocrinol. 67, 29-39 (1989). ZEGERS, N. D., CLAASEN, E., NEELEN, c., et al. Epitope prediction and confirmation for the human androgen receptor: Generation of monoclonal antibodies for multi-assay performance following the synthetic peptide strategy. - Biochim. biophys. Acta (Arnst.) 1073, 23-32 (1991).
2.5 Multi-assay performance of a monoclonal antibody directed against the androgen receptor TH. H. VAN DER KWAST, N.D.ZEGERS, G.JENSTER, W.BOERSMA, A.O.BRINKMANN,
J. TRAPMAN
Department of Pathology and Department of Endocrinology and Reproduction, Erasmus University Rotterdam (The Netherlands), Department of Immunology, TNO Medical Biological Laboratory, Rijswijk (The Netherlands)
Introduction Demonstration of androgen receptors (AR) has thus far relied on ligand binding techniques performed on tissue homogenates or else autoradiography using the specific radiolabeled ligand RI881 (e.g. GORELIC et al. 1987; PETERS and BARRACK 1987). Disadvantages of ligand binding methods performed on tissue homogenates are their sensitivity to proteolysis, their tendency to bind to other steroid binding proteins, and the impossibility of visualizing cellular heterogeneity for AR expression. In addition, these methods require that the ligand be dissociated from the ~eceptor prior to testing. In parallel with the development of immunocytochemical detection methods for other steroid receptors, several research groups have recently focussed their efforts on generating AR specific poly- and monoclonal antibodies that could be applied in various immunochemical assay systems (LUBAHN et al. 1988; CHANG et al. 1989; VAN LAAR et al. 1989; HUSSMAN et al. 1990). Inability to perform a large scale purification of the AR molecule due to its vulnerability to proteolysis and its low tissue content led us to employ the synthetic peptide (SP) strategy for purposes of antibody generation (ZEGERS et al. 1991). Following an alternative approach some laboratories, including our own, were also able to generate antibodies against fusion proteins where a large part of the molecule had been produced by recombinant DNA techniques (CHANG et al. 1989; VAN LAAR et al. 1989). As a consequence the amino acid sequence of the immunoreactive epitope is not known. Obviously, use of these strategies became feasible as a result of the elucidation of the AR cDNA sequence (LUBAHN et al. 1988; TRAPMAN et al. 1988; FABER et al. 1989). Steroid hormone receptors, including AR, consist of three domains: the N-terminal part which is involved in the regulation of gene transcription, a DNA binding domain and a steroid binding domain at the C-terminus (CARSON-JURICA et al. 1990). Interaction of steroid receptors with the specific ligand may lead to transformation of the AR molecule e. g. due to dimerization of AR molecules, conformational changes of the tertiary protein structure, phosphorylation of some amino acid residues, binding to specific DNA sites
62 . Th. H. van der Kwast et al.
and the dissociation of heat shock protein 90 (hsp90) (VAN LAAR et al. 1990). The effects of ligand binding on immunoreactivity using our epitope specific antibody was investigated with immunochemical methods, including immunocytochemistry.
Methods and results Generation of antibodies to SP61 Selection of SP61 was based on analysis of primary and secondary structure parameters such as the hydrophylic nature and expected flexibility of the hAR molecule (ZEGERS et al. 1991). The selected oligopeptide contained a stretch of 20 aminoacids that did not show homology with sequences of other steroid receptors. Homology of SP61 with sequences of other proteins could further be excluded by matching with protein sequences obtained from the protein sequence database (Protein Identification Resource, National Biomedical Research Foundation, Washington, D.C., USA). Immunization of rabbits and mice was performed with a conjugate of SP61 and keyhole limpet hemocyanin. In a direct ELISA using as testing agent SP61 conjugated to the carrier protein BSA and in an agarose precipitation assay antisera were evaluated for their immunoreactivity. Preimmune rabbit sera served as a negative control. After immunization of mice with a conjugate consisting of (glutaraldehyde activated) keyhole limpet hemocyanin and SP61, a fusion was performed of mouse spleen cells with SP2/0 plasmocytoma cells. Out of 768 hybridomas 107 SP61 binding clones were identified by ELISA. In the agarose precipitation assay 15 positive anti-hAR clones were selected. Only one of these clones, designated F39.4, showed the expected nuclear immunoreactivity with prostatic cells in immunohistochemistry (Fig. 1). Surprisingly, some of the hybrid om as produced antibodies with an unexpected immunohistochemical staining pattern (Fig. 2). Seven of the clones with the highest reactivity in immunoprecipitation assay were subcloned inlcuding the clone F39.4 (ZEGERS et al. 1991 ).
Immunohistochemistry The commonly used procedure for immunohistochemical demonstration of the estrogen receptor yielded optimal results. Particularly, incubation with chilled methanol was essential for obtaining an intense nuclear staining. Thus, frozen sections of 5 /lm thickness were fixed immediately for 10 min in 4% buffered formalin, rinsed in phosphate buffered saline and subsequently dehydrated in chilled methanol (4 min, -20°C) and acetone (2 min, -20°C). Sections were then transferred to PBS for 10 min. To minimize nonspecific binding of reagents in subsequent steps, sections were treated with normal goat serum or normal rabbit serum diluted 1: 10 in PBS for 15 min at room temperature. Incubations of the appropriate dilutions of the primary antibody were performed overnight at 4°C. Immunoreactivity was visualized with an indirect conjugated peroxidase method using diaminobenzidine as a substrate. As it was noted that the peroxidase antiperoxidase complex (PAP) yielded better results the latter method was also frequently employed for the visualization of the AR.
Monoclonal antibody against the androgen receptor . 63
Fig. 1. Immunostaining of prostatic tissue with a selective nuclear staining of stromal and epithelial cells by F39.4.
Fig. 2. Unexpected cytoplasmic staining of the basal epithelial cels of prostatic glands with an antibody reactive in ELISA with SP61.
64 . Th. H. van der Kwast et al.
Polyclonal antibodies directed against SP61 were raised in two rabbits. Without the requirement of absorption with putatively AR negative human tissues, both antisera exhibited a predominantly nuclear staining reaction of the secretory epithelial cells of the prostate and seminal vesicle, whereas virtually no reactivity was seen in lymphatic tissues (RUIZEVELD DE WINTER et al. 1989). Preimmune sera also yielded some nuclear staining if applied in high titers; but at dilutions used for immunohistochemical staining with the immune sera this rather faint but definite nuclear staining was diluted out. Using monoclonal antibody F39A, the immunoreactivity pattern of various human tissues was compared with previously reported biochemical ligand binding results obtained on these tissues. Male and female reproductive organs were tested as well as tissues from the digestive, respiratory and urinary tracts, lymphatic tissue, skeletal and myocardial muscle and endocrine organs. In all male and most female reproductive organs F39A immunostaining was found in both epithelial and stromal cells including smooth muscle cells. This staining was predominantly nuclear. Only in some prostatic glands was a dotted cytoplamic staining surrounding the nucleus occasionally seen (Fig. 3). Substantiation of the specificity of the reactivity of the polyclonal anti-AR antibodies as well as monoclonal antibody F39A with SP61 blocking experiments was performed either with the SP61 conjugate or with another AR eDNA derived synthetic peptide conjugate (SP60) as a control. Accordingly, appropriately fixed frozen sections of hyperplastic prostate tissue were incubated with mixtures of primary antibody and variable concentrations of the peptide conjugates. It was found that at a concentration of 0.25 ng SP-KLH/ml a specific blocking of the nuclear immunostaining reaction could be achieved.
Fig. 3. Prostatic ductular structures with perinuclear cytoplasmic staining reaction by F39.4.
Monoclonal antibody against the androgen receptor . 65
Specificity The hAR has a molecular weight of 110 kD. Western blot analysis showed that F39.4 is reactive with a protein (with an apparent size of 110 kD) in a lysate from the AR positive human prostate carcinoma cell line LNCaP. The specificity of F39.4 as a ligand was tested with goat anti-mouse immunoglobulin (GaM) agarose testing using nuclear extracts from LNCaP (androgen receptor), NHIK (glucocorticoid), MCF7 (estrogen receptor) and T47D (progesterone receptor), and radiolabeled R1881, dexamethasone, oestradiol, and RS020. Slight cross-reactivity with oestrogen receptors was seen only at a very high antibody concentration. No cross-reactivity was found with the ligand-bound native progesterone or glucocorticoid receptor (RUIZEVELD DE WINTER et al. 1991).
Effect of ligand binding on F39.4 immunoreactivity Since ligand binding may result in (conformational) changes of the AR molecule an investigation was made of whether ligand binding of the receptor would affect its immunoreactivity with F39.4. Several approaches were used: (l)The AR positive cell line LNCaP was cultured in the presence of the steroid R1881 or in culture medium containing charcoal stripped fetal calf serum; cytospin preparations of these cultured cells were then immunostained with F39.4. Only a slight decrease in immunoreactivity was seen in the LNCaP cells cultured in steroid deprived serum as compared with the androgen (R1881) stimulated culture. Previous experiments on AR RNA expression in androgen stimulated and androgen deprived LNCaP cells had indicated an increase rather than a decrease in AR mRNA after androgen depletion (TRAPMAN et al. 1990). (2) A series of transurethrally resected prostate cancer specimens obtained at the time of progression during androgen ablation therapy were examined with F39.4. Androgen ablation
ANDROGEN INDUCED NUCLEAR LOCALIZATION OF AR - R1881
AR constructs
N
DNA
N
c
+
R1881
c
Fig.4. Induction of nuclear localization of AR in COS cells transiently transfected with expression plasmid containing intact AR gene or AR gene with a deletion in DNA and hinge region.
66 . Th. H. van der Kwast et al.
therapy consisted of bilateral orchiectomy or endocrine therapy. In both cases, a high AR expression was fond in the majority of the tumors (VAN DER KWAST et al. 1991). (3) COS cells were transfected with expression plasmids containing the intact hAR gene or constructs of the AR gene lacking part of the DNA-binding domain and part of the hinge region. The transiently transfected COS cells were cultured for 2 days in the presence of R1881 or in medium containing steroid depleted fetal calf serum. It was found that, in the presence of R1881, F39.4 selectively stained the nuclei of transfected cells, while androgen depletion led to a cytoplasmic staining reaction in addition to (diminished) nuclear staining. Cells transfected with the DNA-domain deleted hAR expression plasmid showed an exclusively cytoplasmic staining reaction in the absence of the ligand, but a nuclear reactivity could be induced by culturing the cells in the presence of R1881 GENSTER et al. 1991; see Fig. 4).
Discussion In this paper we showed that the SP immunization strategy led to the successful generation of a mouse monoclonal antibody exhibiting multi-assay specificity for the human AR. The immunohistochemical screening on cryostat sections of multiple tissues including the prostate proved necessary to eliminate hybridomas producing antibodies exhibiting unwanted immunohistochemical reactivity though they seemed to be specific in ELISA or GAM agarose assay. The specificity of F39.4 as well as rabbit antisera raised against SP61 in respect of the SP was eventually confirmed by the ability to eliminate reactivity by blocking with the specific SP. Furthermore, immunohistochemical analysis of multiple tissues revealed a staining pattern which was largely consistent with the results obtained previously with ligand binding methods (RUIZEVELD DE WINTER et al. 1991). In contrast to the ubiquitous nuclear localization of AR in some ductal structures in prostatic tissue an exclusively speckled perinuclear staining was observed (Fig. 3). Possibly no nuclear transport of the AR can take place in these cells. Transfection experiments showed that the nuclear localization of AR is dependent in part on the presence of the specific ligand GENSTER et al. 1991). In contrast to the results obtained with transfected COS cells a predominant cytoplasmic localization of AR was, however, not found in prostatic carcinoma tissues of patients under androgen ablation therapy (VAN DER KWAST et al. 1991). Interaction of the specific ligand with AR probably leads to phosphorylation and release of hsp 90 as well as dimerization of AR molecules. This may lead to an alteration in the exposure of epitopes. From our results it is apparent that immunoreactivity with F39.4 was not strongly influenced by interaction or depletion of the ligand. Observed variations in intensity of immunostaining in the presence or absence of androgens may therefore be considered as an effect of up- or downregulation of AR expression by androgen.
References BRINKMANN, A. 0., FABER, P. W., VAN ROOI], H. C. et al.: The human androgen receptor: domain structure, genomic organization and regulation of expression. - J. Steroid Biochem. 34, 307-310 (1989). CARSON-JURICA, M. A., SCHRADER, W. T., O'MALLEY, B. W.: Steroid receptor family: structure and functions. - Endocr. Rev. 11, 201-211 (1990).
Monoclonal antibody against the androgen receptor . 67 CHANG, c., CHODAK, G., SARAC, E., TAKEDA, H., LIAO, S.: Prostate androgen receptor: immunohistochemical localization and mRNA characterization. - J. Steroid Biochem. 34,311-313 (1989). FABER, P. W., KUIPER, G. G. J. M., VAN ROOI], H. C. J., VAN DER KORPUT, J. A. G. M., BRINKMANN, A. 0., TRAPMAN, J.: The N-terminal domain of the human androgen receptor is encoded by one, large exon. Mol. cell. Endocrinol. 61, 257-262 (1989). GORELIC, L. S., LAMM, D. L., RAMzy, I., RADWIN, H. M., SHAIN, S. A.: Androgen receptors in biopsy specimens of prostate adenocarcinoma. Heterogeneity of distribution and relation to prognostic significance of receptor measurements for survival of advanced cancer patients. - Cancer 60,211-219 (1987). JENSTER, G., VAN DER KORPUT, J. A. G. M., VAN VROONHOVEN, C. et al.: Domains of the human androgen receptor involved in steroid-binding, transcriptional activation and subcellular localization. Mol. Endrocrinol. 5, 1395-1404 (1991). LUBAHN, D. B., JOSEPH, D. R., SAR, M., TAN, J., HIGGS, H. N., LARSON, R. E., FRENCH, F. S., WILSON, E. M.: The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate. - Mol. Endocrinol. 2, 2165-1275 (1988). HUSSMAN, D. A., WILSON, C. M., MCPHAUL, M. J., TILLEY, W. D., WILSON, J. D.: Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. - Endocrinol. 126, 2359-2368 (1990). PETERS, C. A., BARRACK, E. R.: Androgen receptor localization in the human prostate: demonstration of heterogeneity using a new method of steroid receptor autoradiography. - J. Steroid Biochem. 27,533-541 (1987). RUIZEVELD DE WINTER, J. A., TRAPMAN, J., VERMEY, M., et al.: Androgen receptor expression in human tissues: An immunohistochemical study. J. Histochem. Cytochem. 39,927-936, 1991. VAN DER KWAST, TH. H., SCHALKEN, J., RUIZEVELD DE WINTER, J. A., et al. Androgen receptors in endocrinetherapy-resistant human prostate cancer. Int.-J. Cancer 48, 189-193 (1991). VAN LAAR, J. H., BERREVOETS, C. A., TRAPMAN, J., ZEGERS, N. D., BRINKMANN, A. 0.: Hormone-dependent androgen receptor phosphorylation is accompanied by receptor transformation in human lymph node carcinoma of the prostate cells. - J. bioI. Chern. 266, 3734-3738 (1991). VAN LAAR, J. H., VOORHORST-OGINK, M. M., ZEGERS, N. D., et al.. Characterization of polyclonal antibodies against the N-terminal domain of the human androgen receptor. - Mol. Cell. Endocrinol. 67, 29-39 (1989). ZEGERS, N. D., CLAASEN, E., NEELEN, c., et al. Epitope prediction and confirmation for the human androgen receptor: Generation of monoclonal antibodies for multi-assay performance following the synthetic peptide strategy. - Biochim. biophys. Acta (Arnst.) 1073, 23-32 (1991).
