SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2078603739> ?p ?o ?g. }

Showing items 1 to 100 of ±103 with 100 items per page.

W2078603739 endingPage "15979" @default.
W2078603739 startingPage "15973" @default.
W2078603739 abstract "Promoter interference assay was employed to examine in intact cells the roles of the functional domains of androgen receptor (AR) and the ligand for specific DNA interactions using a cytomegalovirus-(androgen response element)-chloramphenicol acetyltransferase reporter (pCMV-ARE2-CAT). Native rat and human ARs interfered with pCMV-ARE2-CAT expression in a hormone-dependent fashion. Low steroid-independent interference seemed to occur because of the ligand binding domain (LBD), which was transcriptionally inhibitory also in a heterologous context. AR devoid of LBD (rARΔ641–902) decreased pCMV-ARE2-CAT activity by 50%. The rARΔ46–408 mutant devoid of the NH2-terminal transcription activation region exhibited ligand-dependent promoter interference of a similar magnitude. Ligand and DNA binding-deficient mutants (hARM807R and rARC562G, respectively) did not influence pCMV-ARE2-CAT expression, although hARM807R binds to ARE in vitro. Non-steroidal anti-androgens casodex and hydroxyflutamide antagonized agonist-dependent promoter interference, whereas cyproterone acetate, RU 56187, RU 57073, and RU 59063 were partial agonists/antagonists. Collectively, interaction of ARs with ARE in intact cells does not require the presence of the COOH-terminal or NH2-terminal domain and/or their interaction. In the context of native AR, however, the androgen-induced conformational change in LBD is mandatory for generation of a transcriptionally competent receptor that binds to DNA in intact cells. Promoter interference assay was employed to examine in intact cells the roles of the functional domains of androgen receptor (AR) and the ligand for specific DNA interactions using a cytomegalovirus-(androgen response element)-chloramphenicol acetyltransferase reporter (pCMV-ARE2-CAT). Native rat and human ARs interfered with pCMV-ARE2-CAT expression in a hormone-dependent fashion. Low steroid-independent interference seemed to occur because of the ligand binding domain (LBD), which was transcriptionally inhibitory also in a heterologous context. AR devoid of LBD (rARΔ641–902) decreased pCMV-ARE2-CAT activity by 50%. The rARΔ46–408 mutant devoid of the NH2-terminal transcription activation region exhibited ligand-dependent promoter interference of a similar magnitude. Ligand and DNA binding-deficient mutants (hARM807R and rARC562G, respectively) did not influence pCMV-ARE2-CAT expression, although hARM807R binds to ARE in vitro. Non-steroidal anti-androgens casodex and hydroxyflutamide antagonized agonist-dependent promoter interference, whereas cyproterone acetate, RU 56187, RU 57073, and RU 59063 were partial agonists/antagonists. Collectively, interaction of ARs with ARE in intact cells does not require the presence of the COOH-terminal or NH2-terminal domain and/or their interaction. In the context of native AR, however, the androgen-induced conformational change in LBD is mandatory for generation of a transcriptionally competent receptor that binds to DNA in intact cells. Androgen receptor (AR) 1The abbreviations used are: AR, androgen receptor; hAR, human AR; rAR, rat AR; ARE, androgen response element; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; DBD, DNA binding domain; ER, estrogen receptor; ERE, estrogen response element; LBD, ligand-binding domain; MMTV, mouse mammary tumor virus; Nterm, NH2-terminal region of rAR; PR, progesterone receptor; PRE, progesterone response element; SV, simian virus. belongs to the nuclear receptor superfamily of ligand-regulated transcription factors (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6110) Google Scholar, 2Beato M. Herrlich P. Schütz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1639) Google Scholar, 3Truss M. Beato M. Endocr. Rev. 1993; 14: 459-479Crossref PubMed Scopus (588) Google Scholar, 4Katzenellenbogen J.A. O'Malley B.W. Katzenellenbogen B.S. Mol. Endocrinol. 1996; 10: 119-131Crossref PubMed Scopus (526) Google Scholar). Although the interaction of AR with specific hormone-responsive DNA elements is usually required for androgen-dependent transcriptional activation, binding of AR to specific DNA motifs is not always necessary for the receptor's ability to down-regulate gene expression (5Kallio P.J. Poukka H. Moilanen A. Jänne O.A. Palvimo J.J. Mol. Endocrinol. 1995; 9: 1017-1028Crossref PubMed Google Scholar, 6Palvimo J.J. Reinikainen P. Ikonen T. Kallio P.J. Moilanen A. Jänne O.A. J. Biol. Chem. 1996; 271: 24151-24156Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 7Schneikert J. Peterzeil H. Defossez P.-A. Klocker H. de Launoit Y. Cato A.C.B. J. Biol. Chem. 1996; 271: 23907-23913Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). With regard to the ligand requirement for the recognition of specific DNA elements by AR, in vitroelectrophoretic mobility shift assays have yielded conflicting results. AR protein expressed in reticulocyte lysate or produced insect cells is capable of binding to specific androgen response elements (AREs)in vitro even in the absence of androgen (8Palvimo J.J. Kallio P.J. Ikonen T. Mehto M. Jänne O.A. Mol. Endocrinol. 1993; 7: 1399-1407PubMed Google Scholar, 9Kallio P.J. Palvimo J.J. Mehto M. Jänne O.A. J. Biol. Chem. 1994; 269: 11514-11522Abstract Full Text PDF PubMed Google Scholar); however, the ligand requirement for the binding of hAR to AREs in vitro has also been reported (10Wong C. Zhou Z. Sar M. Wilson E.M. J. Biol. Chem. 1993; 268: 19004-19012Abstract Full Text PDF PubMed Google Scholar). Very limited information is available on ARE occupancy by the receptor protein in intact cells.In vivo footprinting of an androgen-dependent enhancer of the mouse slp gene failed to reveal clear protection of hormone response elements by AR (11Scarlett C.O. Robins D.M. Mol. Endocrinol. 1995; 9: 413-423PubMed Google Scholar). While this work was in progress, Kuil and Mulder (12Kuil C.W. Mulder E. Endocrinology. 1996; 17: 1870-1877Crossref Scopus (24) Google Scholar) reported that AR interaction with an “idealized” consensus ARE derived from polymerase chain reaction selection experiments is ligand-dependent in cultured cells. The promoter interference assay developed by Hu and Davidson (13Hu M.C.-T. Davidson M. Cell. 1987; 48: 555-566Abstract Full Text PDF PubMed Scopus (163) Google Scholar) is based on competition of DNA-binding proteins, such as nuclear receptors, for binding with essential transcription factors driving a constitutively active heterologous promoter. The interference is achieved by inserting an attachment sequence between the TATA box and the start site of transcription in the promoter. A promoter interference assay has been employed in a few reports to examine the interaction of estrogen receptor (ER) with its cognate response elements in mammalian and frog cells (14Reese J.C. Katzenellenbogen B.S. Mol. Cell. Biol. 1992; 12: 4531-4538Crossref PubMed Scopus (109) Google Scholar, 15Xing H. Shapiro D.J. J. Biol. Chem. 1993; 268: 23227-23233Abstract Full Text PDF PubMed Google Scholar) and that of AR with AREs in Chinese hamster ovary cells (12Kuil C.W. Mulder E. Endocrinology. 1996; 17: 1870-1877Crossref Scopus (24) Google Scholar). Because of the use of transiently transfected cells, the physiological chromatin environment is not achieved under these conditions. However, using a pCMV-PRE2-CAT construct stably integrated into T47D breast cancer cells, Gass et al. (16Gass, L., Leonhardt, S., Altmann, M., and Edwards, P. (1996)Abstracts of the 10th International Congress of Endocrinology, San Francisco, June 12–15, abstract p2-682.Google Scholar) recently reported that progesterone receptor (PR) is able to interfere with expression of this reporter also in the context of stable chromatin conformation, albeit less well than in transiently transfected cells. In our previous studies, we constructed several NH2-terminal deletion mutants of AR to define domains crucial for the activation functions of this protein (5Kallio P.J. Poukka H. Moilanen A. Jänne O.A. Palvimo J.J. Mol. Endocrinol. 1995; 9: 1017-1028Crossref PubMed Google Scholar, 8Palvimo J.J. Kallio P.J. Ikonen T. Mehto M. Jänne O.A. Mol. Endocrinol. 1993; 7: 1399-1407PubMed Google Scholar, 9Kallio P.J. Palvimo J.J. Mehto M. Jänne O.A. J. Biol. Chem. 1994; 269: 11514-11522Abstract Full Text PDF PubMed Google Scholar). One of the mutants, rARΔ46–408, devoid of a region mandatory for transcriptional activation (8Palvimo J.J. Kallio P.J. Ikonen T. Mehto M. Jänne O.A. Mol. Endocrinol. 1993; 7: 1399-1407PubMed Google Scholar), bound to ARE in vitro with somewhat lower affinity than the wild-type AR (9Kallio P.J. Palvimo J.J. Mehto M. Jänne O.A. J. Biol. Chem. 1994; 269: 11514-11522Abstract Full Text PDF PubMed Google Scholar) but behaved as a dominant negative regulator of the native protein, possibly through forming transcriptionally inactive heterodimers with the latter (8Palvimo J.J. Kallio P.J. Ikonen T. Mehto M. Jänne O.A. Mol. Endocrinol. 1993; 7: 1399-1407PubMed Google Scholar, 9Kallio P.J. Palvimo J.J. Mehto M. Jänne O.A. J. Biol. Chem. 1994; 269: 11514-11522Abstract Full Text PDF PubMed Google Scholar). The ability of rARΔ46–408, or comparable mutants of the glucocorticoid receptor subfamily of nuclear receptors, to interact with specific DNA sequences in intact cells has, however, not been addressed previously. The COOH-terminal ligand binding domain (LBD) provides the means to regulate the function of native AR in a steroid-dependent fashion. Removal of this region generates a constitutively active receptor form (17Rundlett S.E. Wu X.-P. Miesfeld R.L. Mol. Endocrinol. 1990; 4: 708-714Crossref PubMed Scopus (109) Google Scholar, 18Jenster G. van der Korput H.A.G.M. van Vroonhoven C. van der Kwast T.H. Trapman J. Brinkmann A.O. Mol. Endocrinol. 1991; 5: 1396-1404Crossref PubMed Scopus (421) Google Scholar), whose activity is dependent on the promoter context. 2T. Ikonen, J. J. Palvimo, and O. A. Jänne, submitted for publication. However, in vivo DNA binding characteristics of LBD-deficient AR forms or those of other members of the glucocorticoid receptor subfamily have not been examined. In the present work we have used promoter interference assays to investigate whether binding of AR to specific DNA sequences is ligand-dependent in intact CV-1 cells. We have also examined which functional domains of the wild-type receptor, in addition to the DNA binding domain (DBD), are mandatory for specific DNA binding in vivo. To assess the role of the ligand, several non-steroidal and steroidal anti-androgens alone or together with an androgen agonist were used. Finally, immunocytochemical analysis of cultured cells was employed to rule out the possibility that some of the results were compromised by altered subcellular distribution of receptor proteins. Chemicals were obtained from Sigma, Bio-Rad, and Qiagen GmbH (Hilden, Germany). Testosterone was purchased from Makor Chemicals (Jerusalem, Israel). [3H]Acetyl-coenzyme A was obtained from DuPont NEN. DNA-modifying enzymes were purchased from Pharmacia Biotech Inc. and Promega Corp. (Madison, WI). Oligonucleotides were synthesized using Gene Assembler Plus (Pharmacia). Non-steroidal anti-androgens casodex ((2RS)-4′-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3′-(trifuoromethyl)propionanilide) and hydroxyflutamide (4-hydroxy-α,α,α-trifluoro-2-methyl-4′-nitro-m-propionotoluidide) were obtained from Zeneca Pharmaceuticals (Macclesfield, U. K.) and Schering Corp. (Bloomfield, NJ), respectively. Cyproterone acetate (6-chloro-1,2-methylene-17α-hydroxy-4,6-pregnadiene-3,20-dione acetate) was from Schering AG (Berlin, Germany), and RU 56187 (4-(5-oxo-2-thioxo-3,4,4-tri-methyl-1-imidazolidinyl)-2-trifluoromethylbenzonitrile), RU 57073 (4-[4,4-dimethyl-3-(2-hydroxyethyl)-5-oxo-2-thioxo-1-imidazolidinyl]-2-trifluoro-methylbenzonitrile) and RU 59063 (4-[4,4-dimethyl-3-(4-hydroxybutyl)-5-oxo-2-thioxo-1-imidazolidinyl]-2-trifluoromethylbenzonitrile) were donated by Dr. G. Teutsch (Roussel UCLAF, Romainville, France). pCMV-ARE2-CAT reporter was constructed from pCMV-0-CAT (a gift from Dr. B. Katzenellenbogen, University of Illinois, Urbana) by inserting a double-stranded 45-base pair oligonucleotide, 5′-CATAGTACGTGATGTTCTAGGCCTAGTACGTGATGTTCTCGAGCT-3′, containing duplicated high affinity AREs (half-sites underlined) of the C3(1) gene of prostatic binding protein (9Kallio P.J. Palvimo J.J. Mehto M. Jänne O.A. J. Biol. Chem. 1994; 269: 11514-11522Abstract Full Text PDF PubMed Google Scholar, 19De Vos P. Claessens F. Winderickx J. Van Dijck P. Celis L. Peeters B. Rombauts W. Heyns W. Verhoeven G. J. Biol. Chem. 1991; 266: 3439-3443Abstract Full Text PDF PubMed Google Scholar) into theSacI site between the TATA box and the transcription start site of pCMV-0-CAT. pCMV-ERE2-CAT was constructed in the same way except that the inserted 45-base pair oligomer contained two estrogen response elements in lieu of AREs (14Reese J.C. Katzenellenbogen B.S. Mol. Cell. Biol. 1992; 12: 4531-4538Crossref PubMed Scopus (109) Google Scholar). For transcriptional activation experiments, the reporters pARE2-tk-CAT and pMMTV-CAT were used (20Reinikainen P. Palvimo J.J. Jänne O.A. Endocrinology. 1996; 137: 4351-4357Crossref PubMed Scopus (67) Google Scholar). Expression vectors pSGrAR, pSGhAR, pSGrARC562G, pSGhARM807R, pSGrARΔ46–408, pSGrARΔ46–408/C562G, and pSGrARΔ641–9022 have been described previously (5Kallio P.J. Poukka H. Moilanen A. Jänne O.A. Palvimo J.J. Mol. Endocrinol. 1995; 9: 1017-1028Crossref PubMed Google Scholar,8Palvimo J.J. Kallio P.J. Ikonen T. Mehto M. Jänne O.A. Mol. Endocrinol. 1993; 7: 1399-1407PubMed Google Scholar, 21Adeyemo O. Kallio P.J. Palvimo J.J. Kontula K. Jänne O.A. Hum. Mol. Genet. 1993; 2: 1809-1812Crossref PubMed Scopus (37) Google Scholar, 22Ikonen T. Palvimo J.J. Kallio P.J. Reinikainen P. Jänne O.A. Endocrinology. 1994; 135: 1359-1366Crossref PubMed Scopus (155) Google Scholar). pG5SV40CAT containing five GAL4-binding elements upstream of the SV40 promoter and pSV40CAT control vector were gifts from Dr. N. Lehming (Max-Delbrück Laboratory, Max-Plack Institute, Cologne, Germany). GAL4-LBD and GAL-Nterm contained amino acids 641–902 and 5–538 of rAR, respectively, fused in-frame to Saccharomyces cerevisiae GAL4-DBD.2 CV-1 and COS-1 cells were obtained from American Type Culture Collection (Rockville, MD) and maintained in Dulbecco's modified Eagle's medium containing penicillin (25 units/ml) and streptomycin (25 units/ml) supplemented with 10% (v/v) fetal bovine serum (Life Technologies, Inc.). Twenty-six h before transfection, 1.5 × 106 cells were seeded on 10-cm plates. The medium was replaced 2–4 h before transfection with Dulbecco's modified Eagle's medium containing 10% charcoal-stripped fetal bovine serum. The calcium phosphate coprecipitation method was used for transfections as described previously (8Palvimo J.J. Kallio P.J. Ikonen T. Mehto M. Jänne O.A. Mol. Endocrinol. 1993; 7: 1399-1407PubMed Google Scholar). The total amount of DNA was 10 μg/10-cm plate and included the following components: 0.5–2.5 μg of receptor expression vector DNA (pSGrAR, pSGhAR, pSGrARC562G, pSGhARM807R, pSGrARΔ46–408, pSGrARΔ46–408/C562G, and pSGrARΔ641–902), 2 μg of pCMV-ARE2-CAT reporter (or pCMV-ERE2-CAT when indicated), 3 μg of β-galactosidase expression vector (pCH110, Pharmacia) as an internal control, and empty pSG5 DNA as needed. Ten μg of pGEM-3Z DNA was used in mock transfections. In transfections with pG5SV40CAT or pSV40CAT reporter (1 μg/10-cm plate), 5 μg of GAL4-DBD-LBD, GAL4-DBD-Nterm, or GAL4-DBD expression vector was used, and the amount of total DNA was adjusted to 10 μg with pBluescript II SK DNA (Stratagene, La Jolla, CA). Eighteen h after transfection, the cells were switched into fresh medium containing androgens and anti-androgens at concentrations specified in the legends to the figures and Table I. The cells were harvested 30 h later, and CAT and β-galactosidase activities were measured and normalized as described previously (23Eastman A. BioTechniques. 1987; 5: 731Google Scholar, 24Rosenthal N. Methods Enzymol. 1987; 152: 704-720Crossref PubMed Scopus (403) Google Scholar).Table IThe ability of different steroidal and nonsteroidal anti-androgens to confer ARE binding ability on AR as determined by promoter interference assayCompoundpCMV-ARE2-CAT activity%Vehicle85.5 ± 2.9Testosterone (10 nm)46.0 ± 3.5Casodex (1 μm)77.0 ± 3.1Hydroxyflutamide (1 μm)99.5 ± 6.1RU 56187 (1 μm)43.5 ± 4.0RU 57073 (1 μm)48.0 ± 1.5RU 59063 (1 μm)42.5 ± 1.9Cyproterone acetate (1 μm)40.0 ± 1.6CV-1 cells were transfected with 1 μg of pSGrARΔ46–408, 2 μg of pCMV-ARE2-CAT, 3 μg of pCH110 (β-galactosidase), and 4 μg of pSG5 vector DNA. Eighteen h after transfection, the cells received fresh medium with 10 nm testosterone or 1 μmanti-androgen and were collected 30 h later for the measurement of CAT and β-galactosidase activities. The values (mean ± S.E.,n = 3) are expressed relative to that in CV-1 cells transfected without pSGrARΔ46–408. Open table in a new tab CV-1 cells were transfected with 1 μg of pSGrARΔ46–408, 2 μg of pCMV-ARE2-CAT, 3 μg of pCH110 (β-galactosidase), and 4 μg of pSG5 vector DNA. Eighteen h after transfection, the cells received fresh medium with 10 nm testosterone or 1 μmanti-androgen and were collected 30 h later for the measurement of CAT and β-galactosidase activities. The values (mean ± S.E.,n = 3) are expressed relative to that in CV-1 cells transfected without pSGrARΔ46–408. CV-1 cells were seeded on glass slips on 10-cm plastic plates and were transfected with 1 or 2.5 μg of pSG5r/hAR DNA, 2 μg of pCMV-ARE2-CAT, 3 μg of pCH110 and filled to 10 μg of total DNA with empty pSG5. Replacement of medium and exposure to steroids were conducted as in the promoter interference assay. Cells were fixed in methanol at −20 °C for 10 min and washed three times with phosphate-buffered saline (150 mm NaCl, 20 mm Na2HPO4, pH 7.3) at 22 °C for 10 min. The primary antibody K183 (diluted 1:200) is a polyclonal rabbit antiserum raised against full-length rAR purified to homogeneity using preparative polyacrylamide gel electrophoresis under denaturing conditions from Sf9 insect cells infected with a recombinant baculovirus encoding rAR (25Xie Y.-B. Sui Y.-P. Shan L.-X. Palvimo J.J. Phillips D.M. Jänne O.A. J. Biol. Chem. 1992; 267: 4939-4948Abstract Full Text PDF PubMed Google Scholar). In short, the purification procedure was as follows. Two × 107cells were lysed in 2 ml of electrophoresis sample buffer (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), passed five times through a 25-gauge needle, and soluble proteins were separated using Bio-Rad model 491 Prep Cell apparatus with a 37-mm (inner diameter) tube. Electrophoresis was carried out at 50 mA, proteins were eluted with 50 mm Tris/HCl, 5 mmβ-mercaptoethanol, 0.02% (v/v) Nonidet P-40 (pH 8.0) at 0.45 ml/min, and 4.5-ml fractions were collected. Fractions containing rAR were pooled and concentrated using Amicon P-10 membrane (Amicon, Beverly, MA). Rabbits were injected with 50 μg of rAR protein at each immunization. Diluted K183 antiserum (1:200) was incubated on cells for 1 h at 22 °C, and fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Philadelphia) was used (1:200 dilution, 30 min at 22 °C) to detect the antigen. Conditions for immunoblotting were as described previously (8Palvimo J.J. Kallio P.J. Ikonen T. Mehto M. Jänne O.A. Mol. Endocrinol. 1993; 7: 1399-1407PubMed Google Scholar), except that antiserum K183 (diluted 1:1,500) was used as the primary antibody. An AR-dependent promoter interference reporter plasmid, pCMV-ARE2-CAT, was constructed in a fashion similar to that described for ER (14Reese J.C. Katzenellenbogen B.S. Mol. Cell. Biol. 1992; 12: 4531-4538Crossref PubMed Scopus (109) Google Scholar) by inserting a duplicated high affinity ARE sequence between the TATA box and transcription start site of pCMV-0-CAT (Fig. 1 A). Two AREs were inserted, as the presence of more than one palindromic response element facilitates AR-ARE interactions in vitro (9Kallio P.J. Palvimo J.J. Mehto M. Jänne O.A. J. Biol. Chem. 1994; 269: 11514-11522Abstract Full Text PDF PubMed Google Scholar). pCMV-0-CAT devoid of ARE sequences was initially used as the control reporter, but its basal activity in CV-1 cells was several times higher than that of pCMV-ARE2-CAT. This result was in agreement with a previous report (13Hu M.C.-T. Davidson M. Cell. 1987; 48: 555-566Abstract Full Text PDF PubMed Scopus (163) Google Scholar) that insertion of sequences into a similar location (between the TATA box and the start site of transcription) decreases CAT expression. For this reason, we used pCMV-ERE2-CAT reporter (Fig. 1 A) as control to assess the specificity of AR-ARE interaction in intact cells. CV-1 cells were cotransfected with various AR expression plasmids (Fig. 1 B) and pCMV-ARE2-CAT (or pCMV-ERE2-CAT) reporter driven by the constitutively active CMV promoter. Native rAR and hAR interfered with pCMV-ARE2-CAT expression in such a way that both ligand-dependent and ligand-independent inhibition of the reporter activity was seen (Fig. 2, A andB). rAR showed somewhat higher ligand-independent promoter-interfering activity than hAR. Since expression levels of rAR and hAR proteins were not compared in these experiments, the reason for this species difference remains to be elucidated. Both ARs also elicited some inhibition of pCMV-ERE2-CAT expression which was, however, not increased by androgen (Fig. 2, C andD). Even though the weak ligand-independent activity of AR was not specific for the hormone response element inserted in the promoter interference construct, it was specific for the promoter interference assay itself, as a similar androgen-independent activity was not observed in transactivation experiments. It is also worth pointing out that maximal promoter interference and transcriptional activation were achieved with comparable amounts of rAR or hAR expression plasmid (1 μg DNA/plate). 3U. Karvonen, O. A. Jänne, and J. J. Palvimo, unpublished observations. However, ligand-occupied hAR was twice as active as rAR in these latter experiments (110-fold versus 50-fold increase over control, respectively3), which is in contrast to the behavior of apo-hAR and apo-rAR in promoter interference assays (see Fig. 2,A and B). AR forms incapable of DNA or hormone binding (rARC562G and hARM807R, respectively) were used to assess the importance of these regions for specific AR-ARE interaction in vivo. rARC562G with a Cys → Gly substitution at codon 562 does not bind to DNA in vitroand is transcriptionally inactive (22Ikonen T. Palvimo J.J. Kallio P.J. Reinikainen P. Jänne O.A. Endocrinology. 1994; 135: 1359-1366Crossref PubMed Scopus (155) Google Scholar). hARM807R is incapable of hormone binding because of a Met → Arg substitution at codon 807 (21Adeyemo O. Kallio P.J. Palvimo J.J. Kontula K. Jänne O.A. Hum. Mol. Genet. 1993; 2: 1809-1812Crossref PubMed Scopus (37) Google Scholar). In electrophoretic mobility shift assays under cell-free conditions, hARM807R bound to ARE with approximately the same affinity as the wild-type receptor in the absence of androgen (21Adeyemo O. Kallio P.J. Palvimo J.J. Kontula K. Jänne O.A. Hum. Mol. Genet. 1993; 2: 1809-1812Crossref PubMed Scopus (37) Google Scholar), but exposure to androgen did not elicit a conformational change in the LBD of hARM807R in a fashion similar to that in the wild-type LBD (21Adeyemo O. Kallio P.J. Palvimo J.J. Kontula K. Jänne O.A. Hum. Mol. Genet. 1993; 2: 1809-1812Crossref PubMed Scopus (37) Google Scholar,27Kallio P.J. Jänne O.A. Palvimo J.J. Endocrinology. 1994; 134: 998-1001Crossref PubMed Scopus (46) Google Scholar). rARC562G and hARM807R mutants did not interfere with pCMV-ARE2-CAT activity in a steroid-dependent fashion, but they exhibited weak hormone-independent interference, essentially indistinguishable from that of the wild-type apo-AR (Fig.3, A and B). The fact that rARC562G behaved like apo-AR both in the presence and absence of testosterone implies that specific binding of the wild-type AR to DNA in intact cells is totally dependent on the integrity of the first zinc finger and that the conformational change elicited by an active androgen in the LBD does not abolish the ability of this region to interfere weakly with pCMV-ARE2-CAT expression. Previous studies have shown that AR forms devoid of the entire LBD behave as constitutively active transactivators and are capable of binding to specific DNA elements in vitro (17Rundlett S.E. Wu X.-P. Miesfeld R.L. Mol. Endocrinol. 1990; 4: 708-714Crossref PubMed Scopus (109) Google Scholar, 18Jenster G. van der Korput H.A.G.M. van Vroonhoven C. van der Kwast T.H. Trapman J. Brinkmann A.O. Mol. Endocrinol. 1991; 5: 1396-1404Crossref PubMed Scopus (421) Google Scholar).2 The rARΔ641–902 mutant that lacks the LBD reduced expression of pCMV-ARE2-CAT by 50% but did not influence that of pCMV-ERE2-CAT (Fig. 4 A). It is of note that maximal promoter interference was achieved with the lowest amount of pSGrARΔ641–902 expression vector used (0.5 μg of DNA/10-cm plate), and no additional increase in the interference occurred when the amount of plasmid was augmented 5-fold (Fig.4 A) or 10-fold (data not shown). Interaction of rARΔ641–902 with DNA was, as expected, independent of the presence of ligand, whether an agonist (Fig. 4 A) or an antagonist (54 ± 5% of control in cells transfected with 0.5 μg of pSGrARΔ641–902 in the presence of 1 μm casodex). To examine the role of the NH2-terminal region, rARΔ46–408 and rARΔ46–408/C562G mutants were used. rARΔ46–408 expressed in insect cells binds to specific DNA sequences in vitro, albeit with a somewhat lower affinity than the wild-type AR (8Palvimo J.J. Kallio P.J. Ikonen T. Mehto M. Jänne O.A. Mol. Endocrinol. 1993; 7: 1399-1407PubMed Google Scholar, 9Kallio P.J. Palvimo J.J. Mehto M. Jänne O.A. J. Biol. Chem. 1994; 269: 11514-11522Abstract Full Text PDF PubMed Google Scholar). When expressed in CV-1 cells, rARΔ46–408 elicited promoter interference in an androgen-dependent fashion (Fig.4 B). This interference required the presence of ARE sequences, as a similar steroid-dependent inhibition of pCMV-ERE2-CAT expression failed to occur under the same conditions (Fig. 4 B). Likewise, an intact DBD of the receptor was required, as judged by the inability of rARΔ46–408/C562G to interfere with pCMV-ARE2-CAT activity.2 Some hormone-independent inhibition of pCMV-ARE2-CAT activity was observed with rARΔ46–408 when higher amounts of expression plasmid were used in transfections (Fig.4 B). Taken together, interaction of AR with specific DNA sequences in intact cells can take place in the absence of LBD or most of the NH2-terminal region. DNA binding of the receptor, even in the presence of LBD and steroid, is not sufficient to elicit proper physiological responses, as the rARΔ46–408 mutant is transcriptionally inactive (8Palvimo J.J. Kallio P.J. Ikonen T. Mehto M. Jänne O.A. Mol. Endocrinol. 1993; 7: 1399-1407PubMed Google Scholar). Comparison of the dose-response curves between rARΔ641–902 and rARΔ46–408 suggests that LBD is responsible for the weak hormone-independent interference of AR with pCMV-ARE2-CAT or pCMV-ERE2-CAT expression (Fig.4, A and B). The results with full-length AR forms (Fig. 2) were in general agreement with those obtained with rARΔ641–902 and rARΔ46–408 mutants. The ability of LBD to interfere with transcription was examined by additional experiments, in which pG5SV40CAT reporter containing five GAL4 binding sites upstream of the SV40 promoter was used. CV-1 cells were cotransfected using this reporter or an appropriate control reporter (pSV40CAT) and expression vectors encoding GAL4-DBD, GAL4-LBD, or GAL4-Nterm fusion proteins. The NH2-terminal region of AR (residues 5–538) fused to GAL4-DBD did not influence the activity of the latter; however, when the fusion partner was LBD (residues 641–902), repression of transcriptional activity took place (Fig.5 A). It is of note that the ability of ligand-free LBD to repress transcription at a distance exceeded that of the ligand-occupied LBD in the GAL4-DNA-binding construct (Fig.5 A). The repressive action of LBD-containing fusion protein was dependent on its tethering to DNA, as no inhibition was detected in the absence of GAL4-binding elements (Fig. 5 B). Several anti-androgens (casodex, hydroxyflutamide, RU 56187, RU 57073, RU 59063, and cyproterone acetate) were examined by promoter interference assays using rARΔ46–408, as differentiation between ligand-dependent and -independent activities was more clear-cut with this mutant than with native ARs. All other anti-androgens examined but hydroxyflutamide were able to confer some promoter-interfering activity upon rARΔ46–408, with cyproterone acetate and the RU compounds being as potent as an agonist (10 nm testosterone) at the concentration used (1 μm) (Table I). Additional studies with casodex revealed that its low agonist-like activity in the promoter interference assay was also detectable with wild-type rAR and at varying receptor levels (Fig. 6 A). When present concomitantly with androgen, casodex inhibited the agonist-induced promoter interference significantly but not completely (Fig. 6 B). Previous studies have shown that casodex and hydroxyflutamide are completely devoid of inherent transactivation ability (22Ikonen T. Palvimo J.J. Kallio P.J. Reinikainen P. Jänne O.A. Endocrinology. 1994; 135: 1359-1366Crossref PubMed Scopus (155) Google Scholar, 28Jenster G. Trapman J. Brinkmann A.O. Biochem. J. 1993; 293: 761-768Crossref PubMed Scopus (216) Google Scholar), whereas cyproterone acetate is a partial agonist/antagonist (29Kemppainen J.A. Lane M.V. Sar M. Wilson E.M. J. Biol. Chem. 1992; 267: 968-974Abstract Full Text PDF PubMed Google Scholar). Three N-substituted arylthiohydantoin anti-androgens (RU 56187, RU 7073, and RU 59063) were as effective as testosterone in conferring promoter-interfering activity on rARΔ46–408 (TableI). This is in agreement with binding affinities of these compounds for rat and human ARs in vitro, which are 92–300% of that of testosterone (30Teutsch G. Goubet F. Battmann T. Bonfils A. Bouchoux F. Cerede E. Gofflo D. Gaillard-Kelly M. Philibert D. J. Steroid Biochem. Mol. Biol. 1994; 48: 111-119Crossref PubMed Scopus (99) Google Scholar). When CV-1 cells cotransfected with pSGrAR and pMMTV-CAT vectors were exposed to 1 μm of each RU anti-androgen alone, CAT activity was 50% with RU 56187, 28% with RU 57073, and 35% with RU 59063 of that with 10 nmtestosterone. Concomitant exposure of the cells to 10 nmtestosterone and 1 μm RU anti-androgens resulted in reporter gene activities that were 39–66% of those achieved with androgen alone, indicating that the three RU compounds were partial agonists/antagonists under these conditions. As the RU compounds promoted AR-ARE interaction in CV-1 cells as well as testosterone (Table I), conformational requirements on LBD for transcriptional activation and recognition of specific DNA sequences (promoter interference) are not identical in intact cells. The polyclonal antiserum (K183) used in these experiments was specific for AR proteins, as no extra bands were observed in mock-transfected cells, and only the receptor protein was detected in immunoblots of COS-1 cells transfected with some of the AR expression constructs used in promoter interference assays (Fig.7). The antiserum recognized rARΔ46–408 and rARΔ641–902 mutants as efficiently as the native receptor protein and, therefore, was useful for immunological studies of different AR forms in cultured cells. CV-1 cells transfected with 1 μg of pSGrAR DNA showed some immunoreactivity in the cytoplasm in the absence of androgen, whereas unliganded hAR was more clearly nuclear in its localization under the same conditions (Fig. 8, A and C). Both receptors became solely nuclear upon hormone exposure (Fig. 8,B and D). rARC562G and hARM807R exhibited both cytoplasmic and nuclear localization in the absence of testosterone (Fig. 8, E and F). The AR form devoid of most of the NH2-terminal region (rARΔ46–408) showed mainly nuclear localization even in the absence of androgen (Fig.8 G), and it was strictly nuclear after exposure to testosterone (data not shown). Likewise, the LBD-deficient mutant (rARΔ641–902) was nuclear in the absence of the hormone (Fig.8 H). CV-1 cells transfected with pSGhAR expression vector and exposed to casodex or hydroxyflutamide exhibited only nuclear localization of the receptor protein (Fig. 8, I andJ). Taken together, immunocytochemical studies of transfected CV-1 cells expressing wild-type and mutant AR proteins revealed that the receptor forms were mainly nuclear even in the absence of ligand, indicating that differences in subcellular localization do not explain their dissimilar behavior in the promoter interference assay. The results of this work show that sequence-specific DNA binding of the native AR in cultured CV-1 cells requires the presence of ligand. In this respect, our data agree with a recent report by Kuil and Mulder (12Kuil C.W. Mulder E. Endocrinology. 1996; 17: 1870-1877Crossref Scopus (24) Google Scholar), who also used the promoter interference assay to examine the interaction of AR with a synthetic consensus ARE in Chinese hamster ovary cells. Our data show further that neither LBD nor a major part of the NH2-terminal transactivation region of the receptor is needed for its interaction with specific DNA sequencesin vivo. LBD is not only dispensable but also appears to be responsible for the ability of apo-AR to interfere weakly with pCMV-ARE2-CAT expression. In addition, unoccupied LBD fused to GAL4-DBD was capable of repressing transcriptional activity of a heterologous SV40 promoter at a distance. Should AR bind to AREs as a homodimer in intact cells, as it does in vitro (8Palvimo J.J. Kallio P.J. Ikonen T. Mehto M. Jänne O.A. Mol. Endocrinol. 1993; 7: 1399-1407PubMed Google Scholar, 9Kallio P.J. Palvimo J.J. Mehto M. Jänne O.A. J. Biol. Chem. 1994; 269: 11514-11522Abstract Full Text PDF PubMed Google Scholar), the results with rARΔ641–902 illustrate that this dimerization utilizes sequences other than those residing in LBD. Moreover, the interaction between COOH- and NH2-terminal regions of AR, which has been shown to occur in mammalian cells (31Langley E. Zhou Z. Wilson E.M. J. Biol. Chem. 1995; 270: 29983-29990Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar),2 is not mandatory for the ability of the receptor to recognize specific DNA elements in vivo, as shown by appropriate deletion mutants in promoter interference assay. Studies with rARΔ46–408 that lacks most of the NH2-terminal region and with anti-androgens also provided compelling evidence that mere DNA binding in vivo is not sufficient for the AR to become a competent transcriptional activator. The hARM807R mutant is incapable of hormone binding, totally inactive in transactivation assays, and the reason for complete androgen insensitivity of a patient (21Adeyemo O. Kallio P.J. Palvimo J.J. Kontula K. Jänne O.A. Hum. Mol. Genet. 1993; 2: 1809-1812Crossref PubMed Scopus (37) Google Scholar). It could be speculated that inappropriate folding of this mutant's LBD may render it free of intracellular chaperones such as heat shock proteins (32Veldscholte J. Berrevoets C.A. Zegers N.D. van der Kwast T.H. Grootegoed J.A. Mulder E. Biochemistry. 1992; 31: 7422-7430Crossref PubMed Scopus (116) Google Scholar), thereby permitting its binding to AREs even in vivo. However, our present data and those of Kuil and Mulder (12Kuil C.W. Mulder E. Endocrinology. 1996; 17: 1870-1877Crossref Scopus (24) Google Scholar) demonstrate that specific DNA binding in living cells is not achieved without a ligand-induced allosteric change in the LBD. The findings that hARM807R binds well to AREs in vitro (21Adeyemo O. Kallio P.J. Palvimo J.J. Kontula K. Jänne O.A. Hum. Mol. Genet. 1993; 2: 1809-1812Crossref PubMed Scopus (37) Google Scholar) and is localized in nuclei of transfected cells (this study) are interpreted to mean that the steroid-induced change in LBD conformation is primarily required for the release of AR from associated inhibitory protein complexes, rather than for generation of a receptor form capable of ARE recognition. The mutant rARΔ46–408, which has a region mandatory for transcriptional activation deleted (8Palvimo J.J. Kallio P.J. Ikonen T. Mehto M. Jänne O.A. Mol. Endocrinol. 1993; 7: 1399-1407PubMed Google Scholar), recognized specific DNA sequences in CV-1 cells in a fashion indistinguishable from that of the wild-type receptor, implying that the NH2 terminus is not essential for this interaction. This result is in agreement with our previous studies on AR-ARE interaction in vitro, in that NH2-terminal internal deletions compromised the affinity of rAR for AREs only to a moderate extent; the K D of interaction increased from 0.5–1.0 nm to 3.0 nm (8Palvimo J.J. Kallio P.J. Ikonen T. Mehto M. Jänne O.A. Mol. Endocrinol. 1993; 7: 1399-1407PubMed Google Scholar, 9Kallio P.J. Palvimo J.J. Mehto M. Jänne O.A. J. Biol. Chem. 1994; 269: 11514-11522Abstract Full Text PDF PubMed Google Scholar). AR protein produced in insect cells has been suggested to require intracellular hormone exposure to overcome the inhibition imposed by the NH2-terminal domain on dimerization and DNA binding of the aporeceptor (10Wong C. Zhou Z. Sar M. Wilson E.M. J. Biol. Chem. 1993; 268: 19004-19012Abstract Full Text PDF PubMed Google Scholar). The present experiments showed, however, that unliganded rARΔ46–408 was incapable of sequence-specific DNA binding in CV-1 cells (Fig.4 B), implying that a major part of the NH2-terminal region is not critical for maintaining the receptor in a non-DNA-binding form. Anti-androgens that were examined in this work can be divided into two groups on basis of their ability to modulate the binding of AR to AREs. The first group is not capable of converting the receptor to a form that binds to specific DNA sequences in CV-1 cells, or it does this poorly. When present concomitantly with an agonist, these compounds inhibit the agonist-dependent promoter interference. Non-steroidal anti-androgens casodex and hydroxyflutamide belong to this group. The second group includes the steroidal anti-androgen cyproterone acetate and three N-substituted arylthiohydantoin anti-androgens (RU 56187, RU 57073 and RU 59063). These compounds, at least at the concentrations used, converted AR to a form capable of ARE binding in CV-1 cells. Moreover, the compounds of the second group behaved as partial agonists/antagonists in transactivation experiments. Kuil and Mulder (12Kuil C.W. Mulder E. Endocrinology. 1996; 17: 1870-1877Crossref Scopus (24) Google Scholar) have also reported that in Chinese hamster ovary cells, casodex and hydroxyflutamide inhibited DNA binding of AR, but cyproterone acetate failed to do so. Ligand-free human and rat ARs exhibited weak promoter-interfering activity. In this respect, our results on AR differed form those reported by Reese and Katzenellenbogen (14Reese J.C. Katzenellenbogen B.S. Mol. Cell. Biol. 1992; 12: 4531-4538Crossref PubMed Scopus (109) Google Scholar) and Xing and Shapiro (15Xing H. Shapiro D.J. J. Biol. Chem. 1993; 268: 23227-23233Abstract Full Text PDF PubMed Google Scholar) on human and frog ERs, respectively. In these latter cases, unoccupied wild-type ER showed moderate to high interfering activity, and some ligand-free ER mutants were even more potent than the estrogen-bound wild-type ER (14Reese J.C. Katzenellenbogen B.S. Mol. Cell. Biol. 1992; 12: 4531-4538Crossref PubMed Scopus (109) Google Scholar, 15Xing H. Shapiro D.J. J. Biol. Chem. 1993; 268: 23227-23233Abstract Full Text PDF PubMed Google Scholar). Likewise, apo-PR elicited strong interference with pCMV-PRE-CAT expression, which was almost 50% of that of the progestin-bound PR (16Gass, L., Leonhardt, S., Altmann, M., and Edwards, P. (1996)Abstracts of the 10th International Congress of Endocrinology, San Francisco, June 12–15, abstract p2-682.Google Scholar). The observed differences in the behavior of nuclear receptors in promoter interference assays could result, at least in part, from dissimilar affinities of their LBDs for auxiliary proteins communicating with transcription machinery. A COOH-terminal region of PR has recently been reported to contain a transcriptional repressor domain that functions through a putative corepressor (33Xu J. Nawaz Z. Tsai S.Y. Tsai M.-J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12195-12199Crossref PubMed Scopus (79) Google Scholar); however, a similar function has not so far been described for the COOH terminus of AR. The use of a heterologous GAL4 system supported the notion that LBD is responsible for the weak ligand-independent promoter-interfering activity of AR. The activity of this LBD in the context of GAL4 fusion protein was not dependent on the ligand-induced conformational change; rather, ligand-free GAL4-LBD bound to elements upstream of SV40 promoter exhibited stronger repressive action than the ligand-occupied form, suggesting that apo-LBD is the favored conformation in recruitment of putative repressor proteins. In conclusion, the results of this work indicate that interaction of AR with specific DNA sequences in CV-1 cells does not involve COOH-terminal or NH2-terminal regions of the receptor, even though the NH2-terminal domain contains sequences required for transcriptional activation. In the context of wild-type AR, the androgen-induced conformational change in the COOH terminus is, however, mandatory for generation of a transcriptionally competent receptor protein that binds to ARE sequences in intact cells, even though mere DNA binding in vivo is not sufficient for the AR to acquire competence in transcriptional activation. And finally, LBD appears to be responsible for the weak promoter-interfering activity of apo-AR. We thank Dr. Ismo Virtanen for help in immunocytochemistry and Drs. Benita Katzenellenbogen and Norbert Lehming for plasmids." @default.
W2078603739 created "2016-06-24" @default.
W2078603739 creator A5016359992 @default.
W2078603739 creator A5062665892 @default.
W2078603739 creator A5083561452 @default.
W2078603739 creator A5089068068 @default.
W2078603739 date "1997-06-01" @default.
W2078603739 modified "2023-09-29" @default.
W2078603739 title "Interaction of Androgen Receptors with Androgen Response Element in Intact Cells" @default.
W2078603739 cites W1484666375 @default.
W2078603739 cites W1510583609 @default.
W2078603739 cites W1559820839 @default.
W2078603739 cites W1588136567 @default.
W2078603739 cites W1610648155 @default.
W2078603739 cites W1674624604 @default.
W2078603739 cites W1967013096 @default.
W2078603739 cites W1967641240 @default.
W2078603739 cites W1987183940 @default.
W2078603739 cites W1992235715 @default.
W2078603739 cites W2002791165 @default.
W2078603739 cites W2009948262 @default.
W2078603739 cites W2037365073 @default.
W2078603739 cites W2050567539 @default.
W2078603739 cites W2076466919 @default.
W2078603739 cites W2079196098 @default.
W2078603739 cites W2082528130 @default.
W2078603739 cites W2093952109 @default.
W2078603739 cites W2100837269 @default.
W2078603739 cites W2143917623 @default.
W2078603739 cites W2153042859 @default.
W2078603739 cites W2158349558 @default.
W2078603739 cites W2172256092 @default.
W2078603739 cites W7532581 @default.
W2078603739 cites W90893581 @default.
W2078603739 doi "https://doi.org/10.1074/jbc.272.25.15973" @default.
W2078603739 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9188499" @default.
W2078603739 hasPublicationYear "1997" @default.
W2078603739 type Work @default.
W2078603739 sameAs 2078603739 @default.
W2078603739 citedByCount "77" @default.
W2078603739 countsByYear W20786037392012 @default.
W2078603739 countsByYear W20786037392013 @default.
W2078603739 countsByYear W20786037392014 @default.
W2078603739 countsByYear W20786037392015 @default.
W2078603739 countsByYear W20786037392016 @default.
W2078603739 countsByYear W20786037392017 @default.
W2078603739 countsByYear W20786037392019 @default.
W2078603739 countsByYear W20786037392020 @default.
W2078603739 countsByYear W20786037392021 @default.
W2078603739 countsByYear W20786037392023 @default.
W2078603739 crossrefType "journal-article" @default.
W2078603739 hasAuthorship W2078603739A5016359992 @default.
W2078603739 hasAuthorship W2078603739A5062665892 @default.
W2078603739 hasAuthorship W2078603739A5083561452 @default.
W2078603739 hasAuthorship W2078603739A5089068068 @default.
W2078603739 hasBestOaLocation W20786037391 @default.
W2078603739 hasConcept C121608353 @default.
W2078603739 hasConcept C126322002 @default.
W2078603739 hasConcept C134018914 @default.
W2078603739 hasConcept C170493617 @default.
W2078603739 hasConcept C185592680 @default.
W2078603739 hasConcept C2777911890 @default.
W2078603739 hasConcept C2780192828 @default.
W2078603739 hasConcept C55493867 @default.
W2078603739 hasConcept C61367390 @default.
W2078603739 hasConcept C71315377 @default.
W2078603739 hasConcept C71924100 @default.
W2078603739 hasConcept C86803240 @default.
W2078603739 hasConcept C95444343 @default.
W2078603739 hasConceptScore W2078603739C121608353 @default.
W2078603739 hasConceptScore W2078603739C126322002 @default.
W2078603739 hasConceptScore W2078603739C134018914 @default.
W2078603739 hasConceptScore W2078603739C170493617 @default.
W2078603739 hasConceptScore W2078603739C185592680 @default.
W2078603739 hasConceptScore W2078603739C2777911890 @default.
W2078603739 hasConceptScore W2078603739C2780192828 @default.
W2078603739 hasConceptScore W2078603739C55493867 @default.
W2078603739 hasConceptScore W2078603739C61367390 @default.
W2078603739 hasConceptScore W2078603739C71315377 @default.
W2078603739 hasConceptScore W2078603739C71924100 @default.
W2078603739 hasConceptScore W2078603739C86803240 @default.
W2078603739 hasConceptScore W2078603739C95444343 @default.
W2078603739 hasIssue "25" @default.
W2078603739 hasLocation W20786037391 @default.
W2078603739 hasOpenAccess W2078603739 @default.
W2078603739 hasPrimaryLocation W20786037391 @default.
W2078603739 hasRelatedWork W2004261902 @default.
W2078603739 hasRelatedWork W2014676068 @default.
W2078603739 hasRelatedWork W2058317585 @default.
W2078603739 hasRelatedWork W2068395526 @default.
W2078603739 hasRelatedWork W2072661483 @default.
W2078603739 hasRelatedWork W2075574887 @default.
W2078603739 hasRelatedWork W2103250319 @default.
W2078603739 hasRelatedWork W2121679333 @default.
W2078603739 hasRelatedWork W2136266902 @default.
W2078603739 hasRelatedWork W2171723716 @default.
W2078603739 hasVolume "272" @default.
W2078603739 isParatext "false" @default.