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Home , Myc, Receptor (biochemistry)

( RESEARCH 54, 1566-1573, March 15. 1

John Kokontis, Kenji Takakura, Nissim Hay, and Shutsung Liao2

The Ben May Institute ¡J.K., K. T., N. H., S. L.¡,Departments of and Molecular [S. L.j and and Physiology [N. H/, The University of Chicago, Chicago. Illinois 60637

ABSTRACT cellular events probably occur which allow proliferation to bypass the androgen requirement. Loss of AR expression may, therefore, Proliferation of LNCaP 104-S cells, a donai subline of the human either drive the selection for such events or occur secondarily (4). prostate cancer cell line, was very slow in androgen-depleted medium but The androgen-responsive human prostatic carcinoma cell line LN increased 10-13-fold in the presence of 0.1 UMof a synthetic androgen, KISSI. This induction of proliferation was diminished at higher concen CaP (5, 6) has been used extensively as an in vitro model for exam trations of KISSI, indicating the biphasic nature of the androgen effect. ining the role of the AR in the control of prostatic carcinoma cell After 20-30 passages in androgen-depleted medium, these cells progressed proliferation. LNCaP cells express large amounts of AR mRNA and to 104-1 cells, which exhibited much lower proliferative sensitivity to 0.1 . The levels of AR mRNA but not protein appear to be under IIMRISSI. After another 20-30 passages, LNCaP 104-1 cells gave rise to moderate negative control by androgen (7-9). ARs in LNCaP cells 104-R cells, which proliferated rapidly without additional androgen. Pro possess a in the androgen binding domain which alters the liferation of 104-R cells was induced 2-fold by 0.01 UMR1881 but was specificity of binding and steroid-induced transactivation repressed by 0.1 MMR1881 and above. Thus, androgen induction and (10, 11) and is probably responsible for the aberrant proliferative repression of proliferation could be seen at lower concentrations of an response of LNCaP cells to (12-14). drogen as the cells progressed. During the transition of 104-S cells to 104-R cells, the androgen mRNA level increased 2.5-fold whereas We were interested in determining whether changes might occur in the protein level increased 15-fold in the absence of the proliferative response of LNCaP cells to androgen when cells are androgen. Androgen receptor transcriptional activity, measured by andro cultured over a long of time in androgen-depleted medium and gen induction of prostate-specific antigen mRNA and chloramphenicol if so, to identify the accompanying molecular changes. If androgen acetyltransferase activity in transfected cells, increased up to 20-fold dur sensitivity could be altered in a clonal isolate of androgen-sensitive ing the progression. LNCaP cells, therefore, appear to be able to adapt to LNCaP cells by long-term androgen deprivation, it should be helpful reduced androgen availability by increasing their sensitivity to androgen, in the study of molecular and/or cytogenetic changes which occur raising questions concerning the therapeutic strategies used against pros concomitantly with the observed changes in proliferative response. A tate cancer. Androgen induction of c-myc expression in 104-R cells occurred at a 10-fold lower concentration (0.01 nvi) than in 104-S cells demonstration that changes in specific gene expression take place over (0.1 IIMI.In all stages, and c-myc expression were re time in a clonal isolate would support the idea that prostate tumor cells pressed by androgen at a high concentration (20 IIMi, but the repression of are able to adapt to lowered androgen concentration in their environ cell proliferation was blocked by retroviral overexpression of c-myc. ment and that androgen-independent cells do not necessarily arise during androgen ablation therapy through selection of a preexisting, androgen-independent subpopulation (15). The use of a clonal subline also simplifies the interpretation of androgen response in a heterog- INTRODUCTION enous parental cell population. We were also particularly interested in Prostate cancer is currently the most common malignancy and is the changes in the expression of c-myc, because expression of this critical second leading cause of cancer death among males in the United growth-related protooncogene has been reported to be negatively States (1). The mechanism of prostatic and tumori- regulated by high concentrations of androgen and positively correlated genesis likely involves a multistep progression from precancerous with proliferation in LNCaP cells (16, 17). Deregulated expression of cells to cells which proliferate and metastasize. The growth and de c-myc has been observed in a variety of neoplasias and has been velopment of prostate cancer appears to be androgen dependent ini implicated in neoplastic development (for reviews, see Refs. 18-21). tially, making it vulnerable to androgen ablation and Elevated c-myc expression has been observed in benign prostatic therapies (2). Most often, however, prostate cancer cells lose androgen hyperplasia and prostatic carcinoma (22, 23). In addition to having a dependency and responsiveness during the progression to malignant role in the induction of cell proliferation, c-myc also appears to func stages, and tumor cells which are resistant to endocrine therapy ulti tion under certain circumstances (overexpression during mately proliferate. While loss of AR3 expression may accompany loss withdrawal) in (24-27). of androgen dependency and responsiveness (3), other prerequisite In this paper, we report on the results of experiments with an androgen-responsive LNCaP 104 clonal subline subcultured in andro

Received 10/23/93; accepted 1/28/94. gen-depleted medium for about 60 passages. As the 104 cells pro The cosls of publication of this article were defrayed in part by Ihe payment of page gressed, they became more sensitive to low concentrations of andro charges. This article must therefore be hereby marked advertisement in accordance with gen, and proliferative response of this subline to R1881 at 0.1 UM 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by NIH Grams DK41670, DK 37694. and CA 58073. concentration changed from positive to negative. LNCaP 104 cells 2 To whom requests for reprints should be addressed, at The Ben May Institute, Box appeared to adapt to lowered androgen levels by increasing AR ex MC6027, The University of Chicago. 5841 South Maryland Avenue, Chicago, IL 60637. 1The abbreviations used are: AR. androgen receptor: RI881, 17ß-hydroxy-17-methyl- pression and transcriptional activity. These results show that androgen estra-4.°..ll-trien-3-one; ß;-MG.ß2-microglobulin: DMEM, Dulbecco's modified Eagle's regulation of c-myc expression may play an important role in the medium: DHT. Sor-dihydrotestoslerone; FBS. fetal bovine serum; CS-FBS, dextran-coated charcoal-stripped FBS; CAT, chloramphenicol acetyltransferase: PSA, prostate-specific apparent androgen sensitivity of prostate cancer cells. These findings antigen: cDNA, complementary DNA; PBS, phosphate-buffered saline; SDS-PAGE, so additionally suggest that in designing a therapeutic approach to the dium dodccyl sulfate-polyacrylamide gel electrophoresis; MMTV-LTR. mouse mammary later stages of prostate cancer that show apparent loss in androgen- tumor long terminal repeat; RSV LTR, Rous sarcoma virus long terminal repeat; DMNT, mibolerone or 7a,17a-dimethyl-19-nortestosterone; EGF. epidermal growth dependent proliferation, it is necessary to consider the underlying factor. mechanism and the usefulness of hormonal intervention. 1566 ANDROfiEN RECEPTOR ACTIVITY IN PROSTATE CANCER CELLS

MATERIALS AND METHODS untreated FBS was equivalent to the activity of 0.05 HMR1881. LNCaP 104-R cells derived from 104-1 cells were also maintained in DMEM supplemented Materials. The LNCaP cell line was obtained from Dr. T. M. Chu at the with 10% CS-FBS. One passage consisted of 7 days of growth to confluency, Roswell Park Memorial Institute (Buffalo, NY), and the androgen-insensitive starting from a 1:9 dilution of a trypsinized cell suspension. Responsiveness of prostale cancer PC-3 cell line was obtained from American Type Tissue Col cell proliferation to various concentrations of R1881 was measured by deter lection. [a-32P]dCTP (3000 Ci/mmole), [a-12P]UTP (800 Ci/mmole), and the mining the cell density of trypsinized cell suspensions with a hemocytometer Multiprime Random Primer Kit were purchased from Amersham. Restriction after 6 days of growth. Cells were plated in triplicate in 12-well dishes (3 X endonucleases and other were purchased from Bochringcr Mann IO4 cells/well) in DMEM supplemented with 10% CS-FBS in the presence or heim, Stratagcne, BRL, and Promcga. AN-21, a polyclonal rabbit absence of R1881. In instances of cell clumping, pelleted cells were treated raised against a 21-amino acid peptidc corresponding to the NH;-terminus of with a solution of 0.4% Nonidet P-40, 50 min Tris, and 5 mM MgCK, pH 7.5, human and rat AR (28, 29) was prepared in this laboratory by Drs. Ching Song and the density of free nuclei was determined with a hemocytometer. LNCaP and Richard Hiipakka using methods described by Tsang and Wilkins (30). An 104 sublines were infected with retrovirus generated from the pMV7 retroviral enhanced chemiluminescence detection was purchased from Amersham. A vector (32) using the procedures of Brown and Scott (33). The pMV7 vector lucifcrase assay system kit was purchased from Promega. R1881 was from contains the gene encoding ncomycin phosphotransferase, which confers se New England Nuclear, and other steroids were from Steraloids. The MMTV- lectable resistance to geneticin (G418; Gibco). A 1.5-kilobase ¿V»RI-////idlII CAT vector was derived from pMSG-CAT (Pharmacia) by the excision of the human c-myc cDNA fragment was inserted into £roRI-////idlll-digested 2-kilobase BamW fragment containing the gpt gene (11). pMV7, which was transfected into the mouse i|/2 packaging cell line using the LNCaP Subline Isolation, Cell Culture, and Retroviral Infection. The LNCaP 104-S subline was isolated with 11 other sublines by cloning individual calcium phosphate precipitation technique. After 2 weeks of selection in me colonies after seeding serially diluted LNCaP cells. LNCaP sublines were then dium containing 0.4 mg/ml geneticin. ecotrophic viral progeny produced from geneticin-resislant cells were used lo infect mouse PA317 cells, which pro maintained in DMEM (Gibco) supplemented with 1 n\i DHT and 10% FBS (Upstate Biotechnology, Inc.). LNCaP I04-I cells were derived from the LN duced amphotrophic retrovirus capable of infecting human cells. Retrovirally CaP 104-S subline by continuous passage, starting at passage 40 in DMEM infected LNCaP cells were maintained in DMEM supplemented with 10% supplemented with 10% CS-FBS (31), and were subsequently maintained in untreated FBS and 0.5 mg/ml geneticin. the same medium (Fig. \A ). As an assay of residual androgenic activity in Anchorage-independent Growth in Agarose. Low melting point agarose CS-FBS, CAT activity in PC-3 cells incubated in 10% CS-FBS and transiently (FMC Bioproducts) was suspended at 0.75% in water, autoclaved, and cooled transfected with MMTV-CAT and pSG5-LNCaP AR (11) expression vectors to 50°C.One-tenth volume of 10 x DMEM and 0.1 volume of either CS-FBS was about 20% of the level in PC-3 cells treated with 0.01 nui R1881. By the or untreated FBS were added to make a final agarose concentration of 0.6%. same assay, androgenic activity present in medium supplemented with 10% Three ml was dispensed into 6-cm tissue culture plates. LNCaP cells (2 x IO4) were suspended in 1 ml of DMEM supplemented with either 10% CS-FBS or 10% FBS and with R1881 at a concentration 2-fold higher than the final CS-FBS concentration. One ml of the 0.6% agarose-DMEM-FBS solution was added to the cells, and the cell suspension was poured over the previously gelled agarose layers. After 13 days, colonies on duplicate plates were randomly scored as less 104-S 104-1 104-R than or greater than 200 /j.m in diameter along the largest dimension until 100 colonies in either size category were counted. RNA Analysis. Cells were grown in the presence or absence of R1881 to 20 40 60 80 100 80% confluency (4-5 days). Four h prior to harvesting, culture medium was Passage Number replaced with fresh medium and R1881. Total RNA was isolated from scraped cells using the guanidinium lysis-LiCl precipitation technique (34). Ribonuclease protection assay was performed as described by Zinn et al. (35) and Hay et al. (36). Samples (20 f¿gof total RNA plus 32P-labcIed RNA probes) were denatured at 80°Cfor 7 min and were hybridized at 56°Cfor 14-16 h. RNase treatment was with 35 /xg/ml RNase A and 1000 units/ml Tl (Sigma) at 31°Cfor 50 min. followed by the addition of 125 ng/ml proteinase K (Merck) for 10 min at 37°C.After phenol- extraction and ethanol precipitation, RNase-resistant fragments were analyzed on 5% denaturing polyacrylamidc gels followed by autoradiography and quantitation with an AMBIS radioanalytic imaging system (AMBIS Systems, San Diego, CA). For detection of AR mRNA, a 210-base pair Kpnl-Sacl fragment spanning a portion of the DNA-binding domain (28) was inserted into pBlueScript SK+ for in vitro transcription of '2P-labeled antisense RNA with T3 RNA polym- erase. For detection of PSA mRNA, a 77-base pair fragment specific to PSA was generated by polymerase chain reaction amplification of a 2-)xl aliquot of a AZAP LNCaP cDNA library using the two 22-base primers, 5'-AGCCTA- GAGAAGGCTGTGAGCC-3' and 5'-AGTCCCTCTCCTTACTTCATCC-3' (37). The 77-base pair fragment was inserted into the EcoKV site of pBlue Script SK+ for transcription of '2P-labeled antisense RNA with T3 RNA polymerase. The PSA sequence and fragment orientation were confirmed by 0 .001 .01 0.1 1.0 10 100 dideoxy sequence analysis. For detection of c-myc mRNA, a 1.5-kilobase EcoRI-Wi/idIII cDNA fragment inserted into pGEM-3 was used for transcrip R1881 (nM) tion of 32P-labeled antisense RNA with SP6 RNA polymerase (38). A 600-base pair probe was generated by in vitro transcription after linearization of this Fig. \.A, strategy of androgen deprivation in LNCaP 104 cells. Starting at passage 40, plasmid with Cla\. A 144-base pair Pstl-Hincll fragment at the 5' terminus of 104-S cells were grown in DMEM supplemented with 10% CS-FBS. I04-I and 104-R cells arose al the indicated passage numbers. B, dose-response curve of LNCaP 1()4 cell human ß2-MG(39) inserted into SP65 was used for transcription of 12P-labeled proliferation over a range of RI8S1 concentration. Cells (3 x 104/well) were plated in anlisense RNA with SP6 RNA polymerase. Inclusion of ß2-MGantisense RNA triplicale wells in a I2-well multiwell plate and grown in DMEM supplemented with 10% CS-FBS and R188I at the indicated concentrations. Cell densities were determined after probe in hybridizations served as an internal standard for the amount of RNA 6 days of growth. LNCaP 104-S, 104-1, and 104-R cells were tested at passages 16, 65, present. The results of RNase protection assays shown are representative of at and 95, respectively. least three independent experiments. 1567 ANDROGEN RECEPTOR ACTIVITY IN PROSTATE CANCER CELLS

mented with 10% CS-FBS over passages 40-70, the basal rate of proliferation in 10% CS-FBS increased while stimulation of prolif eration by 0.1 nM R1881 diminished. However, stimulation of prolif eration by 0.01 nM R1881 was higher for 104-1 cells than for 104-S cells. Cells at this stage were designated "104-1." At about passages 80-100, the basal rate of proliferation increased to a maximum, and proliferation of these cells, designated "104-R," was induced about 2-fold by 0.01 nM R1881 but was significantly repressed by R1881 at concentrations of 0.1 nM and above. Two distinct patterns of pro liferative response to androgen were exhibited by 104-S and 104-R cells, and an intermediate pattern of response was exhibited by 104-1 65 92 14 65 92 cells. The change in androgen sensitivity was dependent on the ad LNCaP 104 passage number aptation of these cells to androgen-depleted medium. The androgen Fig. 2. Effect of 0.1 nu R1881 on anchorage-independent growth of LNCaP 104 cells sensitivity profile of 104-S cells maintained for over 80 passages in in agarose. The percentage of colonies greater than 200 ;im in diameter was determined after 13 days of growth in DMEM supplemented with either 10% CS-FBS or 10% medium supplemented with 1 nM DHT and 10% untreated FBS was untreated FBS in the presence or absence of 0.1 nM R1881. the same as that of the parent 104-S cells. The inhibition of 104-R cell proliferation by 0.1 nM R1881 was AR Immunoblotting. LNCaP 104-S, LNCaP 104-1, 104-R, and PC-3 cells specific to androgen and steroids which exhibit androgenic activity in were grown in the presence or absence of R1881 for 4 days in DMEM LNCaP cells. LNCaP cells do not express receptors for progestins or supplemented with 10% CS-FBS. Cells were washed in PBS, trypsinized, and estrogens. However, progesterone and 17ß-,which have been resuspended in DMEM supplemented with 10% CS-FBS. After counting, shown to use LNCaP AR in gene transactivation (10, 11), also exhib aliquots containing IO6 cells were centrifuged. Cell pellets were resuspended in ited some inhibitory activity. The relative inhibitory activities of ste 100 /xl of 2X SDS-PAGE loading buffer and heated at 95°Cfor 5 min. roids at 0.1 nM concentration were, in decreasing order, R1881 = present in 10-fj.l aliquots (IO5 cells) were separated on 10% SDS-PAGE gels and electroblotted onto nitrocellulose filters. Nonfat dry milk-blocked filters were successively incubated with 0.1 fig/ml affinity-purified AN-21 polyclonal A. BP rabbit anti-AR antibody, and a 1:2000 dilution of goat anti-rabbit IgG conju gated with horseradish peroxidase (Amersham) in PBS containing 0.2% Tween 396- 20 and 5% nonfat dry milk. Each incubation was for l h at room temperature. 344- Unbound antibody was removed after each incubation by washing for l h (four 298- changes) in PBS with 0.2% Tween 20 al room temperature. The enhanced chemiluminescence reagent was prepared and used according to the instruc 220- tions of the supplier. The relative amount of AR staining was quantified by 201- densitometric scanning of film after a 10-s exposure to filters immediately after the chemiluminescence reaction was initiated. MMTV-CAT Transactivation. The MMTV-CAT vector (20 /*g) contain 154- ing the MMTV-LTR linked with the CAT gene was cotransfected with a 134- pRSVL vector (2 ng) into LNCaP 104-S, LNCaP 104-1, and LNCaP 104-R cells on 10-cm plates (5 plates/treatment) using the calcium phosphate pre cipitate method. The pRSVL vector contains the luciferase gene linked to the RSV LTR (40). Cells, incubated in DMEM supplemented with 10% CS-FBS, R1881 °55 "•°.°2 °5 5 " °.°2 »¿°- 2 °¿°^° were treated with l nM R1881 or ethanol at the time of transfection. Cells were (nM) harvested 60 h later. Cell extracts were isolated using reporter lysis buffer i E ii__f i (25 HIMbicine-0.05% Tween 20-0.05% Tween 80, pH 7.6; Promega), and 104-S 104-R luciferase activity was assayed according to the procedure supplied by Pro- B. 2.0 mega. Cell extracts were normalized within cell line groups for luciferase activity, and aliquots containing equivalent luciferase activity were used in 4-h og CAT reactions at 37°C.Acetylation of [14C]chloramphenicol was quantified after thin layer chromatography using the AMBIS radioanalytic imaging N< 1.5 system.

RESULTS •5= 1.0 S° Isolation of 104-1 and 104-R Cell Lines Exhibiting Altered Pro- liferative Response to Androgen. Cells making up the LNCaP prostate carcinoma cell line population display a heterogenous pro- II °'5 liferative response to androgen, indicative of variable changes taking CE n

mRNA, AR mRNA levels in 104-R cells were 2.5-fold higher than the levels detected in LNCaP 104-S cells grown in the absence of andro- KDa gen (Fig. 3). Incubation of 104-S cells in increasing concentrations of 215- R1881 resulted in a gradual reduction of AR mRNA level, while 105- -AR incubation of 104-R cells in 0.1 nin R1881 caused a sharp drop in AR mRNA level when the level of AR mRNA was normalized to the level 70- of ß2-MGmRNA. AR mRNA was not detected in the androgen- insensitive PC-3 cells (data not shown). Immunoblot analysis of AR content in LNCaP whole cell extracts 43- normalized by cell number showed that the level of the M, 100,000 AR in LNCaP 104-1 and 104-R cells grown in medium with R1881 o e (nM) androgen-depleted CS-FBS was about 3-fold and 15-fold higher, re 104-S 104-1 104-R spectively, than that of 104-S cells (Fig. 4). Growth of 104-S cells over a range of R1881 concentration resulted in a dose-dependent increase LNCaP PC-3 of AR protein content with maximal enhancement of nearly 5-fold B. observed with 10 nM R1881. This induction of AR protein level by androgen in 104-S cells is consistent with findings of Krongrad et al. l 16 (8), but the scale of the induction in 104-S cells is much greater. i R1881 treatment of 104-1 cells produced a maximal 2- to 3-fold E increase. Treatment of 104-R cells with R1881, on the other hand, 12 resulted in no increase over the already high basal level when 0.01 and 0.1 nM R1881 were used and reduced AR levels by 50% when 1 and o

A. BP

220- 201- O 0.01 0.1 1.0 10 R1881 (nM) 154- Fig. 4. A, ¡mmunoblotof total cellular proteins extracted from l X IO5 LNCaP 104 or 134- -P2-MG PC-3 cells separated on a 10% SDS-PAGE gel. Cells were grown for 4 days in DMEM supplemented with 10% CS-FBS and R1881 at the indicated concentrations. AR was incubated with rabbit polyclonal antibody AN-21 raised against the NH^-terminal 21 amino acids of human AR and was visualized using an enhanced chemiluminescence procedure (30-s exposure). Prestained protein molecular weight markers were from BRL. B, graphical rcperesentation of AR staining intensity relative to the intensity of the AR 75- band from 104-S cells grown in the absence of R1881. AR was quantified by optical densitometry of an autoradiograph obtained after a 10-s exposure. Proteins were extracted -PSA from LNCaP 104-S. 104-1, and 104-R cells at passages 20, 66, and 97, respectively, and PC-3 cells were extracted by the procedure described in "Materials and Methods."

DMNT > 5a-DHT » progesterone > 17ß-estradiol,whereas dexa- R1881 i- O O O o methasone had no effect. (nM) The proliferative response of LNCaP 104-S, 104-1, and 104-R cells 104-S 104-R to 0.1 nM R1881 was also examined by measuring anchorage-inde pendent colony growth in agarose in the presence of CS-FBS or untreated FBS (Fig. 2). While 0.1 nM R1881 was essential for signifi cant growth of 104-S colonies in 10% CS-FBS, R1881 increased only by about 75% the growth of 104-1 colonies and almost completely inhibited the growth of 104-R colonies. In the presence of untreated FBS that obviously contained endogenous androgens, addition of R1881 to media did not affect the growth of LNCaP colonies at any stage. Endogenous androgens may have been sufficient for maximal stimulation of the proliferation of 104-S and 104-1 cells and inhibition of 104-R cell proliferation. AR Expression in LNCaP 104 Cell Lines. To determine whether changes in AR expression and activity were concomitant with changes in the proliferative response of LNCaP 104 cells to androgen, AR mRNA level, protein level, and androgen induction of PSA mRNA .001 .01 0.1 1.0 10 100 level and MMTV activity were measured. Northern blot R1881 (nM) analysis of total RNA from LNCaP 104-S and 104-R revealed the Fig. 5. A, autoradiogram of RNase protection assay using 32P-labeled antisense PSA presence of the major form of AR mRNA, approximately 10 kilobases and ß2-A/CRNA probes to measure the induction of PSA mRNA expression in LNCaP in length (data not shown). For more sensitive measurement of AR 104-S and 104-R cells by R1881. Total RNA was isolated from LNCaP 104-S cells (passage 16) and 104-R cells (passage 95) grown in DMEM supplemented with 10% mRNA levels, RNase protection assay was performed using a 210- CS-FBS in the presence of R1881 at the indicated concentrations. B, graphical represen base antisense RNA probe. When normalized to the amount of ß2-MG tation of protected PSA probe normalized to the amount of protected ßz-MGprobe. 1569 ANDROGEN RECEPTOR ACTIVITY IN PROSTATE CANCER CELLS

0.20 12-fold in 104-1 cells, and 272-fold in 104-R cells. It is unclear why 104-1 cells exhibited the lowest induction of CAT activity, in light of 11 the results with PSA expression. It is possible that the apparent low induction by androgen in 104-1 cells reflects differences in the activity E£ SE of other factors involved in the basal and/or induced transcriptional <= C activity of the MMTV LTR. LNCaP 104-1 cells also exhibited the «i 0.10 lowest level of luciferase activity (Table 1). IS Effect of Androgen on c-myc Expression in LNCaP 104 Cell Lines. Androgen regulation of c-myc expression in LNCaP 104 cells 0.05 was examined to determine whether expression of this critical growth- related gene changed concurrently with the increase in AR expression and transcriptional activity and with the change in the proliferative R1881 - + + •f + response to androgen. Based on RNase protection analysis of c-myc <°-1nM) 104-S 104-1 104-R PC-3 mRNA levels, 0.1 nM R1881 induced the c-myc mRNA level over Fig. 6. RNase prelection assay using -'2P-labeled antisense PSA and ß2-MGRNA 2-fold in 104-S cells but reduced the c-myc mRNA level to less than probes to measure the induction of PSAmRNAexpression in LNCaP 104-S. 104-1,104-R. 20% of the control value in 104-R cells (Fig. 7). Treatment of 104-R and PC-3 cells by 0.1 HMR1881. Total RNA was isolated from LNCaP 104-S cells (passage 15), 104-1cells (passage 60), 104-R cells (passage 84), and PC-3 cells grown in cells with 0.01 nm R1881 caused a 25% increase over the basal c-myc DMEM supplemented with 10%CS-FBS in the absence or presence of 0.1 DMR1881. The mRNA level, which was nearly 2-fold higher than the basal level of protected PSA probe was normalized to the amount of protected ßi-MGprobe. c-myc expression in 104-S cells. Concentrations of R1881 higher than 0.1 nMreduced c-myc mRNA levels in 104-S cells, consistent with the Table 1 Induction of CAT activity in LNCaP 104 cells diminished effect of high R1881 concentration on 104-S cell prolif CAT activity, expressed as percentage conversion of [l4C]chloramphenicol to acety- eration. Expression of c-myc in intermediate stage 104-1 cells was lated forms, in LNCaP 104cells at different passage numbers (indicated by subscript) was measured after cotransfection of MMTV-CAT and pRSVL expression vectors. conversionCell104-S29 % BP1018-506' induction luciferase HMR18816.79(1.99)"1 by R188137.7 activity76(11.8)* -c-myc

UM4»104-R97-R18810.18(0.03)°0.12(0.02) 1.48(0.29) 12.3272Relative 1 (0.22) 396- 0.13(0.03)+ 35.3 (2.79)Fold 14(1.84)A. 344- " Numbers in parentheses, SE; n ~ 5. 298- '' Numbers ¡nparentheses, SE; n = 10.

220- 10 nM R1881 were used. AR was not detectable in PC-3 cells by 201- immunoblot analysis. AR Activity in LNCaP 104 Cell Lines. AR transcriptional activity in LNCaP 104 cells was assessed by two methods: (a) induction by 154- -fc-MG androgen of PSA mRNA level as measured by RNase protection 134- assay; and (b) induction by androgen of CAT activity in cells tran siently transfected with MMTV-CAT, an expression vector carrying the CAT gene linked with the androgen response element-containing R1881 *• T- T" O O O O *~ i~ f O O O MMTV-LTR (41, 42). The 5' regulatory region of the PSA gene, like o (nM) °dd-"° o the MMTV-LTR, has been shown to contain a functional androgen O II 104-S 104-R response element (43). PSA mRNA levels were responsive to R1881 B. over a range of concentration in LNCaP 104-S and 104-R cells (Fig. 5). However, the induction of PSA mRNA level by R1881 was up to 21-fold higher (with 0.1 nM R1881) in 104-R cells than in 104-S 0.5 cells. The level of PSA mRNA induction by androgen in 104-S cells co < is comparable to the induction by androgen of PSA mRNA in LNCaP 0.4 cells reported by others (37, 44, 45). Induction of PSA mRNA level by ¡i 0.1 nM R1881 in LNCaP 104-1 cells was intermediate with respect to 11 0.3 the induction observed in 104-S and 104-R cells (Fig. 6). The basal level of PSA mRNA expression was slightly higher in untreated 104-1 oc 2 0.2 and 104-R cells than in untreated 104-S cells, possibly reflecting the E.y sensitivity of AR in the later stage 104 cells to residual androgen in the i 0.1 medium. AR-mediated induction of PSA mRNA level, however, is clearly androgen-dependent in 104-1 and 104-R cells. Expression of PSA mRNA was not observed in PC-3 cells. O .001 .01 0.1 1.0 10 100 An increase in AR activity in LNCaP 104-R cells but not in 104-1 R1881 (nM) cells was observed in LNCaP 104 cells transiently transfected with the Fig. 7.A, RNase protection assay using 32P-labeIedantisense c-myc and ß?-MGRNA MMTV-CAT expression vector (Table 1). To normalize within a cell probes to measure the inductionor repression of c-mycmRNAexpression in LNCaP 104-S type for plate-to-plate variation in transfection efficiency, cell number, and 104-R cells over a range of R1881 concentration. Total RNA was isolated from and harvesting efficiency, cells were cotransfected with pRSVL, a LNCaP 104-S cells (passage 16) and 104-R cells (passage 95) grown in DMEM supple mented with 10% CS-FBS ¡nthe presence of R1881 at Ihe indicated concentrations. B. vector in which expression of the luciferase gene is driven by the RSV graphical representation of protected c-myc probe normalized to the amount of protected LTR (40). R1881 induced CAT activity nearly 38-fold in 104-S cells, Bi-MG probe. 1570 ANDROGEN RECEPTOR ACTIVITY IN PROSTATE CANCER CELLS

virus carrying the c-myc gene linked with the enhancer/promoter of 104-S 104-R the Moloney murine sarcoma virus long terminal repeat. Cell prolif eration of infected and uninfected cells in the presence or absence of 20 nM R1881 in 10% untreated FBS was compared (Fig. 9). In unin fected LNCaP 104-S, 104-1, and 104-R cells, cell numbers were reduced to about 50% of the control values by R1881. In cells over- expressing c-myc, R1881 inhibition of proliferation was not observed. The protection from growth inhibition was c-myc dependent since proliferation of LNCaP 104-S cells infected with retrovirus not ex pressing c-myc was reduced to a similar level by 20 nM R1881 as in uninfected 104-S cells. In comparison with 104-S cells, cells at the later stages of passage (104-1 and 104-R), uninfected or retrovirally R1881 FBS infected, exhibited a progressive decline in proliferation rate when 0.1 nM - C-treated grown in medium supplemented with 10% untreated FBS. In unin 20 nM - +1, Untreated 104-S 104-R fected cells, this decline may have been due to the fact that the cells at the later passages were more sensitive than 104-S cells to endog Fig. 8. RNase protection assay using 12P-labeled antisense c-myc and ß2-MGRNA probes to measure the induction or repression of c-myc expression in LNCaP 104-S and enous androgens present in untreated FBS. However, because this 104-R cells by 0.1 nM R1881 (in 10% CS-FBS) and 20 nu R1881 (in 10% untreated FBS). progressive decline in proliferation is also observed in cells overex- Total RNA was isolated from LNCaP 104-S cells (passage 14) and 104-R cells (passage pressing c-myc, the phenomenon may not be mediated through c-myc. 84) grown in the presence of R1881 at the indicated concentrations. The amount of protected c-myc probe was normalized to the amount of protected ß2-MGprobe. Changes in Morphology of LNCaP 104 Cells. LNCaP 104 cells underwent a change in morphology in the transition from 104-S to 104-1 and 104-R stages. LNCaP 104-1 and 104-R appeared progres sively more rounded and less spindle-shaped than 104-S cells, which Uninfected MV7 MV7-c-myc resembled the morphology of the parental LNCaP population. In addition, 104-1 and especially 104-R cells tended to grow as rounded clusters or islands of cells instead of a uniform monolayer. In this T "o 6 respect, they are similar to the LNCaP FGC clone 22 cells, which were T- described as an androgen-independent subline by van Steenbrugge et X al. (6). However, LNCaP 104-1 and 104-R cell clusters do not exhibit 1« the "flattened character" of clone 22 cells.

DISCUSSION It is well recognized that proliferation of LNCaP cells in steroid- R1881 depleted medium is induced at an optimal androgen concentration, - + - 4- - + 20 nM above which diminished proliferation is observed (5, 12, 47). Prolif LNCaP 104-S I I eration in untreated FBS-supplemented medium is repressed by mod Fig. 9. Repression of LNCaP 104 cell proliferation by 20 nM R1881 and blockage of erate to high concentrations of androgen (16), particularly when meta- repression by retroviral c-myc overexpression. Cells were plated in triplicate wells in bolically stable androgens such as R1881 and DMNT are used. The DMEM supplemented with 10% untreated FBS, and cell densities were determined after 6 days of growth in the presence or absence of 20 nM R1881. LNCaP 104-S, 104-1, and use of metabolically stable R1881 in this study made it possible to 104-R cells were tested at passages 14, 71, and 95, respectively. LNCaP 104-S MV-7 cells analyze androgen concentration-dependent changes in the biphasic were infected with retrovirus derived from pMV7 vector containing no insert. LNCaP 104-S. 104-1, and 104-R MV7-c-myr cells were infected at passages 15, 62, and 88, response (6, 47, 48) over the period of androgen deprivation. The fact respectively, with retrovirus-derived pMV7 vector containing a human c-myc cDNA that the concentration of R1881 required for induction and repression insert. of the proliferation of LNCaP 104 cells gradually decreased as the cells were passaged in steroid-depleted medium suggests that these negligibly affected by 0.1 DMR1881 (data not shown), mirroring the cells gradually increased their sensitivity to lower concentrations of pattern of R1881 regulation of LNCaP 104 cell proliferation. There androgen (Figs. 1 and 2). It is important to note that although optimal fore, the altered proliferative response to androgen and increase in AR induction of proliferation of 104-R cells by R1881 was less than that expression and transcriptional activity in LNCaP 104 cells during the of 104-S cells (2-fold compared with 10- to 13-fold), optimal induc transition from 104-S to 104-R stages were accompanied by a shift to tion was observed at a 10-fold lower concentration of R1881. Addi higher sensitivity in the pattern of androgen regulation of c-myc ex tionally, the basal proliferation rate of untreated 104-R cells was much pression. When 104-S and 104-R cells were grown in the presence of higher than that of 104-S cells. The levels of AR mRNA and protein untreated FBS, R1881 at high concentration (20 nM) reduced the level were significantly higher in 104-R cells than in 104-S cells. LNCaP of c-myc mRNA to about 25-30% of the level of untreated 104-S and 104-R cells, therefore, can be considered to have enhanced prolifera 104-R cells (Fig. 8). This result again is consistent with the inhibitory tive sensitivity to androgen. Enhanced androgen sensitivity is sup effect of R1881 at high concentrations on LNCaP cell proliferation ported by the progressively increased induction of androgen respon (16, 46). Southern blotting of genomic DNA isolated from 104-S, sive genes, PSA, and the MMTV-CAT reporter gene in 104-R cells 104-1, and 104-R cells revealed neither rearrangement nor amplifica compared with that in 104-S and 104-1 cells. Enhanced sensitivity to tion of the c-myc gene (data not shown). R1881 may be the additive result of increased expression of the AR Effect of Retroviral c-myc Expression on Androgen Control of mRNA and protein (Figs. 3 and 4) and increased transcriptional ac LNCaP 104 Cell Proliferation. To determine the effect of nonregu- tivity of AR in 104-R cells. The approximately 5-fold increase in the lated overexpression of c-myc on androgen control of LNCaP 104 cell AR protein level of 104-R cells over 104-S cells in the presence of proliferation, 104-S, 104-1, and 104-R cells were infected with retro- R1881 probably cannot account for the 21-fold higher induction of 1571 ANDROGEN RECEPTOR ACTIVITY IN PROSTATE CANCER CELLS

PSA mRNA (Fig. 5) observed in 104-R cells. Therefore, other possi possible that androgen affects c-myc mRNA levels in LNCaP cells bilities should also be considered. For example, these cells could have through multiple pathways. changes in AR phosphorylation (49-51), in the activity of transcrip The mechanism by which the synthetic androgen R1881 can elicit tion factors or AR-associated factors (52-54), and in androgen binding opposite proliferative responses at different concentrations is not pres properties which could alter intranuclear localization and the half-life ently understood. However, the idea that the LNCaP cell line is a rare of AR (51,55). example of a type of prostate tumor, the growth of which is only The increase in AR expression and activity may be viewed as an inhibited by androgen (16), is not consistent with our observation that adaptive response of LNCaP 104 cells to exceedingly low concentra during the adaptation to low androgen concentration, LNCaP 104-S tions of available androgen (15). This stands in contrast to the loss of and 104-1 cells progress to 104-R cells characterized by increased AR androgen sensitivity in Shionogi 115 mouse mammary tumor cells expression and activity and enhanced sensitivity to androgen. If an after androgen withdrawal (4, 56). The increased expression of AR drogen exerted only negative selective pressure, cells deprived of mRNA in 104-R cells after long-term androgen deprivation is consis androgen probably would not increase their sensitivity to it over time. tent with the repressive effect of androgen on AR mRNA levels in If the type of adaptation in AR expression and activity exhibited by LNCaP cells observed after short-term androgen treatment (Fig. 3; LNCaP 104 cells is a general phenomenon, the clinical implications Refs. 7, 9, and 45). This adaptive response of LNCaP cells to low are significant. Some prostatic tumor cells under the selective pressure androgen concentration offers no information about how some pros of antiandrogen and/or androgen ablation therapy may be able to adapt tate tumor cells and established cell lines such as PC-3 and DU 145 to lower androgen concentration (such as the level supplied by adre have lost AR expression. It is possible that loss of AR expression nals in castrated patients) by increasing the transcriptional activity or steroid affinity of AR and thereby overcome the therapy. Additionally, occurs after alterations in the expression of specific genes involved in adaptation to lower androgen availability may allow cancer cells to control of cell proliferation take place which make AR expression nonessential for and survival. For example, PC-3 cells survive and proliferate during therapy until other events occur which enable cells to bypass the androgen requirement altogether. Prolifera have been shown to lack expression of (57), and DU 145 cells tion of prostate cancer cells which are hypersensitive to very low express an inactive form of the retinoblastoma gene product (57, 58). concentrations of androgen may in fact be repressed by moderate Recently, Yuan et al. (59) reported that the proliferation of PC-3 cells concentrations of androgen. The molecular mechanisms involved in stably transfected with an AR expression vector is inhibited by an the transition from 104-S to 104-R stages and the effects of changes drogen. This inhibition may be analogous to the inhibition by andro in AR expression and activity on regulation of c-myc and other critical gen of LNCaP 104-R cell proliferation described in this paper. growth-related genes in LNCaP cells are currently under investigation. The induction and repression of cell proliferation by androgen in LNCaP 104 cells appear to be mediated chiefly, although perhaps not exclusively, through a pathway involving the regulation of c-myc ACKNOWLEDGMENTS expression. Induction and repression of c-myc expression in LNCaP 104-S and 104-R cells by 0.1 nM R1881 correlated directly with the We thank Ching Song and Richard Hiipakka for assistance in the prepara tion and purification of the AN-21 antibody. induction and repression of cell proliferation by R1881 at the same concentration, with the exception that no induction of c-myc mRNA REFERENCES level was observed in 104-R cells treated with 0.01 nM R1881. Re 1. Carter, H. B.. and Coffey, D. S. The prostate: an increasing medical problem. Prostale. pression of cell proliferation by 20 nin R1881 was effectively over 16: 39-48, 1990. come by exogenous retroviral overexpression of c-myc. The mecha 2. Muggins. C, and Hodges, C. V. Studies on prostate cancer. I. The effect of castration, nism by which androgen receptor regulates c-myc mRNA level in of estrogen, and androgen injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res., 1: 293-297, 1941. LNCaP cells is not understood. 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