Mibolerone and Processed for Either Mitogens Activate the Various Sequential Players in the Cell Cycle Fluorescence-Activated Cell Sorting Or Western Analysis

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[CANCER RESEARCH 56, I5.W-1544. April I. 1 Androgens Regulate the Expression of Proliferating Cell Nuclear Antigen Posttranscriptionally in the Human Prostate Cancer Cell Line, LNCaP Jaime E. Perry and Donald J. Tindall1

Departments of Urology Research Â¡J.E. P.. D. J. T.I and Biochemistry/Molecular Biology [D. J. TJ. Mayo Clinic Foundation. Rochester. Minnesota 55905

ABSTRACT for DNA polymerase 5-driven DNA synthesis (7, 8). This ubiquitous protein is cell cycle regulated. PCNA synthesis reaches a maximum Proliferating cell nuclear antigen (PCNA) expression is required for during the S phase of the cell cycle (9) and is required for DNA DNA replication. Because androgens are critical for prostate cell prolif synthesis and cell division ( 10). PCNA. as an index of tumor proliferation, we investigated the effects of androgen on PCNA expression in the erative activity, is used to gain insight into the process of neoplastic prostatic cancer cell line LNCaP. Flow cytometric analysis was used to measure cellular DNA content with dual labeling of PCNA. Semiconfluent progression and clinical patient management (II). LNCaP cells were grown in serum-free medium containing varying con Thus far, little is understood about the mechanisms by which centrations of the synthetic androgen mibolerone and processed for either mitogens activate the various sequential players in the cell cycle fluorescence-activated cell sorting or Western analysis. Supplementation events necessary for cell growth. The regulation of PCNA expression of serum-free medium with androgens resulted in dose-dependent changes appears to be cell type specific; epidermal growth factor and platelet- in PCNA immunorcactivity, with maximum stimulation (2-fold) being derived growth factor in BALB/c3T3 cells induce PCNA mRNA (12). achieved at 48 h with IO "Mmibolerone. Non-androgenic steroids did not whereas in thyroid epithelial cells, PCNA protein expression is in change PCNA immunoreactivity compared with untreated controls, and duced by thyroid-stimulating hormone via cAMP (13). the antiandrogen, casodex, inhibited the mibolcrone-stimulated increase In this paper, we present evidence for the androgenic regulation of in PCNA immunoreactivity, suggesting that the androgenic induction of the expression of PCNA protein in LNCaP cells. Our data suggest that PCNA is mediated through the androgen receptor. The presence of a non-consensus androgen response element in the promoter region of the it is unlikely that this regulation involves transcriptional events, since PCNA gene led us to investigate whether androgen responsiveness of the no change was observed in steady-state mRNA for PCNA in LNCaP PCNA gene in LNCaP cells might be mediated at the transcriptional level. cells with androgen treatment. We show that androgen treatment No change in steady-state mRNA for PCNA with androgen administration increases the expression of PCNA protein by at least two mechanisms: was observed. However, an investigation of the androgenic regulation of (a) an increase in its half-life as assessed by immunoprecipitation of PCNA protein stability indicated that androgen treatment increased the radioactively-labeled PCNA with androgen treatment; and (/;) an half-life of J5S-labeled PCNA protein. In addition, polysome run-off trans increase in the translational efficiency of PCNA as assayed by poly- lation assays demonstrated an increase in PCNA protein after a 6-h stimulation of LNCaP cells with 10~*M mibolerone. These data suggest some run-off translation. The regulation of PCNA protein expression by androgens in LNCaP cells likely occurs through the interaction of that androgen induction of prostate cell proliferation may be mediated, at androgens with the androgen receptor through posttranscriptional least in part, through PCNA at the posttranscriptional level. mechanisms.

INTRODUCTION MATERIALS AND METHODS Prostate cancer ranks among the leading causes of death in men. Cells and Culture Conditions. LNCaP cells (ATCC, Rockville, MD) were Since both secretory functions and growth of the adult prostate are maintained in RPMI 1640 with glulamine and sodium bicarbonate. The me androgen regulated, the mechanism by which androgens support the dium was supplemented with the antibiotics penicillin and streptomycin, which growth of prostatic tissue in benign and malignant pathologies is an were added to a final concentration of 50 /j.g/ml and PCS (Biofluids. Rockville. important area of focus. Changes in the growth process between MD) to 5%. Cells were used between passages 25 and 50 after attaining about normal and malignant prostatic cells may provide important clues as 70% confluence. Cells were incubated in serum-free medium for 3 days prior to the best possible treatments for metastatic disease or even prevent- to treatment. Treatments were added with a change of serum-free medium. ative strategies. Treatments included mibolerone (DuPont/New England Nuclear. Boston. The androgen-sensitive cell line LNCaP. derived from a lymph MA), casodex (ICI 176.334: Zeneca Pharmaceuticals, Wilmington, DE). R5020 (New England Nuclear, Boston, MA) . diethylstilbestrol, dihydrotest- node carcinoma of the prostate ( 1), retains many of the characteristics osterone. 17-ÃŸ-estradiol, and dexamethasone (all from Steraloids. Wilton. of prostatic epithelial cells. LNCaP cells respond to androgen stimu NH). Control cells were treated with the volume of ethanol used to deliver the lation by transcriptionally activating the genes for, and subsequently secreting, prostate-specific glandular kallikreins (PSA2 and hK2: steroids (final concentration, 0.01%). Flow Cytometric Analysis. Flow cytometry was performed on LNCaP Refs. 2-4), and by increasing cell proliferation (5). This cell line cells as follows: cells were collected, washed, and dispersed in PBS. Disper provides a useful transition between normal prostatic epithelial cells sion of the cells into a single-cell suspension was accomplished by drawing the and other established prostate cancer cell lines that no longer express cells in PBS 10 times through a 26-gauge. blunt-ended needle. An aliquot of the androgen receptor and are. therefore, androgen insensitive. the cells was set aside for cell counts using a Coulter counter (Coulter Two DNA polymerases, a and 8, are essential for DNA synthesis Electronics, Hialeah. FL). The rest of the cells were fixed in \c/c paratormal- (6) and cell proliferation. PCNA serves as a requisite auxiliary protein dehyde in PBS with 30 /j.g/ml lysolecithin (ICN Biochemicals. Cleveland. OH) added to permeabilize the cells. The cells were washed in buffer A (3% BSA in PBS with 0.05% Tween 20), split into two sets, and incubated overnight Received 6/26/95; accepted 1/31/96. with either anti-PCNA (PCIO clone; 1:10; DAKO, Carpenteria, CA) or anti- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked tulvi'rlisemenl in accordance with PSA (1:100; Hybritech, San Diego. CA) diluted in buffer A. After washing in 18 U.S.C. Section 1734 solely lo indicate this faci. buffer A, the cells were incubated for l h with a FITC-conjugated second 1To whom requests for reprints should be addressed, at Department of Urology antibody (GAM-FITC; 1:100; Organon Technika/Cappel. West Chester. PA) Research. Mayo Clinic Foundation. Rochester. MN 55905. Phone: (507) 284-8553; Fax: (507) 284-2384; E-mail: [email protected]. diluted in buffer A. washed, and treated with RNase (1 mg/ml: Sigma Chem 2 The abbreviations used are: PSA. prostate-specific antigen; PCNA, proliferating cell ical Co., St. Louis, MO) for 15 min at 37Â°C.Cells were suspended in buffer A nuclear antigen; GAPDH. glyceraldehyde-3-phosphate dehydrogenase. with 0.1 mg/ml final concentration of propidium iodide and analyzed on a 1539

FACStar Plus flow cytometer (Beclon Dickinson. Sunnyvale, CA) at a wave Polysome Run-Off Translation Assay. LNCaP cells at 60-70% conflu length of 488 nm with a 620-nm pass filter. ence were placed in serum-free medium for 48 h. The medium was changed; Northern Analysis. Northern analysis was performed on RNA isolated by then 24 h later, mibolerone treatments were added for 12 h. Polysome isolation the lithium chloride/urea method (14). LNCaP cells were disrupted in 3 M was performed according to the method of Taylor and Schimke (17) with lithium chloride and 6 M urea. The DNA was sheared with an Ultra-Turrax minor modifications. Cells were collected and homogenized in cold buffer B homogenizer (24.000 rpm; Janke and Kunkel. Staufen, Germany). The solution (50 mM Tris-Cl, 25 mM NaCI. and 5 mM MgCU, pH 7.7; I ml/TI75 flask at was left overnight at 4Â°C, and RNA was separated by centrifugaron at 70% confluence) containing 0.25 M sucrose and 500 /xg/ml sodium heparin 107,000 X g. The pellet containing the RNA was dissolved in 0.1% SDS and (Sigma; 170 USP units/mg). The homogenate was centrifuged at 9,500 rpm (14,600 X Â£max)for 10 min at 4Â°C.One-tenth volume of 10% Triton X-100/ 0.2 mM EDTA and extracted with phenol:chloroform (1:1) and chloroform. The RNA was precipitated with 0.16 M sodium acetate in ethanol. Purified, 10% SDS was added to the supernatant and mixed in a homogenizer. Five ml total RNA (20 jig/lane) was separated in a 1% denaturing agarose gel and of supernatants were overlain on a discontinuous sucrose gradient of 2 ml 2.5 transferred to Hybond N blotting membrane (Amersham. Arlington Heights. M sucrose. 4 ml 1.0 M sucrose, and 0.6 ml 0.5 M sucrose, where each sucrose IL). Blots were cross-linked using an automatic UV Stratalinker (Stratagene. solution was made with buffer B and 500 /ng/rn' sodium heparin. Tubes were La Jolla. CA), prehybridized for I h at 42Â°Cin prehybridization solution (45% centrifuged at 41.(XX)rpm (2(X),(XX)X Aav)for 90 min at 4Â°C.Polysomes were deionized formamide, 5X SSC. 0.5% SDS, 10% Denhardt's solution, 10 mM collected at the 2.5 Msucrose interface in an opalescent band that was aspirated phosphate buffer, 15% dextran sulfate, and 100 mg/ml herring sperm DNA). in 0.8 ml using a syringe and needle placed 5 mm below the interface. The Randomly [12P]-labeled probes prepared from cDNAs to PCNA, PSA, or polysome fraction was diluted with 4 volumes of buffer B and microfuged for 5 min at 4Â°C.The polysomes were resuspended in buffer B. and the A,W,:A,KO GAPDH were added to the blots in prehybridization solution and incubated overnight at 42Â°Cin a shaking waterbath. Blots were washed twice in 1x SSC ratio was measured (approximately 1.9). containing 0.25% SDS at 42Â°Cand once at 55Â°Cbefore being exposed to The polysome run-off assay was performed with minor modifications to the X-ray film X-Omat AR (Kodak. Rochester. NY). Films were scanned for a method of Srivastava (18). The wheat germ translation system from Amersham two-dimensional assessment of the relative expression of each RNA. PCNA was used for the in vitro translation. The reaction was performed in a total volume of 50 /j.1containing 1 ^.1 1 M KCI. 3.4 /Â¿I1 mM amino acid mixture and PSA mRNA data were normalized to GAPDH. (minus methionine). 25 /il wheat germ extract, 2.5 A26agonist, mibolerone. At androgen concen twice with cold PBS before unlabeled medium was replaced as a "chase." The trations of 10~9 Mand greater, the percentage of cells staining positive chase was performed in the presence or absence of 1 nM mibolerone and was with PCNA antibody by flow cytometric analysis was significantly allowed to proceed for various lengths of time before cells were collected, and increased (2-fold) over nontreated cells at 48 h. as seen in Fig. 1. An PCNA was immunoprecipitated as follows. Cells were washed twice with PBS. Cold lysis buffer [50 nM Tris-HCI (pH 7.4). 150 mM NaCI, 20 nM EDTA. increase in PCNA immunoreactivity per microgram of cell homoge 0.5% NP40, 1 mM phenylmethylsulfonyl fluoride, 25 ng/ml leupeptin, 25 nate was demonstrated by Western blot analysis at lower concentra tions of mibolerone, with an increase in PCNA expression detectable j^g/ml aprotinin, 1 mM benzamidine, and 10 ng/ml trypsin inhibitor] was with mibolerone treatment as low as 10~'Â°M (0.1 nM; Fig. 2Â«).The added, and the cells were lysed by rotation in this buffer for 20 min. Nuclei were removed by microfuging for 5 min in the cold. The lysates were difference in effective doses demonstrated in Figs. 1 and 2u may result precleared by incubating with normal rabbit serum and protein G-agarose from a difference in the sensitivity of one technique versus the other. (Boehringer. Mannheim. Germany) for 30 min with rotation in the cold, Flow cytometry measures immunoreactivity in each cell by fluores followed by a 10-minute microfugation. During this incubation. anti-PCNA cence, whereas Western analysis measures immunoreactivity in an (PC 10 clone; DAKO) was diluted 1:5 in lysis buffer and precoupled to protein homogenate of cells separated by gel electrophoresis. Fig. 2b demon A-agarose (Boehringer) with rotation. Precleared lysates were incubated over strates the specificity of the PC 10 anti-PCNA monoclonal antiserum night with the protein A-agarose precoupled to anti-PCNA. Negative controls for PCNA. Preabsorption of the antiserum with a peptide fragment included omitting the anti-PCNA antiserum and adding PCNA peptide frag derived from the PCNA protein corresponding to the epitope recog ments to the overnight incubations. PCNA was immunoprecipitated by mi nized by the monoclonal antiserum specifically eliminated detection crofuging the lysates for 10 min. The immunoprecipitates were washed three times with lysis buffer at room temperature, then resuspended in SDS sample of the M, 36.000 PCNA band by Western analysis. buffer and separated on 10% SDS-polyacrylamide gels. Dried gels were The Androgenic Effect on LNCaP PCNA Expression Is Steroid exposed overnight on Phosphorlmager screens (Molecular Dynamics. Sunny Specific. The dose-dependence of the PCNA response suggested that vale, CA). Radioactive bands were analyzed within the linear range of the the increase in PCNA protein with androgenic stimulation of the cells Phosphorlmager 425 (Molecular Dynamics, Sunnyvale. CA). may be mediated through the androgen receptor. The androgen recep- 1540

100-, Additional corroboration that the induction of PCNA may be me diated via the androgen receptor is demonstrated by the ability of an anti-androgen to block androgen-induced PCNA protein expression (Fig. 4). The anti-androgen, casodex, at concentrations of 1 and 2 /AM 80- had no effect on PCNA protein expression in LNCaP cells when administered alone (Fig. 4a). However, casodex administered in com bination with 0.1 nM mibolerone prevented the stimulation of PCNA 60- protein expression by mibolerone as demonstrated by Western anal ysis (Fig. 4Â¿>). The Increase in PCNA Expression Correlates with Cell Num ber and PSA Secretion. Further evidence that the increase in PCNA 40- expression in LNCaP cells is mediated through the androgen receptor is presented in Fig. 5a-c. Flow cytometric analysis of PCNA expres sion (Fig. 5a) correlated with an increase in cell number (Fig. 5b) and 20- with the secretion of the androgen-regulated protein, PSA (Fig. 5c) after androgen administration. In Fig. 5a, the time course of PCNA expression with mibolerone treatment is shown. The number of PCNA-positive cells as measured by flow cytometric analysis was

0.0 0.01 0.1 1.0 10.0 100.0

Mibolerone Concentration (nM) 100-,

Fig. 1. The androgen dose-dependence of LNCaP cell PCNA expression. Flow cyto metric analysis of PCNA immunoreactivity in androgen-treated LNCaP cells. LNCaP cells were treated for 48 h with 0-100 nM mibolerone (KL = 1 nM) and then were 80- collected, fixed, and processed for flow cytometric analysis. *, significant differences in the numbers of cells staining for PCNA compared with untreated controls (P s 0.02; n = 4; one way ANOVA with Duncan's procedure). 60- a. b. O .01 .1 l 10 10 [Mibolerone] nM 40- 69 -

46 - 20- PCNA 30 -

21 - Control Mib DHT E2 R5020 DES Dex Fig. 2. Western analysis demonstrating the androgen dose-dependence of LNCaP cell PCNA expression. LNCaP cells were treated for 24 h with doses of mibolerone ranging from 0 to 10 nM (KL = I nM). Cells were collected and homogenized in the presence of Treatment protease inhibitors. Twenty /Â¿gprotein from cell homogenates were loaded into each lane Fig. 3. Steroid specificity of PCNA expression in LNCaP cells. Flow cytometric and separated by PAGE. Two lanes are shown for each concentration of mibolerone used analysis of PCNA immunoreactivity in LNCaP cells treated for 48 h with 1 nM concen to stimulate PCNA expression. In a, PC-10 antiserum was used at a dilution of 1:500. The trations of mibolerone (Mib), dihydrotestosterone (DHT), 17-ÃŸ-estradiol, R5020, dieth blot shown in b shows a side-by-side preparation that was incubated with PC-10 antiserum ylstilbestrol (DES), dexamethasone (Dex), or vehicle (Control). Bars, SEM of four that had been exposed to a peptide fragment from the PCNA molecule for l h prior to experiments. *. significant increase over control group (P s 0.01 ; one-way ANOVA with dilution and exposure to the blot. The positions of molecular weight markers are indicated Duncan's procedure). by bars to the left of the blot and are given in kilodaltons. Arrow, the position of the 36-kDa PCNA protein band. a. 0.1 0 00 [Mibolerone] nM tor in LNCaP cells contains a mutation in the steroid binding domain O O 1 2 [Casodex] uM (19, 20) that effectively broadens its specificity for steroids, notably estrogen and progesterone (21), although the receptors for estrogens and progestins are lacking in LNCaP cells (22). However, certain steroid agonists (e.g., R5020, diethylstilbestrol, and dexamethasone) are unable to bind and activate the mutated LNCaP androgen receptor. b. o.i 0 0.1 0.1 [Mibolerone] nM Therefore, a series of steroid agonists was used to determine whether 0 0 1 2 [Casodex] |iM the increase in PCNA expression in LNCaP cells is steroid specific. Flow cytometric analysis confirmed that mibolerone, dihydrotestosterone, and 17-|3-estradiol significantly increase the percentage of PCNA-positive cells (Fig. 3), whereas neither the progesterone ago Fig. 4. Western blot analysis of casodex inhibition of PCNA induction by mibolerone. nist R5020, the estrogen agonist diethylstilbestrol, nor the glucocor- LNCaP cells were treated for 20 h with 0. 1, or 2 /IM casodex or 0.1 nM mibolerone (a) ticoid agonist dexamethasone increased the percentage of cells stain or with the above concentrations of casodex in combination with 0.1 nM mibolerone (b). Twenty jag of protein were loaded per lane. PCNA was detected with the monoclonal ing positively for PCNA above that seen in control cells treated with anti-PCNA serum (PC10) at 1:500 using the ECL reaction (dilution of second antibody vehicle alone. was 1:10,000). 1541

strates that the androgenic stimulation of PSA secretion into the culture medium stimulated by mibolerone followed a similar time course. Androgens Do Not Affect Steady-State mRNA Levels of PCNA. Our observations that the androgenic stimulation of PCNA protein expression in LNCaP cells is: (a) mediated through the androgen + Mibolerone receptor, a known transcription factor; and (b) correlated with the - Mibolerone secretion of PSA, a protein transcriptionally regulated by androgens, led us to investigate the possibility that PCNA mRNA expression may be under androgenic regulation. We used Northern analysis to study the effect of androgens on PCNA mRNA expression. A partial cDNA corresponding to the 850-bp 3' fragment of the full-length 1.4-kb 1 2 3 PCNA cDNA was used to probe Northern blots. Full-length PSA and GAPDH cDNA probes were used as positive treatment controls and Days of Treatment loading controls, respectively, on blots prepared side-by-side. Fig. 6 shows representative Northern blots of RNA collected from LNCaP cells following 9 h of treatment with 0-100 nM mibolerone. Similar blots were prepared from LNCaP cells treated with mibolerone over the course of 24 h. A graph summarizing data collected from Northern blots demonstrates that concentrations of mibolerone as low as 10" " M at 24 h induced at least a doubling of the PSA mRNA in LNCaP cells (Fig. 7); however; at no time did any concentration of mibo- + Mibolerone - Mibolerone o o o IH [Mibolerone] nM â€¢¿ o o 123 PCNA Days of Treatment PSA

{ | GAPDH s Fig. 6. Androgen-independence of PCNA mRNA expression in LNCaP cells. LNCaP T? + Mibolerone g cells were treated for 9 h with 0-100 nM mibolerone prior to RNA isolation by lithium - Mibolerone chloride extraction. Twenty fig total RNA were loaded in each lane of a denaturing gel. a blotted, and probed for expression of PCNA. PSA. and GAPDH with specific cDNA probes. Androgen regulation of PSA expression is evident; PCNA and GAPDH mRNA levels are not androgen regulated.

Dose of _ Days of Treatment Mibolerone us â€”¿D 0.01 nM Fig. 5. Time course of androgen-stimulated responses in LNCaP cells. LNCaP were treated with 1 nM mibolerone for up to 3 days. Cells were collected at each 24-h interval, â€”¿â€¢â€”0.1nM assessed for PCNA immunoreactivity by flow cytometric analysis (a) and counted to "3â€”¿ assess cell proliferation (b}\ media was analyzed for secreted PSA (c). Bars, SEM of three ' â€”¿Oâ€”1.0 nM experiments. *, significant differences (P ^ 0.01; one-way ANOVA with Duncan's procedure) compared with cells treated for 0 days. â€”¿â€¢ 10.0 nM Z K â€”¿A 100.0 nM significantly increased within 1 day of treatment with the androgen agonist, mibolerone. At 2 days, the androgen-stimulated increase in PCNA-positive cells reached a maximum with a doubling in the number of cells staining. These results are mirrored by the data shown in Fig. 5b, the time course of mibolerone-stimulated cell proliferation. Time (Hours) Mibolerone significantly increased LNCaP cell numbers within 1 day Fig. 7. Summary of Northern analyses for the time course and dose-responsiveness of of treatment. The numbers of cells collected at 1,2, and 3 days PSA steady-state mRNA levels to androgen treatment. Graphic representation of data scanned from blots such as that depicted in Fig. 6. Data were normalized to GAPDH following initiation of mibolerone treatment were significantly in expression as a loading control and expressed in terms of fold induction over untreated creased compared with time-matched control cells. Fig. 5c demon control. The androgen-independence of PSA steady-state mRNA is shown. 1542

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1996 American Association for Cancer Research. ANDROGENS REGULATE PCNA IN LNCaP CELLS lerone tested alter the steady-state level of PCNA mRNA in the Table 1 Quamitaiion ofi!tS-labeled PCNA protein Â¡mmunoprecipilated following a polysome run-off experiment LNCaP cells. In vitro translation using |35S]methionine was performed on polysomes collected from The Increase in PCNA Expression with Androgen Stimulation either untreated cells or cells treated with 10~9 M mibolerone (Mib) for 12 h. M, 36.000 Results Partly from an Increase in Protein Stability. Having ruled radioreactive bands were quantified using a Phosphorlmager and are expressed as vol umes of 35S-labeled PCNA bands. Data were analyzed by one-way ANOVA with out an increase in mRNA as the mechanism by which androgens Duncan's procedure; P s 0.001. increase the expression of PCNA in LNCaP cells, we investigated the Treatment"Control136814181281124311851259Ave* possibility that androgens alter the half-life of the PCNA protein. The half-life of PCNA was measured in the presence and absence of ''MMÃ¬h1939205818711964190420031956 10~9 Mmibolerone by labeling LNCaP cells with ['"'Slmethionine and cysteine for 8 h. Cells were harvested at different time periods after replacement of the labeled medium with unlabeled medium, and 35S-labeled PCNA was immunoprecipitated. The results suggested that androgen treatment of LNCaP cells labeled with [15S]methionine Â±SE 1292Â±3510 Â±28 and cysteine did affect the rate of clearance of radiolabeled, immu- " Volume of ~ S. noprecipitable PCNA. The PCNA band cleared with a tlf2 of 23.1 h in ''Average fold increase in translated PCNA with 10' M mib = 1.5. the absence of mibolerone, whereas treatment with mibolerone during clearance extended the ti/2 to 32.7 h (Fig. 8). Analysis of the slopes of the lines by regression analysis demonstrates a significant difference treatment appears to be mediated through the androgen receptor since in clearance rates (P < 0.005). the response: (a) is inhibited by an antiandrogen; (b) is dose depend The Increase in PCNA Expression with Androgen Stimulation ent; (c) is steroid specific; and (d) follows the same time course as Results Partly from an Increase in the Translational Efficiency of other androgen-dependent processes that require the presence of a PCNA. The increase in PCNA half-life does not account for the functional androgen receptor. This is the first report to our knowledge observed 2-fold increase in PCNA expression with androgen treat of the androgenic regulation of the expression of any cyclically ment of LNCaP cells; therefore, we suspected the involvement of expressed protein known to be involved in cellular proliferation. other posttranscriptional mechanisms. We tested the hypothesis that We addressed the question of how the increase in PCNA protein is an increase in translational efficiency of PCNA within the androgen- accomplished through androgenic stimulation. The androgen receptor stimulated LNCaP cells may play a role in the increase in PCNA is a known transcription factor that acts through androgen response expression. An in vitro translation assay, in which polysome-associ- elements in the promoter regions of regulated genes (23). Transcrip- ated mRNA was translated in the presence of [35S]methionine, was tional regulation of PCNA expression by androgens through such an performed on polysomes collected from either untreated cells or cells element was possible since a non-consensus androgen-response ele treated with IO"9 M mibolerone for 12 h. Quantitation of the PCNA- ment is located 300 bp upstream of the proposed cap site (24). specific bands from the polysome run-off translation assay demon However, our data suggest that androgenic regulation of PCNA ex strated an average 1.5-fold increase in LNCaP cells treated with pression in LNCaP cells does not appear to be at either the level of mibolerone for 12 h (Table 1). Volumes of 35S radioreactive bands transcription or mRNA stability, since there was no change in steady- were significantly different (P Â£0.001) between untreated and mi state PCNA mRNA levels with androgen treatment. We do know that bolerone treated cells. PCNA specificity was confirmed by immuno- these cells have the capability of responding to androgens since staining a twin blot for PCNA protein (data not shown). androgen treatment greatly enhanced expression of PSA mRNA. In addition to the role androgens play in the transcriptional regu DISCUSSION lation of certain proteins, androgens have long been understood to regulate protein expression through posttranslational mechanisms. These data demonstrate that PCNA, a cell growth requisite protein, The posttranscriptional cleavage of two secretory protein precursors is regulated by androgens in an androgen-sensitive prostatic cancer into four major secretory products in the seminal vesicle has been cell line. The increase in PCNA protein expression with androgen shown to be under androgenic control (25). In many cell types, androgen receptor protein increases following short term treatment (1-2 days) with androgens, while at the same time a decrease in androgen receptor mRNA is often observed (26, 27). The increase in androgen receptor is suspected to be related to a decrease in turnover, possibly involving a change in the phosphorylation state of the pro tein. Androgens appear to regulate the expression of nucleolin, a protein involved in rRNA synthesis in the prostate, in a similar fashion (28). We tested the ability of androgens to indirectly affect the expres sion of PCNA through posttranscriptionul mechanisms. Alterations in protein turnover were studied by testing the clearance rate of meta- O (-) Mibolerone bolically labeled PCNA. The half-life of PCNA in LNCaP cells not treated with androgen is 23.1 h. In NIH/3T3 cells, the reported â€¢¿(+) Mibolerone half-life of this protein is 20 h (29), whereas the reported half-life of PCNA in HeLa cells is 8 h (30). These differences may relate to the 8 12 16 20 24 doubling times of the cell lines. LNCaP cells have a doubling time of Time (Hours) about 40 h, whereas the doubling time of HeLa cells is about 12 h and that of NIH/3T3 cells is about 24 h. LNCaP cells, like prostatic Fig. 8. Semilog plots summarizing the clearance of metabolically labeled PCNA in the carcinomas in general, are relatively slow growing: therefore, any absence (O) or presence (â€¢)of androgen. The half-life of the PCNA under each of the two treatment conditions is calculated from the slope of the line of each graph and is given at the perturbation that increases the potential for these cells to replicate, top of each graph. The slopes of the lines are significantly different (P ^ 0.005; n = 6). such as increasing the half-life of a cyclically expressed protein 1543

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1996 American Association for Cancer Research. ANDROGENS REGULATE PCNA IN LNCaP CELLS required for protein synthesis, may contribute significantly to cell cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replica growth. In addition, we tested the effect of androgens on translational tion in viva. Nature (Lond.), 326: 471-475, 1987. 9. Bravo. R.. and Celis. J. E. A search for differential polypeplide synthesis throughout efficiency by looking at the ability of androgens to affect PCNA the cell cycle of HeLa cells. J. Cell Biol.. 84: 795-802, 1980. translation on polysomes collected from androgen-treated cells. We 10. Jaskulski, D.. deRiel. J. K.. Mercer, W. E.. Calabretta. B.. and Baserga. R. Inhibition have demonstrated that translational efficiency is another posttran- of cellular proliferation by antisense oligodeoxynucleotides to PCNA cyclin. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1996 American Association for Cancer Research. Androgens Regulate the Expression of Proliferating Cell Nuclear Antigen Posttranscriptionally in the Human Prostate Cancer Cell Line, LNCaP

Jaime E. Perry and Donald J. Tindall

Cancer Res 1996;56:1539-1544.

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