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 ¡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 prolif- eration, 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-ß-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 37°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 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1996 American Association for Cancer Research. ANDROGENS REGULATE PCNA IN LNCaP CELLS 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 4°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 £max)for 10 min at 4°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 42°Cin prehybridization solution (45% centrifuged at 41.(XX)rpm (2(X),(XX)X Aav)for 90 min at 4°C.Polysomes were deionized formamide, 5X SSC.