Establishment of Prostatic Cell Line ‘‘Pro9ad’’From a p53-Deficient Mouse

Makoto Hanazono,1 Eriko Nakagawa,2 Shinichi Aizawa,3 andYasuhiro Tomooka2*

1Department of Clinical Research, Ichihara Hospital, School of Medicine, Teikyo University,Chiba, Japan

2Department of Biological Science and Technology, Science University of Tokyo,Chiba, Japan

3Laboratory of Morphogenesis, Institute of Molecular Embryology and Genetics, KumamotoUniversity School of Medicine, Kumamoto, Japan

BACKGROUND. We demonstrated that p53-deficiency is sufficient for immortalization offetal uterine cells. In the present study, we further extended our previous observations toprostate tissues from a young p53-deficient adult mouse.METHODS. Cell lines were established from the ventral prostate of a p53-deficient malemouse and maintained in medium containing 10% heat-inactivated fetal calf serum supple-mented with insulin (10 mg/ml), transferrin (10 mg/ml), cholera toxin (10 ng/ml), and sele-nium (10−8 M).RESULTS. Pro9ad, one of the lines established, exhibits a typical epithelial morphology inculture. Despite the possession of androgen receptors, the growth of Pro9ad was not stimu-lated by 5a-dihydrotestosterone. Hepatocyte growth factor (HGF) slightly stimulated prolif-eration, whereas fibroblast growth factor-1 (FGF-1), keratinocyte growth factor (KGF), andplatelet-derived growth factor AB (PDGF-AB) had no stimulating effect on growth. However,FGF-2, epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) acceleratedproliferation in a dose-dependent manner. EGF and IGF-1 additively stimulated growth.CONCLUSIONS. These results suggest that Pro9ad shares characteristics in common withprimary prostatic epithelial cells despite p53-deficiency, and that p53-deficiency alone allowsestablishment of clonal cell lines of the prostate epithelium. Furthermore, the prostates ofp53-deficient mice are useful sources for obtaining cell lines. Prostate 36:102–109, 1998.© 1998 Wiley-Liss, Inc.

KEY WORDS: p53-deficiency; ventral prostate; cell line

INTRODUCTION

The rodent ventral prostate is composed of at leastthree major cell populations: luminal epithelium, basalepithelium, and stromal cells [1–3]. The luminal epi-thelium expresses high levels of androgen receptors(ARs) [4–7]. Castration causes apoptotic cell death anddramatic regression of the epithelium [8,9]; the regres-sion is reversible by androgen replacement. The mo-lecular mechanisms involved in the control of andro-gen-dependent proliferation are not understood, al-though several studies tried to understand it on anumber of prostatic cell lines [10–16].

Recently, Metz et al. [17] reported that p53-deficientcells bearing both myc and raf readily yielded celllines, and they also suggested that p53-deficiency isinsufficient for immortalization. However, we demon-strated that deficiency is sufficient for immortalization

Contract grant sponsor: Agency of Science and Technology of Japan.*Correspondence to: Yasuhiro Tomooka, Department of BiologicalScience and Technology, Science University of Tokyo, 2641Yamazaki Noda, Chiba 278, Japan. E-mail: tomoylab@rs.noda.sut.ac.jpReceived 1 September 1997; Accepted 23 January 1998

The Prostate 36:102–109 (1998)

© 1998 Wiley-Liss, Inc.

of fetal uterine cells [18]. Therefore, we tried to estab-lish epithelial cell lines from the ventral prostate ofp53-deficient mice. We report that p53-deficiencyalone allows establishment of clonal cell lines of theprostate epithelium. The methodology for culturingPro9ad, one of these lines, and the profile, are reportedherein.

MATERIALS AND METHODS

Cell Culture, Cloning, and Passaging

The prostate was dissected from a young p53-deficient adult mouse (hybrid between C57BL/6 andCBA) [19] and placed in ice-cold Ca++- and Mg++-freephosphate-buffered saline (PBS) and minced, andplated on 100-mm tissue culture dishes. The mediumused was a 1:1 mixture of Dulbecco’s modified Eagle’smedium and Ham’s nutrient mixture F-12 (DMEM/F12) without phenol red (Sigma Chemical Company,St. Louis, MO) containing 10% heat-inactivated fetalcalf serum (FCS; Commonwealth Serum Laboratories,Melbourne, Australia), supplemented with insulin (I:10 mg/ml, Sigma Chemical Company), selenium (Se:10−8 M, Sigma Chemical Company), cholera toxin (CT:10 ng/ml, Sigma Chemical Company), transferrin(Trf: 10 mg/ml, Sigma Chemical Company), penicillin(31 mg/ml, Sigma Chemical Company), streptomycin(50 mg/ml, Sigma Chemical Company), and Fungizone(2.5 mg/ml, Life Technologies, Grand Island, NY).

Several cell populations with distinct morphologywere observed in the confluent monolayer developedfrom minced prostatic tissues. Several areas of epithe-lial cells with homogeneous morphology were iso-lated, using the tip of a micropipette under a phase-contrast microscope. They were then separately trans-ferred into 12-multiwell culture plates and cultured inDMEM/F12 containing 10% FCS supplemented withI, Se, CT, and Trf (10 + ALL). To obtain clones, the cellswere dispersed in 500 ml 0.05% trypsin-0.53 mMEDTA (Life Technologies), and small aliquots of thecell suspension (1/500 or 4/500) were seeded into 100-mm culture dishes. They were cultured in the mediumas described above until colony formation was ob-served. The colonies were again isolated and culturedas described above. The procedure was repeated, ifnecessary, until the cells were morphologically judgedas a homogeneous population. Several clonal cell lineswere established by this procedure. One of them, des-ignated ‘‘Pro9ad,’’ exhibited a typical epithelial mor-phology. When cells became confluent, one tenth ofthem were passaged. They were continuously cul-tured and passaged more than 50 times, and the esti-mated cell division is at least 150 times. Cells used inthe present study were at passages between 23–50.

Immunocytochemical Staining

Cells in a small volume of suspension were seededon Lab Tek tissue culture chamber slides (Miles Labs,Naperville, IL) and incubated in 10 + ALL for 3 days.Cells on the slides were fixed in 95% ethanol contain-ing 1% acetic acid at 0°C for 1 hr. After rinsing threetimes with ethanol (99%), the slides were placed in95% ethanol at 0°C for 1 hr and then immersed in PBSfor several hours at 4°C. For AR examination, cellswere incubated in DMEM/F12 containing 10% dex-tran-coated charcoal (DCC; dextran 0.025%, charcoal0.25%)-treated serum (DCCFCS) [20] with or without10−8 M 5a-dihydrotestosterone (DHT; Sigma ChemicalCompany) for 2 days. Cells on the slides were fixed in2% paraformaldehyde for 15 min [21]. The slides wererinsed three times with cold PBS.

After preincubation for 2 hr in PBS containing 5%normal goat serum (NGS; Zymed Laboratories, SanFrancisco, CA) and 1% bovine serum albumin (BSA;Sigma Chemical Company) to block nonspecific bind-ing of antibodies, cells were covered with primary an-tibodies, and incubated at room temperature for 1 hr.The primary antisera were anti-cytokeratin (reactingwith both acidic and basic forms, 1/100; BiomedicalTechnologies, Stoughton, MA) and anti-AR (PG-21, 1/50; Affinity Bioreagents, Golden, CO) polyclonal anti-bodies and anti-vimentin monoclonal antibody (1/10;Boehringer Mannheim GmbH, Mannheim, Germany).After washing with cold PBS, cells were incubatedwith fluorescein isothiocyanate (FITC)-conjugatedanti-rabbit IgG serum (Biosource International, Ca-marillo, CA) or FITC-conjugated anti-mouse IgG se-rum (Biosource International) at room temperature for1 hr. On control slides, incubation with the primaryantibody was omitted. Cells on slides were washed inPBS, and mounted in PBS containing 10% glycerol.Immunoreactivity was examined with a fluorescencemicroscope (BH2-RFK, Olympus Optical Ind., Tokyo),using a blue-light excitation filter system (DM500 +0515/20EY455).

Immunoblot Analysis

When cells became confluent, the culture mediumwas removed and dishes were washed twice in PBS.The cells were incubated in trypsin solution at roomtemperature for 2–3 min. Cells were collected in a tubeand washed once in PBS. Then 2 ml 0.5 M Tris buffer(pH 7.6) containing 1% Nonident P-40 (Sigma Chemi-cal Company), 0.5% deoxycholic acid (Sigma Chemi-cal Company), and 10% sodium dodecyl sulfate (SDS;Sigma Chemical Company) were added to the tubeand incubated at 0°C for 20 min. The tube was centri-fuged for 10 min at 3,000 rpm and the supernatant was

Prostate Cell Line From a p53−/− Mouse 103

collected as the cytoplasmic fraction. The pellet(nuclear fraction) was further incubated in 50 ml 0.5 MTris buffer (pH 7.6) containing 100 mg/ml DNase(Worthington), 0.1 M MgCl2, and 0.01 M PMSF (SigmaChemical Company) at 0°C for 3 hr.

An adult CD-1 male mouse was killed by cervicaldislocation, and the prostate and skeletal muscle weredissected out as described previously [22]. In brief, thetissues (0.025–0.1 g) were minced and suspended in 1ml 0.08 M Tris buffer (pH 6.9) containing 0.11 M SDS,and 0.1 M dithiothreitol (Sigma Chemical Company).The suspension was homogenized with an ice-coldglass homogenizer. The homogenate was sonicated for10 sec and immediately incubated at 95°C for 5 min.Then the homogenate was centrifuged at 2 × 105 g for30 min and the supernatant was collected. Protein con-centrations were determined using the BioRad ProteinAssay (Bio-Rad Laboratories, Richmond, CA). Ali-quots of all samples were stored at −80°C until analy-sis.

The samples were electrophoresed in SDS poly-acrylamide-slab gel and electrophoretically trans-ferred onto polyvinylidine difluoride membranes(Millipore Co., Bedford, MA). Membranes were rinsedwith Tris-buffered saline (TBS; pH 7.6) for 15 min. Themembranes were blocked with 5% skim milk in TBS-0.05% Tween-20 (TBS-T) at 4°C overnight and incu-bated with anti-AR antibody (1/50; Affinity Biore-agents). The antibody was diluted with 3% skim milkin TBS-T. After washing in TBS-T containing 3% skimmilk, the membranes were again incubated withhorseradish peroxidase-conjugated antibody (Bio-source International) for 1 hr. They were washed twicein TBS-T containing 3% skim milk and once in TBS-T,and incubated in a 1:1 mixture of enhanced chemilu-minescence (ECL; Amersham International plc, Buck-inghamshire, UK) reaction solution at room tempera-ture for 1 min. Autographs of chemiluminescencewere prepared by exposing the membranes to X-rayfilm (Kodak) for 30 sec at room temperature.

Effects of Androgen on Growth

Pro9ad cells were trypsinized, collected, andwashed three times in PBS. The cells were plated onto96-multiwell dishes (1 × 103 cells/well) in DMEM/F12containing 10% DCCFCS supplemented with I, Se, CT,and Trf. DHT was dissolved in absolute ethanol andadded to the medium at concentrations ranging from10−7–10−12 M. Control cultures received ethanol ve-hicle (0.01%). Media were changed every 2 days.These experiments were also performed on cells pre-cultured in DMEM/F12 containing 10% DCCFCS or inserum-free DMEM/F12 for 48 hr before adding DHT.At 1, 3, 5, and 7 days, media were removed and cells

were incubated in 100 ml DMEM/F12 containing 10%alamar Blue™ (Biosource) for 3 hr [23]. Cell numbersper well were estimated by the cellular reduction ofthe alamar Blue™. The absorbance of the reducedproducts, which was measured spectrophotometri-cally by a microplate reader, was proportional to thenumber of cells (r2 = 0.991). Student’s t-test was usedfor statistical analysis.

Effects of Growth Factors in Serum-Free Culture

The serum-free medium (SFM) was DMEM/F12supplemented with Trf (10 mg/ml), Se (10−8 M), and0.1% BSA. Growth factors tested were IGF-1 (ToyoboCo., Osaka, Japan), KGF (Prepro Tec. Inc., Rocky Hill,NJ), HGF (R&D Systems, Minneapolis, MN), PDGF-AB (R&D Systems), and FGF-1 and FGF-2 (BoehringerMannheim). All factors were added to SFM at finalconcentrations of 0.3, 1, 3, 10, and 30 ng/ml. EGF wastested at additional concentrations of 0.03 and 0.01ng/ml. Cells were seeded at 103/well and cultured for3 days. In a separate experiment, DHT (10−8 M) wasadded to SFM containing EGF (10 ng/ml) or IGF-1 (10ng/ml). Cell numbers per well were estimated by thealamar Blue assay.

RESULTS

Cell Morphology and Immunocytology

Pro9ad cells are typically epithelial, with a polygo-nal shape in a dense confluent monolayer which de-velops a honeycomb pattern (Fig. 1A).

After 72 hr in culture on Lab Tek tissue culturechamber slides, the cells were stained with indirectimmunofluorescent methods. The epithelial nature ofPro9ad was confirmed by specific immunofluorescentdetection of cytokeratin fibers forming a network sur-rounding the nucleus (Fig. 1B-a). They were negativefor anti-vimentin monoclonal antibody (Fig. 1B-b).

Localization of AR

Immunocytochemical staining confirmed that onlythe cytoplasm contained detectable AR. Addition ofDHT induced no change of AR localization. Negativecontrol without the primary antibody did not showany staining (data not shown). In immunoblot analy-sis, AR in Pro9ad cells was detected only in the cyto-plasmic extract as a strong single band at 110 kDa,whereas the nuclear extract had no specific immuno-reactive bands (Fig. 2). A strong band and a weak oneat 110 kDa were detected in prostate and skeletalmuscle samples, respectively (Fig. 2), confirming aprevious study [22].

104 Hanazono et al.

Androgen Sensitivity

To examine DHT stimulation of Pro9ad cell prolif-eration, growth rates were monitored in medium con-taining 10% DCCFCS with I, Trf, CT, and Se. DHT atall concentrations examined (10−7–10−9 M, in Fig. 3;10−10–10−12 M, data not shown) did not stimulategrowth throughout the culture period (Fig. 3).

We also repeated these experiments on cells precul-tured in serum-free DMEM/F12 for 24 hr before add-ing DHT. Again, DHT showed no growth stimulation.Furthermore, we tested antiandrogens, flutamide, andcyproterone acetate, to know whether Pro9ad cells aresensitive to them. However, these reagents at 10−7 Mhad no significant effect on growth (data not shown).

Effects of Growth Factors in Serum-Free Culture

Effects of growth factors on Pro9ad cells were ex-amined in SFM. KGF, PDGF-AB, and FGF-1 did notstimulate cell proliferation at any concentrations ex-amined. HGF had no significant effect on growth ex-cept at 30 ng/ml, at which it slightly, but significantly,stimulated proliferation. Growth was markedly stimu-lated by FGF-2 (Fig. 5B), IGF-1 and EGF (Fig. 4A,B); allfactors showed a dose-dependent stimulation. Themaximal response to FGF-2 was 122% over the control(P < 0.01) at 30 ng/ml. In contrast, EGF maximally

Fig. 1. Typical epithelial morphology (A) and immunocyto-chemical staining (B) or Pro9ad cells. The epithelial nature ofPro9ad was confirmed by the immunofluorescent detection ofcytokeratin, forming a fiber network in the cytoplasm (B-a).Pro9ad cells were not stained with vimentin monoclonal antibody(B-b). Bars: A, 50 µm; B, 20 µm.

Fig. 2. Detection of AR in Pro9ad cells by Western blotting. ARwas detectable as a single band at 110 kDa (arrowhead). Lane 1,mouse ventral prostate (30 µg/lane); lane 2, mouse skeletalmuscle (30 µg/lane); lanes 3 and 4, cytoplasmic extract (lane 3,100 µg/lane 4, 200 µg/lane); lanes 5 and 6, nuclear extract (lane5, 100 µg/lane; lane 6, 200 µg/lane).

Fig. 3. Androgen sensitivity of Pro9ad cells. To examine DHTstimulation of Pro9ad cell proliferation, growth rates were moni-tored in 10% DCCFCS containing medium supplemented with I,Trf, CT, and Se. Cells were cultured with increasing concentra-tions of DHT (10−7–10−9 M) or vehicle. Data are expressed aspercentage of control on day 1 (mean ± SEM, standard errorswere less than 5%, n = 5). *P < 0.01 vs. control for each day.

Prostate Cell Line From a p53−/− Mouse 105

stimulated proliferation at 0.3 ng/ml (Fig. 4B, 128%over the control, P < 0.01), and EGF induced a dose-dependent proliferation at concentrations between0.03–0.3 ng/ml. Maximal proliferation was caused byIGF-1. IGF-1 stimulated proliferation by 155% over thecontrol (P < 0.01) at 1 ng/ml, and by 370% over thecontrol (P < 0.01) at 30 ng/ml (Fig. 4A).

Estimation of Additive Effects of EGF, IGF-1,and DHT

The effect of DHT with growth factors was exam-ined on Pro9ad cells in SFM. EGF (10 ng/ml) alonestimulated growth 140% over the control, and IGF-1(10 ng/ml) alone stimulated growth 240% over thecontrol. EGF and IGF-1 together stimulated thegrowth 380% over the control. These results clearly

indicate that the two factors additively stimulatedgrowth (Fig. 6A). DHT (10−8 M) had no significantstimulation of growth even in the presence of EGF orIGF-1 (Fig. 6B).

Fig. 4. Effects of EGF and IGF-1 in serum-free medium. Cellswere cultured in either nonsupplemented SFM (as control) or SFMsupplemented with IGF-1 (A: 0.3–30 ng/ml) or EGF (B: 0.03–10ng/ml) for 3 days. Data are expressed as percentage of control(mean ± SEM, n = 8). *P < 0.01 vs. control.

Fig. 5. Effects of HGF, PDGF-AB (A), FGF-1, FGF-2 (B), andKGF (C) in serum-free medium. Cells were cultured in eithernonsupplemented SFM (as control) or SFM supplemented withvarious growth factors (0.3–30 ng/ml) for 3 days. Data are ex-pressed as percentage of control (mean ± SEM, n = 8). *P < 0.01vs. control.

106 Hanazono et al.

DISCUSSION

A simple method was described to establish pros-tate cell lines from a p53-deficient mouse. Several celllines were obtained with this method, and one of themwith epithelial morphology, designated ‘‘Pro9ad,’’was analyzed in this study.

Recently, Metz et al. [17] reported that p53-deficientcells transfected with both myc and raf readily yieldedcell lines, and suggested that p53-deficiency was in-sufficient for immortalization. However, Tsukada etal. [19] preliminarily reported that p53-deficiencyalone was sufficient for establishment of clonal celllines from various organs, such as seminal vesiclesand mammary glands, while cells from wild-typemice (p53+/+) did not proliferate over 1 month. Wedemonstrated that p53-deficiency gave rise not only to

clonal but also to immortalized cell lines from otherfetal tissues [18]. More recently, lines of smoothmuscles [24] and osteoblast-like cells [25] have beenalso established from p53-deficient mice in other in-dependent laboratories without any special skills. Inthe present study, we further extended our observa-tions to prostate tissues from a young adult mouseand succeeded in establishing cell lines.

Pro9ad was strongly positive for cytokeratin poly-clonal antibody, but negative for vimentin monoclonalantibody, as previously reported for both luminal andbasal epithelial cells of the ventral prostate [1–3].However, the two types of epithelial cells are differentin their AR content: luminal epithelial cells arestrongly AR-positive, whereas basal epithelial cells areAR-negative [4]. Basal cells of the human prostate alsolack immunoreactive AR [26]. Prins et al. [4] proposedthat the absence of AR in basal cells may be a featurecommon to many species. In the present study, bothimmunocytochemistry and immunoblot analysis dem-onstrated that Pro9ad cells are AR-positive. Althoughthese results suggest the possibility that Pro9ad is ofluminal epithelial origin, further information isneeded to specify the origin.

AR is localized in the nuclei of proliferating luminalcells [4,6,7]. Cytoplasmic AR is apparent after castra-tion [6,7], and androgen replacement stimulates ARreappearance in the nuclei [7]. Thus, the ability to re-tain AR within the nuclei may be impaired by castra-tion, leading to accumulation of AR within the cyto-plasm [6,7]. Pro9ad was established in medium with-out the supplementation of androgen; therefore, it ispossible that AR might accumulate in the cytoplasm.However, addition of DHT to the culture medium didnot result in AR accumulation in the nuclei, indicatingthat Pro9ad may lack the ability to retain AR in thenuclei, possibly as a consequence of p53-deficiency.The absence of p53 may result in an increased rate ofgenetic mutations [27]. In the human prostatic cancercell line LNCaP, the nature of AR was changed by apoint mutation [28,29]. Jenster et al. [30] reported thatmutated AR, which lacks the DNA-binding domain,was located predominantly in cytoplasm even in thepresence of androgen. The loss of the ability to retainAR in the nuclei of Pro9ad may reflect a mutation inAR. In the present study, DHT did not stimulatePro9ad proliferation in either serum-supplemented orserum-free conditions. The altered AR system may ex-plain the hormonal unresponsiveness.

Many growth factors are involved in growth regu-lation of epithelial cells. PDGFs have a wide range ofbiological activities and stimulate epithelial cells incertain conditions, such as wound healing [31–33].However, PDGF-AB failed to stimulate the prolifera-tion of Pro9ad in serum-free culture. On the other

Fig. 6. Additive effects of EGF, IGF-1, and DHT. Cells werecultured either in nonsupplemented SFM (as control) or in thepresence of EGF (10 ng/ml), or IGF-1 (10 ng/ml), or both (A) for3 days. Data are expressed as percentage of control (mean ± SEM,standard errors were less than 5%, n = 8; a, P < 0.01 vs. control;b, P < 0.01 vs. EGF alone; c, P < 0.01 vs. IGF-1 alone). Cells werecultured in the presence of EGF (10 ng/ml), or IGF-1 (10 ng/ml)with or without 10−8 M DHT (B) for 3 days. Data are expressedas percentage of control (mean ± SEM, standard errors were lessthan 5%, n = 8; a, P < 0.01 vs. control and DHT alone; b, P < 0.01vs. EGF alone).

Prostate Cell Line From a p53−/− Mouse 107

hand, FGF-1 and KGF stimulated the proliferation ofprostate epithelial cells in vitro [34–37]. In the presentstudy, FGF-1 and KGF did not stimulate the prolifera-tion of Pro9ad. FGF-2 and HGF slightly induced pro-liferation. Sensitivity to FGF-2 and HGF seems to beassociated with the progression of prostate carcinoma[38–40]. In addition, the progression of human pros-tate cancer is correlated with p53 genetic alteration[41]. Therefore, Pro9ad might be a good model forstudying the progression of prostate cancer.

Prostatic epithelial cells in primary culture expresstype 1 IGF receptor, and IGF-1 stimulates proliferationdose-dependently [42]. EGF receptor is expressed innormal ventral prostate [43], and EGF stimulates theproliferation of epithelial cells in primary culture [44].Our results demonstrated that EGF and IGF-1 stimu-lated the growth of Pro9ad, and that their growth ef-fects were dose-dependent and additive. EGF andIGF-1 do not share common signaling pathways[45,46]. Pro9ad cells appear to express receptors forboth EGF and type 1 IGF, and to retain an ability torespond to these ligands.

CONCLUSIONS

Although Pro9ad shows altered sensitivity to sev-eral growth factors, it still retains characteristics incommon with primary prostatic epithelial cells. There-fore, Pro9ad may provide a useful model for studyingmechanisms of growth and differentiation in the pros-tate epithelium and also may allow us to analyze theprogression to carcinoma accompanying loss of p53function. Furthermore, the prostates of p53-deficientmice are useful sources for obtaining cell lines.

ACKNOWLEDGMENTS

We thank Prof. N. Shimizu, Third Department ofInternal Medicine, and Associate Prof. M. Masai, De-partment of Urology, Teikyo University School ofMedicine, Ichihara Hospital, for their ongoing help,encouragement, and valuable advice. We also thankProf. Howard A. Bern, University of California atBerkeley, for his critical reading of this manuscript.This work was supported by Special CoordinationFunds for Promoting Science and Technology of theAgency of Science and Technology of Japan (to Y.T.).

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