The Prostate 62:40 ^ 48 (2005)

Characterizationof ProstaticNeuroendocrineCell Line Established FromNeuroendocrine

Carcinomaof TransgenicMouseAllograftModel

Kohsuke Uchida, Naoya Masumori,* Atsushi Takahashi,Naoki Itoh, and Taiji Tsukamoto

DepartmentofUrologic SurgeryandAndrology, SapporoMedical University SchoolofMedicine, Sapporo, Japan

BACKGROUND. The role of neuroendocrine (NE) cells in prostate cancer remains unclear. Ausefulmodel is necessary to study the biology ofNE cells.We herein describe the establishmentand characterization of an immortalized cell line fromanNE-10 allograft ofmurine prostaticNEcarcinoma.METHODS. A novel cell line, designated NE-CS, was developed from an NE-10 allograft thatwas established from the ventral prostate of the LPB-T-antigen (Tag) transgenic mouse, line 10(12T-10). We investigated the growth, karyotype, electron microscopic findings, expression ofTag and androgen receptor (AR), and tumorigenesis of the cells in athymic mice.RESULTS. The immortal cell line NE-CS was maintained in vitro for more than 2 years. TheNE-CS cells had dendritic-like extensions with dense core granules in the cytoplasm andproduced serotonin and somatostatin in conditioned medium. The cells expressed neither Tagnor AR. They showed androgen-independent growth in vitro and a hypotetraploid karyotypesimilar to the original NE-10 allograft. The NE-CS cells, which were subcutaneously inoculatedinto athymic mice, formed tumors with the NE phenotype. The tumors exhibited acceleratedgrowth compared to the original NE-10 allograft.CONCLUSIONS. The established cell line has characteristics of NE differentiation andtumorigenic ability. This cell line may be a promising model to understand the molecularmechanisms associated with the acquisition of hormone refractory prostate cancer. Prostate 62:40–48, 2005. # 2004 Wiley-Liss, Inc.

KEY WORDS: prostatic cancer cell line; neuroendocrine

INTRODUCTION

Although most prostate cancers are originallyandrogen-dependent, androgen-independent tumorcells eventually emerge during androgen ablation,leading to clinical relapse [1]. In spite of recent trialsusing combined androgen ablation with novel chemo-therapeutic agents and steroids, there is no effectivetherapeutic option for hormone-refractory prostatecancer. Therefore, identification of the mechanism bywhich a tumor escapes from androgen regulationwould obviously be of significant importance forplanning of treatment strategies. Several studies in-vestigating the mechanisms have shown that numer-ous molecules are involved in this phenomenon [2].However, the mechanism of acquisition of hormone

refractory cancer during androgen retrieval therapystill remains unclear.

Malignant cells expressing a neuroendocrine (NE)phenotype have been identified within conventionaladenocarcinoma of the prostate [3,4]. A number of

Grant sponsor: Stiftelsen Japanese-Swedish Cooperative Founda-tion; Grant sponsor: Japanese Ministry Of Education, Science, Sportsand Culture (grant-in aid); Grant number: 13671660.

*Correspondence to: NaoyaMasumori, MD, Department of Urology,Sapporo Medical University School of Medicine, S-1, W-16, Chuo-Ku, Sapporo 060-8543, Japan. E-mail: masumori@sapmed.ac.jpReceived 11 December 2003; Accepted 15 March 2004DOI 10.1002/pros.20111Published online 27 April 2004 in Wiley InterScience(www.interscience.wiley.com).

� 2004 Wiley-Liss, Inc.

clinical studies suggested that NE cells might play arole in prostate cancer development or progression[5,6]. It has been speculated that a variety of peptidessecreted from NE cells stimulate the growth of thesurrounding cells in paracrine fashion through anandrogen-independent pathway [7]. However, there isno direct evidence on whether NE cells influence thedevelopment and progression of prostate cancer orandrogen independence.

Wepreviously developed a transgenicmousemodelof rapidly evolving prostate cancer composed of NEcarcinoma as well as adenocarcioma with advancingage, using a recombinant gene expressing anSV40 largeT-antigen (Tag) transforming sequence under the re-gulatory control of the rat large probasin promoter [8].To establish a model for further studies in vivo, theallograft was established from a ventral lobe of theprostate [9]. This allograft, designated NE-10, main-tains the expression of Tag and exhibits NE featuresdefined histologically by small cell carcinoma andimmunohistologically by chromogranin A, a NEmark-ers. The NE-10 allograft is negative for androgenreceptor (AR) and shows androgen-independentgrowth. TheNE-10 allograft subcutaneously implantedinto athymicmicemetastasizes to the liver and the lungat 12th week after implantation. We consider thisallograft to be a useful in vivomodel to study biologicalbehavior involved in theprogressionof prostate cancer.

Human specimens of prostate cancer are extremelydifficult to propagate in vitro [10]. The development ofeffective therapies for treatment of hormone refractoryprostate cancer has been limited due to the lack ofappropriate animal models/cell lines. In vitro cellculture models are invaluable for defining themechan-ism of progression of prostate cancer and for testingpreventive and therapeutic regimens. If it is possible toestablish cell lines from the allograft, it will be easier toinvestigate the properties of NE cells in addition to theinteraction between NE cells and adenocarcinomacells. In this study, we established and characterizedan in vitro cell line developed from theNE-10 allograft.

MATERIALSANDMETHODS

Establishmentof Cell Line

Tissue used to establish the cell line was obtainedfrom the NE-10 allograft subcutaneously inoculatedinto backs of male athymic mice. The obtained tissueswere well washed with phosphate-buffered saline(PBS) supplemented with gentamicin (100 mg/ml,Gibco BRL, Breda, The Netherlands) and then theywere gently cut into small pieces, 1–2 mm in size. Thepieces were transferred to 150 ml of dissociationmixture that consisted of RPMI-1640 medium (GibcoBRL) supplemented with 5% fetal bovine serum (FBS,

ICN Biomedicals, Costa Mesa, CA), gentamicin, col-lagenase 1 (225 IU/ml, Sigma, St. Louis, MO) andhyaluronidase (125 IU/ml, Sigma). They were gentlystirred at 378C for 24 hr in 5% CO2 in a humidifiedincubator. At the end of this period they were trans-ferred to 75-cm2 flask (Becton Dickinson, FranklinLakes, NJ). After 3 days, the cell colonywhich grew outand attached to the flask was identified. The derivedcell line, designated NE-CS, was maintained in RPMI-1640 medium containing 10% FBS, MEM non-essentialamino acid (10 ml/L, Gibco BRL), MEM sodiumpyruvate, penicillin–streptomycin (10 ml/L, GibcoBRL), and 7.5% NaHCO3 in 5% CO2 in a humidifiedincubator. The culture medium was changed every 3–4 days. When the cells reached approximately 80%confluence, they were dispersed using 0.1% trypsinwith 0.4% ethylendiaminetetraacetic acid (EDTA) solu-tion and used for subsequent passage. Morphologicalfeatures of the cells were observed by inverted phase-contrast light microscopy.

As a comparative cell linewe used LNCaP cells withpassage numbers between 44 and 49, obtained from theAmerican Type Culture Collection. The cells were pro-pagated in the same medium used for NE-CS.

Cytogenetic Study

For cytogenetic analysis, NE-CS cells at the 12thpassage were used when they reached 80% confluencein a 75-cm2flask.After removal of themedium, the cellswere rinsed with PBS and detached by 0.1% trypsin–EDTA solution. Then they were centrifuged and resus-pended in the medium. Chromosomes were analyzedusing standard hypotonic pretreatment proceduresand Giemsa staining.

Neuropeptides in ConditionedMedium

Nueropeptides secreted from the NE-CS cells intothe medium that had homology between the mouseand human were determined. The NE-CS cells atpassage 10 were suspended in 12 ml of RPMI-1640with 10% FBS and then incubated at 378C for 3 days.Next, the medium was replaced by serum-free med-ium. After 3 days of incubation, the supernatant wascollected and stored at �208C until the analysis. Theconcentration of serotonin was measured by high-performance liquid chromatography (SRL, Tokyo,Japan). The concentrations of vasoactive intestinalpeptide (VIP), parathyroid hormone-related protein(PTHrP), somatostatin, and calcitonin in the mediumwere measured by radioimmunoassay (SRL).

GrowthCurve

The growth curve of NE-CS cells was determined byculturing the cells in a 6-well culture plate (Cornin,

ProstaticNeuroendocrineCell Line InVitro 41

Corning, NY). The cells (2� 104) were suspended in2 ml of RPMI-1640 supplemented with normal FBS orcharcoal-stripped serum (Biowest, Nuaille, France),and plated in each well. The number of cells per wellwas counted every 2 days for 6 days after incubation at378C, based on triplicate wells per each time point. Thedoubling time of growing cells was calculated fromthe exponential growth phase of the growth curve. Thegrowth curve of LNCaP was also determined by thesame procedure as for NE-CS.

ReverseTranscription (RT)-PCR

Total RNA was extracted using an RNeasy kit(Qiagen,Valencia, CA) according to themanufacturer’sinstructions. A total of 5 mg of total RNA was reversetranscribed in a thermal cycler (Perkin-Elmer, Nor-walk, CT) using SuperScript II (Invitrogen, Carlsbad,CA) and oligo(dT)12–18 primers according to themanufacturer’s instructions for 1 hr at 428C in a 20-mlreaction mixture. Resulting cDNA (1 ml) was amplifiedwith Taq polymerase and one set of oligonucleotideprimers. Sampleswere denatured for 5min at 948Candthen amplified for 25 cycles at 948C for 30 sec, 558C for30 sec, and728Cfor 1min.Aliquots (2ml) fromeachPCRsample were then analyzed by agarose gel electrophor-esis. Forward and reverse primer sequences were asfollows: Tag (50-TCCAGAGATTTGCCTTCAGGTCA-GGG, 50-ACCTCTACAAATGTGGTATGGC), b-actin(50-GATTCCTATGTGGGCGACGAG, 50-CCATCTCT-TGCTGGAAGTCC).

Western BlotAnalysis

The expression of Tag and AR was examined byWestern blotting as previously described [9]. As posi-tive controls for Tag and AR, the NE-10 allograft andLNCaP cell line were used, respectively. Equalamounts of lysate protein (10 mg) in RIPA buffer wererun on 10% SDS–PAGE gels and electrophoreticallytransferred to a polyvinylidene fluoride membrane(Immobilin-P, Millipore, MA). The membrane wasblocked in 10% non-fat dry milk in TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.2% Tween-20) over-night at 48C. After washing with TBS-T, the blot wasincubated for 1 hr at room temperature with anantibody to SV40 Tag (1:100, Oncogene ResearchProducts, Boston, MA) or AR (1:200, Sigma). Themembranewaswashed and then incubated with a hor-seradish peroxidase-conjugated anti-mouse or -rabbitsecondary antibody (Amersham Pharmacia Biotech,Piscataway, NJ) for 45 min at room temperature. Theimmunoreactive proteins were visualized by enhanc-ed chemiluminenscence and exposed to Hyperfilm(Amersham Pharmacia Biotech).

ElectronMicroscopic Examination

NE-CS cells at 80% confluence were trypsinized,pelleted, and washed with PBS. The tissue sampleswere cut into small pieces after excision from theathymic mice. Those samples were fixed with 2.5%glutaraldehyde and post-fixed with 1% osmium tetr-oxide in phosphate buffer, dehydrated with ethanol,and embedded in Epon812. The sections were thenstained with uranyl acetate and lead citrate, andexamined under an electron microscope (Hitachi,Tokyo, Japan).

Tumorigenicityof Cell Line

NE-CS at the 10th passage was examined fortumorigenicity. First, the cells were grown to 95%confluence, washed with serum-free medium, trypsi-nized to release cells, and resuspended in serum-freemedium. The cells, 1� 107 suspended in 75 ml of serum-freemedium,weremixedwith 75ml ofMatrigel (BectonDickinson, Sunnyvale, CA), and then subcutaneouslyinjected into the backs of 6-week-old athymic mice(Balb/c, nu/nu, Sankyo Labo, Tokyo, Japan). Thegrowth of the tumors was observed every week afterinjection. When the tumors grew large enough, up to2 cm in diameter, they were excised and fixed forhistopathological examination. To examine the growthproperties of the derived tumors compared to the NE-10 allograft, 50 mg of tumor was subcutaneouslyinoculated into the backs of male athymic mice at6 weeks of age, and they were followed weekly for11 weeks after inoculation. The volumes (mm3) werecalculated by the formula 0.523� long diameter(mm)2� short diameter (mm). Themicewere sacrificedand the lung and liver were removed. The tissues werefixed in 10% formalin and embedded in paraffin. The5 mm-thick paraffin-embedded material was routinelyprocessed for hematoxylin and eosin staining.

Immunohistochemistry

Immunohistochemistry for Tag and cytokeratin wasperformed according to the procedure reported pre-viously [8]. Immunohistochemistry for vimentin wasperformed using 1:200 vimentin, LN-6 (Sigma) as theprimary antibody. Antigen retrieval for vimentin wasachieved by microwaving in 0.01 M citrate buffer for30 min.

RESULTS

Establishmentof aNewCell Line

We established a new cell line, designated NE-CS,from an NE-10 allograft with NE differentiation. Sincethe NE-CS cells have been maintained in vitro for over

42 Uchida et al.

2 years through more than 20 passages, we consideredthem to represent an established immortal cell line.This cell line morphologically consisted of polygonalcells with irregular dendritic-like extensions and,occasionally, multiple nucleoli. Long processes wereobserved in some cells (Fig. 1). When the cells grewbeyond confluence, they piled up on top of each other(loss of contact inhibition). In serum-free mediumincubated with NE-CS cells, 20.6 ng/ml and 26.0 pg/ml of serotonin and somatostatin were detectable,respectively, while VIP was under the detection level(5 pg/ml). Under the same conditions, the detectableneuropeptides in the supernatant of LNCaP cells wereserotonin at 1.5 ng/ml, VIP at 15.0 pg/ml, andsomatostatin at 21.0 pg/ml.

Cell Growth

We investigated in vitro growth properties of thiscell line under regular and steroid-reduced cultureconditions. The growth rate of NE-CS cells was slowerthan that of LNCaP cells in the medium with FBS(Fig. 2). The doubling times of the NE-CS and theLNCaP cells were 33 and 24 hr, respectively. Thenumber of LNCaP cells was significantly reduced atday 6 in the medium with charcoal-stripped FBS. Onthe other hand, NE-CS cells cultured in the mediumwith charcoal-stripped FBS showed a growth ratesimilar to those cultured in the medium with FBS.

ChromosomeAnalysis

Cytogenetic analysis by G-banding was performedonNE-CS cells at passage 10. The chromosome numberfor a normal mouse cell is 40. Although chromosomalnumbers ranged from 38 to 78, most of the NE-CS cells

were approximately hypotetraploid. This chromo-somal pattern was consistent with that of the NE-10allograft showing near hypotetraploid DNA content[9].

ElectronMicroscopic Findings

Electron microscopic study demonstrated that NE-CS cells had dense core granules in the cytoplasm as acommon feature of NE differentiation (Fig. 3A). Thegranules were pleomorphic, round, oval, or elongated,and occasionally had a lucent halo. Most of the NE-CScells had irregular, large bizarre nuclei. Microvilli thatvaried in number and length were observed.

Expression of SV40 Tag andAndrogen Receptor

OnWestern blot analysis, the expression of Tag wasdetected in the extract of the NE-10 allograft but not inthe NE-CS cells/tumor or the LNCaP cells (Fig. 4A). Inaddition, the mRNA for Tag was not detected in theNE-CE cells/tumor by RT-PCR assay (Fig. 5). Theseresults indicated that the Tag transgene was lost afterthe cells were established in culture. As expected, ARwas undetectable in the NE-CS cells/tumor as in theNE-10 allograft, in contrast to the LNCaP cells withclear expression of AR (Fig. 4B).

Tumorigenicityof Cell Line

Measurable tumors were identified on day 14 afterinoculation of NE-CS cells. The tumorswere excised onday 28 and used for histological examination. The NE-CS tumor was composed of poorly differentiated cells.Although the majority of the tumor showed a solidpattern composed of spindle-shaped cells, a small partof it appeared sheet-like in composition with vaguemargins.Manynuclei had apunctuate appearance. Theappearance of the pleomorphic NE-CS tumor wassomewhat different from the originalNE-10 allograft inH–E staining (Fig. 6A,B). However, electron-micro-scopy demonstrated that the NE-CS tumor consisted ofcells with dense-core neurosecretory-type granules(Fig. 3B), like the NE-CS cell line, showing the typicalfeatures of NE cells (Fig. 3A). Thus, the ultrastructuralfindings for theNE-CS tumorwere comparable to thosefor the NE-CS cell line. In immunohistochemical stain-ing, theNE-10 allograft had expression of Tag, whereasthe NE-CS tumor was negative for Tag (Fig. 6C,D).Immunostaining for cytokeratin was positive in theNE-CS tumor, though itwas fainter than for the originalNE-10 allograft (Fig. 6E,F). On the other hand, nostaining for vimentin was observed in the NE-10allograft or the NE-CS tumor (Fig. 6G,H).

The growth rate and metastatic ability of the NE-CStumor were investigated. Since the NE-CS tumor grew

Fig. 1. Phase-contrast photomicrograph of NE-CS cells at 10thpassage. Magnification �100. [Color figure can be viewed in theonlineissue,whichis availableatwww.interscience.wiley.com.]

ProstaticNeuroendocrine Cell Line InVitro 43

at two-fold the rate of theNE-10 allograft (Fig. 7), it wasimpossible to keep the mice with NE-CS tumors aliveuntil 11 weeks. All mice bearing the NE-10 allograftdeveloped metastases of the liver and, in four of fivemice, the lung at 11 weeks after inoculation. On theother hand, no mice bearing NE-CS tumors developedmetastasis although they were sacrificed at 7 weeksafter inoculation due to the life-threatening hugetumors.

DISCUSSION

NE cells have been identified in benign and malig-nant prostatic tissues. In particular, 30–100% of pros-tatic adenocarcinomas contain NE cells [3,4]. NE cellsare considered to be important regulators for cellgrowth and differentiation in the prostate as well asin respiratory and gastrointestinal organs. It has beendemonstrated that the clusters of prostatic cells aroundNE cells exhibit increasing expression of the prolifera-tion marker Ki-67 and augmented expression of theantiapoptotic regulator Bcl-2 [11,12], which indicatesthat NE cells promote growth of the surrounding carci-noma cells in a paracrine fashion. Clinically, it has been

reported that NE differentiation correlates with theresponse to endocrine therapy for prostate cancer [13]and NE differentiation in prostate cancer is associatedwith an unfavorable prognosis through androgen-independent progression [13–15]. Thus, NE cells mayplay an important role in the transition from androgendependence to independence in prostate cancer. How-ever, the role of NE cells in the progression of prostatecancer remains to be understood. If there is a modelrepresenting the NE phenotype that make it easy toinvestigate the interaction between prostate adenocar-cinomamodel, the questions related to the possible roleof NE differentiation will be resolved.

We established a new prostatic NE cell line, namedNE-CS, derived from anNE-10 allograft in the athymicmouse. This cell line has the features of NE differentia-tion similar to the NE-10 allograft except for Tagexpression. The presence of microvilli, positive immu-nostaining for cytokeratin, and negative immunostain-ing for vimentin suggest an epithelial origin for thecells. This new cell line may be a promising modelhelping to understand the mechanism of prostatecancer progression, and open up new approaches totherapeutic intervention.

Fig. 2. Growth curves for LNCaP (A) and NE-CS lines (B). Six-well plates were seeded with 2�104 cells/well in RPMI-1640 with FBS orcharcoal-stripped FBS.Cells were trypsinized and countedwith a hemocytometer every 2 days (in triplicate).Blackcircles and open squaresindicate the cells cultured in themediumwith FBS and charcoal-stripped FBS, respectively.Each timepointrepresents an average of 3-wells.Errorbarsindicate standarddeviation.

44 Uchida et al.

NE cells are best characterized by expression ofNE markers such as chromogranin A, serotonin, andneuron-specific enolase. In addition, they lack Bcl-2expression although their proliferative activity is low[16]. They have irregular dendritic-like extensions thatallow them to contact neighboring epithelial cells over a

distance of several cell diameters [17]. Electron micro-scopy demonstrates that NE cells have dense coregranules in the cytoplasm. The variation in the ultra-structural morphology of the granules indicates theheterogeneityofNEcells [17].NEcells are considered tobe androgen unresponsive because of lack of androgenreceptor. In this report, we demonstrated that our newcell line (NE-CS) had typical features for NE cellsrepresented by growth in charcoal-stripped serum,lack of AR expression, and dense core granules in thecytoplasm.

NE cells secrete a variety of neuropeptides andbiogenic amines in a paracrine or autocrine manner[3,5]. The most frequently identified biologically activeproduct of NE cells is serotonin. In concordance withthe functions of NE cells in other epithelial organs, ithas been suggested that prostatic NE cells play a rolein the maintenance of homeostasis and regulation ofprostatic fluid [3]. We found that the NE-CS cell lineproduced serotonin and somatostatin in the super-natant. Serotonin may play a role as an autocrine/paracrine growth factor in prostate cancer similar tothat postulated in carcinogenesis of intestinal carci-noma [18,19]. It has been reported that serotonin-uptake inhibitors suppress the growth of humanprostate tumor cells in vitro and in vivo in immune-deficient mice [20]. Somatostatin is immunohisto-chemically detected in a subset of NE cells. It has beendemonstrated that somatostatin had antiproliferativeactions against normally dividing cells and malignanttumor cells through the induction of cell-cycle arrestand apoptosis [21]. Thus, NE-CS cells that produceserotonin and somatostatin may influence prostateadenocarcinoma cells. The balance between substanceshaving proliferative activity such as serotonin andthose having antiproliferative activity such as soma-tostatinmaydetermine the growthproperties of adeno-carcinoma cells.

The NE-CS cell line and the tumors derived fromNE-CS cells did not express Tag. A similar finding hasbeen reported in cell lines established from the TRAMP

Fig. 3. Ultrastructural appearance of NE-CS cell line demon-strates numerous pleomorphic dense core granules in the cyto-plasm.A: NE-CS cell line.B:The tumor derived fromNE-CS cellsthatwere subcutaneously inoculated into athymicmice.Magnifica-tion,�60,000.

Fig. 4. Westernblot analysis forT-antigen (Tag) andAR.A: SV40largeT-antigen (86kDa).B: AR(110kDa).C:b-Actin (43kDa).

Fig. 5. RT-PCR forTag.A: SV40 largeT-antigen (633 base pairs),(B):b-Actin (532basepairs).

ProstaticNeuroendocrineCell Line InVitro 45

model that is constructed with a short probasin pro-moter linked to SV40 early regions [22]. Expression ofTag in vivo disappeared in established in vitro cell lineseven though they maintained growth and tumorigeni-city. Thus, Tag expression is indispensable to initiate

transformation while continual Tag expression is notrequired for the maintenance of the transformed statein vitro. Since Tag acts as a potent immunogen, a cellline not expressing Tag may be convenient forimmunotherapeutic approaches to prostate cancer [22].

Fig. 6. Histologyandimmunohistochemistry forTagofNE-10allograft (A,C,E,G)andthe tumorderivedfromNE-CScells (B,D,F,H).A,B:H^E staining.C,D: Immunohistochemistry forTag. E, F: Immunohistochemistry for cytokeratin.G,H: Immunohistochemistry for vimentin.Magnification,�400.

46 Uchida et al.

Although many SV40 immortalized cell lines do notform tumors in athymic mice [23], the NE-CS cells aretumorigenic in vivo. The NE-CS tumor showed muchmore aggressive growth behavior than the originaltumor, the NE-10 allograft. Only NE-CS cells withprominent growth ability may have been selectedduring establishment of the cell line and raised in theclone along with passages. On the other hand,metastases to the lung and liver were not identified inmice bearing the NE-CS tumor. This does not necessa-rily mean that the NE-CS tumor lost metastaticpotential, because the observation period was shorterthan that in mice with the original NE-10 allograft.However, it is a clear that metastatic ability was notconsistent with the growth of cancer cells as reportedpreviously [24]. Further investigation is needed todetermine themetastatic potential of the NE-CS tumor.

We successfully established an immortal cell linewith the properties of NE cells. There are a fewlimitations to this study. The LNCaP cell line may notbe ideal for a comparativemodel, since these cells are ofhuman origin and have the ability to transform into anNE-like phenotype upon androgen withdrawal [25].Regardless of these limitations, we believe that the cellline will contribute to investigation of the relationshipbetween NE cells and prostatic adenocarcinoma cellsand identification of the mechanism of androgenindependence.

CONCLUSIONS

We described a promising new cell line named NE-CS that showed typical features of NE cells. Thesuccessful establishment of a murine prostatic NE cellline would be amajor breakthrough for prostate cancerresearch. The properties of the cells that secrete mea-surable NE peptides with homology to those of thehuman may contribute to investigation of the interac-tion with other cells even though it is derived fromhuman specimens. Thus, this primary NE cell line maybe useful as an in vitro model to study various bio-chemical and molecular biological pathways involvedin the pathogenesis and development of prostatecancer.

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48 Uchida et al.