Research Article Mouse Prostate Epithelial Luminal Cells ...based label-retaining assays demonstrate that mouse pro static epithelial stem cells are concentrated in the proximal T

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 913179 8 pageshttpdxdoiorg1011552013913179

Research ArticleMouse Prostate Epithelial Luminal Cells Lineage Originate inthe Basal Layer Where the Primitive StemEarly Progenitor CellsReside Implications for Identifying Prostate Cancer Stem Cells

Jianjun Zhou12 Lionel Feigenbaum3 Carole Yee4 Hongbin Song2 and Clayton Yates1

1 Department of Biology and Center for Cancer Research Tuskegee University Tuskegee AL 36088 USA2 Institute of Disease Control and Prevention Chinese Academy of Military Medical Sciences Beijing 100071 China3 Science Applications International Corporation-Frederick National Cancer InstituteFrederick Cancer Research and Development Center Frederick MD 21702 USA

4Dermatology Branch National Cancer Institute NIH Bethesda MD 20892 USA

Correspondence should be addressed to Hongbin Song hongbinsong263net and Clayton Yates cyatestuskegeeedu

Received 25 February 2013 Accepted 3 May 2013

Academic Editor Tiziano Verri

Copyright copy 2013 Jianjun Zhou et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Prostate stem cells are thought to be responsible for generation of all prostate epithelial cells and for tissuemaintenanceThe lineagerelationship between basal and luminal cells in the prostate is not well clarifiedWe developed amousemodel to trace cell fate and amouse model with a slowly cycling cell label to provide insight into this questionThe results obtained indicate that putative mouseprostate stem cells are likely to reside in the basal layer

1 Introduction

The prolonged use of androgen deprivation therapy is associ-ated with a decline in responsiveness to treatment of prostatecancer treatment an effect that generally results in a poorclinical outcome Thus a focus on the origin of cells capableof developing the androgen-independent tumor mass subse-quent to hormonal therapy is of interest

Normal prostatic epithelium is comprised of a stratifiedstructure with secretory luminal cells that are androgen-responsive and an interspersed minor population of highlyproliferative androgen-receptor- (AR-) negative basal cellsTheseAR-negative cells are thought to give rise toAR-positiveluminal cells [1] As determined with human prostate stem-like cells purified from the basal compartment basal cells cangenerate luminal cells in vitro [2] Other markers such asthe keratin expression pattern have also yielded informationregarding a subpopulation of stem cellsprogenitor cellsLuminal and basal populations are identifiable through ex-pression of specific keratins Basal cells express keratins 5 and14 only but if luminal cells develop they lose these keratinsand express keratins 8 and 18 [3ndash5]

The traditional model of prostate epithelial differentia-tion proposes that adult prostate stem cells are containedwithin the basal layer and give rise to progenitorstem cellsand androgen-independent transit-amplifying cells whichcan differentiate into luminal cells This concept is supportedby the isolation of intermediate cells which express bothluminal and basal makers keratin 5 and keratin 18 and bythe differentiation of these cells down the luminal lineage[6] In animal models of castration-induced regeneratingprostate basal cells preferentially survive androgen ablationhowever a subset of luminal cells demonstrate castration-resistance [7] As determined with a prostate-specific anti-gen (PSA) CreERT2-based genetic lineage markingtracingmodel a subset of luminal cells not only survive but they alsoretain capability for regeneration after castration [8] Whilethese studies highlight the regenerative capacity of preexist-ing luminal cells the origin of regenerative luminal cells isstill unclear since both luminal and basal cell populationssurvive after castration

As presently reported two approaches were used to deter-mine the lineage-derived fate of basal cells (1) tracking that

2 BioMed Research International

allows lineage-specific tagging of the keratin 14-expressingpopulation of cells throughout prostate development in adultmice and (2) a keratin 5H2BGFP-label retainingmodel dem-onstrating that slowly cycling cells are represented in only asubfraction of keratin 5-expressing cells after hormonecas-trationmanipulationThese cells have the capacity to give riseto more differentiated luminal cells Results with these mod-els which suggest that the putative adult stem cells whichhave a slowly-cycling feature most likely reside in the basallayer and are positive for keratin 5 and keratin 14 clarify someof the basic aspects of the biology of the rodent prostate gland

2 Materials and Methods

21 Cell Fate Tracing Assay Keratin 14-CreERtam transgen-ic mice (STOCK Tg (KRT14-creEsr1)20EfuJ) LacZ report-er mice (B6129S4-Gt(ROSA)26Sortm1SorJ) and EGFP re-porter mice (B6129-Gt(ROSA)26Sortm2ShoJ) were pur-chased from the Jackson Laboratory Bar Harbor ME USAThe two transgenic mouse lines were crossed and doubletransgenic markers of Cre and LacZ were confirmed by PCRwith genomic DNA isolated from their progenies Tamoxifen(Sigma-Aldrich) was prepared at 100mgmL in ethanol heat-ed to 60∘C to dissolve the powder and then diluted 10 timeswith sunflower oil aided by 2min vortex plus sonication foran additional 30min Tamoxifen was used to induce Cre ac-tivity in gaining its access to the nuclear compartment fromthe cytoplasm The administration to young (3 weeks old oryounger) double-transgenic mice of 05mg tamoxifen wasaccomplished by intraperitoneal injections daily for 5 daysThe double-transgenicmicewere sacrificed after two cycles ofprostate involution (2 weeks) and regeneration by castrationand by supplementation (for 2-3 weeks) with testosteronepellets (125mg sustained release Innovative Research ofAmerica) The prostates were removed and frozen sectionsfrom dorsal and ventral glands were obtained The presenceof 120573-galactosidase was demonstrated with X-gal by use of acommercial kit (Invitrogen)

22The Label-Retaining Assay Transgenic mice expressing aTet-OFF tTA regulatory transactivator under the control of akeratin 5 promoter were generously provided by Adam Glick(National Cancer Institute The Pennsylvania State Universi-ty) These mice were bred to TRETight-H2BGFP transgenicmice that we created in the NCI-Frederick animal facilityExpression of H2BGFP is naturally turned on in the double-transgenic mice (keratin 5-tTATRETight-H2BGFP) follow-ing development and can be turned off in the basal epitheliumof the prostate following the application of doxycycline Awash-out period is required to identify the label-retainingcellsThe prostate epithelial cells that are still H2BGFP+ afterthe wash-out period mark the label-retaining cells whichbecause of their slowly cycling feature should represent pros-tate epithelial stem cells To ensure the observation of thoserare putative stem cells resistant to label dilution due to cellturnover this wash-out period continued throughout themultiple cycling (over 10) of the prostate epithelium betweenregression and expansion by hormonal manipulation Afterthe label-retaining assay of themurine prostate was complete

the animals were anesthetized and the prostates were re-moved for histologic analysis

23 Castration and Hormone Manipulation The hormonalmanipulation consisted of alternating 2-3 week periods ofandrogen deprivation and restoration This manipulationresults in regression of the prostate epithelium to a residualbasal layer (androgen deprivation) followed by expansion toa multilayered epithelium (androgen restoration) Hormonaldeprivation was accomplished by castration and androgenrestoration was achieved by use of implantable testosteronepellets (125mg sustained release Innovative Research ofAmerica) which maintained a serum level of 4 ngmL

24 120573-Galactosidase Staining The 120573-galactosidase stainingkit was used according to the manufactures suggested pro-cedure to demonstrate the activity of LacZ Briefly thawedcryosections of mouse prostates were placed in the fixativesolution for 10min After washing the samples were incu-bated at 37∘C with a solution containing X-gal and the bluecells were assessed within 2 hThe slides were counterstainedwith nuclear fast red solution

25 Immunofluorescence Formarker expression standard in-direct immunofluorescence staining was performed on 6 120583mmouse tissue cryosections Briefly the thawed sections werefixed with 4 paraformaldehyde in PBS for 10 minutes atroom temperature and after washing with PBS blocked with10 donkey whole serum in PBS After washing cells wereincubated sequentially with primary antibody rabbit poly-clonal anti-mousekeratin5 (CovanceResearchProducts Inc)and Alexa Fluor-594 or 546-conjugated donkey-anti-rabbitsecondary antibody (Invitrogen) 410158406-diamidino-2-phenyl-indole (Sigma) with washing after each step and mountingmedium (Vector) Rabbit anti-mouse keratin 18 (clone LE61)was a gift from EB Lane (University of Dundee DundeeUK) Standard direct immunofluorescence was performedsimilar to the above procedures with EGFP detection exceptthat the only antibody used was Alexa Fluor 594 conjugatedrabbit anti-GFP (Invitrogen) The images were acquired andanalyzedwith a Zeiss Axio fluorescence imaging system (CarlZeiss MicroImaging Inc)

3 Results

31 Tracing of Basal Cell Development in AdultMouse Prostatevia Keratin 14 Analysis of keratin expression during prostatedevelopment has been useful in distinguishing undifferent-iated basal cells and during tissue regeneration the moredifferentiated luminal cells To determine if luminal cellswhich constitute the major population (90) in mouse pros-tate epithelia are derived frombasal cells during injury repairthe lineage tracing CreloxP system was utilized to track cellsexpressing keratin 14 (a marker of basal cells) and their dif-ferentiated progenies in the mouse prostateThese transgenicmice contain the tamoxifen-inducible Cre gene (CreER)under the control of the keratin 14 promoter To visualizethe developmental and regenerative pattern of keratin 14

BioMed Research International 3

K14 promoter ROSA promoter Stop codon LacZ

ROSA promoter LacZ

Off

On

LoxP

CreER +

(a)

Cre

LacZ

K14-

CreE

RRO

SA-L

acZ

LacZ

RO

SA-L

acZ

foun

der

K14-

CreE

R fo

unde

r

(b)

Figure 1 Mouse prostate basal lineage development in mice as determined by the cell fate tracking system (a) Schematic representationof the generation of keratin 14-CreERROSA-LacZ mouse model (b) PCR for CRE and LaZ expression in the keratin 14-CreERRosa-LacZconfirmed the successful generation of transgenic mice

expressing-cells keratin 14-CreER mice were crossed withROSA 26-LacZmice ROSA 26 is a universal promoter in thiscase driving a LacZ gene preceded by a floxed stop sequence[9] (Figure 1(a)) For bothCRE and LacZ expression crossingwas verified by PCR (Figure 1(b)) Primers for both CREand LacZ are listed in Table 1 Crossing ROSA 26-LacZ micewith keratin 14-CreER mice and subsequent dosing withtamoxifen led to recombination of the Rosa 26minigene withloss of the stop sequence and expression of the LacZ gene

Before being sacrificed tamoxifen-treated keratin 14-CreERROSA-LacZ mice underwent 2 cycles of androgenmanipulation by castration and testosterone supplementation(Figure 2(a)) Experimental and control mice were stainedwith X-gal to visualize LacZ gene expression The keratin14-CreER genetic-based labeling efficiency was evident intamoxifen-treated mice as no X-gal staining was observedin untreated mice (Figure 2(b)) Tamoxifen-induced keratin14 expressing-basal prostate cells gave rise to a full lineageof cells in the adult prostate nodule LacZ expression wasevident in the luminal cells in both the distal and intermediateregions with essentially no staining in the basal cell layerindicating that keratin 14-positive basal cells gave rise toluminal cells (Figure 2(c)) In contrast LacZ expressionwas observed in the proximal region and expression wascontained with the basal layer of cells Untreated keratin14-CreERROSA-LacZ mice did not exhibit any positivestaining Thus it appears that in the intermediate and distalregions keratin 14-expressing cells adopt a luminal fate andare exclusively retained in the basal layer in the proximalregion

32 Slowly Cycling Cells within the Proximal Region AreKeratin 5+Keratin 18minus The accepted paradigm for prostatedevelopment is that all epithelial cells are derived from acommon embryonic precursor that expresses keratin 5 [10]Experiments involving use of bromodeoxyuridine- (BrdU-)based label-retaining assays demonstrate that mouse pro-static epithelial stem cells are concentrated in the proximal

Table 1 Primers for Genotyping for K14-CreERRosa-LacZ mice

Primers Sequence 51015840 rarr 31015840

LacZ mutant forward GCG AAG AGT TTG TCC TCA ACCLacZ wild typeforward

AAA GTC GCT CTG AGT TGT TAT

LacZ wild typereverse

GGA GCG GGA GAA ATG GAT ATG

Cre forward GCG GTC TGG CAG TAA AAA CTA TC

Cre reverse GTG AAA CAG CAT TGC TGT CAC TT

region of prostatic ducts where keratin 5-expressing basalcells are highly enriched [11] However a major limitation inthe use of BrdU to assess label retention is that the presumedslow turnover of stem cells may prevent these cells fromincorporating the label In addition it is not possible to testthe function of cells prospectively isolated based on theirBrdU content To circumvent these problems we generateda keratin 5-tTA-TRE-H2BGFP mouse strain that allows fortightly controlled ubiquitous doxycycline-inducible expres-sion of an H2B-GFP fusion protein with the keratin 5 pro-moter upstream (Figure 3(a)) Since skin keratinocytes arealso derived from basal keratin 5-expressing cells the ldquogreenskinrdquo can be detected by UV light further demonstrating adouble transgenic individual mouse with H2BGFP-labeledmouse prostate epithelium (Figure 3(b))

Keratin 5 expression was tracked through multiple cyclesof androgen manipulation by castration of young miceand by injection of testosterone pellets into adult mice(Figure 4(a)) Keratin 5-tTA-TRE-H2BGFP mice were sacri-ficed at the end of the chase and full cycles of label wash-out(involution and regeneration of more than 10 cycles) Tissuesections of the proximal region were imaged with sectionsof skin as negative controls (Figure 4(b)) Cells that retainedthe GFP label were rare however all positive cells were


Involution Regeneration

Tamoxifen Castration Testosterone

Cycle2

3 w

(2 + 3w)Cycle 1

(a)

Urogenital organs

Whole view

Tamoxifen-treated Tamoxifen-free control

(b)

Proximal Distal EGFP

Prostate

Intermediate

Tamoxifen-treated

Tamoxifen-free control

(c)

Figure 2 Lineage tracking of keratin 14 expression in developing prostate mouse nodules (a) Schematic representation of the treatmentprotocol (b) 120573-Galactosidase staining or fluorescent imaging of the genetic label controlled by basal cell-specific keratin 14 gene promoteractivity showed up in all cell lineages of mouse prostate nodules before and after treatment of the mice with tamoxifen (c) The moredifferentiated distal prostate region showed a higher density of 120573-galactosidase staining than that in the proximal and intermediate regionsThe magnification of the microscopy was 400x

containedwithin the basal layer (Figure 4(b))TheGFP signalwas specific for immunofluorescence with an antibody toGFP (anti-GFP) demonstrated virtually an identical stainingpattern (Figure 4(b)) To assess the keratin expression patternfurther application of antibodies to keratin 5 and keratin18 markers of basal and luminal cells respectively was alsoconductedAs determinedwith differential color immunoflu-orescence most cells within the basal layer are keratin 5positive (red) however colocalized immune-stained keratin5endogenous keratin 5-GFP cells are still rare (Figure 5(a))This was confirmed by anti-GFP and Alexa 594 antibodies aspositive controls (Figure 5(b)) These cells were also negativefor expression of keratin 18 (Figure 5(c)) which furthersuggested that these slowly cycling cells had features ofstem cells Results with the keratin 5-tet-off-H2BGFP modelwhich were consistent with the conclusion from data derivedwith the model for genetic tracing keratin 14-CreERROSA-LacZ reinforce the concept that although rare the renewable

cells that survive androgen manipulation are found in theproximal region and are derived from the basal layer

4 Discussion

Stem cell biology and tumor biology are intimately relatedAn understanding of the function of prostate stem cells inthe lineage-associated differentiation responsible for main-taining tissue integrity has potential application to prostateregeneration and tumorigenesis Previous attempts to definethese lineage-derived cells have utilized assays performedin vitro and in vivo murine models have been used predom-inately In contrast to human prostate which has a completeprostate tissue unit with a continuous basal and luminal layerthe mouse prostate has four major lobe pairs and is furtherdivided into numerous ducts There is no clear basal layerin the ducts Results from previous investigations utilizingthe label-retaining assay suggest that the proximal region of


BK5 X TRETight H2B-EGFP

pTRE-Tight-H2BGFP

TREmodMini CMV promoter

Amp resistance gene

Col E1 ori

H2B

GFP

SV40 polyA

BamHI (722)

ClaI (1471)HindIII (1476)

NotI (1463)

KpnI (340)

ApaLI (2123)

ApaLI (3369)

AvaI (1689)

EcoRI (533)NcoI (520)

NcoI (739)

XhoI (1689)

AvaI (2)XhoI (2)

EcoRI (324)

3692bp

tetR VP16

(a)

Control mouse in the same litter

The Tg K5-tTA-TRE-H2BGFP

(b)

Figure 3 Generation of double transgenic mice keratin 5-tTA TREtight-H2B-EGFP (a)The transgenic construct was generated by insertingan H2B-GFP fused gene fragment into the pTRE-Tight vector (b) Schematic representation of generation of the double-transgenic mice bycrossing keratin 5-tTA and TREtight-H2B-EGFP mice (c) Epifluorescence of a whole animal under UV light Only the double-transgenicmice with keratin 5-tTA TREtight-H2B-EGFP showed green skin under UV light

the mouse prostate is more likely to contain adult prostatestem cells that express basalmarkers and that the distal regionof the duct contains more differentiated luminal cells withexpression of luminal makers [11 12] In contrast there isevidence to support a luminal origin of cells for normal pros-tate regeneration as well as prostate cancer stem cells [13 14]Nevertheless there remains a lack of agreement as to whichcells are responsible for prostate development

To identify stemprogenitor cells capable of generatingmultiple lineages we utilized a multidirected approach Firstwe were interested in identifying cells within the basal layer

and determining which basal-derived cells were capable ofluminal differentiation Keratin 14 is a specificmarker of basalcells As established with the mouse model of tamoxifen-induced label-retaining keratin 14-CreERROSA-LacZ theinitial keratin 14-mediated LacZ expression was eventuallyrestored in the luminal cells as opposed to basal cells in thedistal and intermediate regions However within the prox-imal region keratin 14 expression was primarily expressedin the basal layer Based on these findings we propose thatduring prostate development especially in the scenario oftissue injury luminal cells are derived from the basal layer



Label chasing with dox treatment for 8ndash10 cycles

Castration Testosterone

Cycle2

3 w

(2 + 3w)Cycle 1

(a)

Prostate Skin

DAPI EGFP Merged DAPI EGFP Merged

Founder TRE-H2BGFP

K5-tTA-TRE-H2BGFP before chase

K5-tTA-TRE-H2BGFP chase2w

K5-tTA-TRE-H2BGFP chase7ndash8w

FVB

(b)

Figure 4 Keratin 5-expressing cells in the proximal region of prostate tissue after multiple cycles of androgen deprivation and restoration (a)Schematic representation of the labeling protocol in Keratin 5-tTA-TRE-H2BGFP mice (b) Mice were sacrificed at the end of the chase andfull cycles of label wash-out (involution and regeneration of more than 10 cycles) and tissue sections from the proximal regions of prostatewere imaged for endogenous GFP expression indicating the K5 positive cells Skin tissue was utilized as a positive control

However since most if not all of the basal cells retainedkeratin 14 expression these cells are likely to be the progenitorcells not the stem cells within the basal layer that give riseto keratin 14-expressing luminal cells Thus our results agreewith other reports that the basal layer is the origin of moredifferentiated luminal cells

Multiple lines of evidence support the hypothesis thatstem cells within the basal layer of prostates in noncastratedand castrated animals are responsible for tubule-formingcapacity and give rise to multipotent progenitor cells withinthe basal layer By use of an artificial tissue injury and repairprocess the presence of living keratin 5-positive stem cellswas tracked in keratin 5-tTA-TRE-H2BGFP mice with thehypothesis that the luminal lineage is derived from a basallineage Furthermore the slowly cycling cells that retain theH2BGFP label after multiple cycles of castrationhormonereplacement would represent the population of adult prostatestem cellsThe keratin 5 GFP-positive cells were only a minorfraction of the cellsThese GFP-positive keratin 5-expressingcells also lacked coexpression of luminal marker keratin 18

This suggests that all other cells including transit-amplifyingcells and possibly some progenitorstem cells entering intothe cell cycle which loseH2BGFP expression are diluted outSince keratin 5 is a marker of basal cells the results indicatethat these cells are basal cells or at least that they are derivedfrom the basal layer

With keratin 5 as a marker others have shown that theproximal cells of mouse prostatic ducts are slow cyclinghave a high proliferative potential and give rise to largebranched glandular structures that produce prostatic secre-tory products [11] Nevertheless we cannot be sure thatkeratin 5+keratin 18minus cells are prostate stem cells since it isimpossible to determine distant lineage with this approachOur evidence does suggest that these cells are basal-restrictedslowly cycling progenitor cells Further evidence is requiredto characterize this population of cells

We present strong evidence that the basal-derived lineageis responsible for prostate development and tissue regen-eration but there are indications that luminal cells retainregenerative capacity after animals are castrated The most


EGFP DAPI K5 Merged

(a)

EGFP signal DAPI Anti-GFP Merged

(b)

EGFP DAPI K18 Merged

(c)

Figure 5 In the basal layer of transgenic prostates H2BGFP-labeled slowly cycling cells are keratin 5-specific not keratin 18-specific (a)Fluorescent microscopy confirmation of H2BGFP tet-off labeling and chase in the prostate of the double transgenic mice Mouse tissuescontaining H2BGFP-labeled slowly cycling cells costained with keratin 5 antibody were specific to the basal layer (b)The H2BGFP label inmouse tissue was confirmed by a GFP-specific antibody (c) In the transgenic prostate H2BGFP-labeled slowly cycling cells were not keratin18-specific as determined by costaining with keratin 18 antibody

recent is a report of experiments involving use of a PSA-CreERT2-based genetic lineage tracing system These resultsdemonstrate that in a mouse model preexisting luminal epi-thelial cells survive and proliferate during cycles of regressionand regrowth of adult prostate [15] Although keratin 5-positive cells were evident by immunostaining none co-localized with PSA-expressing cells that were responsible forAR-positive luminal cells While the different number ofregenerative cycles could be responsible for these divergentresults it is not clear if the observed luminal cell regener-ation is the result of basal-derived luminal progenitor cellsthat become active only during an immediate response toinjuryrepair Furthermore there may be within the basaland luminal compartments stemprogenitor cells with thecapacity to regenerate prostate tubules after the animalsare castrated Our findings that keratin 14-expressing cellswith restricted label are expressed in luminal cells duringmouse development points to a contribution of stem cellprogenitor cells within the basal compartment during devel-opment and regeneration Further evidence derived withdouble-knockout conditional-expression models with basaland luminal differentiation targets is required to determinewhich population of cells is responsible for tissue regenera-tion

The present findings have implications in initiation ofprostate cancer Basal cells from primary benign human

prostate tissue with the cooperative effects of AKT ERG andAR recapitulate the histological and molecular features ofhuman prostate cancer with loss of basal cells and expansionof luminal cells expressing PSA and alpha-methylacyl-CoAracemase in immunodeficient mice [16 17] In PTEN-nullmice there is a preferential expansion of basal cells relative toluminal cells [18] and these cells demonstrate more efficientcapacity for cancer initiation relative to luminal cells A rolefor basal cells in prostate cancer has been implicated duringtreatment as well Most prostate cancers are treated withhormone ablation therapy with eventual relapse of hormone-insensitive cells Thus hormonal therapy may interfere onlywith the bulk population of AR-expressing cells while theAR-negative more primitive tumor initiating cells survivehormonal therapy and give rise to progeny that have devel-oped amechanism to escape hormonal therapyThus furthercharacterization of the population associated with tumorinitiation could lead to more effective therapies

In summary with the use a mouse model for trackingcell fates and a mouse label-retaining assay we determinedthat in the prostate luminal cells are derived from a basallineage and that slowly cycling cells which may representadult prostate stem cells reside in the basal cell compartmentThese observations have implications for the concept ofepithelial cell lineage in the developing human prostate andfor identification of cancer steminitiating cells


Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Jianjun Zhou designed and performed the experiments an-alyzed the data and wrote the paper Jonathan C Vogel de-signed the experiments and analyzed the data Lionel Feigen-baum made the TRETight-H2BGFP transgenic foundermice Carole Yee maintained and genotyped the transgenicmice andoptimized the immunostaining experimentsHong-bin Song and Clayton Yates analyzed the data and wrote thepaper

Acknowledgments

The authors would like to provide a special honor to the lateJonathan C Vogel who was directly involved in this workHis guidance was instrumental This work was support-ed by Grants G12 RR03059-21A1 (NIHRCMI) [CY] U54CA118623 (NIHNCI) [CY] a Department of Defense GrantPC120913 [CY] the National Natural Science Foundationof China (no 81070969 and no 81171554) and the BeijingMunicipal Natural Science Foundation (no 7102126)

References

[1] P E Burger X Xiong S Coetzee et al ldquoSca-1 expression iden-tifies stem cells in the proximal region of prostatic ducts withhigh capacity to reconstitute prostatic tissuerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 20 pp 7180ndash7185 2005

[2] A T Collins F K Habib N J Maitland and D E NealldquoIdentification and isolation of human prostate epithelial stemcells based on 12057221205731-integrin expressionrdquo Journal of Cell Sciencevol 114 part 21 pp 3865ndash3872 2001

[3] H F English R J Santen and J T Isaacs ldquoResponse of glandu-lar versus basal rat ventral prostatic epithelial cells to androgenwithdrawal and replacementrdquo Prostate vol 11 no 3 pp 229ndash242 1987

[4] A S Goldstein J Huang C Guo I P Garraway and O NWitte ldquoIdentification of a cell of origin for human prostate can-cerrdquo Science vol 329 no 5991 pp 568ndash571 2010

[5] A S Goldstein T Stoyanova and O N Witte ldquoPrimitive ori-gins of prostate cancer in vivo evidence for prostate-regenerat-ing cells and prostate cancer-initiating cellsrdquoMolecular Oncolo-gy vol 4 no 5 pp 385ndash396 2010

[6] C Grisanzio and S Signoretti ldquop63 in prostate biology andpathologyrdquo Journal of Cellular Biochemistry vol 103 no 5 pp1354ndash1368 2008

[7] S W Hayward and G R Cunha ldquoThe prostate developmentand physiologyrdquoRadiologic Clinics of North America vol 38 no1 pp 1ndash14 2000

[8] D A Lawson Y Zong S Memarzadeh L Xin J Huang andO N Witte ldquoBasal epithelial stem cells are efficient targets forprostate cancer initiationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 107 no 6 pp2610ndash2615 2010

[9] K G Leong B E Wang L Johnson and W Q Gao ldquoGenera-tion of a prostate from a single adult stem cellrdquoNature vol 456no 7223 pp 804ndash808 2008

[10] J Liu L E Pascal S Isharwal et al ldquoRegenerated luminal epi-thelial cells are derived from preexisting luminal epithelial cellsin adult mouse prostaterdquo Molecular Endocrinology vol 25 no11 pp 1849ndash1857 2011

[11] R BNagleMK Brawer J Kittelson andV Clark ldquoPhenotypicrelationships of prostatic intraepithelial neoplasia to invasiveprostatic carcinomardquo American Journal of Pathology vol 138no 1 pp 119ndash128 1991

[12] E J Robinson D E Neal and A T Collins ldquoBasal cells are pro-genitors of luminal cells in primary cultures of differentiatinghuman prostatic epitheliumrdquo Prostate vol 37 no 3 pp 149ndash1601998

[13] A Tsujimura Y Koikawa S Salm et al ldquoProximal locationof mouse prostate epithelial stem cells a model of prostatichomeostasisrdquo Journal of Cell Biology vol 157 no 7 pp 1257ndash1265 2002

[14] G J L H van Leenders W R Gage J L Hicks et al ldquoInterme-diate cells in human prostate epithelium are enriched in prolif-erative inflammatory atrophyrdquo American Journal of Pathologyvol 162 no 5 pp 1529ndash1537 2003

[15] S Wang A J Garcia M Wu D A Lawson O N Witte andH Wu ldquoPten deletion leads to the expansion of a prostaticstemprogenitor cell subpopulation and tumor initiationrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 103 no 5 pp 1480ndash1485 2006

[16] X Wang M K Kruithof-de Julio K D Economides et al ldquoAluminal epithelial stem cell that is a cell of origin for prostatecancerrdquo Nature vol 461 no 7263 pp 495ndash500 2009

[17] Y Yang J Hao X Liu B Dalkin and R B Nagle ldquoDiffer-ential expression of cytokeratin mRNA and protein in normalprostate prostatic intraepithelial neoplasia and invasive carci-nomardquo American Journal of Pathology vol 150 no 2 pp 693ndash704 1997

[18] B P Zambrowicz A Imamoto S Fiering L A HerzenbergW G Kerr and P Soriano ldquoDisruption of overlapping tran-scripts in the ROSA 120573geo 26 gene trap strain leads to wide-spread expression of 120573-galactosidase in mouse embryos andhematopoietic cellsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 94 no 8 pp 3789ndash3794 1997

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allows lineage-specific tagging of the keratin 14-expressingpopulation of cells throughout prostate development in adultmice and (2) a keratin 5H2BGFP-label retainingmodel dem-onstrating that slowly cycling cells are represented in only asubfraction of keratin 5-expressing cells after hormonecas-trationmanipulationThese cells have the capacity to give riseto more differentiated luminal cells Results with these mod-els which suggest that the putative adult stem cells whichhave a slowly-cycling feature most likely reside in the basallayer and are positive for keratin 5 and keratin 14 clarify someof the basic aspects of the biology of the rodent prostate gland

2 Materials and Methods

21 Cell Fate Tracing Assay Keratin 14-CreERtam transgen-ic mice (STOCK Tg (KRT14-creEsr1)20EfuJ) LacZ report-er mice (B6129S4-Gt(ROSA)26Sortm1SorJ) and EGFP re-porter mice (B6129-Gt(ROSA)26Sortm2ShoJ) were pur-chased from the Jackson Laboratory Bar Harbor ME USAThe two transgenic mouse lines were crossed and doubletransgenic markers of Cre and LacZ were confirmed by PCRwith genomic DNA isolated from their progenies Tamoxifen(Sigma-Aldrich) was prepared at 100mgmL in ethanol heat-ed to 60∘C to dissolve the powder and then diluted 10 timeswith sunflower oil aided by 2min vortex plus sonication foran additional 30min Tamoxifen was used to induce Cre ac-tivity in gaining its access to the nuclear compartment fromthe cytoplasm The administration to young (3 weeks old oryounger) double-transgenic mice of 05mg tamoxifen wasaccomplished by intraperitoneal injections daily for 5 daysThe double-transgenicmicewere sacrificed after two cycles ofprostate involution (2 weeks) and regeneration by castrationand by supplementation (for 2-3 weeks) with testosteronepellets (125mg sustained release Innovative Research ofAmerica) The prostates were removed and frozen sectionsfrom dorsal and ventral glands were obtained The presenceof 120573-galactosidase was demonstrated with X-gal by use of acommercial kit (Invitrogen)

22The Label-Retaining Assay Transgenic mice expressing aTet-OFF tTA regulatory transactivator under the control of akeratin 5 promoter were generously provided by Adam Glick(National Cancer Institute The Pennsylvania State Universi-ty) These mice were bred to TRETight-H2BGFP transgenicmice that we created in the NCI-Frederick animal facilityExpression of H2BGFP is naturally turned on in the double-transgenic mice (keratin 5-tTATRETight-H2BGFP) follow-ing development and can be turned off in the basal epitheliumof the prostate following the application of doxycycline Awash-out period is required to identify the label-retainingcellsThe prostate epithelial cells that are still H2BGFP+ afterthe wash-out period mark the label-retaining cells whichbecause of their slowly cycling feature should represent pros-tate epithelial stem cells To ensure the observation of thoserare putative stem cells resistant to label dilution due to cellturnover this wash-out period continued throughout themultiple cycling (over 10) of the prostate epithelium betweenregression and expansion by hormonal manipulation Afterthe label-retaining assay of themurine prostate was complete

the animals were anesthetized and the prostates were re-moved for histologic analysis

23 Castration and Hormone Manipulation The hormonalmanipulation consisted of alternating 2-3 week periods ofandrogen deprivation and restoration This manipulationresults in regression of the prostate epithelium to a residualbasal layer (androgen deprivation) followed by expansion toa multilayered epithelium (androgen restoration) Hormonaldeprivation was accomplished by castration and androgenrestoration was achieved by use of implantable testosteronepellets (125mg sustained release Innovative Research ofAmerica) which maintained a serum level of 4 ngmL

24 120573-Galactosidase Staining The 120573-galactosidase stainingkit was used according to the manufactures suggested pro-cedure to demonstrate the activity of LacZ Briefly thawedcryosections of mouse prostates were placed in the fixativesolution for 10min After washing the samples were incu-bated at 37∘C with a solution containing X-gal and the bluecells were assessed within 2 hThe slides were counterstainedwith nuclear fast red solution

25 Immunofluorescence Formarker expression standard in-direct immunofluorescence staining was performed on 6 120583mmouse tissue cryosections Briefly the thawed sections werefixed with 4 paraformaldehyde in PBS for 10 minutes atroom temperature and after washing with PBS blocked with10 donkey whole serum in PBS After washing cells wereincubated sequentially with primary antibody rabbit poly-clonal anti-mousekeratin5 (CovanceResearchProducts Inc)and Alexa Fluor-594 or 546-conjugated donkey-anti-rabbitsecondary antibody (Invitrogen) 410158406-diamidino-2-phenyl-indole (Sigma) with washing after each step and mountingmedium (Vector) Rabbit anti-mouse keratin 18 (clone LE61)was a gift from EB Lane (University of Dundee DundeeUK) Standard direct immunofluorescence was performedsimilar to the above procedures with EGFP detection exceptthat the only antibody used was Alexa Fluor 594 conjugatedrabbit anti-GFP (Invitrogen) The images were acquired andanalyzedwith a Zeiss Axio fluorescence imaging system (CarlZeiss MicroImaging Inc)

3 Results

31 Tracing of Basal Cell Development in AdultMouse Prostatevia Keratin 14 Analysis of keratin expression during prostatedevelopment has been useful in distinguishing undifferent-iated basal cells and during tissue regeneration the moredifferentiated luminal cells To determine if luminal cellswhich constitute the major population (90) in mouse pros-tate epithelia are derived frombasal cells during injury repairthe lineage tracing CreloxP system was utilized to track cellsexpressing keratin 14 (a marker of basal cells) and their dif-ferentiated progenies in the mouse prostateThese transgenicmice contain the tamoxifen-inducible Cre gene (CreER)under the control of the keratin 14 promoter To visualizethe developmental and regenerative pattern of keratin 14



ROSA promoter LacZ

Off

On

LoxP

CreER +

(a)

Cre

LacZ

K14-

CreE

RRO

SA-L

acZ

LacZ

RO

SA-L

acZ

foun

der

K14-

CreE

R fo

unde

r

(b)


















Cycle2

3 w

(2 + 3w)Cycle 1

(a)

Urogenital organs

Whole view


(b)


Prostate

Intermediate

Tamoxifen-treated


(c)




4 Discussion




pTRE-Tight-H2BGFP


Amp resistance gene

Col E1 ori

H2B

GFP

SV40 polyA

BamHI (722)


NotI (1463)

KpnI (340)

ApaLI (2123)

ApaLI (3369)

AvaI (1689)


NcoI (739)

XhoI (1689)

AvaI (2)XhoI (2)

EcoRI (324)

3692bp

tetR VP16

(a)



(b)









Cycle2

3 w

(2 + 3w)Cycle 1

(a)

Prostate Skin


Founder TRE-H2BGFP




FVB

(b)








EGFP DAPI K5 Merged

(a)


(b)


(c)











Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



ROSA promoter LacZ

Off

On

LoxP

CreER +

(a)

Cre

LacZ

K14-

CreE

RRO

SA-L

acZ

LacZ

RO

SA-L

acZ

foun

der

K14-

CreE

R fo

unde

r

(b)


















Cycle2

3 w

(2 + 3w)Cycle 1

(a)

Urogenital organs

Whole view


(b)


Prostate

Intermediate

Tamoxifen-treated


(c)




4 Discussion




pTRE-Tight-H2BGFP


Amp resistance gene

Col E1 ori

H2B

GFP

SV40 polyA

BamHI (722)


NotI (1463)

KpnI (340)

ApaLI (2123)

ApaLI (3369)

AvaI (1689)


NcoI (739)

XhoI (1689)

AvaI (2)XhoI (2)

EcoRI (324)

3692bp

tetR VP16

(a)



(b)









Cycle2

3 w

(2 + 3w)Cycle 1

(a)

Prostate Skin


Founder TRE-H2BGFP




FVB

(b)








EGFP DAPI K5 Merged

(a)


(b)


(c)











Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Cycle2

3 w

(2 + 3w)Cycle 1

(a)

Urogenital organs

Whole view


(b)


Prostate

Intermediate

Tamoxifen-treated


(c)




4 Discussion




pTRE-Tight-H2BGFP


Amp resistance gene

Col E1 ori

H2B

GFP

SV40 polyA

BamHI (722)


NotI (1463)

KpnI (340)

ApaLI (2123)

ApaLI (3369)

AvaI (1689)


NcoI (739)

XhoI (1689)

AvaI (2)XhoI (2)

EcoRI (324)

3692bp

tetR VP16

(a)



(b)









Cycle2

3 w

(2 + 3w)Cycle 1

(a)

Prostate Skin


Founder TRE-H2BGFP




FVB

(b)








EGFP DAPI K5 Merged

(a)


(b)


(c)











Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



pTRE-Tight-H2BGFP


Amp resistance gene

Col E1 ori

H2B

GFP

SV40 polyA

BamHI (722)


NotI (1463)

KpnI (340)

ApaLI (2123)

ApaLI (3369)

AvaI (1689)


NcoI (739)

XhoI (1689)

AvaI (2)XhoI (2)

EcoRI (324)

3692bp

tetR VP16

(a)



(b)









Cycle2

3 w

(2 + 3w)Cycle 1

(a)

Prostate Skin


Founder TRE-H2BGFP




FVB

(b)








EGFP DAPI K5 Merged

(a)


(b)


(c)











Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Cycle2

3 w

(2 + 3w)Cycle 1

(a)

Prostate Skin


Founder TRE-H2BGFP




FVB

(b)








EGFP DAPI K5 Merged

(a)


(b)


(c)











Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


EGFP DAPI K5 Merged

(a)


(b)


(c)











Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Acknowledgments


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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