colocalization of basal and luminal cell-type cytokeratins in...

ICANCER RESEARCH 52. 6182-6187. November 15, 1992]

Colocalization of Basal and Luminal Cell-type Cytokeratins in Human

Prostate Cancer

Anja P. M. Verhagen, Frans C. S. Ramaekers, Tilly W. Aalders, H. Ewout Schaafsma, Frans M. J. Debruyne,and Jack A. Schalken1Urological Research Laboratory' [A. P. M. V., T. W. A., F. M. J. D., J. A. SJ and Department of Pathology [H. E. S.J, University Hospital Nijmegen, Nijmegen,the Netherlands, and Department of Molecular Cell Biology and Genetics, University ofLimburg, Maastricht, the Netherlands [F. C. S. R.]

ABSTRACT

In the epithelium of secretory acini of the prostate two different celltypes can be discriminated on the basis of localization, morphology, anddegree of differentiation, the luminal and basal cells. The possibility ofa developmental relationship between basal and luminal cells has beena subject of interest in several studies. According to the stem cell modelat least three cell types, i.e., stem, amplifying, and transit cells, can bediscriminated in the epithelium of prostate secretory acini. We previously reported that in the process of degeneration and regeneration innormal rat prostate a population of cells could be identified as candidates for the amplifying cells. These cells showed a keratin expressionprofile intermediate between those of basal and luminal cells. We nowshow, by using keratin antibodies, that also in normal human prostate atleast three subpopulations of cells can be identified, one of them puta-tively representing amplifying cells as defined in the stem cell model.Furthermore, these antibodies were used to obtain a better insight intothe different cell types involved in the etiology and progression of pro-static carcinoma. Both primary and hormone-independent prostatic tumors were investigated. Our results indicated that the candidate stemcell population was absent in prostatic carcinoma. Unlike earlier reportson the unique presence of cells with luminal characteristics in prostaticcarcinoma, we identified also a population of cells coexpressing basaland luminal cell-type cytokeratins in primary and hormone-independentprostatic carcinoma. Since amplifying cells are defined in the stem cellmodel as precursors of transit (luminal) cells in the hierarchical pathway of prostatic epithelium differentiation, we postulate that on thebasis of the keratin expression profile this subpopulation is most likelythe target for neoplastic transformation.

INTRODUCTION

Prostate cancer, the most common malignancy in the Western male population, is characterized by its androgen responsiveness. Most prostatic cancers retain an androgen dependencefor stimulation of their growth. From this finding, Huggins andHodges (1) concluded that prostatic tumors could be treated byandrogen ablation therapy. Indeed, prostatic cancers appear toretain an androgen responsiveness, yet unfortunately this response is of limited duration and the tumors show progressionto an androgen-independent state. Various studies indicate thatthis unresponsive state is due to the presence of hormone-independent cells at the start of therapy (2, 3). Even though thepresence of such a population is established, it has not beenpossible to elucidate the origin of these cells. Moreover, themolecular processes occurring during normal proliferation anddifferentiation of the prostate are poorly understood, and abetter understanding of these mechanisms is an essential requirement for elucidation of the origin and progressive behaviorof this malignancy. Recently the differentiation pathways innormal and malignant rat prostate have been immunopheno-

Reccived 1/27/92; accepted 9/4/92.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at Urological ResearchLaboratory, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen,The Netherlands.

typed by using antibodies directed against keratins (4, 5). Keratins are major components of the cytoskeleton of epithelialcells and at least 20 different keratins can be distinguished inhuman epithelia. They are differentially expressed in specificepithelial cell types or in specific pathways of epithelial differentiation (6). In general, the epithelium of secretory acini of theprostate can be described as consisting of two different celltypes, i.e., the luminal and basal cells, which differ in localization, morphology, and degree of differentiation. These two celllayers can be easily discriminated by antibodies directed againstkeratins (4, 5, 7-10). A more recent hypothesis postulates theexistence of at least three different cell types, i.e., stem, amplifying, and transit cells (11). Cells that are morphologically intermediate, because they have structural features characteristicof both the basal and luminal cells, have been identified in thenormal prostatic epithelium of monkeys (12), humans (13), anddogs (14); in addition, they are apparent in mice under conditions of androgen manipulation (15). Our study describing theprocess of degeneration and regeneration in normal rat prostaterevealed a population of cells showing a keratin expressionprofile intermediate between those of basal and luminal cells(4). This observation was consistent with the concept, as described by Isaacs (3), that stem cells comprise a subpopulationof the basal cells that subserve a population of transient amplifying cells, which in turn give rise to the secretory tall columnarepithelial (transit) cells. The amplifying cells would then bephenotypically characterized by coexpression of basal and luminal cell-type cytokeratins. Antibodies directed against keratins can, therefore, serve as markers for the different cell typesin the differentiation pathways and are used in this study toobtain more insight into the etiology and progression of prostate cancer.

MATERIALS AND METHODS

Human Prostatic Tumor Tissue. Surgical specimens were categorized into three groups on the basis of the OS2 (16). Primary prostatic

tumors were obtained by transurethral resection or radical prostatectomy from 49 patients (GS 2-3, 22; GS 4, 17; GS 5, 10). Surgicalspecimens from 15 (GS 2-3, 5; GS 4, 5; GS 5, 5) patients with recurrentprostatic cancer and a history of androgen ablation therapy (eitherorchiectomy or hormonal therapy) were obtained by transurethral resection and are referred to as hormone-independent tumors. Thesepatients had complaints caused by locally recurrent tumors. Apart fromthe clinical progression, high KÃŒ67labeling indices indicated that thetumor cells indeed were proliferating, despite the androgen withdrawal(17). The tissues were kept frozen at -200Â°C until use.

Indirect Immunofluorescence and Immunoperoxidase Technique.Frozen sections of the prostatic tissue (6 Â¿im)were cut on a cryostat andair-dried before fixation in acetone for 10 min. After washing in PBSfor 10 min, the tissue sections were incubated with 10% normal goatserum in PBS at room temperature for 30 min and then incubated withthe primary antiserum for 1 h. After repeated washing in PBS (3 times,

2 The abbreviations used are: GS, Gleason score; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PMSC, phenylmethylsulfonyl chloride; SDS,sodium dodecyl sulfate.

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BASAL CELL-TYPE CYTOKERATINS IN PROSTATIC CARCINOMA

10 min) the procedure followed was different for the two techniques, cell population, as summarized in Table 1. Occasionally K19For immunofluorescence detection fluorescein isothiocyanate-conju-gated goat anti-mouse IgG (1:25 in 10% normal goat serum; NordicTilburg, the Netherlands) was applied. After incubation for 30 min andextensive washing in PBS (3 times, 10 min) the sections were mountedin Gelvatol (Monsanto, St. Louis, MO) containing 100 mg/ml 1,4-diazobicyclo[2.2.2]octane (Janssen Pharmaceutica, Beerse, Belgium).For the immunoperoxidase method horseradish peroxidase-labeled rabbit anti-mouse IgG (heavy and light chain specific; Nordic) diluted1:100 in PBS containing 1% BSA was applied. After incubation for 30min and extensive washing in PBS (3 times, 30 min) the peroxidaseactivity was detected with 3,3-diaminobenzidine (0.5 g/liter; Sigma, St.Louis, MO) containing 0.6% imidazole (Sigma) and 0.04% H2O2. Afterwashing with water the reaction was intensified with CuSO4 (0.5% in0.9% NaCl). After washing with water the sections were counterstainedwith hematoxylin. Double-label direct immunofluorescence studiesused the monoclonal antibody RGB 53 directly labeled with biotin incombination with the unlabeled monoclonal antibody RCK 103, following the procedure previously described (4). For each section fiveareas of similar grade were analyzed semiquantitatively, by two independent investigators, for the fraction of cells staining.

Gel Electrophoresis and Immunoblotting. Cryostat sections (20 Â¿im)of prostatic tissue of homogeneous degree of differentiation were extracted with cold, high-salt, detergent buffer containing 0.5% TritonX-100, 1.5 MKC1, 10 mM Tris-HCl, pH 7.4, 5 IHMEDTA, and 0.5 minPMSC (Merck). After extraction (20 min, 0Â°C)the insoluble material

was sedimented (5 min, 800 x g), washed in low-salt detergent buffercontaining 10 m\i Tris-HCl, pH 7.4, 5 min EDTA, and 0.5 mMPMSC,dissolved in SDS sample buffer (2% SDS, 10% glycerol, 5% /J-mercap-toethanol, 0.1 M Tris-HCl, pH 6.8), and finally heated for 2 min at100Â°C.Proteins were separated according to their molecular weight in

13% SDS polyacrylamide gels, as described by Laemmli (18). Transferof the proteins to nitrocellulose was performed in 192 mM glycine, 25min Tris, pH 8.3, 20% methanol, at 50 V/250 mA (overnight). Nonspecific binding sites on the nitrocellulose blot were blocked by incubation of the blot for 1 hr with preincubation buffer containing 3%BSA, 350 mM NaCl, 10 mM Tris-HCl, pH 7.4, and 0.5 mM PMSC.Then the appropriate dilution of the primary antibody in buffer 1,containing 0.3% BSA, 150 mM NaCl, 10 IHMTris-HCl, 0.5% sodiumdeoxycholate (BDH Chemicals Ltd, Poole, England), and 0.1% SDSwas applied for 2 h. After extensive washing in buffer 1 (3 times, 10min) and PBS containing 0.5% Triton X-100 (10 min), the blot wasincubated for 1 hr with horseradish peroxidase-labeled rabbit anti-mouse IgG (heavy and light chain specific; Nordic) diluted 1:500 inPBS containing 0.5% Triton X-100 and 0.5% BSA. Finally, after washing 3 times for 10 min in 0.5% Triton X-100 in PBS the peroxidaseactivity was visualized by incubation of the blot for approximately10-15 min with 0.5 mg/ml 4-chloro-l-naphthol and 0.012% H2O2 in

PBS.Antibodies Directed against Keratins. The following monoclonal an

tibodies were used in this study, (a) RGE 53 (IgGl), indicated in thisstudy as K18, is monospecific for keratin 18 in immunoblotting (19). (b)RCK 103 (IgGl), indicated in this study as Kbasai,stains the basal cellcompartment in human prostate (4, 5, 20). (e) LL002 (IgG3), indicatedin this study as K14, is monospecific for keratin 14 (21). (d) RCK 107is also directed against keratin 14. Because RCK 107 gave results similar to those obtained with LL002, it is also indicated as K14. Theresults with K14 shown in this study are those with LL002. (e) LP2K,indicated in this study as K19, recognizes only keratin 19 in the immunoblotting assay (22).

RESULTS('viola-rutili Immunophenotypes in Nonmalignant Human

Prostatic Secretory Acini. In the nonmalignant human prostatic secretory acini, keratin expression patterns were examinedusing the monoclonal antibodies K,,.,,,.,,,K14, K18, and K19.The keratins recognized by these reagents are all expressed inthe glandular cells (Fig. 1). Kbasaland K14 specifically stain thebasal cell population, while K18 and K19 react with the luminal

reactivity was also detected in the basal cells. Besides the twopredominant immunophenotypes, K14VKbasal+/K18~ andK14~/Kbasar/K18+, a third cell type was detected that showed

a keratin expression pattern intermediate between those ofbasal and luminal cells. These cells can be characterized asK14-/Kbasa|+/K18 . In the morphologically defined basal cell

population the combination of K14 and Khasaienables discrimination of two cell types. Cells positive for K14 were alwayspositive for Kbasa),as shown in Fig. 1. However, a subpopula-tion of cells was detected that was positive for Kbasaibut negative for K14, as shown in Fig. \A and confirmed by double-

immunofluorescence studies (data not shown). This indicatesthat sometimes K18~ cells were found that contained themarker Kbasa, but lacked K14. These are indicated as K14~/Kbasai+/K18~ (see also Table 1). Morphologically defined lumi

nal cells are characterized by the absence of staining for K14and Kbasa|. The luminal cells were positive for K18 and K19,although not all luminal cells were positive for K19 (data notshown). In the morphologically defined luminal cells of thenormal human prostate, occasional cells were found that werepositive for Kbasaibut negative for K14. These cells were alsopositive for K18 and are indicated as K14~/Kbasal+/K18+, as

shown in Fig. \B (see also Table 1).Keratin Expression Patterns in Primary Prostatic Tumors.

Tissue sections of 49 cases of human primary prostatic tumorwere examined for the expression of keratins in the tumor cells.In all cases K14 did not react with any of the tumors, as shownin Figs. 2 and 3 and summarized in Table 2. Kbasaireacted withsome of the tumor cells. Examples of Kbasa,+tumors are shownin Fig. 2, while the Kbasa|~ tumors are illustrated in Fig. 3. Of

all the well differentiated tumors investigated, an average of35% of the cells were positive (see Table 2). The different tumors showed a great variability in reactivity of Kbasai(0-90%).With malignant progression and decrease of degree of differentiation the number of Kbasal+ cells also decreased. In poorly

differentiated tumors fewer cells positive for Kbasalwere found(an average of 25%, with a range of 0-45% of cells staining). Inanaplastic tumors no Kbasai+cells were found, although a few

positive cells were present. The tumor cells were always positivefor K18, while K19 stained 50-75% of the tumor cells (see

Table 2).Keratin Expression Patterns in Hormone-independent Pros

tatic Tumors. Tissue sections of 15 human hormone-independent prostatic tumors were also examined for expression of thevarious keratins. It appeared that in all the cases examined thetumor cells were negative for K14, as summarized in Table 3.Kbasaiexpression was heterogeneous and similar to the patternsdescribed for the primary tumors. In the well differentiated andpoorly differentiated tumors approximately 10-15% of the cellswere positive for Kbasa|(Table 3). Kbasa|+cells were also positive

for K18. Anaplastic tumors were totally negative for Kbasahalthough sometimes a few positive cells were found. K18 wasexpressed in all tumor cells. Kl9 again showed a heterogeneousexpression pattern but definitely decreased from 90% positivefor the well differentiated tumors to 50% positive for the poorlydifferentiated tumors and 25% positive for the anaplastic tumors (Table 3).

Characterization of Keratin Expression by Immunoblotting.The presence and identity of the various keratins were alsocharacterized by immunoblot analysis. In Fig. 4A the immuno-blot pattern of normal human prostate is shown, in which allfour antibodies recognized keratin bands with the expected molecular weights (K14, Mr 50,000; K18, A/r 45,000; K19, M,

6183




K14 K18

f

\ â€¢¿�v

\

B_iÂ»

ÃŽ

Fig. 1. Expression pattern of cytokeratins in nonmalignant secretory acini of the prostate. A, illustration of the predominant staining pattern in the acini. Left,staining of the basal cells, with K,,.,...,,staining all basal cells. Middle, immunohistochemical staining with K14, detecting a subpopulation of basal cells. Right, stainingof all luminal cells with K18. fi. illustration of a staining pattern occasionally observed in the acini, k,,.,,.,, also stains a significant portion of the luminal cells.

Table 1 General keratin expression pattern in the different cell typesin normal prostate

Keratin expression

Stem cells(basal cells)

Amplifyingcells

Transit cells(luminal cells)

KI4

KI8K19

40,000; for Kbasa,a band with an apparent molecular weight of58,000 was observed, which may possibly represent keratin 5).Fig. 4B shows the immunoblot pattern of a lesion of a poorlydifferentiated primary prostatic tumor in which only Kbasa),K18, and K19 showed a positive reaction.

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DISCUSSION

Relatively little is known about the fundamental cell biologyof prostatic epithelium. On the basis of localization and differentiation the prostatic epithelium has been described as consisting of basal and luminal cells. The relationship betweenbasal and luminal cells is still a matter of discussion, althoughrecent observations (23) strongly suggest that cells from thebasal compartment proliferate and differentiate into luminalcells during prostatic development. According to the stem cellmodel (11) the prostatic epithelium consists of at least threedifferent cell types, i.e., stem, amplifying, and transit cells. Inthis hypothesis the three components are arranged in an expanding hierarchy. Stem cells have a high potential for cellproliferation but are normally slow-cycling. On the basis of




K basal K 14 K18 K19

B

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"".-A '**â€¢¿�*tÃ

**%

&

i fi Os

-^^^-^ ^. â€¢¿�Â«*

*40Ã‰|tfifv

" Â»* x â€¢¿�-.

D

'â€¢ V . * ^ "

Â» â€¢¿�

a Â»-

Fig. 2. Human prostate cancers expressing Kbâ€ž.i.From left to rfcAl, staining with Kbas., (RCK 103), K14 (LL002). K18 (RGE 53), and K19 (LP2K). A-D, expressionpattern in four different tumor samples of varying GS (GS 2-4).

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K basal K 14 K18 K19

BFig. 3. Human prostate cancers negative for K,,.,,,,,.From /c/f to rigAt. staining with K,,,,,,.,,(RCK 103), k 14 (LL002), KI8 (RGE 53), and K19 (LP2K). I. example

of a K,..â€ži well differentiated tumor. H. example of a K,,asll] poorly differentiated tumor.

Table 2 Percentage of the tumor cells of all investigated primary prostatictumors that are positive for the various keratins

Keratin-positive tumor cells (%)

GS1-3 GS4 GS5

KI4

KINK19

035

10075

025

10050

00

11III60

Table 3 Percentage of the tumor cells of all investigated recurrent prostatictumors that are positive for the various keratins

Keratin-positive tumor cells (%)

KI4"basalK18

K19GS1-30

1510090GS40

10100

50GS50

0100

25

morphology and location the stem cells are most likely part ofthe basal cell population, although no clear evidence has beenpresented to support this idea unequivocally (24-26). Stem cellsgive rise to more rapidly dividing amplifying cells, which havea limited capacity to proliferate. Characteristic features of theamplifying cells have not yet been described, although inmonkeys, humans, dogs, and mice (12-15) morphologically

intermediate types were identified on the basis of structural

B'

1234 1234

Fig. 4. Western blot analysis of the expression of cytokeratins in nonmalignant(.1) and malignant (B) prostate tissue. Blots were incubated with antibodiesagainst Kba5a,(RCK 103) (lane I), K14 (LL002) (lane 2). K19 (LP2K) (tone 3),and K IX (RGE 53) dnm' 4). Note that a weak but definitely visible expressionof Kbasai is found in the tumor, whereas K14 is absent in the cancer specimen(B, lane 2).

features characteristic of both the basal and the luminal cells.The transit cells are highly differentiated and are referred to asthe functionally active prostatic secretory cells; they are localized luminally. Further identification of the various cell types is,however, still hampered by the lack of suitable markers. The

6186




cell kinetics associated with normal cell growth and differentiation have yet to be elucidated and will be of crucial importancein the understanding of the etiology of prostatic cancer. We,therefore, first focused on normal human prostatic epithelium,to identify specific cell populations. It appeared that, as in rat(4), at least three staining patterns could be discriminatedon the basis of keratin expression. We demonstrated that antibodies K14 and Kbasa) are specific markers for basal cells innormal prostate, suggesting that the Kbasa,+/K14+ cell popula

tion is a candidate for the stem cells. Morphologically definedluminal cells with the characteristics K14~/Kbasar/K18+ colo-

calize with secretory cells and are, therefore, most likely thetransit cells. The Kbasai+/K14~ cells may in fact be candidates

for the hypothetical amplifying cell population, because theyshow a keratin expression pattern intermediate between thoseof the basal and the luminal cells (see Table 1). It is obvious thatthe cell type from which a neoplastic lesion may have arisen isan important factor in the etiology of prostatic cancer development. Therefore, in this study prostatic tumors were analyzedwith the goal of obtaining more insight into the different celltypes of which they are composed. Cells that were positive forboth K14 and Kbasa, could not be detected in human prostatictumors, indicating that the immunophenotype that in the stemcell model was postulated to be associated with the stem cells isnot present in prostatic carcinoma. Cells with an expressionpattern characteristic of transit cells were definitely present inprostatic carcinoma, a phenomenon also found in other studies(7, 8, 27, 28).

Interestingly, a population positive for K18 and Kbasa, wasalso detected in prostatic carcinoma. Cells characterized by aK14~/Kbasal"l"/K18+ keratin profile were also found in the dif

ferentiation pathway induced by androgen administration afterorchiectomy (4) and were considered candidates for the hypothetical amplifying cells. The presence of cells with this keratinphenotype in primary and hormone-independent prostatic tumors suggests the existence in human prostatic carcinoma of asubpopulation of cells that we now associate with amplifyingcells. No difference between primary and hormone-independenttumors could be found in this respect. The fact that we haveidentified in nonmalignant and malignant prostatic glandularepithelial cells a population of cells with a keratin immunophenotype intermediate between those of basal and luminal cells isin agreement with the stem cell model for prostatic epithelialdifferentiation (3), which predicts the existence of such a celltype ('amplifying cells') in prostatic epithelial differentiation.

Moreover, this phenotype was also found in prostatic carcinoma, whereas the keratin expression profile of a subpopula-tion of basal cells that putatively serve as stem cells was not.Since in the hierarchical pathway of prostatic epithelium differentiation the amplifying cells are precursors of the transitcells we postulate that this subpopulation is most likely thetarget for neoplastic transformation.

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1992;52:6182-6187. Cancer Res Anja P. M. Verhagen, Frans C. S. Ramaekers, Tilly W. Aalders, et al. Human Prostate CancerColocalization of Basal and Luminal Cell-type Cytokeratins in

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