ARTICLE |
Correspondence to: David L. Hudson, Inst. of Urology and Nephrology, Research Laboratories, University College London Medical School, 67-73 Riding House St., London WIW 7E7, UK. E-mail: d.hudson@ucl.ac.uk
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Summary |
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The prostate grows slowly throughout adult life, leading to benign prostatic hyperplasia (BPH), which often results in urethral obstruction in later years. The symptoms of BPH are the second most common reason for surgery in men over 65. The aim of this study was to determine the relationship between cell proliferation and cell differentiation in BPH tissue. Using multiple antibodies, simultaneously detected with different fluorophore-conjugated secondary antibodies, several subpopulations of epithelial cells were detected. In addition to K14, basal cells also expressed keratins 15, 17, and 19 in various combinations, and some of the luminal cells also expressed K19 together with K8 and K18. Co-staining for cytokeratins and Ki-67 indicated that 44% of proliferative cells expressed K14 and 36% K19, although the difference was not statistically significant. This report provides a detailed description of the relationship between keratin expression and cell proliferation in the prostate and indicates that K19-positive cells form the link between the basal and luminal layers of the epithelium. (J Histochem Cytochem 49:271278, 2001)
Key Words: prostate, epithelium, keratin, differentiation, stem cell, benign prostatic hyperplasia
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Introduction |
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Hyperplasia of the transition zone of the prostate and the resultant urethral obstruction are an almost inevitable accompaniment to aging in men. Benign prostatic hyperplasia (BPH) is the second most common reason for surgery in men over 65 (
In contrast to other epithelial tissues, such as the epidermis (
The aim of this project was to identify subpopulations of cells in the basal layer of the prostate by examining the relationship between cell proliferation and differentiation in BPH tissue. Monoclonal antibodies to keratin subunits combined with fluorescence conjugated, isotype-specific secondary antibodies allowed simultaneous detection of up to three different target proteins in the same tissue section. On the basis of the observations made, it is suggested that the K19 positive cells play a key role in prostate epithelial cell differentiation, moving between the basal and luminal layers.
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Materials and Methods |
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Tissue Acquisition and Processing
Samples of BPH tissues from patients aged 5877 years were confirmed histopathologically to be free of malignancy. Prostate tissue from patients who underwent transurethral prostatectomy (TURP) was snap-frozen in an isopentane bath over liquid nitrogen and stored at -80C until needed. All samples were processed within 30 min. Frozen sections were cut at a thickness of 5 µm and transferred to Vectabond-coated slides (Vector Laboratories; Peterborough, UK), air-dried for 15 min, and stored at -80C. Routinely processed paraffin-embedded blocks of prostate were selected from UCL Hospitals histopathology archives. The paraffin-embedded tissue was cut at a thickness of 3 µm and mounted on Vectabond-treated slides. A further 11 blocks were selected from patients (aged 4974) undergoing radical prostatectomy for prostate carcinoma. The patients were diagnosed with stages T2aT4 cancer with Gleason scores of 69.
Antibodies and Immunohistochemistry
Indirect multiple immunofluorescence staining was carried out on both frozen and paraffin sections using a panel of mouse monoclonal and rabbit polyclonal monospecific antibodies (Table 1). The following antibodies were generous gifts: BL18, anti-K5, LL002, anti-K14 (
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Frozen sections were thawed and immediately blocked with 10% FCS in PBS (v/v) for 1 hr and then incubated for 1 hr at room temperature with primary antibodies diluted to the appropriate concentration in 10% serum. After washing twice in PBS, the slides were incubated for 45 min with Ig subtype-specific anti-mouse antibodies conjugated to fluorescein isothiocyanate (FITC), tetramethyl rhodamine isothyocyanate (TRITC) (Southern Biotechnology; Birmingham AL), or cyanine, Cy5 (Jackson ImmunoResearch Laboratories; Stratech Scientific, Luton, UK). For double staining with mouse and rabbit primary antibodies, FITC-conjugated anti-mouse and TRITC-conjugated anti-rabbit secondary antibodies were used (Dako). After two washes in PBS the sections were mounted in Gelvatol (Monsanto; St Louis, MO). To stain cell nuclei, sections were incubated before mounting with a 1 µg/ml solution of Hoechst 33258 (Sigma) for 5 min.
Paraffin-embedded sections were heated to 55C for 20 min, dewaxed by two 5-min incubations in xylene, and rehydrated through a series of graded alcohols. Antigen retrieval was carried out by microwave heating for 30 min in a citrate-based buffer (antigen unmasking solution; Vector Laboratories). Slides were allowed to cool, washed for 10 min in PBS, and then incubated for 1 hr in 10% (v/v) FCSPBS to block nonspecific primary antibody binding. The sections were incubated with primary antibodies overnight at 4C. The staining procedure was then identical to that used for frozen sections. Primary antibodies were omitted from negative control samples.
Image Capture and Microscopy
The sections were examined under an Hg-arc Zeiss Axiophot fluorescence microscope fitted with a bandpass filter for optimal FITCTRITCCy5 separation. This was coupled to a Coolview 12 cooled charge-couple device (CCD) camera (1024 x 1024, 12-bit pixels; Photonic Science, Robertsbridge, UK) controlled by Image Pro-Plus software (v3.0; Media Cybernetics, Rockville, MD). The image obtained with each antigen was stored separately as a data file. To generate the coincident multicolored images, files were merged and given computer-generated colors using Adobe PhotoShop (v5.0; Adobe Systems, San Jose, CA).
Quantitation of Keratin Distribution and Cell Proliferation
The extent of keratin staining was assessed for each sample by counting the total number of cross-sections of ducts or acini within the section and then recording the number that stained positively for each individual keratin.
To assess the distribution of proliferative cells in basal and luminal epithelial populations, all cell nuclei were stained with Hoechst. The basal cells were distinguished from the luminal cells by staining for CD44, a marker of basal cells (
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Results |
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Keratin Staining
Ten BPH samples were stained for keratins with a panel of antibodies and the sections scored for distribution of staining. Staining patterns were consistent between frozen and paraffin-embedded tissue, with only the antibody to K18 failing to stain paraffin-embedded tissue. Initial keratin 19 staining was carried out using both LP2K and RCK108. Similar results were obtained for each antibody, although slightly stronger basal cell staining was seen with LP2K and this antibody was used for all the data presented. As a control for damage during BPH tissue collection, sections from 11 radical prostatectomy whole-mount specimens were double stained for K14 and K19.
Heterogeneous Keratin 14 Expression in the Basal Epithelial Cell Layer
Staining for K5, the heterodimer partner of K14, revealed a virtually complete basal cell layer in all samples (Fig 1A). This was clearly separate from the K5-negative luminal layer that was strongly positive for the keratin pair of 8 and 18 (Fig 1B). The presence of two complete and distinct cell layers was also demonstrated by staining with antibodies against CD44 and CD57 which, as described previously (
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K14 was localized exclusively to the basal layer in a similar pattern to its partner K5 (Fig 1A and Fig 1B). There was staining of the majority of cross-sections in seven of 10 samples. In two samples there were lower levels of expression, with over a third of all cross-sections being negative. One sample showed no immunoreactivity for K14 despite being positive for K5. From a total of 616 cross-sections scored from 10 BPH samples, 81% (496) were positive. In most samples there were gaps in K14 staining of the basal layer with cells in these regions expressing other basal keratins (Fig 1). Gaps were often found at branching points, with strongest K14 staining at the tips of the acini and with weaker or absent staining between.
To identify other basal keratins expressed by K14-negative cells, sections were stained for K14 together with antibodies to keratin 15, 17, or 19 (Fig 1C, Fig 1F, Fig 1G, and Fig 1I), or separately on serial sections (Fig 1D and Fig 1E). Co-expression data are summarized in Table 2. The K14-negative regions indicated by arrows stained strongly for K17 (Fig 1B and Fig 1C) and K19 (Fig 1D and Fig 1E). K17 was expressed in 22% of cross-sections in nine of 10 samples (137 of 616) and, in addition to the small K14-negative patches, was also strongly expressed in long K14-negative regions, which may be representative of ducts (Fig 1F). K19 was found in the basal layers of all samples examined but at a lower frequency than K14, with only 55 (14%) of 389 cross-sections counted being positive. K15 was expressed to various degrees in all samples in 138 (35%) of cross-sections and, although it was often co-expressed with K14 (Fig 1G), there were also regions that stained for K15 alone. These K14 weak, K15 strong patches were often at the base of acini (Fig 1G, arrowheads), in a pattern similar to that seen for K17 and K19. In addition to staining the K14-negative regions, 60% of K15, 42% of K19, and 50% of K17 staining was co-expressed with K14 in some of the samples.
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Keratin 19 Is Expressed in Both Basal and Luminal Cells
In addition to the expression of keratins 8 and 18 in all luminal cells, some staining for keratins 7 and 19 was also observed. K7 was detected in occasional cells in half the samples, co-expressed mainly with K19 (data not shown). K19 was detected in luminal cells in nine samples, with distribution ranging from occasional cells to the complete luminal cell layer. Six cases had co-localization with K8, but in the other three occasional K19-positive cells were found that failed to stain for K8 despite being in a luminal location.
Co-expression patterns of K19 are shown in Fig 1H1M. As shown earlier, K19 was often expressed most strongly in areas of the basal layer which are negative for K14 (Fig 1I, asterisks) and weakest in areas with high levels of K14. K19-positive cells were also seen overlying K14 cells but below the luminal cells (arrows in Fig 1I). This is more clearly shown in Fig 1J, where double staining for K19 and K8 shows two layers of K19 expressing cells under the luminal layer (arrowhead). The cells that appear to be leaving the basal layer are larger and more elongated than the small, more triangular K14-expressing cells. K19 can be co-expressed with K15 (Fig 1H), although not all K15-positive cells expressed K19. This figure also shows a distinctive concentration of K19 in a band appearing to link the apical tips of the luminal cells, which also stained for K8 (Fig 1K). Fig 1K1M show a tubular region with K19 expression in both layers, while K8 is present only in luminal cells.
To confirm that keratin staining in the BPH tissues was not affected by the TURP procedure, 11 sections of whole-mount sections from radical prostatectomies were examined. These were double stained for keratins 14 and 19 and the alternating expression patterns reported above were found to be present in eight of the samples. The regions in which the staining was found were classified on the basis of orientation of the urethra, relationship with the boundary of the prostate, and cell morphology, both stromal and epithelial. From this, it was determined that although the K14/19 pattern was found in the transitional zone in all cases, it was also present in the peripheral zone of four samples and the central zone of two.
Proliferation and Keratin Expression
To assess the proliferative status of the basal and luminal compartments, the distribution of expression of the Ki67 antigen in the two cell layers was measured. Sections were triple-stained with anti-Ki67, anti-CD44, and the nuclear stain Hoechst. Every cross-section in each tissue section was examined by fluorescence microscopy and each Ki67-positive epithelial cell compared to the staining for CD44 to determine whether it was basal or luminal.
Between 74 and 85% of cycling cells were in the basal layer, with a mean of 80.7% compared to 19.3% in the luminal layer (Table 3). To take account of the higher proportion of cells in the luminal layer, the proliferative index for each compartment was determined. Five images were captured from each tissue for each of the three markers, and from this the total number of cells in each epithelial compartment within a field of view were counted, along with the number of Ki67-positive cells. Ki67-positive cells in the stroma were also scored. The proliferative index of the basal layer was 1.66%, whereas the luminal layer had an index of only 0.14%, similar to the 0.16% found in the stromal cells. The ratio of luminal to basal cells was 2.7:1.0.
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To determine which keratins are expressed by proliferative cells, sections were double stained for Ki67 with individual keratins. Almost half of all Ki67-positive cells expressed K14 (44%; range 3057%) and a high proportion (35.8%; range 2048%) expressed K19. Cycling cells were also found that were K15- and K17-positive but in lower numbers, at 15% and 25%, respectively. Cells expressing luminal keratins had the lowest level of cell division, with only 9.4 and 12.4% of cycling cells expressing K18 and K8, respectively.
To determine the proliferative index of each keratin-expressing cell type, the number of Ki67-positive cells was determined for each of the basal keratin partners of K5. The results are shown in Table 4. The highest proliferative index was seen to be for K19 at 1.37% and K15 at 1.2% and the lowest for K17, 0.96% and K14, 0.84%. The 95% confidence intervals indicate that, for these samples, the differences in proliferation between keratins are not significant.
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Discussion |
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The prostate is the site of two of the most common diseases in elderly men, benign prostatic hyperplasia and prostate cancer. Both these conditions are disorders of cell differentiation and cell proliferation. Previous studies examining the heterogeneity of prostate epithelial cells have identified phenotypes intermediate between those of the basal and luminal layers, cells that lack K14 expression but still express K5, with some expressing K8 (
In this study we have shown that K19 is also expressed by some of the K14-negative basal population, with continued expression into the luminal layer. It may therefore be a useful marker for the amplifying non-stem-cell basal population. To examine whether these different keratin expression patterns represent cells with differing proliferative abilities, we measured the proliferation rate of the basal and luminal cell compartments and for each basal keratin phenotype. The basal layer was the main proliferative compartment, with a tenfold higher proliferative index than the luminal layer, confirming previous studies (
In addition to the immunohistological data, there is also evidence from in vitro studies for intermediate cell populations.
Previous studies have described K19 distribution in prostate tissue. One study suggested that K19 expression is restricted to luminal cells (
K19 has been implicated in the differentiation of many different epithelial tissues in which there is a transition between differentiated phenotypes (
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There was wide variation in some of the staining patterns, particularly in relation to the extent of staining of particular keratins in individual cross-sections. This may reflect disruption of the normal architecture as a result of BPH. Studies of normal prostates from young men may determine whether any of these variations are symptomatic of disease progression.
Because prostate disease, both malignant and benign, involves inappropriate cell division and differentiation, it is important to identify the stem cell and amplifying populations. All carcinomas express keratin 8 and 18 and most, including prostatic intraepithelial neoplasia (PIN) lesions, express K19 (
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Footnotes |
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1 Laboratory in which work was carried out.
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Acknowledgments |
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DLH was supported by National Institutes of Health Grant AG14960-02, PF by the Wellcome Trust, and ATG by the Covent Garden Cancer Research Trust.
We wish to thank all those who kindly donated antibodies to this study, especially Birgitte Lane, Trish Purkis, and Irene Leigh for keratin antibodies. Thanks also to those who provided tissue, to Constance Parkinson (UCLH Dept. of Histopathology, London, UK) for invaluable advice, Alan Entwistle of the Ludwig Institute for help and advice on microscopy and image capture, and Liz Rugg (Royal London Hospital, London, UK) for helpful discussion.
Received for publication June 13, 2000; accepted October 5, 2000.
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Literature Cited |
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Bonkhoff H, Remberger K (1996) Differentiation pathways and histogenetic aspects of normal and abnormal prostatic growth: a stem cell model. Prostate 28:98-106[Medline]
Bonkhoff H, Stein U, Remberger K (1994a) Multidirectional differentiation in the normal, hyperplastic and neoplastic human prostate: simultaneous demonstration of cell-specific epithelial markers. Hum Pathol 25:42-46[Medline]
Bonkhoff H, Stein U, Remberger K (1994b) The proliferative function of basal cells in the normal and hyperplastic human prostate. Prostate 24:114-118[Medline]
Bui M, Reiter R (1999) Stem cell genes in androgen-independent prostate cancer. Cancer Metast Rev 17:391-399
Cussenot O, Berthon P, CochandPriollet B, Maitland NJ, Le Duc A (1994) Immunocytochemical comparison of cultured normal epithelial prostatic cells with prostatic tissue sections. Exp Cell Res 214:83-92[Medline]
De Marzo AM, Meeker AK, Epstein JI, Coffey DS (1998) Prostate stem cell compartments: expression of the cell cycle inhibitor p27 Kip1 in normal, hyperplastic, and neoplastic cells. Am J Pathol 153:911-919
Fry PM, Hudson DL, O'Hare MJ, Masters JRW (2000) Comparison of marker protein expression in benign prostatic hyperplasia in vivo and in vitro. Br J Urol 85:504-513
Hudson DL, O'Hare M, Watt FM, Masters JRW (2000) Proliferative heterogeneity in the human prostate: evidence for epithelial cells. Lab Invest 80:1243-1250[Medline]
Isaacs JT (1987) Control of cell proliferation and cell death in the normal and neoplastic prostate: a stem cell model. In Rogers CH, Coffey DS, Cunha G, Grayhack JT, Hinman F, Horton R, eds. Benign Prostate Hyperplasia. Vol II. Bethesda, MD: NIH, 85-94
Isaacs JT, Coffey DS (1989) Etiology and disease process of benign prostatic hyperplasia. Prostate Suppl 2:33-50[Medline]
Isacke CM, Sauvage CA, Hyman R, Lesley J, Schulte R, Trowbridge IS (1986) Identification and characterization of the human Pgp-1 glycoprotein. Immunogenetics 23:326-332[Medline]
Jones PH, Harper S, Watt FM (1995) Stem cell patterning and fate in human epidermis. Cell 80:83-93[Medline]
Lane EB (1982) Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization. J Cell Biol 92:665-673[Abstract]
Liu AY, True LD, LaTray L, Nelson PS, Ellis WJ, Vessella RL, Lange PH, Hood L, van den Engh G (1997) Cell-cell interaction in prostate gene regulation and cytodifferentiation. Proc Natl Acad Sci USA 94:10705-10710
McDonnell TJ, Troncoso P, Brisbay SM, Logothetis C, Chung LW, Hsieh JT (1992) Expression of the protooncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer. Cancer Res 52:6940-6944[Abstract]
Nagle RB, Brawer MK, Kittelson J, Clark V (1991) Phenotypic relationships of prostatic intraepithelial neoplasia to invasive prostatic carcinoma. Am J Pathol 138:119-128[Abstract]
Oesterling JE (1995) Benign prostatic hyperplasia. Medical and minimally invasive treatment options. N Engl J Med 332:99-109
Peehl DM, Sellers RG, McNeal JE (1996) Keratin 19 in the adult human prostate: tissue and cell culture studies. Cell Tissue Res 285:171-176[Medline]
Potten CS, Loeffler M (1990) Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development 110:1001-1020[Abstract]
Potten CS, Morris RJ (1988) Epithelial stem cells in vivo. J Cell Sci Suppl 10:45-62
Purkis PE, Steel JB, Mackenzie IC, Nathrath WB, Leigh IM, Lane EB (1990) Antibody markers of basal cells in complex epithelia. J Cell Sci 97:39-50[Abstract]
Reiter RE, Gu Z, Watabe T, Thomas G, Szigeti K, Davis E, Wahl M, Nisitani S, Yamashiro J, Le Beau MM, Loda M, Witte ON (1998) Prostate stem cell antigen: a cell surface marker overexpressed in prostate cancer. Proc Natl Acad Sci USA 95:1735-1740
Robinson EJ, Neal DE, Collins AT (1998) Basal cells are progenitors of luminal cells in primary cultures of differentiating human prostatic epithelium. Prostate 37:149-160[Medline]
Stasiak PC, Purkis PE, Leigh IM, Lane EB (1989) Keratin 19: predicted amino acid sequence and broad tissue distribution suggest it evolved from keratinocyte keratins. J Invest Dermatol 92:707-716[Abstract]
Verhagen AP, Ramaekers FC, Aalders TW, Schaafsma HE, Debruyne FM, Schalken JA (1992) Colocalization of basal and luminal cell-type cytokeratins in human prostate cancer. Cancer Res 52:6182-6187[Abstract]
Waseem A, Dogan B, Tidman N, Alam Y, Purkis P, Jackson S, Lalli A, Machesney M, Leigh IM (1999) Keratin 15 expression in stratified epithelia: downregulation in activated keratinocytes. J Invest Dermatol 112:362-369
Xue Y, Smedts F, Debruyne FMJ, de la Rosette JJ, Schalken JA (1998) Identification of intermediate cell types by keratin expression in the developing prostate. Prostate 34:292-301[Medline]