Differential Expression of CD44 During Human Prostate Epithelial Cell Differentiation
Prostate Cancer Research Centre, Institute of Urology (TNA,IL,JRM,DLH) and Breast Cancer Research Laboratory (MJO), University College London, London, United Kingdom; Department of Histopathology (AF), Royal Free and University College Medical School, London, United Kingdom; and Prostate Stem Cell Laboratory, Institute of Cancer Research (FA), Sutton, United Kingdom
Correspondence and present address: Dr. David L. Hudson, Prostate Stem Cell Laboratory, Institute of Cancer Research, 15 Cotswold Road, Sutton SM2 5NG, UK. E-mail: david.hudson{at}icr.ac.uk
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Summary |
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Key Words: CD44 differentiation epithelium prostate
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Introduction |
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CD44 has been implicated in a number of normal and disease processes, including intercellular adhesion and tumor metastasis. Normal human salivary gland was found to be negative for the v4/5 isoform but positive for variant exons v3 and v6, where they may be involved in the regulation of growth and renewal of the tissue (Fonseca et al. 2000). Keratinocytes express various CD44 isoforms, including forms containing exons v2-v10, v3-v10, v4-v10, v6-v10, and v8-v10 (Brown et al. 1991
; Hudson et al. 1995
). Previous reports of CD44 expression in non-malignant human prostate have shown that basal and intermediate epithelial cells express CD44 (Heider et al. 1995
; Noordzij et al. 1997
; Tran et al. 2002
) but little is known about changes in expression of variant isoforms during different stages of epithelial differentiation.
Expression of variant isoforms has been implicated in various human malignancies, including colorectal (Heider et al. 1993b), stomach (Heider et al. 1993a
), breast (Matsumura and Tarin 1992
), squamous cell carcinoma (Hudson et al. 1996
), and prostate cancer (Nagabhushan et al. 1996
). In the prostate, CD44s and v6 expression is inversely correlated with tumor stage, grade, and ploidy (Nagabhushan et al. 1996
). In addition, there have been reports that loss of CD44s and v6 may be useful prognostic markers in prostate cancer patients treated by radical prostatectomy (Noordzij et al. 1997
; Kallakury et al. 1998
). Further studies have revealed reduced CD44 expression in prostate cancer metastasis as well as in the corresponding primary tumors (Noordzij et al. 1999
). More recently, Iczkowski et al. (2003)
demonstrated an increase in expression of variants v7-v9 in prostate cancer. In rat, expression of the v4-v7 isoform is involved in pancreatic carcinoma and metastasis (Gunthert et al. 1991
).
The aims of this study were to investigate the expression of CD44 and its isoforms during epithelial cell differentiation in the human prostate. We have developed a cell culture model of differentiation using human prostate epithelial cells isolated from a patient with benign prostatic hyperplasia (BPH) (Daly-Burns et al. unpublished data). Primary cells were conditionally immortalized using a temperature-sensitive SV40 large T-antigen to produce the cell line Pre2.8. At the permissive temperature of 33C the cells are able to proliferate, but when switched to 39C proliferation ceases and the cells undergo morphological changes consistent with the early stages of differentiation. At the higher temperature there is an increase in cell size accompanied by decreased keratin (K) 14 and increased expression of K8, K19, and prostate stem cell antigen (PSCA), a marker for prostate epithelial transit-amplifying cells (Tran et al. 2002). The expression of CD44 during differentiation of prostate epithelial cells was investigated in Pre2.8 cells and in prostate tissue and primary prostate epithelial cell cultures.
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Materials and Methods |
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Primary Culture of Human Prostate Epithelial Cells
Human prostate tissue was obtained from patients undergoing transurethral resection of the prostate for BPH. Cells were isolated as described previously (Hudson et al. 2000). Briefly, minced prostate tissue was digested in 0.5 mg/ml collagenase type 1A (Sigma Aldrich; Gillingham, UK) in RPMI 1640 (Invitrogen; Paisley, UK) at 37C for 1820 hr. The digested tissue was centrifuged at 170 x g for 20 sec and the pellet of epithelial acini and ducts digested to a single cell suspension in trypsin/versene (Invitrogen). Epithelial cells were resuspended in PrEGM medium and plated into 6-cm tissue culture dishes at 3000 cells/dish in the presence of a feeder layer of irradiated 3t6 cells.
RT-PCR
Total cellular RNA was isolated from cultured cells or from fresh tissue using the RNEasy Minikit (Qiagen; Crawley, UK) and 1 µg was reverse-transcribed and first-strand cDNA synthesized using Superscript II (Invitrogen) according to the manufacturer's instructions. The reverse transcription reactions were then diluted fivefold and 2 µl was used for PCR amplification. The PCR conditions used were as follows: 94C for 5 min followed by thirty cycles of 94C for 30 sec, 60C for 1 min, and 72C for 1 min 30 sec, with a final extension at 72C for 5 min. PCR products were analyzed by electrophoresis on 1.5% agarose gels stained with ethidium bromide (1 µg/ml). CD44 exon-specific primers were as used previously (Hudson et al. 1995).
DNA Sequencing
PCR products were gel-purified in 1% agarose using a QIAquick PCR Purification Kit (Qiagen). Purified PCR products were sequenced using the dRhodamine Cycle Sequencing Kit (Applied Biosystems; Warrington, UK) on an ABI Prism automated sequencer (PerkinElmer Life Sciences; Beaconsfield, UK). CD44-specific oligonucleotides were used for initiation of sequencing of the purified PCR products.
Western Blotting Analysis
Epithelial cultures were rinsed three times with PBS and incubated on ice for 15 min in the presence of extraction buffer consisting of 1% NP40, 0.4% sodium deoxycholate, 66 mM EDTA, 1 mM phenylmethyl sulphonyl fluoride, and 10 mM TRIS (pH 7.4) (Sigma Aldrich). (Isacke et al. 1990). Protein extracts were centrifuged for 15 min at 4C in an Eppendorf microfuge at 13,000 rpm and the pellets discarded. Total protein concentration was determined using the Bradford assay (Bio-Rad Laboratories; Hemel Hempstead, UK). Protein samples (30 µg) were resolved on a 7.5% polyacrylamide gel and transferred to Immobilon-P membrane (Millipore; Watford, UK). Immunodetection of specific proteins was performed as follows. Nonspecific antibody binding was blocked for 30 min in 5% skimmed milk powder in PBS containing 0.1% Tween-20 (PBST; Sigma Aldrich). The membranes were then incubated for 1 hr with E1/2, anti-CD44, in PBST containing 2% skimmed milk powder, followed by peroxidase-conjugated anti-mouse IgG for 45 min. The membranes were washed in PBST between antibody incubations and antibody binding was detected using the ECL chemiluminescence kit (Amersham Biosciences UK; Chalfont St Giles, UK).
Immunohistochemistry
Prostate tissue from patients undergoing transurethral resection of the prostate (TURP) for the treatment of BPH or from six male cadavers (aged 15 to 36 years; mean 25.2 years) were cut at 3 µm and mounted on Vectabond-treated slides (Vector Laboratories; Peterborough, UK). Antigen retrieval was carried out on deparaffinized tissue sections by boiling in antigen retrieval buffer (Vector Laboratories) for 30 min in a microwave oven. Cultured cells were fixed in formaldehyde on ice for 10 min. Tissue sections or cells were blocked in normal horse serum at room temperature for 30 min before incubation overnight at 4C for tissues or 1 hr for cells, with either E1/2 (a gift from C. Isacke; Isacke et al. 1986), VFF-327 anti-CD44v3, VFF-8 anti-CD44v5, VFF-7 anti-CD44v6, VFF-17 anti-CD44v7/8, VFF-14 anti-CD44v10 (all from Serotec; Oxford, UK (Heider et al. 1993b
; Koopman et al. 1993
), LP2K anti-keratin 19 (a gift from EB Lane; Stasiak et al. 1989
), or LLOO2 anti-keratin 14 (a gift from EB Lane; Purkis et al. 1990
). The CD44 antibodies were used at a dilution of 1:100 and the keratin antibodies were supernatants diluted 1:10. After thorough washing in PBS, sections were incubated with isotype-specific fluoroscein isothiocyanate- (FITC) or tetramethyl rhodamine-isothiocyanate (TRITC)-conjugated secondary anti-mouse antibodies (Southern Biotech; Cambridge Bioscience, Cambridge, UK) at RT for 1 hr. Nuclei were stained by incubation with a 1-µg/ml solution of Hoechst 33,258 (Sigma Aldrich) for 5 min. Primary antibody was omitted from negative control tissue sections or cells. Stained sections and cells were mounted in Gelvatol (Monsanto; St Louis, MO) and examined under an Hg-arc Zeiss Axiophot fluorescence microscope. The microscope 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).
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Results |
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CD44 Protein Expression in Pre2.8 and BPH Cell Cultures
Extracts of Pre2.8 cells and primary cultures of BPH were resolved on SDS-PAGE gels, transferred to Immobilon P membrane, and probed with the antibody E1/2, which recognizes all isoforms of CD44. Equal amounts of protein (30 µg) were loaded in all lanes. Weak expression of the standard 90-kD CD44s was observed in all samples (Figure 2B, arrow), although in some cases it was seen only on longer exposure of the autoradiogram. Three other CD44 isoforms were expressed at 33C by the Pre2.8 cells. The most abundant form has a molecular mass of 160 kD, with the least abundant variant being
250 kD. As the cells differentiate at 39C, there is a shift in the levels of CD44 protein expression, with the largest isoform being more abundant at this temperature, and by day 14 this is the major CD44 variant expressed (Figure 2A). The CD44 isoforms also have an altered electrophoretic mobility, with a lower apparent molecular weight at 39C than at 33C. A comparison of the bands produced by Pre2.8 with primary epithelial cell culture from BPH tissue revealed a similar pattern of isoforms, with three variant bands and the standard form. The variant bands were expressed at similar levels and they had the same apparent molecular weight as Pre2.8 cells at 33C (Figure 2B).
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To confirm that the staining patterns were not due to aberrant growth of cells in BPH, a further six tissue samples were obtained from tissues collected from young male cadavers. Alternating staining patterns for CD44v5 and K14 were found in all cases and in all three glandular zones, central, peripheral, and transition. In both BPH and normal tissue there were areas of basal cell hyperplasia with multiple K14-negative cell layers expressing high levels of CD44 v5 (Figures 3I and 3J). Some cases showed small areas of CD44v5-negative, K14-positive basal cells (Figure 3I), whereas others did not express K14 (Figure 3J). In all cases a single luminal layer could be distinguished overlying the CD44v5-positive cells.
Other K14-negative prostate-associated epithelial tissues also revealed interesting staining patterns. Ejaculatory ducts within the prostate showed strong basal staining for both CD44v5 (Figure 3K) and K19 (Figure 3L), while prostatic urethra stained strongly in all but the outermost cell layer for CD44v5 (Figure 3M, arrowheads) and in all layers for K19 (Figure 3N). Proximal prostatic ducts, adjacent to the urethra, are colonized by the transitional epithelium, composed of several layers of basal cells and a single layer of larger luminal cells (umbrella cells). In these regions CD44v5 is detected in all cells except the luminal cells (Figure 3O, arrowheads), while K19 is expressed by most cells. In areas where there are only two layers, K19 stains only the basal cells (Figure 3N, upper left). The distribution of the CD44 variant isoforms was further examined by staining the normal tissues with antibodies to CD44v3, v6, v7/8, and v10. Anti-v10 failed to stain at cellcell boundaries in any of the tissues with high background nuclear staining. The other three antibodies, however, had staining patterns similar to that seen for CD44v5. K14-positive CD44-negative cells were found with all the antibodies, implying that all variant isoforms are preferentially expressed by more differentiated K14-negative basal cells.
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Discussion |
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CD44 has been implicated in the progression of many types of tumor, including prostate cancer. However, little has been documented about the distribution of the CD44 variant isoforms in non-malignant human prostate tissue (Heider et al. 1995; Noordzij et al. 1997
). The aim of the present study was to investigate the expression of CD44 during normal human prostate epithelial cell differentiation. RT-PCR analysis and DNA sequencing revealed four CD44 isoforms expressed by prostate tissue, BPH and normal, and by both the immortalized Pre2.8 cells and primary cultures of BPH. The shortest isoform corresponds to the standard form of the gene, whereas the three other isoforms represent alternatively spliced CD44 variants. None of these variants expressed exon v1, which is consistent with the report that this exon contains a stop codon (Screaton et al. 1993
). The CD44 transcripts expressed were also observed at the protein level as determined by Western blotting analysis, the largest isoform being expressed most abundantly by day 14 at 39C when Pre2.8 cells are further along the differentiation pathway (Daly-Burns et al. unpublished data). Three major isoforms were expressed at similar levels in primary cultures of BPH epithelial cells. However, these cultures contain a mixed population of proliferating and differentiating cells. Therefore, we used Pre2.8 cells growing at permissive and non-permissive temperatures to provide an in vitro model in which differentiation could be induced.
Although sequencing of RT-PCR products revealed that the CD44 variants expressed by the cells at the two temperatures were all of equal size, an apparent shift in molecular weight was observed by Western blotting analysis. This may be due to differences in the posttranslational modification of the CD44 protein. CD44 contains a number of N- and O-linked glycosylation sites, all of which contribute to further diversity in protein expression. Our results show that the isoforms expressed by Pre2.8 cells grown at the non-permissive temperature have an increased electrophoretic mobility, and therefore a lower molecular mass, compared with those grown at 33C and with the primary BPH cultures. This difference may be the result of reduced glycosylation of the CD44 variants, perhaps caused by aberrant activity of glycosylation enzymes at 39C.
Our in vitro studies suggested that the CD44 v3-v10 isoform might play a role in the differentiation of normal prostate epithelial cells. On switching to the non-proliferative temperature, upregulation of differentiation-associated markers such as keratin 8, p21, and prostate stem cell antigen (PSCA) occurs (Daly-Burns et al. unpublished data). To confirm whether the v5-containing CD44 isoform may also play a role in vivo, we looked at the expression of the v5-containing variant in prostate tissue specimens. Consistent with reports that CD44 isoforms are expressed by the proliferating compartments of epithelia, positive staining for CD44 v5 was seen in basal epithelial cells in BPH tissues (Mackay et al. 1994). However, many v5-positive cells were negative for K14, a marker for the least-differentiated basal cells. In addition, v5-positive staining was seen in epithelial cells located in a position intermediate between the basal and luminal cell layers. These cells were larger and taller than the characteristically small spindle-shaped basal cells, suggesting that the v5-positive cells may be in the process of differentiating and moving from the basal to the luminal layer. We have previously shown that intermediate cells also stain positively for K19 and have postulated that they may represent an epithelial cell population that is in the process of differentiation (Hudson et al. 2001
). Other workers have shown an intermediate population that expresses low levels of K8 together with K5 (van Leenders et al. 2000
) and lacks p27kip1, a protein that is expressed by all other epithelial cells in normal prostate (De Marzo et al. 1998
). Late intermediate cells have also been shown to express prostate stem cell antigen (Tran et al. 2002
), but there have been no cell surface markers for early intermediate cells. Figure 4 shows a hypothetical differentiation pathway for human prostatic epithelial cells that illustrates where the various differentiation markers, including keratins and CD44 isoforms, are expressed (adapted from Hudson et al. 2001
).
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These results provide evidence, of early differentiation-associated changes in CD44 expression during normal prostate epithelial cell differentiation. Little is known of the transition between basal and luminal cells in the prostate, and the findings indicate that the CD44 v3-v10- containing isoform may be a marker for the early stages of this differentiation process. CD44 has been used as a target for separating basal from luminal prostate cells using immunomagnetic bead-based cell sorting (Collins et al. 2001). The identification of a CD44 isoform as a cell surface marker specific for the transit-amplifying population in prostate could provide a valuable tool for the further characterization of the differentiation process.
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Acknowledgments |
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We thank Clare Isacke and Birgitte Lane for kind gifts of antibodies.
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Footnotes |
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Literature Cited |
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