progenitor and intermediate cell types and the origin of human prostate cancer

Differentiation (2005) 73:463–473 DOI:10.1111/j.1432-0436.2005.00047.x

r 2005, Copyright the Authors Journal compilation r 2005, International Society of Differentiation

O RI G INA L AR T I C L E

Erik J. Tokar . Brooke B. Ancrile . Gerald R. Cunha . Mukta M. Webber

Stem/progenitor and intermediate cell types and the origin of human prostate cancer

Received September 30, 2005; accepted in revised form October 1, 2005

Abstract Theories of cell lineage in human prostatic epithelium, based on protein expression, propose that basal and luminal cells: 1) are either independently capable of self-renewal or 2) arise from stem cells expressing a full spectrum of proteins (p63, cytokeratins CK5/14, CK8/18, and glutathione-S-transferase-pi [GST-pi]) similar to cells of the embryonic urogenital sinus (UGS). Such embryonic-like stem cells are thought to give rise to mature basal cells and secretory luminal cells. By single cell cloning of an immortalized, normal human prostate-derived, non-tumorigenic RWPE-1 cell line, we isolated and characterized two epithelial cell types, WPE-stem and WPE-int. WPEstem cells show: i) strong, sixfold greater nuclear expression of p63; ii) nearly twofold greater expression of CK14; iii) threefold less CK18, and iv) low androgen receptor (AR) expression as compared with WPE-int cells. WPE-stem cells are androgen-independent for growth and survival. WPE-int cells express very low p63 and CK5/14, and high CK18. WPE-int cells are androgen-independent for growth and survival but are highly responsive as shown by androgen induction of AR and prostate specific antigen (PSA). Compared with WPE-int cells, WPE-stem cells are smaller and show more rapid . ) Erik J. Tokar
Brooke B. Ancrile
Mukta M. Webber (* Departments of Zoology and Medicine Michigan State University East Lansing, MI 48824-1312 U.S.A. E-mail: [email protected] Gerald R. Cunha Departments of Anatomy and Urology University of California San Francisco San Francisco CA 94143-0452 U.S.A. U.S. Copyright Clearance Center Code Statement:

growth. WPE-stem cells can grow in an anchorage-independent manner in agar with 4.5-fold greater cloning efficiency and as free floating ‘‘prostaspheres’’ in liquid medium; and express over 40-fold higher matrix metalloproteinase-2 activity. These results indicate that WPE-stem cells express several features characteristic of stem/progenitor cells present in the UGS and in adult prostatic epithelium. In contrast, WPE-int cells have an intermediate, committed phenotype on the pathway to luminal cell differentiation. We propose that in normal prostatic epithelium, cells exist at many stages in a continuum of differentiation progressing from stem cells to definitive basal and luminal cells. Establishment and characterization of clones of human prostatic epithelial cells provide novel models for determining cell lineages, the origin of prostate cancer, and for developing new strategies for tumor prevention and treatment. Key words human prostate
differentiation
p63
stem cells
androgen receptor
PSA
cytokeratins

Introduction The prostate can undergo successive rounds of androgen deprivation and replacement without diminishing its ability for continued epithelial regeneration (Isaacs, 1987). This regenerative capacity has been attributed to stem/progenitor cells within adult prostatic epithelium. It was hypothesized that the adult prostate contains stem, transit/amplifying, and postmitotic cells and that stem cells were androgen-independent for survival (Isaacs, 1987). Prostatic epithelium may contain a spectrum of cells expressing a continuum of biological properties, differentiation markers, and variable degrees of androgen dependence. Consequently, androgen deprivation elicits massive loss of androgen-dependent, luminal cells and their androgen-dependent precursors.

0301–4681/2005/7309–463 $ 15.00/0

464

However, stem cells and some transit/amplifying or intermediate cells are thought to be androgen-independent for survival. Thus, androgen-induced prostatic regeneration appears to be a function of androgenindependent stem and intermediate cells. Adult tissue stem cells and their close progeny, thought to reside in the basal cell compartment of stratified and glandular epithelia, are multipotent and have the ability to regenerate the complete complement of cell types characteristic of the fully differentiated tissue. Multipotency, the most unique feature of a stem cell, is unfortunately the most under-studied feature of prostatic stem cell biology. Asymmetric cell division, another characteristic of stem cells, allows self-renewal and maintenance of the stem cell population as well as generation of daughter cells that enter the differentiation pathway. The proliferative, self-replicative, and multipotent characteristics of prostatic epithelial stem cells provide the necessary cellular mechanisms for androgen-induced prostatic regeneration. Stem cells, their immediate progeny, and intermediate cells in the prostatic epithelial lineage are the likely targets of carcinogenesis. To understand the origin of carcinoma cells, it is necessary first to understand how normal prostatic epithelial cell populations are maintained in homeostasis and how homeostasis is altered in carcinogenesis. Adult bone marrow stem cells, whose existence has been known for many years, have the ability to reconstitute all of the cells of the blood (Blau et al., 2001). At least some adult stem cells also possess the developmental plasticity to trans-differentiate into cell types outside of the normal differentiation repertoire of their tissue of origin (Anderson et al., 2001). For example, the ability of adult urinary bladder epithelium to be induced by urogenital sinus mesenchyme to differentiate into prostatic epithelium is presumably because of stem cells within adult bladder epithelium that have the ability to trans-differentiate (Cunha et al., 1987). Interest in adult tissue-specific stem cells is not only relevant to the maintenance of tissue homeostasis and regeneration but also to the origin of cancer. It has been shown that tumors contain a small population of ‘‘cancer stem cells’’ that also have the ability for self-renewal and are responsible for cancer cell expansion (Reya et al., 2001). It is, therefore, important to isolate and characterize populations of stem/progenitor and intermediate cell types, understand the lineage relationships of the various cell types present in normal prostatic epithelium, and identify the anatomical niches in which they reside. In an effort to identify prostatic epithelial stem cells and cells of the epithelial lineage, several functional and molecular markers have been employed, such as the ability for self-renewal, high cloning efficiency, the expression of the nuclear protein p63, and specific patterns of cytokeratin (CK) expression. In this regard, the prostatic epithelium is traditionally considered to be

composed of three major cell types, luminal, basal, and neuroendocrine cells. Luminal cells express CK8/18, androgen receptor (AR), and prostate-specific antigen (PSA), while basal cells express p63 and CK5/14 (Bonkhoff and Remberger, 1996; Wang et al., 2001; Bonkhoff, 2004). The protein p63 shares high amino acid homology with the tumor suppressor protein p53 and exists in at least six different splice variant forms (DiRenzo et al., 2002; Westfall and Pietenpol, 2004). Very high expression of p63 is seen in the basal cell compartment of many stratified and glandular epithelia. Although p63 expression is not restricted to stem cells, the highest expression of p63 occurs in stem cells and p63 has been suggested to be a marker of epithelial stem/progenitor cells (Yang et al., 1999, 2002; Pellegrini et al., 2001; McKeon, 2004). It has now become evident that certain prostatic epithelial cells co-express both basal and luminal cell-associated markers (Verhagen et al., 1992; Hudson et al., 2001; van Leenders and Schalken, 2001; Wang et al., 2001; van Leenders et al., 2003). The challenge now is to relate marker expression with stem cells and cell lineages, and to identify their niches within prostatic epithelium. Based on many lines of circumstantial evidence the consensus is that prostatic epithelial stem cells reside in the basal compartment (van Leenders and Schalken, 2001; Schalken and van Leenders, 2003; van Leenders et al., 2003; Bonkhoff, 2004). If this is true, then luminal cells emerge as descendants of multipotent stem cells in the basal compartment. However, p63-knock-out prostatic tissue lacks basal cells but contains luminal and neuroendocrine cells (Kurita et al., 2004). Whether luminal and basal cells arise from the same or separate stem/progenitor cells requires further investigation. The CK8/18-expressing human prostatic luminal cells are considered to be androgen-dependent, show strong expression of nuclear AR and synthesize and secrete PSA. AR has been reported to be absent, weak, or strongly expressed in CK5/14-positive human prostatic epithelial basal cells (Bonkhoff and Remberger, 1996; Bonkhoff, 2004). In the rat ventral prostate AR has been detected in basal cells as early as day 1 of life (Prins and Birch, 1995). Cells in the basal compartment appear to be relatively androgen-independent for survival but some cells are androgen responsive for proliferation (Bonkhoff and Remberger, 1996; Bonkhoff, 2004). This is supported by the observation that administration of androgen to castrated rats or mice results in the up-regulation of AR in basal cells which then undergo cell proliferation (Evans and Chandler, 1987; Mirosevich et al., 1999). Other investigators propose that p63- and CK5/14-positive basal cells lack AR and PSA (Garraway et al., 2003), or that AR is present in basal cells but at very low levels (van Leenders et al., 2003). In view of these differing reports, we examined the expression of AR and PSA in the parent cell line, RWPE-1, and in two isolated clones designated as

465

WPE-stem and WPE-int, which we believe, represent two distinct cell types within prostatic epithelial cell lineage. Cancer cells share many characteristics with stem cells, e.g., the ability for self-renewal, anchorage-independent growth, high cloning efficiency, and the expression of anti-apoptotic proteins such as bcl-2 and transporter proteins. Many cancer cells also express high levels of matrix metalloproteinases (MMPs) that are associated with their invasive behavior. Studies demonstrating increased expression of MMPs by stem cells are limited primarily to hematopoietic and neural stem cells (Frolichsthal-Schoeller et al., 1999; Janowska-Wieczorek et al., 1999). Therefore, we examined MMP-2 and MMP-9 activity in our two clonally derived, WPE-stem and WPE-int, cell lines. This study reports the isolation and characterization of two cloned cell types isolated from non-tumorigenic RWPE-1 human prostatic epithelial cells derived from the peripheral zone (PZ) of a normal human prostate (Bello et al., 1997). RWPE-1 cells have retained many characteristics of their normal counterparts; they polarize and form acini and ducts when grown in threedimensional matrigel cultures and in the presence of androgen, up-regulate AR and secrete PSA (Bello et al., 1997; Webber et al., 1997; Bello-DeOcampo et al., 2001). We have characterized the two cloned cell lines by examining the expression of p63, CK5/14, CK18, AR, PSA, MMP-2 and MMP-9, and anchorage-dependent and -independent growth. Given the differences in the differentiation states and other properties, we believe that WPE-stem and WPE-int cells represent distinct cell types within the prostatic epithelial cell lineage and may represent prostatic stem/progenitor and intermediate cells, respectively. Further analysis of these cell lines may: 1) provide models for unraveling the composition and lineage of cell populations in human prostatic epithelium; 2) define patterns of differentiation that lead to basal and secretory cells, and 3) shed light on cells from which cancer arises.

Materials and methods Keratinocyte serum free medium (KSFM) with epidermal growth factor (EGF) and bovine pituitary extract (BPE) supplements (No. 17005-042), antibiotic/antimycotic mixture (PSF), and Trypsin/ EDTA from Gibco/Invitrogen (Rockville, MD); donor calf serum (DCS) and fetal bovine serum (FBS) from Intergen (Purchase, NY); Dulbecco’s phosphate-buffered saline Ca21/Mg21-free (D-PBS), piodonitrotetrazolium (INT), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), and monoclonal antibody (MoAb) to CK 18 (clone CY-90, No. C8541) from Sigma (St. Louis, MO); type IV collagen (No. 3410-01) and laminin-1 (No. 3400-10) from Trevigen (Gaithersburg, MD); Bacto agar from Difco (Detroit, MI); MoAb to p63 (4A4: sc-8431) and MoAb to AR (441: sc-7305) from Santa Cruz Biotechnology (Santa Cruz, CA); MoAb to high molecular weight cytokeratins (34bE12, No. M0630) and MoAb to prostate-specific antigen (clone ER-PR8; No. M0750) from Dako

Cytomation (Carpinteria, CA); horse serum, Vectastain ABC Peroxidase kit, and 3,3 0 -diamino-benzidine (DAB) Substrate kit from Vector Laboratories (Burlingame, CA); NuPAGE, 10% Bis-Tris pre-cast gels, NuPAGE MOPS SDS Running Buffer, and NuPAGE Transfer Buffer from Invitrogen Life Technologies (Carlsbad, CA); Immobilon-P transfer membranes 0.45 mm pore size from Millipore Corporation (Bedford, MA); 60 mm plates, 96-well plates, T-25 and T-75 flasks, four-chamber slides (No. 35-4114), and Fibronectin (No. 354008) from Beckton-Dickinson/Falcon (Bedford, MA); NEPER nuclear and cytoplasmic extraction kit, and bovine serum albumin (BSA) standard from Pierce Biotechnology (Rockford, IL).

Cells and cell culture WPE-int and WPE-stem cell lines were cloned from RWPE-1 cells. The RWPE-1, non-tumorigenic human prostatic epithelial cell line, was developed by immortalization of epithelial cells, derived from PZ of a normal human prostate, with a single copy of human papillomavirus-18 DNA (Bello et al., 1997; Webber et al., 1997). The immortalizing viral proteins do not bind to p63 (Strano et al., 2001). The RWPE-2 human prostatic carcinoma cell line was derived from RWPE-1 cells by transformation with Ki-ras (Bello et al., 1997, Webber et al., 1997). The RWPE2-W99 cell line was derived from a clone of RWPE-2 cells grown in agar, and selected for high Ki-ras expression. All cell lines were maintained in complete KSFM medium containing 50 mg/ml BPE, 5 ng/ml EGF, and 1% antibiotic/antimycotic mixture. Medium was changed every 48 hr. Cells were passaged upon confluence and seeded at 1–2  106 cells/T-25 flask.

Cloning of WPE-int and WPE-stem RWPE-1 cells were subjected to single-cell cloning using a cell suspension containing 10 cells/ml. One hundred microliters of this suspension were plated/well in 96-well plates. Cells were not disturbed for 6 days after which the medium was changed and every 48 hr subsequently. Cells from wells containing a single cell were allowed to grow. Based on different cell morphology, two clones were expanded and subjected to a second round of cloning giving rise to WPE-int and WPE-stem cell lines used in the present study.

Maintenance of WPE-stem cells After isolation of this clone, it was observed that although the cells proliferated, they did not attach well or spread on tissue culture plastic surface. Therefore, these cells were maintained in flasks coated with a mixture of type IV collagen and fibronectin (2.5 mg each/cm2). To a T-25 flask, 3 ml of the matrix mixture were added and allowed to remain at 371C in the incubator for 24 hr. The matrix mixture was removed, the flask rinsed twice with D-PBS and then used for plating cells.

Anchorage-dependent growth To assess growth, cells were plated at densities of 625, 1,250, 2,500, 5,000, and 10,000 cells/well using 12 replicate wells/cell number in duplicate 96-well plates, giving 24 replicate wells/cell line. As WPEstem cells require matrix for attachment, all three cell lines were grown in wells coated with a mixture of type IV collagen and fibronectin (2.5 mg each/cm2) as described above. Medium was changed every 48 hr. Cells were stained with MTT, 3, 6, and 9 days after plating as previously described (Webber et al., 2001). Absorbance was measured at 540 nm with a Titertek microplate reader (Huntsville, AL). The absorbance values show a direct relationship with cell number; data are plotted  standard deviation (SD).

466 Anchorage-independent growth A soft agar assay was performed to examine colony-forming efficiency (CFE) according to a previously described procedure (Webber et al., 2001). Five replicate plates/cell line, with 12,500 cells/plate, were incubated at 371C for 21 days. Colonies were then stained with INT for 12 hr at 371C and fixed in 10% buffered formalin. Colonies  0.2 mm were counted using a BioTran II automated colony counter (Edison, NJ), percent CFE was calculated, and plotted  SD. SDS-PAGE zymography for MMP activity Cells were plated in complete KSFM at one million cells/60 mm plate in duplicate for each cell line. After 48 hr the medium was aspirated, cells rinsed 2  with 3 ml of D-PBS, and 2.2 ml basal KSFM (without BPE and EGF) was added per plate for 48 hr at 371C. Conditioned medium was collected and centrifuged to remove cell debris. To the supernatant, 2% Triton-X 100 was added at a final concentration of 0.01%, gently vortexed, aliquoted, and stored at  701C. Protein content of samples was determined by the Lowry high protein assay (Pierce). To examine MMP-2 and MMP-9 activity, SDS-PAGE zymography was performed using 10% gels containing 0.1% gelatin. Conditioned medium samples containing 2 mg protein and 5 ml of Laemmli sample buffer were electrophoresed (Bio-Rad Mini Protean II apparatus, Bio-Rad, Hercules, CA) at 157 V for 1.5 hr (41C) in 1  running buffer (100 mM Tris base, 0.765 M glycine, pH 8.3). Gels were washed on a plate rocker at room temperature 2  , 30 min each, in 2.5% Triton-X 100 in dH2O to remove the SDS. Gels were then incubated (371C) for 18 hr in Tris-HCl soaking buffer (50 mM Tris-HCl, 200 mM NaCl, 5 mM CaCl2, 0.02% Brij-35, pH 7.5) to re-nature the proteases and stained with 0.25% Coomassie brilliant blue R-250 for 20 min followed by  20 min of destaining in methanol:acetic acid:dH2O (4:1:5) solution. The area of lysis bands was measured densitometrically using the NIH ImageJ analysis program. Immunostaining For RWPE-1 and WPE-int, 10,000 cells and for WPE-stem, 20,000 cells were plated per well in 1 ml of complete KSFM in four-well chamber slides. The WPE-stem cells were plated on laminin-1-coated chamber slides (10 mg/cm2) to allow cells to flatten to facilitate examination of nuclear and cytoplasmic staining. Medium was changed every 48 hr. Upon reaching sub-confluence, cells were rinsed 2  with D-PBS, fixed for 2 min in 1:1 mixture of methanol:acetone at room temperature, and stored at  201C. To detect AR and PSA, cells were plated as above, and 48 hr after plating the medium was replaced with fresh medium containing the synthetic androgen mibolerone (5 nM). Cells in medium containing 0.1% ethanol vehicle were used as the control. Cells were fixed after 4 days of treatment. Cytokeratins 5/14, 18, p63, AR, and PSA were visualized by immunostaining with monoclonal antibodies using the Vector avidin–biotin immunoperoxidase protocol as described previously (Bello et al., 1997) using the antibodies listed in the ‘‘Materials and methods’’. Cells exposed only to biotinylated secondary Ab (1:200) served as controls. Antibody dilutions and incubation times were: p63, 1:100 (1 hr), CK5/14, 1:100 (1 hr), CK 18, 1:500 (1 hr), AR, 1:100 (2 hr), PSA, 1:50 (24 hr at 41C).

used to determine protein content. Samples of nuclear extract containing 25 mg protein for p63 and AR, and of cytoplasmic extract containing 10 mg protein for CK 5/14, 2.5 mg for CK18, and 40 mg for PSA were run in NuPAGE 10% Bis-Tris pre-cast gels at 175 V for 1.5 hr at 41C. Proteins were transferred to immobilon-P transfer membranes at 30 V at 41C. For immunoblotting, monoclonal antibodies were used (see ‘‘Materials and methods’’). Blots were stained using avidin–biotin immunoperoxidase Vectastain ABC and DAB kits. To examine the expression of AR and PSA, one million cells were plated/T-25 flask. After 48 hr cells were treated for 4 days with medium containing 5 nM mibolerone, changing medium every 48 hr. Nuclear and cytoplasmic extracts were prepared, as described earlier. Blots were analyzed with the NIH ImageJ analysis program. Statistical analysis Results for anchorage-dependent and -independent growth were plotted as mean with SD. One way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test, was used to compare the growth of the two cloned cell lines with RWPE-1 cells. A two-tailed t-test was used to also compare the growth of the two cloned cell lines to each other. GraphPad Instat version 3.01 was employed; p  0.05 was considered to be statistically significant. All experiments were repeated two to four times.

Results Cell morphology and stem/progenitor cell markers by immunostaining To determine if WPE-stem and WPE-int cells show stem/progenitor cell and intermediate cell phenotypes, respectively, cell morphology, and the expression of p63, CK5/14, and CK18 were first examined. Cultures of all three cell lines show differences in cell morphology (Figs. 1A–1D). RWPE-1 and WPE-int cells show the characteristic, polygonal, epithelial morphology (Figs. 1A,1B) but WPE-stem cells are round and attached loosely to the plastic dish (Fig. 1C). However, when plated on laminin-1-coated plates, WPE-stem cells spread and show an elongated morphology (Fig. 1D). The WPE-stem cells are much smaller than the WPE-int cells. Of the three cell lines, WPE-stem cells show homogeneous strong nuclear staining for p63 (Fig. 1G). In contrast, WPE-int cells show (Fig. 1F) homogeneous but weak staining for p63. The parent RWPE-1 cells show (Fig. 1E) heterogeneous weak-to-strong nuclear staining. The monoclonal antibody 34bE12 recognizes CK5/14 in the normal prostate. WPE-stem cells exhibit strong staining for CK5/14 (Fig. 1J), while RWPE-1 and WPE-int cells show lighter staining for CK5/14 (Figs. 1H,1I). In contrast, while RWPE-1 and WPE-int cells show strong staining for CK 18 (Figs. 1K,1L), WPEstem cells show lighter staining for CK18 (Fig. 1M).

Western blot analysis Cells were plated at one million cells/T-25 flask. Upon reaching confluence, cells were pooled from duplicate flasks and centrifuged. Nuclear and cytoplasmic extracts were prepared using the NE-PER kit according to the manufacturer’s protocol. Extracts were aliquoted and stored at  701C. The Lowry high protein assay was

WPE-stem cells express high levels of p63 and cytokeratin 14 The immunostaining results were confirmed by Western blot analysis for p63, CK5/14, and CK18 (Fig. 2). High

467

WPE-stem cells expressed threefold less CK18 than WPE-int cells (Figs. 2E,2F). Results showed that the tumorigenic RWPE2-W99 cells expressed  twofold less CK18 than RWPE-1 and WPE-int cells. Induction of androgen receptor and PSA

Fig. 1 Cell morphology and immunostaining: (A–D) H&E staining. (A, B) RWPE-1 and WPE-int cells show epithelial, polygonal morphology. (C) When WPE-stem cells are grown on plastic, they are loosely attached and most cells have a round morphology. (D) When WPE-stem cells are grown on a laminin-1-coated surface, they are firmly attached, spread out and have an elongated morphology. (E–G) Nuclear staining for p63. RWPE-1 cells show heterogeneous, WPE-int cells show weak, and WPE-stem cells show strong, uniform nuclear staining for p63. (H–J). RWPE-1 and WPE-int cells stain positive for CK5/14; WPE-stem cells show strong, positive staining for CK5/14. (K–M) RWPE-1 and WPE-int cells show strong, positive staining for CK18 while WPE-stem show positive but weaker staining for CK18; bar 5 20 mm.

p63 expression (Mr  65 kDa) was detected in WPEstem cells and low p63 expression was detected in WPEint cells (Figs. 2A,2B). In Fig. 2, we have included a tumorigenic cell line RWPE2-W99 to explore the possible origin of these malignant cells on the basis of their protein expression. Both RWPE-1 and RWPE2-W99 cells express intermediate level of p63 (Figs. 2A,2B). Densitometric analysis of p63 expression in Western blots showed (Fig. 2B) that WPE-stem cells express sixfold more p63 than WPE-int cells. Similarly, WPEstem cells showed nearly twofold more CK14 than WPE-int cells (Figs. 2C,2D). The tumorigenic RWPE2W99 cells expressed eightfold less CK14 as compared with WPE-stem cells. On the basis of molecular weight, the protein band was determined to be CK14 (Mr  50 kDa) and not CK5, which has a Mr of  58 kDa (Moll et al., 1982, Verhagen et al., 1992).

To determine if WPE-stem and WPE-int cells respond differently to androgen, the effect of the synthetic androgen, mibolerone, on the expression of AR and PSA was examined. Cells were treated with 5 nM mibolerone for 4 days, and immunostaining and Western blot analyses were performed. Results showed (Fig. 3A) that in the absence of androgen RWPE-1, WPE-int and WPEstem cells showed weak staining for AR (Fig. 3Aa–c). Upon treatment with mibolerone, a marked increase in staining for AR occurred in RWPE-1 and WPE-int cells (Fig. 3Ad,e), but WPE-stem cells showed only a small increase (Fig. 3Af). Cytoplasmic staining for PSA was barely or not detectable in all three cell lines in the absence of androgen (Fig. 3Bg–i). However, treatment with mibolerone caused a marked increase in PSA expression in RWPE-1 and WPE-int cells but only a small increase in WPE-stem cells (Fig. 3Bj–l). Western blot analysis confirmed that mibolerone induced higher expression of AR and PSA in RWPE-1 and WPE-int cells than in WPE-stem cells (Fig. 3C). These results indicate that while both WPE-stem and WPE-int cells are androgen-independent for growth and survival, WPE-int are markedly more responsive to androgen than WPEstem cells with regard to AR and PSA expression. WPE-stem cells show greater anchorage-dependent growth than WPE-int cells Anchorage-dependent growth of WPE-stem and WPEint cells was assessed to determine differences in growth. Results in Figure 4A show that WPE-stem cells grow considerably faster than the parent RWPE-1 and WPEint cells when plated at five different densities. WPEstem cells have a significantly (po0.0001) higher growth rate at all cell densities than WPE-int cells when grown in an anchorage-dependent manner on type IV collagen/fibronectin-coated plates. WPE-stem cells show high cloning efficiency in anchorage-independent growth Stem cells are considered to have the ability to grow in an anchorage-independent manner (Tiberio et al., 2002; Dontu et al., 2003a, b). The parental RWPE-1 cells failed to show anchorage-independent growth, confirming our previous observation (Webber et al., 2001). In contrast, WPE-stem cells showed a high CFE of 0.9%, while WPE-int cells had a low CFE of 0.2% (Fig. 4B).

468

Fig. 2 Western blot analysis for protein expression. (A, B) The expression of p63 was examined in nuclear extracts. The WPE-stem cells express sixfold more p63 than WPE-int cells; RWPE-1 and RWPE2-W99 cells show similar expression as shown in the bar graph. (C, D) The expression of CK14 was examined in cytoplasmic extracts. WPE-stem cells show threefold more CK14 as compared with WPE-int cells; the tumorigenic RWPE2-W99 cells show the lowest expression. (E, F). The expression of CK18 was examined in cytoplasmic extracts. WPE-stem cells show threefold less CK18 as compared to WPE-int cells; RWPE-1 cells show high while the tumorigenic RWPE2-W99 cells show lower expression.

The difference between the CFE of the two cell lines is highly significant (po0.0001) with WPE-stem cells having a 4.5-fold greater CFE than WPE-int cells. These results show that while the parental RWPE-1 cell line, consisting of a mixed cell population, is not able to grow in agar two clones with high and low CFE, respectively, have been isolated from it. Plates demonstrating clone formation in agar are shown in Figure 4C. One of the WPE-stem agar clones is shown in Figure 1Da. Furthermore, WPE-stem cells can also grow in liquid medium as free-floating clusters of cells we call ‘‘prostaspheres’’ (Fig. 4Db,c), similar to ‘‘mammospheres’’ (Dontu et al., 2003b). To show that ‘‘prostaspheres’’ are composed of viable cells, single, isolated spheres were plated on laminin-1-coated chamber slides where they form monolayer colonies (Fig. 4Dd).

WPE-stem cells show high levels of MMP activity Zymographic analysis of conditioned medium showed that RWPE-1 cells express both MMP-9 and MMP-2 (Fig. 4E). In WPE-int cells, MMP-9 activity was not detectable and MMP-2 was barely detectable. In contrast, WPE-stem cells expressed both MMP-2 and MMP-9. MMP-2 activity was  10-fold higher in WPE-stem cells than that in RWPE-1 cells and over 40-

fold higher than that expressed by WPE-int cells. These results demonstrate another marked phenotypic difference between WPE-stem and WPE-int cells.

Discussion By single-cell cloning, we have isolated and characterized two human prostatic epithelial cell types from the non-tumorigenic, RWPE-1 cell line derived from the PZ of normal human prostate (Bello et al., 1997; Webber et al., 1997). WPE-stem cells show high expression of p63 and CK14 and low expression of CK18; upon exposure to androgen, they show weak AR and PSA expression. The sixfold higher expression of p63 in WPEstem than in WPE-int cells is consistent with a stem/ progenitor cell phenotype. Another feature of stem cells is their small cell size, which is associated with p63 expression. WPE-stem cells with high levels of p63 are much smaller than WPE-int cells, which express low p63 levels. These results are consistent with the observation that p63-positive prostatic basal cells are significantly smaller than luminal cells (English et al., 1987). In addition, in human keratinocyte cultures the small less differentiated cells show high p63 expression; as these cells differentiate and increase in size, p63 expression decreases and eventually disappears (Parsa et al.,

469

Fig. 3 Immunostaining for the expression of androgen receptor (AR) and prostate specific antigen (PSA) before and after exposure to 5 nM mibolerone for 4 days. (A) AR. (a–c) In the absence of mibolerone, all three cell lines show weak but detectable expression of AR; (d–f) after exposure to mibolerone, RWPE-1 and WPE-int cells show a marked increase in staining for AR while WPE-stem cells show little increase. (B) PSA: (g–i) in the absence of mibolerone, PSA expression was not detectable in all three cell lines; (j–l) after exposure to mibolerone, RWPE-1 and WPE-int cells show a marked increase in staining for PSA, while WPE-stem cells show a small increase. (C) In Western blot analysis, while both RWPE-1 and WPE-int cells show AR and PSA protein expression, WPEstem cells are positive but show low expression after exposure to androgen.

1999). Stem/progenitor cells predominantly express DNp63 isoforms, which appear to be essential in maintaining the stem/progenitor cell population (Signoretti et al., 2000; Pellegrini et al., 2001; DiRenzo et al., 2002; McKeon, 2004). Of the six p63 isoforms the DNp63 is the most common and its Mr has been variously shown to be 65–72 kDa (Hall et al., 2000; Bamberger et al., 2002; DiRenzo et al., 2002). The p63 protein detected in our cell lines was estimated to be  65 kDa which is consistent with a DNp63 isoform (DiRenzo et al., 2002).

Tissue stem cells in vivo are slow cycling, long lived, and divide only if there is loss of the progenitor cell population (Reya et al., 2001; Dontu et al., 2003b). In keratinocytes a decrease in p63 expression is associated with reduced proliferative potential and subsequent terminal differentiation (Westfall and Pietenpol, 2004). We, therefore, compared the proliferative potential of WPE-stem and WPE-int cells in monolayer culture. In the absence of constraints imposed by differentiated cells, the WPE-stem cells show significantly more rapid growth than WPE-int cells. The high p63-expressing WPE-stem cells also formed clones in agar at 4.5 times greater efficiency than WPE-int cells. Embryonic and adult stem cells (e.g., mammary and neural) can similarly proliferate in an anchorage-independent manner and show high CFE attached to a substrate or growing in a free-floating form (Tiberio et al., 2002; Dontu et al., 2003a, b). The expression of DNp63 isoforms is associated with the ability of cells to grow in an anchorageindependent manner (Tiberio et al., 2002; Dontu et al., 2003a). WPE-stem cells were also able to grow suspended in liquid medium, as free floating, ‘‘prostaspheres,’’ which gave rise to cell colonies when plated on laminin-1-coated slides, a characteristic consistent with a stem/progenitor cell phenotype. Thus, by several criteria, WPE-stem cells exhibit features characteristic of the postulated stem/progenitor cells described in the human, mouse, and rat urogenital sinus epithelium, which express the fullest spectrum of marker proteins (p63, basal cell cytokeratins CK5/14, luminal cell cytokerartins CK8/18, and GST-pi). Such putative stem/ progenitor cells have been detected in the basal compartment of the adult prostate, which also contains intermediate and differentiated basal cells (Wang et al., 2001). A small subset of intermediate cells in the basal compartment is negative for CK5/14 but positive for p63, and may be a luminal cell progenitor cell type initiating luminal cell differentiation (Signoretti et al., 2000; Wang et al., 2001). Our second cell line, WPE-int, shows features of a luminal cell progenitor with low levels of p63 and CK5/14, high levels of CK18, and marked induction of AR and PSA by androgen. Thus, WPE-int cells appear to belong to an intermediate cell type undergoing luminal cell differentiation but still expressing p63 at a low level. Such intermediate cells may ultimately give rise to terminally differentiated p63-negative luminal cells (Yang et al., 1999). Constitutive, high p63 expression has been reported in the basal, stem/progenitor cell compartment of stratified (epidermis, cervix, esophagus, urothelium) and glandular epithelia (lacrymal, mammary, and prostate). In stratified epithelia p63 expression is also seen in supra-basal layers (Yang et al., 1999, 2002; Pellegrini et al., 2001; McKeon, 2004). Given the broad expression of p63 in a variety of epithelia, p63-deficient mice show a lack or abnormal development of skin, breast, prostate, and other epithelia and die shortly after birth (McKeon,

470

2004). Thus, p63 has been suggested to play a critical role in the process of epithelial differentiation and maintenance of the regenerative potential of the stem/ progenitor cell population (see McKeon, 2004). The prostate contains p63-positive cells in the basal compartment and p63-negative luminal and neuroendocrine cells. Grafts of p63-null embryonic UGS grown for 1 month in male nude mouse hosts develop prostatic tissue containing neuroendocrine cells and luminal cells that express AR and mouse prostate-specific proteins.

In p63 null prostate, luminal and neuroendocrine cells clearly emerged in the absence of p63-positive basal cells (Kurita et al., 2004). However, this does not mean that in normal mice, p63-positive prostatic stem cells do not reside in the basal compartment. It may be that the critical inductive effects of urogenital sinus mesenchyme and androgen may have invoked an alternative pathway to luminal and neuroendocrine cell differentiation in p63-deficient and basal cell-deficient prostatic grafts. While many human cancers overexpress p63 (Parsa et al., 1999; Westfall and Pietenpol, 2004), most prostate carcinomas and prostate cancer cell lines (DU-145, LNCaP, and PC-3) have been reported to be p63 negative (Signoretti et al., 2000). However, a small subset of p63-positive prostate carcinomas show high Gleason grade (Parsons et al., 2001; Di Como et al., 2002; Westfall and Pietenpol, 2004). The cloned, RWPE2-W99 human prostate cancer cell line, derived from our RWPE-1 cells by transformation with Ki-ras (Bello et al., 1997), expresses p63 and is also positive for CK5/ 14 and CK18. This cell line expressing both luminal and basal cell markers may provide a useful model for examining the origin of prostate cancer. It is likely that true prostatic stem cells or intermediate cells, similar to WPE-int cells on the pathway to secretory differentiation, are the most frequent target for carcinogenesis. This realization may provide insights into better cancer prevention and treatment strategies. The cloning of WPE-int and WPE-stem cell lines in the absence of androgen led to the selection of cell types that are androgen-independent. Under these conditions the main proliferating cell type is the basal cell (Chaproniere-Rickenberg and Webber, 1982). Several cell types within the prostatic epithelial cell lineage are androgen-independent for growth and survival and probably include stem/progenitor, intermediate, and possibly mature basal cells. While AR and PSA are

Fig. 4 (A) A comparison of growth of WPE-stem, WPE-int, and the parental cell line RWPE-1 in monolayer culture. Cells were plated at densities of 625–10,000 cells/well in 96-well plates and allowed to grow for 5 days. Results are plotted as mean of 48 wells/cell line  standard deviation (SD). A comparison shows that the growth of WPE-stem is significantly greater than that of WPE-int cells at all cell densities with po0.0001 (n 5 48). (B, C) Colony forming efficiency (CFE): cells were plated in agar at a density of 12,500 cells/30 mm plate and allowed to grow for 21 days when colonies were stained and colonies  0.2 mm were counted. Results are shown as percent CFE  SD. The difference between CFE of WPE-stem and WPE-int is extremely significant po0.0001. (D) Growth in suspension: WPE-stem cells are able to form colonies in agar (a) and can grow as free-floating ‘‘prostaspheres’’ in liquid medium (b, c, confocal microscopy); to show that cells in prostaspheres are viable, single prostaspheres, when plated on laminin-1coated chamber slides, grow to form colonies (d); bar 5 20 mm. (E) SDS-PAGE zymography showing matrix metalloproteinase (MMP)-9 and MMP-2 activity in all three cell lines where WPEstem cells show over 40-fold higher MMP-2 activity as compared with WPE-int cells.

471

considered to be markers of differentiated, secretory luminal prostatic epithelial cells, our results are consistent with the observations that AR is expressed in basal cells, which also express 5a-reductase, the enzyme required for the conversion of testosterone to 5a-dihydrotestosterone (5a-DHT) and that AR and PSA are expressed in p63- and CK5/14-positive basal cells (Bonkhoff and Remberger, 1996; Garraway et al., 2003; van Leenders et al., 2003; Bonkhoff, 2004). Cells of the basal compartment of prostatic epithelium play an important role in maintaining stromal: epithelial relationships, normal luminal cell differentiation, and prostatic ductal integrity (Cunha et al., 1987; Bonkhoff, 2004; Kurita et al., 2004). In the mammary gland, myoepithelial cells are important in maintaining luminal cell polarity (Gudjonsson et al., 2003); their loss in breast cancer and that of basal cells in prostate cancer appears to be associated with loss of epithelial cell polarity and organization during carcinogenesis. Hence, in regard to maintenance of ductal structure, basal cells in the prostate appear to be functionally equivalent to myoepithelial cells in the mammary gland, even though prostatic basal cells do not serve a contractile function as is the case for myoepithelial cells. Epithelial differentiation has been generally considered to occur in well-defined, distinct steps where each step can be identified by the profile of expressed proteins. Cytokeratins are such marker proteins. Stem cells and their close progeny express CK5/14 and p63, while the terminally differentiated luminal secretory cells express CK8/18. This may be an over simplification as differentiation may occur in a continuum, in which stem/progenitor and intermediate cell populations may gradually shift the proportion of basal and luminal cell markers. Our results and those of others show that the expression of differentiation markers is not an ‘‘all or none’’ phenomenon, but that as epithelial cells proceed through differentiation, many different intermediate cell types may be found with different levels of stem, basal, or luminal cell markers (Signoretti et al., 2000; Wang et al., 2001; Garraway et al., 2003; van Leenders et al., 2003; Bonkhoff, 2004). The two cloned cell lines described herein are cells located in distinctly different positions along this continuum and may facilitate further investigations on prostatic epithelial cell lineages. Cell:cell interactions are extremely important in maintaining homeostasis between the basal and differentiated cell compartments in vivo where differentiated cells regulate cell proliferation in the basal compartment by the production of growth inhibitors, such as, transforming growth factor b (TGF-b) (Barnard et al., 1989, Iversen, 1991). Although WPE-stem cells were isolated from RWPE-1 cells, the RWPE-1 cells did not grow in agar (Webber et al., 2001). This suggests that in the heterogeneous RWPE-1 cell population the more differentiated cells prevent anchorage-independent growth

of undifferentiated cells with a stem/progenitor cell phenotype. Similarly, mixed populations of immortalized, normal human mammary epithelial cells also fail to grow in agar (DiRenzo et al., 2002). RWPE-1 cells express TGF-b (data not shown), which may exert a negative feed back on stem/progenitor cell types. However, WPE-stem cells, cloned from RWPE-1 cells, when grown alone, show high CFE. Such a negative feed back among cells of the prostatic epithelial cell population may have important implications in the treatment of benign and malignant tumors of the prostate. Finasteride and Dutasteride are two 5a-reductase inhibitors currently being used for the treatment of benign hyperplasia and are being tested for the prevention of prostatic cancer. Treatment with 5a-reductase inhibitors can reduce intra-prostatic 5a-DHT levels by as much as  97% (Thompson et al., 2003; Andriole et al., 2004). Under these conditions the majority of androgen-dependent, secretory cells undergo apoptosis and symptoms of BPH decrease. However, in men on Finasteride treatment, the incidence of more aggressive, high Gleason grade cancers is about 15% higher than in untreated men (Thompson et al., 2003). One possible explanation is that because of the loss of androgen-dependent, differentiated cells, their growth regulatory effect by negative feedback on the proliferation of stem and intermediate cells is lost. This loss of homeostasis allows the androgen-independent, undifferentiated cells to proliferate. If some of these cells have the necessary mutations associated with malignant transformation then they are likely to give rise to more aggressive tumors. The expression of high levels of MMPs in cancer cells, in general, is associated with invasion (StetlerStevenson and Yu, 2001), and the expression of MMP-2 is associated with high Gleason score in prostate cancer (Stearns and Stearns, 1996). Very little is known about MMP expression by stem cells. However, human embryonic neuroectodermal and circulating bone marrow CD34(1) stem cells express elevated levels of MMPs (Frolichsthal-Schoeller et al., 1999; JanowskaWieczorek et al., 1999). WPE-int cells show low MMP2 and undetectable MMP-9 activity. In contrast, WPE-stem cells express MMP-9 and over 40-fold higher MMP-2 activity than WPE-int cells. The high MMP2 activity expressed by WPE-stem cells is a phenotypic property that stem/progenitor cells share with cancer cells. We propose that in the normal prostatic epithelium, cells exist at many stages in a continuum of differentiation, beginning from stem/progenitor cells through multiple intermediate cell types along different lineages, to terminally differentiated cells. Malignant transformation can occur in cells at any stage while they still have the ability to proliferate. Additional genetic changes and changes in gene expression during promotion subsequently define the malignant phenotype. The abil-

472

ity to grow in an anchorage-independent manner is a property of many cancer cells that may be shared with stem cells. Solid tumors generally consist of a heterogeneous cell population. When grown in soft agar, only a very small number of cancer cells form colonies, thus, indicating that most cancer cells are not clonogenic and that only rare cancer stem cells may have this ability (Reya et al., 2001; Webber et al., 2001; Kondo et al., 2004). Such clonogenic cells suggest the existence of cancer stem cells, which may, in part, maintain an expanding tumor cell population. It is evident that in the prostate, the traditional, simple division into ‘‘basal cells’’ and ‘‘luminal cells’’ is no longer appropriate because all basal cells and all luminal cells are apparently not alike. We propose that it would be more appropriate to view a basal cell compartment as containing stem/progenitor cells, their intermediate progeny, as well as mature basal cells. Similarly, the luminal cell compartment is likely to contain mature secretory luminal cells as well as intermediate cell types on their way to secretory differentiation. During prostatic differentiation secretory cells appear to emerge by maintaining CK8/18 and losing p63 and CK5/14, while basal cells emerge by maintaining p63 and CK5/14 and losing CK8/18. The putative stem/ progenitor cells in the adult prostate, which express the full spectrum of luminal and basal cell markers, are extremely rare (Wang et al., 2001). Experiments in vivo, such as those described by Hayward et al. (1998), utilizing grafts of recombinants composed of WPE-stem or WPE-int cells plus UGS mesenchyme, may further elucidate the multipotency and lineage relationships of these cell lines. In summary, WPE-stem cells appear to be similar to the UGS-stem/progenitor cells as they co-express a wide range of stem/progenitor as well as luminal cell markers including high levels of p63, CK5/14, and MMP-2, and low levels of CK18 and AR. This is consistent with the expression of a multilineage transcriptosome by stem cells where most differentiation-associated genes are maintained at a low but detectable level of transcription. Transcription of high levels of a more restricted specific set of proteins occurs subsequently as cells differentiate into different cell types (Chiu and Rao, 2003). The WPE-stem cells are anchorage- and androgen-independent for growth and survival. In contrast, WPE-int cells are anchorage-dependent and exhibit an intermediate cell phenotype.WPE-int cells show very low expression of p63 and CK5/14 but high CK18 and are very responsive to androgen as shown by AR and PSA induction. WPE-int cells appear to exhibit an intermediate phenotype en route to luminal differentiation and, thus, may be early luminal progenitor cells. These two cells lines provide novel models for exploring cell lineage relationships in human prostatic epithelium and the origin of prostate cancer. In view of the likely origin of prostate cancer in stem or intermediate cell types,

these cell lines also provide useful models for developing new strategies for the prevention and treatment of tumors of the prostate. Acknowledgments We thank Dr. Shirley Owens for her assistance with the confocal microscopy image, Katherine J. Fabis for her assistance in the laboratory, and Leslie-Ann S. Ovitt and Adam K. Keith for their assistance in the preparation of figures and this manuscript.

References Anderson, D.J., Gage, F.H. and Weissman, I.L. (2001) Can stem cells cross lineage boundaries? Nat Med 7:393–395. Andriole, G.L., Humphrey, P., Ray, P., Gleave, M.E., Trachtenberg, J., Thomas, L.N., Lazier, C.B. and Rittmaster, R.S. (2004) Effect of the dual 5alpha-reductase inhibitor dutasteride on markers of tumor regression in prostate cancer. J Urol 172: 915–919. Bamberger, C., Pollet, D. and Schmale, H. (2002) Retinoic acid inhibits downregulation of DNp63a expression during terminal differentiation of human primary keratinocytes. J Invest Dermatol 118:133–138. Barnard, J.A., Beauchamp, R.D., Coffey, R.J. and Moses, H.L. (1989) Regulation of intestinal epithelial cell growth by transforming growth factor type b. Proc Natl Acad Sci USA 86: 1578–1582. Bello, D., Webber, M.M., Kleinman, H.K., Wartinger, D.D. and Rhim, J.S. (1997) Androgen responsive adult human prostatic epithelial cell lines immortalized by human papillomavirus 18. Carcinogenesis 18:1215–1223. Bello-DeOcampo, D., Kleinman, H.K., DeOcampo, N.D. and Webber, M.M. (2001) Laminin-1 and a6b1 integrin regulate acinar morphogenesis of normal and malignant human prostate epithelial cells. Prostate 46:142–153. Blau, H.M., Brazelton, T.R. and Weimann, J.M. (2001) The evolving concept of a stem cell: entity or function? Cell 105: 829–841. Bonkhoff, H. (2004) Morphogenesis of prostate cancer. In: Sell, S. (ed.) Stem cells handbook. Humana Press, Totowa, pp. 445–453. Bonkhoff, H. and Remberger, K. (1996) Differentiation pathways and histogenetic aspects of normal and abnormal prostatic growth: a stem cell model. Prostate 28:98–106. Chaproniere-Rickenberg, D.M. and Webber, M.M. (1982) A chemically defined medium for the growth of adult human prostatic epithelium. In: Growth of cells in hormonally defined media. Vol. 9, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 1109–1115. Chiu, A.Y. and Rao, M.S. (eds.) (2003) Human embryonic stem cells. Humana Press, Totowa, pp. v–xi. Cunha, G.R., Donjacour, A.A., Cooke, P.S., Mee, S., Bigsby, R.M., Higgins, S.J. and Sugimura, Y. (1987) The endocrinology and developmental biology of the prostate. Endocr Rev 8: 338–362. Di Como, C.J., Urist, M.J., Babayan, I., Drobnjak, M., Hedvat, C.V., Teruya-Feldstein, J., Pohar, K., Hoos, A. and CordonCardo, C. (2002) p63 expression profiles in human normal and tumor tissues. Clin Cancer Res 8:494–501. DiRenzo, J., Signoretti, S., Nakamura, N., Rivera-Gonzalez, R., Sellers, W., Loda, M. and Brown, M. (2002) Growth factor requirements and basal phenotype of an immortalized mammary epithelial cell line. Cancer Res 62:89–98. Dontu, G., Abdallah, W.M., Foley, J.M., Jackson, K.W., Clarke, M.F., Kawamura, M.J. and Wicha, M.S. (2003a) In vitro propagation and transcriptional profiling of human mammary stem/ progenitor cells. Genes Dev 17:1253–1270.

473 Dontu, G., Al-Hajj, M., Abdallah, W.M., Clarke, M.F. and Wicha, M.S. (2003b) Stem cells in normal breast development and breast cancer. Cell Prolif 36(Suppl. 1): 59–72. English, H.F., Santen, R.J. and Isaacs, J.T. (1987) Response of glandular versus basal rat ventral prostatic epithelial cells to androgen withdrawal and replacement. Prostate 11:229–242. Evans, G.S. and Chandler, J.A. (1987) Cell proliferation studies in the rat prostate: II. The effects of castration and androgen-induced regeneration upon basal and secretory cell proliferation. Prostate 11:339–351. Frolichsthal-Schoeller, P., Vescovi, A.L., Krekoski, C.A., Murphy, G., Edwards, D.R. and Forsyth, P. (1999) Expression and modulation of matrix metalloproteinase-2 and tissue inhibitors of metalloproteinases in human embryonic CNS stem cells. Neuroreport 10:345–351. Garraway, L.A., Lin, D., Signoretti, S., Waltregny, D., Dilks, J., Bhattacharya, N. and Loda, M. (2003) Intermediate basal cells of the prostate: in vitro and in vivo characterization. Prostate 55:206–218. Gudjonsson, T., Rnnov-Jessen, L., Villadsen, R., Bissell, M.J. and Petersen, O.W. (2003) To create the correct microenvironment: three-dimensional heterotypic collagen assays for human breast epithelial morphogenesis and neoplasia. Methods 30:247–255. Hall, P.A., Campbell, S.J., O’Neill, M., Royston, D.J., Nylander, K., Carey, F.A. and Kernohan, N.M. (2000) Expression of the p53 homologue p63a and DNp63a in normal and neoplastic cells. Carcinogenesis 21:153–160. Hayward, S.W., Haughney, P.C., Rosen, M.A., Greulich, K.M., Weier, H.-U.G., Dahiya, R. and Cunha, G.R. (1998) Interactions between adult human prostatic epithelium and rat urogenital sinus mesenchyme in a tissue recombination model. Differentiation 63:131–140. Hudson, D.L., Guy, A.T., Fry, P., O’Hare, M.J., Watt, F.M. and Masters, J.R.W. (2001) Epithelial cell differentiation pathways in the human prostate: identification of intermediate phenotypes by keratin expression. J Histochem Cytochem 49:271–278. Isaacs, J.T. (1987) Control of cell proliferation and cell death in the normal and neoplastic prostate: a stem cell model. In: Rodgers, C.H., Coffey, D.S., Cunha, G., Grayhack, J.T., Heinman, F. and Horton, R. eds. Benign prostatic hyperplasia. Vol. II, pp. 85–94 (NIH Publ. No. 87-2881). Iversen, O.H. (1991) The hunt for endogenous growth-inhibitory and/or tumor suppression factors: their role in physiological and pathological growth regulation. Adv Cancer Res 57:413–453. Janowska-Wieczorek, A., Marquez, L.A., Nabholtz, J.M., Cabuhat, M.L., Montano, J., Chang, H., Rozmus, J., Russell, J.A., Edwards, D.R. and Turner, A.R. (1999) Growth factors and cytokines upregulate gelatinase expression in bone marrow CD34(1) cells and their transmigration through reconstituted basement membrane. Blood 93:3379–3390. Kondo, T., Setoguchi, T. and Taga, T. (2004) Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci USA 101:781–786. Kurita, T., Medina, R.T., Mills, A.A. and Cunha, G.R. (2004) Role of p63 and basal cells in the prostate. Development 131: 4955–4964. McKeon, F. (2004) p63 and the epithelial stem cell: more than status quo? Genes Dev 18:465–469. Mirosevich, J., Bentel, J.M., Zeps, N., Redmond, S.L., D’Antuono, M.F. and Dawkins, H.J.S. (1999) Androgen receptor expression of proliferating basal and luminal cells in adult murine ventral prostate. J Endocrinol 162:341–350. Moll, R., Franke, W.W., Schiller, D.L., Geiger, B. and Krepler, R. (1982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11–24. Parsa, R., Yang, A., McKeon, F. and Green, H. (1999) Association of p63 with proliferative potential in normal and neoplastic human keratinocytes. J Invest Dermatol 113:1099–1105. Parsons, J.K., Gage, W.R., Nelson, W.G. and De Marzo, A.M. (2001) p63 protein expression is rare in prostate adenocarcinoma:

implications for cancer diagnosis and carcinogenesis. Urology 58:619–624. Pellegrini, G., Dellambra, E., Golisano, O., Martinelli, E., Fantozzi, I., Bondanza, S., Ponzin, D., McKeon, F. and De Luca, M. (2001) p63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA 98:3156–3161. Prins, G.S. and Birch, L. (1995) The developmental pattern of androgen receptor expression in rat prostate lobes is altered after neonatal exposure to estrogen. Endocrinology 136:1303–1314. Reya, T., Morrison, S.J., Clarke, M.F. and Weissman, I.L. (2001) Stem cells, cancer, and cancer stem cells. Nature 414:105–111. Schalken, J.A. and van Leenders, G. (2003) Cellular and molecular biology of the prostate: stem cell biology. Urology 62(Suppl. 5A): 11–20. Signoretti, S., Waltregny, D., Dilks, J., Isaac, B., Lin, D., Garraway, L., Yang, A., Montironi, R., McKeon, F. and Loda, M. (2000) p63 is a prostate basal cell marker and is required for prostate development. Am J Pathol 157:1769–1775. Stearns, M.E. and Stearns, M. (1996) Immunohistochemical studies of activated matrix metalloproteinase-2 (MMP-2a) expression in human prostate cancer. Oncol Res 8:63–67. Stetler-Stevenson, W.G. and Yu, A.E. (2001) Proteases in invasion: matrix metalloproteinases. Semin Cancer Biol 11:143–152. Strano, S., Rossi, M., Fontemaggi, G., Munarriz, E., Soddu, S., Sacchi, A. and Blandino, G. (2001) From p63 to p53 across p73. FEBS Lett 490:163–170. Thompson, I.M., Goodman, P.J., Tangen, C.M., Lucia, M.S., Miller, G.J., Ford, L.G., Lieber, M.M., Cespedes, R.D., Atkins, J.N., Lippman, S.M., Carlin, S.M., Ryan, A., Szczepanek, C.M., Crowley, J.J. and Coltman, C.A. (2003) The influence of finasteride on the development of prostate cancer. N Engl J Med 349:215–224. Tiberio, R., Marconi, A., Fila, C., Fumelli, C., Pignatti, M., Krajewski, S., Giannetti, A., Reed, J.C. and Pincelli, C. (2002) Keratinocytes enriched for stem cells are protected from anoikis via an integrin signaling pathway in a Bcl-2 dependent manner. FEBS Lett 524:139–144. van Leenders, G.J.L.H., Gage, W.R., Hicks, J.L, van Balken, B., Aalders, T.W, Schalken, J.A. and De Marzo, A.M. (2003) Intermediate cells in human prostate epithelium are enriched in proliferative inflammatory atrophy. Am J Pathol 162: 1529–1537. van Leenders, G.J.L.H. and Schalken, J.A. (2001) Stem cell differentiation within the human prostate epithelium: implications for prostate carcinogenesis. BJU Int 88(Suppl. 2): 35–42. Verhagen, A.P.M., Ramaekers, F.C.S., Aalders, T.W., Schaafsma, H.E., Debruyne, F.M.J. and Schalken, J.A. (1992) Colocalization of basal and luminal cell-type cytokeratins in human prostate cancer. Cancer Res 52:6182–6187. Wang, Y., Hayward, S.W., Cao, M., Thayer, K.A. and Cunha, G.R. (2001) Cell differentiation lineage in the prostate. Differentiation 68:270–279. Webber, M.M., Bello, D., Kleinman, H.K. and Hoffman, M.P. (1997) Acinar differentiation by non-malignant immortalized human prostatic epithelial cells and its loss by malignant cells. Carcinogenesis 18:1225–1231. Webber, M.M., Quader, S.T.A., Kleinman, H.K., Bello-De Ocampo, D., Storto, P.D., Bice, G., DeMendonca-Calaca, W. and Williams, D.E. (2001) Human cell lines as an in vitro/in vivo model for prostate carcinogenesis and progression. Prostate 47:1–13. Westfall, M.D. and Pietenpol, J.A. (2004) p63: molecular complexity in development and cancer. Carcinogenesis 25:857–864. Yang, A., Kaghad, M., Caput, D. and McKeon, F. (2002) On the shoulders of giants: p63, p73 and the rise of p53. Trends Genet 18:90–95. Yang, A., Schweitzer, R., Sun, D., Kaghad, M., Walker, N., Bronson, R.T., Tabin, C., Sharpe, A., Caput, D., Crum, C. and McKeon, F. (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398:714–718.