Cell differentiation lineage in the prostate

Prostatic epithelium consists mainly of luminal and basal cells, which are presumed to differentiate from common progenitor/stem cells. We hypothesize that progenitor/stem cells are highly concentrated in the embryonic urogenital sinus epithelium from which prostatic epithelial buds develop. We further hypothesize that these epithelial progenitor/stem cells are also present within the basal compartment of adult prostatic epithelium and that the spectrum of differentiation markers of embryonic and adult progenitor/stem cells will be similar. The present study demonstrates that the majority of cells in embryonic urogenital sinus epithelium and developing prostatic epithelium (rat, mouse, and human) co-expressed luminal cytokeratins 8 and 18 (CK8, CK18), the basal cell cytokeratins (CK14, CK5), p63, and the so-called transitional or intermediate cell markers, cytokeratin 19 (CK19) and glutathione-S-transferase-pi (GSTpi). The majority of luminal cells in adult rodent and human prostates only expressed luminal markers (CK8, CK18), while the basal epithelial cell compartment contained several distinct subpopulations. In the adult prostate, the predominant basal epithelial subpopulation expressed the classical basal cell markers (CK5, CK14, p63) as well as CK19 and GSTpi. However, a small fraction of adult prostatic basal epithelial cells co-expressed the full spectrum of basal and luminal epithelial cell markers (CK5, CK14, CK8, CK18, CK19, p63, GSTpi). This adult prostatic basal epithelial cell subpopulation, thus, exhibited a cell differentiation marker profile similar to that expressed in embryonic urogenital sinus epithelium. These rare adult prostatic basal epithelial cells are proposed to be the progenitor/stem cell population. Thus, we propose that at all stages (embryonic to adult) prostatic epithelial progenitor/stem cells maintain a differentiation marker profile similar to that of the original embryonic progenitor of the prostate, namely urogenital sinus epithelium. Adult progenitor/stem cells co-express both luminal cell, basal cell, and intermediate cell markers. These progenitor/stem cells differentiate into mature luminal cells by maintaining CK8 and CK18, and losing all other makers. Progenitor/stem cells also give rise to mature basal cells by maintaining CK5, CK14, p63, CK19, and GSTpi and losing K8 and K18. Thus, adult prostate basal and luminal cells are proposed to be derived from a common pleuripotent progenitor/stem cell in the basal compartment that maintains its embryonic profile of differentiation markers from embryonic to adult stages.     

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

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. WPE-stem 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 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.     

Role of p63 and basal cells in the prostate

The prostate contains two major epithelial cell types - luminal and basal cells - both of which develop from urogenital sinus epithelium. The cell linage relationship between these two epithelial types is not clear. Here we demonstrate that luminal cells can develop independently of basal cells, but that basal cells are essential for maintaining ductal integrity and the proper differentiation of luminal cells. Urogenital sinus (UGS) isolated from p63(+/+) and p63(-/-) embryos developed into prostate when grafted into adult male nude mice. Prostatic tissue that developed in p63(-/-) UGS grafts contained neuroendocrine and luminal cells, but basal cells were absent. Therefore, p63 is essential for differentiation of basal cells, but p63 and thus basal cells are not required for differentiation of prostatic neuroendocrine and luminal epithelial cells. p63(-/-) prostatic grafts also contained atypical mucinous cells, which appeared to differentiate from luminal cells via activation of Src. In the response to castration, regression of p63(-/-) prostate was inordinately severe with almost complete loss of ducts, resulting in the formation of residual cystic structures devoid of epithelium. Therefore, basal cells play critical roles in maintaining ductal integrity and survival of luminal cells. However, regressed p63(-/-) prostate did regenerate in response to androgen administration, indicating that basal cells were not essential for prostatic regeneration.     

Quantitative immunohistochemical detection of the molecular expression patterns in proliferative inflammatory atrophy

The proliferative inflammatory atrophy (PIA) is considered as a possible precursor of prostate intraepithelial neoplasia (PIN) or prostate cancer (PCa). In this study we assessed quantitatively the expression of AMACR, p63, COX-2, GST and iNOS in serial paraffin-embedded tissue sections obtained after radical prostatectomy of PCa patients (n = 30). The applicability of these markers to distinguish PIA, PIN and PCa was evaluated. We also compared the immunohistochemical expression profiles of AMACR, COX-2 and GST in the luminal and basal cells in lesions of PIN arisen in PIA or PIA alone. Two different patterns of COX-2 expression according to the p63 status of the basal cells were found. This observation gives us grounds to hypothesize that the diverse COX-2 patterns resulted from an initial basal cell damage which subsequently propagated to its luminal secretory cells progeny.     

Down-Regulation of c-neu Receptors by Nerve Growth Factor in PC12 Cells

Abstract: A small number of p185^{c- neu} receptors have been found on PC12 cells. These receptors show some basal phosphorylation in quiescent cells. When the cells are treated with nerve growth factor (NGF) for a short time, some increase in phosphorylation is seen, mainly on serine and threonine residues, and this is accompanied by a slight shift in the apparent molecular weight. Epidermal growth factor (EGF) also increases the phosphorylation of p185^{c- neu} , in this case on tyrosine residues. Neither heregulin-β1 nor gp30 stimulates the tyrosine phosphorylation of p185^{c- neu} , and neither has a proliferative effect on the cells. Treatment of the cells with NGF for 5 days produces a 70–80% reduction in the number of p185^{c- neu} receptors. This down-regulation does not occur when PC12nnr5 cells, which lack the high-affinity NGF receptor, p140 ^trk , are treated with NGF.The level of p185^{c- neu} mRNA is not altered by NGF treatment, suggesting that the down-regulation is due to either a translational or a posttranslational alteration.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Keratin expression: a measure of phenotypic modulation of human prostatic epithelial cells by growth inhibitory factors

Expression of certain cytokeratins can be indicative of the state of differentiation of epithelial cells. The basal cells in the normal adult human prostatic epithelium are characterized by the expression of cytokeratins 5 and 14, whereas the secretory luminal cells contain cytokeratins 8 and 18. Cells cultured from the prostatic epithelium expressed cytokeratins 5, 8, and 18, and thus had features of both basal and luminal cells. Certain growth-inhibitory conditions altered keratin expression in conjunction with growth modulation. Deletion of peptide factors and hormones from the culture medium induced the expression of cytokeratins 1 and 10, associated with a squamous phenotype. These same squamous keratins were found in very dense, stratified cultures that were maintained at confluency in standard, complete medium for extended periods. Retinoic acid enhanced the expression of secretory luminal cell-associated cytokeratins 8 and 18 in semi-confluent cultures. Other growth inhibitory factors such as suramin, transforming growth factor-, and interferon- had no effect on keratin expression. These observations indicate that the differentiation of prostatic epithelial cells can be directed toward alternate pathways, either squamous or secretory, by different growth-inhibitory conditions. However, not all growth inhibitory factors altered differentiation, demonstrating that growth inhibition in itself is not a sufficient inducer of differentiation.     

Expression of prostate specific membrane antigen (PSMA) in prostatic adenocarcinoma and prostatic intraepithelial neoplasia

The prostatic membrane antigen (PSMA) is a protein that is expressed in the prostatic epithelium. We studied the expression of PSMA in a series of 55 patients with different stages of prostate cancer and we compared the PSMA staining in prostate cancer cells, in high-grade prostatic intraepithelial neoplasia (PIN) and in histologically benign prostatic epithelium for the same specimen. For this purpose archival paraffin-embedded specimens were studied by immunohistochemistry with a monoclonal antibody 7E11-C5.3 against PSMA using the streptavidin-biotin method. The mean percentage of PSMA immunoreactivity was 56.67% in prostate cancer (CaP) cells, and 48.6% in PIN cells, which was significantly higher than benign-appearing prostatic epithelium (5.72%) (for each pair, p<0.001). PSMA expression was greater in CaP with a higher Gleason score (p=0.01), but no relationship was found with serum PSA value. We conclude that PSMA overexpression is detected in high-grade PIN and is associated with a higher Gleason score of prostate cancer. It is a potential marker for studying carcinogenesis and progression of prostate cancer.     

Nuclear volume estimates in prostatic intraepithelial neoplasia

OBJECTIVE: Prostatic intraepithelial neoplasia (PIN), the most likely precursor of prostatic adenocarcinoma, is divided into two grades, low and high. Pathologists may encounter difficulties in applying these criteria in daily practice. In view of the clinical significance of high grade PIN as strong predictor of carcinoma, the separation of low and high grade PIN plays an important role in patient management. The aim of the present study was to evaluate three-dimensional nuclear size estimation in normal prostatic glands, low and high grade PIN, and prostatic adenocarcinoma as an element in their classification. STUDY DESIGN: We studied 31 formalin-fixed, paraffin-embedded, whole-mounted radical prostastectomy specimens that contained foci of normal prostatic glands, low and high grade PIN, and prostatic adenocarcinoma. Hematoxylin-eosin-stained sections were selected for the stereologic estimation of volume-weighted mean nuclear volume by the "point-sampled intercepts" method. On each focus, an average of six fields of vision were systematically chosen. RESULTS: The quantitative results indicate a significant increase in nuclear volume from normal prostatic glands (mean, 209.0 micron 3; SD, 64.6 micron 3) to low grade PIN, high grade PIN and prostatic adenocarcinoma with increments of 49%, 88% and 109%, respectively (F = 29.1, P < .001). Two-group comparisons (Duncan procedure) showed differences between low and high grade PIN and prostatic adenocarcinoma (P < .01). The difference between high grade PIN and prostatic adenocarcinoma was not significant. CONCLUSION: Three-dimensional estimates of nuclear size discriminate low and high grade PIN. Lack of stereologic differences between high grade PIN and prostatic adenocarcinoma further supports high grade PIN as a precursor of prostatic adenocarcinoma.     

Generation of active TGF-beta by prostatic cell cocultures using novel basal and luminal prostatic epithelial cell lines

Two prostatic epithelial lines, one of basal origin and one of luminal origin, were established from the dorsolateral prostates of p53 null mice. The cell lines are nontumorigenic when inoculated subcutaneously under the renal capsule or intraprostatically in syngeneic mice. The luminal cell line (PE-L-1) expresses cytokeratins 8 and 18 and the basal cell line (PE-B-1) expresses cytokeratins 5 and 14. The basal cells require serum for growth, whereas the luminal cells grow only in serum-free medium. Both cell lines require the presence of growth factors for optimal growth in culture, with EGF and FGF-2 having the greatest effect on the growth rate. Both lines express androgen receptor (AR) mRNA and protein. Androgen stimulates growth of the basal cell line, indicating that the ARs are functional, whereas growth of the luminal cells is unaffected by androgens. The luminal line is significantly inhibited by exogenous TGF-beta and produces low levels of endogenous TGF-beta. In contrast, the basal cell line produces significant amounts of TGF-beta and its growth is not influenced by this cytokine. Coculture of luminal cells with prostatic smooth muscle cells results in the generation of increased levels of biologically active TGF-beta, indicating a paracrine mechanism of TGF-beta activation that may be involved in the maintenance of normal prostatic function. To our knowledge this is the first report describing both basal and luminal prostatic cell lines from a single inbred animal species and the first indication that prostatic epithelial and stromal cells interact to generate the biologically active form of TGF-beta. These lines will provide an important model for determining basal/luminal interactions in both in vitro and in vivo assays.     

Localization of annexins I, II, IV and VII in whole prostate sections from radical prostatectomy patients

Annexins (ANXs) represent a family of calcium and phospholipid binding proteins that are involved in several physiological processes e.g. signal transduction, cellular differentiation and proliferation. Since they are known to be dysregulated in a variety of cancers we investigated the immunolocalization of ANXs in whole prostate sections containing benign prostatic epithelium (BPE), benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and prostate cancer (PCa) in order to evaluate their possible role during tumorigenesis. Samples were obtained from 28 patients undergoing radical prostatectomy. Gross sections of whole prostates were examined immunohistochemically for the distribution of ANX I, II, IV and VII. In BPE all ANXs were localized to the cell membranes and the cytoplasm of all gland cells. In BPH the immunoreactivity of ANX I and II was restricted to the basal cells of glands and expression pattern of ANX IV and VII was similar to BPE. In PIN only basal cells expressed ANX II. In PCa ANX II immunoreactivity was absent and weak ANX I and ANX IV immunoreactivity was restricted to the cytoplasm of tumor cells. ANX VII immunoreactivity was seen in some but not all tumor cells. Since ANX IV and VII expression did not show significant changes in PCa compared to non-neoplastic tissue and PIN an essential role during prostate tumourigenesis seems unlikely. In contrast, as progression from PIN to PCa is characterized by a reduction of ANX I and II this suggests that downregulation of these proteins could represent an important event in prostate carcinogenesis.     

High-grade prostatic intraepithelial neoplasia

High-grade prostatic intraepithelial neoplasia is considered the most likely precursor of prostatic carcinoma. The only method of detection is biopsy; prostatic intraepithelial neoplasia (PIN) does not significantly elevate serum prostate-specific antigen concentration and cannot be detected by ultra-sonography. The incidence of PIN in prostate biopsies averages 9% (range, 4%-16%), representing 115,000 new cases of PIN diagnosed each year in United States. PIN has a high predictive value as a marker for adenocarcinoma, and its identification warrants repeated biopsy for concurrent or subsequent invasive carcinoma. Carcinoma will develop in most patients with PIN within 10 years. PIN is associated with progressive abnormalities of phenotype and genotype that are intermediate between normal prostatic epithelium and cancer, indicating impairment of cell differentiation and regulatory control with advancing stages of prostatic carcinogenesis. Androgen deprivation therapy decreases the prevalence and extent of PIN, suggesting that this form of treatment may play a role in chemoprevention.     

Mechanisms regulating norgestomet inhibition of endometrial gland morphogenesis in the neonatal ovine uterus

In many species, endometrial gland adenogenesis occurs neonatally in an ovary- and steroid-independent manner. Chronic exposure of the developing neonatal ovine uterus to norgestomet (NOR) from birth permanently ablates endometrial gland morphogenesis or adenogenesis, creating an adult ovine uterine gland knockout (UGKO) phenotype. This study was conducted to determine the mechanism(s) whereby NOR inhibits adenogenesis in the neonatal ewe. Ewe lambs received no implant or a NOR implant at birth and on postnatal day (PND) 14, and they were necropsied on PND28. Histological analyses of the tracts indicated NOR exposure specifically inhibited endometrial adenogenesis, but no histoarchitectural differences were observed in the oviduct, cervix, or vagina. No effect of NOR treatment was detected on proliferating cell nuclear antigen (PCNA) expression in the endometrial luminal epithelium (LE), stroma, or myometrium. In control (CX) ewes, estrogen receptor alpha (ER-alpha) and progesterone receptor (PR) mRNA and protein were expressed strongly in nascent and proliferating glandular epithelium (GE) but were undetected in epithelium of NOR uteri. Expression of c-met and fibroblast growth factor receptor 2IIIb (FGFR2IIIb) mRNA was detected in the LE and GE of CX uteri. In NOR uteri, c-met was expressed in the LE similar to CX uteri, but FGFR2IIIb mRNA levels were lower than in the LE of CX uteri. Uterine hepatocyte growth factor (HGF), the ligand for c-met, and FGFR2IIIb mRNA expression was substantially lower in NOR ewes, but expression of FGF-7 and FGF-10 mRNAs, ligands for FGFR2IIIb, was unaffected. These results indicate that NOR disrupts endometrial adenogenesis by ablating epithelial ER-alpha expression and altering expression of paracrine growth factors and/or receptors involved in epitheliomesenchymal interactions. Likewise, these mechanisms are proposed to be important regulators of normal uterine gland morphogenesis in the neonate.     

Evidence of progenitor cells of glandular and myoepithelial cell lineages in the human adult female breast epithelium: a new progenitor (adult stem) cell concept

Abstract.  Although experimental data clearly confirm the existence of self-renewing mammary stem cells, the characteristics of such progenitor cells have never been satisfactorily defined. Using a double immunofluorescence technique for simultaneous detection of the basal cytokeratin 5, the glandular cytokeratins 8/18 and the myoepithelial differentiation marker smooth muscle actin (SMA), we were able to demonstrate the presence of CK5⁺ cells in human adult breast epithelium. These cells have the potential to differentiate to either glandular (CK8/18⁺) or myoepithelial cells (SMA⁺) through intermediary cells (CK5⁺ and CK8/18⁺ or SMA⁺). We therefore proceeded on the assumption that the CK5⁺ cells are phenotypically and behaviourally progenitor (committed adult stem) cells of human breast epithelium. Furthermore, we furnish evidence that most of these progenitor cells are located in the luminal epithelium of the ductal lobular tree. Based on data obtained in extensive analyses of proliferative breast disease lesions, we have come to regard usual ductal hyperplasia as a progenitor cell-derived lesion, whereas most breast cancers seem to evolve from differentiated glandular cells. Double immunofluorescence experiments provide a new tool to characterize phenotypically progenitor (adult stem) cells and their progenies. This model has been shown to be of great value for a better understanding not only of normal tissue regeneration but also of proliferative breast disease. Furthermore, this model provides a new tool for unravelling further the regulatory mechanisms that govern normal and pathological cell growth.