Tilly Aalders

Intermediate Cells in Human Prostate Epithelium Are Enriched in Proliferative Inflammatory Atrophy

Within the human prostate epithelium four cell populations can be discriminated based on their expression of keratins (K). Basal cells express high levels of K5 and K14, as well as p63, whereas they have very low levels of androgen receptor, prostate-specific antigen (PSA), K8, and K18. Luminal secretory cells lack p63, K5, and K14 but express high levels of K8, K18, androgen receptor, and PSA. Additionally, cells have been identified with a keratin phenotype intermediate between basal and luminal cells that co-express high levels of K5 and K18 (K5/18) as well as hepatocyte growth factor receptor c-MET. Although intermediate cells have been proposed as precursor cells of prostate cancer, their biology is ill defined. Epithelial cells in proliferative inflammatory atrophy (PIA) appear to be cycling rapidly as indicated by expression of Ki-67, and morphological transitions have been identified between PIA and high-grade prostate intraepithelial neoplasia. Many of the atrophic epithelial luminal cells in PIA are candidates for intermediate cells based in part on weak expression of PSA and androgen receptor, high levels of K8/18, and lack of p63. The objective of this study was to further clarify the phenotype of the proposed intermediate cells in PIA and to quantitatively determine the level in which these intermediate cells preferentially occur in PIA lesions. Intermediate cells were immunohistochemically demonstrated using antibodies to K5, K14, K18, and c-MET. Using radical prostatectomy specimens (n = 15) the area fraction of intermediate cells in normally differentiated prostate epithelium and PIA were quantified by a grid point counting method. Atrophic luminal cells of PIA lesions expressed K5 in 39.2 +/- 7.4% of cells compared to 2.4 +/- 2.3% in normal epithelium (P < 0.00001). By contrast, K14 was only expressed in 3.0 +/- 3.2% of the luminal cells. Previous studies have shown that virtually 100% of these atrophic luminal cells are strongly positive for K8/18. c-MET was present in 44.1 +/- 14.1% of luminal cells in PIA but only in 2.1 +/- 2.8% of luminal cells in normal epithelium (P < 0.00001). To unambiguously determine whether intermediate luminal cells in PIA show increased proliferative activity and decreased p27(kip1) expression, double-staining immunofluorescence of Ki-67 and K5, as well as p27(Kip1) and K5 was performed. Luminal cells in PIA often co-expressed K5 and Ki-67. Although p27(Kip1) was strongly expressed in K5-negative differentiated cells in normal epithelium, p27(Kip1) staining was absent in many of the K5-positive cells in the luminal compartment of PIA. We conclude that cells phenotypically intermediate between basal and secretory cells are enriched in PIA lesions. The finding of a large number of highly proliferating intermediate cells in PIA provides further support that these cells may serve as preferred target cells in prostate carcinogenesis.

Expression of basal cell keratins in human prostate cancer metastases and cell lines.

Within normal human prostate epithelium, basal and luminal cells can be discriminated by their expression of keratins (K). While basal cells express K5/14, luminal cells show expression of K8/18 and an intermediate cell population can be identified by co‐expression of K5/18. Prostate cancer is predominantly composed of luminal and neuroendocrine cells, while a minority of cells have a basal phenotype. In order to distinguish between basal and intermediate cells, and to assess the effects of androgen deprivation on prostate cancer, 56 human prostate cancer metastases and three cancer cell lines were characterized using antibodies to K5, K14, K18, and the neuroendocrine marker chromogranin A (ChA). The staining was performed on paraffin tissue and visualized by the avidin–biotin–peroxidase complex method. Protein expression was quantified as the number of positive cells in 20 high power fields (HPF; 400×). Keratin expression in the prostate cancer cell lines LNCaP, DU145, and PC3 was analysed by immunofluorescence with triple staining and confocal laser scanning microscopy. Prostate cancer metastases were consistently positive for K18 and negative for K14, irrespective of hormonal therapy. K5 expression was displayed in 28.9% of the tumours without treatment, in 75% after androgen deprivation, and in 57.1% of hormone‐escaped prostate carcinomas. After androgen deprivation, the number of K5‐expressing cells increased significantly. While androgen‐dependent prostate cancer showed a median of 0 cells/20 HPF (range 0–50), regressed tumours displayed 22.5 (range 0–65) and hormone‐escaped tumours 7.5 (range 0–361) positive cells/20 HPF. Expression of ChA was observed in 47.4% of the androgen‐dependent tumours. The number of neuroendocrine cells was not significantly affected in regressed or hormone‐escaped disease. The androgen‐dependent cell line LNCaP stained for K18, while the androgen‐independent lines DU145 and PC3 both expressed K5 and 18. Expression of K5 in the absence of K14 identifies the existence of an intermediate cell population in prostate carcinoma. Accumulation of intermediate cells in regressed and hormone‐escaped prostate cancer indicates that for their survival, these cells are androgen‐independent. Copyright

hTERT-Immortalized Prostate Epithelial and Stromal-Derived Cells: an Authentic In vitro Model for Differentiation and Carcinogenesis

Prostate cancer is the most commonly diagnosed type of cancer in men, and there is no available cure for patients with advanced disease. In vitro model systems are urgently required to permit the study of human prostate cell differentiation and malignant transformation. Unfortunately, human prostate cells are particularly difficult to convert into continuously growing cultures. We report here the successful immortalization without viral oncogenes of prostate epithelial cells and, for the first time, prostate stromal cells. These cells exhibit a significant pattern of authentic prostate-specific features. In particular, the epithelial cell culture is able to differentiate into glandular buds that closely resemble the structures formed by primary prostate epithelial cells. The stromal cells have typical characteristics of prostate smooth muscle cells. These immortalized cultures may serve as a unique experimental platform to permit several research directions, including the study of cell-cell interactions in an authentic prostate microenvironment, prostate cell differentiation, and most significantly, the complex multistep process leading to prostate cell transformation.

Quantitative measurement of telomerase reverse transcriptase (hTERT) mRNA in urothelial cell carcinomas

Telomerase reverse transcriptase (hTERT) messenger RNA has been detected in 95% of bladder tumors using RT‐PCR. In this study, we quantified the expression of hTERT in 35 bladder urothelial cell carcinomas and in 6 normal bladder epithelia using a real‐time quantitative PCR assay. hTERT expression was detected in all 35 urothelial cell carcinomas of varying grade and stage, but not in normal tissue samples. An increase in both pathological grade and clinical stage as prognostic parameters correlated with increased hTERT expression. Using different cutoff values for grades and stages, normalized hTERT expression values could discriminate among low, medium, and high grade tumors and between superficial and muscle‐invasive tumors. We conclude that standardized real‐time measurement of hTERT expression can be used for early tumor detection and may be used for determination of prognosis in urothelial cell carcinomas of the bladder. Int. J. Cancer 87:217–220, 2000.

RAPID MICROWAVE-STIMULATED FIXATION OF ENTIRE PROSTATECTOMY SPECIMENS

Conventional fixation of large solid surgical specimens is a slow process. Consequently, autolytic damage to tissues may occur if the fixative does not reach the central part of the specimen in time. However, as there is also a time relationship between formalin fixation and antigen masking, fixation for too long can also be detrimental. In seeking the optimum balance for fixation, microwave irradiation might be of assistance. This study set out to evaluate methods for fixing entire prostate glands within a brief period of time, using microwave‐stimulated formalin fixation. The results show that entire prostates can be optimally fixed if formalin is present throughout the tissue as the temperature is increased by microwave irradiation. This is achieved by injecting the fixative into the prostate at multiple sites immediately following prostatectomy. The technique described ensures standardization of a critical step during tissue processing, leading to uniform microscopic results with both routine and immunohistochemical stains. It is a simple, rapid method, suitable for routine diagnostic use. Using this modified approach, DNA of much larger sizes can be extracted from paraffin‐embedded material, which could expand the possibilities for molecular analysis.

Rational basis for the combination of PCA3 and TMPRSS2:ERG gene fusion for prostate cancer diagnosis

The prostate cancer gene 3 (PCA3) and TMPRSS2:ERG gene fusion are promising prostate cancer (PCa) specific biomarkers. Our aim was to simultaneously quantify the expression levels of PCA3 and TMPRSS2:ERG in a panel of benign prostatic hyperplasia (BPH), normal prostate adjacent to PCa (NP) and PCa tissue samples, to provide a rational basis for the understanding of the false‐positive and false‐negative results of the urine assays.

Biomarkers for the diagnosis of prostatic inflammation in benign prostatic hyperplasia

Chronic prostatic inflammation could be a central mechanism in benign prostatic hyperplasia (BPH) progression. Currently, the histological examination of prostate biopsies remains the only way to diagnose prostatic inflammation. Our objective was to find new noninvasive biomarkers for the diagnosis of prostatic inflammation.

Components of the plasminogen activator system and their complexes in renal cell and bladder cancer: comparison between normal and matched cancerous tissues

To analyse and compare the concentration of plasminogen activator (PA), urokinase‐type PA (uPA), tissue‐type PA (tPA), PA inhibitor (PAI)‐1 and PAI‐2, and the complexes uPA‐PAI‐1 and tPA‐PAI‐1 and calculated uPA and tPA uncomplexed with PAI‐1 (‘free’) in urothelial cell carcinoma and matched benign urothelium, and in renal cell carcinoma (RCC) and matched benign renal tissue.

DC-SCRIPT: AR and VDR regulator lost upon transformation of prostate epithelial cells

Nuclear receptors (NR), including the Androgen Receptor (AR) and the Vitamin D Receptor (VDR), play an important role in prostate cancer etiology. We recently found that DC‐SCRIPT is a prognostic marker in breast cancer and a unique NR coregulator differentially regulating different classes of NRs. Here we investigated the importance of DC‐SCRIPT in prostate cancer.

Identification of androgen-responsive genes that are alternatively regulated in androgen-dependent and androgen-independent rat prostate tumors.

The vast majority of androgen‐dependent prostate tumors progress toward incurable, androgen‐independent tumors. The identification of androgen‐responsive genes, which are still actively transcribed in the tumors of patients who have undergone androgen ablation, may shed light on the molecular mechanisms underlying this phenomenon. To address this question, we chose the Dunning R3327 rat model system, in which the progression from androgen‐dependent to ‐independent tumors is represented by several transplantable prostate‐derived tumors. Gene expression profiles were analyzed in normal rat prostates and in the prostates of rats 14 days after castration by use of microarrays containing approximately 5,000 oligonucleotides, together representing more than 4,800 known rat genes. These expression profiles were compared with similarly obtained expression profiles of androgen‐dependent and androgen‐independent rat prostate tumors. By doing so, a series of known and novel prostate cancer–associated androgen‐responsive genes was identified. Within this series, we were able to identify several clusters of genes that are differentially regulated in the various prostate tumors. These genes may serve as (i) novel prognostic identifiers and (ii) novel therapeutic targets.