Comparative gene and protein expression in primary cultures of epithelial cells from benign prostatic hyperplasia and prostate cancer

Cancer Letters 227 (2005) 213–222 www.elsevier.com/locate/canlet

Comparative gene and protein expression in primary cultures of epithelial cells from benign prostatic hyperplasia and prostate cancer Amy Rose, Yue Xu, Zuxiong Chen, Zengbin Fan, Thomas A. Stamey, John E. McNeal, Mitchell Caldwell, Donna M. Peehl* Department of Urology, Stanford University School of Medicine, Stanford, CA 94305-5118, USA Received 29 December 2004; accepted 25 January 2005

Abstract Primary cultures are widely used to investigate the disease-specific biology of prostate cancer and benign prostatic hyperplasia (BPH). To identify genes differentially expressed between epithelial cells cultured from adenocarcinomas versus BPH tissues, we used probe array technology. Gene expression profiles were evaluated on Affymetrix Human Cancer G110 Array Chips containing w1900 cancer-related genes. After defined statistical analysis, genes that were over-expressed in cancer cultures were identified. Protein expression of four of the differentially expressed genes was measured in immunoblots, and the expression of two other genes was measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). While no gene or protein was consistently over-expressed in all cancer versus BPH cell cultures, cytokeratin 16 protein was highly elevated in several of the cancer cultures, suggesting that a hyperproliferative phenotype may be characteristic of prostate cancer cells. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Prostate; Benign prostatic hyperplasia; Prostate cancer; Cytokeratin 16

1. Introduction

Abbreviations: BPH, benign prostatic hyperplasia; DTT, dithiothriotol; EGF, epidermal growth factor; HSP-70, heat shock protein; K10, keratin 10; K16, keratin 16; K18, keratin 18; MIF, macrophage migration inhibitory factor; ODC, ornithine decarboxylase; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction; TBP, TATA-box binding protein; TGF-a, transforming growth factor-a; UBC, ubiquitin C. * Corresponding author. Tel.: C1 650 725 5531; fax: C1 650 723 4200. E-mail address: [email protected] (D.M. Peehl).

Adenocarcinomas of the prostate are the most common noncutaneous cancers in American men and the second leading cause of death from cancer [1]. Benign prostatic hyperplasia (BPH) is a highly prevalent disease representing the most common cause of urinary obstruction in the aging male population [2]. Clinically, BPH does not predispose to the development of prostate cancer, and the two are considered to have different etiology and cellular

0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.01.037

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origin [3]. Molecular genetic profiling studies in our institution identified genes differentially expressed between Gleason grade 4/5 prostatic adenocarcinomas and BPH tissues [4]. Using the HuGeneFL probe array representing approximately 6800 genes (Affymetrix, Inc., Santa Clara, CA), 86 genes were identified as differentially expressed between grade 4/5 cancers and BPH (P%0.0005). Cluster analysis showed distinct separation between cancer and BPH tissues with 22 genes up-regulated in cancer tissue compared to BPH. These genes may have utility as markers of grade 4/5 cancer or provide new therapeutic targets. Primary cultures of prostatic cells provide a valuable tool with which to investigate the biology of the normal prostate and changes that take place in benign and malignant diseases. Characterization and validation of these cells as representative models of prostate structure, function and pathology are needed. While molecular and cellular differences between primary cultures from adenocarcinomas versus those from BPH or normal tissues have been reported [5], there is no definitive marker to distinguish malignant from benign cells in culture. Taking advantage of array technology similar to that used by Stamey et al. to compare cancer and BPH tissues [4], we evaluated the genetic expression profiles of epithelial cells cultured from BPH and malignant tissues. Although a number of genes were found to be over-expressed in cancer versus BPH cultures by microarray analysis, additional comparative measurements of protein products by immunoblot analysis or of gene expression by realtime reverse transcription-polymerase chain reaction (RT-PCR) failed to identify a single gene or protein that was consistently elevated in all cancer cultures versus those from BPH. Cytokeratin 16 protein, however, was highly over-expressed in a number of cancer cultures, suggesting that the hyperproliferative phenotype associated with cytokeratin 16 expression is characteristic of at least some primary cultures of cancer cells compared to those from BPH.

2. Materials and methods 2.1. Cell culture

according to previously described methods [6]. None of the patients had received prior chemical, hormonal, or radiation therapy. Each cancer tissue from which cultures were established contained O90% malignant epithelial cells and was derived from the peripheral zone. Adenocarcinomas were classified according to the Gleason grading system [7]. BPH tissues were derived from the transition zone and contained no cancer. All cell cultures were grown in serum-free medium [6]. Cells were serially passaged until sufficient numbers were available for analysis (w12–15 population doublings). The nomenclature for the cancer cultures is E-CA-x (the ‘x’ denoting each culture from a separate donor), and E-BPH-x for the cultures from BPH. The cultures and the histopathology of the tissues of origin are listed in Table 1. 2.2. Preparation of cDNA Total RNA was isolated from cells at about 80% confluency, 24 h after feeding fresh medium, using RNeasy kits (Qiagen, Valencia, CA). Table 1 Primary cell cultures used in analyses Cell culture E-BPH-1 E-BPH-2b,c,d E-BPH-3b E-BPH-4b,c,d E-BPH-5a,b,c,d E-BPH-6a,b,c,d E-BPH-7a,b,c,d E-BPH-8a,c,d E-BPH-9a,c,d E-CA-1b E-CA-2b,c,d E-CA-3b E-CA-4b,c,d E-CA-5a,b,c,d E-CA-6a,b,c E-CA-7a,b,c,d,e E-CA-8a,b,c,d E-CA-9a E-CA-10a a b c

Tissues dissected from prostatectomy specimens were processed for primary culture of epithelial cells

Histology of tissue of origin

a,b,c,d,e

d e

Used in Used in Used in Used in Used in

BPH BPH BPH BPH BPH BPH BPH BPH BPH Cancer, Gleason grade 3/4 Cancer, Gleason grade 3/3 Cancer, Gleason grade 3/3 Cancer, Gleason grade 3/3 Cancer, Gleason grade 3/3 Cancer, Gleason grade 3/4 Cancer, Gleason grade 3/4 Cancer, Gleason grade 3/3 Cancer, Gleason grade 4/4 Cancer, small cell carcinoma

microarray analysis. immunoblot analysis. RT-PCR analysis of ODC. RT-PCR analysis of UBC. immunochemistry analysis.

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Enzymes and buffers used in the following reactions were purchased from GIBCO BRL (Invitrogen, Carlsbad, CA). Total RNA (0.5 mg/ml) was incubated with 1 ml of Oligo T7-dT primer at 70 8C for 10 min, quick spun and put on ice for 5 min. Four ml of 5! First Strand Buffer, 2 ml of 0.1 M dithiothriotol (DTT), 1 ml of 10 mM dNTP and l ml of RNase inhibitor mixture were added, mixed well and incubated at 37 8C for 2 min, then superscript reverse transcriptase II (1 ml) was added, mixed well and incubated at 42 8C for 1 h. The First Strand reaction mixture was cooled down on ice, and 91 ml of DEPCtreated water, 30 ml of 5! Second Strand Buffer, 3 ml of 10 mM dNTP, 1 ml of Escherichia coli DNA ligase, 4 ml of E. coli DNA polymerase I, and 1 ml of RNase H were added and mixed well, then incubated at 16 8C for 2 h. Then, 2 ml of T4 DNA polymerase were added, and the mixture was incubated at 16 8C for 10 min. Ten microlitres of 0.5 M EDTA were added to stop the reaction. The reaction mixture of double strand cDNA was added to an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), vortexed and centrifuged in a pre-centrifuged Phaselock-gel tube at full speed for 2 min. The aqueous upper phase of the tube was transferred to a fresh microtube, and 0.5 volumes of 5 M NH4Ac, 1 ml of glycogen and 2.5 volumes of absolute ethanol were added to precipitate cDNA. The cDNA pellet was obtained by centrifuging at full speed for 20 min at room temperature and washing twice with 75% ethanol. 2.3. cRNA synthesis and labeling The cDNA was added to 9 ml of DEPC-treated water, 2 ml of 10! HY reaction buffer, 2 ml of biotinlabeled NTP mixture, 2 ml of 0.1 M DTT, 2 ml of 10! RNase inhibitor mix and 1 ml of T7 RNA polymerase (all from Enzo Biochem, Inc., Farmingdale, NY). The reaction mixture was incubated at 37 8C for 4.5 h with mixing of the contents of the tube every hour during the incubation. Clean up of biotin-labeled cRNA was performed using the Qiagen RNeasy kit according to the manufacturer’s instructions. Fragmentation of the biotin-labeled cRNA was carried out in fragmentation buffer containing 40 mM Tris acetate, 100 mM potassium acetate, 30 mM magnesium acetate, pH

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8.1, at 95 8C for 2 min and immediately cooled down on ice. 2.4. Hybridization, sample quality control and data analysis Array hybridization and scanning were performed by the Protein and Nucleic Acid Core Facility at Stanford University. Each labeled cRNA sample was assessed by agarose gel eletrophoresis to confirm the integrity of the RNA, and spectrophotometry (A260/A280O1.8) was used to examine the quality of the RNA sample. If the RNA sample was intact, small aliquots of total RNA samples were prepared and hybridized to the GeneChipw Test3 Array Chip (Affymetrix, Inc., Santa Clara, CA). Scanning was performed by the same facility at Stanford University. All the samples were checked according to the quality criteria provided by the manufacture’s protocols. Detailed protocols for data analysis and documentation of the sensitivity, reproducibility, and other aspects of the quantitative microarray analysis using Affymetrix technology have been reported [8]. The remainder of each sample was applied to Affymetrix Human Cancer G110 arrays. Hybridization intensities from the GeneChips were transformed into probe values by Affymetrix Microarray Suite software, Version 4. A decision matrix was employed to determine the presence or absence of each transcript (the ‘absolute call’) in each cRNA sample. 2.5. Real-time RT-PCR Total RNA was reverse transcribed as described using Thermoscript RT (Invitrogen, Carlsbad, CA). Real-time PCR was performed with the DNA Engine Opticon 2 Continuous Fluorescence Detection System (MJ Research, San Francisco, CA). Each reaction contained 100 ng of cDNA, 0.3 mM primers and DyNAmo SYBR Green qPCR Master Mix (Finnzymes, Espoo, Finland). Each reaction was done in triplicate to minimize the experimental variation (standard deviation was calculated for each reaction). Transcript levels of TATA-box binding protein (TBP) were assayed simultaneously with ornithine decarboxylase (ODC) and ubiquitin C (UBC) as an internal control to normalize transcript levels. All primers were supplied by Qiagen and the sequences used

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were: ODC fwd 5 0 -AAAAGCTCTCCCTGTGTCA3 0 , ODC rev 5 0 -CAATCCGCAAAACCAACTTT-3 0 ; UBC fwd 5 0 -TCCAAGACAAGGAAGGCATC-3 0 , UBC rev 5 0 -TTTCCCAGCAAAGATCAACC-3 0 ; TBP fwd 5 0 -TGCTGAGAAGAGTGTGCTGGAG3 0 , TBP rev 5 0 -TCTGAATAGGCTGTGGGGTC-3 0 . 2.6. Immunoblot analyses Cells were grown to 80% confluency and fed fresh medium 24 h prior to protein isolation. Cells were suspended by incubation in trypsin/EDTA and centrifuged. The cell pellet was washed in ice-cold phosphate-buffered saline (PBS), resuspended in urea-Tris buffer (9 M urea, 75 mM Tris–HCl, pH 7.5, 0.15 M 2-mercaptoethanol) and sonicated briefly. Protein concentrations were determined by Bio-Rad assay (Bio-Rad, Hercules, CA). Typically 20 mg of protein from each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PDVF membranes (Osmonics, Westborough, MA) and blocked in PBS with 5% non-fat milk. For the macrophage migration inhibitory factor (MIF) assay, proteins were separated by 10% Bis-Tris NuPage electrophoresis with MES running buffer. Anti-species horseradish peroxidase—conjugated secondary antibodies were obtained from DAKO (Carpenteria, CA) and visual detection was performed using the enhanced chemoluminescence method (Amersham, Piscataway, NJ). The antibodies were diluted in PBS containing 0.1% Tween as follows: cytokeratin 16, 1:1000 (US Biological, Swampscott, MA); heat shock protein-70, 1:1,000 (Santa Cruz Biotechnologies, Santa Cruz, CA); MIF, 1:500 (R&D Systems, Minneapolis, MN); b-tubulin, 1:500 (Zymed, South San Francisco, CA); and pan-actin, 1:1000 (Neomarkers, Fremont, CA). All antibodies were incubated overnight at 4 8C with the blots. After exposure, films were scanned using the GS710 Calibrated Imaging Densitometer (Bio-Rad) and the density of the bands quantified using the Quantity One Program (Bio-Rad). The density of each experimental band was normalized to actin in each protein lysate. The ratio of antibody:actin in E-BPH-1 was arbitrarily set to one and relative ratios were calculated. To compare expression levels between BPH and cancer cells, unpaired t-tests were generated using the Stat View, Inc., program (Berkeley, CA).

Results were considered statistically significant at P! 0.05. 2.7. Immunochemistry E-BPH-1 and E-CA-7 cultures were inoculated at 5000 cells per 60 mm dish and fed every 3–4 days until w80% confluent. At several time points, cells were fixed with 2% paraformaldehyde and permabilized with 95% ethanol. Non-specific binding was blocked with 10% horse serum, then cells were incubated with antibodies specific for cytokeratin 18 (1:250, Biogenix, San Ramon, CA), cytokeratin 16 (1:2500, US Biological), and cytokeratin 10 (1:500, Neomarkers). Binding of the primary antibody was detected with biotinylated anti-mouse IgG, the avidin–biotin complex reagent, and the substrate diaminobenzidine as previously described [9].

3. Results 3.1. Gene expression profiles of BPH and cancerderived primary cultures Six primary cultures each of epithelial cells derived from BPH tissues and adenocarcinomas (Table 1) were serially passaged to population doublings of w12–15 (tertiary passage). At this time, cells were grown to w80% confluency and fed fresh medium 24 h prior to isolation of total RNA. cRNA was prepared and hybridized to Affymetrix Human Cancer G110 Array Chips containing w1900 cancer-related genes. Genes whose expression was evaluated as absent (‘absolute call’ of zero) in all six BPH cultures and present in all six cancer-derived cultures were determined. A total of 159 genes fit this criterion. Of these, the 20 genes with the highest numbers for ‘average difference’, a value that is directly related to the level of expression of the transcript in the cancer cultures, are listed in Table 2. 3.2. Immunoblot analyses of selected proteins After identifying genes differentially expressed between BPH and cancer cells based on the results of the microarray analysis, we selected certain genes to investigate further in additional analyses. The genes

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Table 2 Genes over-expressed in cancer compared to BPH epithelial cell cultures, in order of decreasing values for average difference Probe name

Gene target

Average difference

347_s_at 2016_s_at 1366_i_at 151_s_at 895_at 870_f_at 1315_at 1081_at 1179_at 1009_at 1840_g_at 1693_s_at

HUMRSPT Human homolog of yeast ribosomal protein S28 Human Wilm’s tumor-related protein (QM) mRNA Human ubiquitin mRNA HSTUB2 Human mRNA fragment encoding beta-tubulin Homo sapiens macrophage migration inhibitory factor (MIF) gene Human metallothionein-III gene Human mRNA for ornithine decarboxylase antizyme, ORF 1 and ORF 2 Human ornithine decarboxylase gene Heat Shock Protein, 70 Kda Human putative protein kinase C inhibitor (PKCI-1) mRNA Ras-Like Protein Tc4 HUMTIMP Human gene for tissue inhibitor of metalloproteinases; partial sequence Human wild-type p53 activated fragment-1 (WAF1) mRNA Human mRNA for proteasome subunit HC5 Ribosomal Protein S20 Human keratin type 16 gene HUM927A Human interferon-inducible protein 9–27 mRNA Homo sapiens ataxia-telangiectasia group D-associated protein mRNA Human FK506-binding protein (FKBP) mRNA Human GTPase (rhoC) mRNA

130395.6 120464.4 115839.1 97920.03 79510.47 69919.67 62613.5 46984.87 46843.58 45339.52 43031.4 41241.2

2031_s_at 1447_at 326_i_at 601_s_at 676_g_at 1898_at 880_at 1395_at

listed in Table 2 with the highest values for ‘average difference’ in the cancer cells were of most interest, since a high value indicates a high level of expression of the transcript. Among those genes, our selection was based on previous studies that suggested roles for particular genes in prostate cancer, and/or on knowledge of biological functions. Levels of protein encoded by four selected genes—cytokeratin 16 (K16), heat shock protein (HSP)-70, MIF and b-tubulin—were measured by immunoblot analysis. Cells were available from four each of the original BPH and cancer cultures used for the microarray analyses. To supplement our protein studies, we included three additional cell cultures derived from BPH, and four additional cell cultures from adenocarcinomas (Table 1). As for the microarray analyses, these cells were serially passaged, grown to w80% confluency, then fed 24 h prior to isolation of protein. The average relative levels of protein expression, normalized to actin in each culture, in BPH and cancer cells were determined (Table 3). The relative levels of expression of HSP-70, MIF and b-tubulin proteins were not statistically different between the seven BPH and eight cancer cultures. The relative mean level of expression of K16 protein was more than three times

36280.47 35785.33 34364.57 33798.45 31847.73 31539.5 31127.78 29979.65

higher in the cancer than in the BPH cultures (6.90 versus 2.26), but this difference was not statistically significant (PZ0.11). Notably, however, if overexpression is characterized as more than twice the average expression in the BPH cultures, 75% of the cancer cultures over-expressed K16 compared to 28% of the BPH cultures. 3.3. Immunocytochemical analysis of K16 expression in BPH and cancer cells The high level of K16 expression seen in immunoblot analyses in several of the cancer-derived cultures was noteworthy and we decided to examine expression at the cellular level. To determine how the expression of K16 might vary with time and/or cell density, immunostaining of E-BPH-1 and E-CA-7 cells was carried out at days 5, 7 and 14 after inoculating each culture at 5000 cells per 60 mm dish. These cell cultures were chosen because they showed a low and high level of K16 expression, respectively, in the prior immunoblot studies. In addition to K16, cells were also stained for keratin 18 (K18), a marker of differentiated prostatic secretory cells, and keratin 10 (K10), a marker of squamous differentiation.

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Table 3 Relative levels of protein expression in BPH and cancer cell cultures Cells

K16

HSP-70

MIF

b-tubulin

E-BPH-1 E-BPH-2 E-BPH-3 E-BPH-4 E-BPH-5 E-BPH-6 E-BPH-7 Mean E-CA-1 E-CA-2 E-CA-3 E-CA-4 E-CA-5 E-CA-6 E-CA-7 E-CA-8 Mean P-Value

1.0 2.84 1.46 5.3 4.66 0.28 0.25 2.26 2.57 8.55 5.76 20.96 12.95 0.92 3.46 0.05 6.90 0.11

1.0 1.72 1.76 1.72 2.64 0.35 0.28 1.35 1.34 0.31 0.83 0.38 0.44 0.75 0.61 0.95 0.70 0.07

1.0 1.44 1.07 3.11 1.19 0.46 0.8 1.30 2.45 1.07 0.97 0.4 0.04 0.72 NA 0.47 0.87 0.35

1.0 1.22 2.28 2.2 1.3 1.58 0.2 1.40 2.14 0.48 1.49 1.00 0.05 0.22 0.16 0.97 0.81 0.14

Differential expression of K16 between BPH and cancer cells became most pronounced as cell density became high at day 14 (Fig. 1). K18 was equivalently expressed between BPH and cancer cells and the majority of cells were stained in each population (not shown). K10 was not expressed until the cells became dense, and even at that time, K10 was apparent only in a small proportion of cells in each culture (not shown). In spite of the high degree of K16 staining in the cancer cells, the BPH cells also showed a substantial amount of positive staining and thus the use of K16 as a marker to distinguish normal cells from cancer cells in primary cultures would be limited. 3.4. Real-time RT-PCR validation of selected genes We could not identify antibodies against ubiquitin C (UBC) or ornithine decarboxylase (ODC) that were acceptable for use on immunoblots. Therefore, we measured RNA levels of these two genes by real-time RT-PCR in BPH and cancer cultures. Figs. 2 and 3 show relative levels of RNA expression of each of these genes, normalized to levels of TATA-binding box (TBP) RNA in each cell culture. Although relative RNA levels of both genes were higher in certain cell cultures than others, neither ODC nor

UBC proved to be consistently over-expressed in cancer compared to BPH cell cultures.

4. Discussion Using microarray analysis, we identified a number of genes that appeared to be differentially overexpressed in primary cultures of epithelial cells derived from prostatic adenocarcinomas compared to cultures from BPH. Many of these genes were of interest because they had been implicated as prostate cancer-associated markers in previous studies. We selected six of these genes (MIF, HSP-70, b-tubulin, K16, UBC and ODC) to further investigate by realtime RT-PCR and/or immunoblot analysis in an expanded set of cancer- and BPH-derived primary cultures. MIF is a proinflammatory cytokine known to activate macrophages and T cells [10]. MIF expression has been localized to the glandular epithelium of the prostate and this factor stimulates in vitro growth of prostate epithelial cells [11]. It was reported that the DU 145 prostate cancer cell line secreted about twice as much MIF protein as normal prostatic epithelial cells and contained four times

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E-CA-8

E-CA-7

E-CA-5

E-CA-4

E-CA-2

E-BPH-9

E-BPH-8

E-BPH-7

E-BPH-6

E-BPH-5

E-BPH-4

E-BPH-2

100 90 80 70 60 50 40 30 20 10 0 E-BPH-1

the amount of normal MIF mRNA with a longer halflife than the message found in normal cells [12]. We did not find, however, that MIF protein was differentially expressed between primary cultures of cancer versus BPH by immunoblot analysis. Proteomic analysis of human prostate tissues by two-dimensional gel electrophoresis and mass spectrometry demonstrated increased expression of HSP70 in malignant tissue [13]. Expression of HSP-70 is enhanced after transformation by oncogenes, and elevated levels of HSP-70 protect cells from apoptotic death induced by tumor necrosis factor-a and transforming growth factor-b [14]. HSP-70 protein, however, was not confirmed to be consistently

upregulated in primary cultures of cancer cells compared to BPH cells in our analysis. b-tubulin is one of the principal components of microtubules which are essential for spindle formation, cell shape, and cell transport. Chemotherapeutic agents such as paclitaxel that target microtubule assembly are important for the treatment of solid cancers including prostate adenocarcinoma. Evidence suggests that particular isotypes of b-tubulin may be potential markers for distinguishing BPH from malignancy [15]. Furthermore, the increased expression of specific b-tubulin isotypes may confer resistance to the anti-microtubule effects of drugs like paclitaxel [16]. However, in our immunoblot analyses, levels of b-tubulin protein were not significantly different between BPH and cancer cultures. UBC is an integral factor in the process of protein turnover and cell death [17]. In our real-time RT-PCR analyses, UBC levels were relatively consistent among all of the BPH and cancer cultures, with the exception of extremely high levels in one BPH and one cancer culture. We have no explanation for the extraordinary expression of UBC in these particular cultures. ODC is an androgen-responsive gene and is the rate limiting enzyme in the synthesis of polyamines, critical proteins in the process of cell proliferation and cell death [18]. It is also a member of the early response gene family and ODC must be activated if cells are to move from G1 to S phase in the cell cycle. Relative Expression of UBC

Fig. 1. Immunocytochemical labeling of BPH and prostate cancer cells for keratin 16 (K16). Cells were inoculated at 5000 per dish and fed until confluency was reached. Immunochemical staining revealed expression of K16 in a significant number of cells in each population, with expression seemingly present in a larger percentage of cancer compared to BPH cells.

219

Fig. 2. Real-time RT-PCR analysis of relative levels of expression of ubiquitin-C (UBC) RNA in BPH and cancer cultures. Transcript levels of TBP were assayed simultaneously and the relative ratio of UBC:TBP in each cell culture (with the ratio of UBC:TBP in E-BPH-1 set as 1.0) was plotted.

A. Rose et al. / Cancer Letters 227 (2005) 213–222

E-CA-8

E-CA-7

E-CA-6

E-CA-5

E-CA-4

E-CA-2

E-BPH-9

E-BPH-7

E-BPH-8

E-BPH-6

E-BPH-5

E-BPH-4

E-BPH-2

5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 E-BPH-1

Relative Expression of ODC

220

Fig. 3. Real-time RT-PCR analysis of relative levels of expression of ornithine decarboxylase (ODC) RNA in BPH and cancer cultures. Transcript levels of TBP were assayed simultaneously and the relative ratio of ODC:TBP in each cell culture (with the ratio of UBC:TBP in E-BPH-1 set as 1.0) was plotted.

Of particular interest are recent studies showing that ODC activity is significantly higher in prostate cancer than in benign tissue from the same patient [19]. Green tea polyphenols have been shown to reduce testosterone-related induction of ODC by a significant degree both in vitro and in vivo [20] and thus early phase chemoprevention trials are underway to determine if ODC may be a target for cancer prevention and therapy. Despite our initial finding of overexpression of the ODC gene in primary cultures of prostate cancer compared to BPH cells in microarray analyses, we were unable to confirm this by real-time RT-PCR. Instead, there was a wide range in levels of expression of the ODC gene among both types of cell cultures. Of the genes and proteins that we investigated, K16 demonstrated the highest degree of upregulation in cancer cells by immunoblot analysis, although not to a statistically significant degree. Nevertheless, the mean relative level of K16 protein was more than 3-fold higher in cancer than in BPH cultures, and several cancer cultures showed extraordinarily high levels of K16. When we evaluated K16 expression by immunocytochemistry in growing cell cultures over time, it was apparent the K16 expression is not constant but rather varies with cell density and perhaps other environmental factors. K16 expression was most pronounced in cells approaching confluency compared to low-density

cells. Subjective analysis of immunocytochemical staining suggested a modest increase in positive staining in cancer compared to BPH cells at high density, but certainly the degree of differential expression was not sufficient to advocate the use of K16 as a marker to differentiate malignant cells from BPH cells in primary culture. Traditionally, K16 has been viewed as a marker of squamous differentiation and thus not particularly relevant to prostate cancer as squamous differentiation is not commonly observed in the human prostate. New evidence, however, suggests that K16 may be more biologically relevant than suspected. Epidermal growth factor (EGF) and transforming growth factor (TGF)-a were shown to induce K16 expression in normal human epidermal keratinocytes [21]. The same group also identified an EGF response element located in the promoter of the K16 gene. Recently, another group validated the finding that EGF stimulated the expression of K16 in a spontaneously immortalized epidermal keratinocyte cell line and demonstrated that this expression occurred in both mRNA and protein in a time-dependent fashion [22]. It has long been recognized that enhanced expression of growth factors is an important event in the development and progression of prostate cancer. It is possible that the high expression of K16 that we observed in several cancer-derived primary cultures is due to elevated expression of endogenous EGF and/or TGFa. The fact that K10 was not similarly elevated in cancer cultures suggests that squamous differentiation was not the basis for increased expression of K16, and that elevated K16 is more likely associated with hyperproliferation. The fact that immunoblot or real-time RT-PCR analyses did not confirm the differential expression of genes identified in the microarrays or their protein products in primary cultures derived from cancer versus BPH may be due to several factors. Protein and RNA were isolated from different cell culture replicates or, in some cases, from different primary cultures. While we tried to keep the passage number, cell density and feeding schedule as consistent as possible, these factors are impossible to control precisely. The change in K16 expression in growing cultures over time illustrates the impact that culture conditions can have on protein expression. In addition, it is well established that

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RNA expression does not always correlate with protein expression, and gene expression identified in microarrays is not always validated in subsequent analyses of protein or even by RT-PCR analysis of gene expression. Comparing our microarray results with primary cultures to those from the previous study by members of our group with BPH and cancer tissues [4], we did not find the same set of genes to be upregulated in cultured cancer cells as found in cancer tissues. This finding is reminiscent of that from comparative microarray analysis of gene expression in LNCaP and DU 145 prostate cancer cell lines and malignant prostate tissues, which showed only a few genes to be commonly upregulated in both [23]. This disparity between tissues and cell cultures possibly reflects the fact that most changes in gene expression in malignancies are epigenetic rather than genetic, and are controlled in part by environmental conditions. Also, certain differences in gene expression between normal and malignant tissues may be based in nonepithelial cells and therefore may not be demonstrated in epithelial cell cultures. Despite our inability to identify a single gene or protein that was consistently over-expressed in primary cultures of cancer cells compared to those from BPH, there is substantial evidence to suggest that cultures derived from adenocarcinomas have distinctive properties compared to those from normal tissues or BPH. Cancer-derived primary cultures uniquely exhibit chromosomal abnormalities, differential expression of estrogen receptors, altered responses to growth regulatory factors, altered expression of metabolic enzymes, expression of avb3 integrin, elevated expression of hyaluronan, and other properties [5]. Additional studies may therefore identify a marker that will be useful for specifically selecting cancer cells from tissues for culture, or specifically distinguishing cancer from BPH or normal cells in cultured populations.

5. Conclusion Microarray analysis of primary cultures of prostatic epithelial cells from BPH and cancer identified genes that were over-expressed in cancer compared to

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BPH cultures. Additional analyses by immunoblot and real-time RT-PCR failed to confirm consistent over-expression at the protein and/or RNA levels in all cancer versus BPH cultures. However, cytokeratin 16 expression was elevated in many cancer cultures compared to those from BPH and perhaps merits further study as a marker of a hyperproliferative phenotype.

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