Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is overexpressed in prostate cancer, but the mechanism by which MIF exerts effects on tumor cells remains undetermined. MIF interacts with its identified membrane receptor, CD74, in association with CD44, resulting in ERK 1/2 activation. Therefore, we hypothesized that increased expression or surface localization of CD74 and MIF overexpression by prostate cancer cells regulated tumor cell viability. Prostate cancer cell lines (LNCaP and DU-145) had increased MIF gene expression and protein levels compared with normal human prostate or benign prostate epithelial cells (p < 0.01). Although MIF, CD74, and CD44 variant 9 expression were increased in both androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells, cell surface of CD74 was only detected in androgen-independent (DU-145) prostate cancer cells. Therefore, treatments aimed at blocking CD74 and/or MIF (e.g., inhibition of MIF or CD74 expression by RNA interference or treatment with anti-MIF- or anti-CD74- neutralizing Abs or MIF-specific inhibitor, ISO-1) were only effective in androgen-independent prostate cancer cells (DU-145), resulting in decreased cell proliferation, MIF protein secretion, and invasion. In DU-145 xenografts, ISO-1 significantly decreased tumor volume and tumor angiogenesis. Our results showed greater cell surface CD74 in DU-145 prostate cancer cells that bind to MIF and, thus, mediate MIF-activated signal transduction. DU-145 prostate cancer cell growth and invasion required MIF activated signal transduction pathways that were not necessary for growth or viability of androgen-dependent prostate cells. Thus, blocking MIF either at the ligand (MIF) or receptor (CD74) may provide new, targeted specific therapies for androgen-independent prostate cancer.     

Neuroendocrine peptides stimulate adenyl cyclase in normal and malignant prostate cells

Elevations of intracellular cAMP in human prostate cancer cells have been shown to increase invasiveness and to promote neuronal differentiation. Since neuroendocrine peptides capable of activating adenyl cyclase are present in prostatic nerves and epithelial neuroendocrine cells, we investigated normal and malignant human prostate cells for changes in intracellular cAMP in response to the prostatic peptides vasoactive intestinal peptide (VIP), calcitonin (CT), and calcitonin gene-related peptide (CGRP). Normal prostate epithelial cells and LNCaP prostate cancer cells exhibited, respectively, 6- and 30-fold increases in intracellular cAMP in response to VIP. ALVA-31 and PPC-1 prostate cancer cells demonstrated 20- to 200-fold increases in cAMP in response to CGRP, while normal epithelial cells and LNCaP cells exhibited smaller (2- to 6-fold) responses. Only DU-145 cells increased cAMP substantially in response to CT. VIP receptor mRNA was identified by Northern blot analysis only in those cells that responded to VIP. CT receptor mRNA was identified only in DU-145 cells by polymerase chain reaction and Southern blot analysis. These results suggest that VIP and possibly CGRP receptors are likely to be present in both normal and malignant prostate cells. VIP or CGRP may regulate secretion of proteases by normal or prostate cancer cells and may influence epithelial cell differentiation.     

Inhibition of cell survival signal protein kinase B/Akt by curcumin in human prostate cancer cells

Although curcumin has been shown to inhibit prostate tumor growth in animal models, its mechanism of action is not clear. To better understand the anti-cancer effects of curcumin, we investigated the effects of curcumin on cell survival factor Akt in human prostate cancer cell lines, LNCaP, PC-3, and DU-145. Our results demonstrated differential activation of Akt. Akt was constitutively activated in LNCaP and PC-3 cells. Curcumin inhibited completely Akt activation in both LNCaP and PC-3 cells. The presence of 10% serum decreased the inhibitory effect of curcumin in PC-3 cells whereas complete inhibition was observed in 0.5% serum. Very little or no activation of Akt was observed in serum starved DU-145 cells (0.5% serum). The presence of 10% serum activated Akt in DU-145 cells and was not inhibited by curcumin. Results suggest that one of the mechanisms of curcumin inhibition of prostate cancer may be via inhibition of Akt. To our knowledge this is the first report on the curcumin inhibition of Akt activation in LNCaP and PC-3 but not in DU-145 cells.     

Prostate tumor CXC-chemokine profile correlates with cell adhesion to endothelium and extracellular matrix

Though chemokines of the CXC family are thought to play key roles in neoplastic transformation and tumor invasion, information about CXC chemokines in prostate cancer is sparse. To evaluate the involvement of CXC chemokines in prostate cancer, we analyzed the CXC coding mRNA of both chemokine ligands (CXCL) and chemokine receptors (CXCR), using the prostate carcinoma cell lines PC-3, DU-145 and LNCaP. CXCR proteins were further evaluated by Western blot, CXCR surface expression by flow cytometry and confocal microscopy. The expression pattern was correlated to adherence of the tumor cells to an endothelial cell monolayer or to extracellular matrix components. Based on growth and adhesion capacity, PC-3 and DU-145 were identified to be highly aggressive tumor cells (PC-3>DU-145), whereas LNCaP belonged to the low aggressive phenotype. CXCL1, CXCL3, CXCL5 and CXCL6 mRNA, chemokines with pro-angiogenic activity, were strongly expressed in DU-145 and PC-3, but not in LNCaP. CXCR3 and CXCR4 surface level differed in the following order: LNCaP>DU-145>PC-3. The differentiation factor, fatty acid valproic acid, induced intracellular CXCR accumulation. Therefore, prostate tumor malignancy might be accompanied by enhanced synthesis of angiogenesis stimulating CXC chemokines. Further, shifting CXCR3 and CXCR4 from the cell surface to the cytoplasm might activate pro-tumoral signalling events and indicate progression from a low to a highly aggressive phenotype.     

MicroRNA-Dependent Regulation of <Emphasis Type="Italic">IGF1R</Emphasis> Gene Expression in Hormone-Sensitive and Hormone-Resistant Prostate Cancer Cells

Using multiple parallel sequencing on Illumina platform, we identified eight microRNAs that showed significant opposite changes of gene expression in cells of the hormone-sensitive LNCaP prostate cancer cell line and in cells of the hormone-resistant DU-145 cell line, in comparison to the microRNA expression in the normal prostate tissue cells. We found that the insulin-like growth factor 1 receptor (IGF1R) gene is a target of five microRNAs whose expression is increased in LNCaP cells and reduced in DU-145 cells.     

Similarities and differences between effects of angiotensin III and angiotensin II on human prostate cancer cell migration and proliferation

Proliferation plays a critical role in tumor growth when cell migration is essential to invasion. The effect of Ang III and Ang II was evaluated on these important processes. Changes in the migration potential of prostate cancer cells were investigated using Wound Healing Test and a Transwell Migration Chamber with a 3μm pore size. Cell proliferation was measured with a BrdU Assay and Countess Automated Cell Counter, thus determining the influence of angiotensins on hormone-dependent (LNCaP) and hormone-independent (DU-145) human prostate cancer lines. The influence of Ang III and Ang II on classic receptors may be inhibited by Losartan or PD123319. Test peptide modulation of the AT1 and AT2 receptors was examined by Western Blot and fluorescent immunocytochemistry. The results indicate that Ang III promotes the migration of both LNCaP and DU-145 lines, whereas Ang II stimulates this process only in androgen-independent cells. Both angiotensin peptides can induce prostate cancer cell proliferation in a time- and dose-dependent manner. The obtained results show that Ang III and Ang II can modify the expression of classic receptors, particularly AT2. These results suggest that the investigated peptide can modulate cell migration and proliferation in prostate cancer cells. Angiotensins probably have a greater influence on proliferation in the early-stage prostate cancer model than hormone-independent cell lines. Assume also that Ang II can enhance the migration tendency aggressive prostate cancer cells, while Ang III does so more effective in non-metastatic cells.     

beta-Lapachone-induced apoptosis in human prostate cancer cells: involvement of NQO1/xip3.

beta-Lapachone (beta-lap) induces apoptosis in various cancer cells, and its intracellular target has recently been elucidated in breast cancer cells. Here we show that NAD(P)H:quinone oxidoreductase (NQO1/xip3) expression in human prostate cancer cells is a key determinant for apoptosis and lethality after beta-lap exposures. beta-Lap-treated, NQO1-deficient LNCaP cells were significantly more resistant to apoptosis than NQO1-expressing DU-145 or PC-3 cells after drug exposures. Formation of an atypical 60-kDa PARP cleavage fragment in DU-145 or PC-3 cells was observed after 10 microM beta-lap treatment and correlated with apoptosis. In contrast, LNCaP cells required 25 microM beta-lap to induce similar responses. Atypical PARP cleavage in beta-lap-treated cells was not affected by 100 microM zVAD-fmk; however, coadministration of dicoumarol, a specific inhibitor of NQO1, reduced beta-lap-mediated cytotoxicity, apoptosis, and atypical PARP cleavage in NQO1-expressing cells. Dicoumarol did not affect the more beta-lap-resistant LNCaP cells. Stable transfection of LNCaP cells with NQO1 increased their sensitivity to beta-lap, enhancing apoptosis compared to parental LNCaP cells or vector-alone transfectants. Dicoumarol increased survival of beta-lap-treated NQO1-expressing LNCaP transfectants. NQO1 activity, therefore, is a key determinant of beta-lap-mediated apoptosis and cytotoxicity in prostate cancer cells.     

Metastasis-associated alterations in phospholipids and fatty acids of human prostatic adenocarcinoma cell lines.

Metastatic variants of human prostatic adenocarcinoma cell lines (DU-145, LNCaP, and ND-1) were studied by using soft agar colony forming efficiency, nude mice tumorigenicity, in vitro invasion assay, and type IV collagenase assay. The DU-145 and ND-1 cell line showed higher metastatic potential than LNCaP. Lipids from DU-145, ND-1, and LNCaP cells were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. The major lipids were phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, fatty acids, and cholesterol. The sphingomyelin level was significantly higher in highly metastatic cells (DU-145 and ND-1) compared with the lower metastatic variant (LNCaP). The increase in the synthetic pathway and decrease in degradation pathway of sphingomyelin in microsomal fractions was sufficient to account for the measured increase in sphingomyelin in DU-145 cells compared with LNCaP cells. The major fatty acids of these lipids were palmitic (16:0), stearic (18:0), oelic (18:1), and arachidonic acid (20:4). The arachidonic acid level was significantly decreased in DU-145 and ND-1 compared with LNCaP cells. Electron microscopic studies showed no significant changes in the morphology of DU-145, ND-1, and LNCaP cells. The results of these investigations demonstrate for the first time that sphingomyelin and arachidonic acid contents are different in high and low metastatic variants of human prostatic adenocarcinoma cell lines.     

Time-lapse confocal reflection microscopy of collagen fibrillogenesis and extracellular matrix assembly in vitro

The development of the next generation of biomaterials for restoration of tissues and organs (i.e., tissue engineering) requires a better understanding of the extracellular matrix (ECM) and its interaction with cells. Extracellular matrix is a macromolecular assembly of natural biopolymers including collagens, glycosaminoglycans (GAGs), proteoglycans (PGs), and glycoproteins. Interestingly, several ECM components have the ability to form three-dimensional (3D), supramolecular matrices (scaffolds) in vitro by a process of self-directed polymerization, "self-assembly". It has been shown previously that 3D matrices with distinct architectural and biological properties can be formed from either purified type I collagen or a complex mixture of interstitial ECM components derived from intestinal submucosa. Unfortunately, many of the imaging and analysis techniques available to study these matrices either are unable to provide insight into 3D preparations or demand efforts that are often prohibitory to observations of living, dynamic systems. This is the first report on the use of reflection imaging at rapid time intervals combined with laser-scanning confocal microscopy for analysis of structural properties and kinetics of collagen and ECM assembly in 3D. We compared time-lapse confocal reflection microscopy (TL-CRM) with a well-established spectrophotometric method for determining the self-assembly properties of both purified type I collagen and soluble interstitial ECM. While both TL-CRM and spectrophotometric techniques provided insight into the kinetics of the polymerization process, only TL-CRM allowed qualitative and quantitative evaluation of the structural parameters (e.g., fibril diameter) and 3D organization (e.g., fibril density) of component fibrils over time. Matrices formed from the complex mixture of soluble interstitial ECM components showed an increased rate of assembly, decreased opacity, decreased fibril diameter, and increased fibril density compared to that of purified type I collagen. These results suggested that the PG/GAG components of soluble interstitial ECM were affecting the polymerization of the component collagens. Therefore, the effects of purified and complex mixtures of PG/GAG components on the assembly properties of type I collagen and interstitial ECM were evaluated. The data confirmed that the presence of PG/GAG components altered the kinetics and the 3D fibril morphology of assembled matrices. In summary, TL-CRM was demonstrated to be a new and useful technique for analysis of the 3D assembly properties of collagen and other natural biopolymers which requires no specimen fixation and/or staining.Copyright 2000 John Wiley & Sons, Inc.     

Pro-apoptotic activity of ergosterol peroxide and (22E)-ergosta-7,22-dien-5α-hydroxy-3,6-dione in human prostate cancer cells

With the aim of identifying novel agents with antigrowth and pro-apoptotic activity on prostate cancer cells, we assayed the effect of ergosterol peroxide and (22E)-ergosta-7,22-dien-5α-hydroxy-3,6-dione, a semisynthetic compound, against androgen-sensitive (LNCaP) and androgen-insensitive (DU-145) human prostate cancer cells. Our results indicate that after 72 h of incubation, ergosterol peroxide and (22E)-ergosta-7,22-dien-5α-hydroxy-3,6-dione at micromolar concentrations exhibited an inhibitory effect on LNCaP and DU-145 cell growth (MTT assay), but the semisynthetic compound was the most active. In addition, our results indicate that apoptotic cell demise is induced in LNCaP and DU-145 cells. In fact, a significant increase of caspase-3 activity, not correlated to LDH release, marker of membrane breakdown, was observed in both cell lines treated with ergosterol peroxide and the semisynthetic compound. With respect to genomic DNA damage, determined by COMET and TUNEL assays, the results obtained show a significant increase in DNA fragmentation when compared with the untreated control.In conclusion, the results obtained in this study, demonstrating that ergosterol peroxide and (22E)-ergosta-7,22-dien-5α-hydroxy-3,6-dione attenuate the growth of prostate cells, at least in part, triggering an apoptotic process, permit to confirm the use of mushrooms as origin of compounds to be used as novel therapeutic agents for prostate cancer treatment, or as models for molecules more active and selective.     

Antiproliferative activity of methylated analogues of E- and Z-resveratrol

The stilbenoids E-resveratrol (E-3,5,4'-trihydroxystilbene, 1), E-3,5,4'-trimethoxystilbene (2), E-3,4,4'-trimethoxystilbene (3) and E-3,4'-dimethoxy-5-hydroxystilbene (4) were converted by photoisomerization to their corresponding Z-isomers 5-8. Compounds 1-8 were subjected to antiproliferative activity bioassays towards a set of four different human cancer cell lines, namely DU-145 (androgen not responsive human prostate tumor), LNCaP (androgen responsive human prostate tumor), M-14 (human melanoma) and KB (human mouth epidermoid carcinoma). The methylated analogues of 1 are more active than the natural lead in the majority of bioassays. The most active compound was Z-3,5,4'-trimethoxystilbene (6), which showed against DU-145 and LNCaP cells GI50 values close to those of the anticancer drug vinorelbine; 6 resulted more active than its E-isomer 2 towards DU-145, LNCaP and especially KB cell lines. A number of methylated Z-isomers displayed a higher activity than their E-isomers, but E-resveratrol (1) was more active than Z-resveratrol (5) towards all the tested cell lines.     

Differential effects on growth, cell cycle arrest, and induction of apoptosis by resveratrol in human prostate cancer cell lines.

Epidemiologic studies have suggested that nutrition plays an important role in carcinogenesis and that 30% of cancer morbidity and mortality can potentially be prevented with proper adjustment of diets. Resveratrol, a polyphenol present in red wines and a variety of human foods, has recently been reported to exhibit chemopreventive properties when tested in a mouse skin cancer model system. In this study, we investigated the effects of resveratrol on growth, induction of apoptosis, and modulation of prostate-specific gene expression using cultured prostate cancer cells that mimic the initial (hormone-sensitive) and advanced (hormone-refractory) stages of prostate carcinoma. Androgen-responsive LNCaP and androgen-nonresponsive DU-145, PC-3, and JCA-1 human prostate cancer cells were cultured with different concentrations of resveratrol (2. 5 x 10(-5)-10(-7) M). Cell growth, cell cycle distribution, and apoptosis were determined. Addition of 2.5 x 10(-5) M resveratrol led to a substantial decrease in growth of LNCaP and in PC-3 and DU-145 cells, but only had a modest inhibitory effect on proliferation of JCA-1 cells. Flow cytometric analysis showed resveratrol to partially disrupt G1/S transition in all three androgen-nonresponsive cell lines, but had no effect in the androgen-responsive LNCaP cells. In difference to the androgen-nonresponsive prostate cancer cells however, resveratrol causes a significant percentage of LNCaP cells to undergo apoptosis and significantly lowers both intracellular and secreted prostate-specific antigen (PSA) levels without affecting the expression of the androgen receptor (AR). These results suggest that resveratrol negatively modulates prostate cancer cell growth, by affecting mitogenesis as well as inducing apoptosis, in a prostate cell-type-specific manner. Resveratrol also regulates PSA gene expression by an AR-independent mechanism.     

The effect of chelerythrine on cell growth, apoptosis, and cell cycle in human normal and cancer cells in comparison with sanguinarine

We compared the effects of chelerythrine (CHE) and sanguinarine (SA) on human prostate cancer cell lines (LNCaP and DU-145) and primary culture of human gingival fibroblasts. CHE and SA treatment of cell lines for 24 h resulted in (1) inhibition of cell viability in a dose-dependent manner in all tested cells (as evaluated by MTT test and bromodeoxyuridine incorporation assay); (2) dose-dependent increase in DNA damage in all tested cells (as evaluated by DNA comet assay); (3) changes in apoptosis (assessed by western blot analysis and TUNEL assay); and (4) significant induction of cyclin kinase inhibitors p21^Waf1/Cip1 and p27^Kip1 in prostate cancer cells (identified by western blot analysis). Our study demonstrates that CHE had significant cytotoxic effect, independent of p53 and androgen status, on human prostate cancer cell lines. Normal gingival fibroblasts and DU-145 cells were more sensitive to the treatment with both alkaloids than were LNCaP cells. CHE and SA may be prospective natural molecules for use in the treatment of prostate cancer owing to their involvement in apoptosis and cell cycle regulation.     

A novel artificial substrate for cell culture: Effects of substrate flexibility/malleability on cell growth and morphology

Summary Gels of glyoxyl agarose (GA) are evaluated as a novel flexible substrate for cell culture with physical properties comparable to extracellular matrix (ECM) gels. We show here that cells adhere well to pure GA gels; in addition, specific interactions involving matrix receptors can be studied when individual matrix molecules are bound to the gel covalently. When cells are grown on such substrates, morphology is comparable to that observed on “natural” matrix gels (reconstituted gels of collagen type I or of Matrigel): rather than being flattened as in monolayer cultures on tissue culture plastic the cells assume a rounded morphology and tend to form tissue-like aggregates. The effects of the artificial matrix gels are discussed in the context of previous publications on cell interactions with the extracellular matrix, suggesting that in addition to specific recognition of matrix molecules the physical properties of ECM by themselves can be decisive for cell differentiation. We conclude that gels of glycoxyl agarose a) provide a useful model to mimic the physical properties of matrix gels without the presence of specific adhesion factors; b) may be useful as a general, non-specific ECM allowing cells to be cultured in vitro under conditions favorable for differentiation; and c) allow to design a variety of “synthetic” ECM models composed of a chemically defined gel matrix, which can be supplemented with covalently bound molecules to be recognized by cell surface receptors.     

Calcium signals in prostate cancer cells: specific activation by bone-matrix proteins

Cancer of the prostate commonly metastasizes to bony sites where cells acquire an aggressive, rapidly proliferating, androgen-independent phenotype. The interaction between bone and prostate, thus, becomes a key factor in disease progression. Fluctuations in intracellular ionized Ca2+ [Ca2+]i are rapid, regulated signal transduction events often associated with cell proliferation. Hence, Ca2+ signals provide a convenient measure of early events in cancer cell growth. This study developed single cell fluorescent imaging techniques to visualize Ca2+ signals in Fura-2 loaded prostatic cancer cell lines of various metastatic phenotypes. Solubilized bone fractions containing extracellular matrix and associated proteins were tested for the ability to trigger Ca2+ signals in prostate cancer cell lines. Fractions representing the complete repertoire of non-collagenous proteins present in mineralized bone were tested. Results demonstrated that two bone fractions termed D3b- and D4a-triggered Ca2+ signals in prostate cancer cells derived from bone (PC-3), but not brain (DU-145) metastases of prostate cancer. Lymph-node derived LNCaP cells also did not produce a Ca2+ signal in response to addition of soluble bone matrix. No other bone fractions produced a Ca2+ signal in PC-3 cells. It is of interest that bone fractions D3b and D4a contain a number of non-collagenous matrix proteins including osteonectin (SPARC) and osteopontin (OPN), as well as prothrombin. Moreover, antibody LM609 that recognizes the alpha v beta 3 integrin, blocks the ability of OPN to trigger a Ca2+ transient in PC-3 cells. These studies support a conclusion that bone-matrix proteins play a role in the growth and progression of metastatic prostate cancer, and that prior growth in bone may be associated with development of a bone-matrix-responsive phenotype.     

Olfactory ensheathing glia and Schwann cells: two of a kind?

Prostatic carcinoma affects 1 in 11 men and targets bone with sclerotic metastases. The study of prostate carcinoma growth in bone has been hampered by the lack of suitable animal models. We have developed an in vivo model of prostate carcinoma growth in bone by inoculating three human prostate carcinoma cell lines (PC-3, DU-145, and LNCaP) into the tibia of congenitally athymic mice. Developing tumors were analyzed by radiographic, histologic, immunohistochemical, and in situ hybridization examination. Seven of the nine PC-3 inoculated mice and all (9/9) of the DU-145 inoculated mice developed tumors in the injected limb. In contrast, inoculation with LNCaP cells failed to produce tumors (0/9). Radiologically, the tumors had a mixed sclerotic/lytic appearance with extracortical extension. All the PC-3 tumors invaded the bone marrow cavity, cortical bone, and surrounding soft tissue. The DU-145 tumors were confined to the bone marrow cavity in 7/9 animals. CK18 and Ki67 localization identified the human tumor cells and their proliferative activity, respectively. The PC-3- and DU-145-induced tibial tumors expressed alpha(1)I procollagen and osteopontin mRNA, to varying degrees. All the tumors demonstrated an up-regulation of osteoclasts at the bone/tumor interface compared with the control limbs. Thus, this is a reliable and reproducible in vivo model of prostate carcinoma growth in bone enabling the study of the interactions that occur between prostate cancer cells and bone at an important part of the metastatic cascade, namely, growth and invasion at a distant site.     

Synthesis and biological evaluation of novel cytotoxic phospholipids for prostate cancer

We describe herein synthesis, SAR, and biological evaluation of a novel series of cytotoxic serine amide phosphates (SAPs) for prostate cancer. These compounds were tested for their cytotoxicity in five human prostate cancer cell lines (DU-145, PC-3, LNCaP, PPC-1, and TSU), and in CHO and RH7777 cells (negative controls). Comparison of anticancer effects of these compounds with a standard chemotherapeutic agent 5-fluorouracil shows that they are very effective in killing prostate cancer cells with low micromolar cytotoxicity and provide us a new lead for the development of drugs for prostate cancer.     

DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: implications for the androgen receptor functions and regulation

The majority of human prostate cancer cell lines, including the two "classical" cell lines DU-145 and PC-3, are reported to be androgen receptor (AR)-negative. However, other studies have provided evidence that the DU-145 and PC-3 cell lines express AR mRNA. These contradictory observations prompted us to investigate whether DU-145 and PC-3 cell lines express the androgen receptor. Using antipeptide antibodies directed against three distinct regions of the human AR protein and an improved method to detect AR protein in immunoblotting, we report that DU-145 and PC-3 cell lines express AR protein. We found that the relative levels of the AR mRNA and protein that were detected in DU-145 and PC-3 cell lines were lower than the LNCaP, an AR-positive cell line. Moreover, the antibody directed against the non-variant region (amino acids 299-315), but not the variant N- or C-terminal region (amino acids 1-20 and 900-919, respectively) of the human AR protein, detected the expression of AR in all prostate cancer cell lines. Notably, treatment of these cell lines with dihydrotestosterone (DHT) resulted in measurable increases in the AR protein levels and considerable nuclear accumulation. Although, treatment of DU-145 and PC-3 cells with DHT did not result in stimulation of the activity of an AR-responsive reporter, knockdown of AR expression in PC-3 cells resulted in decreases in p21(CIP1) protein levels, and a measurable decrease in the activity of the p21-luc-reporter. Our observations demonstrate the expression of AR protein in DU-145 and PC-3 prostate cancer cell lines.