Increases in NMR-visible lipid and glycerophosphocholine during phenylbutyrate-induced apoptosis in human prostate cancer cells

DU145 human prostatic carcinoma cells were treated with the differentiating agents phenylacetate (PA) and phenylbutyrate (PB) and examined in perfused cultures by diffusion-weighted 1H and 31P nuclear magnetic resonance spectroscopy (NMR). PA and PB (10 mM) induced significant (>3-fold) time-dependent increases in the level of NMR-visible lipids and total choline in 1H spectra, and glycerophosphocholine levels in the 31P spectra, with the increases being greater for PB. These effects were accompanied by significant increases in cytoplasmic lipid droplets and intracellular lipid volume fraction as observed by morphometric analysis of Oil Red O-stained cells. PB treatment caused cell cycle arrest in the G1 phase and induction of apoptosis. In contrast, PA-treated DU145 cells showed an accumulation of cells in G2/M and no evidence of apoptosis. These results demonstrate that significant differences exist in the mechanism of PA and PB activity, although both compounds cause similar, but graded alterations in lipid metabolism. The simultaneous accumulation of mobile lipid and glycerophosphocholine suggests that PB and PA induce phospholipid catabolism via a phospholipase-mediated pathway. The mobile lipid accumulation following the induction of either apoptosis and cytostasis by related differentiating agents indicate that the presence of NMR-visible lipids may not be a specific event causally resulting from the induction of apoptosis.     

Interaction of p14ARF with Brca1 in cancer cell lines and primary breast cancer

We report an association between p14ARF and Brca1 in which both proteins co-immunoprecipitate (co-IP) in DU145 cells. The N-terminal 64 residues of p14ARF encoded by exon 1beta are sufficient for this association. Inside the cell, ectopic p14ARF co-localizes with ectopic and endogenous Brca1 in A375 cells. Endogenous p14ARF co-localizes with endogenous Brca1 in DU145 cells but not in H1299 cells. Since p14ARF interacts with B23 in the nucleolus, Brca1 co-localizes with B23 in DU145 but not in H1299 cells. While ectopic ARF potently inhibited DU145 cell proliferation, it had no effect on the proliferation of H1299 cells, suggesting that the interaction between ARF and Brca1 contributes to ARF-mediated tumor suppression. Consistent with this notion, ectopic p14ARF modulates endogenous Brca1 expression in MCF7 breast cancer cells and p14ARF co-localizes with Brca1 in normal breast epithelial cells. This co-localization is enhanced in primary breast cancer. Taken together, the results show that p14ARF associates with Brca1, which may play a major role in tumor suppression.     

Enhanced radiosensitivity of androgen-resistant prostate cancer: AZD1152-mediated Aurora kinase B inhibition

Aurora kinase B (AURKB) is critical to the process of mitosis, aiding in chromosome condensation by phosphorylating histone H3. We investigated the effects of AZD1152, an AURKB inhibitor, on radiosensitivity of androgen-insensitive prostate cancer cells. The goal of this study was to test whether AZD1152 increases the susceptibility of hormone-refractory prostate cancer cells to radiation-induced DNA damage and to determine the conditions of AZD1152 treatment that maximize radiosensitization. PC3 and DU145 cells were treated with various AZD1152 doses for various durations to elucidate the conditions that yielded maximal increases in G(2)/M-phase and polyploid cells. To assess DNA damage, γ-H2AX phosphorylation was quantified for cells grown under radiosensitizing conditions and subjected to either no radiation or 5 Gy radiation. Radiosensitivity was determined by clonogenic assays. Cell cycle effects in both cell lines were maximized by treatment with 60 nM AZD1152 for 48 h. AZD1152-treated cells exhibited significantly increased DNA damage 30 min postirradiation (PC3: 100% compared to 68%, P = 0.035; DU145: 100% compared to 69%, P = 0.034), with additional DNA damage 6 h postirradiation (PC3: 85% compared to 15%, P = 0.002; DU145: 67% compared to 21%, P = 0.012). Radiosensitivity was increased in both cell lines, with dose enhancement ratios of 1.53 for PC3 cells (P = 0.017) and 1.71 for DU145 cells (P = 0.02). This study identifies the optimal AZD1152 treatment conditions to maximize the radiosensitization of PC3 and DU145 cells. These results suggest a major role for DNA damage and impairment of DNA repair mechanisms in AZD1152-induced radiosensitization of prostate cancer cells.     

The human cyclin B1 protein modulates sensitivity of DNA mismatch repair deficient prostate cancer cell lines to alkylating agents

DNA damage caused by alkylating agents results in a G2 checkpoint arrest. DNA mismatch repair (MMR) deficient cells are resistant to killing by alkylating agents and are unable to arrest the cell cycle in G2 phase after alkylation damage. We investigated the response of two MMR-deficient prostate cancer cell lines DU145 and LNCaP to the alkylating agent MNNG. Our studies reveal that DU145 cancer cells are more sensitive to killing by MNNG than LNCaP. Investigation of the underlying reasons for lower resistance revealed that the DU145 cells contain low endogenous levels of cyclin B1. We provide direct evidence that the endogenous level of cyclin B1 modulates the sensitivity of MMR-deficient prostate cancer cells to alkylating agents.     

Index

Anticancer role of oxindole compounds is well documented. Here, we synthesized new derivatives of 3-hydroxy-2-oxindole functionalized at position 3 (1a–f) which are expected to have antiproliferative activity in cancer cells. Human prostate cancer cell line (DU145) was treated with the synthesized derivatives at 40-μM concentration for 24, 48, and 72 h. Compounds 1-ethyl-3-hydroxy-1,1′,3,3′-tetrahydro-2H,2′H-3,3′-biindole-2,2′-dione (1d), 5-bromo-1-ethyl-3-hydroxy-1,1′,3,3′-2H,2′H-3,3′-biindole-2,2′-dione (1e), and 5-chloro-1-ethyl-3-hydroxy-1,1′,3,3′-tetrahydro-2H,2′H-3,3′-biindole-2,2′-dione (1f) were found to significantly reduce DU145 cell viability at 48 and 72 h whereas no significant changes were observed up to 24 h. The compounds 1e and 1f showed the most cytotoxicity effect and had a similar antiproliferative activity on DU145 cell line. They have halogen and ethyl substitutions at positions 5 and 1, respectively. The IC₅₀ of compound 1e for DU145 and A375 cells at 48 h was determined. The apoptotic effects and cell cycle progression of compound 1e at 1/2 × IC₅₀ (55 μM) concentration in DU145 cells were investigated by nuclei staining, comet assay, flow cytometry, and scanning electron microscopy (SEM). The results obtained showed that this compound increased the percentage of tail DNA, increased the occurrence of the sub-G1 phase, and induced G2M arrest and apoptosis in DU145 cells after exposure for 48 h to a 55-μM concentration. The SEM images revealed cell contraction at 24 h, cell condensation, plasma membrane blebbing, and formation of apoptotic bodies at 48 and 72 h. These observations suggest that the antiproliferative activity of compound 1e may be to induce apoptosis in DU145 cells.     

Detergent-resistant membrane fractions contribute to the total 1H NMR-visible lipid signal in cells.

Leukocytes and other cells show an enhanced intensity of mobile lipid in their 1H NMR spectra under a variety of conditions. Such conditions include stimulation, which has recently been shown to involve detergent-resistant, plasma membrane domains (DRMs) often called lipid rafts. As there is much speculation surrounding the origin of cellular NMR-visible lipid, we analysed subcellular fractions, including DRMs, by NMR spectroscopy. We demonstrated that DRMs isolated by density gradient centrifugation from lymphoid (CEM-T4, stimulated Jurkat cells), and monocytoid (THP-1) cells produced NMR-visible, lipid signals. Large scale subfractionation of THP-1 cells determined that while cytoplasmic lipid droplets constituted much of the total NMR-visible lipid, the contribution of DRMs was significant. Qualitative and quantitative lipid analyses revealed that DRMs and lipid droplets differed in their lipid composition. DRMs were enriched in cholesterol and ganglioside GM1, and contained relatively unsaturated fatty acids compared with the lipid droplets. Both lipid droplets and DRMs contained neutral lipids (triacylgycerols, cholesterol ester, fatty acids in THP-1 cells) that could, in addition to phospholipids, contribute to the NMR-visible lipid. The lipid droplets also exhibited different protein profiles and contained 500-fold less protein than DRMs, confirming that DRMs and droplets were fractionated as separate entities. The NMR-visible lipid in DRMs is therefore unlikely to be a contaminant from lipid droplets. We propose a micropartitioning of the NMR-visible mobile lipid of whole cells between intracellular lipid droplets, where most of this lipid resides, and detergent-resistant plasma membrane domains.     

Inhibition of tetraphenylphosphonium-induced NMR-visible lipid accumulation in human breast cells by chlorpromazine

The effects of chlorpromazine (CPZ) on the lipid accumulation induced by the cationic lipophilic compound tetraphenylphosphonium chloride (TPP) were examined using proton nuclear magnetic resonance spectroscopy (NMR), lipid extraction and thin layer chromatography (TLC), and electron microscopy (EM). Chlorpromazine at concentrations of 12 or 25 microM significantly reduced the NMR-visible lipid accumulation induced by a 48-h treatment with 6.25 microM TPP in the human breast cell line, HBL-100, without affecting cell viability. TPP caused threefold increases in whole-cell triglyceride levels that were attenuated by the addition of CPZ. Electron micrographs of TPP-treated HBL-100 cells showed that the destruction of mitochondria was accompanied by the accumulation of lipid droplets and myelinoid bodies. The addition of CPZ to TPP-treated cells reduced the occurrence of lipid droplets but not of mitochondrial destruction. Treatment with CPZ, in the presence or absence of TPP, resulted in large cytoplasmic inclusions indicating the inhibition of lysosomal metabolism. The induction and attenuation of NMR-visible lipids in conjunction with concomitant changes in both intracellular lipid droplets and whole-cell triglyceride levels provides evidence that NMR-visible lipids arise from cytoplasmic neutral lipid droplets. The observation that CPZ, a known inhibitor of lysosomal and cytosolic lipid metabolism, attenuates the formation of neutral triglycerides indicates that lysosomal processing may be a major step in the accumulation of NMR-visible lipid in breast cell lines.     

Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells

We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well.     

Comparative 31P and 1H NMR studies on rat astrocytes and C6 glioma cells in culture.

Rat astroglial cells in primary culture (95% enrichment) and C6 glioma cells were adapted to grow on microcarrier beads. In vivo 31P NMR spectra were collected from cell-covered beads perfused in the NMR tube. The NMR-visible phosphorylated metabolite contents of both cell types were determined using saturation factors calculated from the values of longitudinal relaxation times determined for C6 cells using progressive saturation experiments. On the other hand, the amounts of phosphorylated metabolites in cells were determined from proton decoupled 31P NMR spectra of cell perchloric acid extracts. The results indicate that the NTP and Pi contents of the normal and tumoral cells were similar, whereas the PCr level was higher in C6 cells and the NDP and phosphomonoester levels higher in astrocytes. The comparison of 1H NMR spectra of cell perchloric acid extracts evidenced larger inositol and alanine contents in C6 cells, whereas larger taurine and choline (and choline derivatives) contents were found in astrocytes. The Glu/Gln ratio was very different, 3.5 and 1 in C6 cells and astrocytes, respectively. In both cases, the more intense resonance in the 1H NMR spectrum was assigned to glycine. Based on the comparison of the metabolite content of a tumoral and a normal cell of glial origin, this work emphasizes the usefulness of a multinuclear NMR study in characterizing intrinsic differences between normal and tumoral cells.     

High-resolution proton NMR measures mobile lipids associated with Triton-resistant membrane domains in haematopoietic K562 cells lacking or expressing caveolin-1

High-resolution proton NMR spectra of intact tumour cells generally exhibit intense signals due to isotropically mobile lipids (MLs) of still uncertain nature and origin. NMR studies performed on intact wild-type and caveolin-1-infected haematopoietic K562 cells showed that, under our experimental conditions, part of the ML signals are due to lipid complexes resistant to extraction in Triton X-100 at 4 degrees C. This evidence suggests that a portion of NMR-visible lipid structures are compatible with Triton-resistant membrane rafts and therefore biophysically distinct from NMR-visible Triton-soluble lipid bodies. Similarly to lipid rafts and caveolae, the organization of the Triton-insoluble ML domains could be compromised by treatment with beta-octylglucoside or methyl-beta-cyclodextrin. Exposure to exogenous sphingomyelinase caused an increase in ML NMR visibility, indicating the possible involvement of ceramides in ML formation. The mobility of these lipids was found to be temperature sensitive, suggesting a transition in cells going from 4 degrees C to 25-37 degrees C. These new results are here discussed in the light of possible contributions of plasma membrane microdomains to NMR-visible ML signals.     

Phenylbutyrate-induced apoptosis and differential expression of Bcl-2, Bax, p53 and Fas in human prostate cancer cell lines

OBJECTIVE: To assess the mechanisms of action of phenylbutyrate (PB), an investigational chemotherapeutic agent for prostate cancer (PCa), in apoptosis induction in PCa cell lines in vitro. STUDY DESIGN: We analyzed the differential expression of different apoptosis modulators, Bcl-2, Bax, p53 and Fas, for their potential role in PB-induced apoptosis using relative quantitative flow cytometry (FCM). Both androgen-dependent (LNCaP) and androgen-independent (C-4-2, PC-3-PF and DU145) human PCa cell lines were examined. RESULTS: PB induced apoptosis in PCa cell lines in a dose-dependent manner. Fifty percent apoptosis could be induced by 5-10 mM PB. Bcl-2 was down-regulated 30-75% and the Bax:Bcl-2 ratio elevated in apoptotic PCa cell lines regardless of their androgen dependency or p53 status. FCM revealed a heterogeneous stimulatory effect on the expression of Bax and Bcl-2 in PC3-PF cells at 0.5-2.5 mM PB. In a p53-positive cell line (DU145), p53 was repressed by 70% and Fas elevated sixfold with 10 mM PB. CONCLUSION: Our data show that PB-induced PCa apoptosis is associated with the relative repression of Bcl-2 and with up-regulation of Bax and Fas proteins and that this PB-induced apoptosis is independent of p53 and androgen-dependency status of PCa cell lines.     

Lovastatin reversed the enhanced sphingomyelin caused by 27-hydroxycholesterol in cultured vascular endothelial cells

Statins have pleiotropic properties which are involved in inhibiting the thrombogenic response. In this study, the effects of lovastatin on two phospholipids, phosphatidylcholine and sphingomyelin, were studied in cultured endothelial cells in the presence of an oxysterol, 27-hydroxycholesterol. After the cells were cultured with 50 nM of lovastatin for 60 h, lovastatin was found to decrease the incorporation of [³H]choline into phosphatidylcholine and sphingomyelin, inhibited CTP: phosphocholine cytidylyltransferase (CT) activity without altering the activity of sphingomyelin synthase and neutral sphingomyelinase. And lovastatin was not found to have a direct inhibitive effect on activity of CT. Exogenous mevalonic acid or cholesterol reversed the reduction of cholesterol concentration that was caused by lovastatin, but had no significant effect on the diminished [³H]sphingomyelin by lovastatin. The increase of [³H]sphingomyelin by 27-hydroxycholesterol was not detected in the presence of lovastatin. These findings suggest that (1) lovastatin can reduce sphingomyelin content by means of inhibiting phosphatidylcholine synthesis; and (2) The decrease in sphingomyelin is not related to the diminished cholesterol concentration or mevalonate-derived intermediates. This inhibitive effect of lovastatin on sphingomyelin may benefit cellular calcification caused by sphingomyelin.     

Anti-proliferative effects of evodiamine on human prostate cancer cell lines DU145 and PC3

Prostate carcinoma is one of the most common malignant tumors and has become a more common cancer in men. Previous studies demonstrated that evodiamine (EVO) exhibited anti-tumor activities on several cancers, but its effects on androgen-independent prostate cancer are unclear. In the present study, the action mechanisms of EVO on the growth of androgen-independent prostate cancer cells (DU145 and PC3 cells) were explored. EVO dramatically inhibited the growth and elevated cytotoxicity of DU145 and PC3 cells. The flow cytometric analysis of EVO-treated cells indicated a block of G2/M phase and an elevated level of DNA fragmentation. The G2/M arrest was accompanied by elevated Cdc2 kinase activity, an increase in expression of cyclin B1 and phosphorylated Cdc2 (Thr 161), and a decrease in expression of phosphorylated Cdc2 (Tyr 15), Myt-1, and interphase Cdc25C. TUNEL examination showed that EVO-induced apoptosis was observed at 72 h. EVO elevated the activities of caspase 3, 8, and 9 in DU145 cells, while in PC3 cells only the activities of caspase 3 and 9 were elevated. EVO also triggered the processing of caspase 3 and 9 in both DU145 and PC3 cells. We demonstrate that roscovitine treatment result in the reversion of G2/M arrest in response to EVO in both DU145 and PC3. However, inhibitory effect of roscovitine on EVO-induced apoptosis could only be observed in DU145 rather than PC3. In DU145, G2/M arrest might be a signal for initiation of EVO-triggered apoptosis. Whereas EVO-triggered PC3 apoptosis might be independent of G2/M arrest. These results suggested that EVO inhibited the growth of prostate cancer cell lines, DU145 and PC3, through an accumulation at G2/M phase and an induction of apoptosis.     

西达本胺对人前列腺癌DU145及PC3细胞凋亡的影响

目的:研究西达本胺对前列腺癌DU145、PC3细胞生长抑制及凋亡调控的作用。方法:设置西达本胺浓度分别为4、8、16、32和64μmol/L的5个处理组和对照组(未加西达本胺),分别处理DU145、PC3细胞不同时间,采用MTT法检测细胞的增殖抑制情况,用倒置显微镜观察细胞形态,用流式细胞仪进行细胞凋亡分析,用免疫印迹法检测细胞内Bax、Bcl-2、caspase-3、caspase-9的表达水平。结果:与对照组比较,西达本胺处理组可使细胞明显变圆、体积缩小、脱壁细胞增多;MTT法检测显示,随着西达本胺浓度的增加和作用时间的延长,对DU145、PC3细胞的增殖抑制作用增强,并呈时间、剂量正相关(P<0.01);流式细胞术显示,对照组和16、64μmol/L西达本胺组细胞凋亡率分别为42.24%、50.23%;随着西达本胺浓度的增加,Bcl-2的表达呈下调趋势,Bax、caspase-3、caspase-9的表达呈上调趋势,呈剂量正相关。结论:西达本胺对前列腺癌DU145、PC3细胞生长具有明显的抑制作用,作用机制可能与Bax、Bcl-2、caspase-3及caspase-9凋亡信号通路有关。     

Parathyroid Hormone Related-Protein Promotes Epithelial-to-Mesenchymal Transition in Prostate Cancer

Parathyroid hormone-related protein (PTHrP) possesses a variety of physiological and developmental functions and is also known to facilitate the progression of many common cancers, notably their skeletal invasion, primarily by increasing bone resorption. The purpose of this study was to determine whether PTHrP could promote epithelial-to-mesenchymal transition (EMT), a process implicated in cancer stem cells that is critically involved in cancer invasion and metastasis. EMT was observed in DU 145 prostate cancer cells stably overexpressing either the 1-141 or 1-173 isoform of PTHrP, where there was upregulation of Snail and vimentin and downregulation of E-cadherin relative to parental DU 145. By contrast, the opposite effect was observed in PC-3 prostate cancer cells where high levels of PTHrP were knocked-down via lentiviral siRNA transduction. Increased tumor progression was observed in PTHrP-overexpressing DU 145 cells while decreased progression was observed in PTHrP-knockdown PC-3 cells. PTHrP-overexpressing DU 145 formed larger tumors when implanted orthoptopically into nude mice and in one case resulted in spinal metastasis, an effect not observed among mice injected with parental DU 145 cells. PTHrP-overexpressing DU 145 cells also caused significant bone destruction when injected into the tibiae of nude mice, while parental DU 145 cells caused little to no destruction of bone. Together, these results suggest that PTHrP may work through EMT to promote an aggressive and metastatic phenotype in prostate cancer, a pathway of importance in cancer stem cells. Thus, continued efforts to elucidate the pathways involved in PTHrP-induced EMT as well as to develop ways to specifically target PTHrP signaling may lead to more effective therapies for prostate cancer.     

Multinuclear NMR spectroscopy of the cellular slime mold Polysphondylium pallidum. Monitoring of the encystment and excystment processes.

Polysphondylium pallidum microcysts and amoebae have been investigated by 31P- and natural-abundance proton-decoupled 13C-NMR spectroscopy. Microcysts have been found to contain as prominent metabolites a phosphomonoester, inositol hexakisphosphate (1.4 mM), two phosphodiesters [glycerophosphocholine (5.5 mM) and glycerophosphoethanolamine (2.6 mM)], as well as nucleoside triphosphates (3 mM) and polyphosphates (greater than 10 mM), the polyamines 1,3-diamino-propane (3.5 mM), putrescine (16 mM) and spermidine (3 mM) and the sugar trehalose (31 mM). In vivo 31P-NMR has shown that the level of nucleoside triphosphates in microcysts was maintained metabolically and that the pH of their cytosol, deduced from the chemical shift of cytosolic Pi was 7.2. The absence of trehalose, glycerophosphocholine and glycerophosphoethanolamine in P. pallidum amoebae was the most remarkable difference from microcysts. Microcyst germination (excystment), induced by reduction of the ionic strength of the microcyst bathing medium, was monitored non-invasively by 31P- and 13C-NMR spectroscopy. The major modifications observed during excystment were the progressive disappearance of trehalose used as energy source, of glycerophosphocholine and glycerophosphoethanolamine used as membrane phospholipid precursors, and, finally, the appearance of NMR-visible polyphosphates and of cellobiose. As a mirror situation, P. pallidum amoebae responded to a high-ionic-strength stress by production of trehalose, glycerophosphocholine, and glycerophosphoethanolamine, and induction of an encystment process.     

Human osteocalcin: a strong promoter for nitric oxide synthase gene therapy, with specificity for hormone refractory prostate cancer

BACKGROUND: Gene therapy has been identified as a promising treatment strategy for hormone refractory prostate cancer (HRPC). We report, for the first time, the use of the human osteocalcin (hOC) promoter to control inducible nitric oxide synthase (iNOS) transgene expression in HRPC. METHODS: Human prostate carcinoma cells (PC3, DU145, LNCaP), colon cancer cells (HT29) and human microvascular endothelial cells (HMEC-1) were transfected in vitro with constitutively driven CMV/iNOS or hOC/iNOS plasmid DNA by cationic lipid vector. End points of these experiments were Western blotting, NO(.) generation using the Greiss test to measure accumulated nitrite, and clonogenic assay. RESULTS: Transfection of the hOC/iNOS plasmid increased iNOS protein and total nitrite levels in PC3 and DU145 cells, but not LNCaP or HT29. Transfection with CMV/iNOS or hOC/iNOS resulted in no additional cytotoxicity in androgen-dependent LNCaP cells or in the non-prostate cell lines. However, transfection with either construct resulted in a greatly reduced cell survival (to 10-20%) in the androgen-independent PC3 and DU145 cell lines. CONCLUSIONS: Utilising the tumour-type specific properties of the hOC promoter in tandem with the iNOS gene, we have demonstrated target cell specificity, and transgene activation, in the androgen-independent prostate cancer cell lines (PC3 and DU145), an effect absent in normal and androgen-dependent cells. Furthermore, the levels of NO(.) generated are comparable with those seen generated with constitutively (CMV)-driven iNOS. The data obtained from this study provide a basis for future development of hOC/iNOS gene therapy.