The immunosuppressive cytokine interleukin-4 increases the clonogenic potential of prostate stem-like cells by activation of STAT6 signalling.

Interleukin-4 plays a critical role in the regulation of immune responses and has been detected at high levels in the tumour microenvironment of cancer patients, where concentrations correlate with the grade of malignancy. In prostate cancer, interleukin-4 has been associated with activation of the androgen receptor, increased proliferation and activation of survival pathways such as Akt and NF-κB. However, its role in therapy resistance has not yet been determined. Here we investigate the influence of interleukin-4 on primary epithelial cells from prostate cancer patients. Our data demonstrate an increase in the clonogenic potential of these cells when cultured in the presence of interleukin-4. In addition, a Phospho-Kinase Array revealed that in contrast to previously published work, signal transducer and activator of transcription6 (STAT6) is the only signalling molecule activated after interleukin-4 treatment. Using the STAT6-specific inhibitor AS1517499 we could confirm the role of STAT6 in increasing colony-forming frequency. However, clonogenic recovery assays revealed that interleukin-4 does not rescue the effects of either irradiation or docetaxel treatment. We therefore propose that although the interleukin-4/STAT6 axis does not appear to be involved in therapy resistance, it does play a crucial role in the colony-forming abilities of the basal cell population in prostate cancer. IL-4 may therefore contribute to disease relapse by providing a niche that is favourable for the clonogenic growth of prostate cancer stem cells.

miR-25 Modulates Invasiveness and Dissemination of Human Prostate Cancer Cells via Regulation of αv- and α6-Integrin Expression.

Altered microRNA (miRNA; miR) expression is associated with tumor formation and progression of various solid cancers. A major challenge in miRNA expression profiling of bulk tumors is represented by the heterogeneity of the subpopulations of cells that constitute the organ, as well as the tumor tissue. Here, we analyzed the expression of miRNAs in a subpopulation of epithelial stem/progenitor-like cells in human prostate cancer [prostate cancer stem cell (PCSC)] and compared their expression profile to more differentiated cancer cells. In both cell lines and clinical prostate cancer specimens, we identified that miR-25 expression in PCSCs was low/absent and steadily increased during their differentiation into cells with a luminal epithelial phenotype. Functional studies revealed that overexpression of miR-25 in prostate cancer cell lines and selected subpopulation of highly metastatic and tumorigenic cells (ALDH(high)) strongly affected the invasive cytoskeleton, causing reduced migration in vitro and metastasis via attenuation of extravasation in vivo. Here, we show, for the first time, that miR-25 can act as a tumor suppressor in highly metastatic PCSCs by direct functional interaction with the 3'-untranslated regions of proinvasive αv- and α6-integrins. Taken together, our observations suggest that miR-25 is a key regulator of invasiveness in human prostate cancer through its direct interactions with αv- and α6-integrin expression.

Low-temperature plasma treatment induces DNA damage leading to necrotic cell death in primary prostate epithelial cells.

BACKGROUND: In recent years, the rapidly advancing field of low-temperature atmospheric pressure plasmas has shown considerable promise for future translational biomedical applications, including cancer therapy, through the generation of reactive oxygen and nitrogen species. METHOD: The cytopathic effect of low-temperature plasma was first verified in two commonly used prostate cell lines: BPH-1 and PC-3 cells. The study was then extended to analyse the effects in paired normal and tumour (Gleason grade 7) prostate epithelial cells cultured directly from patient tissue. Hydrogen peroxide (H2O2) and staurosporine were used as controls throughout. RESULTS: Low-temperature plasma (LTP) exposure resulted in high levels of DNA damage, a reduction in cell viability, and colony-forming ability. H2O2 formed in the culture medium was a likely facilitator of these effects. Necrosis and autophagy were recorded in primary cells, whereas cell lines exhibited apoptosis and necrosis. CONCLUSIONS: This study demonstrates that LTP treatment causes cytotoxic insult in primary prostate cells, leading to rapid necrotic cell death. It also highlights the need to study primary cultures in order to gain more realistic insight into patient response.

Phorbol ester stimulates ethanolamine release from the metastatic basal prostate cancer cell line PC3 but not from prostate epithelial cell lines LNCaP and P4E6.

BACKGROUND: Malignancy alters cellular complex lipid metabolism and membrane lipid composition and turnover. Here, we investigated whether tumorigenesis in cancer-derived prostate epithelial cell lines influences protein kinase C-linked turnover of ethanolamine phosphoglycerides (EtnPGs) and alters the pattern of ethanolamine (Etn) metabolites released to the medium. METHODS: Prostate epithelial cell lines P4E6, LNCaP and PC3 were models of prostate cancer (PCa). PNT2C2 and PNT1A were models of benign prostate epithelia. Cellular EtnPGs were labelled with [1-(3)H]-Etn hydrochloride. PKC was activated with phorbol ester (TPA) and inhibited with Ro31-8220 and GF109203X. D609 was used to inhibit PLD (phospholipase D). [(3)H]-labelled Etn metabolites were resolved by ion-exchange chromatography. Sodium oleate and mastoparan were tested as activators of PLD2. Phospholipase D activity was measured by a transphosphatidylation reaction. Cells were treated with ionomycin to raise intracellular Ca(2+) levels. RESULTS: Unstimulated cell lines release mainly Etn and glycerylphosphorylEtn (GPEtn) to the medium. Phorbol ester treatment over 3h increased Etn metabolite release from the metastatic PC3 cell line and the benign cell lines PNT2C2 and PNT1A but not from the tumour-derived cell lines P4E6 and LNCaP; this effect was blocked by Ro31-8220 and GF109203X as well as by D609, which inhibited PLD in a transphosphatidylation reaction. Only metastatic PC3 cells specifically upregulated Etn release in response to TPA treatment. Oleate and mastoparan increased GPEtn release from all cell lines at the expense of Etn. Ionomycin stimulated GPEtn release from benign PNT2C2 cells but not from cancer-derived cell lines P4E6 or PC3. Ethanolamine did not stimulate the proliferation of LNCaP or PC3 cell lines but decreased the uptake of choline (Cho). CONCLUSIONS: Only the metastatic basal PC3 cell line specifically increased the release of Etn on TPA treatment most probably by PKC activation of PLD1 and increased turnover of EtnPGs. The phosphatidic acid formed will maintain a cancer phenotype through the regulation of mTOR. Ethanolamine released from cells may reduce Cho uptake, regulating the membrane PtdEtn:PtdCho ratio and influencing the action of PtdEtn-binding proteins such as RKIP and the anti-apoptotic hPEBP4. The work highlights a difference between LNCaP cells used as a model of androgen-dependent early stage PCa and androgen-independent PC3 cells used to model later refractory stage disease.

DNA hypermethylation in prostate cancer is a consequence of aberrant epithelial differentiation and hyperproliferation.

Prostate cancer (CaP) is mostly composed of luminal-like differentiated cells, but contains a small subpopulation of basal cells (including stem-like cells), which can proliferate and differentiate into luminal-like cells. In cancers, CpG island hypermethylation has been associated with gene downregulation, but the causal relationship between the two phenomena is still debated. Here we clarify the origin and function of CpG island hypermethylation in CaP, in the context of a cancer cell hierarchy and epithelial differentiation, by analysis of separated basal and luminal cells from cancers. For a set of genes (including GSTP1) that are hypermethylated in CaP, gene downregulation is the result of cell differentiation and is not cancer specific. Hypermethylation is however seen in more differentiated cancer cells and is promoted by hyperproliferation. These genes are maintained as actively expressed and methylation-free in undifferentiated CaP cells, and their hypermethylation is not essential for either tumour development or expansion. We present evidence for the causes and the dynamics of CpG island hypermethylation in CaP, showing that, for a specific set of genes, promoter methylation is downstream of gene downregulation and is not a driver of gene repression, while gene repression is a result of tissue-specific differentiation.

HDAC inhibitor confers radiosensitivity to prostate stem-like cells.

BACKGROUND: Radiotherapy can be an effective treatment for prostate cancer, but radiorecurrent tumours do develop. Considering prostate cancer heterogeneity, we hypothesised that primitive stem-like cells may constitute the radiation-resistant fraction. METHODS: Primary cultures were derived from patients undergoing resection for prostate cancer or benign prostatic hyperplasia. After short-term culture, three populations of cells were sorted, reflecting the prostate epithelial hierarchy, namely stem-like cells (SCs, α2ß1integrin(hi)/CD133(+)), transit-amplifying (TA, α2ß1integrin(hi)/CD133(-)) and committed basal (CB, α2ß1integrin(lo)) cells. Radiosensitivity was measured by colony-forming efficiency (CFE) and DNA damage by comet assay and DNA damage foci quantification. Immunofluorescence and flow cytometry were used to measure heterochromatin. The HDAC (histone deacetylase) inhibitor Trichostatin A was used as a radiosensitiser. RESULTS: Stem-like cells had increased CFE post irradiation compared with the more differentiated cells (TA and CB). The SC population sustained fewer lethal double-strand breaks than either TA or CB cells, which correlated with SCs being less proliferative and having increased levels of heterochromatin. Finally, treatment with an HDAC inhibitor sensitised the SCs to radiation. INTERPRETATION: Prostate SCs are more radioresistant than more differentiated cell populations. We suggest that the primitive cells survive radiation therapy and that pre-treatment with HDAC inhibitors may sensitise this resistant fraction.

Retinoic acid represses invasion and stem cell phenotype by induction of the metastasis suppressors RARRES1 and LXN.

The mouse haematopoietic stem cell (SC) regulator Latexin (LXN) is the only known homologue of the retinoic acid receptor responder 1 (RARRES1) gene. Both genes lie adjacent on chromosome 3 and differ mostly by the presence of a transmembrane domain in RARRES1. Despite their homology, it is not known whether they possess similar regulatory mechanisms, cellular localization and function. Here, we identified RARRES1 and LXN as highly significantly downregulated genes in human prostate SCs, whose expression was induced by the pro-differentiation agent all-trans retinoic acid (atRA). AtRA induced expression in the most differentiated cells compared with the SC fraction, suggesting that this subpopulation was less responsive to atRA. Small interfering RNA suppression of RARRES1 and LXN enhanced the SC properties of primary prostate cultures, as shown by a significant increase in their colony-forming ability. Expression of both RARRES1 and LXN was co-ordinately repressed by DNA methylation in prostate cancer cell lines and inhibition of RARRES1 and LXN increased the invasive capacity of primary prostate cultures, which also fully rescued an inhibitory effect induced by atRA. Moreover, we showed that RARRES1 and LXN reside within different sub-cellular compartments, providing evidence that RARRES1 is not a plasma membrane protein as previously supposed but is located primarily in the endoplasmic reticulum; whereas LXN was detected in the nucleus of prostate epithelial cells. Thus, LXN and RARRES1 are potential tumour suppressor genes, which are co-ordinately regulated, SC-silenced genes functioning to suppress invasion and colony-forming ability of prostate cancer cells; yet the proteins reside within different sub-cellular compartments.

A transductionally retargeted adenoviral vector for virotherapy of Her2/neu-expressing prostate cancer.

The efficacy of adenovirus (Ad)-based gene therapy of solid tumors, such as prostate cancer, is limited. One of the many problems is that the virus infects many different cell types in the body, resulting in high toxicity, whereas the target cancer cells are often less prone to wild-type Ad infection. Our aim was to develop genetically de- and retargeted Ad vectors to reduce off-target effects and increase target infection for prostate cancer. We have previously reported an Ad5 vector specific for the cancer-associated receptor Her2/neu, created by inserting Her2/neu-reactive Affibody(®) molecules (ZH) into the HI loop of a coxsackievirus and adenovirus receptor binding-ablated fiber (Ad[ZH/1]). In addition to virus retargeting to Her2/neu, this virus was further modified from wild-type Ad by changing the RGD motif in the penton base to EGD and by substitution of the KKTK motif in the third shaft repeat to RKSK, resulting in the vector Ad[ZH/3]. The ZH-containing vectors could be produced to high titers and were specific for their target, resulting in efficient infection and killing of Her2/neu-positive androgen-dependent PC346C prostate cancer cells in vitro. Here we show that the oncolytic Ad[ZH/3] vector significantly prolonged survival time and reduced serum prostate-specific antigen levels in an orthotopic prostate tumor model in nude mice to the same extent as wild-type Ad5. Our results show that Her2/neu targeting using Ad-based vectors for prostate cancer is feasible and may serve as a basis for the development of gene therapy of human prostate cancer as well as other Her2/neu-expressing cancers.

In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus.

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.

Human prostate cell lines from normal and tumourigenic epithelia differ in the pattern and control of choline lipid headgroups released into the medium on stimulation of protein kinase C.

BACKGROUND: Expression of protein kinase C alpha (PKCα) is elevated in prostate cancer (PCa); thus, we have studied whether the development of tumourigenesis in prostate epithelial cell lines modifies the normal pattern of choline (Cho) metabolite release on PKC activation. METHODS: Normal and tumourigenic human prostate epithelial cell lines were incubated with [(3)H]-Cho to label choline phospholipids. Protein kinase C was activated with phorbol ester and blocked with inhibitors. Choline metabolites were resolved by ion-exchange chromatography. Phospholipase D (PLD) activity was measured by transphosphatidylation. Protein expression was detected by western blotting and/or RT-PCR. Choline uptake was measured on cells in monolayers over 60 min. RESULTS: Normal prostate epithelial cell lines principally released phosphocholine (PCho) in contrast to tumourigenic lines, which released Cho. In addition, only with normal cell lines did PKC activation stimulate Cho metabolite release. Protein kinase C alpha expression varied between normal and tumourigenic cell lines but all showed a PKCα link to myristoylated alanine-rich C kinase substrate (MARCKS) protein. The five cell lines differed in Cho uptake levels, with normal PNT2C2 line cells showing highest uptake over 60 min incubation. Normal and tumourigenic cell lines expressed mRNA for PLD1 and PLD2, and showed similar levels of basal and PKC-activated PLD activity. CONCLUSIONS: The transition to tumourigenesis in prostate epithelial cell lines results in major changes to Cho metabolite release into the medium and PKC signalling to phosphatidylcholine turnover. The changes, which reflect the metabolic and proliferative needs of tumourigenic cells compared with untransformed cells, could be significant for both diagnosis and treatment.

Use of the PSA enhancer core element to modulate the expression of prostate- and non-prostate-specific basal promoters in a lentiviral vector context.

Composite promoters combining the prostate-specific antigen (PSA) enhancer core element with promoter elements derived from gene coding for human prostate-specific transglutaminase gene, prostate-specific membrane antigen gene, prostate-specific antigen, rat probasin or phosphoglycerate kinase were characterized for their ability to specifically express the enhanced green fluorescent protein (EGFP) gene in prostate versus non-prostate cancer cell lines when transferred with a human immunodeficiency virus-1-based lentiviral vector. By themselves minimal proximal promoter elements were found to inefficiently promote relevant tissue-specific expression; in all the vectors tested, addition of the PSA enhancer core element markedly improved EGFP expression in LnCaP, a cancer prostate cell line used as a model for prostate cancer. The composite promoter was inactive in HuH7, a hepatocarcinoma cell line used as a model of neighboring non-prostate cancer cells. Among the promoters tested, the combination of the PSA enhancer and the rat probasin promoter showed both high specificity and a strong EGFP expression. Neither a high viral input nor the presence of the cPPT/CTS sequence affected composite promoter behavior. Our data suggest that composite prostate-specific promoters constructed by combining key elements from various promoters can improve and/or confer tissue specific expression in a lentiviral vector context.

Gene therapy for prostate cancer: current strategies and new cell-based approaches.

Prostate cancer is the most commonly diagnosed cancer in adult males in the Western world. It accounts for one in ten cancer cases and is the second leading cause of cancer death in men, after lung cancer. A number of curative treatments are available for patients with localized prostate cancer such as radical prostatectomy, radiotherapy, or brachytherapy. However, a proportion of these men will develop progressive disease, and some will present de novo with advanced and metastatic prostate cancer, which is amenable to palliation only with androgen-withdrawal therapy. Most of these patients will eventually develop hormone refractory disease which is incurable, and for whom gene therapy, if feasible may develop as an alternative treatment option. In this review we discuss the gene therapy vectors and strategies that are currently in use, new cell-based approaches, discuss their advantages and disadvantages, and review the potential or proven pre-clinical and clinical efficacy in prostate cancer models/patients.

Prostate cancer stem cells: a target for new therapies.

Prostate cancer is now a common disease in men over 50 years of age. Medical therapies for prostate cancer are based on discoveries from the mid-twentieth century, and in the long term are rarely curative. Most treatments are directed towards an androgen receptor-expressing, highly proliferative target cell, which does indeed form the vast majority of cells in a prostate tumour. However, by invoking the existence of a cancer stem cell which, like normal epithelial stem cells in the prostate, does not express androgen receptor and is relatively quiescent, the observed resistance to most medical therapies can be explained. The phenotype of the prostate cancer stem cells is that of a basal cell and cultures derived from cancers, but not benign tissues, express a range of prostate cancer-associated RNAs. Furthermore, stem cells purified on the basis of alpha2beta1 high integrin and CD133 cell surface antigen expression, from an established culture of Gleason 4 (2+2) prostate cancer (P4E6), were able to form multiple intraprostatic tumours in nude mice when grafted orthotopically in a matrigel plug containing human prostatic stroma. The final tumours reexpressed androgen receptor and displayed a histology similar to that of a Gleason 4 cancer.

Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues.

Extracellular proteases of the matrix metalloproteinase (MMP) and serine protease families participate in many aspects of tumour growth and metastasis. Using quantitative real-time RT-PCR analysis, we have undertaken a comprehensive survey of the expression of these enzymes and of their natural inhibitors in 44 cases of human prostate cancer and 23 benign prostate specimens. We found increased expression of MMP10, 15, 24, 25 and 26, urokinase plasminogen activator-receptor (uPAR) and plasminogen activator inhibitor-1 (PAI1), and the newly characterised serine proteases hepsin and matriptase-1 (MTSP1) in malignant tissue compared to benign prostate tissue. In contrast, there was significantly decreased expression of MMP2 and MMP23, maspin, and the protease inhibitors tissue inhibitor of metalloproteinase 3 (TIMP3), TIMP4 and RECK (reversion-inducing cysteine-rich protein with Kazal motifs) in the cancer specimens. The expression of MMP15 and MMP26 correlated positively with Gleason score, whereas TIMP3, TIMP4 and RECK expression correlated negatively with Gleason score. The cellular localisation of the expression of the deregulated genes was evaluated using primary malignant epithelial and stromal cell cultures derived from radical prostatectomy specimens. MMP10 and 25, hepsin, MTSP1 and maspin showed predominantly epithelial expression, whereas TIMP 3 and 4, RECK, MMP2 and 23, uPAR and PAI1 were produced primarily by stromal cells. These data provide the first comprehensive and quantitative analysis of the expression and localisation of MMPs and their inhibitors in human prostate cancer, leading to the identification of several genes involved in proteolysis as potential prognostic indicators, in particular hepsin, MTSP1, MMP26, PAI1, uPAR, MMP15, TIMP3, TIMP4, maspin and RECK.

Clusterin overexpression in both malignant and nonmalignant prostate epithelial cells induces cell cycle arrest and apoptosis.

Expression of the castration-induced clusterin protein is incompatible with the survival of human prostate cancer cells in tissues and in cell culture. To investigate the fate of human prostate epithelial cells, when engineered to maintain expression of clusterin protein, we have used an IRES-hyg vector and hygromycin selection. PC-3 prostate tumour cells were substantially more sensitive to clusterin expression than nonmalignant PNT1a cells, showing multiple phenotypic changes including cell cycle arrest and increased apoptosis. The results strengthen the hypothesis that clusterin expression is proapoptotic. Expression of exogenous clusterin in both cell types resulted in its relocation from the cytoplasm and a nuclear accumulation of the protein, as was also seen in the same cells when apoptosis was induced by etoposide treatment. To survive clusterin expression, the PC-3 tumour cells developed apoptosis-inhibitory properties. This could have significance for the resistance of prostate cancers to chemo/radiotherapy, where clusterin overexpression is observed.

Stromal-epithelial interactions influence prostate cancer cell invasion by altering the balance of metallopeptidase expression.

Perturbations of stromal-epithelial interactions in the developing tumour can contribute to cancer invasion and metastasis. The structurally related metallopeptidases endothelin-converting enzyme (ECE) and neutral endopeptidase (NEP) contribute sequentially to the synthesis and inactivation of ET-1, a mitogenic peptide that has been shown to affect tumour behaviour. This study has investigated the interaction between metastatic tumour epithelial cells, which lack NEP, and stromal cells, which we have shown to express ECE-1 (stromal-epithelial interactions), using Matrigel invasion chambers. The epithelial cell lines utilised in this study include androgen-sensitive LNCaP, androgen-independent PC-3, Du145 and recently established PNT-1a, PNT2-C2 and P4E6 prostate cell lines. Specific inhibition of endogenous ECE-1 activity in stromal cells reduced PC-3 and Du145 invasion by 70 and 50%, respectively. Addition of recombinant NEP to inactivate endogenous mitogenic peptides resulted in 50 and 20% reductions in invasion in PC-3 and Du145 cells, respectively. Neutral endopeptidase effects were reversed in the presence of thiorphan, a specific NEP inhibitor. Supplementation of defined media with bradykinin and ET-1 significantly increased PC-3 invasion by 40 and 50%, respectively. Du145 cell invasion increased by approximately 100% on adding ET-1. These studies implicate the metallopeptidases NEP and ECE-1 as mediators of prostate cancer invasion via a stromal/epithelial interaction.

Targeting gene therapy for prostate cancer.

Gene therapy is rapidly emerging as a viable clinical strategy to treat prostate cancer. New developments, such as targeted expression of therapeutic genes, and viruses that are designed to selectively replicate in prostate cancer cells have led to vectors with improved safety, even in elderly male patients. This review describes the various different viral and non-viral strategies employed to date, with a summary of current clinical trials. The main focus of the review is a discussion of the need, and the potential methods that can be used for targeted expression of the therapeutic gene specifically to prostate tumours and metastases. This includes methods of abrogating vector transduction of non-specific tissues, enhancement of transduction into prostate tumour tissue, transcriptional control of the therapeutic gene and some examples of prostate cancer-specific therapeutic genes. We also consider the future of prostate cancer gene therapy and the factors that should be taken into account when designing clinical trials, in a field that is expected to impact on clinical management of a common tumour type.

Expression of a model gene in prostate cancer cells lentivirally transduced in vitro and in vivo.

In a preclinical model for prostate cancer gene therapy, we have tested lentiviral vectors as a practical possibility for the transfer and long-term expression of the EGFP gene both in vitro and in vivo. The human prostate cancer cell lines DU145 and PC3 were transduced using experimental conditions which permitted analysis of the expression from a single proviral vector per cell. The transduced cells stably expressed the EGFP transgene for 4 months. After injection of the transduced cell populations into Nod-SCID mice a decrease in EGFP was only observed in a minority of cases, while the majority of tumors maintained transgene expression at in vitro levels. In vivo injection of viral vector preparations directly into pre-established subcutaneous or orthotopic tumor masses, obtained by implantation of untransduced PC3 and DU145 cells led to a high transduction efficiency. While the efficiency of direct intratumoral transduction was proportional to the dose of virus injected, the results indicated some technical limitations inherent in these approaches to prostate cancer gene therapy.

Structure of the human S100A12-copper complex: implications for host-parasite defence.

S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins. Together with S100A8 and S100A9, it belongs to the calgranulin subfamily, i.e. it is mainly expressed in granulocytes, although there is an increasing body of evidence of expression in keratinocytes and psoriatic lesions. As well as being linked to inflammation, allergy and neuritogenesis, S100A12 is involved in host-parasite response, as are the other two calgranulins. Recent data suggest that the function of the S100-family proteins is modulated not only by calcium, but also by other metals such as zinc and copper. Previously, the structure of human S100A12 in low-calcium and high-calcium structural forms, crystallized in space groups R3 and P2(1), respectively, has been reported. Here, the structure of S100A12 in complex with copper (space group P2(1)2(1)2; unit-cell parameters a = 70.6, b = 119.0, c = 90.2 A) refined at 2.19 A resolution is reported. Comparison of anomalous difference electron-density maps calculated with data collected with radiation of wavelengths 1.37 and 1.65 A shows that each monomer binds a single copper ion. The copper binds at an equivalent site to that at which another S100 protein, S100A7, binds zinc. The results suggest that copper binding may be essential for the functional role of S100A12 and probably the other calgranulins in the early immune response.

Macrophages in gene therapy: cellular delivery vehicles and in vivo targets.

The appearance and activation of macrophages are thought to be rapid events in the development of many pathological lesions, including malignant tumors, atherosclerotic plaques, and arthritic joints. This has prompted recent attempts to use macrophages as novel cellular vehicles for gene therapy, in which macrophages are genetically modified ex vivo and then reintroduced into the body with the hope that a proportion will then home to the diseased site. Here, we critically review the efficacy of various gene transfer methods (viral, bacterial, protozoan, and various chemical and physical methods) in transfecting macrophages in vitro, and the results obtained when transfected macrophages are used as gene delivery vehicles. Finally, we discuss the use of various viral and nonviral methods to transfer genes to macrophages in vivo. As will be seen, definitive evidence for the use of macrophages as gene transfer vehicles has yet to be provided and awaits detailed trafficking studies in vivo. Moreover, although methods for transfecting macrophages have improved considerably in efficiency in recent years, targeting of gene transfer specifically to macrophages in vivo remains a problem. However, possible solutions to this include placing transgenes under the control of macrophage-specific promoters to limit expression to macrophages or stably transfecting CD34(+) precursors of monocytes/macrophages and then differentiating these cells into monocytes/macrophages ex vivo. The latter approach could conceivably lead to the bone marrow precursor cells of patients with inherited genetic disorders being permanently fortified or even replaced with genetically modified cells.