Identification of genes differentially expressed as result of adenovirus type 5- and adenovirus type 12-transformation

Background: Cells transformed by human adenoviruses (Ad) exhibit differential capacities to induce tumours in immunocompetent rodents; for example, Ad12-transformed rodent cells are oncogenic whereas Ad5-transformed cells are not. The E1A gene determines oncogenic phenotype, is a transcriptional regulator and dysregulates host cell gene expression, a key factor in both cellular transformation and oncogenesis. To reveal differences in gene expression between cells transformed with oncogenic and non-oncogenic adenoviruses we have performed comparative analysis of transcript profiles with the aim of identifying candidate genes involved in the process of neoplastic transformation. Results: Analysis of microarray data revealed that a total of 232 genes were differentially expressed in Ad12 E1- or Ad5 E1-transformed BRK cells compared to untransformed baby rat kidney (BRK) cells. Gene information was available for 193 transcripts and using gene ontology (GO) classifications and literature searches it was possible to assign known or suggested functions to 166 of these identified genes. A subset of differentially-expressed genes from the microarray was further examined by real-time PCR and Western blotting using BRK cells immortalised by Ad12 E1A or Ad5 E1A in addition to Ad12 E1- or Ad5 E1-transformed BRK cells. Up-regulation of RelA and significant dysregulation of collagen type I mRNA transcripts and proteins were found in Ad-transformed cells. Conclusion: These results suggest that a complex web of cellular pathways become altered in Ad-transformed cells and that Ad E1A is sufficient for the observed dysregulation. Further work will focus on investigating which splice variant of Ad E1A is responsible for the observed dysregulation at the pathway level, and the mechanisms of E1A-mediated transcriptional regulation

Retinoic acid and androgen receptors combine to achieve tissue specific control of human prostatic transglutaminase expression: a novel regulatory network with broader significance

In the human prostate, expression of prostate-specific genes is known to be directly regulated by the androgen–induced stimulation of the androgen receptor (AR). However, less is known about the expression control of the prostate-restricted TGM4 (hTGP) gene. In the present study we demonstrate that the regulation of the hTGP gene depends mainly on retinoic acid (RA). We provide evidence that the retinoic acid receptor gamma (RAR-G) plays a major role in the regulation of the hTGP gene and that presence of the AR, but not its transcriptional transactivation activity, is critical for hTGP transcription. RA and androgen responsive elements (RARE and ARE) were mapped to the hTGP promoter by chromatin immunoprecipitation (ChIP), which also indicated that the active ARE and RARE sites were adjacent, suggesting that the antagonistic effect of androgen and RA is related to the relative position of binding sites. Publicly available AR and RAR ChIP-seq data was used to find gene potentially regulated by AR and RAR. Four of these genes (CDCA7L, CDK6, BTG1 and SAMD3) were tested for RAR and AR binding and two of them (CDCA7L and CDK6) proved to be antagonistically regulated by androgens and RA confirming that this regulation is not particular of hTGP

Engineered expression of the Coxsackie B and adenovirus receptor (CAR) in human dendritic cells enhances recombinant adenovirus-mediated gene transfer

Dendritic cells (DCs) are key antigen-presenting cells (APCs) that act as central modulators of cellular immune responses. Genetic modification of DCs has considerable therapeutic potential in the treatment of a wide spectrum of diseases, including cancer and persistent viral infection. In this report, we show that pre-treatment of DCs with a recombinant adenovirus encoding the major adenovirus receptor, Coxsackie B and adenovirus receptor (CAR), significantly increased the uptake of recombinant adenoviruses (Ads) by primary immature monocyte-derived DCs. This could be correlated with CAR mRNA and surface protein expression. Transduction of DCs by recombinant adenoviruses did not significantly alter cellular viability. Therefore, we propose that pre-treatment of DCs with Ad5-CAR is one strategy to increase the susceptibility of DCs to transduction by recombinant Ads

Evaluating Baculovirus as a Vector for Human Prostate Cancer Gene Therapy

<div><p>Gene therapy represents an attractive strategy for the non-invasive treatment of prostate cancer, where current clinical interventions show limited efficacy. Here, we evaluate the use of the insect virus, baculovirus (BV), as a novel vector for human prostate cancer gene therapy. Since prostate tumours represent a heterogeneous environment, a therapeutic approach that achieves long-term regression must be capable of targeting multiple transformed cell populations. Furthermore, discrimination in the targeting of malignant compared to non-malignant cells would have value in minimising side effects. We employed a number of prostate cancer models to analyse the potential for BV to achieve these goals. <i>In vitro</i>, both traditional prostate cell lines as well as primary epithelial or stromal cells derived from patient prostate biopsies, in two- or three-dimensional cultures, were used. We also evaluated BV <i>in vivo</i> in murine prostate cancer xenograft models. BV was capable of preferentially transducing invasive malignant prostate cancer cell lines compared to early stage cancers and non-malignant samples, a restriction that was not a function of nuclear import. Of more clinical relevance, primary patient-derived prostate cancer cells were also efficiently transduced by BV, with robust rates observed in epithelial cells of basal phenotype, which expressed BV-encoded transgenes faster than epithelial cells of a more differentiated, luminal phenotype. Maximum transduction capacity was observed in stromal cells. BV was able to penetrate through three-dimensional structures, including <i>in vitro</i> spheroids and <i>in vivo</i> orthotopic xenografts. BV vectors containing a nitroreductase transgene in a gene-directed enzyme pro-drug therapy approach were capable of efficiently killing malignant prostate targets following administration of the pro-drug, CB1954. Thus, BV is capable of transducing a large proportion of prostate cell types within a heterogeneous 3-D prostate tumour, can facilitate cell death using a pro-drug approach, and shows promise as a vector for the treatment of prostate cancer.</p></div

BV transduction of cells growing in three dimensions.

<p>(<b>A</b>) PC346C spheroids transduced with Bv-[CMV-EGFP] (or mock transduced) at 3 days post-infection. (<b>B</b>) PC346C tumours harvested from xenografted mice at 3 days post-infection with BV at 3×10⁷ (one representative image shown) or 1×10⁸ (two representative images shown) pfu per inoculation. Red fluorescence shows cell nuclei (Hoescht), while BV-driven EGFP expression is shown in green. Arrows indicate the site of intratumoural BV injection.</p

BV transduction of prostate cell lines.

<p>(<b>A</b>) Percentage of EGFP-positive cells following transduction of a panel of high grade malignant (red), low grade malignant (black) or non-malignant (blue) prostate cell lines with BV-[CMV-EGFPCAT] at MOI = 500 for 48 h. Error bars depict −/+ one standard deviation. (<b>B</b>) Relative expression levels of EGFP following transduction with BV-[CMV-EGFPCAT] at a saturating MOI (500 for LNCaP, PC3 and PNT1A, 1000 for PNT2C2). The percentage of EGFP-positive cells was normalised to the levels achieved following 48 h incubation in the presence of virus for each cell type (set to 1). Malignant cell lines: PC3 (▴), LNCaP (▪); Non-Malignant Cell Lines: PNT2C2 (♦), PNT1A (▾). Error bars depict −/+ one standard deviation. (<b>C</b>) Confocal microscopy images (single slice or Z-stack) of BV-transduced LNCaP, PC3 or PNT1A cells at 8 h post-transduction (MOI = 500). Red fluorescence indicates BV capsid (anti-vp39), nuclear staining in blue (DAPI) and BV-driven EGFP expression in green.</p

BV transduction of primary prostate epithelial cells.

<p>(<b>A</b>) Percentage of EGFP-positive cells at 48 h post-transduction with Bv-[CMV-EGFPCAT] at MOI = 500 for increasing lengths of time in three primary malignant prostate epithelial samples (Gleason 6, 7 or 8/9), maintained in a basal state by culture in KSFMsup (▪), or cultured in differentiating conditions in DH10 (▴). Error bars depict −/+ one standard deviation. (<b>B</b>) Localisation of BV capsids in primary epithelial cells from a Gleason 8/9 tumour grown in a bilayer, either 1 h or 16 h after incubation with Bv-[CMV-EGFPCAT] at MOI = 250. Red fluorescence indicates BV capsids detected with anti-vp39, nuclear DAPI staining is shown in blue and BV-driven EGFP expression in green. Confocal images of the upper layer of cells (luminal-like; at an average z-distance of 14.58 µm from the ventral position) and lower layer (basal-like; at an average z-distance of 6.47 µm from the ventral position) are shown. (<b>C</b>) Frequency (normalised to mock = 1%) or mean fluorescence intensity of EGFP-positive primary stromal cells derived from malignant or benign biopsies 24 h following transduction with Bv-[CMV-EGFPCAT] at MOI = 500.</p

Use of macrophages to target therapeutic adenovirus to human prostate tumors

New therapies are required to target hypoxic areas of tumors as these sites are highly resistant to conventional cancer therapies. Monocytes continuously extravasate from the bloodstream into tumors where they differentiate into macrophages and accumulate in hypoxic areas, thereby opening up the possibility of using these cells as vehicles to deliver gene therapy to these otherwise inaccessible sites. We describe a new cell-based method that selectively targets an oncolytic adenovirus to hypoxic areas of prostate tumors. In this approach, macrophages were cotransduced with a hypoxia-regulated E1A/B construct and an E1A-dependent oncolytic adenovirus, whose proliferation is restricted to prostate tumor cells using prostate-specific promoter elements from the TARP, PSA, and PMSA genes. When such cotransduced cells reach an area of extreme hypoxia, the E1A/B proteins are expressed, thereby activating replication of the adenovirus. The virus is subsequently released by the host macrophage and infects neighboring tumor cells. Following systemic injection into mice bearing subcutaneous or orthotopic prostate tumors, cotransduced macrophages migrated into hypoxic tumor areas, upregulated E1A protein, and released multiple copies of adenovirus. The virus then infected neighboring cells but only proliferated and was cytotoxic in prostate tumor cells, resulting in the marked inhibition of tumor growth and reduction of pulmonary metastases. This novel delivery system employs 3 levels of tumor specificity: the natural “homing” of macrophages to hypoxic tumor areas, hypoxia-induced proliferation of the therapeutic adenovirus in host macrophages, and targeted replication of oncolytic virus in prostate tumor cells