Expression of p85α in bladder tumours and cell lines.

<p>A. Analysis of p85α mRNA expression levels in 105 bladder tumour samples and 52 normal bladder samples. Data from Sanchez-Carbayo et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084411#B30" target="_blank">30</a>]. B. Quantitative analysis of p85α immunoblotted protein samples from bladder cancer cell lines normalized to tubulin and shown relative to pooled normal human urothelial cells (NHU-pool).</p

Distribution of <i>PIK3R1</i> mutations identified in UC.

<p>Distribution of <i>PIK3R1</i> mutations identified in UC.</p

Position of mutations in the p85α protein.

<p>A. Ribbon diagram of the structure of the complex of p110α with the niSH2 region of p85α (PDB code 2RD0) showing residues mutated in UC. B. Relationship of p85α nSH2 to p110α C2 domain showing proximity of N377 in nSH2 to C2 domain residue E365. C. Relationship of the iSH2 domain of p85α with the C2 domain of p110α showing proximity of R557 to N345 in C2. D. Space-filling model showing R481 and R557 residues in iSH2 of p85α in contact with C2 of p110α. E. Structure of p85 dimer (PDB code 1PBW) showing position of PIK3R1 point-mutated residues E137, R262 and K288 and the region deleted (W237-Y242) in UC in relation to residues involved in p85 dimerization (M176, dark green/light cyan; L161, I177 and V181, light green/cyan). The position of residue R274 (magenta), which is implicated in Rab-GAP activity is also shown. In addition, three glutamic acid residues (E266, E284 and E291) that interact with R262 are indicated, with E291 also interacting with K288. </p

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