Down-regulation of endogenous AR expression by AR shRNA in prostate cancer cells

<p><b>Copyright information:</b></p><p>Taken from "A promoting role of androgen receptor in androgen-sensitive and -insensitive prostate cancer cells"</p><p></p><p>Nucleic Acids Research 2007;35(8):2767-2776.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885678.</p><p>© 2007 The Author(s)</p> () LNCaP cells were infected with either the GFP adenovirus or the different AR shRNA adenovirus at an MOI of 40. Whole-cell lysates were prepared after 48 h of viral infection, and then analyzed by western blotting. Specific antibodies used to detect protein expression are labeled in the figure. () Identical experiments performed in LAPC4 cells. () LNCaP cells were infected with either the GFP adenovirus or AR shRNA3 adenovirus at an MOI of 40. Cells were fixed and immunostained 72 h after viral infection. Representative confocal laser scanning microscopy images of cells are shown. () Identical experiments performed in LAPC4 cells

Reduction of AR expression inhibits tumor xenograft formation in athymic mice

<p><b>Copyright information:</b></p><p>Taken from "A promoting role of androgen receptor in androgen-sensitive and -insensitive prostate cancer cells"</p><p></p><p>Nucleic Acids Research 2007;35(8):2767-2776.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885678.</p><p>© 2007 The Author(s)</p> () LAPC4 cells were transduced with the AR shRNA or GFP lentiviruses at a MOI of 3 for 24 h. Cells were harvested, resuspended in PBS and mixed with an equal volume of Matrigel ECM. Here, 100 μl of cell suspension (1 × 10 cells/ml) was injected subcutaneously in opposite lateral flanks of 6–8-week-old athymic male mice. Mice were monitored twice weekly. Tumors were measured in two dimensions with calipers, and tumor volume (mm) was calculated with the formula  = (length × width)/2. ‘Asterisk’ indicates a significant difference

Down-regulation of AR expression inhibits the growth of androgen-sensitive prostate cancer cells

<p><b>Copyright information:</b></p><p>Taken from "A promoting role of androgen receptor in androgen-sensitive and -insensitive prostate cancer cells"</p><p></p><p>Nucleic Acids Research 2007;35(8):2767-2776.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885678.</p><p>© 2007 The Author(s)</p> () LNCaP cells were seeded into 96-well plates in media with or without DHT after 3 h adenovirus infection at an MOI of 10. Cell growth was measured every other day by MTS assay. The data represent the mean ± SD of three independent experiments. () Identical experiments performed in LAPC4 cells. () LNCaP cells were seeded into 24-well plates at 400 cells/well after 3 h adenovirus infection at an MOI of 10. Cells were cultured with the media in the presence or absence of DHT for 14 days and colonies were fixed and stained with crystal violet. () Similar experiments performed in LAPC4 cells

Optimized assay parameters.

<p>The best-matched pair with resulting dynamic range, reference range, and dilution factors are shown for the 7 biomarker assays as well as representative intra-assay variation values.</p

Detection Scheme.

<p>A. The c.Ab. is covalently bound to a magnetic bead that is incubated with sample and fluorescein-tagged d.Ab. This sample is introduced into a flow cell capped with a piezo-electric membrane coated with an anti-fluorescein antibody. An electromagnet above the membrane is controlled with embedded software. B. Upon magnetic perturbation, all beads (black circles) move towards the membrane (Bi), and beads with a completed immunocomplex (black circles coated with red dots) bind to the anti-fluorescein antibody (Bii). After the field is removed and flow restored, only beads with a completed immunocomplex remain bound (Biii). These beads alter the oscillation of the membrane (represented as orange sinusoidal curve), which is interpreted as signal in arbitrary units [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139484#pone.0139484.ref036" target="_blank">36</a>]. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139484#pone.0139484.s003" target="_blank">S1 Video</a>. In B, the solid blue line labeled Pos refers to beads in the presence of antigen, and the dotted red line labeled Neg refers to beads in the absence of antigen.</p

Assay Construction.

<p><b>A)</b> Optimal antibody pairs were identified with a “checkerboard” assay that evaluated different antibody pairs as well as isotype controls. Metric plotted here is signal difference between the negative control and 1 ng/mL recombinant PAP. The different antibodies are defined in the Materials and Methods section. <b>B)</b> A full calibration curve illustrates the different sensitivities of different antibody pairs for PAP. Pair 1: Cos2 c.Ab.; Cos1 d.Ab. Pair 2: RDPo c.Ab.; RDMo. d.Ab. The red error bars represent the standard deviation of at least three replicate measurements. <b>C)</b> Reproducibility from day to day is <8%. <b>D)</b> The piezo-based approach shows 3 log orders improvement in analytical sensitivity versus direct ELISA when identical antibody pairs are used (Cos1/Cos2).</p

Serum Data.

<p>Raw values with means for BPH, CaP, and post-surgery (PS) samples. Biomarkers include tPSA (A), fPSA(B), CA1 (C), PAP (D), IL6-sr (E), SPARC (F), and SPON2 (G). Horizontal lines indicate mean values.</p

Serum Validation.

<p><b>A)</b> Percent recovery for three representative biomarkers. Matrix effects can either dampen signal (CA1, PAP) or inflate signal (SPARC). Ideal dilution factors were dependent on sensitivity of the assay and reference range of the biomarker (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139484#pone.0139484.t001" target="_blank">Table 1</a>). Black dashed line indicates 100% spike recovery. <b>B)</b> Bland-Altman plot [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139484#pone.0139484.ref038" target="_blank">38</a>] validating specimen integrity of a subset (N = 35) of the clinical samples with high sample volumes. Red dashed line indicates 95% confidence interval.</p

Clinical Data Analysis.

<p>A) Mean values with standard deviation for the seven biomarkers with BPH, CaP, and post-surgery [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139484#pone.0139484.ref039" target="_blank">39</a>] samples. Units are ng/mL. The p value reported here is the significance between the BPH and CaP samples. AUC values are also given and report discrimination between CaP and BPH. Lower panel presents ROC curves for PSA, SPON2, and PSA OR SPON2. The OR operator increases the AUC to 0.84 from 0.80 for PSA alone.</p

Analysis of PSA recurrence-free survival.

a<p>Log rank test (univariate analysis) or Wald test (multivariate analysis).</p>b<p>Analyzed as a continuous variable.</p>c<p>Stratification based on limited representation of Gleason 6 and 4+4 cases.</p>d<p>Stratifies pathologic stage based on organ confinement.</p