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

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

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

Upk3a^GCE/+ drives conditional expression of the human <i>AR</i> transgene in mouse bladder urothelium.

<p>(<b>A)</b> Schematic depicting the Cre/LoxP-mediated recombination, resulting in deletion of the STOP cassette from the <i>R26AR transgene</i> allele. (<b>B)</b> Experimental set-up for tamoxifen-induced human <i>AR</i> transgene expression. (<b>C)</b> Genomic PCR analyses of recombination events with specific primers for the human <i>AR</i> transgene allele (see Fig 2A) with bladder urothelium tissue samples of <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i> and <i>R26AR</i>^{<i>LoxP/+</i>} mice. The AR trangene allele was detected in the samples of both <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i> and <i>R26AR</i>^{<i>LoxP/+</i>} mice (black arrow), but TM induced recombination on the AR transgene allele was only observed in <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i> mice (white arrow). (<b>D-F’)</b> Immunohistochemical staining of the bladder urothelium of TM injected <i>R26AR</i>^{<i>LoxP/+</i>} mice using either the antibody that is reactive to both mouse and human AR protein (m&hAR), <b>or</b> the antibody that is reactive to only human AR (hAR) (n = 9). <b>(F-G’)</b> Immunohistochemical staining of the bladder urothelium with the two different AR antibodies as described above using the samples of TM-injected <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i> mice (n = 12). (<b>H-K”)</b> Single channel and merged immunofluorescent images of bladder tissues of TM-injected <i>R26AR</i>^{<i>LoxP/+</i>} and <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i> mice stained with antibodies against CK5 p63, and the human AR (in red) (n = 6).</p

The effect of androgens on tumor development in <i>R26AR</i>^{<i>LoxP/+</i>} and <i>R26AR</i>^{<i>LoxP/+</i>}<i>Upk3a</i>^<i>GCE/+</i>mice.

<p>(<b>A)</b> Schematic depicting experimental design for castration of male mice and testosterone pellet insertion of female mice. (<b>B)</b> Analysis of the androgen effect on different genotypes of male and female mice with castration and testosterone pellet insertion. (<b>C)</b> Quantification of Ki67-positive cells in castrated and intact male mice and female mice with and without testosterone pellet (n = 4).</p

Upk3a^GCE/+ targets Cre recombination to bladder urothelium.

<p>(<b>A)</b> Schematic demonstrating Cre-mediated labeling of Upk3a^GCE/+-positive cells with the mT/mG reporter system. (<b>B)</b> Schedule of tamoxifen (TM) injection to induce Cre recombination of <i>R26</i>^{<i>mTmG/+</i>}:<i>Upk3a</i>^<i>GCE/+</i> mice. (<b>C-C’)</b> Lower and higher magnification images of labeled mT, mtd-Tomato, (red) or mG, mGFP, (green) in the bladder urothelium of <i>R26</i>^{<i>mTmG/+</i>}:<i>Upk3a</i>^<i>GCE/+</i> prior to TM injection (n = 4). (<b>D-D’)</b> Lower and higher magnification images of labeled mT (red) or mGFP (green) in the bladder urothelium in <i>R26</i>^{<i>mTmG/+</i>}:<i>Upk3a</i>^<i>GCE/+</i> following TM injection (n = 6). (<b>E-G)</b> Immunofluorescent images of bladder urothelium of TM injected <i>Upk3a</i>^<i>GCE/+</i><i>R26</i>^{<i>mTmG/+</i>} mice co-stained with antibodies against GFP (E-G”) with p63 (E’-E”), CK5 (F’-F”), or CK8 (G’-G”) (n = 6).</p

Pathological Abnormalities in the Prostates of Targeted <i>Pten</i> Knockout mice.

<p>Pathological Abnormalities in the Prostates of Targeted <i>Pten</i> Knockout mice.</p

Deletion of <i>Pten</i> induces cell proliferative advantage in prostate tissues.

<p><b>A–I.</b> Cell proliferation was examined by immunostaining for Ki-67. Prostate sections isolated from 4-week-old <i>Pten^loxP/loxP</i>, <i>Pten^loxP/+:Osr1-Cre,</i> and <i>Pten^loxP/loxP Osr1-Cre</i> mice were stained for Ki-67. A total of 1000 epithelial cells in each lesion from three different lesions from three mice of each genotype were evaluated for Ki-67 immunoreactivity. <b>J.</b> The positive immunoreactive cells for Ki-67 in <i>Pten^loxP/loxP</i>:<i>Osr1-Cre+</i> mice are significantly greater than both <i>Pten^loxP/loxP</i> control mice and <i>Pten^loxP/wt:Osr1-Cre</i> mice, P<0.01.</p

Development of PIN lesion in <i>Pten^loxP/loxP:Osr1-Cre</i> mice.

<p><b>A–C.</b> H&E staining for 2 to 4 week old <i>Pten^loxP/loxP Osr1-Cre</i> mice. Representative images of mouse PIN lesions are shown from different prostate lobes of 2 week old <i>Pten^loxP/loxP</i>:<i>Osr1-Cre</i> mice; AP, anterior; D/LP, dorso/lateral, and VP, ventral prostate lobes. Immunohistochemical analyses of these samples show the PIN lesions with concurrent absence of <i>Pten</i> expression (please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053476#pone.0053476.s001" target="_blank">Figure S1</a>). The typical high-grade PIN lesions are mainly cribriform or stratified in nature, and often distended the prostate glandular unit profile and/or completely filled the prostate glandular unit lumen. Mitoses were rare. The fibromuscular stroma remained intact, with overall glandular unit profiles being recognizable. <b>D–G.</b> Histological analyses of the prostate tissues isolated from different age groups of <i>Pten^loxP/loxP</i>:<i>Osr1-Cre</i> mice. However, the nature and extent of the high grad PIN in <i>Pten^loxP/loxP:Osr1-Cre</i> mice did not appear to change with advancing age, as evidence by the similarities of lesions in the 4-week old mice as compared to their 2-, 7-, and 12-month of cohorts.</p

Generating and analyzing <i>Pten Osr1-Cre</i> conditional knockout mice.

<p><b>A.</b> Diagram for <i>Pten</i> knockout strategy. In <i>Pten^loxP/loxP</i> mice, <i>LoxP</i> recognition sequences were inserted into the endogenous <i>Pten</i> locus flanking exon 5 as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053476#pone.0053476-Wang2" target="_blank">[15]</a>. Two different Cre transgenic lines carrying <i>Osr1-Cre</i> or <i>PB-Cre4</i> transgene were crossed to <i>Pten^loxP/loxP</i> mice for the generation of <i>Pten^loxP/loxP:Osr1-Cre</i> and <i>Pten^loxP/loxP;PB-</i>Cre4 mice. <b>B and C.</b> Genomic PCR analyses for <i>Pten^loxP/loxP</i>:<i>Osr1-Cre</i> and <i>Pten^loxP/loxP</i>:<i>PB-Cre4</i> mice. PCR was performed using genomic DNA samples harvested from various organs in <i>Pten^loxP/loxP</i>:<i>Osr1-Cre</i> and <i>Pten^loxP/loxP</i>:<i>PB-Cre4</i> mice. Primers in the <i>Pten</i> locus were used to detect either the <i>loxP</i> allele or the <i>Cre</i>-recombined allele. The 0.65 kb fragment of the <i>Pten loxP</i> allele (solid arrows) was observed in all 15 organs from both <i>Pten^loxP/loxP</i>:<i>Osr1-Cre</i> and <i>Pten^loxP/loxP</i>:<i>PB-Cre4</i> mice. The 0.3kb Cre-recombined fragment (open arrows) was detected only in the prostate of <i>Pten^loxP/loxP:PB-Cre4</i> mice and in the prostate, urinary bladder, brain, lung, kidney, stomach, and ovary of <i>Pten^loxP/loxP</i>:<i>Osr1-Cre</i> mice. <b>D.</b> Western blotting to detect <i>Pten</i> expression in <i>Pten^loxP/loxP</i>:<i>Osr1-Cre</i> and <i>Pten^loxP/loxP</i>:<i>PB-Cre4</i> mice. Whole cell lysates isolated from prostate lobes were analyzed by Western blot with the PTEN antibody. b-actin was used as a loading control. AP, anterior; DLP, dorsolateral and VP, ventral prostate lobes.</p