Additional file 2: of Mosaic genome-wide maternal isodiploidy: an extreme form of imprinting disorder presenting as prenatal diagnostic challenge

Results from quantitative fluorescence-polymerase chain reaction (QF-PCR) for chromosomes 13, 18, 21, X, and Y including peak areas and allele ratios. AF: uncultured amniotic fluid cells; AFc: cultured amniotic fluid cells; P: placenta tissue; M: mother, F: father, n.a. not applicable. (PDF 79 kb

Stilbene Induced Inhibition of Androgen Receptor Dimerization: Implications for AR and ARΔLBD-Signalling in Human Prostate Cancer Cells

<div><p>Background</p><p>Advanced castration resistant prostate cancer (CRPC) is often characterized by an increase of C-terminally truncated, constitutively active androgen receptor (AR) variants. Due to the absence of a ligand binding domain located in the AR-C-terminus, these receptor variants (also termed ARΔLBD) are unable to respond to all classical forms of endocrine treatments like surgical/chemical castration and/or application of anti-androgens.</p><p>Methodology</p><p>In this study we tested the effects of the naturally occurring stilbene resveratrol (RSV) and (E)-4-(2, 6-Difluorostyryl)-N, N-dimethylaniline, a fluorinated dialkylaminostilbene (FIDAS) on AR- and ARΔLBD in prostate cancer cells. The ability of the compounds to modulate transcriptional activity of AR and the ARΔLBD-variant Q640X was shown by reporter gene assays. Expression of endogenous AR and ARΔLBD mRNA and protein levels were determined by qRT-PCR and Western Blot. Nuclear translocation of AR-molecules was analyzed by fluorescence microscopy. AR and ARΔLBD/Q640X homo-/heterodimer formation was assessed by mammalian two hybrid assays. Biological activity of both compounds <i>in vivo</i> was demonstrated using a chick chorioallantoic membrane xenograft assay.</p><p>Results</p><p>The stilbenes RSV and FIDAS were able to significantly diminish AR and Q640X-signalling. Successful inhibition of the Q640X suggests that RSV and FIDAS are not interfering with the AR-ligand binding domain like all currently available anti-hormonal drugs. Repression of AR and Q640X-signalling by RSV and FIDAS in prostate cancer cells was caused by an inhibition of the AR and/or Q640X-dimerization. Although systemic bioavailability of both stilbenes is very low, both compounds were also able to downregulate tumor growth and AR-signalling <i>in vivo</i>.</p><p>Conclusion</p><p>RSV and FIDAS are able to inhibit the dimerization of AR and ARΔLBD molecules suggesting that stilbenes might serve as lead compounds for a novel generation of AR-inhibitors.</p></div

RSV and FIDAS down-regulate activity of AR-dependent reporter-gene activity in LNCaP and 22Rv1 cells.

<p>AR-positive LNCaP and 22Rv1 cells were transfected with the reporter-gene plasmids pARE(2x)-luc and pRT-TK, as described in Material and Methods. Subsequently cells were incubated with RSV or FIDAS in presence/absence of 5 nM DHT for 24 hours. AR-reporter-gene activity was determined using the Dual-Luciferase Reporter Assay System (Promega) according to the manufactures instructions. Results are expressed in % (AR_{DHT stimulated}/AR_{basal activity}) which was set at 100% for RSV or FIDAS untreated cells (controls). Each value represents the mean of at least 3 independent experiments ± standard deviation, (p-values compared to corresponding DHT-treated control *p<0.01, **p<0.05)</p

Effects of RSV and FIDAS on the nuclear translocation of AR and Q640X.

<p>Prostate cancer cells (PC-3) were transfected with expression plasmids coding for green fluorescent AR-EosFP or Q640X-EGFP fusion proteins. Nuclear localization of AR or Q640X fusion proteins was analyzed by fluorescence microscopy. Data present the percentage of fluorescent cells exhibiting a predominantly nuclear fluorescence.</p

Effects FIDAS on prostate cancer micro-tumors growing on the CAM.

<p>CAM assays were performed with PC-3 (AR negative) and LNCaP (AR-positive) as described in Material and Methods. Proliferation of PC cells was determined by nuclear staining of KI67. AR-activity was analyzed by PSA-staining.</p

Effects of RSV and FIDAS on AR- and AR-V7-mRNA-expression in prostate cancer cell lines.

<p>RNA isolation and qRT-PCR were performed as described in Material and Methods. Data are expressed in % of RSV or FIDAS untreated controls which were at 100% (ctrl). Each value represents the mean of at least 3 independent experiments ± standard deviation (p-values compared to untreated controls, *p<0.05 and **p<0.01)</p

Primers and Probes for qRT-PCR.

<p>Primers and Probes for qRT-PCR.</p

RSV and FIDAS do not inhibit nuclear translocation of AR and Q640X.

<p>PC-3 cells were transiently transfected with expression plasmids coding for green fluorescent AR-EosFP or Q640X-EGFP fusion proteins as described in Material and Methods. Subsequently cells were treated with/without 5 nM DHT in presence/absence of 100 µM RSV for 120 minutes. Nuclear localization of fluorescent AR or Q640X proteins was analyzed by fluorescence microscopy.</p

Effects of RSV and FIDAS on the dimerization of AR and Q640X.

<p>Formation of AR and Q640X homo/hetero-dimers was analyzed 24 hours after RSV or FIDAS treatment using a M2H as described in Material and Methods. Within this time frame RSV as well as FIDAS did not exhibit a significant <i>in vitro</i> toxicicity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098566#pone.0098566.s002" target="_blank">Table S1</a>). <b><i>AR/AR dimers</i></b>: Formation of AR/AR homodimers was analyzed in PC-3 cells grown under androgenic stimuli (5 nM DHT) in presence/absence of RSV or FIDAS (FIDAS/RSV untreated + DHT  = 100%). <b><i>AR/Q640X and Q640X/Q640X dimers</i></b>: Formation of AR/Q640X heterodimers (AR-VP16/ACT and Q640X-GAL4/BIND) or Q640X/Q640X homodimers was analyzed in the absence of DHT in RSV/FIDAS treated/untreated PC-3 cells (FIDAS/RSV untreated was set at 100%), *p<0.05.</p

Effects of RSV and FIDAS on on PCa cells <i>in vivo</i>.

<p>PC-3 and LNCaP cells were seeded onto the CAM to form tumors as described Material and Methods. Tumor grafts were allowed to grow for 48 hours. Subsequently, 50 µl of RSV (100 µM) or FIDAS (50 µM) were injected into a CAM-vein allowing the systemic spread of the compound. After 48 hours tumor xenografts were fixed, paraffin embedded and serially sectioned. Microtumors were stained for KI67 and PSA. Data are expressed in % of KI67 or PSA positive cells, grown in the absence of RSV or FIDAS ± standard deviation of at least 3 independent experiments (ctrl  =  RSV/FIDAS untreated  = 100%).</p