Induction of RAR and RXR subtypes by RA, BMP-4 and RA/BMP-4 double treatment in WERI-Rb1 cells as revealed by Real-time PCR.

<p>Messenger RNA expression levels at the beginning of the treatment (0h) were used as a reference and set as 1. The housekeeping gene GAPDH was used as an endogenous control. Data confirm an additive induction of <i>RARα</i> (A), <i>RARß</i> (B) and <i>RXRγ</i> (D) by RA/BMP-4 double treatment, whereas the up-regulation of <i>RARγ</i> (C) is solely attributable to RA. Values are means from four to five independent assays ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 statistical differences calculated by one way Annova and Newman-Keuls Post test.</p

Additive Effects of Retinoic Acid (RA) and Bone Morphogenetic Protein 4 (BMP-4) Apoptosis Signaling in Retinoblastoma Cell Lines

<div><p>Retinoids have been shown to serve promising therapeutic agents for human cancers, e.g. the treatment of neuroblastoma. Synthetic retinoids, specific for particular retinoic acid (RA) receptors, are tested as new therapy strategies. In the present study, application of recombinant retinoic acid (RA) lowers retinoblastoma (RB) cell viability and induces apoptosis in RB cell lines. Combined treatment of RA and bone morphogenetic protein 4 (BMP-4) increases the pro-apoptotic effect of RA in the RB cells lines WERI-Rb1, Y-79, RB355, RBL-30 and RBL-15, indicating an additive effect. We could show that in WERI-Rb1 cells RA/BMP-4 mediated cell death is at least partially caspase-dependent, whereby RA and BMP-4 additively increased (i) Apaf-1 mRNA levels, (ii) caspase-9 cleavage activity and (iii) the number of activated, cleaved caspase-3 positive cells. Compared to single application of RA and BMP-4, combined RA/BMP-4 treatment significantly augments mRNA levels of the retinoic acid receptors (RARs) <i>RARα</i> and <i>RARß</i> and the retinoic X receptor (RXR) <i>RXRγ</i> suggesting an interaction in the induction of these RA receptor subtypes in WERI-Rb1 cells. Agonist studies revealed that both, RARs and RXRs are involved in RA/BMP-4 mediated apoptosis in WERI-Rb1 retinoblastoma cells. Employing specific RAR subtype antagonists and a <i>RXRß</i> and <i>RXRγ</i> knockdown, we proved that RA/BMP-4 apoptosis signaling in WERI-Rb1 cells requires the RA receptor subtypes RARα, RARß, RXRß and RXRγ. Deciphering signaling mechanisms underlying apoptosis induction of RA and BMP-4 in WERI-Rb1 cells, our study provides useful starting-points for future retinoid-based therapy strategies in retinoblastoma.</p></div

<i>RXRß</i> knockdown and <i>RXRß/RXRγ</i> double knockdown studies.

<p>WERI-Rb1 cells with an <i>RXR</i>ß knockdown (sh RXRß) and/or an <i>RXRγ</i> knockdown (sh RXRγ<i>)</i> and non-silencing controls were treated for 24 h with a combination of RA and BMP-4 in the presence (+) or absence (-) of a RARα antagonist (ER50891) and a RARß antagonist (LE135). Values are means ± SEM. *P < 0.05; ***P < 0.001 statistical differences calculated by one way Annova and Newman-Keuls Post test. n.s.: no significant statistical difference.</p

Involvement of caspases in RA and BMP-4 mediated apoptosis induction in WERI-Rb1 cells.

<p>A, Blockage experiments with a broad spectrum caspase inhibitor (Boc-D-fmk) in the presence (+) or absence (-) of RA and BMP-4. B, Quantification of cleaved (= activated) caspase-3 positive cells after treatment with RA, BMP-4 or both factors. C-H, Immunocytochemical caspase-3 staining. I, Up-regulation of caspase-9 cleavage activity following administration of RA, BMP-4 or a combination of both. Untreated cells, revealing the endogenous caspase-9 cleavage activity, served as a standard and were set as 1. Cells treated with the solvent for RA and BMP-4 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131467#sec002" target="_blank">material and method</a>), respectively, served as controls (ctr.). J, Induction of <i>Apaf-1</i> upon application of RA, BMP-4 and RA+BMP-4 as revealed by Real-time PCR. Messenger RNA expression levels at the beginning of the treatment (0h) were used as a reference and set as 1. Values are means from 3 independent experiments ± SEM. *P < 0.05;**P < 0.01; ***P < 0.001 statistical differences calculated by Student`s <i>t</i>-test (A) or statistical differences calculated by one way Annova and Newman-Keuls Post test (B,I,J).</p

Effect of RA treatment on cell viability, cell proliferation and cell death in the retinoblastoma cell line WERI-Rb1.

<p>Cells were grown in medium containing indicated concentrations of RA or ethanol (control; set as 100%). Application of RA decreased the viability of WERI-Rb1 cells as determined by WST-1 assays (A). Cell counts from BrdU incorporation experiments (B) and counts of DAPI-positive, pycnotic nuclei (C) were performed to determine proliferation and apoptosis rates, respectively. Application of increasing amounts of RA had no significant effect on the number of BrdU-positive, proliferating cells (B), but significantly induced apoptosis in WERI-Rb1 cells (C).*P < 0.05; **P < 0.01 significant statistical differences compared to the control group calculated by Student`s <i>t</i>-test. n.s.: no significant statistical difference.</p

Application scheme.

<p>^aBOC-D-fmk: caspase inhibitor</p><p>^bTTNPB: RAR agonist</p><p>^cmethoprene acid: RXR agonist</p><p>^dER50891: RARα antagonist</p><p>^eLE135: RARß antagonist.</p><p>Application scheme.</p

FACS analyses of WERI-Rb1 cells.

<p>WERI-Rb1 cells were treated for 48 h with two fix concentrations of RA (0.5μM and 5μM; A,B) or BMP-4 (10 ng/ ml and 40 ng/ml; C,D) and rising concentrations of BMP-4 (1–40 ng/ml) or RA (0.05–10 μM), respectively. Under these conditions, RA and BMP-4 additively and dose-dependently induced an increase in subG0/G1, representing apoptotic cells. Gray bars: untreated control cells; white bars: cells treated (+) or not (-) with equivalent concentrations of HCl/BSA, the solvent for BMP-4, ethanol, the solvent for RA, or both. Values are means from 3–4 independent assays ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 statistical differences calculated by one way Annova and Newman-Keuls Post test.</p

RAR and RXR agonist studies.

<p>Using 5 μM TTNPB, a stable RAR agonist (A) and 5 μM methoprene acid, a RXR agonist (B) in the presence (+) or absence (-) of RA and BMP-4 revealed the involvement of both RA receptor subtypes in RA and RA/BMP-4 mediated apoptosis induction in WERI-Rb1 cells. Values are means from two to three independent experiments ± SEM. **P < 0.01; ***P < 0.001 statistical differences calculated by one way Annova and Newman-Keuls Post test.</p

<i>RARα</i> and <i>RARß</i> antagonist (A) and <i>RXR</i>γ knockdown (B) studies.

<p>A, Application (+) of ER50891, a specific RARα antagonist or LE 135, a selectively RARß antagonist for 24 h disclosed the involvement of both RAR subtypes in RA/BMP-4 mediated apoptosis induction in WERI-Rb1 cells. B, WERI-Rb1 cells with a <i>RXR</i>γ knockdown (sh RXRγ) and non-silencing controls were treated for 24 h with RA or a combination of RA and BMP-4 in the presence (+) or absence (-) of a RARα antagonist (ER 50891) and a RARß antagonist (LE 135). Values are means from 3 independent assays ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 statistical differences calculated by one way Annova and Newman-Keuls Post test.</p

Dance training is superior to repetitive physical exercise in inducing brain plasticity in the elderly

<div><p>Animal research indicates that a combination of physical activity and sensory enrichment has the largest and the only sustaining effect on adult neuroplasticity. Dancing has been suggested as a human homologue to this combined intervention as it poses demands on both physical and cognitive functions. For the present exploratory study, we designed an especially challenging dance program in which our elderly participants constantly had to learn novel and increasingly difficult choreographies. This six-month-long program was compared to conventional fitness training matched for intensity. An extensive pre/post-assessment was performed on the 38 participants (63–80 y), covering general cognition, attention, memory, postural and cardio-respiratory performance, neurotrophic factors and–most crucially–structural MRI using an exploratory analysis. For analysis of MRI data, a new method of voxel-based morphometry (VBM) designed specifically for pairwise longitudinal group comparisons was employed. Both interventions increased physical fitness to the same extent. Pronounced differences were seen in the effects on brain volumes: Dancing compared to conventional fitness activity led to larger volume increases in more brain areas, including the cingulate cortex, insula, corpus callosum and sensorimotor cortex. Only dancing was associated with an increase in plasma BDNF levels. Regarding cognition, both groups improved in attention and spatial memory, but no significant group differences emerged. The latter finding may indicate that cognitive benefits may develop later and after structural brain changes have taken place. The present results recommend our challenging dance program as an effective measure to counteract detrimental effects of aging on the brain.</p></div