Identification of the Axis ß-Catenin-BTK in the Dynamic Adhesion of Chronic Lymphocytic Leukemia Cells to Their Microenvironment.

In the microenvironment, cell interactions are established between different cell types to regulate their migration, survival and activation. ß-Catenin is a multifunctional protein that stabilizes cell-cell interactions and regulates cell survival through its transcriptional activity. We used chronic lymphocytic leukemia (CLL) cells as a cellular model to study the role of ß-catenin in regulating the adhesion of tumor cells to their microenvironment, which is necessary for tumor cell survival and accumulation. When co-cultured with a stromal cell line (HS-5), a fraction of the CLL cells adhere to stromal cells in a dynamic fashion regulated by the different levels of ß-catenin expression. In non-adherent cells, ß-catenin is stabilized in the cytosol and translocates into the nucleus, increasing the expression of cyclin D1. In adherent cells, the level of cytosolic ß-catenin is low but membrane ß-catenin helps to stabilize the adhesion of CLL to stromal cells. Indeed, the overexpression of ß-catenin enhances the interaction of CLL with HS-5 cells, suggesting that this protein behaves as a regulator of cell adhesion to the stromal component and of the transcriptional regulation of cell survival. Inhibitors that block the stabilization of ß-catenin alter this equilibrium and effectively disrupt the support that CLL cells receive from the cross-talk with the stroma.

A comparative study of Caesalpinia bonduc (L.) Roxb. root extracts on sexual behaviour in male Wistar rats.

Caesalpinia bonduc is among the traditionally used plant in Benin, for its enhancement of male sexual activity. This study was undertaken to investigate the potential effect of C. bonduc root extracts on sexual behaviour of male Wistar rats. For that, thirty-six rats were allocated into six groups and orally treated with dimethyl sulfoxide (control), Sildenafil citrate (standard) and C. bonduc root extracts (hexane, ethyl acetate, ethanol and methanol) orally for twenty-eight days. Sexual behaviour parameters such as intromission frequency, intromission latency, mount latency and mount frequency were evaluated on day 0, 14 and 28. After the study completion, the body and reproductive organ weights as well as testicular histology and testosterone level were recorded. C. bonduc root extracts treatments had no significant effect on the body weight of rats. Enhancement in sexual behaviour was observed in ethanolic extract treated rats. An significant increase in mount frequency and intromission frequency as well as significant reduction in mount latency and intromission latency were noticed for ethanolic extract. The same extract caused an improvement in testosterone levels, relative testes weight and histological architecture. The findings demonstrate the aphrodisiac potential of C. bonduc root and lend support to the folkloric use as aphrodisiac.

High Glucose Level Impairs Human Mature Bone Marrow Adipocyte Function Through Increased ROS Production.

Bone marrow adipocytes (BMAds) accumulate in aging, menopause, and metabolic diseases such as Type 2 diabetes. These osteoporotic conditions are associated with oxidative stress and hyperglycemia which are both considered as critical factors underlying bone fragility. Glucose excess and reactive oxygen species (ROS) are known to favor adipogenesis over osteoblastogenesis. In this study, we investigated whether high glucose exposure could determine dysfunction of mature BMAds, specifically through ROS production. The effects of low (LG, 5 mM) or high glucose (HG, 25 mM) concentrations were examined using human bone mesenchymal stromal cells (hBMSCs) in the time course of differentiation, and, up to 21 days once adipocytes were mature. HG did not alter the adipocyte differentiation process of hBMSCs. Yet, after 21 days under HG exposure, PPARG, CEBPA, and adiponectin mRNA expressions were decreased. These alterations were also observed following adipogenic inducer withdrawal as well as in adipocytes fully differentiated in LG then cultured in HG for the last 11 days. Without inducers, HG condition also led to decreased leptin mRNA level. Importantly, intracellular and extracellular ROS concentrations measured using Amplex Red were significantly raised by 50% under HG exposure. This rise was observed once adipocytes ended differentiation and was reproduced within the different cell culture settings without any cytotoxicity. Among genes involved in ROS metabolism, the mRNA level of the H2O2 generating enzyme NOX4 was found upregulated in the presence of HG. Following cell separation, mature BMAds were shown to overproduce ROS and to display the gene alterations in contrast to non-lipid-laden cells. Finally, a non-lethal treatment with a pro-oxidant agent under LG condition reduces the mRNA levels of PPARG, adiponectin, and leptin as the HG condition does in the absence of inducers, and amplifies the effect of glucose excess on gene expression. HG concentration drives mature BMAds toward altered expression of the main adipokines and transcriptional factors. These perturbations are associated with a rise in ROS generation likely mediated through enhanced expression of NOX4. Mature BMAds are thus responsive to changes in glucose and ROS concentrations, which is relevant regarding with their phenotype and function in age- or metabolic disease-related osteoporosis.

MECHANISMS IN ENDOCRINOLOGY: Bone marrow adiposity and bone, a bad romance?

Bone marrow adipocytes (BMA-) constitute an original and heterogeneous fat depot whose development appears interlinked with bone status throughout life. The gradual replacement of the haematopoietic tissue by BMA arises in a well-ordered way during childhood and adolescence concomitantly to bone growth and continues at a slower rate throughout the adult life. Importantly, BM adiposity quantity is found well associated with bone mineral density (BMD) loss at different skeletal sites in primary osteoporosis such as in ageing or menopause but also in secondary osteoporosis consecutive to anorexia nervosa. Since BMA and osteoblasts originate from a common mesenchymal stem cell, adipogenesis is considered as a competitive process that disrupts osteoblastogenesis. Besides, most factors secreted by bone and bone marrow cells (ligands and antagonists of the WNT/ß-catenin pathway, BMP and others) reciprocally regulate the two processes. Hormones such as oestrogens, glucocorticoids, parathyroid and growth hormones that control bone remodelling also modulate the differentiation and the activity of BMA. Actually, BMA could also contribute to bone loss through the release of paracrine factors altering osteoblast and/or osteoclast formation and function. Based on clinical and fundamental studies, this review aims at presenting and discussing these current arguments that support but also challenge the involvement of BMA in the bone mass integrity.

Ascorbic acid alters cell fate commitment of human neural progenitors in a WNT/ß-catenin/ROS signaling dependent manner.

BACKGROUND: Improving the neuronal yield from in vitro cultivated neural progenitor cells (NPCs) is an essential challenge in transplantation therapy in neurological disorders. In this regard, Ascorbic acid (AA) is widely used to expand neurogenesis from NPCs in cultures although the mechanisms of its action remain unclear. Neurogenesis from NPCs is regulated by the redox-sensitive WNT/ß-catenin signaling pathway. We therefore aimed to investigate how AA interacts with this pathway and potentiates neurogenesis. METHODS: Effects of 200 µM AA were compared with the pro-neurogenic reagent and WNT/ß-catenin signaling agonist lithium chloride (LiCl), and molecules with antioxidant activities i.e. N-acetyl-L-cysteine (NAC) and ruthenium red (RuR), in differentiating neural progenitor ReNcell VM cells. Cells were supplemented with reagents for two periods of treatment: a full period encompassing the whole differentiation process versus an early short period that is restricted to the cell fate commitment stage. Intracellular redox balance and reactive oxygen species (ROS) metabolism were examined by flow cytometry using redox and ROS sensors. Confocal microscopy was performed to assess cell viability, neuronal yield, and levels of two proteins: Nucleoredoxin (NXN) and the WNT/ß-catenin signaling component Dishevelled 2 (DVL2). TUBB3 and MYC gene responses were evaluated by quantitative real-time PCR. DVL2-NXN complex dissociation was measured by fluorescence resonance energy transfer (FRET). RESULTS: In contrast to NAC which predictably exhibited an antioxidant effect, AA treatment enhanced ROS metabolism with no cytotoxic induction. Both drugs altered ROS levels only at the early stage of the differentiation as no changes were held beyond the neuronal fate commitment stage. FRET studies showed that AA treatment accelerated the redox-dependent release of the initial pool of DVL2 from its sequestration by NXN, while RuR treatment hampered the dissociation of the two proteins. Accordingly, AA increased WNT/ß-catenin signaling output i.e. MYC mRNA level, whereas RuR attenuated it. Moreover, AA improved neurogenesis as much as LiCl as both TUBB3-positive cell yield and TUBB3 mRNA level increased, while NAC or RuR attenuated neurogenesis. Markedly, the neurogenesis outputs between the short and the full treatment with either NAC or AA were found unchanged, supporting our model that neuronal yield is altered by events taking place at the early phase of differentiation. CONCLUSIONS: Our findings demonstrate that AA treatment elevates ROS metabolism in a non-lethal manner prior to the NPCs commitment to their neuronal fate. Such effect stimulates the redox-sensitive DVL2 activation and WNT/ß-catenin signaling response that would enhance the ensuing neuronal cell differentiation.

Bone Marrow Adipose Tissue: To Be or Not To Be a Typical Adipose Tissue?

Bone marrow adipose tissue (BMAT) emerges as a distinct fat depot whose importance has been proved in the bone-fat interaction. Indeed, it is well recognized that adipokines and free fatty acids released by adipocytes can directly or indirectly interfere with cells of bone remodeling or hematopoiesis. In pathological states, such as osteoporosis, each of adipose tissues - subcutaneous white adipose tissue (WAT), visceral WAT, brown adipose tissue (BAT), and BMAT - is differently associated with bone mineral density (BMD) variations. However, compared with the other fat depots, BMAT displays striking features that makes it a substantial actor in bone alterations. BMAT quantity is well associated with BMD loss in aging, menopause, and other metabolic conditions, such as anorexia nervosa. Consequently, BMAT is sensed as a relevant marker of a compromised bone integrity. However, analyses of BMAT development in metabolic diseases (obesity and diabetes) are scarce and should be, thus, more systematically addressed to better apprehend the bone modifications in that pathophysiological contexts. Moreover, bone marrow (BM) adipogenesis occurs throughout the whole life at different rates. Following an ordered spatiotemporal expansion, BMAT has turned to be a heterogeneous fat depot whose adipocytes diverge in their phenotype and their response to stimuli according to their location in bone and BM. In vitro, in vivo, and clinical studies point to a detrimental role of BM adipocytes (BMAs) throughout the release of paracrine factors that modulate osteoblast and/or osteoclast formation and function. However, the anatomical dissemination and the difficulties to access BMAs still hamper our understanding of the relative contribution of BMAT secretions compared with those of peripheral adipose tissues. A further characterization of the phenotype and the functional regulation of BMAs are ever more required. Based on currently available data and comparison with other fat tissues, this review addresses the originality of the BMAT with regard to its development, anatomy, metabolic properties, and response to physiological cues.

Oxidative stress does not play a primary role in the toxicity induced with clinical doses of doxorubicin in myocardial H9c2 cells.

The implication of oxidative stress as primary mechanism inducing doxorubicin (DOX) cardiotoxicity is still questionable as many in vitro studies implied supra-clinical drug doses or unreliable methodologies for reactive oxygen species (ROS) detection. The aim of this study was to clarify whether oxidative stress is involved in compliance with the conditions of clinical use of DOX, and using reliable tools for ROS detection. We examined the cytotoxic mechanisms of 2 µM DOX 1 day after the beginning of the treatment in differentiated H9c2 rat embryonic cardiac cells. Cells were exposed for 2 or 24 h with DOX to mimic a single chronic dosage or to favor accumulation, respectively. We found that apoptosis was prevalent in cells exposed for a short period with DOX: cells showed typical hallmarks as loss of anchorage ability, mitochondrial hyperpolarization followed by the collapse of mitochondrial activity, and nuclear condensation. Increasing the exposure period favored a shift to necrosis as the cells preferentially exhibited early DNA impairment and nuclear swelling. In either case, measuring the fluorescence lifetime of 1-pyrenebutyric acid or the intensities of dihydroethidium or amplex red showed a consistent pattern in ROS production which was a slight increased level far from representative of an oxidative stress. Moreover, pre-treatment with dexrazoxane provided a cytoprotective effect although it failed to detoxify ROS. Our data support that oxidative stress is unlikely to be the primary mechanism of DOX cardiac toxicity in vitro.

Spatio-temporal model of endogenous ROS and raft-dependent WNT/beta-catenin signaling driving cell fate commitment in human neural progenitor cells.

Canonical WNT/ß-catenin signaling is a central pathway in embryonic development, but it is also connected to a number of cancers and developmental disorders. Here we apply a combined in-vitro and in-silico approach to investigate the spatio-temporal regulation of WNT/ß-catenin signaling during the early neural differentiation process of human neural progenitors cells (hNPCs), which form a new prospect for replacement therapies in the context of neurodegenerative diseases. Experimental measurements indicate a second signal mechanism, in addition to canonical WNT signaling, being involved in the regulation of nuclear ß-catenin levels during the cell fate commitment phase of neural differentiation. We find that the biphasic activation of ß-catenin signaling observed experimentally can only be explained through a model that combines Reactive Oxygen Species (ROS) and raft dependent WNT/ß-catenin signaling. Accordingly after initiation of differentiation endogenous ROS activates DVL in a redox-dependent manner leading to a transient activation of down-stream ß-catenin signaling, followed by continuous auto/paracrine WNT signaling, which crucially depends on lipid rafts. Our simulation studies further illustrate the elaborate spatio-temporal regulation of DVL, which, depending on its concentration and localization, may either act as direct inducer of the transient ROS/ß-catenin signal or as amplifier during continuous auto-/parcrine WNT/ß-catenin signaling. In addition we provide the first stochastic computational model of WNT/ß-catenin signaling that combines membrane-related and intracellular processes, including lipid rafts/receptor dynamics as well as WNT- and ROS-dependent ß-catenin activation. The model's predictive ability is demonstrated under a wide range of varying conditions for in-vitro and in-silico reference data sets. Our in-silico approach is realized in a multi-level rule-based language, that facilitates the extension and modification of the model. Thus, our results provide both new insights and means to further our understanding of canonical WNT/ß-catenin signaling and the role of ROS as intracellular signaling mediator.

Ca2+-mediated mitochondrial reactive oxygen species metabolism augments Wnt/ß-catenin pathway activation to facilitate cell differentiation.

Emerging evidence suggests that reactive oxygen species (ROS) can stimulate the Wnt/ß-catenin pathway in a number of cellular processes. However, potential sources of endogenous ROS have not been thoroughly explored. Here, we show that growth factor depletion in human neural progenitor cells induces ROS production in mitochondria. Elevated ROS levels augment activation of Wnt/ß-catenin signaling that regulates neural differentiation. We find that growth factor depletion stimulates the release of Ca(2+) from the endoplasmic reticulum stores. Ca(2+) subsequently accumulates in the mitochondria and triggers ROS production. The inhibition of mitochondrial Ca(2+) uptake with simultaneous growth factor depletion prevents the rise in ROS metabolism. Moreover, low ROS levels block the dissociation of the Wnt effector Dishevelled from nucleoredoxin. Attenuation of the response amplitudes of pathway effectors delays the onset of the Wnt/ß-catenin pathway activation and results in markedly impaired neuronal differentiation. Our findings reveal Ca(2+)-mediated ROS metabolic cues that fine-tune the efficiency of cell differentiation by modulating the extent of the Wnt/ß-catenin signaling output.

Adriamycin dose and time effects on cell cycle, cell death, and reactive oxygen species generation in leukaemia cells.

We investigate the relative importance of the different mechanisms of Adriamycin, an anthracycline, and their interrelations, in particular the link between cell cycle arrest, cell death, and generation of reactive oxygen species (ROS) that is suspected to be the origin of cardiotoxic side-effects. We introduced a lifetime fluorescence based technology and used videomicrofluorometry, two efficient analytical methods. We show that depending on the doses and time after incubation, ADR will not reach the same compartments (nucleus, mitochondria, cytosol) in the cells, having consequences on the production of ROS, growth arrest pathways and cell death pathways.

New method for the detection of reactive oxygen species in anti-tumoural activity of adriamycin: a comparison between hypoxic and normoxic cells.

Tumour hypoxia plays a role in chemoresistance in several human tumours. However, how hyperbaric oxygen leads to chemotherapeutic gain is unclear. This study investigates the relation of reactive oxygen species (ROS) generation with anti-tumoural effect of adriamycin (ADR) on CCRF-CEM cells under hypoxic (2% O(2)) and normoxic (21% O(2)) conditions. A new method was used to measure intracellular ROS variations through the fluorescence lifetime of 1-pyrenebutyric acid. At 24 h, ADR, probably via semiquinone radical, enhances ROS levels in normoxic cells compared to hypoxic cells. Long-term studies show that ROS are also generated by a second mechanism related to cell functions perturbation. ADR arrests the cell cycle progression both under hypoxia and normoxia, indicating that oxygen and ROS does not influence the DNA damaging activity of ADR. The findings reveal that moderate improvement of ADR cytotoxicity results from higher ROS formation in normoxic cells, leading to elevated induction of cell death.

Variation of 1-pyrenebutyric acid fluorescence lifetime in single living cells treated with molecules increasing or decreasing reactive oxygen species levels.

1-Pyrenebutyric acid (PBA) is a fluorescent probe whose fluorescence lifetime depends on local oxygen and free radical concentrations. We propose to use PBA fluorescence lifetime to quantify reactive oxygen species (ROS) in biological samples. Time-resolved microfluorimetry was used to record the fluorescence decay of single living cells loaded with this probe. We measured intracellular PBA fluorescence lifetimes and reduced nicotinamide adenine dinucleotide phosphate intensities under various oxygen concentrations. To confirm the feasibility of the new method, CCRF-CEM cells were treated with drugs that are known to increase or decrease ROS production. After treatment with adriamycin, we observed a decrease of PBA fluorescence lifetime. This corresponded to an increase of ROS concentration (80%). After treatment with cysteamine, we observed a reduction of the ROS concentration by 67%. Moreover, addition of exogenous H(2)O(2) solution resulted in a decrease of PBA fluorescence lifetime due to a raising of the intracellular ROS concentration. These results support our hypothesis linking a part of PBA fluorescence lifetime variations to intracellular fluctuation of ROS.

Effect of adriamycin treatment on the lifetime of pyrene butyric acid in single living cells.

We investigated the fluorescence lifetime of pyrene butyric acid (PBA) using various O2 concentrations in cells. Both in living and freshly fixed cells, PBA lifetime decreased with oxygen concentration. We recorded decay curves in single cells and measured PBA lifetime and NAD(P)H intensity values. Under nitrogen atmosphere, the probe lifetime differences (199 and 209 ns in living and freshly fixed cells, respectively) suggest a supplemental pathway for the deactivation of the probe when the cell functions are not stopped. We propose reactive oxygen species (ROS) to be the additional quenchers that cause this decrease. We further studied the effect of drugs generating ROS the anthracycline doxorubicin (adriamycin). For living cells, PBA lifetime decreased after adriamycin (ADR) treatment (200 and 1000 ng/ml). This supports our hypothesis that under nitrogen atmosphere and for freshly fixed cells, PBA lifetimes increase to an unchanging value due to absence of quenchers.