Mohamed M. Bekhite

Mimicry of a constitutively active pre–B cell receptor in acute lymphoblastic leukemia cells

Pre–B cells undergo apoptosis unless they are rescued by pre–B cell receptor–dependent survival signals. We previously showed that the BCR-ABL1 kinase that is expressed in pre–B lymphoblastic leukemia bypasses selection for pre–B cell receptor–dependent survival signals. Investigating possible interference of BCR-ABL1 with pre–B cell receptor signaling, we found that neither SYK nor SLP65 can be phosphorylated in response to pre–B cell receptor engagement. Instead, Brutons tyrosine kinase (BTK) is constitutively phosphorylated by BCR-ABL1. Activated BTK is essential for survival signals that otherwise would arise from the pre–B cell receptor, including activation of PLCγ1, autonomous Ca2+ signaling, STAT5-phosphorylation, and up-regulation of BCLX L. Inhibition of BTK activity specifically induces apoptosis in BCR-ABL1 + leukemia cells to a similar extent as inhibition of BCR-ABL1 kinase activity itself. However, BCR-ABL1 cannot directly bind to full-length BTK. Instead, BCR-ABL1 induces the expression of a truncated splice variant of BTK that acts as a linker between the two kinases. As opposed to full-length BTK, truncated BTK lacks kinase activity yet can bind to BCR-ABL1 through its SRC-homology domain 3. Acting as a linker, truncated BTK enables BCR-ABL1–dependent activation of full-length BTK, which initiates downstream survival signals and mimics a constitutively active pre–B cell receptor.

Regulation of the multidrug resistance transporter P-glycoprotein in multicellular prostate tumor spheroids by hyperthermia and reactive oxygen species.

Hyperthermia is an important component of many cancer treatment protocols. In our study the regulation of the multidrug resistance (MDR) transporter P‐glycoprotein by hyperthermia was studied in multicellular prostate tumor spheroids. Hyperthermia treatment of small (50–100 μm) tumor spheroids significantly increased P‐glycoprotein and mdr‐1 mRNA expression with a maximum effect at 42°C, whereas only moderate elevation of P‐glycoprotein was found in large (350–450 μm) tumor spheroids. Hyperthermia caused an elevation of intracellular reactive oxygen species (ROS). Inhibition of ROS generation with NADPH‐oxidase inhibitors diphenylen iodonium (DPI) and 4‐(2‐aminoethyl)benzenesulfonyl fluoride (AEBSF) abolished P‐glycoprotein expression but did not affect its transcript levels following heat treatment. This indicates that P‐glycoprotein levels are controlled by regulating its translation rate or stability. Hyperthermia incubation resulted in a differential activation of p38 mitogen‐activated protein kinase (MAPK), extracellular regulated kinase 1,2 (ERK1,2), and c‐jun N‐terminal kinase (JNK) immediately, 4 hr and 24 hr after treatment. Furthermore, upregulation of hypoxia‐inducible factor 1α (HIF‐1α) was observed. Elevation of HIF‐1α and P‐glycoprotein expression following hyperthermia treatment were abolished upon coadministration of the p38 inhibitor SB203580. In contrast the JNK inhibitor SP600125 and the ERK1,2 inhibitor UO126 resulted in increase of HIF‐1α and P‐glycoprotein in the control as well as the hyperthermia‐treated samples, indicating negative regulation of intrinsic HIF‐1α and P‐glycoprotein expression by ERK1,2 and JNK signaling cascades. In summary our data demonstrate that hyperthermia‐induced upregulation of P‐glycoprotein and HIF‐1α is mediated by activation of p38, whereas ERK1,2 and JNK are involved in repression of P‐glycoprotein and HIF‐1α under control conditions.

VEGF-mediated PI3K class IA and PKC signaling in cardiomyogenesis and vasculogenesis of mouse embryonic stem cells.

VEGF-, phosphoinositide 3-kinase (PI3K)- and protein kinase C (PKC)-regulated signaling in cardiac and vascular differentiation was investigated in mouse ES cells and in ES cell-derived Flk-1+ cardiovascular progenitor cells. Inhibition of PI3K by wortmannin and LY294002, disruption of PI3K catalytic subunits p110α and p110δ using short hairpin RNA (shRNA), or inhibition of p110α with compound 15e and of p110δ with IC-87114 impaired cardiac and vascular differentiation. By contrast, TGX-221, an inhibitor of p110β, and shRNA knockdown of p110β were without significant effects. Antagonists of the PKC family, i.e. bisindolylmaleimide-1 (BIM-1), GÖ 6976 (targeting PKCα/βII) and rottlerin (targeting PKCδ) abolished vasculogenesis, but not cardiomyogenesis. Inhibition of Akt blunted cardiac as well as vascular differentiation. VEGF induced phosphorylation of PKCα/βII and PKCδ but not PKCζ. This was abolished by PI3K inhibitors and the VEGFR-2 antagonist SU5614. Furthermore, phosphorylation of Akt and phosphoinositide-dependent kinase-1 (PDK1) was blunted upon inhibition of PI3K, but not upon inhibition of PKC by BIM-1, suggesting that activation of Akt and PDK1 by VEGF required PI3K but not PKC. In summary, we demonstrate that PI3K catalytic subunits p110α and p110δ are central to cardiovasculogenesis of ES cells. Akt downstream of PI3K is involved in both cardiomyogenesis and vasculogenesis, whereas PKC is involved only in vasculogenesis.

Hypoxia, leptin, and vascular endothelial growth factor stimulate vascular endothelial cell differentiation of human adipose tissue-derived stem cells.

The plasticity of human adipose tissue-derived stem cells (hASCs) is promising, but differentiation in vitro toward endothelial cells is poorly understood. Flow cytometry demonstrated that hASCs isolated from excised fat tissue were positive for CD29, CD44, CD70, CD90, CD105, and CD166 and negative for the endothelial marker CD31, and the hematopoietic cell markers CD34 and CD133. hASCs differentiated into adipocytes after cultivation in adipogenic medium. Exposure of hASCs for 10 days under hypoxia (3% oxygen) in combination with leptin increased the percentage of CD31(+) endothelial cells as well as CD31, VE-Cadherin, Flk-1, Tie2, von Willebrand factor, and endothelial cell nitric oxide synthase mRNA expression. This was enhanced on co-incubation of vascular endothelial growth factor (VEGF) and leptin, whereas VEGF alone was not sufficient. Moreover, hASCs cultured on a matrigel surface under hypoxia/VEGF/leptin, showed a stable branching network. Hypoxic conditions significantly decreased apoptosis as evaluated by cleaved caspase-3, and increased prolyl hydroxylase domain 3 mRNA expression. Hypoxia increased expression of VEGF as well as leptin transcripts, which were significantly inhibited on co-incubation with either VEGF or leptin or a combination of both. Furthermore, leptin treatment of hypoxic cells increased the expression of the long/signaling form of the leptin receptor (ObRL), which was augmented on co-incubation with VEGF. The observed endothelial differentiation was dependent on the Akt pathway, as co-administration with Akt inhibitor abolished the observed effects. In conclusion, our data demonstrate that hASCs can be efficiently differentiated to endothelial cells by mimicking the hypoxic and pro-angiogenic microenvironment of adipose tissue.

Static Electromagnetic Fields Induce Vasculogenesis and Chondro-Osteogenesis of Mouse Embryonic Stem Cells by Reactive Oxygen Species-Mediated Up-Regulation of Vascular Endothelial Growth Factor

Electromagnetic fields (EMFs) are used to treat bone diseases. Herein, the effects of static EMFs on chondroosteogenesis and vasculogenesis of embryonic stem (ES) cells and bone mineralization of mouse fetuses were investigated. Treatment of differentiating ES cells with static EMFs (0.4-2 mT) stimulated vasculogenesis and chondro-osteogenesis and increased reactive oxygen species (ROS), which was abolished by the free radical scavengers trolox, 1,10-phenanthroline (phen), and the NAD(P)H oxidase inhibitor diphenylen iodonium (DPI). In contrast, EMFs of 10 mT field strength exerted inhibitory effects on vasculogenesis and chondro-osteogenesis despite robust ROS generation. EMFs of 1 mT and 10 mT increased and decreased vascular endothelial growth factor (VEGF) expression, respectively, which was abolished by DPI and radical scavengers. EMFs activated extracellular-regulated kinase 1/2 (ERK1/2), p38, and c-jun N-terminal kinase (JNK), which was sensitive to DPI treatment. The increase in VEGF by EMFs was inhibited by the ERK1/2 inhibitor U0126 but not by SB203580 and SP600125, which are p38 and JNK inhibitors, respectively, suggesting VEGF regulation by ERK1/2. Chondroosteogenesis and vasculogenesis of ES cells was blunted by trolox, DPI, and the VEGF receptor-2 (flk-1) antagonist SU5614. In mouse fetuses 1 mT EMFs increased and 10 mT EMFs decreased bone mineralization, which was abolished in the presence of trolox. Hence, EMFs induced chondro-osteogenesis and vasculogenesis in ES cells and bone mineralization of mouse fetuses by a ROS-dependent up-regulation of VEGF expression.

Redox Stimulation of Cardiomyogenesis Versus Inhibition of Vasculogenesis Upon Treatment of Mouse Embryonic Stem Cells with Thalidomide

Thalidomide [α-(N-phthalimido)-glutarimide] exerts antiangiogenic properties and causes cardiac malformations in embryos. Herein the effects of thalidomide on cardiovascular differentiation were investigated in mouse embryonic stem (ES) cell-derived embryoid bodies. Thalidomide inhibited the formation of capillary-like blood vessels and decreased tumor-induced angiogenesis in confrontation cultures of embryoid bodies and multicellular prostate tumor spheroids, but stimulated cardiomyogenesis of ES cells. The number of CD31- and CD144-positive endothelial cells was not impaired, suggesting that thalidomide acted on vascular tube formation and cell migration rather than endothelial differentiation. Thalidomide increased reactive oxygen species generation, which was abolished by the NADPH oxidase inhibitor VAS2870 and the complex I respiratory chain inhibitor rotenone. Conversely, thalidomide decreased nitric oxide (NO) generation and endothelial NO synthase activity. VAS2870 abrogated thalidomide stimulation of cardiomyogenesis, whereas inhibition of vasculogenesis persisted. In NOX-1 and NOX-4 shRNA gene-inactivated ES cells, cardiomyogenesis was severely impaired and thalidomide failed to stimulate cardiac cell commitment. The NO donor S-nitrosopenicillamine reversed the antiangiogenic effect of thalidomide and increased capillary structure formation, whereas scavenging NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and inhibition of endothelial NO synthase by N(G)-nitro-l-arginine methyl ester decreased cardiovascular differentiation. Our data demonstrate that thalidomide causes an imbalance of reactive oxygen species/NO generation, thus stimulating cardiomyogenesis and impairing vascular sprout formation.

Antibacterial Capacity of Differentiated Murine Embryonic Stem Cells During Defined In Vitro Inflammatory Conditions

Embryonic stem (ES) cells are a powerful model for the development of cells responsible for the cellular immune response. Therefore, we analyzed the defense and phagocytic capacity of embryoid bodies (EBs) derived from ES cells using in the vitro inflammatory conditions caused by Escherichia coli. Further, we used this phagocytic activity to purify activated immune cells. Our data show that spontaneously differentiated 18-day-old EBs of the cell line CGR8 contained immune cells, which were positive for CD45, CD68, CD11b, F4/80, and CD19. Exposure of these EBs to E. coli with defined infection doses of bacterial colony-forming units (CFUs) led to a significant time-dependent reduction of CFUs, indicating the immune responses exerted by EBs. This was paralleled by an upregulation of inflammatory cytokines, that is, IL-1β and TNF-α. Western blot analysis of infected EBs indicated an upregulation of CD14 and cytochrome b-245 heavy chain (NOX2). Silencing of NOX2 significantly reduced the antibacterial capacity of EBs, which was partially explained by reduction of F4/80-positive cells. To identify, isolate, and further cultivate phagocytic active cells from differentiated EBs, a cocultivation assay of differentiated ES cells with green fluorescent protein (GFP)-labeled E. coli was established. Colocalization of GFP-labeled E. coli with cells positive for CD45, CD68, and F4/80 revealed time-dependent phagocytotic uptake, which was underlined by colocalization with the LysoTracker-Red(®) dye as well as preincubation with cytochalasin D. In conclusion, a primitive immune response with efficient phagocytosis was responsible for the antibacterial capacity of differentiated EBs.

Embryonic Stem Cells for Tissue Biocompatibility, Angiogenesis, and Inflammation Testing

Aim: To introduce embryoid bodies derived from mouse embryonic stem (ES) cells, which differentiate blood vessel-like structures and leukocytes, as a novel in vitro model system for biocompatibility, inflammation, and angiogenesis studies. Methodology/Results: Punched spherical discs of bioabsorbable polymers (ε-caprolactone and L-lactide in different compositions) with a diameter of 2 mm and a thickness of 0.2 mm were inoculated with embryoid bodies for cocultivation. As reference material for biocompatible, nonbioabsorbable, and bioincompatible materials, polymer punched discs of petriPERM (PP) membrane (polytetrafluoroethylene) as well as polyvinylchloride (PVC) were used. Tissue outgrowth on the polymer discs decreased and cell toxicity increased upon confrontation on bioabsorbable biomaterials and PVC. Bioabsorbable polymers as well as PVC decreased the branching points and total tube length of CD31-positive vascular structures in embryoid bodies. With the exception of PP, all applied materials increased the differentiation of CD68-positive macrophages and the generation of reactive oxygen species, which is indicative of proinflammatory processes upon contact of tissue with biomaterials. Consequently, cocultivation with polymers increased secretion of the cytokines interleukin-6, monocyte chemotactic protein-1, and tumor necrosis factor-α. Conclusion: Three-dimensional tissues cultivated from ES cells are well-suited for testing the biocompatibility, the vascular response, and the inflammatory reaction towards bioabsorbable and nonbioabsorbable polymers.