Biocompatible Au nanoparticles with surfaces modified by PEG (polyethylene glycol) were developed in view of possible applications for the enhancement of radiotherapy. Such nanoparticles exhibit preferential deposition at tumor sites due to the enhanced permeation and retention (EPR) effect. Here, we systematically studied their effects on EMT-6 and CT26 cell survival rates during irradiation for a dose up to 10 Gy with a commercial biological irradiator (E(average) = 73 keV), a Cu-Kalpha(1) x-ray source (8.048 keV), a monochromatized synchrotron source (6.5 keV), a radio-oncology linear accelerator (6 MeV) and a proton source (3 MeV). The percentage of surviving cells after irradiation was found to decrease by approximately 2-45% in the presence of PEG-Au nanoparticles ([Au] = 400, 500 or 1000 microM). The cell survival rates decreased as a function of the dose for all sources and nanoparticle concentrations. These results could open the way to more effective cancer irradiation therapies by using nanoparticles with optimized surface treatment. Difficulties in applying MTT assays were also brought to light, showing that this approach is not suitable for radiobiology.

Recent studies using insulin-like growth factor I (IGF-I) knockout mice demonstrate that IGF-binding protein (IGFBP)-5, an important bone formation regulator, itself is a growth factor with cellular effects not dependent on IGFs. Because IGFBP-5 contains a nuclear localization sequence that mediates transport of IGFBP-5 into the nucleus, we propose that IGFBP-5 interacts with nuclear proteins to affect transcription of genes involved in bone formation. We therefore undertook studies to identify proteins that bind to IGFBP-5 using IGFBP-5 as bait in a yeast two-hybrid screen of a U2 human osteosarcoma cDNA library. Five related clones that interacted strongly with the bait corresponded to the FHL2 gene, which contains four and a half LIM domains. Co-immunoprecipitation studies with lysates from U2 cells overexpressing FHL2 and IGFBP-5 confirmed that interaction between IGFBP-5 and FHL2 occurs in whole cells. In vitro interaction studies revealed that purified FHL2 interacted with IGFBP-5 but not with IGFBP-3, -4, or -6. Northern blot analysis showed that FHL2 was strongly expressed in human osteoblasts. Nuclear localization of both FHL2 and IGFBP-5 was evident from Western immunoblot analysis and immunofluorescence. The role of FHL2 as an intracellular mediator of the effects of IGFBP-5 and other osteoregulatory agents in osteoblasts will need to be verified in future studies.

Mutations of ephrin B1 in humans result in craniofrontonasal syndrome. Because little is known of the role and mechanism of action of ephrin B1 in bone, we examined the function of osteoblast-produced ephrin B1 in vivo and identified the molecular mechanism by which ephrin B1 reverse signaling regulates bone formation. Targeted deletion of the ephrin B1 gene in type 1␣2 collagen-producing cells resulted in severe calvarial defects, decreased bone size, bone mineral density, and trabecular bone volume, caused by impairment in osterix expression and osteoblast differentiation. Coimmunoprecipitation of the TAZ complex with TAZspecific antibody revealed a protein complex containing ephrin B1, PTPN13, NHERF1, and TAZ in bone marrow stromal (BMS) cells. Activation of ephrin B1 reverse signaling with soluble EphB2-Fc led to a time-dependent increase in TAZ dephosphorylation and shuttling from cytoplasm to nucleus. Treatment of BMS cells with exogenous EphB2-Fc resulted in a 4-fold increase in osterix expression as determined by Western blotting. Disruption of TAZ expression using specific lentivirus small hairpin RNA (shRNA) decreased TAZ mRNA by 80% and ephrin B1 reverse signaling-mediated increases in osterix mRNA by 75%. Knockdown of NHERF1 expression reduced basal levels of osterix expression by 90% and abolished ephrin B1-mediated induction of osterix expression. We conclude that locally produced ephrin B1 mediates its effects on osteoblast differentiation by a novel molecular mechanism in which activation of reverse signaling leads to dephosphorylation of TAZ and subsequent release of TAZ from the ephrin B1/NHERF1/TAZ complex to translocate to the nucleus to induce expression of the osterix gene and perhaps other osteoblast differentiation genes. Our findings provide strong evidence that ephrin B1 reverse signaling in osteoblasts is critical for BMS cell differentiation and bone formation.Osteoporosis is a common disease characterized by an agedependent decrease in bone mineral density (BMD) and a microarchitectural deterioration of bone tissue, with a consequent increase in the risk of developing fragility fractures of the hip, spine, and other skeletal sites (19). The decrease in bone mass occurs when the body fails to form enough new bone to replace the amount of old bone resorbed leading to reduced bone strength. There are two major known causes of osteoporosis: low peak BMD, which is typically achieved by around age 30, and high bone loss rate, which occurs particularly after menopause and during the natural process of aging. The accumulation of peak bone mass depends on bone growth during early skeletal development and the balance between osteoblastic bone formation and osteoclastic bone resorption during the postnatal growth period. Therefore, understanding the regulatory factors that govern bone development, bone size, bone mineralization, and bone quality during active growth periods as well as bone homeostasis during menopause and aging is essential for development of therapeutics to prevent osteoporosi...

The purpose of this work was to evaluate the efficacy of labeling human mesenchymal stem cells (hMSCs) by ionic superparamagnetic iron oxide (SPIO) without a transfection agent and verifying its capability to be detected with clinical 1.5 T magnetic resonance (MR) at the single-cell level. Human hMSCs were incubated for 24 h with an ionic SPIO, Ferucarbotran. The labeling efficiency of hMSCs was determined by iron content measurement spectrophotometrically, and the influence of labeling on cell behavior was ascertained by examination of cell viability using the trypan blue exclusion method, cell proliferation analysis using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, mitochondrial membrane potential (MMP) change, differentiation capacity, and reactive oxygen species (ROS) production measured by dichlorofluorescein diacetate (