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Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce nonspecific effects. Here, we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of matrix metalloproteinase 9 (MMP9) and altered fibronectin deposition patterning. A Cdc42 mutant rescue experiment found that downstream of Cdc42, p21-activated kinase (PAK), but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation, and vesicle trafficking, Cdc42 is essential in ECM remodeling in three dimensions.

In this work, we numerically and experimentally demonstrate that all-angle negative refraction can be obtained with the acoustic gradient metasurface of subwavelength thickness. The coiling labyrinthine structures are utilized to build the desired gradient metasurface, and the apparent negative refraction occurring beyond the critical incident angle has been validated by simulations and experimental measurements, which agrees well with the theoretical predictions given by the revised generalized law of refraction while taking the contribution of the Bragg scattering into account. This work provides the solution to manipulate the acoustic waves and shows good promise in building functional diffractive acoustic elements.

The estrogen receptor-alpha (ERalpha) is a critical transcription factor that regulates epithelial cell proliferation and ductal morphogenesis during postnatal mammary gland development. Tissue recombination and transplantation studies using the first generation of ERalpha knockout (ERKO) mice suggested that this steroid hormone receptor is required in the mammary stroma that subsequently exerts its effect on the epithelium through additional paracrine signaling events. A more detailed analysis revealed that ERKO mice produce a truncated ERalpha protein with detectable transactivation activity, and it is likely that this functional ERalpha variant has masked the biological significance of this steroid receptor in the mammary epithelium. In this article, we describe the generation a Cre-lox-based conditional knockout of the ERalpha gene to study the biological function of this steroid receptor in the epithelial compartment at defined stages of mammary gland development. The mouse mammary tumor virus (MMTV)-Cre-mediated, epithelial-specific ablation of exon 3 of the ERalpha gene in virgin mice severely impaired ductal elongation and side branching. The conditional knockout resulted in ablation of the ERalpha protein, and the progesterone receptor (PR), whose expression is under the control of ERalpha, was largely absent. The whey acidic protein (WAP)-Cre-mediated deletion of ERalpha during successive gestation cycles resulted in a loss of ductal side-branching and lobuloalveolar structures, ductal dilation, and decreased proliferation of alveolar progenitors. These abnormalities compromised milk production and led to malnourishment of the offspring by the second lactation. These observations suggest that ERalpha expression in the mammary epithelium is essential for normal ductal morphogenesis during puberty and alveologenesis during pregnancy and lactation.

A pixilated anode design of a high pressure xenon gamma-ray spectrometer is investigated. This design is composed of a single planar cathode and four anode pixels embedded in the center of a “non-collecting” anode plate. This design removes the Frisch grid which can suffer from excessive micro-phonics in real-world applications. To investigate the energy resolution of this design, the charge collecting efficiency and waveform of induced charge on each anode were calculated via tracking electron cloud drifting.

Estrogen receptor–α (ERα) activity in the brain prevents obesity in both males and females. However, the ERα-expressing neural populations that regulate body weight remain to be fully elucidated. Here we showed that single-minded–1 (SIM1) neurons in the medial amygdala (MeA) express abundant levels of ERα. Specific deletion of the gene encoding ERα (Esr1) from SIM1 neurons, which are mostly within the MeA, caused hypoactivity and obesity in both male and female mice fed with regular chow, increased susceptibility to diet-induced obesity (DIO) in males but not in females, and blunted the body weight–lowering effects of a glucagon-like peptide-1–estrogen (GLP-1–estrogen) conjugate. Furthermore, selective adeno-associated virus-mediated deletion of Esr1 in the MeA of adult male mice produced a rapid body weight gain that was associated with remarkable reductions in physical activity but did not alter food intake. Conversely, overexpression of ERα in the MeA markedly reduced the severity of DIO in male mice. Finally, an ERα agonist depolarized MeA SIM1 neurons and increased their firing rate, and designer receptors exclusively activated by designer drug–mediated (DREADD-mediated) activation of these neurons increased physical activity in mice. Collectively, our results support a model where ERα signals activate MeA neurons to stimulate physical activity, which in turn prevents body weight gain.

Bisphenol-A (BPA) shows proliferative actions in uterus and mammary glands and may influence the development of male and female reproductive tracts in utero or during early postnatal life. Because of its ability to function as an estrogen receptor (ER) agonist, BPA has the potential to disrupt normal endocrine signaling through regulation of ER target genes. Some genes are regulated by both estradiol (E2) and BPA, but those exclusive to either agent have not been described. Using a yeast strain incorporating a vitellogenin A2 ERE- Lac Z reporter gene into the genome, we found that BPA induced expression of the reporter in colonies transformed with the ER α expression plasmid, illustrating BPA-mediated regulation within a chromatin context. Additionally, a reporter gene transiently transfected into the endometrial cancer (Ishikawa) cell line also showed BPA activity, although at 100-fold less potency than E2. To compare global gene expression in response to BPA and E2, we used a variant of the MCF-7 breast cancer cell line stably expressing HA-tagged ER α . Cultures were treated for 3 h with an ethanol vehicle, E2 (10 −8 M), or BPA (10 −6 M), followed by isolation of RNA and microarray analysis with the human U95A probe array (Affymetrix, Santa Clara, CA, USA). More than 300 genes were changed 2-fold or more by either or both agents, with roughly half being up-regulated and half down-regulated. A number of growth- and development-related genes, such as HOXC1 and C6, Wnt5A, Frizzled, TGF β -2, and STAT inhibitor 2, were found to be affected exclusively by BPA. We used quantitative real-time PCR to verify regulation of the HOXC6 gene, which showed decreased expression of approximately 2.5-fold by BPA. These results reveal novel effects by BPA and E2, raising interesting possibilities regarding the role of endocrine disruptors in sexual development.

The major heat shock protein, Hsp70, can protect against cell death by directly interfering with mitochondrial apoptosis pathways. However, Hsp70 also sensitizes cells to certain apoptotic stimuli like TNF. Little is known about how Hsp70 enhances apoptosis. We demonstrate here that Hsp70 promotes TNF killing by specifically binding the coiled-coil domain of I kappa B kinase gamma (IKK gamma) to inhibit IKK activity and consequently inhibit NF-kappa B-dependent antiapoptotic gene induction. An IKK gamma mutant, which interacts with Hsp70, competitively inhibits the Hsp70-IKK gamma interaction and relieves heat-mediated NF-kappa B suppression. Depletion of Hsp70 expression with RNA interference rescues TNF-mediated cell death. Although TNF may or may not be sufficient to trigger apoptosis on its own, TNF-triggered apoptosis was initiated or made worse when Hsp70 expression increased to high levels to disrupt NF-kappa B signaling. These results provide significant novel insights into the molecular mechanism for the pro-apoptotic behavior of Hsp70 in death-receptor-mediated cell death.

With excellent angular resolution, good energy resolution, and a broad energy band, the Chandra Advanced CCD Imaging Spectrometer (ACIS) is the best instrument for studying the X-ray halos around some Galactic X-ray point sources caused by the dust scattering of X-rays in the interstellar medium. However, the direct images of bright sources obtained with the ACIS usually suffer from severe pileup. Making use of the fact that an isotropic image could be reconstructed from its projection in to any direction, we can reconstruct the images of the X-ray halos from the data obtained with the High Energy Transition Grating Spectrometer (HETGS) and/or in continuos clocking (CC) mode. These data have no or less serious pileup and enable us to take full advantage of the excellent angular resolution of Chandra. With the reconstructed high-resolution images, we can probe the X-ray halos as close as 1" to their associated point sources. Applying this method to Cygnus X-1 observed with the Chandra HETGS in CC mode, we derived an energy-dependent radial halo flux distribution and concluded that in a circular region (2' in radius) centered a the point source: (1) relative to the total intensity, the fractional halo intensity is about 15% at keV and drops to aboout 5% at approximately 6 keV (2) about 50% of the halo photons are within the region of a radius less than 40 inches and (3) the spectrum of the pooint source is slightly distorted by the halo contamination.

The bone marrow niche is an important milieu where hematopoietic stem and progenitor cells (HSPCs) are maintained to ensure their lifelong contribution to hematopoiesis. Recent evidence has highlighted the critical importance of the perivascular bone marrow (BM) niche as the key host and regulator of HSPCs. Bone marrow endothelial cells (BMECs) are major components of the vascular niche. While studies have shown that an alteration in a component of the niche can affect hematopoiesis and promote the development of myeloproliferative disorders/myelodysplastic syndromes, it remains unclear how altered BMECs can impact hematopoiesis. To this end, we have generated a Tamoxifen (TAM)-inducible Tie2-CreER/LSL-KRasG12D;tdTomato mouse model to introduce an oncogenic KRas mutation specifically in adult endothelial cells. The tdTomato reporter overlaps with the CD31 and vascular endothelial growth factor receptor 2 (VEGFR2) endothelial cell markers and shows no detectable leakage into the adult hematopoietic compartment. To evaluate changes in hematopoiesis, we performed complete blood counts at 12 weeks post TAM injection and found that the Tie2-CreER/LSL-KRasG12D mice (KRasG12D mice) had significantly more leukocytes (p=0.031) and neutrophils (p=0.002) than controls. Flow cytometry analysis confirmed that the KRasG12D mice had a significantly higher percentage of myeloid cells with concurrent decrease in lymphocyte percentage in the peripheral blood (p=0.016). At 16 weeks post TAM injection, a significant decrease in B cells could also be noted in the blood of KRasG12D mice (p=0.028). Compared to controls, the KRasG12D mice displayed splenomegaly (p=0.025) and their spleens had a higher percentage of myeloid cells (p=0.002). There was an increase in the common myeloid progenitor compartment in the spleen and a significant increase in the granulocyte macrophage progenitor compartment (p=0.014) of KRasG12D mice. These mice also had an increase in the short-term hematopoietic stem cell (ST-HSC) compartment both in the BM and spleen. Colony forming assays revealed that KRasG12D mice had a higher number of total colonies formed from BM (p=0.044), spleen (p=0.007) and blood cells (p=0.56). Genotyping PCR showed no KRasG12D activation in hematopoietic cells, confirming that the observed phenotypes were due to an effect in BMECs. To complement our native inducible mouse model, we transplanted BM cells from syngeneic BoyJ mice into lethally irradiated Tie2-CreER;KRasG12D or KRasWT recipients. The endothelial KRasG12D recipientsdied between 75-200 days post transplantation (p=0.0079) while the KRasWT recipients remained alive. The KRasG12D recipients also displayed splenomegaly (p=0.004). Competitive transplant studies with donor cells from KRasG12D or KRasWT mice with competitor cells from syngeneic mice (CD45.1) showed that BM cells from the KRasG12D mice (CD45.2) outcompeted cells from KRasWT mice with a significantly higher percentage of CD45.2 donor chimerism in all blood lineages examined. To uncover any molecular events underpinning these hematopoietic changes, we performed quantitative real-time polymerase chain reaction. Our preliminary experiments from total BM RNA of KRasG12D or KRasWT mice indicate that there is a significant increase in VEGFα and a decrease in transforming growth factor β in KRasG12D mice, accompanying the above noted increase in the ST-HSC population. Collectively, our data provide strong evidence that an abnormal vascular niche caused by oncogenic insults in BMECs can disrupt normal hematopoiesis and promote a myeloproliferative phenotype, thereby implicating abnormal BMECs as novel contributors to blood pathogenesis. Studies are underway to further assess the molecular contributions from the disrupted vascular niche and the resulting HSPCs. Uncovering the mechanism of how altered BMECs can remodel hematopoiesis holds the exciting promise of better therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

Estrogen receptor alpha (ERα), a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER’s ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER’s transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.

Background: Targeting T cells alone has yielded limited success in the prevention of graft-versus-host disease (GvHD) following allogeneic blood and marrow transplantation (BMT). Dendritic cells (DCs) play a central role in alloreactivity and therefore represent a suitable target. Proteasome inhibitors (PI), with their ability to inhibit the function and maturation of DC, have prompted investigators to examine their potential role in the prevention of GvHD. The investigational PI, ixazomib (IXZ), dissociates rapidly from 20S and is therefore truly reversible. It is also orally bioavailable. Our aim in this study was to explore its effect on healthy volunteer peripheral blood dendritic and T cells and in a pre-clinical GvHD mouse model. Methods: To characterize the effects of IXZ on healthy volunteer peripheral blood DCs, DCs were isolated using EasySep Pan-DC Pre-Enrichment Cocktail with purity over 90% (STEMCELL Technologies). DCs were then treated with IXZ at different concentrations (10-40nM) for 4 hrs and then stimulated with lipopolysaccharide (LPS) for 16 hrs. After this treatment, DCs were surface stained with antibodies against maturation markers and analyzed by flow cytometry. DC survival was evaluated with 7AAD staining and FACS analysis. To assess the effect of IXZ on the production of pro-inflammatory cytokines, DCs were incubated with IXZ at increasing concentration before or after the addition of LPS. Total pro-inflammatory cytokines in the supernatant of tissue culture were measured using EMD Millipore cytokine arrays. Standard mixed lymphocyte reaction and T cell proliferation assays were used to evaluate T cell function. At a minimum, all experiments were done in triplicate. Unpaired t test was used for statistical analysis. A p-value < 0.05 was considered significant. The B6 → BALB/c pre-clinical GvHD model was adopted to evaluate the effect of IXZ on GvHD development. Mice were transplanted in 3 groups. Group 1 received a lethal dose of total body irradiation (TBI), donor bone marrow (BM) cells, and IXZ, group 2 received TBI, donor BM cells donor splenocytes, and a vehicle, and group 3 received TBI, donor BM cells, donor splenocytes, and IXZ. The dose of BM cells and splenocytes was 5 X 106 each. IXZ was given at 1.5 mg/kg subcutaneously. Two dosing schedules were tested in 2 separate experiments: day-1 and +2 or day +1 and +4. Results: IXZ inhibited the expression of 6 DC maturation markers including CD40, CD54, CD80, CD83, CD86 and CD197 (CCR-7). The inhibition started at a concentration of 10nM and was dose-related. IXZ also decreased the percentage of total DCs simultaneously expressing multiple markers. DCs viability remained unchanged in comparison to control at a concentration of 10nM and dropped to 68% and 43%, on average with concentrations of 20nM and 40nM, respectively. IXZ significantly decreased DC production of IL-6, IL-12, and IL-23 starting at the concentration of 20nM. IL-1β was decreased at the concentration of 40 nM. Importantly, there was no significant change in the cytokine production by DCs when IXZ was added 4 hrs after LPS except for IL-1β which increased at 30nM. Starting at the concentration of 10nM, IXZ dose-dependently inhibited T cell proliferation. At 40nM IXZ abolished T cells. In our in vivo study IXZ improved GvHD scores on days +7 and +11 in group 3 in comparison to group 2 when it was given on days -1 and +2. Conversely, when IXZ was given on day +1 and +4, group 3 mice had higher scores of GvHD and worse survival outcomes when compared to group 2. There was no noticeable drug toxicity in group 1 mice. Conclusion: In summary: 1) IXZ inhibits DC maturation with relative preservation of cell viability and inhibits pro-inflammatory cytokine production in DCs when added before LPS stimulation; 2) IXZ inhibits T-cell proliferation; 3) IXZ affects GvHD development in a schedule-dependent fashion with early administration improving and late administration worsening GvHD. Additional analysis of tissue and serum samples is in progress. These results provide background for careful design of clinical trials using IXZ for the prevention of GvHD. Disclosures Al-Homsi: Millennium Pharmaceuticals: Research Funding.

Rho GTPases including RhoA, Rac1, and Cdc42 are a class of intracellular signaling proteins critical for the regulation of cytoskeleton organization, adhesion, and migration. Molecular mechanisms of mammalian cell migration were first revealed in fibroblasts where RhoA, Rac1, and Cdc42 facilitate in the multistep process including establishment and maintenance of polarity, formation of actin-rich protrusions, remodeling of adhesive contacts, and generation of force. In hematopoietic stem/progenitor cells, Rho GTPases relay signals from chemokines and cytokines such as SDF-1α and SCF to the actin and microtubule cytoskeleton through effector kinases and/or adaptor molecules that affect adhesion or transcription. Comprehensive use of murine conditional gene knockout technology combined with biochemical approaches in recent studies allows for physiologically relevant investigations of the involvement of Rho GTPases in hematopoietic stem/progenitor cell migration, providing important mechanisms for the stem/progenitor maintenance.

Rho GTPases including RhoA, Rac1, and Cdc42 are a class of intracellular signaling proteins critical for the regulation of cytoskeleton organization, adhesion, and migration. Molecular mechanisms of mammalian cell migration were first revealed in fibroblasts where RhoA, Rac1, and Cdc42 facilitate in the multistep process including establishment and maintenance of polarity, formation of actin-rich protrusions, remodeling of adhesive contacts, and generation of force. In hematopoietic stem/progenitor cells, Rho GTPases relay signals from chemokines and cytokines such as SDF-1α and SCF to the actin and microtubule cytoskeleton through effector kinases and/or adaptor molecules that affect adhesion or transcription. Comprehensive use of murine conditional gene knockout technology combined with biochemical approaches in recent studies allows for physiologically relevant investigations of the involvement of Rho GTPases in hematopoietic stem/progenitor cell migration, providing important mechanisms for the stem/progenitor maintenance.

A new method for calculating the equivalent circuit of a quasi-static region based on scattered fields is presented. Two problems with a quasi-static region are chosen to validate the procedure. It is then shown that the geometry in the quasi-static region can be modeled accurately in Mininec as an equivalent circuit and that the computation times with the equivalent circuit are significantly less.

LaBr3(Ce) scintillators have a high atomic number, high light yield, and fast decay. Thus, detectors with LaBr3(Ce) crystal can achieve high energy resolution with a significantly improved efficiency over Nal(Tl) scintillators of the same geometry. In this work, LaBr3 was investigated for use in a Compton camera with a two-plane array design, as the material for both the scattering and absorbing detector arrays. We calculated and modeled the following properties of a LaBr3 Compton Camera: 1) The probability of single Compton scatter; 2) Doppler broadening effects; 3) The relationships between intrinsic efficiency and angular uncertainty; 4) Optimized efficiency for a required angular resolution by selecting various geometries, such as the thickness of detectors and distance between detectors. Our study showed that Doppler broadening effects induced an intrinsic limitation of ~0.05 radian angular uncertainty for Compton cameras with LaBr3 as the scattering detector. In comparison of LaBr3 with other traditional scatter detector materials such as Si, we found that below thickness of ~2 cm LaBr3 has a higher efficiency for observation of single Compton scattering events, including the hits of the recoil electron and scattered photons, than that of Si. Based on these investigations, a prototype of Compton imaging system was proposed and evaluated with Monte Carlo simulation (Geant4) also.

The energy resolution and position sensing with two types of pixelated anode designs of high pressure xenon gamma-ray spectrometer were investigated. The first design is composed of a single planar cathode and four anode pixels embedded in the center of a "non-collecting" anode plate. The second design is composed of a cylindrical surface cathode and seven pixelated central cylinder anodes. These designs remove the Frisch grid which can suffer from excessive micro-phonics in real-world applications. The distribution of electron cloud created by interaction between gamma-rays and Xe was obtained with Monte Carlo (GEANT4) simulations and shows the dimension of electron clouds increasing from approximately 0.1 mm to ~100 mm for gamma-ray energy range from 50 keV to 10 MeV in Xe at ~50 atm pressure (~0.5g/cm 3 density). The charge collecting efficiency and waveform of induced charge on each anode were obtained by tracking electron cloud drifting. The position sensing based on both position dependence of weighting potential and the distribution of induced charge among anode pixels were also probed.

Gypenosides (GPs) were tested for their ability to protect primary cultures of immature cortical cells against oxidative glutamate toxicity. In immature neural cells, glutamate cytotoxicity is known to be mediated by the inhibition of cystine uptake, leading to depletion of intracellular glutathione (GSH). The depletion of GSH impairs cellular antioxidant defenses resulting in oxidative stress and cell death. We found that pretreatment with GPs (100-400 microg/ml) significantly protected cells from glutamate-induced cell death. It was therefore of interest to investigate whether GPs protect cortical cells against glutamate-induced oxidative injury through preventing GSH depletion. Results show that GPs significantly up-regulated mRNAs encoding gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione reductase (GR) and enhanced their activities for GSH synthesis as well as recycle. Furthermore, GPs lowered the consumption of GSH through decreased accumulation of intracellular peroxides, leading to an increase in the intracellular GSH content. GPs were also found to prevent lipid peroxidation and reduce the influx of Ca(2+) which routinely follows glutamate oxidative challenge. GPs treatment significantly blocked glutamate-induced decrease in levels of Bcl-2 and increase in Bax, leading to a decrease in glutamate-induced apoptosis. Thus, we conclude that GPs protect cortical cells by multiple antioxidative actions via enhancing intracellular GSH, suppressing glutamate-induced cytosolic Ca(2+) elevation and blocking glutamate-induced apoptosis. The novel role of GPs implies their remarkable preventative and therapeutic potential in treatment of neurological diseases involving glutamate and oxidative stress.

Closed form expressions are provided for currents induced in small loop antennas and the magnetic fields created (e.g., scattered) by those induced currents. The accuracy of the expressions has been verified with MININEC, a commonly-used electromagnetics software code. Applications of this work are noted, one of which is a new way of thinking about the operation of inductivelycoupled RFID (radio frequency identification) systems. As an alternative to viewing such systems in terms of the coupling coefficient (as commonly done with transformertype systems) we view it from an induced-current/scatteredfield perspective. The closed-form expressions developed here can be used as tools for EMC and RFID personnel to estimate the fields scattered from RFID loop. The results can also be used to improve general understanding of inductively-coupled noise, and provide estimates of the voltage and current induced in small “sniffer” probes. Keywords-component; RFID systems, loops; induced currents; scattered fields; incident plane wave, sniffer probes.

RFID systems will soon become ubiquitous. This paper provides insight into one way of viewing the operation of high-frequency RFID systems that use passive tags. The relationship between the impedance of the tag circuit and the fields scattered by the tag are highlighted using two indepen- dent numerical codes. In addition, analytical expressions and numerical results for the scattered electric field are provided for a dipole with an assumed sinusoidal current distribution. Such results provide EMC engineers with an approximate method of determining the fields scattered from passive, high-frequency RFID tags.

Although surface mount components are widely used in high frequency circuit design, the distortion of the characteristics of these devices at high frequencies should be considered during analog circuit design. The work reported in this paper employs a novel combination of measurement results and fast computational tools to provide the circuit response in both time and frequency domains. In particular, this method allows very fast computation of the circuit response (in the time or frequency domain) for systems composed of a variety of elements - including those for which the equivalent circuit is not known. This technique allows the designer to carefully (and quickly) investigate the contribution of individual components to the overall response. In this paper a brief overview is provided of the methods employed.

Xenoestrogens such as bisphenol-A (BPA) can mimic endogenous 17beta-estradiol (E2) in vitro and in vivo through binding the estrogen receptor (ER), and modulating target gene expression. In the present study, we compared global gene regulation by BPA and E2 in estrogen responsive (ERalpha-HA) human breast cancer cells derived from the MCF-7 cell line. The ERalpha-HA cells (stably over-expressing ERalpha) were exposed to E2 (10(-8)M) or BPA (10(-6)M), for 3h followed by analysis of global gene expression. More than 40 transcripts were significantly changed in ERalpha-HA cells, with many being unique to BPA. At least 15 genes were modulated by BPA in the ER-null C4-12 cell line, indicating ER independent activity. Utilizing quantitative reverse transcription-polymerase chain reaction (RT-PCR), we confirmed BPA and E2 mediated regulation of four selected genes. A consensus Alu-type estrogen responsive element (ERE) was found in the Wiskott-Aldrich syndrome protein (WASP) gene, which conferred responsiveness to BPA and E2 in a reporter gene assay. Significant stimulation was seen only in ERalpha expressing cells, thus indicating a functional ERE. Taken together these data illustrate novel gene regulation by BPA and E2, which has implications for in vivo actions and previous reports of additive and synergistic effects on breast cancer cell growth.

Understanding the properties of the hot corona is important for studying the accretion disks in black hole X-ray binary systems. Using the Monte-Carlo technique to simulate the inverse Compton scattering between photons emitted from the cold disk and electrons in the hot corona, we have produced two table models in the $XSPEC$ format for the spherical corona case and the disk-like (slab) corona case. All parameters in our table models are physical properties of the system and can be derived from data fitting directly. Applying the models to broad-band spectra of the black hole candidate XTE J2012+381 observed with BeppoSAX, we estimated the size of the corona and the inner radius of the disk. The size of the corona in this system is several tens of gravitational radius, and the substantial increase of the inner disk radius during the transit from hard-state to soft-state is not found., Accepted version, appear on ApJ 20 January 2005 issue

For an accretion disk around a black hole, the strong relativistic effects affect every aspect of the radiation from the disk, including its spectrum, light-curve, and image. This work investigates in detail how the images will be distorted, and what the observer will see from different viewing angles and in different energy bands.

Estrogen 17β-estradiol (E2) rapidly modulates several signaling pathways related to cell growth, preservation, and differentiation. The physiological role of these nongenomic effects with regard to downstream outcomes, and the relationship with transcriptional estrogen activity are unclear. Furthermore, the ability of selective estrogen receptor modulators (SERMs) to trigger nongenomic actions is largely unknown. To determine whether estrogen receptor (ER) ligands exert nongenomic activity in endometrial adenocarcinoma cells, and whether this activity affects transcription and DNA synthesis, we challenged human Ishikawa cells with E2 or partial ER agonists 4-hydroxytamoxifen (OHT) and raloxifene (ral). Serum-starved Ishikawa cells exposed for 5 min to 0.1 nm E2 showed induced phosphorylation of MAPK (ERK1/2). Ral and 4-OHT each at 1 nm also stimulated ERK in a rapid transient manner. E2 and 4-OHT induced proto-oncogene c-fos mRNA expression in Ishikawa cells within 30 min, but ral had no effect. In contrast to nongenomic action, only E2 stimulated expression of an estrogen response element (ERE)-driven luciferase (LUC) reporter gene. To examine DNA synthesis, [3H]-thymidine incorporation was measured in serum-starved cultures exposed to E2 or partial agonists for 2 d. E2 at 1 nm stimulated thymidine uptake in an ERK-dependent manner, but 1 nm 4-OHT, 1 nm ral, and 0.1-nm concentrations of E2 had no significant effects. Taken together, these data indicate that both nongenomic and direct transcriptional ER effects are likely required to promote DNA synthesis.

Mutation of the functionally redundantHoxa 11/Hoxd 11genes gives absent or rudimentary kidneys resulting from a dramatic reduction of the growth and branching of the ureteric bud. To understand better the molecular mechanisms ofHoxa 11/Hoxd 11function in kidney development, it is necessary to identify the downstream target genes regulated by their encoded transcription factors. To this end, we conducted a screen forHoxa 11-responsive genes in two kidney cell lines.HEK293cells, which usually do not expressHoxa 11, were modified to allow inducibleHoxa 11expression. ThemK10cells, derived specifically for this study fromHoxa 11/Hoxd 11double-mutant mice, were also modified to give cell populations with and withoutHoxa 11expression. Differential display, Gene Discovery Arrays, and Affymetrixgenechipprobe arrays were used to screen for genes up- or down-regulated byHoxa 11. Nine genes,PDGF A,Cathepsin L,annexin A1,Mm.112139,Est2 repressor factor,NrCAM,ZNF192,integrin-associated protein, andGCM1, showed reproducible 3-fold or smaller changes in gene expression in response toHoxa 11. One gene, theIntegrinα8, was up-regulated approximately 20-fold afterHoxa 11expression. TheIntegrinα8gene is expressed together withHoxa 11in metanephric mesenchyme cells, and mutation ofIntegrinα8gives a bud-branching morphogenesis defect very similar to that observed inHoxa 11/Hoxd 11mutant mice.In situhybridizations showed a dramatic regional reduction inIntegrinα8expression in the developing kidneys ofHoxa 11/Hoxd 11mutant mice. This work suggests that theIntegrinα8gene may be a major effector ofHoxa 11/Hoxd 11function in the developing kidney.

Bone marrow endothelial cells (BMECs), the cells forming bone marrow vasculature, have been implicated as a critical constituent of bone marrow microenvironment essential for the maintenance of normal and malignant hematopoietic stem cells. They are a part of BM niche regulating the self-renewal and differentiation of hematopoietic stem cells (HSCs) and the homing and progression of pre-leukemia and leukemia cells. It has been realized that pathological angiogenesis such as increased bone marrow microvessel density is associated with a spectrum of human diseases including MDS, MPD and leukemia. Recent discovery of oncogenic mutations of BCR-ABL and JAK2V617F in BMECs and spleen ECs primes the hypothesis that pathological angiogenesis from oncogenic insults may promote or facilitate abnormal hematopoiesis leading to the development and maintenance of blood malignancies. Clinical studies have linked germline mutations of Ras signaling components (e.g. NF1, SHP2, CBL and K-RAS) with JMML, a subgroup of MPD/MDS that is difficult to cure and for which bone marrow transplantation often results in relapses and graft failure. Studies in animal models have shown that activation of oncogenic K-Ras in hematopoietic stem cells could partially recapitulate the symptoms of these MPD/MDS patients, suggesting a HSC intrinsic role of oncogenic transformation. It remains to be seen if deregulated K-Ras signaling in the BM microenvironment, BMECs in particular, may also contribute to the hematologic abnormalities and disease progression, and whether pathologicial BMECs provide an addition therapeutic opportunity to limit transplant-associated relapse. To this end, we recently established a new mouse model that allows endothelial lineage-specific, inducible genetic manipulation of BMECs in adult animals in vivo and a cell culture system to select, expand and functional assay freshly isolated murine BMEC activities in vitro. A strain of Tie2-CreER transgenic mice crossed with loxP-stop-loxP fluorescence reporters (EGFP or Td-tomato) is sensitive to Tamoxifen induction in specifically turning on Cre activity in endothelial cells in 4-8 week old animals with over 80% efficiency in BMECs. Our culture conditions select and expand mouse primary BMECs in a large scale without immortalization or genetic manipulation, and the resulting BMECs retain endothelial lineage markers and are fully functional in 2-demensional and 3-demensional capillary formation assays as well as Matrigel implant plug assay. Using this inducible model, we generated Tie2CreER/LSL-K-Ras G12D mice, to specifically turn on K-Ras G12D in endothelial cells to test if an oncogenic K-Ras insult in EC compartment can affect hematopoiesis. Three consecutive Tamoxifen injections to the mice readily activated K-Ras G12D expression in the BMECs, but not in the blood lineages. The mice bearing endothelial K-Ras G12D died 4-6 months after Tamoxifen induction. An analysis of the K-Ras G12D mice revealed that the oncogenic insult caused a significant change in BM vascular structure and abnormal hematopoiesis. K-Ras G12D mutation in the ECs led to pathological angiogenesis in the bone marrow as manifested in the K-Ras G12D mice an increased colony-forming unit EPC activity, drastically increased vessel permeability, and increased BMEC proliferation. The pathological angiogenesis in the bone marrow was associated with abnormal blood phenotypes in K-Ras G12D mice, showing a significant increase of WBC and lymphocyte counts in peripheral blood, splenomegaly, severe anemia, and a significant increase of CFU-C activity in the blood and spleen. Ongoing transplant experiments will address whether the oncogenic K-Ras expression in endothelial cells is sufficient to fully promote blood cell transformation. In parallel, the oncogenic K-Ras expressing BMECs in primary culture are being examined to determine whether abnormal Ras signaling including RAF-MEK and/or PI3K-AKT pathways are involved in mediating BMEC proliferation and angiogenesis. Our study suggests a novel concept that pathologic bone marrow microenvironment, BMECs in particular, can play an important role in blood cell transformation, and implies that rational targeting of oncogenic pathways in the BMECs may provide an additional therapeutic avenue for blood malignancies, besides direct suppression of abnormal hematopoietic cells, to restore normal hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 617 Self-renewal, differentiation, and proliferation of hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) are maintained in a complex microenvironment of the adult bone marrow (BM). BM endothelial cells (ECs) have been proposed to be a key component of HSC and LSC niche. However, in contrast to the well-developed culture system of human ECs, current work of murine BM endothelial cells is hindered by a lack of mouse bone marrow endothelial cell primary culture and suitable assay methods to clearly define murine BMEC functionality in vivo and in vitro, which limits genetic and mechanistic studies by using mouse models. To establish an in vivo approach to study the EC function in adult mice, a strain of Tie2-CreER transgenic mice was generated to allow conditional and inducible manipulation of BMECs by Cre recombinase expression under the Tie2 promoter. In vivo lineage tracing was achieved in a Tie2-CreER/TD-tomato or -EGFP reporter mouse strain. Upon a four day Tamoxifen injection regimen, TD-tomato or EGFP reporter was readily visualized in bone marrow vasculature that colocalizes with CD31+ ECs as determined by immunostaining. FACS analysis of Tie2-CreER/EGFP reporter mice showed that the EGFP+ cells in the BM were exclusively in the CD45- VEGFR2+ and CD31+ cell fraction, with no EGFP+ cells being detectable in the CD45+ hematopoietic lineages or osteoblast/stroma cell fractions, suggesting that the Tie2-driven CreER expression is limited to the endothelial lineage in the adult BM. Next, we developed an in vitro method to culture and assay the mouse BMECs functionally. An in vitro selection process allowed us to establish a primary BM cell culture condition that permitted functional expansion and maintenance of mouse BMECs in long-term tissue culture, yielding homogenous CD45- cells expressing endothelial markers CD31, CD34 and VEGFR2. These cells formed capillary-like structures in 2-demensional and 3-demensional tubes/capillaries, and showed TD-tomato reporter color when derived from the Tamoxifen induced Tie2-CreER/TD-tomato mouse BM. They showed expected adhesion and migration activities and morphology of ECs. Lineage chasing assays using isolated CD45+ and CD45- BM cells from the Tie2-CreER/Td-tomato mice demonstrated that the BMECs in our culture system, bearing the Tie2-promoter driven TD-tomato color and CD31+ marker, were exclusively derived from CD45- non-hematopoietic lineage. Taken together, we have established a faithful assay method for studying murine BM EC functions in vivo and in vitro, allowing the tracking and genetic manipulation of adult BM ECs in mice and in culture. The method can be useful for delineating molecular and cellular mechanisms of BMEC regulation and EC-mediated BM niche function, and may have value in testing anti-angiogenic activities of anticancer drugs in animal models. Disclosures: No relevant conflicts of interest to declare.

Abstract 508 Adult hematopoietic stem/progenitor cells (HSPCs) are maintained in the unique microenvironment of bone marrow (BM) niche to sustain life-long blood production of mammals. Several types of cells in the BM, including endothelial cells (ECs), have been shown to contribute to the niche components in regulating the homeostasis of HSPCs as well as leukemia stem cells in the BM. However, because of the current technical limitations in studying BM ECs in mice, particularly the lack of an EC-specific inducible gene targeting method in adult mice and controversies surrounding the suitable murine EC culture conditions in vitro, the signaling pathways that regulate the putative BMEC niche remain poorly understood. Here we have utilized several complementary mouse models to achieve an inducible, BMEC-specific deletion of Cdc42 to unveil an essential role of this Rho family GTPase in regulating BMEC morphogenesis and proliferation, and in maintaining hematopoietic homeostasis of adult mice. First, by using the interferon-inducible Mx1-Cre;Cdc42lox/lox and the tamoxifen inducible Rosa26-CreER;Cdc42lox/lox mice engrafted with syngeneic WT HSPCs, we found that both conditions of inducible deletion of Cdc42 in the BM microenvironment, including BMECs, caused sinusoidal dilation, a drastic decline of endothelial capillary density, and a loss of endothelial progenitor activity and mature endothelial cells, in the bone marrow. Significantly, the BMEC phenotypes of these non-specific Cre-driver genetic models could be recapitulated by using a mouse model of Tie2-CreER;Cdc42lox/lox that allows inducible endothelial lineage-specific deletion of Cdc42 in the BM in response to tamoxifen, which we have shown rigorously by lineage tracing to be a suitable model for BMEC characterizations (Feng et al., ASH abstract, 2012). Second, we have established an in vitro culture system that expands and maintains primary BMECs in culture, allowing a direct assessment of the proliferation, survival, adhesion, migration, tube formation, as well as capillary assembly in 3D, activities of the BMECs. Deletion of Cdc42 by in vivo tamoxifen induction or in vitro Cre transduction of the BM cells of Tie2-CreER; Cdc42lox/lox genotype resulted in a complete loss of angiogenesis activity of BMECs that was associated with a drastic reduction of adhesion, tube-formation, and 3D capillary assembly capabilities of BMECs. In addition, endothelial cell proliferation was also compromised, as seen by a decrease of CyclinD1 expression and a diminished BrdU incorporation. These genetic results were phenocopied by findings using a Cdc42 specific inhibitor, CASIN, as CASIN treatment inhibited BMEC adhesion, diminished BM endothelial colony-forming activity, and suppressed BMEC tube-formation. A rescue experiment using retroviral expression of a panel of effector-coupling defective Cdc42 mutants and/or constitutively active PAK or AKT mutant in the Cdc42 knockout BMECs identified AKT and p21 PAK as the major downstream effectors of Cdc42 in BMEC maintenance. Third, using the three inducible mouse models, we found that Cdc42 in BMECs and microenvironment is required for maintaining hematopoietic homeostasis. Deletion of Cdc42 in BMECs caused a mobilization of HSPCs to the peripheral organs and a subsequent reduction of wild-type HSPCs in the BM, as assayed by genotypic FACS analysis, colony-forming activity and competitive transplantation of BM cells from endothelial Cdc42-null or control mice. Taken together, our results demonstrate that the Rho GTPase Cdc42 in BMECs mediates critical signaling cascade through PAK and Akt in maintaining the BMEC angiogenesis and morphogenesis. Cdc42 in BMECs could serve as a potential therapeutic target for bone marrow diseases including leukemia and lymphoma. Disclosures: No relevant conflicts of interest to declare.

Abstract 1273 The maintenance and differentiation of hematopoietic stem cells (HSC) are critical for blood cell homeostasis, which is tightly regulated by a variety of factors. In spite of extensive investigation of HSC biology, however, the mechanism of regulation of HSC and progenitor cell division, particularly the unique molecular events controlling the mitosis process during HSC differentiation, remains unclear. RhoA GTPase is a critical intracellular signaling nodal that has been implicated in signal transduction from cytokines, chemokines, wnt/notch/shh, and adhesion molecules to impact on cell adhesion, migration, cell cycle progression, survival and gene expression. Recent mouse genetic studies in keratinocytes and embryonic fibroblast cells showed that RhoA is a key regulator of mitosis. By using an interferon-inducible RhoA conditional knockout mouse model (Mx-cre;RhoAlox/lox), we have made the discovery that RhoA plays an indispensible role in primitive hematopoietic progenitor differentiation through the regulation of mitosis and survival. RhoA deficient mice die at ∼10 days because of hematopoietic failure, as evidenced by a loss of bone marrow, splenocyte and PB blood cells. Syngenic as well as reverse transplant experiments demonstrate that these effects are intrinsic to the hematopoietic compartment. RhoA loss results in pancytopenia associated with a rapid exhaustion of the lin−c-kit+ (LK) phenotypic progenitor population (within 4 days after two polyI:C injections). Meanwhile, the lin−c-kit+sca1+ (LSK) primitive cell compartment is transiently increased in BM after RhoA deletion due to a compensatory loss of quiescence and increased cell cycle. Interestingly, we find that within the LSK population, there is a significant accumulation of LSKCD34+Flt2− short-term HSCs (ST-HSC) and a corresponding decrease in frequency of LSKCD34+Flt2+ multipotent progenitors (MPPs). Consistent with these phenotypes, the LK and more differentiated hematopoietic cell populations of RhoA knockout mice show an increased apoptosis while the survival activities of LSK and more primitive compartments of WT and RhoA KO mice remain comparable. These data suggest that RhoA plays an indispensible role in the step of ST-HSCs differentiation to MPP cells, possibly through the regulation of MPP cell survival. This hypothesis is further supported by a competitive transplantation experiment. Deletion of RhoA in a competitive transplantation model causes an extinction of donor derived (CD45.2+) differentiated cells (myeloid, erythroid, T and B cells) in the peripheral blood. Interestingly, bone marrow CD45.2+ LSK cells are only marginally affected by deletion of RhoA and RhoA−/− LSK cells are able to engraft into 2nd recipient, whereas CD45.2+ LK and more differentiated cells are mostly eliminated after RhoA deletion. This effect is associated with a decrease in the survival of CD45.2+ RhoA−/− LK, but not LSK cells. Further in vitro culture of isolated lin− progenitors demonstrates that RhoA deficiency results in a failure of cytokinesis, causing an accumulation of multinucleated cells, further suggesting that RhoA is essential for the cytokinesis of hematopoietic progenitors. Surprisingly, the well-defined Rho downstream target, actomyosin machinery, does not appear to be affected by RhoA knockout. We are further exploring the mechanism of RhoA contribution to the differentiation of HSCs by dissecting the signaling and functional relationship of RhoA regulated survival activity and cell cycle mitosis in early hematopoietic progenitors. Disclosures: No relevant conflicts of interest to declare.

Abstract 2109 In addition to maintaining steady state hematopoiesis in the adult life, bone marrow (BM) derived cells contain endothelial progenitor cell (EPC) activity and may play a role in promoting vascular regeneration upon injury or angiogenesis during tumor progression. Whether hematopoietic stem/progenitor cells (HSPCs) in the BM can give rise to functional EPCs and if they contribute significantly to endothelial cell regeneration remain controversial questions. Cdc42 is a member of the Rho GTPase family that has been shown to play essential and unique roles in multiple lineages of blood cell regulation including HSPC proliferation and cytoskeleton organization. In addition, Cdc42 is found to regulate blood vessel permeability and endothelial lumen formation [1, 7]. By using a conditional knockout (KO) and BM transplant mouse model (i.e. Mx1-cre; Cdc42loxP/loxP mice and transplant recipients) that allows specific, inducible deletion of cdc42 gene in the bone marrow compartment and a small molecule Cdc42 activity-specific inhibitor (CASIN), we have examined the hypothesis that Cdc42 critically regulates the BM-derived EPC and/or angiogenic supporting cell production and function. First, we found that inducible Cdc42 KO in BM cells inhibited colony-forming activity of total BM cells by ~10-fold and ~40-fold in two CFU-EPC assays that have been used in the published literature - in EGM2 medium containing VEGF, FGF2, IGF1 and EGF [6] and a modified EGM2 medium containing VEGF, FGF2 and IGF1 [8], respectively. The colonies formed from Cdc42 KO BM gave a distinct small round morphology. Concomitantly, Cdc42 deletion resulted in a ~3-fold enhancement of hematopoietic colony-forming CFU-C activity of the BM cells, consistent with our previous report on hematopoietic regulation by Cdc42. Both effects on EPC and HSPC activities can be attributed to hematopoietic cell regulation by Cdc42, since similar reduction of CFU-EPC activity and increase in CFU-C activity were observed in Mx-cre; Cdc42lox/lox transplant recipient mice after polyIC induction. Second, given the controversies in the literature about cell markers of BM EPCs, FACS analysis using 5 different sets of putative EPC markers was carried out to examine the effect of Cdc42 knockout on BM EPC compartment. PolyIC induced Cdc42 deletion led to ~4-fold reduction of CD45lo/CD11b-/VE-Cad+ cells [2], ~3-fold reduction of CD45-/PDGFRa+ [5], ~3 fold reduction of CD45-/Lin-/Flk1+ [3, 4], 2-fold reduction of CD45+/Lin-/Flk1+ and 3-fold decrease of CD45-/CD31+ EC population [9]. Similar results were also obtained from the BM Cdc42 deleted transplant recipients. Third, to further distinguish EPCs of blood lineage from those of non-blood lineage in the BM cells, purified CD45+ or CD45- cells were isolated from BM cells by double FACS sorting, and their abilities to produce EC-like cells and to express endothelial markers CD31, PDGFRa and vWF in a matrigel culture, were analyzed. Cdc42 deletion led to a drastic reduction of the tube-forming and CD31+/PDGFRa+/vWF+ cells by over 100-fold, in both the CD45+ and CD45- BM cell culture. Fourth, treatment of WT BM cells with CASIN abolished colony-forming activity of BM cells in the CFU-EPC assays, mimicking that of Cdc42 knockout. Upon CASIN withdrawn, the cells could continue differentiation into adherent EC-like colonies. Finally, Cdc42 knockout resulted in a 3-fold reduction of VEGFR2 cell surface presentation, but not expression, in BM cells, suggesting Cdc42 may regulate VEGFR2 localization to impact endothelial lineage commitment. While experiments to dissect the signaling mechanisms and the requirement of Cdc42 for angiogenesis and endothelial or endothelial cell-supporting function are ongoing, these results indicate that Cdc42 is essential for BM derived EPC or EPC-like activities. Targeting Cdc42 may provide a novel strategy in modulating BM-EPC and EC regeneration. References: 1. Broman et al. Circ Res 98, 73–80 (2006) 2. Gao et al. Science 319, 195–8 (2008) 3. Chakroborty et al. JCI 118, 1380–9 (2008) 4. Ciarrocchi et al. PloS One. 2, e1338 (2007) 5. Morikawa et al. JEM 206, 2483–96 (2009) 6. Pitchford et al. Cell Stem Cell 4, 62–72 (2009) 7. Sacharidou et al. Blood 115, 5259–69 (2010) 8. Wary et al. Stem Cells 27, 3112–20 (2009) 9. Yoder et al. Blood 109, 1801–9(2007) Disclosures: No relevant conflicts of interest to declare.

Abstract 2618 Rho family small GTPases are critical regulators of multiple functions of blood cells, including adhesion, migration, proliferation, survival and gene expression. Elucidation of their roles in hematopoiesis could be fundamental for understanding the mechanisms of various blood diseases and improving therapeutic outcomes for hematopoietic abnormalities. The Rho GTPases Rac1, Rac2 and Cdc42 have been found essential in maintaining hematopoietic stem cell (HSC) niche residency and regulating myelopoiesis and lymphopoiesis in previous mouse gene targeting studies. Using a dominant-negative mutant overexpression approach, an earlier study showed that suppression of RhoA activity enhanced HSC proliferation and engraftment potential; however, the bone fide role of RhoA in blood development remains unknown given limitations of such a mutant overexpression approach in specificity, dosage effect, and physiological relevance. Here, we stringently define the function of RhoA in HSC maintenance and hematopoiesis using an interferon inducible RhoA conditional knockout mouse model (Mx-cre+; RhoAloxp/loxp). Systematic deletion of RhoA caused lethality of the mice 7 days post polyIC induction due to hematopoietic failure that was accompanied by drastically decreased bone marrow (BM) cellularity (to ∼1/3 of wild type controls), a loss of splenocytes, and a significant reduction of cell counts of most cell lineages in peripheral blood, suggesting RhoA is required for multiple blood cell lineage differentiation and production. Syngenic transplant experiments yielded similar results, demonstrating that these effects are intrinsic to the hematopoietic compartment. The observed cytopenia resulting from RhoA loss was associated with the exhaustion of BM phenotypic HSPC (Lin−Sca1+c-kit+, LSK) and the hierarchical progenitor cells (Lin−c-kit+, LK) in number and frequency, a complete loss of colony forming activities, and a total engraftment failure. In addition, BrdU chase labeling and Annexin V/7-AAD staining revealed that RhoA deletion caused a transient increase of proliferation (1.7 fold increase in S phase) and reduction of survival (16.8 fold reduction in Annexin V− 7-AAD−) of remaining LSK in the BM. These results indicate that RhoA plays an indispensible, cell autonomous role in HSPC maintenance and hematopoiesis. In a competitive transplantation model where Mx-cre+;RhoAloxp/loxp or Mx-cre−;RhoAloxp/loxp CD45.2+ BM cells and WT CD45.1+ competitor cells were transplanted at 1:1 ratio into syngenic CD45.1+ recipients prior to polyIC induction, RhoA deletion also caused a complete extinction of donor derived (CD45.2+) Mac1+Gr1+ myeloid cells, B220+ B cells, as well as CD3+ T cells, in the peripheral. Interestingly, distinct from these more differentiated lineages, BM CD45.2+ LSK population was only marginally affected (88.9 % of pre-polyIC induction level) while the CD45.2+ LK cells and later hierarchical lineages were rapidly eliminated after RhoA deletion in this competitive transplant model. This was associated with increased apoptosis in CD45.2+ RhoA−/− LK, but not LSK, cells, suggesting a specific requirement of RhoA in the myeloid progenitor cell survival. Further, the CD45.2+ RhoA−/− LSK and LSKCD150+ cell populations, not differentiated donor-derived progenitors, from the primary competitive transplant BM, were able to be maintained in secondary transplant recipients 5 months post-transplantation, indicating that RhoA serves as a key regulator at an early progenitor differentiation step. Interestingly, RhoA deletion did not affect lin− cell p-MLC and p-cofilin contents and in vitro expansion in response to SCF stimulation, suggesting that RhoA is not required for actomyosin signaling nor SCF induced proliferation. Taken together, our results implicate RhoA as a unique and essential regulator of multipotent progenitor differentiation and survival that controls multi-lineage hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Cyclophosphamide's lack of hematopoietic stem cell toxicity and its unique effects on the immune system have prompted several investigators to explore its potential for the prevention of graft-versus-host disease (GVHD). In haploidentical hematopoietic stem cell transplants, post-transplant cyclophosphamide together with standard prophylaxis reduces the incidence of GVHD to acceptable rates without the need for T cell depletion. In matched related and unrelated donor settings, cyclophosphamide alone has produced encouraging results. In particular, the low incidence of chronic GVHD is noteworthy. Here, we present a review of the current understanding of the mechanism of action of post-transplant cyclophosphamide and summarize the clinical data on its use for the prevention of GVHD.