Expression Of Membrane Receptors Research Articles

Role of TREM2 and CD200R in modulating microglial response in astrocyte targeted IL10Tg mice following perforant pathway transection.

One of the endogenous mechanisms regulating inflammatory cell activation following brain injury is the expression of modulator and/or inhibitor membrane receptors. Recent studies demonstrate that regulatory receptor TREM2-mediated phagocytic function of microglia/macrophages (MM) is required for debris clearance and inhibitory receptor CD200R-mediated deactivation of microglia is necessary for maintenance of CNS tissue homeostasis. Also, anti-inflammatory Interleukin-10 (IL-10) has been demonstrated in activated astrocytes and microglia after injury in the CNS but its specific role still remains ambiguous.

Abstract 4238: Exploring the therapeutic potential of IR and IGF-1R/IR (co-)targeting in ovarian cancer

Abstract Ovarian cancer is the most lethal gynecological malignancy. Despite high response rates to platinum based therapies most patients relapse with resistant disease. For these patients novel therapeutic strategies are needed. Hyper-activation of the insulin-like growth factor receptor (IGF-1R) has been implicated in the development, progression and platinum resistance of ovarian cancer. However, targeting the IGF-1R has shown limited clinical benefit. The insulin receptor (IR), highly homologous to the IGF-1R, confers resistance to IGF-1R targeted therapies. Furthermore, in ovarian cancer alternative splicing results in the expression of IR-A & IR-B isoforms, while in normal adult tissues IR-B is the predominant isoform. Therefore targeting the IR or IR-A may be of clinical importance. In this study we determined the therapeutic potential of IR targeting in ovarian cancer. First, the expression levels of the IR isoforms, IGF-1R and stimulatory ligands IGF-I & II were determined in a panel of ovarian cancers and ovarian cancer cell lines of different histological subtype (endometrioid, clear cell, serous) using RT-PCR. IR isoforms and IGF-1R were expressed in 96% (43/44) and 78% (35/44) of the ovarian cancers compared to 89% (8/9) and 100% (9/9) of the ovarian cancer cell lines. IGF-I and IGF-II were expressed in all the ovarian cancers compared to 11% (1/9) and 0% (0/9) of the ovarian cancer cell lines, indicating the presence of an autocrine loop. In addition, all cell lines showed membrane receptor expression and functional downstream signaling upon IGF-I, IGF-II & insulin, via PI3K as determined by western blot and flow cytometry. Subsequently, we investigated which splicing factors may be involved in the IR alternative splicing. Expression of splicing factors (hnRNPA1A, hnRNA2B1, CELF1, SRSF1, hnRNPH, hnRNPF) were determined by qRT-PCR. However, expression levels did not correlate with IR isoform levels, indicating a more complex regulation of IR splicing. Finally, we determined the effect of IR, IGF-1R and IR/IGF-1R (co-)targeting by S961 (1000nM), MAB391 (20µg) and OSI906 (10µM), respectively on ovarian cancer cell survival by MTT and clonogenic assays. IR inhibition by S961 resulted in a decrease in cell viability up to 40% in 7/9 ovarian cancer cell lines. Preliminary data showed similar results upon IGF-1R/IR inhibition with OSI906. IGF-1R inhibition by MAB391 did not affect cell viability. Furthermore a reduction in clonogenic capacity (75%) by OSI906 is observed in 2/3 ovarian cancer cell lines tested. In conclusion, in ovarian cancer functional IR and IGF-1R signaling as well as the presence of a possible autocrine loop may contribute to ovarian cancer pathogenesis and progression. Targeting IR signaling with the IR-antagonist S961 and co-targeting IR/IGF-1R signaling by OSI906 reduced ovarian cancer cell viability, indicating its therapeutic potential in ovarian cancer. Funded by the Dutch Cancer Foundation: Grant RUG 2011-5231 Citation Format: Jolanda A.L. Visser, Roelien A.M. Meijering, Anne K.L. Reyners, Ate G.J. van der Zee, Steven de Jong. Exploring the therapeutic potential of IR and IGF-1R/IR (co-)targeting in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4238. doi:10.1158/1538-7445.AM2014-4238

Photoperiodic regulation of melatonin membrane receptor (MT1R) expression and steroidogenesis in testis of adult golden hamster, Mesocricetus auratus

Photoperiodic modulation of melatonin membrane receptor (MT1R) expression in testis has never been reported for any seasonal breeder. Thus, the aim of the present study was to investigate the expression dynamics of MT1R in testis and its interaction with testicular steroidogenesis in a long-day breeder, Mesocricetus auratus. Hamsters were exposed to different photoperiodic conditions i.e. critical- (CP; 12.5L:11.5D); short-day- (SD; 8L:16D) and long-day- (LD; 16L:8D) for 10weeks wherein testicular steroidogenesis, local melatonin synthesis and the expression of MT1R were analyzed. SD induced melatonin suppressed testicular steroidogenesis as evident from regressed testicular histoarchitecture, decreased expression of AR, StAR, LH-R, P450SCC and enzyme activities of 3β- and 17β-HSD. Differential photoperiodic regulation of MT1R expression in testis suggests its involvement in photoperiodic signal transduction for seasonal adjustment of reproduction. Increased S-NAT (Serotonin N-acetyl transferase) activity and local testicular melatonin under SD condition suggest an inhibitory effect of the local melatonergic system on testicular steroidogenesis. Completely opposite responses were recorded for all the parameters analyzed when hamsters were exposed to CP or LD conditions. In conclusion, we may suggest that photoperiod via regulating circulatory and local melatonin level as well as MT1R expression in testes fine tunes the steroidogenesis and thereby, the reproductive status of male golden hamster.

P01.17 * ROLE OF PHOSPHATYDILCHOLINE-SPECIFIC PHOSPHOLIPASE C IN MODULATION OF CXCL12/CXCR4 AXIS IN A HUMAN GLIOMA CELL LINE

BACKGROUND: The CXCR4 chemokine receptor and its ligand CXCL12 are one of the most well studied chemokine systems in tumor biology. In gliomas CXCL12/CXCR4 axis regulates multiple mechanisms that sustain tumor growth as migration, proliferation, angiogenesis and is emerging as a novel biologically relevant target for brain tumor therapy. Previous studies, in our laboratory highlights a role of the phosphatydilcholine-specific phospholipase C (PC-PLC) in the regulation and expression of growth factor membrane receptors, suggesting a relationship between the metabolism of phosphatydilcholine (PC) and cell receptor status. AIM: Investigate the crosstalk between the chemokine receptor CXCR4 and PC-PLC and its implications in glioma cells growth. MATERIALS AND METHODS: U87MG, a glioblastoma cell line was treated with a conventional CXCR4 antagonist, AMD3100 (10 µM), or with a PC-PLC inhibitor, D609 (Xantate) (100 µM). Confocal Laser Scanning Microscopy (CLSM) and Western Blotting (WB) experiments were performed on cells to investigate protein expression and intracellular localization. Cell metabolism on both treated or untreated U87MG cells was evaluated by in vitro Nuclear Magnetic Resonance Spectroscopy (MRS). RESULTS: CLSM analyses highlighted for the first time an extensive co-localization of CXCR4 and PC-PLC on the plasma membrane of U87MG cells. Furthermore we observed a down-modulation of CXCR4 from cell membrane in response to both inhibitors, AMD3100 and D609, however D609 only induced a decrease in CXCR4 total protein content. Moreover, D609 treatment reduced PC-PLC expression, induced a reduction of pAKT and exerted a significative anti-proliferative effect on U87MG cells (49% inhibition). MRS analyses revealed alterations on U87MG metabolism, showing in D609-treated cells an increase of choline-containing metabolites (Glycerophosphocholine, GPCho; Choline, Cho) (increase of 77%) that could be correlated with activation of lipases and phospholipases that can induce an accumulation of neutral lipids associated with the inhibition of cell proliferation. D609 treatment also induced a significative decrease of intracellular concentration of Lactate (38%), and preliminary analyses on lactate deydrogenase (LDH) showed a decrement of the enzyme activity (30%) suggesting that inactivation of PC-PLC could impaired the glycolysis pathway in U87MG. CONCLUSIONS: In this study we identified for the first time an interaction between the chemokine receptor CXCR4 and the enzyme PC-PLC on a glioma cell line. The specific inhibition of PC-PLC modulates CXCR4 receptor localization on cellular membrane and protein expression. Our findings suggest that the inhibition of PC-PLC could represent a new molecular approach in CXCR4-overexpressing glioma cells.

Proton receptor GPR68 expression in dendritic-cell-like S100β-positive cells of rat anterior pituitary gland: GPR68 induces interleukin-6 gene expression in extracellular acidification

S100β-positive cells, which do not express the classical pituitary hormones, appear to possess multifunctional properties and are assumed to be heterogeneous in the anterior pituitary gland. The presence of several protein markers has shown that S100β-positive cells are composed of populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. Recently, we succeeded in separating S100β-positive cells into round-cell (dendritic-cell-like) and process-cell types. We also found the characteristic expression of anti-inflammatory factors (interleukin-6, Il-6) and membrane receptors (integrin β-6) in the round type. Here, we further investigate the function of the subpopulation of S100β-positive cells. Since IL-6 is also a paracrine factor that regulates hormone producing-cells, we examine whether a correlation exists among extracellular acid stress, IL-6 and hormone production by using primary cultures of anterior pituitary cells. Dendritic-cell-like S100β-positive cells notably expressed Gpr68 (proton receptor) and Il-6. Furthermore, the expression of Il-6 and proopiomelanocortin (Pomc) was up-regulated by extracellular acidification. The functional role of IL-6 and GPR68 in the gene expression of Pomc during extracellular acidification was also examined. Small interfering RNA for Il-6 up-regulated Pomc expression and that for Gpr68 reversed the down-regulation of Il-6 and up-regulated Pomc expression by extracellular acidification. Thus, S100β-positive dendritic-like cells can sense an increase in extracellular protons via GPR68 and respond by the production of IL-6 in order to suppress the up-regulation of Pomc expression.

Multicolor imaging of cancer cells with fluorophore-tagged aptamers for single cell typing.

The discrimination of the type of cancer cells remains challenging due to the subtle differences in their expression of membrane receptors. In this work, we developed a multicolor cell imaging method for distinguishing the type of cancer cells with fluorophore-tagged aptamers. We found that the interaction between aptamers and cancer cells was affected by both of the sequence of aptamers and the labeled dyes. As the co-ownership of biomarkers for different cancer cell lines, the fluorophore-tagged aptamers interacted with different cancer cell lines in different degree, resulting in a distinct color to discriminate the type of cancer cells at single cell level. Taking advantage of the cross-reactive ability of the fluorophore-tagged aptamers, we could not only distinguish the cancerous cells quickly from large quantities of noncancerous cells, but also identify the type of the cancerous cells. This work has potential application for cancer diagnostic and therapy in the future.

An inside job: how endosomal Na(+)/H(+) exchangers link to autism and neurological disease.

Autism imposes a major impediment to childhood development and a huge emotional and financial burden on society. In recent years, there has been rapidly accumulating genetic evidence that links the eNHE, a subset of Na+/H+ exchangers that localize to intracellular vesicles, to a variety of neurological conditions including autism, attention deficit hyperactivity disorder (ADHD), intellectual disability, and epilepsy. By providing a leak pathway for protons pumped by the V-ATPase, eNHE determine luminal pH and regulate cation (Na+, K+) content in early and recycling endosomal compartments. Loss-of-function mutations in eNHE cause hyperacidification of endosomal lumen, as a result of imbalance in pump and leak pathways. Two isoforms, NHE6 and NHE9 are highly expressed in brain, including hippocampus and cortex. Here, we summarize evidence for the importance of luminal cation content and pH on processing, delivery and fate of cargo. Drawing upon insights from model organisms and mammalian cells we show how eNHE affect surface expression and function of membrane receptors and neurotransmitter transporters. These studies lead to cellular models of eNHE activity in pre- and post-synaptic neurons and astrocytes, where they could impact synapse development and plasticity. The study of eNHE has provided new insight on the mechanism of autism and other debilitating neurological disorders and opened up new possibilities for therapeutic intervention.

Prognostic value of immunohistochemical marker HER2/neu in patients resected for gastric cancer stage IIIA

IntroductionPatients with the same stage of malignancy often have different life-span, which makes it necessary to search for new prognostic factors.The high incidence and low survival rate of patients with gastric cancer also cause an increased interest in studying the immunohistochemical profile of tumors of the stomach. Recently, researchers are studying the prognostic significance of immunohistochemical markers such as: EGFR [10], VEGF [12,15,18], HER2/neu [8,17,19], expression of betakatenin [1], hERG1 [7], p53 [14], PCNA [14,20]Of particular interest is the HER2/neu. Overexpression of HER2/neu promotes tumor growth and plays an important role in the pathogenesis of some human malignancies [17].Nevertheless, the impact of HER2/neu status on the prognosis of patients with gastric cancer remains controversial [6,11,19]. Nakajima Garcia and colleagues believe that the increased expression of HER2/neu in patients with gastric cancer correlates with poor prognosis and more aggressive disease [11]. At the same time, Begnami found that life expectancy in gastric cancer patients with HER-2/neu-positively stained tumors was significantly shorter than in patients with HER-2/neu-negatively stained tumors (17 months vs. 40 months, respectively, p=0.023) [5].The aim of researchStudy the prognostic significance of immunohistochemical marker HER-2/neu in patients resected for gastric cancer stage IIIA.Materials and methodsMaterials for the study were histologic surgical specimens of the primary tumor in 21 patients who had surgery in the Zaporozhye Regional Clinical Oncology Center due to stomach cancer stage IIIA, including 14 men (67%) and 7 females (33%).Patients underwent surgery in the volume: gastrectomy and distal subtotal resection, 86% of patients received adjuvant chemotherapy of standard schemes.Immunohistochemical study of HER2/neu (erbB-2) used a rabbit polyclonal antibody. Immunohistochemical reaction was evaluated as negative in the absence of expression of membrane receptors or HER2/neu coloring immunopositive less than 5% of tumor cells ("-" - no reaction) as low positive - in the presence of 6-10 % of tumor cells immunopositive ("+"), moderately positive - 10-50% ("++") and high pozitive - 50% positive tumor cells ("+++"). For further comparative immunohistochemical study identified two main levels of immunoreactivity: low or reduced protein expression (-/+) and high level or overexpression of the marker (++/+++).To assess survival used the Kaplan - Meier. Comparison of survival curves was performed using the Cox-F test.ResultsThe largest group of patients with gastric cancer stage IIIA consisted of patients with a negative reaction to the presence of receptor HER2/neu (47,6%), the second place is a group of patients with moderately positive reaction - 23.8%, patients with low positive and high pozitive reactions were equally divided - by 14.3%.Median survival of patients with gastric cancer stage IIIA was 19.8 months.The study two groups of patients with a low level or reduced protein expression (-/+) and high level or overexpression of the marker (++/+++) showed a higher survival rate in the former group (median survival was 50 months) compared to the second group, where the median was equal to 16 months (p<0.05).Conclusion1. Median survival of patients with gastric cancer operated stage IIIA was on average 19.8 months.2. High level or overexpression of marker HER2/neu (++/+++) is bad prognostic sign for life expectancy of patients with stage IIIA (p<0.05).3. The prospect of further study is research of the prognostic value of marker HER2/neu in patients with gastric cancer stages I and II.

Experimentally induced stress, oxidative load and changes in immunity in a tropical wild bird, Perdicula asiatica: involvement of melatonin and glucocorticoid receptors

Throughout the year, birds encounter various environmental challenges such as extreme temperatures, rainfall and shortage of food. Here we report on the effect of stress on the general immunity of wild birds as measured by several assays including melatonin, an anti-stress hormone. We selected Perdicula asiatica, a wild tropical bird, and exposed them to experimental stressors such as water deprivation, food deprivation and immobilization, i.e., stressors that they would encounter in a natural environment. We measured the oxidative load in the spleen in terms of superoxide dismutase (SOD), catalase activity and thiobarbituric acid-reactive substance (TBARS) levels. The immune status was judged by total leukocyte count (TLC), heterophil/lymphocyte ratios (H/L) and percent stimulation ratio of splenocytes (%SR). The peripheral levels of melatonin and corticosterone were also determined and correlated with the expression of melatonin (Mel1a/Mel1b) and glucocorticoid receptors. Our results showed a significant (p<0.05) decrease in splenic SOD and catalase activity, while a significant (p<0.05) increase in TBARS and a corticosterone level was observed. Stressful conditions also decreased the immune status as reflected by the low values of H/L ratios, TLC and %SR. In contrast, melatonin pretreatment significantly (p<0.05) reduced the oxidative stress and improved the immune parameters when compared to untreated control birds. This suggests that melatonin prevents/alleviates oxidative damage and suppresses the immune status induced by stressful conditions via its membrane receptor expression (Mel1a and Mel1b) in P. asiatica.

Experimental Screening mRNA Targets by Reverse Transcription for Ribozyme to GPA Expression Interference

An oligodeoxynucleotide library which the sequence were defined at 5 prime and randomized at 3 prime was employed to screen mRNA accessible targets,in reverse transcription and PCR after hybridized the library with mRNA. The mRNA of Glycophorin A(GPA),type I transmembrane glycoprotein,was screened and obtained 4 targets sequences. Accordingly 4 antisense nucleic acids designed respectively,the binding efficiency of every target were verified by using RNase H with antisense nucleic acids. Among them 2 targets showed better effects on binding and cutting. Designed 2 ribozymes to these targets,packaged in lentivirus system,then infected K562 cells(human erythroid leukemia line),the down-regulation effect of gene expression was validated by Real Time RT-PCR and by Western Blot. It was found that the screened targets showed the best effective knocking down effects on gene expression. The study provided a reference for mRNA targets screening and Ribozyme design,and was helpful in membrane receptor expression interference,since GPA is a transmembrane protein.

Flavonoids on Allergy

Flavonoids are secondary products of plants that include thousands of compounds classified in several classes. Preclinical studies mainly carried out in rodents suggest that they may have a role in the prevention of immunoglobulin E (IgE) synthesis and mast cell degranulation. Interestingly, using animal models with allergic asthma, it can be concluded that preventive treatment with particular flavonoid classes can reduce airway hyperresponsiveness, which is accompanied by lowered inflammatory mediators such as histamine and cytokines, and cell infiltration. In addition, there are some clinical trials in patients with allergic asthma or rhinitis that offer promising results with regard to these natural compounds. On the other hand, the dissection of cellular mechanisms that have interacted with flavonoids allow their effectiveness to be understood. Among these mechanisms there is a lower expression of IgE receptor or other membrane receptors, the modulation of calcium influx, and the downregulation of particular signaling pathways that eventually produces lower primary and secondary mediator release. In conclusion, some particular flavonoids could be an alternative or complementary therapy in the prevention and treatment of some allergies. Nevertheless, an increased number of clinical trials is required in order to confirm the therapeutic role of flavonoids.

Giant vesicles functionally expressing membrane receptors for an insect pheromone

To date, biochemical approaches to membrane receptors have been limited to the following methods: knockout or overexpression of membrane receptors by gene introduction and genome engineering or extraction of membrane receptor-surfactant complexes from innate cells and their introduction into model biomembranes. Here, we describe the development of a third method involving gene expression using cell-free in situ protein synthesis inside model biomembrane capsules. We verified this method by synthesizing olfactory receptors from the silkmoth Bombyx mori inside giant vesicles and found that they were excited in the presence of their ligand the Bombyx mori sex pheromone.

Abstract P2-09-15: Effect of [4-valine 5-glutamine] desmopressin, a novel vasopressin analog, on human breast cancer cell growth in two-dimensional (2D) and three-dimensional (3D) cultures

Abstract Background: Desmopressin (dDAVP), a synthetic nonapeptide derivative of arginine vasopressin, is a safe hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor (V2R). This receptor is expressed in endothelium and also in some tumor cells. V2R stimulation is associated with increased levels of cyclic AMP and cyclic AMP-dependent protein kinase activation. We previously reported that dDAVP can inhibit progression of residual metastatic cells in preclinical models. Among other mechanisms, the compound induces an agonist effect on V2R present in tumor cells. This nonapeptide was substituted in positions 4 and 5 searching for compounds with improved biological activity. Such positions belong to the conformational peptide loop which has a key role in ligand-receptor interaction. After an extensive in vitro and in vivo characterization, novel peptide compound [4-valine 5-glutamine] dDAVP ([V4Q5]dDAVP) exerted a remarkable and improved antitumor effect in comparison to parental peptide dDAVP. [V4Q5]dDAVP inhibited tumor growth in both syngeneic F3II and xenogeneic MDA-MB-231 breast cancer models, modulating tumor aggressiveness and vasculature. In this study, to further understand direct cytostatic action of this novel vasopressin analog on tumor cells, we assessed the effect of [V4Q5]dDAVP on clonogenic and three dimensional growth of V2R-expressing human breast carcinoma cell lines. Methods and results: We used MCF7 and triple negative MDA-MB-231 human breast cancer cell lines to conduct the experiments. Vasopressin V2 membrane receptor expression in tumor cell lines was assessed by RT-PCR and immunofluorescence. We first examined peptide ability to modify in vitro low density cell growth by the clonogenic growth assay. Incubation with [V4Q5]dDAVP (100-1500 nM) during one week significantly reduced the number of tumor cell colonies showing a concentration dependant manner and an IC50 value of 1135 nM. To assess the effect of novel analog on three dimensional growth of cancer cells we used two different assays. The first one evaluates the ability of basement membrane extract (BME)-embedded single cells to proliferate and form spheroids. The second assay evaluates growth of single fully established spheroids (Approx. 300 mm diameter) transplanted individually on a non-adherent surface. [V4Q5]dDAVP, at a concentration of 1000 nM, was able to significantly reduce the number of BME-embedded spheroids formed from single cells after 14 days of treatment. In a similar manner, incubation of established and isolated spheroids with [V4Q5]dDAVP using the same concentration for 9 days, caused a decrease in multicellular spheroids final diameter. Cellular metabolic activity analysis by the MTS assay showed no significant differences between treated and control groups. Conclusions: We report for the first time the inhibitory effect of [V4Q5]dDAVP on colony and spheroid formation and growth of human breast carcinoma cells. Our results using three dimensional models suggest that V2R stimulation in cancer cells may affect early stages of microtumor establishment. Further analysis of involved signaling pathways in treated 3D cultures is needed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-15.

A Novel Therapeutic Approach In Acute Myeloid Leukemia (AML): In Vivo Preclinical Pharmacokinetic (PK), Pharmacodynamic (PD) and Antileukemia Activities Of Synthetic 2'-O-Methylphosphorothioate Mir-29b

AML is one of the most common types of leukemia. Despite recent progress in understanding the mechanisms of leukemogenesis, molecular risk assessment and risk-adapted treatments, only 40% of younger (<60 years) and <10% of older (>60 years) AML patients achieve long-term complete remission, indicating the need for novel therapeutic approach. microRNAs (miRs) are short non-coding RNAs and have been found deregulated in leukemia. In AML, aberrant downregulation of miR-29b leads to upregulation of Sp1 which complexes with NFκB. This complex transactivates oncogenes such as DNMTs (DNA methyltransferases) that mediate DNA hypermethylation and epigenetic gene silencing, and FLT3 and KIT that encode receptor tyrosine kinases which mediate cell proliferation and survival. Thus, restoring normal levels of miR-29b with synthetic RNA oligonucleotides may downregulate the aforementioned oncogenes and represent a novel therapeutic approach in AML. In this study, we determined the PK and PD activities of a synthetic single-strand miR mimic, 2'-O-methylphosphorothioate (2'-MeOPS) miR-29b and demonstrated its effective dose and schedule to achieve antileukemia activity in vivo.PK studies of 2'-MeOPSmiR29b were performed at different dose levels (7.5, 16, 24, and 32 mg/kg) administered as intravenous (i.v.) or intraperitoneal (i.p.) dosing bolus in healthy CD2F1 mice. Plasma and major organ tissues were collected from 5 minutes to 48 hours following miR administration for determination of the miR concentrations. 2'-MeOPS miR-29b displayed a three-compartmental plasma PK profile with C5min of 29.5 µM, area-under-the-curve (AUC0-∞) of 574.3 min*μM, alpha-half-life of 3.8 minutes, beta-half-life of 98.8 minutes, gamma-half-life of 21.5 hours, and a total body clearance of 0.007 L/min*kg when given i.v. at 24 mg/kg. I.v. and i.p. administrations showed similar PK behaviors. Tissue miR distribution showed slow elimination rate and substantially high concentrations throughout 48 hours in all tissues, including bone marrow (BM) and spleen, but not in brain. Plasma PK and tissue distribution lack dose-proportionality. Multiple daily dosing resulted in tissue accumulation. After four daily doses, BM and spleen miR-29b concentrations were twice higher than that of a single dose 6 or 24 hours following the last administration (p<0.05).PD studies showed time-dependent downregulation of DNMTs and Sp1 expression by 2'-MeOPSmiR-29b in BM of MV4-11 cells-engrafted NOD/SCID/Gamma mice, an aggressive murine model for AML. Those proteins were reduced ∼50% and ∼80%, respectively, 24 or 48 hours after a single bolus of 2'-MeOPSmiR-29b at 24 mg/kg. Based on the PK-PD data, two administration routes and schedules were tested one week after MV4-11 cells transplantation and followed longitudinally for disease progression and survival: (a) a dailyx4 i.v. doses in week 1 alternating with dailyx4 i.p. doses in week 2 which was repeated 3 cycles; (b) a dailyx4 i.p. doses every week for 8 weeks. 2'-MeOPSmiR-29b significantly improved the survival of AML mice. The median survival (ms) of saline and scrambled (sc) miR-treated AML mice were 28 and 29 days, respectively, post-transplantation (similar for a and b regimen) vs. 44 and 37 days of miR-29b-treated AML mice given a and b regimen, respectively (p = 0.001).In order to increase the specificity of the synthetic miR delivery to leukemia cells in turn minimizing the potential host toxicity, we formulated a transferrin (Tf)-conjugated nanoparticle (NP) to encapsulate the 2'-MeOPSmiR to target MV4-11 cells that with enriched expression of Tf membrane receptor. In vivo i.p. administration of 2.4 mg/kg Tf-NP-2'MeOPSmiR-29b with b regimen prolonged the survival of the MV4-11-engrafted leukemic mice (ms = 43 days, p=0.01) as compared to the Tf-NP-2'MeOPS sc miR-treated mice (ms=30 days). Thus, the therapeutic activity of the Tf-NP-formulated 2'MeOPSmiR-29b was similar to that of naked 2'-MeOPSmiR-29b given at a 10-fold higher dose (2.4 vs. 24 mg/kg).To our knowledge, this is the first synthetic miR mimic to be evaluated for PK, PD activities and antileukemic efficacy in a murine leukemia model. The correlation of the PK and PD findings will facilitate the development a PK-PD model for designing optimal dose and schedule for future preclinical investigation and rapid transition to the clinic. Disclosures:Caligiuri:Innate Pharma: Membership on an entity's Board of Directors or advisory committees.

Melatonin enhances chondrogenic differentiation of human mesenchymal stem cells

Intramembranous ossification and endochondral ossification are two ways through which bone formation and fracture healing occur. Accumulating amounts of evidence suggests that melatonin affects osteoblast differentiation, but little is known about the effects of melatonin on the process of chondrogenic differentiation. In this study, the effects of melatonin on human mesenchymal stem cells (MSCs) undergoing chondrogenic differentiation were investigated. Cells were induced along chondrogenic differentiation via high-density micromass culture in chondrogenic medium containing vehicle or 50nm melatonin. Histological study and quantitative analysis of glycosaminoglycan (GAG) showed induced cartilage tissues to be larger and richer in GAG, collagen type II and collagen type X in the melatonin group than in the untreated controls. Real-time RT-PCR analysis demonstrated that melatonin treatment significantly up-regulated the expression of the genes involved in chondrogenic differentiation, including aggrecan (ACAN), collagen type II (COL2A1), collagen type X (COL10A1), SRY (sex-determining region Y)-box 9 (SOX9), runt-related transcription factor 2 (RUNX2) and the potent inducer of chondrogenic differentiation, bone morphogenetic protein 2 (BMP2). And the expression of melatonin membrane receptors (MT) MT1 and MT2 were detected in the chondrogenic-induced-MSCs by immunofluorescence staining. Luzindole, a melatonin receptor antagonist, was found to partially block the ability of melatonin to increase the size and GAG synthesis of the induced cartilage tissues, as well as to completely reverse the effect of melatonin on the gene expression of ACAN, COL2A1, COL10A1, SOX9 and BMP2 after 7days of differentiation. These findings demonstrate that melatonin enhances chondrogenic differentiation of human MSCs at least partially through melatonin receptors.

Nonhematopoietic cells represent a more rational target of in vivo hedgehog signaling affecting normal or acute myeloid leukemia progenitors

Recent work has shown that leukemic stem cell self-renewal in chronic myeloid leukemia is dependent on cell-intrinsic hedgehog (Hh) signaling, and early clinical trials suggest that targeting this pathway is also therapeutic in patients with acute myeloid leukemia (AML). In this study, we aimed to better understand Hh signaling in normal hematopoiesis and AML by molecularly and functionally analyzing more than 200 primary human AML patient samples compared with nonleukemic controls. Gene expression analysis indicated that Hh pathway transcripts were similarly regulated in AML and nonleukemic controls, regardless of whether samples were purified based on primitive phenotypes. Consistent with these results, pharmacologic inhibition of Smoothened (SMO) did not preferentially reduce invitro colony formation of AML versus normal progenitors. Using a unique analytic approach, messenger RNA expression of membrane receptor SMO was found to be unexpectedly rare within all hematopoietic samples analyzed, which is indicative of heterogeneity at the level of Hh signaling machinery. In contrast, abundant SMO expression could be readily detected in the nonhematopoietic fraction of human and murine bone marrow (BM) cells. Our predictions of increased SMO(+) cell frequencies within nonhematopoietic BM fractions were further supported by single-cell protein analyses. Although we did not find support for cell-autonomous sensitivity of AML cells to Hh pathway inhibition, we alternatively suggest that nonhematopoietic BM cells represent an indirect target through which primitive normal and leukemic cells can be modulated. These findings suggest current approaches to applying Hh inhibition should be carefully reevaluated to account for BM niche cell regulation that might be selectively Hh responsive.

Investigation of Free Fatty Acid Associated Recombinant Membrane Receptor Protein Expression in HEK293 Cells Using Raman Spectroscopy, Calcium Imaging, and Atomic Force Microscopy

G-protein-coupled receptor 120 (GPR120) is a previously orphaned G-protein-coupled receptor that apparently functions as a sensor for dietary fat in the gustatory and digestive systems. In this study, a cDNA sequence encoding a doxycycline (Dox)-inducible mature peptide of GPR120 was inserted into an expression vector and transfected in HEK293 cells. We measured Raman spectra of single HEK293 cells as well as GPR120-expressing HEK293-GPR120 cells at a 48 h period following the additions of Dox at several concentrations. We found that the spectral intensity of HEK293-GPR120 cells is dependent upon the dose of Dox, which correlates with the accumulation of GPR120 protein in the cells. However, the amount of the fatty acid activated changes in intracellular calcium (Ca(2+)) as measured by ratiometric calcium imaging was not correlated with Dox concentration. Principal components analysis (PCA) of Raman spectra reveals that the spectra from different treatments of HEK293-GPR120 cells form distinct, completely separated clusters with the receiver operating characteristic (ROC) area of 1, while those spectra for the HEK293 cells form small overlap clusters with the ROC area of 0.836. It was also found that expression of GPR120 altered the physiochemical and biomechanical properties of the parental cell membrane surface, which was quantitated by atomic force microscopy (AFM). These findings demonstrate that the combination of Raman spectroscopy, calcium imaging, and AFM may provide new tools in noninvasive and quantitative monitoring of membrane receptor expression induced alterations in the biophysical and signaling properties of single living cells.

Cell-free protein expression systems in microdroplets: Stabilization of interdroplet bilayers

Cell-free protein expression with bacterial lysates has been demonstrated to produce soluble proteins in microdroplets. However, droplet assays with expressed membrane proteins require the presence of a lipid bilayer. A bilayer can be formed in between lipid-coated aqueous droplets by bringing these into contact by electrokinetic manipulation in a continuous oil phase, but it is not known whether such interdroplet bilayers are compatible with high concentrations of biomolecules. In this study, we have characterized the lifetime and the structural integrity of interdroplet bilayers by measuring the bilayer current in the presence of three different commercial cell-free expression mixtures and their individual components. Samples of pure proteins and of a polymer were included for comparison. It is shown that complete expression mixtures reduce the bilayer lifetime to several minutes or less, and that this is mainly due to the lysate fraction itself. The fraction that contains the molecules for metabolic energy generation does not reduce the bilayer lifetime but does give rise to current steps that are indicative of lipid packing defects. Gel electrophoresis confirmed that proteins are only present at significant amounts in the lysate fractions and, when supplied separately, in the T7 enzyme mixture. Interestingly, it was also found that pure-protein and pure-polymer solutions perturb the interdroplet bilayer at higher concentrations; 10% (w/v) polyethylene glycol 8000 (PEG 8000) and 3 mM lysozyme induce large bilayer currents without a reduction in bilayer lifetime, whereas 3 mM albumin causes rapid bilayer failure. It can, therefore, be concluded that the high protein content of the lysates and the presence of PEG polymer, a typical lysate supplement, compromise the structural integrity of interdroplet bilayers. However, we established that the addition of lipid vesicles to the cell-free expression mixture stabilizes the interdroplet bilayer, allowing the exposure of interdroplet bilayers to cell-free expression solutions. Given that cell-free expressed membrane proteins can insert in lipid bilayers, we envisage that microdroplet technology may be extended to the study of in situ expressed membrane receptors and ion channels.

Activation of the EfnB1/EphB1 Forward Signaling Promotes Cell Cycle Arrest in Acute Myeloid Leukemia Cells

AbstractAbstract 4719Ephrin signaling has been shown to contribute to the pathogenesis of many solid tumors with respect to tumor growth, tumor cell survival, angiogenesis, and metastasizing capacity (Cytokine Growth Factor Rev. Dec;15(6):419-33, et al. Neuro Oncol. 2012). Recently, an aberrant DNA methylation status of ephrin receptors and ligands was described to be associated with outcome in acute lymphoblastic leukemia (Blood.2010 Mar 25;115(12):2412-9). In acute myeloid leukemia (AML), we found an intriguing heterogeneity in membrane receptor expression levels of EphB1. Therefore, we challenged to evaluate the role of EphB1 receptor forward signaling in AML.We investigated the influence of the EphB1 receptor forward signaling in THP-1 (EphB1high), HL60 (EphB1int), and MOLM13 (EphB1low) AML cell lines through exogenous stimulation with the EphB1 ligand; EfnB1. EfnB1 stimulation of the AML cell lines demonstrated to reduce AML growth solely in the EphB1high and EphB1int cell lines (Fig. 1A,P = 0.001 and P =.056). In addition, EfnB1 stimulation induced apoptosis most pronounced in the EphB1high cells (Fig. 1B). Interestingly, MGG stained cytospins of EfnB1 treated THP-1 showed multinucleation of AML cells (Fig. 1C). We hypothesized that these phenotypic effects could be assigned to cell cycle arrest in THP-1 cells. Additionally, cell cycle regulatory proteins CDC2 and CyclinB1 were evaluated by immunoblotting of EfnB1 stimulated THP-1 cells. Phosphorylation of the inactivating CDC2 Tyr15-site demonstrated to be up-regulated in EfnB1 stimulated THP-1 cells, which might be initiated by the increased total CDC2 protein levels that we found (Fig. 1D). CyclinB1 displays enhanced protein expression in EfnB1 treated THP-1 cells. Moreover, quantitative RT-PCR analysis showed that the expression of cell cycle inhibitor p21 is significantly induced by 3-fold in EfnB1 stimulated THP-1 cells, via increasing levels of p53 (Fig. 1E, both P = <0.001).To verify whether the EfnB1 induced cell cycle arrest is EphB1 specific, we enforced EphB1 expression in HL60 EphB1int and MOLM13 EphB1low AML cells by introducing a GFP fused EphB1 overexpression construct. EphB1 overexpression increased EphB1 protein expression levels sufficiently in both AML cell lines, as confirmed by flowcytometric analysis and immunoblots. Exogenous EfnB1 stimulation further increased the apoptosis in EphB1 overexpressing cells in both AML cell lines (Fig. 1F). Again, we found increasing levels of phospho-CDC2Tyr15 and CyclinB1 by immunoblots.From this study, we conclude that AML cells with high EphB1 expression can be forced into a cell cycle arrest upon ligand binding in vitro, while AML cells lacking EphB1 expression have a proliferative and anti-apoptotic survival advantage. The clinical significance and exploitation of EphB1 induced cell cycle arrest in AML will be analyzed in the near future. [Display omitted] (A) Absolute cell counts represent the growth inhibitory effects of EfnB1 ligand stimulation in AML cell lines THP-1 and HL60. (B) Flowcytometric Annexin V/PI apoptosis assay displayed the induction of apoptosis as a result of EfnB1 ligand stimulation in THP-1 and MOLM-13 AML cells. (C) MGG stained cytospins of EfnB1 treated THP-1 cells promotes the induction of multinucleated cells due to cell cycle arrest. (D) Immunoblots showed an enhanced apoptotic BAX/BCL2 ratio, in synergy with an upregulation of cell cycle inactivating checkpoint kinase CDC2Tyr15 upon EfnB1 stimulation in THP-1. (E) qRT-PCR confirms cell cycle inhibition by a 3-fold upregulation of p21 and a 1.5-fold induced expression of p53 in THP-1 EfnB1 stimulated cells. (F) The flowcytometric Annexin V/PI apoptosis assay showed that EfnB1 ligand induced apoptosis is even further induced in EphB1 overexpressing HL60 and MOLM-13 cells. Disclosures:No relevant conflicts of interest to declare.

Enhanced early expression of membrane receptors with the Rous sarcoma virus promoter in baculovirus‐infected insect cells

The effect of the Rous sarcoma virus (RSV) long terminal repeat enhancer/promoter on expression levels of complementary DNAs (cDNAs) encoding seven transmembrane receptors was studied using the baculovirus expression vector system. Expression of the human α(2B)-adrenoceptor (AR) cDNA under the control of the polyhedrin (POL) promoter produced up to 7.6 pmol/mg protein at 28 H post infection (p.i.) in Sf9 cells. The addition of the RSV promoter increased the expression to 11.6 pmol/mg protein. Dramatic increases in expression levels at early times were also obtained with the α(2A)-AR, the M₁ and M₄ muscarinic receptors, and the orexin OX₁ receptor. Analysis of the time-dependent expression revealed that expression driven by the RSV promoter reaches almost maximum 24 H p.i. and that this promoter is superior to the often used POL promoter at early times p.i. when functional studies need to be performed. Functional enhancement of signaling as a result of early expression is demonstrated with the α(2B)-AR and the OX₁ receptor. Finally, enhanced green fluorescent protein fluorescence in living cells was used to monitor expression by various viral promoters. The results verified the early transcriptional activity of the RSV promoter, whereas the cytomegalovirus promoter was found to be poorly active in Sf9 cells.