Erythropoietin-Induced Activation of the JAK2/STAT5, PI3K/Akt, and Ras/ERK Pathways Promotes Malignant Cell Behavior in a Modified Breast Cancer Cell Line

Abstract Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.

The 67-kd laminin receptor is preferentially expressed by proliferating retinal vessels in a murine model of ischemic retinopathy.

Endothelial cell association with vascular basement membranes is complex and plays a critical role in regulation of cell adhesion and proliferation. The interaction between the membrane-associated 67-kd receptor (67LR) and the basement membrane protein laminin has been studied in several cell systems where it was shown to be crucial for adhesion and attachment during angiogenesis. As angiogenesis in the pathological setting of proliferative retinopathy is a major cause of blindness in the Western world we examined the expression of 67LR in a murine model of hyperoxia-induced retinopathy that exhibits retinal neovascularization. Mice exposed to hyperoxia for 5 days starting at postnatal day 7 (P7) and returned to room air (at P12) showed closure of the central retinal vasculature. In response to the ensuing retinal ischemia, there was consistent preretinal neovascularization starting around P17, which persisted until P21, after which the new vessels regressed. Immunohistochemistry was performed on these retinas using an antibody specific for 67LR. At P12, immunoreactivity for 67LR was absent in the retina, but by P17 it was observed in preretinal proliferating vessels and also within the adjacent intraretinal vasculature. Intraretinal 67LR immunoreactivity diminished beyond P17 until by P21 immunoreactivity was almost completely absent, although it persisted in the preretinal vasculature. Control P17 mice (not exposed to hyperoxia) failed to demonstrate any 67LR immunoreactivity in their retinas. Parallel in situ hybridization studies demonstrated 67LR gene expression in the retinal ganglion cells of control and hyperoxia-exposed mice. In addition, the neovascular intra- and preretinal vessels of hyperoxia-treated P17 and P21 mice labeled strongly for 67LR mRNA. This study has characterized 67LR immunolocalization and gene expression in a murine model of ischemic retinopathy. Results suggest that, although the 67LR gene is expressed at high levels in the retinal ganglion cells, the mature receptor protein is preferentially localized to the proliferating retinal vasculature and is almost completely absent from quiescent vessels. The differential expression of 67LR between proliferating and quiescent retinal vessels suggests that this laminin receptor is an important and novel target for future chemotherapeutic intervention during proliferative vasculopathies.

Reduced Nitro-oxidative Stress and Neural Cell Death Suggests a Protective Role for Microglial Cells in TNFα−/− Mice in Ischemic Retinopathy

Purpose. Neovascularization occurs in response to tissue ischemia and growth factor stimulation. In ischemic retinopathies, however, new vessels fail to restore the hypoxic tissue; instead, they infiltrate the transparent vitreous. In a model of oxygen-induced retinopathy (OIR), TNFa and iNOS, upregulated in response to tissue ischemia, are cytotoxic and inhibit vascular repair. The aim of this study was to investigate the mechanism for this effect.

Methods. Wild-type C57/BL6 (WT) and TNFa-/- mice were subjected to OIR by exposure to 75% oxygen (postnatal days 7–12). The retinas were removed during the hypoxic phase of the model. Retinal cell death was determined by TUNEL staining, and the microglial cells were quantified after Z-series capture with a confocal microscope. In situ peroxynitrite and superoxide were measured by using the fluorescent dyes DCF and DHE. iNOS, nitrotyrosine, and arginase were analyzed by real-time PCR, Western blot analysis, and activity determined by radiolabeled arginine conversion. Astrocyte coverage was examined after GFAP immunostaining.

Results. The TNFa-/- animals displayed a significant reduction in TUNEL-positive apoptotic cells in the inner nuclear layer of the avascular retina compared with that in the WT control mice. The reduction coincided with enhanced astrocytic survival and an increase in microglial cells actively engaged in phagocytosing apoptotic debris that displayed low ROS, RNS, and NO production and high arginase activity.

Conclusions. Collectively, the results suggest that improved vascular recovery in the absence of TNFa is associated with enhanced astrocyte survival and that both phenomena are dependent on preservation of microglial cells that display an anti-inflammatory phenotype during the early ischemic phase of OIR.

Gene delivery using dimethyldidodecylammonium bromide-coated PLGA nanoparticles

In this present work we describe a poly(lactic-co-glycolic acid) (PLGA) nanoparticle formulation for intracellular delivery of plasmid DNA. This formulation was developed to encapsulate DNA within PLGA nanoparticles that combined salting out and emulsion evaporation processes. This process reduced the requirement for sonication which can induce degradation of the DNA. A monodispersed nanoparticle population with a mean diameter of approximately 240 nm was produced, entrapping a model plasmid DNA in both supercoiled and open circular structures. To induce endosomal escape of the nanoparticles, a superficial cationic charge was introduced using positively charged surfactants cetyl trimethylammonium bromide (CTAB) and dimethyldidodecylammonium bromide (DMAB), which resulted in elevated zeta potentials. As expected, both cationic coatings reduced cell viability, but at equivalent positive zeta potentials, the DMAB coated nanoparticles induced significantly less cytotoxicity than those coated with CTAB. Fluorescence and transmission electron microscopy demonstrated that the DMAB coated cationic nanoparticles were able to evade the endosomal lumen and localise in the cytosol of treated cells. Consequently, DMAB coated PLGA nanoparticles loaded with a GFP reporter plasmid exhibited significant improvements in transfection efficiencies with comparison to non-modified particles, highlighting their functional usefulness. These nanoparticles may be useful in delivery of gene therapies to targeted cells. (C) 2010 Elsevier Ltd. All rights reserved.

Optical transmission properties and electric field distribution of interacting 2D silver nanorod Arrays

Silver nanorods have been grown by electrodeposition into thin film porous alumina templates (AAO). Optical transmission measurements using p-polarized incident white light shows clear plasmon resonance extinction peaks. We successfully model the dependence on angle in incidence of extinction peak height and position using a multiple-multipoles (MMP) approach with the different spectral features being clearly associated with the effective electric field distribution and coupling between individual nanorods.

Pim2 cooperates with PML-RAR alpha to induce acute myeloid leukemia in a bone marrow transplantation model

Although the potential role of Pim2 as a cooperative oncogene has been well described in lymphoma, its role in leukemia has remained largely unexplored. Here we show that high expression of Pim2 is observed in patients with acute promyelocytic leukemia (APL). To further characterize the cooperative role of Pim2 with promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha), we used a well-established PML-RAR alpha (PR alpha) mouse model. Pim2 coexpression in PR alpha-positive hematopoietic progenitor cells (HPCs) induces leukemia in recipient mice after a short latency. Pim2-PR alpha cells were able to repopulate mice in serial transplantations and to induce disease in all recipients. Neither Pim2 nor PR alpha alone was sufficient to induce leukemia upon transplantation in this model. The disease induced by Pim2 overexpression in PR alpha cells contained a slightly higher fraction of immature myeloid cells, compared with the previously described APL disease induced by PR alpha. However, it also clearly resembled an APL-like phenotype and showed signs of differentiation upon all-trans retinoic acid (ATRA) treatment in vitro. These results support the hypothesis that Pim2, which is also a known target of Flt3-ITD (another gene that cooperates with PML-RAR alpha), cooperates with PR alpha to induce APL-like disease. (Blood. 2010; 115(22): 4507-4516)

Activated oncogenic pathways and therapeutic targets in extranodal nasal-type NK/T cell lymphoma revealed by gene expression profiling

We performed comprehensive genome-wide gene expression profiling (GEP) of extranodal nasal-type natural killer/T-cell lymphoma (NKTL) using formalin-fixed, paraffin-embedded tissue (n = 9) and NK cell lines (n = 5) in comparison with normal NK cells, with the objective of understanding the oncogenic pathways involved in the pathogenesis of NKTL and to identify potential therapeutic targets. Pathway and network analysis of genes differentially expressed between NKTL and normal NK cells revealed significant enrichment for cell cycle-related genes and pathways, such as PLK1, CDK1, and Aurora-A. Furthermore, our results demonstrated a pro-proliferative and anti-apoptotic phenotype in NKTL characterized by activation of Myc and nuclear factor kappa B (NF-kappa B), and deregulation of p53. In corroboration with GEP findings, a significant percentage of NKTLs (n = 33) overexpressed c-Myc (45.4%), p53 (87.9%), and NF-kappa B p50 (67.7%) on immunohistochemistry using a tissue microarray containing 33 NKTL samples. Notably, overexpression of survivin was observed in 97% of cases. Based on our findings, we propose a model of NKTL pathogenesis where deregulation of p53 together with activation of Myc and NF-kappa B, possibly driven by EBV LMP-1, results in the cumulative up-regulation of survivin. Down-regulation of survivin with Terameprocol (EM-1421, a survivin inhibitor) results in reduced cell viability and increased apoptosis in tumour cells, suggesting that targeting survivin may be a potential novel therapeutic strategy in NKTL. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

RUNX3 protein is overexpressed in human basal cell carcinomas

Basal cell carcinomas (BCC), which are the most common form of skin malignancy, are invariably associated with the deregulation of the Sonic Hedgehog (Shh) signalling pathway. As such, BCC represent a unique model for the study of interactions of the Shh pathway with other genes and pathways. We constructed a tissue microarray (TMA) of 75 paired BCC and normal skin and analysed the expression of beta-catenin and RUNX3, nuclear effectors of the wingless-Int (Wnt) and bone morphogenetic protein/transforming growth factor-beta pathways, respectively. In line with previous reports, we observed varying subcellular expression pattern of beta-catenin in BCC, with 31 cases (41%) showing nuclear accumulation. In contrast, all the BCC cases tested by the TMA showed RUNX3 protein uniformly overexpressed in the nuclei of the cancer cells. Analysis by Western blotting and DNA sequencing indicates that the overexpressed protein is normal and full-length, containing no mutation in the coding region, implicating RUNX3 as an oncogene in certain human cancers. Our results indicate that although the deregulation of Wnt signalling could contribute to the pathogenesis of a subset of BCC, RUNX3 appears to be a universal downstream mediator of a constitutively active Shh pathway in BCC.

Role of endoreduplication and apomeiosis during parthenogenetic reproduction in the model brown alga Ectocarpus

The filamentous brown alga Ectocarpus has a complex life cycle, involving alternation between independent and morphologically distinct sporophyte and gametophyte generations. In addition to this basic haploid–diploid life cycle, gametes can germinate parthenogenetically to produce parthenosporophytes. This article addresses the question of how parthenosporophytes, which are derived from a haploid progenitor cell, are able to produce meiospores in unilocular sporangia, a process that normally involves a reductive meiotic division.
We used flow cytometry, multiphoton imaging, culture studies and a bioinformatics survey of the recently sequenced Ectocarpus genome to describe its life cycle under laboratory conditions and the nuclear DNA changes which accompany key developmental transitions.
Endoreduplication occurs during the first cell cycle in about one-third of parthenosporophytes. The production of meiospores by these diploid parthenosporophytes involves a meiotic division similar to that observed in zygote-derived sporophytes. By contrast, meiospore production in parthenosporophytes that fail to endoreduplicate occurs via a nonreductive apomeiotic event.
Our results highlight Ectocarpus’s reproductive and developmental plasticity and are consistent with previous work showing that its life cycle transitions are controlled by genetic mechanisms and are independent of ploidy.

Out-of-field cell survival following exposure to intensity-modulated radiation fields

PURPOSE:
To determine the in-field and out-of-field cell survival of cells irradiated with either primary field or scattered radiation in the presence and absence of intercellular communication.
METHODS AND MATERIALS:
Cell survival was determined by clonogenic assay in human prostate cancer (DU145) and primary fibroblast (AGO1552) cells following exposure to different field configurations delivered using a 6-MV photon beam produced with a Varian linear accelerator.
RESULTS:
Nonuniform dose distributions were delivered using a multileaf collimator (MLC) in which half of the cell population was shielded. Clonogenic survival in the shielded region was significantly lower than that predicted from the linear quadratic model. In both cell lines, the out-of-field responses appeared to saturate at 40%-50% survival at a scattered dose of 0.70 Gy in DU-145 cells and 0.24 Gy in AGO1522 cells. There was an approximately eightfold difference in the initial slopes of the out-of-field response compared with the a-component of the uniform field response. In contrast, cells in the exposed part of the field showed increased survival. These observations were abrogated by direct physical inhibition of cellular communication and by the addition of the inducible nitric oxide synthase inhibitor aminoguanidine known to inhibit intercellular bystander effects. Additional studies showed the proportion of cells irradiated and dose delivered to the shielded and exposed regions of the field to impact on response.
CONCLUSIONS:
These data demonstrate out-of-field effects as important determinants of cell survival following exposure to modulated irradiation fields with cellular communication between differentially irradiated cell populations playing an important role. Validation of these observations in additional cell models may facilitate the refinement of existing radiobiological models and the observations considered important determinants of cell survival.

Microcomputed tomography imaging in a rat model of delayed union/non-union fracture

We aimed to develop a clinically relevant delayed union/non-union fracture model to evaluate a cell therapy intervention repair strategy. Histology, three-dimensional (3D) micro-computed tomography (micro-CT) imaging and mechanical testing were utilized to develop an analytical protocol for qualitative and quantitative assessment of fracture repair. An open femoral diaphyseal osteotomy, combined with periosteal diathermy and endosteal excision, was held in compression by a four pin unilateral external fixator. Three delayed union/non-union fracture groups established at 6 weeks-(a) a control group, (b) a cell therapy group, and (c) a group receiving phosphate-buffered saline (PBS) injection alone-were examined subsequently at 8 and 14 weeks. The histological response was combined fibrous and cartilaginous non-unions in groups A and B with fibrous non-unions in group C. Mineralized callus volume/total volume percentage showed no statistically significant differences between groups. Endosteal calcified tissue volume/endosteal tissue volume, at the center of the fracture site, displayed statistically significant differences between 8 and 14 weeks for cell and PBS intervention groups but not for the control group. The percentage load to failure was significantly lower in the control and cell treatment groups than in the PBS alone group. High-resolution micro-CT imaging provides a powerful tool to augment characterization of repair in delayed union/non-union fractures together with outcomes such as histology and mechanical strength measurement. Accurate, nondestructive, 3D identification of mineralization progression in repairing fractures is enabled in the presence or absence of intervention strategies. (c) 2007 Orthopaedic Research Society.

pcDNA3.1tdTomato is superior to pDsRed2-N1 for optical fluorescence imaging in the F344/AY-27 rat model of bladder cancer

PURPOSE: Animal models are important for pre-clinical assessment of novel therapies in metastatic bladder cancer. The F344/AY-27 model involves orthotopic colonisation with AY-27 tumour cells which are syngeneic to F344 rats. One disadvantage of the model is the unknown status of colonisation between instillation and sacrifice. Non-invasive optical imaging using red fluorescence reporters could potentially detect tumours in situ and would also reduce the number of animals required for each experiment.

MATERIALS AND METHODS: AY-27 cells were stably transfected with either pDsRed2-N1 or pcDNA3.1tdTomato. The intensity and stability of fluorescence in the resultant AY-27/DsRed2-N1 and AY-27/tdTomato stable cell lines were compared using Xenogen IVIS®200 and Olympus IX51 systems.

RESULTS: AY-27/tdTomato fluorescence intensity was 60-fold brighter than AY-27/DsRed2-N1 and was sustained in AY-27/tdTomato cells following freezing and six subsequent sub-cultures. After sub-cutaneous injection, fluorescence intensity from AY-27/tdTomato cells was threefold stronger than that detected from AY-27/DsRed2-N1 cells. IVIS®200 detected fluorescence from AY-27/tdTomato and AY-27/DsRed2-N1 cells colonising resected and exteriorised bladders, respectively. However, the deep-seated position of the bladder precluded in vivo imaging. Characteristics of AY-27/tdTomato cells in vitro and in tumours colonising F344 rats resembled those of parental AY-27 cells. Tumour transformation was observed in the bladders colonised with AY-27/DsRed2-N1 cells.

CONCLUSIONS: In vivo whole-body imaging of internal red fluorescent animal tumours should use pcDNA3.1tdTomato rather than pDsRed2-N1. Optical imaging of deep-seated organs in larger animals remains a challenge which may require proteins with brighter red or far-red fluorescence and/or alternative approaches.

Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions

Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. we have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha 2 beta 1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where 0 is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.

Relative quantification of the proteomeic changes associated with the mycotoxin zearalenone in the H295R steroidogenesis model

Zearalenone (ZEN) is a mycotoxin with endocrine disrupting effects having vast economic implications in e.g. pig farming. Structurally, ZEN resembles 17b-estradiol, and thus is able to bind to estrogen receptors (ER) in target cells. Because of this, it is also classified as a non-steroidal estrogen, a phytoestrogen, a mycoestrogen, and a growth promoter. Quantitative proteomic analysis was undertaken using stable-isotope labeling by amino acids in cell culture (SILAC) upon exposure of the steroidogenesis cell model H295R with ZEN to elucidate its effect on protein regulation. ZEN significantly regulated 21 proteins, including proteins with known endocrine disrupting effects and several oncogenes. In addition, network analysis using Ingenuity Pathway Analysis showed that ZEN affected the oxidative phosphorylation pathway and the mitochondrial dysfunction pathway, both previously reported to be involved in endocrine dysfunction.

Artemisinins act through at least two targets in a yeast model

Artemisinin and related compounds are potent and widely used antimalarial drugs but their biochemical mode of action is not clear. There is strong evidence that ATP-dependent calcium transporters are a key target in the malarial parasite. However, work using Saccharomyces cerevisiae suggests that disruption of mitochondrial function is critical in the cell killing activity of these compounds. Here it is shown that, in the absence of reducing agents, artemisinin and artesunate targeted the S. cerevisiae calcium channels Pmr1p and Pmc1p. Both compounds affected the growth of yeast on fermentable and nonfermentable media. This growth inhibition was not seen in a yeast strain in which the genes encoding both calcium channels were deleted. In the presence of reducing agents, which break the endoperoxide bridge in the drugs, growth inhibition was only observed in nonfermentable media. This inhibition could be partially relieved by the addition of a free radical scavenger. These results suggest that the drugs have two biochemical modes of action - one acting by specific binding to calcium channels and one involving free radical production in the mitochondria.