Enhanced survival of lethally irradiated adenosine A3 receptor knockout mice. A role for hematopoietic growth factors?

Adenosine A₃ receptor knockout (A₃AR KO) mice and their wild-type (WT) counterparts were compared from the point of view of their abilities to survive exposures to lethal doses of γ-radiation belonging to the range of radiation doses inducing the bone marrow acute radiation syndrome. Parameters of cumulative 30-day survival (experiment using a midlethal radiation dose) or cumulative 11-day survival (experiment using an absolutely lethal radiation dose), and of mean survival time were evaluated. The values of A₃AR KO mice always reflected their higher survival in comparison with WT ones, the P values being above the limit for statistical significance after the midlethal radiation dose and standing for statistical significance after the absolutely lethal radiation dose. This finding was considered surprising, taking into account the previously obtained findings on defects in numbers and functional properties of peripheral blood cells in A₃AR KO mice. Therefore, previous hematological analyses of A₃AR KO mice were supplemented in the present studies with determination of serum levels of the granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. Though distinct differences in these parameters were observed between A₃AR KO and WT mice, none of them could explain the relatively high postirradiation survival of A₃AR KO mice. Further studies on these mice comprising also those on other than hemopoietic tissues and organs can help to clarify their relative radioresistance.     

促红细胞生成素对血液透析患者血浆脂联素水平及左心室肥厚的影响

目的：促红细胞生成素（EPO）在维持性血液透析患者中具有保护心血管的作用,本文旨在探讨EPO在血液透析病人中对脂联素（ADPN）水平及左心室肥厚的影响。方法：46名维持性血液透析患者用EPO治疗8周,分别在治疗前,治疗第2、4、8周后测定血红蛋白（Hb）,红细胞压积（Hct）,血浆白蛋白（ALB）,体重指数（BMI）,C反应蛋白（CRP）,血清铁蛋白（SF）,血清铁饱和度（TAST）,ELISA测定ADPN水平;彩色多普勒超声测定左心室心肌重量（LVM）及计算左心室心肌重量指数（LVMI）。结果：血液透析患者血清ADPN水平高于正常人;用EPO治疗2周后即出现ADPN水平升高,第4周后ADPN水平进一步升高,第8周与第4周比较无明显变化;应用EPO可降低CRP、SF水平;ADPN是LVMI的重要影响因子,EPO可改善左心室肥厚与功能。结论：EPO可提高血液透析患者ADPN水平,降低CRP、SF水平,减轻左心室肥厚。     

Production and characterization of long-acting recombinant human albumin–EPO fusion protein expressed in CHO cell

A long-lasting recombinant human albumin-linker-erythropoietin (EPO) is a human albumin gene fused to the N-terminal of EPO with a (GGSGG)_n-repeated linker inserted between albumin and EPO. Albumin–EPO fusion genes were co-transfected with the dhfr gene. Albumin–EPO fusion protein has three kinds of sub-types (IALE, AD2LE, AD1LE). Albumin–EPO fusion protein was quantified with human EPO ELISA. The in vitro efficacy of albumin–EPO fusion protein was estimated using F-36E cell, and in vivo efficacy of albumin–EPO fusion protein was estimated using normocythemic mice (B6D2F1). We also determined the in vivo half-life in a Sprague–Dawley rat. A PLA program analysis result demonstrated that the albumin–EPO fusion protein IALE is about 7.8-fold more potent than rHuEPO in increasing the hematocrit of normal mice.     

Gab1 transduces PI3K-mediated erythropoietin signals to the Erk pathway and regulates erythropoietin-dependent proliferation and survival of erythroid cells

In this study, we examined the biological functions of Gab1 in erythropoietin receptor (EPOR)-mediated signaling in vivo. Knockdown of Gab1 by the introduction of the Gab1 siRNA expression vector into F-36P human erythroleukemia (F-36P-Gab1-siRNA) cells resulted in a reduction of cell proliferation and survival in response to EPO. EPO-induced activation of Erk1/2 but not of Akt was significantly suppressed in F-36P-Gab1-siRNA cells compared with mock-transfected F-36P cells. The co-immunoprecipitation experiments revealed an EPO-enhanced association of Gab1 with the Grb2–SOS1 complex and SHP-2 in F-36P cells. A selective inhibitor of phosphatidylinositol 3-kinase (PI3K) LY294002 and short interfering RNA (siRNA) duplexes targeting the p85 regulatory subunit of PI3K (p85-siRNA) independently suppressed tyrosine phosphorylation of Gab1; its association with Grb2, SHP-2 and p85; and the activation of Erk in EPO-treated F-36P cells. LY294002 inhibited EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2 in human primary EPO-sensitive erythroid cells. The co-immunoprecipitation experiments using the Jak inhibitor AG490 or siRNA duplexes targeting Jak2 and in vitro binding experiments demonstrated that Jak2 regulated Gab1-mediated Erk activation through tyrosine phosphorylation of Gab1. Taken together, these results suggest that Gab1 couples PI3K-mediated EPO signals with the Ras/Erk pathway and that Gab1 plays an important role in EPOR-mediated signal transduction involved in the proliferation and survival of erythroid cells.     

人促红细胞生成素基因和GFP共表达的输卵管特异性表达载体的构建

本实验从蛋鸡输卵管基因组DNA中扩增出约1.3kb的卵清蛋白5＇端调控区（OV）,并从质粒扩增出人促红细胞生成素（hEPO）基因组DNA。将hEPO亚克隆入pEGFP-C1载体的多克隆位点,命名为pEGFP-hEPO,然后将OV片段亚克隆入经Ase I和Vsp I双酶切的切口处,替换掉CMV启动子,经测序和酶切鉴定正确,成功构建了鸡卵清蛋白5＇端调控区调控人促红细胞生成素的共表达载体pOV-GFP-hEPO。并利用脂质体转染法转染鸡输卵管上皮细胞,用荧光倒置显微镜观测绿色荧光蛋白的表达,结果显示共表达载体pOV-GFP-hEPO能够在鸡输卵管上皮细胞定位表达。为制备生产人促红细胞生成素的鸡输卵管生物反应器奠定了基础。     

缺氧培养对大鼠神经干细胞体外增殖影响及其机制的研究

目的：研究应用缺氧对体外培养的大鼠神经干细胞增殖的影响。方法：将细胞分为4小时缺氧组、12小时缺氧组、促红细胞生成素（EPO）中和抗体组、IgG组以及对照组．测定大鼠神经干细胞经缺氧培养后的各组细胞克隆形成率以及EPO的表达变化。结果：单细胞培养条件下,与对照组相比,4小时缺氧组和IgG组克隆形成率明显增高;中和抗体组无明显变化;12小时组克隆形成率降低。但无统计学意义。缺氧4小时后,EPO蛋白在预处理后即刻出现表迭,4h达高峰,8h仍有部分表达。结论：短时间缺氧可以促进神经干细胞增殖．长时间缺氧则作用相反。缺氧对NSCs增殖作用的影响可能是由EPO介导产生。     

Human Ubiquitin C Promoter Based Expression of Erythropoietin in CHO K1 Cell Lines: A Simple Transfectants Screening Approach

Erythropoietin (EPO), a glycoprotein hormone that regulates the production of erythrocytes in the human body, is of clinical importance in the treatment of anemia. Low expression levels of this recombinant hormone and time-consuming screening methods have made its commercial production expensive. Cloning of human EPO gene in a shuttle vector pUB6/V5-HisB driven by human ubiquitin C promoter and its transfection in CHO K1 cell lines by electroporation resulted in a moderate level of EPO expression. The limiting-dilution screening method required several months to obtain high expression stable transfectants but needed only short duration for selection in contrast to the present screening strategy. The supernatants of stably transfected cells were found to be biologically active by in vitro erythroid cluster forming activity.     

EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα⁺) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.     

The effects of erythropoietin signaling on telomerase regulation in non-erythroid malignant and non-malignant cells

Treatment with erythropoietin (EPO) in several cancers is associated with decreased survival due to cancer progression. Due to the major importance of telomerase in cancer biology we hypothesized that some of these effects may be mediated through EPO effect on telomerase. For this aim we explored the possible effects of EPO on telomerase regulation, cell migration and chemosensitivity in non-erythroid malignant and non-malignant cells.     

Design,synthesis, and activity evaluation of novel erythropoietin mimetic peptides

The approval of the erythropoietin (EPO) mimetic peptide drug peginesatide in 2012 was a breakthrough for the treatment of secondary anemia. However, due to severe allergic reactions, peginesatide was recalled a year later. In this study, 12 novel peptides were designed and synthesized by substituting specific amino acids of the monomeric peptide in peginesatide, with the aim of obtaining new EPO mimetic peptides with higher activities and lower side effects than the parent compound. Their cell proliferation activities were evaluated, and the structure-activity relationships were analyzed. Five compounds had equal cell proliferation activity to the control peptide. Among them, one compound showed a higher in vivo activity than the control peptide, with no obvious side effects.