EPO And Its Receptor Expression Research Articles

Role of Erythropoietin in Cerebral Glioma: An Innovative Target in Neuro-Oncology

Erythropoietin (EPO) is a cytokine primarily involved in the regulation of erythropoiesis. In response to hypoxia-ischemia, hypoxia-inducible factor 1 induces EPO production, which, in turn, inhibits apoptosis of erythroid progenitor cells. By the same mechanism and acting through other signaling pathways, EPO exerts neuroprotective effects. Increased resistance to hypoxia and decreased apoptosis are thought to be important mechanisms for tumor progression, including malignant glioma. Because recent studies have demonstrated that EPO and its receptor (EPOR) are expressed in several tumors and can promote tumor growth, in the present study, we investigated EPO and EPOR expression in human glioma and the effect of EPO administration in a rat model of glioma implantation. Using Western blotting and immunohistochemical analysis, we examined the expression of EPO, EPOR, platelet endothelial cell adhesion molecule, and Ki-67 in human glioma specimens and experimentally induced glioma in rats. In the experimental setting, a daily dose of recombinant human EPO (rHuEPO) or saline solution were administered for 21 days in Fischer rats subjected to 9L cell line implantation. In both human and animal specimens, we found an increase in EPOR expression as long as the lesion presented with an increasing malignant pattern. A significant direct correlation was found between the expression of EPOR and Ki-67 and EPOR and platelet endothelial cell adhesion molecule in low- and high-grade gliomas. The rats treated with rHuEPO presented with significantly larger tumor spread compared with the saline-treated rats. The results of our study have shown that the EPO/EPOR complex might play a significant role in the aggressive behavior of high-grade gliomas. The larger tumor spread in rHuEPO-treated rats suggests a feasible role for EPO in the aggressiveness and progression of malignant glioma.

Erythropoietin Increases GABAA Currents in Human Cortex from TLE Patients

Erythropoietin (EPO) is a hematopoietic growth factor that has an important role in the erythropoiesis. EPO and its receptor (EPO-R) are expressed all over in the mammalian brain. Furthermore, it has been reported that EPO may exert neuroprotective effect in animal models of brain disorders as ischemia and epilepsy. Here, we investigate whether EPO could modulate the GABA-evoked currents (IGABA) in both human epileptic and non-epileptic control brain tissues.Therefore, we transplanted in Xenopus oocytes cell membranes obtained from autoptic and surgical brain tissues (cortex) of seven temporal lope epilepsy (TLE) patients and of five control patients. Two microelectrodes voltage-clamp technique has been used to record IGABA. Moreover, qRT-PCR assay was performed in the same human tissues to quantify the relative gene expression levels of EPO/EPO-R. To further confirm experiments in oocytes, we performed additional experiments using patch-clamp recording in slices obtained from rat cerebellum.We show that exposure to EPO significantly increased the amplitude of the IGABA in all the patients analyzed. No differences in the expression of EPO and EPO-R in both TLE and control patients have been found. Notably, the increase of IGABA has been recorded also in rat cerebellar slices. Our findings show a new modulatory action of EPO on GABAA receptors (GABAA-Rs). This effect could be relevant to balance the GABAergic dysfunction in human TLE. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Abstract 3464: The effect of EPO-receptor in estrogen receptor positive breast cancer

Abstract 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, recent clinical trials have indicated that rhEPO treatment might promote tumor progression and have 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 in ERα+ breast cancer cells and diminished ERα+ phosphorylation on Ser118, results that further support a mechanism by which EPOR affects proliferation via ERα+ mediated mechanisms. In conclusion, we show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation in ERα+ expressing breast cancer cells, in part via its capacity to positively modulate ERα activity. Citation Format: Marica C. Vaapil, Susann Reinbothe, Anna-Maria Larsson, Caroline Wigerup, Jianmin Sun, Annika Jögi, Drorit Neumann, Lars Rönnstrand, Sven Påhlman. The effect of EPO-receptor in estrogen receptor positive breast 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 3464. doi:10.1158/1538-7445.AM2014-3464

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.

Expression of erythropoietin and its receptor in renal tissues of rats with chronic cyclosporine A nephrotoxicity

Objective To examine whether cyclosporine A (CsA)treatment can induce anemia and changes in expression of erythropoietin(EPO)and its receptor(EPOR)in renal tissues of rats with chronic CsA nephrotoxicity.Methods Sprague- Dawley rats were treated daily with subcutaneous injection of vehicle(olive oil,1mL/kg,control group)or CsA (15mg/kg, CsA nephrotoxicity model group)for 4weeks.The renal function and hemoglobin (Hb)and hematocrit(Hct)levels were analyzed by Automatic Biochemistry Analyzer in the two groups.Masson Trichrome staining was used to examine the extent of tubulointerstitial fibrosis.Immunohistochemistry and Western blotting analysis were used to examine the expression of EPO and EPOR protein.And cell apoptosis was observed by TUNEL assay and electron microscope.Results Compared with the control group,CsA nephrotoxicity group showed renal insufficiency,tubulointerstitial fibrosis,and significantly more apoptotic cells (P0.01).Meanwhile,CsA nephrotoxicity group also had anemia,manifested by significantly decreased Hb and Hct levels (P0.01).Immunohistochemistry and Western blotting analysis showed that CsA treatment significantly decreased EPO expression and significantly increased EPOR expression compared with the control group (P0.01).Furthermore,we also found that EPO protein expression was negatively associated with tubulointerstitial fibrosis(r=-0.729,P0.001)and the number of TUNEL-positive cells(r= -0.841,P0.001).Conclusion Renal EPO expression is decreased in rats with chronic CsA nephrotoxicity,which resulting in anemia;and tubular epithelial cell apoptosis induced by CsA is related to decrease of EPO protein.

Prognosis of patients with locally advanced squamous cell carcinoma of the head and neck

This study investigated the prognostic role of tumor cell expression of erythropoietin (EPO) and its receptor (EPO-R) in patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN) treated with surgery plus radiotherapy. The impact of EPO, EPO-R, and 11additional factors on locoregional control (LRC), metastases-free survival (MFS), and overall survival (OS) was retrospectively evaluated in 144patients. Additional factors were age, gender, performance status, preradiotherapy (pre-RT) hemoglobin levels, tumor site, histologic grade, T category, N category, human papillomavirus (HPV) status, extent of resection, and chemotherapy. Univariate analyses were performed with the Kaplan-Meier method and the log-rank test, multivariate analyses with the Cox proportional hazard model. On multivariate analysis, improved LRC was significantly associated with no EPO expression (risk ratio [RR] 3.72; 95 % confidence interval [CI] 1.35-15.42; p = 0.008), lower T category (RR 1.60; 95 %CI 1.14-2.32; p = 0.005), and oropharynx or larynx cancer (RR 1.23; 95 %CI 1.02-1.49; p = 0.033). Improved MFS was significantly associated with no EPO expression (RR 5.45; 95 %CI 1.13-97.81; p = 0.031), lower T category (RR 1.66; 95 %CI 1.11-2.65; p = 0.013), lower N category (RR 2.44; 95 %CI 1.04-6.66; p = 0.039), HPV positivity (RR 3.14; 95 %CI not available; p = 0.034), and oropharynx or larynx cancer (RR 1.28; 95 %CI 1.01-1.61; p = 0.041). Improved OS was significantly associated with no EPO expression (RR 4.77; 95 %CI 1.63-20.68; p = 0.003), no EPO-R expression (RR 2.36; 95 %CI 1.22-4.92; p = 0.010), lower T category (RR 1.44; 95 %CI 1.04-2.04; p = 0.027), oropharynx or larynx cancer (RR 1.30; 95 %CI 1.08-1.57; p = 0.007), and pre-RT hemoglobin ≥ 12g/dl (RR 1.94; 95 %CI 1.03-3.65; p = 0.042). EPO expression of tumor cells was an independent prognostic factor for LRC, MFS, and OS. EPO-R expression was an independent prognostic factor for OS.

Does Erythropoietin and Its Receptor may Play a Role in Human Intervertebral Disk Degeneration?

IntroductionDegeneration of lumbar intervertebral discs is considered to be a cause of low back pain and radiculopathy. Many studies have shown that intervertebral disk inflammation and axonal growth of afferent fibers innervating the disk degeneration have a principal role in this pathological process. Both mechanical compression and chemical factors within nucleus pulposus (NP) are associated with the occurrence of radiculopathy. NP contains various proinflammatory cytokines that play important roles in the chemical pathogenesis of NP-induced peripheral nerve injury. The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) is a key trigger in the pathogenesis of NP-induced inflammation and apoptosis and its inhibition can reduce NP degeneration. A class of MAPKs and p38 MAPK is phosphorylated by stress signals such as inflammatory cytokineso, smotic shock, and ischemia. Spinal cord and peripheral nerve injury cause the activation of p38 in dorsal root ganglion (DRG) neurons and spinal cord microglia. Furthermore, TNF-α phosphorylates p38 and activated p38 MAPK upregulates the biosynthesis of TNF- α. TNF and p38 are involved in neuronal cell apoptosis in peripheral nerve injuries. Erythropoietin (EPO) is a pleiotropic cytokine originally identified for its role in erythropoiesis. Recent studies have demonstrated that EPO and its receptor (EPO-R) are expressed in the central nervous system, where EPO exerts neuroprotective functions.Materials and MethodsThe aim of the present study was to investigate whether the resident EPO and EPO-R network is present and functional in the NP of intervertebral disk in different grade of degeneration. In this study, we analyzed 20 human NP obtained following microdiscectomy. The expressions of p-p38, TNF- α, EPO, and EPOR were assessed by immunohistochemical and immunoblotting analysis. Data were analyzed by unpaired Student t-test and Dunnett t-test (significance level, p < 0.05).ResultsIn barely degenerated disk, significantly weak expression of EPO and EPO-R was observed (p < 0.05). Accordingly, the expression of both p-p38 and TNF in the NP was significantly lower than in well-degenerated disk (p < 0.05). In clearly degenerated disc, a marked increase of expression of EPO and EPO-R was observed (p < 0.05) and a consensual increase of both p-p38 and TNF in the NP was observed (p < 0.05).ConclusionThese observations suggest that the local EPO and EPO-R system is markedly engaged in the degeneration of intervertebral disk by a feasible cross-talk with network of proinflammatory cytokine such as TNF- α. These data may increase our knowledge on mechanisms underlying intervertebral disk degeneration and strongly support the possible usefulness of a therapeutic approach based on exogenous EPO administration.I confirm having declared any potential conflict of interest for all authors listed on this abstractNoDisclosure of InterestNone declared

Does Erythropoietin and Its Receptor may Play a Role in Human Intervertebral Disk Degeneration?

Introduction Degeneration of lumbar intervertebral discs is considered to be a cause of low back pain and radiculopathy. Many studies have shown that intervertebral disk inflammation and axonal growth of afferent fibers innervating the disk degeneration have a principal role in this pathological process. Both mechanical compression and chemical factors within nucleus pulposus (NP) are associated with the occurrence of radiculopathy. NP contains various proinflammatory cytokines that play important roles in the chemical pathogenesis of NP-induced peripheral nerve injury. The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) is a key trigger in the pathogenesis of NP-induced inflammation and apoptosis and its inhibition can reduce NP degeneration. A class of MAPKs and p38 MAPK is phosphorylated by stress signals such as inflammatory cytokineso, smotic shock, and ischemia. Spinal cord and peripheral nerve injury cause the activation of p38 in dorsal root ganglion (DRG) neurons and spinal cord microglia. Furthermore, TNF-α phosphorylates p38 and activated p38 MAPK upregulates the biosynthesis of TNF- α. TNF and p38 are involved in neuronal cell apoptosis in peripheral nerve injuries. Erythropoietin (EPO) is a pleiotropic cytokine originally identified for its role in erythropoiesis. Recent studies have demonstrated that EPO and its receptor (EPO-R) are expressed in the central nervous system, where EPO exerts neuroprotective functions. Materials and Methods The aim of the present study was to investigate whether the resident EPO and EPO-R network is present and functional in the NP of intervertebral disk in different grade of degeneration. In this study, we analyzed 20 human NP obtained following microdiscectomy. The expressions of p-p38, TNF- α, EPO, and EPOR were assessed by immunohistochemical and immunoblotting analysis. Data were analyzed by unpaired Student t-test and Dunnett t-test (significance level, p &lt; 0.05). Results In barely degenerated disk, significantly weak expression of EPO and EPO-R was observed ( p &lt; 0.05). Accordingly, the expression of both p-p38 and TNF in the NP was significantly lower than in well-degenerated disk ( p &lt; 0.05). In clearly degenerated disc, a marked increase of expression of EPO and EPO-R was observed ( p &lt; 0.05) and a consensual increase of both p-p38 and TNF in the NP was observed ( p &lt; 0.05). Conclusion These observations suggest that the local EPO and EPO-R system is markedly engaged in the degeneration of intervertebral disk by a feasible cross-talk with network of proinflammatory cytokine such as TNF- α. These data may increase our knowledge on mechanisms underlying intervertebral disk degeneration and strongly support the possible usefulness of a therapeutic approach based on exogenous EPO administration. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared

88P Epo-Independent Functional Epo Receptor in Breast Cancer Enhances Estrogen Receptor Activity and Promotes Cell Proliferation

ABSTRACT The main function of erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis, and recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. Recent clinical trials have indicated that rhEPO treatment can have a negative effect on tumor progression and patient survival. In addition, EPOR expression has been detected in different cancer forms. Today the presence and function of EPOR in different cancer forms are still controversial, in part because the specificity of existing anti-EPOR antibodies has been questioned. Here we report an in-house polyclonal antibody that reliably detects the full-length isoform of the EPOR and we show that breast cancer cell lines as well as breast cancer tissue express the receptor protein. However, rhEPO stimulation of EPOR expressing breast cancer cells did not result in increased proliferation or activation of EPOR (receptor phosphorylation) or phosphorylation of JAK2/STAT5/AKT/ERK, mediators of the classical EPOR signaling pathway. A function of the receptor was however demonstrated in EPOR knockdown experiments resulting in decreased proliferation in estrogen receptor positive (ER+) cancer cells, while these effects were not seen in ER- cells. EPOR knockdown decreased ER activity in ER+ breast cancer cells, which could explain the EPOR effect on proliferation. Also, EPOR knockdown improved tamoxifen effects in ER+ breast cancer cells. This is in accordance with our previous clinical observation that high EPOR expression in tumors correlates to impaired tamoxifen response in patients with ER+ breast cancer (Clin Cancer Res 2009;15(17);5552-9). In conclusion we show that EPOR protein is expressed in breast cancer cells where it appears to act as a novel EPO-independent proliferation factor in ER expressing breast cancer cells, possibly via its capacity to modulate ER activity. Disclosure All authors have declared no conflicts of interest.

Expression of Erythropoietin and Erythropoietin Receptor in Human Dental Pulp

Introduction In addition to the involvement in erythropoiesis, erythropoietin (EPO) and its receptor (EPO-R) have been shown to be expressed in various nonhematopoietic organs and tissues with diverse biological effects. The purpose of this study was to evaluate the expression patterns of EPO and EPO-R in healthy and inflamed human dental pulp tissues. To gain insight into the possible mechanisms involved in the regulation of EPO and EPO-R expression, we further investigated the hypothesis that their expression in cultured human dental pulp cells (DPCs) may be regulated upon hypoxia, an important factor involved in dental pulp inflammation. Methods Samples of healthy and inflamed dental pulp tissues were obtained from patients undergoing surgical or orthodontic treatment. The protein localization and messenger RNA levels of EPO and EPO-R in the pulp tissues were detected by immunohistochemistry and quantitative real-time polymerase chain reaction (PCR), whereas the EPO and EPO-R expressions in DPCs in vitro were evaluated by Western blotting and quantitative real-time PCR. Results EPO and EPO-R proteins were detected in inflamed dental pulp, whereas no obvious EPO expression was detected in healthy dental pulp. The EPO messenger RNA level was significantly up-regulated in inflamed pulps compared with healthy pulps. Moreover, the messenger RNA and protein levels of EPO and EPO-R were up-regulated in DPCs under hypoxia in vitro. Conclusions The up-regulation of EPO might be involved in dental pulp inflammation, which is probably attributed to hypoxia. Further studies are needed to investigate the potential role of EPO and EPO-R in dentin-pulp repair and regeneration.

Neuroprotective role of erythropoietin by antiapoptosis in the retina

Erythropoietin (EPO) stimulates red blood cell production, in part by inhibiting apoptosis of the red blood cell precursors. The erythropoietic effects of EPO are circadian stage dependent. Retinal injury due to light occurs through oxidative mechanisms and is manifest by retinal and retinal pigment epithelium (RPE) cells apoptosis. The visual cycle might be circadian coordinated as a means of effectively protecting the retina from the detrimental effects of light-induced, oxygen-dependent, free radical-mediated damage, especially at the times of day when light is more intense. We show that the retinal expression of EPO and its receptor (EPOR), as well as subsequent Janus kinase 2 (Jak2) phosphorylations, are each tightly linked to a specific time after oxidative stress and in anticipation of daily light onset. This is consistent with physiological protection against daily light-induced, oxidatively mediated retinal apoptosis. In vitro, we verify that EPO protects RPE cells from light, hyperoxia, and hydrogen peroxide-induced retinal cell apoptosis, and that these stimuli increase EPO and EPOR expression in cultured RPE cells. Together, these data support the premise that EPO and its EPOR interactions represent an important retinal shield from physiologic and pathologic light-induced oxidative injury.

Erythropoietin expresson during the postnatal development of the rat retina

Event Abstract Back to Event Erythropoietin expresson during the postnatal development of the rat retina Arnold Szabó1*, Dora Vegvari1, Attila Magyar1, Gabor Deak2, Akos Lukats1, Agnes I. Berta1 and Agoston Szel1 1 Department of Human Morphology and Developmental Biology, Semmelweis University, Hungary 2 Eye Clinic, Semmelweis University, Hungary Both the erythropoietin (EPO) and its receptor (EPO-R) are expressed in the retina, however, their cellular distribution and involvement in retinal development is not known. In the present study, we examined developing postnatal and adult rat retinas in order to identify the temporal and spatial onset of their expression. In the adult retina both the EPO and its receptor were expressed in the photoreceptor inner segments, in the inner nuclear, ganglion, and both plexiform layers. Their expression patterns changed similarly. At birth, we found strong immunoreactivities in the ganglion cell layer and in the inner cell rows of the neuroblast layer and weak staining in the outer part of the neuroblast layer. During the following days the immunoreactivity of the ganglion cells and presumptive amacrine cells became more prominent. The cells of the ganglion cell layer showed a cytoplasmatic staining to the anti-erythropoietin antibody. From the second half of the first postnatal week strongly stained horizontal cells were observed by both antibodies at the level of the separating outer plexiform layer. During the second week a transient EPO-R expression was found in some cells of the outer nuclear layer, which followed a centroperipheral gradient. At the level of the developing photoreceptor inner and outer segments exclusively the inner segments were positively stained by both antibodies. We assume that the EPO is produced locally by the ganglion cells and its main targets are the amacrine, horizontal and photoreceptor cells and it may act as a regulating factor during the retinal development. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Developmental neurobiology and subcortical functions Citation: Szabó A, Vegvari D, Magyar A, Deak G, Lukats A, Berta AI and Szel A (2009). Erythropoietin expresson during the postnatal development of the rat retina. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.076 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Mar 2009; Published Online: 03 Mar 2009. * Correspondence: Arnold Szabó, Department of Human Morphology and Developmental Biology, Semmelweis University, Budapest, Hungary, szabar@ana2.sote.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Arnold Szabó Dora Vegvari Attila Magyar Gabor Deak Akos Lukats Agnes I Berta Agoston Szel Google Arnold Szabó Dora Vegvari Attila Magyar Gabor Deak Akos Lukats Agnes I Berta Agoston Szel Google Scholar Arnold Szabó Dora Vegvari Attila Magyar Gabor Deak Akos Lukats Agnes I Berta Agoston Szel PubMed Arnold Szabó Dora Vegvari Attila Magyar Gabor Deak Akos Lukats Agnes I Berta Agoston Szel Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Erythropoietin and erythropoietin receptor coexpression is associated with poor survival in stage I non-small cell lung cancer

7655 Background: The role of erythropoietin (EPO) and its receptor (EPO-R) in tumor biology is under active investigation. This study was designed to evaluate the prognostic impact of EPO and EPO-R expression in stage I non-small cell lung cancer (NSCLC) patients. Methods: EPO and EPO-R expression in 158 tumor samples from resected stage I NSCLC was evaluated using immunohistochemistry and tissue-array technology. Disease specific survival time was calculated from the date of surgery to death from cancer-related causes. Results: EPOR and EPO were highly expressed in 20.9% and 35.4% of the tumors respectively. High EPO-R compared to negative or low level of expression was associated with a poor 5-year disease specific survival (60.6% versus 80.8%, logrank test: p=0.01). High EPO compared to negative and low level of expression was associated with a trend toward a poor 5-year disease specific survival (69.6% versus 80.4%, logrank test: p=0.13). High level of EPO-R and EPO coexpression compared to other groups of patients was associated with a poor 5- year disease specific survival (50.0% versus 80.0% of survival at the end of follow-up, logrank test: p=0.005). In multivariate analysis for disease specific survival, high level of EPO-R and EPO coexpression was an independent prognostic factor for disease specific survival (HR 2.214, 95% CI: 1.012–4.848, p=0.046). Conclusions: These results establish for the first time the pejorative prognostic value of EPO and EPO-R expression in early stage resected NSCLC and suggest a potential paracrine and/or autocrine role of endogenous EPO in NSCLC aggressiveness. No significant financial relationships to disclose.

Survival and invasiveness of astrocytomas promoted by erythropoietin

The hypoxia-inducible pleiotropic hormone, erythropoietin (EPO), has recently been found to promote the development and survival of neurons and astrocytes. Since hypoxia has been implicated in the malignant progression of some human cancers, the authors investigated whether EPO signaling influenced the malignant properties of human astrocytoma cells. Reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemical studies were used to measure EPO and its receptor (EPOR). Cell viability, Matrigel invasion assays, metalloprotease assays, EPO neutralizing antibodies, and EPOR overexpression were used to study the biological actions of EPO. Expression of both EPO and EPOR was observed in the hypoxic regions and invasive margins of glioma specimens obtained at biopsy, and expression of EPOR correlated with the stage of the tumor. The EPOR was also functionally upregulated by hypoxia in cultured glioblastoma multiforme (GBM) cells. Both hypoxia and EPO protected cultured GBM cells from cisplatin cytotoxicity and promoted the invasiveness of GBM cells through Matrigel by potentiating metalloprotease activity. Hypoxia-enhanced cell invasion was attenuated in cells that overexpressed a nonfunctional EPOR. Hypoxia-inducible autocrine and paracrine EPO signaling participates in the malignant progression of GBMs.

The expression of erythropoietin receptor in the spinal cord after acute traumatic injury in adult rats

目的 探讨急性脊髓损伤后促红细胞生成素(EPO)及其受体(EPO-R)在脊髓内的表达.方法 Wistar大鼠69只,随机分为三组:正常对照组(5只,不手术,作为后两组的对照)、假手术组(仅行椎板切除术)和脊髓损伤组.根据术后时间点不同后两组又分为1 h、6 h、12 h、24 h、3 d、7 d、14 d和28 d八个亚组(每组4只).采用RT-PCR、Western blot免疫印迹法和免疫组织化学染色法检测EPO及EPO-R的表达.结果 正常对照组、假手术组和脊髓损伤组在各时相点均未发现有EPO表达.正常对照组、假手术组未发现EPO-R的表达,脊髓损伤组在伤后1 h未见EPO-R mRNA和蛋白的表达,6 h开始有表达,12 h有明显的表达,至24 h达到高峰,3 d和7 d时仍维持在高表达水平,未见减弱,14 d开始下降,至28 d时仍有EPO-R蛋白的表达.免疫组化显示EPO-R阳性细胞主要位于神经元、少突胶质细胞、血管内皮细胞和脊髓中央管内室管膜细胞.结论 大鼠急性脊髓损伤后脊髓内大量表达EPO-R,这是外源性EPO与EPO-R结合产生神经保护作用的分子基础。

The Expression of Erythropoietin and Its Receptor in Breast Cancer Is Associated with In Vivo Tumor Hypoxia.

Erythropoietin (EPO) is primarily produced in the adult kidney where hypoxia is the major stimulus for EPO expression under the control of an oxygen-sensing mechanism. A series of previous studies in our laboratories and others have demonstrated the expression of EPO and its receptor (EPOR) in breast cancer cells. In previous work, we found that the administration of antagonists of EPO-EPOR signaling was associated with delayed tumor growth in a rodent syngeneic breast cancer model, suggesting the presence of a functional EPO-EPOR system in cancer. The mechanisms of EPO and EPOR expression in tumor cells requires further study. In vitro experiments using monolayer cultures of breast cancer cells have suggested that EPO and EPOR expression in tumor cells may be hypoxia-regulated. The objective of our current studies was to determine the relationship between the expression of EPO, EPOR and tumor hypoxia in breast cancer to test the hypothesis that EPO or EPOR expression in malignant cells may be associated with the presence of in vivo tumor hypoxia, an important factor involved in resistance to radiation treatment, tumor aggressiveness and poor prognosis. Thirty-eight patients with primary breast cancer were enrolled in a tumor hypoxia study under a research protocol approved by the Institutional Review Board at the University of North Carolina Hospitals. All patients provided signed informed consent. The patients received an intravenous infusion of the hypoxia marker pimonidazole hydrochloride (Hypoxyprobe-1™) prior to tumor biopsy. Two or more biopsies were obtained from 33 tumors and 5 primary tumors had a single biopsy available. Contiguous sections from a total of 93 biopsies were analyzed by immunohistochemistry using monoclonal antibodies for the expression of EPO and EPOR and pimonidazole binding. We found EPO expression in tumor cells in 94% of the biopsies. Focal EPO expression pattern was observed in many tumors. EPOR expression was present in 93% of biopsies. The pattern of EPOR immunoreactivity was predominantly cytoplasmic but was found to be localized to the membrane in some sections. In many tumors, co-localization of EPO and EPOR expression in tumor cells was present when contiguous sections were examined. Tumor hypoxia was detected in 83% of the biopsies at variable levels and did not always co-localize with EPO or EPOR expression in tumor cells. Semi-quantitative analyses for EPO immunostaining and tumor hypoxia on a section-by-section basis revealed a significant positive correlation between levels of micro-regional EPO expression and pimonidazole binding (r = 0.6, P < 0.0001, n=93 by two-tailed Spearman's rank correlation analysis). A similar significant positive correlation was found between levels of EPOR expression and pimonidazole binding (r = 0.63, P < 0.0001, n=93). In addition, there was a significant association between the semi-quantitative EPO score in tumor cells and the EPOR score (r = 0.6, P < 0.0001, n=93). We also determined the micro-vessel density (MVD), a marker of tumor angiogenesis, in 35 biopsies using factor VIII immunostaining. There was no correlation between EPO or EPOR expression and MVD in these samples. In conclusion, these data demonstrate for the first time that EPO and EPOR expression in breast cancer cells correlates with in vivo tumor hypoxia in clinical specimens of primary breast cancer.

Erythropoietin Receptor Expression in Canine Mammary Tumor: An Immunohistochemical Study

Erythropoietin (EPO) is a cytokine primarily involved in the regulation of the erythropoiesis. Recently, it has been demonstrated that EPO and its receptor (EPOR) are expressed in several neoplastic cell lines and solid tumors. Furthermore, in vitro and in vivo studies have shown that EPO could promote human breast carcinoma growth by means of the binding with its receptor, although a clear function for EPO in this setting has not been yet established. While the human medical literature has been accumulating strong evidence on EPO's role in oncogenesis, to date, there are no veterinary reports focusing on such an issue. The aim of the present study was to investigate the immunohistochemical expression of EPOR in canine mammary gland dysplastic and neoplastic lesions. Our results show a weak to moderate EPOR expression in dysplastic glands, being immunoreactivity enhanced as the lesion shows an increasing malignant pattern. On the basis of these findings, this study describes, for the first time, the evidence for EPOR expression in canine mammary gland tumor and suggests a feasible EPO's role for canine mammary tumor progression.

Expression patterns of erythropoietin and its receptor in the developing spinal cord and dorsal root ganglia

Recombinant human erythropoietin (EPO) is neuroprotective in animal models of adult spinal cord injury, and reduces apoptosis in adult dorsal root ganglia after spinal nerve crush. The present work demonstrates that spinal cord and dorsal root ganglia share dynamic expression patterns of EPO and its receptor (EPOR) during development. C57Bl mice from embryonic days (E) 8 (E8) to E19 were studied. In spinal cord and dorsal root ganglia, EPOR expression in all precursor cells preceded the expression of EPO in subsets of neurons. On E11, EPO-immunoreactive spinal motoneurons and ganglionic sensory neurons resided adjacent to EPOR-expressing radial glial cells and satellite cells, respectively. From E12 onwards, EPOR-immunoreactivity decreased in radial glial cells and, transiently, in satellite cells. Simultaneously, large-scale apoptosis of motoneurons and sensory neurons started, and subsets of neurons were labelled by antibodies against EPOR. Viable neurons expressed EPO and EPOR. Up to E12.5, apoptotic cells were EPOR-immunopositive, but variably EPO-immunonegative or EPO-immunopositive. Thereafter, EPO-immunonegative and EPOR-immunopositive apoptotic cells predominated. Our findings suggest that EPO-mediated neuron-glial and, later, neuron-neuronal interactions promote the differentiation and/or the survival of subsets of neurons and glial cells in central as well as in peripheral parts of the embryonic nervous system. Correspondingly, expression of phospho-Akt-1/protein-kinase B extensively overlapped expression sites of EPO and EPOR, but was absent from apoptotic cells. Identified other sites of EPO and/or EPOR expression include radial glial cells that transform to astrocytes, cells of the floor plate and notochord as well as neural crest-derived boundary cap cells at motor exit points and cells of the primary sympathetic chain.

Erythropoietin and Erythropoietin Receptor Expression after Experimental Spinal Cord Injury Encourages Therapy by Exogenous Erythropoietin

Erythropoietin (EPO) is a pleiotropic cytokine originally identified for its role in erythropoiesis. Recent studies have demonstrated that EPO and its receptor (EPO-R) are expressed in the central nervous system, where EPO exerts neuroprotective functions. Because the expression of the EPO and EPO-R network is poorly investigated in the central nervous system, the aim of the present study was to investigate whether the resident EPO and EPO-R network is activated in the injured nervous system. A well-standardized model of compressive spinal cord injury in rats was used. EPO and EPO-R expression was determined by immunohistochemical analysis at 8 hours and at 2, 8, and 14 days in the spinal cord of injured and noninjured rats. In noninjured spinal cord, weak immunohistochemical expression of EPO and EPO-R was observed in neuronal and glial cells as well as in endothelial and ependymal cells. In injured rats, a marked increase of expression of EPO and EPO-R was observed in neurons, vascular endothelium, and glial cells at 8 hours after injury, peaking at 8 days, after which it gradually decreased. Two weeks after injury, EPO immunoreactivity was scarcely detected in neurons, whereas glial cells and vascular endothelium expressed strong EPO-R immunoreactivity. These observations suggest that the local EPO and EPO-R system is markedly engaged in the early stages after nervous tissue injury. The reduction in EPO immunoexpression and the increase in EPO-R staining strongly support the possible usefulness of a therapeutic approach based on exogenous EPO administration.

Mitogen-activated Protein Kinase Plays an Essential Role in the Erythropoietin-dependent Proliferation of CTLL-2 Cells

Erythropoietin (EPO) and its receptor (EPOR) are required for development of erythrocytes. It has been shown that the ectopic expression of EPOR confers EPO-dependent proliferation on an interleukin 3 (IL3)-dependent cell line, Ba/F3, whereas the IL2-dependent T cell line, CTLL-2 expressing the EPOR (T-ER), fails to proliferate in response to EPO. However, the molecular basis of the EPO unresponsiveness in CTLL-2 has not been clarified. We found that the expression level of JAK2 in T-ER cells was much lower than that in Ba/F3 cells. Therefore, we examined the effects of forced expression of JAK2 in T-ER cells. In T-ER transformants expressing JAK2 (T-JER), EPO induced tyrosine phosphorylation of the EPOR, JAK2, and STAT5, and consequently STAT5-responsive genes including bcl-X and cis1 were normally induced. Furthermore, T-JER cells were resistant to apoptosis until at least 72 h after switching from IL2 to EPO. Although T-JER cells could not continuously proliferate in the presence of EPO, additional expression of JAK2 in T-JER (T-JJER) to a level similar to that in Ba/F3 cells supported long term proliferation in response to EPO. JAK2 was equally co-immunoprecipitated with the EPOR among T-JER, T-JJER, and Ba/F3 cells expressing the EPOR (BF-ER). However, EPO-dependent mitogen-activated protein (MAP) kinase activation was observed in T-JJER and BF-ER cells but not in T-JER cells. EPO-dependent long term proliferation of T-JER cells was conferred by expression of the constitutively activated form of MEK1. Our results suggest that MAP kinase activation is, at least in part, an important component for mitotic signal from the EPOR, and CTLL-2 cells probably lack signaling molecule(s) in JAK2 and the Ras-MAP kinase pathway.