P14arf metilasyonunun nöroblastom minimal rezidüel hastaliktaki rolü

Nöroblastom, primordial nöral krest hücrelerinden köken alan ve tüm çocukluk çağı kanserlerinin %8-10'unu oluşturan bir tümördür. Bir yaş altındaki çocukların en sık görülen tümörüdür. Tümörün biyolojik davranışındaki değişkenlik nedeniyle, nöroblastom spontan regresyonlar, benign transformasyon ya da agresif seyir gösterebilmekte, bu durum hastalığın prognozunu belirlemede ve tedavisinde sorunlara yol açmaktadır. Epigenetik değişiklikler, tümörün oluşumunda ve progresyonunda önemli rol oynamaktadır. Bu değişikliklerin sonucunda görülen malign hücre genomu, spesifik histon modifikasyonlarının azalması ve bazı genlerin metilasyonu veya demetilasyonu ile karakterizedir. p14ARF metilasyonunun oluşturduğu tümöral etki, N-Myc gibi onkogenler tarafından aktive olduktan sonra p53 tümör baskılayıcı gen ekspresyonunu bloke ederek hücre çoğalmasını negatif yönde düzenleyen faktörleri inhibe etmek şeklindedir. Bu çalışmada, Kelly (Nöroblastik, N-Myc +) ve SH-SY5Y (Nöroblastik, N-Myc -) insan nöroblastom hücre hatlarında p14ARF geni demetile edici bir ajan ile bloke edilerek Türk Pediatrik Onkoloji Grubu (TPOG) kemoterapi protokolünde kullanılan ilaçların tümör hücreleri üzerindeki sitotoksik etkileri değerlendirilmiştir. Ayrıca, p14ARF geninin N-Myc amplifikasyonu olan ve olmayan hücre hatlarındaki etkisi değerlendirilerek tümörün prognozunu ne yönde etkilediği ve sitotoksik etki değişikliği ile prognoz arasında bir ilişki bulunup bulunmadığı araştırılmıştır. İlaçların hücre canlılığı ve hücre hasarına etkisi tripan mavisi boyaması ile değerlendirilmiştir. Nöroblastom hücre hatlarında sitotoksik etki farklılığı saptanmış, bu etkinin mekanizmalarını incelemek için p14ARF geninin metilasyon ve ekspresyon düzeyleri ile birlikte N-Myc expresyon düzeyleri hedef olarak seçilmiştir. p14ARF ve N-Myc mRNA ekspresyonu real-time PCR ile, protein ekspresyonu ELISA yöntemi ile değerlendirilmiştir. Projemizde, nöroblastomdaki minimal rezidüel hastalık modelinde p14ARF geninin N-Myc amplifikasyonu ile olan ilişkisi ve bu ilişkinin rutin kemoterapi protokolünde kullanılan ilaçlarla olan etkileşiminde demetilasyonun rolü, bugüne kadar henüz incelenmeyen epigenetik tedavi yaklaşımı sınırlarında geniş kapsamlı olarak araştırılmış, metilasyon ilişkili tedavi ile tümörün prognozu arasında bir ilişki olup olmadığına dair önemli ipuçları elde edilmiş ve bu ilişkinin yönü ortaya konmuştur. Sonuç olarak, bu öncü çalışmada nöroblastomda minimal rezidüel hastalığın önlenmesindeæ gerek tedavinin kesilmesinden sonra karşımıza çıkan erken ya da geç dönemdeki relapsların tedavisine yönelik yeni bir yaklaşım önerisi elde edilmiş, gerekse de rutin kemoterapi protokolünde kullanılan ilaçların p14ARF gen düzeyleri üzerindeki etkileri geniş bir perspektifte incelenmiştir. İleriye yönelik olarak, p14ARF gen düzeyleri ile ilişkili transkripsiyon faktörlerinin prognostik öneminin araştırılması uygun bir yaklaşım olacaktır. Yine ekspresyon düzeyinin yüksek olduğu hücre serilerinde bu faktörlere karşı geliştirilmiş inhibitörlerin etkinliği de tedavide bu ekspresyonların önemini ortaya koyacaktır. Neuroblastoma originating from primordial neural crest cells constitutes 8-10 % of whole childhood cancers. It is the most frequently diagnosed tumor of infancy. Because of the variations in its biological behavior, neuroblastoma may show spontaneous regression, benign transformation or agressive progression and this uncertainity leads to difficulties in predicting prognosis and treatment. Epigenetic changes has a significant role in the occurence and progression of the tumor and the resultant malignant cell genome is characterized by the reduction of specific histone modifications and methylation or demethylation of genes. Tumorigenesis effect of p14ARF methylation depends upon inhibiting factors that impairs cell proliferation via blocking p53 gene expression due to its N-Myc mediated activation. In the present study, cytotoxic effects of blocked p14ARF by demethylating agent on Kelly (Neuroblastic, N-Myc +) and SH-SY5Y (Neuroblastic, N-Myc -) human neuroblastoma cell lines assigned according to whether Turk Pediatric Oncology Group (TPOG) chemotherapy protocol drugs are added. Moreover, with the evaluation of the effects of p14ARF gene on cell lines with or without N-Myc amplification a probable change in prognosis and any relation between cytotoxic effects and prognosis were investigated. Drug induced effects on cell viability, cell damage and apoptotic cell death ratio were assessed with trypan blue dying. The difference of cytotoxic effects were observed in neuoroblastoma cell lines. For to investigate the mechanisms of this effect, p14ARF gen methylation and expression levels, and N-Myc expression levels were targeted. Expressions of mRNA and protein will be determined with real-time PCR and ELISA, respectively. So that, in this project relationship between p14ARF gene and N-Myc amplification, as well as the role of gene methylation on chemoterapeutic agents in epigenetic treatment approach which has not been studied yet in minimal residual disease model of neuroblastoma, were investigated in detail and a possible relation between this treatment model and the prognosis and its direction were demonstrated. In conclusion, in this pioneering study we have demonstrated that not only new therapeutic approach for early or late relapses following the ending of treatment were found but also effects of chemoterapeutic agents on p14ARF gene on this model were investigated in detail. Regarding our results, investigation of transcription factors with related p14ARF gene levels may help to determine prognosis in neuroblastoma. Moreover, development of targeted therapies against these transcriptional factors may be a therapeutic approach in the near future

In vitro učinci selena na stanične linije humanog glioblastoma multiforme: preliminarno istraživanje

Glioblastoma multiforme (GBM) is caused by the central nervous system-derived glial cells, and represents the most common (50%-60%) form of primary brain tumors. The aim of this study was to investigate the in vitro effects of selenium on human GBM cells. In the present study, GMS-10 and DBTRG-05MG human GBM cell lines were used as a model to examine selenium entering the cell, cell proliferation, cytotoxicity, DNA fragmentation and Ki-67 protein expression in selenomethionine treated and non-treated groups. Seleno-L-methionine (SeMet) as the organic source of selenium exerted effects on cell proliferation and cytotoxicity, as assessed with WST-1 and lactate dehydrogenase (LDH) tests, respectively. Apoptosis was assessed by DNA fragmentation with an enzyme-linked immunosorbent assay. Ki-67 protein expression was determined by Western blotting, while selenium measurements were performed in the supernatants and lysates by using Graphite Furnace Atomic Absorption Spectrometry. Th is is the first study to examine the effects of SeMet on cell proliferation and death in GMS-10 and DBTRG-05MG cells. Both GBM cell lines responded to SeMet in a dose- and time-dependent manner. WST-1 test showed that low-dose SeMet treatment (50 and 100 μM) increased cell proliferation. Analysis of intracellular SeMet levels by using AAS showed results consistent with viability and cytotoxicity tests. SeMet treatment for 72 h caused increased DNA fragmentation in both cell lines. In conclusion, our results suggest that SeMet induces cell death at high doses, while increasing cell proliferation at low doses. In the view of the data obtained in this investigation, further studies focusing on the possibility of using SeMet against different types of GBM and in combination with prospect synergic compounds are considered to be worthwhile.Glioblastom multiforme (GBM) uzrokuju glijalne stanice podrijetlom iz središnjega živčanog sustava i to je najčešći (50%-60%) oblik primarnog tumora mozga. Cilj ovoga istraživanja bio je ispitati in vitroučinke selena na stanice humanog GBM. Rabili smo stanične linije GMS-10 i DBTRG-05MG humanog GBM kao model za ispitivanje ulaska selena u stanicu, stanične proliferacije, citotoksičnosti, fragmentacije DNA i izraženosti proteina Ki-67 u skupini tretiranoj selenometioninom i skupini bez takve obrade. Seleno-L-metionin (SeMet) kao organski izvor selena utjecao je na staničnu proliferaciju i citotoksičnost, što je procijenjeno pomoću testova WST-1 odnosno laktat dehidrogenaze (LDH). Apoptoza je procijenjena fragmentacijom DNA testom ELISA. Izražajnost proteina Ki-67 utvrđena je Western blotingom, dok su mjerenja selena provedena u supernatantima, a lizati primjenom GFAAS. Ovo je prvo istraživanje u kojem su se ispitivali učinci SeMet na staničnu proliferaciju i smrt u stanicama GMS-10 i DBTRG-05MG. Obje stanične linije GBM pokazale su o dozi i vremenu ovisan odgovor na SeMet. Test WST-1 pokazao je da je tretman niskom dozom SeMet (50 i 100 μM) pove-ćao proliferaciju stanica. Rezultati analize unutarstaničnih razina SeMet pomoću AAS bili su sukladni rezultatima testova stanične životnosti i citotoksičnosti. Tretman pomoću SeMet kroz 72 h uzrokovao je povećanu fragmentaciju DNA u objema staničnim linijama. U zaključku, naši rezultati ukazuju na to da SeMet u visokim dozama izaziva staničnu smrt, dok u niskim dozama povećava staničnu proliferaciju. U svjetlu podataka dobivenih u ovom istraživanju smatramo da bi bilo opravdano daljnja istraživanja usredotočiti na moguću primjenu SeMet kod različitih tipova GBM i u kombinaciji s mogućim sinergističnim spojevima

Erythropoietin drives breast cancer progression by activation of its receptor EPOR

YesBreast cancer is a leading cause of cancer-related deaths. Anemia is common in breast cancer patients and can be treated with blood transfusions or with recombinant erythropoietin (EPO) to stimulate red blood cell production. Clinical studies have indicated decreased survival in some groups of cancer patients treated with EPO. Numerous tumor cells express the EPO receptor (EPOR), posing a risk that EPO treatment would enhance tumor growth, but the mechanisms involved in breast tumor progression are poorly understood. Here, we have examined the functional role of the EPO-EPOR axis in preclinical models of breast cancer. EPO induced the activation of PI3K/AKT and MAPK pathways in human breast cancer cell lines. EPOR knockdown abrogated human tumor cell growth, induced apoptosis through Bim, reduced invasiveness, and caused downregulation of MYC expression. EPO-induced MYC expression is mediated through the PI3K/AKT and MAPK pathways, and overexpression of MYC partially rescued loss of cell proliferation caused by EPOR downregulation. In a xenotransplantation model, designed to simulate recombinant EPO therapy in breast cancer patients, knockdown of EPOR markedly reduced tumor growth. Thus, our experiments in vitro and in vivo demonstrate that functional EPOR signaling is essential for the tumor-promoting effects of EPO and underline the importance of the EPO-EPOR axis in breast tumor progression.Cancer Research UK - C10141/A9977 (TRL, MET, KKC). European Commission FP7 (EpoCan) 282551 (TRL, KBM); Invest NI RD0914223 (TRL, KBM)

Low oxygen tension primes aortic endothelial cells to the reparative effect of tissue-protective cytokines

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared to 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis

A nationwide multicentre study in Turkey for establishing reference intervals of haematological parameters with novel use of a panel of whole blood

IntroductionA nationwide multicentre study was conducted to establish well-defined reference intervals (RIs) of haematological parameters for the Turkish population in consideration of sources of variation in reference values (RVs). Materials and methodsK2-EDTA whole blood samples (total of 3363) were collected from 12 laboratories. Sera were also collected for measurements of iron, UIBC, TIBC, and ferritin for use in the latent abnormal values exclusion (LAVE) method. The blood samples were analysed within 2 hours in each laboratory using Cell Dyn and Ruby (Abbott), LH780 (Beckman Coulter), or XT-2000i (Sysmex). A panel of freshly prepared blood from 40 healthy volunteers was measured in common to assess any analyser-dependent bias in the measurements. The SD ratio (SDR) based on ANOVA was used to judge the need for partitioning RVs. RIs were computed by the parametric method with/without applying the LAVE method. ResultsAnalyser-dependent bias was found for basophils (Bas), MCHC, RDW and MPV from the panel test results and thus those RIs were derived for each manufacturer. RIs were determined from all volunteers’ results for WBC, neutrophils, lymphocytes, monocytes, eosinophils, MCV, MCH and platelets. Gender-specific RIs were required for RBC, haemoglobin, haematocrit, iron, UIBC and ferritin. Region-specific RIs were required for RBC, haemoglobin, haematocrit, UIBC, and TIBC. ConclusionsWith the novel use of a freshly prepared blood panel, manufacturer-specific RIs’ were derived for Bas, Bas%, MCHC, RDW and MPV. Regional differences in RIs were observed among the 7 regions of Turkey, which may be attributed to nutritional or environmental factors, including altitude

Effects of erythropoietin on depressive symptoms and neurocognitive deficits in depression and bipolar disorder

<p>Abstract</p> <p>Background</p> <p>Depression and bipolar disorder are associated with reduced neural plasticity and deficits in memory, attention and executive function. Drug treatments for these affective disorders have insufficient clinical effects in a large group and fail to reverse cognitive deficits. There is thus a need for more effective treatments which aid cognitive function. Erythropoietin (Epo) is involved in neuroplasticity and is a candidate for future treatment of affective disorders. The investigators have demonstrated that a single dose of Epo improves cognitive function and reduces neurocognitive processing of negative emotional information in healthy and depressed individuals similar to effects seen with conventional antidepressants. The current study adds to the previous findings by investigating whether repeated Epo administration has antidepressant effects in patients with treatment resistant depression and reverses cognitive impairments in these patients and in patients with bipolar disorder in remission.</p> <p>Methods/design</p> <p>The trial has a double-blind, placebo-controlled, parallel-group design. 40 patients with treatment-resistant major depression and 40 patients with bipolar disorder in remission are recruited and randomised to receive weekly infusions of Epo (Eprex; 40,000 IU) or saline (NaCl 0.9%) for 8 weeks. Randomisation is stratified for age and gender. The primary outcome parameters for the two studies are: depression severity measured with the Hamilton Depression Rating Scale 17 items (HDRS-17) <abbrgrp><abbr bid="B1">1</abbr></abbrgrp> in study 1 and, in study 2, verbal memory measured with the Rey Auditory Verbal Learning Test (RAVLT) <abbrgrp><abbr bid="B2">2</abbr><abbr bid="B3">3</abbr></abbrgrp>. With inclusion of 40 patients in each study we obtain 86% power to detect clinically relevant differences between intervention and placebo groups on these primary outcomes.</p> <p>Trial registration</p> <p>The trial is approved by the Local Ethics Committee: H-C-2008-092, Danish Medicines Agency: 2612-4020, EudraCT: 2008-04857-14, Danish Data Agency: 2008-41-2711 and ClinicalTrials.gov: NCT 00916552.</p

Erythropoietin Couples Hematopoiesis with Bone Formation

It is well established that bleeding activates the hematopoietic system to regenerate the loss of mature blood elements. We have shown that hematopoietic stem cells (HSCs) isolated from animals challenged with an acute bleed regulate osteoblast differentiation from marrow stromal cells. This suggests that HSCs participate in bone formation where the molecular basis for this activity is the production of BMP2 and BMP6 by HSCs. Yet, what stimulates HSCs to produce BMPs is unclear.In this study, we demonstrate that erythropoietin (Epo) activates Jak-Stat signaling pathways in HSCs which leads to the production of BMPs. Critically, Epo also directly activates mesenchymal cells to form osteoblasts in vitro, which in vivo leads to bone formation. Importantly, Epo first activates osteoclastogenesis which is later followed by osteoblastogenesis that is induced by either Epo directly or the expression of BMPs by HSCs to form bone.These data for the first time demonstrate that Epo regulates the formation of bone by both direct and indirect pathways, and further demonstrates the exquisite coupling between hematopoiesis and osteopoiesis in the marrow

Evaluation of Functional Erythropoietin Receptor Status in Skeletal Muscle In Vivo: Acute and Prolonged Studies in Healthy Human Subjects

BACKGROUND: Erythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle. METHODOLOGY/PRINCIPAL FINDINGS: The protocols involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1-10 hours, and 2) a separate study with prolonged administration for 16 days with biopsies obtained before and after. The presence of erythropoietin receptors in muscle tissue as well as activation of Epo signalling pathways (STAT5, MAPK, Akt, IKK) were analysed by western blotting. Changes in muscle protein profiles after prolonged erythropoietin treatment were evaluated by 2D gel-electrophoresis and mass spectrometry. The presence of the erythropoietin receptor in skeletal muscle was confirmed, by the M20 but not the C20 antibody. However, no significant changes in phosphorylation of the Epo-R, STAT5, MAPK, Akt, Lyn, IKK, and p70S6K after erythropoietin administration were detected. The level of 8 protein spots were significantly altered after 16 days of rHuEpo treatment; one isoform of myosin light chain 3 and one of desmin/actin were decreased, while three isoforms of creatine kinase and two of glyceraldehyd-3-phosphate dehydrogenase were increased. CONCLUSIONS/SIGNIFICANCE: Acute exposure to recombinant human erythropoietin is not associated by detectable activation of the Epo-R or downstream signalling targets in human skeletal muscle in the resting situation, whereas more prolonged exposure induces significant changes in the skeletal muscle proteome. The absence of functional Epo receptor activity in human skeletal muscle indicates that the long-term effects are indirect and probably related to an increased oxidative capacity in this tissue

The neuroprotective and neurorescue effects of carbamylated erythropoietin Fc fusion protein (CEPO-Fc) in a rat model of Parkinson's disease

Parkinson's disease is characterized by progressive degeneration of dopaminergic neurons. Thus the development of therapeutic neuroprotection and neurorescue strategies to mitigate disease progression is important. In this study we evaluated the neuroprotective/rescue effects of erythropoietin Fc fusion protein (EPO-Fc) and carbamylated erythropoietin Fe fusion protein (CEPO-Fc) in a rat model of Parkinson's disease. Adult female Sprague-Dawley rats received intraperitoneal injection of EPO-Fc, CEPO-Fc or PBS. Behavioral evaluations consisted of rota-rod, cylinder and amphetamine-induced rotation tests. In the neuroprotection experiment, the CEPO-Fc group demonstrated significant improvement compared with the EPO-Fc group on the amphetamine-induced rotation test throughout the four-week follow-up period. Histologically, significantly more tyrosine hydroxylase (TH)-positive neurons were recognized in the substantia nigra (SN) pars compacta in the CEPO-Fc group than in the PBS and EPO-Fc groups. In the neurorescue experiment, rats receiving CEPO-Fc showed significantly better behavioural scores than those receiving PBS. The histological data concerning striatum also showed that the CEPO-Fc group had significantly better preservation of TH-positive fibers compared to the PBS and EPO-Fc groups. Importantly, there were no increases in hematocrit or hemoglobin levels in the CEPO-Fc group in either the neuroprotection or the neurorescue experiments. In conclusion, the newly developed CEPO-Fc might confer neuroprotective and neurorescue benefits in a rat model of Parkinson's disease without the side effects associated with polycythemia. CEPO-Fc might be a therapeutic tool for patients with Parkinson's disease