Pseudihypoxia in renal cell carcinoma

Циљ: Тумор реналних ћелија (RCC) је високо васкуларизовани тумор са израженом способношћу деобе ћелија захваљујући смањеној периферној концентрацији кисеоника. Цео систем хипоксија индуцибилних протеина представља значајан патогенетски механизам у настанку и промоцији тумора бубрега, као и настанку повећане отпорности на терапију са тирозин киназним инхибиторима (TKИ) и блокаторима васкуларног ендотелног фактора раста (VEGF) и његових рецептора. Главни циљеви рада су: 1) секвенцирање von Hippel-Lindau (VHL) гена и утицај на активност хипоксија активираних гена као што су хипоксија индуцубилни фактор (HIF-1α), који следствено активира еритропоетин (ЕРО) и VEGF и њихове рецепторе. 2) испитивање регулације VHL активности кисеоник зависним (пролил хидроксилазе-PHD) и независним путевима (“heat shock протеин-Hsp”). 3) поређење митоген активишуће протеин киназе (МАРК) и фосфатидилинозитол-3 киназе (PI-3), пролиферативних путева у туморском ткиву и здравом ткиву, као и активност „Janus kinazе” и “Signal Transducer and Activator of Transcription” (JAK2-STAT5) пута. 4) експресија гена и сигналних путева у ендотелијалним ћелијама гајених у нормалним и хипоскичним условима као модела развоја крвних судова у туморском ткиву. Методологија: У студију је укључено 50 пацијената који су били индиковани за хируршки захват тј. парцијалну или тоталну нефректомију. Локални етички комитет Клиничког центра Србије је одобрио студију. Пацијенти су били упознати са студијом, тако да се пре почетка операције узимала крв у цитрату за изолацију ДНК и након операције узимао се мали исечак туморског и здравог ткива за изолацију РНК, ДНК и протеина. Коришћен модел ендотелијалних ћелија које су гајене у нормалним и хипоксичним условима и касније изолована РНК и протеини за анализу...Objective: Renal cell carcinoma (RCC) is highly vascularized and proliferative tumor in relation to reduced oxygen tension, The entire system of hypoxia-inducible proteins represents a relevant pathogenetic mechanism in the initiation and promotion of renal tumors as well as development of enhanced therapy resistance to anti-angiogenic drugs and tyrosine kinase inhibitors. The aims of this study were: 1) to sequence von Hippel-Lindau (VHL) gene and to examine the influence of mutations in VHL gene on hypoxia activated genes, like hypoxia inducible factor 1 (HIF-1α) together with erythropoietin (ЕРО) and vascular endothelial growth factor (VEGF) and their receptors. 2) to estimate the regulation of VHL activity by oxygen dependent prolil hydroxylases (PHD) and independent heat shock protein (Hsp) pathway. 3) to compare two major proliferative pathways МАРК (mitogen activated protein kinase) and PI-3 (phosphatidylinositol 3-kinase) in tumor and healthy tissue, and activity of Janus kinazе and Signal Transducer and Activator of Transcription (JAK2-STAT5) pathway. 4) to identify activated genes and signaling pathways in endothelial cells under low and normal oxygen tension, as a model for oxygen regulation and proliferation of endothelial cells in tumor tissue. Methodology: In our study we analyzed 50 renal tumor and surrounding normal tissue samples of patients after radical nephrectomy, for DNA, RNA and protein analysis. Together with tissues, blood samples were collected for DNA isolation. This study was approved by the local comity of Clinical Center of Serbia. Primary endothelial cells and endothelial cell lines were cultured under low and normal oxygen tension and used for RNA and protein extraction. Results: With direct sequencing and multiplex ligation-dependent probe amplification (MLPA) methods of VHL gene, in tumors and surrounding healthy tissues, somatic mutations in VHL gene were present in 58% of all tumor samples

Stvaranje azot-monoksida izazvano bradikininom nije dovoljno za indukciju gama-globina

Introduction Hydroxycarbamide, used in therapy of hemoglobinopathies, enhances nitric oxide (NO) production both in primary human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cell line (TrHBMEC). Moreover, NO increases γ-globin and fetal hemoglobin levels in human erythroid progenitors. Objective In order to find out whether simple physiologic stimulation of NO production by components of hematopoietic microenvironment can increase γ-globin gene expression, the effects of NO-inducer bradykinin were examined in endothelial cells. Methods The study was performed in co-cultures of human erythroid progenitors, TrHBMEC and HUVECs by ozone-based chemiluminescent determination of NO and real-time quantitative RT-PCR. Results In accordance with previous reports, the endogenous factor bradykinin increased endothelial cell production of NO in a dose- and time-dependent manner (0.1-0.6 μM up to 30 minutes). This induction of NO in HUVECs and TrHBMEC by bradykinin was blocked by competitive inhibitors of NO synthase (NOS), demonstrating NOS-dependence. It has been shown that bradykinin significantly reduced endothelial NOS (eNOS) mRNA level and eNOS/s-actin ratio in HUVEC (by twofold). In addition, bradykinin failed to increase γ-globin mRNA expression in erythroid progenitors only, as well as in co-culture studies of erythroid progenitors with TrHBMEC and HUVEC after 24 hours of treatment. Furthermore, bradykinin did not induce γ/β globin ratio in erythroid progenitors in co-cultures with HUVEC. Conclusion Bradykinin mediated eNOS activation leads to short time and low NO production in endothelial cells, insufficient to induce γ-globin gene expression. These results emphasized the significance of elevated and extended NO production in augmentation of γ-globin gene expression.Uvod Hidroksikarbamid, koji se koristi u lečenju hemoglobinopatija, podstiče stvaranje azot-monoksida (NO) kako u primarnim ljudskim endotelnim ćelijama pupčane vene (HUVEC), tako i u izmenjenoj endotelnoj ćelijskoj liniji poreklom iz koštane srži (TrHBMEC). Štaviše, NO povećava stvaranje γ-globina i fetalnog hemoglobina u ljudskim progenitorima eritropoeze. Cilj rada Da bismo ustanovili da li jednostavna fiziološka stimulacija stvaranja NO od komponenti mikrosredine hematopoeze može povećati ekspresiju γ-globinskog gena, ispitivali smo efekte bradikinina, već poznatog stimulatora stvaranja NO. Metode rada Studija je izvedena u zajedničkim kulturama ljudskih progenitora eritropoeze sa TrHBMEC ili HUVEC i ispitivana hemiluminiscentnim merenjem NO posredstvom ozona, kao i primenom kvantitativnog RT-PCR na genskom nivou. Rezultati U skladu s prethodnim izveštajima, pokazali smo da endogeni faktor bradikinin povećava stvaranje NO u endotelnim ćelijama na dozno i vremenski zavisan način (0,1-0,6 μM do 30 minuta). Ovo stvaranje NO u HUVEC i TrHBMEC izazvano bradikininom blokirano je od strane konkurentskih inhibitora NO-sintaze (NOS), pokazujući NOS-zavisnost. Utvrdili smo da bradikinin značajno smanjuje stvaranje iRNK endotelne forme NOS (eNOS), kao i odnos eNOS i β-aktina u HUVEC (dvostruko manje). Pored toga, bradikinin ne povećava ekspresiju iRNK γ-globinskog gena ni u zasebnim progenitorima eritropoeze, niti u zajedničkim kulturama progenitora eritropoeze sa TrHBMEC ili HUVEC posle 24 sata tretmana. Bradikinin ne menja ni odnos γ i β globina u zajedničkim kulturama progenitora eritropoeze sa HUVEC. Zaključak Aktivacija eNO_ izazvana bradikininom dovodi do kratkog i malog povećanja NO u endotelnim ćelijama, što je nedovoljno da podstakne ekspresiju gena za γ-globin. Ovi rezultati naglašavaju važnost povećanog i produženog stvaranja NO radi stimulacije ekspresije γ-globina

Proinflammatory Cytokine IL-6 and JAK-STAT Signaling Pathway in Myeloproliferative Neoplasms

The recent JAK1/2 inhibitor trial in myeloproliferative neoplasms (MPNs) showed that reducing inflammation can be more beneficial than targeting gene mutants. We evaluated the proinflammatory IL-6 cytokine and JAK-STAT signaling pathway related genes in circulating CD34(+) cells of MPNs. Regarding laboratory data, leukocytosis has been observed in polycythemia vera (PV) and JAK2V617F mutation positive versus negative primary myelofibrosis (PMF) patients. Moreover, thrombocytosis was reduced by JAK2V617F allele burden in essential thrombocythemia (ET) and PMF. 261 significantly changed genes have been detected in PV, 82 in ET, and 94 genes in PMF. The following JAK-STAT signaling pathway related genes had augmented expression in CD34(+) cells of MPNs: CCND3 and IL23A regardless of JAK2V617F allele burden; CSF3R, IL6ST, and STAT1/2 in ET and PV with JAK2V617F mutation; and AKT2, IFNGR2, PIM1, PTPN11, and STAT3 only in PV. STAT5A gene expression was generally reduced in MPNs. IL-6 cytokine levels were increased in plasma, as well as IL-6 protein levels in bone marrow stroma of MPNs, dependent on JAK2V617F mutation presence in ET and PMF patients. Therefore, the JAK2V617F mutant allele burden participated in inflammation biomarkers induction and related signaling pathways activation in MPNs

Stimulated stromal cells induce gamma-globin gene expression in erythroid cells via nitric oxide production

Objective. We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). We have now expanded those studies to demonstrate that stimulation of gamma-globin gene expression is also mediated by NOS induction in stromal cells within the bone marrow microenvironment. Materials and Methods. Using NO analyzer, we measured NO production in endothelial and macrophage cell cultures. In coculture studies of erythroid and stromal cells, we measured globin gene expression during stimulation by NO induers. Results. Hydroxyurea (30 - 100 mu M) induced NOS-dependent production of NO in human macrophages (up to 1.2 mu M). Coculture studies of human macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to threefold) in the presence of hydroxyurea. NOS-dependent stimulation of NO by lipopolysaccharide (up to 0.6 mu M) has been observed in human macrophages. We found that lipopolysaccharide and interferon-gamma together increased gamma-globin gene expression (up to twofold) in human macrophage/erythroid cell cocultures. Coculture of human bone marrow endothelial cells with erythroid progenitor cells also induced gamma-globin mRNA expression (2.4-fold) in the presence of hydroxyurea (40 mu M). Conclusion. These results demonstrate an arrangement by which NO and fetal hemoglobin inducers may stimulate globin genes in erythroid cells via the common paracrine effect of bone marrow stromal cells

Microarray and Proteomic Analyses of Myeloproliferative Neoplasms with a Highlight on the mTOR Signaling Pathway

The gene and protein expression profiles in myeloproliferative neoplasms (MPNs) may reveal gene and protein markers of a potential clinical relevance in diagnosis, treatment and prediction of response to therapy. Using cDNA microarray analysis of 25,100 unique genes, we studied the gene expression profile of CD34(+) cells and granulocytes obtained from peripheral blood of subjects with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The microarray analyses of the CD34(+) cells and granulocytes were performed from 20 de novo MPN subjects: JAK2 positive ET, PV, PMF subjects, and JAK2 negative ET/PMF subjects. The granulocytes for proteomic studies were pooled in 4 groups: PV with JAK2 mutant allele burden above 80%, ET with JAK2 mutation, PMF with JAK2 mutation and ET/PMF with no JAK2 mutation. The number of differentially regulated genes was about two fold larger in CD34(+) cells compared to granulocytes. Thirty-six genes (including RUNX1, TNFRSF19) were persistently highly expressed, while 42 genes (including FOXD4, PDE4A) were underexpressed both in CD34(+) cells and granulocytes. Using proteomic studies, significant up-regulation was observed for MAPK and PI3K/AKT signaling regulators that control myeloid cell apoptosis and proliferation: RAC2, MNDA, S100A8/9, CORO1A, and GNAI2. When the status of the mTOR signaling pathway related genes was analyzed, PI3K/AKT regulators were preferentially up-regulated in CD34(+) cells of MPNs, with down-regulated major components of the protein complex EIF4F. Molecular profiling of CD34(+) cells and granulocytes of MPN determined gene expression patterns beyond their recognized function in disease pathogenesis that included dominant up-regulation of PI3K/AKT signaling

Pseudihypoxia in renal cell carcinoma

Циљ: Тумор реналних ћелија (RCC) је високо васкуларизовани тумор са израженом способношћу деобе ћелија захваљујући смањеној периферној концентрацији кисеоника. Цео систем хипоксија индуцибилних протеина представља значајан патогенетски механизам у настанку и промоцији тумора бубрега, као и настанку повећане отпорности на терапију са тирозин киназним инхибиторима (TKИ) и блокаторима васкуларног ендотелног фактора раста (VEGF) и његових рецептора. Главни циљеви рада су: 1) секвенцирање von Hippel-Lindau (VHL) гена и утицај на активност хипоксија активираних гена као што су хипоксија индуцубилни фактор (HIF-1α), који следствено активира еритропоетин (ЕРО) и VEGF и њихове рецепторе. 2) испитивање регулације VHL активности кисеоник зависним (пролил хидроксилазе-PHD) и независним путевима (“heat shock протеин-Hsp”). 3) поређење митоген активишуће протеин киназе (МАРК) и фосфатидилинозитол-3 киназе (PI-3), пролиферативних путева у туморском ткиву и здравом ткиву, као и активност „Janus kinazе” и “Signal Transducer and Activator of Transcription” (JAK2-STAT5) пута. 4) експресија гена и сигналних путева у ендотелијалним ћелијама гајених у нормалним и хипоскичним условима као модела развоја крвних судова у туморском ткиву. Методологија: У студију је укључено 50 пацијената који су били индиковани за хируршки захват тј. парцијалну или тоталну нефректомију. Локални етички комитет Клиничког центра Србије је одобрио студију. Пацијенти су били упознати са студијом, тако да се пре почетка операције узимала крв у цитрату за изолацију ДНК и након операције узимао се мали исечак туморског и здравог ткива за изолацију РНК, ДНК и протеина. Коришћен модел ендотелијалних ћелија које су гајене у нормалним и хипоксичним условима и касније изолована РНК и протеини за анализу...Objective: Renal cell carcinoma (RCC) is highly vascularized and proliferative tumor in relation to reduced oxygen tension, The entire system of hypoxia-inducible proteins represents a relevant pathogenetic mechanism in the initiation and promotion of renal tumors as well as development of enhanced therapy resistance to anti-angiogenic drugs and tyrosine kinase inhibitors. The aims of this study were: 1) to sequence von Hippel-Lindau (VHL) gene and to examine the influence of mutations in VHL gene on hypoxia activated genes, like hypoxia inducible factor 1 (HIF-1α) together with erythropoietin (ЕРО) and vascular endothelial growth factor (VEGF) and their receptors. 2) to estimate the regulation of VHL activity by oxygen dependent prolil hydroxylases (PHD) and independent heat shock protein (Hsp) pathway. 3) to compare two major proliferative pathways МАРК (mitogen activated protein kinase) and PI-3 (phosphatidylinositol 3-kinase) in tumor and healthy tissue, and activity of Janus kinazе and Signal Transducer and Activator of Transcription (JAK2-STAT5) pathway. 4) to identify activated genes and signaling pathways in endothelial cells under low and normal oxygen tension, as a model for oxygen regulation and proliferation of endothelial cells in tumor tissue. Methodology: In our study we analyzed 50 renal tumor and surrounding normal tissue samples of patients after radical nephrectomy, for DNA, RNA and protein analysis. Together with tissues, blood samples were collected for DNA isolation. This study was approved by the local comity of Clinical Center of Serbia. Primary endothelial cells and endothelial cell lines were cultured under low and normal oxygen tension and used for RNA and protein extraction. Results: With direct sequencing and multiplex ligation-dependent probe amplification (MLPA) methods of VHL gene, in tumors and surrounding healthy tissues, somatic mutations in VHL gene were present in 58% of all tumor samples

Transcriptional profiling of erythroid progenitors from G-CSF mobilized and nonmobilized peripheral blood

Purpose: The purpose of this study was to examine the gene expression profile of granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood (mPB)-derived progenitors, used in transplantation. Methods: We correlated gene expression patterns of highly enriched steady-state peripheral blood (PB)- and mPB-derived CD71(+) cells by microarray and ingenuity pathway analyses, to identify the transcriptional program during in vitro erythroid differentiation. Results: The gene expression was more than doubled in mPB-derived (4180 genes) compared to PB-derived erythroid progenitors (1667 genes) while PB-and mPB-derived erythroid progenitors shared 1534 common genes. Comparative analysis of transcript levels showed differential expression of 54 genes between cultured erythroid progenitors of PB and mPB origin, where we identified common 13 downregulated and 30 upregulated genes. The most significant genes in mPB-derived erythroid progenitors were P4HB, DDIA3, ARPC2 and ATP5G3. Regarding G-CSF stimulation the G-CSF receptor CSF2RB (1.1-fold) was linked via STAT3 to erythroid-specific ALAS2 (2.9-fold) and GATA2 (1.3-fold) factors, all upregulated in mPB-derived erythroid progenitors, coupled to common upregulated NUDC gene involved in the proliferation of erythroid cells. Conclusion: This report provides an extensive transcriptional profile of cultured erythroid progenitors and leads to a better understanding of diversity among the progenitor sources

The statistically significant genes (p<0.01) among MPNs in CD34⁺ cells determined by microarray analysis.

<p>The negative values represent down-regulated genes, while positive values represent up-regulated genes compared to HuURNA. BG—Between groups significance with p<0.01, MD—Maximal mean difference of corresponding BG.</p><p>The statistically significant genes (p<0.01) among MPNs in CD34⁺ cells determined by microarray analysis.</p