Uticaj reaktivnih vrsta kiseonika na aktivnost mTOR signalnog puta kod pacijenata sa mijeloproliferativnim neoplazmama

common characteristic of malignancies is an increase of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In such circumstances macromolecules are damaged and the cell structure and function are disrupted. The oxidative DNA damage is considered to be the initial event in the developement of carcinogenesis. ROS affects the function of hematopoietic stem cells, changes the cell cycle progression, accelerаtes aging and induces hematological malignancies. The BCR/ABL oncogene and JAK2V617F mutation induces the formation of ROS in hematopoietic stem cells that leads to genomic instability and DNA damage. In the myeloproliferative neoplasm (MPN) patients, JAK2V617F mutation and BCR/ABL oncogene are associated with the constitutive activation of the PI3K/AKT/mTOR signaling pathway, which stimulates the growth and proliferation of cells. As ROS and RNS may affect the activity of mTOR, we have examined the parameters of antioxidant protection and oxidative damage of lipids and proteins in circulation as well as the AKT/mTOR signaling pathway activity in the MPN granulocytes and human erythroleukemia (HEL) cell line with JAK2V617F mutation. Methods: The study includes 110 de novo MPN patients: 30 polycythemia vera (PV), 24 essential thrombocythemia (ET), 34 primary myelofibrosis (PMF) as well as 22 patients with the diagnosis of chronic myeloid leukemia (CML) and 10 healthy subjects. Markers of oxidative and nitrosative stress in plasma and erythrocyte lysates were determined by the colorimetric method. The intracellular production of ROS was determined by fluorogenic reagent H2DCF-DA. The levels of nitrotyrosine, inducible NO synthase (iNOS) and the activation rate of AKT/mTOR/p70S6K kinases were determined in granulocytes of MPN patients using immunocytochemistry and immunoblotting methods. The influence of hydrogen peroxide (H2O2) and 2, 2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) oxidants on AKT/mTOR signal pathway, cell cycle and survival were tested by Western blot, FACS and MTT test in HEL cells...Zajednička karakteristika maligniteta je povećana koncentracija reaktivnih vrsta kiseonika (ROS) i reaktivnih vrsta azota (RNS) koje mogu da prouzrokuju oštećenja makromolekula u ćeliji i dovedu do narušavanja njene strukture i funkcije. Oksidativno oštećenje DNK smatra se inicijalnim događajem u razvoju kancerogeneze. ROS utiču na funkciju hematopoetskih matičnih ćelija, dovode do promena ćelijskog ciklusa, ubrzanog starenja ćelija ili do nastanka hematoloških maligniteta. BCR/ABL onkogen i JAK2V617F mutacija indukuju stvaranje ROS u hematopoetskim matičnim ćelijama što vodi genomskoj nestabilnosti i oštećenju DNK. Pod uticajem JAK2V617F mutacije i BCR/ABL onkogena kod hroničnih mijeloproliferativnih neoplazmi (HMN) dolazi do konstitutivne aktivacije PI3K/AKT/mTOR signalnog puta, koji reguliše metabolizam i stimuliše rast i proliferaciju ćelija. S obzirom da ROS i RNS mogu uticati na aktivnost mTOR kinaze, ispitani su parametri antioksidativne zaštite i oksidativno izmenjenih lipida i proteina u cirkulaciji, kao i stepen aktivacije AKT/mTOR signalnog puta u granulocitima bolesnika sa HMN i humanoj eritroleukemijskoj ćelijskoj liniji (eng. Human erythroleukemia cell line, HEL) sa JAK2V617F mutacijom. Metode: U studiju je uključeno 110 de novo HMN bolesnika: 30 sa policitemijom verom (PV), 24 sa esencijalnom trombocitemijom (ET), 34 sa primarnom mijelofibrozom (PMF), 22 bolesnika sa dijagnozom hronične mijeloidne leukemije (HML) i 10 zdravih ispitanika. Markeri oksidativnog i nitrozativnog stresa u plazmi i lizatima eritrocita određeni su kolorimetrijskom metodom. Količina intracelularne produkcije ROS određena je upotrebom fluorogenog reagensa H2DCF-DA. Nivo nitrotirozina, inducibilne NO sintaze (iNOS) i stepen aktivacije AKT/mTOR/p70S6K kinaza određeni su u granulocitima bolesnika sa HMN imunocitohemijskom metodom i metodom imunoblota. U in vitro uslovima ispitan je uticaj vodonik peroksida (H2O2) i N- acetil cisteina na aktivaciju mTOR i ekspresiju iNOS i nitrotirozina. Uticaj dva različita oksidansa, H2O2 i 2,2'-azobis(2-amidinopropan) dihidrohlorida (AAPH), na aktivaciju AKT/mTOR signalnog puta, preživljavanje i ćelijski ciklus HEL ćelija, u in vitro uslovima, ispitan je metodom imunoblota, FACS metodom i MTT testom..

The impact of reactive oxygen species on the mTOR signaling pathway activity in patients with myeloproliferative neoplasms

Заједничка карактеристика малигнитета је повећана концентрација реактивних врста кисеоника (ROS) и реактивних врста азота (RNS) које могу да проузрокују оштећења макромолекула у ћелији и доведу до нарушавања њене структуре и функције. Оксидативно оштећење ДНК сматра се иницијалним догађајем у развоју канцерогенезе. ROS утичу на функцију хематопоетских матичних ћелија, доводе до промена ћелијског циклуса, убрзаног старења ћелија или до настанка хематолошких малигнитета. BCR/ABL онкоген и JAK2V617F мутација индукују стварање ROS у хематопоетским матичним ћелијама што води геномској нестабилности и оштећењу ДНК. Под утицајем ЈАК2V617F мутације и BCR/ABL онкогена код хроничних мијелопролиферативних неоплазми (ХМН) долази до конститутивне активације PI3K/AKT/mTOR сигналног пута, који регулише метаболизам и стимулише раст и пролиферацију ћелија. С обзиром да ROS и RNS могу утицати на активност mTOR киназе, испитани су параметри антиоксидативне заштите и оксидативно измењених липида и протеина у циркулацији, као и степен активације AKT/mTOR сигналног пута у гранулоцитима болесника са ХМН и хуманој еритролеукемијској ћелијској линији (енг. Human erythroleukemia cell line, HEL) са ЈАК2V617F мутацијом. Методе: У студију je укључено 110 de novo ХМН болесника: 30 са полицитемијом вером (ПВ), 24 са есенцијалном тромбоцитемијом (ЕТ), 34 са примарном мијелофиброзом (ПМФ), 22 болесника са дијагнозом хроничне мијелоидне леукемије (ХМЛ) и 10 здравих испитаника. Маркери оксидативног и нитрозативног стреса у плазми и лизатима еритроцита одређени су колориметријском методом. Количина интрацелуларне продукције ROS одређенa је употребом флуорогеног реагенса H2DCF-DA. Ниво нитротирозина, индуцибилне NO синтазе (iNOS) и степен активације АKT/mTOR/p70S6K киназа одређени су у гранулоцитима болесника са ХМН имуноцитохемијском методом и методом имуноблота. У in vitro условима испитан је утицај водоник пероксида (H2O2) и N- ацетил цистеина на активацију mТОR и експресију iNOS и нитротирозина. Утицај два различита оксиданса, H2O2 и 2,2'-азобис(2-амидинопропан) дихидрохлорида (AAPH), на активацију AKT/mTOR сигналног пута, преживљавање и ћелијски циклус HEL ћелија, у in vitro условима, испитан је методом имуноблота, FACS методом и МТТ тестом...common characteristic of malignancies is an increase of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In such circumstances macromolecules are damaged and the cell structure and function are disrupted. The oxidative DNA damage is considered to be the initial event in the developement of carcinogenesis. ROS affects the function of hematopoietic stem cells, changes the cell cycle progression, accelerаtes aging and induces hematological malignancies. The BCR/ABL oncogene and JAK2V617F mutation induces the formation of ROS in hematopoietic stem cells that leads to genomic instability and DNA damage. In the myeloproliferative neoplasm (MPN) patients, JAK2V617F mutation and BCR/ABL oncogene are associated with the constitutive activation of the PI3K/AKT/mTOR signaling pathway, which stimulates the growth and proliferation of cells. As ROS and RNS may affect the activity of mTOR, we have examined the parameters of antioxidant protection and oxidative damage of lipids and proteins in circulation as well as the AKT/mTOR signaling pathway activity in the MPN granulocytes and human erythroleukemia (HEL) cell line with JAK2V617F mutation. Methods: The study includes 110 de novo MPN patients: 30 polycythemia vera (PV), 24 essential thrombocythemia (ET), 34 primary myelofibrosis (PMF) as well as 22 patients with the diagnosis of chronic myeloid leukemia (CML) and 10 healthy subjects. Markers of oxidative and nitrosative stress in plasma and erythrocyte lysates were determined by the colorimetric method. The intracellular production of ROS was determined by fluorogenic reagent H2DCF-DA. The levels of nitrotyrosine, inducible NO synthase (iNOS) and the activation rate of AKT/mTOR/p70S6K kinases were determined in granulocytes of MPN patients using immunocytochemistry and immunoblotting methods. The influence of hydrogen peroxide (H2O2) and 2, 2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) oxidants on AKT/mTOR signal pathway, cell cycle and survival were tested by Western blot, FACS and MTT test in HEL cells..

Neutrophil Death in Myeloproliferative Neoplasms: Shedding More Light on Neutrophils as a Pathogenic Link to Chronic Inflammation

Neutrophils are an essential component of the innate immune response, but their prolonged activation can lead to chronic inflammation. Consequently, neutrophil homeostasis is tightly regulated through balance between granulopoiesis and clearance of dying cells. The bone marrow is both a site of neutrophil production and the place they return to and die. Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders characterized by the mutations in three types of molecular markers, with emphasis on Janus kinase 2 gene mutation (JAK2V617F). The MPN bone marrow stem cell niche is a site of chronic inflammation, with commonly increased cells of myeloid lineage, including neutrophils. The MPN neutrophils are characterized by the upregulation of JAK target genes. Additionally, MPN neutrophils display malignant nature, they are in a state of activation, and with deregulated apoptotic machinery. In other words, neutrophils deserve to be placed in the midst of major events in MPN. Our crucial interest in this review is better understanding of how neutrophils die in MPN mirrored by defects in apoptosis and to what possible extent they can contribute to MPN pathophysiology. We tend to expect that reduced neutrophil apoptosis will establish a pathogenic link to chronic inflammation in MPN

Nitric Oxide Synthase Dependency in Hydroxyurea Inhibition of Erythroid Progenitor Growth

Hydroxyurea (HU) causes nitric oxide (NO) bioactivation, acting as both a NO donor and a stimulator of NO synthase (NOS). To examine whether HU effects are NO mediated by chemical degradation or enzymatic induction, we studied human and mouse erythroid cells during proliferation, apoptosis, and differentiation. The HU and NO donor demonstrated persisted versus temporary inhibition of erythroid cell growth during differentiation, as observed by γ-and β-globin gene expression. HU decreased the percentage of erythroleukemic K562 cells in the G2/M phase that was reversed by N-nitro l-arginine methyl ester hydrochloride (L-NAME). Besides activation of endothelial NOS, HU significantly increased apoptosis of K562 cells, again demonstrating NOS dependence. Administration of HU to mice significantly inhibited colony-forming unit-erythroid (CFU-E), mediated by NOS. Moreover, burst-forming-units-erythroid (BFU-E) and CFU-E ex vivo growth was inhibited by the administration of nitrate or nitrite to mice. Chronic in vivo NOS inhibition with L-NAME protected the bone marrow cellularity despite HU treatment of mice. NO metabolites and HU reduced the frequency of NOS-positive cells from CFU-E and BFU-E colonies that was reverted by NOS inhibition. HU regulation of the G2/M phase, apoptosis, differentiation, cellularity, and NOS immunoreactive cells was NOS dependent. Inhalation of NO therapy as well as strategies to increase endogenous NO production could replace or enhance HU activity

Neuronal Nitric Oxide Synthase Mediates the Effect of Ethanol on IgA

Aims: We showed previously that the acute effect of ethanol on intestinal immunoglobulin A (IgA) expression might be mediated by endogenous nitric oxide (NO). To extend these findings, this study was designed to investigate a possible role of neuronal NO synthase (nNOS) in the observed phenomenon, using 7-nitroindazole (7-NI), a selective inhibitor of its activity. Methods: Adult male Wistar rats were treated with: (a) ethanol (4 g/kg, intraperitoneally, i.p.), (b) 7-NI (25 mg/kg, i.p.) followed by ethanol (4 g/kg, i.p.) 30 min later and (c) 7-NI (25 mg/kg, i.p.) followed by saline 30 min later. Untreated rats were used as controls. The concentrations of serum and intestinal IgA were measured by enzyme-linked immunosorbent assay, while the expression of nNOS was determined using western blot and immunohistochemistry. Results: Acute ethanol treatment significantly increased the concentration of IgA in the ileal extracts, whereas it decreased its serum level. Inhibition of nNOS activity by 7-NI abolished this action of alcohol on IgA. Additionally, western blot analysis revealed that the acute alcohol administration induced an increase in the expression of intestinal nNOS. Furthermore, nNOS-immunoreactive cells, observed within the lamina propria of small intestine, were numerous in ethanol-treated rats. Conclusion: Taken together, these results extended our previous findings suggesting that nNOS mediates the acute effect of ethanol on IgA and supported an immunomodulatory role of this enzyme isoform

Nitric Oxide Mediation in Hydroxyurea and Nitric Oxide Metabolites’ Inhibition of Erythroid Progenitor Growth

In several systems, hydroxyurea has been shown to trigger nitric oxide (NO) release or activation of NO synthase (NOS). To elucidate this duality in its pharmacological effects, during myelosuppression, we individually examined hydroxyurea’s (NO releasing agent) and NO metabolites’ (stable NO degradation products) effects on erythroid colony growth and NOS/NO levels in mice using NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). Hydroxyurea and nitrite/nitrate decreased the bone marrow cellularity that was blocked by PTIO only for the NO metabolites. Hydroxyurea inhibition of colony-forming unit-erythroid (CFU-E) formation and reticulocytes was reversed by PTIO. Moreover, hydroxyurea, through a negative feedback mechanism, reduced inducible NOS (iNOS) expressing cells in CFU-E, also prevented by PTIO. Nitrate inhibition of burst-forming units-erythroid (BFU-E) colony growth was blocked by PTIO, but not in mature CFU-E. The presented results reveal that NO release and/or production mediates the hydroxyurea inhibition of mature erythroid colony growth and the frequency of iNOS immunoreactive CFU-E

Idiopatski i sekundarni stečeni megakolon kod pasa udruženi su sa smanjenom vip-inervacijom u oštećenom kolonu

It is well established that megacolon in carnivores, including both cats and dogs, is a common finding. Megacolon occurs more often in the cat that the dog. Based on current data idiopathic megacolon is a common cause of constipation in cats (62% of constipated cats are affected by diopathic megacolon). There is no evidence of idiopathic megacolon in dogs and publications about this disease in this species is very scarce. We investigated the enteric nervous system in the dilated portion (DP) of the colon in dogs with idiopathic aquired (n=7) or secondary aquired megacolon (n=21) and compared the results with a normal colon in control dogs (n=3). Colonic sections of surgical specimens were investigated by conventional and immunohistochemical methods, including pan-neuronal markers (NSE, synaptophisin, and neurofilament) and VIP, as well as S-100 protein for detection of ganglionic glial cells. Compared to controls, the two megacolon groups showed no changes of density of enteric neurons in both submucosal and myenteric nervous plexuses in DP of the colon and of enteric glial cells. However, compared to controls and dogs with secondary megacolon, there was a significant decrease in the density of NFP-ir nerve fibers in the longitudinal muscle layer in dogs with idiopathic acquired megacolon. In addition, dogs with idiopathic megacolon display decreased VIP-ir in the myenteric plexus and lamina propria mucosae, and absence of VIP-ir neurons in the submucosal plexus of DP of the colon. Similar alterations, although of lesser severity, may be found in dogs with secondary aquired megacolon. We consider that both idiopathic and secondary aquired megacolon might occur on the basis of a dysplastic changes of VIP-ir enteric neurons.Poznato je da se magakolon javlja kod mesojeda, uključujući mačke i pse, pri čemu je ovo oboljenje daleko učestalije kod mačaka. Na osnovu dosadašnjih saznanja, idiopatski megakolon je čest uzročnik konstipacije kod mačaka i 62% mačaka sa konstipacijom ima idiopatski megakolon. Istovremeno, podaci o psima sa idiopatskim megakolonom veoma su oskudni. U ovom radu je proučavan enterični nervni sistem u dilatiranom delu kolona kod 7 pasa sa idiopatskim megakolonom i 21 psa sa sekundarnim stečenim megakolonom, a rezultati su upoređeni sa normalnim kolonom kod 3 kontrolne zdrave životinje. Tkivni preseci kolona bojeni su klasičnim histološkim i imunohistohemijskim metodama, pri čemu su primenjeni pan-neuronski markeri (NSE, sinaptofizin i neurofilament) i VIP, kao i S-100 protein za detekciju glijalnih ćelija u enteričnim ganglijama. Nisu otkrivene razlike u gustini enteričnih neurona u submukoznom i mijenteričnom pleksusu kod životinja sa megakolonom, kao ni razlike u gustini glijalnih ćelija enteričnih ganglija, u odnosu na kontrolnu grupu životinja. Međutim, u odnosu na kontrolnu grupu, kod životinja sa idiopatskim megakolonom dokazana je smanjena VIP-imunoreaktivnost (ir) u mienteričnom pleksusu i krznu mukoze, kao i kompletno odsustvo VIP-ir neurona u submukoznom pleksusu dilatiranog dela kolona. Slične promene, ali u manjem stepenu, postojale su kod pasa sa sekundarnim stečenim megakolonom. Može da se zaključi da u patogenezi idiopatskog i sekundarnog stečenog megakolona značajnu ulogu imaju displastične promene u VIP-ergičkim neuronima enteričkog nervnog sistema

BRCA1 and TOP2A gene amplification and protein expression in four molecular subtypes of breast cancer

We studied TOP2A amplification (using FISH methods), and TOP2A and BRCA1 protein overexpression (immunohistochemistry) in four molecular subtypes of breast cancer. Of 53 patients, 32 showed TOP2A and 38 showed BRCA1 overexpression. The highest percentage of TOP2A amplification (47.8%) and deletion (13%) was detected in Luminal B subtypes. Of 11 Luminal B tumors with TOP2A amplification, 9 (81.8%) overexpressed TOP2A. BRCA1 protein overexpression showed significant positive correlation with TOP2A protein expression. BRCA1 and TOP2A proteins exhibited similar patterns of expression in Luminal B and triple-negative breast cancer, suggesting the same prognosis in those patients

Ghrelin endocrine cells in the human stomach during prenatal and early postnatal development

The aim of this study was to investigate the appearance, localization and density of ghrelin cells in the human stomach during prenatal development. For this purpose the antrum and corpus of embryos, fetuses and infants are stained immunohistochemically by the streptavidin-biotin technique. The presence of P/D1 cells at 11 weeks of fetal development, their highest density during the first detection and higher density in the corpus than in antrum, and their localization in the glandular base of stomach gland, all suggest that ghrelin plays a major role in the early stages of the developing stomach

Hydroxyurea Induces Bone Marrow Mesenchymal Stromal Cells Senescence and Modifies Cell Functionality In Vitro

Hydroxyurea (HU) is an antineoplastic agent that functions as an antimetabolite compound by inhibiting the ribonucleotide reductase. HU acts mainly as a cytostatic drug that through DNA replication stress may trigger a premature senescence-like cell phenotype, though its influence on bone marrow-derived mesenchymal stem/stromal cell (BMMSC) functions has not elucidated yet. Our results indicate that HU inhibits the growth of human BMMSC alongside senescence-like changes in both morphology and replicative potential, provokes cell cycle arrest at the S phase without affecting cellular viability and induces the expression of senescence-associated β-galactosidase and p16INK4. Moreover, HU-induced senescent BMMSC, although they did not change MSC markers expression, exhibited reduced capacity osteogenic and adipogenic differentiation. Conversely, HU treatment increased immunoregulatory functions of BMMSC compared with untreated cells and determined by T-cell proliferation. Interestingly, HU did not influence the capacity of BMMSC to induce monocytic myeloid-derived suppressor cells. Thus, these results suggest that HU improves the BMMSC functions on the T-cell inhibition and preserves their interaction with myeloid cell compartment. Mechanistically, BMMSC under HU treatment displayed a downregulation of mTOR and p38 MAPK signaling that may explain the reduced cell differentiation and increased immunomodulation activities. Together, the results obtained in this investigation suggest that HU by inducing senescence-like phenotype of BMMSC influences their cellular differentiation and immunoregulatory functions