CXCR7 Is Highly Expressed in Acute Lymphoblastic Leukemia and Potentiates CXCR4 Response to CXCL12

Recently, a novel CXCL12-binding receptor, has been identified. This CXCL12-binding receptor commonly known as CXCR7 (CXC chemokine receptor 7), has lately, based on a novel nomenclature, has received the name ACKR3 (atypical chemokine receptor 3). in this study, we aimed to investigate the expression of CXCR7 in leukemic cells, as well as its participation in CXCL12 response. Interesting, we clearly demonstrated that CXCR7 is highly expressed in acute lymphoid leukemic cells compared with myeloid or normal hematopoietic cells and that CXCR7 contributed to T-acute lymphoid leukemic cell migration induced by CXCL12. Moreover, we showed that the cellular location of CXCR7 varied among T-lymphoid cells and this finding may be related to their migration capacity. Finally, we hypothesized that CXCR7 potentiates CXCR4 response and may contribute to the maintenance of leukemia by initiating cell recruitment to bone marrow niches that were once occupied by normal hematopoietic stem cells.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)INCT-SangueUniv Estadual Campinas, Ctr Hematol & Hemoterapia, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, São Paulo, BrazilWeb of Scienc

Gene expression profiles of CD34+ and stromal cells from patients with myelodysplastic syndrome

Orientador: Sara Teresinha Olalla SaadTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: As síndromes mielodisplásicas (SMDs) constituem um grupo heterogêneo de desordens hematopoéticas, caracterizadas por exibirem hematopoese ineficaz com evidências de displasia da medula óssea resultando em citopenias no sangue periférico. Tecnologia de microarranjo tem permitido o refinado mapeamento da atividade transcricional do genoma humano. RNAs não codificadores (ncRNAs) transcritos de regiões intrônicas de genes conhecidos estão envolvidos em vários processos relacionados com controle transcricional e pós-transcricional da expressão gênica, interações com cromatinas, modificação de histonas e também estão se tornando evidentes em vários tipos de cânceres. Caracterização de ncRNAs em células progenitoras e células estromais de pacientes com SMD representa uma estratégia aparentemente importante para o entendimento da regulação gênica nesta doença. Neste estudo, o perfil de expressão gênica de células CD34+ e estromais de pacientes com SMD do subgrupo anemia refratária com sideroblastos em anel (ARSA) foi comparado com o de indivíduos saudáveis, usando oligoarranjos de 44 kilobases contendo íntrons e éxons, o qual incluiu sequências para genes codificadores, RNAs sense e antisense totalmente e parcialmente intrônicos. Em células CD34+ de pacientes com SMD-ARSA, 216 genes foram diferencialmente expressos (q-value ? 0,01) em comparação com indivíduos saudáveis, dos quais 65 (30%) eram transcritos não codificadores. O gene DMT1, um transportador de ferro, foi encontrado hiperexpresso em células CD34+ de SMD-ARSA. Em medula óssea total de 34 pacientes, a expressão foi mais evidente no subgrupo de pacientes com SMD de baixo risco/INT-1. Ensaios de imuno-histoquímica corroboram os dados encontrados na análise de expressão gênica e demonstram que DMT1 se encontra mais expresso nas células eritroblásticas. A hiperexpressão de DMT1 pode estar relacionada com o homeostase do ferro anormal nas SMDs. Em células estromais de SMD-ARSA, 12 genes foram diferencialmente expressos (q-value ? 0,05) em comparação com indivíduos saudáveis, dos quais 3 (25%) eram transcritos não codificadores. O gene SEMA3A, um membro secretado da família das semaforinas, foi encontrado hiperexpresso em células estromais de SMD-ARSA e na medula óssea total de 34 pacientes; sua hiperexpressão foi mais evidente em pacientes com SMD de alto risco/INT-2 e em pacientes com leucemia mieloide aguda (n=19). Ensaios funcionais demonstraram que SEMA3A está envolvido com aumento da adesão, diminuição da diferenciação e apoptose de células leucêmicas cocultivadas com células estromais HS27 hiperexpressando SEMA3A e age de maneira parácrina sobre as células precursoras. Pela primeira vez, o perfil diferencial de ncRNA em células CD34+ e células estromais entre SMD-ARSA e indivíduos saudáveis foi demonstrado, sugerindo que ncRNA pode ter um importante papel durante o desenvolvimento das síndromes mielodisplásicasAbstract: Myelodysplastic syndromes (MDS) are a group heterogeneous of hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Noncoding-RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and chromatins interaction, and histone modification and they are becoming evident in several cancers. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. In this study, gene expression profiles of CD34+ and stromal cells of MDS patients with refractory anemia with ringed sideroblasts (RARS) subgroup were compared those of healthy individuals, using 44 kilobases combined introns and exons oligoarrays, which included probes for protein-coding genes, for sense and antisense strands of totally and partially intronic noncoding RNAs. In CD34+ cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value ? 0.01) in comparison to healthy individuals, of which 65 (30%) were noncoding transcripts. The DMT1 gene, an iron-transporter, was found up-regulated in CD34+ cells of MDS-RARS. In the total bone marrow of 34 patients, the expression of DMT1 was more evident in the subgroup of low risk/INT-1 MDS patients. The immunohistochemistry assay confirms the data obtained in the gene expression assay and show that DMT1 is more expressed in erythroid cells. The higher expression of DMT1 can be related with abnormal iron homeostasis in MDS. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value ? 0.05) in comparison to healthy individuals, of which 3 (25%) were noncoding transcripts. The SEMA3A gene, a secreted member of the semaphorins family, was found up-regulated in stromal cells of MDS-RARS and in the total bone marrow of 34 patients; further, the higher expression was more evident in high risk/ INT-2 subgroup of MDS patients and acute myeloid leukemia patients (n = 19). Functional assays demonstrated that SEMA3A is related to adhesion increase, differentiation decrease, and apoptosis of leukemia cells cocultivated with HS27 stromal cells higher expressing SEMA3A, also acting in a paracrine fashion in the precursors cells. These results demonstrated, for the first time, in CD34+ cells and stromal cells the differential ncRNA expression profile between MDSRARS and healthy individuals, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromesDoutoradoMedicina ExperimentalDoutor em Fisiopatologia Medic

Band 3tambaú: A De Novo Mutation In The Ae1 Gene Associated With Hereditary Spherocytosis. Implications For Anion Exchange And Insertion Into The Red Blood Cell Membrane.

Hereditary spherocytosis (HS) is attributed to red blood cell membrane protein defects, caused by mutations in ankyrin, spectrin, band 3 and protein 4.2. In this study, the presence of band 3 mutations was investigated in a patient presenting mild HS and band 3 deficiency. Using single strand conformation polymorphism analysis, a shift in exon 16 of the band 3 gene was found. DNA sequencing revealed a point mutation 2102 T>C, changing methionine at position 663 to lysine. The M663K substitution was not found in either the parents or in the siblings, and the restriction fragment length polymorphism analysis of 100 alleles from a random Brazilian population did not reveal this mutation, suggesting that this gene defect is more likely to be a de novo mutation, causing HS. Flow cytometry of eosin-5-isothiocyanate (EITC)-labelled erythrocytes showed, in the patient, 54% of band 3 protein content vs. 78% based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, suggesting that flow cytometry is a more sensitive method and may be used as a diagnostic tool in membrane disorders related to band 3 deficiency. The characterisation of novel AE1 mutations is helpful to improve the understanding of the role of band 3 protein in cell physiology.74396-40

Improved photodynamic action of nanoparticles loaded with indium (III) phthalocyanine on MCF-7 breast cancer cells

Indium (III) phthalocyanine (InPc) was encapsulated into nanoparticles of PEGylated poly (D,L-lactide-co-glycolide) (PLGA-PEG) to improve the photobiological activity of the photosensitizer. the efficacy of nanoparticles loaded with InPc and their cellular uptake was investigated with MCF-7 breast tumor cells, and compared with the free InPc. the influence of photosensitizer (PS) concentration (1.8-7.5 mu mol/L), incubation time (1-2 h), and laser power (10-100 mW) were studied on the photodynamic effect caused by the encapsulated and the free InPc. Nanoparticles with a size distribution ranging from 61 to 243 nm and with InPc entrapment efficiency of 72 +/- 6 % were used in the experiments. Only the photodynamic effect of encapsulated InPc was dependent on PS concentration and laser power. the InPc-loaded nanoparticles were more efficient in reducing MCF-7 cell viability than the free PS. for a light dose of 7.5 J/cm(2) and laser power of 100 mW, the effectiveness of encapsulated InPc to reduce the viability was 34 +/- 3 % while for free InPc was 60 +/- 7 %. Confocal microscopy showed that InPc-loaded nanoparticles, as well as free InPc, were found throughout the cytosol. However, the nanoparticle aggregates and the aggregates of free PS were found in the cell periphery and outside of the cell. the nanoparticles aggregates were generated due to the particles concentration used in the experiment because of the small loading of the InPc while the low solubility of InPc caused the formation of aggregates of free PS in the culture medium. the participation of singlet oxygen in the photocytotoxic effect of InPc-loaded nanoparticles was corroborated by electron paramagnetic resonance experiments, and the encapsulation of photosensitizers reduced the photobleaching of InPc.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Federal Institute of Espirito SantoFed Inst Espirito Santo, BR-29192733 Aracruz, ES, BrazilUniv Fed Espirito Santo, Hlth Sci Ctr, Biotechnol Program RENORBIO, BR-29040090 Vitoria, ES, BrazilUniversidade Federal de São Paulo, Dept Exact Sci & Earth, BR-09972270 Diadema, SP, BrazilUniv Estadual Campinas, Dept Cellular Biol, BR-13083863 Campinas, SP, BrazilUniv Fed Espirito Santo, Dept Pharmaceut Sci, BR-29040090 Vitoria, ES, BrazilUniv Estadual Campinas, Inst Chem, Dept Phys Chem, BR-13083970 Campinas, SP, BrazilUniversidade Federal de São Paulo, Dept Exact Sci & Earth, BR-09972270 Diadema, SP, BrazilWeb of Scienc

Hematopoietic Cell Kinase (HCK) Is Highly Expressed in Myelodysplastic Syndrome (MDS) and Regulates Proliferation, Cell Cycle Progression and Hematopoietic Differentiation

54th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH)1202

CXCR7 silencing decreases MOLT4 and Jurkat cell migration.

<p>Cell migration toward either RPMI with 0.1% BSA and RPMI or 0.1% BSA containing CXCL12 (200 ng/mL) used as negative control and chemoattractant, respectively. After 4 h, the number of migrated cells was counted and was expressed as a percentage of the input, i.e., the number of cells applied directly to the lower compartment in parallel wells. The migration of cells was normalized to 100% +/− sd of triplicates. (A) The CXCR7 silencing resulted in significant changes in MOLT4 chemotactic response (<i>P = 0.0159</i>). The inhibition of CXCR4-dependent chemotaxis by its antagonist AMD3100 (1.25 µg/mL) promoted a similar effect (<i>P = 0.0159</i>). Moreover, the silencing of CXCR7 plus the treatment with AMD3100 exhibited a synergistic effect in cell chemotactic capacity (<i>P = 0.0086</i>). (B) The same effect was observed with Jurkat cells. The CXCR7 silencing (<i>P = 0.0366</i>) or the inhibition of CXCR4-dependent chemotaxis by its antagonist AMD3100 (<i>P = 0.019</i>) reduced Jurkat chemotactic response. The simultaneous silencing of CXCR7 and treatment with AMD3100 also exhibited a synergistic effect upon cell chemotactic capacity (<i>P = 0.0191</i>); <i>Mann-Whitney</i> test.</p

Clinical characteristics of patients.

<p>RCDU indicates refractory cytopenia with unilineage dysplasia; RCMD, refractory cytopenia with multilineage dysplasia; RARS, refractory anemia with ring sideroblasts, RAEB1/2, refractory anemia with excess blasts 1/2.</p

Flow cytometry analysis of CXCR7 expression in acute leukemia cell lines.

<p>Panel A. Myeloid cell lines. Panel B. Lymphoid cell lines. Results are presented as percent of positive cells.</p

Higher expression of CXCR7 in T-acute lymphoid leukemia lines MOLT4 and Jurkat.

<p>A) Western blot analysis of CXCR7 protein levels in myeloid (U937, P39, K562 and KG-1), B-lymphoid (Daudi, Raji) and T-lymphoid (MOLT4 and Jurkat) cell lines. Total cell extracts were blotted with antibodies against CXCR7 (42 kDa), CXCR4 (42 kDa) or β-actin (42 kDa), as a control for equal sample loading, and developed with the ECL Western Blot Analysis System. CXCR7 protein was detectable in all acute leukemia cell lines; however CXCR7 was more expressed in the T-acute lymphoid cell lines MOLT4 and Jurkat when compared to other cell lines. CXCR4 proteins levels were homogeneous in all cell lines analyzed. B) Quantitative expression of <i>CXCR7</i> mRNA in leukemic cells lines. mRNA expression levels of <i>CXCR7</i> were normalized by <i>HPRT</i> and <i>GAPDH</i> endogenous control. <i>CXCR7</i> mRNA was more expressed in T-acute lymphoid cell lines MOLT4 and Jurkat when compared to other cell lines.</p