Background Chronic myeloproliferative disorders (MPDs) are clonal haematopoietic stem cell malignancies characterised by an accumulation of mature myeloid cells in bone marrow and peripheral blood. Deregulation of the apoptotic machinery may be associated with MPD physiopathology. Aims To evaluate expression of death receptors' family members, mononuclear cell apoptosis resistance, and JAK2 allele burden. Subjects and Methods Bone marrow haematopoietic progenitor CD34 cells were separated using the Ficollhypaque protocol followed by the Miltenyi CD34 isolation kit, and peripheral blood leukocytes were separated by the Haes-Steril method. Total RNA was extracted by the Trizol method, the High Capacity Kit was used to synthesise cDNA, and real-time PCR was performed using SybrGreen in ABIPrism 7500 equipment. The results of gene expression quantification are given as 2ÀDDCt . The JAK2 V617F mutation was detected by real-time allelic discrimination PCR assay. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-hypaque protocol and cultured in the presence of apoptosis inducers. Results In CD34 cells, there was mRNA overexpression for fas, faim and c-flip in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), as well as fasl in PMF, and dr4 levels were increased in ET. In leukocytes, fas, c-flip and trail levels were increased in PV, and dr5 expression was decreased in ET. There was an association between dr5 and fasl expression and JAK2V617F mutation. PBMCs from patients with PV, ET or PMF showed resistance to apoptosis inducers. Conclusions The results indicate deregulation of apoptosis gene expression, which may be associated with MPD pathogenesis leading to accumulation of myeloid cells in MPDs.

No abstract

As previously shown, higher levels of NOTCH1 and increased NF-κB signaling is a distinctive feature of the more primitive umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), as compared to bone marrow (BM). Differences between BM and UCB cell composition also account for this fi nding. The CD133 marker defi nes a more primitive cell subset among CD34+ HSC with a proposed hemangioblast potential. To further evaluate the molecular basis related to the more primitive characteristics of UCB and CD133+ HSC, immunomagnetically purifi ed human CD34+ and CD133+ cells from BM and UCB were used on gene expression microarrays studies. UCB CD34+ cells contained a signifi cantly higher proportion of CD133+ cells than BM (70% and 40%, respectively). Cluster analysis showed that BM CD133+ cells grouped with the UCB cells (CD133+ and CD34+) rather than to BM CD34+ cells. Compared with CD34+ cells, CD133+ had a higher expression of many transcription factors (TFs). Promoter analysis on all these TF genes revealed a signifi cantly higher frequency (than expected by chance) of NF-κB-binding sites (BS), including potentially novel NF-κB targets such as RUNX1, GATA3, and USF1. Selected transcripts of TF related to primitive hematopoiesis and self-renewal, such as RUNX1, GATA3, USF1, TAL1, HOXA9, HOXB4, NOTCH1, RELB, and NFKB2 were evaluated by real-time PCR and were all signifi cantly positively correlated. Taken together, our data indicate the existence of an interconnected transcriptional network characterized by higher levels of NOTCH1, NF-κB, and other important TFs on more primitive HSC sets.

Apoptosis deregulation might have a role in the pathophysiology of polycythemia vera (PV). This study evaluated Bcl-2 molecule expression in CD34+ cells and leukocytes in 12 PV patients. Gene expression was investigated by real time PCR using SybrGreen Quantitect kit and protein expression was evaluated by western-blotting. JAK2 V617F mutation was detected according to Baxter et al (2005). CD34+ cells from PV patients presented higher levels of A1 and Mcl-1 expression (median: 22.6 and 5.2, respectively) in comparison with controls (0.9 and 0.5, p=0.004 and p=0.020); while Bcl-2 and Bcl-xL expression decreased in PV patients (0.18 and 1.19) compared with controls (1.39 and 2.01, p=0.006 and p=0.020). CD34+ cells in PV patients showed an elevated Bid expression (14.4) in comparison with healthy subjects (1.0; p=0.002). Patients' leukocytes showed an A1 augmentation (7.41, p=0.001) and a reduced expression of Bax (0.19; p=0.040) and Bad (0.2; p=0.030). There was no correlation between JAK2 V617F allele burden and molecular expression. PV patients showed alterations in Bcl-2 members' expression, which may interfere with control of apoptotic machinery and contribute to disease pathogenesis.
A desregulação da apoptose parece participar da fisiopatologia da policitemia vera (PV). Este estudo avaliou a expressão das moléculas da família Bcl-2 em células hematopoéticas CD34 + e leucócitos de 12 pacientes com PV. Foram realizados: a quantificação da expressão gênica por PCR em tempo real utilizando kit Sybrgreen Quantitect, avaliação da expressão de proteínas por western-blot e detecção da mutação JAK2 V617F segundo Baxter et al. (2005). Células CD34 + dos pacientes com PV apresentaram maior expressão de A1 e Mcl-1 (mediana: 22,6 e 5,2, respectivamente) em comparação com controles (0,9 e 0,5, p = 0,004 e p = 0,020) e expressão de Bcl-2 e Bcl-xL diminuída nestes pacientes (0,18 e 1,19) em relação aos controles (1,39 e 2,01, p = 0,006 e p = 0,020). Células CD34 + dos pacientes com PV mostraram expressão elevada de bid (14,4) em comparação aos controles (1,0; p = 0,002). Leucócitos dos pacientes mostraram aumento de A1 (7,41, p = 0,001) e expressão reduzida do Bax (0,19; p = 0,04) e Bad (0,2; p = 0,030). Não houve correlação entre percentagem de alelos JAK2 V617F mutados e expressão molecular. Pacientes com PV apresentaram alterações na expressão de moléculas Bcl-2 que podem interferir no controle da apoptose e contribuir para a patogênese da doença