pSTAT5 and ERK exhibit different expression in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic progenitor cell disorders characterized by the proliferation of one or more hematopoietic lineages. The classical MPNs include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) entities. These disorders are characterized by bone marrow morphology typical for each disease, and by the presence of JAK2V617F mutation in the marrow and blood. However, JAK2V617F cannot account for the phenotypic heterogeneity of MPNs because approximately half of all cases of ET and PMF show no evidence of this molecular marker. Therefore, the search for novel markers of these diseases is necessary to improve pathomorphological and molecular diagnostics. This study aimed to investigate the changes in expression patterns of the proteins STAT5 (the signal transducers and activators of transcription 5) and ERK (extracellular signal-regulated kinase) in bone marrow trephine specimens, derived both from patients with wild-type and mutant (V617F) forms of JAK2 kinase. Furthermore, the changes in STAT5 and ERK2 gene expression levels in the same patients were also investigated. The results of our immunohistochemical, immunoblotting and RT-qPCR studies revealed at least four major unique features of three types of MPNs. These include: i) more pronounced expression of phosphoSTAT5 protein in patients with JAK2V617F mutation compared to patients with wild-type of JAK2 kinase ii) different expression pattern of pSTAT5 in the nucleus and the cytoplasm of megakaryocytes and other bone marrow cells; iii) approximately 5-fold higher expression level of STAT5a gene in PV in comparison to patients with PMF and approximately 2-fold higher than in ET patients; iv) different, intracellular expression patterns of ERK2 and ERK1/2 antigens allowed to distinguish each subtype of MPN. These abnormalities in expression patterns of STAT5 and ERK proteins and genes provide some novel molecular features of MPNs and brings us closer to explaining the pathogenesis of MPNs.

Analysis of expression of MHC class I molecules and TAP genes in malignant human cell lines.

TAP proteins (transporters associated with antigen processing) take part in the transport of oligopeptides created in proteasomes from cytoplasm into endoplasmic reticulum. In the endoplasmic reticulum those oligopeptides are bound to MHC class I molecules and transported to the cell surface. TAP proteins consist of two subunits: TAP1 and TAP2. It has been previously shown that TAP protein expression can be decreased in malignant cells, followed by reduced protein expression or complete lack of MHC class I antigens on the cell surface. The aim of the study was to characterize of MHC class I protein expression and TAP mRNA synthesis in twenty human malignant tumor cell lines. MHC class I protein expression was examined by immunohistochemistry and flow cytometry. Expression of TAP genes was studied using RT-PCR and real-time PCR. All tested cell lines expressed MHC class I molecules. Flow cytometry showed different expression of MHC class I protein in tested cell lines. Molecular analysis revealed the presence of TAP1 and TAP2 gene transcripts in all cell lines examined. Quantitative real time PCR analysis showed differences of gene expression among cell lines tested.

Flow cytometric analysis of CD55 and CD59 expression on blood cells in paroxysmal nocturnal haemoglobinuria.

PNH is a rare clonal disorder of hematopoietic stem cells, therefore all blood cells lineages are involved. The main feature is an increased sensitivity of erythrocytes to complement-mediated cell lysis due to deficiency of membrane-bound GPI (glycosylphosphatidylinositol)-anchored proteins which normally function as inhibitors of reactive hemolysis. In the present study, we performed flow cytometric analysis using monoclonal antibodies against CD55 and CD59 for the detection of PNH-type clone in the blood of 50 patients (28 females and 22 males, age range 7-67 yrs). In one patient only we found a large population (95%) of granulocytes with decreased expression of both CD55 and CD59 molecules (type I PNH) and in two others with partial loss of CD55 expression (type II PNH). The expression was determined chiefly on granulocytes which in the control group showed reliable and high expression of CD55 and CD59.