Gene Expression Profiling and Proteomic Analysis in Erythrocytosis and Thrombocytosis (I)

Project: National Science and Technology CouncilNational Science and Technology Council Academic Grants

Project Details

Abstract

Background: Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative disorders (MPD), they are complex and heterogeneous, either clinically or biologically. PV and ET share some pathophysiologic and clinical similarities but both lack specific clonal or molecular markers. Currently, their diagnosis is based on a set of clinical criteria and made mainly by exclusion. The molecular etiology of PV and ET remains unknown, the definite goal in the investigation of PV and ET is to elucidate the molecular aberrations or cellular regulatory dysfunction responsible for these disorders. Microarray-based gene expression profiles may serve to identify genes related to pathogenesis of MPD. The evolving field of proteomics should further identify distinct protein expression patterns and/or identify differentially expressed protein for diagnostic and prognostic discrimination among subtypes of MPD. Purpose: This project will be conducted to determine the gene expressing profiling (This part of works will be done in Dr. Koeffler’s laboratory) and to find a distinct protein expression patterns for discriminating among patients with erythrocytosis and thrombocytosis. We also attempt to characterize these proteins if identified. Study design and methods: Serum/plasma and bone marrow or peripheral blood cells are obtained with informed consent from patients presenting with erythrocytosis or thrombocytosis, and cells will be further fractionated into non-adherent non-T (NANT) cells, granulocytes, T-cells, platelets, and RBC. Protein lysates from each fractionated cells and serum/plasma will be applied to SELDI compatible system (ClinProt/Magnetic beads/Bruker Daltonics) through capillary liquid chromatography and then analyzed on the MALDI-TOF-TOF mass spectrometry (MS). That will link to a high-order analytical approach using samples from well-characterized PV and ET patients to define an optimal discriminatory proteomic pattern between PV and ET (first and second year). Two-dimensional gel electrophoresis (2DE) system will be used to examine and compare the protein expression patterns in each fraction of cells obtained from PV and ET. The differentially expressed proteins will be excised and then identified by MALDI-TOF MS. We then use this model to test the blinded test samples (second year). We will also use 18O isotope labeling and 2D-HPLC-MS/MS technology (18O MudPIT) for samples with scanty materials in order to get better protein identification and relative quantitation. In the 3rd year, culture condition medium obtained from serum-free suspension culture of NANT cells will be harvested at different time points, concentrated and then subjected to proteomic analysis using SELDI-compatible TOF MS and 2DE/MALDI-TOF-TOF MS. Western blot analysis will be used to validate the identified protein and ELISA assay will be applied for screening. Finally, the results of different protein patterns will be analyzed and correlated with clinical course as well as with X-chromosome inactivation patterns for female patients, endogenous erythroid colony growth, expression level of PRV-1 gene and gene expression profiling in patients with various subtypes of MPD, with special reference on idiopathic erythrocytosis or ET patients who have a PV evolution later. Significance: Global gene expression profiling and differential protein patterns may help to refine disease classification and outcome prediction in patients presenting with erythrocytosis and thrombocytosis. The results will potentially provide insights into the biology of the MPD subgroups, which in turn, will improve the understanding of the pathogenesis of various subgroups of MPD.

Project IDs

Project ID:PC9408-1843
External Project ID:NSC94-2314-B182-043
StatusFinished
Effective start/end date01/08/0531/07/06

Keywords

  • erythrocytosis
  • thrombocytosis
  • gene expression profiling
  • proteomic analysis
  • protein profiling

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