Polycythemia vera (PV) is an acquired clonal hematopoietic disorder of stem cells characterized by erythrocytosis, granulocytosis and thrombocytosis. In PV there are normal erythropoietin (EPO)-dependent erythroblasts and a fraction of abnormal EPO-independent erythroid progenitors. The molecular targets responsible for these abnormal EPO- independent erythroid progenitors to proliferate and differentiate into mature red cells are still unclear. To identify the genes expressed differently in these two populations of erythroid cells, we used a two phase liquid erythroid culture system to culture and separate the EPO- dependent and EPO-independent erythroid cells from 12 phlebotomy-only treated PV patients. The polymerase chain reaction-based differential display method (DDRT-PCR) was used to identify mRNAs differentially expressed in these two subpopulation. Sixty-three mRNA transcripts display differential expression in EPO-dependent and independent groups. After using reverse dot blot to confirm and quantitate the intensity of these mRNA transcripts, twenty transcripts with downregulated or upregulated expression in EPO-independent erythroid cells compared with EPO-dependent group were cloned and sequenced. Eleven of these transcripts have high homology with already-known sequences, including B3 (beta-globin gene), B7 (ferritin heavy subunit mRNA), A3 ( Nrf1, NF-E2-related transcription factor), B1 (human nuc2 homologue), D1(human translationally controlled protein), and are downregulated in EPO-independent erythroid cells, with 26, 19, 18, 11, 7 fold lower expression in EPO-independent than in EPO-dependent groups, respectively. It has been reported that the CNC-basic leucine family member Nrf1 (A3) plays an essential role during fetal liver hematopoiesis; mice homozygous for the Nrf1 mutation suffer from anemia as a result of abnormal fetal liver hematopoiesis. The human homologue of yeast nuc2 (B1) has been reported to interacts with the Rb protein in a specific manner. The expression of A3, B1 and D1 in 12 PV patients was confirmed by Northern blot. The role of A3, B1 and D1 in PV is under investigation. Nine of twenty transcripts have no homology with known sequences. Three of nine unknown transcripts were upregulated in the EPO-independent group. By Northern blot analysis, their expression and size were confirmed. Their full cDNA sequence is being obtained by using a human cDNA library. Investigation of both downregulated and upregulated cDNA transcripts in EPO-independent erythroid cells may provide insights into the pathogenesis of and possible therapeutic targets in PV.
