Differential display of precursor mRNA was used to study potential novel genes which are associated with hematopoietic lineage commitment and explore new lineage specific markers for monitoring lineage differentiation. In vitro culture systems for erythroid, myeloid and megakaryocytic lineages were established by stimulating peripheral blood derived progenitors in the presence of the lineage specific cytokines erythropoietin (EPO), granulocyte (G-CSF) and thrombopoietin (TPO) respectively. Early precursors of three lineages were enriched and isolated by Glycophorin A, CD13 and CD61 antibodies coupled with FACS and were subjected to differential display analysis. A total of 48 combinations of oligo dT primers and arbitrary decamers primers were used. One hundred and thirty differentially expressed bands confirmed by triplicate independent sample preparations were reamplified. Ten cDNA fragments, confirmed by reverse dot blot were cloned and subjected to further analysis. Sequence of these clones showed that one clone was highly expressed in erythroid lineage and was identical to the human ferritin heavy subunit mRNA. After Northern blot analysis, three clones were further characterized. Clone 1.2, preferentially expressd in erythroid and megakarycytic lineages, showed 94% homology with mouse X16 mRNA gene thought to be involved in RNA processing. It is highly expressed in K562 erythroleukemia and Molt-4 cell lines by a factor of 3, when compared to a megakaryocytic (HL-60) or myeloid (Mega-01) cell line. It is ubiquitously expressed in all 23 tissues tested, with highest expression in thyroid, heart and spleen. Clone 112.9 is, specifically expressed in normal myeloid lineages, and is not expressed in K562, HL-60, Mega-04 and Molt-4 cell lines. Forced expression of 112.9 in these cell lines leads to a decrease in their proliferation rate. It is differentially expressed in multiple tissues, most strongly in prostate, small intestine, and colon, moderately in bone marrow and stomach, absent in other tissues. Its high expression in epithelial tissues suggests its potential role in cell proliferaion. Clone128.6, exclusively expressed in megakaryocytic lineage, is 95% homologous to multiple ESTs. It is highly expressed in K562, HL-60, Meg-01 and Molt-4 cells, more than its expression in normal lineages and all other tissues tested perhaps an indication of its unmature nature. Full lengths of the above three clones have been achieved by 5RACE and 3RACE. We expect to express these cDNA encoded protein in prokaryocytic cells and further characterize their functions.During the course of our experiments in megakaroycyte differentiation in the two-phase liquid culture system, we found unexpectedly that TPO alone stimulate erythroid cell proliferation and differentiation, demonstrated by erythroid colony formation, production of benzidine+ cells and hemoglobin synthesis. Monoclonal anti-human EPO antibody and anti-human EPO-R antibody completely abrogated the erythroid differentiative ability of TPO. This implied that binding of EPO and EPO-R is essential of erythropoiesis and the following signal transduction may be augmented by the signals emanating from TPO-c-Mpl interaction. These results establish that TPO affects not only the proliferation of erythroid progenitors but also the differentiation of erythroid progenitors to mature erythroid cells.
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