Microarray Analysis of Thrombopoietin Induced Lineage Commitment of Bi-Potential Cell Line HU-3.

HU-3 is a cytokine dependent cell line derived from a patient with megakaryoblastic leukemia. The cells have

1. a strong megakaryocytic phenotype when cultured in GM-CSF and

2. undergo terminal erythropoiesis when switched to stem cell factor and erythropoietin. (Morgan, 1997).

HU-3 cells are equally responsive to thrombopoietin (TPO), but no longer can be induced into the erythroid pathway nor do they advance along the megakaryocyte differentiation pathway. Microarray analysis of the bi-potential and unipotential cells using arrays with genome-wide coverage and bioinformatics software identified a panel of genes associated with the loss of erythroid potentiality and with an apparent commitment to the megakaryocytic lineage. Down-regulation of erythroid-specific genes expressed in the bi-potential cells was the predominant effect of the TPO microenvironment.

HU-3 (TPO) did not show an enhanced megakaryocyte phenotype nor increased expression of CD41 (GpIIb), CD42 (Gp Ib) CD61 (GpIIIa), CD62 (P-selectin); Gp V, Gp IX, VEGF, and PDGF; however, several megakaryocyte-linked genes were up-regulated: pro-collagen lysine, annexin A1, angiopoietin 2, beta 1 integrin, and galactin 1 among others. Interleukin 8 expression increased approximately 30-fold in the TPO cultures. AML1 (RUNX1), GATA-1, and SPi-1/PU.1 are reported to be required for megakaryocyte and erythroid maturation, but were expressed at low levels and were not up-regulated by TPO. This observation may explain the block in the ability of HU-3 to enter the maturation pathway once unipotentiality had been achieved. The HU-3 cell line is a biologically relevant in vitro system for exploring the genetic program at the onset of megakaryocyte commitment and identifying genes necessary for entry into the maturation pathway.