Establishment and characterization of the thrombopoietin-dependent megakaryocytic cell line, UT-7/TPO

Abstract

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (EPO) for growth and survival. We investigated the effect of thrombopoietin (TPO), the ligand for the receptor encoded by c-mpl proto-oncogene, on the proliferation and differentiation of UT-7 and its sublines. We found that UT-7/GM, which is a subline of UT-7, but neither UT-7 nor UT-7/EPO, can proliferate in response to TPO. The subline, UT-7/TPO, was established from UT-7/GM by culture at lower concentrations of TPO. UT-7/TPO cells had morphologically mature megakaryocytic characteristics such as developed demarcation membrane in the cytoplasm and multinucleated appearance. This was also confirmed by the high expression of platelet factor-4 and glycoprotein IIb at the mRNA levels and by the high level of DNA content. UT-7/TPO can be maintained by TPO alone, with a doubling time of 24 hours in log growth phase. In the absence of TPO, the majority of the cells died within a few days. Thus, UT-7/TPO has an absolute dependence on TPO for growth and survival and has mature megakaryocytic features. The mRNA for c-mpl was detected in UT-7/TPO and, to a lesser degree, in UT-7/GM. The mRNA level of NF- E2 p45, reported to be an erythroid-specific transcription factor, was upregulated in UT-7/TPO, whereas it was down-regulated in the erythroid subline, UT-7/EPO. There were no significant differences in GATA-1 and GATA-2 mRNA levels among UT-7 and its sublines. Not only EPO but also TPO induced the tyrosine phosphorylation of JAK2 tyrosine kinase and STAT5-related protein. These findings indicate that UT-7/TPO would be a useful model with which to analyze the gene regulation of megakaryocytic maturation- associated proteins and to study the specific actions of TPO.

The human megakaryocytic cell line UT-7/TPO responds to platelet agonists with intracellular Ca2+elevation and P-selectin expression

“…Recently, a number of megakaryocytic cell lines were established, and it is considered that some of them have platelet-like functions and signal transduction pathways (Nagano et al, 1993;Somasundaram et al, 1997). In those cell lines, UT-7/TPO has mature megakaryocytic properties (Komatsu et al, 1996), functional GPVI signal and TX generation by arachidonic acid stimulation (Kawaguchi et al, 2010). We also confirmed that the generated TX acted on its receptor as an autocrine effect and caused [Ca 2+ ]i elevation.…”

“…UT-7/TPO, a human megakaryocytic cell line established from UT-7/GM (Komatsu et al, 1996), expresses thrombopoietin receptor and shows thrombopoietin-dependent growth and survival. The character of the cell shows megakaryocytic features such as a developed demarcation membrane in the cytoplasm, expression of megakaryocyte/platelet-specific proteins (e.g.…”

The production of tissue inhibitors of metalloproteinases (TIMPs) in megakaryopoiesis: possible role of platelet‐ and megakaryocyte‐derived TIMPs in bone marrow fibrosis

“…Cell lines. The following cell lines were used: myeloid: KG1, HL60 and NKM1; monocytoid: U937 and Nomo1; lymphoid: Raji, RPMI 8402, SKLY16, MT1, Jurkat, Molt 4, Ara10, NALM1, NALM6 and BALL-1; and erythroleukaemia and megakaryocytic leukaemia: HEL, K562, TF1 (Kitamura et al, 1989), Meg-01 (Ogura et al, 1985), HU-3 (Morgan et al, 1994), MB-02 (Morgan et al, 1991) and UT7/TPO (Komatsu et al, 1996). Myeloid, monocytoid and lymphoid cell lines were provided by Dr T. Naoe (Nagoya University School of Medicine, Branch Hospital, Nagoya, Japan) (Ichihashi et al, 1993).…”

A journey to produce platelets in vitro

“…Using this information, the Src family of kinases was targeted with the inhibitor SU6656 in the UT-7/TPO, a human megakaryoblastic leukemia cell line, 42 and in developing primary MKs. The results showed enhanced polyploidization of culture-derived MKs in the presence of SU6656.…”