Establishment and characterization of the thrombopoietin-dependent megakaryocytic cell line, UT-7/TPO.
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.
Komatsu
N
N
Division of Hematology, Department of Medicine, Jichi Medical School, Tochigi, Japan.
Kunitama
M
M
Yamada
M
M
Hagiwara
T
T
Kato
T
T
Miyazaki
H
H
Eguchi
M
M
Yamamoto
M
M
Miura
Y
Y
D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival; D004273:DNA, Neoplasm / Q000032:analysis; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D050981:Erythroid-Specific DNA-Binding Factors; D004921:Erythropoietin / Q000494:pharmacology; D050982:GATA1 Transcription Factor; D050989:GATA2 Transcription Factor; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D053614:Janus Kinase 2; D007947:Leukemia, Megakaryoblastic, Acute / Q000473:pathology*; D008533:Megakaryocytes / Q000187:drug effects* / Q000378:metabolism; D008894:Milk Proteins; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D011003:Ploidies; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D050799:STAT5 Transcription Factor; D013926:Thrombopoietin / Q000494:pharmacology*; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D050989:GATA2 Transcription Factor; C494711:GATA2 protein, human; D007377:Interleukin-3; C000715470:MAS1 protein, human; D008894:Milk Proteins; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; D009363:Neoplasm Proteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D014157:Transcription Factors; D004921:Erythropoietin; C082218:MPL protein, human; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013926:Thrombopoietin; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2