TEL/ETV6 accelerates erythroid differentiation and inhibits megakaryocytic maturation in a human leukemia cell line UT-7/GM

Abstract

TEL/ETV6 accelerates erythroid differentiation in the murine erythroleukemia cell line. To clarify the effects of TEL on megakaryocytic maturation as well as erythroid differentiation, we chose the human leukemia cell line UT-7/GM that differentiates into the erythroid and megakaryocytic lineages by treatment with erythropoietin and thrombopoietin, respectively. Upon erythropoietin exposure, overexpressed TEL stimulated hemoglobin synthesis and accumulation of the erythroid differentiation-specific transcripts such as gamma-globin, delta-aminolevulinic acid synthase-erythroid, and erythropoietin receptor. Moreover, the glycophorin A(+)/glycoprotein IIb(-) fraction appeared more rapidly in the TEL-overexpressing cells. Interestingly, overexpression of TEL was associated with lower levels of the megakaryocytic maturation-specific glycoprotein IIb and platelet factor 4 transcripts under the treatment with thrombopoietin. Consistently, the glycophorin A(-)/glycoprotein IIb(+) fraction increased more slowly in the TEL-overexpressing cells. Finally, expression of endogenous TEL proteins in UT-7/GM cells was down-regulated following erythropoietin and thrombopoietin exposure. All these data suggest that TEL may decide the fate of human erythrocyte/megakaryocyte common progenitors to differentiate towards the erythroid lineage and against the megakaryocytic lineage.

Figure 1

Establishment of UT‐7/GM sublines overexpressing FLAG‐tagged TEL proteins. Clones T‐5 and T‐6 were obtained from UT‐7/GM cells that were transfected with pCXN2‐FLAG‐TEL and selected by G418 resistance. Clones M‐1 and M‐4 were established from UT‐7/GM cells that were transfected with the empty pCXN2 vector and selected by G418 resistance. Expression of FLAG‐tagged TEL proteins was confirmed by western analysis with anti‐FLAG antibody. An arrow indicates overexpressed FLAG‐TEL proteins.

Figure 2

TEL accelerates hemoglobin synthesis induced by treatment with erythropoietin (EPO) in the UT‐7/GM clones. The mock (M‐1 and M‐4) and TEL‐overexpressing (T‐5 and T‐6) UT‐7/GM clones were cultured in the presence of EPO (10 U/mL). Hemoglobin synthesis was evaluated by the proportions of benzidine‐positive cells and their averages in three independent experiments were indicated with standard deviations.

Figure 3

Erythroid lineage‐specific gene transcription in the UT‐7/GM clones under treatment with erythropoietin (EPO). The mock (M‐1 and M‐4) and TEL‐overexpressing (T‐5 and T‐6) UT‐7/GM clones cultured in the presence of EPO (10 U/mL) were harvested at each time point indicated (days 0, 2, 4, 7). Total mRNA was extracted and subjected to northern analysis with γ‐globin (A), ALAS‐E (B), EPO‐R (C) and GAPDH (D) probes.

Figure 4

Erythroid and megakaryocytic lineages‐specific surface antigen expression in the UT‐7/GM clones under the treatment with erythropoietin (EPO). The mock (M‐1 and M‐4) and TEL‐overexpressing (T‐5 and T‐6) UT‐7/GM clones cultured in the presence of EPO (10 U/mL) were harvested at each time point indicated (days 0, 4, 7) and subjected to flow‐cytometric analysis. GPIIb on X axis and glycophorin A on Y axis were megakaryocyte‐ and erythrocyte‐specific markers, respectively.

Figure 5

Morphology and megakaryocytic lineage‐specific gene transcription in the UT‐7/GM clones under treatment with thrombopoietin (TPO). The mock (M‐1 and M‐4) and TEL‐overexpressing (T‐5 and T‐6) UT‐7/GM clones cultured in the presence of TPO (100 ng/mL) were harvested at each time point indicated (days 0, 7, 10, 14, 28). (a) Cytospin preparations of M‐4 and T‐5 at day 28. Wright‐Giemsa staining, ×100. (b–d) Total mRNA was extracted and subjected to northern analysis with GPIIb (b), PF 4 (c) and GAPDH (d) probes. Signal ratios between day 0 and the indicated time points were quantified and presented below each lane.

Figure 6

Erythroid and megakaryocytic lineage‐specific surface antigen expression in the UT‐7/GM clones under the treatment with thrombopoietin (TPO). The mock (M‐1 and M‐4) and TEL‐overexpressing (T‐5 and T‐6) UT‐7/GM clones cultured in the presence of TPO (100 ng/mL) were harvested at each time point indicated (days 0, 4, 7, 14) and subjected to flow‐cytometric analysis. GPIIb on X axis and glycophorin A on Y axis were megakaryocyte‐ and erythrocyte‐specific markers, respectively.

Figure 7

TEL represses ultrastructural platelet peroxidase (PPO) reactions after 14 days of treatment with thrombopoietin (TPO) in the UT‐7/GM clones. The mock (M‐1 and M‐4) and TEL‐overexpressing (T‐5 and T‐6) UT‐7/GM clones were cultured in the presence of TPO (100 ng/mL) for 14 days. PPO reactions were evaluated by electron microscopic analysis.

Figure 8

Expression of endogenous TEL proteins in parental UT‐7/GM cells. (a) Expression of endogenous TEL proteins in parental UT‐7/GM cells was confirmed under the presence of GM‐CSF (1 ng/mL) by western analysis (lane 3) or immunoprecipitation assay (lane 1) with anti‐TEL antibody. Overexpressed FLAG‐tagged TEL proteins in clone T‐5 were shown in lane 4. An arrow indicates endogenous TEL or overexpressed FLAG‐tagged TEL proteins; (b,c) Parental UT‐7/GM cells cultured in the presence of erythropoietin (10 U/mL); (b) or thrombopoietin (100 ng/mL); (c) were harvested at each time point indicated (days 0, 1, 2, 3, 5, 10, 14, 21, 28). Cell lysates were extracted and subjected to western analysis with anti‐TEL antibody. Arrows indicate endogenous TEL proteins.