Lineage Shift of a Megakaryocytic-Erythroid Leukemia Cell Line by Adhesion to Fibronectin.

Acute megakaryocytic leukemia (AMgL) occurs in about 1% of AMLs and has poor prognosis in adults. Research of AMgL has been hampered by this rarity. To overcome this obstacle, the established immortal AMgL cell lines are useful materials for the study of megakaryocytic malignancies. A few cell lines with the characteristics of megakaryocytes have been established, but half of the cell lines are derived from blastic crisis of CMLs. In normal hematopoiesis, erythroblasts and megakaryocytes have a common precursor. And erythroid and megakaryocytic leukemias frequently share their phenotypes simultaneously. However, the mechanisms of lineage shift between the two remain unknown. We recently established an AMgL cell line named JAS-R. Megakaryocytic-erythroid JAS-R cells showed the phenotypic change by adhesion to culture dishes. Adherent JAS-R cells (named JAS-RAD) show megakaryocytic phenotype concomitant with erythropoietin secretion and MDR1 expression, while suspension cells (JAS-REN) show erythroid characters. Because the adhesion requires bivalent cation and is inhibited by RGDS tetrapeptide, it is presumably mediated by integrins. As for ligands, fibronectin was found to be the most powerful, using dishes coated with collagens (Type I and type IV), laminin, fibronectin and poly-D-lysine. Next we studied the phenotypic changes of JAS-REN cells along with the adhesion to fibronectin. JAS-REN cells were seeded on fibronectin-coated dishes and cultured for 48 hours. Then adherent and floating cells were harvested, separately. Cells were also treated with phorbol ester (TPA). Flow cytometry analysis disclosed that adherent cells had megakaryocytic phenotype with increased CD41a and CD61, and with decreased CD235a. The gene expression profile revealed that FLI1, RUNX1 and GFI1 were increased in fibronectin-adherent cells while GATA1, FOG1 and NFE2 were not changed. These characteristics of fibronectin-adherent cells resembled JAS-RAD. Similar results were observed in TPA-treated cells. Among these transcription factors, the induction of FLI1 was prominent. The promoter activity of FLI1 studied by the luciferase assay was higher in JAS-RAD than JAS-REN. These data suggest the substantial change of AMgL blast cells depending on the growth milieu in vivo. These changes might influence the result of chemotherapy