TERMINAL MEGAKARYOCYTIC DIFFERENTIATION OF TF-1 CELLS IS INDUCED BY PHORBOL ESTERS AND THROMBOPOIETIN AND IS BLOCKED BY EXPRESSION OF PML RAR-ALPHA FUSION PROTEIN/

We have analyzed the differentiation program of growth factor-dependen t TF-1 erythroleukemia cells as well as clones with inducible expressi on of the APL-specific PML/RAR alpha protein. We have shown that TF-1 cells may be induced to megakaryocytic differentiation by phorbol este r (phorbol dibutyrate, PDB) addition, particularly when combined with thrombopoietin (Tpo), RT-PCR studies showed that Tpo induces Tpo recep tor (TpoR or c-mpl), whose expression was further potentiated by PDB a ddition. When the cells are induced with both PDB and Tpo erythropoiet in receptor (EpoR) expression was inhibited. in the absence of Zn2+-in duced PML/RAR alpha expression, PDB and Tpo induced megakaryocytic dif ferentiation of TF-1 MTPR clones as observed in 'wild-type' TF-I cells . Conversely, when PML/RAR alpha expression was induced by Zn2+, PDB a nd Tpo treatment of these clones caused only a reduced level of megaka ryocytic differentiation. These observations indicate that: (1) TF-1 c ells as well as other erythroleukemic cells, possess the capacity to d ifferentiate to megakaryocytic cells when grown in the presence of pro tein kinase (PKC) activators and more efficiently when combined with T po; (2) the PML/RAR alpha gene has a wide capacity to interfere with t he program of hematopoietic differentiation, including megakaryocytic differentiation. Finally, we also observed that PML/RAR alpha expressi on in TF-1 cells induces an up-modulation of interleukin-3 receptor, c -kit and c-mpl, a phenomenon which may offer these cells a growth adva ntage.