Effect of RA on RARα2 during myelomonocytic differentiation and on the expression of RARα2 by EPO.

RA potentiates induction of RARα2 during myelomonocytic differentiation and reverses inhibition of its expression by EPO. (A) Semiquantitative RT-PCR analysis of RARα1 and α2 expression in FDCP-mixA4 cells after a 2-day treatment with the indicated GFs and/or retinoids. The recombinant GFs were used at 1000 U/mL (G-CSF) and 50 U/mL (GM-CSF). Concentration of RA was 10⁻⁶ M. G-CSF/GM-CSF indicates standard FDCP-mixA4 myelomonocytic differentiation with the use of conditioned medium.15,39Erythroid differentiation was induced by EPO (1000 U/L [1 U/mL]) and resulted in a decrease of RARα1 and RARα2 expression (lane 9) versus untreated control (lane 8). These data are consistent with the results shown in Figure 1B, which were derived from a different passage of FDCP-mixA4 cells. Addition of RA partially reversed this effect (lane 10). Glyceraldehyde phosphate dehydrogenase (GAPDH) levels were used as a control. (B) Frequency of hematopoietic cells representing different lineages and maturation stages in cultures from which RNA was isolated and used in the above RT-PCR analysis. Note that at day 2 the majority of cells in each culture retain the blast cell–line morphology characteristic of undifferentiated FDCP-mixA4 cells. Therefore, RA-mediated enhancement of the RARα2 expression precedes any morphological changes.