Antisense mybInhibition of Purified Erythroid Progenitors in Development and Differentiation Is Linked to Cycling Activity and Expression of DNA Polymerase α

These studies aimed to determine the expression and functional role of c-mybin erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic-fetal liver (FL) were treated with either c-mybantisense oligomers or 3H-thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myboligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-mybon the number of BFU-E colonies was 28.3% ± 15.8% in PB, 53.4% ± 9.3% in BM, and 68.2% ± 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited (73.2% ± 10.4% and 74.2% ± 12.7%). Using highly purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% ± 4.2% in PB, 29.4% ± 6.5% in BM, and 40.1% ± 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% ± 7.9% and 60.98% ± 6.6%, respectively. We then investigated the kinetics of c-mybmRNA level during the erythroid differentiation of highly purified adult PB and FL BFU-E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-mybmRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-mybmRNA and protein in differentiating embryonic precursors peaked 2 days earlier. In both cases, c-mybmRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-mybantisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase. Under the same experimental conditions a progressive increase of the mRNA level of DNA polymerase α was observed. These observations suggest that in early erythroid differentiation c-mybactivation is associated with the progression of progenitors into the S phase of the cell cycle, as well as to the synthesis of DNA polymerase α