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Antisense myb inhibition of purified erythroid progenitors in development
and differentiation is linked to cycling activity and expression of DNA
polymerase alpha
M Valtieri, D Venturelli, A Care, C Fossati, E Pelosi, C Labbaye, G Mattia, AM Gewirtz, B Calabretta and C Peschle
Department of Hematology-Oncology, Istituto Superiore di Sanita, Rome,
Italy.
These studies aimed to determine the expression and functional role of
c-myb in 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-myb antisense oligomers or 3H-thymidine (3H-TdR). A direct correlation
was always observed between the inhibitory effect of anti-myb oligomers and
the level of cycling activity. Thus, the inhibitory effect of antisense
c-myb on 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-myb mRNA 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-myb mRNA from day 4 through day 8 of culture and a
sharp decrease at later times, whereas the expression of c-myb mRNA and
protein in differentiation embryonic precursors peaked 2 days earlier. In
both cases, c-myb mRNA 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-myb antisense 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 alpha was
observed. These observations suggest that in early erythroid
differentiation c-myb activation is associated with the progression of
progenitors into the S phase of the cell cycle, as well as to the synthesis
of DNA polymerase alpha.
Volume 77,
Issue 6,
pp. 1181-1190,
03/15/1991
Copyright © 1991 by The American Society of Hematology
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