title: Antisense myb inhibition of purified erythroid progenitors in development and differentiation is linked to cycling activity and expression of DNA polymerase alpha.
abstract: 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.
authors.affiliation: Department of Hematology-Oncology, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Valtieri
authors.forename: D
authors.initials: D
authors.lastname: Venturelli
authors.forename: A
authors.initials: A
authors.lastname: Caré
authors.forename: C
authors.initials: C
authors.lastname: Fossati
authors.forename: E
authors.initials: E
authors.lastname: Pelosi
authors.forename: C
authors.initials: C
authors.lastname: Labbaye
authors.forename: G
authors.initials: G
authors.lastname: Mattia
authors.forename: A M
authors.initials: AM
authors.lastname: Gewirtz
authors.forename: B
authors.initials: B
authors.lastname: Calabretta
authors.forename: C
authors.initials: C
authors.lastname: Peschle
mesh_terms: D016375:Antisense Elements (Genetics); D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015672:Erythroid Precursor Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000502:physiology; D020598:Proto-Oncogene Proteins c-myb; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D013936:Thymidine / Q000494:pharmacology; D014316:Tritium
chemical_list: D016375:Antisense Elements (Genetics); D007377:Interleukin-3; D016376:Oligonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D014316:Tritium; D012194:RNA-Directed DNA Polymerase; D004257:DNA Polymerase II; D013936:Thymidine