Oligodeoxynucleotides antisense to c-abl specifically inhibit entry into
S-phase of CD34+ hematopoietic cells and their differentiation to
granulocyte-macrophage progenitors
V Rosti, G Bergamaschi, C Lucotti, M Danova, C Carlo-Stella, F Locatelli, L Tonon, G Mazzini and M Cazzola
Department of Internal Medicine, University of Pavia, Italy.
A number of experimental observations suggest that the proto-oncogene c-
abl participates in the regulation of hematopoietic cell growth. We used an
antisense strategy to study the relationship between c-abl expression and
hematopoietic cell proliferation and differentiation. Purified normal human
bone marrow-derived CD34+ cells were obtained by immunomagnetic selection
and incubated with 18-base-unmodified antisense oligodeoxynucleotides
complementary to the first six codons of the two alternative first exons of
c-abl, la and lb. At the end of incubation, an aliquot of cells was assayed
for clonogenic growth and the remainder was used for flow cytometric
analyses. Cell kinetics were evaluated by means of both single parameter
DNA and bivariate DNA/bromodeoxyuridine (BrdU) flow cytometry. Apoptosis
was routinely studied by DNA flow cytometric analysis and, in some cases,
also through DNA agarose gel electrophoresis for detection of
oligonucleosomal DNA fragments. Expression of differentiation markers was
studied by flow cytometry. Exposure to antisense oligonucleotides
specifically inhibited the accumulation of c-abl mRNA in CD34+ cells.
Preincubation with the c-abl antisense oligomers reduced the proportion of
cells in S-phase from 19% +/- 5% (mean +/- SD) to 7% +/- 4% (P < .05),
and BrdU labeling from 13% +/- 6% to 6% +/- 3% (P < .05). Flow cytometry
and DNA agarose gel electrophoresis showed that treated CD34+ cells
accumulated in the G0/G1 region of the DNA histogram with no evidence of
either differentiation or apoptosis. By contrast, both growth factor
deprivation and exposure of CD34+ cells to the tyrosine kinase inhibitor
tyrphostin AG82 clearly induced apoptosis. When cells were preincubated
with antisense oligonucleotides and then plated for evaluation of colony
formation, this resulted in a significant inhibition of colony forming unit
granulocyte-macrophage growth (from 44 +/- 15 to 22 +/- 9; P < .01) but
had no effect on burst-forming unit erythroid growth (24 +/- 11 v 21 +/-
11; P < .05). These results suggest that c-abl expression is critical
for entry of human CD34+ hematopoietic cells into S-phase and for their
differentiation to granulocyte-macrophage progenitors. They also indicate
that other tyrosine kinases besides p145c-alb are active in the prevention
of apoptosis, so that inhibition of c-abl RNA accumulation arrests CD34+
cells in G0/G1 without activating programmed death.
Volume 86,
Issue 9,
pp. 3387-3393,
11/01/1995
Copyright © 1995 by The American Society of Hematology
