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A functional analysis of protooncogene Vav's role in adult human
hematopoiesis
SM Luger, J Ratajczak, MZ Ratajczak, WI Kuczynski, RS DiPaola, W Ngo, CV Clevenger and AM Gewirtz
Department of Pathology and Laboratory Medicine, University of Pennsylvania
School of Medicine, Philadelphia 19104, USA.
The Vav protooncogene is expressed almost exclusively in hematopoietic
cells, but its role in regulating adult human hematopoietic cell
development remains uncertain. To analyze Vav function in adult blood cell
formation, we used antisense (AS) oligodeoxynucleotides (ODN) to disrupt
its expression in normal and malignant human hematopoietic cells. Bone
marrow or peripheral blood mononuclear cells (MNC) were obtained from
consenting normal donors and patients with acute or chronic myelogenous
leukemia (AML and CML, respectively) and polycythemia vera (PV). Adherent
and T-cell-depleted (A-T-) MNC or CD34+ MNC were exposed to unmodified
sense, antisense, or scrambled sequence ODN corresponding to codons 2-7 of
Vav's mRNA sequence. Cells were then assayed for Vav mRNA expression by
reverse transcription- polymerase chain reaction and Vav protein expression
by Western binding. After showing that Vav-targeted AS ODN could
specifically diminish Vav mRNA and protein expression, we assessed the
ability of Vav-deficient cells to form myeloid and erythroid colonies in
methyl- cellulose cultures. When normal CD34+ MNC were exposed to Vav AS
ODN, no effect on colony-forming unit-granulocyte-macrophage (CFU-GM) or
CFU- megakaryocyte colony formation was observed. In contrast erythroid
colony growth was inhibited by a mean +/- SD of 62% +/- 16%. In patients
with hematopoietic malignancies. Vav-targeted AS ODN inhibited CFU-GM
colony formation in a sequence-specific and dose-dependent manner in 1 of 3
AML, 13 of 17 CML, and 2 of 2 PV patients. At the highest concentration
used, the Vav AS ODN inhibited CFU-GM colony formation from 66% to 81% when
compared with control cell colony growth. Burst-forming unit-erythroid
(BFU-E) colony-formation was also assessed in 7 PV patients. The
Vav-targeted AS ODN inhibited BFU-E colony formation in all by a mean +/-
SD of 81% +/- 4%. These findings suggest that Vav function may not be
easily complemented in a significant subset of normal adult erythroid
progenitor cells and may also be necessary for myeloid progenitor cell
growth in a variety of hematopoietic malignancies.
Volume 87,
Issue 4,
pp. 1326-1334,
02/15/1996
Copyright © 1996 by The American Society of Hematology
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