TY - JOUR
T1 - A functional analysis of protooncogene Vav's role in adult human hematopoiesis
AU - Luger, Selina M.
AU - Ratajczak, Janina
AU - Ratajczak, Mariusz Z.
AU - Kuczynski, Wojech I.
AU - DiPaola, Robert S.
AU - Ngo, Winnie
AU - Clevenger, Charles V.
AU - Gewirtz, Alan M.
PY - 1996/2/15
Y1 - 1996/2/15
N2 - 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 ray protein expression by Western blotting. 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 methylcellulose 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.
AB - 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 ray protein expression by Western blotting. 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 methylcellulose 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.
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U2 - 10.1182/blood.v87.4.1326.bloodjournal8741326
DO - 10.1182/blood.v87.4.1326.bloodjournal8741326
M3 - Article
C2 - 8608221
AN - SCOPUS:0030049003
SN - 0006-4971
VL - 87
SP - 1326
EP - 1334
JO - Blood
JF - Blood
IS - 4
ER -