A Functional Analysis of Protooncogene Vav's Role in Adult Human Hematopoiesis

Abstract

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.