Abstract

The Wilms’ Tumor 1 (WT1) gene is highly expressed in bone marrow progenitor cells, and is downregulated during the differentiation towards mature blood cells. Several lines of evidence suggest that WT1 plays an important role in leukemia development. WT1 overexpression can be detected in more than 80% of acute leukemia’s and an inverse correlation has been found between the expression levels of WT1 and the overall survival of patients. In addition, in about 9% of AML cases and 3% of ALL cases, WT1 is mutated and the presence of these mutations may have an adverse effect on the survival of the patients. So far, little is known about the biological activities of the wildtype and the WT1-mutant proteins during hematopoiesis and the presence of different isoforms with different biological activities has hampered a clear interpretation of the results so far. In a comprehensive study, we have investigated the function of all four major WT1 isoforms in primary mouse bone marrow cells using in-vitro and in-vivo assays. In addition, we have studied for each isoform the effect of mutations on the biological activity.

In-vitro studies: 4 wildtype WT1 isoforms, 6 mutants and an empty vector control were retrovirally transduced into primary murine bone marrow cells. Subsequently, the transduced cells were FACS-sorted and used for various assays. WT1 inhibited the in-vitro colony formation (CFU-GM) by 60–95%, depending on the expressed isoform. In contrast, expression of the corresponding WT1 mutant proteins had no effect on colony formation.

To study the underlying mechanism, we cultured the WT1-transduced bone marrow cells and analyzed the cells each day for proliferation (Cell count & DNA histograms), differentiation (Mac1, Gr1) and apoptosis (Annexin V) using FACS analysis. In agreement with the colony assays, the expression of all 4 wildtype WT1 isoforms induced growth arrest and resulted in accelerated differentiation.

Target genes: To investigate which genes may be involved in the observed phenotypes, we quantitatively analyzed the expression levels of 34 putative WT1-target genes in the transduced murine bone marrow cells. Briefly, primary mouse bone marrow cells were retrovirally transduced with 4 different wildtype WT1 isoforms, 4 different mutant isoforms or an empty vector control. Sixteen hours after transduction, the transduced cells were FACS-sorted and RNA was extracted for quantitative real-time RT-PCR analysis. We have identified a number of putative WT1-target genes that are differentially regulated by the 4 wildtype WT1 isoforms but not by the WT1 mutant proteins: E-cadherin, syndecan, NGF-receptor, Egr-1, TGF-b, c-Myc, Vitamin D-receptor, insulin-receptor thrombospondin and the taurine transporter.

In-vivo studies: To study the effect of WT1 on more immature bone marrow stem cells, we have transplanted WT1-transduced primary mouse CD45.2 bone marrow cells together with empty-vector-transduced primary mouse CD45.1 bone marrow cells into ablatively irradiated syngenic CD45.1/CD45.2 heterozygous mice. Six weeks after transplantation, 5-colour FACS analysis of peripheral blood indicated that the expression of WT1 promotes myeloid differentiation (Mac1 & Gr1) and inhibits the formation of B- (IgM/B220) and T-cells (CD4 & CD8).

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