Effective treatment of leukemic cell lines with wt1 siRNA.

The expression of wt1 and bcl-2 is considered to have a proliferating and survival supporting effect in leukemia blast cells. Here we describe the use of siRNA against wt1 and bcl-2 in leukemic cell lines for successful growth inhibition. We have used two different sequences designated as siRNA-A and siRNA-B corresponding to positions within the wt1 coding sequence to downregulate wt1 and a commercially available siRNA kit to downregulate bcl-2. WT1 and bcl-2 gene expression in transfected leukemic cell lines were evaluated with RT-PCR and western blot analyses. MTT assay was used to measure the cell viability and flow cytometry using annexin V/PI-staining for apoptosis. K562 and HL-60 cell lines transfected with siRNA-A targeted to wt1 had greatly decreased levels of both wt1 mRNA and protein expression. In contrast, siRNA-B and control siRNA led almost to no effect on wt1 mRNA and protein expression. siRNA-A-reduced wt1 mRNA expression was associated with a decreased cell proliferation and increased number of apoptotic cells in K562 and HL-60 cells by 24 and 48 h after transfection. Combined treatment with wt1 siRNA and bcl-2 siRNA simultaneously was not able to override the cell growth and apoptosis effects compared to single treatment with wt1 siRNA. siRNAs targeted against human wt1 might be a valuable tool as antiproliferative agent against wt1 expressing leukemic cells.