WT1 and BCR-ABL specific small interfering RNA have additive effects in the induction of apoptosis in leukemic cells.
OBJECTIVE
The Wilms' tumor gene (WT1) is aberrantly over-expressed in leukemic cells. Therefore, we wanted to study the effect of small interfering (siRNA) targeting WT1 in leukemic cells and normal CD34-positive cells with regard to proliferation, induction of apoptosis, and cell differentiation. Furthermore, we wanted to evaluate whether the additional use of BCR-ABL siRNA could increase the anti-leukemic effects of WT1 siRNA in chronic myeloid leukemia (CML) cells.


METHODS
We measured WT1 expression by reverse transcription polymerase chain reaction (RT-PCR) in various cell lines and in leukemic cells from patients, then transfected the cells with WT1-specific and BCR-ABL-specific siRNA before carrying out microarray analysis. We used the tunnel assay to measure apoptotic cells.


RESULTS
We observed a reduction of WT1 gene expression, measured by real-time RT-PCR, in all studied cell lines: K-562, Kasumi-1, MV 4-11 and NB-4, as well as in cells of AML and CML patients. The results also demonstrated that WT1 siRNA significantly induced apoptosis and inhibited proliferation in MV4-11 cells, NB-4 cells, Kasumi-1 cells (p<0.01) and in K-562 cells (p<0.02) versus controls. In normal CD34-positive cells, the proliferation was only slightly inhibited (by about 20%) and no induction of apoptosis was found. Combined transfection with WT1 and BCR-ABL siRNA together in K-562 cells increased the inhibition of the rate of proliferation and the rate of induced apoptosis compared to transfection with BCR-ABL siRNA or WT1 siRNA alone (p<0.01). We found that most genes involved in cell signaling and protein metabolism were regulated by the WT1 gene in K-562 cells in a microarray analysis.


CONCLUSIONS
In conclusion, WT1 might be a suitable target for new therapeutic strategies using siRNAs in leukemic cells.
Elmaagacli
Ahmet H
AH
Department of Bone Marrow Transplantation, University Hospital of Essen, Hufelandstr. 55, 45122 Essen, Germany. ahmet.elmaagacli@uni-essen.de
Koldehoff
Michael
M
Peceny
Rudolf
R
Klein-Hitpass
Ludger
L
Ottinger
Helmut
H
Beelen
Dietrich W
DW
Opalka
Bertram
B
D017209:Apoptosis / Q000187:drug effects*; D004357:Drug Synergism; D016044:Fusion Proteins, bcr-abl / Q000187:drug effects / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007938:Leukemia / Q000188:drug therapy* / Q000473:pathology; D034741:RNA, Small Interfering / Q000494:pharmacology* / Q000627:therapeutic use; D014407:Tumor Cells, Cultured; D025721:WT1 Proteins / Q000187:drug effects* / Q000235:genetics
D034741:RNA, Small Interfering; D025721:WT1 Proteins; D016044:Fusion Proteins, bcr-abl