Additive Effects of Anti-Leukemia Capacity by Gfi-1B- and Bcr-Abl-Specific siRNA In Advanced Chronic Myeloid Leukemic Cells

Growth factor independent-1B (Gfi-1B) is a transcription factor with six C2H2 zinc fingers and one unique N-terminal SNAG (Snail/Gfi-1) transcriptional repressor domain. The Gfi-1B gene was mapped to chromosome 9q34.13 and is so located downstream the bcr-abl translocation in chronic myeloid leukemia (CML) cells. Selective inhibition of the BCR-ABL tyrosine kinase by RNA interference has been demonstrated in leukemic cells. Therefore, we evaluated the specific bcr-abl small inferring RNA (siRNA) and gfi-1B siRNA silencing in K562 cells and in primary CML cells with myeloid blast crises.

For in vitro transfection with gfi-1B siRNA, a total of 175 pM gfi-1B siRNA, No. AF 081946 was added to 1×10E5 cells and transfection was performed as previously described by us. Sequences of siRNA directed against the BCR-ABL transcript were published previously by Scherr (Blood 2003) and transfection with 54 pM bcr-abl siRNA were preformed as described for gfi-1B siRNA. As control we used two or more non-silencing siRNA (mismatched or scrambled siRNA) from Qiagen.

Using fluorescence marked non-silencing siRNA (Qiagen) we evaluated either the transfection rate in K562 cells and in primary CML cells. At mean (± standard deviation) we found a transfection rate of 69.4 ± 5.7% in K562 cells and 45.8 ± 12.8% in primary CML cells. The bcr-abl siRNA or the combination with both siRNAs revealed the strongest effects in reducing the viability of K562 cells by 28.5 to 5.3 % (mean) compared to controls (controls were set up to 100%). The gfi-1B siRNA reduced the viability of these cells by 49.8 % after the start of transfection. To confirm that cell growth is dependent on the timing of siRNA silencing, we next investigated the time course of the cotransfection of bcr-abl siRNA and gfi-1B siRNA induced effects in K562 cells. A strong reduction of cell growth to 21.9 + 4.5% compared to the rate of spontaneous viable cells in K562 cells was observed after 24 h of siRNA transfection, which was statistically significant (p<0.0001). We further observed a strong inhibition of cell growth to 5.3 + 1.8% (p<0.0001) compared to the cell growth rate after 48 h of cotransfection with these above siRNAs. After 72 h of the cotransfection of bcr-abl siRNA and gfi-1B siRNA no further reduction of cell growth was measured in K562 cells, in fact cell growth increased to 8.6 + 3.2% compared to the spontaneous cell growth rate. The combination of both bcr-abl siRNA and gfi-1B siRNA reduced the bcr-abl mRNA level to 2 fold higher comparing the amount after bcr-abl siRNA alone (p<0.0001). Cotransfection with bcr-abl siRNA and gfi-1B siRNA resulted in an exaggerated decrease of proliferation rate to 9.9 + 1.7% compared to controls. The inhibitory efficiency of bcr-abl siRNA and gfi-1B siRNA has shown a 3-fold increasing result comparing to bcr-abl siRNA alone and a 5-fold increasing effect comparing to gfi-1B siRNA alone. Concordantly, we observed an additive effect on induction of apoptosis by cotransfection of bcr-abl siRNA and gfi-1B siRNA in K562 cells. The rate of induced apoptosis increased from 1 + 0.09 (controls set to 1.0) to 3.3 + 0.2, whereas the use of either bcr-abl siRNA alone or gfi-1B siRNA alone was again less effective. In order to verify the above anti-leukemic results, we assessed bcr-abl siRNA and gfi-1B siRNA transfection in primary cells from patients with blast crisis of CML. We found either a reduction of the bcr-abl mRNA- and of the gfi-1B mRNA amount in these advanced CML patients compared to untreated controls. Furthermore we found in primary CML cells a strongly decreased level of c-myc gene expression after cotransfection with bcr-abl siRNA and gfi-1B siRNA to 65% compared to controls.

Our data suggested that silencing by both bcr-abl siRNA and gfi-1B siRNA may be associated with an additive antileukemic activity against K562 cells and in primary CML cells in myeloid blast crises, and might be a suitable target to treated advanced CML.

No relevant conflicts of interest to declare.