Abstract

Background

The activation of Notch is crucial for the growth of T-ALL cells with NOTCH1 mutations. The precise roles of Notch signaling in AML cells are not fully understood, although the growth of AML cells is diversely affected by Notch ligand stimulation as we previously reported. We also reported the effects of γ-secretase inhibitors (GSIs), which block Notch activation, on the growth of leukemia cells. However, these effects might not necessarily be due to Notch inhibition as GSIs also exhibit some off-target effects. To elucidate the roles of Notch signaling in leukemia cells, we examined the effects of small interfering RNA (siRNA)-mediated knockdown of NOTCH1 and NOTCH2on cell proliferation and down-stream signaling pathways such as mTOR signaling in T-ALL and AML cell lines.

Methods

Two T-ALL cell lines (DND-41 and KOPT-K1) and 2 AML cell lines (THP-1 and TMD7) were used in this study. The cells were transfected with siRNAs targeting NOTCH1 (siN1), NOTCH2 (siN2), or control siRNA by using the pipette tip chamber-based electroporation system. The effects of siN1 and siN2 transfection on cell proliferation and induction of apoptosis were examined using a colorimetric WST-8 assay and by observing cytospin preparations of the harvested cells, respectively. The effects of the siRNA-transfection on the mRNA and protein expression were examined by quantitative RT-PCR and immunoblotting, respectively.

Results

Transfection with siN1 and siN2 selectively suppressed the expression of Notch1 and Notch2 mRNA and protein, respectively. In T-ALL cell lines, NOTCH1 knockdown as well as NOTCH2 knockdown suppressed cell proliferation and induced apoptosis. Immunoblot analysis showed that Myc expression was downregulated in NOTCH1-knockdown cells but not affected in NOTCH2-knockdown cells. In AML cell lines, cell proliferation was not significantly affected by NOTCH siRNAs. NOTCH2 knockdown increased the level of cleaved Notch1 fragment without increasing Notch1 expression. The knockdown of NOTCH1 and NOTCH2 reduced the expression and phosphorylation of mTOR protein in THP-1 cells. To confirm this finding, we examined the effects of activation of Notch by the recombinant Notch ligands, Jagged1 and Delta1, on the expression of mTOR protein. The activation of Notch resulted in an increase in the level of the mTOR protein and its phosphorylation in THP-1 cells. Thus, siRNA-transfection and ligand stimulation of Notch showed contrasting effects. Phosphorylation of Akt, 4E-BP1, and S6K was also induced after the stimulation.

Discussion

Using siRNA-mediated knockdown experiments, we found that Notch2 signaling plays a role in the growth of T-ALL cells, independent of Myc expression. The autonomous activation of Notch signaling in AML cells had little effect on the proliferation of these cells. Notch2 protein seemed to be involved in the activation of Notch1 in AML cells. Regarding the crosstalk between Notch and mTOR signaling, it is known that the Notch-induced Hes1 protein suppresses PTEN transcription, resulting in the promotion of Akt phosphorylation. We found an alternative pathway linking Notch and mTOR signaling in THP-1 cells in which the PTEN gene is homozygously deleted. In THP-1 cells, Notch activation promotes the expression and phosphorylation of the mTOR protein and the activation of mTOR signaling. These findings would contribute to the development of effective Notch-targeted therapy against leukemia.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.