Search

Background Aberrantly activated Notch signaling has been found in more than 50% of patients with T-cell acute lymphoblastic leukemia (T-ALL). Current strategies that employ γ-secretase inhibitors (GSI) to target Notch activation have not been successful. Many limitations, such as non-Notch specificity, dose-limiting gastrointestinal toxicity and GSI resistance, have prompted an urgent need for more effective Notch signaling inhibitors for T-ALL treatment. Human four-and-a-half LIM domain protein 1C (FHL1C) (KyoT2 in mice) has been demonstrated to suppress Notch activation in vitro, suggesting that FHL1C may be new candidate target in T-ALL therapy. However, the role of FHL1C in T-ALL cells remained unclear. Methods Using RT-PCR, we amplified full-length human FHL1C, and constructed full-length and various truncated forms of FHL1C. Using cell transfection, flow cytometry, transmission electron microscope, real-time RT-PCR, and Western blotting, we found that overexpression of FHL1C induced apoptosis of Jurkat cells. By using a reporter assay and Annexin-V staining, the minimal functional sequence of FHL1C inhibiting RBP-J-mediated Notch transactivation and inducing cell apoptosis was identified. Using real-time PCR and Western blotting, we explored the possible molecular mechanism of FHL1C-induced apoptosis. All data were statistically analyzed with the SPSS version 12.0 software. Results In Jurkat cells derived from a Notch1-associated T-ALL cell line insensitive to GSI treatment, we observed that overexpression of FHL1C, which is down-regulated in T-ALL patients, strongly induced apoptosis. Furthermore, we verified that FHL1C-induced apoptosis depended on the RBP-J-binding motif at the C-terminus of FHL1C. Using various truncated forms of FHL1C, we found that the RBP-J-binding motif of FHL1C had almost the same effect as full-length FHL1C on the induction of apoptosis, suggesting that the minimal functional sequence in the RBP-J-binding motif of FHL1C might be a new drug candidate for T-ALL treatment. We also explored the molecular mechanism of FHL1C overexpression-induced apoptosis, which suppressed downstream target genes such as Hes1 and c-Myc and key signaling pathways such as PI3K/AKT and NF-κB of Notch signaling involved in T-ALL progression. Conclusions Our study has revealed that FHL1C overexpression induces Jurkat cell apoptosis. This finding may provide new insights in designing new Notch inhibitors based on FHL1C to treat T-ALL. [ABSTRACT FROM AUTHOR]

The Notch signaling pathway has been implicated in the development of several leukemia and lymphoma. In order to investigate the relationship between Notch signaling and acute myeloid leukemia (AML), in this study, we expressed a recombinant Notch ligand protein, the DSL domain of the human Jagged1 fused with GST (GST-Jag1). GST-Jag1 could activate Notch signaling in the human promyelocytic leukemia cell line HL60, as shown by a reporter assay and the induced expression of Notch effector gene Hes1 and Hes5. However, GST-Jag1 had no effect on the proliferation and survival of HL60 cells. HL60 cells expressed both Notch ligands and receptors, and had a potential of reciprocal stimulation of Notch signaling between cells. We, therefore, blocked Notch signaling in cultured HL60 cells using a γ-secretase inhibitor (GSI). We found that GSI inhibited the proliferation of HL60 cells significantly by blocking the cell-cycle progression in the G1 phase. Furthermore, GSI induced remarkably apoptosis of HL60 cells. These changes in GSI-treated HL60 cells correlated with the down-regulation of c-Myc and Bcl2, and the low phosphorylation of the Rb protein. These results suggested that reciprocal Notch signaling might be necessary for the proliferation and survival of AML cells, possibly through the maintenance of the expression of c-Myc and Bcl2, as well as the phosphorylation of the Rb protein. [ABSTRACT FROM AUTHOR]