Si-Yong Huang

Myc induced miR-144/451 contributes to the acquired imatinib resistance in chronic myelogenous leukemia cell K562

Imatinib resistance remains the big hurdle for CML therapy. Previous study reveals that c-myc is important for bcr-abl CML cell proliferation, while its role in imatinib resistance is largely unknown. In this study, we first found that c-myc expression is upregulated in imatinib resistant K562R cells, which in turn enhances the expression of miR-144/451. Knockdown of c-myc or restoration of miR-144/451 in the K562R cells sensitizes K562R cells to imatinib therapy. Our study here reveals an regulatory pathway between myc and miR-144/451 and highlights that targeting either myc or miR-144/451 might be valuable for eliminating the imatinib resistant CML cells.

Notch signaling maintains proliferation and survival of the HL60 human promyelocytic leukemia cell line and promotes the phosphorylation of the Rb protein

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.

Knockdown of SOD1 sensitizes the CD34+ CML cells to imatinib therapy

Leukemia stem cell is thought to be one of the leading causes of imatinib resistance and the resultant relapse of chronic myelogenous leukemia (CML). Eradicating the leukemia stem cells holds the promise of CML treatment. In this study, we found that the CD34+ subpopulation in the CML cell line K562 had a higher expression of SOD1 than that in the CD34 negative cells. Knockdown of SOD1 in CD34+ cells had no significant effects on cell survival and growth, while it sensitized the CD34+ cells to imatinib therapy. N-acetyl-L cysteine (NAC) blocked the pro-apoptotic effects of SOD1 knockdown, suggesting the antioxidant effects of SOD1 was essential for the resistance of CD34+ cells to imatinib therapy. In summary, our results suggest that antagonizing the enhanced endogenous antioxidant activity in leukemia stem cells sheds lights on CML therapy.

FHL1C induces apoptosis in notch1-dependent T-ALL cells through an interaction with RBP-J

BackgroundAberrantly 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 (GSIs) 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.MethodsUsing 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.ResultsIn 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.ConclusionsOur 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.