title: FHL1C induces apoptosis in Notch1-dependent T-ALL cells through an interaction with RBP-J.
abstract: 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 (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.
  
  
  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.
authors: Wei
authors: W
authors: Fu
authors: Kai
authors: K
authors: Wang
authors: Jun-Long
authors: JL
authors: Zhao
authors: Heng-Chao
authors: HC
authors: Yu
authors: San-Zhong
authors: SZ
authors: Li
authors: Yan
authors: Y
authors: Lin
authors: Liang
authors: L
authors: Liang
authors: Si-Yong
authors: SY
authors: Huang
authors: Department of Hematology, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, People's Republic of China. liangym@fmmu.edu.cn.
authors: Ying-Min
authors: YM
authors: Liang
authors: Hua
authors: H
authors: Han
authors: Hong-Yan
authors: HY
authors: Qin
mesh_terms: D020816:Amino Acid Motifs; D017209:Apoptosis / Q000235:genetics*; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D018450:Disease Progression; D015870:Gene Expression; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D060588:LIM Domain Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D054218:Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D051881:Receptor, Notch1 / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: C101149:FHL1 protein, human; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D009124:Muscle Proteins; C497570:RBPJ protein, human; D051881:Receptor, Notch1