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. 2013 Apr 11;13(1):34.
doi: 10.1186/1475-2867-13-34.

Notch1 signaling is involved in regulating Foxp3 expression in T-ALL

Xiaodan Luo  1 , Huo Tan, Yueqiao Zhou, Tiantian Xiao, Chunyan Wang, Yangqiu Li
Affiliations

Notch1 signaling is involved in regulating Foxp3 expression in T-ALL

Xiaodan Luo et al. Cancer Cell Int. .

Abstract

Background: T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy. Immune tolerance induced by CD4+CD25+ regulatory T cells (Tregs) with high expression of Foxp3 is an important hypothesis for poor therapy response. Notch1 signaling is thought to be involved in the pathogenesis of this disease. Crosstalk between Notch and Foxp3+Tregs induced immune tolerance is unknown in T-ALL. We studied Foxp3 and Notch1 expression in vivo and in vitro, and analyzed the biological characteristics of T-ALL cell line systematically after Notch inhibition and explored the crosstalk between Notch signaling and Foxp3 expression.

Methods: In vivo, we established T-ALL murine model by Jurkat cells transplantation to severe combined immunodeficiency (SCID) mice. Notch1 and Foxp3 expression was detected. In vitro, we used γ-secretase inhibitor N-S-phenyl-glycine-t-butyl ester (DAPT) to block Notch1 signaling in Jurkat cells. Notch1, Hes-1 and Foxp3 genes and protein expression were detected by PCR and western blotting, respectively. The proliferation pattern, cell cycle and viability of Jurkat cells after DAPT treatment were studied. Protein expression of Notch1 target genes including NF-κB, p-ERK1/2 and STAT1 were determined.

Results: We show that engraftment of Jurkat cells in SCID mice occurred in 8 of 10 samples (80%), producing disseminated human neoplastic lymphocytes in PB, bone marrow or infiltrated organs. Notch1 and Foxp3 expression were higher in T-ALL mice than normal mice. In vitro, Jurkat cells expressed Notch1 and more Foxp3 than normal peripheral blood mononuclear cells (PBMCs) in both mRNA and protein levels. Blocking Notch1 signal by DAPT inhibited the proliferation of Jurkat cells and induced G0/G1 phase cell cycle arrest and apoptosis. Foxp3 as well as p-ERK1/2, STAT1 and NF-κB expression was down regulated after DAPT treatment.

Conclusions: These findings indicate that regulation of Foxp3 expression does involve Notch signaling, and they may cooperatively regulate T cell proliferation in T-ALL.

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Figures

Figure 1
Figure 1
Morphology of cells of bone marrow smear and liver. Morphology of cells of bone marrow was studied under microscopy (Wright-Giemsa, ×1000) and liver was inspected for signs of leukemic infiltration (HE, ×400). Neoplastic cells were found in T-ALL group.
Figure 2
Figure 2
Foxp3 gene expression. We assessed Notch1 and Foxp3 expression in PB in T-ALL mice and the control by RT-PCR. Both Notch1 and Foxp3 were detected in T-ALL group, while in the control group, Notch1 was not detected. The expression of Foxp3 in T-ALL group was significantly higher than the control group (P < 0.05).
Figure 3
Figure 3
Notch1 and Foxp3 protein expression in tumor tissues. Tumor tissues in T-ALL mice were collected immunohistochemical assay. Samples were treated with rabbit polyclonal anti-Notch1 and anti-Foxp3. Both Notch1 and Foxp3 protein were detected in T-ALL mice and Foxp3 protein was detected mostly around tumor tissues. Image-pro plus was used to evaluate the expressions of Notch1 and Foxp3 through immunohistochemical staining. Protein expression was measured in IOD. Notch1 and Foxp3 protein expression in T-ALL mice were significantly higher than the control (P < 0.05).
Figure 4
Figure 4
Foxp3 expression in Jurkat cells and PBMCs. The expression of Foxp3 in Jurkat cells and PBMCs was analyzed by flow cytometry. Foxp3-expressing jurkat cells were much more than Foxp3-expressing PBMCs (P < 0.05).
Figure 5
Figure 5
Jurkat cell viability after DAPT treatment. Jurkat cell viability assay was performed by CCK8 method and the inhibition rate was analyzed. Jurkat cells were treated with increasing concentrations of DAPT (1, 2.5, 5, 10, 20 μM). The proliferation rate of Jurkat cell increased as the concentration of DAPT increased after 24 and 48 hours stimulation, especially the 48-hour time point. The inhibition was in a concentration-dependent manner with the greatest effect observed at 20 μM DAPT.
Figure 6
Figure 6
Jurkat cells cycle after DAPT treatment. The effect of DAPT on cell cycle was determined by flow cytometric analysis. Jurkat cells were treated with increasing concentrations of DAPT (1, 5, 10, 20 μM) for 48 hours. The percentage of Jurkat cells in the sub-G0/G1 phase increased significantly while in S and G2/M phase decreased compared to the cells alone and DMSO control (P < 0.05).
Figure 7
Figure 7
Apoptosis rate of Jurkat cells after DAPT treatment. The effects of DAPT on apoptosis were analyzed by annexin V/PI staining. Jurkat cells were treated with increasing concentrations of DAPT (1, 5, 10, 20 μM) and the results showed that the early and late apoptosis rate increased as DAPT concentrations increased compared to the cells alone and DMSO control (P < 0.05).
Figure 8
Figure 8
Expression of Hes1, Notch1-Cleaved and Hes-1 protein after DAPT treatment. A: Real-Time PCR was used to assess Hes-1 gene expression. Hes1 was down regulated in Jurkat cells treated with 10 μM DAPT for 24, 48 and 72 hours, especially for 48 hours. B: At 48 hours time point, DAPT had the greatest effect on Hes1 expression when its concentrations were 10 μM. C: Notch1-Cleaved and Hes-1 protein expression was assessed by western blot. At 48-hours treatment with 10 μM DAPT, Notch1-Cleaved and Hes-1 protein expression was lower than the control group (P < 0.05).
Figure 9
Figure 9
Foxp3 gene expression after DAPT treatment. A: Foxp3 gene expression was down regulated as the concentrations of DAPT increased compared to the control. DAPT had the greatest effect on Foxp3 expression when DAPT was 10 μM. B: Jurkat cells were treated with DPAT at 10 μM for 24, 48 and 72 hours and DAPT had the greatest effect at 48-hours time point. After 72 hours, Foxp3 expression was up regulated.
Figure 10
Figure 10
p-ERK1/2, STAT1 and NF-κB protein expression. p-ERK1/2, STAT1 and NF-κB protein expression assessed by western blot. Similar to Foxp3 protein expression, p-ERK1/2, STAT1 and NF-κB protein expression was down regulated when Jurkat cells were treated with 10 μM DAPT for 48 hours.
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References

    1. Ferrando A. The role of NOTCH1 signaling in T-ALL. Hematology Am Soc Hematol Educ Program. 2009;2009:353–361. doi: 10.1182/asheducation-2009.1.353. - DOI - PMC - PubMed
    1. Palomero T, Ferrando A. Therapeutic targeting of NOTCH1 signaling in T-ALL. Clin Lymphoma Myeloma. 2009;9(Suppl 3):S205–S210. - PMC - PubMed
    1. Aster JC, Blacklow SC, Pear WS. Notch signalling in T-cell lymphoblastic leukaemia/lymphoma and other haematological malignancies. J Pathol. 2011;223:262–273. - PMC - PubMed
    1. Real PJ, Ferrando AA. NOTCH inhibition and glucocorticoid therapy in T-cell acute lymphoblastic leukemia. Leukemia. 2009;23:1374–1377. doi: 10.1038/leu.2009.75. - DOI - PMC - PubMed
    1. Trioche P, Nelken B, Michel G, Pellier I, Petit A, Bertrand Y, Rohrlich P, Schmitt C, Sirvent N, Boutard P, Margueritte G, Pautard B, Ducassou S, Plantaz D, Robert A, Thomas C, Desseaux K, Chevret S, Baruchel A. French “real life” experience of clofarabine in children with refractory or relapsed acute lymphoblastic leukaemia. Exp Hematol Oncol. 2012;1:39. doi: 10.1186/2162-3619-1-39. - DOI - PMC - PubMed