Overexpression of Notch1 Inducing T-ALL Phenotype and Promoting Angiogenesis in Zebrafish

Aim: Abnormal activated Notch1 gene is familiar to us as a poor prognostic factor in T cell acute lymphoblastic leukemia (T-ALL). This in vivo study aimed to establish a stable line of zebrafish overexpressing the human Notch1 gene under the control of a heat stress-inducible promoter, and explore its roles in etiology of T-ALL, and in tumor-related angiogenic process.

Methods: Rag2-ICN1(intracellular domain Notch1)-RFP Plasmid was constructed and injected into the embryos Tg (fli1-EGFP) in the one cell stage to construct the double-fluorescence tracer and double transgenic zebrafish, named as Tg(ICN1:RFP/fli1:EGFP). Expression of ICN1 gene and its influence on zebrafish were assayed through fluorescence microscope, RT-PCR, western-blot, FCM, blood smear, RFQ-PCR and drug treatment.

Results: Twenty of the 867 (2.3%) mosaic F0 zebrafish embryos were identified to be the germline transgenic zebrafish, including 9 males and 11 females. FCM and peripheral blood smears suggested a significant reduction in the proportion of red blood cells and a increasing of lymphocyte ratio in transgenic zebrafish as compared to control group. RFQ-PCR shows that lymphocytic regulatory factor ikaros, rag1 and lck were significantly reduced, indicates obvious differentiation obstacle in lymphoid lineages. These results were absolutely consistent with the results of FCM and peripheral blood smear. Quercetin suppressed the proliferation activity of T-ALL cell lines and induced apoptosis in a time- and dose-dependent way, meanwhile, quercetin can inhibited Notch1, Hes1, Dll4 and VEGF gene and these proteins expression in A3 and Molt-4 cells (T-ALL cell line). Furthermore, quercetin displayed a significantly reduction in the length of ISV and SIVs in Tg(rag2-ICN1-RFP/fli1-EGFP) zebrafish).

Conclusions: The above results induced by overexpression of Notch1 gene closely resemble the main aspects of human T-ALL, suggesting that Notch1 plays a role in the etiology of AML. Some key lymphocytic regulatory factors were apparently changed, which indicates obvious differentiation obstacle in lymphoid lineages and accompanied with the increasing small lymphocytes and precursors. Quercetin inhibited the proliferation and induced apoptosis of T-ALL cell lines as well as inhibition of zebrafish angiogenesis by downregulating the expression of Dll4/Notch1 and VEGF pathways. Therefore, this Notch1 transgenic zebrafish model facilitates dissection of Notch1-mediated signaling in vivo, and enables high-throughput scale screens to identify the potential therapeutic targets.