DAPT Affects The Notch Signaling In Burkitt’s Lymphoma Cell Line(Raji Cell)

Objective: To explore Notch signaling pathway in Raji cells; and to analyze the impact of DAPT on the survival and Notch signaling of Raji cells; and to evaluate the regulation of NF-κB signaling by Notch, and to reveal possible mechanisms of cross-talk between Notch and NF-κB signaling.Methods: Notch1 transmembrane subunit(NTM) expression and Hes1 m RNA were examined by Western blot assay and q RT-PCR respectively, with PBMC of health donors as negative controls and T-ALL cell line Jurkat cell as a positive control. Raji cells were cultured with various concentrations of DAPT for 24 h,48h and 72 h, then CCK-8 test was used to measure cell proliferation. The apoptosis and cell cycle of Raji cell after DAPT treatment were examined by Flow cytometry. NTM protein as well as the p65 and p50 proteins in nucleus and cytoplasm were detected by Western blot after 48 h treatment with DAPT. At the same time, Notch1, Hes1, c-myc, p65 and p50 m RNA expression were detected by q RT-PCR after 48 h treatment of DAPT.Results: 1. Compared with PMBC, Raji cells express more NTM protein, but the protein in both of them are lower than that in Jurkat cells. According to q RT-PCR results, Raji cells and Jurkat cells express significant amounts of Hes1 m RNA. 2. The proliferation of Raji cells are inhibited by DAPT with a concentration and time dependent manner. 3. The apoptosis of Raji cells is induced significantly by DAPT. 4. DAPT induces S phase arrest in Raji cells. 5. DAPT mediates a slight increase in NTM protein and decreases Hes1 m RNA expression of the cells. 6. The proteins of p50 and p65 in nucleus increase after DAPT treatment, and so do the c-myc m RNA of the cells.Conclusion: 1. Raji cells express high amounts of NTM protein and Hes1 m RNA. 2. DAPT inhibits the proliferation, induced apoptosis and caused S phase arrest of Raji cells. 3. The possible mechanism of the phenomenon above mentioned may be due to the decrease of Hes1 m RNA expression through inhibiting the proteolytic cleavage of NTM, and the increase of the NF-κB activ ity.