Study On Relationship Between Expression Of Syk And Suppressive Effect Of AS₂O₃ To B Lymphoma Cells

Objective: Syk is a nonreceptor protein tyrosine kinase. It is widely expressed in hemopoietic cells and plays a critical role in the differentiation and activation of lymphocyte. Recent findings reveal that syk appears to be involved in the development of various malignant cancers, but the mechamism is uncertain. The experiment detected the expression of syk in various B lymphoma cells, assayed the sensitivity to AS₂O₃ of various B lymphoma cells that has different expression of syk and examined the effect of AS₂O₃ to the expression of syk to investigate the relationship between expression of syk and suppressive effect of AS₂O₃ to B lymphoma cells.Methods: 1 Detect expression of syk in various B lymphoma cells. Using immunocytochemestry staining and Western blot to detect the expression of syk protein in Raji, Ramos and Namalwa cells.The expression of syk mRNA was examined by semiquantitative RT-PCR technique.2 Use MTT assay to detect the suppressive effect of AS₂O₃ among different concentration and different time to Raji, Ramos and Namalwa cells. According to the result of MTT, choose the proper drug concentration and action time to assay the influence of syk on the suppressive effect of AS₂O₃.3 The cell cycle of Raji and Ramos cells treated with AS₂O₃ under different concentration(1μmol/L,2μmol/L,4μmol/L)was detected by flow cytometry. Expression of syk and cell cycle associated gene p21^WAF1/CIP1 mRNA and protein was assessed by semiquauntitative RT-PCR and FCM.4 Flow cytometry was used to measure apoptosis percentage of Raji and Ramos cells treated with different concentration drugs for 72h. The effects of AS₂O₃ on the expression of apoptosis associated genes bcl-2 mRNA were detected by semiquantitative RT-PCR.5 Silenced the expression of syk in Raji cells by RNAi and then assessed the effects of AS₂O₃ on the expression of cell-cycle related gene p21^WAF1/CIP1 by semiquauntitative RT-PCR and FCM after RNAi.Results: 1 Among different B Lymphoma cells, Raji and Namalwa cells express syk (514bp) mRNA, but Ramos cells lack expression of syk mRNA. Immunocytochemistry staining results: the staining of syk was positive in both Raji and Namalwa cells and the buffy stain can be seen in endochylema, but the endochylema of Ramos cells has no the buffy stain. Western blot assay results: the specific expression of syk protein can be detected in both Raji and Namalwa cells but can not be detected in Ramos cells.2 With concentration-dependent and time dependent, AS₂O₃ can obviously inhibit proliferation of Raji, Ramos and Namalwa cells. The inhibitory ratio was (78.3±3.1)%, (69.3±3.9)% and (47.6±4.6)% respectively with the concentration of 4μmol/L and 72h. For Raji cells, AS₂O₃ has more inhibitive activity (p<0.01).3 Compared to control group, the number of G0/G1 phase cells of Raji and Ramos cells increased markedly, but the number of S and G2/M phase cells decreased, after treatment with AS₂O₃ for 72h, the difference was significant(p<0.01). Expression of syk and p21^WAF1/CIP1 protein increased significantly in Raji cells after treatment with AS₂O₃ compared to control group, the difference showed significantly different(p<0.01). syk and p21^WAF1/CIP1 mRNA expression also increased obviously in Raji cells exposed to AS₂O₃, compared with control group the difference was significant (p<0.01). However, Ramos cells still lack expression of syk and p21^WAF1/CIP1 mRNA after exposure to AS₂O₃.4 The apoptosis rate of Raji and Ramos cells treated with different concentration AS₂O₃ for 72h increased significantly compared to the control group(p<0.01). Expression of bcl-2 mRNA decreased markedly in Ramos cells exposed to AS₂O₃, compared with control group the difference was significant(p<0.01). However, in Raji cells, there was no significantly difference of expression of bcl-2 mRNA between before and after treatment.5 RNAi effectively silence expression of syk mRNA and protein in Raji cells. In the Raji cells that expression of syk was silenced by RNAi, there was no significant difference in expression of p21^WAF1/CIP1 mRNA and protein between before and after treatment with different concentration AS₂O₃ (P>0.05).Conclusions: 1 Expression of syk mRNA and protein is positive in both Raji and Namalwa cells, while, which is negative in Ramos cells.2 AS₂O₃ has very strong inhibitory effect on the proliferation of Raji,Namalwa and Ramos tumor cells, especially to Raji cells in which expression of syk is positive.3 AS₂O₃ could hinder the cell cycle of Raji and Ramos cells at G0/G1 phase. AS₂O₃ can up-regulate mRNA and protein expression of syk and p21^WAF1/CIP1 in Raji cells, but it has no influence on expression of syk and p21^WAF1/CIP1 in Ramos cells. The result suggested that AS₂O₃ could block the cell cycle of Raji cells by elevating expression of p21^WAF1/CIP1.4 AS₂O₃ can induce apoptosis in both Raji and Ramos cells and down-regulate expression of bcl-2 mRNA in Ramos cells, but it has no effect on the expression of bcl-2 mRNA in Raji cells. The results suggested that AS₂O₃ maybe realize its activity by down-regulation expression of bcl-2 in Ramos cells. 5 The mechanisms of anti-tumor effect in vitro by AS₂O₃ associate with blocking cell cycle and inducing cellular apoptosis. AS₂O₃ maybe elevated expression of p21^WAF1/CIP1 in Raji cells via the expression of syk.