Kawazoe N, Watabe M, Masuda Y, Nakajo S, Nakaya K
Laboratory of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Oncogene. 1999 Apr 15;18(15):2413-21. doi: 10.1038/sj.onc.1202555.
Bufalin, a component of the Chinese medicine chan'su, induces apoptosis in various lines of human tumor cells, such as leukemia HL60 and U937 cells, by altering the expression of apoptosis-related genes, for example, bcl-2 and c-myc. In this study, we characterized a gene that is involved in bufalin-induced apoptosis by the differential display (DD) technique. The partial nucleotide sequence of one of the differentially expressed clones obtained after treatment with bufalin was identical to that of the human gene for Tiam1. When U937 cells were treated with 10(-7) M bufalin, expression of both Tiam1 mRNA and the protein was induced 1 h after the start of the treatment. The increase of Tiam1 mRNA was transient but the level of Tiam1 protein continued to increase at least for 6 h. In addition, the activities of Rac1 and p21-activated kinase (PAK) were also stimulated by bufalin treatment. To evaluate the role of Tiam1 in the apoptotic process, we examined the effects of the expression of sense and antisense RNA for Tiam1 in U937 cells. Apoptosis was strongly induced by bufalin in cells that expressed sense RNA for Tiam1 as compared to apoptosis in control cells treated with bufalin only. Cells expressing antisense RNA for Tiaml were significantly more resistant than the control bufalin-treated cells to induction of DNA fragmentation in response to bufalin. Moreover, sense transformants had elevated activities of PAK and c-Jun NH2-terminal kinase (JNK). These results suggest that Tiaml might play a critical role in bufalin-induced apoptosis through the activation of Rac1, PAK, and JNK pathway.
蟾酥灵是中药蟾酥的一种成分，它通过改变凋亡相关基因（如bcl-2和c-myc）的表达，诱导多种人类肿瘤细胞系（如白血病HL60和U937细胞）发生凋亡。在本研究中，我们利用差异显示（DD）技术鉴定了一个参与蟾酥灵诱导凋亡的基因。用蟾酥灵处理后获得的一个差异表达克隆的部分核苷酸序列与人类Tiam1基因的序列相同。当用10^(-7) M蟾酥灵处理U937细胞时，处理开始1小时后Tiam1 mRNA和蛋白的表达均被诱导。Tiam1 mRNA的增加是短暂的，但Tiam1蛋白的水平至少持续增加6小时。此外，蟾酥灵处理还刺激了Rac1和p21激活激酶（PAK）的活性。为了评估Tiam1在凋亡过程中的作用，我们检测了U937细胞中Tiam1正义和反义RNA表达的影响。与仅用蟾酥灵处理的对照细胞相比，在表达Tiam1正义RNA的细胞中，蟾酥灵强烈诱导凋亡。表达Tiam1反义RNA的细胞比对照蟾酥灵处理细胞对蟾酥灵诱导的DNA片段化具有显著更高的抗性。此外，正义转化体的PAK和c-Jun NH2末端激酶（JNK）活性升高。这些结果表明，Tiam1可能通过激活Rac1、PAK和JNK途径在蟾酥灵诱导的凋亡中起关键作用。
