Tiam1 is involved in the regulation of bufalin-induced apoptosis in human leukemia cells

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.