| Summary: | 碩士 === 中山醫學大學 === 醫學分子毒理學研究所 === 95 === A novel mixed lineage kinase-like protein ZAK, containing a kinase domain(KD), a leucine-zipper (LZ) and a sterile-alpha motif(SAM). The molecular weight of this protein is 91 kDa and functions as MAP kinase kinase kinase(MAP3K). Overexpression of the ZAK gene could induce apoptosis of Hep3B hepatoma cells. The expression of ZAK in mammalian cells specifically leads to the activation of the JNK/SAPK pathway through MKK7. Expression of wild-type ZAK increases the cell population in the G2/M phase of the cell cycle, which may indicate G2/M phase arrest. In this research, we try to investigate the role of ZAK gene in lung cancer cells. First of all, we found that ZAK gene has higher expression levels in normal lung tissue than in lung tumor tissue by real time PCR. Next, when we used ZAK shRNA to suppress ZAK expression in A549 cells, it would increase cell growth. On the other hand, when we used wild-type ZAK gene to promote ZAK expression in H460 cells, it would decrease cell growth. We observed cell cycle by flow cytometry, the percentage of sub-G1, S and G2/M phase was decreased and the percentage of G1 phase was increased in ZAK shRNA transfected cell. The percentage of sub-G1, S and G2/M phase was increased and the percentage of G1 phase was decreased in wild-type ZAK gene transfected cell. Then, we observed the expression levels of cell cycle regulated proteins by Western blot, the expression levels of CDK4 and CDK6 were obviously decreased and the expression level of cyclin A was obviously increased in ZAK shRNA transfected cell. The expression levels of cyclin A, cyclin B1, cyclin E, CDK4, Cdc2 and p21 were dramatically elevated in wild-type ZAK gene transfected cell. Morover, we observed the activities of MAPKs by Western blot. We found that the expression levels of p-JNK and p-p38 were suppressed and the expression levels of p-ERK was increased in ZAK shRNA transfected cell. The expression levels of p-JNK and p-ERK were enhanced in wild-type ZAK gene transfected cell. Finally, the increased ability of tumor growth in ZAK shRNA transfected cell and the decreased ability of tumor growth in wild-type ZAK gene transfected cell by animal models. In conclusion, we assume that ZAK may be a tumor suppressor gene in some lung cancer cell lines.
|
|---|
