Role of bcl-2 family members in 4-hne induced apoptosis
Yılmaz, Sinem (2008) Role of bcl-2 family members in 4-hne induced apoptosis. [Thesis]
Official URL: http://192.168.1.20/record=b1276343 (Table of Contents)
In this study we have investigated the role of Bcl-2 protein family members in response to 4-HNE induced apoptosis by using endothelial cells and monocytes as models for atherosclerosis. Cell viability and Annexin-V staining experiments demonstrated that 4-HNE was cytotoxic for both human umbilical vein endothelial cells (huv-ec-c's) and human leukemic monocyte lymphoma cell line (U937 cells) and induced apoptosis in a dose dependent manner. In order to gain further insight into the apoptotic mechanism, the protein levels of Bcl-2 family members were investigated by immunoblot analysis. In 20 μM 4-HNE treated huv-ec-c's, Bcl-XL and Bax were upregulated within 4 hours of treatment, followed by a sharp decrease in the prosurvival Bcl-2 protein. Among proapoptotic members of the Bcl-2 family; Bid truncation occurred prior to Bcl-2 down-regulation. In 20μM of 4-HNE treated U937 cell line for 12 hours, Bcl-XL was downregulated at an early time point of 2 hours, then restored back to control level at the 4th hour, whereas prosurvival Bcl-2 had a sharp decrease in a time dependent manner. Contrary to Bcl-2 and BC1-XL, the protein level of Mcl-1 did not change in 12 hours time after 4-HNE treatment. The proapoptotic member of Bcl-2 family, Bax was upregulated however Bak, responsible for pore formation on the mitochondria membrane, was downregulated after the 4-HNE treatment in 2 hours time. In 10μM of 4-HNE treated U937 cell line for 24 hours, Bcl-XL down-regulation took place between 2 to 8 hours and Bcl-2 was downregulated at 2 to 12 hours then Bcl-XL and Bcl-2 protein levels were reestablished at 12 hours and 24 hours respectively. Mcl-1 and Bak protein levels did not change. Bax was also upregulated similar to high dose treatment. Protein levels of Bim and Bid were also studied by immunoblotting and were not found to change In our experimental system, several cell lines (huv-ec-c's and U937 cells) and several Bcl-2 family proteins' levels (Among prosurvival; Bcl-2, Bcl-XL and Mcl-1, among proapoptotics; Bax, Bak, Bid, Bim) were studied in response to 4-HNE treatment. Shortly, Bcl-2 proteins mainly responded in cell specific manner. Among these proteins, in huv-ec-c's BC1-XL and Bax were upregulated, Bcl-2 was downregulated. In U937 cells, Bcl-XL and Bcl-2 were downregulated at early time points, Mcl-1 level remained the same whereas Bax was upregulated. Only Bak protein responded in dose dependent manner in monocytes. In order to enlighten the role of Bak in the apoptotic mechanism, further investigations are necessary. Finally, these results indicate the involvement of mitochondrial pathway and sequential activation of Bcl-2 family proteins in high and low doses of 4-HNE induced apoptosis in two different cell lines.
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