|M.Sc Student||Mohadeb Odelya|
|Subject||The Interaction between TSPO and p53 in Relation to Cell|
Death, Implication for Lung Cancer Cells
|Department||Department of Medicine||Supervisors||Professor Rafael M. Nagler|
|Professor Emeritus Moshe Gavish|
|Full Thesis text|
Previous studies in our lab have shown that the mitochondrial 18 kDa Translocator Protein (TSPO) is a prerequisite for the mitochondrial apoptosis pathway involved reactive oxygen species (ROS) generation, cardiolipin oxidation, and collapse of mitochondrial membrane potential (Δψm). In addition to the TSPO, it was shown that the tumor suppressor protein p53 interacts with the Bax, Bak and voltage-dependent anion channel (VDAC) to initiate the mitochondrial apoptosis pathway including Δψm collapse, and caspases 3 and 9 activation. Therefore, we pose the question whether p53 is part of the apoptotic mechanism induced by TSPO.
To address this question we used the pro-apoptotic agent (CoCl2), which modulates mitochondrial activity related to initiation of apoptosis by the TSPO. For our study we induced mitochondrial apoptosis by CoCl2 in H1299 - ts/p53Val135 cells which express functional p53 at 32ºC and non-functional p53 at 37ºC. In addition, we used the original H1299 - wt cells lacking p53 as control cells.
In order to detect changes in the cells viability, including apoptosis induced by the CoCl2, the following assays were performed, including Trypan blue dye exclusion and Propidium Iodide (PI) labeling. Cell proliferation kit (XTT based) and JC-1 labeling assays were also performed in order to detect changes in mitochondrial activity and mitochondrial membrane potential (ΔΨm), respectively. NAO labeling was used in order to detect mitochondrial ROS generation. Western blots and binding analysis were performed in order to detect changes in the expression and binding characteristics of TSPO.
Furthermore, we studied the effects of the TSPO specific ligands, PK 11195 and FGIN-1-27 after application of CoCl2 in the presence and absence of functional p53.
In this study we found that p53 has no effect on TSPO expression and TSPO binding characteristics. We also found that CoCl2 induced cell death and decreased mitochondrial activity including cardiolipin oxidation and ΔΨm collapse, regardless of p53. In addition, we showed that PK 11195 prevented the lethal effects of CoCl2 regardless of p53, unlike FGIN-1-27 which protected the cells from CoCl2 in the absence of p53 but failed to protect the cells in the presence of p53.
In conclusion, p53 by itself has no influence on TSPO induced apoptosis. However, p53 apparently interferes with interactions between FGIN-1-27 and the TSPO, preventing FGIN-1-27 to exert its inhibitory functions regarding cell death induction by the TSPO i.e. FGIN-1-27 sets TSPO free to exert its pro cell death function.