טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentZeno-Sizikov Sivan
SubjectThe Role of TSPO and ROS in Mediating Cell Death Induced
by CoCl2 and PPIX
DepartmentDepartment of Medicine
Supervisors Professor Menashe Zaaroor
Professor Emeritus Moshe Gavish
Full Thesis textFull thesis text - English Version


Abstract

This thesis was designed to understand the effect of environmental changes on the expression and function of the 18kDa Translocator Protein (TSPO).

One aspect of the study was to change cells’ environment by growing them in medium supplied with CSF (cerebrospinal fluid) instead of the commonly used serum, fetal calf serum (FCS). This change affected TSPO expression, and also caused changes in cells morphology.

TSPO has been reported to be closely associated with the mitochondrial permeability transition pore (MPTP).  It is considered that TSPO exerts pro-apoptotic functions via modulation of MPTP opening. 

Appling a more specific approach, we also affected the cells’ environment by adding cobalt chloride (CoCl2) to the medium. This also affected TSPO expression in a concentration depended way.

CoCl2, which is sometimes used as a hypoxia mimicking agent, is also known to be able to induce apoptosis. We wanted to know whether CoCl2 may induce apoptosis via the TSPO.  We used the U118MG human glioblastoma cell line. We applied the specific TSPO ligand, PK 11195, as well as TSPO knockdown with siRNA, and studied their influence on the effects of CoCl2 on cell death. Our studies show that activation of TSPO by CoCl2 application is required for ROS generation leading to cardiolipin oxidation (indication for ROS generation).

To better understand the effect of TSPO expression and function on cell death caused by ROS generation induced by CoCl2, we treated TSPO siRNA transfected cells and their Scr control cells with protoporphyrin IX (PPIX). PPIX is a putative endogenous TSPO ligand. Co is known to interact with PPIX. As it is known that PPIX interacts with the TSPO, we decided to study the role of TSPO in cell death induction by PPIX. CoCl2 treatment of cells may cause increase in PPIX levels that in turn may cause TSPO activation that eventually lead to ROS generation. We exposed TSPO siRNA transfected cells and their Scr control cells to PPIX and measured cell death as well as PPIX accumulation in whole cells.

Our study shows that ROS apparently generated due to TSPO activation can have very different effects: it may lead to cell death; it may also lead to degradation of macromolecules potentially detoxifying the cell. The main conclusion of this thesis is that changing cell environment affects TSPO expression. Those changes in TSPO expression may be part of a mechanism whereby cells deal with challenges posted by the environment.