|M.Sc Student||Scher Keren|
|Subject||The Peripheral-Type Benzodiazepine Receptor in MCF-7 Cells|
as a Model for Breast Cancer and a Potential
Target for Gene Therapy
|Department||Department of Biotechnology||Supervisor||Professor Emeritus Moshe Gavish|
The Peripheral Type Benzodiazepine receptor (PBR) is not only widely expressed throughout the body, but it is also genetically conserved from bacteria to humans. Many functions have been attributed to it, but its primary role remains a puzzle. In the current study, we have investigated the relative importance of PBR as a regulator of apoptosis and cell proliferation in MCF-7 cells. Using the antisense knockout approach, we stably transfected cultures of MCF-7 cells with either control or 18kDa-PBR antisense knockout plasmids. The antisense knockout vector was driven by the CMV promoter, which is very effective in the human MCF-7 cell line. This construct induces 18kDa-IBP (Isoquinoline Binding Protein) antisense RNA, which should down regulate endogenous 18kDa-IBP RNA levels. As IBP is an essential subunit of the PBR, the result of this antisense knockout should be a knockdown expression of the PBR. Control and knockout MCF-7 cell lines were then compared at the level of receptor binding. On knockout, the PK 11195 treated, tritium labeled PBR ligand binding was reduced by almost 50% compared to control cells, while ligand affinity to the receptors remained unaltered. Additionally, we investigated the effect of PBR ligands and the combination of PBR ligands and Arsenic trioxide on cell viability and apoptosis using PBR knockout cells and control MCF-7 cells. PBR ligands and Arsenic trioxide reduce cell viability in both PBR knockout cells as well as the control cells. Arsenic trioxide induces apoptosis only in the control cells. On the otherhand, the combination of PBR ligands and Arsenic trioxide did not affect cell viability in both PBR knockout cells and the control cells, compared to Arsenic trioxide alone. We conclude that Arsenic trioxide works down stream to PBR ligands. We also found that Arsenic trioxide may increase PBR density in MCF-7 cells.
The PBR gene can be exploited for gene therapy. By making malignant cells over- express the PBR gene, hopefully it will be possible to achieve very selective and effective treatment for cancer.