טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentHeld-Kuznetsov Viktoria
SubjectAdenine Nucleotide Transporter (ANT) Knockdoen with Short
Interfering RNA and Tumorigenic Characteristics of
The C6 Glioma Cell Line
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Moshe Gavish


Abstract

The mitochondrial PBR, which is considered to play a role in tumorigenicity, consists of the 18-kDa isoquinoline binding protein (IBP) and the 32-kDa voltage dependent anion channel (VDAC), located on the outer mitochondrial membrane; and the 30-kDa ANT located on the inner mitochondrial membrane. In human, three ANT isoforms (ANT1, ANT2, ANT3) have been identified so far, and in rat, two isoforms (ANT1, ANT2). ANT1 and ANT3 exchange cytosolic ADP for intramitochondrial ATP generated by oxidative phosphorylation. In contrast, ANT2 may exchange intramitochondrial ADP with extramitochondrial ATP resulting from glycolytic metabolism. The reverse ADP/ATP translocation by ANT2 is thought to be required to maintain mitochondrial integrity and cancer cell viability. We generated ANT2 knockdown C6 rat glioma cells with the aid of ANT2 specific short interfering RNA. ANT knockdown led to decreased in vitro tumorigenicity, as determined with anchorage independent cell proliferation, which showed a 100% abolition of the normal formation of large colonies. The ANT2 knockdown also resulted in a 50% decrease in cell proliferation rate. In addition, ANT2 knockdown led to a 70-90% reduction in the normal induction of apoptosis by the antineoplastic agent, Erucylphosphocholine. However, ANT2 knockdown did not affect expression levels of IBP and VDAC, and also binding of the PBR ligand [H3]PK 11195 remained unaffected. Thus, ANT2 appears to be required for the normal proliferation of C6 glioma cells, and also for the induction of apoptosis by at least some anticancer agents.