UC San Diego

Platinum (Pt)-based chemotherapeutics are widely used, polar molecules that do not readily diffuse across the plasma membrane. Most patients develop resistance to Pt drugs. Pt resistance is not fully understood, however one common phenotype is decreased Pt accumulation inside the cells. As such, an understanding of how Pt molecules enter the cell is essential. The copper (Cu) pathway has been shown to be important for Pt to enter the cell. The overall goal of the experiments described in this dissertation was understanding the role of the main copper transporter (CTR1) in the accumulation of Pt drugs inside the cell, and understanding the domains of CTR1 required for Pt transport. This was accomplished by studying cell lines that express wild type CTR1 (CTR1⁺/⁺), cells that have a homozygous knockout of CTR1 (CTR1⁻/⁻) and cells that have been transduced to express mutant variations of CTR1, targeted to key regions of the CTR1 molecule. It was discovered that CTR1 is a key player in the accumulation of not only cisplatin (cDDP), but also carboplatin (CBDCA) and oxaliplatin (L-OHP) ; as studied by 0 second and 5 minute drug accumulation. It was also shown that the isomer transplatin is not transported by CTR1. It was demonstrated that CTR1 is a key to sensitivity to cDDP in vitro. Importantly, CTR1 was shown to be vital to cDDP response in vivo. The role of CTR1 was demonstrated by re- expressing CTR1 in the CTR1⁻/⁻ cells and a restoration of cDDP sensitivity and accumulation was seen. Building on these observations, mutant variants of CTR1 were expressed in CTR1⁻/⁻ cells. Analysis of the N-terminal region of CTR1 revealed a role in the accumulation of cDDP and in sensitization, but not in controlling down-regulation. Analysis of the CTR1 pore demonstrated that M150,154 residues play a role in controlling the influx of cDDP. Finally, it was demonstrated that the Y103 residue is important for controlling the influx of cDDP, sensitivity to the drug and the ability to down-regulate CTR1 in the presence of the drug. It was also discovered that C189 is important for CTR1 functionality for cDDP accumulation and sensitivity