The aim of this project is to elucidate the mechanism for entry of the anti-cancer drug cis-diamminedichloroplatinum II (cisplatin) into cancer cells and to determine how drug resistance affects the subcellular distribution of cisplatin. Drug-sensitive and drug-resistant liver and epidermoid carcinoma cells were incubated with cisplatin at concentrations up to 400 micromolar for periods up to 4 hours. After using trypsin to release the cells from the culture dishes and adding serum to stop the trypsin activity, the cells were allowed to recover their sodium/potassium gradient. The cell suspension was then transferred into 200-micrometer diameter plastic capillary tubes, frozen at 2000 bar in a high-pressure freezing machine, freeze-substituted in acetone containing glutaraldehyde fixative, and embedded at low temperature with Lowicryl resin to minimize elemental re-distribution. Plastic sections of thickness 200-300 nanometers were mounted on gold grids and imaged at 300 kV beam voltage in a transmission electron microscope before being sent for x-ray microprobe analysis to the Advanced Photon Source at Argonne National Laboratory. Elemental x-ray maps were correlated with ultrastructure obtained by electron microscopy. In drug-sensitive cells, we have shown that platinum accumulates in vesicular structures within the cytoplasm. Data are being analyzed to determine how this distribution changes in drug-resistant cells. Additionally, immuno electron microscopy is being performed to determine differences in localization of specific membrane proteins between sensitive and resistant cell lines.
