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. The enzyme trypsin was used to release the cells gently from the culture dishes. Serum was subsequently added to stop the trypsin activity and to allow the cells to recover their sodium/potassium gradient. Cells were pelleted and either rapidly frozen in liquid ethane and cryosectioned to perform elemental microanalysis, or they were transferred into 200-micrometer plastic capillary tubes, high-pressure frozen, freeze substituted, plastic-embedded and sectioned to perform morphological and immunolabeling studies. The cryosections were cryo-transferred into a 100 kV scanning transmission electron microscope equipped with a field-emission source and an energy-dispersive x-ray spectrometer for electron probe x-ray microanalysis. Physiologically normal Na/K gradients were found across the plasma membrane of cisplatin treated cells. In sensitive cells, platinum was detected in membrane-bound structures within the cytoplasm, but no platinum was detected in resistant cells. Experiments are now in progress to correlate the platinum-containing organelles with cellular morphology and the localization of specific proteins in the freeze-substituted preparations.
