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 then pelleted, rapidly frozen in liquid ethane and cryosections cut to a thickness of 100 nm. Sections were picked up on copper grids and stored under liquid nitrogen prior to analysis. Specimens were cryo-transferred into a FEI CM120 transmission electron microscope or a VG Microscopes HB501 STEM equipped with energy-dispersive x-ray spectrometers for electron probe x-ray microanalysis. Cells prepared in this way were found to have normal Na/K gradients across the plasma membrane, even after cisplatin treatment. In sensitive cells, platinum was detected in membrane-bound structures within the cytoplasm, but no platinum was detected in resistant cells. Specimens have being sent to the Advanced Photon Source at Argonne National Laboratory for elemental mapping with the synchrotron-based x-ray microprobe at higher sensitivity. In addition, experiments are being performed on high-pressure frozen, sensitive and resistant cells to search for differences in ultrastructure between resistant and sensitive cells and to correlate these with platinum uptake.
