Immunocytochemical labelling of aerobic and hypoxic mammalian cells using a platinated derivative of EF5

The monoclonal antibody ELK3-51 was previously developed to detect adducts of the 2-nitroimidazole EF5. Direct immunofluorescence was used to detect adducts of EF5 or of a platinated derivative cis-[PtCl2(NH3)EF5] in SCCVII cells treated under aerobic or hypoxic conditions. Fluorescence measurements of these cells using both image and flow cytometric methods were compared, giving similar profiles. Platination significantly decreased immunofluorescence levels (approximately 4-fold less than EF5) after 3 h in hypoxia, but also increased levels after exposure in air (approximately 1.5 x) such that the hypoxic ratio decreased from approximately 50 to approximately 13. Platinated EF5 also showed significantly greater cytotoxicity than its parent in both aerobic and hypoxic cells. These results are consistent with targeting of EF5 to DNA, which was confirmed qualitatively by confocal microscopy.