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Dansyl lysine, a new probe for assaying heat-induced cell killing and thermotolerance in vitro and in vivo.

Heat induces an increase in the fraction of cells staining with the nontoxic fluorescent membrane dye dansyl lysine (DL). The fraction of cells excluding DL can, under certain circumstances, be closely correlated to the fraction of cells surviving a heat treatment. Dansyl lysine has previously been shown to select for cholesterol-free membrane domains. We now describe the use of DL to provide rapid estimates of the kinetics of thermotolerance development and decay in vitro and in vivo. Following a 45 degree C-10-min heat shock, Chinese hamster ovary cells develop resistance to the lethal effects of a second heat treatment. This thermotolerance, as measured by both clonogenicity and resistance to DL staining, is maximal at approximately 12 h and gradually decays with a t1/2 of 2 to 3 days. DL staining also has utility in predicting the heat survival response in vivo. Radiation-induced fibrosarcomas grown in C3H mice were heated to 43 degrees C for 30 min. From 1 to 3 days later the tumors were excised and a single cell suspension was prepared. Tumor cells were then heated in vitro and assayed for survival or scored microscopically for DL staining. The two assays again yielded similar results showing maximal resistance by 19 h which decreased toward control heat sensitivity by 49 h. The evaluation of intrinsic heat sensitivity and induced thermotolerance is critical to rational treatment design in clinical hyperthermia. DL staining is rapidly quantitated by flow cytometry and can be applied to biopsy samples to provide estimates of heat sensitivity within hours.