Radiation sensitivity of tumour cells stained in vitro or in vivo with the bisbenzimide fluorochrome Hoechst 33342

Abstract

The DNA-binding bisbenzimide fluorochrome Hoechst 33342 is being used routinely in radiobiological studies to assess cell kinetic parameters and tumour blood flow. However, there are reports in the literature which indicate that exposure to this compound can affect the radiation sensitivity of tumour cell populations. In this investigation, it was found that staining murine tumour cells in vitro with H33342 at concentrations > 0.1 .mu.M before irradiation resulted in radioprotection. The protection factor calculated for fibrosarcoma cells stained with 10 .mu.M H33342 was 1.7. Varying the time between radiation treatment and exposure to the fluorochrome demonstrated that the effect rapidly changed to radiosensitisation when staining was performed subsequent to irradiation. Cells in transplanted KHT tumours were stained in vivo by intravenous administration of H33342 to determine whether the radiation sensitivity of these populations might also be modified. Flow cytometric analysis of suspensions prepared from tumours stained in this manner revealed that recovered cells exhibited a > 100-fold range in fluorescence intensities. These suspensions were irradiated in vitro and the cells were then fractionated according to fluorochrome content using cell sorting. Little evidence for a radioprotective effect was observed when these subpopulations were assessed for survival, even when tumour-bearing mice were given doses of H33342 which approached the LD50. Further analysis demonstrated that insufficient amounts of the fluorochrome were taken up by cells during in vivo staining to attain levels required for radioprotection. However, our results indicate that the amount of H33342 accumulated by cells may affect the radiation sensitivity of populations exposed to high concentrations of this fluorochrome, such as those required to achieve stoichiometric binding to DNA.