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(PP368) Using Hoechst 33342 to target radioactivity to the cell nucleus.
Yasui, Linda*,1, Chen, Kai2, Jones, T. Patrick1, Caldwell, James1, Guse, Diana1, Kassis, Amin2, 1 Department of Biological Sciences, DeKalb, IL, USA2 Department of Radiology, Boston, MA, USA
ABSTRACT- Hoechst 33342 (H33342) enters cells and targets DNA at AT-rich regions of the minor groove. This ability can be exploited to specifically irradiate DNA in cells when a radioactive atom (125I) is covalently attached to the molecule. The radiosynthesis of 125I-H33342 has recently been optimized, making this agent a useful tool for targeting DNA. H33342 uptake and radioprotection were examined in CHO cells. The uptake and binding to DNA were visualized by a fluorescence emission shift of the dye when bound to DNA. Considerable membrane blebbing and ruffling occurred when micromolar concentrations of H33342 was present in the cell culture medium. Within minutes after its addition, blue vesicles were apparent in the cell cytoplasm and, at 30 min, the nuclei were stained dark blue. Consequently, a 30-min incubation time was used for all subsequent experiments. H33342 was found to exhibit slight radioprotection against gamma radiation. Uptake appears to be an active process because incubation of cells with H33342 at 4oC removed its radioprotective effect. These promising initial uptake and radioprotection studies provided impetus to investigate cell killing and DNA damage induction by 125I-H33342. Cells were incubated with 125I-H33342 for 30 min at 37oC and then frozen for decay accumulation. At various time points, the cells were quickly thawed and immediately assessed for cell clonogenicity or DNA double-strand- break (dsb) damage induction using the gamma H2AX foci assay. Cells were readily killed by the decay of 125I-H33342, and the monoexponential survival curve generated had a D0 = 167 125I decays per cell. Cell death can be attributed to induction of DNA dsbs as indicated by the gamma H2AX foci assay (which also confirms the DNA targeting of 125I-H33342) where the number of foci increases linearly at low numbers of accumulated 125I decays.
Key words: Hoechst 33342, 125I, gamma H2AX foci, cell killing
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