Cellular uptake, nuclear localization and cytotoxicity of 125I-labelled DNA minor groove binding ligands in K562, human erythroleukaemia cells

Full text: Iodine-125 decays by orbital electron capture and internal conversion resulting in the emission of numerous Auger electrons which produce a highly localised radiochemical damage in the immediate vicinity of the site of decay. Given the requirement to deliver 125I to the nuclear DNA, a minor groove binding bibenzimidazole, 125I-iodoHoechst 33258 was investigated. It has been noted that this analogue may be prone to de-iodination in vitro and in vivo, given the presence of an orthoiodophenol moiety which is analogous to that in thyroxins. Therefore, an 125I -iodoHoechst analogue without the hydroxyl group was also studied. The 125I -iodoHoechst 33258 analogue was prepared by direct iodination of Hoechst 33258 and 125I iodoHoechst was prepared by demetallation of a trimethylstannyl precursor. DNA binding studies indicated that both iodo-analogues bind to calf thymus DNA, KD = 89 ± 30nM, n = 0.018 bp-1 for iodoHoechst 33258 and KD = 121 ± 31nM, n = 0.024 bp-1 for iodoHoechst. Similarly, nuclear localization following incubation with 5μM of either ligand at 37 deg C was observed in K562 cells by fluorescence microscopy. Flow cytometry was used to investigate the kinetics of drug uptake and efflux in K562 cells. The results indicated that when 106 cells were incubated with 5μM ligand at 37 deg C, the uptake reached a plateau at approximately 43 minutes for iodoHoechst 33258 and approximately 52 minutes for iodoHoechst. Ligand efflux results indicated two-phase kinetics. The initial phase which involves 50-60% of drug was characterised by a half-life time (t1/2) of 55.4 minutes for efflux of iodoHoechst 33258 and a t1/2 of 10.3 minutes for efflux of iodoHoechst, at 37 deg C. Furthermore, the results suggested that the DNA binding sites in a 106 cell/ml suspension were saturated by incubation with 3μM iodoHoechst 33258 and 5μM iodoHoechst. In the initial cytotoxicity experiments using 125I-iodoHoechst 33258, K562 cells were incubated for 1 hour at 37 deg C with three different concentrations (1nM, 5nM and 5μM) and specific activities of the drug to provide different ligand:DNA binding ratios. Following incubation the cells were washed to remove extracellular ligand and 125I -decays were accumulated at 5 deg C in the presence and absence of a radical scavenger, dimethyl sulphoxide (10% v/v). Flow cytometric analysis indicated that under these conditions there was no efflux of the ligand from the cells and thin layer chromatography indicated that the ligand was stable during accumulation of 125I -decays. The results of the survival assays indicated increased sensitivity to 125I -induced cytotoxicity and decreased protection by dimethyl sulphoxide resulting from improved nuclear delivery of the ligand at higher ligand concentrations. The cytotoxicities of 125I -iodoHoechst 33258 and 125I -iodoHoechst were compared by incubating l.5x106 K562 cells with 5nM and 5μM of either ligand with varying specific activities at 37 deg C for 20 hours. The specific activities in parallel 5nM and 5μM samples were adjusted to provide equivalent rates of 125I -decay. The results indicated a radiation dose-dependent increase in cytotoxicity. Also, an increase in toxicity was observed in 5μM samples compared to 5nM samples due to increased chemotoxicity of the unlabelled ligands. Furthermore, the results indicated an increase in cytotoxicity following incubation with 125I -iodoHoechst 33258 compared to 125I -iodoHoechst