A mammalian cell mutant with enhanced capacity to dissociate a bis-benzimidazole dye-DNA complex

Abstract

The bis-benzimidazole dyes (specifically Hoechst 33258 and the more lipophilic derivative Hoechst 33342) are non-intercalating AT base pair-specific ligands which bind to cellular DNA by non-covalent association with the minor groove. The interaction of dye with cellular DNA is thought to be the principal pathway for the cytotoxic, mutagenic and DNA-damaging properties of such agents. Upon binding and near UV light excitation, dye molecules exhibit fluorescence enhancement such that dye/DNA association and dissociation in individual cells can be monitored by flow cytometry. We have studied dye uptake and the DNA--dye dissociation characteristics of a Hoechst 33258-resistant mouse cell line (HoeR415) compared to the response of the parental cell line Ltk-. HoeR415 was found to show similar levels of cross resistance (approximately 10-fold) to Hoechst 33258 and Hoechst 33342 compared to parental responses except that the more lipophilic ligand was approximately 30-fold more toxic. Estimates of Hoechst 33342 uptake using flow cytometry or radiolabelling methods indicated that resistance could not be attributed to reduced cellular uptake, low initial levels or different modes of DNA binding. Both cell lines showed similar initial levels of dye-induced DNA strand-breakage. However, Hoechst 33342 resistance did correlate with an enhanced capacity (10-fold) of HoeR415 to remove dye from cellular DNA compared with the relatively long retention (T 1/2 300 min) of ligand by the parental cell line. Our results are consistent with the view that ligand persistence rather than indirect DNA damage is a more important factor in the cytotoxicity of non-intercalating DNA-binding ligands. A model is presented of the cellular processes of DNA damage recognition and surveillance for ligands which interact with the minor groove of DNA.

Overlapping Substrate and Inhibitor Specificity of Human and Murine ABCG2

“…The Ltk-mouse cell line was developed from a subline of the BrdU-resistant strain of the L-M mouse line (Kit et al, 1963). The Ltk-drug-selected subline (Ltk-HoeR415) has been shown to have increased Abcg2 expression and function (Smith et al, 1988(Smith et al, , 2009 mice, and the MEF3.8 M32 subline were provided by Alfred H. Schinkel (Netherlands Cancer Institute). The human ABCG2-expressing sublines used (H460 MX20 and MCF7 FLV500) have been previously characterized (Robey et al, 2001b(Robey et al, , 2004.…”

Intrinsic Conformational Preferences of the Hoechst Dye Family and their Influence on DNA Binding

“…In this field, recent NMR spectroscopic studies demonstrated that moving the phenolic -OH group of Hoechst 33258 from the para to the meta position provides an additional hydrogen bond between the m -OH and DNA (Parkinson et al, 1994). Controversially, the ethoxy derivative of the phenolic -OH group of Hoechst 33258, named Hoechst 33342, has a lower affinity for DNA than the parent drug (Smith et al, 1988).…”

Kinetic analysis of intracellular Hoechst 33342—DNA interactions by flow cytometry: Misinterpretation of side population status?

“…Emission spectrum analysis using narrow band‐pass filters (centered at 400, 450, 500, 555, and 600 nm wavelengths) reveals that dye–DNA dissociation during the incubation of mammalian cells in dye‐free medium results in preferential loss of red versus violet fluorescence. The reverse spectral shift is accelerated when overall dye efflux is enhanced 10‐fold by specific overexpression of ABCG2 (19, 54). This kinetic relationship can be exploited to identify moribund cells which show an “instantaneous” and distinct red‐biased Hoechst 33342–DNA emission signature (55) due to ready access of dye to DNA binding sites.…”

“…Such an approach is useful for the FACS isolation of rare SP cells that may require extended instrument run‐times. However, an early study has shown ongoing efflux in SP‐like cells has an initial T ½ of significantly <30 min for the loss of red fluorescence, allocating non‐SP cells to an SP fraction, highlighting the need to control the potential for on‐going dye wash‐out (54).…”