A mammalian cell mutant with enhanced capacity to dissociate a bis-benzimidazole dye-DNA complex.
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.
Smith
P J
PJ
MRC Clinical Oncology and Radiotherapeutics Unit, MRC Centre, Cambridge, UK.
Lacy
M
M
Debenham
P G
PG
Watson
J V
JV
D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism* / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism*; D051379:Mice; D009154:Mutation
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D004247:DNA; C017807:bisbenzimide ethoxide trihydrochloride