DNA ligands as radioprotectors: molecular studies with Hoechst 33342 and Hoechst 33258.
Following earlier reports of radioprotection of cells by Hoechst 33342, we have investigated radioprotection of isolated DNA by the minor groove binders Hoechst 33258 and Hoechst 33342. Analysis of radiation-induced single strand breakage in plasmid DNA (pBR322) showed concentration-dependant protection, up to a dose-modifying factor of 9.3 for 25 microM Hoechst 33258, at which the ligand: bp ratio was 0.67. Since the ligands bind at discrete sites along DNA, sequencing gel analysis was used to investigate the radioprotective effects of the ligands both at and between the ligand-binding sites. These experiments showed that although protection was more pronounced at the binding sites, there was also some reduction in strand-breakage between binding sites. Detailed analysis at a particular site, the EcoR1 site in a 3'-32P-endlabelled 100bp restriction fragment from pBR322, showed that protection was most pronounced at the 'inner T': GAATTC. Irradiation of a synthetic oligodeoxynucleotide containing a single ligand-binding site, and labelled at the 5'-end, gave the expected doublet bands in high resolution gels, corresponding to fragments with 3'-phosphoryl- and 3'-phosphorylglycollate terminii. In the Hoechst 33258-protected sample, the 3'-phosphorylglycollate band was preferentially suppressed within the binding site. These results, together with published crystal structure data for a Hoechst 33258/dodecamer complex, suggest that the site-specific radioprotection may be due to H-atom donation from the benzimidazole NH groups in the ligand to radiation-induced radicals on 4'-deoxyribosyl carbons. In contrast to the experiments with purified DNA, in which the two ligands yielded similar results, Hoechst 33342 was a much more active radioprotector in experiments with intact cells. For 20 microM Hoechst 33342, the dose-modifying factor was 1.7 at 1% survival and 1.3 at 10% survival, whereas the same level of Hoechst 33258 yielded barely detectable protection, perhaps due to a demonstrably lower cellular uptake. Presumably the radioprotection of cells by Hoechst 33342 is due to suppression of DNA strand breakage, and further investigation of the protection mechanism(s) should enable development of improved radioprotectors.
Denison
L
L
Peter MacCallum Cancer Institute, Melbourne, Australia.
Haigh
A
A
D'Cunha
G
G
Martin
R F
RF
D001483:Base Sequence; D001562:Benzimidazoles / Q000494:pharmacology*; D006690:Bisbenzimidazole / Q000494:pharmacology*; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000528:radiation effects*; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids; D011837:Radiation-Protective Agents / Q000494:pharmacology*
D001562:Benzimidazoles; D004277:DNA, Single-Stranded; D008024:Ligands; D011837:Radiation-Protective Agents; D006690:Bisbenzimidazole; C017807:bisbenzimide ethoxide trihydrochloride