Hoechst 33342-induced apoptosis is associated with decreased immunoreactive topoisomerase I and topoisomerase I-DNA complex formation.
Hoechst 33342, but not Hoechst 33258, induces apoptosis and inhibits topoisomerase 1 activity in vivo. Topoisomerase I relaxes superhelical DNA through a single strand breakage/rejoining reaction in which the active site tyrosine links covalently to a 3' phosphate at the break site, forming a transient intermediate called a cleavable complex. The fate of cellular topoisomerase 1 in Hoechst 33342-induced apoptosis is unknown. We analyzed the binding capacity of topoisomerase 1 to 32P-labeled plasmid pCI DNA, the immunoreactive topoisomerase 1 concentration and topoisomerase 1 activity in BC3H-1 myocytes and HL-60 cells treated with Hoechst 33342 and Hoechst 33258 by using covalent transfer of 32P radioactivity from plasmid DNA to topoisomerase 1, Western blotting and topoisomerase 1-mediated plasmid relaxation assay, respectively. Hoechst 33342, but not Hoechst 33258, induced topoisomerase 1 dysfunction in both BC3H-1 myocytes and HL-60 cells measured by (1) a decrease in the topoisomerase 1 to DNA binding capacity or cleavable complex formation; (2) a decrease in intracellular concentration of immunoreactive topoisomerase 1; and (3) an inhibition of nuclear endogenous topoisomerase 1 activity. These results suggest that destruction of immunoreactive topoisomerase 1 and topoisomerase 1-DNA complexes or cleavable complexes results in inhibition of topoisomerase 1 activity, a key step in the Hoechst 33342-induced apoptotic process.
Zhang
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Department of Clinical Pathology, William Beaumont Hospital, 3601 West 13 Mile Road, Royal Oak, Michigan 48073-6769, USA.
Kiechle
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F
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001323:Autoantibodies / Q000097:blood; D001562:Benzimidazoles / Q000494:pharmacology*; D006690:Bisbenzimidazole / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D051379:Mice; D009132:Muscles; D010761:Phosphorus Radioisotopes; D010957:Plasmids / Q000235:genetics; D012595:Scleroderma, Systemic / Q000276:immunology
D001323:Autoantibodies; D001562:Benzimidazoles; D004278:DNA, Superhelical; D010761:Phosphorus Radioisotopes; D004247:DNA; D004264:DNA Topoisomerases, Type I; D006690:Bisbenzimidazole; C017807:bisbenzimide ethoxide trihydrochloride