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Hoechst 33342-Induced Apoptosis is Associated with Decreased Immunoreactive Topoisomerase I and Topoisomerase I-DNA Complex Formation

Address correspondence to Frederick Kiechle, M.D., Ph.D., Department of Clinical Pathology, William Beaumont Hospital, 3601 West 13 Mile Road, Royal Oak, Michigan 48073-6769, USA; tel 248 551 8020; fax 248 551 3694; e-mail fkiechle{at}beaumont.edu.

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.

Keywords: Apoptosis, Hoechst 33342, Hoechst 33258, topoisomerase I, topoisomerase I-DNA complex

Abbreviations: TOP1, topoisomerase I; TOP2, topoisomerase II; H342, Hoechst 33342; H258, Hoechst 33258; FBS, fetal bovine serum; MEME, minimum essential medium Eagle; BCA, bicinchoninic acid; SDS, sodium dodecyl sulfate

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