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Mitochondrial Membrane Potential Change Induced by Hoechst 33342 in Myelogenous Leukemia Cell Line HL-60

Address correspondence to Timothy P Singleton, M.D., Flow Cytometry Laboratory, William Beaumont Hospital, 3601 West 13 Mile Road, Royal Oak, MI 48073-6769; tel 248 551 2935; fax 248 551 3694; e-mail tsingleton{at}beaumont.edu.

Hoechst 33342’s effects on apoptosis and mitochondrial membrane potential (delta psi) were investigated in a myelogenous leukemia cell line, HL-60. Delta psi was detected with 2 lipophilic cationic fluorochromes: 3,3’-dihexyloxacarbocyanine iodide [DiOC₆(3)] or 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Mitochondrial mass was measured with nonyl acridine orange (NAO). Protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized mitochondria in control experiments. Cell viability was determined by propidium iodide uptake. Hoechst 33342 at 10–20 mg/L decreased fluorescence for DiOC₆(3) at 0.5 hr. The fluorescence partially normalized at 3 hr and then progressively decreased at 5–24 hr, resulting in cell shrinkage and death. Mitochondrial mass decreased 40–70% by 1 hr and 70–90% at 24 hr. A lower concentration of Hoechst 33342, 5 mg/L, reduced the delta psi at 0.5 hr, but delta psi returned to control values after 3 hr. Mitochondrial mass decreased 30–40% and then partially normalized, and cell viability was >92% at 24 hr. Protonophore carbonyl cyanide m-chlorophenylhydrazone lowered delta psi with little cell death. Thus, at high concentration, Hoechst 33342 induces depolarization of delta psi and subsequent apoptosis. Lack of apoptosis at low concentration of Hoechst 33342, despite depolarization of delta psi, indicates that mitochondrial membrane depolarization alone is insufficient to induce apoptosis.

Keywords: apoptosis, Hoechst 33342, mitochondrial membrane potential, myelogenous leukemia

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