Mitochondrial membrane potential change induced by Hoechst 33342 in myelogenous leukemia cell line HL-60
Jenn C Chen1, Xinbo Zhang, Timothy P Singleton
1Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Michigan 48073-6769, USA.
Abstract:
Abstract. 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.
Insights
High concentrations of Hoechst 33342 induce apoptosis in leukemia cells by disrupting mitochondrial membrane potential (delta psi). Lower concentrations cause transient delta psi changes without significant cell death, indicating depolarization alone is insufficient for apoptosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Apoptosis, or programmed cell death, is crucial for development and disease.
- Mitochondrial membrane potential (delta psi) plays a key role in regulating apoptosis.
- Hoechst 33342 is a fluorescent dye with potential cytotoxic effects.
Purpose of the Study:
- To investigate the effects of Hoechst 33342 on apoptosis and mitochondrial membrane potential (delta psi) in HL-60 myelogenous leukemia cells.
- To determine the concentration-dependent effects of Hoechst 33342 on cell viability and mitochondrial function.
- To assess whether mitochondrial membrane depolarization alone is sufficient to induce apoptosis.
Main Methods:
- HL-60 cells were treated with varying concentrations of Hoechst 33342.
- Mitochondrial membrane potential (delta psi) was measured using lipophilic cationic fluorochromes DiOC6(3) and JC-1.
- Mitochondrial mass was assessed using nonyl acridine orange (NAO).
- Cell viability was determined by propidium iodide uptake.
- Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control for mitochondrial depolarization.
Main Results:
- High concentrations (10-20 mg/L) of Hoechst 33342 significantly decreased delta psi and mitochondrial mass, leading to cell shrinkage and death within 24 hours.
- Lower concentrations (5 mg/L) caused a transient reduction in delta psi and mitochondrial mass, with cell viability remaining high (>92%).
- Protonophore CCCP induced mitochondrial depolarization but minimal cell death, supporting the role of other factors in apoptosis.
Conclusions:
- High-dose Hoechst 33342 induces apoptosis in HL-60 cells through significant and sustained mitochondrial membrane potential depolarization.
- Transient mitochondrial depolarization at lower Hoechst 33342 concentrations does not lead to apoptosis, suggesting it is not the sole trigger.
- These findings highlight the critical role of sustained mitochondrial dysfunction in Hoechst 33342-induced apoptosis.
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