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Annals of Clinical & Laboratory Science 34:458-466 (2004)
© 2004 Association of Clinical Scientists

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

Jenn C. Chen, Xinbo Zhang, Timothy P. Singleton and Frederick L. Kiechle
Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Michigan

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.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.

(received 3 May 2004; accepted 24 May 2004)

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


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mitochondria maintain a physiologic proton gradient across the inner and outer membranes, with a negative charge within the mitochondria. This potential energy is harnessed for ATP metabolism [1,2]. Lipophilic cationic dyes, eg 3,3’-dihexyloxacarbocyanine iodide [DiOC6(3)] and 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) [3–8], are concentrated within mitochondria and released during mitochondrial membrane depolarization. In the early phase of apoptosis, disruption of mitochondrial membrane potential (delta psi) and resulting mitochondrial permeability transition may be important for the generation of reactive oxygen species, release of cytochrome C into the cytosol, and ultimate nuclear DNA fragmentation [1, 9–13]. The release of cytochrome C leads to activation of caspases that generate reactive oxygen species by disrupting oxygen consumption by mitochondrial complex I and II substrates [12]. The depolarization of mitochondrial membrane potential usually occurs prior to phosphatidylserine externalization during apoptotic pathways [11]. Delta psi may be decreased without progression to apoptosis. Apoptosis may occur in the absence of any decrease in delta psi [13].

Bisbenzimides, (eg, Hoechst 33342 (H33342 [GenBank] ) and Hoechst 33258 (H33258 [GenBank] )) are DNA dyes that bind to the adenine-thymine region in the minor groove of DNA. We have previously shown that H33342 [GenBank] (but not H33258 [GenBank] ) induces apoptosis in a variety of cell lines [14–16], including myelogenous leukemic cells (HL-60). In HL-60 cells, H33342 [GenBank] induced membrane phosphatidylserine externalization and accumulation of hypodiploid DNA content [14]. Both cellular changes were observed 1 hr after exposure to H33342 [GenBank] . This paper describes the effects of H33342 [GenBank] and H33258 [GenBank] on the early apoptotic changes of mitochondrial membrane potential and mitochondrial mass in HL-60 cells.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture.  The human promyelocytic leukemia cell line HL-60 was grown in RPMI medium 1640 with 10% fetal bovine serum (GIBCO/Invitrogen, Grand Island, NY). The cells were harvested in log growth phase and resuspended at 109 cell/L in fresh minimum essential medium (Cellgro, Herndon, VA) supplemented with 2% fetal bovine calf serum.

Mitochondrial membrane potential and mitochondrial mass.  The cells were incubated with or without H33342 [GenBank] or H33258 [GenBank] in 0.5 ml of 109 cell/L HL-60 suspension in 12x75 mm plastic tubes (Becton-Dickinson, Franklin Lakes, NJ) at 37°C with 5% CO2. After incubation of cultured cells at various times with different amounts of H33342 [GenBank] or H33258 [GenBank] , the delta psi or mitochondrial mass were measured with DiOC6(3) or nonyl acridine orange (NAO, Molecular Probes, Eugene, OR), respectively, added to achieve a final concentration of 20 nM and incubated for 5 min at room temperature. Stock solutions of H33342 [GenBank] , H33258 [GenBank] , 4 µM DiOC6(3), or 4 µM NAO were dissolved in dimethyl-sulfoxide (DMSO, Sigma Chemical Co, St Louis, MO). Finally, 2.5 µL of propidium iodide (PI) (1.0 mg/L stock) was added for the last 30 sec of incubation to assess cell viability. List mode files were collected for 25,000 events with a Coulter XL flow cytometer (Beckman Coulter, Miami, FL).

Additional experiments were performed with JC-1 to measure delta psi. The cells were incubated with 30 µM JC-1 at 37°C for 30 min, and washed twice with PBS before analysis by a FACScan cytometer [17]. At high concentrations, JC-1 monomers form aggregates. The monomers emit green fluorescence, and the aggregates emit red fluorescence. Decrease of delta psi was determined by the decrease of red fluorescence emission [7,8]. Decrease of monomeric fluorescence may suggest the loss of mitochondrial mass [18]. For a positive control, 100 µM of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Sigma) was added as an uncoupler of mitochondrial oxidation. The JC-1 and CCCP stock solutions were dissolved in DMSO.

Statistics.  Flow cytometric data were analyzed with the WinList software (version 5.0, Verity Software, Topsham, ME). Statistical analyses were performed using Statmost software (version 2.01, DataMost Corp, Salt Lake City, UT) and Excel (Microsoft, Redmond, WA). Student’s unpaired t-test was used to compare the difference between 2 populations. A p value <0.05 was considered significant.


   Results
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 Abstract
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 Materials and Methods
 Results
Discussion
 References
 
H33342 decreased delta psi, mitochondrial mass, and cell shrinkage.  Fig. 1Go, which is representative of 3 experiments, shows that treatment of HL-60 cells for 1 hr with 10 mg/L H33342 [GenBank] induced decreased delta psi, detected by decreased fluorescence for DiOC6(3), in 17% of cells (third quadrant of histogram D), compared to 5% of control cells lacking H33342 [GenBank] (third quadrant of histogram B). These low delta psi cells were heterogeneous but concentrated among smaller cells with low forward light scatter and low side scatter (histogram C). Dead cells with bright fluorescence for PI were rare and had low forward scatter and high side scatter (histogram C). H33342 [GenBank] induced decreased NAO in 40% of cells, compared to 5% of controls lacking H33342 [GenBank] (histograms F and H).



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  		Fig. 1. HL-60 cells after one hr incubation, with (C, D, G, H) or without (A, B, E, F) 10 mg/L H33342 [GenBank] . Cells were stained with propidium iodide and either DiOC6(3) (A, B, C, D) or NAO (E, F, G, H).

 
Dose and time course of H33342-induced decrease of delta psi and mitochondrial mass.  Fig. 2Go shows that H33342 [GenBank] induced depolarization of delta psi in a small population of the viable cells, as detected by decreased DiOC6(3) fluorescence. The low delta psi cells appeared as early as 0.5 hr of incubation. Treatment with 5 mg/L of H33342 [GenBank] decreased DiOC6(3) fluorescence in 16, 10, 4.3, 2.4, and 12.5% of viable cells at 0.5, 1, 3, 5, and 24 hr of incubation, respectively. There was <3% cell death (cells with bright fluorescence for PI) until 24 hr of incubation, at which time there was 13% cell death. Increasing H33342 [GenBank] to 15 mg/L decreased delta psi in 14, 12, and 1% of the viable cells and induced cell death in 2, 3, and 21% of total cells at 0.5, 1, and 3 hr, respectively. The incidence of cell death was 52% at 5 hr and 89% at 24 hr.



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  		Fig. 2. Delta psi depolarized with different doses of H33342 [GenBank] (mg/L, top) over time (right, hours). Cells were stained with DiOC6(3) and propidium iodide. Histograms are representative of 2 separate experiments.

 
The delta psi change was investigated with JC-1, which may be less affected by changes in plasma membrane potential [19]. One hr incubation with 5 or 10 mg/L H33342 [GenBank] resulted in concomitant loss of JC-1 aggregate and JC-1 monomer (Fig. 3Go, histograms D and F compared to B, representative of 2 experiments). As in the experiment with DiOC6(3), H33342 [GenBank] treated cells shrank in size (low forward scatter) and side scatter (Fig. 3Go, histograms C, E). As a positive control, 100 µM CCCP, an uncoupler of mitochondrial oxidation, lowered the delta psi in 98% of viable cells (Fig. 3Go, histogram H). CCCP slightly increased the size of cells (Fig. 3Go, panel G) [20].



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  		Fig. 3. Delta psi was detected by JC-1 in HL-60 cells after 3 hr incubation with 0, 5, and 10 mg/L H33342 [GenBank] (A and B, C and D, E and F, respectively). CCCP replaced H33342 [GenBank] in control experiments (G and H).

 
The effect of Hoechst H33342 [GenBank] on mitochondrial mass was measured by the uptake of NAO, a dye that binds to cardiolipin in the outer leaflet of the inner mitochondrial membrane [21]. Fig. 4Go shows that H33342 [GenBank] reduced mitochondrial mass (left shift of the NAO fluorescence peak) in a dose- and time-dependent manner. The decrease in mitochondrial mass was marked in dead cells, corresponding to 24 hr with 5 mg/L H33342 [GenBank] or after 1 hr with 15–20 mg/L H33342 [GenBank] . Viable cells had less tendency to lose mitochondrial mass (5 mg/L H33342 [GenBank] for 0–5 hr).



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  		Fig. 4. Mitochondrial mass was measured with different doses of H33342 [GenBank] (mg/L, top) over time (right, hr). Plots show fluorescence for NAO and the number of events/cells.

 
H33258 induced little loss of delta psi or mitochondrial mass.  H33258, the parent compound of H33342 [GenBank] , induced little or no change of delta psi or mitochondrial mass at equivalent doses (data not shown).

Decreased delta psi in cells after H33342.  Since H33342 [GenBank] might cause changes in the delta psi of all cells or just in a subset of the cells, we analyzed the relative delta psi changes of all viable cells (Fig. 5AGo) and the percentage of viable cells that have decreased delta psi (Fig. 5BGo). Fig. 5AGo shows that H33342 [GenBank] induced a significant decrease of delta psi in all viable cells after one hr incubation compared to the initial time readings (Student’s t-test, p <0.05, p < 0.002, p <0.01, and p <0.002 for 5, 10, 15, and 20 mg/L, respectively). The decreased immunofluorescence for DiOC6(3) was reversed at 3 hr, when it was not different from the initial time (p >0.05 for all H33342 [GenBank] concentrations). After 5 hr of incubation, the delta psi reduced for 10, 15, and 20 mg/L treated cells (p <0.05), whereas the 5 mg/L H33342 [GenBank] treated cells had delta psi similar to the initial time reading (p >0.3).



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  		Fig. 5. The time course (hr) of H33342 [GenBank] effects at various doses: solid square, 0 mg/L; open square, 5 mg/L; open triangle, 10 mg/ L; solid diamond, 15 mg/L; and solid circle, 20 mg/L. Charts show relative delta psi of all viable cells as measured by DiOC6(3) and as compared to control with 0 mg/L (A), relative mitochondrial mass of all viable cells as measured by NAO and as compared to control with 0 mg/L (B), percentage of viable cells with low DiOC6(3) (C), percentage of viable cells with low NAO (D), and percentage of dead cells (E). Data points are arithmetic mean +/– SD from 3-6 experiments.

 
Percentage of cells with decreased delta psi after H33342.  The percentages of viable cells with low DiOC6(3) after 1 hr of H33342 [GenBank] treatment ranged from 8% to 28% and were significantly increased from initial times (Fig. 5BGo, p <0.02, <0.002, <0.001, <0.002 for 5, 10, 15, and 20 mg/L, respectively). The percentage of low DiOC6(3) cells at 3 hr decreased to values not different from initial times (p >0.5 for all H33342 [GenBank] concentrations). From 5 to 24 hr, the percentage of low DiOC6(3) viable cells increased again, except for the 5 mg/L H33342 [GenBank] -treated cells (Fig. 5BGo).

Time course of H33342’s effect on mitochondrial mass and cell death.  Since H33342 [GenBank] might cause changes in the mitochondrial mass of all cells or just in a subset of cells, we analyzed the mitochondrial mass of all viable cells and the percentage of viable cells with decreased mitochondrial mass. The mitochondrial mass was reduced after one hr in a dose dependent manner (p <0.001 for all doses). The reduction of mitochondrial mass moderated at 3 hr (Fig. 5CGo). Over time, the NAO fluorescence continued to decline, except for the 5 mg/L-treated cells which gradually rose to about -20% of control (Fig. 5CGo). The percentage of viable cells with low NAO followed a similar trend (Fig. 5DGo).

Time course of H33342’s effect on cell death.  For 15 and 20 mg/L of H33342 [GenBank] the percentage of dead cells increased in a dose- and time-dependent manner (Fig. 5EGo). At 24 hr an average of 86 to 97% of cells were dead for 15 to 20 mg/L H33342 [GenBank] , respectively. For 5 and 10 mg/L of H33342 [GenBank] , cell death was <10% for up to 5 hr. A marked increase in cell death was observed at 24 hr for 10 mg/L. Fewer than 12% dead cells were observed with 5 mg/L H33342 [GenBank] , even at 24 hr.

Effect of CCCP on the H33342-induced changes in delta psi.  We investigated whether decreased mitochondrial membrane potential would enhance H33342 [GenBank] -induced cell death. HL-60 cells were incubated with or without 20 µM CCCP, an uncoupler of mitochondrial electron transport, for 1 hr at 37°C, followed by addition of 5 or 15 mg/L H33342 [GenBank] for another 5 hr. Fig. 6Go, which is representative of 3 separate experiments, shows that CCCP increased the percentage of cells with low delta psi from 11% (Fig. 6BGo, no CCCP, 5 mg/L H33342 [GenBank] ) to 59% (Fig. 6DGo, 20 µM CCCP, 5 mg/L H33342 [GenBank] ), but the percentage of dead cells did not increase (about 2%). At 15 mg/L H33342 [GenBank] , addition of CCCP increased cell death (from 33 to 47%, Figs. 6F and 6HGo) and increased the percentage of viable cells with low delta psi (28 to 94%).



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  		Fig. 6. HL-60 cells were incubated with (C, D, G, H) or without (A, B, E, F) CCCP followed by 5 mg/L (A, B, C, D) or 15 mg/L (E, F, G, H) H33342 [GenBank] . Cells were stained with DiOC6(3) and propidium iodide.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that H33342 [GenBank] induces lowering of delta psi as early as 0.5 hr after incubation. This is earlier than externalization of phosphatidylserine from the inner to the outer plasma membrane or the appearance of a hyperdiploid fraction on a DNA histogram [14]. Some cells decreased delta psi and shrank in size but maintained plasma membrane integrity to exclude PI, much like the preapoptotic cells described by Mignotte and Vayssiere [22]. Dead cells (bright fluorescence for PI) have smaller size (lower forward scatter) and increased cellular complexity (higher side scatter) than the low delta psi cells (Fig. 1Go). This suggests that H33342 [GenBank] induced apoptosis by an early decrease of mitochondrial membrane potential followed by shrinkage of cell size and loss of membrane ability to exclude PI.

The results from JC-1, DiOC6(3), and NAO studies indicate that H33342 [GenBank] has an early effect of lowering mitochondrial mass, in addition to mitochondrial delta psi. NAO binds to cardiolipin on the outer leaflet of the inner mitochondrial membrane [23,24]. Rapid loss of NAO fluorescence suggests alteration of mitochondrial membrane structure and mass, although uptake of NAO by mitochondria might also be affected by drugs altering the mitochondrial membrane potential [25]. Electron microscopic studies have shown that NAO staining signifies an alteration of mitochondrial structure [21,26]. H33342 [GenBank] also reduced JC-1 monomer fluorescence (Fig. 3Go), consistent with decreased mitochondrial mass [18].

The percentage of viable cells with low mitochondrial mass (Fig. 5DGo) is higher than the percentage of viable cells with low delta psi (Fig. 5BGo). This indicates that some cells with low mitochondrial mass may not necessarily have decreased delta psi. Although it is conceivable that cells with low mitochondrial mass might have temporarily decreased delta psi in the past, the results also suggest heterogeneity of HL-60 cells or mitochondria in their susceptibility to H33342 [GenBank] . Salvioli et al [27] showed that in staurosporine-treated HL-60 cells, there were cells that were apoptotic with no change in delta psi, while others changed delta psi without being apoptotic. At the single cell level, some mitochondria depolarized while others did not [27,28]. At higher H33342 [GenBank] doses cells with highest reduction of NAO fluorescence or loss of delta psi irreversibly progressed to nuclear apoptosis and cell death, as in the current study (Fig. 5EGo).

At a low dose (5 mg/L), H33342 [GenBank] decreased DiOC6(3) and NAO fluorescence initially, but the fluorescence then increased and resulted in little cell death (Fig. 5Go). With low dose H33342 [GenBank] and CCCP, an uncoupler of mitochondrial respiration, the percentage of low delta psi cells increased, but this resulted in little cell death (Fig. 6Go). The cells shrank in size but maintained membrane integrity to exclude PI. Although it is possible that a small subset of cells decreased delta psi and proceeded to apoptosis, low dose H33342 [GenBank] -induced decrease in delta psi may not necessarily lead to apoptosis. Depolarization of delta psi does not lead inexorably to apoptosis in experimental models [20,29]. Depolarization of delta psi is not required for apoptosis [13,30]. Other factors, as found with high dose H33342 [GenBank] , may be necessary for apoptosis to proceed. For example, high dose (15 mg/L) but not low dose (5 mg/L) H33342 [GenBank] has been found to inhibit topoisomerase I in HL-60 cells [14]. The topoisomerase I poisons can induce novel protein-DNA crosslinks in intact cells, cause subcellular damage that alters DNA regulatory pathways, and eventually lead to cell death [31,32].

In summary, high dose H33342 [GenBank] depolarized delta psi, decreased mitochondrial mass, and induced apoptosis (Fig. 5Go). Low dose H33342 [GenBank] depolarized delta psi and decreased mitochondrial mass, but caused minimal cell death. These findings suggest that depolarization of the mitochondrial membrane potential is insufficient to initiate the apoptotic pathway. High dose H33342 [GenBank] has other effects, in addition to changes in delta psi, that promote apoptosis.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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