©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Peroxynitrite-induced Apoptosis in HL-60 Cells (*)

King-Teh Lin , Ji-Yan Xue , Miguel Nomen , Bernd Spur , Patrick Y-K Wong (§)

From the (1)Department of Cell Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Stratford, New Jersey 08084

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Peroxynitrite (ONOO), an anion and a potent oxidant, generated by the interaction of nitric oxide (NO) and superoxide is able to induce apoptosis in HL-60 human leukemia cells in a time- and concentration-dependent manner. Characteristic morphology of apoptosis can be observed 3 h after HL-60 cells are exposed to 10 µM ONOO. Treatment of HL-60 cells with increasing concentrations of ONOO from 1 to 100 µM confirms the concentration dependence of apoptosis as evidenced by: 1) degradation of nuclear DNA of these cells into integer multiples of approximately 200 base pairs; 2) colorimetric DNA fragmentation assay; and 3) evidence of condensation of chromatin and nuclear fragmentation shown by propidium iodide staining. Under the same conditions, peroxynitrite causes apoptosis in another transformed cell line, U-937 cells, but is ineffective at inducing apoptosis in normal endothelial cells derived from human umbilical cord and normal human peripheral blood mononuclear cells. This direct evidence of peroxynitrite inducing apoptosis implicated a new function of this potent oxidant.


INTRODUCTION

Peroxynitrite is a strong oxidant generated from the interaction of nitric oxide (NO)()and superoxide (O). The in vivo formation of this compound has recently been demonstrated in macrophages and other cellular systems (1-3). The production of ONOO has been demonstrated to be associated with the activation and expression of inducible NO synthase (4, 5) and is implicated in the pathophysiology of such diseases as acute endotoxemia(4) , inflammatory bowel disease(5, 6) , neurological disorders(7) , and atherosclerosis(8, 9) . An active role for ONOO in inflammatory diseases is further supported by the detection of immunoreactive nitrotyrosine on proteins at sites of inflammation(5, 8) . Furthermore, the inhibitory effects of superoxide dismutase, an O scavenger, on NO-mediated cytotoxicity suggest that ONOO may contribute to the NO-mediated biological effects(7, 10) . NO is a diffusible messenger that is known to exhibit a variety of biological activities including vasorelaxation, neurotoxicity, bacteriostasis, and inhibition of tumor cell growth(7, 11, 12, 13) . Recently, nitric oxide has been reported to mediate apoptosis in murine macrophages, and activated murine macrophages can induce apoptosis in tumor cells through a NO-dependent or independent mechanism(14, 15) . Macrophages, neutrophils, and endothelial cells have the ability to generate both NO and superoxide, from which peroxynitrite can be formed in large amounts(2, 3, 10) . Although NO has been reported to be involved in the process of apoptosis(14, 15) , the possible involvement of peroxynitrite in this process has not been reported. In this communication we demonstrate, for the first time, that peroxynitrite can induce apoptosis in two transformed cell lines, i.e. HL-60 and U-937, in a concentration- and time-dependent manner but fails to have any effect on normal endothelial cells and peripheral blood mononuclear cells under the conditions utilized.


EXPERIMENTAL PROCEDURES

Cell Culture

The human promyelocytic leukemia HL-60 cell line and human monocytic tumor cell line U-937 were purchased from ATCC and maintained at 0.2-1 10 cells/ml in RPMI medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.) and antibiotics/antimycotics (Sigma). Cells were maintained in a culture incubator at 37 °C under a 5% CO humidified atmosphere and were used for experiments during the exponential phase of growth. Cell counts were performed routinely to maintain low population density and were assayed for viability by their ability to exclude trypan blue dye.

Human umbilical vein endothelial cells (HUVECs) were obtained and cultured in M-199 medium with Earle's salts (Life Technologies, Inc.) as described(16) . Cell apoptosis assays were performed on second passage endothelial cells. Endothelial cells were maintained at confluence for 2-3 days prior to experiments.

Human peripheral blood mononuclear cells (PBMCs) were recovered from heparin-anticoagulated blood of healthy volunteers by centrifugation of mixed blood sample with Ficoll-Paque plus (Pharmacia Biotech Inc.) as described in the manufacturer's instructions. Freshly isolated PBMC were washed twice with phosphate-buffered saline (PBS) before experiments. Cells were kept in minimal essential medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal bovine serum and antibiotics/antimycotics and were maintained in a culture incubator after experimental treatments.

Preparation of Peroxynitrite

Peroxynitrite was prepared by the reaction of 1 eq of HO with 1 eq of 2-methoxyethyl nitrite (17) under basic conditions as described(18) . Briefly, 10 ml of 160 mM peroxynitrite was prepared by adding 1.5 ml of HO (0.1 M) to 1.5 ml of 2 N sodium hydroxide and diluted with 7 ml of distilled HO. To this solution 0.15 ml (1.6 mM) of 2-methoxyethyl nitrite was added at room temperature. The UV spectrum of the solution after 15 min showed a quantitative formation of peroxynitrite after all HO and nitrite were consumed ( = 302 nm; = 1670 ± 50 liters/mol cm). The light yellow solution of peroxynitrite (150-200 mM) can be stored at -80 °C for an extended time. Aliquots of peroxynitrite were monitored spectroscopically at 302 nm to accurately determine the concentration before and after each experiment.

In order to avoid any possible indirect effects to the cells resulting from interaction of ONOO with components of cell medium and to prolong the half-life of ONOO, a Tris-PBS pH 8.7 (50 mM Tris, pH 8.7, D-PBS without Ca) buffer system was used. It did not adversely affect the growth of the cells used in this study. Cells were suspended in this Tris-PBS buffer rather than in cell medium when treated with ONOO. This Tris-PBS buffer also served to offset any increase in pH caused by the addition of 5 µl of 0.5 N NaOH used as the vehicle, which might have damaged the cells resulting in an artifactual apoptosis.

HL-60 cells, U-937 cells, and PBMCs were centrifuged, washed twice with PBS (Ca/Mg free), and resuspended in 5 ml of Tris-PBS (1 10 cells/ml). Various stock concentrations of ONOO were freshly prepared in 0.5 N NaOH. Five microliters of each stock were added to separate cell suspensions and incubated for 10 min at 37 °C. The cells were washed, centrifuged, resuspended in culture medium, and maintained in a culture incubator for the additional time required for each experiment. Five microliters of Tris-PBS buffer and 0.5 N NaOH were used in the control and vehicle, respectively. Cell density and viability were examined by trypan blue exclusion. In endothelial cells, 2.5 µl of various concentrations of ONOO and 0.5 N NaOH were added into 2.5 ml of Tris-PBS buffer under the same experimental conditions mentioned above.

Cell Fixation and DNA Labeling

Cells for morphological examination were washed twice with PBS, fixed in 1 ml of cold 70% ethanol at 4 °C for 60 min, centrifuged, washed again, and resuspended in 0.5 ml of PBS. RNase (type I-A, Sigma, 250 µg/ml) was added, followed by propidium iodide (PI, Sigma, 25 µg/ml)(19) . After 15 min in the dark at 25 °C, the cells were photographed under an Olympus IMT2-RFL fluorescence microscope.

DNA Fragmentation and Quantitation Assay

The extent of DNA fragmentation was determined by a modified method of Sellins and Cohen (20). The cells were harvested and washed at the indicated time by centrifugation at 250 g for 5 min. The pellet was lysed with 0.5 ml of hypotonic lysing buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 0.5% Triton X-100), and the lysates were centrifuged at 13,000 g for 20 min to separate intact and fragmented chromatin. Both pellet and supernatant were precipitated overnight at 4 °C with 12.5% trichloroacetic acid. The precipitates were sedimented at 13,000 g for 20 min. The DNA precipitate was heated to 90 °C for 10 min in 80 µl of 5% trichloroacetic acid and was quantitated by diphenylamine reactions(21) . The percentage of fragmentation was calculated as the ratio of DNA in the supernatants to the total DNA.

DNA Extraction and Electrophoresis

At each time point, cells were harvested and washed, and the DNA was extracted with phenol/chloroform (1:1)(22) . The effect of ONOO on DNA fragmentation was examined by gel electrophoresis as described(19) . Briefly, the pellet was resuspended in a lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 10 mM EDTA, 100 µg/ml proteinase K, 1% SDS) and incubated at 37 °C; proteinase K was added every few hours until the mixture became clear. The DNA was extracted with an equal volume of phenol/chloroform, precipitated overnight in -20 °C ethanol containing 0.3 M final concentration of sodium acetate (pH 5.2), and centrifuged for 30 min, 4 °C, at 13,000 g. The pellet was resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). RNase (100 µg/ml) was added to each sample and incubated for 1 h at room temperature. The DNA samples were mixed with loading buffer and loaded onto a 1% agarose gel. A HindIII digest of -DNA was applied to each gel to provide molecular size markers of 23.5, 9.6, 6.6, 4.3, 2.2, 2.1, and 0.5 kilobase pairs. Electrophoresis was carried out in TBE buffer (2 mM EDTA, 89 mM Tris, 89 mM boric acid), and the DNA was visualized by ethidium bromide staining and photographed on Polaroid type 667 (3000 ASA) film.


RESULTS AND DISCUSSION

Treatment with various concentrations of peroxynitrite resulted in a time-dependent decrease in cell density of HL-60 cells (Fig. 1). Control and vehicle-treated cells demonstrated a normal growth pattern. However, cells treated with peroxynitrite resulted in a concentration-dependent loss of cell density at an effective concentration of 10 µM. Cell viability was the same in the control, vehicle, and treated groups up to 6 h as determined by trypan blue exclusion. Morphological changes (blebbing and shrinkage) were detected 3 h after treatment with 10 µM peroxynitrite (data not shown), and the time-dependent decrease in cell density was clearly distinct at 12 h. In order to investigate whether this phenomenon was caused by apoptosis, HL-60 cells treated with 10 µM ONOO were harvested at different times. Apoptosis of HL-60 cells was recognized by a characteristic alteration in cell morphology and the appearance of DNA fragments equivalent to approximately 200 base pairs and multiples thereof (Fig. 2A and Fig. 4C). A ladder pattern of the DNA fragmentation, as detected by electrophoresis, appeared as early as 3 h after cells were exposed to peroxynitrite (Fig. 2A, lane4) and was time-dependent. However, no ladder pattern was observed, under identical conditions, in the control and vehicle-treated groups even after 5 h (Fig. 2A, lanes1 and 2). Alternatively, HL-60 cells incubated with sodium azide (0.1%), a substance that induces cell death through a non-apoptotic mechanism(23) , showed no apoptotic ladder pattern (Fig. 2A, lane6). To further characterize this phenomenon, various concentrations of peroxynitrite, up to 100 µM, were added, and cells were harvested 5 h after exposure to peroxynitrite. The apoptotic ladder DNA bands were clearly detected at 10 µM and higher concentrations (Fig. 2B, lanes 4-6), whereas neither control, nor vehicle, nor 1 µM peroxynitrite demonstrated any of the characteristic ladder pattern (Fig. 2B, lanes 1-3). This concentration-dependent response to peroxynitrite was also quantitated by colorimetric DNA fragmentation assay, as shown in Fig. 3, and further substantiated by the morphological changes (Fig. 4). As described under ``Experimental Procedures,'' propidium iodide was used to assess changes in cell morphology. We observed that 5 h after the initial 10-min exposure to peroxynitrite, there was chromatin condensation and nuclear fragmentation (Fig. 4, compare C and D with A and B). A number of intact cell fragments (apoptotic bodies), which are small membrane-bound bodies containing fragments of highly condensed DNA, appeared after 100 µM ONOO treatment (Fig. 4D). Plasma membrane integrity, however, was intact for several hours following the initial changes in chromatin appearance. To ascertain that this apoptotic process is induced by peroxynitrite and not by its decomposed products, supernatant from each incubation was collected and incubated with freshly prepared HL-60 cells. No apoptotic ladder of DNA fragmentation was detected from those cells exposed to pretreated supernatant previously containing up to 100 µM ONOO (data not shown). The apoptotic process induced by peroxynitrite was not blocked but, actually, enhanced by the protein synthesis inhibitor, cycloheximide (50 µg/ml) (Fig. 2B, lane7). Induction of apoptosis was also observed in U-937 cells exposed to different concentrations of peroxynitrite (Fig. 2C). However, the response to peroxynitrite in U-937 cells was slightly weaker than that of HL-60 cells. This was evident by a lower percentage of DNA fragmentation (data not shown). Unlike HL-60 cells, U-937 cells were not affected by 10 µM ONOO (Fig. 2C, lane4), but at higher concentrations (50 µM and 100 µM), the ladder pattern of DNA fragmentation (Fig. 2C, lanes5 and 6) as well as chromatin condensation was observed (data not shown). These results suggested that apoptosis of transformed leukemia cells can be effectively induced with micromolar concentrations of peroxynitrite.


Figure 1: Effect of various concentrations of ONOO on in vitro proliferation of HL-60. Exponentially grown HL-60 cells were exposed to 0.03 µM (▾), 0.1 µM (), 1 µM (), and 10 µM () ONOO under experimental conditions as described under ``Experimental Procedures.'' After treatment with ONOO, cells were seeded at 2 10/ml and were cultured under standard conditions. Cell number and viability were determined using a hemocytometer chamber by trypan blue dye exclusion at the indicated time. , vehicle; , control.




Figure 2: Time and dose-dependent effects of ONOO on DNA fragmentation. A, agarose gel electrophoresis of DNA extracted from HL-60 cells 1 h (lane3), 3 h (lane4), and 5 h (lane5) after cells were exposed to 10 µM ONOO as described under ``Experimental Procedures.'' Both control (lane1) and vehicle (lane2) experiments were carried out under the same conditions for 5 h. 0.1% sodium azide was added to the HL-60 cells culture medium for 5 h of incubation as a negative control. No DNA fragments were seen in sodium azide-treated HL-60 cells (lane6). B, HL-60 cells were exposed to various concentrations of ONOO and were harvested 5 h after treatment as described under ``Experimental Procedures.'' Lanes 1-6 are: control, vehicle, 1 µM, 10 µM, 50 µM, 100 µM, respectively. Cycloheximide were added into the cell culture medium (50 µg/ml) 30 min before cells were exposed to 100 µM ONOO and thereafter coincubated with cells for 5 h (lane7). Lane8 is a HindIII digest of -DNA providing molecular size markers. C, U-937 cells were exposed to various concentrations of ONOO under the same conditions as described in B above. Lanes 1-7 are control, vehicle, 1 µM, 10 µM, 50 µM, 100 µM, and HindIII-digested -DNA marker, respectively.




Figure 4: Fluorescence microscopy appearance of ethanol-fixed, PI-stained HL-60 cell nuclei 5 h after ONOO treatment. PanelA, untreated control; panelB, vehicle. The majority of ONOO-treated HL-60 cell nuclei show a marked condensation of chromatin at 10 µM (panelC) and 100 µM (panelD) ONOO.




Figure 3: Dose-dependent DNA fragmentation of HL-60 cells induced by various concentrations of ONOO. DNA fragmentation was quantitated by diphenylamine assay as described under ``Experimental Procedures.'' The ratio of DNA cleavage products to total DNA is given as a percentage.



In order to investigate whether peroxynitrite can induce apoptosis in normal cells, we performed the treatment under identical conditions to normal HUVECs and PBMCs. No characteristic apoptotic morphology or ladder pattern was observed after 5 h of peroxynitrite treatment (up to 100 µM) (Fig. 5).


Figure 5: Effects of ONOO on human normal cells. Human umbilical vein endothelial cells and human peripheral blood mononuclear cells were harvested after exposure to various concentrations of ONOO as described in the Fig. 2B. A, fluorescence microscope appearance of ethanol-fixed, PI-stained HUVECs nuclei in vehicle (panela) and 100 µM (panelb) ONOO-treated groups showed no difference. DNA extracted from various dose-treated HUVECs (B) and PBMCs (C) were performed on agarose gel electrophoresis. Lanes 1-7 are: HindIII-digested -DNA marker, control, vehicle, 1 µM, 10 µM, 50 µM, 100 µM, respectively.



Apoptosis is a highly regulated process of cell death and appears to be an essential and critical mechanism used by biological organisms to maintain homeostasis. The loss of the ability by the cell to undergo spontaneous apoptosis rather than increasing cell proliferation rates has been suggested to be one of the mechanisms involved in some tumor malignancies(24, 25) . It has been reported that macrophages can induce tumor cell death through nitric oxide-mediated induction of apoptosis (15). NO itself is a relatively weak oxidant; however, when it reacts with O (k = 6.7 10M s)(26) , it generates a strong oxidizing agent, peroxynitrite anion. Many NO-mediated cytotoxic effects have been shown to be mediated through the interaction of ONOO with proteins and membrane lipid (27, 28). We demonstrate here, for the first time, that ONOO can induce apoptosis in HL-60 and U-937 cell lines but not in normal HUVECs and PBMCs. Since peroxynitrite can cross the cell membrane and is sufficiently stable for several seconds in the buffer used during the experiment, an more efficient defense mechanism may exist in the two normal cells. However, the exact mechanism still needs further investigations.

The cytotoxicity of ONOO has been suggested to be involved in the initiation of lipid peroxidation(29) , oxidation of sulfhydryls(1) , and inactivation of enzymes in the mitochondria electron transport chain(30) . Different from NO, ONOO has specific abilities to cause tyrosine nitration and, thus, alter the protein activities(2, 31) . Tyrosine phosphorylation, a critical modification in cellular signal transduction, is an integral aspect of cellular growth and transformation(32) . It has been shown that nitration of tyrosine will inhibit the phosphorylation of the nitrated tyrosine molecule(33) . Thus, the disruption of the signal transduction cascade resulting from the nitration of tyrosine residues may be a critical pathological event caused by peroxynitrite. However, the exact mechanism of ONOO to elicit the apoptotic effect on transformed cells remains to be established. In addition, the observation that the protein synthesis inhibitor, cycloheximide, failed to inhibit HL-60 cell apoptosis after induction by ONOO suggests that new protein synthesis may not be required for the ONOO-induced apoptotic response. Whether or not modification of certain functional residues of the proteins by peroxynitrite contributes to this apoptotic effect is now under investigation.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants R01-25316-15, 41747, and P01-43203 (to P. Y-K W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Stratford, NJ 08084. Tel.: 609-566-6078; Fax: 609-566-6195.

The abbreviations used are: NO, nitric oxide; ONOO, peroxynitrite; O, superoxide; PBMC, human peripheral blood mononuclear cell; HUVEC, human umbilical vein endothelial cell; PBS, phosphate-buffered saline; PI, propidium iodide.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.