From the
Peroxynitrite (ONOO
Peroxynitrite is a strong oxidant generated from the interaction
of nitric oxide (NO)
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.
In order to avoid any possible
indirect effects to the cells resulting from interaction of
ONOO
HL-60 cells, U-937 cells, and PBMCs were centrifuged, washed twice
with PBS (Ca
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
The cytotoxicity of 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.
(
)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.
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.
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 H
O
(0.1 M) to 1.5 ml of 2 N sodium hydroxide and diluted with
7 ml of distilled H
O. 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 H
O
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.
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.
/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.
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 10
M
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.
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.
,
peroxynitrite; O, superoxide; PBMC, human peripheral blood mononuclear
cell; HUVEC, human umbilical vein endothelial cell; PBS,
phosphate-buffered saline; PI, propidium iodide.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.