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Lysis of Human Monocytic Leukemia Cells by Extracellular Adenosine
Triphosphate: Mechanism and Characterization
of the Adenosine Triphosphate Receptor
By Emma Spranzi, Julie Y. Djeu, Susan L. Hoffman, P.K. Epling-Burnette, and D. Kay Blanchard
The present study shows that extracellular adenosine triphosphate (ATP) has the capacity to mediate dose-dependent lysis of the monocytic leukemia cell line THP-1. The
lysis, assessed by 5‘Cr release, was found to be selective
for ATP, because adenosine diphosphate (ADP) or other
nucleotides were less effective in their ability to lyse the
cells. The amount of “Cr released was particularly enhanced by the stimulation of the cells with 1,000 U/mL of
interferon gamma (IFN-7) for 3 days, and the sensitivity
was time and dose dependent. Analysis of the mechanism
of lysis indicated that the fully ionized form, A T P - , me-
diated the lysis, because the addition of cation chelators or
the absence of the divalent cations, Ca2+ and Mg2+, in the
culture medium of a 6-hour 61Cr release assay increased
the percent specific lysis. Therefore, the ATP receptors on
THP-1 cells were classified as P, purinoceptors. Moreover, it is shown here that the Caz+/calmodulin complex
plays a role in the regulation of the lysis by extracellular
ATP of THP-1 cells, because antagonists of this complex,
such as trifluoperazineor KN-62, were found to inhibit the
ATP-mediated cell lysis.
0 1993 by The American Society of Hematology.
T
rinergic” receptors recognizing ATP are classified as surface
P2 purinoceptors, and they have been subdivided on the
basis of their selectivity for various nucleotides as P2x,P2y,
and P, purinergic receptors.839In the present report, we
have attempted to define, through the measurement of responses induced by purine nucleotide agonist and antagonist, the receptor type for ATP involved in the cell lysis of
IFN-y-treated and granulocyte-macrophagecolony-stimulating factor (GM-CSF)-treated THP- 1 cells, as well as the
pathway that is activated on binding ofthe ATP molecule to
the cell membrane.
In examining the mechanism of lysis of THP- 1 cells by
ATP, it was discovered that metabolic inhibitors of calmodulin-linked processes were able to block the lytic effect of
this purine nucleotide. Calmodulin is known to activate at
least 20 different enzymes, and it plays an important regulatory role in cell proliferation and activation.” The present
study also shows a role for this calcium-binding protein in
the signal transduction mechanism of ATP-mediated cell
death.
HE PROFOUND EFFECT of adenosine triphosphate
(ATP) on cell physiology has been described over a
period of years in several laboratories. In addition to physiologic modulation, ATP has recently been found to be lytic
for mouse lymphocytes derived from the thymus and
spleen,’ B16 melanoma cells? transformed 3T3 cells,2 and
YAC-1 and P-815 tumor cell^.^,^ The results observed in
these murine cells prompted us to examine whether ATP
might mediate lysis of human cells. We have detected that
human monocyte-derived macrophages are subject to lysis
by ATP as measured by ”Cr release over a period of 6 hours,
and, more importantly, we showed the ability of interferony (IFN-y) to enhance the sensitivity of macrophages to
ATP.5Therefore, we hypothesized that human myeloid leukemic cells might be sensitive to ATP because of the susceptibility of human macrophages. Furthermore, it might be
possible to increase their sensitivity to ATP by treatment
with IFN-y, with the potential of developing new therapeutic modalities. These studies were consequently performed
using the THP- 1 cell line, an established leukemic cell line
that displays monocytic properties, including immunologic
function,6.’ and we have examined the lytic effect of ATP on
these target cells and their modulation of susceptibility by
IFN-7.
Because extracellular ATP does not cross the plasma
membrane of viable cells, the nucleotide must therefore interact with specific receptors on cellular surfaces. The “pu-
From the Department of Medical Microbiology dt Immunology
and the Department ofBiochemistry andMolecular Biology, University of South Florida College of Medicine, H. Lee M ~ f i t tCancer
Center and Research Institute, Tampa, FL.
Submitted December 21, 1993; accepted May 5, 1993.
Supported in part by National Cancer Institute Grant No. CA46820.
Address reprint requests to D. Kay Blanchard, PhD, University of
South Florida College of Medicine, H. Lee Mofitt Cancer Center dt
Research Institute, 12902 Magnolia Dr, Tampa, FL 33612.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 I993 by The American Society of Hematology.
0006-4971/93/8205-0030$3.00/0
157%
MATERIALS AND METHODS
Materials. ATP (tissue culture grade), adenosine diphosphate
(ADP), uridine triphosphate (UTP), cytosine triphosphate (CTP),
thymidine triphosphate (TTP), guanosinetriphosphate (GTP), inosine triphosphate (ITP), 2’-3’-0-(4-benzoylbenzoyI)adenosine
5’41%
phosphate (BzATP), ATP-& trifluoperazine (TFP; in 70% ethanol), 4,4‘-diisothiocyanatostilbene-2,2’-disulfonicacid (DIDS;
solubilizedin 70%ethanol), and 5’-pfluorosulfonylbenzoyl-adenosine (FSBA) were obtained from Sigma Chemical Co (St Louis,
MO). N-(6-aminohexil)-5-chloro- I -naphthalenesulfonamide(W-7;
solubilizedin 95%ethanol) and KN-62 (dissolvedin dimethyl sulfoxide [DMSO]) were obtained from Calbiochem (La Jolla, CA),
and 2-methyl thio-adenosine(2MeSATP)was from ICN Biochemicals (Cleveland, OH). Human recombinant IFN--y was generously
provided by Genentech Corp (South San Francisco, CA) and human recombinant GM-CSF was a generous gift from Immunex
(Seattle, WA). All plasticware was obtained from COSTAR (Cambridge, MA).
Cell culture. THP-I cells (American Type Culture Collection,
Rockville, MD) were cultured in RPMI-I640 supplemented with
10%fetal calf serum (FCS) (Hyclone Labs, Logan UT), 2 mmol/L
L-glutamine, 100 U/mL penicillin, 100 pg/mL streptomycin, 5
mmol/L HEPES buffer, and 5 X IO-’ mmol/L 2-mercaptoethanol,
and will subsequently be referred to as complete medium. The cell
density was 1 X IO6 cells/mL at the initiation of culture and was
Blood, Vol82, No 5 (September l ) , 1993: pp 1578.1585
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1579
ATP LYSES HUMAN LEUKEMIC CELLS
incubated at 37°C in a 5%CO, atmosphere for 3 days unless noted.
All media and reagents contained less than 0. I ng/mL of endotoxin
as determinedby the Limulus lysate assay (MA Biologics, Walkersville, MD).
Measurement of ATP-mediated cyfotoxicify. A 6-hour "Cr-release assay was used to measure the effect of ATP on cytokinetreated THP-1 cells, and was performed essentially as previously
described.' THP-1 cells were labeled with sodium [5'Cr]chromate
(Amersham, Arlington Heights, IL) for 1 hour in 0.5 mL of medium, washed, and then added to serial dilutions of ATP in microtiter wells at 1 X IO4 cells/well in a final volume of 0.2 mL in each
well. All determinationswere done in triplicate,and the SEM of all
assays was calculated and was typically 5% of the mean or less.
Student's t-tests were performed to identify significant differences
between treatments.
RESULTS
Cytotoxic effectof extracellular ATP. THP- 1 cells were
tested for sensitivity to lysis by a wide range of concentration of ATP in a 6-hour "Cr release assay (Fig 1). In addition to cells cultured in medium alone, THP-1 cells were
incubated in GM-CSF or IFN-y for 3 days and were also
tested for sensitivity to ATP-mediated lysis. The effect of
ADP on cytokine-treated and untreated THP-1 cells was
included as a control for specificity. As shown, a marked
increase in the release of radioactivity was observed for the
IFN-y-treated cells in the presence of 1.25 mmol/L ATP
(58.2% k 4.0% specific lysis) as compared with the GMCSF-treated cells (3.1% 2 0.9% specific lysis) or cells cultured in medium alone (8.2% & 2.2% specific lysis). The
reaction appeared selective for ATP, because neither GMCSF-treated cells nor the medium control cells responded
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80
60
50
40
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0.16 0.32 0.62
1.25
2.5
5.0
CONCENTRATION (mM)
Fig 1. ATP/ADP dose-response for THP-1 cells. THP-1 cells
were incubated for 3 days in medium alone, with 1,000 U/mL of
IFN-y or with 1,000U/mL of GM-CSF before being used as target
cells in a 6-hour 6'Cr release in the presence of increasing concentrations of ATP or ADP. The mean of triplicate cultures is represented, and the SEM was typically within 5%. Data are representative of three experimentsthat were performed with similar results.
(A),medium ATP; (A),medium ADP; (0),lFNg ATP; (01,
lFNg
ADP; (0).GM-CSF
ATP; (M), GM-CSF
ADP.
+
+
+
+
+
+
"I
/
50
0
.16
.32
.62
1.25
2.5
5
CONCENTRATION OF ATP 0
Fig 2. Effect of IFN-y concentration on the sensitivity of THP-1
cells to ATP. THP-1 cells were incubated in medium alone or with
the indicated concentrationof IFN-y for 3 days before being used as
target cells in a 6-hour "Cr release assay. Numbers are means of
triplicate determinations and the data are representative of three
experiments that were performed with similar results. (A), medium; (m), ? U/mL: (A), 1 0 U/mL: @), 100 U/mL; (O), 1,000 U/mL.
to ADP. Only IFN-y-treated cells showed a slight sensitivity (10.4% k 0.2% specific lysis) to ADP at 1.25 mmol/L,
which was unlike human IFN-treated macrophages in
which no susceptibility to ADP-mediated lysis was noted.5
Time course of IFN-y-induced efects. The timing of
the addition of IFN-y to the cultures was found to be a
factor in the increased sensitivity to ATP-mediated lysis.
The lysis in a 6-hour "Cr release assay, induced by 1.25
mmol/L ATP, of THP- 1 cells cultured with 1,000 U/mL of
IFN-y or GM-CSF for the indicated length of time was assessed. IFN-y was able progressivelyto increase the sensitivity of THP- 1 cells to ATP-mediated lysis from the baseline
level of 12% k 2% specific lysis, to 26% k 2% at day 2, to
36% k 3% at day 3, and increasing to 55% k 4% at day 4 of
incubation. However, the parallel incubation of THP- 1 cells
with GM-CSF for 4 days did not increase the sensitivity of
these cells to ATP, and even appeared to be slightly, but not
significantly, decreased with 9% & 1% specific lysis. For further experimentation, 3 days of incubation with cytokines
was selected as the optimal incubation time, as the viability
of the cells begins to decrease to approximately 80%by day
4, presumably because of the antiproliferative effect of
IFN-y .
Dose of IFN-y required to induce sensitivity. The effect
of different concentrations of IFN-y on the percent lysis was
studied to identify the optimal concentration of cytokine
needed to induce ATP-mediated lysis. THP- 1 cells were cultured in the presence of the indicated concentration of IFNy (Fig 2) for 3 days before being tested for ATP sensitivity in
a 6-hour "Cr-release assay. As shown, increasing concentrations of IFN-y induced increasing susceptibility of THP-1
to lower concentrations of ATP; ie, treatment of cells with
100 U/mL of IFN-y resulted in 11% f 2% specific lysis,
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1580
SPRANZI ET AL
whereas 1,000 U/mL induced 42% f 2% specific lysis at the
same ATP concentration of 1.25 mmol/L. Therefore, for
our subsequent experiments, the dose of IFN-y used was
1,000 U/mL.
In studies using human macrophages,' significantly less
IF"-y was required to induce ATP sensitivity (maximal
activation was found using 100 U/mL as compared with
1000 U/mL for THP-1 cells). To show that the response of
THP- I was specific for IFN-y, THP- 1 cells were stimulated
with (1) medium alone; (2) 1,000 U/mL IFN-y; (3) 1,000
U/mL of IFN-y that had been neutralized using excess antiIFN-y monoclonal antibodies (MoAbs) (Genzyme Corp,
Cambridge, MA) at 37°C for 1 hour before its addition to
THP-1; and (4) anti-IFN-y alone. Cultures were then incubated for 3 days and used as target cells in a 2-hour "Cr
release assay. The specific lysis of each of these cultures in
the presence of 1.25 mmol/L ATP was 6% f 1%, 45% f 3%,
8%k 1%,and 8% f 1%, respectively, indicating that sensitization of THP- 1 tumor cells by IFN-y was specific for this
cytokine. Finally, incubation of THP-1 cells with up to
1,000 U/mL of GM-CSF for 3 days did not differ significantly from a control culture incubated in the presence of
medium alone (data not shown). Thus, the remaining experiments will be performed by comparing IFN-y-treated
cells with GM-CSF-treated THP- 1 cells to compare the effects of different cytokines on these monocytic cells.
Time course of "Cr releasefrom ATP-stimulated THP-1
cells. We next investigated the early events that occurred
after the exposure of THP- 1 cells to extracellular ATP. The
supernatants were collected at the indicated time points
from IFN-y-treated or GM-CSF-treated cells after stimulation with 1.25 mmol/L ATP (Fig 3). GM-CSF-treated
THP- I showed little 51Crrelease after 2 hours of incubation
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30
20
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2
4
6
LENGTH OF I W A T I O N
24
h)
Fig 3. Kinetics of 5'Cr release from THP-1 cells after stimulation
with ATP. THP-1 cells were cultured for 3 days in medium alone,
with IFN-y, or with GM-CSF before being used as target cells in a
51
Cr release assay with 2.5 mmol/L ATP. Supernatants were
harvested at the indicated time points. Numbers are means f SEM
of triplicate determinations and data are representative of three
experimentsthat were performed with similar results. (R),medium;
1, +GM-CSF; (a),
+IFNg.
Table 1. Effect of Various Nucleotides on Cr Release
of THP-1 Target Cells
Percent Specific Lysis & SEM
(% of ATP Control)
Purine nucleotide triphosphates
ATP
GTP
ITP
Pyrimidine nucleotide triphosphate
UTP
CTP
ATP analogues
ATPrS
Adenosine
57 f 0.6 ( 1 0 0 )
-5 f 0.7 (0)
5 f 1.o (9)
1 f 0.8 (2)
2 f 1.4 (4)
29 f 1.5 (51)
1 f 0.7 (2)
THP-1 cells were incubatedfor 3 days in the presence of 1,000 U/mL
of IFN-y before being used as target cells in a 6-hour "Cr release assay
using 2.5 mmol/L concentration of each compound as the lytic agent.
The mean of triplicate cultures f SEM is shown. Data are representative
of two experiments that were performed with similar results. The numbers in parentheses indicate percentage of ATP control.
with ATP and required 4 hours to show significant lysis,
which steadily climbed to 26% by 24 hours. Further incubation up to 30 hours did not increase the maximal lysis of
these cells by ATP (data not shown). IFN-y-treated THP- 1
cells, however, rapidly released the radioactivity into the
supernatant fluids, which was maximal after the first 2
hours. The percent of "Cr released after 24 hours was not
significantly different from the percent released at 2 hours.
Thus, there was a major difference between IFN-y-treated
and GM-CSF-treated THP- 1 cells in the rapidity of the radiolabel release. Moreover, this difference in the sensitivity
to ATP is maintained over time, as a longer incubation does
not result in a more complete killing of GM-CSF-treated
cells, which showed 26% specific lysis after 24 hours as compared with 72% in IFN-y-treated cells incubated with ATP
for the same length of time.
Effect of other nucleotides on THP-1 cells. To determine
the specificity of the ATP stimulation, various nucleotides
at a 2.5 mmol/L concentration were assessed for their efficacy against IFN-y-treated THP-1 cells in a 5-hour "Cr
release assay (Table I). Of all the nucleotides tested, as well
as adenosine, only ATP caused lysis greater than lo%, indicating that the ligand selectivity is the same as that which
causes the lysis of normal human macrophages, as described
previou~ly.~
ATPyS, a poorly hydrolyzable ATP analogue,
was half as effective as ATP, causing 29% specific lysis as
compared with 57% lysis by ATP. These results indicate
that the hydrolysis of ATP is not indispensable for the lysis
of THP- 1 cells.
Effect of divalent cations on ATP-mediated lysis of THP-1
cells. A growing body of data suggests the existence of specific cell surface receptors for ATP, and classification of
these purinoceptors has been based on selective binding of
agonists and antagonists of ATP." The presence of the PZ
purinoreceptor has been described in rat mast cells,'* in
mouse macrophage^,'^ and in transformed cell lines,I4 and
has been shown to mediate the permeabilizing effect of ATP
on these cells. The observation that only ATP, but not aden-
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1581
ATP LYSES HUMAN LEUKEMIC CELLS
osine, ADP, UTP, ITP, CTP, or GTP, can induce the lysis
of THP-I cells could suggest that the receptor for ATP on
THP- 1 cells belongs to the P,, class. Moreover, it has been
shown that the ligand that activates the P2z receptor is the
tetrabasic ion, ATP", which is present as a minor equilibrium component in solutions containing divalent cations,
to which ATP is normally ~ o m p l e x e d . ~ Because
~.~~,'~
of the
high affinity of ATP- for MgZf and Ca2+,the amount of
ATP- required to induce lysis depends on the divalent cation concentration in the medium. To determine the influence of divalent cations in ATP-mediated lysis of THP-1
cells, we measured the percent lysis of these cells by 2.5
mmol/L ATP in various concentrations of Ca" and Mg2+.
The cytotoxicity assay was performed in a buffered salt solution comprising 135 mmol/L NaCI, 5 mmol/L KCI, 30
mmol/L HEPES, pH 7.4, containing 0.2% bovine serum
albumin (BSA) and 2-mercaptoethanol, and lacking Ca2+
and Mg2+.Increasing amounts of cations were then added
to the medium in the form of CaC1, and MgC1,. As shown
in Table 2, increasing the concentration of Ca2' and Mg2+
from 1 to 4 mmol/L caused decreased release of "Cr in the
supernatant. Thus, as the effective concentration of ATPwas decreased, the percent lysis of THP-1 was concomitantly lowered. Conversely, in the absence of micromolar
concentrations of the cations, the percent specific lysis increased along with the higher concentration of the AT€-'
ionic species. The same effect was observed with THP-1
cells treated with GM-CSF, although the percent specific
lysis for the GM-CSF-treated cells (Table 2), in the presence
of micromolar concentrations of cations, was lower than
that of IFN-y-treated cells.
Next, we evaluated the effect of chelating agents on ATPmediated lysis of THP- I . The lytic effect of ATP was increased by the addition in the incubation medium of EDTA
Table 2. Effect of Divalent Cations on ATP-Mediated Lysis of
THP-1 Cells
% Specific Lysis ? SEM in the Presence of
IFN-y-treated THP-1
mmol/L Ca2+)
0
1
2
4
GM-CSF-treated THP-1
(mmoi/L Ca2+)
0
1
2
4
0 mmol/L
Mg2+
1 mmol/L
Mg2+
2 mmol/L
Mg2+
4 mmol/L
MgZ+
46+2
38+2
0+1
0+1
28+1
13+1
4+1
1+1
13+1
10+1
3+1
0+1
3+1
12+1
3+1
0+1
13+1
1+1
0+1
0+1
6+1
3+1
1+1
121
0
0
0
0
2 1
+1
2 1
+1
I t 1
lil
1+1
0+1
THP-1 cells were incubated for 3 days in the presence of 1,000
U/mL of IFN-y or GM-CSF before being used as target cells in a 6-hour Cr
release assay using 1.25 mmol/L ATP as the lytic agent. The indicated
concentrations of CaCI, and MgCI, were included in the assay medium (a
buffered salts solution lacking Ca2+and Mg2+). Data are representative
of three experiments performed with similar results.
from 1 to 4 mmol/L, which chelated the extracellular free
divalent ions and subsequently increased the relative concentration of ATP-. The addition of EDTA increased the
lysis of THP-I , an effect that was most prominent at lower
concentration of ATP. As little as 1 mmol/L of EDTA increased the lysis ofTHP-I cells from 6.3% ? 0.8% to 19.1%
f 1.7%, with 0.31 mmol/L ATP. Increasing the concentration of EDTA to 4 mmol/L augmented the sensitivity to this
concentration of ATP to 68.7% f 1.7%,confirming that the
active form of ATP was the tetrabasic ionic species, and
defines the permeabilizing ATP receptor on THP- 1 cells as
the PZzpurinoceptor.
A TP-degrading activity on the THP-1 cell surface. Filippini et all7have shown the existence of ATPases on the cell
surface of cytolytic T lymphocytes. The difference in percent lysis of IFN-y-treated, or GM-CSF-treated cells after
exposure to ATP could be caused by a difference in activity
of the ecto-ATPases on the surface of these cells, and it was
possible that GM-CSF-treated THP-I cells were more resistant to ATP because of elevated levels of surface ecto-ATPase activity. Because FSBA has been found to be a very
efficient inhibitor of ecto-ATPases in the lymphocytes,"
THP-I cells were pretreated for 30 minutes at 37°C with 10,
100, and 1,000pmol/L FSBA before incubating for 4 hours
in the presence of 2.5 mmol/L ATP. No difference in the
percent 51Crrelease was seen between the untreated THP- 1
cells and the cells pretreated with FSBA. For example, the
percent specific lysis for GM-CSF-treated THP- 1 cells, in
the presence of 2.5 mmol/L ATP, was 20.8% k 0.9% with
pretreatment of the cells with 1000 pmol/L FSBA, as compared with 2 1.9% +. 2.2% lysis of cells incubated similarly
but without FSBA. In addition, FSBA had no effect on the
ATP-mediated lysis of IFN-y-treated THP- 1 cells. These
results suggest that differences in ecto-ATPase activity was
not the cause of the difference between IFN-y-treated cells
and GM-CSF-treated cells.
Efect of DIDS,a purinoceptor antagonist, on ATP-mediated lysis of THP-I. DIDS is an irreversible cross-linking reagent that inhibits anion exchange, and it has been
shown to block the effects of ATP on all the physiologic
responses by preventing the binding of ATP to a site on the
plasma membrane." In particular, DIDS can selectively
block the purinergic receptor for the ATP- form.20In view
of the significance of these findings, we investigated the effect of DIDS on ATP binding in our system. GM-CSFtreated and IFN-y-treated THP- 1 cells were incubated for
I5 minutes at 37°C with 100, 200, and 300 pmol/L DIDS
before being washed and incubated for 4 more hours in the
presence of 1.25 mmol/L ATP. The lysis of both types of
cells was significantly blocked in a dose-dependent manner
by DIDS (Fig 4). The lysis of IFN-y-treated cells was 69%
2%,41% & 2%, 23% k 1%, and 19%& 1% in the presence of
0, 100, 200, and 300 pmol/L DIDS, respectively. At higher
concentrations of DIDS, direct toxicity of this compound
on THP-1 cells was noted. Similar inhibition of ATP-mediated lysis by DIDS was seen using GM-CSF-treated THP1 cells, although lysis by ATP was not as pronounced. These
results suggest that ATP may act on a common DIDS-sensi-
*
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1582
SPRANZI ET AL
100
100
80
80
8
6 0
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60
40
40
20
20
P
%-
in
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s
0
0
0
Fig 4. Dose-response relationship characterizing the inhibitow
action of DlDS on ATP-induced lysis of THP-1 cells. THP-1 cells
were cultured for 3 days with the indicated cytokines and used as a
target cells in a 6-hour 5'Cr release assay. Before adding 1.25
mmol/L ATP, the cells were incubated with the indicated concentration of DlDS for 15 minutes. Numbers are means f SEM of
triplicate determinations and data are representative of three experiments that were performed with similar results. (O), GM-CSF;
fH), IFNg.
tive purinergic receptor present on the surface membrane of
THP-I cells.
Effect of ATP analogues. Recent studies on the classification of the PZzreceptor has been done using analogues of
ATP as agonists." P,, receptors are characterized by a potent agonist, 2MeSATP, commonly considered the key analogue in the definition of P,, receptor, whereas the compound BzATP is an effective agonist of the Pzzpurinoceptor
on mouse fibroblast and rat parotid acinar cells.21.22
Therefore, we attempted to explore the nature of the purinergic
receptor responsible for the lysis of THP- 1 cells in the presence of ATP by comparing the effect of BzATP, 2MeSATP,
and ATP on IFN-y-treated cells. Figure 5 shows the high
lytic effect of BzATP, which is similar to the potency of
ATP, whereas the cells were completely unresponsive to
2MeSATP. The percent of specific lysis stimulated by
BzATP at 1.25 mmol/L was 48% f 2.5%, whereas the specific lysis induced by 2MeATP was considerably lower at
2.5% k 1.5%. These findings suggest that the involvement of
the Pzz receptor in the lysis of THP-I cells is more likely
than the involvement of the P,, receptor, although other P,
receptors also respond to BzATP.
Eflect of Cd+/culmodulininhibitors. The assignment of
a specific role for the Caz+/calmodulin complex in the regulation of permeabilization by external ATP has been described in the murine transformed cell line 3T6 and B16
melanoma cell lines.z,23.24
In those studies, extracellular
ATP drastically increased membrane permeability to Na+
and K+ leading to a decrease in plasma membrane potential
and an increase in nonselective permeability, and all these
events were regulated by the Ca'+/calmodulin complex.
Other evidence in our laboratory suggested a role for the
0.16
0
100
200
300
CONCENTRATION OF DlDS (chm
0.31
0.62
1.25
CONCENTRATION (mM)
2.5
Fig 5. Effect of ATP analogues on lysis of THP-1 cells. THP-1
cells were cultured with IFN-y for 3 days before being used as target cells in the presence of the indicated concentration of ATP or
analogues. The mean f SEM of triplicate determination is shown.
Data are representative of three experiments that were performed
with similar results. (0).
ATP; (+), 2MeSATP; (e), BzATP.
Ca2+/calmodulin in the signal transduction pathway leading to the cell death of human macrophages (manuscript in
preparation). Based on these data, we examined the role of
Ca*+/calmodulin in the cellular processes of THP- 1 cells
after exposure to ATP. Therefore, inhibitors ofCa'+/calmodulin-linked enzymes were assessed for their ability to
block ATP-mediated lysis. There are several mechanisms
by which drugs might act to inhibit the action of Caz+/calmodulin. For example, W7 and TFP act by binding to the
complex and modifying its activity.25Figure 6 shows that
Q
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80
I
0
1.25
2.5
5.0
10.0
CONCENTRATION OF TFP (&VI)
Fig 6. Effect of TFP on ATP-mediated lysis of THP-1 cells. Cytokine-treated THP-1 cells were incubated 15 minutes with the indicated concentration of TFP before being used as target cells in a
5'Cr release assay with 1.25 mmol/L ATP. Numbers are means 2
SEM of triplicate determinations and data are representative of
three experiments that were performed with similar results. (El),
GM-CSF; (B),
IFNg.
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1583
ATP LYSES HUMAN LEUKEMIC CELLS
TFP greatly inhibited the ATP-mediated lysis of THP-1
cells treated with IFN-y or GM-CSF. For these assays, we
used a concentration of 1.25 mmol/L ATP, which caused a
specific lysis of 72% f 3% for IFN-y-treated cells and 24% k
2% for GM-CSF-treated cells. Different concentrations of
TFP were added for 15 minutes at 37°C to the cells before
adding ATP for 5 more hours. TFP appeared to be more
effective in inhibiting the ATP-mediated lysis of GM-CSFtreated THP-1 cells than IFN-y-treated cells, because 2.5
pmol/L TFP suppressed lysis of the former cells by 64%,
whereas 5.0 pmol/L concentration was required to elicit the
same level of inhibition in the latter cells. Surprisingly, the
other calmodulin antagonist, W7, was unable to block lysis
induced by ATP of both GM-CSF-treated and IFN-ytreated THP-1 cells (data not shown). Because the calmodulin antagonists TFP and W7 have been shown to interact
with multiple enzyme systems, a third, more selective compound, KN-62, was also used in these assays. Although W7
and TFP inhibit most calmodulin-dependent enzyme activities by interacting with the Caz+/calmodulincomplex, KN62 has been reported to be a selective inhibitor of Ca*+/calmodulin kinase I1 enzyme, because it binds directly to this
enzyme.26We found that KN-62 was able completely to
block lysis of both IFN-y-treated and GM-CSF-treated
THP-1 cells (Fig 7). Serial dilutions of KN-62 were added to
the cells for 15 minutes at 37°C before adding 1.25 mmol/L
ATP and incubating for 6 hours. As little as 0.3 pmol/L of
KN-62 was able significantly to inhibit the lysis of both
GM-CSF-treated and IFN-y-treated cells, with almost
complete inhibition being noted at 2.5 pmol/L. It should be
noted that there was no direct toxicity of these inhibitors
against THP-I cells at the concentrations used in these experiments. Additionally, the effect of TFP cells and KN-62
on the ATP-mediated lysis of THP-1 cells that were not
irI
80
8Q. 6 0
uf
8
p
40
0
20
a\o
0
0
0.3
0.6
1.2
2.5
5.0
CONCENTRATION OF KN-62 (W)
Fig 7. Effect of KN-62 on ATP-mediated lysis of THP-1 cells.
Cytokine-treated cells were incubated 1 5 minutes with the indicated concentration of KN-62 before being used as target cells in
”Cr release assay with 1 2 5 mmol/L ATP. Numbers are means ?
SEM of triplicate determinations and data are representative of
three experiments that were performed with similar results. (0).
GM-CSF; (EA), IFNg.
stimulated with cytokines was not significantly different
from the effect of these inhibitors on GM-CSF-stimulated
cells (data not shown). Thus, these findings suggest that
there is an involvement of Caz+/calmodulinin the ATP-mediated lysis of THP-1 cells whether or not these cells were
activated with cytokines.
DISCUSSION
The present investigation was designed to characterize
the mechanism of ATP-mediated lysis ofthe monocytic cell
line THP- I . The lysis induced by ATP has been described
for various cell types, although the mechanism were not
characterized. The experiments reported here showed that
ATP has potent cytotoxic effect on THP- 1 tumor cells. This
result appeared to be selective for ATP, because adenosine,
ADP, UTP, CTP, GTP, and ITP produced no lytic effects.
For IFN-y-treated cells, the ATP-mediated lysis was proportional to the concentration of this purine nucleotide in
the medium and was maximal when the concentration of
ATP was 2.5 mmol/L or more. IFN-y, in fact, greatly increased the sensitivity of the cells to ATP.
The sensitization of THP- 1 cells in the presence of IFN-y
was time and concentration dependent, as the maximal sensitivity to ATP was reached after 3 days with 1,000 U/mL
IFN-y. Not only the sensitivity to ATP was increased but
also the kinetics of the lysis was faster, as more than 70% of
the 5’Cr was released from IFN-y-treated THP-1 cells
within 2 hours after the addition of ATP. For GM-CSFtreated cells, the release of W r in the supernatant was gradual and the maximal lysis after 5 hours was under 30%.
These differences could be explained by a relative decrease in the expression of ecto-ATPase activity on the cell
surface after treatment with IFN-y or by a differential expression of ATP receptors either in the number or in their
affinity for ATP. From our data, it seemed to be unlikely
that the ecto-ATPases can degrade the lytic agent, as the
addition of FSBA, which is known to inhibit the ecto-ATPase activity of cytotoxic T lymphocyte (CTL),I7to the culture medium of GM-CSF-stimulated THP- l cells did not
increase the percent of lysis, ie, 18%k 1% in the presence of
2.5 mmol/L ATP, to the level observed for IFN-y-treated
THP-1 cells, which was 72% f 2% at the same concentration of ATP. Thus, it seems more likely that a surface receptor for ATP was involved in the increased sensitivity of IFNy-treated THP- 1 cells.
The presence of specific surface ATP receptors has been
described by numerous reports in the literature in the past
several years. As summarized in a review by D ~ b y a k , ~ ’
there are at least three classes of ATP-specific receptors
grouped on the basis of their selectivity for various nucleotides and their interaction with agonists or antagonists. P,
is the class of receptors responsible for the formation of
nonselective pores permeable to ions and metabolites up to
1 Kd in mass and leading to cell death, and it is reported to
be the ligand that mediates the lysis of J774 mouse macrophages” and of EL4 thymoma and P8 I5 mastocytoma cell
lines.I7This receptor has been shown to bind the tetrabasic
form.
anion ATPFrom our studies, the same anionic form seems to be the
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SPRANZI ET AL
1584
ligand of the ATP receptor of THP-1 cells, which leads to
cell lysis. By modulating the concentration of the ATP'form in the culture medium with chelating agents or with
Ca2+-freeand Mg2+-freemedium, the lysis of THP-I cells
can be regulated. The observation that the lysis was modulated equally in both GM-CSF-treated or IFN-y-treated
cells supports the suggestion that the ATP'- form was recognized by the same or similar receptors in both cell types.
Moreover, the suppressive effect of DIDS on THP-1 cell
lysis by ATP and the ability of DIDS to block P,,-type purinoceptors in rat parotid acinar cells'' seem to confirm that
the anionic form of ATP is the active receptor ligand for the
purinoceptor on THP- 1 cells.
The effect of ATP analogues, such as 2MeSATP, which
binds the P,, receptor with high affinity," and BzATP,
which is recognized by both P,, and P,, receptors,22provide
support that P,, purinoceptors are the receptors involved in
the ATP-mediated lysis of THP- 1 cells. Their insensitivity
to 2MeSATP compared with their high levels of lysis caused
by the analogue BzATP provide evidence for the central
role of PZzreceptors in our system.
The molecular pathway that mediates ATP-induced cell
death is unknown. We investigated the role of calmodulin
in the signal transduction mechanism leading to cell death
by using the metabolic inhibitors TFP and W7, which have
been widely used to study Ca'+/calmodulin-dependent enzyme system^.^^^^^ The efficacy of the calmodulin antagonist
TFP to block the lysis of THP-1 cells suggests the involvement of calmodulin protein in the ATP-mediated lysis, although the inefficacy of W7 brings to our attention the possibility that these inhibitors could interact with calmodulin
via more than one mechanism. Moreover, it has been
shown that calmodulin inhibitors, such as W7 and TFP,
have different selectivity for the hydrophobic sites on calmodulin and might therefore interact differently with this
calcium-binding p r ~ t e i n . ~ ~ , ~ '
The efficacy of the more selective inhibitor KN-62, which
binds directly to the enzyme Ca2+/calmodulinkinase I1 and
not to calmodulin alone,26in blocking THP- 1 cell lysis provides further evidence that calmodulin is involved in the
ATP-mediated lysis of both IFN-y-treated and GM-CSFtreated cells. Obviously, the involvement of this enzyme in
the pathway leading to cell death warrants further investigation.
In conclusion, there were many similarities between the
lytic effect of ATP on human macrophages5 and the monocytic cell line THP-1. Such findings prompt us to speculate
that, in our model, the physiologic role of ATP may be the
downregulation of macrophages that have been stimulated
with IFN-y during an immune response. It would be interesting to determine the relative expression of the P,, purinoceptor from the late myeloblast stage through the terminally
differentiated stages represented by circulating monocytes
and how stimulation with IFN-y might modulate this receptor. From the data presented in this article, it is tempting
to speculate that IFN-y treatment of THP- 1 cells increases
the expression of the putative ATP receptor. This is supported by the observation that little difference, other than
increased sensitivity to ATP lysis, was noted between IFN-
treated and INF-untreated cells; ie, all cells responded similarly to the range of inhibitors used in this study. However,
until the PZzpurinoceptor can be quantitated, the relationship between IFN-y and the ATP receptor on myeloid cells
will remain unidentified.
REFERENCES
1. Pizzo P, Zanovello P, Bronte V, Di Virgilio F: Extracellular
ATP causes lysis of mouse thymocytes and activates a plasma
membrane ion channel. Biochem J 274: 139, 1991
2. Kitagawa T, Amano F, Akamatsu Y: External ATP-induced
passive permeability change and cell lysis of cultured transformed
cells: Action in serum-containing growth media. Biochim Biophys
Acta 941:257, 1988
3. Di Virgilio F, Bronte V, Collavo D, Zanovello P Responses of
mouse lymphocytes to extracellular adenosine 5'-triphosphate
(ATP). Lymphocytes with cytotoxic activity are resistant to the permeabilizing effect of ATP. J Immunol 143:1955, 1989
4. Zanovello P, Bronte V, Rosato A, Pizzo P, Di Virgilio F Responses of mouse lymphocytes to extracellular ATP. 11. Extracellular ATP causes cell type-dependent lysis and DNA fragmentation. J
Immunol 145:1545, 1990
5. Blanchard DK, McMillen S, Djeu JY: IFN-y enhances sensitivity of human macrophages to extracellular ATP-mediated lysis. J
Immunol 147:2579, 1991
6. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno
T, Tada K: Establishment and characterization of a human acute
monocyte leukemia cell line (THP-I). Int J Cancer 26: 171, 1980
7. Krakauer T, Oppenheim JJ: Interleukin 1 production by a
human acute monocytic leukemia cell line. Cell Immunol 80:223,
1983
8. Burnstock G, Kennedy C: Is there a basis for distinguishing
two types of P, purinoceptor? Gen Pharmacol 16:433, 1985
9. Gordon J L Extracellular ATP: Effects, sources and fate. Biochem J 233:309, 1986
IO. Means AR, VanBerkum MFA, Bagchi I, Lu KP, Rasmussen
CD: Regulatory functions of calmodulin. Pharmacol Ther 50:255,
1991
1 1. OConnor SE, Dainty IA, Leff P Further subclassification of
ATP receptors based on agonist studies. Trends Pharmacol Sci
12:137, 1991
12. Tatham PER, Cusack NJ, Gomperts B D Characterization
of the A T P - receptor that mediates permeabilization of rat mast
cells. Eur J Pharmacol 147:13, 1988
13. Steinberg TH, Newman AL, Swanson JA, Silverstein SC:
ATP'- permeabilizes the plasma membrane of mouse macrophages
to fluorescent dyes. J Biol Chem 262:8884, 1987
14. Heppel LA, Weisman FA, Friedberg I: Permeabilization of
transformed cells in culture by external ATP. J Membrane Biol
86:189, 1985
15. Cockcroft S, Gomberts BD: The ATP'- receptor of rat mast
cells. Biochem J 188:789, 1980
16. Steinberg TH, Silverstein SC: Extracellular ATP'- promotes
cation fluxes in the 5774 mouse macrophage cell line. J Biol Chem
262:3118, 1987
17. Filippini A, Taffs RE, Agui T, Sitkovsky MV: Ecto-ATPase
activity in cytolytic T-lymphocytes. J Biol Chem 265:334, 1990
18. Ohta T, Nagano K, Yoshida M: The active site structure of
Na+/K+-transporting ATPase: Location of the 5'-(p-fluoro-sulfony1)benzoyladenosine binding site and soluble peptides released
by trypsin. Proc Natl Acad Sci USA 83:207 1, 1986
19. McMillian MK, Soltoff SP, Lechleiter JD, Cantley LC, Talamo BR: Extracellular ATP increases free cytosolic calcium in rat
parotid acinar cells. Biochem J 255:291, 1988
20. Soltoff SP, McMillian MK, Cragoe EJ, Cantley LC, Talamo
From www.bloodjournal.org by guest on August 1, 2017. For personal use only.
A T P LYSES HUMAN LEUKEMIC CELLS
BR: Effects of extracellular ATP on ion transport systems and
[Ca2+],in rat parotid acinar cells. J Gen Physiol 95:3 19, 1990
21. Erb L, Lustig KD, Ahmed AH, Gonzalez FA, Weisman GA:
Covalent incorporation of 3'-0-(4-benzoyl)benzoyl-ATP into a Pz
purinoceptor in transformed mouse fibroblasts. J Biol Chem
265:7424, 1990
22. Soltoff SP, McMillian MK, Talamo BR: ATP activates a
cation-permeable pathway in rat parotid acinar cells. Am J Physiol
262(Cell Physiol 3 1):C934, 1992
23. De BK, Weisman G A The role of calcium ions in the permeability changes produced by external ATP in transformed 3T3 cells.
Biochim Biophys Acta 775:381, 1984
24. Gonzalez FA, Ahmed AH, Lustig KD, Erb L, Weisman GA:
Permeabilization of transformed mouse fibroblasts by 3'-0-(4-benzoy1)benzoyl adenosine 5'-triphosphate and the desensitization of
the process. J Cell Physiol 139:109, 1989
25. Prozialeck WC: Structure-activity relationships of calmodulin antagonists. Ann Reports Medical Chem 18:203, 1983
26. Tokumitsu H, Chijiwa T, Hagiwara M, Mizutani A, Tera-
1585
sawa M, Hidaka H: KN-62, l-[N,O-bis(5-isoquinolinesulfonyl)-Nmethyl-L-tyrosyl]-4-phenylpiperazine,
a specific inhibitor of Ca2+/
calmodulin-dependent protein kinase 11. J Biol Chem 265:43 15,
1990
27. Dubyak GR: Signal transduction by P,-purinergic receptors
for extracellular ATP. Am J Respir Cell Mol Biol4:295, 199 I
28. Greenberg S, Di Virgilio F, Steinberg TH, Silverstein SC:
Extracellular nucleotides mediate Ca2+fluxes in 5774 macrophages
by two distinct mechanisms. J Biol Chem 263:10337, 1988
29. Schulman H: The multifunctional Ca2+/calmodulin-dependent protein kinase. Adv Second Messenger Phosphoprotein Res
22:39, 1988
30. Schaeffer P, Luginer C, Follenius-Wund A, Gerard D, Stoclet JC: Comparative effects of calmodulin inhibitors on calmodulin's hydrophobic sites and on the activation of cyclic nucleotide
phosphodiesterase by calmodulin. Biochem Pharmacol 36: 1989,
1987
3 1. Zimmer M, Hofmann F Differentiation of the drug-binding
sites of calmodulin. Eur J Biochem 164:411, 1987
From www.bloodjournal.org by guest on August 1, 2017. For personal use only.
1993 82: 1578-1585
Lysis of human monocytic leukemia cells by extracellular adenosine
triphosphate: mechanism and characterization of the adenosine
triphosphate receptor
E Spranzi, JY Djeu, SL Hoffman, PK Epling-Burnette and DK Blanchard
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