[CANCER RESEARCH 43. 4402-4406.
September 1983)
Growth Inhibition of Human Tumor Cells in Soft-Agar Cultures by
Treatment with Low Levels of Adenosine 5'-Triphosphate
Eliezer Rapa port,' Robert F. Fishman, and Cicek Gercel2
Department ol Microbiology and the Hubert H. Humphrey Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118
ABSTRACT
Treatment of a variety of human tumor cells in monolayer
cultures with low levels (40 to 80 UM) of adenosine 5'-diphosphate (ADP) or adenosine 5'-triphosphate (ATP) was recently
shown to produce arrest of cellular growth in the S phase of the
cell cycle (E. Rapaport, J. Cell. Physiol., 774: 279-283, 1983).
We now demonstrate that exposure of two well-characterized
colonie adenocarcinoma (HT-29 and SW-620) and two pancreatic
adenocarcinoma (CAPAN-1 and PANC-1) cell lines in soft-agar
cultures to exogenously supplied 5 to 20 ßMATP results in
substantial inhibition of cellular growth. Exposure of the cells to
5 to 20 U.M ADP produces slightly smaller growth-inhibitory
effects, while 20 UM adenosine 5'-monophosphate or adenosine
have marginal effects on cellular proliferation in these systems.
Successful demonstration of these effects requires the use of
heat-inactivated fetal bovine serum, since normal fetal bovine
serum possesses enzymatic activities which catalyze the rapid
degradation of adenine nucleotides. Tumor cell growth was
assayed by the well-established colony formation assay as well
as by [3H]thymidine incorporation into acid-insoluble material.
[3H]Thymidine incorporation is performed 4 to 14 days after
tide pools, we concluded that the observed effects are the result
of en bloc incorporation of small amounts of ADP and ATP into
the cellular acid-soluble nucleotide pools (23). Substantially
higher levels (0.2 to 0.5 mw) of ATP were recently reported to
perturb the plasma membrane of a variety of transformed cells,
causing release of cellular pools of nucleotides and phosphate
esters into the medium within minutes after treatment (18, 26).
These effects have been suggested to depend on the phosphorylation of a membrane protein by externally added ATP (4) and
could not be reproduced by the use of ADP (13).
Since ATP levels of about 1 ^M have been identified in human
plasma under certain physiological conditions (7, 19), studies of
the chemotoxicity of 5 to 20 U.MATP towards human tumor cells
are of particular interest with regard to the possible selective
killing of tumor cells in a host.
Human tumor cell growth was monitored by tumor colony
formation assay and by a modification of a [3H]thymidine incor
phatase catalyzing the dephosphorylation of ATP and ADP, the
effective levels of ATP that inhibit the growth of human tumor
cells in this system, which is widely claimed to predict the in vivo
response of a tumor, are lower than the 5 to 20 UM which are
exogenously supplied. The two previously characterized, welldifferentiated pancreatic and colonie tumor cell lines (CAPAN-1
and HT-29) were shown to exhibit higher chemosensitivity to
wards treatment with ATP and ADP than did the lesser-differ
entiated pancreatic and colonie tumor cell lines (PANC-1 and
SW-620).
poration procedure (30) in cells cultured in soft agar. The human
tumor stem cell assay developed by Hamburger eÃ-al. (9, 27) is
commonly used for the screening of potential antineoplastic
drugs (31) as well as for the selection of therapy for individual
patients (32). The predictive value of this system for clinical
response (31) has, however, been questioned (28). We have
utilized a cytostatic assay which is based on continuous expo
sure of human tumor cells in soft-agar cultures to ATP and ADP
in order to test the growth-inhibitory properties of these agents.
We demonstrate here that low levels (5 to 20 ^M) of exoge
nously supplied ATP and ADP yield significant inhibition of growth
of established human tumor cell lines in soft-agar cultures. The
effective concentrations which lead to substantial inhibition of
tumor cell growth are well below 5 to 20 UMATP or ADP supplied
to the medium because of ectoenzymatic activities associated
with the cells which catalyze the degradation of adenine nucleo
tides.
INTRODUCTION
MATERIALS AND METHODS
We have recently demonstrated that the plasma membranes
of several lines of human tumor cells but not of untransformed
human cells in culture are permeable to low levels of ATP and
ADP (23). Treatment of human tumor cells in monolayer cultures
with low levels of exogenously supplied ATP or ADP yielded
arrest of these cells in the S phase of their cycle followed by cell
death. Since the rapid conversion of cellular ADP to ATP in tumor
cells is known (21) and since none of the known breakdown or
modification products of ATP or ADP could produce similar
effects on cellular growth or on total cellular acid-soluble nucleo-
Materials. Nucleotides (as sodium salts) were purchased from Sigma
Chemical Co., St. Louis, Mo. Highest purity grade was obtained and
used without further purification (23). Agarose (SeaKem) was purchased
from FMC Corporation, Rockland, Maine. Cells were cultured in modified
MEM3 (containing 4-fold the amounts of vitamins and amino acids
' To whom requests for reprints should be addressed.
2 Supported by Training Grant CA-09423 from the National Cancer Institute.
3 The abbreviations used are: MEM, modified minimum essential medium; PBS.
phosphate-buffered saline (8 g NaCI. 0.2 g KCI, 1.15 g NaHPO«7H2O. and 0.2 g
KH2PO4 per liter); i.a., intraarterial.
plating and correlates well with results obtained by colony for
mation assays. Due to the ectoenzymatic activities of the cells
which include adenosinetriphosphatase
and adenosinediphos-
Received March 17, 1983; accepted June 10. 1983.
4402
specified for MEM) from Grand Island Biological Company, Grand Island,
N. Y. Media were supplemented with antibiotics (100 units penicillin per
ml and 100 fig streptomycin per ml), L-glutamine (2 rriM), 10% fetal bovine
serum for routine culture procedures and 15% heat-inactivated fetal
bovine serum (20 hr at 58°)for soft-agar cultures.
Cell Lines. The cell lines used in this study were: CAPAN-1, a
CANCER
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VOL. 43
Inhibition of Tumor Cell Growth by ATP
pancreatic adenocarcinoma (14) obtained from Dr. J. Fogh of the SloanKettering Institute for Cancer Research, Rye, N. Y.; PANC-1 a pancreatic
44Jr
HT 29
adenocarcinoma (17) obtained from the Naval Bioscience Laboratory,
Naval Supply Center, Oakland, Calif.; SW-620, a colonie adenocarcinoma
(15) obtained from the American Tissue Culture Collection; and HT-29,
20
a colonie adenocarcinoma (6) obtained from the Naval Bioscience Lab
oratory. All cell lines were of epithelioid morphology and were shown to
be free of Mycoplasma contamination.
Preparation of Soft-Agar Cultures. Cells were cultured in soft agar
using a modification of the procedure of Hamburger and Salmon (9).
Layers of 1 ml of 0.5% agarose in complete medium (modified MEM
containing 15% heat-inactivated fetal bovine serum, antibiotics, and
glutamine) were prepared in 35-mm Petri dishes. Cells were prepared by
trypsinization and suspended in 1 ml of 0.3% agarose (total volume) in
complete medium and plated on top of the agar underiayer. The final cell
concentrations were 5 x I04/plate. The appropriate concentrations of
nucleotides were included in both layers by using stock solutions of 2
m« concentrations of nucleotides in modified MEM (without serum).
Plates were inspected for the presence of good single-cell suspensions
and were incubated in a 5% CO2 humidified incubator at 37°.In order to
obtain continuous exposure to nucleotides, an appropriate concentration
of nucleotides in 200 /¿Iof modified MEM without serum was added
every 24 hr as well as 200 n\ of modified MEM to the control dishes.
These additions were performed starting 24 hr after plating, for 10 days
(or 6 days for SW-620 cells). Colony counts were performed 17 to 20
days after plating, using an inverted microscope. Only colonies containing
aggregates of more than 30 cells were counted.
Incorporation of [3H]Thymidine into Acid-insoluble Material. |:|H|-
100
80
60
40
20
10O
-80
60
-40
-20
PANC-1
10
E
d
100
80
60
4O
20
20
10
5
10
15
20
UHI
80
60
40
20
5
10
15'
17
Time After Plating (days)
Chart 1. Inhibition of [3H]thymidine incorporation and tumor cell colony formation
by treatment of human tumor cells in soft-agar cultures with adenine nucleotides.
, time course of [3H]thymidine incorporation. Tumor colony formation assays
are recorded by symbols only at the right side of each panel. Each point is the
average of 2 experiments, x, control (untreated) dishes; O, 20 /JM adenosine; •,
20 MMAMP; G, 20 MMADP; •20 M" ATP; A, 10 MMATP; A, 5 MMATP.
Thymidine (5 ¿iCi;specific activity, 16 Ci/mmol) in 0.5 ml of MEM was
carefully layered over the agar layers, and incorporation was allowed to
proceed for 20 hr. The excess medium was then removed, and 2 ml of
3% glutaraldehyde in Hanks' balanced salt solution were added. The
poration into acid-insoluble material were performed on Days 6,
10, and 14 after plating, while colony formation assays were
conducted on Days 17 or 20. Whereas the colony counts provide
an overall assessment of the growth-inhibitory effects of the
nucleotides, the [3H]thymidine incorporation assays yield data
dishes were incubated at room temperature for 30 min, the liquid was
carefully removed from the top of the agar, and the dishes were then
placed at -20° for 30 min. After warming to room temperature, both
relating to the time course of the inhibitory effects and suggest
mechanistic aspects of the inhibitory activities. In all cases, the
[3H]thymidine incorporation data correlate well with the clono-
agar layers were removed with the aid of boiling PBS (6 ml) into 15-ml
genic assays. The 2 well-differentiated tumor lines, CAPAN-1
and HT-29, were extremely sensitive to treatment with low levels
of ATP or ADP. Colony-forming efficiencies and [3H]thymidine
plastic centrifuge tubes placed in a boiling water bath. Unlabeled cells
(106) in 1 ml of PBS were then added to each tube. These cells were
used as unlabeled carriers for the effective centrifugaron and pelleting
of the labeled cells. The cells were then centrifuged at 800 x g at room
temperature for 5 min, and the supernatant was aspirated. The cell pellet
was suspended in 10% trichloroacetic acid containig 0.1 M pyrophos
phate and precipitated onto glass fiber filters which were washed,
digested with NCS, and counted in a toluene-based scintillation cocktail.
Determination of Adenine Nucleotides. At the times indicated, 50 /il
of media were removed from the soft-agar dishes, and the levels of ADP
and ATP were determined by high-pressure liquid chromatography ac
cording to our published procedures (22, 24).
Determination of Ecto-S'-nucleotidase
Activity. Log-phase monolayer cultures 4 days after plating [in order to allow the ecto-5'-nucleotidase activity which was partially destroyed by trypsin treatment enough
time to recover (12)] in 35-mm Petri dishes were washed 3 times with
MEM without serum and incubated with 20 ^M AMP in MEM. Determi
nation of ecto-5'-nucleotidase
activity was according to published pro
cedures (20). Aliquots of the media were removed at a variety of time
points, and the rate of degradation of AMP was determined by high-
incorporation on the 10th and 14th day after plating were dra
matically reduced by exposure of the cells to low levels of ATP
or ADP (Chart 1; Table 1; Figs. 1 and 3). ATP was more effective
than were comparable concentrations of ADP. Whereas adeno
sine or AMP were not cytostatic towards CAPAN-1, they inhib
ited the growth of HT-29 cells, although less than ADP or ATP.
It is thus suggested that the growth-inhibitory effects of ATP and
ADP are not due to their degradation products, AMP or adeno
sine. Inhibition of tumor cell colony formation by the 2 lessdifferentiated cell lines was also observed. In this case, the
inhibition was much more pronounced with ATP than with ADP,
AMP, or adenosine (Chart 1). The [3H]thymidine incorporation
into CAPAN-1 cells 6 days after plating (Chart 1) and into HT-29
cells 4 days after plating (data not shown) suggests the mecha
nism of the cellular response to ATP and ADP treatment. The
higher levels of [3H]thymidine incorporation into the treated cells
Inhibition of Human Tumor Cell Proliferation by Low Levels
of Exogenously Supplied ATP and ADP. The significant inhibi
tion of human tumor cell proliferation in soft-agar cultures
versus the control (untreated) cells, which are similar to results
obtained in monolayer cultures (23), suggest that at this point
significant populations of growing cells are accumulated in the S
phase of their cycle. This interpretation is based on the findings
that, although DNA synthesis (elongation) is slowed down in the
treated cells, resulting in increased population of cells in the S
phase of their cycle, the specific activity of the precursor [3H]-
achieved by treatments with low (5 to 20 ßM)levels of ATP or
ADP is demonstrated in Chart 1. Assays of [3H]thymidine incor-
dTTP pool, which is markedly elevated in S phase, leads to the
observed increases in [3H]thymidine incorporation (23). The time
pressure liquid chromatography
(22, 24).
RESULTS
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1983
4403
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E. Rapaport et al.
course of [3H]thymidine incorporation (Chart 1) indicates that a
10-day exposure to ATP and ADP (6 days in the case of SW-
Table 2
ATP levels in the media of soft-agar cultures of human tumor cells
ExogenousATP (20 ^M final concentration)was added every 24 hr, after removal
620) is sufficient to provide irreversible inhibition of growth of
of a samplefor the assay of ATP and ADP by high-pressureliquid chromatography.
CAPAN-1 and HT-29 cells. There is less growth-inhibitory activity
Concentration in the medium (^M)at following times
after plating8
in the cases of PANC-1 and SW-620, and the inhibition may not
be totally irreversible. However, treatment of all the cell lines with
ATP for 10 days produced significant reduction in colony for
days33.4 days3.9 days0.167.42.76
lineSW-620HT-29PANC-1CAPAN-1NucleotideADP
Cell
mation even 5 weeks after plating (data not shown).
i16.6
37.711.8 15.116.3
ATPADP
7.511.4
Treatment of these 4 human tumor cell lines in monolayer
cultures with 20 ^M 0,7-imidoadenosine 5'-triphosphate and a,ßmethyleneadenosine
5'-triphosphate
did not inhibit cellular
growth, whereas 20 /JM ATP produced marked inhibition of
growth under the same conditions. The inhibitory effects of ATP
did not depend on its source [equine muscle, synthetic ATP
produced by phosphorylation of adenosine, or ATP from a variety
of sources that was further purified by chromatography on
DEAE-cellulose columns (23)], while 20 MMAMP did not produce
similar inhibition of growth regardless of its source (data not
shown). We thus conclude that inhibition of human tumor cell
growth by ATP is not likely to be the result of a contaminating
agent.
Degradation of Adenine Nucleotides by Ectoenzymatic Ac
tivities of Cells Growing in Soft-Agar Cultures. The concentra
tions of ATP and ADP in the medium of soft-agar cultures
supplied with 20 »MATP (final concentration) every 24 hr were
determined. Due to the ectoenzymatic ATPase and ADPase
activities of the cells (29), levels of ATP or ADP to which the cells
were actually exposed are substantially lower than the 20, 10,
or 5 UM supplied in the medium. The data outlined in Table 2
demonstrate the levels of ATP and ADP in the medium just
before the additions of 20 U.MATP every 24 hr. Since AMP and
adenosine (produced by ecto-5'-nucleotidase activity), which are
the products of this enzymatic degradation, were shown to
exhibit low growth-inhibitory effects, we conclude that the effec
tive levels of ATP and ADP capable of producing the observed
growth-inhibitory effects are much lower than the levels which
were actually supplied in the medium. Considered together, the
data in Tables 1 and 2 suggest that inhibition of tumor cell colony
formation is directly related to the levels of ATP and ADP (formed
in situ as a result of ecto-ATPase activities) to which the cells
are exposed within the first 10 days after initiation of treatment.
The ecto-5'-nucleotidase activities of the 4 cell lines utilized were
Table 1
efficiencies in soft-agar cultures after treatment with
adenine nucleotides
Cells were cultured in 35-mm Retri dishes. Cells were plated at 5 x 104 per dish,
Tumor cells colony-forming
and colonies were counted 17 to 20 days after plating. Only colonies composed of
more than 30 cells were counted.
Cloning efficiency8
Nucleotide added to the
(MM)NoneAdenosine
medium
ATPADP
1.13.1
1.52.0
2.16.2
ATPADP
0.65.6
0.89.9
2.110.1
4.5b
11.40.3
b4.51.9100.6b6.014
b°i
4.26
ATP1day4.4 0.52days14.625.117.9
2.83days22.6
a ATP was added every 24 hr for a total of 6 days in the case of SW-620 cells
or for a total of 10 days in the case of the other cell lines. Determinations are the
average of 2 experiments.
0 Below the level of detection.
determined to be (nmol AMP metabolized per min per 106 cells):
SW-620, 0.02; HT-29, 1.89; PANC-1, 3.46; CAPAN-1, 3.76.
Thus, only the poorly differentiated SW-620 cell line (15) exhibits
very low levels of ecto-5'-nucleotidase activity.
DISCUSSION
It has been demonstrated recently that a variety of human
tumor cells are permeable to low levels of exogenously supplied
adenine nucleotides and that micromolar quantities of ATP or
ADP, but not their enzymatic degradation or modification prod
ucts, are capable of significantly inhibiting the growth of these
cells in monolayer cultures (23). We now report studies utilizing
a cytostatic assay which employ continuous exposures to ATP
and ADP of human tumor cells in soft-agar cultures. We have
modified the assays of [3H]thymidine incorporation into cells
cultured in soft agar, and the data obtained by this procedure
supplement the well-established tumor colony formation assay.
Four cell lines were chosen for this study because they are wellcharacterized human tumor lines, and each pair of colonie or
pancreatic adenocarcinomas was composed of a well-differen
tiated tumor line and a lesser-differentiated tumor line. CAPAN1 cells (14), contrary to PANC-1 (17) cells, aggregate in monolayer cultures to form glandular-like structures. CAPAN-1 has
been characterized as a "well-differentiated pancreatic adenocarcinoma of ductal origin" (14). Its levels of CEA production,
which have been viewed as an indicator of the degree of tumor
differentiation, are higher than those of PANC-1." The 2 human
colonie tumor lines have been classified as a well-differentiated
line (HT-29) and a dedifferentiated line (SW-620) based on their
cytogenetics
and morphological
features (15). The dedifferen
tiated SW-620 cell line was shown here to exhibit very low ecto(20)AMP
5'-nucleotidase activity, which is in agreement with published
(20)ADP
(20)ADP
reports (29).
(10)ADP
(5)ATP
The data demonstrate that treatment with low levels of ATP
(20)ATP
or ADP affords substantial inhibition of growth of these human
(10)ATP
tumor cells in soft-agar cultures. Adenosine or AMP does not
(5)SW-6200.460.450.380.420.430.460.190.320.38HT-2914.096.473.970.030.090.420.040.100.42PANC-19.549.639.339.869.079.372.714.775.89CAPAN-16.636.166.390.412.194.440.420
' Number of colonies per plate divided by the number of plated tumor cells x
100. Data are the average of 2 experiments.
4404
4H. Z. Kupchik, personal communication.
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VOL. 43
Inhibition of Tumor Cell Growth by ATP
exhibit any cytostatic effects under the same conditions except
in the case of HT-29 cells, which are inhibited by these com
pounds but to a much lesser degree than by ATP or ADP.
Comparable levels of ATP are more effective than ADP, and in
the present study the well-differentiated human tumor lines are
inhibited by ATP or ADP to a much greater extent than the lesser
differentiated cell lines. The cytostatic effects of ATP and ADP
on human tumor cells may not have been observed earlier due
to the presence of specific and nonspecific phosphomonoesterase and phosphodiesterase activities in sera from a variety of
animals (12, 23). These activities lead to rapid degradation of
ATP and ADP. The use of heat-inactivated fetal bovine serum
(20 hr at 58°)drastically reduces the serum phosphomonoesterase and phosphodiesterase activities, enabling the exposure of
human tumor cells to low levels of ATP and ADP and assessment
of their chemosensitivity. Cellular ectoenzymatic ATPase and
ADPase activities contribute to the reduction in ATP and ADP
levels supplied to the medium and require the addition of nucleotides every 24 hr during the first 6 to 10 days after plating. In
this regard, it is important to note that nonspecific phosphomonoesterase activities of human tissues are much lower than the
same activities of comparable mouse tissues (10, 16). Thus, it
may be easier to maintain continuous exposure of human cells
to ATP or ADP as compared with animal cells. The maintenance
of continuous drug exposure for the inhibition of tumor cell colony
formation has recently been emphasized (1).
Assays of ATP and ADP levels actually present in the medium
indicate that the effective concentrations of ATP and ADP which
exhibit the growth-inhibitory effects are much lower than the
initial levels of 5,10, or 20 ßM,respectively, even though nucleotides were added in the media every 24 hr up to the 10th day
after plating. At this point, however, the inhibitory effects pro
duced by low ATP levels can easily be observed by the [3H]thymidine incorporation assay. The [3H]thymidine incorporation
assays correlate well with the colony formation capabilities of
the cells, which were assessed 7 to 10 days later. Whether the
higher chemosensitivity of CAPAN-1 and HT-29 cells towards
ATP as compared with PANC-1 and SW-620 cells is related to
their degree of tumor differentiation or to their reduced capabili
ties of catalyzing the degradation of ATP and ADP over a 10- to
14-day period or is due to other properties of the cells and their
plasma membranes cannot be ascertained from the present
study.
In conclusion, although a variety of transformed animal cells
(4,13,18, 26) and lymphoid cells (8,11) have been affected in a
variety of ways by much higher levels of exogenously supplied
ATP (200 to 500 MM),this is the first demonstration of cytostatic
effects exerted by low (below 10 MMconcentrations of actual in
situ levels) concentrations of ATP and ADP on human tumor
cells. The importance of the observations reported here can be
viewed in accordance with earlier findings that humans and
animals can tolerate i.v. and i.a. injections or infusions of small
amounts of ATP (3, 5, 25). In effect, ATP has been identified in
human plasma (7, 19). Plasma ATP levels, under certain phys
iological conditions, are only one order of magnitude below the
ATP levels which are demonstrated in this report to effectively
inhibit human tumor cell growth.
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Figs. 1 to 4. Human tumor cell lines in sott-agar cultures 3 weeks after plating, x 50. A, control (untreated) cultures; B, cultures exposed to 20 /¡uATP. Fig. 1, HT29; Fig. 2. SW-620; Fig. 3, CAPAN-1 ; Fig. 4. PANC-1 cells.
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VOL. 43
Growth Inhibition of Human Tumor Cells in Soft-Agar Cultures
by Treatment with Low Levels of Adenosine 5 ′-Triphosphate
Eliezer Rapaport, Robert F. Fishman and Cicek Gercel
Cancer Res 1983;43:4402-4406.
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