(Received for publication, March 17, 1995)
From the
Our previous studies have shown that exogenous ATP induces cell growth inhibition in transformed mouse fibroblasts, 3T6 cells, whereas the growth of their nontransformed counterparts, Swiss 3T3 cells, is only slightly affected. In this study a similar selective, ATP-induced growth inhibition was found in Balb/c SV40-3T3 cells and in primary cultures of adenovirus-transformed murine fibroblasts. The inhibitory activity was found in the conditioned media of ATP-treated cultures. Several lines of evidence have shown that ectoprotein kinase (ecto-PK) plays a major role in the ATP-induced growth inhibition. (a) There is a good correlation between the activity of ecto-PK and the ability of ATP to induce cell growth inhibition. (b) The removal of the ecto-PK from the cell surface prevents the ATP-induced growth inhibition. (c) Addition of the removed enzyme to the cell culture reconstitutes the ability of ATP to induced growth inhibition. (d) Serum-containing, or serum-free, conditioned media from untreated cultures gain an inhibitory activity after their phosphorylation, and dephosphorylation of conditioned media from ATP-treated cultures results in the loss of the inhibitory activity. (e) Growth medium by itself does not inhibit cell proliferation after its phosphorylation. The findings described in d and e indicate, as well, that the ATP-induced growth inhibitor is produced by the cells. The putative inhibitor was found to be a protein, with an apparent molecular mass of 13 kDa. The selectivity of the inhibition for transformed cells is due to the higher level of ecto-PK in these cells, as well as to their higher susceptibility to the inhibitor, as compared with their nontransformed counterparts.
The pioneering study of Drury and Szent-Gyorgyi (1) indicated that purine nucleotides and nucleosides may serve
as signaling molecules. This possibility has been established by
numerous studies in which extracellular ATP (and other purine
nucleotides) was found to affect various physiological processes,
including neurotransmission, histamine secretion, platelet aggregation,
vasoconstriction and dilatation, heart function, and many
others(2, 3, 4, 5, 6) .
These effects were found to be mediated by receptors for nucleotides
and nucleosides, the purinergic receptors (or purinoceptors). Receptors
that interact with ATP (and certain ATP analogs) were designated
P-purinoceptors, whereas the P
-purinoceptors
respond to adenosine and AMP (and some of their derivatives). The two
main purinoceptor families were divided to subtypes, according to their
interactions with various ligands, and the physiological effects
induced by the activated receptors. The variety of the ATP-induced
biological effects is attributed to the receptor diversity in different
cells(4, 5, 6, 7, 8) .
Recent studies have shown that ATP serves as a fast neurotransmitter in
the peripheral and the central nerve systems(9, 10) .
ATP was found to be released from neurons, blood platelets, and chromaffin cells by exocytosis. In many cases, ATP is co-released with neurotransmitters or hormones, such as acetylcholine, serotonin, catecholamines, and enkephalins(11, 12, 13, 14) . Under stress conditions, such as hypoxia, ATP is released from erythrocytes, heart muscle, and blood vessel cells by an unknown mechanism, which might include membrane channel proteins(15) . Recent studies have shown that overexpression of the mdr1 product, the multidrug resistance protein, as well as the cystic fibrosis transmembrane protein (both are ATP-activated ``anion pumps''), result in release of ATP from the relevant cells (16, 17) . Another source of ecto-ATP is cell damage, like injury. The local, temporary, ecto-ATP level could be rather high, since ATP concentration in the cytosol is 3-5 mM and in the storage vesicles is in the range of 0.1-1 M(2) .
Extracellular ATP has a dual effect on the proliferation of mouse fibroblasts, Swiss 3T3 cells, and their transformed derivatives, 3T6 cells. At low ATP concentrations (<0.05 mM) ATP serves as a co-mitogen, which markedly promotes growth factor-induced cell proliferation. The co-mitogenic effect of ATP was found to be mediated by receptors for ATP, related to the signal transduction system of the inositol phospholipids (18, 19, 20, 21, 22) At higher ATP concentrations (>0.1 mM), however, ATP induces cell growth inhibition in the transformed mouse fibroblasts, 3T6 cells, but not in their nontransformed counterparts, 3T3 cells(23, 24, 25, 26) . Similarly, external ATP was found to inhibit the growth of cells originated from human pancreas, colon, and breast carcinomas and from Friend erythroleukemia(27, 28, 29, 30, 31, 32, 33) . The receptors involved in mitogenesis were found to be desensitized at elevated concentrations of ATP(34) , and thus it is unlikely that they are involved in the ATP-induced growth inhibition.
Several
mechanisms have been suggested for the ATP-induced inhibition: (i) ATP
is taken up by the cells, affects the balance in the cellular
nucleotide pool, and inhibits DNA synthesis in adenocarcinoma cells (27) ; and (ii) external ATP induces potassium ion efflux from
erythroleukemia cells(33) . These effects, however, were not
detected in mouse fibroblasts, at submillimolar concentrations of
ATP(25, 35) . We have shown that slow hydrolysis of
ATP to adenosine, and the continuous uptake of the latter, result in
alterations in the cellular nucleotide pool and induce growth
inhibition(26) . This mechanism, however, can explain the
initiation of the inhibition, but not its continuation, since both
adenine nucleotides and adenosine are metabolized within 1
day(26) . Preliminary experiments indicate that the inhibitory
activity is maintained in the conditioned medium of ATP-treated
cultures (designated CM+), ()after ATP and its
hydrolysis products are metabolized(24) . These findings might
indicate that ATP activates a latent, putative inhibitor in the
CM+. Preliminary observations indicated that ecto-PK has a role in
the ATP-induced cell growth inhibition(36) .
It has been established that ecto-PK is present on the cell surface of various cells and mediates the phosphorylation of certain membrane and extracellular proteins(37, 38) . Most of this activity can be removed from the cell surface by gentle washing with buffer containing a substrate protein(39, 40) . The ecto-PK was found to be serine/threonine protein kinase, similar to the intracellular casein kinase(37, 41) .
In this study we show that ecto-PK activity is necessary for the activation of a latent, putative cell growth inhibitor in the conditioned medium of ATP-treated cultures of transformed mouse fibroblasts. The ecto-PK has a role, as well, in the selectivity of the ATP-induced inhibition for transformed and cancerous cells.
Conditioned media from cultures treated with ATP (CM+) were prepared as follows. ATP was added to one day old culture to the final concentration of 0.3 mM. After an additional day (or 2 days) the conditioned medium was centrifuged to remove cell debris and either used immediately or stored at -70 °C. Concentrated CM+ was prepared by filtration of CM+, using an Amicon ultrafiltration device, equipped with a 5-kDa cut-off filter. Serum-free conditioned media from ATP-treated cultures (SFCM+) were similarly prepared: cells were grown for 1 day in serum-containing medium. Then the cells were washed three times and incubated with serum-free medium, at 37 °C. After 20 min ATP was added to the final concentration of 0.3 mM, and the cells were incubated for an additional day. Then the medium was collected and treated as described for CM+. Conditioned media from cultures that were not exposed to ATP, CM-, and SFCM-, respectively, were prepared using the same procedures, but without ATP.
Cells were
counted in either hemeocytometer or Coulter counter, after their
detachment by trypsinization, centrifugation at 800 g,
and resuspension in growth medium.
The relative growth of cell
cultures was calculated according to the equation: relative growth
= N - N
/N
- N
, where N
is the cell number
per dish (or well) when additions were made; N
is
the cell number in the treated culture, and N
is
the cell number in the untreated one.
Purified CKII from human placenta has been found to be similar to ecto-PK (41) and was utilized for phosphorylation under the same conditions used for determination ecto-PK activity.
Figure 1:
Exogenous ATP selectively inhibits the
growth of transformed mouse fibroblasts. Transformed mouse fibroblasts,
3T6 cells (A), their nontransformed counterparts, Swiss 3T3
cells (B), SV40-Balb/c 3T3 cells (C), and their
nontransformed counterparts, Balb/c 3T3 cells (D), were
inoculated into dishes (3.3 cm) at indicated cell densities (0 h).
After 1 day, ATP (0.3 mM) was added to some of the cultures
(), whereas the other cultures remained untreated (
). Cell
proliferation was determined during the following two days. Data are
mean of six experiments for Swiss cells and two for Balb/c cells, each
done in duplicates. S.D. is less than 15%.
Figure 2:
Effect of conditioned media on cell
proliferation. A, conditioned media from ATP-treated 3T6 cell
cultures, CM+, and from untreated ones, CM-, were prepared
as describe under ``Experimental Procedures.'' The CM-
() and the CM+ (
) were transferred to 1-day-old
cultures of 3T6 cells. ATP (0.3 mM) was added to parallel
cultures (
), and other cultures were not treated (
). Cell
number was determined for 2 additional days. B, conditioned
media from ATP-treated cultures of 3T3 or 3T6 cells were added to
1-day-old cultures of either 3T3 or 3T6 cells. After 2 additional days
the cells were counted, and the relative growth was calculated as
described under ``Experimental Procedure.'' C,
CM+ was concentrated 10-fold by ultrafiltration, using a 5-kDa
cut-off filter. The concentrated CM+ was added to cultures of 3T6
cells, to various final concentrations. After an additional 2 days the
cells were counted, and the relative growth was calculated. D,
serum-free conditioned media from 3T6 cell cultures were prepared as
described under ``Experimental Procedures.'' The SFCM+,
and SFCM-, were concentrated 10-fold by ultrafiltration and added
to 1-day-old cultures of 3T6 cells, to the final concentration
equivalent to the original one. After 2 additional days the cells were
counted, and the relative growth was calculated. Data are the mean of
two to four experiments, each done in duplicates. S.D. is in the range
4-20%.
Conditioned media were prepared in 3T3 and 3T6 cells cultures and were applied to either 3T3 or 3T6 cell cultures. The 3T6-CM+ markedly inhibits the growth of 3T6 cells, but not 3T3 cells (Fig. 2B). The 3T3-CM+ induces small inhibition in 3T6 cells and a moderate inhibition in 3T3 cells. These findings indicate that the selectivity of the ATP-induced inhibition is due not only to the ability of 3T6 cells to produce an inhibitory CM+, but also to the higher susceptibility of 3T6 cells to the 3T6-CM+. The moderate inhibition induced by 3T3-CM- on 3T3 cell growth might be attributed to growth inhibitors involved in the density-dependent growth inhibition that were found to be selective for the nontransformed cells(42, 43) . The CM+ from 3T6 cell cultures was concentrated 10-fold by ultrafiltration and then samples were added to 3T6 cells at the indicated final concentration (Fig. 2C). The inhibitory activity was found to increase with the increase of 3T6-CM+ concentration.
The inhibition induced by SFCM+ was found to be similar to that of CM+, and the slight inhibition obtained in the presence of SFCM- is about the same as induced by CM- (Fig. 2D). Thus, the ATP-activated cell growth inhibitor seems to be a cell product, not a serum component. The inhibitory activity is maintained in CM+ and in SFCM+ after their concentration by ultrafiltration, using a 5-kDa cut-off filter, indicating that the molecular weight of the inhibitor is higher than the filter porosity.
Figure 3:
Correlation between ecto-PK activity and
the ability of ATP to induce cell growth inhibition. A, 3T3
and 3T6 cells were inoculated at various cell densities in the range of
10 to 3
10
cells/dish (3.3 cm). After 1
day the activity of ecto-PK was determined in 3T3 (
) and 3T6
(
) cells. ATP was added to some of the dishes, and after 2
additional days the cells were counted. The relative growth of
ATP-treated 3T3 (
) and 3T6 (
) cells was calculated. The data
are mean of six experiments, each done in duplicates. S.D. is in the
range of 3-10% for cell growth and 7-20% for ecto-PK
activity. B, similar experiments were performed with primary
cultures of MEF and their virally transformed derivatives (VAD). The
ecto-PK activity (
) and the relative growth (
) of
ATP-treated MEF cells was compared with the enzymatic activity (
)
and the growth (
) of ATP-treated VAD cells. Data are mean of two
experiments, each done in duplicate. S.D. is in the range 0-10%
for growth and 0-19% for ecto-PK.
Similar experiments were performed in primary cultures of MEF and
their virally transformed derivatives, VAD (Fig. 3B).
Both ecto-PK activity and ATP-induced cell growth inhibition were found
to be markedly higher in VAD cells as compared with MEF cells. For
example, when ATP was added to 3 10
cells (per
dish) the growth of MEF cells was not inhibited, whereas the growth of
the VAD cells was inhibited by 50%. Even at the density of 10
cells/dish the proliferation of VAD cells was arrested (Fig. 3B). The good correlation between ecto-PK
activity and the ability of ATP to exert cell growth inhibition
suggests that ecto-PK-mediated phosphorylation activates a putative
inhibitor in the conditioned media of ATP-treated cultures. This
suggestion is supported by additional experiments.
Ecto-PK could be removed from the cell surface by washing with a buffer containing a substrate for the kinase (e.g. phosvitin or casein)(39) . The growth of cells washed with a buffer containing either phosvitin or casein was only insignificantly inhibited by ATP (Fig. 4A). The growth of cells washed with buffer containing a nonsubstrate protein, like albumin, was inhibited to the same extent as the control cells, washed with growth medium, or with a protein-free buffer (Fig. 4A). Reconstitution experiments further support the concept that ecto-PK mediates the activation of a latent inhibitor. Cells were washed with a buffer containing phosvitin, and the washings, containing ecto-PK, were concentrated by ultrafiltration. Samples of the concentrated washings were applied to washed cells, to final concentrations in the range of 0.3-3.0-fold, as compared with the original concentration. Than ATP was added to the dishes, and cell proliferation was determined. The ATP-induced inhibition was found to be dependent on the concentration of the washings added, indicating the pivotal role of ecto-PK activity in the ATP-induced cell growth inhibition (Fig. 4B).
Figure 4:
The effects of ecto-PK activity removal
and its re-addition on ATP-induced cell growth inhibition. A,
1-day-old cultures of 3T6 cells were washed three times with either
serum-free growth medium (DMEM) or with P-buffer (see
``Experimental Procedures''). Some of the dishes were washed
with P-buffer containing 1 mg/ml of either albumin, phosvitin, or
casein. Then growth medium was added to the cultures, either with or
without ATP (0.3 mM). After 1 day the cells were counted, and
the relative growth was calculated as the ratio: cell number in
ATP-treated/cell number in untreated ones. B, cultures of 3T6
cell were washed with buffer containing 1 mg/ml phosvitin, three times.
The washing solutions were pooled and concentrated 10-fold by
ultrafiltration, using a 5-kDa cut-off filter. The concentrated washing
solutions were added to 1-day-old cultures of 3T6 cell to the final
concentrations of either 0.3,
1.0, or
3.0, as
compared with the original concentration. ATP (0.3 mM) was
added to some of the cultures, and after 1 day the cells were counted.
The relative growth was calculated as describe in A. Two
experiments were performed, each done in duplicates, with almost
identical results.
The role of protein kinase was further studied, using purified CKII,
which was found to be similar to ecto-PK(41) . The half-life of
the enzymatic activity of the added CKII in cultures of 3T6 or 3T3
cells was found to be about 4 h (Table 2). Cell-free conditioned
media from either 3T3 or 3T6 cells were incubated with either CKII,
ATP, or with CKII and ATP. After 2 days, during which the ATP was
hydrolyzed, and the enzymatic activity was markedly reduced, the media
were added to one day old cultures, and the cells were counted after 2
additional days. Fig. 5A shows that the growth of 3T6
cells was inhibited by conditioned medium preincubated with ATP. A
2-fold increase of the inhibition, however, was obtained by conditioned
medium incubated with ATP and CKII. Conditioned media incubated with
CKII by itself did not inhibit 3T6 cell growth. The finding that
cell-free CM- gains an inhibitory activity after incubation with
ATP indicates that a certain protein kinase activity, probably ecto-PK
activity, is present in the conditioned medium. The activity was
determined and found to be 0.3 pmol of phosphate
mg
protein
15 min
, per
dish. The data are in agreement with the finding of protein kinase
activity in fresh serum(46) . (It should be emphasized that
protein kinase activity was not found in growth media containing
commercially available serum, routinely incubated at 56 °C for half
an hour). The inhibition of 3T3 cells under the same conditions was
much less pronounced than that of 3T6 cells, due to the lower level of
ecto-PK and the lower susceptibility of the cells to the inhibitor (Fig. 5A).
Figure 5:
The effect of casein kinase II and cAMP on
the growth of ATP-treated 3T3 and 3T6 cells. A, conditioned
media from either 3T3 or 3T6 cell cultures were transferred to empty
dishes, and either ATP (0.3 mM), CKII (5 µl), or both,
were added to the dishes. After 2 days of incubation in a CO incubator at 37 °C, the media of 3T3 cells were transferred
to fresh, 1-day-old 3T3 cell cultures and the media from 3T6 cells
transferred to fresh cultures of 3T6 cells. The cell densities were 1
10
and 1
10
cells/dish for 3T3
and 3T6 cells, respectively. After an additional 2 days the cells were
counted, and the relative growth was calculated. Two experiments were
performed with 3T3 cells, and one with 3T6 cells, each done in
duplicates. S.D. was in the range 6-13 percent. B,
1-day-old cultures of 3T6 cell were treated with either ATP, cAMP, or
both, and after 2 days the cells were counted and the relative growth
was calculated. Two experiments were performed, each done in
duplicates, and the S.D. was less than 15%.
In additional experiments serum-free conditioned media were subjected to either phosphorylation or dephosphorylation. SFCM- did not significantly affect cell growth, but after its phosphorylation the medium considerably inhibited cell proliferation. When this medium was dephosphorylated its inhibitory activity disappeared. SFCM+ inhibited cell proliferation, but after its dephosphorylation, the inhibition diminished (Table 3).
An additional protein kinase was found on the cell surface, a cAMP-dependent kinase(44, 47, 48) . Addition of cAMP at low concentration to cultures of 3T6 cells did not affect the ATP-induced inhibition (Fig. 5B). Thus, the cAMP-dependent PK apparently does not play a major role in the ATP-induced growth inhibition.
Figure 6:
Fractionation of SFCM+ from 3T6
cells on gel filtration column. Twenty-fold concentrated SFCM+
from 3T6 cell culture (0.75 ml) was subjected to column chromatography
on a Sephadex G-50 column (50 0.9 cm), pre-equilibrated, and
eluted with 50 mM Tris-HCl buffer, pH = 7.5. The
optical density of the fractions was determined at 280 nm (solid
line). The inhibitory activity of the fractions (dashed
line) was determined as described under ``Experimental
Procedures.'' Trypsin inhibitor, lysozyme, and aprotinin (the
respective molecular masses 21, 14, and 6.5 kDa) were used as molecular
mass markers (arrows).
Figure 7:
Fractionation of SFCM+ and
SFCM- from 3T6 and 3T3 cell cultures on hydrophobic and ion
exchange columns. Conditioned media were prepared as described under
``Experimental Procedures.'' Ammonium sulfate was added to
400-ml batches of SFCMs to the final concentration of 2.0 M.
Each batch was loaded and fractionated on a hydrophobic column
Fractogel TSK-butyl 650 (Merck; 70 1.5 cm), pre-equilibrated
with 2.0 M ammonium sulfate. Fractionation was performed by
using a descending gradient of ammonium sulfate 2.0-0.0 M (400 ml), followed by 50 mM potassium phosphate buffer,
pH = 7.0 (150 ml). The optical density (280 nm) was continuously
determined during the fractionation of SFCM+ (solid line)
and SFCM- (dashed line) from either 3T6 cell cultures (A) or 3T3 cell cultures (B). Ammonium sulfate
concentration was determined by measuring the conductivity of the
fractions (dotted line). C, the inhibitory activity
was determined in the fractions of the hydrophobic column, after salt
removal (see ``Experimental Procedures''). Significant cell
growth inhibition was found only in the fractions of peak 1, from
SFCM+ of 3T6 cells (A, dark arrow). The inhibitory
activities of the parallel peak 2, from SFCM- (A, light
arrow), as well as of the relevant peaks from SFCM+ and
SFCM- from 3T3 cells (B, peaks 3 and 4, respectively)
were negligible. The mean data of three experiments, each done in
duplicate, are presented, and the S.D. is in the range 8-22%. D, fractions of peak 1 (A) were pooled, desalted (see
``Experimental Procedures''), concentrated 10-fold, and
subjected to chromatography on DEAE-cellulose column, using NaCl
gradient (dotted line) of 0.2-1.2 M. Fractions
included in each one of the optical density peaks (solid line)
were pooled, desalted, concentrated, and examined for their ability to
inhibit cell growth. An inhibitory activity was found only in the peak
composed of fractions 51-64 (arrow). Fractions of peak 2 (A) (dashed line) were subjected to the same
procedure, but no inhibitory activity was detected. The mean data of
two experiments, each done in duplicates, are presented. S.D. is in the
range 2-8 percent.
Fractions of either peak 1 or peak 2 were pooled, phosphorylated,
using purified CKII and [-
P]-ATP, and then
subjected to SDS-polyacrylamide gel electrophoresis and
autoradiography. Bands of a 13-kDa phosphorylated protein are shown in
the autoradiogram of the phosphorylated peaks (Fig. 8). The
apparent molecular weight of the phosphorylated protein is the same as
the apparent molecular weight of the inhibitory activity (Fig. 6), suggesting that the a 13-kDa phosphoprotein is the
activated putative inhibitor. A protein band of 13 kDa was not detected
by staining the gel with either coomassie blue or silver stain (not
shown), indicating that the concentration of the inhibitory protein in
the conditioned medium is rather low. The putative ATP-induced cell
growth inhibitor was designated AGI.
Figure 8:
Phosphorylated proteins in conditioned
media and their isolated fractions. SFCM+ and SFCM- were
fractionated on hydrophobic columns, as described in the legend to Fig. 7A. Fractions of either peak 1, or peak 2 (Fig. 7A), were pooled, concentrated by
ultrafiltration, and subjected to phosphorylation in the presence of
[-
P]ATP, either with or without purified
CKII. The samples were subjected to SDS-polyacrylamide gel
electrophoresis and autoradiography, as described under
``Experimental Procedure.'' Lane A, molecular weight
markers. Lanes B and C, peak 1 (Fig. 7A) without or with CKII, respectively. Lanes
D and E, peak 2 (Fig. 7A) without or with
CKII, respectively. Lane F, CKII by itself. Lanes G and H, SFCM+ without or with CKII,
respectively.
The data presented in this study demonstrate that ATP-induced cell growth inhibition in transformed mouse fibroblasts is mediated by a putative, endogenous growth inhibitor, activated by phosphorylation, catalyzed by an ectoprotein kinase. Several lines of evidence support these conclusions. (i) There is a good correlation between the activity of ecto-PK and the ability of ATP to induce cell growth inhibition (Fig. 3; (36) ). (ii) The removal of the ecto-PK from the cell surface prevents the inhibitory activity of ATP (Fig. 4A). Re-addition of the removed enzyme reconstitutes the ATP-induced growth inhibition (Fig. 4B). (iv) Conditioned medium from untreated cultures (SFCM-) gains the ability to inhibit cell growth upon its phosphorylation, and this ability is lost after its dephosphorylation. Similarly, dephosphorylation of SFCM+ results in the loss of the inhibitory activity (Table 3). (v) Growth medium by itself, either with or without serum, does not inhibit cell proliferation after its phosphorylation. Taken together, the findings described in iv and v also indicate that the inhibitor is originated in the cell and not added to the culture with the growth medium.
Three types of ectoprotein kinases were found on the cell surface: CKI (49) , CKII(37, 39, 41, 49) , and cAMP-dependent kinase (47) . Purified CKII was found to activate the inhibitor (Fig. 5). Thus the CKII-like ecto-PK probably plays a major role in the inhibitor activation, whereas the contribution of CKI-like ecto-PK to the activation is unknown. Apparently, the cAMP-dependent protein kinase has no major role in the activation (Fig. 5).
Various data suggest that the putative
inhibitor is a protein. (i) The inhibitor is activated by a protein
kinase. (ii) The inhibitor's activity is lost after its exposure
to protease, but not to nucleotidases (Table 4). (iii) The
molecular mass of the putative inhibitor was found to be about 13 kDa (Fig. 6). (iv) The major phosphoprotein in an inhibitor-enriched
preparation was found to have the same molecular mass (Fig. 8).
(v) In ATP-treated cultures of human breast cancer cells, the
inhibitory activity was found to be in the molecular weight range of
8-24 kDa(32) . Another example for an inhibitor that is
released from the cell in its latent form and activated outside the
cell is the transforming growth factor-(50) .
The ATP-induced cell growth inhibition was found to be selective for transformed mouse fibroblasts (Fig. 1, 2, and 7; (24) ). This selectivity is most likely due to the higher activity of ecto-PK in the transformed cells, which enables the phospho-activation of the inhibitor (Fig. 3). In addition the transformed cells are more susceptible to the inhibitory activity then their nontransformed counterparts ( Fig. 2and Fig. 5). The reason for the higher susceptibility is still obscure. It might be speculated that the effects of the AGI on the cell are mediated by an AGI receptor, preferably present on the surface of the transformed cells. Similar selective, ATP-induced, cell growth inhibition was found in cells originated from human colon cancer as compared with their non-cancerous counterparts(27) .
Taken together, the data obtained suggest that the putative inhibitor AGI is synthesized in the cell, released to the growth medium, and activated by ecto-PK-mediated phosphorylation, in the presence of extracellular ATP. This mechanism implies that ATP is needed for the initiation of the inhibition, but it is not essential for its continuation, whereas according to other suggested mechanisms the presence of ATP is required during the entire inhibition period. It has been suggested that extracellular ATP (or ADP) enters the cells and causes imbalance of the cellular nucleotide pool, inhibition of DNA synthesis, and cell proliferation in human carcinomas of the colon and the pancreas and in melanoma cells(27) . In mouse fibroblasts, however, we did not find an uptake of extracellular ATP(35) . We suggested that the increase in internal ATP in the presence of extracellular ATP is mediated by ATP hydrolysis to adenosine, followed by adenosine uptake and its phosphorylation to adenine nucleotides inside the cells(26) . A similar pathway has been suggested for the increase of ATP pools in erythrocytes and other cells(29) .
In erythroleukemia cells, the inhibition has been attributed to cell
membrane permeabilization by ATP at a relatively high concentration (1
mM) of ATP(33) . Similar ATP-induced permeabilization
was found in mast cells(51) , macrophages(52) , and
certain other cells(4) . In mouse fibroblasts, however, ATP at
submillimolar concentrations does not induce cell membrane
permeabilization in the presence of growth
medium(24, 53, 54, 55) . Thus, the
inhibition induced by ATP at 0.1-0.3 mM is probably not
mediated by membrane permeabilization. The agonist for cell
permeabilization is free ATP (ATP), which serves as
a ligand to the P
-purinoceptor
receptor(4, 51, 56) . It has been suggested
the increase in membrane permeability may lead to considerable increase
of cellular Ca
concentration, followed by
apoptosis(57) . An increase in cytosolic Ca
concentration by either membrane depolarization by ionophores for
monovalent cations (58, 59) or by calcium ionophore (60) was found to induce cytostatic and cytotoxic effects in
nontransformed and in transformed cells, whereas the ATP-induced
effects were detected only in the latter ones. ATP-induced apoptosis
was found in thymocytes and certain tumor cells, after exposure of the
cells to >1.0 mM ATP(61, 62) . Apoptotic
cells were not detected, however, in the cultures of mouse fibroblasts
after the addition of ATP at submillimolar concentrations, in the
presence of growth medium, and apoptosis has been observed only at
concentrations higher than 2.0 mM ATP. (
)It should
be mentioned, however, that ATP-induced cell membrane permeabilization
is not necessarily followed by apoptosis(63, 64) .
It is tempting to speculate that the selectivity of ATP for transformed and cancerous cells could be used for the development of chemotherapeutic agents, either ATP by itself, its derivatives, or the putative inhibitor AGI. ATP has been found to inhibit the development of tumors in experimental animals, to abrogate cachexia, and to improve hepatic function, blood flow, and performance status(28, 29, 30, 65, 66, 67) . Human patients have already been treated with ATP, in cases of coronary artery disease(68, 69) , acute renal failure, multiple organ failure (70) , or cystic fibrosis(71) , without the appearance of significant side effects.