©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of Cell Growth Inhibitor by Ectoprotein Kinase-mediated Phosphorylation in Transformed Mouse Fibroblasts (*)

(Received for publication, March 17, 1995)

Ilan Friedberg (1)(§) Ilana Belzer (1) Orly Oged-Plesz (1) Dieter Kuebler (2)

From the  (1)Department of Cell Research and Immunology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, 69978 Tel Aviv, Israel and the (2)Division of Pathochemistry, The German Cancer Research Center (DKFZ), 69120 Heidelberg, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(2)-purinoceptors, whereas the P(1)-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+), (^1)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.


EXPERIMENTAL PROCEDURES

Materials

ATP, and most other compounds, were purchased from Sigma and were of the highest purity available. [-P]ATP was obtained from DuPont NEN. Plasticware for cell cultures was purchased from Sterilin (Teddington, United Kingdom). Growth media and sera were obtained from Biological Industries (Beit Haemek, Israel).

Cells, Cell Culture, and Conditioned Media

Mouse fibroblasts, 3T3 cells, and their transformed derivatives, 3T6 cells, were grown in plastic dishes, or multiwell plates, on Dulbecco's modified Eagle's medium (DMEM), containing 100 units/ml penicillin, 100 µg/ml streptomycin, supplemented with 10% heat-inactivated newborn calf serum, at 37 °C, in humidified atmosphere, containing 5% CO(2). Cells were inoculated at 5 10^4 cells/cm^2, unless otherwise mentioned. Balb/c 3T3 cells and their virally transformed derivatives, Balb/c SV40-3T3 cells, were grown under similar conditions. Primary cultures of mouse embryo fibroblasts (MEF cells), and their adenovirus-transformed derivatives (VAD; obtained from R. Ehrlich, Tel Aviv University), were grown in DMEM, containing 10% fetal calf serum.

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(e) - N(o)/N(c) - N(o), where N(o) is the cell number per dish (or well) when additions were made; N(e) is the cell number in the treated culture, and N(c) is the cell number in the untreated one.

Determination of Ecto-PK Activity and Protein Phosphorylation

The activity of ecto-PK was determined as described(37, 39) . Briefly, cells were washed with buffer containing 30 mM Tris-HCl, 5 mM KH(2)PO(4), 70 mM NaCl, 5 mM magnesium acetate, 0.5 mM EDTA, and 75 mM glucose, pH 7.5 (designated P-buffer), and incubated with the same buffer containing 1 mg/ml phosvitin, at 30 °C. After 5 min [-P]-ATP was added to the final concentration of 0.5 µM, 2 µCi/ml, and the incubation continued for the indicated time intervals (usually 15 min) with gentle agitation. The supernatant was transferred to a tube and subsequently precipitated with 10% trichloroacetic acid and resuspended in a small volume of 1 N NaOH, in the cold, three times, to remove absorbed ATP. The radioactivity in the samples was then determined, and the specific activity of the phosphorylated protein was calculated. In some experiments the phosphorylated proteins were subjected to SDS-polyacrylamide gel electrophoresis and autoradiography.

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.

Removal of Ecto-PK from the Cells

Ecto-PK was removed from the cell surface as described(39) . Briefly, cells in culture (e.g. in a 3.3-cm dish) were washed twice with 3 ml of P-buffer and then incubated for 8 min, with gentle agitation, in the presence of 1 ml of P-buffer, containing 1 mg/ml phosvitin, at 30 °C. The cells were washed an additional two times, and the washings were pooled and concentrated by ultrafiltration.

Column Chromatography and Gel Electrophoresis

Hydrophobic Column

Fractogel TSK-butyl-650 (Merck, Darmstadt, Germany) columns (70 x 1.5 cm) were equilibrated with 2.0 M ammonium sulfate solution. Solid ammonium sulfate was added to the sample (e.g. 400 ml of conditioned medium) to the final concentration of 2.0 M. Samples were loaded on the column, and then eluted with a descending gradient of 2.0-0.0 M ammonium sulfate (400 ml), followed by 150 ml of 50 mM potassium phosphate buffer, pH 7.0. The optical density of the eluted solution was continuously monitored, at 280 nm wave length, using a flow-through cuvette (Strama, Essex, UK), Spectronic 601 spectrophotometer (Milton Roy, Rochester, NY), and recorder (Pantons, Japan). Fractions of 10 ml were collected, and the salt concentration was determined by measuring the electrical conductivity of the solution by conductometer (model YSI-32, Yellow Spring Instruments, Yellow Springs, OH). The column was washed with 200 ml 0.1 M NaOH.

Ion Exchange Column

DEAE-cellulose column (7 0.9 cm) was equilibrated with 0.2 M NaCl. Samples (e.g. 2.0 ml) were loaded on the column and eluted with NaCl solution, 0.2-1.2 M. The salt concentration was determined by measuring the electrical conductivity of the fractions, as described previously.

Gel Filtration Column

Sephadex G-50 column (50 0.9 cm) was equilibrated with Tris-HCl buffer, pH 7.2. Samples (up to 0.75 ml) were loaded and eluted with the same buffer, 6 ml/h. Fractions of 1.5 ml were collected.

Desalting and Inhibitory Activity Determination in Column Fractions

Fractions obtained from hydrophobic or ion exchange columns were desalted before their inhibitory activity was determined, by either chromatography on Sephadex G-25 column, pre-equilibrated and eluted with DMEM, or by subsequent dilution and concentrations of the fractions in an ultrafiltration device (Amicon) equipped with a 5-kDa cut-off filter. Desalted samples were added to 1-day-old culture, for determination of their effect on cell proliferation. The final concentration of the samples in the culture was about the same as in the original conditioned medium.

Gel Electrophoresis

SDS-polyacrylamide gels were prepared, used, and stained as described previously(37, 39) , according to standard procedures.


RESULTS

Cell Growth Inhibition by Exogenous ATP

Swiss mouse fibroblasts, 3T3 cells, their transformed derivatives, 3T6 cells, Balb/c-3T3 murine fibroblasts, and their virally transformed derivatives, Balb/c SV40-3T3 cells, were inoculated at various cell densities. After 1 day, ATP (0.3 mM) was added to some of the cultures, and cell proliferation was determined during the following days (Fig. 1). Fig. 1A shows that ATP-induced growth inhibition in 3T6 cells increases with the decrease of the cell density. The effect of ATP on the nontransformed counterparts, 3T3 cells, is much less pronounced and expressed only at a very low cell density (Fig. 1B). The growth of the virally transformed Balb/c SV40-3T3 cells is inhibited by extracellular ATP in a cell density-dependent manner (Fig. 1C), like the growth of 3T6 cells. The effect of ATP on the growth of the nontransformed Balb/c 3T3 cells is very low (Fig. 1D), similar to the effect in Swiss 3T3 cells. ATP-induced growth inhibition is partly abrogated by serum (Table 1). Apparently, the growth rate of the cells in ATP-treated cultures is dependent on the inhibitory effect induced by ATP and the stimulatory effect of the growth factors in the serum. Accordingly, the effect of the serum decreased with the increase of ATP concentration (data not shown).


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 (bullet), 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%.





Conditioned Medium from ATP-treated Cultures Inhibits Cell Proliferation

Cultures of 3T6 cells were grown for 1 or 2 days and then the growth medium was replaced with either conditioned medium from ATP-treated cultures (CM+) or with conditioned medium from untreated ones (CM-). The CM+ inhibits cell proliferation, whereas the CM- only slightly affects cell growth (Fig. 2A). The growth inhibiting factor in the CM+ is probably not ATP, since adenine nucleotide are metabolized in the cell culture within 1 day(26) .


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+ (black square) were transferred to 1-day-old cultures of 3T6 cells. ATP (0.3 mM) was added to parallel cultures (bullet), 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.

The Role of Ectoprotein Kinase in ATP-induced Cell Growth Inhibition

Ectoprotein kinase of the CK type was found on the cell surface of various cells (37, 39, 44) including mouse fibroblasts (45) . Our preliminary study suggests that ecto-PK has a role in the ATP-induced growth inhibition(36) . This possibility was further studied. A significant correlation was found between the activity of ecto-PK and the ability of ATP to exert cell growth inhibition (Fig. 3A). Furthermore, the data show that the activity of ecto-PK in 3T6 cells is higher than the activity in 3T3 cells. In parallel, the ability of ATP to induce growth inhibition is more pronounced in 3T6, as compared with 3T3 cells. It should be mentioned that the inhibition of the nontransformed 3T3 cells occurs only at low cell densities that are not used under standard conditions. In routine experiments >10^5 cells are inoculated per dish (3.3 cm), and the growth inhibition is in the range of 0-10%.


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 10^3 to 3 10^5 cells/dish (3.3 cm). After 1 day the activity of ecto-PK was determined in 3T3 () and 3T6 (black square) 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 (bullet) 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 (black square) and the growth (bullet) 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 10^5 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 10^6 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(2) 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 10^4 and 1 10^5 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.

Some Properties of the ATP-activated Growth Inhibitor

The inhibitory activity of SFCM+ was markedly reduced after incubation with a protease, but not by DNase or RNase, indicating that the inhibitor is a protein (Table 4). Concentrated SFCM+ was subjected to molecular sieve chromatography, and the apparent molecular mass of the inhibitory activity peak was found to be 13 kDa (Fig. 6). Only insignificant inhibitory activity was found during the fractionation of SFCM-. The SFCM+ and SFCM- were subjected, as well, to chromatography on a hydrophobic column, which enables the fractionation of relatively large amounts of nonconcentrated conditioned media (Fig. 7). The inhibitory activity of SFCM+ from 3T6 cells (Fig. 7A) was found in peak 1 (fractions 75-85). In parallel, peak 2 from SFCM- was eluted at a lower salt concentration (fractions 85-100), with no inhibitory activity. The relevant peaks, 3 and 4, obtained during the fractionation of SFCM+ and SFCM-, respectively, from 3T3 cell cultures were eluted almost at the same salt concentration (fractions 85-100) and had no inhibitory activity (Fig. 7B). Thus, peak 1, containing the inhibitory activity, was eluted at higher salt concentration than the inactive peaks 2, 3, and 4. This difference might be due to protein phosphorylation that causes alteration of the protein structure and in its affinity to the column. The inhibition induced by the putative inhibitor increased with the increase of its concentration (Fig. 7C). The inhibitory activity in peak 1 (Fig. 7A) was further fractionated on DEAE-cellulose column (Fig. 7D). The inhibitory activity was eluted at relatively high salt concentration, indicating that the putative inhibitor might be negatively charged. No inhibitory activity was detected when fractions included in peak 2 (Fig. 7A) were subjected to the same procedure.




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.




DISCUSSION

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-beta(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. (^2)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.


FOOTNOTES

*
This work was supported in part by grants from the National Council for Research and Development (Israel)/Deutches Krebsforschungzentrum (Heidelberg, Germany); The United States-Israel Binational Scientific Foundation (BSF), Jerusalem; the Israel Cancer Research Fund; and the Israel Cancer Association and the Ela Kodesz Institute for Research on Cancer Development and Prevention. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell Research and Immunology, Faculty of Life sciences, Tel Aviv University, Tel Aviv 69978, Israel.

(^1)
The abbreviations used are: CM+ and CM-, conditioned medium from ATP-treated or untreated cultures, respectively; SFCM+ and SFCM-, serum-free conditioned medium from ATP-treated or untreated cultures, respectively; PK, protein kinase; DMEM, Dulbecco's modified Eagle's medium; MEF, mouse embryo fibroblasts (primary cultures); VAD, MEF transformed by adenovirus; CK, casein kinase; CKI and CKII, casein kinase type I and II, respectively; AGI, ATP-induced growth inhibitor.

(^2)
I. Friedberg, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. W. Pyerin for a generous supply of purified CKII.


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