Vol. 187, No. 3, 1992 September
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages 1486-1492
30, 1992
ADENOSINE MODULATES CELL GROWTH IN HUMAN EPIDERMOID CARCINOMA (A431) CELLS H.B. Tey, H.E. Khoo* and C.H. Tan Department of Biochemistry National University of Singapore Kent Ridge Singapore 0511 Received
August
18,
1992
SUMMARY: Adenosine mediates many physiological functions via activation of extracellular receptors. The modulation of cell growth by adenosine was found to be receptor-mediated. In A431 cells adenosine evoked a biphasic response in which a low concentration (-10 uM) produced inhibition of colony formation but at higher concentrations (up to 100 PM) this inhibition was progressively reversed. Evidence for the involvement of Al (inhibitory) and A2 (stimulatory) adenosine receptors in regulating cell growth of these tumor cells was obtained through plating efficiency studies based on the relative potency of adenosine agonists and antagonists, When both Al and A2 receptors were blocked, colony formation or growth was not inhibited at low concentrations of adenosine but was inhibited at high adenosine concentrations. 0 1992 Academic Press, Inc.
Adenosine current advances
mediates
a wide variety of physiological
have made it possible
to elucidate
functions
in man [i] and
the role played by adenosine
receptors in cellular response [2]. It has been proposed that adenosine-reactive sites can be distinguished into “R” and “P” sites. The extracellular membrane-bound R-site is more responsive
to the ribose ring of adenosine
and its analogs. Occupancy
of the R-
site leads to activation of adenylate cyclase. In contrast, the P-site mediates the inhibition of adenylate cyclase. It is located on the internal side of the cell membrane and has a distinct affinity for the purine moiety [3]. The existence of two distinct external adenosine receptors (Al and A2) rather than just one R-site was later demonstrated in cultured brain cells, mast cells and myocardial cells [4]. The Al receptor mediates inhibition of adenylate cyclase and has a relatively higher affinity for R-N6-phenylisopropyladenosine (R-PIA) than for 5’-N-ethylcarboxamidoadenosine (NECA). For the A2 receptor which generally enhances CAMP production, the order of affinity for the adenosine analogs is reversed, NECA being more effective than R-PIA [2]. *To whom correspondence 0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
should be addressed.
Inc. reserved.
1486
Vol.
187,
No.
3,
BIOCHEMICAL
1992
Cultured human epidermoid
AND
BIOPHYSICAL
carcinoma
in the study of cellular and molecular
RESEARCH
COMMUNICATIONS
(A431) cells have been used as a model
responses
evoked by growth factors and other
mitogens [5, 61. We report here that adenosine causes a biphasic response formation of these cells due to the existence of Al and A2 receptors.
MATERIALS
AND METHODS
A431 cells were grown in Dulbecco’s mented with 10% heat-inactivated at 56OC to destroy adenosine
in colony
Modified Eagle’s Medium (DMEM) supple-
Fetal Bovine Serum (FBS,). FBS was heated for 2 hr
deaminase
(ADA) activity. Since our preliminary
work
(Fig. 1A) showed that the inhibition of ADA by 5 PM erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not have any significant effects on cell growth in response to adenosine, the rest of the experiments
were conducted
with only FBS,. For plating studies,
one hundred cells were seeded in Corning multidishes (surface area 35 mm2) containing DMEM supplemented with 10% FBS,. On the following day, cells were treated with varying concentrations
of adenosine
with and without theophylline,
adenosine
analogs
and antagonists for 24 hours in DMEM containing 1% FBS,. The adenosine-containing medium was then removed and replaced by DMEM supplemented with 10% FBS,. Five days after seeding
, the cells were fixed and stained with Giemsa and the colonies
scored by microscopic
examination
of the dishes.
cells in 10% FBSi was 80 - 90% (designated
The plating efficiency
of 100 A431
as control). The number of colonies
in a
test group is expressed as a percentage of the control number. 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was obtained from Research Biochemicals, Natick, Mass., U.S.A. and EHNA was a gift from Wellcome Research Laboratories,
Beckenham,
U.K. All other
chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Statistical analysis was performed
using the Student’s t-test.
RESULTS AND DISCUSSION Exposure to adenosine response (Fig.lA).
at concentrations
of 0.1-1000
Colony formation was progressively
PM produced
a biphasic
inhibited as the concentration
of
adenosine was increased from 1 uM to 10 pM. At a concentration of 10 PM, adenosine produced almost complete inhibition of colony formation. However, inhibition decreased as the concentration
of adenosine
was further increased from 10 uM to 100 PM. At 100
PM adenosine colony formation was approximately 90% of control values. Further increases in adenosine concentration resulted in progressive inhibition of colony formation. The above observations
suggested
the involvement
of two types of extracellular
adenosine receptors (inhibitory and stimulatory) in these cells. To show that both Al and A2 receptors may be responsible for the biphasic response of A431 cells to adenosine, colony formation was studied in the presence of selected adenosine analogs, RPIA and NECA, and antagonists, theophylline and DPCPX. The dose-response curves 1487
Vol.
BIOCHEMICAL
187, No. 3, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
3 p 100 z 8
60
w 0’ F
60
2E
40
0
0.1
Figure
of the adenosine
1 sence
1
10 AGONIST
ADENOSINE l@d)
100
1000
l@vi)
A. Effect of adenosine on plating efficiency of A431 ceils in the ab(0) and presence of 50 pM EHNA (A). B. Effect of R-PIA (0) and NECA (A) on cofony formation. All points are mean + S.E.M. calculated from 5 independent experiments.
agonists were also biphasic
both R-PIA and NECA inhibited
(Fig. 1 B). At a concentration
colony formation
of 10 PM,
but R-PIA was more potent
than
NECA suggesting the presence of Al receptors. On the other hand, 100 uM NECA was more effective than 100 pM R-PIA in supporting colony formation thus indicating the presence of A2 receptors. Since R-PIA and NECA are not metabolized by adenosinemetabolizing enzymes (7), these results support the hypothesis that the effects of adenosine and these agonists on cell growth are modulated via extracellular
receptors.
Xanthines such as theophylline, caffeine, isobutylmethylxanthine and 8-phenyltheophylline are non-selective adenosine antagonists that block the binding of adenosine to both Al and A2 receptors could be substantially
[8]. The biphasic
overcome by 0.1 uM theophylline
response
induced
by adenosine
(Table 1). Thus, this concentra-
tion of theophylline was able to increase colony formation in the presence of 10 PM adenosine from 9% to 88% of the control value by blocking the inhibitory Al receptors. It was also able to decrease
colony formation
in the presence
of 100 uM adenosine
from 92% to 49% reflecting a block of the stimulatory A2 receptors by theophylline. Fig. 2A shows colony formation in response to increasing concentrations of theophylline in the absence and presence of 10 and 100 PM adenosine. A low concentration of theophylline (0.1 pM) by itself had little or no toxic effect but it reversed the inhibitory and stimulatory effects of 10 and 100 uM adenosine respectively. When the concentration of theophylline was increased (1-l 00 FM), colony formation decreased due to a toxic effect of theophylline itself. However, 0.1-10 uM adenosine was able to reduce the toxicity of theophylline while higher concentrations of adenosine increased the toxicity of theophylline (Fig. 28). Therefore, when both Al and A2 receptors had been blocked by theophylline (l-100 PM), 10 uM adenosine was able to promote growth while 100 PM adenosine inhibited colony formation. Although theophylline has been widely used as an antagonist of adenosine receptors, it is also an inhibitor of phosphodiesterase (PDE) (9). In order to confirm that 1488
Vol.
BIOCHEMICAL
187, No. 3, 1992 Table 1.
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Effect of adenosine, adenosine agonists and antagonists alone and in combination on colony formation Colony formation (% control) (mean + S.E.M.)* in the presence of adenosine
Additions
OPM
100
10pM
100 pM
9fl
92+6
Theophylline
(0.1 PM)
99+5
88k6
49 f 6
Dipyridamole
(100 FM)
73f7
10&l
75+5
9&l
76+6
Ro20-1724
(100 PM)
78 f 4
Uridine
(0.5 FM)
92+7
27f3
Uridine
(5 PM)
63 f 5
11 +2
85 ck 3
91 f6
89 k 6
14f2
72+2
Theophylline (0.1 PM) + Uridine (0.5 FM) Dipyridamole (100 PM) + Uridine (0.5 PM)
l
Calculated
theophylline
71 *lo
from 3 to 5 independent
is acting
through
102f
10
experiments.
its binding
to the adenosine
receptors,
colony
formation
in
of other PDE inhibitors (Ro20-1724) (10) and dipyridamole, (9) which do not block adenosine receptors were studied. The addition of Ro20-1724 (100 PM) and dipyridamole (100pM) had no significant effects on the biphasic response elicited by
the presence
60
THEOPHYLLINE
Figure
2
A.
presence
ADENOSINE
I@4
of theophylline
on colony
formation
I++)
in the absence
(0)
and
in the absence
(0)
and
of 10 PM (A) and 100 pM (m) adenosine. B.
presence
Effect
--
Effect
of adenosine
of 1 PM (A), All points
on colony
formation
10 PM (M) and 100 PM (0) theophylline.
are mean
+ S.E.M.
calculated
1489
from
5 independent
experiments.
Vol.
BIOCHEMICAL
187, No. 3, 1992
0
0.01
0.1
1
10
AND BIOPHYSICAL
0
100
0.1
1
DPCPX i/.&4)
Figure
3
A.
presence
of DPCPX
Effect
on colony
of adenosine
of 0.01 FM (A) and All points
adenosine
formation
in the
100
1000
absence
(0)
and
on colony
formation
are mean f S.E.M.
probably due to its role as an antagonist bition of PDE.
in the absence
(0)
and
10 FM (U) DPCPX. calculated
from
4 independent
(Table 1). Thus, the blockage by theophylline
Dipyridamole
10
ADENOSINE (@dI
of IO PM (A) and 100 pM (m) adenosine. 0.
presence
Effect
RESEARCH COMMUNICATIONS
of the adenosine
is not only a PDE-inhibitor
experiments.
of the effects of adenosine was receptors rather than its inhi-
but also a strong blocker of adenosine
uptake into cells (9). We have also demonstrated that 100 PM dipyridamole can inhibit the uptake of adenosine (0.1-100 PM) by about 70% in A431 cells (data not shown). Since the addition of dipyridamole did not affect the biphasic response elicited by adenosine, it is therefore unlikely that the biphasic response is due to the intracellular action of adenosine but it is more likely to be mediated by the adenosine Fig. 3A shows that the presence
receptors.
of DPCPX, which is a specific Al -antagonist
(11) abolished the inhibitory effect of 10 PM adenosine but did not affect the stimulatory effect of 100 uM adenosine. Subsequently, it was shown that 0.01 PM and 10 f.r,M DPCPX reversed the inhibitory effect of 10 PM adenosine by approximately 30% and back to the control value respectively (Fig. 3B). Therefore, the binding of DPCPX to the Al receptors substantially blocked the inhibitory effects of adenosine. These observations indicate the existence of both Al and A2 receptors in A431 cells which mediate inhibition of colony formation over the range l-l 0 PM but promotion of colony growth over the range 20-l 00 uM adenosine respectively. The presence of A2 receptors which enhance CAMP accumulation in A431 cells has also been previously reported [5]. A previous study (12) has also shown a biphasic response of a baby hamster kidney (BHK) cell line to adenosine. It was postulated that the toxic effect of low concentrations of adenosine may be due to the depletion of intracellular pyrimidine nucleotide pools. The addition of exogenous adenosine to 3T6 fibroblasts and a human lymphoblastoid cell line (MGL-5) led to a rapid elevation of adenine nucleotide levels, while uridine and cytidine nucleotide pools were drastically depleted [13]. However, inhibition of growth at higher concentrations of adenosine was suggested to be due to inhibition of 1490
Vol.
187,
No.
3,
BIOCHEMICAL
1992
S-adenosylmethionine-mediated adenosine
AND
methylation
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
reactions by S-adenosylhomocysteine
- an
metabolite [14].
In the light of these reports, we investigated Table 1, the inhibitory effects of 1 OuM adenosine
the effects of uridine. As shown in could be partially reversed by 0.5 pM
uridine (p c.0001) but not by a higher concentration
(5pM) of uridine which was already
inhibitory on its own. The toxic effect of 100 uM adenosine in the presence of 0.1 uM theophylline could also be reversed with the addition of 0.5 PM uridine (Table 1). It is known that adenosine can enter the cell and be phosphorylated, and that elevated levels of intracellular adenine nucleotides can inhibit pyrimidine nucleotide biosynthesis [15]. The ability of uridine to overcome partially some of the effects of adenosine supports the hypothesis that the growth-inhibitory interference
action of adenosine
may in part be due to
with pyrimidine nucleotide synthesis [16]. However, when dipyridamole
used to block adenosine
was
uptake into the cells (Table l), uridine could no longer reverse
the inhibitory effects of adenosine,
thus indicating that the biphasic response to adeno-
sine is largely mediated by the extracellular receptors. In summary, we propose that the presence of Al receptors as well as A2 receptors in A431 cells allows inhibition of growth at low concentrations
of adenosine,
medi-
ated by the Al receptors, but promotion of cell growth at high adenosine concentrations, mediated by the A2 receptors. When Al and A2 receptors were blocked by the adenosine antagonist,
theophylline,
been growth-promoting
the high concentration
of adenosine
became toxic while the low concentration
which had previously of adenosine
which
was previously toxic was able to support growth. When Al receptors were selectively blocked by DPCPX, the toxic effect of 10 uM adenosine was overcome. Since the Alreceptor is inhibitory,
a specific activator of Al receptors that does not activate the A2
receptors may be able to inhibit cell multiplication. Elucidation of the roles played by Al and A2 adenosine receptors together with growth factors in modulating cell growth appears critical to a better understanding
of the regulation of cell proliferation
and car-
cinogenesis.
Acknowledgments cal assistance
- This work was supported
by NUS Grant RP890359.
The techni-
of Miss F.H. Ng is gratefully acknowledged.
REFERENCES 1. 2.
Newby, A.C. (1984) Trends in Biochemical Science 9, 42-44. Burnstock, G.(1989) In: Adenosine Receptors in the Nervous System (Ribeiro JA, ed.), pp. l-l 4. Taylor & Francis, London, 1989.
3. 4.
Londos, C. and Wolff, J. (1977) Proc. Natl. Acad. Sci. USA 74, 5482-5486. Londos, C., Cooper, D.M.F. and Wolff, J. (1980) Proc. Natl. Acad. Sci. USA 77,
5.
2551-2554. Huang, N.N., Wang, D.J., Gonzalez, F.A. and Heppel, L.A. (1991) J. Cell Physiol. 146, 483-494. 1491
Vol.
6. 7.
187,
No. 3, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Wrann, M.M. and Fox, C.F. (1979) J. Biol. Chem. 254, 8083-8086. Schwabe, U. (1985) in: Adenosine - Receptors and Modulation of Cell Function (Stefanovich,
8.
BIOCHEMICAL
V., Rudolphi,
K. and Schubert,
Oxford. Daly, J.W. (1985) In: Adenosine: (Stefanovich,
V., Rudolphi,
Receptors
P., eds.) ~~15-30, and Modulation
K. and Schubert,
IRL Press,
of Cell Function
P., eds.), pp. 31-46.
IRL Press,
Oxford. 9.
Clark, R.B., Gross, R., Su, Y.F. and Perkins, 52964303.
10. 11.
Rozengurt,
E. (1982) Exp. Cell Res. 139, 71-78.
Schwabe,
U. (1991) In : Role of Adenosine
Biological System (Imai, S. and Nakazawa, Publishers, B.V., New York.
J.P. (1974) J. Biol. Chem. 249,
and Adenine
Nucleotides
in the
M., eds.) pp 59-69, Elsevier Science
12.
Juranka, P., Meffe, F., Guttman, S., Archer, S.M. and Chan, V.L. (1984) Mutation Res. 129, 397-402.
13. 14.
Green, H. and Chan, T.S. (1973) Science 182, 836-837.
15.
Fox, H.I. and Kelly, W.N. (1970) Ann. Rev. Biochem. 47, 655-686.
16.
Ishii, K. and Green, H. (1973) J. Cell Sci. 13, 429-439.
Kredich, N.M. and Martin, D.W. (1977) Cell 12, 931-938.
1492
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages 1486-1492
30, 1992
ADENOSINE MODULATES CELL GROWTH IN HUMAN EPIDERMOID CARCINOMA (A431) CELLS H.B. Tey, H.E. Khoo* and C.H. Tan Department of Biochemistry National University of Singapore Kent Ridge Singapore 0511 Received
August
18,
1992
SUMMARY: Adenosine mediates many physiological functions via activation of extracellular receptors. The modulation of cell growth by adenosine was found to be receptor-mediated. In A431 cells adenosine evoked a biphasic response in which a low concentration (-10 uM) produced inhibition of colony formation but at higher concentrations (up to 100 PM) this inhibition was progressively reversed. Evidence for the involvement of Al (inhibitory) and A2 (stimulatory) adenosine receptors in regulating cell growth of these tumor cells was obtained through plating efficiency studies based on the relative potency of adenosine agonists and antagonists, When both Al and A2 receptors were blocked, colony formation or growth was not inhibited at low concentrations of adenosine but was inhibited at high adenosine concentrations. 0 1992 Academic Press, Inc.
Adenosine current advances
mediates
a wide variety of physiological
have made it possible
to elucidate
functions
in man [i] and
the role played by adenosine
receptors in cellular response [2]. It has been proposed that adenosine-reactive sites can be distinguished into “R” and “P” sites. The extracellular membrane-bound R-site is more responsive
to the ribose ring of adenosine
and its analogs. Occupancy
of the R-
site leads to activation of adenylate cyclase. In contrast, the P-site mediates the inhibition of adenylate cyclase. It is located on the internal side of the cell membrane and has a distinct affinity for the purine moiety [3]. The existence of two distinct external adenosine receptors (Al and A2) rather than just one R-site was later demonstrated in cultured brain cells, mast cells and myocardial cells [4]. The Al receptor mediates inhibition of adenylate cyclase and has a relatively higher affinity for R-N6-phenylisopropyladenosine (R-PIA) than for 5’-N-ethylcarboxamidoadenosine (NECA). For the A2 receptor which generally enhances CAMP production, the order of affinity for the adenosine analogs is reversed, NECA being more effective than R-PIA [2]. *To whom correspondence 0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
should be addressed.
Inc. reserved.
1486
Vol.
187,
No.
3,
BIOCHEMICAL
1992
Cultured human epidermoid
AND
BIOPHYSICAL
carcinoma
in the study of cellular and molecular
RESEARCH
COMMUNICATIONS
(A431) cells have been used as a model
responses
evoked by growth factors and other
mitogens [5, 61. We report here that adenosine causes a biphasic response formation of these cells due to the existence of Al and A2 receptors.
MATERIALS
AND METHODS
A431 cells were grown in Dulbecco’s mented with 10% heat-inactivated at 56OC to destroy adenosine
in colony
Modified Eagle’s Medium (DMEM) supple-
Fetal Bovine Serum (FBS,). FBS was heated for 2 hr
deaminase
(ADA) activity. Since our preliminary
work
(Fig. 1A) showed that the inhibition of ADA by 5 PM erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not have any significant effects on cell growth in response to adenosine, the rest of the experiments
were conducted
with only FBS,. For plating studies,
one hundred cells were seeded in Corning multidishes (surface area 35 mm2) containing DMEM supplemented with 10% FBS,. On the following day, cells were treated with varying concentrations
of adenosine
with and without theophylline,
adenosine
analogs
and antagonists for 24 hours in DMEM containing 1% FBS,. The adenosine-containing medium was then removed and replaced by DMEM supplemented with 10% FBS,. Five days after seeding
, the cells were fixed and stained with Giemsa and the colonies
scored by microscopic
examination
of the dishes.
cells in 10% FBSi was 80 - 90% (designated
The plating efficiency
of 100 A431
as control). The number of colonies
in a
test group is expressed as a percentage of the control number. 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was obtained from Research Biochemicals, Natick, Mass., U.S.A. and EHNA was a gift from Wellcome Research Laboratories,
Beckenham,
U.K. All other
chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Statistical analysis was performed
using the Student’s t-test.
RESULTS AND DISCUSSION Exposure to adenosine response (Fig.lA).
at concentrations
of 0.1-1000
Colony formation was progressively
PM produced
a biphasic
inhibited as the concentration
of
adenosine was increased from 1 uM to 10 pM. At a concentration of 10 PM, adenosine produced almost complete inhibition of colony formation. However, inhibition decreased as the concentration
of adenosine
was further increased from 10 uM to 100 PM. At 100
PM adenosine colony formation was approximately 90% of control values. Further increases in adenosine concentration resulted in progressive inhibition of colony formation. The above observations
suggested
the involvement
of two types of extracellular
adenosine receptors (inhibitory and stimulatory) in these cells. To show that both Al and A2 receptors may be responsible for the biphasic response of A431 cells to adenosine, colony formation was studied in the presence of selected adenosine analogs, RPIA and NECA, and antagonists, theophylline and DPCPX. The dose-response curves 1487
Vol.
BIOCHEMICAL
187, No. 3, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
3 p 100 z 8
60
w 0’ F
60
2E
40
0
0.1
Figure
of the adenosine
1 sence
1
10 AGONIST
ADENOSINE l@d)
100
1000
l@vi)
A. Effect of adenosine on plating efficiency of A431 ceils in the ab(0) and presence of 50 pM EHNA (A). B. Effect of R-PIA (0) and NECA (A) on cofony formation. All points are mean + S.E.M. calculated from 5 independent experiments.
agonists were also biphasic
both R-PIA and NECA inhibited
(Fig. 1 B). At a concentration
colony formation
of 10 PM,
but R-PIA was more potent
than
NECA suggesting the presence of Al receptors. On the other hand, 100 uM NECA was more effective than 100 pM R-PIA in supporting colony formation thus indicating the presence of A2 receptors. Since R-PIA and NECA are not metabolized by adenosinemetabolizing enzymes (7), these results support the hypothesis that the effects of adenosine and these agonists on cell growth are modulated via extracellular
receptors.
Xanthines such as theophylline, caffeine, isobutylmethylxanthine and 8-phenyltheophylline are non-selective adenosine antagonists that block the binding of adenosine to both Al and A2 receptors could be substantially
[8]. The biphasic
overcome by 0.1 uM theophylline
response
induced
by adenosine
(Table 1). Thus, this concentra-
tion of theophylline was able to increase colony formation in the presence of 10 PM adenosine from 9% to 88% of the control value by blocking the inhibitory Al receptors. It was also able to decrease
colony formation
in the presence
of 100 uM adenosine
from 92% to 49% reflecting a block of the stimulatory A2 receptors by theophylline. Fig. 2A shows colony formation in response to increasing concentrations of theophylline in the absence and presence of 10 and 100 PM adenosine. A low concentration of theophylline (0.1 pM) by itself had little or no toxic effect but it reversed the inhibitory and stimulatory effects of 10 and 100 uM adenosine respectively. When the concentration of theophylline was increased (1-l 00 FM), colony formation decreased due to a toxic effect of theophylline itself. However, 0.1-10 uM adenosine was able to reduce the toxicity of theophylline while higher concentrations of adenosine increased the toxicity of theophylline (Fig. 28). Therefore, when both Al and A2 receptors had been blocked by theophylline (l-100 PM), 10 uM adenosine was able to promote growth while 100 PM adenosine inhibited colony formation. Although theophylline has been widely used as an antagonist of adenosine receptors, it is also an inhibitor of phosphodiesterase (PDE) (9). In order to confirm that 1488
Vol.
BIOCHEMICAL
187, No. 3, 1992 Table 1.
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Effect of adenosine, adenosine agonists and antagonists alone and in combination on colony formation Colony formation (% control) (mean + S.E.M.)* in the presence of adenosine
Additions
OPM
100
10pM
100 pM
9fl
92+6
Theophylline
(0.1 PM)
99+5
88k6
49 f 6
Dipyridamole
(100 FM)
73f7
10&l
75+5
9&l
76+6
Ro20-1724
(100 PM)
78 f 4
Uridine
(0.5 FM)
92+7
27f3
Uridine
(5 PM)
63 f 5
11 +2
85 ck 3
91 f6
89 k 6
14f2
72+2
Theophylline (0.1 PM) + Uridine (0.5 FM) Dipyridamole (100 PM) + Uridine (0.5 PM)
l
Calculated
theophylline
71 *lo
from 3 to 5 independent
is acting
through
102f
10
experiments.
its binding
to the adenosine
receptors,
colony
formation
in
of other PDE inhibitors (Ro20-1724) (10) and dipyridamole, (9) which do not block adenosine receptors were studied. The addition of Ro20-1724 (100 PM) and dipyridamole (100pM) had no significant effects on the biphasic response elicited by
the presence
60
THEOPHYLLINE
Figure
2
A.
presence
ADENOSINE
I@4
of theophylline
on colony
formation
I++)
in the absence
(0)
and
in the absence
(0)
and
of 10 PM (A) and 100 pM (m) adenosine. B.
presence
Effect
--
Effect
of adenosine
of 1 PM (A), All points
on colony
formation
10 PM (M) and 100 PM (0) theophylline.
are mean
+ S.E.M.
calculated
1489
from
5 independent
experiments.
Vol.
BIOCHEMICAL
187, No. 3, 1992
0
0.01
0.1
1
10
AND BIOPHYSICAL
0
100
0.1
1
DPCPX i/.&4)
Figure
3
A.
presence
of DPCPX
Effect
on colony
of adenosine
of 0.01 FM (A) and All points
adenosine
formation
in the
100
1000
absence
(0)
and
on colony
formation
are mean f S.E.M.
probably due to its role as an antagonist bition of PDE.
in the absence
(0)
and
10 FM (U) DPCPX. calculated
from
4 independent
(Table 1). Thus, the blockage by theophylline
Dipyridamole
10
ADENOSINE (@dI
of IO PM (A) and 100 pM (m) adenosine. 0.
presence
Effect
RESEARCH COMMUNICATIONS
of the adenosine
is not only a PDE-inhibitor
experiments.
of the effects of adenosine was receptors rather than its inhi-
but also a strong blocker of adenosine
uptake into cells (9). We have also demonstrated that 100 PM dipyridamole can inhibit the uptake of adenosine (0.1-100 PM) by about 70% in A431 cells (data not shown). Since the addition of dipyridamole did not affect the biphasic response elicited by adenosine, it is therefore unlikely that the biphasic response is due to the intracellular action of adenosine but it is more likely to be mediated by the adenosine Fig. 3A shows that the presence
receptors.
of DPCPX, which is a specific Al -antagonist
(11) abolished the inhibitory effect of 10 PM adenosine but did not affect the stimulatory effect of 100 uM adenosine. Subsequently, it was shown that 0.01 PM and 10 f.r,M DPCPX reversed the inhibitory effect of 10 PM adenosine by approximately 30% and back to the control value respectively (Fig. 3B). Therefore, the binding of DPCPX to the Al receptors substantially blocked the inhibitory effects of adenosine. These observations indicate the existence of both Al and A2 receptors in A431 cells which mediate inhibition of colony formation over the range l-l 0 PM but promotion of colony growth over the range 20-l 00 uM adenosine respectively. The presence of A2 receptors which enhance CAMP accumulation in A431 cells has also been previously reported [5]. A previous study (12) has also shown a biphasic response of a baby hamster kidney (BHK) cell line to adenosine. It was postulated that the toxic effect of low concentrations of adenosine may be due to the depletion of intracellular pyrimidine nucleotide pools. The addition of exogenous adenosine to 3T6 fibroblasts and a human lymphoblastoid cell line (MGL-5) led to a rapid elevation of adenine nucleotide levels, while uridine and cytidine nucleotide pools were drastically depleted [13]. However, inhibition of growth at higher concentrations of adenosine was suggested to be due to inhibition of 1490
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No.
3,
BIOCHEMICAL
1992
S-adenosylmethionine-mediated adenosine
AND
methylation
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
reactions by S-adenosylhomocysteine
- an
metabolite [14].
In the light of these reports, we investigated Table 1, the inhibitory effects of 1 OuM adenosine
the effects of uridine. As shown in could be partially reversed by 0.5 pM
uridine (p c.0001) but not by a higher concentration
(5pM) of uridine which was already
inhibitory on its own. The toxic effect of 100 uM adenosine in the presence of 0.1 uM theophylline could also be reversed with the addition of 0.5 PM uridine (Table 1). It is known that adenosine can enter the cell and be phosphorylated, and that elevated levels of intracellular adenine nucleotides can inhibit pyrimidine nucleotide biosynthesis [15]. The ability of uridine to overcome partially some of the effects of adenosine supports the hypothesis that the growth-inhibitory interference
action of adenosine
may in part be due to
with pyrimidine nucleotide synthesis [16]. However, when dipyridamole
used to block adenosine
was
uptake into the cells (Table l), uridine could no longer reverse
the inhibitory effects of adenosine,
thus indicating that the biphasic response to adeno-
sine is largely mediated by the extracellular receptors. In summary, we propose that the presence of Al receptors as well as A2 receptors in A431 cells allows inhibition of growth at low concentrations
of adenosine,
medi-
ated by the Al receptors, but promotion of cell growth at high adenosine concentrations, mediated by the A2 receptors. When Al and A2 receptors were blocked by the adenosine antagonist,
theophylline,
been growth-promoting
the high concentration
of adenosine
became toxic while the low concentration
which had previously of adenosine
which
was previously toxic was able to support growth. When Al receptors were selectively blocked by DPCPX, the toxic effect of 10 uM adenosine was overcome. Since the Alreceptor is inhibitory,
a specific activator of Al receptors that does not activate the A2
receptors may be able to inhibit cell multiplication. Elucidation of the roles played by Al and A2 adenosine receptors together with growth factors in modulating cell growth appears critical to a better understanding
of the regulation of cell proliferation
and car-
cinogenesis.
Acknowledgments cal assistance
- This work was supported
by NUS Grant RP890359.
The techni-
of Miss F.H. Ng is gratefully acknowledged.
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