[CANCER RESEARCH 38, 2357-2362,
0008-5472/78/0038-OOOOS02.00
August
1978]
Cytotoxic and Metabolic
Lymphoblasts1
Effects of Adenosine
and Adenine on Human
Floyd F. Snyder,2 Michael S. Hershfield,3 and J. Edwin Seegmiller
Department of Medicine, University of California, San Diego, La Jolla, California 92093
and further studies have shown that cytotoxic concentra
tions of adenosine reduce pyrimidine ribonucleotides (12,
The metabolic and growth inhibitory effects of adeno17, 21, 31) and NAD+ (17, 31) and cause an accumulation of
sine toward the human lymphoblast line WI-L2 were po
orotic acid (12, 31). Adenosine also increases cellular ade
tentiated by the adenosine deaminase inhibitors erythronine nucleotides (12, 17, 31), including a transient increase
9-(2-hydroxy-3-nonyl) adenine (EHNA) and coformycin.
in cAMP4 concentration (34, 35). In addition to studies in
EHNA, 5 nu, or coformycin, 3.5 ¿iM,
at concentrations that cultured cells, increased adenine ribonucleotide pools have
inhibited adenosine deaminase activity more than 90%,
been found in erythrocytes (1, 25) and lymphocytes (28) of
had little effect on cell growth or the metabolic parameters
adenosine deaminase-deficient, immune-defective patients.
studied. Adenosine, 50 /¿M,
plus EHNA, 5 JUM,arrested cell The selective toxicity of adenosine to dividing lymphoid
growth in both parent and adenosine kinase-deficient
cells, including inhibition of both the response of human
lymphoblasts, implicating the nucleoside as the mediator
peripheral blood lymphocytes to mitogen and the growth of
of the cytostatic effect. Adenosine, 50 /KM,in combination
lymphoblastoid cell lines, is considered a possible basis for
with the adenosine deaminase inhibitors reduced 14C02
the severe combined immunodeficiency disease associated
generation from [1-14C]glucose by 38%, depleted 5-phoswith a hereditary absence of adenosine deaminase activity
phoribosy 1-1-pyrophosphate by more than 90%, and re
(11,24).
duced pyrimidine ribonucleotide concentrations. Uridine,
It has been suggested that the toxic effects of adenosine
10 or 100 ftM, reversed adenosine plus EHNA growth are caused by an increased cellular adenine nucleotide
inhibition in WI-L2 but not in adenosine kinase mutants.
pool, but some evidence has suggested a mechanism(s) of
Adenine, 500 //M, which may be converted to the same toxicity that does not require conversion of adenosine or
intracellular nucleotides as adenosine, reduced the adenine to nucleotides. Certain analogs of adenosine that
growth rate by 50% in both parent and adenine phospho- cannot be phosphorylated are cytotoxic (19, 32), and we
ribosyltransferase-deficient lymphoblasts. Although ade
have found the toxicity of adenosine to persist in adenosine
nine also depleted cells of 5-phosphoribosyl-l-pyrophoskinase-less mutants of the WI-L2 human splenic lympho
phate and reduced pyrimidine ribonucleotide by 50%, the blast line; mutants lacking adenine phosphoribosyltransfermechanisms of adenine and adenosine toxicity differ. In ase were as sensitive as was their parent line to growth
contrast to the ability of uridine to reverse adenosine
inhibition by adenine (16). We now report more detailed
cytostasis, growth inhibition by adenine was not reversed studies on the biochemical effects of adenosine and ade
by uridine, indicating that pyrimidine ribonucleotide de
nine on these lymphoblast lines. In these studies we have
pletion is not the primary mechanism of adenine toxicity.
used 2 potent inhibitors of adenosine deaminase, coformy
cin (26) and EHNA (27). We have compared the effects of
INTRODUCTION
adenosine and adenine since both purines can be con
verted to the same intracellular nucleotides (Chart 1) and
The mechanism(s) of cytoxicity for the naturally occurring
might therefore be expected to have similar mechanisms of
nuceoside, adenosine, and its related base adenine is not toxicity if their effects are related only to expanded adenine
understood but remains important, with growing interest in nucleotide pools.
the use of adenosine deaminase inhibitors in combination
chemotherapy. Both adenosine (9, 10, 18, 30) and adenine
MATERIALS AND METHODS
(20) block mitogen-induced transformation of human lym
phocytes. Adenine toxicity toward mouse L-cells was par
Biochemicals. Radiochemicals were purchased from
tially overcome by certain pyrimidines (2), and adenosine
Amersham/Searle Corp., (Arlington Heights, III.): [8toxicity was reversed by uridine in normal (12) but not in
14C]adenine, 59 mCi/mmol; [8-14C] adenosine, 59 mCi/
adenosine deaminase-deficient fibroblasts (4). Hilz and
mmol; [8-14C]hypoxanthine, 59 mCi/mmol; [1-14C]glucose,
Kaukel (17) first documented a decrease in intracellular
9.37 mCi/mmol; [6-14C]glucose, 5.0 mCi/mmol; and sodium
DTP concentration in adenosine-inhibited HeLa cells (21), [14C]formate, 59 mCi/mmol. Adenine, adenosine, hypoxanthine, uridine, and PP-ribose-P (sodium salt) were pur
1This work was supported by Grants AM-1362, AM-5646, and GM-17702
chased from P-L Biochemicals (Milwaukee, Wis.). EHNA
from NIH and grants from the National Foundation and the Kroc Foundation.
was provided by Wellcome Research Labs (Research Tri2 Present address: Division of Pediatrics and Medical Biochemistry, Fac
ABSTRACT
ulty of Medicine, The University of Calgary, Calgary, Alberta T2N 1N4,
Canada. To whom requests for reprints should be addressed.
3 Present address: Department of Medicine, Duke University Medical
Center, Durham, N. C. 27710.
Received December 13, 1977; accepted May 11,1978.
AUGUST 1978
4 The abbreviations used are: cAMP, cyclic adenosine 3':5'-monophosphate; EHNA, eryr/ii-o-9-(2-hydroxy-3-nonyl) adenine; PP-ribose-P, 5-phosphoribosyl-1 -pyrophosphate.
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F. F. Snyder et al.
ATP
II
ADP
II
EHNA, Cofonnycln
/If
(ADENOSINE
«
(AD
DAP \
INOSINE '
t
A
ADENINE
? TG
HYPOXANTHINE
Chart 1. Purine interconversion
showing sites of selected lymphoblast
mutation
and enzyme inhibition.
DAP, 2,6-diaminopurine
resistant and
deficient
in adenine phosphoribosyltransferase;
MTV, 6-methylthioinosine
resistant and deficient in adenosine kinase; TG . 6-thioguanme
resistant and
deficient
in hypoxanthine-guanine
phosphoribosyltransferase;
EHNA and
coformycin,
inhibitors of adenosine deaminase; AMPS, adenylosuccinate.
angle Park, N. C.), and coformycin was provided by Dr. H.
Umezawa, Institute for Microbial Chemistry, Tokyo, Japan.
Lymphoblasts. The human splenic lymphoblast line WlL2 (23) was grown in suspension culture supplemented with
2 nriM glutamine and 10% horse or fetal calf serum (Flow
Laboratories,
Rockville, Md.) as previously described (16,
31). The isolation and characterization
of clonal lympho
blast lines derived from WI-L2 deficient in adenosine kinase
(EC 3.7.1.20) (MTI), both adenosine kinase and hypoxan
thine-guanine phosphoribosyltransferase
(EC 2.4.2.8) (MTITG), and adenine phosphoribosyltransferase
(EC 2.4.2.7)
(DAP) were previously described (16). Cell counts were
measured by a Model ZB, Coulter counter.
Metabolic Studies. Adenosine kinase, adenine phospho
ribosyltransferase,
and hypoxanthine-guanine
phosphori
bosyltransferase
were assayed in lymphoblast extracts as
previously described (16, 30). Adenosine metabolism was
studied in the intact lymphoblast as previously described
(30). Intracellular
PP-ribose-P concentrations
(15, 16, 31)
and de novo purine synthesis (15) were measured as before.
cAMP concentrations
were measured according
to the
method of Wastili ef al. (33). Intracellular nucleotides were
measured by high-pressure liquid chromatography
as pre
viously described (6, 31).
The metabolism of [1-l4C]glucose
to 14C02 was examined
as a measure of hexose monophosphate
shunt activity.
After 24 hr of incubation with test compounds,
lymphoblasts were harvested by centrifugation,
120 x g for 3 min,
and resuspended in fresh medium lacking glucose with 12
mW sodium phosphate, pH 7.2, and the original concentra
tion of test compounds. To 0.5-ml suspensions of 0.5 x 106
cells in stoppered 15- x 80-mm tubes, a final concentration
of 2.5 mW [1-14C]glucose or [6-14C]glucose, 5.0 mCi/mmol,
was added. After 30 min of incubation
at 37°, reactions
were stopped by injection of 0.5 ml of 5 N H2SO„;
then 0.25
ml of Hyamine hydroxide (New England Nuclear, Boston,
Mass.) was added to center wells (Kontes Glass, Berkeley,
Calif.) containing fluted filter paper. After a further 60-min
shaking, trapped 14CO2 was counted at 70% efficiency by
placing center wells in 10 ml Bray's solution (5).
RESULTS
Effects of Adenosine on Human Lymphoblast Growth
and Metabolism. The human lymphoblast line WI-L2 and
derived mutants used in studies of adenosine toxicity were
2358
conditioned
to more than 3 months of growth in medium
supplemented
with 10% horse serum. This serum was
previously shown to deaminate less than 5 /¿molof 50 /XM
adenosine per 25 hr (30). The growth rate of WI-L2 lymphoblasts in this medium was inhibited 50% by approximately
200 /¿Madenosine, and 5000 /UM adenosine completely
arrested growth (Chart 2). The adenosine deaminase inhib
itors EHNA, 5 /iM, and coformycin,
3.5 /¿M(1 pig/ml),
inhibited lymphoblast adenosine deaminase activity more
than 95% in either extracts or whole cells but had little
effect on growth. WI-L2 grew at approximately
90% of the
normal growth rate for 8 weeks in the presence of 5 /¿M
EHNA with weekly subculturing
and addition of EHNA.
EHNA increased the sensitivity of WI-L2 lymphoblasts
to
growth inhibition by adenosine greater than 10-fold, such
that 100 ^M adenosine completely arrested growth in the
presence of 5 /¿MEHNA (Chart 2). Complete growth inhibi
tion by 50 t¿Madenosine and 5 /¿MEHNA occurred after 24
hr of exposure or approximately
1 cell doubling (Chart 3/4)
and was reversible for at least 72 hr of culture (Chart 30),
demonstrating
retention of cell viability despite growth
arrest. The combination
of 50 /J.M adenosine and 3.5 /¿M
coformycin
also arrested growth of WI-L2 lymphoblasts
after 24 hr.
In studies with lymphoblast extracts, we found the appar
ent Km's of adenosine kinase and adenosine deaminase for
adenosine to be approximately
2 to 4 and 40 to 50 /¿M,
respectively. The maximal velocity of the deaminase was
approximately
10-fold greater than that of the kinase. The
metabolism of extracellular adenosine by lymphoblasts ap
peared to be governed by the characterisitics
of these 2
enzymes. Thus deamination
was the principal
route of
adenosine metabolism for intact WI-L2 exposed to 80 /¿M
adenosine; the rates of deamination and phosphorylation
were 1035 and 74 pmol/106 cells/min,
respectively. At a
lower concentration
of adenosine, 4 ¿¿M
or approximately
twice the Kmfor adenosine kinase, phosphorylation
was the
principal route of adenosine metabolism; the rates of deam
ination and phosphorylation
were 33 and 65 pmol/106 cells/
min, respectively. The products of deamination,
¡nosine or
hypoxanthine (500 /¿M),did not inhibit lymphoblast growth.
io-
IO'4
IO"
IO"'
ADENOSINE I molar I
Chart 2. Potentiation of adenosine-mediated
growth inhibition by EHNA.
WI-L2 lymphoblasts were cultured in the absence or presence of adenosine
( O) or adenosine plus 5 /¿MEHNA (•).Each point represents the reciprocal
fraction of the mean doubling time of duplicate adenosine-treated
cultures
measured over a 4- to 5-day growth period with cell number measured every
24 hr, compared to the mean doubling time in the absence of additions.
Cells treated with 5 fiM EHNA had a reciprocal relative doubling time of 0.92.
Cells were incubated for 30 min with EHNA prior to addition of adenosine.
The initial cell density was 0.5 to 1.0 x 105 cells/ml.
CANCER
RESEARCH
VOL.
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38
Adenosine and Adenine Toxicity in Lymphoblasts
Table 1
Effects of adenosine, adenine, hypoxanthine, and uridine on
lymphoblast nucleotide concentrations
Additions were made to logarithmically growing cultures at a
density of 2.5 to 4.0 x 10s cells/ml 24 hr prior to harvesting,
extraction, and high-pressure liquid chromatography as described
in "Materials and Methods." In Experiments A and B, cells were
grown in medium containing 10% horse serum, and in Experiment
C cells were grown in medium containing 10% fetal calf serum.
AMP and orotic acid were not separated by the Chromatographie
system, and peak area was calculated for AMP standard [in a
previous report we have shown this area to be >90% AMP for
untreated WI-L2 cultures (31)]: IAXP = AMP + ADP + ATP; 2GXP
= GMP + GDP + GTP; SUXP = UMP + UDP + UTP + UDP-sugars.
Results are the average of duplicate determinations with the
following range for control cultures: 2AXP, ±5%;2GXP, ±10%;
SUXP, ±9%.
Nucleotide concentration (nmol/10*
cells)
24
48
72
96
120
HOURS
Chart 3. Growth inhibition by adenosine in parent and adenosine kinasedeficient lymphoblasts. Lymphoblasts were cultured with no additions (•)
or
5 /iM EHNA plus 50 /IM adenosine (O), as described for Chart 2. A. 10 (A) or
100 /¿M
(V) und ine was added to EHNA-plus-adenosine-treated lymphoblasts
at 24 hr; B, lymphoblasts exposed to EHNA plus adenosine were resuspended in fresh medium without additions at 24, 48, or 72 hr (A); C', EHNAplus-adenosine-treated, adenosine kinase-deficient (AK~)lymphoblasts (MTI)
were cultured in the absence (O) and presence (A) of 100 ^M uridine; D.
EHNA and adenosine were added to adenosine kinase-hypoxanthine-guanine phosphoribosyltransferase-deficient (AK~, HGPRT~) lymphoblasts (MTITG), which were subsequently cultured in the absence (O) and presence (A)
of 100 ¿¿M
uridine.
Thus the adenosine deaminase inhibitor blocks the major
route for metabolism of substantial amounts of exogenously supplied adenosine and could permit excessive
conversion of adenosine to nucleotides or simply increase
the half-life of adenosine, either of which may be a possible
basis for the potentiation by the inhibitor of adenosine
toxicity.
We examined the question of adenosine growth inhibition
being mediated by the nucleoside or some phosphorylated
product by using adenosine kinase-deficient lymphoblasts.
Adenosine kinase-deficient mutants were selected for re
sistance to 2 ^M 6-methylthioinosine and were designated
MTI (16). A mutant deficient in both adenosine kinase and
hypoxanthine-guanine phosphoribosyltransferase was de
rived from MTI by selection for resistance to 10 /¿M6thioguanine and was designated MTI-TG. The specific activ
ities of adenosine kinase and hypoxanthine-guanine phosphoriboxyltransferase in extracts of mutant lymphoblasts
were less than 0.1 and 2%, respectively, of control WI-L2
(16). Both adenosine kinase-deficient lines showed inhibi
tion of growth by the combination of 50 ^M adenosine and
5 /nM EHNA (Chart 3, C and D); complete growth arrest
required 48 hr. Adenosine was therefore growth inhibitory
in the absence of appreciable metabolism via phosphorylation or deamination.
The effect of adenosine plus EHNA on WI-L2 and MTI
lymphoblast ribonucleotide concentrations was determined
more directly by high-pressure liquid chromatography of
cell extracts (6, 31). At the time of harvesting, cell growth
was not completely arrested because the initial concentra
tion of lymphoblasts exposed to adenosine plus EHNA for
AUGUST 1978
Additions
A. WI-L2
None
Adenosine, 0.05 mM, +
EHNA, 0.005 mM
AMP
2AXP
2GXP
2UXP
0.17
0.13
5.6
9.6
1.5
1.4
3.1
1.1
B. MTI
None
Adenosine, 0.05 mM, +
EHNA, 0.005 mM
0.18
0.14
4.8
5.2
0.9
0.6
2.0
1.2
C. WI-L2
None
Hypoxanthine, 0.5 mw
Adenine, 0.5 mM
Adenine, 0.5 mM, +
uridine, 1.0 mM
0.07
0.15
0.05
0.10
4.3
4.4
6.2
4.9
1.3
1.1
1.3
0.8
2.9
1.4
1.5
7.0
nucleotide analysis (Table 1) was 2.5- to 5-fold greater than
that for the growth studies of Charts 2 and 3. Adenosine, 50
fj.M, plus EHNA, 5 /¿M,
depleted total uracil ribonucleotides
to 35% of control, and adenine ribonucleotides, essentially
ATP, increased (Table 1), presumably from the phosphorylation of adenosine. Exposure of MTI cells to adenosine plus
EHNA also reduced uracil and guanine ribonucleotides,
whereas total adenine plus guanine ribonucleotide concen
trations were essentially unchanged. Adenosine plus EHNA
treatment reduced the concentration of CTP to 80 and 40%
of untreated cells for WI-L2 and MTI, respectively. Thus in
adenosine kinase-deficient cells adenosine plus EHNA de
creased pyrimidine nucleotides under conditions where no
significant change in purine ribonucleotide concentrations
had occurred. At a later stage of complete but reversible
growth inhibition of WI-L2, the nucleotide concentrations
in 'adenosine-treated'5 cells compared to control were:
UTP, 0.05; CTP, 0.17; UDP-sugars, 0.35; NAD*, 0.52; GTP,
1.14; and ATP, 1.62 (31). The extensive depletion of pyrimi
dine nucleotides was accompanied by an accumulation of
intracellular orotic acid (31). The growth inhibitory effect of
adenosine and EHNA was overcome by addition of 10 or
100 /U.Muridine (Chart 3A). Uridine, 100 /*M, in contrast to
its effect on parental cells, failed to reverse adenosine6 WI-L2 lymphoblasts were incubated for 10 hr with 500 ßMcAMP, which
was shown to be slowly converted extracellularly to adenosine by horse
serum activities.
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F. F. Snyder et al.
14C02 generation.
Table 2
Adenine
produced
less than 10% inhibi
Effect of adenosine in combination with adenosine deaminase
inhibitors upon lymphoblast PP-ribose-P concentration
tion, either alone or in combination with coformycin, indi
cating a specificity for inhibition by the nucleoside adeno
Parent and adenosine kinase-deficient
(MTI) lymphoblasts
were
sine. Thus an adenosine-mediated
inhibition
of pentose
cultured for 24 or 48 hr in the absence or presence of 3.5 /¿M
phosphate
synthesis
could
account
in
part
for
the
reduction
coformycin,
5 ¿iMEHNA, or 50 ¡J.Madenosine, and PP-ribose-P
in lymphoblast PP-ribose-P concentration.
The generation
concentrations
were measured as described
in "Materials
and
Methods."
Initial cell density was 0.5 to 0.8 x 105 cells/ml. In the
of 14CO2from [6-14C]glucose, 2.5 mM, was less than 5% of
absence of additions, PP-ribose-P concentrations
(pmol/106 cells)
the rate of 14CO2generation from [1-14C]glucose, indicating
at 24 and 48 hr, respectively,
and 190 for MTI.
were 133 and 290 for WI-L2 and 140
concentration
a low rate of glycolytic metabolism in the amino acid-rich
medium.
Effects of Adenine on Lymphoblast Growth and Metab
olism. Adenosine and adenine, although not directly inter-
hr1.00
hr1.00
convertable
in the human lymphocyte
(29, 30), may be
converted to the same intracellular
nucleotides (Chart 1),
and we have therefore compared their effects on WI-L2
0.84
1.08
0.21
0.01
0.07MTI24
0.94
1.04
0.15
0.29
0.2448
Relative PP-ribose-P
WI-L2AdditionsNone
hr1.00
Coformycin
EHNA
Adenosine
Coformycin
+ adenosine
EHNA + adenosine24
induced growth inhibition
1.28
0.93
0.32
<0.01
0.0748
in the adenosine
lymphoblasts (Chart 3, C and D).
The adenosine-mediated
depletion
hr1.00
1.59
0.88
0.22
<0.01
<0.01
kinase-deficient
of pyrimidine
nucleo-
tides and accumulation
of orotic acid (31) suggested a
possible deficiency of PP-ribose-P, which is required for
further metabolism of orotate to pyrimidine nucleotides.
The PP-ribose-P concentrations
of lymphoblasts cultured in
the presence of adenosine and inhibitors
of adenosine
deaminase activity were examined (Table 2). Despite inhibit
ing lymphoblast adenosine deaminase activity by more than
95%, coformycin or EHNA alone had little effect on either
cell growth or PP-ribose-P concentration.
Adenosine, 50
¿IM,in combination
with either coformycin
of EHNA re
duced the PP-ribose-P concentration
by more than 90% in
both parent and adenosine kinase-deficient
lymphoblasts.
Complete PP-ribose-P depletion in MTI cells ocurred at 48
hr, when the increase in cell density had ceased (Chart 3C).
We have attempted to elucidate the biochemical site of
adenosine-mediated
reduction
in pyrimidine
nucleotides
and PP-ribose-P. Adenosine, 50 to 500 (J.M, in the absence
or presence of EHNA, 5 /LC.M,has no significant effect on
orotate phosphoribosyltransferase
(F. F. Snyder, unpub
lished data) or PP-ribose-P synthetase (31) activities in Wll_2 extracts. Because our results suggest that growth inhi
bition is mediated by adenosine and since this nucleoside
is known to increase cAMP concentrations
of lymphoid
cells (34, 35), cAMP concentrations
were measured. No
significant change in lymphoblast cAMP concentration
was
produced by 24 hr of culture in the absence or presence of
50 UM adenosine plus 5 ¡J.MEHNA; control cells had 0.4
pmol of cAMP per 106 cells.
The oxidative branch of the pentose phosphate pathway
is an important
route for PP-ribose-P synthesis in the
mammalian cell (13). As an estimate of oxidative pentose
phosphate synthesis from glucose, the generation of '"CO.,
from [1-14C]glucose was measured after 24 hr of exposure
of lymphoblasts to adenosine or adenine in the absence
and presence of coformycin or EHNA (Table 3). The growth
inhibitory
combination
of adenosine and coformycin
or
adenosine and EHNA produced the greatest inhibition of
2360
lymphoblasts.
Approximately
0.6 mM adenine produced
50% growth inhibition of lymphoblasts,
whereas 0.5 IDM
hypoxanthine
was not growth inhibitory
(16). Cell lines
selected from WI-L2 that were resistant to 200 /xM 2,6diaminopurine,
designated DAP, compared to 50% growth
inhibition of the parental strain at 5 /J.Mdiaminopurine,
had
less than 0.5% of the parental adenine phosphoribosyl
transferase activity in lymphoblast extracts (16). In intact
adenine phosphoribosyltransferase-deficient
lymphoblasts,
the rate of nucleotide synthesis from 0.5 mM [14C]adenine
was <0.1% of that of parental cells (16). Despite an inability
to metabolize adenine, the DAP mutant showed no in
creased resistance or sensitivity to the growth inhibitory
effects of adenine (16). Thus adenine, like adenosine,
remains growth inhibitory without being converted to intra
cellular nucleotides.
The effect of adenine and hypoxanthine
on PP-ribose-P
concentration,
de novo purine synthesis, and ribonucleotide concentrations
was examined. Adenine, 100 /¿M,which
just begins to slow growth, and hypoxanthine,
500 /¿.M,
which does not inhibit growth, both strongly inhibit de novo
purine synthesis throughout
log-phase growth and cause
prolonged reduction in the intracellular
concentration
of
PP-ribose-P (Table 4). The effects of 500 /¿Madenine or
Table 3
Effect of adenine, adenosine, and coformycin on [ifC]glucose
metabolism in lymphoblasts
Lymphoblasts,
4 x 105 cells/ml, were cultured for 24 hr in the
absence or presence of 50 /J.M adenine, 50 /¿Madenosine, 3.5 ^M
coformycin,
or 5 ¿tMEHNA; harvested; and resuspended in fresh
medium with additions but lacking glucose. After 30 min of incu
bation in fresh medium, either [1-14C]glucose or [6-14C]glucose,
2.5 mM (5.0 mCi/mmol),
was added, and 14CO2 generation
was
measured as described in "Materials and Methods." In the absence
of further additions, the generation of 14CO2was 53, 940, and 1500
cpm/106 cells/30 min from [1-14C]glucose and [6-14C]glucose, re
spectively.
Additions
Relative 14CO2generation
from [1-14C]glucose
1.00
0.92
0.93
0.88
0.79
0.86
0.94
0.62
None
Adenine
Adenosine
EHNA
EHNA + adenosine
Coformycin
Coformycin + adenine
Coformycin + adenosine
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Adenosine and Adenine Toxicity in Lymphoblasts
adenosine by lymphoid cells is insufficient to account for
Table 4
immune dysfunction in adenosine deaminase-deficient pa
Effects of growth in adenine or hypoxanthine on purine synthesis
de novo and PP-ribose-P concentration in parent and adenine
tients. Other metabolic (7) and immunological (8) evidence
phosphoribosyltransferase-deficient lymphoblasts
also supports these conclusions. The potentiation of aden
Parallel cultures of WI-L2 and a DAP clone (<0.1% adenine
osine effects by adenosine deaminase inhibitors may be
phosphoribosyltransferase activity) were incubated at 37°ingrowth
understood in terms of their having blocked the principal
medium (10% dialyzed fetal calf serum) containing the indicated
additions. After 26 hr of exposure, the rate of labeling of ¡ntracel- route for converting exogenously supplied adenosine to
lular purines with sodium [14C]formate (2.17 mw, 4.6 ¿tCi//¿mol) nontoxic metabolites.
and the concentration of PP-ribose-P were determined as "Materi
Pyrimidine nucleotide depletion was postulated to be the
als and Methods."
primary cause of adenosine toxicity on the basis of adenosine-mediated growth inhibition being reversed by uridine
(12). In adenosine kinase-deficient lymphoblasts, however,
uridine
failed to reverse adenosine toxicity in the pres
AdditionsNone
lineWI-L2
ence of EHNA (Chart 3). The inability of uridine to reverse
DAPWI-L2
4590160
384<232 adenosine plus EHNA growth inhibition in the adenosine
kinase-deficient cells may be interpreted as follows. Under
conditions of adenosine-mediated PP-ribose-P depletion,
Adenine, 0.5 rriMCell
DAPPurine
1040PP-ribosecells may have limited PP-ribose-P-dependent synthesis of
both purine and pyrimidine nucleotides, and this view is
WI-L2
215
Hypoxanthine, 0.5
supported
by the observed decrease in pyrimidine and
mw
guanine nucleotides. The inability of MTI cells to phosphorylate adenosine may further limit purine nucleotide synthe
hypoxanthine in the absence and presence of 1 rriM uridine
sis in these cells as compared to WI-L2. Although there may
on intracellular concentrations of adenine, guanine, and
be other effects of adenosine in adenosine deaminase and
uracil ribonucleotides were examined (Table 1). Both ade
adenosine kinase-deficient cells, limitation of PP-ribose-Pnine and hypoxanthine cause a 50% reduction in the con
centration of uracil ribonucleotides, but neither causes an dependent reactions is sufficient to account for growth
inhibition caused by adenosine in the adenosine kinaseaccumulation of orotic acid. In the presence of 1 nriM
deficient
cell.
uridine, pyrimidine nucleotide concentrations were greater
Adenine
remained toxic to mutant lymphoblasts deficient
than in the control cells (Table 1); nevertheless, uridine had
activity. The bio
virtually no effect on the growth inhibition caused by in adenine phosphoribosyltransferase
chemical
effects
of
growth
inhibitory
concentrations
of
adenine (16). These experiments indicate that: (a) dimin
adenosine
and
adenine
on
human
lymphoblasts
are
quali
ished PP-ribose-P concentration per se is not growth inhib
tatively similar in that both decrease pyrimidine nucleotides
itory since adenine, which is toxic, and hypoxanthine,
and PP-ribose-P concentrations. The mechanism by which
which is not toxic, both decreased PP-ribose-P concentra
adenosine reduced pyrimidine nucleotide concentrations
tion to the same extent; and (o) diminished pyrimidine
appears to differ from that of adenine and hypoxanthine in
nucleotide concentration is not the sole basis of adenine
that
adenosine (31) but not the purine bases (Table 1)
toxicity since hypoxanthine causes a similar decrease in
causes an accumulation of orotic acid despite nearly equiv
intracellular pyrimidine ribonucleotides, and, more directly,
alent depression of PP-ribose-P concentration (Tables 2
repletion of these pools with uridine does not alter inhibi
and 4). Also, uridine reversed adenosine but not adenine
tion of growth by adenine.
growth inhibition in WI-L2. The PP-ribose-P-dependent
conversion of purine bases to nucleotides can deplete PPribose-P, whereas adenosine in the presence of adenosine
DISCUSSION
deaminase inhibitors is not readily converted to purine
Both the growth inhibitory and metabolic effects of aden- bases (Chart 1) and therefore appears to deplete PP-riboseosine persisted when adenosine metabolism via phosphoP by another mechanism, perhaps by inhibiting PP-riboseP formation. Thus in a previous report we found no evi
rylation or deamination was blocked by the adenosine
dence for adenosine-mediated inhibition of PP-ribose-P
kinase mutation and the adenosine deaminase inhibitors.
Thus growth inhibitory combinations of adenosine and synthetase activity in lymphoblast extracts (31), but adeno
EHNA or coformycin depleted PP-ribose-P and pyrimidine
sine plus coformycin or EHNA partially inhibited pentose
ribonucleotide concentrations in WI-L2 and adenosine ki- phosphate synthesis under conditions of incomplete
nase-deficient mutants. These observations are important
growth arrest (Table 3). Adenine at a very high concentra
in understanding the mechanism of adenosine toxicity. For tion (0.5 rriM) and after 26 hr of incubation (Table 4) did
lower PP-ribose-P concentration in adenine phosphoriboexample, effects analogous to those of adenosine in reduc
ing PP-ribose-P concentrations have been reported for
syltransferase-deficient cells, but we have not determined
2',5'-dideoxyadenosine
(32) and S'-deoxyadenosine (19), whether this occurred by a mechanism involving pentose
which cannot be phosphorylated to the 5'-nucleotide. EHNA shunt inhibition. However, because growth inhibition by
also inhibits PP-ribose-P-dependent nucleotide synthesis
adenine does not produce as severe depletion of pyrimidine
(14). The minimal effect of low concentrations of the aden
ribonucleotides as does adenosine at comparable growth
osine deaminase inhibitors alone in these and other studies
inhibition, adenine and hypoxanthine appear to depress
primarily the steady state concentration of PP-ribose-P
(3, 9, 22, 30) suggest that the endogenous formation of
syn
thesis (cpm/
30min/106
P (pmol/
cells)236
cells)4575 10«
AUGUST 1978
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2361
F. F. Snyder et al.
without completely blocking its availability for nucleotide
synthesis. Our studies suggest that pentose phosphate
precursors other than glucose should be examined as
alternatives to uridine in counteracting adenosine toxicity.
ACKNOWLEDGMENTS
We thank the laboratory of Dr. S. E. Mayer for cAMP assays and Inga
Jansen and Michael Cruikshank for excellent technical assistance.
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CANCER
RESEARCH
VOL. 38
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Cytotoxic and Metabolic Effects of Adenosine and Adenine on
Human Lymphoblasts
Floyd F. Snyder, Michael S. Hershfield and J. Edwin Seegmiller
Cancer Res 1978;38:2357-2362.
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