[CANCER RESEARCH 32, 2034-2041,
October 1972]
Biochemical Mechanisms for the Synergism between 6-Thioguanine
and 6-(Methylmercapto)purine Ribonucleoside in Sarcoma 180
Cells1
J. Arly Nelson2 and R. E. Parks, Jr.
Section of Biochemical Pharmacology, Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island 029] 2
SUMMARY
The synergism between 6-(methylmercapto)purine
ribonucleoside (MMPR) and 6-thioguanine was studied in the mouse
Sarcoma 180 ascites tumor. The 5'-monophosphate nucleotide
of MMPR forms rapidly to levels of 1 to 2 mM in the tumor
cells, and the steady-state
levels of 5-phosphoribosyl
1-pyrophosphate increase 4- to 5-fold in 6 to 12 hr. This
permits a significantly greater synthesis of 6-thioguanosine
monophosphate (6-thioGMP) after injection of 6-thioguanine.
These findings are similar to those in previous reports
concerning the synergism between 6-mercaptopurine
and
MMPR. In addition, MMPR increases the biological f1/2 of
6-thioGMP from about 7 hr to 10 hr. The concentrations of
ATP and GTP decrease by 50% or greater after MMPR and the
concentration of UTP in the cell doubles, probably as the
result of greater 5-phosphoribosyl 1-pyrophosphate availability
for pyrimidine biosynthesis. With a 6-thioguanine-resistant cell
line, the t1/2 of 6-thioGMP is only 3 hr but increases to about
7 hr after MMPR pretreatment. Azaserine produces effects on
endogenous nucleotide pools and 6-thioGMP formation similar
to those of MMPR, but it is without effect on the r1/2 of
6-thioGMP. The synergism between MMPR and other
thiopurines may involve effects of MMPR on catabolism as
well as synthesis of the analog nucleotides and sequential
blockade of purine biosynthesis.
MMPR causes a significant elevation of the steady-state levels
of PRPP in tissues (25), probably as a result of
pseudofeedback
inhibition of PRPP amidotransferase
by
MMPR-5'-P (10, 11, 13, 17). The increased availability of
PRPP results in enhanced synthesis of the analog nucleotide,
6-mercaptopurine ribonucleoside 5'-phosphate in cells treated
with 6-MP (25, 33). A marked synergism was also observed
when 6-MP and MMPR were examined for their effects on the
steady-state levels of adenine nucleotides of S-180 cells (33).
Although an earlier report from this laboratory dealt
primarily with the interaction of MMPR and 6-MP, it was
reported that potentiation also occurred with MMPR and 6-TG
in prolonging the survival time of mice bearing the S-180
tumor (33). The present study explores in greater detail the
mechanism of this synergism, and the results indicate a
mechanism that is similar to that seen with MMPR and 6-MP.
In addition to enhanced formation of 6-thioGMP, it has been
observed that pretreatment
of S-180 cells with MMPR
significantly increases the half-life of 6-thioGMP in the cells.
Pretreatment with MMPR also causes a marked increase in the
steady-state levels of UTP, which suggests that when the de
novo pathway of purine biosynthesis
is blocked by
MMPR-5'-P, the pathway of pyrimidine nucleotide synthesis is
stimulated. Preliminary
been presented (22,23).
MATERIALS
INTRODUCTION
Striking synergism occurs in antitumor effects when
MMPR3 is administered in combination with other thiopurines
such as 6-MP or 6-TG. Several biochemical observations offer
insights into the mechanism of this synergism. Treatment with
'This work was supported by USPHS Grant GM 16538 and Grant
07340 of the National Cancer Institute of the USPHS.
'Present address:
Biochemistry Research, Southern Research
Institute, Birmingham, Ala. 35205.
'The abbreviations used are: MMPR, 6-(methylmercapto)purine
ribonucleoside; 6-MP, 6-mercaptopurine; 6-TG, 6-thioguanine; PRPP,
5-phosphoribosyl 1-pyrophosphate; MMPR-5'-P, 6-(methylmercapto)purine ribonucleoside 5'-phosphate; S-180, Sarcoma 180; 6-thioGMP,
6-thioGDP, and 6-thioGTP, the 5'-mono di-, and triphosphates of
6-thioguanosine; S-180/TG, Sarcoma 180 cells resistant to 6-thio
guanine; 6-TGR, 6-thioguanosine.
Received March 30, 1972; accepted June 8, 1972.
2034
reports of portions of this work have
AND METHODS
Treatment of Cells. The S-180 and S-180/TG cells were
kindly supplied by Dr. A. C. Sartorelli of Yale University.
They were maintained by the i.p. transplantation of 5 to 6 X
IO6 tumor cells each week in female CD1 mice (Charles River
Laboratories, North Wilmington, Mass.). Drugs, in 0.9% NaCl
solution, were given i.p. to mice 4 to 6 days after implantation
of the tumor cells. Nucleotide levels and 6-TG-3 5S metabolites
were measured in acid-soluble extracts of the cells. For
preparation of the extract, the cells were collected from the
peritoneal cavity and washed by centrifugation at room
temperature; approximately 3 X IO7 tumor cells in a volume
of 1 ml were then added to 0.5 ml of 12% perchloric acid at
0°.As determined by microscopic examination, the washed
tumor cells were virtually free of other cells, such as
erythrocytes and granulocytes. Any tumor cell samples that
were visibly contaminated with blood were-discarded. After
CANCER RESEARCH
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6-TG andMMPR Synergism
glucose-1-P and inorganic pyrophosphatase
(Chart
1).
Omission of glucose-1-P in the incubation was necessary to
demonstrate specificity for UTP, since some hydrolysis of ATP
and GTP occurred in the presence or absence of glucose-1-P,
possibly due to ATPase activity in the enzyme preparations
used. The incubation was performed at room temperature for
90 min with 0.5 ml of neutralized cell extract, 0.20 mM
glucose-1-P, 5 mM MgCl2, 0.15 MM unit of uridyl transferase
(UDP-glucose-pyrophosphorylase,
EC 2.7.7.9), 1 /uM unit of
inorganic pyrophosphatase (EC 3.6.1.1), and 80 mM Tris-HCl
at pH 8.5 in a final volume of 1 ml.
Other Procedures. Radioactivity was measured in a Packard
Tri-Carb liquid scintillation spectrometer with a dioxane
cells) was measured. The cells from each mouse were analyzed scintillation solution (4). Tumor cells were counted with a
separately. Metabolites of 6-TG-35S in the acid-soluble Model B Coulter counter, Coulter Electronics, Inc., Hialeah,
material were also determined with the LCS-1000. For this Fla. PRPP was determined as described by Henderson and
purpose, fractions of the eluting material were collected over Khoo (12) with S-180 cells instead of Ehrlich ascites cells as a
consecutive 2-min intervals (about 0.4 ml/fraction), and the source of AMP pyrophosphorylase activity.
radioactivity in each fraction was measured. Identities of
Reagents and Chemicals. Azaserine was purchased from
Chromatographie peaks were determined by comparison of Calbiochem, Los Angeles, Calif. MMPR, 6-TG, and 6-thiouric
experimental retention times to those of authentic material, acid were obtained from Sigma Chemical Co., St. Louis, Mo.
Dr. Shih-Hsi Chu of this laboratory prepared the 6-TG-35 S by
enzymatic peak shift (5), and/or thin-layer chromatography.
Acid-soluble extracts from S-180 cells treated with 6-TG-3 s S an isotope exchange reaction (21) using rhombic 35S obtained
removal of the insoluble material by centrifugation,
the
acid-soluble extract was neutralized with KOH (for details, see
Ref. 33). The wash medium was composed of 40 mM Tris-HCl,
20 mM KC1, 88 mM NaCl, 2 mM MgCl2, 5.5 mM glucose, and
22 mM potassium phosphate buffer at a final pH of 7.4. This
medium was also used in the in vitro incubation of S-180 cells.
Chromatographie
Procedures
and
Identification
of
6-TG-35S Metabolites. Nucleotide levels were determined in
the acid-soluble extracts by high pressure liquid chromatography, with a VarÃ-anAerograph LCS-1000 instrument, exactly
as described by Brown (5). Generally, 20 /ul of the extract
were placed on the column; therefore, the nucleotide content
of about 4 X 10s cells (approximately 2 mg, wet weight, of
yielded a peak of radioactivity with a retention time of 35
min, a time that corresponds to that of authentic 6-thioGMP.
The radioactivity of this peak also cochromatographed with
6-thioGMP on polyethyleneimine cellulose thin-layer plastic
sheets (Gel 300PEI, Brinkmann Instruments, Inc., Westbury,
N. Y.) when developed with l N NH4OH (RF 0.62), twice with
l M LiCl (RF 0.50), or with a stepwise formate buffer system
(Ref. 8; RF 0.30). Furthermore, incubation of the acid-soluble
extract with guanylate kinase (ATP:GMP phosphotransferase,
EC 2.7.4.8) for 12 hr at room temperature shifted all the
radioactivity with a retention time of 35 min to a peak with a
retention time near 80 min (6-thioGTP; see below). The
reaction did not stop at the nucleoside diphosphate level
(6-thioGDP), probably because the conditions of incubation
are favorable for nucleoside diphosphokinase activity, and
small amounts of this enzyme would lead to the formation of
6-thioGTP. The incubation mixture contained 200 n\ of
concentrated acid-soluble extract, 4 mM ATP, 125 mM KC1,
12.5 mM MgCl2, and 1 ;uM unit of guanylate kinase in a final
volume of 262 jul. The analog nucleotides, 6-thioGDP and
6-thioGTP, had retention times of about 60 and 80 min,
respectively. In extracts of S-180 cells treated with 6-TG-35S,
succinate thiokinase (succinate:CoA ligase-GTP, EC 6.2.1.4)
shifted the radioactivity eluting at 80 min to a peak with a
retention time of 60 min. It has previously been shown that
6-thioGTP can replace GTP in the succinate thiokinase
reaction (7). The incubation
was performed at room
temperature for 30 min with 100 n\ of concentrated extract,
50 mM Tris-succinate, 0.1 mM CoA, and 1 juM unit of
succinate thiokinase in a final volume of 161 /ul.
Identification of UTP in S-180 Cell Extracts. An enzymatic
peak shift (5) was performed to verify the presence of UTP in
acid-soluble extracts of S-180 cells. UTP, with a retention time
of 50 min on the high-pressure liquid Chromatograph, was
quantitatively converted to UDP-glucose (retention time, 25
min) by the action of uridyl transferase in the presence of
from New England Nuclear, Boston, Mass. Dr. K. C. Agarwal
synthesized the 6-thioGMP by an enzymatic method (19). Mr.
David Baccanari provided the succinate thiokinase, which he
had purified from pig heart to a specific activity of
approximately 20 fiM units /mg protein (through Stage 1 in
Table 1 of Ref. 7). Dr. K. C. Agarwal supplied the partially
purified guanylate kinase (specific activity, l //M unit/mg
protein) from human erythrocytes (1). Uridyl transferase
(Grade 3 from Baker's yeast ; specific activity, 45 ¿/Munits/mg
protein) and inorganic pyrophosphatase (Type 3 from Baker's
yeast, 500 p.M units/mg protein) were products of Sigma.
Presentation of Data. Results are expressed as mean value ±
standard error of the mean unless otherwise specified.
Comparison of sample means by Student's t test and the
regression analyses were performed as described by Goldstein
(9). Values for metabolite concentrations are given per ml of
packed tumor cells without correction for extracellular space.
In these experiments, 1 ml of packed cells was equivalent to
approximately 2.2 X IO8 cells.
RESULTS
Synergistic Effects of MMPR and 6-TG on Cellular Growth.
In survival studies reported earlier, mice bearing S-180 cells
were treated for 6-day periods with combinations of MMPR
and 6-TG, and a marked synergism was observed with some
long-term survivors (33). In survival studies in which daily
treatments are given over a period of days, an element of
pretreatment is introduced. This is especially true of a drug
such as MMPR, since it has been shown that the high
intracellular concentrations of the metabolite, MMPR-5'-P, are
maintained in tumor cells for relatively long periods of time,
i.e., t1/2 of 24 hr or more (6, 11, 16, 25). Therefore,
substantial amounts of this analog nucleotide may remain at
the end of each 24-hr period. For evaluation of the effect of
OCTOBER 1972
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2035
J. Arly Nelson and R. E. Parks, Jr.
004
0.04
- 0.03
00 3 •¿
"0.02
-
- 0.02
o.o i -
- 001
30
40
RETENTION TIME (MIN)
Chart 1. Identification of UTP in S-180 cell extract by enzymatic peak shift. Acid-soluble extracts of S-180 cells removed from 4 mice were
pooled and incubated with uridyl transferase in order to convert UTP to UDP-glucose (see Materials and Methods). The mice had received MMPR,
4 mg/kg, 15 hr earlier. The chromatograms were obtained with the high-pressure liquid Chromatograph and with the extract from about 5 mg of
cells.
MMPR pretreatment of the action of 6-TG, mice bearing
approximately
1 X IO6 S-180 cells were given single
treatments with 6-TG and MMPR alone or in combination. Six
days later the numbers of tumor cells present in the peritoneal
cavities of the animals were counted (Table 1). Treatment with
MMPR or TG alone had little effect, a finding which is
consistent with the results of earlier survival time studies (33),
and single, simultaneous treatment with MMPR and 6-TG
showed slight additive effects. However, when the mice were
pretreated with a single dose of MMPR followed by an
injection of 6-TG 6 hr later, a marked decrease in cell numbers
was observed.
Effect of MMPR Pretreatment on 6-TG-35S Uptake. The
effect of MMPR pretreatment on 6-TG uptake into S-180 cells
was examined by administration of 6-TF-35S i.p. to mice
bearing the ascites tumor. The amount of acid-soluble
radioactivity in the tumor cells and the radioactivity in the
peritoneal fluid was then determined over a 1-hr period. There
was a rapid disappearance of radioactivity from the peritoneal
fluid (Chart 2). Only 10 to 20% of the administered
radioactivity remained after 5 min, and after 1 hr this level was
reduced to only 1 to 2%. The disapperance of radioactivity
from the peritoneal fluid appeared to be more rapid in animals
pretreated with MMPR. The uptake of radioactivity into the
tumor cells was increased by MMPR pretreatment, the levels of
radioactivity reached a plateau after about 20 min. The
increased levels of radioactivity in the cells of animals
pretreated with MMPR could not be accounted for by higher
concentrations of labeled 6-TG in the bathing fluid, since at all
time periods the amount of radioactivity in the peritoneal
fluid was less in the animals that received MMPR. In
experiments not shown, the uptake of 6-TG-3 s S by S-180 cells
in vitro was also increased in tumor cells which were removed
2036
Table 1
Effect of MMPR pretreatment on the inhibition of Sarcoma 180
cell growth by 6-TG
The first treatment was given 24 hr after implantation of 1.6 X 10'
tumor cells. The second treatment was given 6 hr later. The cells were
quantitatively removed and counted 6 days after implantation.
First treatment0
NaClMMPR0.9%
0.9%
NaClMMPR
6-TGMMPR0.9%
+
Second treatment
NaCl0.9%
NaCl6-TG0.9%
NaCl6-TG14.6
No. of cells/mouse X IO7
(8)b13.2
±1.4
±0.7(10)12.3
1.2(11)9.8
±
(9)1.8C±0.2
±0.8
(9)
G Doses in mg/kg were MMPR, 4 ; 6-TG, 1.
°No. of mice.
c Significantly different from all other values (p < 0.01).
from mice treated with MMPR in vivo. These results strongly
suggest that the increased uptake of 6-TG is due to an effect of
MMPR on the tumor cells rather than an effect on the host.
Effect on MMPR on PRPP, 6-thioGMP,
and 6-thioGTP Levels
Earlier studies (25, 33) demonstrated
that prior or
concurrent treatment with MMPR causes a significant increase
in the rate and quantity of 6-thioIMP synthesized after 6-MP
administration. In studies with Ehrlich ascites cells, marked
increases in the PRPP levels occurred after MMPR treatment
(25). These PRPP increases became pronounced after a lag of
about 6 hr and lasted for at least 96 hr. In experiments not
shown, similar results were obtained with S-180 cells in
response to treatment with MMPR alone or in combination
with 6-TG. Administration of MMPR caused a 4- to 5-fold
increase in PRPP concentrations to levels of about 2.5 mM
CANCER RESEARCH
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VOL. 32
6-TG and MMPR Synergism
50
60
20-
-20
15o
Q
10-
30
40
TIME AFTER 6-TG-35S,
90
60
min
Chart 2. Loss of 6-TG-35S from peritoneal fluid and entrance into
ascites cells after i.p. injection to mice. Mice bearing 4-day implants of
S-180 cells were pretreated for 12 hr with MMPR, 4 mg/kg, before
injection of 6-TG-35S, 3.6 mg/kg (specific activity, 525 cpm/nmole).
The acid-soluble radioactivity in the cells and the radioactivity in the
peritoneal fluid (P.P.) was determined for each of 2 to 4 mice at the
indicated times after 6-TG-3SS administration. The average volume of
cells per mouse were 0.40 ± 0.02 and 0.54 ±0.04 ml in
MMPR-pretreated mice and in mice not pretreated with MMPR,
respectively.
after a lag of 6 to 12 hr, and these levels remained elevated for
at least 48 hr. In this tumor line, the t1/2 of MMPR-5'-P was
about 30 hr. A marked increase in 6-thioGMP and 6-thioGTP
synthesis occurred concurrently with the elevated PRPP levels,
which supports the hypothesis that the greater availability of
PRPP increases the function of the purine salvage enzyme,
hypoxanthine-guanine phosphoribosyltransferase
(25, 33).
6-thioGMP in these cells, the observed increase in half-life is
consistent with the possibility that the high concentration of
MMPR-5'-P impedes the metabolism of 6-thioGMP. The
combination of enhanced synthesis and impeded degradation
of 6-thioGMP results in an approximately
3-fold greater
amount of this metabolite in the cells after a 24-hr period.
Effects of MMPR and 6-TG on Nucleotide Pools. After a
single dose of MMPR, the levels of ATP and GTP in S-l 80 cells
were reduced by as much as 50% of control values in 5 to 6 hr,
and the levels of UTP were increased more than 2-fold (Chart
4). These effects lasted as long as 48 hr after administration of
the drug. The reductions in ATP and GTP were similar except
for an apparently greater sensitivity of GTP to the drug seen at
1 hr. The effect of MMPR on ATP and GTP levels is much like
the reduction in total adenine pools produced by azaserine,
another inhibitor of purine biosynthesis de novo, in S-180 cells
(see Fig. l in Ref. 30). It appears that the effect of MMPR
(presumably via inhibition of de novo purine biosynthesis) is
to lower total purine nucleotide pools rather than to shift the
energy charge (2), since no marked increase in ADP, GDP,
AMP, or GMP occurred in these experiments.
The effect of MMPR on UTP levels may be independent of
the effect of the drug on ATP and GTP, since there is a partial
recovery of UTP levels to control values between 24 and 48 hr,
whereas the ATP and GTP levels are reduced to the same
degree at these times after giving the drug (Chart 4).
Simultaneous administration of 6-TG added to the effect of
MMPR on ATP and GTP levels and prevented the increase in
UTP observed 1 hr after a dose of MMPR alone (Table 2).
Unlike MMPR or azaserine, 6-TG did not increase UTP levels
at any time studied. This might indicate that the effect of
12
phosphate nucleotides are utilized. Evidence suggesting that
this occurs was obtained when the intracellular half-life of
6-TG-35S in S-180 cells was determined in control animals and
in animals pretreated with MMPR (Chart 3). The disappear
ance of radioactivity from the tumor cells followed Ist-order
kinetics in both control and MMPR-pretreated animals, which
implies that throughout the time course studied a single
rate-limiting step predominated. The half-time of disappear
ance of the intracellular radioactivity in control animals was
about 7 hr, whereas in MMPR-pretreated animals the half-time
was increased about 38% to approximately 10 hr. Since the
acid-soluble
radioactivity
consists
predominantly
of
OCTOBER
o
o
•¿
•¿
NO MMPR
MMPR
•¿3
100 E
-100
t '/, = 9.9hr
- 50
50
15 to 30 min. Also, MMPR pretreatment was shown to
interfere with the conversion of 6-MP to MMPR-5'-P, and it
was suggested that the high levels of MMPR-5'-P interfere with
an S-methylation reaction (33). Because of this observation, it
appeared likely that the high levels of MMPR-5'-P might also
interfere with other enzymatic reactions in which 5'-mono-
24
200
Effect of MMPR on the Intracellular Half-life of 6-TG-3 s S
Earlier studies have shown that when S-180 cells are
incubated with MMPR there is a marked accumulation of
MMPR-5'-P to levels in the range of 1 to 2 mM in a period of
18
200
t '/, = 7.2hr
20 -
- 20
IO
IO
24
TIME AFTER
6-TG
.35.S,hf
Chart 3. Effect of MMPR pretreatment on the biological half-life of
6-TG-35S metabolites in S-180 cells in vivo. Mice bearing 4-day
implants of S-180 cells were given MMPR, 4 mg/kg, 12 hr before the
administration of 6-TG-3SS, 2.2 mg/kg (specific activity, 610
cpm/nmole). The cells were removed, and the radioactivity in the
acid-soluble extracts was determined at the times shown. Each point
represents the mean value determined from 5 mice. The slopes of the 2
lines are significantly different (p < 0.02).
1972
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research.
2037
J. Arly Nelson and R. E. Parks, Jr.
6-TG on ATP and GTP levels is not due to inhibition of purine
biosynthesis de novo via pseudofeedback inhibition of PRPP
amidotransferase by 6-thioGMP (10, 13, 17, 31, 32). Also,
6-TG alone was without effect on ATP and GTP levels 0.5,3,
6, or 24 hr after the drug, and the effect of a simultaneous
dose of 6-TG and MMPR was the same as MMPR alone at these
times (data not shown). A possible explanation for the effect
of 6-TG at 1 hr is that partial inhibition of purine (and
possibly pyrimidine) biosynthesis de novo occurs shortly after
administration
of 6-TG due to utilization of PRPP for
6-thioGMP formation. Since the drug disappears rapidly from
the injection site (see Chart 2), the utilization of PRPP for
6-thioGMP formation may be transitory. Utilization of PRPP
by 6-TG may also account for the prevention of the increase in
UTP levels caused by MMPR. Specifically, UTP levels increased
from 0.27 to 0.50 /¿mole/ml cells after MMPR alone, but
i//-Atr"r
i
i
l
l
l
//
24
l
48
-200-150
\*'
\r!
_Jotr
"""--A---"
^
UTP
\•
Aa^..^
i
\
-100-50-O-i
tzOuu.oi«250
increased only to 0.34 jumole/ml cells after MMPR and 6-TG
(Table 2).
The effects of azaserine to lower ATP and GTP levels and to
increase UTP levels are similar to those of MMPR seen 12 to
13 hr after the drugs (Table 2; Chart 4). Administration of a
high dose of 6-TG 12 hr after azaserine had been given did not
enhance the effects of azaserine on the nucleotide pools.
Since the decreased
concentrations
of the natural
nucleotides in response to drug administration could have been
an artifact resulting from the death of significant numbers of
cells, the technique of fluorochromasia (28) was applied to
determine whether the tumor cells were in a viable state at the
time when the nucleotide measurements were made. Cells were
examined 12 and 36 hr after treatment with MMPR alone and
after simultaneous or sequential treatment with MMPR plus
6-TG. By the use of this technique, no significant loss of
cellular viability was detected at the time of measurement.
Approximately 90 to 95% of the cells in the treated and
control groups appeared viable. Although a large percentage of
these cells are destined to die in response to combined
treatment with MMPR and 6-TG, a significant degree of
cellular lysis had not occurred at the time of biochemical
measurements performed in these studies.
6-TG-3 5S Metabolites and Nucleotide Pools in S-180 Cells
The nucleotide profile of S-180 cells is shown in the upper
figure of Chart 5. The adenine and guanine nucleotides were
present primarily in the form of ATP and GTP whether or not
the cells were treated with MMPR, 6-TG, or the combination
of 6-TG and MMPR. The effect of 6-TG on ATP, GTP, or UTP
f-250-200-150-IOO-50_n
1 hr after giving the drug was insignificant or less than the
4
6
48
effect of MMPR at 13 hr (Chart 4; Table 2); therefore, the
TIME AFTER MMPR, hr
dramatic effect of MMPR to lower the ATP and GTP levels
Chart 4. Effects of MMPR on nucleotide levels in S-180 cells. MMPR,
while increasing the UTP level is well demonstrated in Chart 5.
4 mg/kg, was administered to mice 4 to 5 days after implantation of the
Since these samples were chromatographed
before the
tumor cells. At the times shown the cells were removed, and the
installation of a new column on the LCS-1000 instrument, the
nucleotide levels were measured in the acid-soluble extracts. Each point
resolution is not typical of samples for which nucleotide
represents the mean of values determined separately for cells removed
concentrations
were actually determined or as previously
from each of 4 to 7 mice. All mean values 1 hr or later were
described for the instrument (5).
significantly different (p < 0.05) from simultaneous controls, except
Almost all the acid-soluble radioactivity present in the
for the ATP level measured 1 hr after MMPR. Control values are given
S-180 cells 1 hr after administration of 6-TG-3SS is in the
in Table 2.
ATP
N>~~~~~^--
--^r^^f"f
// ^fjj
Table 2
Effects of 6-TG, MMPR, and azaserine on nucleotide pools in S-180 cells
The first treatment, if given, was administered 4 to 5 days after implantation of the tumor cells. The
second treatment was given 12 hr later. One hr after the second treatment, the cells were removed and
extracted for nucleotide analyses. Results are expressed as jumólesnucleotide per ml cells.
First
treatment
Second
treatment
ATP
GTP
UTP
0.181.98±
±
0.070.64
±
0.040.24
±
0.05o0.63
±
mg/kg6-TG,
1
0.201.922.321.561.111.070.240.070.08b0.09b0.02b0.95
0.050.19
±
0.1360.62
±
mg/kgMMPRC6-TG,
6.5
NaClNoneNoneAzaserinecAzaserineNone6-TG,
0.030.50
±
0.04b0.34
±
0.05"0.42
±
b0.43±0.05
MMPR0.9%
1 mg/kg +
0.030.50
±
0.06b0.49
±
0.03b0.57
±
NaCl6-TG,
±
0.03ba636633
±
0.01
b0.27
6.5 mg/kg2.57
NoneNone0.9%
"Value determined for 39 mice in 7 individual experiments. Other values of n refer to numbers of
mice.
b Significantly different from no treatment controls (p < 0.05).
c Doses in mg/kg were MMPR, 4; azaserine, 2.
2038
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6-TG and MMPR Synergism
IO
0.3
senting the extract of about 9 mg of the
same cells. TU, 6-thiouric acid; TGMP,
TGDP, and TGTP, 6-thioGMP, 6-thioGDP, and 6-thioGTP, respectively.
50
40
30
60
70
60
90
0.3
—¿
NO MMPR
-- MMPR
Chart 5. High-pressure liquid Chro
matograph of acid-soluble material from
S-180 cells. Cells were removed from
mice 1 hr after administration of
6-TG-35S, 6.5 mg/kg (specific activity,
500 cpm/nmole). MMPR, 4 mg/kg, was
given 12 hr before 6-TG-35S to one
group of 5 mice, and another group of 5
mice was not pretreated. The combined
acid-soluble extracts of the tumor cells
from each group were concentrated and
applied to the column. Upper tracing,
nucleotide profile of the extract of
about 4.5 mg of cells; lower tracing,
radioactivity eluting from the LCS-1000
instrument
at 2-min intervals (see
"Materials and Methods"), and repre
20
in
esJO.2
0.2
U
U
m o.i
ce
ai
O
co
m
r o
n
•¿ •¿
NO MMPR
o—-o MMPR
700
700
500
E 500
o.
U
300
TGR.TG
TGDP
TU
TGTP
y \
100
o
300
!
•¿ !
IO
20
I
I
30
40
50
100
Jï^o^.
!
60
70
O
90
80
RETENTION TIME (min)
form of 6-thioGMP and 6-thioGTP (Chart 5, lower tracing).
Since the acid-soluble material from about 9 mg of cells is
shown, the levels of 6-thioGMP were of the order of 0.45 and
0.10 ptmole/ml cells in MMPR-pretreated and control samples,
respectively. The corresponding levels of 6-thioGTP were of
the order of 0.20 and 0.04 ¿¡mole/ml
cells. The acid-soluble
metabolites shown in Chart 4 were from cells of animals
treated with a high dose of 6-TG. However, qualitatively
similar results were obtained 1 to 24 hr after a dose of 6-TG of
only 1 mg/kg (data not shown) and in previous studies of the
metabolism of 6-TG (3, 20).
6-TG-35S Metabolism by S-180 Cells Resistant to 6-TG. The
prolongation of the biological i1/2 of 6-TG-35S by MMPR
pretreatment
(Chart 3) indicated that MMPR-5'-P may
interfere with the catabolism of 6-thioGMP. Dr. A. C.
Sartorelli and his colleagues have developed a S-180 cell line
that is resistant to 6-TG due to an increase in the rate of
catabolism of 6-thioGMP by a nonspecific phosphohydrolase
(3, 34). This tumor provided a model to test whether or not
MMPR-5'-P interferes with the breakdown of 6-thioGMP. The
t1/2 of 6-TG-35 S acid-soluble metabolites is only about 3 hr in
24
l
200
100-
-,
o
o
•¿
.
A
50-
200
MMPR
CONTROL
-100
A AZASERINE
-50
6.6hr
-20
20-1
3.2 hr
•¿IO
IO
O
6
12
18
24
TIME AFTER 6-TG-35S,hr
Chart 6. Biological half-life of 6-TG-'5 in S-180/TG cells. Cells were
removed 1 to 24 hr after administration of 6-TG-35 S, 5 mg/kg, to mice
the resistant cell line (Chart 6), compared to a tl/2 of about 7
hr in the sensitive tumor (Chart 3). Pretreatment with MMPR
increased the f1/2 to greater than 6 hr in the resistant tumor.
Although azaserine pretreatment, like MMPR, increased the
uptake of 6-TG-3 s S by the tumor cells, azaserine pretreatment
did not increase the f1/2. The r1/2 of 6-thioGMP was the same
as that for the total acid-soluble radioactivity in the control
and MMPR-pretreated
samples, i.e., 3.4 and 7.1 hr,
respectively, by high-pressure liquid chromatography of the
samples shown in Chart 6. These data strongly indicate that
MMPR-5'-P interferes with the catabolism of 6-thioGMP via
that had been pretreated for 12 hr with MMPR, 4 mg/kg, or azaserine, 2
mg/kg. The number associated with each line is the t\¡-¿
for that
treatment.
inhibition of a phosphohydrolase in the tumor cells.
The acid-soluble metabolites of 6-TG-3SS in the S-180/TG
determined by thin-layer chromatography (polyethyleneiminecellulose with water as the solvent), there were about equal
cells are shown in Table 3. There is a greater amount of 6-TG
and 6-TGR in the resistant cells than in the sensitive tumor
(Chart 5). This finding is consistent with a lower concentration
of 6-thioGMP maintained by an increased phosphohydrolase
activity in this cell line (34). MMPR pretreatment increased
the level of 6-thioGMP and reduced the levels of 6-TG and
6-TGR in the cells, a result which would be expected from
inhibition of the phosphohydrolase
by MMPR-5'-P. As
OCTOBER 1972
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research.
2039
/. Arly Nelson and R. E. Parks, Jr.
amounts of 6-TG and 6-TGR present in the samples shown in
Table 3. MMPR pretreatment also increased the levels of
6-thioGTP about 3-fold in the S-180/TG tumor.
DISCUSSION
The mechanism by which MMPR potentiates the effect of
6-TG in S-180 cells may be similar to that from the MMPR and
6-MP synergism described previously (24, 25, 33). Specifically,
MMPR increases the steady-state levels of PRPP, thereby
permitting an increased formation of 6-thioGMP from 6-TG or
of 6-thioIMP from 6-MP. In the present report it was also
noted that MMPR protects 6-thioGMP from metabolic
degradation. It appears likely that an alternative substrate
effect occurs. The high concentrations of MMPR-5'-P found in
the tumor cells may compete for a 5'-nucleotidase or
phosphohydrolase that degrades 6-thioGMP. Since the product
of MMPR-5'-P cleavage, MMPR, is a poor substrate for purine
nucleoside phosphorylase (14), MMPR can recycle by reacting
with adenosine kinase. This may account for the maintenance
of high MMPR-5'-P levels in the cell for long periods of time.
There was a marked fall in purine nucleotides after MMPR
and 6-TG treatment, with a greater effect on GTP than ATP
(Table 2). The opposite was found with MMPR and 6-MP,i.e.,
a greater effect on ATP than GTP (33). The greater inhibitory
effect of 6-TG on guanine metabolism and the accumulation
of high levels of 6-thioGMP in S-180 cells (Chart 5) are
consistent with a metabolic block at the level of guanylate
kinase (18, 19). The concentrations of 6-thioGMP in the
tumor cells, especially when the 6-TG and MMPR combination
is given, exceed that of GMP by 1 to 2 orders of magnitude.
The presence of 6-thioGTP in cells treated with 6-TG is in
accord with the earlier observations that 6-thioGMP is an
alternative substrate with a low maximal velocity for guanylate
kinase (19) and that 6-TG is incorporated into the DNA of
various tumors (15). Thus, the 6-thioGTP:6-thioGMP ratio
strongly favors the 5'-monophosphate nucleoside, whereas the
opposite is true with the GTP:GMP ratio (Chart 5). Also, the
low level of 6-thioGDP implies that the 6-thioGDP to
6-thioGTP conversion is rapid relative to the 6-thioGMP to
6-thioGDP conversion.
Although a decrease in adenine and guanine nucleotides
occurs as would be predicted from the postulated sites of
enzymatic blockade by MMPR and 6-TG, it is not possible to
conclude without further evidence that this decrease is directly
Table 3
Acid-soluble metabolites of 6-TG-3 *S in S-180/TG cells
Cells were removed 1 hr after treatment of mice bearing the tumor
with 5 mg of 6-TG-3 sS per kg. One group of mice received 0.9% NaCl,
and another group received 4 mg of MMPR per kg 12 hr before
administration of 6-TG-3SS. The metabolites were determined by
high-pressure liquid chromatography of the pooled acid-soluble extracts
of the cells from 5 mice in each group. Values are expressed as
nmoles/ml cells.
Pretreatment
6-TG, 6-TGR
6-thioGMP
0.9% NaCl
MMPR
17
7
32
92
2040
6-thioGDP
6-thioGTP
13
37
related to lethal effects. This matter has been discussed in
detail in an earlier report (33).
Azaserine, like MMPR-5'-P, is an inhibitor of the de novo
pathway of purine biosynthesis and also increases the
formation of 6-thioGMP from 6-TG (29). When tested in the
S-180/TG tumor, a marked increase in 6-thioGMP synthesis
occurred as expected, but azaserine did not increase the t1/2
of 6-thioGMP as did MMPR (Chart 6). Thus, it appears that
azaserine made more PRPP available for 6-thioGMP synthesis
but did not interfere with the degradation of 6-thioGMP. In
contrast, MMPR treatment of the S-180/TG tumor increased
both the synthesis of 6-thioGMP and its biological half-life.
This observation is consistent with the hypothesis that
MMPR-5'-P protects 6-thioGMP by competing with the
phosphohydrolase
that appears responsible for the rapid
degradation of 6-thioGMP in this resistant tumor line (34).
The increase in UTP levels observed following MMPR
treatment might be explained by an increased availability of
PRPP for orotic acid phosphoribosyltransferase
and resultant
enhancement
of pyrimidine
nucleotide
synthesis. The
possibility exists that MMPR might also potentiate the action
of 5-fluorouracil since a pyrimidine phosphoribosyltransferase
has been shown to be responsible for 5-fluorouridine
5'-phosphate formation (26, 27). Furthermore, if MMPR-5'-P
proves to be a potent alternative substrate for 5'-nucleotidases
or phosphohydrolases
as indicated above, perhaps the
degradation of analog nucleotides other than 6-thioGMP will
be inhibited. These speculations are the subject of continued
investigation.
REFERENCES
1. Agarwal, R. P., Scholar, E. M., Agarwal, K. C, and Parks, R. E., Jr.
Identification and Isolation on a Large Scale of Guanylate Kinase
from Human Erythrocytes. Effects of Monophosphate Nucleotides
of Purine Analogs. Biochem. Pharmacol., 20: 1341-1354, 1971.
2. Atkinson, D. E. Regulation of Energy Metabolism. Exploitable
Molecular Mechanisms and Neoplasia. The University of Texas M.
D. Anderson Hospital and Tumor Institute at Houston, pp.
397-413. Baltimore: The Williams & Wilkins Co., 1969.
3. Bieber, A. L., and Sartorelli, A. C. The Metabolism of Thioguanine
in Purine Analog-resistant Cells. Cancer Res., 24: 1210-1215,
1964.
4. Bray, G. A. A Simple Efficient Liquid Scintillator for Counting
Aqueous Solutions in a Liquid Scintillation Counter. Anal.
Biochem., /: 279-285, 1960.
5. Brown, P. R. The Rapid Separation of Nucleotides in Cell Extracts
Using High-Pressure Liquid Chromatography. J. Chromatog., 52:
257-272, 1970.
6. Caldwell, I. C., Henderson, J. F., and Paterson, A. R. P. The
Enzymic Formation of 6-(Methylmercapto)purine Ribonucleoside
5'-Phosphate. Can. J. Biochem., 44: 229-245, 1966.
7. Cha, S., Cha, C.-J. M., and Parks, R. E., Jr. Succinic Thiokinase. IV.
Improved Method of Purification, Arsenolysis of Guanosine
Triphosphate, Succinate-dependent
Guanosine Triphosphatase
Activity, and Some Other Properties of the Enzyme. J. Biol.
Chem.,242: 2577-2581, 1967.
8. Crabtree, G. W., and Henderson, J. F. Rate-limiting Steps in the
Interconversion of Purine Ribonucleotides in Ehrlich Ascites
Tumor Cells in Vitro. Cancer Res., 31: 985-991, 1971.
CANCER RESEARCH
VOL. 32
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research.
6-TG andMMPR Synergism
9. Goldstein, A. Biostatistics: An Introductory Text. New York: The
Macmillan Co., 1964.
10. Henderson, J. F. Feedback Inhibition of Purine Biosynthesis in
Ascites Tumor Cells by Purine Analogues. Biochem. Pharmacol.,
72:551-556,
1963.
11. Henderson, J. F., Caldwell, I. C., and Paterson, A. R. P. Decreased
Feedback Inhibition in a 6-(Methylmercapto)purine Ribonucleoside-resistant Tumor. Cancer Res., 27: 1773-1778, 1967.
12. Henderson, J. F., and Khoo, M. K. Y. Synthesis of
5-Phosphoribosyl l-Pyrophosphate from Glucose in Ehrlich Ascites
Tumor Cells in Vitro. Ì.
Biol. Chem., 240: 2349-2357, 1965.
13. Hill, D. L., and Bennett, L. L., Jr. Purification and Properties of
5-Phosphoribosyl Pyrophosphate Amidotransferase from Adenocarcinoma 755 Cells. Biochemistry, 8: 122-130, 1969.
14. Krenitsky, T. A., Elion, G. B., Henderson, A. M., and Hitchings, G.
H. Inhibition of Human Furine Nucleoside Phosphorylase: Studies
with Intact Erythrocytes and the Purified Enzyme. J. Biol. Chem.,
245:2876-2881,1968.
15. LePage, G. A. Incorporation of 6-Thioguanine into Nucleic Acids.
Cancer Res., 20: 403-408, 1960.
16. Loo, T. L., Luce, J. K., Sullivan, M. P., and Frei, E. Clinical
Pharmacologie Observations on 6-Mercaptopurine and 6-Methylthiopurine Ribonucleoside. Clin. Pharmacol. Therap., 9: 180-194,
1968.
17. McCollister, R. J., Gilbert, W. R., Jr., Ashton, D. M., and
Wyngaarden, J. B. Pseudo-feedback Inhibition of Purine Synthesis
by 6-Mercaptopurine Ribonucleotide and Other Purine Analogues.
J. Biol. Chem., 239: 1560-1563, 1964.
18. Miech, R. P., Parks, R. E., Jr., Anderson, J. H., Jr., and Sartorelli,
A. C. An Hypothesis on the Mechanism of Action of
6-Thioguanine. Biochem. Pharmacol., 16: 2222-2227, 1967.
19. Miech, R. P., York, R., and Parks, R. E., Jr. Adenosine
Triphosphate-Guanosine
5'-Phosphate Phosphotransferase.
II.
Inhibition by 6-Thioguanosine 5'-Phosphate of the Enzyme
Isolated from Hog Brain and Sarcoma 180 Ascites Cells. Mol.
Pharmacol., 5: 30-37, 1969.
20. Moore, E. C., and LePage, G. A. The Metabolism of 6-Thioguanine
in Normal and Neoplastic Tissues. Cancer Res., 18: 1075-1083,
1958.
21. Morávek,J., and Nejedly, Z. Labelling of 6-Mercaptopurine and
6-Mercaptopurine Riboside by an Exchange Reaction with
Elementary Sulphur-35S. Chem. Ind. London, 530, 1960.
22. Nelson, J. A., and Parks, R. E., Jr. Effects of 6-(Methylmercapto)purine Nucleoside on Purine Nucleotide Pools and 6-Thioguanine
Metabolism in Sarcoma 180 Cells. Pharmacologist, 13: 210, 1971.
23. Nelson, J. A., and Parks, R. E., Jr. Mechanism of the Synergism
between
6-Thioguanine
and
6-(Methylmercapto)purine
Ribonucleoside. Proc. Am. Assoc. Cancer Res., 13: 90, 1972.
24. Paterson, A. R. P., and Moriwaki, A. Combination Chemotherapy:
Synergistic Inhibition of Lymphoma L5178Y Cells in Culture and
in Vivo with 6-Mercaptopurine and 6-(Methylmercapto)purine
Ribonucleoside. Cancer Res., 29: 681-686, 1969.
25. Paterson, A. R. P., and Wang, M. C. Mechanism of the Growth
Inhibition
Potentiation
Arising
from
Combination
of
6-Mercaptopurine with 6-(Methylmercapto)purine Ribonucleoside.
Cancer Res., 30: 2379-2387, 1970.
26. Reyes, P. The Synthesis of 5-Fluorouridine-5'-Phosphate
by a
Pyrimidine Phosphoribosyltransferase
of Mammalian Origin. I.
Some Properties of the Enzyme from P1534J Mouse Leukemic
Cells. Biochemistry, 8: 2057-2062, 1969.
27. Reyes, P., and Hall, T. C. Synthesis of 5-Fluorouridine-5'-
28.
29.
30.
31.
32.
33.
34.
Phosphate by a Pyrimidine Phosphoribosyltransferase of Mam
malian Origin. II. Correlation between the Tumor Levels of the
Enzyme and the 5-Fluorouracil-Promoted Increase in Survival of
Tumor-Bearing Mice. Biochem. Pharmacol., 18: 2587-2590, 1969.
Rotman, B., and Papermaster, B. W. Membrane Properties of Living
Mammalian Cells as Studied by Enzymatic Hydrolysis of
Fluorogenic Esters. Proc. Nati. Acad. Sei. U. S., 55: 134-141,
1966.
Sartorelli, A. C. Approaches to the Combination Chemotherapy of
Transplantable Neoplasms. Progr. Exptl. Tumor Res., 6: 228-288,
1965.
Sartorelli, A. C., and Booth, B. Inhibition of the Synthesis of
Thy mine Nucleotides by Azaserine. Mol. Pharmacol., 3: 71-80,
1967.
Sartorelli, A. C., and LePage, G. A. Metabolic Effects of
6-Thioguanine. II. Biosynthesis of Nucleic Acid Purines in Vivo and
in Vitro. Cancer Res., 18: 1329-1335, 1958.
Sartorelli, A. C., Upchurch, H. F., Bieber, A. L., and Booth, B. A.
Some Metabolic Effects Exerted by Azaserine and Purine Analogs
in Vivo. Cancer Res., 24: 1202-1209, 1964.
Scholar, E. M., Brown, P. R., and Parks, R. E., Jr. Synergistic
Effect of 6-Mercaptopurine and 6-Methylmercaptopurine Ribo
nucleoside on the Levels of Adenine Nucleotides of Sarcoma 180
Cells. Cancer Res., 32: 259-269, 1972.
Wolpert, M. K., Damle, S. P., Brown, J. E., Sznycer, E., Agrawal,
K. C., and Sartorelli, A. C. The Role of Phosphohydrolases in the
Mechanism of Resistance of Neoplastic Cells to 6-Thiopurines.
Cancer Res., 31: 1620-1626, 1971.
OCTOBER 1972
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2041
Biochemical Mechanisms for the Synergism between
6-Thioguanine and 6-(Methylmercapto)purine Ribonucleoside in
Sarcoma 180 Cells
J. Arly Nelson and R. E. Parks, Jr.
Cancer Res 1972;32:2034-2041.
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