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(CANCER RESEARCH 37, 911-917, March 1977]
Uridine Triphosphate Deficiency, Growth Inhibition, and Death
in Ascites Hepatoma Cells Induced by a Combination of
Pyrimidine Biosynthesis Inhibition with Uridylate
Trapping1
Dietrich 0. R. Keppler
Biochemisches Institut, Universitat Freiburg im Breisgau, 0 7800 Freiburg, Germany
SUMMARY
some circumstances. The consequences of nucleotide defi
ciency depend not only on the type or group of nucleotides
involved, but also on the extent and the time period of their
depletion (9, 13). The mechanisms available for an induc
tion of nucleotide deficiency include (a) inhibition of de
A selective deficiency of umidine triphosphate (UTP) was
induced in AS-30D matascites hepatoma cells by the synem
gistic action of D-galactosamine and 6-azaunidine. The me
sistance of these hepatoma cells to low concentrations of D novo synthesis,
, by analogs (4, 36); (b) acceleration of
galactosamine (<2 mM) was due to their active de novo
catabolism, e.g., by phosphate trapping (10, 40); (c) inter
pynimidine synthesis which compensated the trapping of fenence with the generation of high-energy phosphate
umidylate in the form of umidine diphosphate-amino sugars
bonds (10, 29, 33); (d) trapping of the nucleoside mono
derived from D-galactosamine. The additional blockage of phosphate moiety (20, 21); and (e) trapping of the nucleo
de novo pynimidine synthesis led to noncompensated unidy
side portion of nucleotides (10).
late trapping with a UTP content of less than 0.05 mmole/kg
A selective depletion of the UTP pool leading to cell
of cell wet weight as compared to the control level of 0.66 necrosis has been observed in liven after a galactosamine
mmole/kg. The induction of UTP deficiency by incubating
induced trapping of unidylate (8, 16, 20). In ascites hepa
the cells with low concentrations of D-galactosamine and 6- toma cells, however, galactosamine concentrations higher
azaunidine (0.5 mM each) was not accompanied by signifi
than 2 mM were required for a depression of cellular UTP to
cant changes in the content of adenine and guanine nucleo
levels below a critical concentration range onto a content of
tides, unidine diphosphate glucose, and unidine diphos
less than 0.1 mmole/kg (23). In addition, a marked depres
phate galactose. The depletion of UTP pools could be me sion of the ATP level was associated with this reduction of
versed within 10 mm by the addition of unidine; orotate or the UTP pool (23). The approach of this study was the
umacilwere completely ineffective in these hepatoma cells. combination of a blockage ofde novo pymimidine nucleotide
A UTP content in the range of 0.1 to 0.4 mmole/kg,
biosynthesis with an amino sugar-induced trapping of un
induced by either 6-azaumidine or D-galactosamine, was as
dylate. This procedure made possible the use of low galac
sociated with a reversible depression of cell growth in sus
tosamine concentrations and resulted in a severe and selec
pension culture. A UTP content below 0.05 mmole/kg led to tive deficiency of UTP in hepatoma cells. The combination
irreversible growth inhibition and to necrocytosis in culture,
of an inhibition of nucleotide biosynthesis with a trapping of
as well as to a loss of transplantability in vivo. Unidine nucleotides induced by sugar analogs can serve as a tool by
reversal studies indicated that the percentage of cells able which selective nucleotide deficiencies can be induced in
to resume growth in culture decreased with an increasing
various cells or tissues. The choice of the inhibitor and of
time period of UTP deficiency. The deficiency period me the sugar analog enables cell specificity of nucleotide de
quired for irreparable or lethal damage in these hepatoma
pletion. A preliminary report on part of this work was given
cells ranged from 3 to 20 hr.
earlier (16).
The principle of noncompensated unidylate trapping can
be extended to other inhibitors of nucleotide synthesis
combined with various nucleotide-trapping sugar analogs.
MATERIALS AND METHODS
Noncompensated nucleotide trapping may be useful for an
induction of selective nucleotide deficiencies in tumor cells.
Ascltes Hepatoma Cells. The transplantable rat ascites
hepatoma AS-30D (37) was carried in 7- to 10-week-old
INTRODUCTION
female Spnague-Dawley mats(Voss, Tuttlingen, Germany).
The tumor cells were transplanted at 7-day intervals by i.p.
injection of 0.2 ml of ascitic fluid collected under sterile
A depletion of nucleotide pools can serve as an efficient
tool to inhibit cellular growth and to induce cell death under
conditions. Transplant generations 315 to 365 were used in
this study.
Chemicals, Enzymes, and Isotopes. D-Galactosa
Bonn-Bad Godesberg , through “
Forschergruppa Labarerkrankungen ,“
Frei
mine HCI was purchased from C. Roth, Kanlsmuhe, Gem
burg, Germany.
many; 6-azaunidine, umidine, all other nucleosides, nucleo
Received August 17, 1976; accepted December 14, 1976.
,This
work
was
supported
by
the
Deutsche
Forschungsgemeinschaft,
MARCH 1977
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911
0. 0. R. Kepp!er
tides, and cofactors were from Boehmingen Mannheim,
Mannheim, Germany. Powdered Swim's S-77 medium and
Swim's 67-G medium were from Grand Island Biological
Company, Grand Island, N. Y. UDP-galactose 4-epimerase
and lactate dehydrogenase were purchased from Sigma
Chemical Co. , St. Louis, Mo. All other enzymes used in this
investigation were from Boehninger Mannheim, Mannheim,
Germany. [2-14C]Uridine and D-[1-‘4C]galactosam
me were
from Amersham Buchler, Bmaunschweig, Germany.
Incubation of Hepatoma Cells. The AS-30D ascites hepa
toma cells (37) were collected on the 7th day after trans
plantation and were washed and suspended in standard
medium as described previously (23). Incubations for up to
6 hr were performed at cell concentrations of 1.5 to 2.5 x
109/Iiten in closed Erlenmeyer flasks under COIaim (1/20) on
a gyratory shaker at 130 rpm. The temperature of the sus
pension was 37 ±0.4°;the pH was kept between 7.45 and
7.25, if necessary, by addition of small amounts of solid
NaHCO3. Duplicate 10-mi aliquots were removed for cell wet
weight determinations (23) at least twice, usually at 30 and
150 mm afterthe beginningof incubation.
The procedure
for cooling, freezing, and deproteinization of the cells for
nucleotide and metabolite analyses has been described
(23).
Suspension Culture of AS-30D Cells. Cells were col
lected under sterile conditions on the 5th or 6th day after
transplantation and were suspended at a cell concentration
of 2 to 4 x 108/liter in Swim's 67-G medium containing
pancreatic autolysate (1/25 by volume) (31). This medium
was supplemented with 2 mM glutamine and ascitic fluid (1/
50 by volume) sterilized by filtration of AS-30D ascites hepa
toma fluid . The cells were grown during the late exponential
phase at 37°on a gyratory shaken at 120 rpm in siliconized
Erlenmeyen flasks under CO@/aim(1/20). Proliferation and
growth were monitored by cell counts in a blood-counting
chamber and by measurement of the absombance of the
suspensions at 623 nm; cellular DNA (5) and protein con
tents (1) were determined at 24-hr intervals. Morphological
examinations, including the exclusion of trypan blue by
these cells, were performed by phase-contrast microscopy.
EnzymatIc Analyses of Nucleotides. UTP (18), UDP-glu
cose, and UDP-galactose (22) were determined spectropho
tometmically using UDP-glucose dehydrogenase as indicator
enzyme. The lUMP2 was measured as UMP after snake
venom phosphodiesterase hydrolysis of 5'-nucleotides (15).
6-Azauridine 5'-monophosphate did not interfere with the
specific assay for UMP. The enzymatic determination of
@GMP
and ICMP has been described (15). ATP was mea
sured with yeast hexokinase and glucose-6-phosphate de
hydrogenase (26). Pyruvate kinase and adenylate kinase
were used for the analysis of ADP and AMP (14).
Metabolism of D-Galactosamine in AS-30D Cells. The
AS-30D ascites hepatoma line has kept the capacity to me
abbreviations
used
are:
lUMP,
sum
of
all
acid-soluble
uracil
5'-
nucleotides; @GMP,
sum of all acid-soluble guanine 5'-nucleotidas; @CMP,
sum of all acid-soluble cytosine 5'-nucleotides; UDP-hexosamines, sum of
UDP-galactosamine and UDP-glucosamina.
912
Table 1
Galactosamine-induced
change of the labeling
pattern
of uracil
nucleotides
109/Iiterat
AS-30Dcells were incubatedat a concentration of 2.5 x
[2-14C]uridine
37°in standard medium (23) supplemented with 0.35 mM
mMD-galactosamine.
(0.8 mCi/liter)
60mm;
and, in the experimental
flask, with 2
The cells were collected and frozen (23)after
Uracilnucleotides
nucleotideswere extractedwith 0.6 M perchlonicacid.
differentsolvents
were separatedby paperchromatographyin 2
thecounting
(11). The relative radioactivities were determined by
asthe of I -cm segmentsof the paper(11) and wereexpressed
Meanvalues
percentage of total radioactivity in uracil nucleotides.
from 3 experiments ±S.D.Radioactivity
in uracil nucleo
tides(%
of
total)Uracil
RESULTS
2 The
tabolize D-galactose. The galactose elimination matewas 9.1
mmoles/kg cells x hr when the cells were incubated in the
presence of 1 mM galactose. Enzymes of the galactose
pathway also metabolize the galactose analog D-galactosa
mine (for review, see Refs. 8 and 9). A marked accumulation
of galactosamine 1-phosphate to a cellular content of more
than 8 mmoles/kg was observed when AS-30D cells were
incubated in the presence of 2 mM galactosamine (16). The
formation of UDP-hexosam ines (UDP-galactosam me +
UDP-glucosamine) and the accumulation of UDP-N-acetyl
hexosamines were demonstrated by biosynthetic labeling of
these amino sugar nucleotides with [1 @1
4C]galactosam me
(16) and with [2-―C]unidine(Table 1). The diversion of the
labeled unidine to the synthesis of UDP-hexosamines and
UDP-N-acetylhexosamines was associated with a strongly
reduced label in UTP (Table 1). The accumulation of UDP
amino sugars caused an increase in LUMP when the cells
were incubated or grown in the presence of galactosamine
(Chart1).
The Role of de Novo Pyrimidine Biosynthesis in the
Galactosamine-induced Depression of UTP Content. The
trapping of umidylate by formation and accumulation of
UDP-amino sugars derived from galactosamine can be
counterbalanced by an increased synthesis of unidylate
either de novo or on the salvage pathway (16, 20). An active
de novo pymimidine nucleotide biosynthesis in AS-30D hep
atoma cells was indicated by the increase of @UMP
at a mate
of 0.65 mmole/kg cells/hr between 1 and 4 hr after addition
of a low galactosamine concentration (0.5 mM) to the me
dium (Chart 1). This rise of @UMPwas completely sup
pressed after a selective inhibition of the formation of umidy
late from omotidine 5'-phosphate (7) by use of 0.1 to 1 mM 6azaumidine (Chart 1). The increased de novo synthesis of
uridylate was elicited by only a limited depression of the
UTP content to about 0.45 mmole/kg cells (Chart 2B). The
rise of LUMP in AS-30D cells showed a lag phase of about
30 mm, however, in comparison with the drop in the UTP
level (Charts 1 and 2). Higher galactosamine concentrations
GalactosamineUTP
nucleotide
Control
25±4UDP
68±8
8±2UMP
8±2
3±1UDP-glucuronate
4±1
1UDP-glucose
1UDP-hexosamines
+ UDP-galactose
4UDP-N-acetylhexosamines
7 ±2
2 ±1
0
5 ±
2 ±
30 ±
11 ±3
27 ±5
CANCER
RESEARCH
VOL. 37
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Noncompensated Uridylate Trapping in Hepatoma Cells
the UTP pool induced by this “
noncompensated unidylate
trapping.―
Selectivity of UTP Deficiency in Hepatoma Cells. The
depletion of UTP pools in AS-30D cells was associated with
a general depression of pumine and pyrimidine nucleotide
content when galactosamine concentrations higher than 1
mM were used (23). This lack of selectivity in the depletion
of nucleotide pools was no longer observed when the con
centration of galactosamine was lowered to 0.5 mM. Adeno
sine phosphates, the adenylate energy charge, and @GMP
remained in the control range, and the @CMP
decreased to
about 70% as compared to the controls when UTP dropped
to less than 5% (Chart 3). The low levels of UDP-glucose and
UDP-galactose in AS-30D cells (23) remained, in contrast to
liver (21), within the control range when the low concentra
tion of galactosamine was used (Charts 3 and 4). This con
dition made possible a discrimination between the conse
quences of a deficiency of UDP-hexoses and of UTP, me
spectively, in these hepatoma cells.
Induction of UTP Deficiency for Defined Time Periods. A
reversal of UTP deficiency within 10 mm was accomplished
a'
a'
a'
.x
0
E
E
0.
I
D
INCUBATION TIME (hr)
@
Chart 1. Acid-soluble uracil nucleotides and the changes in them induced
by galactosamine and 6-azauridine. The incubation of AS-30D cells and the
determination of cell wet weights ware performed as described under “Mate
rials and Methods.―the WMP was determined anzymatically as UMP after
snake venom phosphodiasterase hydrolysis of call extracts (15). Each point
represents the mean from 6 to 8 separate experiments with standard devia
tions of less than l2% of the mean. The concentrations of D-galactosamine
and 6-azaunidina were 0.5 mM each. 0, control;
6-azauridine; A, 6a.zauridina + galactosamina; •,galactosamine.
by means of unidine (Chart 4, left). The early matesof uptake
®
0.7
@
@
0.5
@
@
@E@EE@
__________
0.3
0.3
E
@:::
p
8.
; o.i
•
8.
______________
0.1
A
@
U
2
3
4
INCUBATIONTIME(he)
5
U
I
I
.5
4
INCUBATIONTIME (he)
Chart 2. Induction of UTP deficiency and adjustment of UTP levels in
ascitas hepatoma cells. AS-30D cells were incubated at 37―
in standard
medium (23)supplemanted with 2 (A) or 0.5 (B) mM o-galactosamine, 1 (A) or
(B) 0.5 mM 6-azaunidina, and 1 mM unidine (A ). Freezing of the cells in liquid
nitrogen (23) preceded the enzymatic analysis of UTP content (18). Mean
values ware from 4 to 8 separate experiments with all standard deviations at
lass than 20% of the mean. The same changes in UTP content were found
when tissue culture medium (Swim's 67-G) instead of standard medium (23)
was used. 0, controls; 0, 6-azaunidina; A, 6-azaunidine + galactosamina; S,
galactosamine; •,6-azauridine + galactosamine + unidine.
induced a stronger depression of the UTP content (Chart
2A) and a higher rate of de novo uridylate formation (16). It
should be noted, in comparison to liver (20, 21), that the rise
in LUMP occurred in the absence of significant changes of
the levels of UDP-glucose and UDP-galactose. Inhibition of
de novo pyrimidine nucleotide biosynthesis by 6-azaumidine
(28) was followed by a slight decrease in LUMP (Chart 1)
and by a depression of the UTP content to 0.13 mmole/kg
cells (Chart 2B). Neither the inhibition of uridylate biosyn
thesis nor a trapping of unidylate induced by galactosamine
(<2 mmoles/liter) resulted in a depletion of UTP pools to
less than 0.1 mmole/kg cells. Only a combination of both
mechanisms led to a severe deficiency of UTP. Under this
condition, a UTP content of less than 0.04 mmole/kg cells
was reached after 1 and 2 hr when galactosamine concen
trations of 2 and 0.5 mM were used, respectively (Chart 2). A
major difference in the effects of these galactosamine con
centrations was thus related to the time course of induction
of UTP deficiency rather than to the extent of depletion of
MARCH
and phosphorylation of 0.5 mM unidine were measured by
the increase of @UMPwith time (Chart 4, right) and
amounted to 0.6 and 6.0 mmoles/kg cells x hr in untreated
and UTP-deficient AS-30D cells, respectively. @UMP
contin
ued to rise at a lower mateof 0.8 mmole/kg cells x hr after
replenishment of the UTP pools (Chart 4). This rise come
sponded to the formation of UDP-amino sugars derived
from galactosamine. Addition of umidine to UTP-deficient
cells resulted in an increase of UDP-glucose to levels above
the control values (Chart 4, left). Uptake and phosphoryla
tion of unidine were not inhibited to a significant extent by
equimolam concentrations of 6-azaunidine. This is consistent
with the low Km/Ki ratio for the inhibition of unidine phos
phorylation in whole cells by 6-azaunidine (25). Cytidine also
served as a source of uracil nucleotides in UTP-deficient
hepatoma cells. A linear increase of LUMP at a nate of 0.5
mmole/kg cells x hr in the presence of 0.5 mM cytidine
(Chart 4, right) indicated that the deamination was rate
)8
I'
U
a,
I
INCUBATIONTIME(hr)
Chart 3. Nucleotide content and energy charge (E.C. @)
during the induc
tion of UTP deficiency in AS-30D cells. The concentrations of D-galactosa
mine and 6-azauridina in the incubation medium were 0.5 mM each. Each
point represents the mean from 4 to 10 separate experiments; all standard
deviations were below 18% of the mean.
1977
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913
D.
0.
R.
Kepp!er
Table 2
Effect of UTP deficiency on transplantability and in vivo growth of
AS-30D cells
The cells were incubated for 6 hr as described under “Materials
and Methods―
in standardmedium(23)supplementedwith 2 mMD
galactosamine,
1 mM 6-azauridine,
1 mM uridine, or combinations
of these compounds. The cells were collected by centrifugation
for
5 mm at 120 x g and resuspended at a concentration of 10―cells/
liter
in cold
Swim's
S-77 medium
supplemented
with
25 mM
Na2HPO4,
pH 7.45. The UTPdeficiency period in the cells treated
with galactosamineand 6-azauridinewas much longer than 6 hr,
since the intracellular
metabolites
of the drugs were still present.
FemaleSprague-Dawleyrats, weighing 210 ±30 g and 82 ±8 days
of age,were given i.p. injections of 3 x 10@
cells/animal. The mean
TIMEAFTERGo(N+ 6-AzoUrd(hr)
TIMEAFTERGoLN+ 6-AzaUrd(hr)
Chart 4. Reversal of UTP deficiency by unidine. AS-30D cells were incu
bated as described under “Materials
and Methods.―After a praincubation
period of 15 mm, D-galactosamine (Gal N) and 6-azaunidine (6-AzaUrd) were
added (1/200 by volume) to give a final concentration of 0.5 mM for each.
Two hr later, unidine, cytidine, or uracil ware added at final concentrations of
0.5 mM.Eachpoint representsthe meanfrom 4to 5 separateexperiments.A,
UTP, and UDP-glucose
(UDP-Glc)
contents;
B, changes
in LUMP.
limiting since @CMPincreased about 6 times faster than
LUMP under this condition. Cytidine was insufficient, how
ever, for a complete reversal of UTP deficiency within 2 hr.
Pynimidine precursors without any detectable effect on the
uracil nucleotide content included umacil(Chart 4, right) and
orotate. The lack of increase in pynimidine nucleotide con
tent after addition of 0.5 mM orotate to the medium was
observed in both untreated and galactosamine-treated AS
30D cells. This is in contrast to liver (22) and may be useful
for a selective protection of the liver against UTP deficiency
(8).
Loss of Transplantability and Tumor Growth in Vivo. The
induction of UTP deficiency in AS-30D cells by galactosa
mine + 6-azaumidine (Chart 2A) caused a loss of transplanta
bility and ascites hepatoma growth (Table 2). This effect
was not observed when UTP deficiency was prevented by
addition of unidine to the incubation medium. The limited
depression of the UTP content by 6-azauridine or by galac
tosamine alone and the intracellular accumulation of galac
tosamine metabolites had no effect on transplantability un
der the conditions studied (Table 2).
InhibitIon of Cell Proliferation In Suspension Culture by
Reversible UTP Deficiency. Cell number, cell protein, and
DNA content doubled in 21 hr when AS-30D cells were
grown in primary suspension cultures at cell concentrations
between 1 and 9 x 108/litem(Charts 5 and 6). This rate of cell
growth and proliferation was depressed by less than 13% in
suspensions supplemented with low concentrations of ga
lactosamine (@0.5 mM) or with uridine + galactosamine +
6-azaumidine (Chart 5). A significant depression of cell
growthwas thusnotcausedby thelimited
reduction
ofUTP
pools in the presence of galactosamine alone (Chart 2B), or
by an intracellular formation of galactosamine metabolites,
or by 6-azaunidine 5'-monophosphate as the metabolite of
6-azauridine (25, 28). A reduction in the cellular UTP con
tent to about 0.13 mmole/kg induced by 6-azaumidine alone
(Chart 2B) led to a marked depression of cell proliferation
(Chart 5). The increase in cellular protein during the initial
24 hr of a culture in the presence of 6-azaumidine was
reduced by 32% when compared to the gain in protein of an
untreated cell suspension. Uridine readily reversed the in
914
survival time of rats with ascites tumor growth was 23 days. Ascites
tumor growth is expressedby the number of tumor-bearing rats as
compared
cells.
to the number
of rats receiving
injections
of AS-30D
survi
vors
Addition to incubation mediumAscitestumor
growth60-day
(%)None15/166Galactosamine13/1476-Azaunid
6-azauridine0/14100Galactosamine
+
6-azauridine12/120+
+
uridine
LU
8-
-a
U,
..a
LU
U
6
TIME (hr)
Chart 5. Effect of different UTP levels on hepatoma cell proliferation in
suspension culture. Cultures of 50 ml each were grown as described under
“Materials
and Methods.―Points represent the mean from cell counts of at
least 2 flasks kept simultaneously under the same conditions. The cell num
bars include viable, nonviable, and necrotic cells. The concentrations of D
galactosamine, 6-azauridine, and uridine were 0.5 mM each. 0, control (no
addition); L@,6-azaunidine; 0, galactosamine; U, galactosamine + 6-azauri
dine; •,galactosamine + 6-azauridine + unidine.
hibitomy effects of 6-azaumidine on AS-30D cell growth.
A complete cessation of cell proliferation induced by ga
lactosamine + 6-azaunidine (Charts 5 and 6) was associated
with a lack of increase in cellular protein and DNA at 24 and
48 hr. Examination of these cells by phase-contrast micros
copy revealed a loss of all cell clusters after 24 hr. The
number of single cells that were altered by a rupture of the
plasma membrane with cytoplasmic protrusions and cell
shrinkage increased between 16 and 48 hr. The appearance
of necrotic cells was associated with an increasing amount
of cellular debris. Prevention of UTP deficiency by addition
CANCER
RESEARCH
VOL. 37
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Noncompensated
Uridylate
Trapping
in Hepatoma
Ce!!s
DISCUSSION
A severe and selective depletion
LU
of the UTP pool can be
induced only if the mateof unidylate-consuming processes
clearly exceeds the rate of umidylate production by de novo
synthesis and regeneration (20). The unidylate-trapping ac
tion of galactosamine was compensated for largely by the
active de novo pynimidine nucleotide biosynthesis of the
-I
LU
U
P0
AS-30D ascites hepatoma
TIME (hr)
Chart 6. Reversal by unidine of the growth inhibition induced by UTP
deficiency in AS-30D cells. The experimental conditions and drug concentra
tions werethe sameasdescribedfor Chart5. •,
control cells (no addition);
cells when concentrations
of the
amino sugar below 2 mM were used (Charts 1 and 2). The
additional blockage of de novo pynimidine nucleotide bio
synthesis led to a noncompensated umidylate trapping with
severe UTP deficiency as the primary consequence. The
selective inhibition of uridylate biosynthesis by 6-azaunidine
has been shown to cause a reversible depression of cell
growth (34) (Chart 5). The depression of UTP content (Chart
2B) and the inhibition of nucleic acid synthesis by 6-azaumi
dine (25) in AS-30D and Novikoff hepatoma cells in suspen
sion were only moderate in comparison with the synergistic
effect of the noncompensated umidylate trapping. It is me
markable that the blockage of de novo pynimidine nucleo
tide biosynthesis by 6-azaumidine in liver in vivo was com
pletely compensated for, probably by salvage pathway syn
thesis, and there is no depression of hepatic UTP content
(21). The rate of net umidylate biosynthesis in the presence
of galactosamine is only one-third as high in liver as in AS
., galactosamine
+6-azaunidine.
Open
symbols,
cultures
inthepresence
of
galactosamine + 6-azaunidine with unidine added at the following times: 0, 6
hr; 0, 9 hr; L@,16 hr; 0, 20 hr.
of uridine completely protected against necrosis of AS-30D
cells. Necrocytosis was also prevented by the addition of
cytidine.
The point of irreversible or irreparable cell damage was
Studied by means of UTP deficiency periods of different
lengths. Uridine was added after 3, 5, 6, 9, 16, 20, and 24 hr,
respectively, to separate cell cultures containing galactosa
mine + 6-azaunidine. The potential to resume exponential
cell growth was lost after about 20 hr (Charts 6 and 7).
Reversal of UTP deficiency after 3 to 16 hr resulted in an
increasing amount of necrotic cells and cellular debris as
sociated with a decreasing number of cell clusters and
viable cells when examined between 24 and 48 hr (Chart 6).
The comparison of the growth rates at a given cell concen
tration with the slope of the control curve indicated that the
percentage of cells able to resume growth after unidine
addition decreased with an increasing length of the UTP
deficiency period (Chart 7).
(U
30D cells (16). It is consistent
that an additional
inhibition
of
unidylate synthesis is not required in liver for an induction of
severe UTP deficiency by galactosamine. The blockage of
pynimidine biosynthesis in hepatoma cells, on the other
hand, allows the use of a galactosamine concentration (0.5
mM) which is lower than that required for a necrogenic
action in hepatocytes [1 mM (24)].
The inhibitory effects of D-galactosamine and D-glucosa
mine on various tumor lines have been studied extensively
(2, 27, 32, 38). The effective amino sugar concentrations
used in most studies were between 20 and 125 mM (2, 38).
These concentrations would cause severe toxicity in vivo , at
least with galactosamine, which induces a spotty necrotic
hepatitis at a dose of 1 to 2 mmoles/kg body weight (8, 19).
The available evidence indicates that the cytotoxicity of
galactosamine and of glucosamine, at least at low concen
trations, is mediated by an interference of uracil nucleotide
metabolism with subsequent induction of UTP deficiency
(3, 9, 21). An additional
general
loss and depression
of
nucleotides caused by phosphate trapping in the form of
amino sugar monophosphates has been observed only at
high concentrations of galactosamine (23) and glucosamine
(3, 30). One may expect that the tumor growth-inhibitory
DEFICIENCYPERIOD(UTP<aO5 mmote/kg)
(hr)
Chart 7. Late consequences of different time periods of UTP deficiency.
The experimental conditions ware the same as those described for Chart 6.
Uridine was added to cultures containing D-galactosamine + 6-azauridine
(0.5 mM each) after 3, 5, 6, 9, 16, and 20 hr; this corresponds to periods of
depression of UTP content below 0.05 mmole/kg of 1, 3, 4, 7, 14, and 18 hr,
respectively (see Chart 28). The growth rates were monitored by measure
ments of the absorbanca of the suspensions at 623 nm. Growth rates be
twean 42 and 48 hr were expressed as percentage of the growth rate in the
control culture at the respective cell concentration.
effects of the umidylate-trapping amino sugars can be in
duced with relatively low concentrations (<1 mM) when the
sugar analogs are combined with an inhibitor of unidylate
synthesis. This relationship has been established for the
effect of galactosamine in AS-30D ascites hepatoma cells
(Charts 5 and 6); a complete inhibition of cell growth by
galactosamine alone required a concentration of about 8
mM (not shown).
The selective and reversible adjustment of UTP levels in
ascites hepatoma cells (Charts 2 to 4) allowed an analysis of
the extent and time period of UTP depression as related to
the growth-inhibitory on the lethal consequences to the
MARCH 1977
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915
D.
0.
A. Keppler
cells. A UTP content of between 0.4 mmole/kg and the
control mange(0.66 ±0.05 mmole/kg) was without a signifi
cant effect on AS-30D cell growth, viability, or transplanta
bility (Charts 2 and 5; Table 2). A UTP content
of between
0.1 and 0.4 mmole/kg was associated with a reversible
depression of cell growth (Charts 2 and 5), but transplanta
bility was not affected
(Table 2). A UTP content
of below
0.05 mmole/kg (Charts 2 and 3) led to lethal cell damage
indicated by an irreversible growth inhibition (Chart 7), nec
rocytosis, and a loss of transplantability (Table 2). The time
period of UTP deficiency required for irreparable or lethal
damage in the hepatoma cells was not in a similar mangefor
all cells in the nonsynchronized suspension culture (Chart
7). The uridine reversal studies indicated that the lethal UTP
deficiency period may be as short as about 3 hr in some
cells and as long as about 20 hr in others. These observa
tions would be compatible with a cell cycle-dependent sen
sitivity of the cells to UTP deficiency. Alternatively, one
could also consider a varying susceptibility of the cells to
the induction of UTP deficiency. A comparable time-de
pendent increase in the percentage of killed cells has been
described after exposure of LS178Y cells to cytosine amabi
noside for different periods of time (6).
The present study with ascites hepatoma cells supports
the view (9, 16) that neither the metabolites of galactosa
mine nor the low levels of UDP-glucose and UDP-galactose
are growth inhibitory or cytotoxic (Charts 3 to 5). The events
leading from UTP deficiency to growth inhibition most likely
include inhibition of ANA and protein synthesis, both of
which have been studied in detail as consequences of he
patic UTP deficiency (9, 20, 35). Our understanding of the
sequence of events leading from UTP deficiency to cellular
deathisincompleteatpresent(17).
The differential
effect
of
this selective substrate deficiency on the various forms of
ANA-polymerases may result in a lethal imbalance of pro
teinsynthesis.
The combination of galactosamine-induced
umidylate
trapping with 6-azaunidine-induced inhibition of the de novo
synthesis of uridylate demonstrates a new principle for an
induction of nucleotide deficiency in tumor cells (Chart 8).
Replacement of galactosamine by glucosamine as the un
dylate-tnapping sugar analog alters the cell specificity. In
analogy to noncompensated unidylate trapping, a noncom
pensated trapping of guanylate or cytidylate may be an
effective means for tumor cell kill as well as for inhibition of
viral multiplication . Noncompensated guanylate trapping
can be induced by the combination of an inhibitor of inosi
nate dehydrogenase (12, 39) with guanylate-trapping ana
logs of D-mannose or L-fucose. The noncompensated nu
cleotide trapping may be useful in the design of drug com
binations inducing selective nucleotide deficiencies by a
metabolic synergism. The intensity and duration of nucleo
tide deficiency can be further influenced by prevention or
inhibition of nucleoside uptake or by supply of precursors
on the salvage pathway. The latter possibility makes possi
ble the reversal of a nucleotide depletion after a defined
time period.
ACKNOWLEDGMENTS
The author is grateful to Ute Stumpp-Grigat for her excellent technical
assistance
andtoDr.James
J.Starling
forhiscriticalreading
ofthemanu
script.
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917
Uridine Triphosphate Deficiency, Growth Inhibition, and Death
in Ascites Hepatoma Cells Induced by a Combination of
Pyrimidine Biosynthesis Inhibition with Uridylate Trapping
Dietrich O. R. Keppler
Cancer Res 1977;37:911-917.
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