(Received for publication, April 3, 1997)
From the Otsuka Department of Clinical and Molecular Nutrition, School of Medicine, The University of Tokushima, 3-18-15, Kuramoto-cho, Tokushima-city, 770, Japan
Factors controlling relative flux rates of the
de novo and salvage pathways of purine nucleotide
biosynthesis during animal cell growth are not fully understood. To
examine the relative role of each pathway for cell growth, three cell
lines including CHO K1 (a wild-type Chinese hamster ovary fibroblast
cell line), CHO ade A (an auxotrophic cell line deficient
of amidophosphoribosyltransferase (ATase), a presumed rate-limiting
enzyme of the de novo pathway), and CHO ade
A
transfected with human ATase cDNA (
A+hATase)
resulting in 30-350% of the ATase activity of CHO K1, were cultured
in purine-rich or purine-free media. Based on the enzyme activities of
ATase and hypoxanthine phosphoribosyltransferase, the metabolic rate of
the de novo and salvage pathways, the rate of cell growth
(growth rate) in three cell lines under various culture conditions, and
the effect of hypoxanthine infusion on the metabolic rate of the
de novo pathway in rat liver, we concluded the following.
1) In
A+hATase transfectants, ATase activity limits the
rate of the de novo pathway, which is closely linked with
the growth rate. 2) Purine nucleotides are synthesized preferentially
by the salvage pathway as long as hypoxanthine, the most essential
source of purine salvage, can be utilized, which was confirmed in rat
liver in vivo by hypoxanthine infusion. The preferential
usage of the salvage pathway results in sparing the energy expenditure
required for de novo synthesis. 3) The regulatory capacity
of the de novo pathway (about 200%) was larger than that
of the salvage pathway (about 20%) with constant hypoxanthine
phosphoribosyltransferase activity.
Purine nucleotides synthesized via de novo and salvage
pathways are indispensable for cell growth through DNA and RNA
syntheses and for the ATP energy supply. Interference of purine
metabolism has thus been the target of antineoplastic drugs. However,
it is unclear which of the two pathways is more important for the supply of purine nucleotides during cell growth. Purine nucleotide synthesis catalyzed by HPRT,1 the key
enzyme of the purine salvage pathway, was reported to be more active
than that catalyzed by ATase, the presumed rate-limiting enzyme of the
de novo pathway, in many tissues and malignant cells (1, 2).
The increased metabolic rate via the de novo pathway and
ATase activity are, however, more strongly linked with cell growth and
malignant transformation than the metabolic rate via the salvage
pathway and HPRT activity (1, 3-7). To interpret this enigma, we
investigated the mutual regulation of purine biosynthesis between the
de novo and salvage pathways. The cDNA cloning of rat
and human ATase (hATase) in our laboratory (8, 9), an ATase-deficient
auxotrophic Chinese hamster ovary fibroblast cell line of CHO ade
A, and CHO ade
A transfected with hATase
cDNA driven by the cytomegalovirus promoter (
A+hATase) enabled us to explore the rate-limiting
property of ATase and to study the relationship between the two
pathways in three cell lines in two purine-free or two purine-rich
media relative to their contribution to cell growth. Furthermore, the
effect of continuous Hx supplement on the metabolic rate of de
novo synthesis was examined in rat liver.
CHO K1 (wild type), CHO ade A
(10), which were gifts from Dr. David Patterson (Eleanor Roosevelt
Institute for Cancer Research, CO), and
A+hATase were
cultured at 37 °C in a CO2 incubator in the following media: (I) Ham's F-12 purine-rich medium containing 30 µM Hx with 10% fetal calf serum (FCS); (II) Ham's F-12
with 10% FCS treated with 1.25 mg (0.9 unit)/liter xanthine oxidase
(XO) from buttermilk (Sigma) at 37 °C overnight, serving as a
purine-free medium; (III) RPMI 1640 purine-free medium supplemented
with 10% purine-free FCS; (IV) RPMI 1640 with 10% purine-free FCS and
30 µM Hx, serving as a purine-rich medium. In media I and
IV, both the de novo and salvage pathways function in CHO K1
and
A+hATase, but only the salvage pathway functions in
CHO ade
A. In media II and III, only the de
novo pathway functions in CHO K1 and
A+hATase, and
neither of the two pathways functions in CHO ade
A.
Purine-free FCS was prepared by dialysis against 100-fold volumes of
0.9% NaCl with several exchanges of the dialysis solution at 4 °C
for 24 h. The complete removal of Hx in media II and III was
confirmed first by the disappearance of the Hx peak in the reversed
phase high performance liquid chromatography analysis through a C18
column in 50 mM potassium phosphate buffer (pH 4.6) with a
gradient from 0 to 5% of methanol for 20 min at the flow rate of 1.5 ml/min (data not shown), and second by no growth of CHO ade
A in these media (Table I).
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hATase cDNA (2.2 kilobase pairs) was inserted into
the cloning site downstream of the cytomegalovirus promoter in
pBCMGSNeo (14.5 kilobase pairs), which includes the replication origin
of bovine papilloma virus (BPV) leading to 10-500 copies per cell in
mammalian cells (11, 12). Five micrograms of this plasmid construct
purified with the Plasmid Maxi Kit (QIAGEN, Hilden, Germany) were mixed
with Transfectam solution (BioSepra Inc., Marlborough, MA) according to
the manufacturer's protocol, and overlaid onto CHO ade A
(2 × 106 cells/90 mm tissue culture plate) in 5 ml of
Opti-MEM (Life Technologies, Inc.). After the incubation at 37 °C
for 6 h in a CO2 incubator, the DNA-containing medium
was replaced with 10 ml of Ham's F-12 with 10% FCS and 800 µg/ml
G418 to select transfectants with a high expression of the BPV vector
(12). Several clones of
A+hATase were isolated after G418
selection for 7 days. To select clones with high ATase activity,
A+hATase was further cultured in Ham's F-12 + XO medium
for 7 days. The culture in this purine-free medium positively selected the clones with high ATase activity associated with a high growth rate.
The number of copies in transfectants was determined by Southern blot
analysis (12-14).
In a 35-mm dish, 1 × 105 cells were plated in 2 ml of medium and incubated at 37 °C in a CO2 incubator. After culturing for 24, 48, 72, or 96 h, the cells were counted after trypsinization with 0.1% trypsin and 0.02% EDTA, using an improved Neubauer hemocytometer. The doubling time (hours) of cultured cells was determined from cell counts during the logarithmic growth phase and its reciprocal was defined as the growth rate.
Determination of Metabolic Rates of de Novo and Salvage PathwaysThe metabolic rates of de novo and salvage pathways were, respectively, determined by the incorporation of [14C]glycine and [14C]Hx in acid-soluble purines in comparison to the acid-insoluble fraction (15, 16). In a 90-mm tissue culture plate, 2 × 106 cells were plated. After the recovery of cell functions from plating by 18 h of culture, [14C]glycine or [14C]Hx was added to the medium at the final concentrations of 200 µM (185 kBq/ml) and 50 µM (37 kBq/ml), respectively. The cells were cultured in radioactive medium for 30 min, washed three times with 10 ml of ice-cold phosphate-buffered saline, and harvested with a rubber policeman. Purine ribonucleotides were extracted from the cells in 1 ml of 2 N perchloric acid at 100 °C for 60 min. The extract was cooled on ice for 5 min and centrifuged at 35,000 × g at 4 °C for 5 min. The supernatant was applied to a column (0.5 × 2 cm) of AG-50W-X8 (Bio-Rad) equilibrated with 0.1 N HCl. After washing with 5 ml of 1 N HCl, the acid-soluble purines were eluted with 5 ml of 6 N HCl, and counted in a scintillation counter. The amount of purines was determined by the absorbance of the eluate at 260 nm. To measure the incorporation of [14C]glycine in protein, the acid-insoluble precipitate after the last centrifugation was washed three times with 10% trichloroacetic acid, dissolved in 200 µl of 0.3 N NaOH, and the radioactivity was counted in a scintillation counter. The protein concentrations were assayed by Bradford's (17) method. The metabolic rate of the de novo pathway was defined as the ratio of the dpm incorporated into purines per 1 OD at 260 nm to the disintegrations/min incorporated into 1 mg of protein, while the rate of the salvage pathway was defined as the disintegrations/min incorporated into purines per 1 OD at 260 nm.
Assay of ATase and HPRT ActivitiesCells detached with trypsin and EDTA were centrifuged at 500 × g at 4 °C for 5 min. The cell pellet was washed with 1 ml of phosphate-buffered saline in a microtube. After centrifugation again at 500 × g at 4 °C for 5 min, the cell pellet was resuspended in 100 µl of sonication buffer containing 50 mM potassium phosphate buffer (pH 7.4), 5 mM dithiothreitol, 100 µg/ml phenylmethylsulfonyl fluoride, and 2 µg/ml aprotinin. Sonication was performed by the Sonifier 250 (Branson Ultrasonics Corp. Danbury, CT) with a 90% duty cycle at the output dial setting of 3 for 15 s. These sonication conditions were selected because they yielded the highest extraction of ATase activity (data not shown). The supernatant after the centrifugation at 35,000 × g at 4 °C for 20 min was used for the assay of both ATase and HPRT. The complete breakdown of the cell membrane was confirmed by microscopic observation of the pellet.
The method of ATase assay was the same as reported previously (18). In brief, the cell lysate was incubated in 50 mM potassium phosphate buffer (pH 7.4) containing 5 mM 5-phosphoribosyl 1-pyrophosphate (PRPP), 5 mM MgCl2, 1 mM dithiothreitol, and 5 mM [14C]glutamine (5.55 kBq/µmol) at 37 °C for 1 h. Formed [14C]glutamate was separated from [14C]glutamine by high voltage paper electrophoresis at 800 W for 15 min and counted in a scintillation counter. The PRPP-dependent hydrolysis of glutamine to glutamate was regarded as representing ATase activity.
To assay HPRT activity, the cell lysate was incubated in 50 mM Tris-Cl (pH 7.4) containing 1.5 mM PRPP, 5 mM MgCl2, and 5 mM [14C]Hx (27.8 kBq/µmol) in the assay volume of 50 µl at 37 °C for 20 min. To stop the reaction, the assay mixture was cooled on ice, and 5 µl of 0.5 M EDTA were added. The formed [14C]IMP was separated from [14C]Hx by high voltage paper electrophoresis at 800 W for 15 min, and the [14C]IMP spot on the filter paper was cut out under a UV lamp. The radioactivity of this spot was counted in a scintillation counter.
Northern Blot Analysis of HPRTBecause the HPRT activity in
CHO fibroblasts was nearly constant under all of the experimental
conditions in this study (see Table VI), mRNA levels of HPRT were
examined by Northern blot analysis using rat HPRT cDNA as a probe.
For the preparation of the probe, total RNA was extracted from rat 3Y1
fibroblasts by ISOGEN (Nippon Gene, Osaka, Japan) and
reverse-transcribed to cDNA with the avian myeloblastosis virus
reverse transcriptase first-strand cDNA synthesis kit (Life
Sciences, Inc., St. Petersburg, FL). Rat HPRT cDNA in 697 base
pairs (a sense primer; 5-GACCGGATCCGTCATGTCGACCCT-3
and an antisense
primer; 5
-CTTGTGAATTCAACTTGCCGCTGT-3
) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA in 621 base pairs (a sense primer; 5
-TGGCGTCTTCACCACCATGGAGAA-3
, and an antisense primer;
5
-TTGTCATTGAGAGCAATGCCAGCC-3
) were amplified by the polymerase chain
reaction using the primer pairs shown in parentheses and cloned into a
pCR II vector (Invitrogen Corp., San Diego, CA). The base sequence of
the cloned polymerase chain reaction products was confirmed to be
identical to the reported sequence by the fluorescent automated DNA
sequencer (ABI 377, Perkin-Elmer, Division of Applied Biosystems,
Foster City, CA). Northern blot analysis was performed by the standard
method (14).
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To examine the effect of the continuous supply of Hx on the metabolic rate of the de novo pathway in rat liver in vivo, 5.0 µmol/200 g of body weight/12 h of Hx were continuously infused for 12 h via an intravenous cannula into the jugular vein of 8-week-old male Wistar rats according to the method previously published (19). Thirty minutes before the end of infusion, 185 kBq/44-45 nmol [14C]glycine was administered intravenously. The rat liver was sampled exactly 30 min after the injection by freeze-clamping, and the liver sample pulverized under liquid nitrogen was placed in 4 ml of 2 N perchloric acid. The metabolic rate of the de novo pathway was then determined by the procedures mentioned above. To examine the effect of the continuous supply of Hx on the increased rate of the de novo pathway by glucagon in liver (19), 0.5 mg of glucagon/200 g of body weight/12 h with or without Hx of 5.0 µmol/200 g of body weight/12 h was similarly infused, and the metabolic rate of the de novo pathway in liver was determined. The stimulative effect of glucagon on the de novo purine synthesis in liver has been reported (19, 20) and is applied to glucagon and insulin therapy for acute liver failure.
Statistical AnalysisAll data were presented as means ± S.D. The number of repetitive determinations for each value or point is as follows: six to eight for Table I, three for Table IV, three to four for Tables V and VI and Fig. 3, and two to four for Figs. 1 and 2. For comparison of data, Student's paired or unpaired t test was used. A probability of less than 0.05 was considered statistically significant.
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To determine the
necessary concentrations of purine bases for the maximal growth rate of
CHO fibroblasts, ATase-deficient CHO ade A cells were
cultured in purine-free RPMI medium supplemented with Hx, adenine, or
guanine at various final concentrations of up to 50 µM.
Of the three purine bases, Hx most effectively increased the growth
rate, while no cell growth was observed with guanine as the only source
of the salvage pathway. Because Hx at a concentration of more than 30 µM increased the growth rate of CHO ade
A
fibroblasts to the maximal plateau level, and because adenine in
addition to 30 µM Hx inversely decreased the growth rate
(data not shown), it was considered that Hx at a concentration over 30 µM drives the flux through the salvage pathway at its
maximum in CHO ade
A fibroblasts. These observations
demonstrated that Hx is the principal source for the salvage pathway in
CHO ade
A fibroblasts.
Six clones of
A+hATase obtained after G418 selection in purine-free medium were
cultured in purine-rich medium. The level of ATase expression among
these clones ranged from about 30 to 350% of that in CHO K1. The level
of ATase expression (plotted on the x axis) strongly
correlated with the metabolic rate of the de novo pathway
and with the growth rate (both plotted on the y axis) (Fig.
1), with correlation formulas of y = 0.00047x 0.53 (r = 0.83) and
y = 0.0045x
3.8 (r = 0.95), respectively. The metabolic rate of the de novo
pathway also strongly correlated with the growth rate
(r = 0.93, data not shown). It is notable that the
doubling time of the
A+hATase clone with the highest
ATase expression among the 6 transfectants examined was shortened down
to 10.5 h, i.e. 9.5 × 10
2/h of
growth rate, from the initial 16.8 h for wild-type CHO K1 cells.
This correlation does not appear to be saturated even at the highest
ATase activity of 350% of that in CHO K1. These results obtained from
A+hATase clearly demonstrated that ATase is a
rate-limiting enzyme of the de novo pathway. The levels of
ATase activity in
A+hATase clones closely correlated with
their growth rate even under conditions where purine nucleotide
synthesis by means of the salvage pathway was available in purine-rich
medium (Fig. 1).
The growth rate of A+hATase clones gradually increased
during their positive selection in purine-free medium. The
A+hATase clone with the lowest ATase activity was
cultured in purine-free medium up to passage number 30, where its
growth rate reached the maximal plateau. The number of copies of the
hATase transgene, the ATase activity, the metabolic rate of the
de novo pathway, and the growth rate were periodically
determined up to passage number 30 (Fig. 2). With the
gradual increase in the number of copies of the transgene from 10 to 40 per cell, as shown by the increase in the intensity of the radioactive
bands in Southern blot analysis, the ATase activity, the metabolic rate
of the de novo pathway, and the growth rate increased in
parallel, and all of these simultaneously reached the plateau level at
passage number 24. The rate-limiting quality of ATase for the de
novo pathway was thus demonstrated both by the correlation of
ATase activity with the metabolic rate of the de novo
pathway among six clones of
A+hATase (Fig. 1) and during
the long term culture of one clone of
A+hATase (Fig. 2).
In addition to the rate-limiting property of ATase for the de
novo synthesis, these observations demonstrated that ATase
activity closely correlated with the growth rate under these
conditions.
Three cell lines including CHO K1, CHO ade
A, and the
A+hATase clone stably expressing
ATase activity at the same level as CHO K1 were used. The doubling time
and the growth rate of these three lines of CHO fibroblasts under the
various culture conditions tested in this study are summarized in
Tables I and II, respectively. In
purine-free medium, CHO ade
A did not proliferate at all.
The growth rate under conditions where both purine biosynthetic
pathways were functioning were significantly higher than those under
conditions where either the de novo or the salvage pathway
was functioning. The difference in the growth rate between the two
different conditions was calculated and presented as the increase in
the growth rate in Table III. The independent
contribution of the de novo pathway in the absence of the
salvage pathway was significantly larger than that of the salvage
pathway in the absence of the de novo pathway (4.2 versus 3.4 × 10
2/h). The additive
contribution of the de novo pathway to the salvage pathway
was also significantly greater than that of the salvage pathway to the
de novo pathway (1.8 versus 1.0 × 10
2/h). The growth rate of 5.2 × 10
2/h achieved by the participation of both pathways was
lower than the sum of 7.6 × 10
2/h obtained by
adding 4.2 × 10
2/hr through the de novo
pathway alone and 3.4 × 10
2/h through the salvage
pathway alone. To interpret the mechanism of this nonadditive
antagonism, the metabolic rates of both pathways and the enzyme
activities of ATase and HPRT, the key enzymes of the de novo
and the salvage pathways, respectively, were determined.
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To explain the antagonism between
two purine biosynthetic pathways, the metabolic rates of the de
novo and the salvage pathways were assayed by the incorporation of
[14C]glycine and [14C]Hx, respectively
(Table IV). When both pathways were functioning, the
metabolic rate of the de novo pathway was suppressed by 69 and
64% in CHO K1 and
A+hATase, respectively, compared
with that when only the de novo pathway was functioning. On
the other hand, the salvage pathway activity was modestly suppressed
through the additive availability of the de novo pathway
only by
17 and
24% in CHO K1 and
A+hATase,
respectively, compared with that when only the salvage pathway was
functioning. These results indicate that purine nucleotides are
synthesized preferentially via the salvage pathway as long as CHO
fibroblasts are allowed to use the salvage pathway. Moreover, the
preferential utilization of the salvage pathway with the concomitant suppression of the de novo pathway led to a large reserve
capacity of de novo synthesis. From a comparison between the
metabolic rates of CHO K1 cultured in Ham's F-12 and Ham's F-12 + XO,
the reserve capacity of de novo synthesis of CHO K1 in
Ham's F-12 was estimated at 218% ((0.89
0.28)/0.28 × 100), whereas the reserve capacity of the salvage pathway of CHO K1 in
Ham's F-12, compared with CHO ade
A in Ham's F-12, was
estimated at only 20% ((2349
1962)/1962 × 100).
The enzyme activities of ATase and HPRT under four
culture conditions were summarized in Table V for the
ATase of CHO K1 and the A+hATase clone (no ATase activity
was detected in CHO ade
A), and Table VI
for the HPRT of CHO K1, CHO ade
A, and the
A+hATase. In both CHO K1 and
A+hATase, the
ATase activity was higher when both pathways were functioning than that
when only the de novo pathway was functioning. ATase
activity under eight experimental combinations of two cell lines and
four culture conditions strongly correlated with the growth rate (Fig.
3A). In contrast to ATase activity, HPRT
activity remained at a constant level irrespective of 12 different
experimental conditions. Even when the growth of CHO ade
A fibroblasts was arrested by culturing them in
purine-free medium, the HPRT activity changed only by
2%, compared
with the other growing conditions examined. The HPRT activity thus
remained at a constant level and did not correlate with the growth rate
(Fig. 3B).
Although little
change of HPRT activity was observed in CHO fibroblasts during growth
arrest (Table VI), Northern blot analysis of HPRT showed an obvious
decrease in growth-arrested CHO ade A in purine-free
medium (Fig. 4). The stability of the HPRT enzyme activity despite its decreased mRNA is probably due to the long half-life of the HPRT protein rather than constant transcriptional activity or mRNA stability.
Animal Study
Hx infusion through a cannula into the jugular
vein of 8-week-old male Wistar rats for 12 h suppressed the
metabolic rate of the de novo pathway from 0.19 ± 0.02 (n = 4) with saline infusion to 0.09 ± 0.02 (n = 4), or 53%. Furthermore, the increased
metabolic rate of the de novo pathway by 295% by glucagon
infusion (0.56 ± 0.05, n = 5) was completely
canceled by the concomitant infusion of Hx (0.17 ± 0.06, n = 4). Thus, the preferential utilization of the
salvage pathway with the suppressed metabolic rate of the de
novo pathway observed in CHO fibroblasts was ascertained by the
decrease in the metabolic rate of the de novo pathway in
liver during Hx infusion in this animal study. Like CHO K1 fibroblasts cultured in Ham's F-12, the large reserve capacity of de
novo synthesis in rat liver before glucagon infusion was estimated to be as high as 195% ((0.56
0.19)/0.19 × 100).
Hx, guanine, and
adenine incorporated into cells are converted to IMP, GMP, and AMP,
respectively, by the catalysts of HPRT and adenine
phosphoribosyltransferase. In cells, IMP is converted to GMP and AMP as
required. However, interconversion between GMP and AMP through IMP is
often insufficient for a balance of these two purine nucleotides (21).
Therefore, generally Hx is the most important source, and HPRT is the
most important enzyme for the salvage pathway. Indeed, of the three
purine bases, Hx had the greatest effect on the growth rate of CHO ade
A fibroblasts. Hx at a concentration of more than 30 µM increased the growth rate of CHO ade
A
fibroblasts to its maximum, but a further addition of adenine decreased
the maximal growth rate. In mitogen-stimulated human T cells and a
human B lymphoblast cell line of WI-L2, under the inhibition of
de novo synthesis with aminopterin, the addition of 30 µM Hx was reported to restore the growth rate and DNA and protein syntheses to normal levels (22). The inhibitory effect of
adenine observed in our study is compatible with the reported inhibitory effect of adenine and adenosine on the growth of WI-L2, although the mechanism is unknown (23).
The molecular
cloning of mammalian ATase made it possible to test the hypothesis of
the rate-limiting property of ATase for de novo synthesis
utilizing an ATase-deficient auxotrophic cell line of CHO ade
A. After the transfection of CHO ade
A
fibroblasts with hATase cDNA, the ATase activity, the metabolic rate of the de novo pathway, and the growth rate were
closely correlated for six clones of
A+hATase (Fig. 1)
and for different passage points of one clone of
A+hATase, with an increase in ATase activity along with
its passage (Fig. 2). These results for the first time provided
molecular evidence that ATase limits the rate of the de novo
purine synthetic pathway. ATase was linked with the growth rate even in
a purine-rich medium where the salvage pathway was also functioning. It
should be noted that the increase in the growth rate was not saturated even with an ATase activity as high as 350% of the level in CHO K1
(Fig. 1). The doubling time of the
A+hATase clone with
the highest ATase expression was as short as 10.5 h. Such rapid
cell growth was hardly observed even in undifferentiated neoplasms.
ATase may function as a progression factor for malignant cells, a
transforming factor, or a product of proto-oncogene. The transforming
ability of ATase could not, however, be examined in our system because
we used already transformed CHO fibroblasts and the BPV vector with the
low oncogenic potential (13). Furthermore, the ATase activity, the
metabolic rate of the de novo pathway, and the growth rate
of
A+hATase gradually increased in purine-free medium
with an increasing number of copies of the transgene (Fig. 2). The
number of copies of the BPV vector was reported to be maintained
constant in each transfectant, although it differed for the various
transfectants (12, 13). The number of copied of the BPV vector in one
transfectant in this study was, however, demonstrated to gradually
increase from 10 to 40 per cell during its maintenance through 24 passages in the purine-free medium. The purine-free medium was
considered to have served as a positive selective pressure for the high
ATase activity associated with the high growth rate.
Although the de novo pathway contributed to the increase in the growth rate more significantly than the salvage pathway, an antagonism between the two pathways was observed when both pathways were functioning (Table III). The determination of the metabolic rates of both pathways under various conditions revealed that the inhibition of the de novo pathway by the salvage pathway is much greater than that of the salvage pathway by the de novo pathway (Table IV). Purine nucleotides were thus shown to be synthesized preferentially via the salvage pathway with the concomitant suppression of de novo synthesis, as long as the salvage pathway can be utilized. This regulation in CHO fibroblasts results in a sparing of the energy expenditure inevitably required for the de novo pathway, in which many ATP molecules are required for purine nucleotide synthesis.
Regulation of ATase and HPRT ActivitiesATase expression is regulated by means of the purine repressor in bacteria (24, 25). The purine repressor with a purine corepressor, either Hx or guanine, specifically binds its 16-base pair operator site, and suppresses ATase expression. Through this mechanism, purine nucleotides in bacteria are synthesized preferentially via the salvage pathway with the concomitant suppression of de novo synthesis, as long as substrates for the salvage pathway are available. To determine whether a repressor-type mechanism is functioning in CHO fibroblasts, the enzyme activities of ATase and HPRT were assayed (Tables V and VI). Because ATase and HPRT are fully activated under the assay conditions in this study, the enzyme activities directly reflect the amount of the corresponding enzymes. Although the metabolic rate of the de novo pathway was distinctly suppressed when both pathways were functioning (Table IV), the ATase activity under this condition was even higher than that when only the de novo pathway was functioning (Table V). The mechanism by which the salvage pathway inhibits the de novo pathway is thus not due to the decrease in ATase expression, unlike the repressor mechanism in bacteria. The suppression of the metabolic rate of the de novo pathway by the salvage pathway probably results from 1) the increased feedback inhibition of ATase by purine nucleotides produced via the salvage pathway and 2) the consumption of PRPP, a common substrate for both pathways and an activator of ATase, through the salvage pathway. The close correlation of ATase expression with the growth rate irrespective of the activity of the salvage pathway (Fig. 3A) suggested that ATase expression is linked with the signal for cell growth rather than the concentrations of PRPP and purine nucleotides.
Because the HPRT activity, unlike the ATase activity, was nearly constant (Table VI), the modest suppression of the salvage pathway by the de novo pathway (Table IV) is not due to a decrease in HPRT expression, but is presumably due to PRPP consumption via the de novo pathway. A large consumption of PRPP by de novo synthesis was suggested by the 4-fold increase in PRPP concentration in murine leukemia cells treated with 6-mercaptopurine and methotrexate to inhibit de novo synthesis (26).
Regulation of HPRT ActivityEven after the growth arrest of
CHO ade A fibroblasts in purine-free medium for 48 h, HPRT activity did not decrease (Table VI), while the HPRT mRNA
level was remarkably decreased (Fig. 4), suggesting that HPRT is a
relatively stable protein. In human peripheral lymphocytes, the
half-life of the HPRT protein was reported to be more than 48 h,
in contrast to an estimated half-life of HPRT mRNA of only 5.1 h (27).
Intravenous infusion of Hx to 8-week-old male
Wistar rats for 12 h suppressed the metabolic rate of the de
novo pathway by 53% and canceled its increase by glucagon
stimulation. Thus, the preferential utilization of the salvage pathway
with the suppression of the de novo pathway was observed not
only in CHO fibroblasts, but also in rat liver in vivo. A
preference for the salvage pathway is probably due to the following
factors: 1) In many tissues and cultured cells, the specific activity
of HPRT is much higher than that of ATase (1). For example, the ratio
of HPRT activity to ATase activity is 6.6 in rat liver (2), or about 10 in CHO fibroblasts (Tables V and VI). 2) The affinity of HPRT to PRPP, a substrate shared by both enzymes, is markedly higher than that of
ATase; the Km of HPRT to PRPP is 4 to 40 µM and that of ATase is 400-900 µM (1, 2,
7). 3) Purine nucleotides produced via the salvage pathway
allosterically inhibit ATase, whereas HPRT is not affected. It was
reported that physiological concentrations of Hx strongly suppress
de novo synthesis in human bone marrow in vivo
(28).
Although the salvage pathway is preferentially utilized, its reserve capacity for purine synthesis during cell growth was small because of the nearly constant activity of HPRT (Table VI). In contrast, the reserve capacity of de novo synthesis for changing its metabolic rate was relatively high, and could be attained through several regulatory mechanisms including the allosteric activation or inhibition of ATase activity (29, 30), the cell cycle-dependent expression of the ATase gene (31), and the relatively short half-life of the ATase protein.2 Resting human T lymphocytes were reported to meet their metabolic demands via the salvage pathway except during cell growth, while intact de novo synthesis is essential for the proliferation of phetohemagglutinin-stimulated T lymphocytes (32). The large capacity of de novo synthesis has been shown in patients with Lesch-Nyhan's syndrome (complete deficiency of HPRT) and in cultured cells from patients with this syndrome. Although these patients are affected with severe lesions in the central nervous system, the growth and development of other organs are almost normal due to compensation by de novo synthesis and, in part, the salvage pathway by adenine phosphoribosyltransferase. Fibroblasts from these patients showed a normal PRPP concentration and nucleotide pools were compensated by an increased de novo synthesis (30). Also, the proliferation of T lymphocytes from the patients in response to mitogenic and antigenic stimulation was normal (33). In the leukocytes from two gouty patients affected with a partial deficiency of HPRT, de novo synthesis was accelerated to more than 13 times that of normal controls (34).
The regulatory factors of ATase gene expression, which is essential for the high capacity control of de novo synthesis, include tissue specificity (1), cell cycle (31), cell density,2 malignant transformation (1, 2, 6, 7), and differentiation (35). Biochemical and molecular biological studies are underway in our laboratory to investigate the regulatory mechanism of ATase gene expression and its activity.
In conclusion, the following concepts were supported in this study using CHO fibroblasts and rat liver. First, the ATase activity in CHO transfectants with hATase cDNA, which ranges from 30 to 350% of that of wild-type CHO K1, limits the metabolic rate of de novo synthesis and strongly correlates with the growth rate. This was confirmed by a parallel increase in the ATase activity, the metabolic rate of the de novo pathway, and the growth rate of one CHO transfectant with hATase cDNA relative to its passage in purine-free medium. Second, purine nucleotides are synthesized preferentially through the salvage pathway not only in cultured fibroblasts, but also in rat liver in vivo, as long as a source of purine salvage is available, with the concomitant suppression of the de novo pathway. This mechanism results in the sparing of energy expended by de novo synthesis. Last, the complex regulatory mechanisms of the gene expression and enzyme activity of ATase compared with the relatively constant enzyme activity of HPRT provide for a larger reserve capacity of de novo synthesis than of salvage synthesis, and reflect the molecular basis for meeting the metabolic demands required for the increased rate of cell growth.