(Received for publication, July 5, 1995; and in revised form, September 28, 1995)
From the
Sensitive high performance liquid chromatography techniques,
which differentiate between purine and pyrimidine ribonucleoside and
deoxyribonucleoside triphosphates, were used to quantify pools in
phytohemagglutinin-stimulated T-lymphocytes (98% CD4 and CD8
) from healthy volunteers. The importance
of de novo synthesis and salvage was evaluated by incubating
the cells with
C-radiolabeled precursors (40
µM), azaserine (20 µM; a glutamine
antagonist), and ribavirin (50 µM; an IMP dehydrogenase
inhibitor). We confirmed that resting T-lymphocytes meet their
metabolic requirements by salvage.
Noteworthy observations were as follows. First, nucleotide pool expansion over 72 h is disproportionate, with that for purines (ATP and GTP) being 2-fold compared with up to 8-fold for pyridine (NAD) or pyrimidine (UTP, UDP-Glc, and CTP) pools. This supports an additional role for the latter in membrane lipid biosynthesis, protein glycosylation, and strand break repair. Second, intact de novo pathways are essential for such expansion. Azaserine not only inhibited purine synthesis (confirmed by N-formylglycinamide polyphosphate accumulation), but also reduced expansion of pyrimidine and NAD pools by 70%. Ribavirin depleted GTP pools by 40% and reduced pyrimidine pool expansion by 40% at 72 h.
These findings underline the importance of pyrimidine ribonucleotide availability as well as GTP synthesis de novo to proliferating T-lymphocytes. They also demonstrate an absence of coordinate regulation between de novo purine and pyrimidine biosynthesis.
Triggering of resting T-lymphocytes under physiological
conditions by antigenic peptides associated with the major
histocompatibility complex is a multistep process. Stimulation leads to
the induction of biochemical signals that are transmitted sequentially
from the cell surface to the nucleus, resulting in gene
transcription(1, 2, 3) . Replication of
activated T-cells requires additional signals mediated by other
costimulatory receptors or accessory molecules (such as CD4, CD8, CD2,
leukocyte function-associated antigen, CD28, and CD45) or binding of
growth-promoting cytokines (such as interleukin-1, -6, and -2). Failure
to produce these costimulatory signals or the associated biochemical
responses could lead to unresponsiveness or cell death instead of
replication(1, 2) . The activation of T-cells in
vivo is usually mimicked in vitro by the addition of
mitogens such as phytohemagglutinin (PHA) ()or by activation
with anti-CD3 or anti-T-cell receptor antibodies(1) . PHA
causes the transformation of resting lymphocytes into rapidly dividing
lymphoblasts, with the synthesis of DNA beginning at 24 h and peaking
around 48 h, preceded by an increase in the rate of synthesis of RNA
and protein(4, 5) .
The importance of intact purine pathways to the normal immune response was highlighted by the discovery of the two potentially fatal genetic immunodeficiencies (purine-nucleoside phosphorylase deficiency and adenosine deaminase deficiency) associated with severe T-lymphocyte or T- and B-lymphocyte depletion and impaired mitogen responses(6, 7) . This led to the development of novel adenosine deaminase and purine-nucleoside phosphorylase inhibitors for the treatment of hematological malignancies as well as immunosuppressive agents(3, 6, 8) . Inhibitors targeting de novo pyrimidine biosynthesis or GTP synthesis at the level of IMP dehydrogenase (Fig. 1), an enzyme associated with T-cell activation and malignancy(3) , have likewise been the focus of strategies aimed at overcoming allograft rejection(3, 9) .
Figure 1: Pathways for purine and pyrimidine metabolism in human cells that synthesize the intermediates essential for cellular growth-related activities (membrane biosynthesis (CDP- and UDP-linked lipid intermediates) and protein glycosylation (GDP-sugars and UDP-sugars)) as well as for DNA and RNA synthesis. Glutamine is an essential substrate in both the purine and pyrimidine de novo pathways, and glycine in the purine de novo pathway. Note that purine salvage takes place at the base (hypoxanthine) level and pyrimidine salvage at the nucleoside (uridine) level in human cells. Not shown is the dCMP deaminase reaction, which, in most mammalian cells studied, represents the principal route for formation of dUMP. IMPDH, IMP dehydrogenase; FAICAR, 5-formamidoimadazole-4-carboxamide ribotide; AICAR, 5-aminoimadazole-4-carboxamide ribotide; SAICAR, N-succino-5-aminoimidazole-4-carboxamide ribotide; CAIR, 4-carboxy-5-aminoimidazole ribotide; AIR, 5-aminoimadazole ribotide; FGAM, N-formylglycineamidine ribotide; FGAR, N-formylglycineamide ribotide; GAR, glycineamide ribotide; PRA, 5-phosphoribosyl-1-amine; OMP, orotidine 5`-monophosphate; OA, orotic acid; DHOA, dihydroorotic acid; N-CA, N-carbamoyl L-aspartate; PP-rib-P, 5-phosphoribosyl-1-pyrophosphate; CP, carbamoyl phosphate.
In the majority of in vitro studies, evaluation of the events following PHA stimulation has been indirect, based on the incorporation of tritiated thymidine, uridine, or leucine(3, 4, 5, 7, 10, 11) . Data documenting ribonucleotide pools in resting T-lymphocytes and the actual changes occurring over 72 h in stimulated T-lymphocytes (an integral component of the above cascade (Fig. 1) that enables an immunocompetent host to respond to mitogenic agents in vivo) are scant(10, 11, 12) . A number of studies have compared ribonucleotide pools in resting T-lymphocytes with pools in malignant or virally infected cells (reviewed in (13, 14, 15, 16, 17, 18) ). Others have focused on lymphocyte deoxyribonucleotide pool changes in response to PHA. However, these have been measured usually by indirect assay, and although a 10-fold expansion has been reported following PHA stimulation in peripheral blood lymphocytes, concentrations are extremely low compared with ribonucleotide pools(19, 20, 21) .
Only recently have the
concentrations of both pyrimidine and purine ribonucleotides been
compared in resting and stimulated T-lymphocytes and equated with the
incorporation of C-radiolabeled precursors(12) .
No study on human T-lymphocytes to our knowledge has coupled
measurement of the actual changes occurring in both purine and
pyrimidine ribonucleotide pools over 72 h following mitogen stimulation
with the evaluation of the effect of de novo synthesis
inhibitors of both pathways on the ability of ribonucleotide pools in
T-lymphocytes to expand in response to PHA stimulation.
To address
these points, we separated T-lymphocytes (CD4 and
CD8
) from healthy donors, stimulated them with PHA,
and followed changes in ribonucleotide concentrations over 72 h using
highly sensitive techniques (high performance liquid chromatography
(HPLC) with in-line photodiode array and radiodetection)(22) .
The HPLC method used was originally developed by us to demonstrate the
erythrocyte dATP accumulation associated with ATP depletion in
inherited adenosine deaminase deficiency and the GTP depletion and dGTP
accumulation in purine-nucleoside phosphorylase
deficiency(23) . More important, the method separates purine
ribonucleotides from pyrimidine ribonucleotides as well as from their
corresponding deoxyribonucleotides and sugar derivatives. The method is
in daily use as a diagnostic tool (22) and for monitoring the
decrease in dATP in adenosine deaminase-deficient patients treated with
polyethylene glycol adenosine deaminase (6) .
The experiments included the use of radiolabeled substrates and T-lymphocytes that were incubated in the presence of azaserine or ribavirin, known inhibitors of specific steps of de novo purine synthesis(15, 18) . The objective was to determine how stimulated T-cells switch on their de novo synthetic pathways to provide the additional ribonucleotide precursors necessary for (a) RNA and DNA synthesis and (b) the other processes associated with cell division: repletion of energy stores (ATP) and the formation of cofactors (NAD), second messenger precursors, and the purine and pyrimidine sugars for membrane lipid synthesis and glycosylation (Fig. 1). The relative roles of salvage of preformed purines and pyrimidines in these processes was also evaluated to enable us to define the normal metabolic response of both pathways to mitogens and thereby identify the precise regulatory steps that might be impaired in lymphocytes of an immunocompromised host following human immunodeficiency virus infection(1, 2, 38) .
In this study, we show that de novo synthesis is essential to provide not only new RNA and DNA, but also the pyrimidine sugars necessary to support the massive expansion in membrane biosynthesis. Clear differences are also evident in the responses of pyrimidine biosynthesis to the inhibitory effects of ribavirin and azaserine in stimulated T-lymphocytes compared with those reported for malignant lymphoblasts(15, 24) .
Figure 2: HPLC chromatogram recorded over 30 min at 254 nm (A and B, lower panels), with automatic scaling by the photodiode array detector to show the individual spectra monitored from 230 to 310 nm (A and B, upper panels), also indicating the peak maxima. A, the separation of mono-, di-, and triphosphates following injection of 150 µl of an extract of T-lymphocytes 72 h after PHA stimulation as described in ``Materials and Methods''; B, the separation of 17 authentic standards. Note the clear separation of CTP and dCTP, ATP and dATP, and GTP and dGTP shown in B and the absence of any detectable deoxynucleotides eluting with these retention times after CTP, ATP, or GTP in A. ADPR, ADP-ribose; UDPG, UDP-Glc; GDPS, GDP-sugar; UDPGA, UDP-glucuronic acid.
Figure 3:
Mean
purine (A and B) and pyrimidine (C and D) ribonucleotide concentrations (picomoles/10 cells) in T-lymphocytes from five healthy donors (left
bars) evaluated on day 1 (D1) and at 24 h (D2),
48 h (D3), and 72 h (D4), respectively, after PHA
stimulation. Mean concentrations of NAD and ADP-ribose (ADPR)
are shown in E. The reduced response (or depletion) of purine,
pyrimidine, and pyridine pools following PHA stimulation and incubation
with the de novo synthesis inhibitors azaserine (center
bars) and ribavirin (right bars) is evident. UDPG, UDP-Glc.
In the HPLC method used, dCTP, dATP, and
dGTP have retention times between 0.7 and 1 min later than the
corresponding ribonucleoside triphosphate (limit of detection, 1
pmol/10 cells) (Fig. 2B), although dTTP
elutes on the front of GTP. The chromatogram showing the different
nucleotides present in healthy lymphocytes at 72 h (Fig. 2A) was automatically autoscaled to the highest
peak (ATP) to show the individual UV spectra by in-line photodiode
array. However, peaks were quantified at 0.02 absorbance units full
scale. Thus, any significant increments in dNTPs in cellular lymphocyte
extracts following PHA stimulation, as reported by others using enzyme
assays(19, 20, 21, 25) , should have
been detectable. The advantages and disadvantages of HPLC compared with
the enzyme assay have been reviewed(20) . The absence of
detectable increments in dNTP pools here, coupled with the fact that
DNA synthesis increases markedly after stimulation, suggests that these
pools must be highly localized in order to be at sufficiently high
concentrations to drive DNA synthesis. The findings support studies
postulating the existence of multienzyme complexes that both synthesize
dNTPs and channel them to sites of DNA replication(26) .
Figure 4:
Incorporation of
[C]glycine (40 µM) into adenine
nucleotides (A), guanine nucleotides (B), de novo intermediates (C), and cell pellet (D) in
control cells incubated either alone (left bars) or with
azaserine (center bars) or ribavirin (right bars).
Cells were pulse-labeled for 2 h at the times indicated after
stimulation: 0 h (D1), 24 h (D2), 48 h (D3),
and 72 h (D4). Azaserine inhibition of purine de novo synthesis at the level of formylglycine-amidine synthetase is
evident from the accumulation of the radiolabel in formylglycinamide
ribotide (FMP) and its di- and triphosphates (FDP and FTP) (C).
The linear increment in the
incorporation of radiolabel into the cell precipitate (Fig. 4D) confirms accelerated synthesis of RNA, DNA,
and protein over 72 h in control cells (left bars), which is
arrested at 24 h by azaserine (center bars) and at 48 h by
ribavirin (right bars). The evident reduction in radiolabel
incorporation into the cell precipitate after 24 h in cells incubated
with azaserine and after 48 h with ribavirin compared with the control
is consistent with cell cycle arrest. This interpretation is in accord
with reports showing inhibition of DNA synthesis in the presence of
these antagonists at the G and G
/early S
transition phases, respectively(3, 15, 18) .
This assumption is supported by the finding that although both drugs
inhibited cell growth in our experiments, cell viability (trypan blue
exclusion) was unaffected.
Figure 5:
Incorporation of precursors (40
µM) of the purine salvage pathway
([C]hypoxanthine) into adenine (A) and
guanine (B) nucleotides and of the pyrimidine salvage pathway
([
C]uridine) into uridine (C) and
cytidine (D) nucleotides and sugars by T-lymphocytes from
healthy donors at the times indicated after PHA stimulation. D1, 0 h; D2, 24 h; D3, 48 h; D4, 72
h. UDPG, UDP-Glc.
In this study, we demonstrate that initiation of blast transformation in T-lymphocytes from healthy subjects requires extensive activation of housekeeping genes coding for key purine and pyrimidine enzymes involving both salvage and particularly the de novo synthetic routes of ribonucleotide formation (Fig. 1). There are several noteworthy observations.
First, the anticipated
expansion in both pyrimidine and purine ribonucleotide pools is evident
at 72 h following PHA stimulation, but the relative increment in
pyrimidine pools is much greater than for purines. The disproportionate
increment in pyrimidine ribonucleotide concentrations in stimulated
T-lymphocytes in this study occurred in the absence of exogenous
pyrimidine ribonucleosides and included the UDP-sugars. This finding
supports an additional role for de novo pyrimidine synthesis
in T-lymphocyte proliferation, namely, to provide the extra pyrimidine
ribonucleotides necessary for the massive expansion in membrane
biosynthesis. The mechanism underlying this stimulation could be
enhanced expression of the multifunctional protein CAD (which contains
the activities of carbamoyl-phosphate synthetase II, aspartate
carbamoyltransferase, and dihydroorotase), as reported for stimulated
murine B-cells(27) . In that study(27) , modest
increases over 12 h preceded a profound increase from 24 to 60 h, which
correlated with movement from the G to the S phase of the
cycle. Metabolic experiments by others using labeled mannose and
choline in activated murine B-lymphocytes have shown a dramatic
phase-specific induction of dolichol-linked lipid intermediate
synthesis, protein N-glycosylation activity, and phospholipid
synthesis(28, 29) . Interestingly, the incorporation
of radiolabeled choline was reduced drastically by an inhibitor of
CDP-choline synthetase(30) . Obviously, any clinical situation
reducing the supply of CTP (the essential substrate for this enzyme),
as in the inhibitor studies discussed below or in the human
immunodeficiency virus type-1
T-lymphocyte experiments
in the accompanying paper(38) , would have a similar effect.
Second, the radiotracer studies comparing the routes of ribonucleotide formation confirm that resting lymphocytes meet their metabolic requirements by salvage, with the de novo purine pathway being virtually inactive(11, 12) . Uridine salvage, although relatively inactive in resting lymphocytes, is also stimulated, particularly at 48 h, confirming uridine kinase as another enzyme induced during the S phase(31) . The limited conversion of uridine into CTP accords with the recent report that uridine salvage is not a significant source of CTP in stimulated lymphocytes, although it is the preferred route in malignant human T-lymphocytic cells(32) . Other studies demonstrating active uptake of exogenous dCyd and Cyd into phosphatidylinositol, via dCDP-choline and CDP-choline and the corresponding diacylglycerol intermediates, imply a special role for pyrimidine salvage in phosphatidylinositol synthesis(33) . These observations are in agreement with earlier reports proposing the existence of independent pyrimidine pools (5, 26
Third, the considerable expansion in the NAD pool evident here in stimulated T-lymphocytes reflects the requirement of dividing lymphocytes for an additional supply of NAD for a number of NAD-dependent reactions, such as the repair of spontaneous DNA strand breaks(34, 35) . Poly(ADP-ribose) polymerase works only in the presence of NAD(16) . The inhibitory effect of azaserine on NAD synthesis supports inhibition of the glutamine-dependent NAD synthetase. The importance of NAD to proliferating lymphocytes is underlined by the fact that NAD depletion, activated by DNA strand breaks, has been implicated in the lymphotoxicity in adenosine deaminase deficiency(6, 8) .
Fourth, the finding that azaserine clearly inhibits expansion of both purine and pyrimidine nucleotide pools provides the first direct evidence of the importance of intact de novo synthetic pathways to blasting lymphocytes. However, the inhibitory effect on both pathways is contrary to the belief that potent inhibition of either de novo purine or pyrimidine biosynthesis is accompanied by a complementary stimulation of the flux through the other pathway(15) . Inhibition of purine synthesis de novo at the level of formylglycine-amidine synthetase is confirmed by the glycine incorporation studies. The radiolabel accumulated in the substrate for formylglycine-amidine synthetase, formylglycinamide ribotide, together with its di- and triphosphates, as also noted in mouse leukemia cells(15) . The reduction in uridine and cytidine ribonucleotide pools is consistent with a block in pyrimidine de novo synthesis at the level of carbamoyl-phosphate synthetase II and CTP synthetase (Fig. 1), as reported for the anti-glutamine agent acivicin in malignant cells in vitro(15) . The inhibition of pyrimidine de novo synthesis by azaserine contrasts with the studies in cell extracts by Jayaram et al.(36) , who found no effect on carbamoyl-phosphate synthetase II. As discussed by Lyons et al.(15) , activities in cell extracts are not reliable indicators of the situation in intact cells in vivo. However, our results are also the converse of the studies in cultured mouse leukemia cells by Lyons et al.(15) , who reported no inhibition of NAD or CTP synthetase by azaserine and a complementary stimulation of pyrimidine biosynthesis.
Interestingly, the studies in stimulated T-lymphocytes preincubated with the purine de novo synthesis inhibitor ribavirin produced the same discordant result compared with studies in mouse lymphoma cells(18, 24) . Although the GTP depletion induced here is similar to that reported by Zimmerman and Deeprose(24) , the blunted response in pyrimidine pool expansion over 72 h is in direct contrast to the almost 2-fold stimulation of pyrimidine biosynthesis noted by them. Some of the inhibitory effects on pyrimidine biosynthesis here may be secondary to GTP depletion, although a direct effect of phosphorylated ribavirin (a GTP analogue(18) ) on GTP-dependent reactions such as CTP synthetase cannot be ruled out. Nevertheless, the observation that a second antagonist of de novo purine synthesis restricts, rather than stimulates, de novo pyrimidine biosynthesis in activated human T-lymphocytes is curious(15, 24) . Allosteric regulation of pyrimidine biosynthesis in T-lymphocytes may thus, as for purines(3, 37) , differ from that in other cell types, particularly malignant cells.
The vital role of IMP dehydrogenase in GTP synthesis for blasting T-lymphocytes, evident from the depletion induced by ribavirin, is well documented for other IMP dehydrogenase inhibitors(3, 9, 16, 18) . GTP depletion could also impair functions other than DNA synthesis in proliferating T-lymphocytes, e.g. the activity of G-proteins involved in signal transduction or formation of the GDP-mannose and GDP-fucose intermediates essential for the glycosylation of adhesion molecules. Such a dual effect may explain the enhanced efficacy of IMP dehydrogenase inhibitors, currently in clinical trial for organ transplantation and cancer chemotherapy(3, 16) .
The results presented here question the concept of coordinate regulation of purine and pyrimidine biosynthesis, which putatively equalizes the rates of formation of purine and pyrimidine nucleotides for nucleic acid synthesis. The findings support the suggested metabolic channeling of dNTPs (26) and highlight the particular importance of pyrimidine ribonucleotide availability to mitogen-stimulated T-lymphocytes. The significance of this in vivo for human immunodeficiency virus type-1-infected lymphocytes is discussed in the accompanying paper(38) . The marked differences evident here in the regulation of nucleotide biosynthesis between dividing human T-lymphocytes and malignant lymphoid cells(15, 24) suggest new avenues that could be exploited therapeutically.