Viperin interacts with the kinase IRAK1 and the E3 ubiquitin ligase TRAF6, coupling innate immune signaling to antiviral ribonucleotide synthesis.

Virus-inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) is a radical SAM enzyme that plays a multifaceted role in the cellular antiviral response. Viperin has recently been shown to catalyze the SAM-dependent formation of 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), which inhibits some viral RNA polymerases. Viperin is also implicated in regulating Lys-63-linked polyubiquitination of interleukin-1 receptor-associated kinase-1 (IRAK1) by the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) as part of the Toll-like receptor-7 and -9 (TLR7/9) innate immune signaling pathways. In these pathways, the poly-ubiquitination of IRAK1 by TRAF6 is necessary to activate IRAK1, which then phosphorylates downstream targets and ultimately leads to the production of type I interferons. That viperin is a component of these pathways suggested that its enzymatic activity might be regulated by interactions with partner proteins. To test this idea, we have reconstituted the interactions between viperin, IRAK1, and TRAF6 by transiently expressing these enzymes in HEK 293T cells. We show that IRAK1 and TRAF6 increase viperin activity ∼10-fold to efficiently catalyze the radical-mediated dehydration of CTP to ddhCTP. Furthermore, we found that TRAF6-mediated ubiquitination of IRAK1 requires the association of viperin with both IRAK1 and TRAF6. Ubiquitination appears to depend on structural changes in viperin induced by SAM binding, but, significantly, does not require catalytically active viperin. We conclude that the synergistic activation of viperin and IRAK1 provides a mechanism that couples innate immune signaling with the production of the antiviral nucleotide ddhCTP.

"Pinching" the ammonia tunnel of CTP synthase unveils coordinated catalytic and allosteric-dependent control of ammonia passage.

Molecular gates within enzymes often play important roles in synchronizing catalytic events. We explored the role of a gate in cytidine-5'-triphosphate synthase (CTPS) from Escherichia coli. This glutamine amidotransferase catalyzes the biosynthesis of CTP from UTP using either l-glutamine or exogenous NH3 as a substrate. Glutamine is hydrolyzed in the glutaminase domain, with GTP acting as a positive allosteric effector, and the nascent NH3 passes through a gate located at the end of a ~25-Å tunnel before entering the synthase domain where CTP is generated. Substitution of the gate residue Val 60 by Ala, Cys, Asp, Trp, or Phe using site-directed mutagenesis and subsequent kinetic analyses revealed that V60-substitution impacts glutaminase activity, nucleotide binding, salt-dependent inhibition, and inter-domain NH3 transport. Surprisingly, the increase in steric bulk present in V60F perturbed the local structure consistent with "pinching" the tunnel, thereby revealing processes that synchronize the transfer of NH3 from the glutaminase domain to the synthase domain. V60F had a slightly reduced coupling efficiency at maximal glutaminase activity that was ameliorated by slowing down the glutamine hydrolysis reaction, consistent with a "bottleneck" effect. The inability of V60F to use exogenous NH3 was overcome in the presence of GTP, and more so if CTPS was covalently modified by 6-diazo-5-oxo-l-norleucine. Use of NH2OH by V60F as an alternative bulkier substrate occurred most efficiently when it was concomitant with the glutaminase reaction. Thus, the glutaminase activity and GTP-dependent activation act in concert to open the NH3 gate of CTPS to mediate inter-domain NH3 transport.

A naturally occurring antiviral ribonucleotide encoded by the human genome.

Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies 1 . Viperin, a member of the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus 2 and HIV3,4. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins3,4. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes.

Resistance to the nucleotide analogue cidofovir in HPV(+) cells: a multifactorial process involving UMP/CMP kinase 1.

Human papillomavirus (HPV) is responsible for cervical cancer, and its role in head and neck carcinoma has been reported. No drug is approved for the treatment of HPV-related diseases but cidofovir (CDV) exhibits selective antiproliferative activity. In this study, we analyzed the effects of CDV-resistance (CDVR) in two HPV(+) (SiHaCDV and HeLaCDV) and one HPV(-) (HaCaTCDV) tumor cell lines. Quantification of CDV metabolites and analysis of the sensitivity profile to chemotherapeutics was performed. Transporters expression related to multidrug-resistance (MRP2, P-gp, BCRP) was also investigated. Alterations of CDV metabolism in SiHaCDV and HeLaCDV, but not in HaCaTCDV, emerged via impairment of UMP/CMPK1 activity. Mutations (P64T and R134M) as well as down-regulation of UMP/CMPK1 expression were observed in SiHaCDV and HeLaCDV, respectively. Altered transporters expression in SiHaCDV and/or HeLaCDV, but not in HaCaTCDV, was also noted. Taken together, these results indicate that CDVR in HPV(+) tumor cells is a multifactorial process.

Effects of Zidovudine Treatment on Heart mRNA Expression and Mitochondrial DNA Copy Number Associated with Alterations in Deoxynucleoside Triphosphate Composition in a Neonatal Rat Model.

The prevention of mother-to-child transmission (MTCT) of HIV is a crucial component in HIV therapy. Nucleoside reverse transcriptase inhibitors (NRTIs), primarily 3'-azido-3'-thymidine (AZT [zidovudine]), have been used to treat both mothers and neonates. While AZT is being replaced with less toxic drugs in treating mothers in MTCT prevention, it is still commonly used to treat neonates. Problems related to mitochondrial toxicity and potential mutagenesis associated with AZT treatment have been reported in treated cohorts. Yet little is known concerning the metabolism and potential toxicity of AZT on embryonic and neonatal tissues, especially considering that the enzymes of nucleoside metabolism change dramatically as many tissues convert from hyperplastic to hypertrophic growth during this period. AZT is known to inhibit thymidine phosphorylation and potentially alter deoxynucleoside triphosphate (dNTP) pools in adults. This study examines the effects of AZT on dNTP pools, mRNA expression of deoxynucleoside/deoxynucleotide metabolic enzymes, and mitochondrial DNA levels in a neonatal rat model. Results show that AZT treatment dramatically altered dNTP pools in the first 7 days of life after birth, which normalized to age-matched controls in the second and third weeks. Additionally, AZT treatment dramatically increased the mRNA levels of many enzymes involved in deoxynucleotide synthesis and mitochondrial biogenesis during the first week of life, which normalized to age-matched controls by the third week. These results were correlated with depletion of mitochondrial DNA noted in the second week. Taken together, results demonstrated that AZT treatment has a powerful effect on the deoxynucleotide synthesis pathways that may be associated with toxicity and mutagenesis.

An in vivo large-scale chemical screening platform using Drosophila for anti-cancer drug discovery.

Anti-cancer drug development involves enormous expenditure and risk. For rapid and economical identification of novel, bioavailable anti-tumour chemicals, the use of appropriate in vivo tumour models suitable for large-scale screening is key. Using a Drosophila Ras-driven tumour model, we demonstrate that tumour overgrowth can be curtailed by feeding larvae with chemicals that have the in vivo pharmacokinetics essential for drug development and known efficacy against human tumour cells. We then develop an in vivo 96-well plate chemical screening platform to carry out large-scale chemical screening with the tumour model. In a proof-of-principle pilot screen of 2000 compounds, we identify the glutamine analogue, acivicin, a chemical with known activity against human tumour cells, as a potent and specific inhibitor of Drosophila tumour formation. RNAi-mediated knockdown of candidate acivicin target genes implicates an enzyme involved in pyrimidine biosynthesis, CTP synthase, as a possible crucial target of acivicin-mediated inhibition. Thus, the pilot screen has revealed that Drosophila tumours are glutamine-dependent, which is an emerging feature of many human cancers, and has validated the platform as a powerful and economical tool for in vivo chemical screening. The platform can also be adapted for use with other disease models, thus offering widespread applications in drug development.

Cyclopentenyl cytosine has biological and anti-tumour activity, but does not enhance the efficacy of gemcitabine and radiation in two animal tumour models.

Cyclopentenyl cytosine (CPEC), targetting the de novo biosynthesis of cytidine triphosphate (CTP), increases the cytotoxicity of gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) alone and in combination with irradiation in several human tumour cells in vitro. We investigated whether CPEC enhances the therapeutic ratio of gemcitabine and irradiation in human pancreatic BxPC-3 xenografts and in rat syngeneic L44 lung tumours. These models were selected because gemcitabine and radiation are used to treat both pancreatic and lung cancer patients and both models differ in growth capacity and in gemcitabine-induced radiosensitisation. A profound dose-dependent CTP-depletion was observed after a single injection of CPEC in both tumour tissue and in normal jejunum. In both models, CPEC alone induced a slight but significant tumour growth delay. The combination of CPEC with gemcitabine, at time intervals that showed CTP-depletion after CPEC, enhanced neither tumour growth delay nor toxicity as compared to gemcitabine alone. In addition, no beneficial effect of CPEC was observed in combination with gemcitabine and radiation. These results suggest that CPEC and gemcitabine alone as well as in combination with radiation target a similar cell population in both tumour models. In conclusion, future clinical development of CPEC as a modulator of gemcitabine combined with radiation is unlikely.

Does saturable formation of gemcitabine triphosphate occur in patients?

AIM: This study aims to determine if intracellular formation of gemcitabine triphosphate (dFdCTP), an active metabolite of gemcitabine, is saturable at doses used for treatment of Asian patients with lung cancer. METHODS: From a phase II trial, plasma concentrations of gemcitabine, its inactive metabolite 2'-2'-difluorodeoxyuridine (dFdU), and mononuclear cell concentrations of gemcitabine-triphosphate were measured in 56 and 33 patients, respectively. The pharmacokinetics of gemcitabine and metabolites were modeled using nonlinear mixed effects modeling (NONMEM). A reduced dataset of ten randomly selected patients was employed to compare first-order and saturable formation of dFdCTP from gemcitabine. RESULTS: The median population clearance estimate for dFdCTP formation with the full dataset was 70.2 L/h/70 kg/1.7 m. Modeling Michaelis-Menten formation of dFdCTP on a reduced dataset estimated K(m) to be 3.6 times higher than the maximum gemcitabine concentration (72.2 microM) measured in this study. CONCLUSIONS: The results showed that first-order and nonsaturable clearance described intracellular dFdCTP formation at clinically applied doses of gemcitabine.

The role of Ureaplasma nucleoside monophosphate kinases in the synthesis of nucleoside triphosphates.

Mollicutes are wall-less bacteria and cause various diseases in humans, animals and plants. They have the smallest genomes with low G + C content and lack many genes of DNA, RNA and protein precursor biosynthesis. Nucleoside diphosphate kinase (NDK), a house-keeping enzyme that plays a critical role in the synthesis of nucleic acids precursors, i.e. NTPs and dNTPs, is absent in all the Mollicutes genomes sequenced to date. Therefore, it would be of interest to know how Mollicutes synthesize dNTPs/NTPs without NDK. To answer this question, nucleoside monophosphate kinases (NMPKs) from Ureaplasma were studied regarding their role in the synthesis of NTPs/dNTPs. In this work, Ureaplasma adenylate kinase, cytidylate kinase, uridylate kinase and thymidylate kinase were cloned and expressed in Escherichia coli. The recombinant enzymes were purified and characterized. These NMPKs are base specific, as indicated by their names, and capable of converting (d)NMPs directly to (d)NTPs. The catalytic rates of (d)NTPs and (d)NDP synthesis by these NMPKs were determined using tritium-labelled (d)NMPs, and the rates for (d)NDP synthesis, in general, were much higher (up to 100-fold) than that of (d)NTP. Equilibrium studies with adenylate kinase suggested that the rates of NTPs/dNTPs synthesis by NMPKs in vivo are probably regulated by the levels of (d)NMPs. These results strongly indicate that NMPKs could substitute the NDK function in vivo.

The role of lysine residues 297 and 306 in nucleoside triphosphate regulation of E. coli CTP synthase: inactivation by 2',3'-dialdehyde ATP and mutational analyses.

Cytidine 5'-triphosphate synthase (CTPS) catalyzes the ATP-dependent formation of CTP from UTP using either NH3 or L-glutamine as the source of nitrogen. To identify the location of the ATP-binding site within the primary structure of E. coli CTPS, we used the affinity label 2',3'-dialdehyde adenosine 5'-triphosphate (oATP). oATP irreversibly inactivated CTPS in a first-order, time-dependent manner while ATP protected the enzyme from inactivation. In the presence of 10 mM UTP, the values of k(inact) and K(I) were 0.054 +/- 0.001 min(-1) and 3.36 +/- 0.02 mM, respectively. CTPS was labeled using (2,8-3H)oATP and subsequently subjected to trypsin-catalyzed proteolysis. The tryptic peptides were separated using reversed-phase HPLC, and two peptides were identified using N-terminal sequencing (S(492)GDDQLVEIIEVPNH(506) and Y(298)IELPDAY(K(306)) in a 5:1 ratio). The latter suggested that Lys 306 had been modified by oATP. Replacement of Lys 306 by alanine reduced the rate of oATP-dependent inactivation (k(inact) = 0.0058 +/- 0.0005 min(-1), K(I) = 3.7 +/- 1.3 mM) and reduced the apparent affinity of CTPS for both ATP and UTP by approximately 2-fold. The efficiency of K306A-catalyzed glutamine-dependent CTP formation was also reduced 2-fold while near wild-type activity was observed when NH3 was the substrate. These findings suggest that Lys 306 is not essential for ATP binding, but does play a role in bringing about the conformational changes that mediate interactions between the ATP and UTP sites, and between the ATP-binding site and the glutamine amide transfer domain. Replacement of the nearby, fully conserved Lys 297 by alanine did not affect NH3-dependent CTP formation, relative to wild-type CTPS, but reduced k(cat) for the glutaminase activity 78-fold. Our findings suggest that the conformational change associated with binding ATP may be transmitted through the L10-alpha11 structural unit (residues 297-312) and thereby mediate effects on the glutaminase activity of CTPS.

Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis. The role of the allosteric activator GTP incoupling between glutamine hydrolysis and CTP synthesis.

CTP synthase catalyzes the reaction glutamine + UTP + ATP --> glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half-reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP-activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP-binding analog adenosine 5'-[gamma-thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself.

Retinoic acid reduces the cytotoxicity of cyclopentenyl cytosine in neuroblastoma cells.

In this paper, it is demonstrated that all-trans, 9-cis and 13-cis retinoic acid (RA) decreased the sensitivity of SK-N-BE(2)c neuroblastoma cells towards the chemotherapeutic agent cyclopentenyl cytosine (CPEC), a potent inhibitor of cytosine-5'-triphosphate synthetase. Retinoic acid attenuated CPEC-induced apoptosis as reflected by a decreased caspase-3 induction. Retinoic acid decreased the accumulation of CPEC, whereas the salvage of cytidine was strongly increased. Metabolic labeling studies using [(3)H]uridine showed a strongly decreased biosynthesis of CTP via CTP synthetase. Retinoic acid likely confers resistance of neuroblastoma cells to CPEC in part by slowing down proliferation, and in part by shifting the synthesis of CTP towards the salvage of cytidine, thereby bypassing CTP synthetase.

Trypanosoma brucei CTP synthetase: a target for the treatment of African sleeping sickness.

The drugs in clinical use against African sleeping sickness are toxic, costly, or inefficient. We show that Trypanosoma brucei, which causes this disease, has very low levels of CTP, which are due to a limited capacity for de novo synthesis and the lack of salvage pathways. The CTP synthetase inhibitors 6-diazo-5-oxo-l-norleucine (DON) and alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) reduced the parasite CTP levels even further and inhibited trypanosome proliferation in vitro and in T. brucei-infected mice. In mammalian cells, DON mainly inhibits de novo purine biosynthesis, a pathway lacking in trypanosomes. We could rescue DON-treated human and mouse fibroblasts by the addition of the purine base hypoxanthine to the growth medium. For treatment of sleeping sickness, we propose the use of CTP synthetase inhibitors alone or in combination with appropriate nucleosides or bases.

Inhibition of Escherichia coli CTP synthase by glutamate gamma-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis.

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography. Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate gamma-semialdehyde. Glutamate gamma-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. Indeed, glutamate gamma-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (K(is) 0.16+/-0.03 mM; K(ii) 0.4+/-0.1 mM) and a competitive inhibitor with respect to ammonia (K(i) 0.39+/-0.06 mM) in the presence of GTP at pH 8.0. The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate gamma-semialdehyde. Although glutamate gamma-semialdehyde exists in solution primarily in its cyclic form, Delta(1)-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Delta(1)-pyrroline-5-carboxylate (L-proline, L-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition. When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate gamma-semialdehyde is decreased approx. 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis.

Induction of gadd153 mRNA by nutrient deprivation is overcome by glutamine.

The growth arrest and DNA damage-inducible (gadd) genes are co-ordinately activated by a variety of genotoxic agents and/or growth-cessation signals. The regulation of gadd153 mRNA was investigated in renal proximal tubular epithelial cells (LLC-PK1) cultured in a nutrient- and serum-deprived medium. The addition of glutamine alone to LLC-PK1 cells cultured in Earl's balanced salt solution (EBSS) is sufficient to suppress gadd153 mRNA expression, and the removal of only glutamine from Dulbecco's modified Eagle's medium (DMEM) is also sufficient to induce gadd153 mRNA expression. Consistent with these findings, the inhibition of glutamine utilization with acivicin and 6-diazo-5-oxo-l-norleucine (DON) in cells grown in a glutamine-containing medium effectively induces gadd153 expression. Glutamine can be used as an energy source in cultured mammalian cells. However, it is unlikely that deficits in cellular energy stores (ATP) are coupled to gadd153 mRNA expression, because concentrations of ATP, UTP and GTP are all elevated in EBSS-exposed cells, and the addition of alpha-oxoglutarate to cells grown in EBSS has no effect on gadd153 mRNA expression. In contrast, concentrations of CTP decline substantially in EBSS and glutamine-deprived DMEM-cultured cells. Glutamine also serves as a precursor for the synthesis of protein and DNA. The addition of glutamine to cells grown in EBSS partly restores CTP concentrations. The addition of pyrimidine ribonucleosides (cytidine and uridine) to LLC-PK1 cells also restores CTP concentrations, in a manner commensurate with their relative abilities to overcome gadd153 expression. Finally, glutamine does not completely suppress DNA damage-induced gadd153 expression, suggesting that multiple signalling pathways lead to the expression of gadd153 mRNA under conditions of nutrient deprivation and DNA damage.

Ribose-enhanced synthesis of UTP, CTP, and GTP from parent nucleosides in cardiac myocytes.

The participation of ribose and its metabolites in some nucleoside salvage reactions is well established. Isolated adult rat cardiac myocytes were used as a model system to determine whether ribose acts as a general stimulant of salvage reactions in cardiac muscle, or whether only certain classes of nucleosides are affected by ribose. Myocytes were incubated with [3H]-adenosine, [3H]-cytidine, [3H]-guanosine, [3H]-thymidine, or [3H]-uridine for 30 or 60 min in the presence or absence of 5 mM ribose. The cells were extracted and the extracts assayed for [3H]-nucleoside and [3H]-nucleotide products. Salvage synthesis of cytosine, guanine and uracil nucleotides from the parent nucleosides was stimulated by ribose. Guanosine and uridine salvage appeared saturated at 50 microM external nucleoside (the dose response of cytidine salvage was not examined). Adenosine salvage was unaffected by ribose addition; the response to increasing external adenosine concentration was non-Michaelis-Menten, showing a peak of activity at 25 microM external nucleoside. Thymidine salvage was also unaffected by ribose, and was saturated at 50 microM external thymidine. These data suggest that adenosine and thymidine are metabolized to their respective nucleotide monophosphates by kinase activity. Cytidine, guanosine, and uridine salvage are stimulated by ribose, and must therefore be metabolized in part by nucleoside phosphorylase and phosphoribosyltransferase activity.

Cytidine triphosphate (CTP) synthetase activity during cell cycle progression in normal and malignant T-lymphocytic cells.

The role of cytidine triphosphate (CTP) synthetase (EC 6.3.4.2.) in the pyrimidine ribonucleotide metabolism of MOLT-3 human T-ALL cell line cells and normal human T lymphocytes during the cell cycle traverse was studied. Highly pure G1-phase samples and samples enriched in S-phase cells were obtained by counterflow centrifugation. The activity of CTP synthetase in situ, measured in pulse-chase experiments, was similar in the G1-phase and S-phase MOLT-3 cells. In contrast, in S-phase T lymphocytes, an increased activity of CTP synthetase was observed compared with G1-phase T lymphocytes. Nevertheless, the MOLT-3 samples showed an increased activity of CTP synthetase in comparison with either G1-phase or S-phase enriched samples of normal T lymphocytes. Therefore, the increased activity of CTP synthetase of MOLT-3 cells is a cell cycle-independent feature, whereas among normal T lymphocytes, the increase in activity of CTP synthetase that arises after a growth stimulus is more prominent in the S-phase.

The roles of uridine-cytidine kinase and CTP synthetase in the synthesis of CTP in malignant human T-lymphocytic cells.

The pattern of incorporation of [14C]uridine showed that in MOLT-3 cells an increased proportion of CTP was synthesized via CTP synthetase, compared to proliferating normal human T lymphocytes at a physiological concentration of cytidine (< 0.5 microM). Furthermore, in the proliferating normal human T lymphocytes similar patterns of incorporation of [14C]uridine were observed in the presence of the physiological concentration of cytidine and after addition of 2 microM of cytidine. In contrast, in the MOLT-3 cells after addition of 2 microM of cytidine the proportion of CTP synthesized by conversion of UTP into CTP was substantially decreased, whereas the salvage of cytidine was proportionally increased. We conclude that the reutilization of uridine is a preferred route in the synthesis of CTP for MOLT-3 cells at physiological concentrations of uridine and cytidine, whereas in proliferating normal human T lymphocytes CTP is largely synthesized through reutilization of cytidine. This difference in salvage of pyrimidine ribonucleosides may be exploited for selective chemotherapy.

Evidence for transformation-related increase in CTP synthetase activity in situ in human lymphoblastic leukemia.

To determine the role of the enzyme CTP synthetase (EC 6.3.4.2) in the synthesis in situ of CTP in normal and in malignant lymphoblastic cells, the metabolism of radiolabeled pyrimidine ribonucleosides was studied in proliferating normal T lymphocytes and was compared with that of proliferating MOLT-3 cell-line cells and differentiated (non-proliferating) MOLT-3 cells. Both the incorporation of [14C]uridine into UTP and CTP and the incorporation of [14C]cytidine in CTP, as well as the fluxes of these labeled nucleosides through the nucleotide pools into nucleic acids, were elevated in proliferating MOLT-3 cells compared to proliferating T lymphocytes. Furthermore, the conversion of UTP into CTP was enhanced in proliferating MOLT-3 cells compared to proliferating T lymphocytes, indicating a higher activity of CTP synthetase in the leukemic cells. In non-proliferating MOLT-3 cells, the pyrimidine ribonucleotide fluxes were decreased compared to proliferating MOLT-3 cells and proliferating T lymphocytes. However, the decreased ratio of uracil/cytosine ribonucleotides that was found in proliferating T lymphocytes and proliferating MOLT-3 cells compared to non-proliferating blood lymphocytes, was preserved in the differentiated MOLT-3 cells. Moreover, although the fluxes had decreased, most CTP was still synthesized by CTP synthetase in the differentiated MOLT-3 cells. Thus, the elevated activity of CTP synthetase in MOLT-3 cells was independent of the cell growth and maturation stage. We conclude that the increased activity of CTP synthetase is associated with the process of malignant transformation in MOLT-3 cells. Therefore, CTP synthetase offers an attractive target for selective therapy in human acute T-lymphoid leukemia.

(6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid effects on nucleotide metabolism in CCRF-CEM human T-lymphoblast leukemia cells.

(6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid [(6R)DDATHF] is a folate antimetabolite with activity specifically directed against de novo purine synthesis, primarily through inhibition of glycinamide ribonucleotide transformylase. This inhibition resulted in major changes in the size of the nucleotide pools in CCRF-CEM cells. After a 4-h incubation with 1 microM (6R)DDATHF, dramatic reductions in the ATP and GTP pools were observed, with almost no effect on CTP, UTP, and deoxyribonucleotide pools. When the incubation was continued in drug-free medium, recovery of ATP and GTP pools was protracted. ATP did not return to normal until 24-36 h, and GTP pools were only partially repleted by 48 h. The ATP and GTP pools were not affected when the initial 4-h incubation with (6R)DDATHF was conducted in the presence of 100 microM hypoxanthine. Addition of hypoxanthine to the medium after a 4-h incubation with (6R)DDATHF caused rapid recovery of the ATP and GTP pools. Similar effects were seen when the purine precursor aminoimidazole carboxamide was used in place of hypoxanthine. The effect of (6R)DDATHF on nucleotide pools and the capability of hypoxanthine or aminoimidazole carboxamide to prevent or reverse this phenomenon correlated directly with the inhibition of cell growth. Presumably as a consequence of the decrease in purine nucleotide triphosphate levels, the conversion of exogenously added uridine, thymidine, and deoxyuridine to nucleotides was markedly decreased. These effects were protracted for almost 48 h and were also reversed by hypoxanthine. Differential repletion of ATP and GTP pools after (6R)DDATHF pre-treatment demonstrated that diminished precursor phosphorylation is primarily a consequence of GTP rather than ATP starvation.