Nucleotide Synthesis Pathway Research Articles

Advances in the Stable Isotope–Mass Spectrometric Measurement of DNA Synthesis and Cell Proliferation

Methods for measuring rates of DNA synthesis, and thus cell proliferation, in humans had not been available until recently. We (D. C. Macallan, C. A. Fullerton, R. A. Neese, K. Haddock, S. S. Park, and M. K. Hellerstein, 1998, Proc. Natl. Acad. Sci. USA 95, 708–713) recently developed a stable isotope–mass spectrometric technique for measuring DNA synthesis by labeling the deoxyribose (dR) moiety of purine deoxyribonucleotides through the de novo nucleotide synthesis pathway. The original analytic approach had limitations, however. Here, we describe technical improvements that increase yield, stability, sensitivity, and reproducibility of the method. The purine deoxyribonucleoside, deoxyadenosine (dA), is directly isolated from hydrolysates of DNA by using an LC18 SPE column. Two derivatives were developed for analyzing the dR moiety of dA alone (without the base), an aldonitrile-triacetate derivative, and a reduced pentose-tetraacetate (PTA) derivative. The PTA derivative in particular exhibited greater stability (no degradation after several weeks), greater GC/MS signal, and much less abundance sensitivity of isotope ratios (i.e., less dependence of mass isotopomer abundances on the amount of material injected into the mass spectrometer source), compared to previous derivatives of dA. The need for complex, multidimensional abundance corrected standard curves was thereby avoided. Using the PTA derivative, dR enrichments from DNA of fully turned over cells of rodents with 2H2O enrichments in body water of 2.2–2.8% were 9.0–9.5%, and less than 1.0 μg DNA (ca. 2 × 105 cells) was required for reproducible analyses. In summary, these methodologic advances allow measurement of stable isotope incorporation into DNA and calculation of cell proliferation and death rates in vivo in humans and experimental animals, with fewer cells, greater reproducibility, and less labor. Many applications of this approach can be envisioned.

The cost effectiveness of mycophenolate mofetil in the first year after living related renal transplantation

REJECTION of transplanted organs results from the ability of lymphocytes to proliferate for the destruction of foreign tissue. Like all human somatic cells, lymphocytes proliferate by mitotic division and need purine and pyrimidine nucleotides for DNA synthesis. There are two major pathways in purine nucleotide synthesis: the de novo pathway and the salvage pathway. Most of human cell types can use salvage pathway for the proliferation if de novo synthesis is blocked; whereas, T and B lymphocytes depend mainly on de novo pathway. Mycophenolate Mofetil (MMF) selectively inhibits the proliferation of T and B lymphocytes because it acts as a reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH), a rate limiting enzyme in the de novo pathway of guanosine nucleotide synthesis. Depletion of guanosine nucleotides with MMF results in impaired DNA synthesis and exerts antiproliferative effect. Since lymphocytes rely on de novo purine synthesis, this antiproliferative effect is selectively on lymphocytes. It is different from azathioprine because of its selective antiproliferative effect on lymphocytes. MMF has also two other effects on immune response: It suppresses antibody formation by B lymphocytes and inhibits the glycosylation of lymphocyte and monocyte glycoproteins. In some studies, MMF, in combination with corticosteroids and cyclosporine, reduced the incidence of renal allograft rejection within the first 6 months after transplantation when compared with triple drug protocols with azathioprine (AZA). In this study, we assessed the cost effectiveness of triple immunosuppressive regimen including MMF in the first year after living related renal transplantation.

Novel role of extracellular carbon dioxide in lymphocyte proliferation in culture.

CO(2)/HCO(3)(-) buffering system is indispensable to maintain the pH of culture media for long-term cell culture. Now-a-days, the zwiterionic hydrogen buffer HEPES is widely used as an additional buffer in the commonly used culture media. There are reports on the successful use of HEPES-buffered media, under CO(2)/HCO(3)(-) free conditions, for long-term cell cultures. However, still CO(2)/HCO(3)(-) buffering system is widely used. We aimed at investigating the reason for this. We found that lymphocytes proliferate in response to concanavalin A only in HCO(3)(-)-buffered medium in the presence of 5% CO(2), but not in the HEPES-buffered medium in the absence of CO(2). However, lymphocyte proliferation was observed in HEPES-buffered medium in the presence of 5% CO(2) and in the absence of HCO(3)(-). On the other hand, a low level proliferation was observed in HEPES-buffered medium supplemented with HCO(3)(-) in the absence of CO(2). Supplementation of the culture medium with TCA cycle intermediates and the precursors for the salvage pathway of nucleotide synthesis did not support the lymphocyte proliferation at all. Based on these findings and other reports, we suggest that extracellular CO(2) plays a novel role in cell proliferation.

Cloning and in vitro expression of the cDNA encoding a putative nucleoside transporter from Arabidopsis thaliana

Nucleoside transporters are integral membrane proteins involved in the uptake or release of nucleosides. Their function constitutes an essential step of the salvage pathway of nucleotide synthesis. In order to study the function of these proteins in higher plants, we cloned the cDNA corresponding to the AtENT1 gene that encodes a putative nucleoside transporter in Arabidopsis thaliana by RT-PCR. The amino acid sequence of the AtENT1 protein deduced from the cloned cDNA shared similarity to those of eukaryotic equilibrative nucleoside transporters. Structure prediction indicated that the deduced AtENT1 protein might possess eleven putative transmembrane domains. Southern hybridization revealed that AtENT1 had one homologue in the Arabidopsis genome. Northern blot analysis showed that AtENT1 might be constitutively expressed in most Arabidopsis organs and in plants at different developmental stages. Two AtENT1 fusion genes, AtENT1-His-tag and GFP-AtENT1-His-tag, were expressed in insect cells. Confocal microscopy demonstrated that the GFP-AtENT1-His-tag fusion protein was targeted specifically to the plasma membrane of insect cells.

Role of Nutrients and Bacterial Colonization in the Development of Intestinal Host Defense

In this introduction to the supplement on the use of pre- and probiotics in the health and disease of pediatric patients, I have summarized factors affecting the initial colonization of the neonatal intestine. The term bacterial-epithelial cross-talk was defined, and examples of the enterocyte response to both pathologic and indigenous flora stimulation illustrated. Immaturities in the human neonatal intestinal response to bacteria and their toxins were reviewed in the context of the pathogenesis of age-specific, bacterial gastrointestinal infectious diseases. Finally, the importance of pre- and probiotics as measures to strengthen the neonate's intestinal host defenses in the prevention and treatment of specific age-related disease were considered.

The evolving role of mycophenolate mofetil in renal transplantation.

Mycophenolate mofetil (MMF) is an ester prodrug with higher bioavailability than the active agent, mycophenolic acid (MPA).1 MPA inhibits inosine monophosphate (IMP) dehydrogenase, an enzyme that facilitates the conversion of IMP to xanthosine monophosphate, a precursor of guanine nucleotides. This is an important step in the de novo pathway of purine nucleotide synthesis on which lymphocytes primarily depend, unlike neutrophils, for example.2 In addition, different isoforms of IMP dehydrogenase exist, and MPA is not only almost five times more potent in inhibiting the type II isoform, that is associated with stimulated rather than resting lymphocytes,3 but is also, to some extent, cell type specific.4 Thus, on theoretical grounds, MMF represents a significant improvement on the antimetabolite azathioprine the use of which is well-established in organ transplantation. MMF is effective in prolonging the survival of mouse and rat cardiac allografts,5,,6 showing an additive effect with cyclosporin, brequinar or tacrolimus.6–,9 It can also reverse rejection of rat cardiac allografts6 and prolong canine hepatic allograft survival10 as well as mouse11–,13 and rat14 pancreatic allograft survival. Finally, in a canine model, it has been shown to prolong renal allograft survival15 and reverse acute rejection.16 Rat allograft studies have reported a role in reducing ischaemia-reperfusion injury.17 An additional experimental observation of potential interest in organ transplantation is the inhibitory effect of MPA on the proliferation of human arterial smooth muscle cells in vitro . Arterial disease plays a central role in chronic graft rejection, holding out hope for additional benefit from the use of MMF.18 In vivo MMF inhibits the graft coronary artery disease of rat cardiac allografts19 and cynomologous monkey-to-baboon cardiac xenografts20 and it attenuates functional, morphological and immunohistochemical changes associated with … Dr A.N. Warrens, Renal Unit, Imperial College School of Medicine, Hammersmith Hospital, L Block, Du Cane Road, London W12 0HS. e-mail: a.warrens{at}ic.ac.uk

Activation-associated necrosis in human immunodeficiency virus infection.

Mitogenic stimulation of lymphocytes from persons infected with human immunodeficiency virus (HIV) resulted in massive cell death. In addition to early apoptosis, a second wave of cell death occurred 48-72 h after stimulation. At that time, the cells were enlarged, leaked content, and had plasma membrane damage-all indicative of necrosis. Furthermore, DNA fragmentation as determined by TUNEL assay was virtually absent. This activation-associated necrosis could not be prevented by interfering with CD95/CD95-ligand interactions or by blocking caspase activity and was unaffected by neutralizing antibodies to tumor necrosis factor-alpha or interferon-gamma. Necrosis was also induced by activation of normal lymphocytes in the presence of ribavirin, which inhibits the de novo pathway of nucleotide synthesis. Lymphocytes from HIV-infected persons are defective in their ability to synthesize nucleotides via this pathway, indicating one possible mechanism for the activation-associated necrosis seen in HIV infection.

RNA-catalysed nucleotide synthesis.

The 'RNA world' hypothesis proposes that early life developed by making use of RNA molecules, rather than proteins, to catalyse the synthesis of important biological molecules. It is thought, however, that the nucleotides constituting RNA were scarce on early Earth. RNA-based life must therefore have acquired the ability to synthesize RNA nucleotides from simpler and more readily available precursors, such as sugars and bases. Plausible prebiotic synthesis routes have been proposed for sugars, sugar phosphates and the four RNA bases, but the coupling of these molecules into nucleotides, specifically pyrimidine nucleotides, poses a challenge to the RNA world hypothesis. Here we report the application of in vitro selection to isolate RNA molecules that catalyse the synthesis of a pyrimidine nucleotide at their 3' terminus. The finding that RNA can catalyse this type of reaction, which is modelled after pyrimidine synthesis in contemporary metabolism, supports the idea of an RNA world that included nucleotide synthesis and other metabolic pathways mediated by ribozymes.

Regulation of human PRS isoform expression.

PRPP is a substrate in the synthesis of virtually all nucleotides1 and is also an important regulator of the de novo pathways of purine2 and pyrimidine3 nucleotide synthesis. Formation of PRPP is catalyzed in eukaryotes by a family of PRPP synthetase (PRS; EC2.7.6.1) isoforms.4–6 The substrates in the reaction are MgATP and Rib-5-P, and Mg2+ and P1 are essential activators. Inhibitors of PRS activity include: purine, pyrimidine, and pyridine nucleotides; reaction products; and 2,3-diphosphoglycerate.4,5,7 Human erythrocyte PRS is composed of a polypeptide subunit of 34.5 kD which undergoes concentration-dependent and effector-mediated reversible aggregation in vitro to active forms of 16 and 32 subunits.4,8 The primary structure and kinetic and physical properties of purified human erythrocyte PRS are those of the purified recombinant PRS 1 isoform.

Basis of pyrimidine nucleotide metabolism in the myocardium.

The metabolism of pyrimidine nucleotides in the myocardium is poorly understood. The turnover of these nucleotides is high, whereas their concentration is rather low. The de novo pathway of synthesis does not seem very efficient, although the utilization of nucleosides could represent the major pathway for pyrimidine nucleotide synthesis. In rat blood, cytidine could be the major precursor for pyrimidine nucleotide synthesis. The precursor, whatever its exact nature (uridine or cytidine), could be species dependent, and the liver could a major role in providing blood nucleosides. Owing to the essential role of pyrimidine nucleotides in the synthesis of macromolecules, acute or chronic alteration of the metabolism of these nucleotides could have crucial consequences on heart viability and function. Providing pyrimidine precursors to the heart, isolated or in situ, induces functional and metabolic effects on the heart. The experimental results suggest that such interventions could be beneficial in clinical situations such as cardioplegia, heart preservation, or recovery from ischemia.

Two activities of the immunosuppressive metabolite of leflunomide, A77 1726: Inhibition of pyrimidine nucleotide synthesis and protein tyrosine phosphorylation

Previous studies have demonstrated that the active metabolite of leflunomide, A77 1726 [ N-(4-trifluoromethylphenyl-2-cyano-3-hydroxycrotoamide)], is capable of inhibiting the activities of tyrosine kinases and dihydroorotate dehydrogenase (DHO-DHase). In the present study, we define the relative contribution of these activities to the ability of A77 1726 to inhibit proliferation of the murine leukemia cell line LSTRA. A77 1726 inhibited LSTRA cell growth and proliferation ( ic 50 ≈ 10–30 μM); this inhibition, however, could be reversed by the addition of exogenous uridine, suggesting that the anti-proliferative activity of A77 1726 may be due to inhibition of de novo pyrimidine nucleotide synthesis. Quantitation of nucleotide levels revealed that A77 1726, at an ic 50 of about 10 μM, selectively inhibited pyrimidine nucleotide but not purine nucleotide synthesis. In vitro enzyme assays confirmed that A77 1726 directly inhibited the activity of DHO-DHase, the fourth enzyme in the de novo pathway of pyrimidine nucleotide synthesis ( ic 50 ≈ 220 nM). LSTRA cells overexpress p56 lck and have elevated levels of tyrosine phosphorylated intracellular proteins. A77 1726 reduced the intracellular levels of tyrosine phosphorylated proteins with relatively high ic 50 values ranging from 50 to 100 μM. A77 1726 also inhibited p56 lck activity in LSTRA membrane preparation and immunoprecipitates; the ic 50 values for inhibition of immunoprecipitated p56 lck autophosphorylation and exogenous substrate histone 2B were 80 and 40 μM, respectively. The anti-tyrosine phosphorylation activity of A77 1726 was not affected by uridine. These studies therefore demonstrate the two activities of A77 1726: inhibition of pyrimidine nucleotide synthesis and interference with tyrosine phosphorylation.

Serum thymidine kinase as a tumor marker of colorectal carcinogenesis induced with 1,2-dimethylhydrazine in rats

Thymidine kinase is the key enzyme in salvage pathway for pyrimidine nucleotide synthesis. High activities of thymidine kinase have been found in rapidly proliferating normal, neoplastic tissues and sera from patients with various diseases including lymphoma, leukemias and small cell lung cancer. We investigated the serum thymidine kinase activities and the colorectal carcinomas in rats treated with 1,2-dimethylhydrazine. The positive correlations between serum thymidine kinase activities and numbers of total and large tumors were observed. These findings suggest the measurement of serum thymidine kinase activity may be clinically valuable in an early stage of the colonic carcinogenesis.

Cytosolic biosynthesis of GTP and ATP in normal rat pancreatic islets

GTP and ATP are necessary for glucose-induced insulin secretion; however, the biosynthetic pathways of purine nucleotides have not been studied in pancreatic islets. The present work examines the cytosolic pathways of purine nucleotide synthesis using intact rat islets cultured overnight in RPMI 1640 medium containing either [ 14C]glycine (to label the de novo pathway) or [ 3H]hypoxanthine (to mark the salvage pathway), with or without mycophenolic acid or l-alanosine (selective inhibitors of cytosolic GTP and ATP synthesis, respectively). Addition of mycophenolic acid decreased total GTP content (mass) by 73–81%; although the incorporation of labeled hypoxanthine into GTP also fell by 87%, the incorporation of glycine did not change. Similarly, l-alanosine decreased ATP mass by 26–33% in the presence of either label; whereas the incorporation of hypoxanthine into ATP fell 59%, the incorporation of glycine was again not significantly decreased. Thus, both the de novo and salvage purine nucleotide biosynthetic pathways are present in rat islets; however, the salvage pathway appears to be quantitatively the more important source of nucleotides. This conclusion was supported by additional studies of the effects on nucleotide content and insulin secretion of various site-specific inhibitors of purine synthesis. These findings have potential relevance to the processes of mitogenesis, cell proliferation and differentiation of islet cells, as well as for the control of insulin secretion.

Gene dosage and expression, and enzyme activity of thymidine kinase and thymidylate synthase in xenografted colorectal adenocarcinomas.

Cytogenetic studies performed on human colorectal tumors have revealed 2 specific patterns of chromosomal anomalies. The major pattern, known as the monosomic type (MT), is characterized by the loss or deletion of chromosomes 18, 17 (short arm 17p) and, less frequently, 1p, 4, 15, 5 (long arm 5q) and 21. The other one, known as the trisomic type (TT), is characterized by the gain of several chromosomes: 7, 12, X, 5 and 8. Losses of chromosome 18 and of the 17p arm never coexist in TT tumors. It was observed that many chromosome losses or deletions involved genes encoding for enzymes of the de novo pathways of nucleotide synthesis. In contrast, gains involved genes encoding for enzymes of the salvage pathways of the same metabolism. This led to the hypothesis that chromosome imbalances corresponded to those of nucleotide synthesis in tumor cells. Such an interrelation was confirmed by the dosage of thymidylate synthase (TS) and thymidine kinase (TK) activities in a series of colorectal grafted tumors. This study has been expanded to a larger series of xenografted tumors (23 cases) in which both TS and TK activities were studied, in parallel with an analysis of mRNA, by Northern blotting. The amount of mRNA was found to correlate with the number of gene copies calculated from cytogenetic data, indicating a direct gene-dosage effect. It also correlated with enzyme activities, but less strongly. This suggests the existence of an efficient post-transcriptional regulation, in particular for TS, whose level of expression varies over a wide range. Such variations may explain the diversity of responses to chemotherapy.

SUPPRESSION OF TUMORIGENESIS AND TUMOR-GROWTH OF MOUSE MAMMARY-GLANDS BY A TRADITIONAL CHINESE HERBAL MEDICINE, SHO-SAIKO-TO

Hyperplastic alveolar nodule (HAN) is a representative preneoplastic state in mammary glands of mice. We investigated the effect of Sho-saiko-to (SST; a Japanese modified traditional Chinese herbal medicine containing seven medicinal plants) on the formation and growth of HAN and mammary tumour growth in SHN virgin mice which showed a high incidence of spontaneous mammary tumours. Chronic oral administration of SST reduced the number and the area of HAN and mammary thymidylate synthetase (TS) activity with slight decrease of serum prolactin level. In the established mammary tumours of mice, 20-day administration of SST reduced the activity of TS and thymidine kinase (TK) with slight reduction of serum prolactin level, though the significant change of mammary-tumour growth was not observed. These results indicate that SST may prevent the mammary tumourigenesis by suppression of the de novo pathway for pyrimidine nucleotide synthesis and the mammary tumour growth by suppression of the salvage pathway as well as the de novo pathway in mice.

Human P53, Mutated at Codon 273, Causes Distinct Effects on Nucleotide Biosynthesis Salvage Pathway Key Enzymes in Rat-1 Cells and in Their Derivatives Expressing Activated ras Oncogene

The introduction of human p53 with mutation at codon 273 into Rat-1 cells induces changes in the salvage pathway of nucleotide synthesis. In cells expressing the mutant p53 the activities of hypoxanthine phosphoribosyl transferase (HPRT) and thymidide kinase (TK) decrease 3.5- and 2-3-fold, respectively, while the activities of adenosine deaminase and uridine kinase, in contrast, increase correspondingly 2.5- and 1.5-fold. On the other hand, in cells transformed by ras oncogene, which causes dramatical reduction in HPRT activity as well as enhancement of TK function, the expression of exogeneous p53 leads to the opposite effects and causes the reversion of activities of both enzymes to the levels found in parental cells.

The synergistic interactions in vitro and in vivo of brequinar sodium with cyclosporine or rapamycin alone and in triple combination.

The rigorous median-effect analysis was used to assess the interactions between cyclosporine and drugs that inhibit nucleotide synthesis pathways. Using in vitro proliferation assays wherein human lymphocytes were triggered by phytohemagglutin, anti-CD3 monoclonal antibody, or mixed lymphocyte reactions, CsA was shown to display additive interactions with 6-mercaptopurine (6-MP), mizorbine (MZB), and mycophenolic acid (MPA), and a synergistic interaction with brequinar (BQR). In the in vitro assays, BQR contributed a further synergistic effect to the double-drug combination CsA/rapamycin (RAPA). Of the four inhibitors of nucleotide synthesis pathways, only BQR noncompetitively inhibited IL-2-stimulated proliferation of the CTLL-2 cell line. Using the in vivo assay of heterotopic Buffalo (BUF, RT-1b) cardiac allografts in Wistar-Furth (WFu, RT-1u) hosts, oral administration of BQR displayed about 100% bioavailability--which, like the bolus intravenous (i.v.) mode, was eight-fold more effective than continuous i.v. infusions. Furthermore median-effect analysis of serial amounts of orally administered BQR demonstrated that it contributes synergistically to the immunosuppressive effects of intravenously delivered CsA/RAPA (0.5/0.01 mg/kg/day). The degree of synergism was proportionate to the extent of the immunosuppression. These findings document the potency of the CsA/RAPA/BQR triple-drug combination and suggest that the synergistic effects may permit dose reductions of each component, thereby mitigating toxicities resulting from the large amounts of individual agents necessary to achieve allo-unresponsiveness.

A defective purine nucleotide synthesis pathway in psoriatic patients.

Purine nucleotide concentrations in skin- and blood-cells of psoriatic patients are abnormal: The increase in the steady state level of cGMP and the decrease in the cAMP concentrations are associated with an enhanced rate of cellular proliferation. Concomitantly we found in the present study decreased ADP and ATP concentrations in blood cells (p less than 0.0001). The change in nucleotide concentrations suggests a defective purine nucleotide synthesis pathway. Stimulation of the Krebs cycle with fumaric acid raises ATP (p less than 0.0001) and most probably cAMP levels and at the same time slows down the purine nucleotide synthesis through end-product inhibition. Both effects can inhibit DNA and protein synthesis activity, which results in inhibition of cellular proliferation. Fumaric acid seems therefore a useful treatment for psoriatic lesions if liver and kidney functions (purine nucleotide and urea cycle) are controlled during treatment.

A defective purine nucleotide synthesis pathway in psoriatic patients.

Purine nucleotide concentrations in skin- and blood-cells of psoriatic patients are abnormal: The increase in the steady state level of cGMP and the decrease in the cAMP concentrations are associated with an enhanced rate of cellular proliferation. Concomitantly we found in the present study decreased ADP and ATP concentrations in blood cells (p less than 0.0001). The change in nucleotide concentrations suggests a defective purine nucleotide synthesis pathway. Stimulation of the Krebs cycle with fumaric acid raises ATP (p less than 0.0001) and most probably cAMP levels and at the same time slows down the purine nucleotide synthesis through end-product inhibition. Both effects can inhibit DNA and protein synthesis activity, which results in inhibition of cellular proliferation. Fumaric acid seems therefore a useful treatment for psoriatic lesions if liver and kidney functions (purine nucleotide and urea cycle) are controlled during treatment.

Relationships between UMPK and PGD activities and deletions of chromosome 1p in colorectal cancers

A deletion involving a large segment of the short arm of chromosome 1(1p−) occurs in about 50% of colorectal cancers. It was previously noticed that, in these tumors, many deletions affect genes encoding for enzymes of the de novo pathway of nucleotide synthesis. The gene for uridine monophosphate kinase (UMPK), mapped on 1p32, is generally involved in del(1p). The activity of the corresponding enzyme was measured and compared to that of 6-phosphogluconate dehydrogenase (PGD), encoded by a gene also mapped on chromosome 1p and frequently deleted, but involved in another metabolism. It was found that a clear relationship exists between activity and the number of chromosome 1p for PGD but not for UMPK, both on primary tumors (PTs) and on tumors grafted into nude mice (GTs). By comparison to corresponding normal mucosae, the activity of PGD was high in PTs and GTs, but this increase was reduced in case of del(1p). The activity of UMPK being increased in PTs but not in GTs, it is assumed that the increase in PTs is due to non-cancerous cells, which are missing in GTs. The fact that no gene dosage effect exists, although the tendency for del(1p) coexists with the relative decrease of UMPK activity in cancerous by comparison to non-cancerous cells, suggests that either mutation or disregulation of UMPK gene occurred early.