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

Abstract

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 ['4C]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 uracilkytosine 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.Previously, we have shown in our laboratories that peripheral blood cells from patients with acute lymphoblastic leukemia, chronic lymphoblastic leukemia and non-Hodgkin lymphoma contain increased amounts of uracil and cytosine nucleotides and contain more uridinediphosphate-sugar compounds with a changed composition than do peripheral blood leukocytes from healthy donors [l -31. Typically, the ratio between uracil and cytosine ribonucleotides is decreased in the leukemic cells [l -31. In proliferating normal T lymphocytes too, an increase in the uracil and cytosine ribonucleotide pools has been observed, with a decreased ratio compared to non-proliferating peripheral blood lymphocytes 141.CTP synthetase catalyses the conversion of UTP into CTP and is the rate-limiting step in the synthesis of cytosine nucleotides from both the de novo and uridine salvage synthesis routes (see Fig. 1). In mouse S 49 T-lymphoma cellline cells the ratio between uracil and cytosine ribonucleotide phate-buffered saline; BrdU, 5-bromo-2'-deoxyuridine.(EC 6.3.4.2); uridine-cytidine kinase (EC 2.7.1.48).pools is balanced by allosteric feedback inhibition of CTP synthetase by the CTP pool [5]. Furthermore, it has been shown by in vitro enzyme analyses that an increased activity of CTP synthetase in tissue homogenates of...

CTP Synthase 1, a Smooth Muscle–Sensitive Therapeutic Target for Effective Vascular Repair

“…11–13 Although two different CTPS isotypes (CTPS1 and CTPS2) are identified in mammals, we found that CTPS1, but not CTPS2, was markedly up-regulated in SMCs by PDGF-BB (Figure 1A–1C, and Supplemental Figure I). PDGF-BB is a well-known SMC mitogen that induces SMC proliferation and neointima formation following vascular injury.…”

Exploring the Potent Inhibition of CTP Synthase by Gemcitabine‐5′‐Triphosphate

“…Most interestingly,w ed iscovered that wild-type CTPS catalyzes the conversion of dF-dUTP into dF-dCTP,f ollowed by significant product inhibition ( Figure S12). Production of dF-dCTP was confirmed by MS ( Figure S13), 15 NNMR spectroscopy (Figure S14), and RP-HPLC analysis( Figure S15). Wild-type CTPS was strongly inhibited by the product-dF-dCTP-when dF-dUTP was used as as ubstrate ( Figure S16), which precluded determination of individual k cat and K m values.…”

“…[1] Because this is the sole route for the de novo biosynthesis of the cytosine base, and also in view of the central role of CTP in the biosynthesis of nucleic acids, [2] phospholipids via the Kennedy pathway, [3,4] and sialic acid, [5] CTPS is an attractive target for the development of antiviral, [6,7] antiprotozoal, [8][9][10][11][12][13][14] and antineoplastic agents. [2,15,16] CTPS is also ar ecognized target for immunosuppressivet herapy due to its importance in T-cell proliferation. [17] CTPS is regulated in ac omplex fashion by many nucleotide effectors.…”

Only One Isoform of Drosophila melanogaster CTP Synthase Forms the Cytoophidium

“…In 1978, Weber and co-workers found that CTP synthase activity in hepatomas was elevated [15]. Subsequent studies demonstrated that unregulated CTP levels and increased CTP synthase activity are features of many forms of cancer such as leukemia, hepatomas, and colon cancer [15], [16], [17], [18], [19], 20,21,22,23,24,25,26,27. Furthermore, CTP synthase is an attractive target for drug development against viral [28] and parasitic disease (e.g.…”