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Behavior of Activities of Thymidine Metabolizing Enzymes in Human Leukemia-Lymphoma Cells1

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Behavior of Activities of Thymidine Metabolizing Enzymes in Human Leukemia-Lymphoma Cells1 [CANCER RESEARCH 49. 1090-1094. March I, 1989] Behavior of Activities of Thymidine Metabolizing Enzymes in Human Leukemia-Lymphoma Cells1 Taiichi Shiotani, Yasuko Hashimoto, Terukazu Tanaka, and Shozo Irino First Department of Internal Medicine, Kagawa Medical School, Ikenobe, Miki, Kagawa, 761-07, Japan ABSTRACT 7). These observations indicate the importance of this catabolic enzyme for dThd utilization. Thymidylate, an important pre The behavior of the activities of thymidine metabolizing enzymes, cursor of DNA synthesis, also may be produced by the de novo dihydrothymine dehydrogenase (EC 1.3.1.2) and thymidine phosphoryl- pathway through dTMP synthase. The correlation between cell ase (EC 2.4.2.4) for thymidine degradation, thymidine kinase (EC proliferation and dThd kinase (3, 4, 6, 8, 9) or dTMP synthase 2.7.1.75) and thymidylate synthase (EC 2.1.1.45) for DNA synthesis, was elucidated in cytosolic extracts from normal human lymphocytes and (8, 10, 11) has been demonstrated. The enhanced capacity for 13 human leukemia-lymphoma cell lines. In the normal human lympho the salvage pathway in leukemia (12, 13) appears to limit the cytes, the activities of dihydrothymine dehydrogenase, thymidine phos- antitumor effectiveness of antimetabolites of de novo pyrimidine phorylase, thymidine kinase, and thymidylate synthase were 6.88, 796, biosynthesis. The enzymic capacities of the de novo and salvage 0.30, and 0.29 nmol/h/mg protein, respectively. In leukemia-lymphoma pathways and dThd catabolism have not been determined si cell lines, the activities of synthetic enzymes, thymidine kinase, and multaneously in human leukemia-lymphoma cells. Therefore, thymidylate synthase, increased two- to 79-fold and 22- to 407-fold of the elucidation of the behavior of the enzymic capacities in the the normal lymphocyte values. In contrast, the activities of the catabolic opposing pathways of dThd metabolism should lead to a deeper enzymes, dihydrothymine dehydrogenase and thymidine phosphorylase, decreased to 5-42% and 3-38% of the values of normal lymphocytes. As insight into the regulation of dThd utilization and may provide suitable targets for selective chemotherapy against lymphopro- a result, the ratio of activities of thymidine kinase/dihydrothymine de hydrogenase was elevated by 7- to 1170-fold, respectively. Thus, recip liferative disorders. rocal behavior in the activities of the opposing enzymes in thymidine In this paper, we present the reciprocal regulation in the metabolism was observed in human leukemia-lymphoma cells. behavior of dThd catabolic enzyme activities (DHT DH and Polyclonal and monoclonal antibodies against dihydrothymine dehy dThd phosphorylase) and those of synthetic capacity for DNA drogenase were prepared and studies on immunotitration of this enzyme (dThd kinase and dTMP synthase) in human leukemia-lym with these antibodies showed that the enzyme protein amount in Jurkat phoma cell lines, and also demonstrate that the decrease in leukemic cells was 36% of that of normal lymphocytes. This was in good DHT DH activity of leukemic cells is due to the decline in the agreement with the decrease in the activity of the enzyme to 32%, amount of enzyme protein. A preliminary report has been indicating that the decrease in activity in the leukemic cells was due to presented (14). the decline in the amount of enzyme protein. The metabolic imbalances in thymidine utilization appear to be char acteristic of human leukemia-lymphoma cells. These observations should MATERIALS AND METHODS confer selective advantages to the lymphoproliferating cells and mark out the catabolic, as well as the synthetic, enzymes as important targets in Chemicals. [Methyl-3H]dThd (20 Ci/mmol), [/m>/A.F/-3H]thymine the design of chemotherapy. (12.6 Ci/mmol), [2-l4C]dThd (58 mCi/mmol), [2-14C]thymine (55.2 mCi/mmol), and iodine-125 (100 mCi/ml, 17 Ci/mg) were purchased from New England Nuclear (Boston, MA). [5-3H]dUMP (8.9 Ci/mmol) INTRODUCTION and ACS-II were obtained from Amersham Corp. (Arlington Heights, IL). RPMI 1640 culture medium was from GIBCO (Grand Island, Despite the use of dThd3 as a chemotherapeutic modality in NY). 2',5'-ADP-Sepharose 4B was obtained from Pharmacia Fine leukemia (1, 2), the behavior of the enzymic capacity of dThd Chemicals Inc. (Piscataway, NJ). All other reagents were of the highest catabolism has received less attention. dThd is salvaged by available analytical grade. dThd kinase, providing an alternate route for dTMP production Cell Culture. 13 human leukemia-lymphoma cell lines were examined for DNA biosynthesis. dThd also may be degraded by dThd for the activities of the dThd-metabolizing enzymes. The T-lymphoblast phosphorylase and through the rate-limiting enzyme of dThd cell lines tested were acute lymphocytic leukemias Molt-4 (15), Jurkat catabolism, DHT DH, for eventual catabolism to CO2 and (16), CCRF-CEM (15), human T-cell leukemia virus I-transformed T- cell MT-2 (17), and malignant lymphoma HPB-MLT (15). The B-cell ammonia (3, 4). Since dThd phosphorylase is an equilibrium lines examined were Burkitt's lymphomas Namalva (18) and Daudi enzyme, the balance of dThd kinase and DHT DH may deter (15), Epstein-Barr virus-transformed B-cell HOR (15), and acute lym mine the metabolic routing of dThd. We purified DHT DH to phocytic leukemia BALL-1 (15). The Non-T- and Non-B-cell lines were homogeneity from rat liver (5). DHT DH activity decreased as chronic myelogenous leukemia in blast crisis K-562 (15), acute pro- the proliferative rate increased in a spectrum of rat hepatomas, myelocytic leukemia HL-60 (15), plasma cell leukemia ARH-77 (15), indicating a significant negative correlation with cell prolifera and histiocytic lymphoma U-937 (19). Molt-4, Jurkat, CCRF-CEM, tion (3, 4, 6). Dihydrothymine dehydrogenase is also responsi Namalva, HL-60, and U-937 cell lines were kindly provided by Dr. ble for the breakdown of the widely used antineoplastic agent Reinhard Kurth (Paul-Ehrlich-Institute, West Germany). HPB-MLT, 5-fluorouracil, thereby limiting its therapeutic effectiveness (5, HOR, Daudi, BALL-1, K-562, and ARH-77 were from Dr. Takeshi Watanabe (Department of Molecular Immunology, Medical Institute Received 8/22/88; revised 11/14/88; accepted 12/1/88. of Bioregulation, Kyushu University School of Medicine, Japan) and The costs of publication of this article were defrayed in part by the payment MT-2 was from Dr. Hajime Ogura (Department of Virology, Okayama of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. University School of Medicine, Japan). Cells were maintained in the 1This investigation was supported by a Grant-in-Aid for Scientific Research logarithmic phase of growth in RPMI 1640 medium supplemented from the Ministry of Education. Science and Culture of Japan (59480265). 2To whom requests for reprints should be addressed. with 10% fetal calf serum in a humidified atmosphere of 5% CO2 in 1The abbreviations used are: dThd. thymidine; dUMP, deoxyuridylate; DHT air. The cultured cells were harvested by centrifugation and washed two DH, dihydrothymine dehydrogenase; PBS. phosphate buffered saline; dTMP, times with PBS. thymidylate. Isolation of Lymphocytes. Lymphocytes were isolated from the hep- 1090 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. RECIPROCAL REGULATION OF THYMIDINE METABOLISM arinized peripheral blood of 15 normal subjects (eight males and seven prepared by the method of Köhlerand Milstein (24). BALB/c mice females, aged from 21 to 39 years) using Ficoll-Hypaque gradient were immunized by the i.p. injection of 100 ¿igpurified DHT DH, method (20), and washed three times in PBS. mixed with an equal volume of complete Freund's adjuvant and boosted Enzyme Assays. Cells were resuspended at 1 to 2 x IO7cells/ml in in the same way three times at 2-week intervals. The mice exhibiting 50 min Tris-HCl (pH 7.4) and disrupted by rapid freeze and thaw in anti-DHT DH titers were sacrificed. Spleen cells (IO8 cells) were fused liquid nitrogen five times. The extracts were centrifuged at 100,000 x with 10* BALB/c mouse myeloma cells with polyethylene glycol 4000. g at 4°Cfor 60 min and the resulting supernatants were immediately Hybridomas were selected in hypoxanthine-amethopterin-thymidine assayed for the enzyme activities as described below. Protein was medium by distributing the cell suspension in 1-ml fractions into 240 determined by a routine method using bovine serum albumin as stand wells of 24-well Costar microplates. Cell growth was supported by a ard (21). Enzyme activity was expressed as nmol of product formed/h/ feeder layer of 105/well of peritoneal macrophages from BALB/c mice. mg protein. All assays were performed in duplicate and activities were Developing clones were cloned and subcloned at 2- to 3-week intervals measured at two enzyme concentrations to ensure the lineality of the by distributing 1 cell/well of a 96-well microtiter plate which contained reaction. IO4peritoneal macrophages as feeders. At 10 to 14 days after cloning, DHT DH. This enzyme was assayed by measuring the conversion of cultures were inspected to count the colony number per well. Half of [mef/ij7-3H]thymine to [merAj7-3H]dihydrothymine with a slight modi the culture medium in each well which contained only I colony was fication of the method of Piper et al. (9). Sodium fluoride and ATP replaced with the same volume of fresh medium. After 2 to 3 days, were omitted from the reaction mixture used by them, because these culture supernatants were screened for specific antibody production. cofactors did not affect the enzyme activity. Thin-layer chromatography Small Scale |'25I|Protein A Binding Assay. The screening of hybrido with polyethyleneimine cellulose sheets (Eastman Kodak, 20 x 20 cm) mas was performed by indirect |'25I)protein A binding assay (25).
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