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 KÃ¶hlerand 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 Â¿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 4Â°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). 20 n\ was employed for the separation of the reaction product from the of antigen solution containing 30 ^g of purified DHT DH was absorbed substrate, instead of paper chromatography with Whatman 3 MM into each well of 60-well Terasaki I microplates (Falcon) by overnight papers. The reaction mixture contained 35 mM Tris-HCl (pH 7.4), 0.24 incubation at 37"C. Wells coated with antigen were subsequently filled mM NADPH, 20 pM [;m>r/i>>/-3Hlthymine(505 mCi/mmol, 0.1 mCi/ with 1% gelatin in PBS and incubated at 37Â°Cfor l h to reduce ml), and enzyme extract in a final volume of 100 /Â¿I.Afterincubation nonspecific antibody binding to plastic. After washing once with PBS, for 30 and 60 min at 37Â°C,the reaction was stopped by boiling for 3 20 uI of 1:200 diluted nonimmunized or immunized serum or undiluted min and then cooling immediately following centrifugation at 10.000 hybridoma culture supernatants were added to each well and incubated at 37Â°Cfor 1 h. The incubation was terminated by another two washes rpm for 5 min. 10 n\ of the supernatants was spotted onto polyethyl eneimine cellulose sheets, which was developed at 21Â°Cby ascending with PBS, followed by the addition of 20 n\ rabbit anti-mouse IgG chromatography for 4 h in the solvent system consisting of f-butylal- antiserum (Miles-Yada Ltd., Rehovot, Israel). The plates were im mersed twice in PBS before 20 n\ (5 x 10" cpm) of |125I]protein A cohol:methylethylketone:water:ammonium hydroxide (4:3:2:1, v/v/v/ solution was distributed to each well and incubated at 37Â°Cfor 1 h. v). The Rf values of standard thymine catabolites with this system were: thymine, 0.55; /i-aminoisobutyric acid, 0.33; /3-ureidoisobutyric acid, After two final washes with PBS, each well was excised with a hot saw and directly counted in a -y-scintillation counter. 0.34; dihydrothymine, 0.78. No significant radioactivity was found at positions other than those of thymine and dihydrothymine. The sheet Immunotitration of DHT DH. The immunotitration of DHT DH was for each sample was cut to 1 x 1 cm and was placed in counting vials carried out by incubation of the cytosol extracts from the normal which contain 1 ml of 70% ethanol. 5 ml of ACS-II scintillation fluid lymphocytes and Jurkat leukemic cells with increasing amounts of mouse anti-DHT DH serum IgG or hybridoma culture medium IgG of were added and counted for radioactivity. dThd Phosphorylase. Thymidine phosphorylase catalyzes the revers clones 1A4 and 2A2 in a final volume of 300 ^1 with 0.9% NaCI for 4 h at 4V. and then the anti-mouse IgG was added in excess. The ible conversion of dThd to thymine. Thus, dThd phosphorylase activity was assayed either by the conversion of [2-'4C]dThd to [2-'4C]thymine incubation was continued for another 4 h at 4Â°C.After centrifugation or formation of [2-'4C]dThd from [2-'4C]thymine as described (22), at 10,000 rpm for 10 min, DHT DH activity remaining in the super with slight modification. After the reaction was terminated by boiling natants was assayed, and compared with the controls without antiserum for 3 min, the reaction mixture was centrifuged at 10,000 rpm for 5 as well as normal serum from nonimmunized mice. min. The reaction products were separated by high-performance liquid chromatography (HLC-803D; TOYO SODA Manufacturing Co. Ltd., RESULTS AND DISCUSSION Japan) instead of paper or thin-layer chromatography, which was used previously. 25 Â¡Aofthe resulting supernatant were applied onto a TSK Comparison of the Activities of dThd Metabolizing Enzymes gel ODS-120A column (4.6 mm ID x 25 cm) and eluted with 5% in Normal Human Lymphocytes. Activities of dThd metaboliz CHjCN with a flow rate of 0.8 ml/min. Fractions were collected with ing enzymes, DHT DH and dThd phosphorylase, for the cata- fraction collector (Pharmacia, Frac-100) at a collection rate of 0.8 ml/ bolic pathway, dThd kinase and dTMP synthase for the syn min. 5 ml of ACS-II scintillation fluid were added and counted for thetic pathway in normal human lymphocytes were 6.88, 796, radioactivity. Thymine and dThd were eluted after 9 and 14 min, 0.30, and 0.29 nmol/h/mg protein, respectively, as presented respectively. dThd Kinase. Thymidine kinase was assayed as described (9), using in Table 1. dThd phosphorylase was also active in the formation [mi>r/i>'/-:lH]dThdas substrate. of dThd from thymine, with an activity of 3912 Â±1191 nmol/ dTMP Synthase. Thymidylate synthase activity was estimated by the h/mg protein (data not shown). Activities of catabolic enzymes amount of tritium released to H2O from [5-3H]dUMP with 100% KOH were several orders of magnitude higher than those of the absorption as reported previously by us (23). synthetic enzymes. The ratio of dThd kinase/DHT DH was Partial Purification of DHT DH from Human Lymphocytes and 0.04. The activity of dThd kinase was similar to that of dTMP Jurkat Leukemic Cells. Dihydrothymine dehydrogenase from lympho synthase. These results suggest that the metabolic balance cytes and Jurkat cells was partially purified by the method reported should favor dThd catabolism and dTMP production might previously (5). Briefly, cytosolic extracts from both cells were treated by 33-65% ammonium sulfate fractionation and passed through 2',5'- equally contribute to the de novo and salvage pathways in normal human lymphocytes. ADP-Sepharose 4B affinity column chromatography (1x5 cm). The Enzymic Programs in dThd Utilization of Leukemia-Lym- specific activities of partially purified DHT DH from lymphocytes and Jurkat cells were 207 and 106 nmol/h/mg protein with purification phoma Cell Lines. Behavior of the activities of dThd metaboliz ing enzymes was examined in 13 leukemia-lymphoma cell lines fold of 33 and 48, recovery of 60 and 70%, respectively. Preparation of Monoclonal Antibody against DHT DH. Dihydrothy (Table 1). The activities of catabolic enzymes (DHT DH and mine dehydrogenase from normal human liver was purified to homo dThd phosphorylase) decreased and those of synthetic enzymes geneity (5). Using purified DHT DH, hybridomas and clones were (dThd kinase and dTMP synthase) increased in all leukemia- 1091

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. RECIPROCALREGULATION OF THVMIDINE METABOLISM Table 1 Activities ofdThd metabolizing enzymes in normal human lymphocytes and human leukemia-lymphoma cells protein)CellsLymphocytesT-Cell Enzyme activities (nmol/h/mg kinaseDHTDH0.040.320.282.338.403.0811.714.0840.973.5117.7646.806.17(1000800f'"700"5,825"21,000"7,700"29,275"10,200"102,425"8,775"44,400"117,000"15,425" phosphorylase0796 kinase0.30 synlhase0.29 Â±95 Â±0.08(10000.93 (1000110.04Â±0.06 linesMolt-4JurkatCCRF-CEMMT-2HPB-MLTB-Cell Â±101 Â±0.41310''"0.61 37,945e'"11Â±7.26 Â±0.721.400.431.061.410.980.37Â±+++++0.450.030.090.500.100.090.63Â±27 Â±0.342033.26 7.90Â±19.5040,655"61.80 Â±13124238236 Â±1.381,087"3.61 21,310"6.30Â±20.28 Â±6420 Â±1.811,203"3.26 2,172"61.80Â±3.06 Â±71 1,087"16.51Â±1.38 21,310"50.34Â±20.28 linesNamalvaHORDaudiBALL-1Non-T, Â±277 Â±2.285,503"4.40 17,359"49.98Â±6.72 1,467"15.16Â±0.98 17,234"57.24Â±6.06 Â±110Â±182 Â±8.655,053"2.21 19,738"22.62Â±10.02 Â±0.051.060.461.38+Â£-t-ND'0.120.080.60(100042e"32"20"6"15"20"14"5"9"15"7"20"TdRÂ±48 737"23.81Â±0.56 7,800"107.82Â±4.86 linesK-562HL-60ARH-77U-937DHTDH6.882.87Â±Â±1.67*0.872.20Non-B cell Â±130 Â±9.877,937"18.83 37,179"91.56Â±21.36 Â±301 +6.296,277"21.53 Â±21.0631,572"3 Â±88 Â±5.377,177"8.51 007"67.201.92Â±6.60 11, Â±81423232122398918(100012Cp"13"3"30"3"9"35"14"23"6"16"38"11"dThdÂ±3.262,837"dTMP Â±19.6223,172"dThd " Enzyme activities, assayed by the conversion of dThd to ilumine, are presented. * Means Â±SE of 15 determinations for lymphocytes and five for leukemia-lymphoma cells. ' Percentage of the lymphocyte values. " Significantly different from values of normal lymphocytes (P < 0.05). ' ND, not detectable. lymphoma cells examined relative to values in normal human in vitro (30, 31), or 5-cyanouracil in vitro (32) enhanced the lymphocytes. Thus, the reciprocal behavior of the activities of utilization of dThd for DNA synthesis or increased antineo- enzymes in the opposing pathways of dThd utilization was plastic activity of 5-fluorouracil. Calculation of the ratio of observed in leukemia-lymphoma cell lines, which is in line with activities of dThd kinase/DHT DH allows a comparison of the that reported in rat hepatomas (3, 4, 6). potential capacity of dThd utilization for DNA synthesis and In the catabolic pathway, DHT DH activity was decreased to degradation to CO2. The ratio of dThd kinase/DHT DH in 5-42% (range from 0.37 to 2.87 nmol/h/mg protein in absolute normal lymphocytes was 0.04, whereas the ratios in leukemia- activity) of normal lymphocytes value. No activity of DHT DH lymphoma cell lines were elevated markedly (from 0.28 to 46.8). was detected in K-562 cells. The activity ofdThd phosphorylase Consequently, the metabolic imbalance of dThd utilization in measured for the catabolic conversion of dThd to thymine also lymphoproliferative cells was profound in the ratio of dThd declined to 3-38% (range from 20 to 301 nmol/h/mg protein) kinase/DHT DH, as reported in rat hepatomas (3, 4). More of the lymphocyte value. The activity of anabolic conversion of over, in leukemic cell lines such as Molt-4 and Jurkat which thymine to dThd (range from 80 to 1664 nmol/h/mg protein) have sufficient ability to degradate dThd through DHT DH (42 was also decreased to 2 to 42%, respectively (data not shown). and 32% of the control activity), the elevation in the activity of The difference in the level of dThd phosphorylase activities dThd kinase (three- and two-fold) was significantly lower than between T- and B-cell leukemias, resulting in different sensitiv that in other cell lines (from 7- to 79-fold). In contrast, dTMP ity for growth inhibition by dThd, has been reported (26, 27). synthase activities were increased markedly (379- and 407-fold) However, no difference in the extent of decrease of the activities over other cell lines (from 22- to 372-fold). These results suggest of dThd phosphorylase or DHT DH was observed between T-, that the de novo pathway might contribute chiefly to DNA B-, and Non-T, Non-B cell lines in our studies. dThd phospho synthesis over the salvage pathway in these cells, because of rylase also could convert thymine to dThd reversely in all relatively decreased utilization of dThd for its phosphorylation leukemia-lymphoma cell lines examined. Therefore, different owing to a sufficient enzymic capacity of dThd degradation. sensitivity to dThd might not be due to the decreased level of Therefore, DHT DH might regulate not only dThd catabolism dThd phosphorylase, as indicated in dThd-resistant CCRF- but also the synthetic enzymic capacity of dThd utilization. CEM cells by Zielke (28). Comparison of Kinetic Properties of DHT DH and dThd In the synthetic pathway ofdThd utilization, the activities of Phosphorylase in Lymphocytes and Jurkat Leukemic Cells. Ki dThd kinase and those of dTMP synthase were increased by 2- netic properties of partially purified DHT DH of normal lym to 79-fold (from 0.61 to 23.8 nmol/h/mg protein) and 22- to phocytes were similar to those of Jurkat leukemic cells. The 407-fold (from 6.3 to 117.9 nmol/h/mg protein) of the lym apparent Kmvalues for thymine and NADPH, and KÂ¡valuefor phocyte value, respectively. The activities of dTMP synthase 5-diazouracil, were 5.0, 63, and 1.6 x 10~6 M for lymphocytes were higher than those of dThd kinase in all leukemia-lym and 4.0, 56, and 0.6 x 10~6M for Jurkat cells. Optimal pH was phoma cells. 7.4 in both cells. These data were similar to those in rat liver The marked decrease in the activities of dThd degradative reported by us (5) and others (7). The apparent Km values for enzymes is as important as the rise in those of DNA synthetic dThd phosphorylase were also similar in both lymphocytes and enzymes, because dThd is channeled not only to DNA synthesis Jurkat cells. The Km values for lymphocytes were 0.25, 0.71, but also to catabolism to CO2 in a competitive manner (3, 4). and 2.27 x IO"3 M for dThd, thymine, and deoxyribose 1- Therefore, the decreased enzymic capacities in dThd catabolism phosphate and 0.33, 0.57, and 1.26 x 10~3 M for Jurkat cells, might favor sparing ofdThd and permit its ready utilization to respectively. Kmvalue for thymine, the substrate for both DHT dTMP by action ofdThd kinase and finally to DNA synthesis. DH and dThd phosphorylase, was two orders of magnitude Indeed, inhibition of DHT DH by 5-diazouracil in vivo (29) and lower in DHT DH (5.0 and 4.0 x IO"6 M for lymphocytes and 1092

Jurkat cells) than that of dThd phosphorylase (0.71 and 0.57 to a decrease in concentration of enzyme protein. x 10~3 M). The specific activity of DHT DH was orders of Biological Significance of the Enzymic Imbalance in dThd magnitude lower than that of phosphorylase (Table 1). These Metabolism in Human Leukemia-Lymphoma Cells. Novel as results indicate that DHT DH is the rate-limiting enzyme and pects of the present study on human leukemia-lymphoma cell a better candidate for determining the enzymic capacity for lines include: (a) demonstration of the reciprocal regulation in dThd catabolism in human leukemia-lymphoma cells. the behavior of the enzyme activities in the opposing pathways Evidence for the Decreased Amount of DHT DH Enzyme of dThd metabolism; (b) demonstration that DHT DH is the Protein in Jurkat Leukemic Cells. Hybridomas prepared from rate-limiting enzyme and a better candidate for determining the purified DHT DH were examined by indirect [I25l]protein A enzymic capacity of dThd catabolism; (c) production of poly binding assay (Table 2). In the clones examined, high radioac clonal and monoclonal antibodies against DHT DH; (d) first tivity was observed in clone number 1A4 (1319 cpm) and 2A2 demonstration of the decreased amount of DHT DH in human (2382 cpm). The subclass of these clones was IgG,l,K, for 1A4 leukemic cells. and IgG, 2b, K,for 2A2. With these clones and anti-DHT DH The increased enzymic activities for the salvage pathway in serum, immunotitration studies were carried out on DHT DH lymphoproliferative disorders as demonstrated here and by in normal lymphocytes and Jurkat cells (Table 3). Dihydrothy- others (13) should limit the antitumor effectiveness of antime- mine dehydrogenase from lymphocytes was neutralized com tabolites of the de novo pyrimidine biosynthesis. Moreover, the pletely by 5.6 mg of polyclonal antibody, anti-DHT DH serum marked decrease in dThd catabolic ability, especially indicated or by 2.5 and 2.3 mg of monoclonal antibodies of 1A4 and by the decline in the activity and amount of DHT DH, should 2A2, respectively. Complete neutralization of DHT DH from favor the utilization of dThd for DNA synthesis. Thus, the Jurkat cells was obtained by the addition of 2.1 mg of anti- balance of anabolism versus catabolism may be a primary factor DHT DH serum or 0.02 and 0.06 mg of 1A4 and 2A2 clones. determining dThd utilization and therapeutic effectiveness by As a result, the amount of antibody required for 100% neutral antimetabolites such as 5-fluorouracil. As the importance of ization for Jurkat cell DHT DH by anti-DHT DH serum was DHT DH on the antitumor effectiveness of 5-fluorouracil and 36% of that for lymphocytes, which was in good agreement its derivatives has been demonstrated (7, 31, 33, 34), biochem with the decrease in the activity of the enzyme of crude extracts ical modulation of dThd catabolism with the combination of to 32% (2.20 versus 6.88 nmol/h/mg protein). The amount of inhibition of both the de novo and salvage pathways should monoclonal antibodies, 1A4 and 2A2, for complete neutrali increase the antitumor effectiveness of antipyrimidine chemo zation for leukemic cells was 1 and 3% ofthat for lymphocytes. therapy. A lesser amount of antibodies was required for complete neu tralization by clone 1A4 and 2A2 than that by polyclonal REFERENCES antibody, suggesting high and specific titers in these clones. No inhibitory effect was observed upon addition of the control 1. O'Dwyer, P. J., King, S. A., Hoth, D. F., and Leyland-Jones, B. Role of nonimmune serum. These results indicate strongly that the thymidine in biochemical modulation: a review. Cancer Res., 47:3911-3919, 1987. decreased activity of DHT DH in Jurkat leukemic cells was due 2. Kufe, D. W., Bearsdsley, P., Karp, D., Parker, L., Rosowsky, A., Canellos, G., and Frei, E. High-dose thymidine infusions in patients with leukemia Table 2 Examination ofhybridoma by indirect fnsl]protein binding assay and lymphoma. Blood, 55: 580-589, 1980. 30 pg of purified DHT DH were allowed to absorb to Terasaki I microplates 3. Weber, G. Biochemical strategy of cancer cells and the design of chemother and subsequently 20 ,il of 1:200 diluted nonimmunized or immunized mouse apy: G. H. A. Clowes Memorial Lecture. Cancer Res., 43:3466-3492, 1983. serum or undiluted hybridoma culture supernatants were added to each well. 4. Weber, G., Shiotani, T., Kizaki, H., Williams, J. C, and Gladstone, N. Positive antigen-antibody binding finally was monitored by the addition of 20 Â«1 Biochemical strategy of the genome as expressed in regulation of pyrimidine of rabbit anti-mouse IgG antiserum followed by incubation of the wells with ' "Ã• metabolism. Adv. Enzyme Regul., 16: 3-19, 1978. labeled protein A solution. The wells were excised and directly counted. 5. Shiotani, T., and Weber, G. Purification and properties of dihydrothymine "llpniii'iii A binding dehydrogenase from rat liver. J. Biol. Chem., 256: 219-224, 1981. 6. Shiotani, T., and Weber, G. Reciprocal regulation of thymidine kinase and Clones (cpm) dihydrothymine dehydrogenase activities in normal and neoplastic prolifer ation. Proc. Am. Assoc. Cancer Res., 19: 53, 1978. Mouse myeloma culture medium 57 7. Tuchman, M., Ramnaraine, M. L. R., and O'Dea, R. F. 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Nucleotide interconversions. IV. Activities of lymphocytes and Jurkat leukemic cells deoxycytidylate deaminase and thymidylate synthetase in normal rat liver and hepatomas. Cancer Res., 21:1421-1426, 1961. The cytosol extracts from lymphocytes and Jurkat cells with increasing amounts of antibodies were incubated at 4"( ' for 4 h and then the antimouse IgG 11. Cadman, E., and Heimer, R. Levels of thymidylate synthetase during normal culture growth of LI 210 cells. Cancer Res., 46: 1195-1198, 1986. was added in excess. The incubation was continued for another 4 h. After 12. Ellims, P. H., Can, T. E., and Van Der Weyden, M. B. Thymidine kinase centrifugation. DHT DH activity remaining in the supernatants was assayed as described in "Materials and Methods." isozymes in chronic lymphocytic leukemia. Br. J. Haematol., 49: 479-481, 1981. (mg)Anti-DHTneutralization titer 13. Can, T. E., Hallam, L., and Van Der Weyden, M. B. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. Behavior of Activities of Thymidine Metabolizing Enzymes in Human Leukemia-Lymphoma Cells

Taiichi Shiotani, Yasuko Hashimoto, Terukazu Tanaka, et al.

Cancer Res 1989;49:1090-1094.

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