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Ecto-Adenosine Triphosphatase Deficiency in Cultured Human T and Null Leukemic Lymphocytes

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Ecto-Adenosine Triphosphatase Deficiency in Cultured Human T and Null Leukemic Lymphocytes Ecto-adenosine triphosphatase deficiency in cultured human T and null leukemic lymphocytes. A biochemical basis for thymidine sensitivity. R M Fox, … , E H Tripp, M H Tattersall J Clin Invest. 1981;68(2):544-552. https://doi.org/10.1172/JCI110286. Research Article Cultured leukemic T and null lymphocytes are highly sensitive to growth inhibition by thymidine, as well as the other deoxynucleosides, deoxyguanosine and deoxyadenosine. By contrast, Epstein-Barr virus-transformed B lymphocytes are relatively resistant to deoxynucleosides. Growth inhibition is associated with the development of high deoxyribotriphosphate pools after exposure to the respective deoxynucleotides. We show that malignant T and null lymphocytes are deficient in ecto-ATPase activity. We show this cell surface enzyme to be of broad specificity, capable of degrading both ribotriphosphates and deoxyribotriphosphates. High levels of this ecto-enzyme are found in deoxynucleoside-resistant, Epstein-Barr virus-transformed B lymphocytes. Ecto-ATPase deficiency may represent a mechanism for increased sensitivity to deoxynucleoside growth inhibition. Find the latest version: https://jci.me/110286/pdf Ecto-Adenosine Triphosphatase Deficiency in Cultured Human T and Null Leukemic Lymphocytes A BIOCHEMICAL BASIS FOR THYMIDINE SENSITIVITY RICHARD M. Fox, SYLVIA K. PIDDINGTON, EDITH H. TRipp, and MARTIN H. N. TATTERSALL, Ludwig Institute for Cancer Research, University of Sydney, New South Wales 2006, Australia A B S T R A C T Cultured leukemic T and null lym- tion by TdR at concentrations two orders of mag- phocytes are highly sensitive to growth inhibition by nitude below millimolar levels. The mechanisms of thymidine, as well as the other deoxynucleosides, this increased sensitivity appear to reflect the ability deoxyguanosine and deoxyadenosine. By contrast, of these leukemic cells to accumulate excess dTTP Epstein-Barr virus-transformed B lymphocytes are at low concentrations of exogenous TdR (5-7). relatively resistant to deoxynucleosides. Growth in- These malignant cells are also similarly sensitive hibition is associated with the development of high to low concentrations of deoxyguanosine (GdR) and deoxyribotriphosphate pools after exposure to the deoxyadenosine (AdR). Again, the mechanisms of respective deoxynucleosides. We show that malignant this inhibition appear to relate to accumulation of the T and null lymphocytes are deficient in ecto-ATPase triphosphates of these deoxynucleosides. An analogy activity. We show this cell surface enzyme to be of has been drawn between the phenomena of deoxy- broad specificity, capable of degrading both ribo- nucleoside-sensitive T and null leukemic cells and triphosphates and deoxyribotriphosphates. High levels the immune-deficiency syndromes associated with of this ecto-enzyme are found in deoxynucleoside- adenosine deaminase (ADA) and purine nucleoside resistant, Epstein-Barr virus-transformed B lympho- phosphorylase (PNP) deficiency. These defects in cytes. Ecto-ATPase deficiency may represent a mecha- nucleoside catabolism result in accumulation of high nism for increased sensitivity to deoxynucleoside levels of deoxyribonucleoside triphosphates (dNTP) growth inhibition. dATP in ADA deficiency and dGTP in PNP deficiency. It has been postulated that these excess dNTP act as INTRODUCTION feedback inhibitors of ribonucleotide reductase, caus- The arrest of cell growth by millimolar concentrations ing inhibition of DNA replication in lymphocyte of thymidine (TdR) is well established. The mecha- precursors, resulting in immune defects (5, 7, 8). Thus, nism of this effect is believed to reflect inhibition these clinical immunodeficiency syndromes asso- of DNA replication by an increase in the cellular ciated with inborn errors of purine catabolism and dTTP pool, which allosterically inhibits ribonucleotide the deoxynucleoside sensitivity of leukemic lym- reductase. This results in depression of the dCTP phocytes indicate a specific vulnerability of lympho- pool below a level critical for continued DNA replica- cytes (particularly T cells) at some stage in their tion (1). differentiation to dNTP intoxication. More recently there has been interest in the clinical We now show that malignant T and null lympho- use of TdR as an antitumor agent following dem- cytes are deficient in ecto- (Ca++,, Mg++)adenosine onstration that TdR could inhibit human tumor xeno- triphosphate y-hydrolase (Ecto-AT'Pase). We have grafts in nude mice (2-4). We and others have reported found this ecto-triphosphatase to be a cell-surface en- that cultured leukemic lymphocytes of T and null zyme of broad specificity capable of degrading both cell type are extremely sensitive to growth inhibi- ribo- and deoxyribonucleoside triphosphates. High levels of this ecto-enzyme are found in deoxynu- Received for publication 6 May 1980 and in revised form cleoside-resistant Epstein-Barr virus (EBV)-transformed 26 February 1981. peripheral blood B lymphocytes. Ecto-ATPase de- 544 J. Clin. Invest. (D The American Society for Clinical Investigation, Inc. * 0021-9738/81/08/0544/09 $1.00 Volume 68 August 1981 544-552 ficiency may represent a mechanism unifying the Erythro-9-(3-[2-hydroxy-nonyl]) adenosine (EHNA) was a gift dNTP accumulation in malignant T and null cells, and from Dr. C. Nichol, Burroughs Wellcome Ltd., Re- ADA and PNP deficiency. search Triangle, N. C. GLOSSARY Enzyme assays ADA Adenosine deaminase TdR kinase. This was assayed as described by Taylor et al. AdR Deoxyadenosine (10). Cells were harvested from log-growth cultures by ADP Adenosine diphosphate centrifugation and resuspended in buffer (0.05 M Tris HCI, CdR Deoxycytidine pH 7.5, 1 mM EDTA, and 5 mM /3-mercaptoethanol) at a dATP Deoxyadenosine triphosphate concentration of 1 x 107/ml. The cells were disrupted by dCTP Deoxycytidine triphosphate sonication and then centrifuged (27,000 g for 10 min at 4°C). dGTP Deoxyguanosine triphosphate Aliquots of the supemate (without prior dialysis), containing DNTP Deoxyribonucleoside triphosphates 10-100 ,ug of protein, were added to a reaction mixture dTDP Thymidine diphosphate (final concentration 0.06 M Tris-HCl pH 7.5, 5 mM MgCl2, dTMP Thymidine monophosphate 5 mM ATP, and 12 ,uM [6-3H]TdR, 1 Ci/mmol. The final dTTP Thymidine triphosphate volume of the assay was 100 Al. The reaction mixtures were EBV Epstein-Barr virus incubated for 30 min at 37°C and terminated by boiling for 4 Ecto-ATPase Ecto-(Ca++, Mg++)adenosine triphosphate- min. 50 M1 of reaction mixture was spotted onto Whatman hydrolase DEAE paper squares, washed four times in ammonium EHNA Erythro-9-(3-(2-hydroxy-nonyl))adenosine formate (0.001 M), then four times in methanol, dried, and GdR Deoxyguanosine counted in a liquid scintillation counter. The assay was linear ID5o Inhibitory dose, 50% to 40 min incubation time and with protein. PEI Polyethyleneimine TdR monophosphate kinase. This assay was performed as PNP Purine nucleoside phosphorylase above with the exception that [methyl-3H]dTMP (0.3 Ci/mmol, TdR Thymidine 30 uM) was used as substrate replacing [3H]TdR. The reac- TLC Thin-layer chromatography tion mixture was incubated for 20 min at 37°C and terminated as above. Aliquots of reaction mixture were spotted onto PEI METHODS TLC plates and chromatographed with 0.4 M LiCl. Markers consisted of cold dTMP, dTDP, and dTTP. After chromatog- Cell lines and culture raphy, the marker areas were identified under ultraviolet light, Cultured human leukemic lymphocytes derived from cut out, and counted in a liquid scintillation counter. patients with various types of acute lymphoblastic leukemia, This assay was linear with time and protein, and dTMP was and lymphocytes transformed by EBV were kindly provided saturating. by Dr. H. Lazarus (Sidney Farber Cancer Institute, Boston, Ecto-ATPase. This was measured using a modification of Mass.), J. Minowada (Roswell Park Memorial Institute, the method of De Pierre and Karnovsky (12). Cells, in log Buffalo, N. Y.), I. Jack (Royal Children's Hospital, Melbourne, growth, were harvested by centrifugation, and 0.8 x 104 cells Victoria), and H. Zola (Flinders Medical Centre, Adelaide, were resuspended unwashed in a reaction mixture of 50-Al South America). volume. Incubations were performed in duplicate at 37°C, The normal peripheral blood lymphocyte lines transformed and the reaction mixture consisted of RPMI 1640 culture by EBV were JE-Tg, WIL, JP, and LAZ 007. The lines derived medium containing 1 mM [8-3H]ATP (0.5 ,Ci) or 1 mM from patients with acute lymphoblastic leukemia were (T cell) [methyl-3H]TTP (0.5 MCi). The mixture was incubated for CCRF-CEM, 8402, CCRF-HSB, HPB-MLT, JM, and MOLT- 30 min, and the reaction terminated by heating at 80°C for 4; and (null cell) Reh and KM-3. The origin and character- 4 min, and the cells pelleted by centrifugation. 5 ,l of super- istics of the malignant cells have been previously described nate was spotted with the appropriate cold triphosphate, and summarized by Minowada (9). All cell lines were grown diphosphate, monophosphate, and deoxynucleoside onto PEI in suspension culture in RPMI 1640 media supplemented TLC plates. The compounds were separated by elution with with 10% fetal calf serum. The lines had approximately LiCl2. The ATP separation required 1 M LiCl2, and for dTTP, similar doubling times (24-30 h). Cells were studied in the 0.6 M LiCl2 was used. This assay was linear with cell number log-phase of growth. and up to 40 min. Ecto-5'nucleotidase. This was measured by a modification Isotopes and chemicals of the method described by Wortmann et al. (7). Incubations were performed in duplicate at 37°C in a reaction volume of [Methyl3H]dTTP (45 Ci/mmol), D-[5-3H]CTP (18.4 Ci/ 50 ,ul containing 0.1-0.2 x 106 intact lymphocytes. The reac- mmol), D-[8-3H]ATP (13.8 Ci/mmol), D-[8-3H]GTP (7.4 Ci/ tion mixture consisted of RPMI 1640 culture medium con- mmol), [8-'4C]AMP (59 mCi/mmol), [8-3H]ATP (22 Ci/mmol), taining 0.5 mM [8-14C]AMP (0.05 MCi) or [8_3H]dTMP (0.05 [methyl-3H]ATP (45 Ci/mmol) and [6-3H]TdR (5.0 Ci/mmol) MCi).
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