Uridine Kinase Activities in Normal and Neoplastic Lymphoid Cells'

[CANCER RESEARCH 37, 1028-1034, April 1977] Uridine Kinase Activities in Normal and Neoplastic Lymphoid Cells'

Neil Greenberg,2 Dorothy E. Schumm, Paul E. Hurtubise,3 and Thomas E. Webb4

Departments of Physiological Chemistiy(N. G., D. E. S., T. E. W.Jand PathologyfP. E. HI, Ohio State University, College of Medicine, Columbus, Ohio 43210

SUMMARY These variations may have relevance to the problem of tumor progression, including the development of resistance Both adult (I) and embryonic (II) forms of uridine kinase to chemotherapeutic analogs that require uridine kinase for have been identified in the transplantable EL-4 leukemia of activation. C57BL/6 mice and in the P815Y mastocytoma of DBA/2 In view of reports that 5-azacytidine induces the regres mice. Only Species I is found in primary tumor cells of sion of acute childhood leukemia (11, 13) and that 5-azacyti lymphoid origin (virus-induced feline lymphosarcoma, hu dine is highly toxic to horse lymphocytes that have been man acute and chronic lymphocytic leukemia) and in nor stimulated with PHA5 (30), it was of particular interest to mal calf thymocytes and porcine peripheral blood lympho investigate the nature of uridine kinase in normal and mito cytes; Species I was induced 4-fold upon stimulation of the gen-stimulated lymphocytes, as well as in neoplastic cells of normal blood lymphocytes with phytohemagglutinmn. The lymphoid origin. An earlier report from this laboratory (26) level of uridine kinase activity in the feline lymphosarcoma established that resting rat peripheral blood lymphocytes of thymus-dependent lymphocyte origin and childhood lym have a low level of uridine kinase, which increases 4-fold phocytic leukemia of possible thymus-dependent lympho within 24 hr after stimulation with the T-cell mitogen, PHA, cyte or null-cell origin was similar to the induced level in while treatment with a B-cell mitogen (dextran sulfate) phytohemagglutinin-stimulated normal lymphocytes, i.e., causes no induction of the enzyme. The induction of uridine thymus-dependent lymphocytes. In contrast lymphocytes of kinase in T-cells is inhibited by pyrimidine ribonucleoside a patient with chronic lymphocytic leukemia of thymus analogs. A significant volume of literature has dealt with the independent lymphocyte origin had a level of uridine kinase differentiation of lymphocyte surface markers and their sig activity comparable to that of the unstimulated normal lym nificance with respect to prognosis in ALL (7, 24, 27, 29). In phocytes or thymocytes. The uridine kinase activity in the cases of ALL in which the cells have the potential to form E EL-4 tumor cells was repressed by acute treatment of the rosettes, a surface marker consistent with a T-ceIl origin of mice with 5-azacytidine. lymphoblasts, the prognosis is often poor (29). Thus, Tsuki moto et al. (29) concluded that a different chemotherapeu INTRODUCTION tic protocol should be considered for patients with lympho blasts that form E-rosettes at diagnosis. If there are high Two forms of uridine kinase (ATP:uridine 5'-phospho Ievelsofuridine kinase in E-rosette-forming Iymphoblastsof transferase, EC 27.1 .48) were previously identified in sev patients with ALL, such cells might, at least initially, exhibit eral rapidly growing transplantable tumors, including the sensitivity to pyrim idme ribonucleoside analogs. Novikoff rat hepatoma (20), the murine 5-37 sarcoma (16), This study is a report on the status of uridine kinase in 2 and the Ehrlich ascites carcinoma (19); more recently the 2 transplantable lymphoid tumors and normal lymphoid tis isozymes have been identified in cultured HeLa cells (N. sues. In addition, uridine kinase was studied in several Greenberg , unpublished observations). Several transplant primary tumors, including 2 virally induced feline lympho able hepatomas with moderate growth rates also contain 2 sarcomas and 1 ALL and 1 CLL of human origin. forms of uridine kinase (16). These uridine kinases have been designated I and II in order of their elution from a MATERIALSAND METHODS Sepharose 6B column, representing the adult and embry onic forms of the enzyme, respectively (20). The concentra Transplantable Tumor Cells. Rats and mice (Laboratory tion of these 2 species varies markedly in transplantable Supply Co., Indianapolis, Ind.) were maintained at 22Â°with tumors during treatment with 5-azacytidine (14, 16), with mechanically controlled light cycles. Animals were main transient increases occurring in 1 or both isoenzymes. tamed on Purina chow and water ad libitum. The Novikoff hepatoma (25) was carried as an ascites tumor in the perito I Supported by USPHS Research Grant CA-13718 from the National Can cer Institute and National Institute of General Medical Sciences Training Grant in Clinical Chemistry GMO-1805. 5 The abbreviations used are: PHA, phytohemagglutinin; T-cells, thymus 2 Present address: Department of Biological Chemistry, Hahnemann Med dependent lymphocytes; B-cells, thymus-independent lymphocytes; ALL, ical College and Hospital, Philadelphia, Pa. 19102. acute lymphocytic leukemia, E-rosette, sheep erythrocyte rosettes; CLL, 3 Present address: Department of Pathology, University of Cincinnati chronic lymphocytic leukemia; Buffer A, 200 mM Tris-HCI (pH 7.4); RPMI Medical Center, Cincinnati, Ohio 45229. 1640, Roswell Park Memorial Institute Tissue Culture Medium 1640; EAC, 4 To whom requests for reprints should be addressed. erythrocyte-antibody complement; Buffer B, 200 mM Tris-HCI (pH 7.4)-20 Received July 12, 1976; accepted December 29, 1976. mM mercaptoethanol-20% (v/v) glycerol.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1977 American Association for Cancer Research. Uridine Kinase Activities in Lymphoid Cells neal cavity of 120- to 140-g female Sprague-Dawley rats. 2 to 3 days in culture under similar conditions (26); how Cells washed with 0.9% NaCI solution were resuspended at ever, a significant induction has already occurred after 36 a concentration of 3 x 10@cells/mI, and 2 ml were inocu hr. The contribution, if any, of the 5 to 10% contaminating lated into each animal. The transplant generation time of monocytes to the uridine kinase activity is not known. the tumor was 7 days, and the yield per animal was approxi Calf thymus gland was obtained from freshly killed ani mately 5 x 10@cells. The EL-4 leukemia (8, 23) of C57BL/6 mals at a local slaughterhouse. The tissue was immediately mice and the P815Y mastocytoma of DBA/2 mice were washed with cold 0.9% NaCI solution and then minced in a obtained from Dr. Sam Waksal, Department of Anatomy, meat grinder. Minced tissue was teased through copper The Ohio State University, Columbus, Ohio. Both tumors gauze, and the resulting cellular suspension was washed 3 were similarly carried as ascites cells by the i.p. inoculation times with cold 0.9% NaCI solution and centrifuged at 150 x of 2.5 x 10@cells/mouse. The transplant generation time for g for 5 mm to obtain the packed cell volume for enzyme these tumors was 7 days under these conditions. fractionation. After completion of these experiments, it was In all cases, harvested cells were suspended in ice-cold determined that the use of copper instead of stainless steel 0.9% NaCI solution and then washed by centrifugation at gauze did not affect uridine kinase activity; i.e. , the passage 150 x g for 5 mm. This procedure was repeated until the of partially purified uridine kinase through copper gauze cells were free of contaminating blood cells or medium. caused no significant reduction in activity. After the final wash, the packed cell volume was deter Supravital Identification of Membrane Markers. The E mined, and the cells were resuspended in 2 volumes of ice rosette assay for identification of human T-cells using sheep cold Buffer A for enzyme fractionation. erythocytes was performed according to the method of Primary Tumor and Normal Lymphoid Cells. Solid lym Jondal et al. (12). The EAC assay for complement receptors phosarcomas induced in cats inoculated with FeLV were of B-cells was performed according to the method of provided by Dr. Edward Hoover, Department of Veterinary Bianco et a!. (1). Immunofluorescent identification of the Pathobiology, The Ohio State University College of Veteri surface membrane immunoglobulmn on B-cells was per nary Medicine, Columbus, Ohio. These tumors were identi formed according to the technique of King et al. (17). fied as T-cell lymphosarcomas on the basis of their ability to Uridine Kinase Fractionation. Uridine kinase was purified form E-rosettes with rodent erythrocytes, as well as by their from all tissues and cells by the method of Krystal and Webb reactivity with antithymic antiserum (E. Hoover, personal (20). All procedures were carried out at 4Â°. Washed cells communication). The tumors were weighed and suspended suspended in 2 volumes Buffer A or homogenized tissues in 2 volumes (1 ml/g) Buffer A and minced with scissors (e.g. , solid tumors) in the same buffer were sonically dis prior to homogenization in a glass homogenizer with 3 persed with three 30-sec bursts at maximum power with the strokes of a tight-fitting Teflon pestle. Biosonik IIA (Bronwill Scientific, Rochester, N. Y.). After Uridine kinase was prepared from the lymphocytes of 1 centrifugation of the sonicate at 105,000 x g for 60 mm (No. patient with ALL and a 2nd patient with CLL. For these 40 rotor; Beckman Instruments, Inc ., Fullerton , Calif.), the human leukemic cell preparations, buffy-coat material ex supernatant was first adjusted to 8.0 mg protein per ml (9) tracted from the peripheral blood of patients by leukapher with Buffer A and then brought to 0.5% streptomycin sulfate esis therapy was diluted with 2 volumes RPMI-1640 (Grand (Sigma) by the addition of an appropriate volume of 10% Island Biological Co., Grand Island, N. V.) and then applied streptomycin sulfate in Buffer A. This suspension was to Ficoll-Hypaque (Winthrop Laboratories, New York, N. Y.) stirred slowly for 30 mm prior to removal of precipitates by gradients (3). The semipurified lymphocytes were then centrifugation at 27,000 x g for 15 mm in the refrigerated washed with ice-cold 0.9% NaCI solution prior to membrane 55-34 rotor of the Sorvall RC-2B centrifuge (Dupont Instru marker identification and enzyme preparation. ments, Sorvall Operations, Newtown, Conn.). The superna Normal peripheral blood lymphocytes were prepared tant was then treated twice with ammonium sulfate from heparinized porcine blood (10 IU heparin per ml) ob (Schwarz/Mann, Orangeburg, N. Y.) for 30 mm, and the tamed from the Department of Animal Science, College of fraction sedimenting at 105,000 x g in 30 mm (Beckman No. Agriculture, The Ohio State University, Columbus, Ohio. 40 rotor) with 30 to 50% ammonium sulfate saturation was Because of the requirement for at least 1.0 ml packed cells suspended in Buffer B at a protein concentration of approx for uridine kinase fractionation, these experiments required imately 30 mg/mI. Ten mg of this protein fraction were the processing of approximately 4 liters whole blood to applied to a 58-cm x 0.635-sq cm column of Sepharose 6B obtain sufficient material. After gravitational sedimentation (Pharmacia Fine Chemicals, Uppsala, Sweden) that was of erythrocytes at room temperature for 50 mm, the lympho equilibrated with Buffer B. The proteins were eluted at a cyte-rich plasma was directly applied to Ficoll-Hypaque gra flowrateof2.0mI/hrwitha pressureheadof35 cm Buffer dients. The concentrated lymphocytes were washed 3 times B. Fractions of 0.85 ml were collected for enzyme assay. with RPMI-1640 prior to culturing for 36 hr at 37Â°.The Uridlne Kinase Assay. Uridine kinase was assayed by a semipurified lymphocytes (containing 90 to 95% small lym modification of the method of Krystal and Webb (20). A 0.1- phocytes) were cultured at a concentration of 1 x 106cells/ ml aliquot of each column fraction containing 40 to 80 @g ml in RPMI-1640 containing 10% porcine plasma, 100 j@g protein was incubated for 20 mm at 23Â°ina total volume of streptomycin sulfate (Sigma Chemical Co., St. Louis, Mo.) 0.2 ml that also contained 150 mM Tris-HCI (pH 7.4), 10 mM per ml, and 100 IU penicillin (Sigma) per ml in the presence 2-mercaptoethanol, 10 mM MgCl2, 1.0 mM ATP, and 0.4 mM or absence (controls) of 100 @gPHA(Grand Island Biologi [2-'4C]uridine (0.05 MCi; New England Nuclear, Boston, cal Co.) per ml. As was shown previously, uridine kinase is Mass.). The reaction was terminated by adding 2.0 ml of 1.0 induced maximally in rat peripheral blood lymphocytes after mM ammonium formate, followed by heating at 95Â°for 2

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mm. The reaction mixture was passed through 10@ M EDTA-activated DEAE-cellulose filter discs (DE-81; Reeve Angel, Clifton , N. J .). Reaction tubes were rinsed once with 1O@M ammonium formate, and the rinse was applied to the filter discs. The discs were then washed with three 5-mI washes of 10@ M ammonium formate and partially dried I (1) under vacuum before counting in liquid scintillant (Scinti Ui @1 sol; Isolaboratory, Inc., Akron, Ohio). Enzyme activity is 0 expressedasnmoles uridinenucleotidesynthesizedperhr z per 0.1-mI aliquot of each column fraction. >. Recovery and Standardization. The recoveries of uridine > kinase activity at each stage of the purification procedure I- havebeen discussedearlier(19,20)and willbesummarized here. The 2 isozymes of uridine kinase are routinely mea sured in the 30 to 50% ammonium sulfate fraction of the streptomycin-treated 105,000 x g supernatant of the cell sonicate. The recoveries are the same whether one begins with a sonicate or the cytosol obtained by careful homoge nization of the cells followed by removal of all subcellular FRACTION NUMBER particles, indicating that essentially all of the uridine kinase Chart 1. Sepharose 6B chromatography of uridine kinase preparations activity is in the soluble fraction of the cell (N. Greenberg, from the Novikoff hepatoma (O), the P815Y mastocytoma ( 0), and the EL-4 unpublished observations). Measurements of several prep leukemia with (U) and without (0) subtherapeutic doses of 5-azacytidine. arations indicate that the 30 to 50% ammonium sulfate The profile for the Novikoff hepatoma represents the average of 3 experi ments Â±SE. and is presented for comparative purposes. All other profiles fraction invariably contains approximately 120% of the ac have been duplicated to within 10%. A 10-mg portion of 30 to 50% saturation tivity present in the whole-cell sonicate due to the removal (NH@),SO4fractionwas applied to a Sepharose 6B column (0.635 sq cm x 58 cm) equilibrated with Buffer B (cf. Materials and Methodsâ€•).Fractions (0.85 of interfering enzymes (cf. 20). Essentially all of this activity ml) were collected, and 0.1-mI aliquots were assayed for uridine kinase is recovered after separation of the isozymes by Sepharose activity (nmoles UMP formed per hr per 0.1 ml of each fraction). 6B chromatography; i.e., in the case of the Novikoff hepa toma celts59%isrecoveredinPeak Iand 41% isrecovered in Peak II. Although it is convenient to express the data as investigate the effect of acute treatment with pyrimidine nmoles uridinenucleotidesynthesizedperhr per 0.1-mI nucleoside analogs on the uridine kinase activity in lymph aliquot of each 0.85-mI column fraction, the data can be oid neoplasms in view of the changes seen after 5-azacyti easily recalculated per 10@cells. Thus the columns were dine treatment of the Novikoff hepatoma (14) or the 5-37 routinely loaded with 10 mg enzyme protein obtained by sarcoma (16). For this experiment, C57BL/6 mice carrying ammonium sulfate fractionation of the sonicate from Ca. i the EL-4 leukemia thought to be of T-cell origin (21, 23) ml packed cells, i.e., 1 x 10@Novikoffhepatoma cells, 2.0 x received a subtherapeutic dose of 5-azacytidine (1.0 mg/kg 108 EL-4 leukemia cells, or 2.5 x 10@P815-Y mastocytoma i.p.) on Days 2, 4, and 6 after inoculation with 2.5 x 10@ cells. tumor cells. The cells were removed for enzyme assay on Day 7. As shown in Chart 1, there is a significant decrease in the activity of both isoenzymes after 5-azacytidine treatment RESULTS in this lymphoid tumor, which is in sharp contrast to the 2- fold increase in the adult isoenzyme after the same treat Since 2 isoenzymes of uridine kinase were found in sev ment of the Novikoff hepatoma (14). This decrease in total eral rapidly growing transplantable ascites tumors of rats uridine kinase activity in the EL-4 leukemia after 5-azacyti and mice (16, 19, 20), it is possible that the presence of 2 dine administration, which was also seen after 2 weeks of isoenzymes is a general property of rapidly growing neo treatment, is analogous to the previously published obser plastic tissues. Chart 1 shows the Sepharose 6B elution vation (30) of the depression of uridine kinase activity in rat profile of uridine kinase purified from rapidly growing neo thymus after treatment with this drug. Since these studies plasms. The profile for the Novikoff hepatoma reported have not been extended beyond 2 weeks, the possibility that earlier (20), with standard errors for 3 separate prepara a biphasic response may occur during prolonged treatment tions, is included only for reference. It is apparent that the has not been ruled out. EL-4 leukemia and the P815-Y mastocytoma contain both The high level of uridine kinase in the EL-4 leukemia is forms of uridine kinase activity, which are similar, if not likewise comparable to the induced level of uridine kinase identical, to the 2 forms of enzyme previously identified in observed in PHA-stimulated lymphocytes (26). However, the the Novikoff hepatoma (20). However, all of these tumors EL-4 leukemia, as well as the other neoplastic cells that have different relative concentrations of the 2 isoenzymes. have been evaluated in these studies thus far, is a trans Such differences may be significant in terms of the growth plantable tumor that has been carried for many generations. rates and relative pyrimidine ribonucleoside analog sensi For clinical correlation it was desirable to assess the status tivity of these different cell lines. of uridine kinase activity in primary tumors as well as nor . With the identification of 2 isoenzymes in the EL-4 leuke mal tissues. For the answer to this question, initial studies mia and the P815-Y mastocytoma, it was of interest to were designed to determine the nature of the induced un

1030 CANCER RESEARCH VOL. 37

dine kinase activity in PHA-stimulated lymphocytes (26). normal lymphocytes (Chart 2), both in form (adult [email protected] Krystal and Webb (20) had previously shown that induct@n zyme) and amount. of unidine kinase in regenerating rat liver results in an in Table 1 lists the membrane marker characteristics of the 2 crease in Species I, Species II being essentially absent. human leukemic cell populations obtained from the pa Shown in Chart 2 are the Sepharose 6B elution profiles from tients before they underwent extensive [email protected] control and PHA-stimulated porcine peripheral blood lym tient A was a 60-year-old male with a WBC count of 19,500/ phocytes. The unidine kinase activity in both control and cu mm (92% lymphocytes). The bone marrow showed PHA-stimulated lymphocytes elutes as a peak close to the marked lymphocytic infiltration. Formation of EAC rosettes position at which the adult isoenzyme is eluted. (Note refer and the presence of monoclonal lgM(K) on the membrane ence arrows at Fractions 23 and 29, respectively.) The 3.5- surface of the lymphocytes isolated from this patient (Table fold increase in the height of the enzyme peak after PHA 1), in addition to the above clinical data, were consistent stimulation is comparable to the 4-fold increase in total with the diagnosis of a B-cell CLL. Patient B was a 12-year unidine kinase activity reported for rat peripheral blood lym old male who pre@entedwith a WBC count of 336,000 (88% phocytes after 24 hr in culture with PHA (5). Thus the induc lymphoblasts). Bone marrow examination showed 90% lym tion of unidine kinase in normal lymphocytes appears to be phoblasts. These data, in addition to the formation of E limited to an increase in the adult isoenzyme, as was pre rosettes and the lack of reactivity of these lymphocytes with dicted from the nature of the induction of unidine kinase in surface membrane immunoglobulin (Table 1) were consiet regenerating liver (20). With reference to the induced en ent with the diagnosis of T-cell ALL. The reason f@r the zyme, it is not clear whether the apparent heterogeneity formation of EAC rosettes by this cell population is not within Peak I, as indicated by the broadness of the peak, is due to the presence of more than 1 isozyme eluting in this region or to the high activity of proteolytic enzymes present in granulocytes that contaminate these preparations of pe nipheral blood lymphocytes. Next, unidine kinase was purified from a primary T-ceIl I2.0 lymphosarcoma that was virally induced in 2 different cats. Shown in Chart 3 is the Sepharose 6B elution profile of unidine kinase purified from 1 of these lymphosarcomas. A I Cl) 2nd tumor gave a similar profile. Shown, in addition, is the Ui -J profile of unidine kinase purified from thymocytes isolated 0 8.0 from normal calf thymus gland. The high activity in the T z cell lymphosarcoma is similar to the level seen in PHA-stim I- ulated peripheral blood lymphocytes (Chart 2). The results > indicate that the adult form of unidine kinase is predominant both in the primary tumor cells and in normal thymocytes, 4 the embryonic form being very low or absent. The unidine 4.0 kinase in normal thymocytes is thus quite similar to that in

4 0@ ,, 8.0 I6 20 24 28 32 I U) FRACTION NUMBER Ui -J Chart 3. Sepharose 6B chromatography of uridine kinase preparations 0 from thymocytes (â€¢)andfrom 1 feline thymic lymphosarcoma (0). Reference z arrows at Fractions 24 and 30 indicate the elution volumes for Peaks I and II, respectively, from the Novikoff hepatoma. Other conditions were as for Chart a- 4.0 > l-@ C.) Table 1 4 MembranemarkerCLLPatientWBC/cucharacteristicSof patients with ALL and

markers markers 0 16 20 24 28 32 pho % sheep FRACTION NUMBER cytes % SmIg % EAC erythro (+)A mmLym (+) (+)T-celIcytes Chart 2. Sepharose 6B chromatography of uridine kinase preparations (%)B-cell from peripheral blood lymphocytes cultured for 36 hr in the presence (0) or (CLL) 95.0 absence (O) of PHA. Cells were cultured at a concentration of 1 x 10/ml in B (ALL)19,500336,00092 8895.2a 1.0 68.04.8 66.0 RPMI-1640 supplemented with 10% porcine plasma, 100 IU penicillin per ml, @ and 100 @Lgstreptomycin sulfate per ml. Reference arrows at Fractions 23 a With fluorescein isothiocyanate-labeled monospecific anti and 29 indicate the elution volumes for Peaks I and II, respectively, from the sara, these cells exhibited a monoclonal lgM(K)on the membrane Novikoff hepatoma. Other conditions were as for Chart 1. surface.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1977 American Association for Cancer Research. N. Greenberg et a!. understood. When this donor of the ALL cells received 1 strategy, particularly if these changes are predictable and course of prednisone and vincnistine, the WBC count can be correlated with clinical responses. dropped from 336,000 to 33,000/cu mm, also consistent Because of the potential importance of unidine kinase in with the diagnosis of ALL. No comparable patients were the treatment ofleukemias with 5-azacytidine (11, 13), initial available for study during the period of this investigation studies with the EL-4 leukemia examined the acute effect of because most, particularly those with childhood ALL, un 5-azacytidine on unidine kinase in this tumor of lymphoid dergo vigorous chemotherapy immediately after diagnosis. origin. The objective was to determine how the unidine Chart 4 shows the Sepharose 6B elution profiles of un kinases in the EL-4 leukemia changed after a short treat dine kinase purified from lymphocytes obtained from the ment with subtherapeutic doses of 5-azacytidine, a protocol patients with ALL. In agreement with previous studies (26), that eventually leads to development of resistance. It was lymphocytes from the patient with a putative B-cell CLL previously reported that the unidine kinase activity in rat showed a low level of unidine kinase that is similar to the liver increased after 5-azacytidine chemotherapy (6) and activity present both in thymocytes (Chart 4) and in control that the adult isoenzyme was the only species present be peripheral blood lymphocytes (Chart 3). fore and after treatment (16). In addition, subtherapeutic doses of 5-azacytidine cause a transient 3-fold increase in the adult isoenzyme of unidine kinase in hepatomas during DISCUSSION the early stages of development of resistance (14). On the other hand, it was also reported that 5-azacytidine causes a The presence of 2 forms of unidine kinase in fetal rat liven decrease in unidine kinase activity in thymus gland (30) and (20), the Novikoff rat hepatoma (20), the munine Ehnlich inhibits the induction of unidine kinase activity in PHA ascites carcinoma (19, 20), and the 5-37 sarcoma (16) has stimulated lymphocytes (26). The decrease in unidine kinase been demonstrated by separation of 2 peaks of activity on activity in the EL-4 leukemia after 5-azacytidine treatment, Sepharose 6B columns under well-defined conditions (16). as shown in Chart 1, supports the conclusion that lymphoid The presence of 2 isoenzymes has also been confirmed by tissue treated with 5-azacytidine will have either a decrease their resolution on sucrose density gradients (19), by poly in unidine kinase activity or a loss of the capacity for induc acrylamide gel electrophoresis, and by DEAE-cellulose tion of the enzyme. This contrasts with findings in hepatic chromatography (15). The present investigation demon tissue, in which unidine kinase activity actually increases strates the existence of 2 isoenzymes in 2 additional trans after 5-azacytidmnetreatment (16). plantable tumors of lymphoid origin, i.e., the P815-Y masto Although the mechanism for the loss of unidine kinase in cytoma of DBA/2 mice and EL-4 leukemia of C57BL/6 mice. the EL-4 leukemia during short 5-azacytidmnetreatment has Because of the important role of unidine kinase in pynimi not been established, cells with a high level of unidine dine nibonucleoside analog chemotherapy, transient kinase may be selectively killed in a heterogeneous tumor changes in the activities of these isoenzymes during the population; such a clonal selection mechanism may also course of chemotherapy (14, 16) may have regulatory sig account for the loss of the enzyme in thymus gland, since it nificance. The Km'Sfor Species I and II in the Novikoff hepa is known that thymocytes constitute a heterogeneous popu toma with unidine as substrate are 100 and 54 @M,respec lation (22). This sensitivity of unidine kinase activity to the tively (14). Consequently, knowledge of these changes in analog offers circumstantial support for the theory that the enzyme activities may be important in planning therapeutic EL-4 leukemia is a T-cell leukemia (21), the assumptions being that leukemic cells with high unidine kinase activity I2.0 have this parameter in common with PHA-stimulated lym phocytes and that this plant mitogen is reasonably specific for T-cells. The lymphocytes from 1 patient with ALL, which showed mixed membrane markers for both B- and T-cells, had a I @;;8.0 level and form (adult isoenzyme) of unidine kinase that was similar to that seen in FeLV-induced T-cell lymphosarcomas 0 (Chart 3) and in PHA-stimulated peripheral blood lympho z cytes, i.e., T-cells (Chart 2). In view of these results, further >. I- correlative studies are warranted to establish whether un > dine kinase activity is a useful biochemical marker for T I-@ 4.0 cells, which can be used as an adjunct to other tests cur nently in use. Because normal lymphocytes show an increase in unidine kinase activity after stimulation with PHA (26) and PHA stimulated lymphocytes are very sensitive to 5-azacytidmne 0 (31), PHA-stimulated lymphocytes might be a useful model l6 20 24 28 32 system for predicting the 5-azacytidine sensitivity of neo FRACTION NUMBER plastically transformed lymphocytes in lymphoblastic leuke Chart 4. Sepharose 6B chromatography of uridine kinase preparations mias. The similarities of the unidine kinase activities in the from an ALL (0) and a CLL (C). Reference arrows at Fractions 24 and 30 indicate elution volumes for Peaks I and II, respectively, from the Novikoff limited sampling of FeLV-induced cat T-cell lymphosar hepatoma. Other conditions were as for Chart 1. coma, the lymphocytes in ALL, and PHA-stimulated peniph

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1977 American Association for Cancer Research. Uridine Kinase Activities in Lymphoid Ce!!s eral blood lymphocytes suggest that all of these cells would REFERENCES be sensitive to pynimidine nibonucleoside analogs if unidine 1. Bianco, C., Patrick, R., and Nussenzweig, V. J. A Population of Lympho kinase activity is indeed the most important determinant in cytes bearing a Membrane Receptor for Antigen-antibody-complement nucleoside analog sensitivity. Therefore, the conrelat@onof Complexes. I. Separation and Characterization. J. Exptl. Med., 132: 702- 705, 1970. the unidine kinase activity in the lymphocytes of patients 2. Bottomly, H. K., Perkins, W. D., and Schwarz, M. R. 3H-Uridine Incorpo with ALL, particularly in the case of T-cell leukemias that are ration by Small Lymphocytes of Tolerant Rats: Relationship to T and B relatively resistant to present therapeutic protocols (4, 24, Lymphocytes. J. Immunol., 115: 648â€”652,1975. 3. Boyum, A. A One-Stage Procedure for Isolation of Granulocytes and 27, 29), with theinsensitivityin vitro to 5-azacytidine, may be Lymphocytes from Human Blood. Scand. J. Clin. Lab. Invest., 21 (Suppl. useful in subsequently predicting the efficacy of 5-azacyti 97):51-76, 1968. dine treatment. 4. Brouet, J. C., Preud'Homme, J-L., and Seligurann, M. Use of B Lympho cytes in Rats and T Membrane Markers in the Classification of Human The question of whether unidine incorporation into acid Leukemias with Special Reference to Acute Lymphoblastic Leukemia. insoluble nucleic acids is a selective marker for 1-cells has Blood Cells, 1: 81-90, 1975. 5. Byrd, W. J., Van Boehmer, H., and Rouse, B. J. The Role of the Thymus received much attention in the literature (2, 10, 18). The in Maturational Development of Phytohemagglutinin and Pokeweed Mi study of unidine kinase activity in relatively pure populations togen Responsiveness. Cellular Immunol., 6: 12-24, 1973. of B- and 1-cells should shed light on this question. The 6. Cihak, A., Vesely, J. , and Harrap, K. R. Enhancement of Rat Liver Uridine Kinase Activity by Various Metabolic Inhibitors. Biochem. Pharmacol., data for a relatively pure population of B-cells (CLL, Chart 4) 23: 1087-1093, 1974. and a similarly pure population of 1-cells (thymocytes, 7. Coleman,M.S.,Greenwood,M. F.,Hutton,J.J., Bollum, F.J.,Lampkin, Chart 3) suggest that resting populations of both B- and 1- B., and Holland, P. Serial Observations on Terminal Deoxynucleotidyl Transferase Activity and Lymphoblast Surface Markers in Acute Lympho cells possess the same low level of unidine kinase activity blastic Leukemia. Cancer Res., 36: 120-127, 1976. and that both populations would be expected to incorporate 8. Gorer, P. A. Studies in Antibody Response of Mice to Tumor Inoculation. Brit. J. Cancer, 4: 372-379, 1950. [3H]unidine to the same extent, unless there is a variation in 9. Gornall, A. G., Bardawill, C. J., and David, M. M. Determination of Serum unidine transport. However, blast transformation of 1-cells Proteins by Means of the Biuret Reaction. J. Biol. Chem., 177: 751-766, causes a marked 4-fold increase in unidine kinase activity, 1948. 10. Gutman, G. A. , and Weissman, I. L. Evidence That Uridine Kinase Incor such that [3H]unidine incorporation by 1-cells would be fa poration Is Not a Selective Marker for Mouse Lymphocyte Subclasses. J. voned. Alternatively, high [3H]unidine incorporation may be Immunol., 115: 739â€”740,1975. a useful marker for T-cell neoplasms, in view of the high 11. Hrodek, 0., and Vesely, J. 5-Azacytidine in Childhood Leukemia. Neo plasma, 18: 403-503, 1971. unidine kinase activity observed in a T-cell and the low 12. Jondal, M., HoIm, G., and Wigzel, H. Surface Markers on Human T and B activity observed in a B-cell neoplasm in the present investi Lymphocytes. I. A Large Population of Immune Lymphocytes Forming Nonimmune Rosettes with SRBC. J. Exptl. Med., 136: 207-215, 1972. gation. These proposals must be regarded as tentative, 13. Karon, M., Seiger, L., Leimbrock, S., Finkelstein, J. Z., Nesbitt, M. E., since even the reliability of the membrane markers used in and Swaney, J. J. 5-Azacytidine: A New Active Agent for the Treatment of the identification of the 1- and B-cells, although gaining the Acute Leukemia. Blood, 42: 359-367, 1973. 14. Keefer, R. C., McNamara, D. J., Schumm, D. E., Billmire, 0. F., and confidence of many investigators, cannot be considered to Webb, T. E. Early Temporal Changes in the Uridine Kinase Isozyme be fully established, and the specificities of the mitogens Profiles of the Novikoff Hepatoma in Response to 5-Azacytidine Treat (PHA and dextran sulfate) are somewhat less certain. In fact ment. Biochem. Pharmacol., 24: 1287-1290, 1975. 15. Keefer, R. C., McNamara, 0. J., and Webb, T. E. The Separation of Two it might be expected that some â€œnullâ€•cell lymphocytic Forms of Uridine Kinase from the Novikoff Hepatoma. Cancer Biochem. leukemias may contain membrane and/on biochemical Biophys.,1: 107-110, 1975. 16. Keefer, R. C., Morris, H. P., and Webb, T. E. 5-Azacytidine-modified markers characteristic of both B- and T-cells. Recent evi Patterns of Uridine Kinase Activities in Normal and Neoplastic Tissues. dence (c.f. Ref. 28) suggests that human leukemia cells Cancer Res., 34: 2260-2265, 1974. bearing B-cell as well as 1-cell characteristic surface 17. King, G. W., Hurtubise, P. E., Sagone, A. L., LoBuglio, A. F., and Metz, E. N. Leukemic Reticuloendotheliosis. A Study of the Origin of the markers resemble immature thymocytes (i.e. , intermediate Malignant Cell. Am. J. Med., 59: 411-416, 1975. cell forms) of 10 to 15 weeks gestation, which have both 18. Klobusicka, M., Babusikova, 0., and Konikova, E. Characterization of types of markers. These intermediate forms may be derived Thymus and Bone Marrow Derived by Means of [3H]-Uridine Incorpora tion. Neoplasma, 22: 631-638, 1975. from cells with B-cell markers, which constitute more than 19. Krystal, G., and Scholefield, P. G. Partial Purification and Properties of 70% of the cells of the fetal human thymus in the 10th to Uridine Kinase from Ehrlich Ascites Tumor Cells. Can. J. Biochem., 51: 379-389, 1971. 20th gestation week. Further, unidine kinase activity mea 20. Krystal, G., and Webb, T. E. Multiple Forms of Uridine Kinase in Normal sured in cellular extracts may not truly reflect the sensitivity and Neoplastic Rat Liver. Biochem. J., 124: 943-947, 1971. of the cells to pynimidine nucleoside analogs due to prefer 21. Lamm, M. E., Boyse, E. A., Old, L. J., Lisowsk-Bernstein, B., and Stockert, E. Modulation of TL (Thymus-Leukemia) Antigens by Fab ential utilization of the de novo synthetic pathway for pynim Fragments of TL Antibody. J. Immunol., 101: 99-103, 1968. idine nucleotide biosynthesis, to differences in the nate of 22. McCaffrey, R., Harrison, T. A., Parkman, R., and Baltimore, D. Terminal nucleic acid biosynthesis, on to differences in the rate of Deoxynucleotidyl Transferase Activity in Human Leukemia Cells and in Normal Human Thymocytes. New EngI. J. Med., 292: 775-780, 1975. cellular uptake of the precursor. As will be shown else 23. Mohit, B., and Fan, K. Hybrid Cell Line from a Cloned Immunoglobulin where,6 comparisons of unidine kinase activity in intact cells Producing Mouse Myeloma and a Nonproducing Mouse Lymphoma. Science, 171: 75â€”77,1971. are complicated by differences in the rate of uptake of the 24. Murphy, S. B., Borella, L., Sen. L., and Mauer, A. Lack of Correlation of nucleoside or nucleoside analog into the cell.The useful Lymphoblast Size with Presence of T-CelI Markers or with Outcome in ness of unidine kinase activity as an adjunct to differential Childhood Acute Lymphoblastic Leukemia. Brit. J. Haematol. 31: 95- 101, 1975. diagnosis and design of chemothenapeutic protocols will 25. Novikoff, A. B. A Transplantable Rat Liver Tumor Induced by 4-Dimethyl require further clinical correlative studies with the animal aminoazobenzene. Cancer Res., 17: 1010-1027, 1957. and, when feasible, the human model systems. 26. Schumm, D. E., and Webb, T. E. Induction of Uridine Kinase in Rat Peripheral Blood Lymphocytes: Dependence on Mitogen Specificity and Effect of Pyrimidine Analogs. Cellular Immunol., 15: 479-483, 1975. 27. Sen, L., and Borella, L. Clinical Importance of Lymphoblasts with T Markers in Childhood Acute Leukemia. New EngI. J. Med., 292: 828-832, 8 N. Greenberg, manuscript in preparation. 1975.

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28. Stein, H., Petersen, W., Gaekicke, G., Lennert, K., and Landbeck, G. 30. Vesely, J., Cihak, A., and Vesela, H. Characterization of Depressed Lymphoblastic Lymphoma of Convoluted or Acid Phosphatase Type-A Uridine Kinase in Rat Thymus following 5-Azacytidine Administration. Tumor of Precursor Cells. Intern. J. Cancer, 17: 292-295, 1976. Collection Czech. Chem. Commun., 39: 899-907, 1974. 29. Tsuklmoto, I., Wong, K. Y;, and Lampkln, B. C. Surface Markers and 31. Zain, B. S., Adams, R. L. P., and Imrie, R. C. Effect of 5-Azacytidine on Prognostic Factors In Acute Lymphoblastic Leukemia. New EngI. J. PHA-stimulated Horse Lymphocytes and Cultured Mouse L-929 Cells. Med.,294:245-248,1976. CancerRes.,33:40-46,1973.

1034 CANCER RESEARCH VOL. 37

Neil Greenberg, Dorothy E. Schumm, Paul E. Hurtubise, et al.

Cancer Res 1977;37:1028-1034.

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