[CANCER RESEARCH 43, 1432-1435, March 1983]0008-5472/83/0043-0000302.00

Cytidine Triphosphate Synthetase Activity in LymphoproliferativeDisorders1

Peter H. Ellims,1 2 T. Eng Gan, and Gabriele MedleyDepartment of Medicine, Monash University, Alfred Hospital, Prahran, 3181, Victoria ¡P.H. £., T. E. G.I, and Division of Anatomical Pathology, Prince Henry's

Hospital, Melbourne. 3004. Victoria [G. M.], Australia

ABSTRACT

Cytidine triphosphate synthetase (CTP synthetase) activitywas measured in extracts from normal and malignant lymphoidcells. A range of enzyme activities was found in the majority ofthe lymphoproliferative disorders examined, with acute lym-

phocytic leukemia, nodular poorly differentiated lymphocyticlymphoma, and diffuse histiocytic (large cell) lymphoma (DHL)types exhibiting the widest ranges. The highest levels occurredin acute lymphocytic leukemia, nodular poorly differentiatedlymphocytic lymphoma transforming to DHL, and DHL. Inchronic lymphocytic leukemia and diffuse well-differentiated

lymphocytic lymphoma, a narrow range was encountered, withindividual values falling within that of the controls. The highestCTP synthetase levels in Hodgkin's disease were found in theclinically aggressive lymphocyte-depleted Hodgkin's disease.

Statistically significant differences were found between thedistributions of CTP synthetase activities in acute lymphocyticleukemia and the chronic leukemias (p < 0.01) and betweenfavorable histological types of non-Hodgkin's lymphoma (dif

fuse well-differentiated, nodular poorly differentiated, and dif

fuse poorly differentiated lymphocytic lymphoma) and the unfavorable DHL category (p < 0.05). It is suggested that CTPsynthetase activity is a biochemical marker of the clinicalaggressiveness of malignant lymphoma. There was not a closecorrelation (r2 = 0.52) between CTP synthetase and thymidine

kinase activities, indicating that other biological factors in addition to cell proliferative rate influence the intracellular CTPsynthetase level. Furthermore, in view of the heterogeneity ofCTP synthetase activity in leukemia and lymphoma, it is likelythat the preexisting enzyme level will be an important determinant of the efficacy of the investigational antileukemic agent 3-

deazauridine, which is thought to act by inhibiting CTP synthetase.

INTRODUCTION

CTP synthetase (UTP:L-glutamine ligase, EC 6.3.4.2) cata

lyzes the formation of CTP from the substrate DTP. The precisebiochemical role of mammalian CTP synthetase activity is uncertain, but the enzyme is thought to play an important part inthe determination of cellular CTP and dCTP pools (19, 25).Recent data suggest that, in addition to this physiological role,CTP synthetase is uniquely important in the pyrimidine metabolism of mammalian tumors (25). It has been established thatthere is a specific increase in the CTP synthetase activity andcontent of rat hepatomas over that found in normal liver (23;25). Furthermore, the degree of increase in CTP synthetase

has been correlated directly with the growth rate of various rathepatomas (23, 25), indicating that the enzyme is a marker ofthe proliferative rate and biological aggressiveness of thesetumors. Preliminary data show that there is an increase in theCTP synthetase activity of other rodent tumors and in humanrenal cell carcinoma, but the tumor specificity of this phenomenon is unclear (25). In addition, CTP synthetase activity isthought to be capable of modulating the cytotoxic effect ofsome pyrimidine antimetabolites. The intracellular CTP levelscan influence the effect of 5-azacytidine by its inhibitory effecton the rate of the uridine-cytidine kinase reaction and bycompetition with the active metabolite 5-azacytidine triphos

phate (12). Similarly, the intracellular dCTP pool opposes theaction of 1-ß-D-arabinofuranosylcytosine triphosphate, the active metabolite of ara-C3 (11), and 5-azadeoxycytidine triphos

phate, the active metabolite of 5-azadeoxycytidine (18). Re

cently, a mutant type of CTP synthetase has been associatedwith expanded intracellular dCTP and CTP pools and, as aconsequence, resistance to ara-C and thymidine (19). Finally,

inhibition of CTP synthetase by the metabolite deazauridinetriphosphate is believed to be the primary mode of action ofthe investigational antileukemic agent DAU (17).

In view of these data, a study was undertaken of the profileof CTP synthetase activity in lymphoproliferative disorders.

MATERIALS AND METHODS

Materials. [5,6-3H]UTP (43 Ci/mmol) and [6-3H]thymidine (5 Ci/

mmol) were purchased from the Radiochemical Centre, Amersham,England. Nucleotides and Dowex 50W-X8 (200 to 400 mesh) were

obtained from Sigma Chemical Co., St. Louis, Mo. Polygram CEL 300PEI/UV254 thin-layer chromatography plates were supplied by Brink-

mann, Inc., New York, N. Y. PCS scintillation counting fluid wasobtained from Amersham, Amersham, England. All other chemicalsused were of analytical grade and were obtained from commercialsources.

Patients Studied. Peripheral blood lymphocytes were obtained froma control group, which contained 10 normal volunteers and 9 patientswith nonneoplastic disorders. Seven lymph nodes obtained at surgicalbiopsy and showing normal histology or minimal reactive changesformed a second control group.

Patients with lymphoproliferative disorders were all previously untreated. They included 11 patients with B-CLL,4 all of whom exhibited

an absolute peripheral blood lymphocytosis (range 15,800 to 69,0007

' The investigation was supported in part by grants from the National Health

and Medical Research Council of Australia.2 Applied Health Fellow of the National Health and Medical Research Council

of Australia. To whom requests for reprints should be addressed.Received April 6, 1982; accepted December 3, 1982.

3 The abbreviations used are: ara-C, 1-/8-D-arabinofuranosylcytosine; DAU, 3-deazauridine; B-CLL, bone marrow-derived chronic lymphocytic leukemia; CLL,chronic lymphocytic leukemia; T-CLL, thymus-derived chronic lymphocytic leukemia; HCL, hairy cell leukemia; ALL, acute lymphocytic leukemia; Thy-ALL, T-cell acute lymphocytic leukemia; C-ALL, common-ALL antigen ALL; null-ALL,null-cell ALL; NHL, non-Hodgkin's lymphoma; DWDLL, diffuse well-differentiated

lymphocytic lymphoma; NPDLL, nodular poorly differentiated lymphocytic lymphoma; DPDLL, diffuse poorly differentiated lymphocytic lymphoma; DHL, diffusehistiocytic (large cell) lymphoma; HD, Hodgkin's disease.

4 Lymphocyte immunological typing studies were kindly performed by mem

bers of the Clinical Immunology Unit, Peter MacCallum Clinic, Melbourne, Australia.

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CIP Synthetase of Lymphoproliferative Disorders

/il) with the majority of cells exhibiting faint immunofluorescence formonoclonal IgM or IgM-lgD and mouse erythrocyte receptor positivity.Two additional patients with CLL were identified as T-CLL by the ability

of the majority of peripheral blood lymphocytes (range 30,960 to75,800//il) to spontaneously bind sheep erythrocytes. Anti-T mono

clonal antibody studies showed that the majority of peripheral bloodlymphocytes expressed the phenotype of helper T-lymphocytes (OKT4)in one patient with T-CLL, whereas, in the second patient, the peripheralblood lymphocytes exhibited the phenotypic features of suppressor T-

lymphocytes (OKT8). One patient had HCL with more than 80% of theperipheral blood lymphocytes (32,350/jul) being abnormal and exhibiting tartrate-resistant acid phosphatase activity. Seven patients hadALL. Lymphoblasts were the predominant peripheral blood mononu-

clear cell in all 7 patients. Peripheral blood lymphoblasts from 2patients (range 8,430 to 22,100//¿I) were identified as Thy-ALL by

their ability to form spontaneous rosettes with sheep erythrocytes.Peripheral lymphoblasts from 4 patients (range 4,380 to 12,100 fil) didnot show T- or B-lymphocyte surface markers but did express the C-ALL antigen and were classified as C-ALL. One patient was consideredto have null-ALL on the basis that the peripheral blood lymphoblasts'6,760/jul) did not express T- or B-cell markers or the C-ALL antigen.

Lymph nodes were obtained at surgical biopsy, and the histologicalfindings were independently reviewed by G. Medley. In 41 patients,the histological findings were those of NHL, which were categorizedaccording to the modified Rappaport classification (2). Individual categories and numbers were: DWDLL, 5; NPDLL, 14; DPDLL, 6; andDHL, 16. Lymph node biopsies from 8 patients exhibited histologicalfeatures of HD with subtypes (15) and numerical distribution beingnodular sclerosing HD, 4; mixed-cellularity HD, 1 ; and lymphocyte-

depleted HD, 3.The Wilcoxon rank sum test was used to compare the CTP synthe-

tase activity among the various types of lymphoproliferative disorders.Correlation between CTP synthetase and thymidine kinase activitieswas assessed by construction of a scatter diagram and linear regression analysis of the data.

Sample Preparation. Normal and leukemic peripheral blood lymphocytes were obtained from 10 ml of heparinized venous blood bycentrifugation on Ficoll-Hypaque (4). The lymphocytes were suspendedat 107 cells/ml of 50 mM Tris-HCI buffer, pH 8.5, containing 5 mM

dithiothreitol and 10 mM L-glutamine (Buffer A) and lyzed by rapidfreeze-thawing twice in liquid nitrogen. The cell extract was thencentrifuged at 10,000 x g at 4°for 15 min, and the supernatant was

assayed for CTP synthetase activity. Samples used to measure thymidine kinase activity were prepared as described previously (9).

Lymph nodes obtained at biopsy were divided into sections forhistological examination, special investigations including immunologi-

cai tests, and CTP synthetase determination. After being rinsed twicewith cold 0.9% NaCI solution, samples (0.1 to 0.5 g) were suspendedin 2 volumes (w/v) of Buffer A and subjected to Dounce homogeniza-tion. The homogenate was centrifuged at 10,000 x g at 4°for 15 min,

and the supernatant was assayed for CTP synthetase. Samples usedto measure thymidine kinase activity were prepared as describedpreviously (9).

CTP Synthetase Assay. The enzyme assay contained, in a totalvolume of 0.1 ml, 50 mM Tris-HCI (pH 8.5), 0.25 mM UTP (5/¿Ci//umol),5 mM ATP, 0.5 mM GTP, 10 mM MgCI2, 10 mM L-glutamine, 10 mM

dithiothreitol, and the enzyme preparation (50 /il). A pH of 8.5 waschosen as the human enzyme has a broad pH optimum between pH8.4 and 9.4.5 Reactions were carried out at 37° for 45 min. The

reaction was terminated by adding 10 pi of 60% perchloric acid to thereaction on ice. Precipitated protein was removed by centrifugation at10,000 x g at 4°for 10 min. The CTP formed was determined by acidhydrolysis of [3H]CTP and [3H]UTP to the nucleoside monophosphates(21). [3H]CMP was then separated by chromatography on Dowex 50-H+ ion-exchange resin (21). The identity of the reaction product and

the validity of this procedure was confirmed by polyethyleneiminecellulose thin-layer chromatography (26). Under the experimental conditions used, 82 to 87% of [3H]CTP was recovered as [3H]CMP. The

reaction was linear with respect to protein concentration and for up to75 min. Enzyme activity was expressed as nmol of CTP formed per hrper mg of protein.

Thymidine Kinase Assay. Thymidine kinase was assayed as previously described using [6-3H]thymidine as the radiolabeled substrato (8,

9).Protein was determined by the method of Bradford (5) using bovine

serum albumin as the standard.

RESULTS

CTP Synthetase Activity in Control Blood Lymphocytesand Lymph Nodes. Peripheral blood lymphocytes from normalindividuals and patients with nonneoplastic medical disordershad a mean CTP synthetase activity of 0.91 ± 0.20 (S.D.)nmol/hr/mg of protein (range, 0.78 to 1.30). Tissue extractsfrom normal lymph nodes and those exhibiting minimal reactivechanges had a mean enzyme activity of 1.18 ±0.19 nmol/hr/mg of protein (range, 0.95 to 1.40).

CTP Synthetase Activity in ALL and Chronic Leukemias.The distribution of CTP synthetase activity in the ALL andchronic leukemias is shown in Chart 1. There was a statisticallysignificant difference (p < 0.01) in the distribution of enzymeactivities between ALL and the chronic leukemias.

CTP synthetase activity in ALL was 3.33 nmol/hr/mg ofprotein, 3.70-fold the mean control level. There was consider

able variation in individual levels, with CTP synthetase activitiesranging from 1.30 to 6.70 nmol/hr/mg of protein. The highestvalues were found in 3 examples of C-ALL, but elevated CTP

synthetase activity was also found in lymphoblast extracts from2 examples of Thy-ALL. One of the patients with C-ALL andboth with Thy-ALL had disease resistant to current antileukemicagents. The 2 other C-ALL patients with high CTP synthetase

levels achieved complete remission following therapy with avincristine-prednisolone-Adriamycin regimen of treatment (28),as did the one patient with null-ALL and one patient with C-ALL

who showed CTP synthetase activity within or close to thecontrol range.

The mean CTP synthetase activity in B-CLL of 0.85 nmol/

hr/mg of protein closely approximated that of the control

5 P. H. Ellims, unpublished observations.

Chart 1. CTP synthetase activity in leukemia. A: •,Thy-ALL; T, C-ALL; V,null-ALL. B: ».B-CLL; A, T-CLL; O, HCL. Each point represents the mean of 3determinations. , range in control lymphocyte extracts; , mean enzymeactivity in each category (2 = B-CLL only).

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P. H. Ellims et al.

group, and the range of individual enzyme levels fell within ornear that of the controls. Similarly, the CTP synthetase activityof a single example of HCL (1.10 nmol/hr/mg of protein) fellwithin the control range. Two examples of T-CLL had CTP

synthetase activities greater than the upper control range, withthe individual values being 1.60 and 1.75 nmol/hr/mg ofprotein, respectively.

CTP Synthetase Activity in NHL and HD. The distribution ofCTP synthetase activities in NHL grouped according to themodified Rappaport scheme (2) and in the subtypes of HD (15)is shown in Chart 2.

Statistically significant differences were not observed in thedistribution of CTP synthetase activities in the various categories of NHL. However, when enzyme values for tumorsexhibiting histological progression to DHL were excluded fromthe analysis, a statistically significant difference (p < 0.05)was found between the distribution of enzyme activities in eachof the favorable categories (DWDLL, NPDLL, and DPDLL) andthat in the unfavorable DHL category. In DWDLL, the meanenzyme activity (1.06 nmol/hr/mg of protein) and range ofactivities fell within that of the controls. There was considerableheterogeneity in the CTP synthetase activities of the other NHLcategories examined, with individual enzyme levels rangingfrom values below the control range to almost 4-fold the mean

control activity. The NPDLL category exhibited the widest rangeof activities, with values ranging from 0.42 to 4.10 nmol/hr/mg of protein. The mean enzyme activity of 1.34 nmol/hr/mgof protein was above the control range. Two of the NPDLLpatients with high CTP synthetase activity showed histologicalevidence of transformation to the unfavorable DHL category.The mean enzyme activity and range for DPDLL were 1.67(range, 0.80 to 3.30) nmol/hr/mg of protein. In this category,the highest CTP synthetase activity occurred in lymph nodeextracts from a patient who had previously been documentedto have NPDLL and for whom there was morphological evidence of further progression to DHL. The DHL category exhibited the highest mean enzyme activity (2.25 nmol/hr/mg ofprotein), together with a range of individual levels, with values

Table 1

CIP synthetase activity in controls and lymphoproliferative disorders

CTP synthetase activity(nmol/hr/mg protein)

DWDLL NPDLL DPDLL DHL

Chart 2. CTP synthetase activity in NHL and HD. A: NHL categorized according to the modified Rappaport classification (2). B: A, nodular sclerosing HD; •mixed-cellularity HD; O, lymphocyte-depleted HD. Each point represents themean of 3 separate determinations. , range in control lymph node extracts;

, mean enzyme activity in each category. /. histological evidence ofprogression to DHL.

Peripheral blood lymphocytesControls (19)a

B-CLL(11)T-CLL (2)HCLd)ALL (7)0.91

b (0.78-1.30)°

0.85 (0.50-1.60)1.68 (1.60-1.75)1.103.33 (1.30-6.70)

LymphoidtissueControls(7)NHLDWDLL

(5)NPDLL(14)DPDLL(6)DHL

(16)HD(8)1.181.061.341.672.251.26(0.95-1.40)(0.90-1.25)(0.42-4.10)(0.80-3.30)(0.50-3.80)(0.45-3.00)

Numbers in parentheses, number of samples.6 Mean CTP synthetase activity.c Numbers in parentheses, range of enzyme activities.

occurring below the control range to greater than 3-fold the

mean control activity. The mean enzyme activity in HD was1.26 (range, 0.45 to 3.00) nmol/hr/mg of protein. The rangeof CTP synthetase activities in HD correlated with the histological subtypes of HD. The individual enzyme activities in nodularsclerosing HD and mixed-cellularity HD fell within or below the

control range, while the CTP synthetase activity [2.25 (range,1.75 to 3.00) nmol/hr/mg of protein] was above the controlrange in all 3 examples of the clinically aggressive lymphocyte-

depleted HD.The mean values and range of CTP synthetase activities in

the controls and the various types of lymphoproliferative disorders are summarized in Table 1.

Relationship between CTP Synthetase and Thymidine Ki-nase Activities. Thymidine kinase activity was determined in28 patients, including 4 with B-CLL, 2 with T-CLL, 4 with ALL,

4 with HD, 2 with DWDLL, 4 with NPDLL, 3 with DPDLL, and 5with DHL. When the individual values for CTP synthetase andthymidine kinase activity were compared by a scatter diagramand subjected to linear regression analysis, a close correlationwas not found (r2 = 0.52).

DISCUSSION

The determination of enzymes in the lymphoproliferativedisorders has been largely carried out using cytochemicalidentification as an ancillary aid in the establishment of the cellorigin of the neoplastic clone (16). More recently, it has beenestablished that the activity of a number of purine and pyrimi-

dine enzymes differs substantially among some types of lymphoproliferative disorders (3, 9). Both in vitro and in vivo studieshave demonstrated that these biochemical differences are exploitable (8, 10, 20, 27). In this study, the activity of thepyrimidine enzyme CTP synthetase was measured in lymphoproliferative disorders, and the enzyme level correlated withhistological features and in some types with the ¡mmunologicalcharacteristics and thymidine kinase activity.

Although the mean CTP synthetase activity in ALL was almost4-fold the mean control level, the profile of enzyme activity in

this category showed considerable heterogeneity. The highestCTP synthetase activity occurred in C-ALL lymphoblasts, but

the variation in enzyme levels did not correlate with ¡mmunological typing. However, in 3 of 5 examples, a high enzyme

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CTP Synthetase of Lymphoproliferative Disordeis

level was associated with clinical resistance to current antileu-kemic agents. In this situation, expansion of the dCTP pool asa result of increased CTP synthetase activity could contributeto clinical ara-C resistance (1). CTP synthetase in B-CLL

showed a narrow range of activities within that of the controlperipheral blood lymphocytes, which is consistent with thewell-differentiated appearance and low proliferative rate of B-CLL cells (9). In contrast, T-CLL cells exhibited an increase inCTP synthetase activity, suggesting that these leukemic cellshave a higher proliferative rate than do B-CLL cells.

The stepwise increase in mean CTP synthetase activity ofthe Rappaport NHL types DWDLL, NPDLL, DPDLL, and DHLcorrelates with the morphological and biological transformationthat is expressed in this classification of NHL (2, 16). Clinicalrelevance for these findings is suggested by the finding of asignificant difference between the enzyme activities in favorable histological categories and in the unfavorable DHL type.The narrow range of enzyme activities found in DWDLL wasclosely similar to the findings in its leukemic counterpart B-CLL, but marked heterogeneity was an outstanding feature ofthe profile of CTP synthetase activity in the other types of NHLexamined. The widest range was found in NPDLL, a type longconsidered to be relatively homogeneous but one which recentdata indicate is heterogeneous, particularly with respect to cellproliferation (6, 7, 9). Among the high enzyme levels in favorable categories were 2 examples of NPDLL and one instanceof DPDLL which showed histological progression to the aggressive DHL type. The wide variation found in the CTP synthetase activities of DHL is consistent with the known morphological, immunological, kinetic, biochemical, and clinical heterogeneity of this category (9, 13, 22, 24). The profile of CTPsynthetase activity in NHL is very similar to that of thymidinekinase, with the stepwise increase in mean enzyme activity ofthe Rappaport categories broadly correlating with the knownbiological aggressiveness of each category (9). This finding isamplified by the data for HD, in which the highest CTP synthetase activities occurred in the most malignant subtype, lymphocyte-depleted HD.

These data suggest that, like rat hepatoma CTP synthetase(25) and human lymphoid thymidine kinase (8, 9), CTP synthetase levels in human malignant lymphomas reflect the proliferative rate of individual tumors and hence clinical aggressiveness. However, the lack of a strong correlation between tumorthymidine kinase and CTP synthetase activities indicates thatother as yet undetermined biological factors also influence theintracellular CTP synthetase level.

Inhibition of CTP synthetase activity is thought to be theprimary mode of action of the investigational antimetaboliteDAD (17). Recent evidence which shows CTP synthetase to bean important contributor to intracellular CTP and dCTP pools(19, 25) and the experimental synergism between DAU and theantileukemic agents 5-azadeoxycytidine (18) and ara-C (14)

suggest that there is therapeutic value to be gained in inhibitingCTP synthetase. The considerable heterogeneity documentedin the CTP synthetase activity of the various types of lympho-proliferative disorders indicates that the preexisting CTP synthetase level will be a significant determinant of DAU cytotox-icity.

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