Cytidine Triphosphate Synthetase Activity in Lymphoproliferative Disorders1

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[CANCER RESEARCH 43, 1432-1435, March 1983] 0008-5472/83/0043-0000302.00 Cytidine Triphosphate Synthetase Activity in Lymphoproliferative Disorders1

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

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

ABSTRACT has been correlated directly with the growth rate of various rat hepatomas (23, 25), indicating that the enzyme is a marker of Cytidine triphosphate synthetase (CTP synthetase) activity the proliferative rate and biological aggressiveness of these was measured in extracts from normal and malignant lymphoid tumors. Preliminary data show that there is an increase in the cells. A range of enzyme activities was found in the majority of CTP synthetase activity of other rodent tumors and in human the lymphoproliferative disorders examined, with acute lym- renal cell carcinoma, but the tumor specificity of this phenom phocytic leukemia, nodular poorly differentiated lymphocytic enon is unclear (25). In addition, CTP synthetase activity is lymphoma, and diffuse histiocytic (large cell) lymphoma (DHL) thought to be capable of modulating the cytotoxic effect of types exhibiting the widest ranges. The highest levels occurred some pyrimidine antimetabolites. The intracellular CTP levels in acute lymphocytic leukemia, nodular poorly differentiated can influence the effect of 5-azacytidine by its inhibitory effect lymphocytic lymphoma transforming to DHL, and DHL. In on the rate of the uridine-cytidine kinase reaction and by chronic lymphocytic leukemia and diffuse well-differentiated competition with the active metabolite 5-azacytidine triphos lymphocytic lymphoma, a narrow range was encountered, with phate (12). Similarly, the intracellular dCTP pool opposes the individual values falling within that of the controls. The highest CTP synthetase levels in Hodgkin's disease were found in the action of 1-ÃŸ-D-arabinofuranosylcytosine triphosphate, the ac tive metabolite of ara-C3 (11), and 5-azadeoxycytidine triphos clinically aggressive lymphocyte-depleted Hodgkin's disease. phate, the active metabolite of 5-azadeoxycytidine (18). Re Statistically significant differences were found between the cently, a mutant type of CTP synthetase has been associated distributions of CTP synthetase activities in acute lymphocytic with expanded intracellular dCTP and CTP pools and, as a leukemia and the chronic leukemias (p < 0.01) and between favorable histological types of non-Hodgkin's lymphoma (dif consequence, resistance to ara-C and thymidine (19). Finally, inhibition of CTP synthetase by the metabolite deazauridine fuse well-differentiated, nodular poorly differentiated, and dif triphosphate is believed to be the primary mode of action of fuse poorly differentiated lymphocytic lymphoma) and the un the investigational antileukemic agent DAU (17). favorable DHL category (p < 0.05). It is suggested that CTP In view of these data, a study was undertaken of the profile synthetase activity is a biochemical marker of the clinical of CTP synthetase activity in lymphoproliferative disorders. aggressiveness of malignant lymphoma. There was not a close correlation (r2 = 0.52) between CTP synthetase and thymidine kinase activities, indicating that other biological factors in ad MATERIALS AND METHODS dition to cell proliferative rate influence the intracellular CTP Materials. [5,6-3H]UTP (43 Ci/mmol) and [6-3H]thymidine (5 Ci/ synthetase level. Furthermore, in view of the heterogeneity of mmol) were purchased from the Radiochemical Centre, Amersham, CTP synthetase activity in leukemia and lymphoma, it is likely England. Nucleotides and Dowex 50W-X8 (200 to 400 mesh) were that the preexisting enzyme level will be an important determi obtained from Sigma Chemical Co., St. Louis, Mo. Polygram CEL 300 nant of the efficacy of the investigational antileukemic agent 3- PEI/UV254 thin-layer chromatography plates were supplied by Brink- deazauridine, which is thought to act by inhibiting CTP synthe mann, Inc., New York, N. Y. PCS scintillation counting fluid was tase. obtained from Amersham, Amersham, England. All other chemicals used were of analytical grade and were obtained from commercial sources. INTRODUCTION Patients Studied. Peripheral blood lymphocytes were obtained from CTP synthetase (UTP:L-glutamine ligase, EC 6.3.4.2) cata a control group, which contained 10 normal volunteers and 9 patients with nonneoplastic disorders. Seven lymph nodes obtained at surgical lyzes the formation of CTP from the substrate DTP. The precise biopsy and showing normal histology or minimal reactive changes biochemical role of mammalian CTP synthetase activity is un formed a second control group. certain, but the enzyme is thought to play an important part in Patients with lymphoproliferative disorders were all previously un the determination of cellular CTP and dCTP pools (19, 25). treated. They included 11 patients with B-CLL,4 all of whom exhibited Recent data suggest that, in addition to this physiological role, an absolute peripheral blood lymphocytosis (range 15,800 to 69,0007 CTP synthetase is uniquely important in the pyrimidine metab olism of mammalian tumors (25). It has been established that 3 The abbreviations used are: ara-C, 1-/8-D-arabinofuranosylcytosine; DAU, 3- deazauridine; B-CLL, bone marrow-derived chronic lymphocytic leukemia; CLL, there is a specific increase in the CTP synthetase activity and chronic lymphocytic leukemia; T-CLL, thymus-derived chronic lymphocytic leu content of rat hepatomas over that found in normal liver (23; kemia; HCL, hairy cell leukemia; ALL, acute lymphocytic leukemia; Thy-ALL, T- cell acute lymphocytic leukemia; C-ALL, common-ALL antigen ALL; null-ALL, 25). Furthermore, the degree of increase in CTP synthetase null-cell ALL; NHL, non-Hodgkin's lymphoma; DWDLL, diffuse well-differentiated lymphocytic lymphoma; NPDLL, nodular poorly differentiated lymphocytic lym ' The investigation was supported in part by grants from the National Health phoma; DPDLL, diffuse poorly differentiated lymphocytic lymphoma; DHL, diffuse histiocytic (large cell) lymphoma; HD, Hodgkin's disease. and Medical Research Council of Australia. 2 Applied Health Fellow of the National Health and Medical Research Council 4 Lymphocyte immunological typing studies were kindly performed by mem of Australia. To whom requests for reprints should be addressed. bers of the Clinical Immunology Unit, Peter MacCallum Clinic, Melbourne, Aus Received April 6, 1982; accepted December 3, 1982. tralia.

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/il) with the majority of cells exhibiting faint immunofluorescence for the validity of this procedure was confirmed by polyethyleneimine monoclonal IgM or IgM-lgD and mouse erythrocyte receptor positivity. cellulose thin-layer chromatography (26). Under the experimental con Two additional patients with CLL were identified as T-CLL by the ability ditions used, 82 to 87% of [3H]CTP was recovered as [3H]CMP. The of the majority of peripheral blood lymphocytes (range 30,960 to reaction was linear with respect to protein concentration and for up to 75,800//il) to spontaneously bind sheep erythrocytes. Anti-T mono 75 min. Enzyme activity was expressed as nmol of CTP formed per hr clonal antibody studies showed that the majority of peripheral blood per mg of protein. lymphocytes expressed the phenotype of helper T-lymphocytes (OKT4) Thymidine Kinase Assay. Thymidine kinase was assayed as previ in one patient with T-CLL, whereas, in the second patient, the peripheral ously described using [6-3H]thymidine as the radiolabeled substrato (8, blood lymphocytes exhibited the phenotypic features of suppressor T- 9). lymphocytes (OKT8). One patient had HCL with more than 80% of the Protein was determined by the method of Bradford (5) using bovine peripheral blood lymphocytes (32,350/jul) being abnormal and exhibit serum albumin as the standard. ing tartrate-resistant acid phosphatase activity. Seven patients had ALL. Lymphoblasts were the predominant peripheral blood mononu- RESULTS clear cell in all 7 patients. Peripheral blood lymphoblasts from 2 patients (range 8,430 to 22,100//Â¿I) were identified as Thy-ALL by CTP Synthetase Activity in Control Blood Lymphocytes their ability to form spontaneous rosettes with sheep erythrocytes. and Lymph Nodes. Peripheral blood lymphocytes from normal Peripheral lymphoblasts from 4 patients (range 4,380 to 12,100 fil) did not show T- or B-lymphocyte surface markers but did express the C- individuals and patients with nonneoplastic medical disorders ALL antigen and were classified as C-ALL. One patient was considered had a mean CTP synthetase activity of 0.91 Â± 0.20 (S.D.) to have null-ALL on the basis that the peripheral blood lymphoblasts nmol/hr/mg of protein (range, 0.78 to 1.30). Tissue extracts '6,760/jul) did not express T- or B-cell markers or the C-ALL antigen. from normal lymph nodes and those exhibiting minimal reactive Lymph nodes were obtained at surgical biopsy, and the histological changes had a mean enzyme activity of 1.18 Â±0.19 nmol/hr/ findings were independently reviewed by G. Medley. In 41 patients, mg of protein (range, 0.95 to 1.40). the histological findings were those of NHL, which were categorized CTP Synthetase Activity in ALL and Chronic Leukemias. according to the modified Rappaport classification (2). Individual cat The distribution of CTP synthetase activity in the ALL and egories and numbers were: DWDLL, 5; NPDLL, 14; DPDLL, 6; and chronic leukemias is shown in Chart 1. There was a statistically DHL, 16. Lymph node biopsies from 8 patients exhibited histological significant difference (p < 0.01) in the distribution of enzyme features of HD with subtypes (15) and numerical distribution being nodular sclerosing HD, 4; mixed-cellularity HD, 1; and lymphocyte- activities between ALL and the chronic leukemias. depleted HD, 3. CTP synthetase activity in ALL was 3.33 nmol/hr/mg of The Wilcoxon rank sum test was used to compare the CTP synthe- protein, 3.70-fold the mean control level. There was consider tase activity among the various types of lymphoproliferative disorders. able variation in individual levels, with CTP synthetase activities Correlation between CTP synthetase and thymidine kinase activities ranging from 1.30 to 6.70 nmol/hr/mg of protein. The highest was assessed by construction of a scatter diagram and linear regres values were found in 3 examples of C-ALL, but elevated CTP sion analysis of the data. synthetase activity was also found in lymphoblast extracts from Sample Preparation. Normal and leukemic peripheral blood lym 2 examples of Thy-ALL. One of the patients with C-ALL and phocytes were obtained from 10 ml of heparinized venous blood by both with Thy-ALL had disease resistant to current antileukemic centrifugation on Ficoll-Hypaque (4). The lymphocytes were suspended at 107 cells/ml of 50 mM Tris-HCI buffer, pH 8.5, containing 5 mM agents. The 2 other C-ALL patients with high CTP synthetase dithiothreitol and 10 mM L-glutamine (Buffer A) and lyzed by rapid levels achieved complete remission following therapy with a freeze-thawing twice in liquid nitrogen. The cell extract was then vincristine-prednisolone-Adriamycin regimen of treatment (28), centrifuged at 10,000 x g at 4Â°for 15 min, and the supernatant was as did the one patient with null-ALL and one patient with C-ALL assayed for CTP synthetase activity. Samples used to measure thymi who showed CTP synthetase activity within or close to the dine kinase activity were prepared as described previously (9). control range. Lymph nodes obtained at biopsy were divided into sections for The mean CTP synthetase activity in B-CLL of 0.85 nmol/ histological examination, special investigations including immunologi- hr/mg of protein closely approximated that of the control cai tests, and CTP synthetase determination. After being rinsed twice with cold 0.9% NaCI solution, samples (0.1 to 0.5 g) were suspended in 2 volumes (w/v) of Buffer A and subjected to Dounce homogeniza- tion. The homogenate was centrifuged at 10,000 x g at 4Â°for 15 min, and the supernatant was assayed for CTP synthetase. Samples used to measure thymidine kinase activity were prepared as described previously (9). CTP Synthetase Assay. The enzyme assay contained, in a total volume of 0.1 ml, 50 mM Tris-HCI (pH 8.5), 0.25 mM UTP (5/Â¿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 was chosen as the human enzyme has a broad pH optimum between pH 8.4 and 9.4.5 Reactions were carried out at 37Â° for 45 min. The reaction was terminated by adding 10 pi of 60% perchloric acid to the reaction on ice. Precipitated protein was removed by centrifugation at 10,000 x g at 4Â°for 10 min. The CTP formed was determined by acid hydrolysis 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 Chart 1. CTP synthetase activity in leukemia. A: â€¢,Thy-ALL; T, C-ALL; V, null-ALL. B: Â».B-CLL; A, T-CLL; O, HCL. Each point represents the mean of 3 determinations. , range in control lymphocyte extracts; , mean enzyme 5 P. H. Ellims, unpublished observations. activity in each category (2 = B-CLL only).

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1983 American Association for Cancer Research. P. H. Ellims et al. group, and the range of individual enzyme levels fell within or Table 1 near that of the controls. Similarly, the CTP synthetase activity CIP synthetase activity in controls and lymphoproliferative disorders of a single example of HCL (1.10 nmol/hr/mg of protein) fell CTP synthetase activity within the control range. Two examples of T-CLL had CTP (nmol/hr/mg protein) Peripheral blood lymphocytes synthetase activities greater than the upper control range, with Controls (19)a b (0.78-1.30)Â° the individual values being 1.60 and 1.75 nmol/hr/mg of B-CLL(11) 0.85 (0.50-1.60) protein, respectively. T-CLL (2) 1.68 (1.60-1.75) HCLd) 1.10 CTP Synthetase Activity in NHL and HD. The distribution of ALL (7)0.91 3.33 (1.30-6.70) CTP synthetase activities in NHL grouped according to the modified Rappaport scheme (2) and in the subtypes of HD (15) LymphoidtissueControls (7)NHLDWDLL is shown in Chart 2. Statistically significant differences were not observed in the (5)NPDLL distribution of CTP synthetase activities in the various cate (14)DPDLL (6)DHL gories of NHL. However, when enzyme values for tumors (16)HD exhibiting histological progression to DHL were excluded from (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) the analysis, a statistically significant difference (p < 0.05) Numbers in parentheses, number of samples. 6 Mean CTP synthetase activity. was found between the distribution of enzyme activities in each c Numbers in parentheses, range of enzyme activities. of the favorable categories (DWDLL, NPDLL, and DPDLL) and that in the unfavorable DHL category. In DWDLL, the mean occurring below the control range to greater than 3-fold the enzyme activity (1.06 nmol/hr/mg of protein) and range of mean control activity. The mean enzyme activity in HD was activities fell within that of the controls. There was considerable 1.26 (range, 0.45 to 3.00) nmol/hr/mg of protein. The range heterogeneity in the CTP synthetase activities of the other NHL of CTP synthetase activities in HD correlated with the histolog categories examined, with individual enzyme levels ranging from values below the control range to almost 4-fold the mean ical subtypes of HD. The individual enzyme activities in nodular sclerosing HD and mixed-cellularity HD fell within or below the control activity. The NPDLL category exhibited the widest range control range, while the CTP synthetase activity [2.25 (range, of activities, with values ranging from 0.42 to 4.10 nmol/hr/ 1.75 to 3.00) nmol/hr/mg of protein] was above the control mg of protein. The mean enzyme activity of 1.34 nmol/hr/mg range in all 3 examples of the clinically aggressive lymphocyte- of protein was above the control range. Two of the NPDLL depleted HD. patients with high CTP synthetase activity showed histological The mean values and range of CTP synthetase activities in evidence of transformation to the unfavorable DHL category. the controls and the various types of lymphoproliferative dis The mean enzyme activity and range for DPDLL were 1.67 orders are summarized in Table 1. (range, 0.80 to 3.30) nmol/hr/mg of protein. In this category, Relationship between CTP Synthetase and Thymidine Ki- the highest CTP synthetase activity occurred in lymph node nase Activities. Thymidine kinase activity was determined in extracts from a patient who had previously been documented 28 patients, including 4 with B-CLL, 2 with T-CLL, 4 with ALL, to have NPDLL and for whom there was morphological evi 4 with HD, 2 with DWDLL, 4 with NPDLL, 3 with DPDLL, and 5 dence of further progression to DHL. The DHL category ex with DHL. When the individual values for CTP synthetase and hibited the highest mean enzyme activity (2.25 nmol/hr/mg of thymidine kinase activity were compared by a scatter diagram protein), together with a range of individual levels, with values and subjected to linear regression analysis, a close correlation was not found (r2 = 0.52).

DISCUSSION

The determination of enzymes in the lymphoproliferative disorders has been largely carried out using cytochemical identification as an ancillary aid in the establishment of the cell origin of the neoplastic clone (16). More recently, it has been established that the activity of a number of purine and pyrimidine enzymes differs substantially among some types of lym phoproliferative disorders (3, 9). Both in vitro and in vivo studies have demonstrated that these biochemical differences are ex ploitable (8, 10, 20, 27). In this study, the activity of the pyrimidine enzyme CTP synthetase was measured in lympho proliferative disorders, and the enzyme level correlated with histological features and in some types with the Â¡mmunological characteristics and thymidine kinase activity. DWDLL NPDLL DPDLL DHL Although the mean CTP synthetase activity in ALL was almost 4-fold the mean control level, the profile of enzyme activity in Chart 2. CTP synthetase activity in NHL and HD. A: NHL categorized accord ing to the modified Rappaport classification (2). B: A, nodular sclerosing HD; â€¢ this category showed considerable heterogeneity. The highest mixed-cellularity HD; O, lymphocyte-depleted HD. Each point represents the CTP synthetase activity occurred in C-ALL lymphoblasts, but mean of 3 separate determinations. , range in control lymph node extracts; , mean enzyme activity in each category. /. histological evidence of the variation in enzyme levels did not correlate with Â¡mmuno progression to DHL. logical typing. However, in 3 of 5 examples, a high enzyme

1434 CANCER RESEARCH VOL. 43

level was associated with clinical resistance to current antileu- Credie, K. In vivo cellular kinetic and pharmacologie studies of 1-/3-D- arabinofuranosylcytosine and 3-deazauridine chemotherapy for relapsing kemic agents. In this situation, expansion of the dCTP pool as acute leukemia. Cancer Res., 41: 1227-1235, 1981. a result of increased CTP synthetase activity could contribute 2. Berard, C. W., and Dorfman, R. F. Histopathology of malignant lymphoma. to clinical ara-C resistance (1). CTP synthetase in B-CLL Clin. Haematol., 3. 39-76, 1974. 3. Blatt, J., Reaman, G., and Poplack, D. G. Biochemical markers in lymphoid showed a narrow range of activities within that of the control malignancy. N. Engl. J. Med., 303: 918-922, 1980. peripheral blood lymphocytes, which is consistent with the 4. Boyum, A. Separation of leucocytes from blood and bone marrow. Scan. J. well-differentiated appearance and low proliferative rate of B- Clin. Lab. Invest., 21 (Suppl. 97): 77-89, 1968. 5. Bradford, M. M. A rapid and sensitive method for the quantitation of micro- CLL cells (9). In contrast, T-CLL cells exhibited an increase in gram quantities of protein utilizing the principle of protein-dye binding. Anal. CTP synthetase activity, suggesting that these leukemic cells Biochem., 72:248-254, 1976. 6. Come, S. E., Jaffe, E. S., Andersen, J. C., Mann. R. B., Johnson. B. L.. De have a higher proliferative rate than do B-CLL cells. Vita, V. T., Jr., and Young, R. C. Non-Hodgkin's lymphomas in leukemic The stepwise increase in mean CTP synthetase activity of phase: clinicopathologic correlations. Am. J. Med., 69: 667-674, 1980. 7. Costa, A., Bonadonna, G., Vicca, E., Vacagussa, P., and Silvestrini, R. the Rappaport NHL types DWDLL, NPDLL, DPDLL, and DHL Labeling index as a prognostic marker in non-Hodgkin's lymphoma. J. Nati. correlates with the morphological and biological transformation Cancer. Inst., 66: 1-5. 1981. that is expressed in this classification of NHL (2, 16). Clinical 8. Ellims, P. H., Gan, T. E., Medley, G., and Van Der Weyden, M. B. Prognostic relevance of thymidine kinase isozymes in adult non-Hodgkin's lymphoma. relevance for these findings is suggested by the finding of a Blood, 58: 926-930, 1981. significant difference between the enzyme activities in favora 9. Ellims, P. H., Van Der Weyden, M. B., and Medley, G. Thymidine kinase ble histological categories and in the unfavorable DHL type. isoenzymes in human malignant lymphoma. Cancer Res., 41: 691-695. The narrow range of enzyme activities found in DWDLL was 1981. 10. Fox, R. M., Piddington, S. K., Tripp, E. H., Dudman, N. P.. and Tattersall, M. closely similar to the findings in its leukemic counterpart B- H. N. Thymidine sensitivity of cultured leukemic lymphocytes. Lancet, 2: CLL, but marked heterogeneity was an outstanding feature of 391-393, 1979. 11. Furth, J. J., and Cohen, S. S. Inhibition of mammalian DMA polymerase by the profile of CTP synthetase activity in the other types of NHL the 5'-triphosphate of 1-/8-r>arabinofuranosylcytosine and the 5'-triphos- examined. The widest range was found in NPDLL, a type long phate of 9-/8-D-arabinofuranosyladenine. Cancer Res., 28: 2061-2067, considered to be relatively homogeneous but one which recent 1968. 12. Grant, S., Rauscher, F., III. Jakubowski, A., and Cadman, E. Effect of N- data indicate is heterogeneous, particularly with respect to cell (phosphonacetyl)-L-aspartate on 5-azacytidine metabolism in P388 and proliferation (6, 7, 9). Among the high enzyme levels in favor L1210 cells. Cancer Res., 47:410-418, 1981. 13. Hansen, H., Koziner, B., and Clarkson, B. Marker and kinetic studies in the able categories were 2 examples of NPDLL and one instance non-Hodgkin's lymphomas. Am. J. Med., 71: 107-123, 1981. of DPDLL which showed histological progression to the ag 14. Lauzon, G. J., Yang, S-E., and Paterson, A. R. Cell cycle arrest as a basis gressive DHL type. The wide variation found in the CTP syn of enhancement of 1-/8-D-arabinofuranosylcytosine anabolism in human lym- phoblastoid RPMI 6410 cells cultured with 3-deazauridine. Biochem. Phar- thetase activities of DHL is consistent with the known morpho macol., 30: 1889-1894, 1981. logical, immunological, kinetic, biochemical, and clinical het 15. Lukes, R. J., and Butler, J. J. The pathology and nomenclature of Hodgkin's erogeneity of this category (9, 13, 22, 24). The profile of CTP disease. Cancer Res., 26: 1063-1081, 1966. 16. Mann, R. B., Jaffe, E. S., and Berard, C. W. Malignant lymphomas. A synthetase activity in NHL is very similar to that of thymidine conceptual understanding of morphologic diversity. Am. J. Pathol., 94:105- kinase, with the stepwise increase in mean enzyme activity of 174, 1979. the Rappaport categories broadly correlating with the known 17. McPartland, R. P., Wang, M. C., Bloch, A., and Weinfeld. H. Cytidine 5'- triphosphate synthetase as a target for inhibition by the antitumor agent 3- biological aggressiveness of each category (9). This finding is deazauridine. Cancer Res., 34: 3107-3111, 1974. amplified by the data for HD, in which the highest CTP synthe 18. Momparler, R. L., Vesely, J., Momparler, L. F., and Rivard, G. E. Synergistic action of 5-aza-2'-deoxycytidine and 3-deazauridine on L1210 leukemic tase activities occurred in the most malignant subtype, lympho cells and EMT6 tumor cells. Cancer Res.. 39: 3822-3827, 1979. cyte-depleted HD. 19. Robert de Saint Vincent, B.. and Buttin, G. Studies on 1-/j-oarabinofura- These data suggest that, like rat hepatoma CTP synthetase nosylcytosine-resistant mutants of Chinese hamster fibroblasts. IV. Altered regulation of CTP synthetase generates arabinosylcytosine and thymidine (25) and human lymphoid thymidine kinase (8, 9), CTP synthe resistance. Biochim. Biophys. Acta, 670. 352-359, 1980. tase levels in human malignant lymphomas reflect the prolifer 20. Russell, N. H., Prentice, H. G., Lee, N., Piga, A., Ganeshaguru, K., Smyth, ative rate of individual tumors and hence clinical aggressive J. F., and Hoffbrand, A. V. Studies on the biochemical sequelae of therapy in Thy-acute lymphoblastic leukaemia with the adenosine deaminase inhib ness. However, the lack of a strong correlation between tumor itor 2'deoxycoformycin. Br. J. Haematol., 49: 1-9, 1981. thymidine kinase and CTP synthetase activities indicates that 21. Savage, C. R., and Weinfeld, H. Purification and properties of mammalian other as yet undetermined biological factors also influence the liver cytidine triphosphate synthetase. J. Biol. Chem., 245: 2529-2535, 1970. intracellular CTP synthetase level. 22. StrÃ¤uchen, J. A., Young, R. C., De Vita, V. T., Jr., Anderson, T., Fantone, J. Inhibition of CTP synthetase activity is thought to be the C., and Berard, C. W. Clinical relevance of the histopathological subclassi fication of diffuse "histiocytic" lymphoma. N. Engl. J. Med., 299: 1382- primary mode of action of the investigational antimetabolite 1384, 1978. DAD (17). Recent evidence which shows CTP synthetase to be 23. Tzeng, D. Y., Sakiyama, S.. Kizaki, H., and Weber, G. Increased concentra an important contributor to intracellular CTP and dCTP pools tion of CTP synthetase in hepatoma 3924A: immunological evidence. Life Sci., 28: 2537-2543, 1981. (19, 25) and the experimental synergism between DAU and the 24. Warnke, R., Miller, R.. Grogan, T., Pederson, M., Dilley, J., and Levy. R. antileukemic agents 5-azadeoxycytidine (18) and ara-C (14) Immunologie phenotype in 30 patients with diffuse large-cell lymphoma. N. suggest that there is therapeutic value to be gained in inhibiting Engl. J. Med., 303: 293-300, 1980. 25. Williams, J. C., Kizaki, H., Weber, G., and Morris, H. P. Increased CTP CTP synthetase. The considerable heterogeneity documented synthetase activity in cancer cells. Nature (Lond.), 277: 71-73, 1978. in the CTP synthetase activity of the various types of lympho- 26. Williams, J. C., Kizaki, H., Weiss, E., and Weber, G. Improved radioisotopic assay for cytidine 5'-triphosphate synthetase (EC 6.3.4.2). Anal. Biochem., proliferative disorders indicates that the preexisting CTP syn 97:46-59, 1978. thetase level will be a significant determinant of DAU cytotox- 27. Wortmann, R. L., Mitcell, B. S., Lawrence, E. N., and Fox, I. H. Biochemical icity. basis for differential deoxyadenosine toxicity to T- and B lymphoblasts. Role for 5'-nucleotidase. Proc. Nati. Acad. Sei. U. S. A., 76: 2434-2438, 1979 28. Zighelboim, J. Treatment of acute lymphoblastic leukemia, pp. 762-765. In: REFERENCES M. J. Cline (moderator). Acute leukemia: biology and treatment. Ann. Intern. 1. Barlogie, B., Plunkett, W., Raber, M., Latreille, J., Keating, M., and Mc- Med., 97: 758-773, 1979.

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Peter H. Ellims, T. Eng Gan and Gabriele Medley

Cancer Res 1983;43:1432-1435.

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