Sphingolipid Metabolism in Leukemic Leukocytes1

[CANCER RESEARCH 27, 1312-1315,July 1967] Sphingolipid Metabolism in Leukemic Leukocytes1

JOHN P. KAMPINE, ROSCOE O. BRADY,- RONALD A. YANKEE, JULIAN N. KANFER, DAVID SHAPIRO, AND ANDREW E. GAL Laboratory of Neurochemislry, National Institute of Neurological Diseases and Blindness, the Medicine Branch, National Cancer Institute, Bethesda, Maryland Â¿0014,and Department of Organic Chemistry, Weizmann Institute of Sciences, Rehovoth, Israel

SUMMARY It has been shown that white blood cells contain sphingolipids (18), and recently we have discovered that leukocytes contain The activity of several enzymes in white blood cells, which catalyze the hydrolysis of sphingolipids and of p-nitrophenyl- very active enzymes which catalyze the hydrolysis of several sphingolipids (14). Attenuation of the activity of glucocerebro phosphate, was determined in leukocyte preparations obtained side-cleaving enzyme in various tissues has been identified as the from hematologically normal individuals and was contrasted metabolic defect in Gaucher's disease (6) and sphingomyelin- with the level of activity of these enzymes in preparations from cleaving enzyme in Niemann-Pick disease (7). The observation patients with acute and chronic leukemia and other diseases. A marked increase in the activity of glucocerebroside-cleaving en that these enzymatic deficiencies are also present in circulating leukocytes has formed the basis of a recently developed diagnos zyme was observed in leukocyte preparations from the patients tic test for sphingolipodystrophies (14). with acute and chronic myelogenous leukemia compared with the controls and patients with lymphocytic leukemia. Sphingo- In view of the presence of these enzymes in leukocyte prepara myelin-cleaving enzyme activity was elevated in leukocytes ob tions, it was considered of interest to examine the level of ac tivity of these sphingolipid hydrolases in leukocyte preparations tained from some of the patients with myelogenous leukemia. Galactocerebroside-cleaving enzyme activity and acid phos- from patients with acute and chronic myelogenous and lympho cytic leukemia. Striking differences in the level of activity of phatase were not elevated in cells obtained from any of the glucocerebroside-cleaving enzyme, and to a lesser extent, sphingo- patients. The major portion of the glucocerebroside-cleaving myelin-cleaving enzyme were observed in the preparations from activity of white blood cells was associated with the acidphosphatase-rich subcellular granular fraction. patients with myelogenous leukemia compared with leukocytes from normal controls and from patients with various other condi tions. The nature and extent of these changes are described in the INTRODUCTION present communication. Studies of normal and loukemic white blood cells have shown that their metabolic activities vary with the different morphologic MATERIALS AND METHODS cell types (23) and can be influenced by phagocytosis (16) and by various hormones and drugs (2, 9, 17). Alterations in enzyme Leukocytes were obtained from normal volunteers and patients activities of leukemic white blood cells have been described for with acute and chronic leukemia and other diseases at the Clini enzymes involved in phosphate, carbohydrate, lipid, protein, and cal Center of the NIH. Circulating leukocyte counts ranged from nucleic acid metabolism (9). Altered alkaline phosphatase ac 1,100 to 320,000 cells per cubic millimeter. tivity of leukocytes in chronic myelogenous leukemia has been re Preparation of Leukocytes. Ten-nil samples of venous blood lated to a change in enzyme content of individual neutrophils were mixed with 2 ml of a solution containing 5% Dextran (9). More recently, studies of specific cell types have shown that (w/v), 0.7% sodium chloride (w/v), and 0.5 mg of heparin per changes in the activity of DNA polymerase and of various en ml. The cells were allowed to settle for 45 minutes at room tem zymes involved in the metabolism of glucose may be related perature, and the upper phase consisting mainly of white blood primarily to changes in the differential count (19). cells and plasma was transferred with a capillary pipette and Complex lipids represented by the sphingolipids have been re centrifuged at 600 X g for 10 minutes. The leukocyte pellet was ported to play an important role in cell structure (22). Some resuspended in 1 ml of 0.85% (w/v) sodium chloride solution and members of this class of lipids have been implicated as receptors residual red cells were lysed by the addition of 3 volumes of dis for drug action (24). Other studies have indicated that specific tilled water. After 90 seconds, 1 volume of 3.6% sodium chloride sphingolipids may be involved in cellular immune reactions (3, was added to return the cell suspension to isotonicity (12). The 13), especially in rapidly growing normal cells and cancer cells suspension was then centrifuged and the pellet washed twice with (20,21). 0.85% sodium chloride solution. Whole leukocyte suspensions in saline were used in the assays for glucoeerebroside and galactocerebroside-cleaving enzyme activity. The suspensions were ad 1A portion of this work was performed under Section 104 (K) justed to a final concentration of leukocytes ranging from 1-6 X of Public Law 480, 83rd Congress, Agreement No. 42515. IO7leukocytes per ml as determined with a Coulter counter. The *Address requests for reprints to Dr. Roscoe O. Brady, NIH, Bethesda, Md. 20014. activity of glucocerebroside-cleaving enzyme is proportional to Received January 4, 1967; accepted March 28, 1967. the white cell concentration in this range (14).

LSI2 CANCER RESEARCH VOL. 27

In order to determine the subcellular distribution of the gluco m/imoles of sphingomyelin-14C, 50 /ig of sodium cholate, 25 cerebroside-cleaving enzyme, white blood cells were homogenized /imoles of potassium acetate buffer (pH 5.0), and water in a final in 5 volumes of 0.34 Msucrose using a motor-driven homogenizer volume of 0.18 ml. The mixtures were incubated for 1 hour at with a Teflon pestle. Subcellular fractions were prepared by 37Â°Cand the amount of phosphorylcholine-14C cleaved was de differential centrifugation according to Cohn and Hirsh (10) ex termined in the manner indicated for the assay of the cerebroside- cept that the second centrifugation was carried out at 8700 X g. cleaving enzymes. Sphingomyelin-cleaving enzyme activity was assayed in a solu The acid phosphatase activity of various leukocyte prepara ble enzyme preparation obtained by sonicating leukocytes sus tions and subcellular fractions was determined according to Cohn pended in 5% (w/v) sodium cholate solution according to the and Hirsch (10) except that p-nitrophenylphosphate was em method described previously (14). ployed as the substrate. Enzymatic Assays. The enzymes which catalyze the hy drolysis of glucocerebroside, galactocerebroside, and sphingo- RESULTS myelin were assayed using 14C-labeled substrates. The synthesis of glucocerebroside and galactocerebroside labeled in carbon 1 of The level of activity of sphingolipid-hydrolyzing enzymes and the hexose portion of the molecule and sphingomyelin labeled in acid phosphatase in leukocytes obtained from normal donors and methyl carbon atoms has been described in earlier communica patients with various diseases is shown in Table 1. The activity tions (5, 8, 15). The enzymatic hydrolysis of the cerebrosides was of these enzymes in patients with acute and chronic myelogenous determined by incubating 50- to lOO-^ilaliquots of the leukocyte leukemia is indicated in Table 2. Of particular interest is suspensions with 125 m/mioles of labeled cerebroside, 300 pg of the higher level of activity of the glucocerebroside-cleaving en sodium cholate, 200 /Â¿gof Cutscum (isooctylphenoxypolyoxy- zyme in the leukemic leukocyte preparations. The mean value of ethanol, Fisher Chemical Co.), and either 15 /miÃ³lesof potassium glucocerebroside-cleaving activity in the control series was phosphate buffer (pH 6.0) for glucocerebroside-cleaving enzyme 4.6 m/iinoles of substrate hydrolyzed per mg of leukocyte protein or 15 //moles of potassium acetate buffer (pH 5.0) for galacto- per hour. Whereas, in preparations from patients with myelog cerebroside-cleaving enzyme. The final volume was brought to enous leukemia, both acute and chronic, the mean value for the 0.15 ml with water. After incubation for 1 hour at 37Â°Cin air, activity of glucocerebroside-cleaving enzyme was 12.8 m/umoles. the amount of glucose-l-I4C or galactose-l-14C cleaved from the This difference is highly significant (P < 0.001). The increased respective substrates was determined by liquid scintillation spec- glucocerebroside-cleaving enzyme activity in the leukemic leuko trometry according to the method described previously (7). cytes was not associated with an increased activity of the galacto- The enzymatic hydrolysis of sphingomyelin was determined on cerebroside-cleaving enzyme. 10- to 50-/il aliquots of the cholate extracts of sonicated leuko The mean level of activity of the sphingomyelin-cleaving en cyte preparations (14). The incubation mixtures contained 70 zyme was also higher in leukocytes from patients with acute and

TABLE 1 Level of Sphingolipid Hydrolascs and Acid Phosphatase in Leukocyte Preparations from Normal Human Controls and Patients with Various Conditions

PatientL.J.B.L.B.W.T.O.H.l).R.Y.R.S.J.K.R.R.P.J.F.S.E.N.Meanbrosidase5.03.44.94.74.74.15.54.34.04.74.95.64.6cerebrosidase5.32.94.03.84.23.12.23.92.73.63.6

melanomaParoxysmal

nocturnalhe-moglobinuria

Renal cell car cinomaHodgkin's dis easeGlucocere-

Â±S.E.Age(years)4138214434323031252827DiseaseNormalNormalNormalNormalNormalNormalNormalNormalMalignantÂ±0.18Galacto-Â±0.28Sphingo-myelinase3.85.75.84.44.14.04.16.64.63.43.74.5Â±0.31Acidphosphatase3.54.25.44.23.35.04.85.24.14.9Â±0.24

The condition of incubation and assay procedure are described in the text. The values are ex pressed in m/imoles of substrate hydrolyzed per mg of protein per hour except for acid phosphatase which is AmÃ³lesper mg of protein per hour.

JULY 1967 1313

TABLE 2 TABLE 4 Leukocyte Enzymes in Myelogenous Leukemia Subcellular Distribution of Leukocyte Glucocerebroside-cleaving Enzyme and Acid Phosphatase PatientV.M.E.E.M.T.Y.B.B.C.M.G.S.H.M.S.E.R.M.S.G.R.A.W.J.S.W.B.L.M.G.I.A.K.E.L.L.M.A.G.Age(years)4316461258444463212741451142361712242240DiseaseAMLÂ»AMLAMLAMLAMLAMLCMLCMLCMLCMLCMLCMLCMLCMLCMLCMLCMLCMLCMLCMLMeanitigo brosidase14.2'8.415.46.36.59.017.06.814.99.319.78.413.512.519.09.714.815.319.115.312.8Â±cerebrosidase2.03.92.92.53.83.64.33.23.03.32.04.22.9Â±niye linase5.28.711.85.64.84.714.14.07.03.413.86.17.37.58.27.5Â±phosphatase6.43.43.45.63.55.43.34.64.23.44.02.84.2Â± hourGlucocere-of substrate hydrolyzed per

FractionWBC broside"m/imoles8.62.25.65.30.4%1002665625p-Nitrophenyl-phosphate6mamÃ³les893153507%1003560568

homogeriateResuspended pellet480 480 X g X gsupernatantResuspended pellet8700 8700 X g X g supernatantAmount 0 The incubation mixtures contained 0.1 ml of homogenized leukocytes or an aliquot of the various subcellular fractions resuspended in a volume of sucrose equivalent to that of the original homogenate. The conditions of incubation and assay procedure are described in the text. 6For the assay of acid phosphatase, 0.01-ml aliquota of the various fractions were employed.

significant (P < 0.01). The mean level of activity of the sphingo- Â±S.E.P myelin-cleaving enzyme in leukocytes from patients with acute 0.98<0.001Galacto-0.340.13Sph0.87<0.01Acid0.320.10 and chronic lymphocytic leukemia was 3.2 m/umoles, and al (comparedtocontrolsGlucocere- though slightly less than the control series, this is not statistically significant (P = 0.09). There was no appreciable difference in " Abbreviations used: AML, acute myelogenous leukemia; acid phosphatase activity between the control series and the pa CML, chronic myelogenous leukemia. tients with either lymphocytic or myelogenous leukemia. 6The measurement of enzymatic activity and conditions of The increases in activity of glucocerebroside-cleaving enzyme incubation are the same as in Table 1. and sphingomyelin-cleaving enzyme appears to be specifically associated with changes in the granulocytic cell series. This is sub TABLE 3 stantiated by the lack of significant changes in those diseases Leukocyte Enzymes in Lymphocytic Leukemia where lymphocyte proliferation markedly alters the granulo- cyte/lymphocyte ratio as shown in Table 3.

PatientU.V.H.D.T.S.R.K.P.N.D.C.J.O.w.s.L.D.Age(years)752423194236271DiseaseALLÂ«ALLALLALLALLALLALLCLLCLLMeanbrosidase1.0Â»2.26.25.87.11.04.05.35.64.2Â±brosidase3.42.02.85.22.02.83.2Â±myelinase1.01.03.43.64.52.04.03.67.13.2Â±phosphatase5.46.23.55.46.27.25.2Â±An attempt was made to relate cell maturation with gluco cerebroside-cleaving enzyme activity. In all of the patients with myelogenous leukemia, the percentage of granulocytic cells less mature than the band and segmented neutrophils were compared with the level of this enzymatic activity. No correlation was evi dent either with the percentage of immature cells in the periph eral blood smear (r = 0.19) or in the final leukocyte suspensions (r = 0.36). The distribution of the enzymes which catalyze the hydrolysis of glucocerebroside and nitrophenylphosphate was examined on subeellular fractions of homogenized leukocytes. Most of the Â±S.E.P hydrolytic activity towards both of these substrates was found 0.770.62Galactocere-0.440.45Sphmgo-0.650.09Acid0.580.64 (compared to in the fraction sedimenting between 480 and 8700 X g (Table 4). controls)Glucocere- Thus it appears that these enzymes are primarily associated with subeellular particles. " Abbreviations used: ALL, acute lymphocytic leukemia; CLL, chronic lymphocytic leukemia. DISCUSSION 6The measurement of enzymatic activity and conditions of incubation are the same as in Table 1. The present study has demonstrated that there are significant changes in at least 2 sphingolipid hydrolases in myelogenous leu- chronic myelogenous leukemia. The average sphingomyelinase kemic cells compared with nonleukemic white cells. Previous re activity in the leukemic preparations was 7.5 mamÃ³lesof sphingo- ports have indicated that "foam cells" occur in myelogenous leu myelin cleaved per mg of leukocyte protein per hour, as compared kemia (1) and that lactosylceramide may accumulate in certain with 4.5 injumolcs in the normal control group. This difference is types of tumor cells (20). Thus, it seems quite possible that dis-

1314 CANCER RESEARCH VOL. 27

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1967 American Association for Cancer Research. Sphingolipid Metabolism in Leukemic Leukocytes turbances in sphingolipid metabolism may be fundamental cocerebroside-cleaving Enzyme from Spleen Tissue. J. Biol. features of some types of neoplastic disorders. Chem., 240: 39^43, 1965. Glucocerebroside-cleaving enzyme activity was highest in the 9. Cline, M. J. Metabolism of the Circulating Leukocyte. Physiol. Rev., 45: 674-720, 1965. subcellular fraction which contained most of the acid phosphatase 10. Cohn, Z. A., and Hirsch, J. G. The Isolation and Properties activity. In other tissues, acid phosphatase activity has been used of Specific Cytoplasmic Granules of Rabbit Polymorpho- as a marker enzyme for lysosomes, and it has been shown in nuclear Leukocytes. J. Exptl. Med., 11%:983-992, 1960. granulocytes that this enzyme is present in the cell fraction con 11. Cohn, Z. A., Hirsch, J. G., and Wiener, E. Lysosomes and taining enzymes usually associated with lysosomes (11). There Endocytosis. The Cytoplasmic Granules of Phagocytic Cells fore, it seems likely that glucocerebroside-cleaving enzyme and and the Degradation of Bacteria. In: A. V. S. de Retick and probably other sphingolipid hydrolases are lysosomal enzymes as M. P. Cameron (eds.), Ciba Foundation Symposium on Ly- in solid tissues (5). somes. Boston: Little Brown & Co., 1963. Although marked changes in glucocerebroside- and sphingo- 12. FallÃ³n, H. J., Frei, E., Davidson, J. D., Trier, J. S., and Burk, myelin-cleaving enzyme activity were demonstrated for a type of D. Leukocyte Preparations from Human Blood: Evaluation disorder primarily involving neutrophils, the level of activity of of Their Morphologic and Metabolic State. J. Lab. Clin. Med., 69: 779-791, 1962. this enzyme did not correlate with the predominant type of neu- 13. Graf, L., and Rapport, M. M. Immunochemical Studies of trophil present in differential counts. Since mixed cell suspensions Organ and Tumor Lipids. XVI. Gel diffusion Analysis of were used for the assays, it is impossible to relate changes in en the Cytolipin K System. Intern. Arch. Allergy, 28: 171-177, zyme activity to specific cell types or to changes in enzyme con 1965. tent of individual cell types. Further studies are necessary to 14. Rampine, J. P., Brady, R. O., Kanfer, J. N., Feld, M., and elucidate this matter, as well as to determine the effects of treat Shapiro, D. The Diagnosis of Gaucher's Disease and Nie ment on glucocerebroside- and sphingomyelin-cleaving enzyme mann-Pick Disease Using Small Samples of Venous Blood. activity. Science, 155: 86-88, 1967. Because of the fact that more complex sphingolipids such as 15. Kanfer, J. N., Young, O. M., Shapiro, D., and Brady, R. O. globoside and gangliosides may be the natural precursors of The Metabolism of Sphingomyelin. I. Purification and Proper ties of a Sphingomyelin-cleaving Enzyme from Rat Liver. J. glucocerebroside (4), it is tempting to speculate that there may be Biol. Chem., Â«4/:1081-1084, 1966. still other qualitative and quantitative differences in sphingo 16. Karnovsky, M. L. Metabolic Basis of Phagocytic Activity. lipid metabolism in leukemic leukocytes. These aspects will be Physiol. Rev., 42: 143-168, 1962. investigated in a subsequent study. 17. Martin, S. P., McKinney, G. R., Green, R., and Becker, C. Effect of Glucose, Fructose, and Insulin on the Leukocytes of REFERENCES Healthy and Diabetic Subjects. J. Clin. Invest., 32: 1171-1179, 1. Albrecht, M. "Gaucher-Zellen" bei chronisch myeloischer 1953. leukamie. Blut, 13: 169-179, 1966. 18. Miras, C. J., Mantzos, J. D., and Levis, G. M. The Isolation and Partial Characterization of Glycolipids of Norman Hu 2. Bisset, S. K., and Alexander, W. D. The Effect of Intravenous man Leukocytes. Biochem. J., 96: 782-786, 1966. Injections of Triiodothyroacetic Acid and L-Triiodothyronine 19. Rabinowitz, Y. DNA Polymerase and Carbohydrate Metab on the Oxygen Consumption of Circulating Leukocytes. Quart. J. Exptl. Physiol., 46: 50-56, 1961. olizing Enzyme Content of Normal and Leukemic Glass Column Separated Leukocytes. Blood, 27: 470-481, 1966. 3. Brady, R. O. Immunological Properties of Glycolipids. J. Am. Oil Chemist's Soc., 43: 67-69, 1966. 20. Rapport, M. M., Graf, L., Skipski, V., and Alonzo, N. F. Immunochemical Studies of Organ and Tumor Lipids. VI. 4. Brady, R. O. Medical Progress: The Sphingolipidoses. New Isolation and Properties of Cytolipin. H. Cancer, 18: 438-445, Engl. J. Med., 275: 312-318, 1966. 1959. 5. Brady, R. O., Gal, A. E., Kanfer, J. N. and Bradley, R. M. 21. Tal, C. Nature of the Cell Membrane Receptor for the Ag The Metabolism of Glucocerebrosides. III. Purification and glutination Factor Present in Sera of Tumor Patients and Properties of a Glucosyl- and Galactosylceramide-cleaving Pregnant Women. Proc. Nati. Acad. Sei. U. 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John P. Kampine, Roscoe O. Brady, Ronald A. Yankee, et al.

Cancer Res 1967;27:1312-1315.

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