Biochemical Pharmacology, Vol. 22, pp. 2763-2765. Pergamon Press, 1973. Printed in Great Britain.
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`Short communications
`
`2763
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`Deamination of 5-azacytidine by a human leukemia cell cytidine deaminase
`
`(Received 7 April 1973; accepted 7 May 1973)
`
`CYTOSINE ARABINOSIDE has become an important agent in the treatment of human leukemia, but its
`effectiveness in some cases appears to be limited by the induction of elevated levels of the catabolic
`enzyme cytidine deaminase.‘ Recently, a new cytidine antimetabolite, 5-azacytidine, has demon-
`strated activity in acute myelocytic leukemia.? The anti-tumor activity of 5-azacytidine has been
`attributed to its metabolism to the nucleotide form and incorporation into RNA. Studies of the
`pharmacology of 5-azacytidine in mice have disclosed excretion of unaltered drug, as well as 5-
`azauracil and various ring-cleavage metabolites.* However, the enzymatic deamination of 5-azacyti-
`dine by human leukemic cells has not been demonstrated previously and is the subject of this report.
`Unlabeled 5-azacytidine (NSC No. 1028165), tetrahydrouridine (NSC No. 112907) and cytosine
`arabinoside (NSC No. 63878) were obtained from the Division of Drug Research and Development,
`National Cancer Institute. Cytidine was obtained from Schwartz-Mann, Orangeburg, N.Y., and
`cytidine-2-'*C from ICN Isotope & Nuclear Division, Cleveland, Ohio. Cytosine arabinoside-2-37H
`was purchased from New England Nuclear Corp., Boston, Mass. Glutamic dehydrogenase from
`bovine liver and in ammonia-free solution was obtained from CalBiochem.
`Human leukemic granulocytes were obtained from untreated patients with chronic granulocytic
`leukemia in the chronic phase of the disease. Heparinized peripheral blood was allowed to sediment
`after addition of dextran,° and the leukocyte-rich plasma removed after 30-120 min. Cells were
`concentrated by centrifugation at 500 g for 15 min, and the cell pack was taken up in 5 ml of 0-15 M
`NaCl. Red blood cells were lysed by the addition of 3 vol. of cold distilled water, and isotonicity was
`restored after 30 sec by addition of | vol. of 0-6 M NaCl. Cells were recentrifuged and resuspended
`in 0-05 M Tris, pH 7:5. Granulocytes were lysed by three cycles of freeze-thawing, followed by 10
`light strokes with a Dounce homogenizer. The supernatant, containing cytidine deaminase activity
`of 5-10 x 10? units*/ml and sp. act. 0-5 units/mg of protein, was stored at 4° until use.
`Deamination of cytidine and of cytosine arabinoside was assayed as previously described,' separat-
`ing product from substrate by ion-exchange chromatography on Dowex-50-H* resin. Deamination
`of 5-azacytidine was assayed by measuring the rate of ammonia production accompanying the
`conversion of 5-azacytidine to 5-azauridine. In the assay, 100-200 yg of supernatant protein was
`incubated with substrate in 0-05 M Tris, pH 7:5, in a total volume of 0-4 ml at 37°. NH4* production
`was determined by addition of the incubation solution to a cuvette containing glutamic dehydro-
`genase, 24 units; alpha-keto-glutarate, 17-0 zmole; EDTA, 1-0 zmole; NADH,0-3 pmole and sodium
`phosphate buffer, pH 7-5, 50 »mole, in a total volume of 1 ml. Theinitial velocity of glutamic dehydro-
`genase activity, as measured by the decrease in absorbance at 340 nm, was directly proportional to
`NH,* generated in the deaminase reaction. By this method deamination of 5-azacytidine was shown
`to be linear with supernatant protein concentrations of 100-500 pg/0-4 ml reaction volume and, in
`the presence of excess 5-azacytidine, to be linear with time for 60 min.
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`3.0
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`=1G. 1. Double reciprocal plot of 5-azacytidine concentration vs reaction velocity. A K, value of
`4-3 x 10°-* M wasdetermined from this plot.
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`* Units of cytidine deaminase = nmoles of substrate consumed perhr.
`CELGENE 2009
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`2764
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`Short communications
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`x
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`220
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`Fic. 2. Sequential u.v. absorption spectra of deamination of 5-azacytidine. See
`experimental conditions.
`
`text
`
`for
`
`The deamination of 5-azacytidine was also observed by u.v. spectrophotometric scanning using a
`Cary, model 15, The reaction solution consisted of 5-azacytidine, 0-4 pmole; Tris, pH 7-5, 0-15
`m-mole and 420 yg of supernatant protein in a total volume of 3 ml.
`Deamination of 5-azacytidine by the extract from human leukemic leukocytes was linear with time
`and protein concentration. A double reciprocal plot of substrate concentration versus NH,* pro-
`duction (Fig. 1) yielded a K,, value of 4-3 x 10-* M which was 20-fold higher than the corresponding
`K,, value for cytidine, 2:2 x 1075 M, and 4-fold higher than the value for cytosine arabinoside, 1-1
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`3.0 _
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`-50 -40 -30 ~-20
`-I0
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`70
`[cYTIDINE}~! x 10%
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`Fic. 3. Competitive inhibition of the deamination of cytidine by 5-azacytidine, 6-7 » 10°* M.
`Cytidine-2-!*C was incubated with enzyme in the presence of unlabeled S-azacytidine, and uridine-
`2-1*C was isolated as described in text. (O-—-O) = cytidine-2-!*C; (@——-@) = cytidine-2-'4C
`with 5-azacytidine.
`
`

`

`Short communications
`
`2765
`
`RATE
`%CONTROL
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`1077
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`io-§
`
`1975
`[THU]
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`1074
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`1073
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`Fic. 4. Inhibition of the deamination of 5-azacytidine, 2 mM (O- — —Q) and cytidine-2-'*C, 0:7 mM
`(@——-@) by tetrahydrouridine. Assays were performed as described in text, except
`that
`tetrahydrouridine was added to reaction solution in concentration indicated 5 min prior to addition
`of substrate.
`
`10-+ M. A marked changein the u.v. absorption of the reaction solution was observed as the reaction
`proceeded. Sequential tracings, as shown in Fig, 2, revealed disappearance of the pyrimidine ring
`peak at 242 nm. The decline in absorption at 242 nm wasdirectly proportional to the NH4* generated
`in the reaction, and was not accompanied by the appearance of a new absorption maximum,as seen
`with the conversion of other cytidine derivatives to their uridine analogs. This was likely due to the
`instability of the deamination product, 5-azauridine, which has been shownto rapidly break down to
`form ribosyl-N-formy] biuret and ribosyl-N-biuret.®
`Further experiments were directed at determining whether the deamination of 5-azacytidine was
`carried out by the same enzymecatalyzing deamination of cytosine arabinoside. Thus, 5-azacytidine
`was found to be a competitive inhibitor of the deamination of both cytidine and cytosine arabinoside
`(Fig. 3). The K, values for 5-azacytidine were 3-2 x 10~* M with cytosine arabinoside as substrate,
`and 2:55 x 10~* M with cytidine as substrate, both of which values agreed well with the previously
`determined X,, value for 5-azacytidine of 4-3 = 107+ M. In addition, tetrahydrouridine, a potent
`inhibitor of the deamination of cytidine and of cytosine arabinoside,’ also inhibited deamination of
`5-azacytidine (Fig. 4). At substrate concentrations 35-40 times the K,, values for each substrate, the
`Isq for tetrahydrouridine inhibition of the deamination of both 5-azacytidine and cytosine arabinoside
`was 5 x 107° M. These results are compatible with the hypothesis that both cytidine analogs are
`deaminated by the same enzyme as has been suggested by Camiener® in his study of human liver
`cytidine deaminase.
`It is thus apparent that 5-azacytidine is subject to enzymatic deamination by peripheral leukemic
`leukocytes, and that
`increased levels of the enzyme cytidine deaminase may compromise the
`effectiveness of 5-azacytidine in treating human leukemia.It is possible that inhibitors of pyrimidine
`deamination such as tetrahydrouridine may be helpful in preventing catabolism of this antineoplastic
`agent.
`
`Laboratory of Chemical Pharmacology,
`National Cancer Institute,
`National Institutes of Health,
`Bethesda, Md. 20014, U.S.A.
`
`Bruce A. CHABNER
`JAMES C. DRAKE
`Davip G. JoHNS
`
`REFERENCES
`
`1. C. D. Steuart and P. J. BURKE, Nature New Biol. 233, 109 (1971).
`2. K. B. McCrepiz, G. P. Bopey, M. A. BurGess, V. RODRIGUEZ, M. P. SULLIVAN and E. J.
`Freireicu, Blood 40, 975 (1972).
`3. M. Jurovcix, K. Raska, A. Sormovaand F, Sorm, Colin Czech. chem. Commun. Engl. Edn 30
`3370 (1965).
`. K. RASKA, Jr., M. JuRovcik, Z. SonMovaA and F. Sor, Colln Czech. chem. Commun. Engl. Edn
`30, 3001 (1965).
`. J. R. BertINo and G. FISCHER, Meth. med. Res. 10, 297 (1964).
`. A, CIHAK, J. SKODA and F. Sorm, Collin Czech. chem. Commun. Engl. Edn 29, 814 (1964).
`. G. W. CAMIENER, Biochem. Pharmac. 17, 1981 (1968).
`. G, W. CaAmIENER, Biochem. Pharmac. 16, 1691 (1967).
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`CO~INA
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