[CANCER RESEARCH 27 Part 1, 535-540, March 1967]

Inhibition of DNA Synthesis by Hydroxyurea: Structure-Activity Relationships1

CHARLES W. YOUNG, GERALD SCHOCHETMAN, SADIE HODAS, AND M. EARL BALIS Divisions of Clinical Chemothirapy and Biological Chemistry, Sloan-Ketlering Institute for Cancer Research, New York, New York 10021

SUMMARY allosteric effectors, altering substrate specificity and the rate of The following comix>unds inhibited incorporation of thy- the overall reaction (12, 13). The molecular mechanism by which midine-3H into the DNA of HeLa cells without significantly hydroxyurea inhibits deoxyribonucleotide synthesis is not known. impairing cellular incorjwration of uridine-3H into RNA or Substrate attack is unlikely because the drug does not impair loucine-3H into protein; the}' are listed in order of potency. synthesis of RNA; an attack ui>on the product of the reaction Dihydro.xyurea > jV-methylhydroxyurea > .V-acetylhydroxy- (deoxyribonucleotides) could not be demonstrated in ex|>eriments urea = hydroxyurea = A'-hydroxyguanidine = N-hydroxyure- with cell-free extracts (26). Because of the lack of structural thane = AT-ethylhydroxyurea > 3-phenyl-l-hydroxyurea = analogy between hydroxyurea and ribonucleotides or deoxyribo formamidoxime > Ar-mcthylacetohydroxamic acid = Ar-methyl- nucleotides, competition by the drug for the enzymatic substrate and/or allosteric-effector sites seems improbable. In an effort to hydroxylamine > acetohydroxamio acid. Hydroxylamine, JV-hydroxyglycine amide and 3-phenyl-l-hydroxy-2-thiourea clarify the relationship between molecular structure and inhibi tory activity, we have studied the effects of various hydroxyl- inhibited incori>oration of all three labeled precursors. Methoxy- amine, methoxyurea, and Ar-methylmethoxyurea had no effect amine and hydroxamic acid derivatives u)xjn protein and nucleic acid metabolism in HeLa cells. The results of our experiments upon thymidine uptake. Addition of three deoxyribonucleosides, deoxyadenosine (0.1 HIM),deo.xyguanosine (0.1 min), and deoxy- are recorded in this communication. cytidine (1.0 /ÕM),to the culture medium provided partial protection against the inhibitory effects of hydroxyurea, hy- MATERIALS AND METHODS droxyurethane, acetohydroxamic acid, hydroxyguanidine, formamidoxime, and Ar-methylhydroxylamine. The data suggest Materials. HeLa cells were obtained from Microbiological Associates and maintained in monolayer culture by weekly that all of these agents inhibit a reaction in the biosynthesis of subdivision. Eagle's minimal essential medium, supplemented deoxyribonucleotides from ribonucleotides. The relationship with calf serum (to 10%), )>enicillin,and streptomycin was used between chemical structure and inhibitory activity is discussed for maintenance of cells. 3H- and 14C-labeled thymidine, uridine, and some ]X)ssibleinhibitory mechanisms examined. and leucine were purchased from the New England Nuclear Corp., as was A^-hydroxyurethane. The following were generously INTRODUCTION provided : A7-hydroxyurea, Ar-ethylhydroxyurea, Ar-acetylhydrox- yurea, methoxyurea, 1-methyl-l-methoxyurea (The Squibb Hydroxyurea inhibits synthesis of deoxyribonucleic acid Institute for Medical Research, New Brunswick, New Jersey); without altering rates of formation of ribonucleic acid or A'-hydroxyglycine, (Merck & Co., Rahway, New Jersey); dihy- protein in mammalian cells (10, 23, 25, 26), echinoderm droxyurca, A^-methylhydroxyurea, 3-phenyl-l-hydroxyurea (Dr. embryos (28), and bacteria (11, 22). The observed decrease in H. Wuest, Sloan-Kettering Institute); sodium hyj>onitrite (Dr. synthesis of DNA is occasioned, at least in part, by the inhibitory A. Bendich, Sloan-Kettering Institute); formamidoxime, aceto effect of the drug upon enzymatic conversion or ribonucleotides hydroxamic acid, O.A^-diacetyl-A'-methylhydroxylamine (which to deoxyribonucleotides (1, 9, 18, 26, 28). The substrates of this hydrolyzes almost instantly in aqueous solutions to A'-methyl- reduction are ribonucleotide diphosphates (13) or triphosphates acetohydroxamic acid), 3-phenyl-l-hydroxy-2-thiourea (E. (12). At least four distinct proteins, Mg++, and NADPH2 are Falco, Sloan-Kettering Institute); A'-hydroxyglycineamide required (13, 17, 19, 20); adenosine triphosphate and the four (Southern Research Institute, Birmingham, Alabama) and N- naturally occurring deoxyribonucleoside triphosphates act as hydroxyguanidine (Dr. T. J. Delia, Sloan-Kettering Institute). Methods. Drug effects upon the protein and nucleic acid me 1This work was supported in part by NCI Grants CA-07800 and tabolism of monolayers of HeLa cells were assayed by means CA-08748. 2 The following abbreviations are used: TCA, trichloracetic of a sequential isotope technic described in detail elsewhere acid; NADP+, NADPH, oxidized and reduced nicotinamide- (27). Monolayers in log-phase of growth received an initial 15- to adenine dinucleotide phosphate, respectively; LADH, horse-liver 30-minute exposure, without drugs, to a precursor labeled with alcohol dehydrogenase; NAD+, oxidized nicol ¡numide adenine 14C, followed by an experimental incubation of 30 minutes' dinucleotide. duration, with drugs, in the presence of the same precursor now Received August 17, I960; accepted November 1, I960. labeled with tritium. The precursors used were: thymidine-2-14C

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TABLE I Inhibitory Effects of Hydroxyurea and Related Compounds upon Incorporation of Labeled Precursors into DNA, RNA, and Protein of HeLa Monolayers°

oí:TdR'93094679209196899209209189l'K80000002200000000Ixu93472001504000000000InhibitoryInhibition* oíincorjioration

testedHydroxylamineMethoxyamineWCompound potency* for DN'Asynthesis7080.93602381000133091111

HHH,CO—

HHHO—N—

lamine0-Methylhydroxy CH,HH,CO—N—

,W-DimethylhydroxylamineFormamidoximeAf-HydroxyglycineW-Hydroxyglycine CH,HO—N— N=CH—NH,H 01 II HO—N—CH,C—OHH 01 amideAcetohydroxamic II HO—NCH,C—NH,H O acidAT-Methylacetohydroxamic 1 II HO—N—C—CH,H,C 01 acidHydroxyureaMethoxyurea#-Methylhydroxyurea1-Methoxy-l-methylureajV-EthylhydroxyureaN-AcetylhydroxyureaFormulaHO-N—II HO—N—C—CH,HO—

N—CNH,H 0H,CO— N—CNH,H,C 01 II HO—N—C—NH,H,C OH,CO— NH,H5C,N—C— 01 NH,CH,—1!HO—N—C— COOHO— N—C—NH,% " All values represent the average of at least two experiments performed in triplicate. Incorporation periods were thirty minutes; drugs and tritium-labeled precursors were present at zero time. 6At the drug concentration in each case which produced approximately 90% inhibition of incorpora tion of thymidine-'H. If no effect on thymidine incorporation was detected, a concentration of 10 HIM was employed. % inhibition = 1- W"C Experimental X 100 'H/"C Control 0 inhibition implies that maximal deviation from control values $ ±14%. cAt drug concentration which inhibited incorporation of thymidine-'H by 50% (I.C.so), potency = LC- Hydroxyurea I.C. soTest drug The I.C.so of hydroxyurea was 0.1 HIM. dTdR, thymidine; UR, uridine; Leu, leucine.

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TABLE I—Continued

of:TdR'919492880910ÃœR000025560Leu000290770InhibitoryInhibition1 of incor|Miration tested3-Phenyl-l-hydroxyureaDihydroxyureaA^-HydroxyurethaneAf-HydroxyguanidineGuanidine3-Phenyl-l-hydroxy-2-thioureaNaCompound potency' for DNAsynthesis38108921000300

OHHO— N—C—Ir—/\=yHÖHN'

OHHO-N—HO—N—C—N—

C—0-C,H5H

NH HO—N-C—NH,NHII

NH,?H,N—C— F_/-\HON— j>HO—C—NH—¿

hyponitriteFormulaH N—N—O~Na+%

(0.025 MC/ml,0.83 MM)and thymidine-[methyl-3H] (0.42 /ic/ml, TABLE 2 0.07 MM)for DNA; uridine-2-HC (0.016 MC/ml, 0.53 MM)and Inhibitory Effects of —NOH-containing Compounds Upon 3H-uridine (0.8 MC/ml, 0.1 MM)for RNA; and L-leucine-l-14C Incorporation of Thymidine-3H by HeLa Cells in the (0.4 MC/ml, 16 MM)Sand i)L-leucine-[4,5-3H] (0.8 MC/ml,0.2 MM)" Presence and Absence of Exogenous for protein. Acid-soluble radioactivity was removed with cold Deoxyribonucleosides 5% TCA, lipids were extracted with ethanol (15 min) and ether Controlincorporation0AdditionsNone88.5878914138100GdR, (10 min) at room temperature. In experiments with leucine, nucleic acids were removed by hot 5% TCA extraction (90°Cfor concen CompoundHydroxylamine 15 min). The acid- and lipid-insoluble residue of each monolayer trations(mil)10131013131.31.341.3% AdR, was solubilized by heating in hydroxide of Hyamine (Packard CdR*10282312.535323992196 Instrument Co.), l M in methanol, at 65°Cfor 2 hours. Scintil lation solution was added and tritium and 14C content in each SOtAr-Methylhydroxylamine sample was determined by the method of Kabara et al. (14) in HC1FormamidoximeA"-Hydroxyglycine• a dual-channel liquid scintillation counter (Packard Instrument Cor])., La Grange, 111.).The 3H/I4C ratio served to quantitate amideAcetohydroxamic precursor incorporation. Deviation from the control 3H/14C ratio acidHydroxyureaAf-Hydroxyguanidine3-Phenyl-l seen in samples which had been exposed to a drug was used as a measure of drug effect. -2-thioureaHydroxyurethaneNoneDrug-hydro xy

RESULTS Drug effects upon rates of incorporation of thymidine, uridine' " Each value represents the mean of at least two experiments and leucine, respectively, into DNA, RNA, and protein of mono- done in triplicate. Drugs, thymidine-3H, and deoxyribonucleo layers of HeLa cells are summarized in Table 1. The relationship sides were present at zero time. The incorporation periods were of the inhibitory effect U]>onDNA synthesis to impairment of 30 minutes. ribonucleotide reduction was assessed in terms of the protection 6 Deoxyguanosine (GdR) (0.1 mM), deoxyadenosine (AdR) given by a mixture of deoxyribonucleosides, i.e., deoxyadenosine (0.1 RIM), and deoxycytidine (CdR) (1 JUM)- (0.1 HIM),deoxyguanosine (0.1 HIM),and deoxycytidine (1.0 MM). The results of these studies are presented in Table 2. Hydroxyl- leucine equally, a finding that is in agreement with the re]>orted amine inhibited incorporation of labeled thymidine, uridine, and effects of the drug upon protein and nucleic acid synthesis in bacteria (21). Inhibition of DNA synthesis by hydroxylamine 3 Eagle's medium lacking leucine, supplemented with calf serum was not altered by addition of exogenous deoxyribonucleosides. to 1%, was vised in experiments with labeled leucine. 0-Methylhydroxylamine (methoxyamine) had no effect upon

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. Charles W. Young, Gerald Schochetman, Sadie Hodas, and M. Earl Balis rates of DNA or RNA synthesis but inhibited protein synthesis. thymidine incort>oration produced by hydroxyurea, hydroxyure .Y-Methvlhydroxylamine, on the other hand, was as |X)tent an thane, acetohydroxamic acid, formamidoxime, .V-hydroxyguani- inhibitor of thymidine uptake as hydroxylamine but had no effect dine, and .V-methylhydroxylamine. By this criterion each of upon incoi'ix>ration of uridine and produced negligible impair these compounds inhibits synthesis of DNA by interfering with ment in cellular uptake of leucine. Further, the inhibitory effect the same, or with closely related, enzymatic reaction(s); pre of JV-methylhydroxylamine u|x>n incorporation of thymidine was sumably, the inhibited reaction is essential for normal reduction lessened by the presence of exogenous deoxyribonucleosides. of ribonucleotides. The —NOH group is required for the inhib O,.V-Dimethylhydroxylamine possessed weak biologic activity itory activity under consideration. The proton on the hydroxyl in this test system, giving 67 % inhibition of thymidine uptake group must be free; i.e., methoxyamine, methoxyurea, and at a concentration of 13 mM; it was roughly 10% as jwtent as AT-methylmethoxyurea are inactive. A compound is less active AT-methylhydroxylamine. if it is largely ionized at neutral pH (acetohydroxamic acid pK Each of nine .V-acyl derivatives of hydroxylamine (hydroxamic 7.55 versus hydroxyurea pK approximately 10.6) (5). Substitu- acids) inhibited incorporation of thymidine without significantly ents which make the hydroxy pK resemble that of an alcohol altering uptake of uridine into RNA or leucine into protein. increase biologic activity; the inhibitory latency of acetohy Alkylation of the hydroxyl (methoxyurea and .V-methylmethoxy- droxamic acid < Ar-methyl-acetohydroxamic acid < hydroxy urea < A"-methylhydroxyurea. A carbonyl group is not a urea) resulted in total loss of biologic activity. On the other hand, alkylation of the hydroxylated nitrogen (Ar-methylhydroxyurea, prerequisite in an inhibitory compound (formamidoxime and A'-methylacetohydroxamic acid, Ar-eth\'lhydroxyurea) or N- AT-methylhydroxylamine), but the most potent derivatives acylation (l-acetyl-1-hydroxyurea) increased potency somewhat contain a carbonyl or an equivalent group (hydroxyurea, or had no effect. The inhibitory potency displayed by the hydroxyguanidine, dihydroxyurea). hydroxamic acids tested, which are of the general formula All of the compounds which inhibit synthesis of DNA in our 0 HeLa system form complexes with metal ions (M). With hy II droxamic acids the complex is felt to be of the form (R—C—NHOH), was significantly influenced by the nature of "R." Thus acetohydroxamic acid (in which R is a methyl group) R—C=N R—C—NH // // was 30-fold less inhibitory than hydroxyurea (R is NH2) or OO OO hydroxyurethane (R is CH3CH2—0—).Mixed deoxyribonucle osides afforded partial protection against the inhibitory effects ÕM! of acetohydroxamic acid and hydroxyurethane in a manner and with hydroxylamines (NH2O)n-Mn+. 0-Methyl derivatives analogous to that observed with hydroxyurea. Therefore, it is of hydroxylamine and hydroxamic acids do not form metal probable that all the hydroxamic acids tested are affecting the complexes and do not inhibit synthesis of DNA. Zn++ has same metabolic site. potentiated the antitumor activity of hydroxyurea in a murine The role of the carbonyl moiety of hydroxyurea was inves experimental tumor system (J. G. Cappuchino, G. S. Tarnowski, tigated by testing A-hydroxyguanidine, 3-phenyl-l-hydroxy-2- and M. E. Balis, unpublished data). Although it seems probable thiourea, and formamidoxime. All three compounds inhibited incorporation of thymidine; the biologic [wtencies of A'-hydroxy- that this ability to react with metals is pertinent to the effect of the active agents upon DNA metabolism in vivo, simple guanidine and 3-phenvl-l-hydroxy-2-thiourea were close to those chelation does not provide a complete explanation. For example, of the corresponding hydroxyureas. Formamidoxime was inter acetohydroxamic acid is a stronger chelating agent than hydroxy mediate in ]K>tencybetween hydroxyurea and .Y-methylhydrox- urea (M. E. Balis, unpublished observation), but it is 30-fold ylamine. A'-Hydroxyguanidine and formamidoxime ]x>ssess less active an inhbitor of DNA synthesis (Table 1). All com- inhibitory specificity similar to that of hydroxamic acids; mixed jwunds which were active in the HeLa test system form com deoxyribonucleosides afford partial protection against them. On plexes with metal ions; but all chelaters were not active, e.g., the other hand, 3-phenyl-l-hydroxy-2-thiourea inhibited uptake N-hydroxyglycine complexed with metal ions but did not inhibit of all three precursors; its effects on DNA synthesis were not synthesis of DNA. altered by exogenous deoxyribonucleosides. Guanidine was Hydroxylamine inhibits synthesis of the three types of macro- slightly inhibitory to leucine uptake but did not alter rates of molecules studied, DNA, RNA, and protein, to a comparable incorporation of thymidine and uridine. degree. It is improbable that a drug-induced inhibitory effect Sodium hyponitrite and Ar-hydroxyglycine were tested because limited to DNA metabolism can be mediated by way of prior they are reported to produce chromosomal abnormalities in cell conversion of said drug to hydroxylamine. In spite of this, two culture and breakdown of isolated DNA in a fashion similar to observations suggest that the hydroxamic acids and the hydrox hydroxylamine and hydroxyurea (2, 3) ; they are inert in our test ylamines have some shared reaction characteristics: (a) The system. A-Hydroxyglycine amide inhibited thymidine uptake biologic activity of hydroxylamine, Ar-methylhydroxylamine, and with a potency about one-fiftieth that of hydroxyurea. N- hydroxyurea is dramatically reduced or ablated by conversion Hydroxyglycine amide also inhibited protein synthesis; its effects of the hydroxy- to a methoxy radical. (6) A^-Methylhydroxyl- on thymidine incori ¡orationwere not decreased by addition of amine and the hydroxamic acids exhibit similar inhibitory deoxyribonucleosides. specificity (Table 1); further, exogenous deoxyribonucleosides partially protect against their inhibitory effect on synthesis of DISCUSSION DNA (Table 2). It seems plausible that Ar-methylation or A mixture of deoxyadenosine, deoxyguanosine, and deoxy- Ar-acylation of hydroxylamine may interfere with some but not cytidine gave partial protection against the impairment of all, of the reactions available to the nonsubstituted molecule;

538 CANCER RESEARCH VOL. 27

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. Hydroxyurea: Structure-Activity Relationships thus consideration of the known reactions of hydroxylamine may 2. Bendich, A., Borenfreimd, E., Korngold, G. C., Krim, M., and be useful in attempting to define the reaction mechanism of Balis, M. E. Amino Acids or Small Peptides as Punctuation in hydroxyurea. the Genetic Code of DNA. In: A. Baselli (ed.), Acidi niicleici e loro funzione biologica, pp. 214-237. Pavia: Tipographic Most of the reactions described for hydroxylamine in purified Successori Fusi, 1964. systems employ drug concentration, temperature, time, and pH 3. Borenfreund, E., Krim, M., and Bendich, A. Chromosomal conditions that differ markedly from those possible in a physi Aberrations Induced by Hyponitrite and Hydroxylamine ologic system. Mutational effects upon T-4 Bacteriophage and Derivatives. J. Nati. Cancer Inst., 32: 667-679, 1964. transforming DNA of Bacillus subtilis, hydrolysis of RNA, and 4. Burton, R. M., and Kaplan, N. O. A Chemical Reaction of cleavage of acyl-esters fall into this category and will not receive Hydroxylamine with Diphosphopyridine Nucleotide. J. Biol. further consideration. The observations of Bendich and co- Chem., 211: 447-463, 1954. workers (2, 3) and of Freese and Freese (6-8) demonstrate that 5. Exner, O., and Simon, W. Acyl Derivatives of Hydroxylamine hydroxylamine, .Y-methylhydroxylamine, hydroxyurea, .V-hy- XII. Dissociation Constants of Hydroxamie Acids and Their droxyurethane, and .V-hydroxyglycine can form an oxidation Functional Derivative. Collection Czech. Chem. Commun., product in dilute, oxygen-containing, aqueous solutions which 30: 4078-4094, 1965. 6. Freese, E., and Freese, E. B. The Oxygen Effect on Deoxyri inactivates transforming DNA, degrades purified DNA, and bonucleic Acid Inactivation of Hydroxylamines. Biochemis produces chromosomal abnormalities in cell culture. However, try, 4: 2419-2433, 1965. some hours are required for the DNA degradative effects to 7. Freese, E. B. Effects of Urethane and Hydroxyurethane on become manifest, whereas inhibition of synthesis of DNA devel Transforming DNA. Genetics, 51: 953-960, 1965. ops within less than five minutes. In addition, A'-hydroxyglycine 8. Freese, E. B., and Freese, E. Two Separate Effects of Hydroxyl and Na hyixmitrite degrade DNA and produce chromosomal amine on Transforming DNA. Proc. Nati. Acad. Sei. U. S., abnormalities (2, 3) but do not inhibit synthesis of DNA (Table 52: 1289-1297, 1964. 1). This phenomenon is thus not pertinent to the rapid inhibition 9. Frenkel, E. P., Skinner, W. N., and Smiley, J. D. Studies on of DNA synthesis produced by many of these same compounds. a Metabolic Defect Induced by Hydroxyurea (NSC-32065). Cancer Chemotherapy Kept., 40: 19-22, 1964. Hydroxylamine inhibits the reactions of LADH by forming a complex with the enzyme and NAD+. Kaplan and co-workers 10. Gale, G. R. Effect of Hydroxyurea on Incorporation of Thy- midine into Ehrlich Ascites Tumor Cells. Biochem. Pharma- (4, 15, 16) suggested that LADH binds and activates hydroxyl col., 13: 1377-1382, 1964. amine analogously to its binding and activation of ethanol. 11. Gale, G. R., Kendall, S. M., McLain, H. H., and DuBois, S. Binding and activation of ethanol is felt to involve a reaction of Effect of Hydroxyurea on Pseudomonas aeruginosa. Cancer the hydroxyl group with an atom of zinc on the surface of the Res., 24: 1012-1019, 1964. enzyme (24). Hydroxylamine complexes with metals; methoxy- 12. Goulian, M., and Beck, W. S. Purification and Properties of amine, which does not form a metal complex, had little inhibitory Cobamide-Dependent Ribonueleotide Reducíase: Evidence effect upon LADH activity (15). This type of hydroxylamine- of the Allosteric Regulation of Substrated Specificity. Federa coenzyme-enzyme interaction may be pertinent to the inhibition tion Proc., 25: 280, 1966. of DNA synthesis produced by hydroxyurea. Formation of a 13. Holmgren, A., Reichard, P., and Thelander, L. Enzymatic hydroxyurea-NADP+-dehydrogenase complex analogous to that Synthesis of Deoxyribonucleotides VIII. The Effects of ATP and dATP in the CDP Reducíase System from E. coli. Proc. of hydroxylamine-NAD+-LADH might interfere with NADPH Nati. Acad. Sei. U. S., 64: 830-836, 1965. synthesis. Because enzymatic reduction of ribonuc leotides is 14. Kabara, J. J., Spafford, N. II., McKendry, M. A., and Free dependent upon NADPH (20), an inhibition of NADPH genera man, N. L. Recent Developments in Simultaneous C14 and tion should result in decreased rates of deoxyribonucleotide Tritium Counting. In: S. Rothchild (éd.),Advances in Tracer production According to this model, reduced lipoic acid and Methodology, Vol. 1, pp. 76-85. New York: Plenum Press, possibly NADPH would prevent hydroxyurea-induced inhibition 1962. of ribonueleotide reduction in experiments utilizing cell-free 15. Kaplan, N. O., and Ciotti, M. M. Direct Evidence for a Di extracts. Since hydroxyurea did not inhibit incorporation of phosphopyridine Nucleotide-hydroxylamine Complex with aceta te-14Cinto the lipids of HeLa cells in vitro or liver cells in Horse Liver Alcohol Dehydrogenase. J. Biol. Chem., 2¡í:431- 445, 1954. vivo (C. W. Young and M. E. Balis, unpublished observation), 16. Kaplan, N. O., Ciotti, M. M., and Stolzenbach, F. E. The it seems clear that the drug does not produce a general depletion Action of Hydroxylamine and Cyanide on Alcohol Dehydro of cellular NADPH. The data suggest that the compounds act genase of Horse Liver. J. Biol. Chem., 211: 419-429, 1954. by complexing with oxido-reducto metalloenzymes of which 17. Laurent, T. C., Moore, E. C. and Reichard, P. Enzymatic LADH is an example. The inhibited enzyme is in the ribonuele Synthesis of Deoxyribonucleotides IV. Isolation and Char otide —>deoxyribonucleotide sequence. The relative potency of acterization of Thioredoxin, the Hydrogen Donor from Esche- various R—NOH derivatives is a function of the ability to richia coli B. J. Biol. Chem., 239: 3436-3444, 1964. chelate on the oxygen and the similarity of the acidity of the 18. Mohler, W. C. Cytotoxicity of Hydroxyurea (NSC-32065) hydroxyl group to that of a primary alcohol. Reversible by Pyrimidine Deoxyribosides in a Mammalian Cell Line Grown In Vitro. Cancer Chemotherapy Rept., 34: 1-6, 1964. REFERENCES 19. Moore, E. C., and Reichard, P. Enzymatic Synthesis of Deoxy ribonucleotides VI. The Cytidine Diphosphate Redactase 1. Adams, H. L. P., Abrams, R., andLieberman, I. Deoxycytidyl- System from Novikoff Hepatoma. J. Biol. Chem., 239: 3453- ate Synthesis and Entry into the Period of Deoxyribonucleic 3456, 1964. Acid Replication in Rabbit Kidney Cells. J. Biol. Chem., 241: 20. Moore, E. C., Reichard, P., and Thelander, L. Enzymatic 903-905, 1966. Synthesis of Deoxyribonucleotides V. Purification and Prop-

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erties of Thioredoxin Reducíase from Escherichia coli B. J. P. D. Boyer, H. Lardy, and K. Myrbäck (eds.), The Enzymes, Biol. Chem., 239: 3445-3452, 1964. Ed. 2, Vol. 7, pp. 25-83. New York: Academic Press, 1963. 21. Rosenkranz, H. S., and Bendich, A. J. Studies on the Bacterio- 25. Yarbro, J. W., Kennedy, B. J., and Barnum, C. P. Hydroxy static Action of Hydroxylamine. Biochim. Biophys. Acta, 87: urea Inhibition of DNA Synthesis in Ascites Tumor. Proc. 40-53. 1964. Nati. Acad. Sei. U. S., 53: 1033-1035, 1965. 22. Rosenkranz, H. S., Garro, A. J., Levy, J. A., and Carr, H. S. 26. Young, C. W., and Hodas, S. Hydroxyurea: Inhibitory Effect Studies with Hydroxyurea I. The Réversible Inhibition of on DNA Metabolism. Science, llß:1172-1174, 1964. Bacterial DNA Synthesis and the Effect of Hydroxyurea on 27. Young, C. W., and Hodas, S. Acute Effects of Cytotoxic Com the Bacteriocidal Action of Streptomycin. Biochim. Biophys. Acta, 114: 501-515, 19(iC. pounds on Incorporation of Precursors into DNA, RNA and protein of HeLa Monolayers. Biochem. Pharmacol., 14-' 205- 23. Schwartz, H. S., Garofalo, M., Sternberg, S. S., and Philips, F. S. Hydroxyurea: Inhibition of Deoxyribonucleic Acid Syn 214, 1965. thesis in Regenerating Liver of Rats. Cancer Res., 25: 1867- 28. Young, C. W., and Karnofsky, D. A. Evidence of Hydroxy- 1879, 1965. urea-Induced Inhibition of Deoxyribonucleotide Formation 24. Sund, H., and Theorell, H. Alcohol Dehydrogenases. In: in Intact Cells. Federation Proc., a5: 195, 1966.

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Charles W. Young, Gerald Schochetman, Sadie Hodas, et al.

Cancer Res 1967;27:535-540.

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