[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 MARCH 1967 535 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 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— NH,HN=CH— 01 II HO—N—CH,C—OHH 01 amideAcetohydroxamic II HO—NCH,C—H,HN O acidAT-Methylacetohydroxamic 1 II HO—CH,H,CN—C— 01 acidHydroxyureaMethoxyurea#-Methylhydroxyurea1-Methoxy-l-methylureajV-EthylhydroxyureaN-AcetylhydroxyureaFormulaHO-N—II HO—N—C—CH,HO— N—CNH,H 0H,CO— CNH,H,CN— 01 II HO—NH,H,CN—C— OH,CO— NH,H5C,N—C— 01 NH,CH,—1!HO—N—C— OHO—CO 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. 536 CANCER RESEARCH VOL. 27 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. Hydroxyurea: Structure-Activity Relationships 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,