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Interstrand Cross-Linking of DNA by 1,3-Bis(2-Chloroethyl)- 1-Nitrosourea and Other 1-(2-Haloethyl)-1-Nitrosoureas

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Interstrand Cross-Linking of DNA by 1,3-Bis(2-Chloroethyl)- 1-Nitrosourea and Other 1-(2-Haloethyl)-1-Nitrosoureas [CANCER RESEARCH 37, 1450-1454, May 1977] Interstrand Cross-linking of DNA by 1,3-Bis(2-chloroethyl)- 1-nitrosourea and Other 1-(2-haloethyl)-1-nitrosoureas Kurt W. Kohn Laboratory of Molecular Pharmacology, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20014 SUMMARY shows that chloroethylnitrosoumeas beaning a single alkylat ing function are active producers of DNA interstrand cross Bifunctional alkylating agents are known to cross-link links, and the mechanism of this effect is investigated. An DNA by simultaneously alkylating two guanine residues lo abstract describing this work has appeared (11). cated on opposite strands. Despite this apparent require ment for bifunctionality, 1-(2-chlonoethyl)-1 -nitrosoureas beaminga single alkylating function were found to cross-link MATERIALS AND METHODS DNA in vitro. Cross-linking was demonstrated by showing inhibition of alkali-induced strand separation. Extensive Nitrosoureas were supplied by Drug Research and Devel cross-linking was observed in DNA treated with 1-(2-chloro opment, Division of Cancer Treatment, National Cancer ethyl)-1 -nitmosourea, 1,3-bis-(2-chlonoethyi)-1 -nitrosoumea, Institute, NIH. The compounds were dissolved in 95% and 1-(2-chlonoethyl)-3-cyclohexyl-1 -nitrosounea. The reac ethanol immediately before use. DNA was isolated from tion occurs in two steps, an initial binding followed by a Escherichia coli by standard procedures using Pronase, second step which can proceed after removal of unbound RNase, and alcohol precipitation. drug. It is suggested thatthe first step is chloroethylation of Reaction mixtures contained DNA (20 @g/ml),0.08 M a nucleophilic site on one strand and that the second step NaCI, 0.01 M NaH2PO4,0.03 M Na2HPO4,0.1 mM EDTA, 5% involves displacement of CI by a nucleophilic site on the ethanol, and the specified concentration of drug. The reac opposite strand, resulting in an ethyl bridge between the tions were carried out at 37°and at pH 7.1. strands. Consistent with this possiblity, 1-(2-fluoroethyl)-3- In postincubation experiments, drug was removed by pre cyclohexyl-1 -nitrosourea produced much less cross-link cipitating the DNA with 2 volumes of ethanol. For facilitation ing, as expected from the known low activity of F, corn of precipitation, the solution was made 0.3 M in NaCI before pared with C1 , as leaving group. 1-Methyl-1-nitrosourea, adding ethanol. The DNA was centrifuged and the pellet which is known to depuninate DNA, produced no detectable was washed with 70% ethanol and medissolved in 1 mM cross-linking. tnisodium EDTA at 0°.Abuffer concentrate was then added to give the same composition used in the initial reaction, and incubation at 37°was resumed. INTRODUCTION For alkali denaturation, 0.1 ml of reaction mixture or postincubation mixture was mixed with 0.2 ml of 0.085 N The 1-alkyl-1 -nitnosouneas, including BCNU,1 CCNU, NaOH-1 mM EDTA. After 3 mm at 23°,the solution was GNU, FCNU, and MNU, can alkylate nucleophilic groups neutralized (to approximately pH 8) by adding 0.2 ml of 0.1 N (reviewed by Wheeler) (25), and are frequently considered citric acid-0.03 M Tnis. After several mm, the solutions were to be a kind of biological alkylating agent. Although stan frozen in dry ice-alcohol and stored at —20°.Undenatured dard alkylating agents such as nitrogen mustards require 2 controls were prepared as above, except that the NaOH and functional groups for antitumon activity, many haloethylni citric acid solutions were mixed before DNA was added, so trosoureas bearing a single alkylating function are ex that the DNA was not exposed to alkali. tremely active antitumon agents (9, 10, 19). The requirement DNA cross-linking was measured by determining the frac for bifunctionality suggests that the cytotoxic lesion pro tion of the DNA that is denaturable by the above alkali duced by alkylating agents such as nitrogen mustards in treatment (12). One cross-link per molecule is sufficient to volves alkylations at 2 neighboring sites and that it perhaps prevent denaturation (12). The fraction of the DNA dena stems from the cross-linking of these sites (21). In particu tured was determined by equilibrium centnifugation in CsCI. Ian,the ability of these bifunctional alkylating agents to form Sample solutions (0.5 ml) were mixed with 0.1 ml of 0.5 M cross-links between opposite DNA strands (7, 8, 12, 14) is an [email protected] Na@CO3-Sarkosyl(0.4 mg/mI), and 0.8 g of attractive possibility for the origin of cytotoxicity. This work CsCI. Clostridium perfringens DNA was added for buoyant density reference. Solutions were adjusted to a refractive I The abbreviations used are: BCNU, 1 ,3-bis(2-chloroethyl)-1-nitnosourea index of 1.3995 by adding small volumes of water. The (NSC 409962); CCNU, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (NSC sodium carbonate buffer increased the separation between 79037); CNU, 1-(2-chlonoethyl)-1 -nitrosourea (NSC 47547); FCNU, 1-(2-fluo native and denatured bands (12, 24), and the detergent roethyl)-3-cyclohexyl-1-nitnosourea (NSC 87974); MNU, 1-methyl-1-nitrosou rea (NSC 23909). prevented selective loss of denatured DNA (12). Samples Received November 23, 1976; accepted February 7, 1977. were placed in 12-mm double-sector analytical cells; the 1450 CANCER RESEARCH VOL. 37 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1977 American Association for Cancer Research. DNA Cross-linking by Nitrosoureas reference sectors were filled with solutions containing CsCI only. Samples were spun at 44,000 rpm for 20 hr at 23°ina A Beckman Model E ultracentnifuge equipped with mono Ref. chrometer and electronic scanner. The areas of the native %@ J L No Drug and denatured bands were measured with a planimeten. @JN RESULTS B HJ\@c LCH?CH, 0 The banding patterns of control native and denatured N-C-NH DNA are shown in Chart 1. It is seen that DNA not exposed N@ 0 to alkali was totally native; it contained no material at dena 2mM CCNU tured density (Chart 1A), whereas DNA exposed to alkali Ji@@ was completely denatured (Chart 1B). A mixture of equal parts of native and alkali-denatured DNA produced clearly C@F CH)CH, 0 \ II separated bands of nearly equal areas (Chart 1C). N-C-NH Q Chart 2 shows the effects of incubation of DNA with N-0 nitrosoureas for 9 hr at 37°.Extensive cross-linking, mdi 2mM FCNU cated by the persistence of DNA banding at native density after alkali treatment, was produced by 2 mM CCNU, BCNU, or GNU; only the result with CCNU is shown (Chart 2B). The DI CH@0 fluoro analog of CCNU, however, produced little cross \ p linking at this concentration and incubation time (Chart 2C). N—C—NH, MNU at 5 mM produced no cross-linking(Chart2D);the N=0 denatured DNA band was measurably broadened, however, 5mM MNU showing that the compound did react with the DNA but produced mainly chain breaks rather than cross-links. Chart 2. Effect of nitrosouneas on DNA denaturability. E. coli DNA was incubated with or without drug for 9 hr at 37°andthen treated with alkali. A, The questionofcross-linkingbyFCNU was pursuedfur nodrug;B,2mMCCNU;C,2mMFCNU;D,5mMMNU. then by extending the reaction time to 21 hr (Chart 3), which yielded small but distinct amounts of cross-linked DNA @ A \\\,@@@_NoDrug (Chart 3, B and C). With MNU, however, the longer reaction time produced further band broadening, but still no detect able cross-linking (Chart 3D). The formation of a covalent connection between 2 DNA strands requires 2 successive reactions: (a) an alkylation or other modification of 1 strand; (b) a reaction of the modified strand with the complementary DNA strand. Since experi B 2mMFCNU ments showed that the extent of cross-linking continued to increase for time periods that exceeded the expected stabil A Ref. Native C B: @ Denatured @ D 5mMMNU @ C Na@ve Chart 3. Effects of prolonged incubation with FCNU or MNU. DNA was : Denatured incubated with or without drug for 21 hr at 37°andthen treated with alkali. A, no drug; B, 2 mM FCNU; C, 5 m@ FCNU; D, 5 mM MNU. ity of the nitrosourea compounds, the possibility was con Chart 1. Equilibrium centnifugation in CsCI-sodium carbonate-Sankosyl. sidered that the rate of cross-link formation was limited by a A, native E. coli DNA; B, alkali-denatured DNA; C, equal parts native and denatured DNA. C. perfringens DNA added for buoyant density reference. slow 2nd step in the reaction sequence. The 2nd step in the Buoyant density increases from left to right. reaction can be isolated by first reacting DNA with drug for a MAY 1977 1451 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1977 American Association for Cancer Research. K. W. Kohn relatively short time and then removing the drug and contin on incubation in the absence of drug after an initial treat uing the incubation of the DNA by itself. Continued cross ment with either CCNU, BCNU, or GNU. Chart 5 shows the link formation during this postincubation would be due to same for FCNU, although cross-linking by this compound completion of the 2nd reaction step. was much smaller in extent and required a longer time to In a test of this idea, DNA was incubated with CCNU, become apparent. BCNU, CNU, or FCNU for 1 hr; alcohol precipitated to Since the cross-linking activity of FCNU is so low com remove drug;redissolved;andincubatedfurtherintheab pared with that of CCNU, the possibility was considered that sence of drug. Chart 4 shows that cross-linking increased the activity of FCNU was due to a trace contamination by a chloroethylnitrosounea.
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