ICANCER RESEARCH 46, 1943-1947, April 19861 DNA Sequence Selectivity of Guanine-N7 Alkylation by Three Antitumor Chloroethylating Agents John A. Hartley,' Neil W. Gibson, Kurt W. Kohn, and William B. Mattes@ L.aboratory ofMolecular Pharmacology. Developmental Therapeutics Program, Division ofCancer Treatment, National Cancer institute, NiH, Bethesda, Maryland 20205 ABSTRACF (4). Both compounds are like C1EtNUs in that they produce The DNA sequenceselectivitiesof guanine-N7alkylation producedby delayed interstrand cross-linking in Mer (guanine-O@-alkyl three chloroethylating antitumor agents, I-(2-chloroethyl)-3-(cis-2-hy transferase deficient) human tumor cell lines, but not in Mer droxy)cyclohexyl-l-nitrosourea(cis-2-OH CCNU), 2-chloroethyl(meth cells, DNA-protein cross-linking in both cell types without ylsulfonyl)methanesulfonate,and 8-carhamoyl-3-(2-chloroethyl)imidazo delay, and selective killing of the Mer cells (5, 6). 15,l-dl-l,2,3,5-tetrazin-4(3H)-one (mitozolomide), were examined using We have recently investigated whether either of these agents a modificationof the Maxam and Gilbert sequencing technique. In a had a simpler alkylation chemistry than the C1EtNUs. One of region ofpBR322 DNA, 2-chloroethyl(methylsulfonyl)methanesulfonate the major alkylation routes of the C1EtNUs is the production produced approximately the same degree of alkylation at all guanines. of hydroxyethyl adducts in DNA (7). Such adducts are thought cis-2-OH CCNU, however,preferentiallyalkylated the middleguanines to have little importance in the expression ofantitumor activity in runs of three or more guanines the intensity of the reaction increased but may contribute to both the mutagenic and carcinogenic with the number of adjacent guanines in the DNA sequence. Mitozolom ide produced the same pattern of preferential alkylation but not as effects of the C1EtNUs (8, 9). In reactions with purified DNA, intensely as cis-2-OH CCNU. Three other nitrosoureas, 1-(2-chloro CIEtSoSo produced only chloroethylation products, whereas a ethyl)-3-cyclohexyl-1-nitrosourea, l-(2-fluorethyl)-3-cyclohexyl-1-nitro C1EtNU produced more hydroxyethylation than chloroethyla sourea, and l-(2-chlornethyl)-1-nifrosoureagave similar patterns of al tion (10). Mitozolomide unexpectedly produced a greater di kylation to that of cis-2-OH CCNU at pH 7.2. The ratio of 7-hydroxy versity ofproducts than did C1EtNU, including chloroethyl and ethylguanine to 7-chloroethylguanine was approximately the same follow hydroxyethyl adducts.4 Thus these three chloroethylating ing treatment of the synthetic polymersdG@•dC.and(dG.dC). with cis agents which possess a similar broad spectrum of antitumor 2-OH CCNU, indicatingthat 7-chloroethylationand 7-hydroxyethylation activity have very different patterns of alkylation. were enhanced similarly by the presence of adjacent guanines. Ethylni In the present study we have examined the base-sequence trosourea produced relatively little alkylation preference. The results suggest that the alkylating intermediates, 2-chloroethyldiazohydroxide selectivity for reaction of these compounds at guanine-N7 po and 2-hydroxyethyldiazohydroxide,tendto react preferentiallywiththose sitions of DNA by a modification of a standard DNA sequenc guanine-N7 positions the electronegativity of which is enhanced by the ing method. The major findings were that both C1EtNUs and presenceof neighboringguanines.This is consistentwith the presenceof mitozolomide exhibited differences in reaction intensities with cationic character in the alkylating centers of these intermediates. 2- different guanines. Especially striking were the disproportion Chioroethyl (methylsulfonyl)methanesulfonate and ethyldiazohydroxide ately strong reactions at runs of 3 or more guanines. C1EtSoSo would not be expected to have strong cationic character, in agreement differed from all other alkylating agents so far studied, in that with their lack of sequence selectivity. the reaction intensities for all guanines were approximately the same. INTRODUCFION C1EtNUs2 are among the most effective classes of compounds MATERIALS AND METHODS so far tested in animal tumor systems (1). These compounds, ClEtSoSo and cis-2-OH CCNU were obtained from the Drug De however, have been disappointing in the clinic (2). With the velopment Branch, National Cancer Institute. [‘4C-eihyIJClEtSoSo(9.6 acceptance that chloroethylation of DNA is the probable cause mCi/mmol) and [‘C-ethyllcis-2-OH CCNU (10.4 mCi/mmol) were of the antitumor action of the CIEtNUs and the appreciation obtained from Research Triangle Institute, NC. Mitozolomide was a of the diversity of the reactions of these compounds, other gift from Professor M. F. G. Stevens, Department of Pharmacy, Aston classes of chloroethylating compounds have been prepared University, England. T4 polynucleotide kinase, Hind III, and pBR322 which might be more confined to the desired reactions. ClEt DNA were obtained from P. L. Biochemicals, EcoRI was from New SoSo prepared by Shealy et a!. (3) is highly effective against England Biolabs, and piperidine was from Fisher. Ultra-pure urea was obtained from B. R. L. mouse tumors. Tests in the National Cancer Institute tumor Determination of Guanine-N7 Alkylation Sites in Defined DNA Se screen showed ClEtSoSo to be as good as C1EtNUs against quences. The basic technique for examining the DNA sequence speci B16 melanoma and Lewis lung carcinoma.3 Mitozolomide, ficity of alkylation is an adaptation of the Maxam and Gilbert chemical another highly effective antitumor compound developed in cleavage technique for DNA sequencing (1 1) and was originally de England which appears to be a pro-drug for chloroethyltriazen scribed by D'Andrea and Haseltine (12). Hind III digested pBR322 oimidazole carboxamide, is undergoing clinical trial in Europe DNA was 32P-labeled at its 5'-ends (1 1) and further cleaved with EcoRI. Following alkylation in 25 mrsitriethanolamine HCI:l mM EDTA, pH Received 8/19/85; revised I2/31/85; accepted 1/2/86. 7.2, at 37T for 2 h, precipitation, and washing, the DNA was treated The costs of publication of this article were defrayed in part by the payment with 1 M piperidine at 90'C for 20 mm to produce breaks specifically of page charges. This article must therefore be hereby marked advertisement in at sites of N7 guanine alkylation. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The 276 base-pair Bam HI-SaIl fragment of pBR322 5'-Iabeled at I To whom requests for reprints should be addressed, at Laboratory of Molec ular Pharmacology, Developmental Therapeutics Program, Division of Cancer the Barn HI site was isolated by preparative electrophoresis on a 0.8% Treatment, Building 37, Room 5A19, 9000 Rockville Pike, Bethesda, MD 20205. agarose gel. The 622 base-pair Hind Ill-Sall fragment of pBR322 5'- 2 The abbreviations used are: CIEtNU, l-(2-chloroethyl)-l-nitrosourea; Cl labeled at the Hind III site was isolated on a 0.6% agarose gel. EtSoSo, 2-chloroethyl (methylsulfonyl)methanesulfonate; cis-2-OH CCNU, l-(2- Alkylation was as described above. chloroethyl)-3-(cis-2-hydroxy)cyclohexyl-l-nitrosourea; mitozolomide, 8-carbam oyl-3-(2-chloroethyl)imidazo(5,I-d@-l ,2.3,5-tetrazin-4(3H)one; HPLC, high-per formance liquid chromatography. 4 J. A. Hartley, N. W. Gibson, K. W. Kohn, and W. B. Mattes, unpublished 3 J. Plowman, personal communication. data. I 943 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research. SEQUENCE SPECIFICITY OF CHLOROETHYLATING AGENTS Electrophoresis of the DNA fragments was on 0.4 mm x 90 cm x 20 and further cleaved with EcoRI, was alkylated with either of cm 6% acrylamide 5 M urea polyacrylamide gels (11). Gels were run the three agents at 37°Cfor 2 h, and the DNA was cleaved with for 3 h at 85 W (approximately 3600 V) to maintain a constant pipendine to produce breaks at sites of guanine-N7 alkylation. temperature of 50°C. Following autoradiography of the gel, relative The pattern of fragments obtained can be seen in Fig. 2. band intensities were determined by microdensitometry using a Beck man DU-8 scanning spectrophotometer with gel scanning accessory. ClEtSoSo (lanes a—c,50—150@sM)produced similar degrees of HPLC of DNA Base Adducts. [email protected]@ and (dG.dC)@ (P. L Bio alkylation at all guanines. On increasing the dose of ClEtSoSo chemicals, Inc., Milwaukee, WI) were dissolved in 0.1 M NaCI, 1 mM up to 1 mM no preferential alkylation of guanines was evident EDTA, 1 mMNaPO@,pH 7 and reacted with ‘4C-ethyl-labeleddrugat (data not shown). In contrast cis-2-OH CCNU (lanes d—f) 37°Cfor 4 h. DNA was precipitated with 95% ethanol at —20T produced occasional hot spots of alkylation. This can be seen overnight and recovered by centrifugation. Depurination was achieved in the guanines at positions 173 and 184 in the pBR322 by reaction with 0.15 Mhydrochloric acid at 95C for 30 mm. Samples sequence, which are the middle guanine in runs of three gua were neutralized with 0.3 MNaOH prior to analysis by reverse-phase nines. In the case of mitozolomide (lanes g—:)the same prefer HPLC in 5-im 4.6 mm x 25 cm C18 ultrasphere columns (Altex Ltd.) ential alkylation was observed but less intensely. on a Beckman model 344 gradient liquid chromatograph. The running Densitometric scans of the autoradiograms from Fig. 2 show buffer was 20 mMammonium formate with a 1—10%acetonitrilelinear the differing alkylation patterns produced by these three agents gradient over 30 mm; acetonitrile was then held at 10% for 10 mm followed by a linear gradient from 10—100%acetonitrileover 30 mm. at an equimolar concentration (Fig. 3). In addition to the The flow rate was maintained at I ml/min. One-mi fractions were visually striking difference observed in the two runs of three collected, 4 ml water and 10 ml Aquassure (New England Nuclear) guanines, some additional, more subtle differences can be seen, scintillation fluid were added, and the samples were counted.