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Experimental Therapeutics |
Department of Chemistry, University of Washington, Seattle, Washington 98195
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The reactions of N-nitrosoureas with DNA in aqueous solution are complex and are further complicated in the haloethyl nitrosoureas. Although the question of the precise chemical nature of the alkylating agent remains a matter of debate (7)
, it is highly probable that one important initial site of alkylation is O6 of G (Fig. 1, 1
; Ref. 8
). In simple model systems, substances of this type have been shown to cyclize (Fig. 1
, 2)
and, in turn, react with an external nucleophile to form an ethylene cross-link (9)
. Enzymatic hydrolysis of the phosphodiester backbone of DNA treated with BCNU has previously afforded the lesion 4 (Fig. 2D)
in which G and C are bridged through their N1 and N3 sites, respectively (9)
. It has been suggested that an alternative structure might be responsible for the interstrand cross-link because 1,2-(diguan-7-yl)ethane (5)
has also been recovered from DNAs treated with BCNU (10)
. This lesion is roughly analogous to the N7-to-N7, dG-to-dG interstrand cross-link formed in DNA by the nitrogen mustard mechlorethanime (11)
. Although both substances 4 and 5 have been identified in BCNU-treated DNA, until now, no direct evidence favored one over the other.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]ATP (New England Nuclear); T4 polynucleotide kinase (USB/Amersham); phosphodiesterase I (crotalus adamanteus venom; Amersham); DNase I (Sigma Chemical Co.); DNase II (Sigma Chemical Co.); alkaline phosphatase (calf intestinal; Boehringer Mannheim); and nuclease S1 (Life Technologies, Inc.). All other chemicals were of commercial origin and used as received. Water was purified using a Millipore Milli-Q deionizer. Oligonucleotides were synthesized on an Applied Biosystems Model 392 synthesizer by the phosphoramidite method on a 1 µmol or 10 µmol scale. DNA was purified by DPAGE and recovered by the crush and soak method; the eluted DNA was subsequently subjected to chromatography on a Waters SepPak C18 column according to the manufacturer’s instructions. Ethanol precipitations were accomplished as described (13)
. After ethanol precipitations, pellets were dried in vacuo. UV Spectra were measured on a Hewlett-Packard 8452A spectrophotometer. Absorbance measurements were made on a Perkin-Elmer Lambda 3A spectrophotometer unless otherwise stated. 5'-Terminal radiolabeling was accomplished, following the T4 polynucleotide kinase manufacturer’s instructions, and stopped by heating at 90°C for 1 min, followed by ethanol precipitation. Radiolabeled DNA was used for all of the following experiments, except for preparative cross-linking and LC-ESI MS of enzymatic hydrolysates. Maxam-Gilbert G reactions were performed as described previously (13)
.
Analytical Scale BCNU Cross-linking of DNA Oligomers.
Analytical scale cross-linking reactions with BCNU used ca. 0.1 OD of total DNA (for self-complementary sequences, 0.001 OD of the radiolabeled strand and 0.1 OD of the unlabeled strand; for nonself-complementary sequences, 0.05 OD of the radiolabeled strand and 0.05 OD of the unlabeled complementary strand) dissolved in 20 µl of 80 mM aqueous sodium chloride/50 mM sodium phosphate buffer (pH 7), heated (90°C, 1 min), and cooled (25°C, 15 min). To the DNA solution, 0.5 µl of BCNU (1 M freshly prepared solution in ethanol) was added. The mixture was incubated 16 h at 37°C, terminated by precipitation, and analyzed by DPAGE. Samples were not thermally denatured before loading. Electrophoresis was performed at 37°C to minimize thermal decomposition of cross-linked DNA. Single-stranded and cross-linked DNA were visualized by autoradiography. Yields of interstrand cross-links were determined by phosphorimagery using a Molecular Dynamics 400A phosphorimager. Data analysis was performed using ImageQuant software (Molecular Dynamics).
Sequence-Random DNA Cleavage.
Iron(II)/EDTA-promoted cleavage reactions were conducted on ca. 10,000 cpm (Geiger counter) of radiolabeled, gel-purified, interstrand cross-linked DNA. Added to the walls of a microfuge tube containing DNA in 6 µl of water and 1 µl of 50 mM Tris buffer (pH 8) were 1 µl of 500 µM (NH4)2Fe(SO4)2/1 µM EDTA, 1 µl of 10 mM sodium ascorbate, and 1 µl of 0.1 M H2O2. The reaction was initiated by centrifugation and stopped after 1 min by the addition of 1 µl of 0.1 M thiourea and vortexing. The resulting solutions were analyzed by DPAGE, followed by drying of the gel, autoradiography, and phosphorimagery.
Preparative Scale BCNU Treatment of GC4G4C.
The preparative cross-linking reaction with BCNU of the DNA containing the central sequence C4G4 was conducted on 250 OD of duplex DNA [16 ml, 80 mM aqueous sodium chloride/50 mM sodium phosphate buffer (pH 7.0)], to which was added BCNU (600 µl of a 1 M freshly prepared solution in ethanol). The reaction was incubated (37°C, 16 h) and ethanol-precipitated. The precipitate was dissolved in water (3–5 ml) and desalted on a Sephadex G-50 column (8 x 0.5 cm) equilibrated with ammonium acetate buffer (10 mM, pH 7.0), and fractions containing DNA were pooled and dried in vacuo. The sample was dissolved in loading solution (600 µl, 1:1 v/v solution of H2O:formamide), and the interstrand cross-linked DNA was separated from residual single strands by DPAGE, as described for DNA purification. The resulting cross-linked DNA (totaling about 1.3 OD) in H2O, was stored at -20°C as a 0.1 OD/10 µl aqueous solution.
Enzymatic Hydrolysis of BCNU Interstrand Cross-linked GC4G4C.
The cross-linked DNA (0.4 OD) was hydrolyzed enzymatically in a total volume of 30 µl including calf intestinal phosphatase (4 µl, 10 units), DNase I (2 µl, 10 units), DNase II (2 µl, 4 units), phosphodiesterase I (0.5 µl, 0.5 units), and nuclease S1 (2 µl, 1800 units) in buffer [50 mM Tris-HCl and 10 mM MgCl2 (pH 8.5)]. Reactions were incubated (3 h, 37°C) and analyzed by LC-MS, as described below.
Synthesis of Compound 4.
The synthesis of compound 4, 1-[N3-deoxycytidyl]-2-[N1-deoxyguanosyl]ethane, was accomplished as described by Bodell and Pongracz (14)
, except that the final purification step was done by HPLC on a Rainin Dynamax, C18, 5 µm x 250 mm x 10 mm column, 5 ml/min, using the following separation conditions. Solvent A was 0.1 M triethylammonium acetate (pH 7.5); solvent B was acetonitrile. The gradient was 0–13% B, linearly over 10 min, followed by a linear gradient from 13–100% B over 2 min. The UV spectrum of the synthetic compound was recorded on a Rainin gradient analytical HPLC system equipped with a Waters 994 photodiode array detector. The purified substance was characterized by its mass spectrum and UV spectrum; MS(ESI): m/z 521.1 ([M+H+]+), 405.2 ([M+H+-deoxyribosyl+]+), 289.1 ([M+H+- 2 x deoxyribosyl+]+; UV [aqueous solution, 10 mM NH4OAc (pH 7.0)]: 
max = 258, 275 nm.
Synthesis of Compound 5.
The synthesis of compound 5, 1,2-(di-guan-7-yl)ethane, was accomplished by a modified procedure of Tong and Ludlum (10)
. A solution of guanosine (100 mg; Aldrich Chemical Co.), 1,2-dibromoethane (100 µl; Aldrich Chemical Co.), and DMSO (1.0 ml; Baker) was heated (37°C, 52 h). Sodium formate [1.5 ml, 0.05 M (pH 4.5)] was added to precipitate most of the excess guanosine. After centrifuging the sample, the supernatant was chromatographed on a 1.5 x 60-cm column of SP-Sephadex C25–120 (Sigma Chemical Co.) using a linear gradient of degassed 0.05–0.5 M sodium formate (pH 4.5) at a flow rate of 
0.75 ml/min. The progress of the chromatography was monitored by UV at 260 nm, collecting 9-ml fractions. The resulting chromatogram exhibited four major peaks, the first three of which were shown, in order of increasing retention time (Rt), to be guanosine, 7-(ß-bromoethyl)guanine, and 7-(ß-bromoethyl)guanosine by their UV and electrospray mass spectra. Fractions corresponding to the last eluting peak were combined and lyophilized to give 2.0 mg of material (16 OD). MS(ES in H2O): m/z 615 (10%), 593 (5%), 461 (35%), and 329 (100%); UV(H2O) max = 258, 282 nm (consistent with N7-to-N7 connectivity). This solution was desalted by isocratic HPLC and lyophilization. A total of 12 injections of the solution were made to a 3.9 mm x 30 cm C18 column (Waters µBondapak) with 2% acetonitrile/0.05 M NH4OAc, pH 5.0, as the mobile phase, at a flow rate of 2.25 ml/min. The collected fractions (retention time 19 min) were pooled and lyophilized to give 4.8 OD (260 nm) of material which was dissolved in water and lyopholized two additional times. The resulting residue was dissolved in aqueous acid (250 µl, 0.1 N hydrochloric acid), and the solution was heated (100°C, 20 min). Analysis of this solution by HPLC, using conditions closely approximating those used by Tong et al. (15)
, gave a major peak at a Rt of 19.2 min. MS(LC-ES): m/z 329, [M+H+]+.
LC-ESI MS of Enzymatic Hydrolysates.
LC-MS analysis was performed on enzymatically hydrolyzed samples, as described below. Injections (20 µl, 0.2 OD) were made to an Alltech Econosphere C18 analytical column attached to a Shimadzu LC-10AD pump system and controller with a dual wavelength UV detector (SPD-10AD, 260 nm). The separation conditions were: solvent A, 10 mM ammonium acetate (pH 7.0); solvent B, acetonitrile (flow rate = 1 ml/min). The gradient was 7 min isocratic at 8% B, 10-min linear gradient to 30% B, 10-min linear gradient to 40% B, 5-min linear gradient to 8% B, and 5-min isocratic at 8% B. The column was allowed to equilibrate for at least 15 min between injections. The LC system was linked (10/90 split) to a VG Quattro II/MassLynx PC DS triple quadrupole mass spectrometer [4000 Da (Q1 + Q3), unit resolution (Q1 + Q3), 100°C, probe voltage 3.4 KV, HV lens 0.9 KV, cone 30 V, data acquisition was 200–2000 Da]. Each substance was analyzed as a combined spectrum, which gave a spectrum that was a summation of the scans recorded for the duration of peak elution that was background subtracted over the regions just before, and trailing, the peak of interest.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows the DNA sequences used in this study. These included a DNA containing no GC bp (G0), as well as a family of oligonucleotides containing from two to eight GC bp isolated from one another by intervening AT bp (G2i-G8i). One sequence (G10i) also contained 10 GC pairs, of which 8 were present at the sequence 5'-d(CG) or 5'-d(GC). Five DNAs had runs of Gs and Cs, sometimes abutted with one another as in the central sequences 5'-d(GC) (G2r), 5'-d(CCGG) (G4r), and 5'-d(GGGCCC) (G6r) and two other DNAs containing 8 or 10 GC pairs in runs of 4 (G8r) or 5 (G10r). Additionally, the sequences of these DNAs were selected to maximize the diversity of the bp immediately flanking each GC pair. Of the 16 possible trinucleotide combinations (N1GN2), 15 are represented at least one time within this family of DNAs. DNA containing a central GC-rich core, designated herein as GC4G4C, was used for characterization of the cross-linking lesion.
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0.6% yield. This residual duplex species could be due either to the presence of end cross-links or an increased Tm (melting temperature) of DNA duplexes containing monoadducts. These residual species were not investigated further. Increasing the total number of GC pairs while keeping them isolated with respect to one another (DNAs G2i through G10i) increased the yield only modestly, with a maximal yield in this series for G8i of 1.7%. DNAs containing runs of GC bp and, particularly, longer runs were severalfold more efficiently cross-linked, with a maximal yield of 3.7% for the DNA containing two runs of five contiguous Gs (G10r).
The actual site of the cross-link within a synthetic DNA duplex was analyzed by sequence random fragmentation of a selectively end-radiolabeled sample. This experiment was conducted on a number of DNA sequences; Fig. 3
shows representative data for the sequence GC4G4C. DNAs were 5'-radiolabeled and treated with BCNU, and the resulting interstrand cross-linked product was purified by DPAGE. The purified material was fragmented using the iron(II)-EDTA-catalyzed decomposition of hydrogen peroxide, under single-hit conditions, which is well-known to result in a relatively sequence-random cleavage of the DNA sugar-phosphate backbone (19)
. Cleavage reactions between the radiolabel and the site of an interstrand cross-link release a fragment comparable in length to that found in the starting (native) DNA. In contrast, cleavage reactions downstream of the cross-link (relative to the position of the radiolabel) leave the radiolabel attached through the cross-link to the opposite strand, greatly retarding the mobility of this cleavage product. If the cross-linked product is structurally homogeneous (cross-linked at a single site within the DNA), DPAGE analysis of the cleavage products will provide a ladder of bands corresponding to the fragmentation ladder for the native DNA, progressing from fragments of high mobility to those of lower mobility, but the ladder of bands will be truncated short of the full-length DNA due to the position of the cross-link. By comparison, noncross-linked DNA will show a ladder of bands of uniform intensity. This procedure has proven useful in elucidating the site of cross-linking with agents such as psoralen (20)
, mitomycin C (21)
, formaldehyde (22)
, and nitrous acid (23)
, which exhibit some sequence specificity in the cross-linking reaction. In contrast, the data for the BCNU interstrand cross-linked DNA (Fig. 3)
exhibit an essentially monotonic decrease in the intensity of bands relative to the intensity of bands in the native cleavage ladder, as one progresses from the radiolabeled to the nonradiolabeled end. This result is strong evidence that the cross-links are uniformly distributed throughout the G-rich region of the DNA.
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revealed primarily the parent mononucleosides and also a more strongly retained peak eluting at 14.9 min, which coeluted with the authentic sample of lesion 4 (data not shown). The mass spectrum of peak 4 (Fig. 2B)
was identical to that of the authentic standard. The UV spectrum of this substance showed the same spectral features as the authentic sample (Fig. 2C)
and, accordingly, this substance has been identified as 1-[N3-deoxycytidyl]-2-[N1-deoxyguanosyl]ethane. Using an estimated molar extinction coefficient of this substance at 260 nm of 19,555 M-1cm-1 (the sum of the extinction coefficients of N1-methyl-deoxyguanosine and N3-ethyl-deoxycytidine, an underestimate given the tendency of covalently tethered nucleic acid bases to stack in solution, resulting in hypochromism), we calculate that this digestion returned a minimum of 0.35 equivalents of this lesion/DNA duplex consistent with peak 4 being responsible for the interstrand cross-link. Enzymatic hydrolysates of DNA recovered from the single-stranded region of the cross-link purification gel showed no evidence of this substance. Thus, peak 4 constitutes at least 35% of the lesions responsible for interstrand cross-linking in the GC4G4C sequence.
Additionally, a trace substance was found in the enzymatic hydrolysate (Rt = 14.0 min), the mass spectrum of which was consistent with the substance 1,2-(diguan-7-yl)ethane, 5 (m/z 329; data not shown). This substance could not be compound 5 because a synthetic sample of compound 5 (10)
eluted at a much different Rt (Rt 
6 min; data not shown) than the trace substance. However, the substance detected as m/z 329 could have been derived from 1,2-di-(deoxyguan-7-yl)ethane (6) that had lost its sugar moieties in the electrospray source, the m/z 329 peak corresponding to the [M + [3H]+-2 x deoxyribosyl+]+ fragment of 6. The UV peak area of this trace substance appeared to be <5% that of 4 and was barely detectable. The vanishingly small quantity of this trace substance prevented its further characterization.
Also present in the enzymatic hydrolysate were trace quantities of substances, the masses of which correspond to monoadducts known to result from BCNU exposure. These included chloroethyldeoxyguanosine (m/z 214.3, Rt 12.4 min) and hydroxyethylguanine (m/z 196.5, Rt 8.3 min). The relative Rts of these substances correspond to those obtained by previous researchers (15)
. These substances occurred in approximately the same proportions (by UV detector peak areas) in the interstrand cross-linked samples as they did in samples from residual single strands of BCNU-treated DNAs. Their presence in the samples of interstrand cross-linked DNAs is evidence for multiple alkylation events on a subpopulation of the interstrand cross-linked DNAs.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is generally accepted that the DNA-DNA interstrand cross-links formed in duplex DNA by BCNU bridge N1 of G and N3 of C residues, a notion that has previously been supported by indirect observations. Specifically, two candidate structures for the interstrand cross-link have been isolated. One of these (structure 4) links G and C via an ethylene bridge, whereas the other (structure 5) links two guanine residues at their N7 positions. It has been noted that O6-alkylguanine-DNA alkyltransferase inhibits the formation of interstrand cross-links by BCNU (4)
and also inhibits the formation of the lesion linking deoxyguanosyl and deoxycytidyl residues (27)
. In contrast, the alkyltransferase has no effect on the impact of N7 alkylation by BCNU. Taken together, these data argue against alkylation at N7 of G as important in DNA interstrand cross-linking, rendering structure 5 unlikely to be the relevant lesion. In contrast, model compounds have clearly shown that O6-2-haloethylguanine derivatives can undergo a cyclization reaction at N1, followed by ring opening to afford an N1-ethyl derivative of G (as in Fig. 1
). This mechanism nicely accounts for the inhibitory impact of the alkyltransferase on cross-linking and points to lesion 4 as the source of the interstrand cross-link. The experimental results described herein directly confirm this hypothesis by the direct isolation of lesion 4 as the major adduct from an interstrand cross-linked synthetic DNA duplex.
It has been previously observed in polynucleotides that the efficiency of monoadduct formation by BCNU in DNA increases with its GC content (17
, 28)
. This effect is qualitatively paralleled by the production of interstrand cross-links in short synthetic DNA duplexes exposed to BCNU. We observed (Table 1)
that the efficiency of interstrand cross-link increases with the ratio of GC bp to total DNA bp. GC content alone, however, is not a good predictor of the efficiency of cross-linking with BCNU because DNAs of similar base composition showed distinct differences in the efficiency of cross-linking. The latter argues for an impact of nucleotide sequence on cross-linking efficiency. We find that DNAs with runs of G in one strand are most efficiently cross-linked. For example, the DNA duplex that contained 10 G residues as two of five runs was some 4-fold more efficiently cross-linked than a similar DNA in which no two G residues were adjacent to one another. This observation is in quantitative agreement with the known greater nucleophilicity of runs of G residues. The observation of enhanced cross-linking efficiency in the DNAs containing runs of dG, likewise, argues against lesion 5, which bridges two G residues as the nucleus of the interstrand cross-link. In this case, one would expect a sequence preference to result, such as 5'-GC, 5'-GNC, or 5'-GNNC, and so on. The literature is replete with instances of these types of sequence preferences for DNA-modifiying agents that alkylate the N7 position of dG (11
, 18
, 29
, 30)
. The most direct evidence that the interstrand cross-link formed by BCNU in synthetic DNA duplexes bridges N1 of G and N3 of C is the isolation of lesion 4 from BCNU-treated interstrand cross-linked DNAs separated from residual single strands. However, the yield of cross-link lesions to interstrand cross-linked DNA duplexes was low and leaves unresolved the issue of whether there is some additional lesion responsible for up to 65% of the interstrand cross-links. The lack of sequence preference of BCNU interstrand cross-linking observed in the iron(II)EDTA cleavage assay argues against the remaining cross-link deriving from compound 6. The aforementioned evidence, when taken together, all point to lesion 4 as the DNA interstrand cross-link: (a) substance 4 is essentially the only cross-link substance found in synthetic interstrand cross-linked samples; (b) cross-linking correlates with GC content in DNA oligos and, particularly, in runs of Gs and does not have a specific sequence preference; (c) O6-alkylguanine-DNA alkyltransferase is known to inhibit interstrand cross-links and to inhibit the formation of dG to dC cross-links in DNA, but does not inhibit formation of N7 alkylated DNAs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant GM-45804. 
2 Present address: Department of Chemistry, Lewis and Clark College, Portland, OR 97219.
3 To whom requests for reprints should be addressed, at Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195-1700.
4 The abbreviations used are: CENU, chloroethylnitrosourea; BCNU, N,N'-bis(2-chloroethyl)-nitrosourea; G, deoxyguanosine; C, deoxycytidine; DPAGE, denaturing PAGE; OD, the quantity of DNA which, when dissolved in 1 ml of water, gives a UV absorbance reading of 1 at 260 nm; LC, liquid chromatography; HPLC high-pressure LC; ESI, electrospray ionization; MS, mass spectrometry.
Received 4/ 1/99. Accepted 7/ 8/99.
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