Antibody Recognition of Melphalan Adducts Characterized Using

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Antibody Recognition of Melphalan Adducts Characterized Using Immobilized DNA: Enhanced Alkylation of G-Rich Regions in Cells Compared to in Vitro Hazel McCartney,† Anthea M. Martin,‡ Peter G. Middleton,§ and Michael J. Tilby*,† Cancer Research Unit and Department of Haematology, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K. Received August 17, 2000

The bifunctional alkylating agent, melphalan, forms adducts on DNA that are recognized by two previously described monoclonal antibodies, MP5/73 and Amp4/42. Immunoreactivity to MP5/73 was lost when alkylated DNA was exposed to alkaline pH, while Amp4/42 only recognized the structures formed after the alkali treatment. Competitive enzyme-linked immunoadsorbent assays (ELISAs) indicated that in 0.01 and 0.1 M NaOH, loss of immunoreactivity to MP5/73 occurred with half-lives that were at least 2-fold longer than half-lives for gain of immunoreactivity to Amp4/42. This supported previously published evidence that Amp4/42 did not simply recognize all the products formed by alkali treatment of adducts that were initially recognized by MP5/73. Adducts recognized by MP5/73 on RNA were considerably more stable at 100 °C and pH 7 than adducts on DNA. This was consistent with the hypothesis that immunorecognition involved N7 guanine adducts and ruled out the involvement of phosphotriesters in immunoreactivity. Synthetic oligodeoxyribonucleotides, covalently immobilized onto 96-well plates, were reacted with melphalan and incubated for various periods with alkali, and then the levels of adducts recognized by each antibody in replicate wells were assayed by a direct binding ELISA. Adducts formed on oligodeoxyguanylic acid were recognized very weakly by Amp4/42, unlike other DNA sequences that were tested. Retention of immobilized DNA during alkali treatment was confirmed by immunoassay of cisplatin adducts. Poor recognition by Amp4/42 of adducts in oligodeoxyguanylic acid was confirmed by a competitive ELISA. Amp4/42, unlike MP5/73, efficiently recognized adducts resulting from alkylation of DNA with chlorambucil. It is concluded that the two antibodies recognized melphalan adducts in different DNA sequence environments and that this explains (a) the different alkali stability of immunoreactive adducts and (b) previous results which showed that, in DNA from melphalan-treated cells, adducts recognized by Amp4/42 formed a smaller proportion of total adducts compared to DNA alkylated in vitro. The results presented here indicate that this was caused by a marked cellular influence on the overall sequence-dependent pattern of DNA alkylation or repair.

Introduction Melphalan (1a) represents a group of drugs based on the bifunctional alkylating agent “nitrogen mustard”. Drugs of this class are long-established, clinically important anticancer agents (1) and are human carcinogens (2). Recently, nitrogen mustards have also been developed in the context of antibody-directed, enzyme-activated prodrug anticancer therapy (3, 4). Consequently, analysis of the interaction of these drugs with DNA is relevant to their mechanisms of cytotoxicity and carcinogenesis as well as to mechanisms of drug resistance. The major nucleic acid-base adducts formed by melphalan have * To whom correspondence should be addressed: Cancer Research Unit, Cookson Building, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K. Telephone: [0] 191 222 5309. Fax: [0] 191 222 7556. E-mail: [email protected]. † Cancer Research Unit, University of Newcastle upon Tyne. ‡ Present address: Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, Dundee DD1 9SY, U.K. § Department of Haematology, University of Newcastle upon Tyne.

been characterized (5-8), and the predominant site of alkylation was shown to be N7 of guanine. Bifunctional alkylation of DNA by melphalan involves guanineguanine and guanine-adenine cross-linkage (6, 8), and the sequence-related basis for interstrand cross-links has been investigated (9).

A monoclonal antibody (MP5/73) raised against adducts formed by melphalan on DNA (10) appeared to

10.1021/tx000178z CCC: $20.00 © 2001 American Chemical Society Published on Web 12/15/2000

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Chem. Res. Toxicol., Vol. 14, No. 1, 2001 Scheme 1. Alkali-Induced Ring Opening of a Guanine Base in DNA, Akylated at N7 by Melphalan

recognize N7 guanine adducts (Scheme 1) (5, 10). Another monoclonal antibody (Amp4/42) appeared to recognize the formamidopyrimidine structure resulting from alkaliinduced ring opening of N7 guanine-melphalan adducts (Scheme 1) (11). These antibodies have been used in past and ongoing studies of the levels of DNA alkylation induced in patients by melphalan (12) and in analyses of individual tumor and bone-marrow stem cells by immunofluorescence (13). The present work was prompted by the observation that immunoreactivity to antibody Amp4/42, of adducts resulting from exposure of DNA to melphalan and then to alkali, was 10-fold lower when the reaction of melphalan with DNA occurred in cells rather than in a solution of purified DNA (11). Since adducts recognized by MP5/73 did not exhibit a comparable effect (12), this observation implied that MP5/73 and Amp4/42 were not recognizing the same adducts before and after ring opening. The same observation also indicated an undefined difference between the properties of adducts formed in cells and those formed in a solution of pure DNA. These implications are relevant to understanding the specificity of the antibodies and hence the interpretation of analyses made with them. They are also relevant to understanding the reaction of melphalan with DNA in cells. This report describes (a) further evidence for the nonequivalence of the initial melphalan-DNA adducts recognized by antibodies MP5/73 and Amp4/42, (b) evidence that both these antibodies recognized N7 modification of guanine, and (c) results supporting the hypothesis that the nonequivalence resulted from differential recognition of the same adduct in different DNA sequence environments. To facilitate this investigation, an apparently new approach was developed for immunological analysis of DNA modifications. This involved covalent immobilization of DNA fragments to the wells of microtiter plates, whereby the effects of exposure to drug, then alkali, and, finally, antibodies were more easily investigated.

Materials and Methods Caution: Melphalan and chlorambucil are human carcinogens (2). Therefore, appropriate precautions must be taken to avoid human exposure. Drugs. Unlabeled melphalan, chlorambucil, and cisplatin were from Sigma. Radioactively labeled drugs were as follows: [ring-3H]melphalan (34 GBq/mmol, Amersham, Bucks, U.K.), [chloroethyl-1,2-14C]melphalan (1.85 GBq/mmol, Moravek Biochemicals Inc.), and [chloroethyl-1,2-14C]chlorambucil (525 MBq/ mmol, National Cancer Institute, Bethesda, MD).

McCartney et al. Nucleic Acids. DNA (highly purified, from calf thymus) and RNA (from yeast, highly polymerized) were from Merck. Oligonucleotides were synthesized using an Applied Biosystems model 392 synthesizer, using conventional phosphoramidite chemistry according to the manufacturer’s instructions. Restriction-digested calf thymus DNA was prepared using HaeIII. Complete digestion was confirmed by agarose gel electrophoresis. Reaction of Nucleic Acids with Melphalan and Chlorambucil. The nucleic acid, dissolved in buffer A [50 mM sodium phosphate (pH 7.0)], was incubated at 37 °C with mixtures of radioactive and nonradioactive drug for 1 or 2 h. Nucleic acids were separated from unbound drug by gel filtration chromatography (Sephadex G-25 or G-75). Elution was with buffer B [50 mM NaCl and 50 mM sodium phosphate (pH 7.0)]. Levels of alkylation were determined from the specific radioactivities of drug and of the alkylated DNA preparations. Treatment of DNA with Alkali. A NaOH solution (4 M volumetric grade solution) was added to the nucleic acid solution in buffer B to give the desired final concentration (when NaOH was added to give a final concentration of 0.01 M, the pH was 11.9). After incubation for the appropriate time, the pH was reduced to approximately 7.8 by addition of 0.5 M sodium phosphate (pH 7). The nucleic acid was concentrated to about 50 µL using centrifugal ultrafiltration devices (Centricon from Amicon, molecular weight cutoff of 10 000 for calf thymus DNA and 3000 for oligodeoxynucleotides) and then diluted to a volume of 500 µL in buffer B. The concentrations of drug adducts and of nucleic acids in the resulting preparations were determined from radioactivity and OD260, respectively. Competitive ELISA.1 The assays using MP5/73 and Amp4/ 42 were performed as described previously (10, 11). In brief, several dilutions of antigen were mixed with a constant amount of diluted antibody and incubated in wells coated with a standard amount of alkylated DNA which, for assays using antibody Amp4/42, had been exposed to alkali. The amount of antibody bound to the wells was determined by a fluorogenic enzyme assay (see below). The quantities of adducts per assay well that caused a 50% reduction of the assay signal (K-value) were determined by a curve-fitting procedure (10). Covalent Attachment of DNA Fragments and Oligonucleotides to 96-Well Plates. The procedure was based upon that of Rasmussen et al. (14) using “Covalink” plates (Nunc Plasticware, Life Technologies, Paisley, Scotland). However, in our hands, the attachment of nucleic acids to the wells was not dependent upon the presence of 5′-terminal phosphate groups. The plastic surfaces of these wells carry covalently attached spacer molecules on each of which a secondary amino group is located near the free end. Aliquots (37.5 µL) of solutions of DNA restriction fragments or synthesized oligonucleotides [10 µg/mL in 10 mM methylimidazole/HCl buffer (pH 7.0)] were added to each well. Then, to each well was added 12.5 µL of a freshly prepared solution of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (20 mM in the methylimidazole buffer described above). After a brief mixing, the plates were incubated overnight at 37 °C in a sealed plastic box. The plates were then washed six times with wash solution [0.75 M NaCl, 0.75 mM Na3 citrate, and 0.075% (w/v) Sarkosyl detergent (pH 7.4)] and then twice with buffer B. The plates, filled with buffer B, were stored at -20 °C. Before use, the wells were incubated with 100 µL of a 0.1 M NaOH solution for 30 min (20 °C) as this reduced the level of background binding of the MP5/73 antibody. They were then washed with PBS (phosphate-buffered saline) [140 mM NaCl and 10 mM sodium and potassium phosphates (pH 7.4)]. Treatment of Immobilized DNA with Melphalan and NaOH. Solutions of melphalan in buffer A were prepared as described above, and 50 µL aliquots were added to the wells carrying immobilized DNA. After incubation (37 °C for 1 h), the 1 Abbreviations: ELISA, enzyme-linked immunoadsorbent assay; PBS, phosphate-buffered saline; K-value, concentration of substance (per assay well) that causes a 50% reduction in the magnitude of the signal of a competitive ELISA.

Immunological Analysis of Melphalan-DNA Adducts

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Figure 1. Time course of changes induced by NaOH to melphalan adducts on denatured DNA (alkylation level, 46 µmol of melphalan/g of DNA) assessed by competitive ELISAs using antibodies MP5/73 (O) and Amp4/42 (2). The NaOH concentration was 0.01 (A and B) or 0.1 M (C and D). (A and C) Points represent the means of at least two determinations of K-values (the quantity of adducts needed to cause a 50% reduction in the magnitude of the assay signal). Dashed lines denote decreases in Amp4/42 K-values from an initial value that was too high to measure. (B and D) Points represent reciprocals of mean K-values, and lines represent equations for one-phase exponential decay or association fitted to the points. Fitted half-life values were as follows: for 0.01 M NaOH, 69 and 30 min for MP5/73 and Amp4/42, respectively; and for 0.1 M NaOH, 9.7 min for MP5/73. drug was removed by washing with buffer A and then plates, containing buffer A, were stored at -20 °C. Wells were washed with water before addition of a 0.1 M NaOH solution (100 µL per well). The wells were sealed and incubated either at 37 °C for 30 min or at 20 °C for various times. The wells were then washed with buffer A and analyzed on the same day by a direct binding ELISA. Immunological Detection of Adducts on Immobilized DNA. Aliquots (50 µL) of solutions of monoclonal antibodies (culture supernatants diluted 500-fold) in buffer C [PBS containing 1% (w/v) BSA and 0.1% (v/v) Tween 20] were added to each well and the wells incubated at 37 °C for 1 h. After the wells had been washed five times with PBST [PBS containing 0.1 % (v/v) Tween 20], 50 µL of biotinylated sheep anti-rat Ig (from Sigma, diluted 2500-fold in buffer C) was added to each well. After incubation (37 °C for 30 min), the plates were washed three times with PBST and 50 µL of the streptavidin-βgalactosidase conjugate (from Boehringer, typically diluted 10000-fold in buffer C) was added to each well. After incubation (37 °C for 30 min), the plates were washed five times with PBST and 50 µL of the substrate solution was added to each well. The substrate solution was PBS containing 0.4 mg/mL 4-methylumbelliferyl-β-D-galactoside and 10 mM MgCl2. After incubation for various periods, the fluorescence in each well was read using a plate reader (Fluoroscan, Labsystems) with excitation at 355 nm and emission at 460 nm.

Results Time Course of Effects of Alkali on MelphalanDNA Adducts. If antibody MP5/73 recognized N7 gua-

nine adducts which were all converted by alkali to ringopened adducts recognized by Amp4/42 (but not by MP5/ 73), then gain of immunoreactivity to Amp4/42 should have occurred at the same rate as the loss of immunoreactivity to MP5/73. DNA was heat denatured before reaction with melphalan to avoid changes in immunorecognition of adducts that would have resulted from alkaliinduced conformational changes to native DNA (10). DNA alkylated with [3H]melphalan was incubated at 37 °C for various times in 0.01 or 0.1 M NaOH. Levels of total adducts in the neutralized and desalted DNA solutions were determined from measurements of radioactivity. For each alkali-treated DNA preparation, the immunoreactivities to each antibody were determined by competitive ELISAs. The effects of the incubation in alkali on the quantities of adduct needed to cause 50% inhibition in the assays (K-values) are plotted in panels A and C of Figure 1 for 0.01 and 0.1 M NaOH, respectively. Both sets of results indicated that decline of MP5/73 immunoreactivity (i.e., increase in the K-value) proceeded more slowly than gain of immunoreactivity to Amp4/42 (i.e., decrease in the K-value). At 0.01 M NaOH, little change in the MP5/73 K-value occurred during the first 30 min despite marked changes in immunoreactivity of the same samples toward antibody Amp4/42. To estimate half-lives for the reactions, the single-exponential decay equation was fitted to reciprocals of the K-values. Reciprocals were used because these values are directly proportional to the

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Figure 3. Effects of heating at 100 °C on melphalan adducts on RNA (O) or denatured DNA (2) assessed by a competitive ELISA using antibody MP5/73. Points represent the means ((SE) of K-values (the quantity of adducts needed to cause 50% inhibition) from three experiments. RNA and DNA preparations were initially alkylated at 100 and 41 µmol of adduct/g of nucleic acid, respectively. The inset shows lines representing one-phase exponential decay equations fitted to the reciprocal of the mean K-values. The half-lives were 68 and 6 min for RNA and DNA, respectively.

Figure 2. Time course of changes induced by NaOH to melphalan adducts on native DNA (alkylation level, 2.4 µmol of melphalan/g of DNA) assessed by competitive ELISAs using antibodies MP5/73 (O) and Amp4/42 (2). The NaOH concentration was 0.01 (A) or 0.1 M (B). Points represent the means of at least two determinations of K-values (the quantity of adducts needed to cause 50% inhibition). Dashed lines denote decreases in Amp4/42 K-values from an initial value that was too high to measure.

levels of immunoreactive adducts. The half-life for loss of adducts recognized by MP5/73 was estimated to be approximately 69 min, while the half-life for gain of adducts recognized by Amp4/42 was approximately 30 min (Figure 1B). In 0.1 M NaOH, the increase in immunoreactivity to Amp4/42 was essentially complete by the first sampling time at 5 min. Adducts not exposed to alkali were not detectably immunoreactive at 68 000 fmol/well (i.e., K-value > 68 000 fmol/well). Hence, the immunoreactivity had increased by more than 10-fold (i.e., more than three half-lives), indicating a half-life of less than 2 min. For the same samples, the K-value for MP5/73 was not significantly altered at the 5 min time point and the estimated half-life for loss of immunoreactivity to MP5/73 was 9.7 min (Figure 1D). In a similar analysis of melphalan adducts formed by reacting 3H-labeled melphalan with native DNA (Figure 2), gain of immunoreactivity to antibody Amp4/42 occurred rapidly, as for denatured DNA; however, immunoreactivity to MP5/73 initially increased. This was followed by a slow decline similar to that observed with denatured DNA. These observations are consistent with an alkali-induced denaturation of the DNA that increased the immunoreactivity of the adducts recognized by MP5/ 73, as expected from previous results (10, 13). This occurred before chemical reactions had a major effect on these adducts. The half-life estimates are approximate, but for several conditions and experiments, the half-life estimated for

the gain of immunoreactivity to Amp4/42 was less than half the value for loss of immunoreactivity to MP5/73. The differences in half-lives were reinforced by the fact that both immunoassays were performed on the same alkali-treated DNA preparations. These differences in rates of alkali-induced change are further evidence that MP5/73 and Amp4/42 do not recognize exactly equivalent adducts. Heat Stability of Melphalan Adducts Recognized by MP5/73. It was a concern that the greater alkali stability of adducts recognized by MP5/73 might have been due to the involvement of phosphotriesters (see Discussion). It was possible to test this by taking advantage of the fact that MP5/73 efficiently recognizes melphalan adducts on RNA as well as DNA (10). Phosphotriesters on RNA are labile (15, 16), while the N-glycosidic bond of N7 alkylguanylic acid is more thermostable than the equivalent bond in the deoxynucleotide (17). The heat stability of immunoreactive adducts was compared in RNA and DNA. DNA, which had initially been heat denatured, and RNA were reacted with radioactive melphalan. They were then each heated at 100 °C in buffer B for various times before analysis using a competitive ELISA with antibody MP5/73. The immunoreactivities were calculated with respect to the total amount of adduct present in the solution of heated DNA. This included alkylated bases released from the DNA; however, these had an insignificant effect on the assay as they have extremely low immunoreactivity (5). The rate of loss of immunoreactivity of adducts on RNA was approximately 10 times slower than the rate for adducts on DNA with half-lives estimated as 68 and 6 min for adducts on RNA and DNA, respectively (Figure 3). Thus, moieties recognized by MP5/73 demonstrated the expected pattern of thermal stability for depurination of alkylated guanine adducts. Pretreatment of the alkylated DNA with 0.1 M NaOH (10 min at 37 °C) caused a large change in the immunoreactivity of adducts to


Figure 4. Effects of brief exposure to alkali on heat stability of melphalan adducts recognized by antibody MP5/73. Denatured calf thymus DNA that had been alkylated with melphalan (41 µmol/g of DNA) was incubated for 10 min at 37 °C either (O) in 0.1 M NaOH or (b) at pH 7.0. These preparations were then heated at 100 °C and neutral pH for various times. The alkali-treated and heat-treated samples were also assayed using antibody Amp4/42 (1).

Amp4/42, but did not influence the thermal lability of adducts recognized by MP5/73 (Figure 4). This supports the independence of the two classes of adducts. Adducts recognized by antibody Amp4/42 showed an increased stability after alkali treatment, as was demonstrated previously (11) and is confirmed in Figure 4. Experiments Using Immobilized DNA. Available evidence (see the Discussion) indicated that the immunoreactive adducts for both antibodies were on guanine N7. Subsequent experiments were aimed at testing the hypothesis that the nonequivalence of adducts recognized by the two antibodies was related to the DNA sequence in which they were located. To facilitate these investigations, an approach was developed in which DNA was covalently immobilized to amino-derivatized microtiter wells. Initial characterization of this method used restriction fragments of calf thymus DNA. Wells coated with DNA were incubated for 1 h with solutions containing melphalan at a range of concentrations. The drug was removed by washing the wells. For each drug concentration, four wells were assayed by a direct binding ELISA using antibody MP5/73 and a further four wells were incubated with 0.1 M NaOH for 60 min (37 °C). These wells were then washed and assayed using antibody Amp4/42. Typical results (Figure 5A) show that, as expected, reaction of melphalan with the immobilized DNA resulted in a concentration-dependent increase in the magnitudes of the fluorescent signals. The immunoassay signals were dependent upon the presence of DNA (data not shown). The relationship between assay signal magnitude and melphalan concentration was not reliably linear, even at the lower drug concentrations. It was concluded that these experiments could not provide accurate kinetic data, but would, nevertheless, be useful for semiquantitative comparisons between different DNA sequences. Synthetic oligodeoxynucleotides were immobilized to wells, and the relationships between melphalan concentration and assay signal magnitude were determined with and without a prior incubation with 0.1 M NaOH (37 °C for 60 min) for antibodies Amp4/42 and MP5/73,


respectively. Oligonucleotides containing guanine showed the formation of adducts recognized by antibody MP5/ 73, while those lacking guanine did not give a significant drug-dependent signal (Figure 5E and data not shown). The overall strengths of the signal differed among oligonucleotides with different sequences, and several factors may have contributed to this variation (see the Discussion). However, for most sequences containing guanine, the magnitude of the signals obtained with antibody Amp4/42 were approximately one-third of the magnitude of the signals obtained using antibody MP5/ 73 for wells exposed to the same drug concentrations. The most interesting finding was that, on oligodeoxyguanylic acid, the adducts were not recognized by Amp4/42 to a significant degree except following exposure to the highest drug concentrations (Figure 5B). Replicate wells were exposed (1 h at 37 °C) to melphalan concentrations from the initial steeply sloping parts of the concentration-response curves. After removal of the unbound drug, these wells were exposed to 0.1 M NaOH for various times at 20 °C and then washed with buffer A (eight replicate wells per time point). The wells were then assayed by direct binding ELISAs, using antibodies MP5/73 and Amp4/42 (four wells for each). The results with calf thymus DNA (Figure 6A) revealed the time-dependent loss of immunoreactivity to MP5/73 and gain of immunoreactivity to Amp4/42. As expected, this method proved to be insufficiently quantitative to show the small difference between the rates of alkali-induced changes detected by the two antibodies. However, the results showed that there were no large differences between the time courses of alkali-induced changes in adducts on different oligonucleotides (Figure 6 and data not shown). Of particular relevance are the results for immobilized oligodeoxyguanylic acid for which the magnitude of the MP5/73 signal declined over a time scale comparable to the other DNA sequences. When oligodeoxyguanylic acid was exposed to a very high concentration of melphalan (200 µg/mL), a weak assay signal was detected using Amp4/42, and this developed in 0.1 M NaOH over the same time scale as for the other sequences. This indicated that the lack of immunorecognition by Amp4/42 of adducts on the oligodeoxyguanylic acid was not due to a markedly slower alkali-induced change. Stability of Immobilized DNA during Alkali Exposures. Plates coated with synthetic oligonucleotides or restriction-digested DNA were exposed to cisplatin, and the resulting adducts were detected using antibody CP9/19 (18). Since cisplatin-DNA adducts are stable in alkali (19), any loss in magnitude of the assay signal during exposure to NaOH would be attributable to loss of DNA from the surface of the wells. Wells coated with denatured restriction-digested DNA or synthetic oligonucleotides were exposed to concentrations of cisplatin that were in the steeply sloping regions of the doseresponse curves. Subsequent exposure to the same alkali conditions that were used for the experiments on melphalan adducts indicated no major loss of either restriction-digested DNA or any of the synthetic oligonucleotides during the time periods that were studied (up to 240 min, data not shown). Monohydroxymelphalan. It has been established that antibodies MP5/73 and Amp4/42 recognize monofunctional adducts of melphalan on DNA as efficiently as the overall adducts formed by melphalan (20). How-

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Figure 5. Effect of melphalan concentration on immunoassay signals using immobilized DNA restriction fragments or synthetic oligodeoxynucleotides. Following drug exposure, the wells were either assayed using antibody MP5/73 (O) or incubated with NaOH (0.1 M for 60 min at 37 °C) and then assayed using antibody Amp4/42 (2): (A) restriction fragments of calf thymus DNA, (B) GGGGGGGGGGGGGGGGGGGGG,(C)CCGCCGCCGCCGCCGCCGCCGCC,(D)TTGTTGTTGTTGTTGTTGTTGTT,(E)TTTTTTTTTTTTTTTTTTTTT, (F) CCCCGCCCCGCCCCGCCCCGCCCC, and (G) TTTTGTTTTGTTTTGTTTTGTTTT. Each point represents the mean of at least three replicate wells minus the reading for wells not treated with melphalan (error bars denote the SD; representative data from one of at least three experiments).

ever, to rule out the possibility that the difference between recognition of adducts on oligo(dG) and the other sequences was related to formation of intrastrand crosslinks between closely spaced guanines, the patterns of immunorecognition were investigated by alkylating immobilized oligonucleotides with monohydroxymelphalan (1b) (20). The pattern of antibody specificity observed was the same as that observed for the bifunctional drug (data not shown). Competitive ELISA Using Synthetic Oligonucleotides. MP5/73 recognized adducts on polyguanylic acid with an efficiency similar to those of adducts on denatured DNA or RNA (Table 1). Synthetic DNA oligonucleotides that had been reacted with radioactive melphalan were exposed to 0.1 M NaOH, neutralized, and desalted by ultrafiltration before the assay using Amp4/42. The adducts on oligodeoxyguanylic acid (dG)21 were 10-fold less immunoreactive than adducts on genomic DNA. Melphalan adducts on oligodeoxyadenylic acid were not detectably immunoreactive to Amp4/42. However, because the ratio of alkylation at N1 to N3 of adenine

generally depends on DNA conformation (6, 17, 21, 22), a double-stranded oligonucleotide consisting of A and T was also investigated. Immunoreactivity to Amp4/42 was undetectable at the maximum concentration tested, but a small degree of immunoreactivity to antibody MP5/73 was detected (Table 1). Thus, for both antibodies, the only strongly immunoreactive adducts involved guanine. Chlorambucil-DNA Adducts. Native and heatdenatured DNA was alkylated with [14C]chlorambucil (2). For analysis with antibody MP5/73, the alkylated native DNA was digested with DNAase I to maximize any immunoreactivity of adducts (12). There was no detectable immunoreactivity toward antibody MP5/73 when analyzed by competitive ELISA (Table 1). The DNA preparations were also incubated in 0.1 M NaOH for various times before analysis by competitive ELISA using antibody Amp4/42. This antibody recognized the alkalitreated chlorambucil adducts with an assay sensitivity similar to that shown for melphalan adducts. The alkaliinduced change, detected by the increase in immunore-


Figure 6. Effects of the time of exposure of alkylated DNA to alkali on the immunoassay signal for antibodies MP5/73 (O) and Amp4/42 (2). DNA restriction fragments or synthetic oligodeoxynucleotides were immobilized, reacted with melphalan, and then exposed to NaOH (0.1 M at 20 °C). The DNA sequences and the melphalan concentrations used in association with each antibody were as follows: (A) restriction-digested calf thymus DNA, 10 µg/mL for MP5/73 and 10 µg/mL for Amp4/4; (B) GGGGGGGGGGGGGGGGGGGGG, 3 µg/mL for MP5/73 and 200 µg/mL for Amp4/42; (C) CCCCGCCCCGCCCCGCCCCGCCCC, 0.3 µg/mL for MP5/73 and 10 µg/mL for Amp4/42; and (D) CCGCCGCCGCCGCCGCCGCCGCC, 0.3 µg/mL for MP5/ 73 and 10 µg/mL for Amp4/42. The 96-well plates were from the same batches of coatings that were used for the experiment whose results are depicted in Figure 5. Each point represents the mean fluorescence intensity of four wells minus the mean reading for wells exposed to alkali but not melphalan (error bars denote the SD; representative data from one of at least three experiments).

activity, appeared to be slower than was observed for melphalan adducts under the same conditions (Figure 7).

Discussion Previous results indicated that antibody MP5/73 recognized melphalan adducts on N7 of guanine (5) and that Amp4/42 recognized the formamidopyrimidine products formed by ring opening of N7-guanine adducts of melphalan (11). The studies presented here were initiated


because of the observation that adducts recognized by antibody Amp4/42 formed a smaller proportion of the total adducts when melphalan had reacted with DNA in cells rather than in a solution of purified DNA (11). This implied that there was a previously unrecognized difference in the nature of the alkylation products formed in cells compared to those formed in vitro. The same observation also implied that antibodies Amp4/42 and MP5/73 did not recognize completely equivalent adducts. This lack of equivalence was supported by the competitive ELISA results depicted in Figures 1 and 2 which show that, under alkaline conditions, loss of immunoreactivity to MP5/73 occurred more slowly than gain of immunoreactivity to Amp4/42. Four hypotheses were considered to explain the nonequivalence of adducts recognized by each antibody. First, one antibody was better able to recognize cross-linked than monofunctional adducts. This was ruled out by the fact that both antibodies recognized DNA modified with monohydroxymelphalan with an efficiency similar to that of DNA modified with melphalan (20) and by the present observation that monohydroxymelphalan gave the same pattern of results with immobilized oligodeoxynucleotides as did melphalan. The second hypothesis was that the antibodies recognized different alkali-labile adducts. There are four characterized alkali-labile products that result from alkylation of DNA: (i) N1 alkyladenine adducts undergo a Dimroth rearrangement to yield the 6-alkylamino derivatives (17, 23); (ii) abasic sites result in hydrolysis of sugar-phosphate bonds; (iii) phosphotriesters hydrolyze to cause DNA strand scission (24-26); and (iv) N7 alkylguanine adducts undergo ring opening to yield formamidopyrimidine derivatives (17, 27). In addition, an alkali-labile alkylation site involving adenine was detected using a DNA sequencing technique (28), but its chemical structure was not identified. Experiments described above failed to detect strong immunoreactivity of adenine adducts to either antibody. The significance of weak immunoreactivity to MP5/73 of adducts on the oligonucleotide consisting of deoxyadenylate and deoxythymidylate is not clear at present. As discussed previously (11), immunorecognition of apurinic sites is not consistent with the heat lability of adducts recognized by the antibodies. Reported rates of alkaline hydrolysis of methylphosphotriesters are slower than rates of ring opening of 7-alkylguanine adducts (26, 29-31). Thus, this difference might, in principle, have explained these observations if Amp4/42 recognized formamidopyrimidine structures while MP5/73 recognized phosphotriesters. Furthermore, the potential involvement of phosphoesters in the recognition of melphalan adducts by MP5/73 might have been suggested by one interpretation of previous experiments. In those experiments, an immunoreactive product was identified which involved one melphalan molecule bound to two molecules of GMP, one of which was alkylated on the phosphate (5). Thus, it is particularly relevant that the following new observations rule out the involvement of phosphotriesters in the immunorecognition of adducts in DNA by MP5/73. First, the half-lives of 69 and 9.7 min for loss of immunoreactivity to MP5/73 at 0.01 and 0.1 M NaOH, respectively, were not consistent with the much slower reported rates for hydrolysis of methyl and ethyl phosphotriesters (26, 29) [half-lives in 0.167 M NaOH at 37 °C were 2-3 and 3-4 h, respectively (26)]. Also, loss of immunoreactivity to

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Table 1. Immunoreactivities of Adducts in Various Nucleic Acids Determined by Competitive ELISAs Using Two Monoclonal Antibodies alkylation level nucleid acid sequence polyguanylic acid yeast RNA oligodeoxyguanylic acid (dG)21 oligodeoxynucleotide TATTATAATA oligodeoxyadenylic acid (dA)21 calf thymus DNA denatured denatured native native (with DNAase I) denatured

concentration of adducts causing 50% inhibition in competitive ELISAs (K-value, fmol/assay well) MP5/73 Amp4/42b

adducts/ kilobase

µmol of adduct/g of nucleic acid

mel mel mel mel mel

3 40 6 1 2

11 100 17 4.3 7.9

60 ( 25 45 ( 2 520d -

mel mel chlor chlor chlor

27 15 3 3 4

82 46 7.9 7.9 11.4

55 ( 14 [9950]e [8950]e

druga

-c 1540 ( 618 [2400]e [8300]e 93 ( 62 41 ( 22 20 ( 9

a mel, melphalan; chlor, chlorambucil. b Determined after exposure of DNA to NaOH (0.1 M for 10 min at 37 °C), except for chlorambucil where 60 min at 37 °C was used. c Not determined. d Mean of two determinations. e No detectable immunoreactivity at this concentration.

Figure 7. Effects of exposure to alkali (0.1 M NaOH at 37 °C) on immunoreactivity of chlorambucil-DNA adducts to Amp4/ 42 assayed using a competitive ELISA: (O) native DNA reacted with chlorambucil, alkylation level of 7.9 µmol/g of DNA; and (2) denatured DNA reacted with chlorambucil, alkylation level of 11.4 µmol/g of DNA. Each point represents the mean of at least three assays ( SE. The inset shows the equation for onephase exponential association fitted to the reciprocals of the K-values for the denatured DNA preparation. The fitted halflife was 21 min.

MP5/73 proceeded only slightly slower than gain of immunoreactivity to Amp4/42. This is not consistent with the large difference between the reported rates for phosphotriester hydrolysis and N7-guanine ring opening (30, 31), although the nature of the alkylating species may affect the stability of the triester (26, 32). A second observation, inconsistent with the involvement of phosphotriesters, is that the adducts recognized by MP5/73 on RNA were considerably more stable at 100 °C than adducts on DNA. This is consistent with the known greater stability of the glycosidic bond of ribose compared to that of deoxyribose nucleotides (17) and would not be expected for phosphotriesters on RNA. The fact that adducts on RNA and polyguanylic acid were detected at all, immunologically (ref 10 and Table 1), is also not consistent with the very unstable nature of phosphotriesters in RNA (15, 16, 33). Thus, the evidence indicates that alkali-labile N7 guanine adducts are involved in the epitopes recognized by both antibodies.

The third hypothesis to explain the nonequivalence of adducts recognized by the two antibodies was that MP5/ 73 recognized the initial adduct together with a hypothetical intermediate structure that formed rapidly in alkali, while Amp4/42 recognized this intermediate together with the final ring-opened structure which formed more slowly. To be able to be detected by an ELISA, any such intermediate would have to be sufficiently stable to persist for several hours after neutralization. There is no evidence for such an intermediate, and its involvement is further ruled out by the observation that a brief alkali treatment increased the thermal stability of adducts recognized by Amp4/42 but not of adducts recognized by MP5/73 (Figure 4). The fourth hypothesis was that one or both antibodies preferentially recognized guanine adducts present in particular DNA sequences. To facilitate investigations of the effects of alkali and drug dose on synthetic DNA oligonucleotides, a method was adopted to covalently bind DNA to amino-derivatized plastic wells. The stability of the bond was confirmed for several different DNA sequences by immunological detection of the alkali-stable adducts formed by cisplatin. In these experiments, the amount of DNA that became bound to the wells was not known and appeared to vary between oligonucleotides and between batches of coated wells. This variation accounts for the slightly lower signal magnitude seen in the data presented (Figure 5) for MP5/73 binding to wells coated with oligodeoxyguanylic acid compared to that of the other guanine-containing sequences. However, important aspects of the experiments were that, for each oligonucleotide, all the wells were coated with the same amount of DNA and, for the same drug exposure, these all carried the same quantity of adducts. Thus, differences in the ratio of the magnitude of the MP5/73 signal to the magnitude of the Amp4/42 signal for the various oligonucleotides revealed differences in sequence preference between the two antibodies. Using the direct binding ELISA and immobilized oligonucleotides, it was discovered that Amp4/42 exhibited very poor binding to melphalan adducts on oligodeoxyguanylic acid. This was not due to a slower rate of alkali-induced change. The failure of Amp4/42 to detect melphalan adducts efficiently in oligodeoxyguanylic acid in the direct binding assay was confirmed by reaction of oligodeoxyguanylic acid with radioactive melphalan,


exposure to alkali, neutralization, desalting, radioactive counting, and a competitive ELISA (Table 1). A preliminary study indicated that cisplatin-modified oligonucleotides as well as calf thymus DNA became adsorbed to “high bind” plastic wells when applied as in the competitive ELISA experiments and remained attached during alkali treatment (4 h with 0.1 M NaOH). However, immunoreactivity of cisplatin adducts on the single-stranded oligonucleotide increased as a result of alkali treatment. The mode of DNA-plastic interaction is not well understood, particularly with regard to the proportion of nucleotides involved in the interaction. Furthermore, the effects of this DNA-plastic interaction on drug-DNA reaction were unknown, and changes in the interaction during alkali exposure could have contributed to changes in immunoreactivity. These factors were likely to be sequence specific and seemed to be particularly relevant for short defined oligonucleotides. Therefore, covalent immobilization of DNA was chosen for this investigation. In the direct binding ELISA experiments, there was a lack of consistent linearity between the assay signal magnitude and drug concentration, even at low concentrations, and the absolute adduct levels could not be directly established. Therefore, contrary to initial expectations, calculation of meaningful reaction rates from the results was not realistic. It is also relevant that differences between results from the direct binding and competitive ELISA techniques probably resulted from the different assay principles. Of particular importance may have been the fact that during later stages of the direct binding assay, the persistence of the bound antibody molecules would have depended upon the affinity constant and the rate of dissociation for the interaction between the antibody and adduct. These parameters may have varied between the different immobilized adducts or DNA sequences. With the competitive ELISA, however, the immobilized DNA to which the antibodies needed to remain bound during later stages of the assay was the same for all the different samples. Consequently, the DNA samples being tested only influenced these assays during the competitive equilibrium stage. A further difference between the two assays was that much higher concentrations of antibodies were used in the direct binding than in the competitive assays. These higher concentrations may have enhanced those associations between antibodies and adducts that formed with lower rate constants. Despite these factors, the method using immobilized DNA did permit investigation of numerous combinations of drug concentrations and alkali times on several different oligonucleotides (Figures 5 and 6 and data not shown). The results indicated which experiments should be carried out by the more laborious approach of using the radioactively labeled drug and competitive ELISA. Recognition of adducts by MP5/73 was strongly influenced by the structure of the substituent on the aromatic ring of the drug molecule since chlorambucil adducts were not detectably immunoreactive to this antibody (compare structures 1 and 2). Binding of Amp4/42 appeared to be much less dependent upon this region of the molecule since it detected chlorambucil adducts with high sensitivity. This difference in specificity is consistent with the hypothesis that the two antibodies recognized different aspects of the molecular structure of adducts, as is


implied by their different dependencies upon the local DNA sequence. The antibodies used in this work were raised against adducts on polymeric DNA. Compared with raising the antibodies against pure alkylated nucleosides, this approach has the disadvantage that it can be more difficult to characterize the recognized structures. However, it has the advantage of being more useful for immunofluorescent quantification of adducts in individual cells. Antibody MP5/73 has been applied in a quantitative immunofluorescent staining [trapped in agarose DNA immunostaining (TARDIS)] technique (13). The effects of alkali on immunostaining in this technique were consistent with the data in Figure 2 showing, for native DNA, an alkali-induced increase in MP5 immunoreactivity before a slow decrease. Overall, there are now three observations that show nonequivalence of the adducts recognized by MP5/73 and Amp4/42. First, adducts recognized by Amp4/42 constituted a smaller fraction of the total adducts when DNA alkylation occurred in cells rather than in vitro. Second, the rates of alkali-induced changes were different for the two classes of adducts. Third, the antibodies showed different abilities to detect adducts in polydeoxyguanylic acid. We conclude that the third observation reveals the mechanism largely underlying the other differences. The differences in rates of alkali-induced changes (Figures 1 and 2) were small. Local DNA sequence has been shown to exert an effect of similar magnitude on the efficiency of alkylation of DNA. This was attributed to variation in local charge density along the DNA (34-36), and it was proposed that the increased electronegative charge associated with guanines favored attack by the positively charged alkylating species. An opposite effect on attack by negatively charged hydroxyl ions might be expected. Preferential alkylation of runs of several guanines occurs during alkylation of pure DNA (34-36). Therefore, this phenomenon does not explain the differences in the proportion of adducts recognized by Amp4/42 in DNA from drug-treated cells and DNA alkylated in vitro. Most direct comparisons of sequence-related patterns of DNA damage in cells compared to DNA in pure solution have, for technical reasons, focused on the highly reiterated R-DNA sequence. Studies using nitrogen mustard compounds (37, 38) and bleomycin (39) showed no differences, while relatively slight differences in the sequence-dependent pattern of reaction were observed for cisplatin (40). A comparison of sequence-dependent patterns of cisplatin adduct formation in a single-copy gene also revealed an overall similarity to the products of reactions on pure DNA. However, at one particular position in the analyzed sequence, adducts were formed at a significant frequency only in vivo (41), and this supports the principle that the organization of DNA into various structures present within chromatin can significantly alter the pattern of attack by reactive chemicals. Previous studies have necessarily compared frequencies of damage formation at sites within specific DNA sequences up to a few hundred base pairs long. This clearly represents a minute proportion of the total genome. A unique aspect of the present immunological comparisons of adduct formation is that the data reflect the properties of the total cellular DNA rather than a specific small region. Therefore, these results could be important if they reflect differences in levels of alkylation that occur in, for example, specific guanine-rich DNA regions. Com-

80


pared to reactivity in other sequences, reactivity in such regions could be enhanced, by DNA organization or proximity to the nuclear membrane or nuclear pore. Such effects would be in addition to the established effect of multiple adjacent guanines. At present, there appears to be no other method available that would reveal global variation in the pattern of adduct formation in relation to DNA sequence. However, other studies have indicated that variation occurs in DNA damage levels between different regions of chromatin (42, 43). The findings presented here provide a further illustration of the potential complication of using antibodies raised against adducts on polymeric DNA, namely, the possibility of effects of local DNA sequence on immunorecognition. However, these findings also indicate that, if properly understood, this effect could provide a useful additional tool for the study of patterns of DNA alkylation in cells. It will be of interest to define more precisely the relationship between the number of contiguous guanines and immunoreactivity to Amp4/42, particularly in relation to known guanine-rich repetitive sequences such as telomeric repeats and by the use of competitive ELISA. It will also be relevant to investigate the distribution of DNA adduct formation and repair in different regions of the genome using alternative methods.

Acknowledgment. This work was funded by the United Kingdom Leukaemia Research Fund and the North of England Children’s Cancer Research Fund. We thank B. T. Golding and P. D. Lawley for numerous valuable discussions and comments on the manuscript. We thank M. Osborne for suggesting and supplying the A+T oligonucleotide and H. Atkins and J. Lunec for synthesis of other oligonucleotides.

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Antibody Recognition of Melphalan Adducts Characterized Using

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