drug-DNA Adducts Research Articles

Structural changes of DNA induced by mono- and binuclear cancer drugs

The structural features of the drug–DNA adducts resulted from treatment of DNA with the platinum based mononuclear drug cisplatin and the binuclear drug [{ trans-PtCl(NH 3) 2} 2H 2N(CH 2) 4NH 2]Cl 2 or bis(platin) have been investigated by atomic force microscopy (AFM). Reduction in the contour length of the DNA fragments has been observed after cisplatin treatment while, compaction and aggregation are found to be the primary structural modifications following treatment with the binuclear drug. The intermolecular interaction upon bis(platin) treatment leads to observation of highly condense aggregates without a distinct sight of single isolated DNA molecule. These differences in drug binding indicate that unlike the mononuclear drug cisplatin, bis(platin) causes extensive interhelical/intermolecular cross-linking through its multiple linking sites. To our knowledge, this is the first report of a comparative AFM study to monitor the effects of a mono- and a binuclear platinum anti-cancer drug on DNA structure. These observations should provide clues towards explaining the distinct biological activities of the two drugs.

Combinatorial Identification of a Novel Consensus Sequence for the Covalent DNA-Binding Polyamide Tallimustine

Many agents successfully used in cancer chemotherapy either directly or indirectly covalently modify DNA. Examples include cisplatin, which forms a covalent adduct with guanines, and doxorubicin, which traps a cleavage intermediate between topoisomerase II and torsionally strained DNA. In most cases, the efficacy of these drugs depends on the efficiency and specificity of their DNA binding, as well as the discrimination between normal and neoplastic cells in their handling of the drug-DNA adducts. While much is known about the chemistry of drug-DNA adducts, little is known regarding the overall specificity of their formation, especially in the context of a whole human genome, where potentially billions of binding sites are possible. We used the combinatorial selection method restriction endonuclease protection, selection, and amplification (REPSA) to determine the DNA-binding specificity of the semisynthetic covalent DNA-binding polyamide tallimustine, which contains a benzoic acid nitrogen mustard appended to the minor groove DNA-binding natural product distamycin A. After investigating over 134 million possible sequences, we found that the highest affinity tallimustine binding sites contained one of two consensus sequences, either the expected distamycin hexamer binding sites followed by a CG base pair (e.g., 5'-TTTTTTC-3' and 5'-AAATTTC-3') or the unexpected sequence 5'-TAGAAC-3'. Curiously, we found that tallimustine preferentially alkylated the N7 position of guanines located on the periphery of these consensus sequences. These findings suggested a cooperative binding model for tallimustine in which one molecule noncovalently resides in the DNA minor groove and locally perturbs the DNA structure, thereby facilitating alkylation by a second tallimustine of an exposed guanine on another side of the DNA.

A Molecular Understanding of Mitoxantrone-DNA Adduct Formation: EFFECT OF CYTOSINE METHYLATION AND FLANKING SEQUENCES

When mitoxantrone is activated by formaldehyde it can form adducts with DNA. These occur preferentially at CpG and CpA sequences and are enhanced 2-3-fold at methylated CpG sequences compared with non-methylated sites. We sought to understand the molecular factors involved in enhanced adduct formation at these methylated sites. This required, first, clarification of factors that contributed to the formation of adducts at CpG sites. For this purpose mass spectrometry of an oligonucleotide duplex (containing a single CpG adduct site) was used to confirm the presence of an additional carbon atom (derived from formaldehyde) on the drug-DNA complex. The effect of 3'-flanking sequences was revealed by electrophoretic analysis of oligonucleotide-drug adducts, and the preferred adduct-forming site was identified as 5'-CGG-3'. Radiolabeled studies of drug-DNA adducts confirmed that the site of attachment involved the exocyclic amino of guanine. Molecular modeling analysis of the relative stability of the intercalated form of mitoxantrone was consistent with observed adduct-forming potential of CG sites with varying flanking sequences. The known preference for adduct formation at methylated CG sites was confirmed by energetics calculations and shown to be due to a shift of equilibrium of the intercalated form of the drug from the major groove (at CG sites) to the minor groove (at methylated CG sites). This increases the relative amount of drug that is located adjacent to the N-2 exocyclic amino of guanine in the minor groove, where covalent linkage is facilitated. These results account for the enhanced covalent binding of mitoxantrone to methylated CG sequences and provide a molecular model of the interactions.

Regulated expression and subcellular localization of HMGB1, a chromatin protein with a cytokine function.

High mobility group box protein 1 (HMGB1) has been considered as a ubiquitous nuclear protein with an architectural function, but even early reports have described its presence outside of the nucleus. Today, we have only started to understand the extranuclear and extracellular functions of HMGB1: we know that it participates in developmental and differentiation processes, triggers and modulates many of the inflammatory cascades in the body, and may even be involved in the metastatic invasion programme of cancer cells. Given such diverse roles, it is important to know which cells express HMGB1, where, and how much. The present review deals with the expression pattern of HMGB1 and provides evidence that, far from being housekeeping, the HMGB1 gene is tightly regulated. This can have implications for therapeutic intervention on inflammatory diseases as well as cancer.

Covalent binding to glutathione of the DNA-alkylating antitumor agent, S23906-1.

The benzoacronycine derivative, S23906-1, was characterized recently as a novel potent antitumor agent through alkylation of the N2 position of guanines in DNA. We show here that its reactivity towards DNA can be modulated by glutathione (GSH). The formation of covalent adducts between GSH and S23906-1 was evidenced by EI-MS, and the use of different GSH derivatives, amino acids and dipeptides revealed that the cysteine thiol group is absolutely required for complex formation because glutathione disulfide (GSSG) and other S-blocked derivatives failed to react covalently with S23906-1. Gel shift assays and fluorescence measurements indicated that the binding of S23906-1 to DNA and to GSH are mutually exclusive. Binding of S23906-1 to an excess of GSH prevents DNA alkylation. Additional EI-MS measurements performed with the mixed diester, S28053-1, showed that the acetate leaving group at the C1 position is the main reactive site in the drug: a reaction scheme common to GSH and guanines is presented. At the cellular level, the presence of GSH slightly reduces the cytotoxic potential of S23906-1 towards KB-3-1 epidermoid carcinoma cells. The GSH-induced threefold reduction of the cytotoxicity of S23906-1 is attributed to the reduced formation of lethal drug-DNA covalent complexes in cells. Treatment of the cells with buthionine sulfoximine, an inhibitor of GSH biosynthesis, facilitates the formation of drug-DNA adducts and promotes the cytotoxic activity. This study identifies GSH as a reactant for the antitumor drug, S23906-1, and illustrates a pathway by which GSH may modulate the cellular sensitivity to this DNA alkylating agent. The results presented here, using GSH as a biological nucleophile, fully support our initial hypothesis that DNA alkylation is the major mechanism of action of the promising anticancer drug S23906-1.

Detection of labile anthracycline-DNA adducts by real-time PCR.

Barminomycin was employed as a model anthracycline that yields thermally stable drug-DNA adducts. Real-time PCR was utilized for the detection of these barminomycin-DNA adducts at drug levels as low as 100 nM in a cell-free assay system, with the lowest level of detection at approximately 20 nM. By contrast, doxorubicin-DNA adducts are heat labile and their levels were underestimated by conventional real-time PCR unless the DNA denaturation temperature was lowered by the addition of glycerol. Doxorubicin-DNA adduct levels of 5.5 per 10 kb were detected by real-time PCR (in the presence of 24% glycerol) following treatment with 0.5 microM doxorubicin (and 2 mM formaldehyde), considerably more sensitive than that detected by a gene-specific Southern-based procedure. Both the absolute fluorescence intensity in the linear PCR amplification range and the crossing point method provided useful dose-dependent estimates of adduct levels. The time required for a complete real-time PCR analysis of drug-induced adduct levels was approximately 40 min, and this may ultimately provide oncologists with a rapid means with which to monitor drug-DNA adduct levels in patients under treatment with anthracyclines. Responses to these drugs could be quickly and efficiently monitored in patients, thereby facilitating optimization of drug dosages as well as early detection of resistance to these agents.

Evidence for a common molecular basis for sequence recognition of N3-guanine and N3-adenine DNA adducts involving the covalent bonding reaction of (+)-CC-1065.

The antitumor antibiotic (+)-CC-1065 can alkylate N3 of guanine in certain sequences. A previous high-field 1H NMR study on the (+)-CC-1065d[GCGCAATTG*CGC]2 adduct (* indicates the drug alkylation site) showed that drug modification on N3 of guanine results in protonation of the cross-strand cytosine [Park, H.-J.; Hurley, L. H. J. Am. Chem. Soc. 1997, 119, 629.]. In this contribution we describe a further analysis of the NMR data sets together with restrained molecular dynamics. This study provides not only a solution structure of the (+)-CC-1065(N3-guanine) DNA duplex adduct but also new insight into the molecular basis for the sequence-specific interaction between (+)-CC-1065 and N3-guanine in the DNA duplex. On the basis of NOESY data, we propose that the narrow minor groove at the 7T8T step and conformational kinks at the junctions of 16C17A and 18A19T are both related to DNA bending in the drugDNA adduct. Analysis of the one-dimensional 1H NMR (in H2O) data and rMD trajectories strongly suggests that hydrogen bonding linkages between the 8-OH group of the (+)-CC-1065 A-subunit and the 9G10C phosphate via a water molecule are present. All the phenomena observed here in the (+)-CC-1065(N3-guanine) adduct at 5'-AATTG* are reminiscent of those obtained from the studies on the (+)-CC-1065(N3-adenine) adduct at 5'-AGTTA*, suggesting that (+)-CC-1065 takes advantage of the conformational flexibility of the 5'-TPu step to entrap the bent structure required for the covalent bonding reaction. This study reveals a common molecular basis for (+)-CC-1065 alkylation at both 5'-TTG* and 5'-TTA*, which involves a trapping out of sequence-dependent DNA conformational flexibility as well as sequence-dependent general acid and general base catalysis by duplex DNA.

DNA repair in resistance to bifunctional alkylating and platinating agents.

Identifying and exploiting metabolic and genetic differences between normal and tumor cells is an important aspect of the development of cancer therapeutic agents. This rationale naturally extends to understanding the properties of tumor cells that make them responsive versus resistant to anticancer drugs. Many factors can contribute to the drug-resistant phenotype of individual tumor cells, and indeed, clinically relevant resistance in a given tumor or cell line often involves more than one mechanism. However, with a specific anticancer drug, one or more mechanisms may dominate. Thus, cyclophosphamide-resistant tumor cells display a high incidence of elevation of aldehyde dehydrogenase (ALDH), an enzyme that eliminates the active metabolite of the drug before it can react with and damage the cells’ DNA’ (see chapter 8 in this volume by Dr. N. Sladek). Similarly, tumor cells that are resistant to bis-chloroethyl nitrosourea (BCNU) display a high incidence of increased activity of the protein 06- alkylguanine DNA alkyltransferase (AGT) which removes mono-alkylated adducts from the 06position of guanine (section 7). With cisplatin and melphalan, the ability of the tumor cells to repair drug-DNA adducts is a frequent contributor to the drug-resistant phenotype2-5. It is the latter mechanism that is the subject of this chapter

The binding affinity of HMG1 protein to DNA modified by cis-platin and its analogs correlates with their antitumor activity

The antitumor activity of cis-platin is believed to result from its interaction with cellular DNA and subsequent processing of DNA adducts by damage recognition proteins. Among them are the high mobility group (HMG) proteins 1 and 2, which have been hypothesized to mediate the effect of cis-platin. One possibility suggests that the tight binding of HMG1 to DNA adducts blocks the repair of damaged DNA. In order to further evaluate such a mechanism, several cis-platinum complexes with known antitumor activity have been used to treat DNA and the affinity of HMG1 to the DNA adduct induced by each drug was determined. The dissociation constants for the complexes of HMG1 with the platinated probe were obtained by gel mobility shift assays. The antitumor activity of the tested platinum compounds was found to correlate with the binding affinity of HMG1 to the respective drug-DNA adduct. These findings support the view that HMG1 contributes to cytotoxicity of cis-platin by shielding damaged DNA from repair. In addition, they offer a fast test for screening new platinum compounds for antitumor activity.

Interstrand cross-linking by adriamycin in nuclear and mitochondrial DNA of MCF-7 cells.

Activation of Adriamycin by formaldehyde leads to the formation of drug-DNA adducts in vitro and these adducts stabilise the DNA to such a degree that they function as virtual interstrand cross-links. The formation of these virtual interstrand cross-links by Adriamycin was investigated in MCF-7 cells using a gene-specific interstrand cross-linking assay. Cross-linking was measured in both the nuclear-encoded DHFR gene and in mitochondrial DNA (mtDNA). Cross-link formation increased linearly with Adriamycin concentration following a 4 h exposure to the drug. The rate of formation of Adriamycin cross-links in each of the genomes was similar, reaching maximal levels of 0.55 and 0.4 cross-links/10 kb in the DHFR gene and mtDNA respectively, following exposure to 20 micro M Adriamycin for 8 h. The interstrand cross-link was short lived in both DNA compartments, with a half-life of 4.5 and 3.3 h in the DHFR gene and mtDNA respectively. The kinetics of total Adriamycin adduct formation, detected using [(14)C]Adriamycin, was similar to that of cross-link formation. Maximal adduct levels (30 lesions/10 kb) were observed following incubation at 20 micro M drug for 8 h. The formation of such high levels of adducts and cross-links could therefore be expected to contribute to the mechanism of action of Adriamycin.

Structural analysis of drug-DNA adducts by tandem mass spectrometry.

The utility of electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) for the characterisation of ligand-oligonucleotide adducts is demonstrated with adducts formed between the oligonucleotide 5'-CACGTG-3' and both a platinating agent, cis-diamminedichloroplatinum(II) (cisplatin), and an alkylating ligand, n-bromohexylphenanthridinium bromide (phenC6Br). We have demonstrated previously that negative ion MS/MS spectra of alkylated oligonucleotides show a highly specific fragmentation pathway that enables the site of binding of the ligand to be readily identified. In comparison, the positive ion ESI-MS/MS spectra reported here also show a single major fragmentation pathway, but the dominant ion is the protonated ligand-base adduct. MS/MS of this ion confirms the site on binding of the ligand to the guanine base. MS/MS spectra of cisplatin adducts show much less specific fragmentation than alkylated adducts, particularly in the negative ion mode. This suggests that the ESI-MS/MS spectra of ligand-DNA adducts are strongly influenced by the extent to which the ligand weakens the glycosidic bond in the residue to which it is bound. For platinating agents, which do not labilise the glycosidic bond, additional experiments involving MS/MS of source-generated product ions were required to enable isomeric adducts to be distinguished.

Retro Models of Pt Anticancer Drug DNA Adducts: Chirality-Controlling Chelate Ligand Restriction of Guanine Dynamic Motion in (2,2'-Bipiperidine)PtG(2) Complexes (G = Guanine Derivative).

Features of cisplatin-type anticancer drug adducts with nucleic acids and their constituents are clouded because they exist as a fluxional mixture of conformers. Retro-model adducts containing the specially designed chiral diamine ligand, Bip = 2,2'-bipiperidine, are dramatically less fluxional. Conformers of BipPtG(2) adducts with R,S,S,R and S,R,R,S asymmetric centers at the N, C, C, and N Bip chelate ring atoms and G = 5'-GMP, 5'-dGMP, 3'-GMP, or 9-ethylguanine are amenable to separate characterization. All possible BipPtG(2) atropisomers (one head-to-head (HH) and Delta and Lambda head-to-tail (HT) atropisomers) were observed by NMR spectroscopy. At equilibrium at low pH, one HT atropisomer dominates. CD spectra, G H8 chemical shifts, and low-pH equilibria of BipPtG(2) and Me(2)()DABPtG(2) (Me(2)()DAB = N,N '-dimethyl-2,3-diaminobutane) are similar when the chelate ring atoms have the same stereochemistries; thus, Bip and Me(2)()DAB are termed chirality-controlling chelates (CCC) since these ligands dictate the absolute conformation of the major HT rotamer. In each case, the HT conformer that cannot form G O6-NH(CCC) hydrogen bonds was dominant, and the G H8 chemical shift indicated that this conformer had less tilted bases, allowing favorable base-base dipole-dipole interactions. For both the R,S,S,R and S,R,R,S Bip chiralities of the BipPt(3'-GMP)(2) complexes, the percentage of DeltaHT rotamer increased near pH 7, a probable consequence of phosphate-cis-G hydrogen bonds accompanied by favorable dipole interactions of less tilted bases. For the 5'-GMP complexes, these factors favor the LambdaHT rotamer near pH 7. When G has a 5'-phosphate group, rotamer distribution is also influenced by phosphate-NH(Bip) hydrogen bonds. At high pH, the nature and/or strength of interactions such as G dipole-G dipole interactions and G O6-NH(Bip) and phosphate-cis-G hydrogen bonding are altered by G N(1)H deprotonation. The features of the complexes at high pH can be largely explained as arising from the net result of these interactions. This information from retro models with a CCC ligand lays the foundation for understanding and evaluating the properties of the highly dynamic adducts of anticancer drugs.

Pharmacokinetically guided dose escalation of carboplatin in epithelial ovarian cancer: Effect on drug-plasma AUC and peripheral blood drug-DNA adduct levels

Platinum based drugs are active agents in epithelial ovarian cancer and increased platinum drug dose intensity is thought to lead to improved survival, because of the largely untested assumption that increased dose intensity results in an increased interaction of the platinum drug with its target, DNA. In a previously reported phase I trial (Lind et al., J Clin Oncol 1996; 14: 800-5), carboplatin dose intensity was increased by the use of G-CSF to support the bone marrow and using pharmacokinetically-guided carboplatin dosing. The objectives of this study were to validate the carboplatin dosing formula during high dose intensity therapy and evaluate the relationship between systemic carboplatin exposure and Pt-DNA adduct levels in peripheral blood leucocytes. A total of 17 patients were studied over four levels of dose intensification. The carboplatin dose was calculated using the 'Calvert formula'. Levels of drug-target interaction in peripheral blood leukocytes were measured using an immunoassay based on a monoclonal antibody that recognises DNA-platinum adducts. Pharmacokinetic measurements were carried out using a previously validated single sample method. The area under the curve of concentration of unbound carboplatin in plasma versus time (AUC) for target AUC values of 5, 7 and 9 mg/ml x min were: 5.6 +/- 1.0, 7.3 +/- 0.7 and 9.8 +/- 0.5 mg/ml x min (mean +/- S.D.). There was a good correlation between target and achieved dose intensities (r2 = 0.899) and the slope of the linear regression line was 0.95 (+/- 0.09 SD) not significantly different to 1.0 (P > 0.6). The levels of immunoreactive DNA adducts were not detectable at a target AUC of 5 mg/ml x min but increased progressively at the higher AUC levels. Accumulation of adducts between courses was not detected. Pharmacokinetically-based carboplatin dosing during high intensity therapy accurately predicted the dose required to achieve a target AUC and resulted in consistent patient exposure to active drug. During the dose escalation study, peripheral blood leucocyte DNA platinum-DNA adduct levels were positively related to drug dose and drug AUC.

Replication block by an enediyne drug-DNA deoxyribose adduct.

Under anaerobic conditions neocarzinostatin chromophore, an enediyne antibiotic, forms a covalent drug-DNA adduct on the 5' carbon of deoxyribose at a specific single site in a 2-nucleotide bulge, rather than strand cleavage, by a mechanism involving general base-catalyzed intramolecular drug activation to a reactive radical species. We have taken advantage of the selectivity of this reaction to prepare a single-stranded oligonucleotide containing a single drug adduct at a T residue and to study its effect on the template properties of the oligonucleotide in replicative synthesis, as followed by 5'-32P-labeled primer extension by several DNA polymerases. With the Klenow fragment of Escherichia coli DNA polymerase I, synthesis stops at the base immediately 3' to the adduct. The same enzyme, but lacking 3' to 5' exonuclease activity, permits synthesis to proceed by one additional nucleotide. This effect is enhanced when Mn2+ is substituted for Mg2+. T4, herpes simplex virus, and cytomegalovirus DNA polymerases all act like Klenow polymerase. Sequenase (exo-minus T7 DNA polymerase) is qualitatively similar to exo-minus Klenow polymerase but is more efficient in inserting a nucleotide opposite the lesion. With the small-gap-filling human DNA polymerase beta, which lacks intrinsic exonucleolytic activity, primer extension proceeds to the nucleotide opposite the lesion. However, when a gap was created opposite the lesion, polymerase beta adds as many as two additional nucleotides 5' to the adduct site. The fidelity of base incorporation opposite the lesion was not impaired, in contrast with adducts on DNA bases.

Abrogation of cisplatin-induced programmed cell death in human breast cancer cells by epidermal growth factor antisense RNA.

Epidermal growth factor receptor (EGF-R) perturbation by receptor ligand(s), e.g., epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), or receptor-specific antibodies accentuates cisplatin-induced toxicity in tumor cells. This sensitization occurs only in tumor cells with high expression of EGF-R but not in those with low expression of EGF-R. Therefore, we have studied the role of EGF-R expression on cisplatin-mediated cytotoxicity. MDA-468 human breast cancer cells were stably transfected with a p-chloramphenicol acetyl transferase (pact[p]-CAT) vector containing a 4.1-kilobase full-length antisense EGF-R complementary DNA. EGF-R content was assessed by 125I-EGF binding and EGF-R immunoblot assays. Cisplatin sensitivity was evaluated by (a) colony-forming assay in vitro, (b) xenograft growth in nude mice, (c) cell cycle distribution of propidium iodide-labeled DNA, (d) DNA fragmentation in agarose gels, and (e) terminal deoxynucleotidyl transferase (Tdt) fluorescence in situ. Cisplatin uptake was measured by atomic absorption spectroscopy, and the levels of drug-DNA intrastrand adducts were determined by a dissociation-enhanced fluoroimmunoassay that utilizes an antibody against cisplatin-modified DNA. Selected clones (MDA-468/AS-EGFR) exhibited more than 90% loss of both 125I-EGF binding and receptor content determined by western blot analysis, whereas clones transfected with the vector alone (MDA-468/p-CAT) had EGF-R levels similar to those of the parent cells. By use of a colony-forming assay, the 1-hour IC50 (i.e., the concentration of drug required for 1 hour to achieve 50% cell kill) for cisplatin was 2 microM or less for parental and vector-transfected clones (n = 4), whereas it was 25 microM or more for all MDA-468/AS-EGFR clones (n = 3). MDA-468/p-CAT clones exhibited internucleosomal DNA fragmentation, enhanced Tdt-end labeling in situ, and G2 arrest 48 hours after a 1-hour incubation with 3-30 microM cisplatin. Under these conditions, apoptosis and G2 arrest were undetectable in all MDA-468/AS-EGFR clones. An MDA-468 subline selected after long-term treatment with a TGF-alpha-Pseudomonas exotoxin A fusion protein 40 lacked EGF binding and also exhibited cisplatin resistance (1-hour IC50: > 30 microM) compared with parental cells. This EGF-R-dependent difference in cisplatin response was confirmed in a nude mouse xenograft model by use of high- and low-EGF-R-expressing cell clones. Total intracellular drug accumulation after a 1-hour cisplatin exposure, as measured by atomic absorption spectroscopy, was identical in both groups of cells. Intrastrand drug-DNA adducts, however, were statistically higher in high EGF-R expressors than in low-EGF-R-expressing clones. These data indicate that a critical level of EGF-R signaling, which is amplified in some common human cancers, is necessary for cisplatin-mediated apoptosis in tumor cells and suggest an inhibitory effect of this pathway on the repair of cisplatin-damaged DNA.

A gel mobility shift assay for probing the effect of drug-DNA adducts on DNA-binding proteins.

A gel mobility shift assay for probing the effect of drug-DNA adducts on DNA-binding proteins.

Oral platinum analogue JM216, a radiosensitizer in oxic murine cells

This study was designed to compare radiosensitization by the oral platinum compound JM216 with cisplatin. RIF1 mouse tumour cells were treated at various doses and at various exposure times with JM216 and irradiated 15 min before the end of drug exposure. The fraction of cells surviving treatment was assessed by colony formation. Results were compared with those for equivalent treatments with cisplatin. JM216 alone showed exponential killing of RIF1 cells, being approximately three times less efficient than cisplatin on a molar basis. For radiosensitization studies, drug doses used gave approximately 50 or 90% cell killing alone. No radiosensitization was seen after 2-h drug exposures, but significant radiosensitization occurred after 1- and 0.5-h exposures (shorter times required proportionally higher drug doses, giving equivalent drug kill). The enhancement ratio and time dependence were similar for the two platinum compounds, reaching 1.5 at the highest concentrations tested. DrugDNA adduct formation was assessed using immunocytochemistry with the NKI-A59 antiserum raised to cisplatin-DNA adducts. The antiserum was shown to recognize JM216-DNA adducts in a dose-dependent manner and maximum nuclear staining was found to be correlated with cell kill for both drugs. However, neither the level of staining at the time of irradiation nor at the time of maximum adducts correlated with radiosensitization, indicating that the number of DNA adducts did not determine radiosensitization. Intracellular glutathione levels were shown to be decreased by the drug, but only by approximately 50%, implying that this was not the cause of the increased radiosensitivity. In summary, JM216 was shown capable of radiosensitizing a platinum-sensitive tumour line to an extent similar to cisplatin. Radiosensitization was exposure-time and drug-concentration dependent, but was not dependent on DNA adduct levels nor glutathione depletion. In contrast, cell kill after drug alone was well correlated with adduct levels. These data suggest that JM216 could replace cisplatin in combined radiotherapy-chemotherapy studies, and also indicate that the NKI-A59 antibody could be used to monitor exposure levels in vivo.

Detection and quantification of melphalan-DNA adducts at the single cell level in hematopoietic tumor cells

Bifunctional alkylating agents, such as melphalan, are widely used in the treatment of hematological malignancies. The effects of these drugs on particular types of hematological cells and the causes of treatment failure are poorly understood. The aim of this work was to establish an ability to measure the extent to which melphalan reacts with the DNA of individual tumor cells, thereby creating new possibilities for molecular pharmacological studies on clinical samples. A novel approach for staining drug-DNA adducts is described in which cells were embedded in agarose and then lysed. The DNA from each cell remained in an ideal state for quantitative immunofluorescent staining using a previously described monoclonal antibody. Immunofluorescence and DNA-Hoechst dye fluorescence were quantified using a cooled slow scan charge coupled device camera and image analysis procedures. Immunofluorescence of drug- treated cells from a human leukemia cell line was partially correlated with DNA content. Mean integrated immunofluorescence of 50 to 100 cells was dependent on drug concentration and was linearly related to adduct levels. In these cells and in chronic lymphocytic leukemia cells obtained from patients, there was considerable intercell heterogeneity in apparent adduct levels. This was also seen in peripheral blood mononuclear cells isolated from a patient after melphalan therapy.

Detection and Quantification of Melphalan-DNA Adducts at the Single Cell Level in Hematopoietic Tumor Cells

Bifunctional alkylating agents, such as melphalan, are widely used in the treatment of hematological malignancies. The effects of these drugs on particular types of hematological cells and the causes of treatment failure are poorly understood. The aim of this work was to establish an ability to measure the extent to which melphalan reacts with the DNA of individual tumor cells, thereby creating new possibilities for molecular pharmacological studies on clinical samples. A novel approach for staining drug-DNA adducts is described in which cells were embedded in agarose and then lysed. The DNA from each cell remained in an ideal state for quantitative immunofluorescent staining using a previously described monoclonal antibody. Immunofluorescence and DNA-Hoechst dye fluorescence were quantified using a cooled slow scan charge coupled device camera and image analysis procedures. Immunofluorescence of drug-treated cells from a human leukemia cell line was partially correlated with DNA content. Mean integrated immunofluorescence of 50 to 100 cells was dependent on drug concentration and was linearly related to adduct levels. In these cells and in chronic lymphocytic leukemia cells obtained from patients, there was considerable intercell heterogeneity in apparent adduct levels. This was also seen in peripheral blood mononuclear cells isolated from a patient after melphalan therapy.

DNA Damage by Anticancer Agents and Its Repair: Mapping in Cells at the Subgene Level with Quantitative Polymerase Chain Reaction

The quantitative polymerase chain reaction (QPCR)-based assay was used to measure DNA damage and repair to a small (523 bp) fragment of the single-copy human N-ras gene in K562 cells. Compared with previous methods DNA preparation from treated cells and the subsequent detection of the radioactive product were considerably simplified. The results demonstrated that and QPCR can be used to measure damage in a small gene segment, caused by cisplatin, nitrogen, and quinacrine mustards. Drug-DNA adducts produced by two novel minor groove binding, sequence-specific molecules (AT-486 and DSB-120) could be detected at physiologically relevant concentrations of drug. For both cisplatin and nitrogen mustard the concentrations required to cause damage in cells were higher than those needed to cause equivalent damage in isolated DNA. In contrast both AT-486 and quinacrine mustard caused more damage at equimolar concentrations in cells than in isolated DNA. DSB-120, which is closely related to AT-486, was found to be 15-fold less effective than the latter at causing damage in treated cells despite similar reactivity with isolated DNA. Repair of damage caused by quinacrine mustard to the same small gene fragment was found to proceed at a constant rate over 24 h. The and QPCR assay presented here is a simple quantitative method to measure damage and repair in subgene functional units such as promoters, introns, and exons.