Neutrophils cause oxidative DNA damage in alveolar epithelial cells.

Inflammation has been recognized as a contributing factor in the pathogenesis of some cancers. In the lung, inflammation is characterized by an influx of polymorphonuclear leukocytes (PMN) that release a variety of reactive oxygen species (ROS). The aim of the present study was to investigate the direct effect of PMN on oxidative DNA damage in lung target cells. Therefore, rat alveolar epithelial cells (RLE) were coincubated with PMN or hydrogen peroxide. Known to be correlated with the incidence of cancer, 7-hydro-8-oxo-2'deoxyguanosine (8-oxodG) was used as an effect marker for oxidative damage. Viability of the RLE, when coincubated with PMN, decreased to 43%, dependent on the ratio between PMN and RLE. After washing off PMN, 8-oxodG levels were significantly increased in RLE, but the highest levels were observed in the washed off PMN fraction. In addition, to avoid washing off procedures, immunohistochemical analysis was used to measure the 8-oxodG levels specifically in the RLE and similar results were obtained. In addition, inhibitor experiments showed that antioxidants ameliorated oxidative DNA damage. Our data provide evidence that ROS released by PMN as well as H2O2, cause oxidative DNA damage in epithelial cells.

Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D.

Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12-24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect. C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53.

cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity.

Polypeptides remaining tightly associated with isolated genomic DNA are of interest with respect to their potential involvement in the topological organization and/or function of genomic DNA. Such residual DNA-polypeptide complexes were used for raising monoclonal antibodies by in vitro immunization. Screening of a murine lambdagt11 cDNA library with these antibodies released a positive cDNA (MC1D) encoding a 16 kDa polypeptide. The cloned homologous human cDNA (HC1D) was identified in the dbest data base by partial sequence comparison, and it was sequenced full length. The cDNA-derived amino acid sequences comprise nuclear location signals but none of the known DNA-binding motifs. However, the recombinantly expressed proteins show in vitro DNA binding affinities. A polyclonal antiserum to the recombinant MC1D protein immunostains sub-nuclear structures, and it detects a residual 16 kDa polypeptide on western blots of DNA digests. These results support the conclusion that the cloned cDNAs reflect mRNAs encoding one of the chemically-resistant polypeptides which can be detected in isolated genomic DNA by sensitive techniques, e.g. by125Iodine labeling and SDS-PAGE.

Formation and persistence of 8-oxoguanine in rat lung cells as an important determinant for tumor formation following particle exposure.

Exposure of rats to quartz (or various other particles) can lead to the development of lung tumors. At the moment, the mechanisms involved in particle-induced tumor formation are not clarified. However, it is suggested that inflammation, in conjunction with the production of reactive oxygen species (ROS) and an enhancement of epithelial cell proliferation, may play a key role in the development of lung tumors. ROS induces 8-oxoguanine (8-oxoGua) and other mutagenic DNA oxidation products, which can be converted to mutations in proliferating cells. Mutation formation in cancer-related genes is a critical event with respect to tumor formation. In this study we investigated the effects of quartz (DQ12) and of the nontumorigenic dust corundum on the induction of 8-oxoGua in the DNA of rat lung cells, as well as on cell proliferation and pulmonary inflammation. Wistar rats were exposed by intratracheal instillation to quartz (2.5 mg/rat) or corundum (2.5 mg/rat) suspended in physiological saline; control animals exposed to physiological saline or left untreated. Measurements were carried out 7, 21, and 90 days after the exposures. 8-oxoGua levels were determined in lung tissue sections at the single cell level by immunocytological assay using a rabbit anti-8-oxoGua antibody. After exposure to quartz, 8-oxoGua levels were significantly increased at all time points of investigation. Additionally, we observed inflammation and an enhanced cell proliferation. Exposure to corundum had no adverse effects on the lung; neither increased 8-oxoGua levels nor enhanced cell proliferation or inflammation were detected. These observations support the suggestion that inflammation associated with increased 8-oxoGua levels in lung cells and increased cell proliferation is an important determinant for particle-induced development of lung tumors in the rat.

Human endothelial culture supernatant selectively promotes IgM-producing hybridomas.

We compared the effect of human endothelial cell culture supernatant (HECS), ESG (Ewing sarcoma growth factor), IL-6 (interleukin-6) and peritoneal macrophages on the recovery of hybridoma cells after fusion with respect to growth, stability and distribution of isotype variants. A selective growth of murine IgM-producing hybridoma cells was observed in the presence of HECS after cell fusion.

Monoclonal antibodies to thymidine glycol generated by different immunization techniques.

Monoclonal antibodies specific for thymidine glycol in oxidized DNA were generated by immunization with thymidine glycol monophosphate (TMP-OH) or thymidine glycol (T-OH), respectively, conjugated to keyhole limpet hemocyanin (KLH) or thyreoglobulin (TG). Forty-five clones (TMP-OH) and 70 clones (T-OH) were examined upon antibody production in ELISA. Four clones secreting IgG1, kappa, were characterized further. In several studies the antibodies derived from the immunization with TMP-OH were inhibited by various inhibitors. In descending order of effectiveness, they were thymidine glycol monophosphate (TMP-OH), thymidine glycol (T-OH), thymidine monophosphate (TMP), and thymidine (Thn). After immunization with T-OH, antibodies were inhibited in following order: T-OH > TMP-OH > TMP > Thn. Inhibition by the bases thymine, adenine, cytosine, and guanine were negligible. In ISB (Immuno Slot Blot) performed with OsO4-treated DNA (Poly-[dA-dT]) and amount of 70 fmol thymidine glycol was detectable. DNA had to be irradiated at a level of at least 20 Gy to detect any damage in ELISA but at a lower level of irradiation (10 Gy) in ISB by one of these antibodies, TPS-1. The antibodies obtained after immunization with hapten-protein are therefore capable of the detection of low frequency lesions in DNA generated by free radicals after radiation or oxidative stress.

Removal of O6-methylguanine from plant DNA in vivo is accelerated under conditions of clastogenic adaptation.

Previously it was shown that the clastogenic efficiency of high doses of alkylating agents in plant root meristems can be reduced significantly by conditioning pretreatment with either a low dose of the same agents, a sublethal heat shock, or heavy metal salts. The molecular mechanisms responsible for these protective effects are still unclear. Here we report on the quantification of O6-methylguanine [O6-MeG] by immuno-slot-blot analysis in DNA of root tip meristems of field bean (Vicia faba) seedlings under conditions of clastogenic adaptation. When root tips were pretreated with a low, conditioning dose of N-methyl-N-nitrosourea (MNU, 10(-4) M, 1 hour) 2 hours before exposure to a high dose of the same clastogen (10(-3) M, 1 hour), the frequency of chromatid aberrations was reduced by more than 50% at a recovery time of 1 B hours, as compared to treatment with the high dose alone. The same was observed when conditioning pretreatment was by a sublethal heat shock [10 minutes, 40 degrees C] or a heavy metal salt (Cd(NO)3, 10(-7) M, 1 hour). The frequency of O6-MeG immediately after exposure to a conditioning and a subsequent challenge treatment was reduced by 43% as compared to treatment with only the high dose. At a recovery time of 18 hours the corresponding frequency of adducts was reduced by 68.3% (related to the initial level) after treatment with the high dose alone, and by 81.3% under adaptive conditions. Sublethal heat shock or heavy metal salt used as conditioning pretreatments also resulted in a decrease of adducts immediately after treatment with the challenge dose. From these data and from prevention of the effects by pretreatment with cycloheximide or O6-benzylguanine we conclude that under conditions of clastogenic adaptation O6-MeG is more efficiently removed from the DNA, presumably by induction of an alkyl acceptor protein such as O6-methylguanine-DNA methyltransferase [MGMT]. This could explain the observed protective effects (clastogenic adaptation.

Formation and repair of O6-methylguanine in recombination hot spots of plant chromosomes.

Mutagen-induced chromatid aberrations are not randomly distributed along the metaphase chromosomes. In the field bean (Vicia faba), defined late-replicating and transcriptionally inactive heterochromatic regions are preferentially involved. After exposure to the alkylating agent N-methyl-N-nitrosourea (MNU) (10(-3) M, 1 hour), 70% of all aberrations are clustered within 6 segments containing tandemly repeated FokI elements of 59 bp, which comprise approximately 10% of the genome. Using immuno-slot-blot analyses, we have studied the frequency of O6-methylguanine (O6-MeG), a mutagenic lesion important for aberration induction, in total genomic DNA as well as in FokI sequences of the field bean after exposure to MNU. In either case, similar numbers of adducts per nucleotide were found immediately after treatment as well as after 18 hours of recovery, when most adducts were removed and significant amounts of chromatid aberrations were detectable. Peculiarities of long FokI element arrays (e.g., formation of specific tertiary structures), resulting in error-prone recombination repair, rather than preferential formation or delayed repair of O6-MeG are apparently responsible for aberration clustering in these hot spot regions.

Binding and repair of O6-ethylguanine in double-stranded oligodeoxynucleotides by recombinant human O6-alkylguanine-DNA alkyltransferase do not exhibit significant dependence on sequence context.

Double-stranded (ds) oligodeoxynucleotides (29mers) containing an O6-ethylguanine (O6-EtGua) flanked 5' and 3' by different bases (5'..TGT..3'; 5'..CGG..3', 5'..GGT..3'; 5'..GGG..3'; 5'..GGA..3') were synthesized to investigate the binding and repair characteristics of recombinant human O6-alkylguanine-DNA alkyltransferase (AT) in vitro. The apparent association constant (KA(app)) of AT to the oligomers and the repair rate constant for O6-EtGua (k) respectively, were determined by gel retardation and a monoclonal antibody-based filter binding assay. When ds- or single-stranded (ss) oligomers with or without O6-EtGua were used, no major differences in KA(app) values were observed with either substrate: KA(app) values for native AT were 7.1 and 8.4 x 10(5) M(-1) respectively, for unmodified and [O6-EtGua]-containing ds-oligomers. The corresponding values for ss-oligomers were 1.0 and 4.9 x 10(5) M(-1). The N-terminal first 56 amino acids of AT only exert a limited influence on DNA binding; the KA(app) values for an N-terminally truncated AT protein (1.1 x 10(5) M(-1)) and native AT were of the same order. Moreover, KA(app) was hardly affected by Cys(145)-methylated AT (2.0 x 10(5) M(-1)). The k-values (6.5-11.5 x 10(6) M(-1)s(-1)) were not significantly dependent on nucleotide sequence. k-values of 5.3 and 4.0 x 10(6) M(-1)s(-1) respectively, were obtained with the N-terminally truncated AT protein and for repair of the postreplicative mispair [O6-EtGua]: T by native AT. The low KA(app), the negligible influence on O6 of ethylation, and the minor modulation KA(app) and k by varying the bases flanking O6-EtGua, all indicate that the binding of AT to DNA is non-specific and mediated mainly by ionic interactions [reduced KA(app) and k-values at increased ionic strength]. Surplus DNA reduces the rate of O6-EtGua repair in ds-oligomers by competitive binding of AT molecules. The reaction mechanism of AT with DNA in vivo requires further investigation.

Urinary excretion of 3-methyladenine and 3-ethyladenine after controlled exposure to tobacco smoke.

The urinary excretion of the DNA alkylation products 3-methyladenine (3-MeAde) and 3-ethyladenine (3-EtAde) after controlled exposure to cigarette smoke over a period of 4 days was determined by competitive radioimmunoassay after separation by HPLC. Twenty-four hour urine samples were collected from five smokers and five non-smokers. Days 1 and 3 (control days) were without smoking, on days 2 and 4 smokers consumed 24 cigarettes each within 8 h in an unventilated room (45 m3) in the presence of non-smokers. Average levels of carbon monoxide during exposure were 15-20 p.p.m., 2.8-3.5 mg/m3 of respirable suspended particles and 75-86 micrograms/m3 of nicotine. Carboxyhemoglobin levels increased by 9.0 and 1.8% in smokers and passive smokers respectively. On control days, urinary excretion of 3-MeAde was similar in smokers and non-smokers (4.7-6.2 micrograms/24 h). Smoking resulted in a significant increase (P < 0.01) in 3-MeAde excretion (13.6-14.8 micrograms/24 h); no change was observed in the average excretion of 3-MeAde by passive smokers (4.8-4.9 micrograms of 3-MeAde/24h). Baseline 3-EtAde excretion on control days was similar in smokers and passive smokers (13.7-32.8 ng/24 h). In smokers, the amount of urinary 3-EtAde was increased > 5-fold (119.3-138.5 ng/24 h) on smoking days; no effect on 3-EtAde excretion was observed on average in passive smokers (18.0-25.2 ng/24 h). The nature of the DNA-reactive agent(s) responsible for the increased urinary excretion of 3-alkyladenines, in particular of the sensitive indicator 3-EtAde, remains to be determined.

Formamidopyrimidine DNA glycosylase in the yeast Saccharomyces cerevisiae.

A DNA glycosylase that excises, 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) from double stranded DNA has been purified 28,570-fold from the yeast Saccharomyces cerevisiae. Gel filtration chromatography shows that yeast Fapy DNA glycosylase has a molecular weight of about 40 kDa. The Fapy DNA glycosylase is active in the presence of EDTA, but is completely inhibited by 0.2 M KCl. Yeast Fapy DNA glycosylase does not excise N7-methylguanine, N3-methyladenine or uracil. A repair enzyme for 7,8-dihydro-8-oxoguanine (8-OxoG) co-purifies with the Fapy DNA glycosylase. This repair activity causes strand cleavage at the site of 8-OxoG in DNA duplexes. The highest rate of incision of the 8-OxoG-containing strand was observed for duplexes where 8-OxoG was opposite guanine. The mode of incision at 8-OxoG was not established yet. The results however suggest that the Fapy- and 8-OxoG-repair activities are associated with a single protein.

Cleavage and binding of a DNA fragment containing a single 8-oxoguanine by wild type and mutant FPG proteins.

A 34-mer oligonucleotide containing a single 7,8-dihydro-8-oxoguanine (8-OxoG) residue was used to study the enzymatic and DNA binding properties of the Fpg protein from E. coli. The highest rates of incision of the 8-OxoG containing strand by the Fpg protein were observed for duplexes where 8-OxoG was opposite C (*G/C) or T (*G/T). In contrast, the rates of incision of duplexes containing 8-OxoG opposite G (*G/G) and A (*G/A) were 5-fold and 200-fold slower. Gel retardation studies showed that the Fpg protein had a strong affinity for duplexes where the 8-OxoG was opposite pyrimidines and less affinity for duplexes where the 8-OxoG was opposite purines. KDapp values were 0.6 nM (*G/C), 1.0 nM (*G/T), 6.0 nM (*G/G) and 16.0 nM (*G/A). The Fpg protein also binds to unmodified (G/C) duplex and a KDapp of 90 nM was measured. The cleavage and binding of the (*G/C) duplex were also studied using bacterial crude lysates. Wild type E. coli crude extract incised the 8-OxoG containing strand and formed a specific retardation complex with the (*G/C) duplex. These two reactions were mediated by the Fpg protein, since they were not observed with a crude extract from a bacterial strain whose fpg gene was inactivated. Furthermore, we have studied the properties of 6 mutant Fpg proteins with Cys-->Gly mutations. The results showed that the 2 Fpg proteins with Cys-->Gly mutations outside the zinc finger sequence cleaved the 8-OxoG containing strand, formed complexes with the (*G/C) duplex and suppressed the mutator phenotype of the fpg-1 mutant. In contrast, the 4 Fpg proteins with Cys-->Gly mutations within the zinc finger motif neither cleave nor bind the (*G/C) duplex, nor do these proteins suppress the fpg-1 mutator phenotype.

N-methyl-N-nitrosourea-induced mutations in human cells. Effects of the transcriptional activity of the target gene.

In this study we addressed the question as to whether the mutagenesis by methylating agents is affected by the transcriptional activity of the damaged gene. An Epstein-Barr virus (EBV)-derived shuttle vector system was developed where the genetic target for mutation analysis, the bacterial gpt gene, is under the control of an eukaryotic inducible promoter in plasmid pF1-EBV and lacks the eukaryotic promoter in plasmid pF2-EBV. Two human cell lines that episomically maintain these shuttle vectors were established. In clone 6NT cells, which contain pF1-EBV plasmid, the gpt gene is actively transcribed and the transcription rate is regulated by zinc ions. In clone 3 cells, which harbor pF2-EBV plasmid, the gpt gene is not transcribed. Following treatment of both cell lines with the potent alkylating carcinogen N-methyl-N-nitrosourea (MNU), G.C to A.T transitions were the major mutagenic event, consistent with the miscoding potential of O6-methylguanine. The mutations were predominantly generated in the non-transcribed DNA strand of the active gpt gene. The same strand-bias was observed when the gpt gene was transcriptionally inactive, indicating that MNU-induced strand-specific formation of mutations is not due to transcription. Our data identify as major determinants of this phenomenon the sequence-specificity of MNU mutagenesis and the conformational properties of the target protein. Differences in mutation distribution were observed between the transcriptionally active and inactive gpt gene. This finding suggests that the organization of active genes in chromatin might modulate DNA alkylation and/or DNA repair.

After X-irradiation a transient arrest of L929 cells in G2-phase coincides with a rapid elevation of the level of O6-alkylguanine-DNA alkyltransferase.

Following X-irradiation of exponentially growing L929 cells two major phenomena have been observed. First, there was a delay in cell division which can be ascribed to the arrest of cells in the G2-phase (G2-block), and, second, the cellular content of the O6-alkylguanine-DNA alkyltransferase (AGT) was markedly increased. Flow cytometrical DNA-measurements revealed that cells began to accumulate in the G2-phase 4 h after irradiation (p.r.) irrespective of the X-ray dose, while both the fraction of cells blocked in G2 and the time period the cells persisted in G2 increased with the radiation dose. About 24 h past release from the G2-block the distribution of cells in the cell cycle was similar to that of untreated control cells. In comparison with control cells the AGT content in irradiated cells (4 Gy) was highest at about 48 h p.r. (3.4-fold increase). The highest ratio of increase in AGT was, however, observed to occur between about 4 and 13 h p.r. (2.6-fold increase). As shown by flow cytometrical measurements using a BrdUrd/DNA double labeling technique, this rapid primary increase in AGT coincides very well with the entrance of cells into the G2-phase. This indicates that the cellular AGT content in X-irradiated (parental) cells started to exceed the basal level at the beginning of the G2-phase, but not before or during the S-phase. Once the AGT level was elevated it continued to increase for 2 to 3 cell doubling times.

Repair of O6-ethylguanine in DNA protects rat 208F cells from tumorigenic conversion by N-ethyl-N-nitrosourea.

O6-Ethylguanine (O6-EtGua) is one of about a dozen different alkylation products formed in the DNA of cells exposed to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea (EtNU). We have evaluated selectively the relative capacity of cells for the specific enzymatic repair of O6-EtGua as a determinant for the probability of malignant conversion. Eleven O6-EtGua-repair-proficient (R+) variant subclones were isolated from the O6-EtGua-repair-deficient (R-) clonal rat fibroblast line 208F by selection for resistance to 1,3-bis-(2-chloroethyl)-1-nitrosourea (frequency, approximately equal to 10(-5). Contrary to the 208F wild-type cells, all variants expressed O6-methylguanine-DNA methyltransferase activity, while both kinds of cells were deficient for repair of the DNA ethylation products O2- and O4-ethylthymine. After exposure to EtNU (less than or equal to 500 micrograms/ml; 20 min), cells were analyzed for the formation of piled-up foci in monolayer culture and of anchorage-independent colonies in semisolid agar medium. Depending on the EtNU concentration, the frequencies of piled-up foci and agar colonies, respectively, in the R+ variants were as low as 1/28th and 1/56th of those in the R- wild type. Contrasting with the cells from R+ variant-derived agar colonies, cells from 208F (R-) agar colonies gave rise to highly malignant tumors when implanted subcutaneously into syngeneic rats. No significant differences in the frequencies of piled-up foci were found between wild-type and variant cells after exposure to the major reactive metabolite of benzo[a]pyrene, (+)-7 beta, 8 alpha-dihydroxy-9,10 alpha-epoxy-7,8,9,10 alpha-tetrahydrobenzo[a] pyrene, for which stable binding to guanine O6 in cellular DNA has not been observed. The relative capacity of cells for repair of O6-alkylguanine is, therefore, a critical determinant for their risk of malignant conversion by N-nitroso carcinogens.

Monoclonal antibody-based immunoassay for the determination of cellular enzymatic activity for repair of specific carcinogen-DNA adducts (O6-alkylguanine).

We describe a rapid and sensitive, monoclonal antibody (mAb)-based immunoassay that permits the quantitation of cellular enzymatic activity for repair of specific carcinogen-DNA adducts. The assay was established on the basis of the observation that complexes between an anti-(O6-ethyl-2'-deoxyguanosine) mAb (mAb ER-6) and O6-ethylguanine (O6-EtGua) residues in double-stranded DNA can be immobilized on nitrocellulose filters. Circular pSV2gpt plasmid DNA was linearized by digestion with EcoRI and reacted with N-ethyl-N-nitrosourea in vitro to a level of about one O6-EtGua residue per DNA molecule, labeled with 32P, and subsequently incubated with extracts prepared from L929 mouse fibroblasts and with mAb ER-6. The amount of repaired O6-EtGua is proportional to the decrease in 32P activity retained on the filters. Maximum O6-alkyl-guanine repair activity requires preparation of the cell extracts in the presence of high (approximately 0.5 M) NaCl concentrations, whereas the repair reaction per se is markedly inhibited by NaCl. Under the conditions used, the detection limit of the assay is approximately 1 x 10(-16) mol of repaired O6-alkylGua. The repair reaction followed second-order kinetics, indicating that O6-EtGua is predominantly repaired by an enzyme activity inactivated by the repair reaction (e.g. via an alkyl group transfer mechanism analogous to that exhibited by the alkyl group accepting cysteine residues of the 'suicidal' bacterial ogt and ada proteins). The data obtained from the assay can be used for computation of the relative enzymatic repair activity of a given cell extract and of the rate constant K (1 x mol-1 x s-1) of the repair reaction.

Distribution of O6-ethylguanine in DNA exposed to ethylnitrosourea in vitro as visualized by electron microscopy using a monoclonal antibody.

Application of a monoclonal antibody (Mab ER-6; Rajewsky et al., 1980) specific for O6-ethyl-2'-deoxyguanosine (O6-EtdGuo), in conjunction with a protein-free spreading procedure for double-stranded DNA molecules and transmission electron microscopy, permits the visualization of antibody molecules complexed to O6-EtdGuo residues formed in DNA upon reaction with the carcinogen N-ethyl-N-nitrosourea (EtNU) (Nehls et al., 1984). To obtain information on the distribution of O6-EtdGuo in native DNA exposed to EtNU in vitro, samples of purified rat brain DNA were briefly incubated with EtNU at concentrations differing by a factor of 8 (0.5 and 4 mg of EtNU/ml, respectively). As determined in DNA hydrolysates by competitive radioimmunoassay, the resulting DNA preparations contained O6-EtdGuo at O6-EtdGuo/2'-deoxyguanosine molar ratios of 15.1 X 10(-5) and 116 X 10(-5), respectively. Interspace distances between Mab-binding sites in both sets of ethylated DNA were determined by electron microscopy both in individual DNA fragments of different size, and in computer-generated, long-thread DNA constructs. Comparative statistical analyses by a newly developed MOLRANDO computer program show a non-random distribution pattern of Mab-binding O6-EtdGuo residues.

Increased cytotoxicity of 1-(2-chloroethyl)-1-nitroso-3(4-methyl)-cyclohexylurea by pretreatment with O6-methylguanine in resistant but not in sensitive human melanoma cells.

Cells from a resistant ("Gr II") and a sensitive ("Str") human melanoma xenograft were incubated in vitro with O6-methylguanine for 2 h, subsequently treated with 1-(2-chloroethyl)-1-nitroso-3(4-methyl)-cyclohexylurea (MeCCNU) for 1 h and then plated in soft agar. In the resistant cells the O6-methylguanine pretreatment (2 mM) yielded an increase in sensitivity towards MeCCNU by a factor of 7.5. In the sensitive melanoma cells pretreatment with O6-methylguanine did not increase cytotoxicity. Human bone marrow cells from three normal donors were similarly pretreated with O6-methylguanine and MeCCNU. There was a clear increase in MeCCNU-induced cytotoxicity. We conclude, that while O6-methylguanine does potentiate the action of MeCCNU in resistant melanoma cells, the therapeutic usefulness of this treatment strategy may be limited.