Formation Of Interstrand Cross-links Research Articles

Acquisition of resistance to 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride in V79 cells through increased removal of O6-alkylguanine.

The molecular mechanism of acquisition of resistance to 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride (ACNU) was investigated using ACNU-resistant clones (ACNUr-1-4) isolated from the V79 cell line. The binding level of alkyl cyanate, a decomposition product of ACNU, to protein in ACNUr-1 cells was not less than that in the parental V79 cells, indicating that the acquired resistance was not due to a reduced intracellular concentration of ACNU. Because O6-chloroethylguanine, an intermediate in cytotoxic interstrand cross-link formation by ACNU, is known to be repaired by the same mechanism as O6-ethyldeoxyguanosine (O6-EtdGuo), we quantitated O6-EtdGuo by radioimmunoassay at various times after exposure of cells to 100 micrograms/ml N-ethyl-N-nitrosourea for 20 min. In V79 cells, elimination of O6-EtdGuo was negligible, but in all four resistant clones, 30 to 59% of the O6-EtdGuo was removed within 24 hr after exposure. This increased removal of O6-EtdGuo among the resistant clones was associated with the activity of O6-alkylguanine DNA alkyltransferase (O6-AGT) determined using cell extracts. The present results indicate that increased removal of O6-chloroethylguanine in ACNU-resistant clones by O6-AGT is mechanistically linked to the acquisition of resistance to ACNU.

UV-induced interstrand cross-linking of d(GT)n.d(CA)n is facilitated by a structural transition.

Photochemical alterations following ultraviolet irradiation of the alternating copolymer d(GT)n.d(CA)n were studied. We found that in solution conditions which produced circular dichroism spectra compatible with B-form or A-form DNA, no interstrand cross-linking or photoproduct formation could be demonstrated. Zimmer et al. (Zimmer, C., Tymen, S., Marck, C., and Guschlbaumer, W. (1982) Nucleic Acids Res. 10, 1081-1091) and Vorlickova et al. (Vorlickova, M., Kypr, J., Sotkrova, S., Sponar, J. (1982) Nucleic Acids Res. 10, 1071-1080) have reported a number of solution conditions which produce a structural transition of this polymer characterized by a negative deviation of the circular dichroism spectrum in the region of 280 nm. The nature of this transition has not yet been elucidated. Following ultraviolet irradiation of d(GT)n.d(CA)n under two conditions which produce this transition (manganese solution or ethanol plus trace salts solution) we found ultraviolet dose-dependent interstrand cross-linking as well as dose-dependent formation of thymine-containing photoproduct. Interstrand cross-linking is demonstrated by two criteria: increase in polymer size as detected by alkaline agarose gel electrophoresis, and generation of intermediate density material in alkaline cesium sulfate isopycnic gradients. The thymine-containing photo-product was demonstrated by thin layer chromatography of acid hydrolysates of the polymer. The photo-product is at least partially photoreversible. These findings suggest that the geometry of the alternative conformation is such that pyrimidines from different strands are closely approximated, allowing for photodimerization.

Mechanisms of cellular resistance to ACNU in rat brain tumor cell line.

9L cells and 9L-2 cells, cell lines derived from the in vivo 9L rat brain tumor model, were treated with 1 (4-amino-2-methyl-5-pyrimidinyl) methyl-3 (2-chloroethyl) -3-nitrosourea hydrochloride (ACNU) and the effect on cell survival, the induction of sister chromatid exchange (SCE), and the formation of DNA interstrand cross-links were compared. Compared to 9L cells, 9L-2 cells were 10-fold more resistant to ACNU and cross-resistant to 1, 3-bis (2-chloroethyl) -1-nitrosourea (BCNU). In contrast to results obtained with chloroethyl nitrosourea, 9L-2 cells were 2-fold more sensitive to nitrogen mustard (HN2) and the amount of cell kill in both 9L and 9L-2 cells treated with cisdiamminedichloroplatinum (II) (cis-Pt) was almost the same. The number of SCEs induced by the treatment of ACNU, BCNU, HN2 and cis-Pt in both cell lines showed a good correlation with cytotoxicity. Compared to 9L cells, 9L-2 cells had 50% fewer DNA interstrand cross-links caused by ACNU and the removals of DNA interstrand cross-links were the same for 9L and 9L-2 cells. These results suggest that DNA interstrand cross-linking may cause cell death, and that it induces the SCEs in cells treated with ACNU and that cellular resistance to chloroethylnitrosoureas, including ACNU, is specific, and that the mechanisms of resistance to ACNU are different from the mechanisms of resistance to other DNA cross-linking agents.

Exposure of HeLa cells to 0 6-alkylguanines increases sensitivity to the cytotoxic effects of alkylating agents

Exposure of HeLa S3 cells to 0.4mM 0 6-methylguanine or 0 6-n-butylguanine for 24 h led to a substantial decrease in the activity of 0 6-alkylguanine-DNA alkyltransferase. Such pretreatment caused a marked increase in the sensitivity of the cells to the cytotoxic effects of the cross-linking alkylating agent 1-(2-chloroethyl)-1-nitroso-3-cyclohexylurea and a smaller increase in the sensitivity to N-methyl-N′-nitro-N-nitrosoguanidine. These results indicate that the repair of DNA by the alkyltransferase plays an important role in the protection of cells from the cytotoxic effects of certain alkylating agents particularly those such as 1-(2-chloroethyl)-1-nitroso-3-cyclohexylurea which ultimately lead to the formation of lethal interstrand cross-links.

A filter incubation method for the determination of potentially crosslinkable sites in DNA in mammalian cells

A method for the determination of DNA monoadducts capable of forming interstrand crosslinks in mammalian cells is described. Such monoadducts were produced by brief treatment of cells with cis-diamminedichloro-Pt(II) ( cis-DDP), 1-(2-chloroethyl)-1-nitrosourea (ClEtNU), l-phenylalanine mustard (L-PAM), or diaziridinylbenzoquinone (AZQ). The method is an alkaline elution procedure in which the DNA from lysed cells is incubated on polycarbonate filters at pH 10 and 37°C. During this incubation, the progressive formation of interstrand crosslinking was observed in drug-treated cells. In the case of ClEtNU and AZQ, DNA strand breaks also formed, due to the presence of labile lesions in the DNA. This made quantitation of interstrand crosslinks difficult for these drugs. For cis-DDP and l-PAM, however, there was no significant production of strand breaks and the assay for interstrand crosslinks was quantifiable.

Comparison of sister-chromatid exchange induction caused by nitrosoureas that alkylate or alkylate and crosslink DNA

We have investigated the induction of sister-chromatid exchanges (SCEs) in 9L rat brain tumor cells treated with the alkylating agent 1-ethyl-1-nitrosourea (ENU) and 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea (ACNU), an agent that both alkylates and crosslinks DNA. Induction of SCEs by ACNU was found to be 143-fold greater than for ENU. However, on an equimolar the alkylation of DNA by 14C-ACNU was approximately 3.2-fold higher than for 14C-ENU. After correction for this difference was made, the induction of SCEs by ACNU was calculated to be 45-fold greater than for ENU. While DNA alkylation products formed by ACNU and ENU are similar, the chloroethyl alkylation product(s) of ACNU can form DNA-interstrand crosslinks; the ethyl alkylation product(s) of ENU cannot. Based on these findings, we propose that the increased induction of SCEs caused by ACNU is a result of the formation of DNA interstrand crosslinks.

Macromolecular DNA-damage in murine and human leukemic and lymphoid cells after in vitro exposure to ASTA Z 7557 (INN mafosfamide).

Cyclophosphamide (CPA) is widely used against leukemic and lymphoproliferative diseases, but in vitro studies on response to this agent so far have been limited to instable derivatives with poor galenic properties. ASTA Z 7557 is a newly synthesized "activated cyclophosphamide" that circumvents the need for hepatic activation and has good stability. The critical cytotoxic lesions after exposure to bifunctional alkylating agents presumably are DNA interstrand crosslinks (ISC). We have, therefore, examined the formation and apparent removal of ISC after in vitro treatment with ASTA Z 7557 by use of the highly sensitive alkaline elution technique. Survival of murine L1210 cells was determined after 1 hour in vitro exposure with a D 37 value of 5.7 micrograms/ml (from the initial shoulder part of the survival curve) and a Do value of 1.5 micrograms/ml (from the exponential part of the curve). Previous labelling of L1210 cells by 125IUdR simplified the alkaline elution procedure but there was some cytotoxicity of the radiochemical itself with a reduction of cloning efficiency from 77% to 61%. The maximum of ISC was observed at 6 h after initiation of treatment with much of the damage apparently removed at 24 h. The simultaneous presence of DNA single strand breaks (SSB), however, confounds the analysis of DNA damage at 24 h and early cytolysis and unaided death of human lymphocytes often preclude the analysis of macromolecular damage at this time. Human peripheral blood cells isolated from patients with leukemic or lymphoproliferative diseases showed a remarkable heterogeneity with regard to the formation of ISC at 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)

The Role of Active Oxygen (102 and 0·2-) Induced by Crude Coal Tar and Its Ingredients Used in Photochemotheraphy of Skin Diseases

Crude coal tar (CCT) and certain photoreactive ingredients of CCT are photosensitizing agents used in the treatment of skin diseases (psoriasis, atopic eczema, etc.). Limited information is available in elucidating the mode of action of CCT in clearing psoriasis or causing skin photosensitization reactions. The production of singlet oxygen (1O2) and superoxide radicals (O(2) or HO2), the formation of interstrand cross-links (ICL) in DNA, and the skin photosensitization reaction caused by CCT or the ingredients present in tar preparations have been examined. Both type I (oxygen-independent) and type II (sensitized reactions requiring molecular oxygen) reactions are induced by CCT. Our data show that CCT and some of the photoreactive ingredients present in CCT produce 1O2, O(2), and ICL in DNA upon exposure to UVA radiation. Based on the equivalent concentration, the efficiency of various agents to produce 1O2 was of the following order: hematoporphyrin greater than phenanthridine greater than acridine greater than methylene blue greater than CCT greater than fluoranthrene greater than anthracene greater than pyrene greater than 8-methoxypsoralen greater than anthralin greater than chloroquine greater than anthralin dimer. The O(2) formation with CCT and its ingredients was also of the same order except for anthracene which was found to be a strong producer of O(2). The therapeutic effectiveness of CCT appears to be due to: (a) its cytotoxic effects, and (b) the production of 1O2, O(2), and ICL by CCT and its photoreactive ingredients. The skin photosensitizing (smarting, edema, and erythema responses) and carcinogenic properties of CCT may also be related to the production of 1O2 and O(2) and the formation of ICL which appear to be responsible for inducing the damage to the DNA and cell membrane.

Pretreatment of human colon tumor cells with DNA methylating agents inhibits their ability to repair chloroethyl monoadducts.

We have recently shown that a variety of human tumor cell lines are capable of preventing chloroethylnitrosourea (CNU)-induced DNA crosslinks, presumably by removing guanine O6-chloroethyl DNA monoadducts before crosslinks can form. Those cells capable of preventing crosslinking were of the Mer+ (Methylation repair) phenotype, and have been shown to be proficient at repair of O6-methyl-guanine adducts by the repair enzyme guanine-O6-methyl-transferase. Mer- tumor cell lines are: deficient at O6-methyl-guanine repair, incapable of preventing CNU interstrand crosslinking, and have recently been shown to lack the repair enzyme O6-methyl-transferase. We wish to report that pretreatment of Mer+ cells (HT-29 human colon carcinoma cells and IMR-90 normal human fibroblasts) with the DNA methylating agent MNNG, under conditions which should inactivate O6-methyl-transferase, apparently saturates the monoadduct repair system, and allows CNU to form interstrand crosslinks in these cells. This effect was also seen when MNU pretreatment was used, but not with MMS or streptozotocin. The formation of CNU-induced interstrand crosslinks following MNNG or MNU pretreatment was coincident with a dramatic increase in cytotoxicity as measured by colony formation assays. In contrast, cytotoxicity was only slightly increased when MMS or streptozotocin pretreatment was used.

Interstrand DNA crosslinks due to AP (apurinic/apyrimidinic) sites

Storage of a solution of DNA containing apurinic sites, even at 4°C leads to the appearance of interstrand crosslinks. Possible biological consequences of these crosslinks, when they appear in cell DNA, are briefly discussed. Formation of interstrand crosslinks in DNA containing tritium-labelled thymine and kept in an aqueous solution might be due, at least partly, to the loss of bases by the autoirradiated DNA.

Effect of 2-chloroethylnitrosoureas on plasmid DNA including formation of strand breaks and interstrand cross-links

Plasmid [3H]pBR 322 was incubated with various alkylating agents including chlorozotocin, N,N'-bis(2-chloroethyl)-N'-nitrosourea (BCNU), N-ethyl-N-nitrosourea (Enu) and dimethylsulfate (DMS). Formation of DNA strand breaks was followed by separation of the various forms of DNA on agarose gels and liquid scintillation counting of the bands. All alkylating agents examined were capable of rapidly producing strand breaks in time and concentration dependent fashion. Bands migrating as relaxed circular and supercoiled forms of the plasmid disappeared, and extensive alkylation resulted in formation of a band that migrated faster than the linear form of DNA. Electron microscopy of this band showed that it consisted of relaxed circles. Prolonged storage of alkylated plasmid resulted in fragmentation of the DNA, possibly due to strand scission at apurinic sites. A new neutral denaturation technique was developed, which allowed for the detection of DNA interstrand cross-links with minimal effects on other potentially labile sites of the alkylated DNA. The level of alkylation was quantitated by incubating [3H]pBR 322 with [2-chloroethyl-U-14C]chlorozotocin and was shown to be independent of DNA concentration but have a linear relationship with drug concentration. Linear and relaxed circular forms of the plasmid were alkylated to a somewhat higher extent than supercoiled DNA. Alkylation of pBR 322 with defined superhelical densities showed no preferential loss in DNA with a specific superhelical density, indicating that alkylation-induced unwinding is independent of superhelicity under the experimental conditions used.

DNA damage, prophage induction and mutation by furazolidone

Ultraviolet absorption data and thermal chromatography through hydroxyapatite (HAP) column revealed that furazolidone treatment of Vibrio cholerae cells produced more than 80% of DNA reversibly bihelical due to the formation of interstrand cross-links and the reaction obeyed a first order relation. Sensitivities of the Escherichia coli strains to the lethal action of the drug were in the order: AB 2480( uvr − rec −) > AB 2463(rec −) > AB 1886( uvr −) > AB 1157(repair proficient) or AB 4401(wild type). Furazolidone was ‘Rec test’ positive, produced dose-dependent prophage induction in E. coli cells and also dose-dependent streptomycin-resistance forward mutation in V. cholerae cells. The quantitative aspect and also the mode of furazolidone action on DNA were discussed.

Cross-linking of DNA induced by chloroethylnitrosourea is presented by O6-methylguanine-DNA methyltransferase.

The DNA repair enzyme O6-methylguanine-DNA methyltransferase has been used as a reagent to analyse the initial reaction sites of alkylating agents such as chloroethylnitrosourea that cross-link DNA. The transferase can be employed for this purpose because it removes substituted ethyl groups from DNA, as shown by its ability to act on O6-hydroxyethylguanine residues in DNA. The enzyme counteracts the formation of interstrand cross-links induced by bis-chloroethylnitrosourea, but not those induced by nitrogen mustard. Once formed, chloroethylnitrosourea-induced cross-links are not broken by the enzyme. In agreement with deductions from experiments with living cells, it is concluded that chloroethylnitrosourea act by forming reactive monoadducts at the O6 position of guanine and/or the O4 position of thymine, which subsequently generate -CH2CH2- bridges to the complementary DNA strand. A new method for quantitating interstrand cross-links in DNA has been employed.

Membrane transport, sulfhydryl levels and DNA cross-linking in Chinese hamster ovary cell mutants sensitive and resistant to melphalan

The mechanism of resistance to the alkylating agent melphalan was investigated in drug-sensitive and -resistant mutants of Chinese hamster ovary cells. Melphalan-resistant cells (Mel R6), selected by a single exposure to melphalan, were 4.5-fold more resistant to drug than sensitive AUXB1 parental cells. Colchicine-resistant cells (CH RC5), which are cross-resistant to melphalan, were 15-fold more resistant than wild type cells. The kinetic parameters for drug influx were not significantly different in sensitive and resistant cells. The steady-state drug level in both Mel R6 and CH RC5 cells was approximately 25 and 35% lower respectively than that in sensitive cells and this difference was accounted for by a more rapid rate of drug efflux from the resistant mutants. However, the level of drug resistance could not be explained entirely by this difference in drug transport. Sulfyddryl group levels were elevated in both Mel R6 and CH RC5 cells relative to sensitive cells and these differences were statistically significant (P < 0.001). Furthermore, DNA interstrand cross-link formation was significantly lower in resistant cells than in sensitive cells. A similar rate of repair of DNA interstrand cross-links was observed in sensitive and resistant cells with the possible exception of a slower rate of repair in Mel R6 cells. A higher level of DNA-protein cross-link activity, which may represent a mechanism for drug inactivation was observed in Mel R6 cells. These studies suggest that resistance to melphalan in Mel R6 and CH RC5 Chinese hamster ovary cell mutants is multifactorial involving lowered steady-state drug levels, enhanced drug efflux, elevated levels of sulfhydryl groups and decreased DNA interstrand cross-linking.

Quantitative aspects of the formation and loss of DNA interstrand crosslinks in Chinese hamster cells following treatment with cis-diamminedichloroplatinum(II) (cisplatin) II. Comparison of results from alkaline elution, DNA renaturation and DNA sedimentation studies

The formation of interstrand crosslinks in the DNA of cis-diamminedichloroplatinum(II) (cisplatin)-treated Chinese hamster cells has been demonstrated by three independent, highly sensitive techniques: namely those of alkaline elution, renaturation of crosslinked DNA and alkaline sucrose gradient velocity sedimentation. Simultaneous measurement of DNA break frequency by the first two methods and a computer model combined with the third enabled the calculation of crosslink frequency after application of low doses of cisplatin. Good agreement was found between the values obtained by the three methods used here, and also with those obtained by direct measurement of the amount of crosslinked hybrid DNA in density-labelled cells treated with higher doses of cisplatin (Roberts, J.J. and Friedlos, F. (1981) Biochim. Biophys. Acta 655, 146–151). When measured by all three methods described here, crosslinking was found to increase during several hours after treatment and then to decrease with a half-life of between 12 and 24 h. For low initial levels of crosslinking, this was largely attributed to an excision repair process, since the formation of breaks in DNA was only minimal.

Quantitative aspects of the formation and loss of DNA interstrand crosslinks in Chinese hamster cells following treatment with cis-diamminedichloroplatinum(II) (cisplatin) I. Proportion of DNA-platinum reactions involved in DNA crosslinking

The presence of interstrand crosslinks in the DNA of cis-diamminedichloroplatinum(II) (cisplatin)-treated Chinese hamster cells was demonstrated by the formation of a species of hybrid-labelled DNA in cells that had been density and radioactively prelabelled with a combination of [ 3H]thymidine and 2′-deoxy-5-bromouridine (BrdUrd) prior to treatment. The extent of crosslinking and of total binding of platinum to the DNA increased linearly with dose over a wide range of concentrations of cisplatin. Simultaneous measurement of the size of co-isolated 14C-labelled DNA from untreated cells permitted an estimate of the size of the crosslinked DNA and hence of the proportion of bound platinum involved in a crosslinking reaction. No increase in the proportion of crosslinking reactions occurred during several hours post treatment incubation of cells when crosslinks were measured by this technique, in contrast to other studies using different techniques to measure DNA interstrand crosslinking in cisplatin treated cells (see following paper, Pera, M.F., Jr., Rawlings, C.J., Shackleton, J. and Roberts, J.J. (1981) Biochim. Biophys. Acta 655, 152–166). The enforced high concentrations of cisplatin required by this method to detect crosslinks precludes studies of their possible repair.

DNA cross-linking and monoadduct repair in nitrosourea-treated human tumour cells.

The 1-(2-chloroethyl)-1-nitrosoureas are potent anti-cancer drugs which produce DNA inter-strand cross-links in a two-step reaction sequence. The first step was proposed to be an addition of a chloroethyl group to a guanine-O6 position of DNA; the second step, which occurs over a period of several hours in the absence of free drug, could then form an interstrand cross-link by the slow reaction of the bound chloroethyl group with a nucleophilic site on the opposite DNA strand. The delay between the formation of chloroethyl monoadducts and the formation of inter-strand cross-links allows time for a DNA repair mechanism, capable of removing the monoadducts, to prevent the cross-linking. We recently proposed this mechanism to account for a difference in inter-strand cross-linking between a normal and a transformed human cell strain. Day and his coworkers (see refs 7, 8 and previous paper) found that some human tumour cell strains (designated Mer- phenotype) are deficient in the ability to repair O6-methylguanine lesions in DNA. We therefore hypothesized that the repair function that removes O6-methylguanine residues from DNA would also remove chloroethyl monoadducts and hence prevent chloroethylnitrosourea-induced inter-strand cross-linking. We now present evidence that supports this hypothesis and indicates also that the O6-methylguanine repair confers resistance to cell killing by chloroethylnitrosourea.

Chemical reaction of DNA with cross-linking agents: Influences of base permutation on chemical reactivity

Certain psoralens can photoreact with the pyrimidine bases of DNA and form interstrand cross-links. In this communication, theoretical mechanism of the photoreaction of DNA with psoralens in the presence of near-ultraviolet light (320–400 nm, u.v.-A) is diagrammatically and quantitatively presented. The number of the receptor binding sites, the theorized photoreaction sites, can be estimated from the base composition of DNA if the extent of the photoreactivity of cytosine and the possibly steric hindrance effects of DNA double helix in photoreactivity can be experimentally determined. The photoreactivity of DNA can be expressed by the number of psoralens photobound. It is predicted that the photoreactivity of DNA is less than, or at most equal to, the number of receptor binding sites depending on the number of cross-links formed during photoreaction. The number of cross-links formed is the consequence of the base permutation which determines the constitution of cross-linking sites, and of the manners of psoralens intercalated in the stacked DNA bases. In addition, the formation of the cross-linking sites is also influenced by the photoreactivity of cytosine which is presently not understood. It is likely that theoretical mechanism of the chemical reaction of DNA with other cross-linking agents can be similarly hypothesized.

An [formula omitted] characterization of interstrand cross-links in DNA exposed to the antitumor drug [formula omitted]-dichlorodiammineplatinum(II)

The cis -isomer of the antitumor drug dichlorodiammineplatinum(II) [ cis -Pt(II)] was tested for its abilty to introduce nicks (single-strand breaks) into supercoiled PM2 DNA. Whereas incubations up to 24 h show no indication of cis -Pt(II)-treated DNA having single-strand breaks, DNA interstrand cross-links were detected in the first 15 min of incubation. Furthermore, the formation of DNA interstrand cross-links was both inhibited and fully reversed after incubation with 2 mM thiourea.

Dictamnine, an alkaloid which crosslinks DNA in the presence of ultraviolet light

In the presence of ultraviolet light, the furoquinoline alkaloid, dictamnine, caused calf thymus DNA to become easily renaturable. The effect was less pronounced than for the furocoumarin, 8-methoxypsoralen. Ease of renaturation is evidence of the formation of interstrand crosslinks in DNA. The mechanism of crosslink formation by this alkaloid may be like that of 8-methoxypsoralen.