The role of human alkyladenine glycosylase in cellular resistance to the chloroethylnitrosoureas.

To investigate the possible role of glycosylase action in causing tumor resistance, a full-length, histidine-tagged human alkyladenine glycosylase has been purified from the cloned human gene contained in a pTrc99A vector propagated in a tag alkA mutant Escherichia coli. This human enzyme releases both 3-methyladenine and 7-methylguanine from methylated DNA but in contrast to previous studies of the bacterial AlkA glycosylase, it does not release any adducts from [(3)H]chloroethylnitrosourea-modified DNA. This finding suggests that the alkyladenine DNA glycosylase-dependent resistance to the toxic effects of the chloroethylnitrosoureas reported previously in the literature may occur by a mechanism other than through direct glycosylase action.

Repair of sulfur mustard-induced DNA damage in mammalian cells measured by a host cell reactivation assay.

DNA damage is thought to be the initial event that causes sulfur mustard (SM) toxicity, while the ability of cells to repair this damage is thought to provide a degree of natural protection. To investigate the repair process, we have damaged plasmids containing the firefly luciferase gene with either SM or its monofunctional analog, 2-chloroethyl ethyl sulfide (CEES). Damaged plasmids were transfected into wild-type and nucleotide excision repair (NER) deficient Chinese hamster ovary cells; these cells were also transfected with a second reporter plasmid containing RENILLA: luciferase as an internal control on the efficiency of transfection.

Role of base excision repair in protecting cells from the toxicity of chloroethylnitrosoureas.

The chloroethylnitrosoureas react extensively with cellular DNA to produce a variety of DNA adducts, including a deoxycytidine-deoxyguanosine (dC-dG) cross-link that is clearly cytotoxic. It is now well established that O6-alkylguanine-DNA-alkyltransferase can prevent formation of this dC-dG cross-link and thereby diminish the toxicity of the chloroethylnitrosoureas. Besides alkyltransferase, DNA glycosylases from various species can also contribute to cellular resistance to the chloroethylnitrosoureas, but the mechanism for this increased resistance has not been established.

Hypothermia causes a reversible, p53-mediated cell cycle arrest in cultured fibroblasts.

Normal human fibroblasts grown in cell culture undergo a reversible growth arrest when incubated at 28 degrees C. During incubation at 28 degrees C, levels of p53 and p21 rise in these cells and cell cycle analysis shows that they have undergone a cell cycle arrest. To examine the importance of p53 in mediating this arrest, mouse embryo fibroblasts that are either wild-type or that are defective in p53 were also subjected to hypothermia.

Fpg protein releases a ring-opened N-7 guanine adduct from DNA that has been modified by sulfur mustard.

Transfection of the Escherichia coli fpg gene into Chinese hamster ovary cells has been reported to enhance survival after exposure to aziridine (C. Cussac and F.Laval, 1996, Nucleic Acids Res.

The chloroethylnitrosoureas: sensitivity and resistance to cancer chemotherapy at the molecular level.

The chloroethylnitrosoureas were developed in a synthetic program that began with the observation that N-methyl-N'-nitro-N-nitrosoguanidine was an effective agent against L1210 cells. The antitumor activity of the chloroethylnitrosoureas is based on their reactions with DNA, especially the formation of a cytosine-guanine crosslink in DNA. Resistance occurs when the enzyme, O6-alkylguanine-DNA alkyltransferase, repairs an intermediate in crosslink formation.

Release of sulfur mustard-modified DNA bases by Escherichia coli 3-methyladenine DNA glycosylase II.

The toxic effects of sulfur mustard have been attributed to DNA modification with the formation of 7-hydroxyethylthioethyl guanine, 3-hydroxyethylthioethyl adenine and the cross-link, di-(2-guanin-7-yl-ethyl)sulfide. To investigate the action of bacterial 3-methyladenine DNA glycosylase II (Gly II) on these adducts, calf thymus DNA was modified with [14C]sulfur mustard and used as a substrate for Gly II. Gly II releases both 3-hydroxyethylthioethyl adenine and 7-hydroxyethylthioethyl guanine from this substrate.

A 32P-postlabeling method for the detection of adducts in the DNA of human fibroblasts exposed to sulfur mustard.

Since the toxicities of sulfur mustard are attributed to DNA alkylation, levels of DNA modification in exposed cells should correlate with the intensity of exposure. We have found that 32P-postlabeling can be used successfully to detect the major adduct, 7-hydroxyethylthio- ethyldeoxyguanosine 5'-phosphate (HETEpdG), that is formed in DNA by sulfur mustard. This method has been used to establish a correlation between exposure and adduct formation in human fibroblasts grown in cell culture and exposed to sulfur mustard concentrations between 2.

A 32P-postlabeling method for detecting unstable N-7-substituted deoxyguanosine adducts in DNA.

Many antitumor agents, including the mustards, form N-7 deoxyguanosine adducts in DNA that are difficult to quantitate by the 32P-postlabeling procedure because of their instability. We have developed a method that is successful for the analysis of such adducts using, as a prototype mustard, 14C-labeled bis(2-chloroethyl)sulfide. This agent forms the unstable product 7-hydroxyethylthioethyldeoxyguanosine in DNA.

Detection of sulfur mustard-induced DNA modifications.

Sulfur mustard is acutely toxic to the skin, eyes, and respiratory tract, and is considered carcinogenic to humans by the IARC. Since all of these toxicities are thought to be initiated by DNA alkylation, the level of DNA damage should serve as a biomarker for exposure. To develop methods of detecting this damage, DNA was modified by [14C]-labeled sulfur mustard and DNA adducts were released by mild acid hydrolysis.

Protection against chloroethylnitrosourea cytotoxicity by eukaryotic 3-methyladenine DNA glycosylase.

A eukaryotic 3-methyladenine DNA glycosylase gene, the Saccharomyces cerevisiae MAG gene, was shown to prevent N-(2-chloroethyl)-N-nitrosourea toxicity. Disruption of the MAG gene by insertion of the URA3 gene increased the sensitivity of S. cerevisiae cells to N-(2-chloroethyl)-N-nitrosourea, and the expression of MAG in glycosylase-deficient Escherichia coli cells protected against the cytotoxic effects of N-(2-chloroethyl)-N-nitrosourea.

Synthesis of the prototype DNA-protein cross-link, 1-(guan-1-yl)-2-(cysteine-S-yl)ethane, and its role in the reactions of the haloethylnitrosoureas.

The haloethylnitrosoureas form a cytotoxic DNA cross-link in a series of reactions which involves initial alkylation of the O6 position of guanine and rearrangement to the intermediate, 1,O6-ethanoguanine; 1,O6-ethanoguanine then reacts with a neighboring cytosine base. O6-Alkylguanine-DNA alkyltransferase can interrupt this process after the initial alkylation step by removing the alkyl group from the O6 position of guanine. Recent evidence suggests that the O6-alkylguanine-DNA alkyltransferase also recognizes 1,O6-ethanoguanine as a substrate, becoming bound to DNA when it interacts with that intermediate.

Identification of the cross-link between human O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea-treated DNA.

Chloroethylnitrosoureas induce reactive O6-guanine adducts in DNA that can form either interstrand cross-links or a covalent complex with the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). To test our hypothesis that these end-products are formed from the common precursor, 1-O6-ethanoguanine, we compared the kinetics of interstrand cross-link formation with those of decay of MGMT complex forming capacity. The half-lives of these processes were identical.

Release of N2,3-ethenoguanine from chloroacetaldehyde-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II.

The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which form N2,3-ethenoguanine in DNA. N2,3-Ethenoguanine is known to cause G----A transitions during DNA replication in Escherichia coli, and its formation may be a carcinogenic event in higher organisms. To investigate the repair of N2,3-ethenoguanine, we have prepared an N2,3-etheno[14C]guanine-containing DNA substrate by nick-translating DNA with [14C]dGTP and modifying the product with chloroacetaldehyde.

The formation and enzymatic repair of DNA modifications caused by the haloethylnitrosoureas and related compounds.

The haloethylnitrosoureas are both useful antitumor agents and known carcinogens. These biological activities are believed to be associated with DNA modification, and some biologically significant lesions have been identified in DNA exposed to these agents. At the same time, DNA repair is a cause of resistance to treatment by these agents, and may also serve as protection against their carcinogenic effects.

Release of N2,3-ethanoguanine from haloethylnitrosourea-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II.

3-Methyladenine DNA glycosylase II (Gly II), purified from Escherichia coli cells which carry the plasmid PYN1000, has been tested for its ability to release N2,3-ethanoguanine from DNA modified by the antitumor agent N-[2-chloroethyl-1,2-14C]-N'-cyclohexyl-N-nitrosourea ([14C]CCNU). Gly II has been shown to release N2,3-ethanoguanine in a protein- and time-dependent manner at a rate that exceeds the rate at which this enzyme releases other alkylated bases from [14C]CCNU-modified DNA. This finding widens the known substrate specificity for Gly II to include a modified base which bears an exocyclic ring structure, a class of modifications caused by a variety of chemical carcinogens.

3-Methyladenine DNA glycosylase activity in a glial cell line sensitive to the haloethylnitrosoureas in comparison with a resistant cell line.

Extracts of a glial cell line (SF-126) which is sensitive to the cytotoxic effect of the haloethylnitrosoureas and of a cell line (SF-188) which is resistant to these agents have been tested for their ability to release methylated bases from a DNA substrate which has been modified with [3H]dimethyl sulfate. In comparison with the sensitive cell line, extracts from the resistant cell line have 2-3-fold higher enzymatic activity. High performance liquid chromatography profiles of the bases which are released by these extracts show that the activity is specific for 3-methyladenine, suggesting that the resistant cells contain elevated levels of 3-methyladenine DNA glycosylase.

Release of 7-alkylguanines from N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA by 3-methyladenine DNA glycosylase II.

Purified bacterial 3-methyladenine DNA glycosylase II releases four 7-alkylguanines from [3H]N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA: 7-(2-hydroxyethyl)guanine,1,2-bis(7-guanyl)ethane, 7-(2-chloroethyl)guanine, and 7-(2-ethoxyethyl)guanine. 7-(2-Ethoxyethyl)guanine, a new compound, is formed as a result of an interaction with ethanol, a common solvent for the 2-haloethylnitrosoureas. Of the four 7-alkylguanines which are released from [3H]N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA, 7-(2-hydroxyethyl)guanine is released at a rate very much slower than the other three.

Development of monoclonal antibodies recognizing 7-(2-hydroxyethyl)guanine and imidazole ring-opened 7-(2-hydroxyethyl)guanine.

7-(2-Hydroxyethyl)guanine (7HEG) is of biological interest because it is formed in vivo by reaction of DNA with ethylene oxide (EO). Furthermore, the major DNA adduct of vinyl chloride, 7-(2-oxyethyl)guanine, can be converted to this adduct by reduction. Two monoclonal antibodies (9E2, 4A5) recognizing 7HEG have been developed from BALB/c mice immunized with the adduct coupled to keyhole limpet hemocyanin.

Formation of N2,3-ethanoguanine in DNA after in vitro treatment with the therapeutic agent, N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea.

HPLC analyses of the bases released by acid from N-(2-chloroethyl)-N-nitrosourea-treated DNA and N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-treated DNA show the presence of a new guanine adduct, N2,3-ethanoguanine. This derivative can be synthesized at the monomer level by treating 2-hydroxyethylguanine with thionyl chloride. The product of this reaction, purified by HPLC, has been shown to have a mol.

Mechanism of action of the nitrosoureas--IV. Synthesis of the 2-haloethylnitrosourea-induced DNA cross-link 1-(3-cytosinyl),2-(1-guanyl)ethane.

The 2-haloethylnitrosoureas have been shown to form the cross-linked structure 1-(3-cytosinyl),2-(1-guanyl)ethane in DNA. This cross-link has now been synthesized by the reaction of O6-(2-fluoroethyl)guanosine with deoxycytidine in dimethyl sulfoxide followed by removal of the sugars by acid hydrolysis. This synthetic route supports the mechanism for cross-link formation in DNA that involves an initial attack on the O6-position of guanine, followed by a rearrangement and subsequent reaction with cytosine.

Release of chloroethyl ethyl sulfide-modified DNA bases by bacterial 3-methyladenine-DNA glycosylases I and II.

Treatment with chloroethyl ethyl sulfide introduces the following modified bases into DNA: 7-ethylthioethylguanine, 3-ethylthioethyladenine, and O6-ethylthioethylguanine. Using the ethylthioethylated bases as models for DNA modifications involving relatively bulky alkyl groups, we have investigated the release of these bases by Escherichia coli 3-methyladenine-DNA glycosylases I and II. 3-Methyladenine-DNA glycosylase I releases only 3-ethylthioethyladenine from chloroethyl ethyl sulfide-modified DNA, but does so at a rate which exceeds the rate of release of 3-methyladenine (m3A) from methyl nitrosourea-modified DNA under these conditions.

Release of 7-alkylguanines from haloethylnitrosourea-treated DNA by E. coli 3-methyladenine-DNA glycosylase II.

Previous studies have related DNA modification by the haloethylnitrosoureas to their antitumor activity. Repair of this damage, particularly by O6-alkylguanine-DNA alkyltransferase, has been linked to tumor resistance by several previous investigations. We report here that E.

Differences in DNA alkylation products formed in sensitive and resistant human glioma cells treated with N-(2-chloroethyl)-N-nitrosourea.

The distribution of alkylated deoxynucleosides and bases has been determined in the DNA of a sensitive and a resistant human glioma-derived cell line exposed to therapeutic levels of [3H]N-(2-chloroethyl)-N-nitrosourea in vitro. The resistant cell line is 5-fold less sensitive to the cytotoxic effects of N-(2-chloroethyl)-N-nitrosourea and 8-fold less sensitive to sister chromatid exchange than the sensitive cell line. In comparison with the sensitive cells, DNA from the resistant cells contains much less of the cross-link, 1-(3-deoxycytidyl),2-(1-deoxyguanosinyl)ethane.