The cytotoxicity of DNA carboxymethylation and methylation by the model carboxymethylating agent azaserine in human cells

M. O'Driscoll, P. Macpherson, Yao-Zhong Xu1 and P. Karran2

Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts EN6 3LD and
1 CRC Nitrosamine-Induced Cancer Research Group, Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carboxymethylating agents are potential sources of endogenous DNA damage that have been proposed as possible contributors to gastrointestinal carcinogenesis. The cytotoxicity of the model DNA carboxymethylating agent azaserine was investigated in human cells. Expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) did not affect sensitivity to the drug in two related Raji Burkitt's lymphoma cell lines. DNA mismatch repair-defective variants of Raji cells which display increased tolerance to DNA methylation damage were not selectively resistant to azaserine. Complementary results were obtained with a second carboxymethylating agent, potassium diazoacetate. In contrast, lymphoblastoid cell lines representative of each of the xeroderma pigmentosum complementation groups, including the variant, were all significantly more sensitive to azaserine than nucleotide excision repair-proficient cells. The hypersensitivity of XP cells was not due to systematic differences in the concentrations of intracellular thiol compounds or related thiol metabolizing enzymes. The data indicate that of the two types of potentially lethal DNA damage which azaserine introduces, carboxymethylated bases and O6-methylguanine, the former are repaired by nucleotide excision repair and are a more significant contributor to azaserine lethality in human cells.

Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; CM, carboxymethyl; DON, 5'-diazo-6-oxonorleucine; GGT, {gamma}-glutamyl transpeptidase; GSH, reduced glutathione; GST, glutathione S-transferase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; KDA, potassium diazoacetate; MGMT, O6-methylguanine-DNA methyltransferase; MMR, mismatch repair; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; MNU, N-methyl-N-nitrosourea; N7-CMGua, N7-carboxymethylguanine; N3-CMAde, N3-carboxymethyladenine; NER, nucleotide excision repair; O6-CMGua, O6-carboxymethylguanine; O6-meGua, O6-methylguanine; S6-CMGua, S6-carboxymethylthioguanine; TG, 6-thioguanine; XP, xeroderma pigmentosum.


    Introduction
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 Abstract
 Introduction
 Materials and methods
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 References
 
Endogenous DNA damage contributes to spontaneous mutagenesis. Methylated DNA bases are likely to be important contributors to intrinsic DNA damage and most cells are protected by DNA repair pathways which remove them (for a review see ref. 1). In human cells, the most significant and selective of these is O6-methylguanine-DNA methyltransferase (MGMT), which directly eliminates O6-methylguanine (O6-meGua) from DNA by in situ demethylation. The absence of MGMT expression, the Mex phenotype, has several known consequences for the cell. Mex cells are very sensitive to methylation-induced mutation and to killing by methylating agents (for a review see ref. 2). In addition, when exposed to methylating agents Mex cells experience a selective pressure to inactivate their DNA mismatch repair (MMR) pathway. The pressure arises because MMR potentiates the cytotoxicity of DNA O6-meGua (3). Mex human cells with defects in essential MMR complexes, such as hMutS{alpha} (46) or hMutL{alpha} (5,7,8) have been isolated by a simple selection for resistance to the methylating agents N-methyl-N-nitrosourea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In addition, cells from MMR-defective human tumours exhibit a similar extensive methylation tolerance (912). Because of the relationship between MMR defects and methylation tolerance, endogenous sources of DNA methylation have been proposed as potential contributors to the emergence of MMR-defective cells in human gastrointestinal or colorectal tumours (13,14).

The precise nature of intrinsic DNA methylating agents remains undefined. Several possible sources have been considered. These include S-adenosylmethionine, which is the intracellular methyl group donor for several enzymatic reactions and which can also act as a non-enzymatic methylating agent (15). Other potential sources include the nitrosation products of abundant naturally occurring compounds such as poly- or monoamines or peptides (16,17). The latter are of particular interest because the bacteria which inhabit the human gastrointestinal tract carry out simple nitrosation reactions of this type (18). Nitrosated amines or peptides introduce O6-meGua into DNA and are selectively mutagenic towards the bacterial counterparts of Mex human cells (17).

Bile salts, composed of amino acids conjugated to bile acids, represent abundant sources of potentially nitrosatable amino acids to which human gastrointestinal cells are exposed (19). It has been known for some time that nitrosated bile salts can introduce DNA damage that is both toxic and mutagenic in bacteria (20). Nitrosated bile salts are carboxymethylating agents which form predominantly N7-carboxymethylguanine (N7-CMGua) when they react with DNA. N3-carboxymethyladenine (N3-CMAde) and O6-carboxymethylguanine (O6-CMGua) (Figure 1Go) are formed as minor products. It has recently been shown that the nitrosated bile acid conjugate N-nitrosoglycocholic acid acts not only as a carboxymethylating agent, but also introduces a significant amount of O6-meGua into DNA (14). This important observation indicates that the abundant bile salts are a potential source not only of CM bases, but also of DNA O6-meGua in cells of the human digestive tract.



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Fig. 1. Metabolism of azaserine and its non-carboxymethylating analogue DON. Azaserine spontaneously decomposes to diazoacetate. This unstable intermediate can then carboxymethylate DNA to form O6-CMGua, N7-CMGua and N3-CMAde as major products. O6-meGua (shown) and the N-7 and N-3 methylpurines (not shown) are formed as minor products. DON is unable to break down to diazoacetate and cannot carboxymethylate or methylate DNA.

 
Nitrosation of amino acids produces N-2-diazoacetylpeptides. Azaserine (O-diazoacetyl-L-serine) (Figure 1Go) is a naturally occurring antibiotic which has the same active diazoacetyl group. Azaserine kills human cells via two known mechanisms. It abolishes de novo purine biosynthesis by inhibiting glutamine aminotransferase (21,22) and it acts as a carboxymethylating agent to introduce N7-CMGua, N3-CMAde and O6-CMGua into DNA (14,23,24). Azaserine resembles N-nitrosoglycocholic acid in that it is also a methylating agent and introduces detectable amounts of O6-meGua into DNA (14). The methylated base introduced by azaserine has demonstrable biological effects and azaserine is selectively mutagenic in bacteria which lack O6-meGua repair (17).

To determine which DNA lesions contribute significantly to the cytotoxicity of this model carboxymethylating agent in human cells, we examined the effect of azaserine on the survival of a number of human cell lines with defined defects in DNA repair. The DNA repair pathways analysed were: MGMT, which directly reverses potentially cytotoxic DNA O6-meGua to guanine; MMR, which converts unrepaired O6-meGua into lethal DNA damage; and nucleotide excision repair (NER), which selectively excises DNA adducts which significantly distort the DNA structure (for a review see ref. 25). MGMT-deficient Mex cells were not significantly more sensitive than related Mex+ cells and the absence of an active MMR pathway did not detectably alter cellular sensitivity to the drug. In contrast, xeroderma pigmentosum (XP) cells were sensitive to azaserine. The predominant cytotoxic DNA lesions introduced by azaserine are therefore the more bulky CM adducts and O6-meGua is, at best, a minor contributor to azaserine lethality.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
All chemicals were obtained from Sigma (St Louis, MO) unless otherwise indicated. Recrystallized MNU, a kind gift of Prof. Peter Swann (University College London, UK), was dissolved in 4 mM potassium acetate, pH 4.0, and aliquots stored at –20°C. 6-Thioguanine (TG), 8-azaguanine and hypoxanthine were each dissolved in 0.1 N potassium hydroxide. Stock solutions of azaserine and 5-diazo-6-oxonorleucine (DON) were made in phosphate-buffered saline. 1-Chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GSH) were each dissolved in water. Ethacrynic acid was dissolved in a minimal volume of ethanol and subsequently diluted in water. [14C]8-hypoxanthine was used at a specific activity of 49.5 mCi/mmol. Potassium diazoacetate (KDA) was kindly provided by Dr David Shuker (MRC Toxicology Unit, University of Leicester, Leicester, UK).

Oligonucleotide synthesis
Duplex oligonucleotides containing G:T, O6-meGua:T or a single unpaired C were prepared as described previously (26,27). The principle for chemical synthesis of the 34mer oligomer containing carboxymethylated thioguanine [5'-AGCTTGGCTGCAGGTXGACGGATCCCCGGGAATT-3', where X is S6-carboxymethylthioguanine (S6-CMGua)] has been described (28). Briefly, a corresponding 34mer containing TG in position X was first prepared as described (29). Two OD A260 units of purified TG-containing 34mer were dissolved in 300 µl of 0.4 M phosphate buffer (pH. 9.2) and 50 µl of 0.1 M sodium 2-iodoacetate in the same phosphate buffer was added. The reaction was monitored by FPLC, by which the starting oligomer (TG oligomer) and the product oligomer (CM-TG oligomer) were well separated (30). The reaction was completed in 4 h. The product peak was isolated by FPLC and desalted with NAP-10 (Pharmacia).

Cells
The Mex TK variant of the Burkitt's lymphoma line Raji, its methylation tolerant counterparts RajiF12 and Raji10 and the assay for MGMT have all been described previously (5,31). One unit of MGMT demethylates 1 pmol O6-meGua in the standard assay. The XP lymphoblastoid cell lines GM2345 (XP group A), GM2498, GM2240, GM2248 (all XPC), GM2485 (XPD) and GM2449 (XP variant) were obtained from the Human Genetic Mutant Cell Repository (Camden, NJ). All cells were maintained in RPMI medium containing 10% fetal calf serum. The response to drugs was determined by monitoring cell growth. After drug treatment, exponentially growing cells (1 ml) at ~2x105 cells/ml were seeded in 24-well dishes. Growth was monitored by daily haemocytometer counting. Cultures were diluted as appropriate to maintain exponential growth. Unless explicitly indicated otherwise, for treatment with azaserine or DON, 1 mM hypoxanthine was included in all cultures for the duration of the experiment. MNU, TG, 8-azaguanine and azaserine were not removed after exposure of the cells. All experiments were repeated two or more times.

Purification of hMutS{alpha} and band shift analysis
hMutS{alpha} was prepared from Raji cells by DNA cellulose and Q-Sepharose chromatography as previously described (27). The Q-Sepharose fraction was further purified by MonoQ FPLC as described by Drummond et al. (32).

Band shift analysis using duplex 32P-labelled oligonucleotides, formed by annealing 5'-end-labelled 34mer oligonucleotides to an excess of the appropriate complementary strand, was carried out as described (27). Briefly, hMutS{alpha} was incubated for 5 min at room temperature with 2 pmol of perfectly matched non-radioactive duplex in 20 µl HEPES–KOH, pH 8.0, 0.5 mM EDTA, 0.1 mM ZnCl2, 10% glycerol, 50 µg poly(dI•dC). The radiolabelled duplex (20 fmol) was then added and incubation continued for a further 20 min. Aliquots were analysed by electrophoresis on 6% polyacrylamide gels and the reaction products detected by autoradiography.

Enzyme assays
Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was assayed in cell extracts (106–107 cells) prepared in 10 mM Tris–HCl, pH 7.4, 10 mM MgCl2, 30 mM KCl, 0.1 mM dithiothreitol and 0.5% Triton X-100. Reactions (50 µl) contained 50 mM Tris–HCl, pH 7.0, 9 mM MgCl2, 2 mM dithiothreitol, 2 mM phosphoribosyl pyrophosphate, 10 µCi/ml [14C]hypoxanthine and 0–25 µg protein. After 15 min at 37°C, reactions were terminated by heating at 70°C for 5 min and cooling on ice. Duplicate 10 µl aliquots were spotted onto DE81 paper (Whatman) which was then washed twice in 50 mM Tris–HCl, pH 8.0, dried and the radioactivity determined by scintillation counting in 5 ml Permablend (Packard). One unit of enzyme activity catalyses the formation of 1 pmol inosine monophosphate/min.

Total cellular acid-soluble thiol content was determined using Ellman's reagent [5,5'-dithio-bis(2-nitrobenzoic acid)]. Aliquots of 5% trichloroacetic acid extracts of 25–500 µg cell sonicates in phosphate-buffered saline were added to 900 µl Ellman's reagent (0.2 mg/ml in 1 M HEPES–KOH, pH 7.8). The A410 was determined and the total thiol content calculated using GSH as a standard.

Total glutathione S-transferase (GST) and GST-{pi} activities were determined by the method of Habig et al. (33). Briefly, cytoplasmic cell extracts (10–200 µg) in a total volume of 1 ml 0.1 mM potassium phosphate, pH 6.5, were incubated with GSH (2 mM for total GST and 0.25 mM for GST-{pi}) and substrate (2 mM CDNB for total GST and 0.2 mM EA for GST-{pi}) for 30 min at 25°C. The reaction products were detected spectrophotometrically by monitoring A340 nm for total GST and A270 nm for GST-{pi}. Total GST product was calculated using the extinction coefficient {varepsilon}340 nm = 9.6/mM/cm and for GST-{pi} {varepsilon}270 nm = 5/mM/cm.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Azaserine cytotoxicity in Raji cells
Purine biosynthesis. A part of the cytotoxicity of azaserine is due to inhibition of de novo purine biosynthesis. A HPRT-deficient subline of Raji Mex which is unable to salvage purine bases, was particularly sensitive to the drug. The HPRT Mex cells were killed by azaserine concentrations as low as 5 µM (Figure 2aGo) and their sensitivity was unaltered by inclusion of 1 mM hypoxanthine in the growth medium. In contrast, the Raji HPRT+ Mexcells were more resistant to azaserine and their resistance was increased further in the presence of hypoxanthine. Thus, growth of Raji HPRT+ Mexcells was inhibited by 300 µM azaserine but cells grew normally in the presence of hypoxanthine following treatment with the same azaserine concentration (Figure 2bGo). In all subsequent experiments, the survival of cells treated with azaserine was determined by monitoring cell growth in the presence of 1 mM hypoxanthine to alleviate the purine deficiency.



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Fig. 2. Sensitivity of HPRT+ and HPRT Raji cells to azaserine. Exponentially growing Raji Mex (a) or their HPRT Mexcounterparts (b) were treated on day 1 with the concentrations of azaserine shown. Hypoxanthine (1 mM) was included in the growth medium as indicated. Cell proliferation was monitored by daily cell counts. A single representative experiment is shown for each variant.

 
DNA damage: the effect of MGMT expression. Raji Mex+ cells express MGMT (0.3 U/mg protein) and are >50-fold more resistant to MNU than their Mex counterparts (<0.05 U/mg protein). In contrast, Raji Mex cells were not detectably more sensitive to azaserine than the Raji Mex+ subline. In the presence of hypoxanthine, growth of Raji Mex and Raji Mex+ cells was unaffected by azaserine concentrations up to 100 µM. Growth inhibition was reproducibly observed at concentrations between 200 and 400 µM (Figure 3a and bGo). We conclude that azaserine is directly cytotoxic towards Raji cells independent of its effect on purine biosynthesis and that MGMT expression does not provide significant protection against cell killing. This suggests that O6-meGua is not a major contributor to the cytotoxic DNA damage induced by the drug.



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Fig. 3. The effect of MGMT expression or mismatch repair defects on the azaserine sensitivity of Raji Mex and Raji Mex+ cells. Exponentially growing Raji Mex (a) or Raji Mex+ (b) were treated on day 1 with the concentrations of azaserine shown. Growth in the presence of 1 mM hypoxanthine was monitored by daily cell counts. The mean values for two independent experiments are shown. Where no error bars are apparent, they are smaller than the symbols. Mismatch repair defects. RajiF12 (c) or Raji10 (d) were treated on day 1 with azaserine as shown. Growth in the presence of hypoxanthine was monitored by daily cell counts. A single representative experiment is shown for each variant.

 
Azaserine cytotoxicity and MMR capacity
Raji Mex cells are proficient in MMR. The methylation-tolerant derivatives of Raji Mex, Raji10 and RajiF12, which retain the Mex phenotype, are both >50-fold resistant to MNU compared with the parental Raji Mex cells (5). Their MMR defects did not significantly affect the azaserine sensitivity of Raji10 and RajiF12. Their growth after treatment was indistinguishable from that of parental Raji Mex cells over a range of azaserine concentrations. In particular, both MMR-defective clones were growth inhibited by exposure to 400 µM azaserine (Figure 3c and dGo). Since Raji10 and RajiF12 are tolerant to the presence of O6-meGua in their DNA, the absence of significant azaserine resistance in these cells is consistent with the absence of a protective effect of MGMT in Raji Mex+ cells. The azaserine sensitivity of Raji10 and RajiF12 provides further evidence that the methylated base does not contribute significantly to the cytotoxicity of the carboxymethylating agent.

In an alternative approach to the question of whether carboxymethylating agents might select for MMR-related DNA damage tolerance, we investigated whether Raji Mex cells which had been selected for azaserine resistance exhibited a methylation-tolerant phenotype. Using a protocol which mimics the selection of methylation-tolerant cells by MNU (5), four independent cultures of Raji Mex were exposed to increasing concentrations of azaserine in the presence of hypoxanthine. The initial concentration of 500 µM azaserine was highly cytotoxic to the cells. Surviving cells were re-treated with 500 µM azaserine and cells which re-grew were exposed to two rounds of treatment with 1 mM azaserine. Cells which withstood this treatment were allowed to recover and treated with 3 mM azaserine. Despite surviving treatment with these highly toxic concentrations of drug, the resulting populations did not display a stable resistance to azaserine following further growth. The MNU and TG resistance of a single clone from each population was investigated. None of the clones displayed the increased MNU and TG resistance which is characteristic of methylation-tolerant cells (data not shown). The absence of a stable tolerance to MNU in the azaserine-exposed cells indicates that, unlike methylating agents, selection for azaserine resistance does not select for a methylation-tolerant phenotype at a detectable frequency.

Similar data were obtained in preliminary studies with KDA. Diazoacetate is a postulated intermediate in the carboxymethylation and methylation of DNA by several agents, including azaserine (Figure 1Go). We found no evidence for selective sensitivity to KDA in Raji Mex compared with Raji Mex+ cells and both were killed by KDA concentrations >200 µM (data not shown). Results with this compound were more variable, however, perhaps reflecting the instability of KDA in biological systems. As we observed with azaserine, selection of KDA-resistant variants of Raji Mex cells by multiple exposures to increasing KDA concentrations from 100 µM to 1 mM did not result in cells with stable resistance to further KDA treatment. In particular, none (of five) of the independent surviving clones exhibited the MNU and TG resistance associated with MMR defects (data not shown). These data are consistent with the O6-meGua produced by these carboxymethylating agents not providing a significant selective pressure for MMR inactivation. This, in turn, indicates that O6-meGua is not an important contributor to their cytotoxic effects.

O6-CMGua binding by a mismatch recognition factor
To investigate whether MMR might recognize O6-CMGua, we used band shift analysis to compare the binding of a highly purified human MutS{alpha} mismatch recognition complex to oligonucleotides containing single defined mispairs or adducts. An oligonucleotide containing a single S6-CMGua was used as a model for O6-CMGua-containing DNA. The thiocarboxymethyl derivative was preferred to O6-CMGua because its synthesis is relatively straightforward. It is likely to be a reasonable model for O6-CMGua–hMutS{alpha} interactions because the mismatch recognition factor is known to bind both O6-meGua and S6-methylthioguanine-containing DNA. Figure 4Go shows that hMutS{alpha} binding to the S6-CMGua-containing substrate is extremely limited and this adduct is recognized poorly compared with a single O6-meGua or a G:T mispair. A single nucleotide loop is the preferred substrate for this mismatch recognition complex, followed by a single base mispair, O6-meGua:T and S6-CMGua:T, respectively.



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Fig. 4. Band shift analysis of hMutS{alpha} mismatch binding. Highly purified hMutS{alpha} as shown was incubated with duplex oligonucleotides radiolabelled to similar specific activities. The duplexes contained a single unpaired C ( /C\ ), a G:T mispair (G:T), an O6-meGua:T base pair (O6meG:T) or an S6carboxymethyl:T base pair (S6CMG:T). Products were separated on a 6% polyacrylamide gel and detected by autoradiography. The arrow denotes the position of the hMutS{alpha}–oligonucleotide complex.

 
The relatively poor interaction between hMutS{alpha} and S6-CMGua is consistent with the unchanged azaserine sensitivity of MMR-defective cells. Together the data indicate that processing of O6-CMGua or O6-meGua by MMR is not likely to be a significant factor in the cytotoxicity of carboxymethylating agents.

Azaserine sensitivity in NER-deficient human cell lines
NER provided considerable protection against the cytotoxicity of azaserine. The XP lymphoblastoid cell line GM2345 is Mex+ and is defective in NER. GM2345 cells belong to XP complementation group A and are essentially completely deficient in the repair of UV photoproducts. They are highly sensitive to killing by UV light. They were also hypersensitive to the toxic effect of azaserine in the presence of hypoxanthine and their growth was completely inhibited by azaserine concentrations as low as 15 µM (Figure 5aGo). This is in contrast to the NER-proficient Raji cells, which were unaffected by azaserine concentrations >10-fold higher.




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Fig. 5. (a) Azaserine sensitivity of XP cells. GM2345, GM2498 or GM2249 were treated with the concentrations of azaserine shown. GM2345 was treated on day 2, the others on day 1. Growth in the presence of 1 mM hypoxanthine was monitored by daily cell counts. (b) Sensitivity of NER-proficient and NER-deficient cells to DON. Raji Mex and GM2345 were treated on day 1 with DON as shown. Growth in the presence of 1 mM hypoxanthine was monitored by daily cell counts.

 
Lymphoblastoid cells from other XP groups were sensitive to similar low concentrations of azaserine. Growth of GM2498, GM2248 (both Mex, XP group C), GM2249 (Mex+, XP group C), GM2252 (Mex+, XP group B), GM2485 (Mex+, XP group D) and GM2449 (Mex+, XP variant) was inhibited by azaserine at concentrations of 10–20 µM (Figure 5aGo and data not shown). All the XP cell lines expressed HPRT (1.4–1.8 U/mg protein measured in cell extracts) and were sensitive to 8-azaguanine, which is selectively toxic towards HPRT+ cells (data not shown). Their hypersensitivity is not therefore due to an inability to utilize hypoxanthine. The general sensitivity of the XP cell lines indicates that the major determinant of azaserine resistance is the ability to remove by NER, or to tolerate, bulky azaserine-induced DNA damage. Since the drug sensitivity of the Mex XP and Mex+ XP cells were indistinguishable, the observations provide additional confirmation that any cytotoxic effects of DNA O6-meGua are negligible or minor.

In a control experiment GM2485, GM2345 and Mex Raji cells were treated with the non-carboxymethylating structural analogue of azaserine, DON (Figure 1Go). This compound inhibits both purine and pyrimidine biosynthesis but does not introduce DNA damage because it cannot be metabolized to the reactive diazoacetate intermediate (21,33). In the presence of hypoxanthine, sensitivity of the XP group A and D cells to the growth inhibitory effects of DON was comparable with that of NER-proficient Raji cells (Figure 5bGo and data not shown). These observations provide independent confirmation that persistent DNA damage induced by azaserine, and not differential inhibition of the synthesis of DNA precursors, is responsible for the selective azaserine sensitivity of XP cells.

Intracellular thiols and azaserine cytotoxicity
The cellular response to azaserine may also be influenced by the extent of DNA modification. The level of intracellular thiol compounds is a potential modulator of azaserine sensitivity (35). {gamma}-Glutamyl transpeptidase (GGT) initiates the breakdown of extracellular GSH and promotes the accumulation of intracellular thiols (for a review see ref. 36). It is known to influence the azaserine sensitivity of some cell lines (35,37). There was a considerable variation in GGT activity in extracts of the cells we used (Figure 6aGo). Values ranged from 40 U/mg protein in the XP variant GM2449 to 5 U/mg protein in GM2485 (XP group D). The values for Raji Mex cells (15 U/mg) and the XP group B GM2252 cells (20 U/mg) were intermediate. The level of GGT activity was not correlated with sensitivity to azaserine and we conclude that the activity of this enzyme is not a limiting factor for the extent of DNA modification by the drug.



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Fig. 6. Thiol metabolizing enzymes and cellular thiol levels determined in cell extracts. (a) GGT activity in cell extracts. (b) GST activity in cell extracts. (c) GST-{pi} activity. (d) Total acid-soluble thiols.

 
The activities of GST in the XP cell lines were closely similar (Figure 6bGo). The equivalent value for Raji Mex cells was considerably lower although the levels of GST-{pi}, which is the form sometimes linked to drug resistance (38,39), were not significantly different between Raji Mex and the XP lines (Figure 6cGo). Since thiols reduce azaserine cytotoxicity, the relative sensitivities of the XP and Raji cells are not consistent with a determining role for intracellular thiols in sensitivity to the drug. In agreement with this, the azaserine resistance of Raji Mex cells was not accompanied by a high intracellular level of total acid-soluble thiol compounds when compared with the XP cells (Figure 6dGo). The data suggest that variations in the levels of intracellular thiols within the limits we observed do not alter azaserine sensitivity to a significant degree. In particular, the NER defect in the XP cell lines overrides any potential effect of thiols on azaserine sensitivity.

In comparison with Raji Mex cells, UV-sensitive XP cell lines, including the XP variant, are all hypersensitive to azaserine. This differential sensitivity is not explained by differences in the metabolism or concentration of intracellular thiols which would provide protection from DNA modification. We conclude that the cytotoxicity of azaserine is overwhelmingly due to the introduction of DNA damage which can be recognized by NER.


    Discussion
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Loss of MMR is a common outcome when cells are exposed to methylating agents. This is a direct consequence of the selective pressure exerted by the lethal processing of persistent DNA O6-meGua by MMR. The possibility that endogenously generated carboxymethylating compounds might elicit loss of MMR during the development of human gastrointestinal cancer was supported by the recent observation that model carboxymethylating compounds are also methylating agents (14). The observation that neither MGMT expression nor the methylation-tolerant phenotype significantly affects the sensitivity of human lymphoma cells to azaserine (or, on less extensive analysis, KDA) suggests, however, that O6-meGua is not a significant contributor to the lethal effect of these model carboxymethylating agents. Analogous carboxymethylating compounds which arise in vivo are therefore unlikely to introduce enough DNA O6-meGua to provide significant selective pressure for the loss of MMR. We note that the ratio of carboxymethylated to methylated bases may vary among different carboxymethylating agents (40). We cannot formally exclude the existence of a carboxymethylating compound for which this ratio is particularly low, which would have properties closer to those of a characteristic methylating agent.

The unchanged resistance of MMR-defective cells and the inability of both azaserine and KDA to elicit a methylation-tolerant phenotype also indicate that MMR does not significantly influence survival by interacting with carboxymethylated bases. The absence of significant interaction between S6-CMGua in the model oligonucleotide substrate and the hMutS{alpha} mismatch recognition complex is in agreement with this and indicates that the abundant O6-CMGua is unlikely to interact with MMR. Recognition by mismatch binding activities is a prerequisite for processing by MMR. This criterion is fulfilled by O6-meGua-containing base pairs (26,41) which are processed into lethal DNA lesions by MMR. Although we cannot exclude the recognition of other CM DNA bases by hMutS{alpha} or an interaction between O6-CMGua and the alternative mismatch recognition complex, hMutSß, both these possibilities appear unlikely. In particular, the normally low levels of hMutSß (42) would be unlikely to influence cytotoxicity to a significant extent. Our observations are not consistent with a significant role for processing of either O6-meGua or O6-CMGua by MMR in the lethality of carboxymethylating agents. The simplest interpretation is that carboxymethylated bases are directly cytotoxic without interacting with the MMR system. We do not rule out the possibility, however, that generation of the highly mutagenic O6-meGua might contribute to the rate of spontaneous mutation in cells exposed in vivo to carboxymethylating agents. Indeed, O6-meGua introduced into DNA by azaserine and other carboxymethylating agents introduces the characteristic G->A transition mutations in O6-meGua-DNA methyltransferase-deficient bacteria (17,20).

The general sensitivity of XP cell lines to azaserine is consistent with the sensitivity of excision repair-defective Escherichia coli UvrA mutants to carboxymethylating agents (43). It suggests that one or more of the bulky CM DNA bases is a significant cytotoxic DNA lesion introduced by this type of agent in human cells. The cytotoxic CM DNA adduct(s) has not been identified. Among the CM bases, the N7-CM and N3-CM purines as well as O6-CMGua and modified pyrimidines are all potential candidates. All are likely to introduce significant distortion into DNA. They would thereby produce potential substrates for NER and would be selectively cytotoxic towards XP cells. There is indirect evidence that the N7-CM and N3-CM purines may be excised by the E.coli AlkA DNA repair enzyme (17,44), which is the counterpart of the human methylpurine-DNA glycosylase (Aag). The human enzyme excises a variety of modified purines, including N7- and N3-methyl, -ethyl and -chloroethyl (45), as well as ethano and deaminated derivatives (46,47). An ability to excise N7-CM and N3-CM purines would certainly be consistent with the general properties of the Aag enzyme. Aag is known not to excise O6-modified guanine bases. Evidence from in vivo studies indicates that O6-CMGua is not recognized to a significant degree by either the bacterial or human O6-meGua-DNA methyltransferases (14,17). O6-CMGua is therefore a plausible candidate for a DNA lesion which is repaired by NER and is selectively toxic towards XP cells independent of their Mex status. The differential lethality of CM DNA lesions in NER-defective XP cells should make it possible to identify precisely the lethal CM bases by analysing the persistence of CM DNA lesions in wild-type and XP cells treated with azaserine or other carboxymethylating agents.

Azaserine is a carcinogen in rats (48). It induces pancreatic and renal tumours. This spectrum of tumours also serves to distinguish the methylating agents from carboxymethylating agents and suggests that the DNA lesions involved in carcinogenesis are different for the two types of agent. The precise reasons for the organotropism of azaserine are unclear. Differential drug uptake by target cells, GGT activity and intracellular thiol concentrations have all been considered as possible mediators of carcinogenesis (35,37). All of these factors may influence the level of reaction with DNA. Our results with cultured human cells indicate that the ability to remove or tolerate bulky CM DNA bases is likely to be one of the predominant factors in the cellular response to carboxymethylating agents like azaserine.


    Acknowledgments
 
M.O'D. and Y.-Z.X. are grateful to Prof. Peter Swann for his encouragement and support. The help of the ICRF Cell Production team at Clare Hall is also gratefully acknowledged.


    Notes
 
2 To whom correspondence should be addressed Email: karran{at}icrf.icnet.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 17, 1999; revised April 15, 1999; accepted May 27, 1999.





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