Pharmacogenetics of drug metabolizing enzyme: thiopurine methyl transferase phenotypes and multidrug resistance 1 gene polymorphism in inflammatory bowel disease.

Inflammatory bowel disease(IBD) is progressing rapidly in developing countries such as Iran. This research is intended to compile the frequency distribution of the drug metabolizing enzyme, thiopurine methyl transferase(TPMT) and the drug transporter, Multi drug resistance(MDR1) which are involved in metabolism of many therapeutics such as thiopurines in inflammatory bowel disease(IBD). Ethnicity is an important variable influencing drug response. The aims of this research were to investigate the association of TPMT phenotypes with MDR1 genotypes. TPMT activity was measured by using a non-extraction HPLC method and genotype for the C3435T polymorphism of MDR1 gene was determined in 215 unrelated IBD patients including of 85 males and 130 females and 212 unrelated healthy individuals consisted of 96 males and 116 females as control group by PCR-RFLP in Iran's western population. TPMT phenotypes demonstrated no frequency for deficient, 2.2% for low and 97.8% for normal activity that is different with results of other studies. Interestingly there were a significant negative correlation between TPMT activities as calculated based on nmol/grHb/h and positive correlation calculated in mU/L with Hb levels in IBD patients and control subjects. Dominant and codominant MDR1 C3435T gene polymorphism increased the risk of IBD by 1.45 and 1.46 times, respectively. IBD patients with MDR1 mutant genotypes C3435T, had lower TPMT activites and Hb concentrations. Using of mU/L is more appropriate than nmol6MTG/grHb/h for expressing TPMT activity. TPMT frequency of deficient and low activity in western Iran is low. The carriers of mutant C3435T MDR1 are not good TPMT methylators.

The identification, analysis and structure-based development of novel inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is an essential enzyme in the microbial folate biosynthetic pathway. This pathway has proven to be an excellent target for antimicrobial development, but widespread resistance to common therapeutics including the sulfa drugs has stimulated interest in HPPK as an alternative target in the pathway. A screen of a pterin-biased compound set identified several HPPK inhibitors that contain an aryl substituted 8-thioguanine scaffold, and structural analyses showed that these compounds engage the HPPK pterin-binding pocket and an induced cryptic pocket. A preliminary structure activity relationship profile was developed from biophysical and biochemical characterizations of derivative molecules. Also, a similarity search identified additional scaffolds that bind more tightly within the HPPK pterin pocket. These inhibitory scaffolds have the potential for rapid elaboration into novel lead antimicrobial agents.

Mucosal improvement in patients with moderate to severe postoperative endoscopic recurrence of Crohn's disease and azathioprine metabolite levels.

BACKGROUND: The value of azathioprine metabolites (6-thioguanine nucleotides [6-TGN]) in monitoring clinical treatment response is still controversially discussed. Data regarding thiopurine metabolite levels and endoscopic improvement are lacking. METHODS: Data were analyzed post hoc from a 1-year, multicenter, double-blind, double-dummy, randomized trial comparing azathioprine 2.0 to 2.5 mg/kg per day versus mesalamine 4 g/d in a subset of 23 postoperative patients with Crohn's disease (CD) treated with azathioprine and having moderate-to-severe endoscopic recurrence according to a modified 6-grade score. Red blood cell (RBC) concentrations of 6-TGN, 6-methyl-mercaptopurine ribonucleotides (6-MMPR), and 6-methyl-thioguanine nucleotides (6-MTGN) were indicated as follows: area under the concentration-time curve, average concentration (C av), and concentration at the final study visit. RESULTS: Overall, 74% of patients showed an improvement in the modified endoscopic score (P = 0.022). Median endoscopic score reduced from 4 at the baseline to 2 at the final visit. Patients with a high C av for 6-TGN (≥ 193 pmol/8 × 10(8) RBC; P = 0.017) or 6-MTGN (≥ 79.2 pmol/8 × 10(8) RBC; P = 0.035) significantly improved in endoscopic score, and the improvement in endoscopic score correlated with C av for 6-TGN (r = -0.51; P = 0.013). For concentration at the final visit, higher values for 6-TGN (≥ 142 pmol/8 × 10(8) RBC; P = 0.017) were associated with a better postoperative score. Sensitivity analysis revealed a significant correlation between 6-TGN (area under the concentration-time curve) and postoperative endoscopic improvement. CONCLUSIONS: Our post hoc analysis from a double-blind, randomized trial suggests that higher RBC 6-TGN levels are associated with endoscopic improvement in patients with severe postoperative endoscopic recurrence of CD. Thus, our study provides first evidence on the utility of monitoring of thiopurine metabolites to achieve mucosal response in CD.

Effects of 6-thioguanine and S6-methylthioguanine on transcription in vitro and in human cells.

Thiopurine drugs are extensively used as chemotherapeutic agents in clinical practice, even though there is concern about the risk of therapy-related cancers. It has been previously suggested that the cytotoxicity of thiopurine drugs involves their metabolic activation, the resultant generation of 6-thioguanine ((S)G) and S(6)-methylthioguanine (S(6)mG) in DNA, and the futile mismatch repair triggered by replication-induced (S)G:T and S(6)mG:T mispairs. Disruption of transcription is known to be one of the major consequences of DNA damage induced by many antiviral and antitumor agents; however, it remains undefined how (S)G and S(6)mG compromise the efficiency and fidelity of transcription. Using our recently developed competitive transcription and adduct bypass assay, herein we examined the impact of (S)G and S(6)mG on transcription in vitro and in human cells. Our results revealed that, when situated on the transcribed strand, S(6)mG exhibited both inhibitory and mutagenic effects during transcription mediated by single-subunit T7 RNA polymerase or multisubunit human RNA polymerase II in vitro and in human cells. Moreover, we found that the impact of S(6)mG on transcriptional efficiency and fidelity is modulated by the transcription-coupled nucleotide excision repair capacity. In contrast, (S)G did not considerably compromise the efficiency or fidelity of transcription, and it was a poor substrate for NER. We propose that S(6)mG might contribute, at least in part, to thiopurine-mediated cytotoxicity through inhibition of transcription and to potential therapy-related carcinogenesis via transcriptional mutagenesis.

6-Thioguanine and S6-methylthioguanine are mutagenic in human cells.

Thiopurines are effective immunosuppressants and anticancer agents. However, the long-term use of thiopurines was found to be associated with a significantly increased risk of various types of cancer. To date, the specific mechanism(s) underlying the carcinogenicity associated with thiopurine treatment remain(s) unclear. Herein, we constructed duplex pTGFP-Hha10 shuttle vectors carrying a 6-thioguanine ((S)G) or S6-methylthioguanine (S6mG) at a unique site and allowed the vectors to propagate in three different human cell lines. Analysis of the replication products revealed that although neither thionucleoside blocked considerably DNA replication in any of the human cell lines, both (S)G and S6mG were mutagenic, resulting in G→A mutation at frequencies of ~8% and ~39%, respectively. Consistent with what was found from our previous study in E. coli cells, our data demonstrated that the mutagenic properties of (S)G and S6mG provided significant evidence for mutation induction as a potential carcinogenic mechanism associated with chronic thiopurine intervention.

LC-MS/MS coupled with stable isotope dilution method for the quantification of 6-thioguanine and S(6)-methylthioguanine in genomic DNA of human cancer cells treated with 6-thioguanine.

Thiopurines, including mercaptopurine (MP), 6-thioguanine ((S)G) and azathioprine, are widely used for the treatment of many human diseases including acute lymphoblastic leukemia (ALL). To exert their cytotoxic effect, these prodrugs need to be metabolically activated to (S)G nucleotides and incorporated into nucleic acids. (S)G in DNA can be methylated spontaneously to S(6)-methylthioguanine (S(6)mG) in the presence of S-adenosyl-l-methionine. It was proposed that S(6)mG, owing to its high miscoding potential (pairing preferentially with thymine), may induce cell death by triggering the postreplicative mismatch repair pathway. Understanding the implications of this pathway in the cytotoxic effect of thiopurine drugs necessitates an accurate measurement of the level of S(6)-methylthio-2'-deoxyguanosine (S(6)mdG) in DNA of cells treated with thiopurine drugs. Here we developed a sensitive HPLC coupled with tandem mass spectrometry (LC-MS/MS) method and measured the level of 6-thio-2'-deoxyguanosine ((S)dG) and S(6)mdG in genomic DNA of four human leukemia cell lines and one human colorectal carcinoma cell line. Our results revealed that, upon treatment with 3 muM (S)G for 24 h, approximately 10, 7.4, 7, and 3% of guanine was replaced with (S)G in Jurkat T, HL-60, CCRF-CEM and K-562 cells, respectively. However, only less than 0.02% of (S)dG was converted to S(6)mdG in the above cell lines. HCT-116 cells had the lowest level (0.2%) of guanine being replaced with (S)G in DNA, and approximately 5 out of 10(4 S)G was converted to its methylated counterpart. This is the first report of the simultaneous and accurate quantification of (S)dG and S(6)mdG in genomic DNA of cultured human cells treated with (S)G. In addition, our results suggested that DNA (S)G might trigger mismatch repair (MMR) pathway without being converted to S(6)mG.

On the analysis of fluorimetric titration curves of purine nucleoside phosphorylase.

The steady-state fluorimetric titration curves for trimeric purine nucleoside phosphorylase (PNP) by two ligands, were analysed using the DynaFit program. Results of this analysis indicate that three binding sites of PNP molecule interact with each other and that the character of this interaction is different for both ligands. The DynaFit program is very useful in studies of oligomeric proteins, but for detection of non-interacting sites some independent tests are necessary.

Determination of leukocyte DNA 6-thioguanine nucleotide levels by high-performance liquid chromatography with fluorescence detection.

A HPLC method for determination of 6-thioguanine nucleotide in DNA was developed. Leukocyte DNA was isolated from peripheral blood, derivatized with chloroacetaldehyde and the formed etheno derivatives N(2),3-etheno 6-thioguanine (epsilon6TG), 1,N(6)-etheno adenine (epsilonA) and N(2),3-etheno guanine (epsilonG) were released from the DNA backbone by hydrolysis at pH 6.0 and 80 degrees C for 60 min. After extraction of epsilon6TG by immobilized metal ion affinity chromatography (IMAC) the sample was analysed by ion-pair reversed-phase HPLC with fluorescence detection. The limit of quantification was 9.0 nM and the intra- and interday precision ranged from 2.8 to 15.5%. In a small cohort of eight children with acute lymphoblastic leukaemia (ALL), a median of one 6-thioguanine base was found for each 3000 normal bases (range 1:2000-1:11000).

Monitoring of thiopurine methyltransferase activity in postsurgical patients with Crohn's disease during 1 year of treatment with azathioprine or mesalazine.

Thiopurine methyltransferase (TPMT) activity determines biotransformation of azathioprine and, thereby, drug efficacy and safety. Evaluation of a possible long-term effect of mesalazine or azathioprine on TPMT activity is of particular clinical importance because both drugs can to be given for several years in inflammatory bowel disease. Monitoring of TPMT activity and three thiopurine metabolites was performed prospectively during a 1 year postoperative period in 21 patients with Crohn's disease randomly assigned to azathioprine (2.0-2.5 mg/kg per day) or mesalazine (4 g/day). TPMT activity did not change significantly within each treatment group during 52 weeks. At any study visit, TPMT activity was not different between 13 patients on azathioprine and eight patients on mesalazine. Concentrations of 6-thioguanine nucleotides (6-TGN, active moiety of azathioprine) and 6-methyl-mercaptopurine ribonucleotides (6-MMPR) did not alter significantly during the observation period, except for a slight decrease in 6-TGN levels when comparing the first with the last visit. In this first report of serial monitoring of 6-methyl-thioguanine nucleotides (6-MTGN) in patients with inflammatory bowel disease taking azathioprine, high levels of 6-TGN were correlated with high levels of 6-MTGN, with the mean 6-TGN:6-MTGN ratio being 2.4. In a well-standardized clinical setting of inflammatory bowel disease, neither mesalazine nor azathioprine significantly affected TPMT activity during a whole year of treatment.

Novel DNA lesions generated by the interaction between therapeutic thiopurines and UVA light.

The therapeutic effect of the thiopurines, 6-thioguanine (6-TG), 6-mercaptopurine, and its prodrug azathioprine, depends on the incorporation of 6-TG into cellular DNA. Unlike normal DNA bases, 6-TG absorbs UVA radiation, and UVA-mediated photochemical damage of DNA 6-TG has potentially harmful side effects. When free 6-TG is UVA irradiated in solution in the presence of molecular oxygen, reactive oxygen species are generated and 6-TG is oxidized to guanine-6-sulfonate (G(SO3)) and guanine-6-thioguanine in reactions involving singlet oxygen. This conversion is prevented by antioxidants, including the dietary vitamin ascorbate. DNA G(SO3) is also the major photoproduct of 6-TG in DNA and it can be selectively introduced into DNA or oligonucleotides in vitro by mild chemical oxidation. Thermal stability measurements indicate that G(SO3) does not form stable base pairs with any of the normal DNA bases in duplex oligonucleotides and is a powerful block for elongation by Klenow DNA polymerase in primer extension experiments. In cultured human cells, DNA damage produced by 6-TG and UVA treatment is associated with replication inhibition and provokes a p53-dependent DNA damage response.

The syntheses and properties of tricyclic pyrrolo[2,3-d]pyrimidine analogues of S6-methylthioguanine and O6-methylguanine.

The syntheses of novel tricyclic pyrrolo[2,3-d]pyrimidine analogues of S6-methylthioguanine are described. The crystal structures and pKa values of these and related O6-methylguanine analogues are reported. All compounds display higher pKa values than O6-methylguanine with the sulfur-containing analogues being the more basic and exhibiting higher stability in aqueous solution. In a standard substrate assay with the human repair protein O6-methylguanine-DNA methyltransferase (MGMT) only the oxygen-containing analogue displayed activity.

Substituted purine analogues define a novel structural class of catalytic topoisomerase II inhibitors.

By screening 1,990 compounds from the National Cancer Institute diversity set library against human topoisomerase IIalpha, we identified a novel catalytic topoisomerase II inhibitor NSC35866, a S6-substituted analogue of thioguanine. In addition to inhibiting the DNA strand passage reaction of human topoisomerase IIalpha, NSC35866 also inhibited its ATPase reaction. NSC35866 primarily inhibited DNA-stimulated ATPase activity, whereas DNA-independent ATPase activity was less sensitive to inhibition. We compared the mode of topoisomerase II ATPase inhibition induced by NSC35866 with that of 12 other substituted purine analogues of different chemical classes. The ability of thiopurines with free SH functionalities to inhibit topoisomerase II ATPase activity was completely abolished by DTT, suggesting that these thiopurines inhibit topoisomerase II ATPase activity by covalently modifying free cysteine residues. In contrast, NSC35866 as well as two O6-substituted guanine analogues, O6-benzylguanine and NU2058, could inhibit topoisomerase II ATPase activity in the presence of DTT, indicating that they have a different mechanism of inhibition. NSC35866 did not increase the level of topoisomerase II covalent cleavable complexes with DNA, indicating that it is a catalytic inhibitor and not a poison. NSC35866 was also capable of inducing a salt-stable complex of topoisomerase II on closed circular DNA. In accordance with these biochemical data, NSC35866 could antagonize etoposide-induced cytotoxicity and DNA breaks in human and murine cancer cells, confirming that NSC35866 also functions as a catalytic topoisomerase II inhibitor in cells.

Reference intervals for thiopurine S-methyltransferase activity in red blood cells using 6-thioguanine as substrate and rapid non-extraction liquid chromatography.

BACKGROUND: Although widely used, thiopurine drugs have a narrow therapeutic index and treatment can result in life-threatening toxicity, the basis being pharmacogenetic variation in thiopurine metabolism by thiopurine S-methyltransferase (TPMT). We recently developed a modified phenotyping assay to determine TPMT activity in red blood cells. Here we describe improvements to the method and establish reference intervals in a large prospective study. METHODS: A modified enzyme assay for TPMT activity is reported. It uses 6-thioguanine as substrate with heat treatment of the incubate to stop the reaction and precipitate protein prior to high-performance liquid chromatographic (HPLC) analysis. Measurement of the reaction product, 6-methylthioguanine (6-MTG), uses HPLC with fluorimetric detection. RESULTS: The assay shows excellent characteristics, with clear discrimination of patients who are deficient in TPMT activity (< 5 nmol 6-MTG per g Hb per h) from heterozygotes (5-24 nmol 6-MTG per g Hb per h) and patients with normal activity (>25 nmol 6-MTG per g Hb per h). CONCLUSION: A modified TPMT assay is described which is suited for routine analysis in a regional centre. The method overcomes the need for extraction and has speeded up the chromatographic determination of 6-MTG, enabling large numbers of samples to be analysed. A prospective study of 1000 individuals has established the distribution of TPMT activity using the assay.

Sequence- and base-specific delivery of nitric oxide to cytidine and 5-methylcytidine leading to efficient deamination.

Nitric oxide (NO) is an important endogenous regulatory molecule, and S-nitrosothiols are believed to play a significant role in NO storage, transport, and delivery. On the basis of their ability to generate NO in vivo, S-nitrosothiols can be used as therapeutic drugs. In this study, we have developed an innovative method for sequence- and base-specific delivery of NO to a specific site of DNA followed by specific deamination. We designed a NO transfer reaction from S-nitroso thioguanine to an imino tautomer of cytosine. Nitrosation of the thioguanosine-containing ODN 1 was carried out with S-nitroso-N-acetylpenicillamine (SNAP) to produce ODN 2. An interstrand NO transfer reaction was performed using ODN 2 and its complementary ODN 3 having dC or dmC at the target site, and a rapid NO transfer reaction was observed. In contrast, a transfer reaction was not observed either with ODN 3 having dT, dA, or dG at the target site or with ODN 5-7 having dC at a nontarget site. In the analysis of deaminated products of the NO-transferred ODN 4, it was found that the transformation ratio from dmC to dT was as high as 42% together with the dmC-diazoate (13%). In conclusion, we have demonstrated the innovative method of sequence- and base-specific delivery of nitric oxide to cytidine and 5-methylcytidine. The selectivity and efficiency of NO transfer followed by deamination exhibited in this study are extremely high compared to those of the conventional methods.

Optimizing treatment with thioguanine derivatives in inflammatory bowel disease.

Thioguanine derivatives, azathioprine and 6-mercaptopurine, represent major drugs in the treatment of chronic active inflammatory bowel disease. They are effective in two-thirds of the patients and safe over the long term in patients who can tolerate them (80-90%). Recent progress in understanding the metabolism of these drugs and its implication in clinical practice have brought up new tools and strategies that are proposed to optimize treatment. In particular, the measurement and characterization of key enzymes and metabolites may have clinical impact. Thus, thiopurine methyl transferase genotyping and activity measurement, as well as erythrocytes, 6-thioguanine nucleotides and 6-methyl mercaptopurine levels, may help in some situations of intolerance or inefficacy with these drugs. Indications for starting and stopping treatment with thioguanine derivatives are also discussed.

Infrared spectra of 6-thioguanine tautomers. An experimental and theoretical approach.

Both amino-thiol N9H and amino-thiol N7H tautomeric forms of 6-thioguanine have been identified in approximately equal abundance in infrared studies of these molecules isolated in the hydrophobic environment of an argon matrix at 12 K. The relative concentrations of the amino-thiol N9H and amino-thiol N7H ([SH, N9H]/[SH, N7H] = K(N9H-N7H) = 1.00 +/- 0.02) are estimated from the observed relative infrared absorbances. From these relative concentrations, the difference in the Gibbs free energy of these two tautomers (deltaG500(N9H-N7H) = -0.012 +/- 0.005 kJ mol(-1) have been estimated. The infrared and Raman spectra of 6-thioguanine in solid state are also discussed in terms of hydrogen bonding and stacking interactions in the crystal which are not considered in the calculation. In an effort to interpret the experimental results, ab initio calculation of the infrared spectrum has been made for the amino-thione N7H tautomer at 3-21G level. Comparison with experimental spectra is of some help in the assignment of the infrared and Raman spectra for 6-thioguanine in the solid state.

Thioguanine administered as a continuous intravenous infusion to pediatric patients is metabolized to the novel metabolite 8-hydroxy-thioguanine.

Thiopurine antimetabolites have been in clinical use for more than 40 years, yet the metabolism of thiopurines remains only partially understood. Data from our previous pediatric phase 1 trial of continuous i.v. infusion of thioguanine (CIVI-TG) suggested that TG was eliminated by saturable mechanism, with conversion of the drug to an unknown metabolite. In this study we have identified this metabolite as 8-hydroxy-thioguanine (8-OH-TG). The metabolite coeluted with the 8-OH-TG standard on HPLC and had an identical UV spectrum, with a lambda(max) of 350 nm. On mass spectroscopy, the positive ion, single quad scan of 8-OH-TG yielded a protonated molecular ion at 184 Da and contained diagnostic ions at m/z 167, 156, 142, and 125 Da. Incubation of TG in vitro with partially purified aldehyde oxidase resulted in 8-OH-TG formation. 8-OH-TG is the predominant circulating metabolite found in patients receiving CIVI-TG and is likely generated by the action of aldehyde oxidase.

Kinetics of the action of thymine DNA glycosylase.

The time course of removal of thymine by thymine DNA glycosylase has been measured in vitro. Each molecule of thymine DNA glycosylase removes only one molecule of thymine from DNA containing a G.T mismatch because it binds tightly to the apurinic DNA site left after removal of thymine. The 5'-flanking base pair to G.T mismatches influences the rate of removal of thymine: kcat values with C.G, T.A, G.C, and A.T as the 5'-base pair were 0.91, 0.023, 0. 0046, and 0.0013 min-1, respectively. Thymine DNA glycosylase can also remove thymine from mismatches with S6-methylthioguanine, but, unlike G.T mismatches, a 5'-C.G does not have a striking effect on the rate: kcat values for removal of thymine from SMeG.T with C.G, T. A, G.C, and A.T as the 5'-base pair were 0.026, 0.018, 0.0017, and 0. 0010 min-1, respectively. Thymine removal is fastest when it is from a G.T mismatch with a 5'-flanking C.G pair, suggesting that the rapid reaction of this substrate involves contacts between the enzyme and oxygen 6 or the N-1 hydrogen of the mismatched guanine as well as the 5'-flanking C.G pair. Disrupting either of these sets of contacts (i.e. replacing the 5'-flanking C.G base pair with a T.A or replacing the G.T mismatch with SMeG.T) has essentially the same effect on rate as disrupting both sets (i.e. replacing CpG.T with TpSMeG.T), and so these contacts are probably cooperative. The glycosylase removes uracil from G.U, C.U, and T.U base pairs faster than it removes thymine from G.T. It can even remove uracil from A.U base pairs, although at a very much lower rate. Thus, thymine DNA glycosylase may play a backup role to the more efficient general uracil DNA glycosylase.

High-performance liquid chromatographic assay of methylthioguanine nucleotide.

This paper describes a specific and sensitive reversed-phase HPLC assay for the measurement of 6-methylthioguanine (methyl-TG) and methyl-TG nucleotides (methyl-TGNs) in red blood cells (RBCs), which is suitable for routine clinical use. Briefly, an ethyl acetate extract of RBCs is evaporated and reconstituted in 0.1 M HCl. The methyl-TG is separated from other thiopurines by reversed-phase HPLC and quantitated using UV detection. For the measurement of methyl-TGNs the free base (methyl-TG) is obtained by acid hydrolysis of the nucleotide back to the parent thiopurine. The intra-assay C.V. over the concentration range of 0.055-1.10 nmol methyl-TG per 4x10(8) (100 microl) RBCs ranged from 2.8 to 8.5%, and the mean recovery of methyl-TG over the calibration range was 61.6% (coefficient of variation, C.V., 3.8%). The lower limit of reproducibility was 0.055 nmol extracted from 100 microl RBCs. Analysis of blood samples from children with leukaemia receiving 6TG chemotherapy, revealed RBC methyl-TGNs at concentrations ranging from 323 to 1365 pmol per 8x10(8) RBCs. No methyl-TG was detected in any of the patient samples.

Cytotoxic mechanism of 6-thioguanine: hMutSalpha, the human mismatch binding heterodimer, binds to DNA containing S6-methylthioguanine.

It has been suggested that the cytotoxicity of 6-thioguanine depends upon (1) incorporation of 6-thioguanine into DNA, (2) methylation by S-adenosylmethionine (SAM) of the thio group to give S6-methylthioguanine, (3) miscoding during DNA replication to give [SMeG] x T base pairs, and (4) recognition of these base pairs by proteins of the postreplicative mismatch repair system. Here we have investigated systematically the ability of proteins present in human cell extracts to bind to DNA containing S6-methylthioguanine. We found that [SMeG] x T base mismatches were bound by the mismatch binding complex, hMutS alpha, and that the level of binding was dependent upon the base 5' to the S6-methylthioguanine in the order G > C = A > T. Extracts from cells that lack either hMSH2 (LoVo cells) or GTBP (HCT-15 cells), two components of the hMutS alpha complex, were unable to bind the [SMeG] x T base pair. We also found that hMutS alpha was able to bind to [SMeG] x C base pairs when the S6-methylthioguanine was in the sequence 5'-Cp[SMeG]. This suggests that miscoding by S6-methylthioguanine residues in DNA during DNA synthesis may not be an absolutely required step in the mechanism of cytotoxicity. Also, since CpG sequences are so important in gene regulation, this result may be of considerable significance.