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O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-alkylguanine residues at different rates depending on the identity of the alkyl group as well as the sequence context. To elucidate the mechanism(s) underlying the differences in rates, we examined the repair of five alkyl groups in three different sequence contexts. The kinact and Km values were determined by measuring the rates of repair of oligodeoxynucleotide duplexes containing the O6-alkylguanine residues with various concentrations of AGT in excess. The time course of the reactions all followed pseudo-first-order kinetics except for one of the O6-ethylguanine substrates, which could be analyzed in a two-phase exponential equation. The differences in rates of repair between the different alkyl groups and the different sequence contexts are dependent on rates of alkyl transfer and not substrate recognition. The relative rates of reaction are in general benzyl>methyl>ethyl>2-hydroxyethyl>4-(3-pyridyl)-4-oxobutyl, but the absolute rates are dependent on sequence. The kinact values between benzyl and 4-(3-pyridyl)-4-oxobutyl range from 2300 to 350000 depending on sequence. The sequence-dependent variation in kinact varied the most for O6-[4-(3-pyridyl)-4-oxobutyl]guanine, which ranged from 0.022 to 0.000016 s(-1). The results are consistent with a mechanism in which the O6-alkylguanine can bind to AGT in either a reactive or an unreactive orientation, the proportion of which depends on the sequence context.

O6-Alkylguanine-DNA alkyltransferase (alkyltransferase) provides an important source of resistance to some cancer chemotherapeutic alkylating agents. Folate ester derivatives of O6-benzyl-2'-deoxyguanosine and of O6-[4-(hydroxymethyl)benzyl]guanine were synthesized and tested for their ability to inactivate human alkyltransferase. Inactivation of alkyltransferase by the gamma-folate ester of O6-[4-(hydroxymethyl)benzyl]guanine was similar to that of the parent base. The gamma-folate esters of O6-benzyl-2'-deoxyguanosine were more potent alkyltransferase inactivators than the parent nucleoside. The 3'-ester was considerably more potent than the 5'-ester and was more than an order of magnitude more active than O6-benzylguanine, which is currently in clinical trials to enhance therapy with alkylating agents. They were also able to sensitize human tumor cells to killing by 1,3-bis(2-chloroethyl)-1-nitrosourea, with O6-benzyl-3'-O-(gamma-folyl)-2'-deoxyguanosine being most active. These compounds provide a new class of highly water-soluble alkyltransferase inactivators and form the basis to construct more tumor-specific and potent compounds targeting this DNA repair protein.

Cisplatin and carboplatin are widely used clinical chemotherapeutic agents, especially against testicular, ovarian, and head and neck cancers. O(6)-Benzylguanine (BG) has been shown to result in enhanced cytotoxicity, apoptosis, and DNA platination when used in conjunction with cisplatin and carboplatin in head and neck cancer cell lines. Microarray expression data indicated overexpression of 19 genes and underexpression of 22 genes specific to treatment with the combination of BG+/-cisplatin compared to cisplatin alone treatment in SQ20b head and neck cancer cells (p<0.05) using the Affymetrix HG-U133A GeneChip((R)). Among the overexpressed probe sets were genes involved in DNA damage and apoptosis, including GADD34, DDIT4, ATF4, and PTHLH. A similarly structured analog, 9-CH(3)-BG, does not enhance cisplatin-induced cytotoxicity or apoptosis nor is there enhanced expression of GADD34 in cisplatin or 9-CH(3)-BG+/-cisplatin-treated cells compared to control cells. Analysis of cells exposed to 9-CH(3)-BG+/-cisplatin allowed us to focus our array list on 32 probe sets specific to BG+cisplatin versus cisplatin, ruling out differentially expressed probe sets common to 9-CH(3)-BG+cisplatin versus cisplatin. Similarly, 14 probe sets were specific to BG+/-cisplatin versus BG, ruling out differentially expressed probe sets common to 9-CH(3)-BG+/-cisplatin versus 9-CH(3)-BG. Quantitative real-time PCR demonstrated a dose dependent increase in GADD34 expression in cells exposed to BG+/-cisplatin with levels approximately >2-fold for cells exposed to BG+cisplatin compared to cisplatin alone. Levels of GADD34 transcripts were determined with both cisplatin and BG+cisplatin at several different time points concomitant with and following drug treatment. At all timepoints, GADD34 transcript levels are approximately two-fold elevated in cells treated with BG+cisplatin compared to cisplatin alone. Furthermore, significant changes in GADD34 expression levels in SQ20b, SCC35, and SCC61 cells, with approximately three-fold, two-fold, and 3.5-fold increases in expression, respectively, upon treatment with BG+/-cisplatin compared with control. Elucidation of these molecular pathways will aid in our goal of synthesizing more powerful modulators to increase efficacy of platinum agents.

Purpose: Modulation of platinating agent cytotoxicity has important clinical implications as a result of their widespread use in the treatment of many different cancers. O6-Benzylguanine (BG) enhances the cytotoxicity of cisplatin against several human tumor lines. The purpose of our work was to elucidate whether BG affects pathways prior to DNA damage (i.e., glutathione, GSH) or following DNA damage (i.e., nucleotide excision repair, NER)., Methods: In efforts to determine the mechanism of enhancement we: (1) evaluated whether different sequences of BG plus cisplatin treatment differed in their ability to enhance cisplatin-induced cytotoxicity and DNA platination; (2) determined the effect of BG on GSH and glutathione S-transferase (GST) activity and; (3) determined whether BG enhanced cisplatin-induced cytotoxicity in cells lacking specific enzymes in the NER pathway. Colony-forming assay, atomic absorption spectroscopy and HPLC were employed to measure tumor cell growth inhibition, quantitate the amount of platinum on DNA, and determine intracellular GSH concentrations, respectively., Results: Increased cytotoxicity and platination of DNA was observed when cells were exposed to BG prior to and/or during cisplatin treatment and not when BG followed cisplatin treatment. BG did not significantly alter GST activity with minimal depletion of GSH. In contrast, buthionine sulfoximine (BSO) caused a much more dramatic decrease in GSH than BG that was not accompanied by a dramatic increase in sensitivity to cisplatin. Furthermore, BG enhanced the cytotoxicity of cisplatin in a series of cell lines deficient in NER., Conclusions: Overall, our results suggest that the mechanism of enhancement involves neither the GSH nor the NER pathways, but triggers an event prior to DNA platination damage that ultimately results in increased cytotoxicity, apoptosis and increased platination levels.

Glucuronic acid linked prodrugs of O(6)-benzylguanine and O(6)-benzyl-2'-deoxyguanosine were synthesized. The prodrugs were found to be quite stable at physiological pH and were more than 200-fold less active as inactivators of O(6)-alkylguanine-DNA alkyltransferase (alkyltransferase) than either O(6)-benzylguanine or O(6)-benzyl-2'-deoxyguanosine. Beta-glucuronidase from both Escherichia coli and bovine liver cleaved the prodrugs efficiently to release O(6)-benzylguanine and O(6)-benzyl-2'-deoxyguanosine, respectively. In combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), the prodrugs were not effective adjuvants for HT29 cell killing. However, as expected, incubation of these prodrugs with beta-glucuronidase in the culture medium led to much more efficient cell killing by BCNU as a result of the liberation of the more potent inactivators, O(6)-benzylguanine and O(6)-benzyl-2'-deoxyguanosine. These prodrugs may be useful for prodrug monotherapy of necrotic tumors that liberate beta-glucuronidase or for antibody-directed enzyme prodrug therapy with antibodies that can deliver beta-glucuronidase to target tumor cells.

Pharmacological inhibitors of cyclin-dependent kinase (CDK)2 are currently in preclinical and clinical development. The purpose of our work was to evaluate a series of guanine derivatives for their ability to inhibit CDK2, affect cell cycle progression, and enhance the cytotoxic and apoptotic effects of cisplatin. A panel of guanine derivatives, including O(6)-benzylguanine (O(6)-BG), S(6)-benzyl-6-thioguanine (S(6)-BG), S(6)-[(cyclohexyl)methyl]-6-thioguanine (S(6)-CMG), O(6)-[(cyclohexyl)methyl]guanine (O(6)-CMG), O(6)-benzyl-9-methylguanine (9-CH(3)-BG), O(6)-[(cyclohexyl)methyl]-9-methyl-guanine (9-CH(3)-CMG), and 7-benzylguanine (N7-BG), exhibited varying degrees of CDK2 inhibition with O(6)-CMG being the most potent and 9-CH(3)-BG, 9-CH(3)-CMG, and N7-BG the least potent compounds. Treatment with S(6)-CMG and O(6)-CMG significantly decreased the percentage of cells in S phase. In SQ20b and SCC61 head and neck cancer cell lines, the most potent CDK2 inhibitor, O(6)-CMG, was also the most effective at enhancing cisplatin-induced cytotoxicity and apoptosis. Cisplatin-induced DNA platination increased in SQ20b cells pretreated with S(6)-BG, S(6)-CMG, and O(6)-CMG. Treatment with both O(6)-BG and trichostatin A, an indirect cell cycle inhibitor, demonstrated additive effects on cisplatin-induced cytotoxicity. In summary, we have identified a group of guanine derivatives that were effective modulators of cisplatin-induced cytotoxicity and apoptosis.

The chemotherapeutic activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine) may be improved by the addition of O6-benzylguanine (O6-BG). The reaction of O6-BG with O6-alkylguanine-DNA alkyltransferase (AGT) prevents the repair of O6-chloroethyl lesions caused by BCNU. In clinics, the combination of O6-BG and BCNU is now being tested for the treatment of brain tumors. However, the effectiveness of this drug regimen may be limited by drug resistance acquired during treatment. To understand the possible mechanisms of resistance of brain tumor cells to the O6-BG/BCNU combination, we generated medulloblastoma cell lines (D283 MED, D341 MED, and Daoy) resistant to the combination of O6-BG and BCNU [O6-BG/BCNU resistant (OBR)]. DNA sequencing showed that all of the parent cell lines express wild-type AGTs, whereas every OBR cell line exhibited mutations that potentially affected the binding of O6-BG to the protein as evidenced previously by in vitro mutagenesis and structural studies of AGT. The D283 MED (OBR), Daoy (OBR), and D341 MED (OBR) cell lines expressed G156C, Y114F, and K165T AGT mutations, respectively. We reported previously that rhabdomyosarcoma TE-671 (OBR) also expresses a G156C mutation. These data suggest that the clonal selection of AGT mutants during treatment with O6-BG plus an alkylator may produce resistance to this intervention in clinical settings.

The tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent pulmonary carcinogen, both methylates and pyridyloxobutylates DNA. Both reaction pathways generate promutagenic O6-alkylguanine adducts. These adducts, O6-methylguanine (O6-mG) and O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG), are repaired by O6-alkylguanine-DNA alkyltransferase (AGT). In this report, we demonstrate that pyridyloxobutyl DNA adducts are repaired by AGT in a reaction that results in pyridyloxobutyl transfer to the active site cysteine. Because minor changes within the binding pocket of AGT can alter the ability of this protein to repair bulky O6-alkylguanine adducts relative to O6-mG, we explored the ability of AGTs from different species as well as several human AGT variants and mutants to discriminate between O6-mG or O6-pobG adducts. We incubated proteins with equal molar amounts of oligodeoxynucleotides containing site specifically incorporated O6-mG or O6-pobG and measured repair. Bacterial AGTs poorly repaired O6-pobG. Mouse and rat AGT repaired both adducts at comparable rates. Wild-type human AGT, variant I143V/K178R, and mutant N157H repaired O6-mG approximately twice as fast as O6-pobG. Human variant G160R and mutants P140K, Y158H, G156A, and E166G did not repair O6-pobG until all of the O6-mG was removed. To understand the role of adduct structure on relative repair rates, the competition experiments were repeated with two other bulky O6-alkylguanine adducts, O6-butylguanine (O6-buG) and O6-benzylguanine (O6-bzG). The proteins displayed similar repair preference of O6-mG relative to O6-buG as observed with O6-pobG. In contrast, all of the mammalian proteins, except the mutant P140K, preferentially repaired O6-bzG. These studies indicate that the rate of repair of O6-pobG is highly dependent on protein structure. Inefficient repair of O6-pobG by bacterial AGT explains the high mutagenic activity of this adduct in bacterial systems. In addition, differences observed in the repair of this adduct by mammalian proteins may translate into differences in sensitivity to the mutagenic and carcinogenic effects of NNK or other pyridyloxobutylating nitrosamines.

Adenocarcinoma of the pancreas is refractory to chemotherapeutic agents, including BCNU and streptozotocin. We have previously shown that drugs, which adduct the O(6)- position of guanine, are ineffective against pancreatic tumor cell lines because of high expression of O(6)-methylguanine-DNA methyltransferase (MGMT). The effect of MGMT inactivation on the resistance of pancreatic tumors to carmustine (BCNU) and to temozolomide (TMZ) was examined in five human pancreatic tumor xenografts in athymic mice. Tumor-bearing mice were treated: (a) with a single i.p. injection of BCNU or TMZ at the maximum-tolerated doses of 75 and 340 mg/m(2), respectively; and (b) with O(6)-benzylguanine (BG) or O(6)-benzyl-2'-deoxyguanosine (dBG) in combination with BCNU or TMZ. Pretreatment with the MGMT inactivators BG or dBG reduced the maximum-tolerated doses of BCNU and TMZ to 35 and 170 mg/m(2), respectively. MIA PaCa-2, CFPAC-1, PANC-1, CAPAN-2, and BxPC-3 having MGMT levels of 890, 1680, 680, 900, and 330 fmol/mg protein, respectively, were unresponsive to BCNU. MIA PaCa-2 and CFPAC-1 were also unresponsive to TMZ, whereas CAPAN-2 responded with a tumor delay of 32 days. BG or dBG sensitized all tumors to both BCNU and TMZ. BG plus BCNU treatment of MIA PaCa-2, CFPAC-1, PANC-1, CAPAN-2, and BxPC-3 induced tumor delays of 18, 16, 12, 14, and 16 days, respectively. In comparison, dBG plus BCNU at doses that were equitoxic to BCNU plus BG yielded tumor delays of 30, 19, 16, 21, and 22 days, respectively. The pancreatic tumors tested displayed functional mismatch repair that, however, may not be always sufficiently restrictive to prevent mutations under alkylation stress. Treatments with either BCNU or TMZ resulted in some degree of mutation in recurring tumors with the exception of CAPAN-2, the only wt-p53 xenograft. dBG, a weak MGMT inactivator in vitro as compared with BG, was markedly more effective than the latter in enhancing the efficacy of BCNU against pancreatic tumor xenografts. Both BG and dBG also enhanced the efficacy of TMZ against pancreatic tumors, possibly because of the repression of MGMT, which cannot be achieved with TMZ treatments alone. These results suggest that pancreatic tumors, which are resistant to DNA alkylating agents, may be sensitized to such agents when pretreated with MGMT inactivators.

O(6)-Benzylguanine (O(6)-BG), a potent inactivator of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT), is presently in clinical trials combined with alkylating agents that modify the O(6) position of DNA guanine residues, i.e., 1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide. Previous work demonstrated that O(6)-BG also enhances the cytotoxicity of cyclophosphamide, ifosfamide, and nitrogen mustards in Chinese hamster ovary cells. We have extended this study to include other clinically relevant agents that form interstrand and intrastrand cross-links including cisplatin and carboplatin. Pretreatment of a series of head and neck tumor cell lines (i.e., SQ20b, JSQ3, SCC25, SCC35, and SCC61), Chinese hamster ovary cells, and HT29 human colon tumor cells with O(6)-BG (100 micro M for 2 h before treatment and 2 h during treatment) resulted in a 2-fold decrease in the ED(50) of cisplatin and a concomitant increase in the percentage of cells undergoing apoptosis. The enhancement was independent of AGT activity. Similar enhancement was observed with carboplatin, but no enhancement was seen in AGT-deficient cell lines with radiation or temozolomide, demonstrating the dependence of the effect on bifunctional, cross-linking agents. Furthermore, levels of platinum on DNA after treatment with cisplatin increased 1.4-fold in SQ20b cells and 4.5-fold in JSQ3 cells immediately after treatment with O(6)-BG plus cisplatin and remained elevated for 48 h. Consistent with greater cytotoxicity and apoptosis is the approximately 2-fold higher amount of DNA damage when cells are treated with O(6)-BG plus cisplatin compared with cisplatin alone. Modulation of cisplatin therapy with O(6)-BG might improve the prognosis of patients with head and neck, ovarian, testicular, or lung cancer who are treated with this drug.

We have previously demonstrated (A. E. Pegg, Cancer Res., 50: 6119-6129, 1990) that O6-benzylguanine (O6-BG) enhances nitrosourea, temozolomide, and cyclophosphamide activity in malignant glioma xenografts growing in athymic nude mice. More recently, we have demonstrated (V. J. Patel et al., Clin. Cancer Res., 6: 4154-4157, 2000; P. Pourquier et al., Cancer Res., 61: 53-58, 2001) that the combination of temozolomide plus irinotecan (CPT-11) displays a schedule-dependent enhancement of antitumor activity secondary to trapping of topoisomerase I by O6-methylguanine residues in DNA. These studies suggested that there might be favorable therapeutic interactions between O6-BG and combinations of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) plus cyclophosphamide or temozolomide plus CPT-11, respectively. Our present results indicate that the combination of cyclophosphamide plus BCNU plus O6-BG produces growth delays modestly-to-markedly-superior to combinations of cyclophosphamide with BCNU. Although the combination of temozolomide and CPT-11 reveals a marked increase in activity compared with either agent used alone, the addition of O6-BG to this combination dramatically increased the growth delay of the O6-alkylguanine-DNA alkyltransferase (AGT)-positive malignant glioma D-456 MG. These results suggest that a Phase I trial of CPT-11 plus temozolomide plus O6-BG in AGT-positive tumors may be an important intervention to maximize the therapeutic benefits of the combination of CPT-11 and temozolomide.

Purpose: We conducted a phase II trial of carmustine (BCNU) plus the O(6)-alkylguanine-DNA alkyltransferase inhibitor O(6)-benzylguanine (O(6)-BG) to define the activity and toxicity of this regimen in the treatment of adults with progressive or recurrent malignant glioma resistant to nitrosoureas., Patients and Methods: Patients were treated with O(6)-BG at an intravenous dose of 120 mg/m(2) followed 1 hour later by 40 mg/m(2) of BCNU, with cycles repeated at 6-week intervals., Results: Eighteen patients were treated (15 with glioblastoma multiforme, two with anaplastic astrocytoma, and one with malignant glioma). None of the 18 patients demonstrated a partial or complete response. Two patients exhibited stable disease for 12 weeks before their tumors progressed. Three patients demonstrated stable disease for 6, 12, and 18 weeks before discontinuing therapy because of hematopoietic toxicity. Twelve patients experienced reversible > or = grade 3 hematopoietic toxicity. There was no difference in half-lives (0.56 +/- 0.21 hour v 0.54 +/- 0.20 hour) or area under the curve values (4.8 +/- 1.7 microg/mL/h v 5.0 +/- 1.3 microg/mL/h) of O(6)-BG for patients receiving phenytoin and those not treated with this drug., Conclusion: These results indicate that O(6)-BG plus BCNU at the dose schedule used in this trial is unsuccessful in producing tumor regression in patients with nitrosourea-resistant malignant glioma, although stable disease was seen in five patients for 6, 12, 12, 12, and 18 weeks. Future use of this approach will require strategies to minimize dose-limiting toxicity of BCNU such as regional delivery or hematopoietic stem-cell protection.

Site-specific mutagenesis by O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine (O(6)-pobGua), a product of DNA pyridyloxobutylation by metabolites of the tobacco-specific nitrosamines N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), was studied in Escherichia coli strain DH10B and human kidney cells (293) when the modified base was incorporated in either a double-stranded or a gapped shuttle vector. In the repair-competent E. coli strain, less than 3% of the colonies produced by double-stranded vectors harboring the modified base were mutant whereas 96% were mutant when DH10B cells were transformed with modified gapped vectors. By contrast, transformation of DH10B cells with plasmids derived from O(6)-pobGua-containing double-stranded and gapped vectors previously replicated in 293 cells produced 7 and 16% mutant colonies, respectively. These percentages increased to 42 and 82%, respectively, when the 293 cells were pretreated with O(6)-benzylguanine to inactivate the O(6)-alkylguanine-DNA alkyltransferase protein. These findings confirm that the adduct is readily repaired by the human O(6)-alkylguanine-DNA alkyltransferase in both double-stranded and gapped vectors and suggest that it is also highly mutagenic in both human cells and E. coli. In the E. coli strain, the adduct produced exclusively G --> A transition mutations although in human 293 cells it also produced G --> T transversions and more complex mutations in addition to G --> A transitions. These data suggest that O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine can contribute significantly to the mutagenic risk posed by exposure to both NNN and NNK in tobacco smoke.

Double-stranded and gapped shuttle vectors were used to study mutagenesis in human cells by O(6)-methyl (m(6)G)-, O(6)-ethyl (e(6)G)-, and O(6)-benzylguanine (b(6)G), and O(4)-methylthymine (m(4)T) when these bases were incorporated site-specifically in the ATG initiation codon of a lacZ' gene. Vectors were transfected into either human kidney cells (293) or colon tumor cells (SO) or into mismatch repair defective human colon tumor cells (H6 and LoVo). Cellular O(6)-alkylguanine-DNA alkyltransferase (alkyltransferase) was optionally inactivated by treating cells with O(6)-benzylguanine prior to transfection. In alkyltransferase competent cells, the mutagenicity of all the modified bases was substantially higher in gapped plasmids than in double-stranded plasmids. Alkyltransferase inactivation increased mutagenesis by the three O(6)-substituted guanines in both double-stranded and gapped plasmids but did not affect m(4)T mutagenesis. In the absence of alkyltransferase, mutagenesis by m(6)G and to a lesser extent e(6)G in double-stranded vectors was higher in the mismatch repair defective H6 and LoVo cells than in SO or 293 cells indicating that e(6)G as well as m(6)G were subject to mismatch repair processing in these cells. The level of mutagenesis by m(4)T and b(6)G was not affected by mismatch repair status. When incorporated in gapped plasmids and in the absence of alkyltransferase, the order of mutagenicity for the modified bases was m(4)T > e(6)G congruent with m(6)G > b(6)G. The O(6)-substituted guanines primarily produced G-->A transitions while m(4)T primarily produced T-->C transitions. However, m(4)T also produced a significant number of T-->A transversion mutations in addition to T-->C transitions in mismatch repair deficient LoVo cells.

O(6)-Benzylguanine (BG) effectively inactivates the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase, and enhances the effectiveness of 1,3-bis(2-chloroethyl)-1-nitrosourea in cells in culture and tumor-bearing animals. BG is presently in phase II clinical trials. In humans, BG is converted to O(6)-benzyl-8-oxoguanine (8-oxoBG), a longer-lived, yet equally potent inactivator. We have isolated and identified the debenzylated product, 8-oxoguanine, in plasma and urine of patients following administration of BG. The purpose of this work was to determine the human liver enzymes responsible for the debenzylation of 8-oxoBG. Therefore, 8-oxoBG was incubated with human liver microsomes and cytosol, and the concentration of 8-oxoguanine was determined. No appreciable product was formed in the cytosol; however, increasing amounts of 8-oxoguanine were formed with increasing concentrations of pooled human liver microsomes. The amount of 8-oxoguanine formed increased with time and substrate concentration. Co-incubation of human liver microsomes with 8-oxoBG and various cytochrome P450 isoform-selective inhibitors suggested the possible involvement of CYP1A2, 2E1, and/or 2A6 in this reaction. Incubation of 8-oxoBG with baculovirus cDNA-overexpressed CYP1A2, 2E1, 2A6, and 3A4 demonstrated that formation of 8-oxoguanine was due mainly to CYP1A2. Debenzylation of 8-oxoBG complied with Michaelis-Menten kinetics with K(m) and V(max) values of 35.9 microM and 0.59 pmol/min/pmol of CYP1A2, respectively. CYP1A2 appears to be mainly responsible for the debenzylation of 8-oxoBG in human liver.

Inactivation of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) enhances tumor cell killing by therapeutic alkylating agents. O(6)-Benzylguanine (b(6)G) can inactivate AGT and is currently in clinical trials to enhance therapy. Short oligodeoxyribonucleotides containing b(6)G are much more effective inactivators, but their use for therapeutic purposes is likely to be compromised by metabolic instability. We have therefore examined the ability to inactivate AGT of an 11-mer oligodeoxyribonucleotide containing b(6)G (11-mpBG) when modified with terminal methylphosphonate linkages to protect it from nucleases. This modification did not reduce the ability to serve as a substrate/inactivator for AGT, and 11-mpBG had an ED(50) value of 1.3 nM, more than 300-fold lower than that for b(6)G. A similar oligodeoxyribonucleotide containing O(6)-methylguanine (m(6)G) was also found to be a good substrate (ED(50) value of 10 nM), but the benzylated form was repaired more rapidly and preferentially. When added to HT29 cell cultures, 5 microM 11-mpBG was able to cause a prolonged inactivation of cellular AGT for at least 72 h and to greatly sensitize the cells to killing by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The 11-mpMG was ineffective at up to 20 microM, suggesting that the benzyl group allows better uptake into the cell. However, even with 11-mpBG, the 1000-fold decrease in potency toward AGT in HT29 cells compared to that toward the protein in vitro suggests that uptake may be a limiting factor. These results suggest that oligodeoxyribonucleotides such as 11-mpBG may prove to be useful drugs for potentiation of alkylating agent chemotherapy if uptake can be improved.

Purpose: The purpose of this study was to determine the usefulness of various 8-substituted O6-benzylguanine (BG) analogs as modulators of the DNA repair protein. O6-alkylguanine-DNA alkyltransferase (AGT). More specifically, the degree of inactivation of AGT in mouse brain, liver, kidney and tumor by O6-benzyl-8-oxoguanine (8-oxoBG), 8-aza-O6-benzylguanine (8-azaBG), O6-benzyl-8-bromoguanine (8-bromoBG) and O6-benzyl-8-trifluoromethylguanine (8-tfmBG) was compared to inactivation by BG, a modulator in phase II clinical trials. BG is converted rapidly to 8-oxoBG in rodents, monkeys and humans. It was reasoned that 8-substituted analogs of BG would exhibit different pharmacological properties compared to BG which could influence tissue bioavailability and, thus, the extent of AGT inactivation in vivo. We compared the tissue distribution of these agents and AGT activity following administration of the 8-substituted analogs., Materials and Methods: At various time points up to 24 h after i.p. administration of the BG analogs, tissues (i.e. brain, liver, kidney), A549 lung tumor xenografts (i.p.) or D456 brain tumor xenografts (i.c.) were harvested from athymic nude mice for AGT analysis. AGT activity was quantified in tissue extracts using a biochemical assay with [3H]methylated DNA as a substrate. In addition, concentrations of BG and 8-oxoBG were determined by HPLC with fluorescence detection in mouse tissues following administration of drug., Results: Each of the 8-substituted analogs of BG demonstrated variable AGT inactivation capabilities that were comparable to or better than those of BG especially in kidney and brain tissues. There was a more pronounced depletion of AGT inactivation in brain and D456 brain tumor xenografts following administration of BG compared to 8-oxoBG that could be explained by a much greater concentration of AGT-inactivating drug (BG plus the metabolite 8-oxoBG for mice treated with BG versus 8-oxoBG for mice treated with 8-oxoBG) present in these tissues. The AUCs for brain, kidney and liver were 3.2, 6.9 and 1 1.8 times greater for BG than for 8-oxoBG., Conclusions: 8-substituted analogs of BG possess unique AGT-inactivation profiles in vivo that are different from that of BG. The AGT-inhibitory activities of BG and its major metabolite, 8-oxoBG, are related to tissue disposition of both drugs.

Purpose: The major mechanism of resistance to alkylnitrosourea therapy involves the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT), which removes chloroethylation or methylation damage from the O(6) position of guanine. O(6)-benzylguanine (O(6)-BG) is an AGT substrate that inhibits AGT by suicide inactivation. We conducted a phase I trial of carmustine (BCNU) plus O(6)-BG to define the toxicity and maximum-tolerated dose (MTD) of BCNU in conjunction with the preadministration of O(6)-BG with recurrent or progressive malignant glioma., Patients and Methods: Patients were treated with O(6)-BG at a dose of 100 mg/m(2) followed 1 hour later by BCNU. Cohorts of three to six patients were treated with escalating doses of BCNU, and patients were observed for at least 6 weeks before being considered assessable for toxicity. Plasma samples were collected and analyzed for O(6)-BG, 8-oxo-O(6)-BG, and 8-oxoguanine concentration., Results: Twenty-three patients were treated (22 with glioblastoma multiforme and one with anaplastic astrocytoma). Four dose levels of BCNU (13.5, 27, 40, and 55 mg/m(2)) were evaluated, with the highest dose level being complicated by grade 3 or 4 thrombocytopenia and neutropenia. O(6)-BG rapidly disappeared from plasma (elimination half-life = 0. 54 +/- 0.14 hours) and was converted to a longer-lived metabolite, 8-oxo-O(6)-BG (elimination half-life = 5.6 +/- 2.7 hours) and further to 8-oxoguanine. There was no detectable O(6)-BG 5 hours after the start of the O(6)-BG infusion; however, 8-oxo-O(6)-BG and 8-oxoguanine concentrations were detected 25 hours after O(6)-BG infusion. The mean area under the concentration-time curve (AUC) of 8-oxo-O(6)-BG was 17.5 times greater than the mean AUC for O(6)-BG., Conclusion: These results indicate that the MTD of BCNU when given in combination with O(6)-BG at a dose of 100 mg/m(2) is 40 mg/m(2) administered at 6-week intervals. This study provides the foundation for a phase II trial of O(6)-BG plus BCNU in nitrosourea-resistant malignant glioma.

O6-Benzylguanine (BG) is a potent, specific inactivator of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, that enhances the sensitivity of tumor cell lines and tumor xenografts to chloroethylnitrosoureas. To search for BG analogues with greater penetration into the cerebrospinal fluid (CSF), we evaluated plasma and CSF pharmacokinetics of BG, 8-aza-O6-benzylguanine (8-azaBG), O6-benzyl-8-bromoguanine (8-BrBG), O6-benzyl-8-oxoguanine (8-oxoBG), O6-benzyl-8-trifluoromethylguanine (8-tfmBG), and O6-benzyl-2'-deoxyguanosine (B2dG) after i.v. administration of 200 mg/m2 of drug through an indwelling Ommaya reservoir in a nonhuman primate model. BG and its analogues were quantified in plasma and CSF using reverse-phase high-performance liquid chromatography assays. The plasma clearances of the four 8-substituted BG analogues were similar (0.04-0.06 l/h/kg), but half-lives ranged from <2 to >24 h. BG was converted to 8-oxoBG, an equally potent O6-alkylguanine-DNA alkyltransferase inactivator, and the elimination of 8-oxoBG was much slower than that of BG. As a result, the plasma area under the curve of 8-oxoBG was 3.5-fold greater than that of BG. B2dG was metabolized to BG and 8-oxoBG, but this pathway accounted for only 20% of B2dG elimination. The CSF penetration percentages (based on the ratio of AUC(CSF): AUCplasma) for BG, 8-azaBG, 8-oxoBG, 8-tfmBG, 8-BrBG, and B2dG were 3.2, 0.18, 4.1, 1.4, <0.3, and 2.0%, respectively. The CSF penetration of BG and its active metabolite 8-oxoBG is greater than the penetration of 8-azaBG, 8-BrBG, 8-tfmBG, and B2dG.

Purpose: O6-Benzylguanine (BG), an O6-methylguanine-DNA methyltransferase (MGMT) inactivator, potentiates the efficacy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and of other DNA chloroethylating and methylating anticancer drugs and is currently undergoing clinical trials. O6-Benzyl-2'-deoxyguanosine (dBG), a less effective MGMT inactivator than BG in vitro, is at least as effective as BG in combination with BCNU against tumor xenografts in athymic mice. In order to identify the mechanism of dBG activation in in vivo systems we tested the metabolism, ability to inactivate MGMT, and efficacy to potentiate BCNU in vivo of two additional 9-substituted derivatives of BG, namely O6-benzyl-9-cyanomethylguanine (CMBG) and O6-benzylguanosine (BGS)., Methods: Metabolism and disposition of these drugs was examined in athymic mice and Sprague-Dawley rats. MGMT suppression was determined in human medulloblastoma (Daoy) tumor xenografts in athymic mice following treatment with BGS, dBG, and CMBG and was compared with the loss of resistance to BCNU as determined by tumor growth delays., Results: Growth delays at 25 mg/m2 BCNU and 133 mg/m2 BG or equimolar doses of CMBG, BGS or dBG were 23.0, 2.5, 21.3 days. and 30.4 days, respectively. The above differences did not correlate with the ED50S of 0.2, 13, 11 microM, and 2 microM determined for the above compounds, respectively, in cell free extracts. Differences in the efficacies of the 9-substituted compounds did correlate, however, with the extent of their metabolic conversion to BG. The maximum concentrations of BG in blood achieved after the administration of equimolar (250 micromol/kg) doses of CMBG, BGS and dBG were 10, 30 microM, and 55 microM, respectively. Although such levels were lower than those achieved in circulation by administration of an equimolar amount of BG, BG levels persisted longer following treatments with BGS or dBG than after treatment with BG itself. Formation of BG was required for continuous and prolonged (> 16 h) suppression of MGMT activity to non-detectable levels (< 5 fmol/mg protein)., Conclusion: Metabolism of BGS and dBG to BG explains the unexpected high efficacy of these compounds in potentiating the antitumor activity of BCNU in the athymic mouse model. The faster and more effective suppression of tumor MGMT by dBG and its greater efficacy, as compared with BGS, also correlates with a more rapid accumulation of BG in blood after dBG than after BGS administration, which results in faster and complete suppression of MGMT in Daov xenografts. Thus, metabolism of dBG and BGS to BG appears to be the determining factor for continuous and prolonged suppression of MGMT activity, and that near complete suppression of such activity during and following BCNU administration is required for the higher efficacy of treatments. Similarly, the failure of CMBG to suppress tumor MGMT to the same extent as BGS, in spite of their similar ED50 values, could be attributed to the metabolism of this compound mainly by pathways other than conversion to BG.

Purpose: To evaluate the role of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) plus O6-benzylguanine (O6-BG) in the treatment of both Mer+ and Mer- tumors., Methods: The effect of pretreatment with O6-BG on the activity of BCNU against Mer- human central nervous tumor xenografts D-54 MG and D-245 MG was evaluated in athymic nude mice., Results: BCNU (1.0 LD10; dose lethal to 10% of treated animals) produced growth delays of 8.9 days and 7.5 days and tumor regressions in six of ten and one of nine animals against D-54 MG, which was derived from a human malignant glioma xenograft. Dose reduction of BCNU to 0.38 LD10 eliminated antitumor activity. The combination of BCNU (0.38 LD10) plus O6-BG produced growth delays of 8.8 days and 7.9 days, with tumor regressions in four of ten and two of nine animals, respectively. BCNU (1.0 LD10) produced a growth delay of 49.8 days and ten of ten tumor regressions against D-245 MG, which was derived from a glioblastoma multiforme. BCNU (0.38 LD10) produced a growth delay of 19.4 days, with nine of ten tumor regressions. The combination of BCNU (0.38 LD10) plus O6-BG produced a growth delay of 65.7 days and seven of eight tumor regressions., Conclusion: These results suggest that the combination of BCNU plus O6-BG may be a rational intervention for both Mer+ as well as Mer- tumors.

Purpose: The major mechanism of resistance to alkylnitrosourea therapy is the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT), which removes chlorethylation or methylation damage from the O6-position of guanine. O6-benzylguanine (O6-BG) is an AGT substrate that inhibits AGT by suicide inactivation. We conducted a phase I trial to define the presurgical dose required for depletion of tumor AGT activity in patients with malignant glioma., Materials and Methods: Patients were to be treated 18 hours before craniotomy with intravenous doses that ranged between 40 and 100 mg/m2 given over 1 hour. Resected tumor was snap-frozen in liquid nitrogen and AGT activity analyzed by high-pressure liquid chromatography (HPLC). Up to 13 patients were treated at a specific dose of O6-BG, with a target end point of > or = 11 of 13 patients with undetectable tumor AGT levels (< 10 fmol/mg protein)., Results: Thirty patients with malignant gliomas were enrolled, with 11 of 11 patients treated at 100 mg/m2 O6-BG demonstrating tumor AGT levels less than 10 fmol/mg protein. No toxicity was noted in any patient treated., Conclusion: These results indicate that 100 mg/m2 of O6-BG can maintain tumor AGT levels less than 10 fmol/mg protein for at least 18 hours after treatment, a time interval in which bis(2-chloroethyl)nitrosourea (BCNU)-induced chloroethyl adducts are fully converted into interstrand cross-links. A 100-mg/m2 dose of O6-BG will be used in combination with BCNU in another phase I trial designed to determine the maximal-tolerated dose of BCNU.

To modulate the bioavailability and perhaps improve the tumor cell selectivity of O6-alkylguanine-DNA alkyltransferase (AGT) inactivators, pivaloyloxymethyl ester derivatives of O6-benzylguanine (BG) were synthesized and tested as AGT inactivators and as substrates for cellular esterases. The potential prodrugs examined were the 7- and 9-pivaloyloxymethyl derivatives of O6-benzylguanine (7- and 9-esterBG), and of 8-aza-O6-benzylguanine (8-aza-7-esterBG and 8-aza-9-esterBG) and the 9-pivaloyloxymethyl derivative of 8-bromo-O6-benzylguanine (8-bromo-9-esterBG). The benzylated purines were all potent inactivators of the pure AGT and of the AGT activity in HT29 cells and cell extracts. Each ester was at least 75 times less potent than the corresponding benzylated purine against the pure human AGT. In contrast, the activities of esters and their respective benzylated purine were similar in crude cell extracts and in intact cells. The increase in potency of esters in cellular extracts could be explained by a conversion of the respective prodrug to the more potent benzylated purine in the presence of cellular esterases. The apparent catalytic activity (Vmax/Km) of liver microsomal esterase for 8-azaBG ester prodrugs was 70-130 times greater than for BG prodrugs and 10-20 times greater than for 8-bromo-9-esterBG. Tumor cell hydrolysis of the esters varied considerably as a function of cell type and prodrug structure. These data suggest that these or related prodrugs may be advantageous for selective AGT inactivation in certain tumor types.

Inactivation of the human DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), by O6-benzylguanine renders tumor cells susceptible to killing by alkylating agents. AGT mutants resistant to O6-benzylguanine can be made by converting Pro140 to an alanine (P140A) or Gly156 to an alanine (G156A). These mutations had a much smaller effect on the reaction with O6-benzylguanine when it was incorporated into a short single-stranded oligodeoxyribonucleotide. Such oligodeoxyribonucleotides could form the basis for the design of improved AGT inhibitors. AGT and mutants P140A and G156A preferentially reacted with O6-benzylguanine when incubated with a mixture of two 16-mer oligodeoxyribonucleotides, one containing O6-benzylguanine and the other, O6-methylguanine. When the 6 amino acids located in positions 159-164 in AGT were replaced by the equivalent sequence from the Escherichia coli Ada-C protein (mutant AGT/6ada) the preference for benzyl repair was eliminated. Further mutation incorporating the P140A change into AGT/6ada giving mutant P140A/6ada led to a protein that resembled Ada-C in preference for the repair of methyl groups, but P140A/6ada did not differ from P140A in reaction with the free base O6-benzylguanine. Changes in the AGT active site pocket can therefore affect the preference for repair of O6-benzyl or -methyl groups when present in an oligodeoxyribonucleotide without altering the reaction with free O6-benzylguanine.

To compare mutagenesis by O6-methylguanine (m6G), O4-methylthymine (m4T) and O6-ethylguanine (e6G), and assess their genotoxicity in Escherichia coli, double-stranded and gapped plasmids were constructed containing a single m6G, e6G or m4T in the initiation codon (ATG) of a lacZ' gene. Modified base induced mutations were scored by the loss of lacZ' activity on X-gal-containing media resulting in formation of white or sectored (mutant) rather than blue (non-mutant) colonies. Genotoxicity experiments with gapped plasmids containing the modified bases indicated that m4T produced a greater number of bacterial colonies than m6G or e6G. m4T was more mutagenic (45% mutant colonies) than m6G (6%) or e6G (11%) in repair competent (w.t.) E. coli when incorporated in double-stranded plasmids. In gapped plasmids, m4T produced 99% mutant colonies (as was observed previously for e6G) in both w.t. E. coli or E. coli deficient in both O6-alkylguanine-DNA alkyltransferases as well as methylation-directed mismatch repair (ada(-)-ogt(-)-mutS[-]). m6G in gapped plasmids produced 62% mutant colonies in w.t. E. coli, but this percentage increased to 94% in the ada(-)-ogt(-)-mutS(-) strain. In double-stranded plasmids both m4T and m6G produced very similar distributions of mutant and non-mutant colonies in the ada(-)-ogt(-)-mutS(-) strain. These observations led to the conclusion that differences in the mutagenicity of m6G and m4T in w.t. E. coli were a result of preferential repair of m6G compared to m4T by alkyltransferase and mismatch repair mechanisms, and did not reflect differences in their respective coding efficiency or their inherent obstructiveness to DNA synthesis as was observed with e6G. The combination of alkyltransferase and mismatch repair was concluded to be primarily responsible for the apparent genotoxicity of m6G compared to m4T in double-stranded plasmids.

Studies were carried out on the inactivation of pure human O6-alkylguanine-DNA alkyltransferase by 9-substituted O6-benzylguanine derivatives in the presence and absence of DNA. The addition of DNA increased the rate of inactivation of the alkyltransferase by O6-benzylguanine and its 9-methyl derivative but had little effect on the rate of inactivation by the 9-cyanomethyl derivative. In contrast, when O6-benzylguanine derivatives with larger 9-substituents such as ribose, 2'-deoxyribose, dihydrotestosterone, or 2-hydroxy-3-(isopropoxy)propyl were used, the addition of DNA was strongly inhibitory to the inactivation. In the case of O6-benzylguanine, O6-benzylguanosine, and O6-benzyl-2'-deoxyguanosine, these results were confirmed by directly measuring the rate of formation by the alkyltransferase of guanine, guanosine, or 2'-deoxyguanosine, respectively. The data indicated that the presence of DNA activated the alkyltransferase, rendering it more reactive with O6-benzylguanine or O6-benzyl-9-methylguanine, but that DNA interferes with the binding of inhibitors with larger 9-substituents, presumably by competing for the same binding site. Since these inactivators readily inactivate alkyltransferase in cells, the amount of cellular alkyltransferase bound to DNA must be small or readily exchangeable with the free form.

O6-Methylguanine is removed from DNA via the transfer of the methyl group to a cysteine acceptor site present in the DNA repair protein O6-alkylguanine-DNA alkyltransferase. The human alkyltransferase is inactivated by the free base O6-benzylguanine, raising the possibility that substantially larger alkyl groups could also be accepted as substrates. However, the Escherichia coli alkyltransferase, Ada-C, is not inactivated by O6-benzylguanine. The Ada-C protein was rendered capable of reaction by the incorporation of two site-directed mutations converting Ala316 to a proline (A316P) and Trp336 to alanine (W336A) or glycine (W336G). These changes increase the space at the active site of the protein where Cys321 is buried and thus permit access of the O6-benzylguanine inhibitor. Reaction of the mutant A316P/W336A-Ada-C with O6-benzylguanine was greatly stimulated by the presence of DNA, providing strong support for the concept that binding of DNA to the Ada-C protein activates the protein. The Ada-C protein was able to repair O6-benzylguanine in a 16-mer oligodeoxyribonucleotide. However, the rate of repair was very slow, whereas the E. coli Ogt, the human alkyltransferase, and the mutant A316P/W336A-Ada-C alkyltransferases reacted very rapidly with this 16-mer substrate and preferentially repaired it when incubated with a mixture of the methylated and benzylated 16-mers. These results show that benzyl groups are better substrates than methyl groups for alkyltransferases provided that steric factors do not prevent binding of the substrate in the correct orientation for alkyl group transfer.

The prognosis for patients with malignant gliomas continues to be dismal. The high degree of resistance of gliomas to nitrosourea-based chemotherapy is one major factor in poor treatment outcome. The identification of O6-alkylguanine-DNA alkyltransferase (AGAT) as a major determinant of nitrosourea resistance has resulted in the development of several agents to inactivate this repair protein and counteract tumor cell resistance. However, a major problem in preclinical trials has been the marked nitrosourea dose limitations imposed by the prior administration of AGAT-depleting agents. We investigated the AGAT depletion and selective enhancement of BCNU activity of intraarterial (i.a.) O6-benzylguanine (O6BG) in the human malignant glioma xenograft D-456 MG growing intracranially (i.e.) in athymic rats. Whereas i.a. O6BG at 2.5 mg/kg produced 100% inhibition of D-456 MG AGAT i.e. activity 8 h after administration, intraperitoneal (i.p.) O6BG at this dose produced only 40% inhibition, requiring dose escalation to 10 mg/kg to produce 100% AGAT depletion. Prior administration of i.p. O6BG (10 mg/kg) and i.a. O6BG (2.5 mg/kg) limited maximum tolerated intravenous (i.v.) BCNU doses (37.5 mg/kg when given alone) to 6.25 and 25 mg/kg, respectively. Higher doses of BCNU alone or in combination with O6BG produced histopathologic evidence of cerebral and hepatic toxicity. Therapy experiments revealed a significantly improved median survival for rats treated with O6BG i.a. (2.5 mg/kg) plus BCNU i.v. (25 mg/kg, days 61 and 59 in duplicate experiments) compared with saline (day 21. P = 0.001). O6BG i.a. or i.p. (days 22 and 23, P = 0.001), BCNU i.v. (37.5 mg/kg, day 29, P = 0.001), and O6BG i.p. (10 mg/kg), plus BCNU i.v. (6.25 mg/kg, day 37, P < 0.001). Therefore, O6BG i.a., by virtue of rapid AGAT depletion and selective uptake into i.c. tumors, offers significant potential for regional chemomodulation of AGAT-mediated nitrosourea resistance in malignant human gliomas with concomitant reduction of systemic toxicity.

To examine the miscoding properties of modified guanine residues bearing increasingly bulky O6-substituents, Rat4 cells, grown in the presence of O6-benzylguanine to deplete the DNA repair protein O6-alkylguanine-DNA alkyltransferase, were transfected with plasmids carrying H-ras genes in which O6-methyl, O6-ethyl- and O6-benzylguanine were substituted for the first, second or both the first and second guanine residues of codon 12 (GGA). DNA from isolated transformed colonies was amplified by PCR and directly sequenced by high-temperature manual and automated methods. The results show that O6-ethylguanine and O6-benzylguanine induced semi-targeted as well as targeted mutations, in contrast to O6-methylguanine, which induced only targeted mutations. When incorporated in place of the first guanine of H-ras codon 12, the targeted mutations induced by all these modified guanines were exclusively G-->A transitions. When incorporated at the second position of codon 12, O6-benzylguanine induced G-->A, G-->T and G-->C mutations. O6-Ethylguanine at the second position induced chiefly G-->A transitions, and O6-methylguanine induced G-->A transitions exclusively. Semi-targeted mutations were strictly G-->A at the base 3' to a position 1 adduct or 5' to a position 2 adduct. The mechanism for induction of targeted mutations probably involves decreasing preference to thymidine incorporation opposite an O6-modified guanine as the size of the O6-substituent increases, while the mechanism for non-targeted mutations may be related to abasic site formation or to translesion synthesis which might be made error-prone by obstructive DNA lesions in this context.

The oxidation of O6-benzylguanine, an inactivator of O6-alkylguanine-DNA alkyltransferase, was examined using human liver cytosol, microsomes, and several P450 isoforms. Incubation of O6-benzylguanine with human liver cytosol resulted in the formation of O6-benzyl-8-oxoguanine, which was inhibited by menadione, a potent inhibitor of aldehyde oxidase. Inhibition by allopurinol, a xanthine oxidase inhibitor, was less dramatic. Oxidation of O6-benzylguanine also occurred with pooled human liver microsomes and was inhibited by both furafylline and troleandomycin, selective inhibitors of CYP1A2 and CYP3A4, respectively. Human P450s CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2E1, and CYP3A4 expressed in Hep G2 hepatoma cells using vaccinia virus vectors were incubated with 10 or 200 microM O6-benzylguanine. At 10 microM, O6-benzylguanine was oxidized primarily by CYP1A2 and to a lesser extent by CYP3A4. However, an appreciable increase in CYP3A4 contribution was noted at 200 microM. CYP1A2 exhibited a more than 200-fold higher relative catalytic activity (Vmax/Km) compared with CYP3A4. Therefore, at therapeutically relevant concentrations of O6-benzylguanine, CYP1A2 could be primarily involved in its oxidation since it shows a much lower Km value (1.3 microM) than CYP3A4 (52.2 microM) and cytosol (81.5 microM). However, one would expect interindividual variation in the extent of oxidation of O6-benzylguanine depending on the levels of aldehyde oxidase, CYP1A2, and CYP3A4.

The ability of a number of compounds that act as inactivators of O6-alkylguanine-DNA alkyltransferase (AGT) to sensitize human tumor cell lines to the effects of N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) were examined. The AGT inactivators tested included O6-benzylguanine (BG) and its 8-aza-, 8-bromo-, 8-methyl-, 8-oxo, and 8-amino-derivatives and O6-[p-(hydroxymethyl)benzyl]guanine. All of these compounds except the 8-amino-derivative were active in greatly increasing the killing of HT29 colon, Du145 prostate, MCF-7 breast and A549 lung tumor cells by BCNU. Their activities were comparable to those of BG. Two pyrimidines, 2,4-diamino-6-benzyloxy-5-nitrosopyrimidine and 2,4-diamino-6-benzyloxy-5-nitropyrimidine, were found to be considerably more potent than BG in enhancing BCNU-induced cell killing. The addition of a steroid group to the 9-position of BG forming either O6-benzyl-9-[3-oxo-4-androsten-17 beta-yloxycarbonyl)methyl]guanine or O6-benzyl-9-[3-oxo-5 alpha-androstan-17 beta-yloxycarbonyl)methyl]guanine also produced compounds effective in enhancing the cytotoxicity of BCNU when added at 10 microM. These results indicate that a range of potent compounds with potentially different pharmacokinetics is available to test the hypothesis that inactivation of AGT overcomes the resistance of many tumor cells to nitrosoureas.

O6-Alkylguanine-DNA alkyltransferase (AGT) is an important DNA repair protein that removes adducts from the O6 position of guanine by transferring them to a cysteine residue within its amino acid sequence. Mammalian AGTs are readily inactivated by incubation with O6-benzyl-guanine (BG), which is an alternative substrate for the protein. To examine this inactivation in more detail and to develop a procedure for the specific labeling of human AGT, we synthesized a BG derivative, O6-(p-hydroxy[3H]methylbenzyl&#95;guanine ([3H]HMBG) and examined its interaction with AGT in HT29 cell extracts and in HT29 cells. Incubation of human AGT with [3H]HMBG led to the incorporation of radioactivity in the protein in a time- and temperature-dependent manner. This reaction was specific, since neither AGT that was first inactivated by reaction with BG nor proteins other than AGT were labeled. Digestion of the labeled AGT with trypsin showed that a single peptide contained the label. Sequencing of this peptide indicated that the label was bound to cysteine-145. These results demonstrate that AGT accepts HMBG as a substrate and becomes inactivated by transfer of a p-hydroxymethylbenzyl residue to the cysteine-145 acceptor site. When [3H]HMBG was added to cultures of HT29 cells which are Mer+ and express active AGT, radioactivity was incorporated in a macromolecule and could be detected by autoradiography. No such labeling occurred with BE or CHO cells, which are Mer- and lack AGT. Examination of the interaction of [3H]HMBG with mutant AGT proteins that differ greatly in their abilities to react with BG showed that there was a strong correlation between the reaction with BG and the labeling with [3H]HMBG. These results indicate that [3H]HMBG is a potentially useful reagent for the detection and localization of AGT activity and for the investigation of its mechanism of action.

Plasmids were constructed with guanine (G) or O6-methyl- (m6G), O6-ethyl-(e6G), or O6-benzyl- (b6G) guanine in the initiation codon (ATG) of the lacZ' gene. Four deoxyuridine residues were incorporated near the modified guanine in the complementary strand. The deoxyuridine-containing plasmids exhibited similarly high transformation efficiencies in ung- Escherichia coli, although the frequency of mutations induced by m6G, e6G, and b6G residues was relatively low. Treatment of the plasmids with uracil-DNA glycosylase (UDG), to remove the uracil residues, or UDG and exonuclease III, to create a gap in the deoxyuridine-containing strand, reduced transformation efficiency for adduct-containing plasmids but did not affect transformation efficiency for control plasmids. However, the same treatments dramatically enhanced mutagenesis by m6G, e6G, and b6G. These results were consistent with blockage of replication by the modified guanines in double-stranded plasmids resulting in preferential replication of the complementary strand. Replication past the modified guanines was forced in the gapped plasmids. The frequency of modified guanine-induced mutations in gapped vectors was similar in strains of E. coli that were proficient in DNA polymerase III but deficient in either DNA polymerase I or II or both polymerase I and II suggesting either that polymerase III was primarily responsible for adduct bypass in all strains or that the probability of base misinsertion during bypass by either polymerase I or II was similar to that for polymerase III. Repair studies with gapped plasmids indicated that m6G was subject to repair by Ada methyltransferase and to postreplication processing by methylation-directed mismatch repair. Neither e6G nor b6G were similarly repaired.(ABSTRACT TRUNCATED AT 250 WORDS)

Several 8-substituted O6-benzylguanines, 2- and/or 8-substituted 6-(benzyloxy)purines, substituted 6(4)-(benzyloxy)pyrimidines, and a 6-(benzyloxy)-s-triazine were tested for their ability to inactivate the human DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT, alkyltransferase). Two types of compounds were identified as being significantly more effective than O6-benzylguanine (the prototype low molecular weight inactivator) at inactivating AGT in human HT29 colon tumor cell extracts. These were 8-substituted O6-benzylguanines bearing electron-withdrawing groups at the 8-position (e.g. 8-aza-O6-benzylguanine and O6-benzyl-8-bromoguanine) and 5-substituted 2,4-diamino-6-(benzyloxy)pyrimidines bearing electron-withdrawing groups at the 5-position (e.g. 2,4-diamino-6-(benzyloxy)-5-nitroso- and 2,4-diamino-6-(benzyloxy)-5-nitropyrimidine). The latter derivatives were also more effective than O6-benzylguanine at inactivating AGT in intact HT29 colon tumor cells. Provided these types of purines and pyrimidines do not exhibit undesirable toxicity, they may be superior to O6-benzylguanine as chemotherapeutic adjuvants for enhancing the effectiveness of antitumor drugs for which the mechanism of action involves modification of the O6-position of DNA guanine residues.

O6-Benzylguanine effectively inactivates the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, leading to an increase in the therapeutic index of 1,3-bis(2-chloroethyl)-1-nitrosourea in nude mouse xenograft studies. To investigate the fate of this inactivator in mammalian systems, we examined its biodistribution and metabolism following i.p. administration of 8-[3H]-O6-benzylguanine to male Sprague-Dawley rats and BALB/c mice. Following administration to rats, there were significantly higher levels of radioactivity in liver than in lung, spleen, kidney, small intestine, and esophagus for up to 24 h. Major urinary metabolites were identified as O6-benzyl-7,8-dihydro-8-oxoguanine, N2-acetyl-O6-benzylguanine, and N2-acetyl-O6-benzyl-7,8-dihydro-8-oxoguanine. Debenzylated metabolites included guanine, 7,8-dihydro-8-oxoguanine, and N2-acetylguanine. In contrast to rat metabolism, acetylated derivatives were not found in mouse urine. However, O6-benzyl-7,8-dihydro-8-oxoguanine was a major metabolite in the mouse. O6-Benzyl-7,8-dihydro-8-oxoguanine was a very effective O6-alkylguanine-DNA alkyltransferase inactivator and exhibited a 50% effective dose in HT29 cell extracts of 0.3 microM compared to 0.2 microM for O6-benzylguanine. The O6-alkylguanine-DNA alkyltransferase depleting activity of N2-acetyl-O6-benzylguanine and N2-acetyl-O6-benzyl-7,8-dihydro-8-oxoguanine were, respectively, 120- and 325-fold lower than O6-benzylguanine in HT29 cell-free extracts.

Plasmids containing a site-specifically incorporated O6-methyl- (m6G), O6-ethyl- (e6G), or O6-benzylguanine (b6G) within the ATG initiation codon of the lacZ' gene were used to transform Escherichia coli that were repair proficient or deficient in one or both of the E. coli O6-alkylguanine-DNA alkyltransferases, the uvr(ABC) excision repair system, the recA-mediated recombination system, or the methylation-directed mismatch repair system. Colonies were scored phenotypically for adduct-induced mutations. With plasmids containing either e6G or b6G, the frequency of adduct-induced mutation was low and independent of the repair proficiency of the strain transformed. Plasmids containing an m6G residue elicited similar responses in all but the mismatch repair-deficient strain. The generally low mutagenicity of all the O6-substituted guanines was interpreted as reflecting an adduct-induced arrest of replication of the modified strand while the unmodified complementary strand was replicated normally. Studies of the involvement of mismatch repair in m6G mutagenesis showed that m6G:T base pairs were more readily processed than m6G:C base pairs, indicating that mismatch repair involving m6G residues occurs after replication. These data support a model in which the E. coli methylation-directed mismatch repair system diverts plasmids containing promutagenic m6G:T base pairs into replication-arrested complexes providing another line of defense against O6-methylguanine mutagenicity in addition to O6-alkylguanine-DNA alkyltransferase repair and excision repair mechanisms.

Many alkylating agents are potent carcinogens and there is considerable evidence that the formation of O6-alkylguanine in DNA can lead to mutations and the initiation of neoplastic growth. The repair of O6-methyl- or O6-ethylguanine in DNA is known to be brought about by the action of a protein termed O6-alkylguanine-DNA alkyltransferase. In order to investigate the role of this activity in the carcinogenic action of methylnitrosourea and ethylnitrosourea, O6-benzylguanine, a potent inhibitor of the alkyltransferase, was used. Groups of 20 female F344 rats were treated with the nitrosourea (0.2 mmol) by gavage in 10 weekly doses and a parallel group was also treated with 4 mg of O6-benzylguanine, 2 h prior to each dose of the nitrosourea. This dose of O6-benzylguanine was sufficient to reduce the alkyltransferase activity to zero in the liver for at least 8 h but activity had returned to about 60% of normal within 24 h. Animals were maintained until they became moribund, when they were killed, or until death related to tumors. The median week of death in the animals receiving methylnitrosourea was reduced from 60 wk to 52 wk by co-treatment with O6-benzylguanine. There was a smaller reduction from 55 to 50 wk in the rats receiving ethylnitrosourea. The treatment with O6-benzylguanine caused no significant change in the incidence of the principal tumors induced by the alkylnitrosoureas and there were no liver tumors produced by the combined treatments. These results show that the level of inactivation of alkyltransferase produced by this dose of O6-benzylguanine was not sufficient to greatly alter the potent carcinogenic effect of these doses of alkylnitrosoureas in this system.

Several new O6-benzylguanine analogs bearing increasingly bulky substituent groups on the benzene ring or at position 9 were tested for their ability to inactivate the human DNA repair protein, O6-alkylguanine-DNA alkyltransferase. Substitution on the benzene ring was well tolerated although activity varied considerably with structural changes in groups attached to position 9. For this site, activity was preserved with large or small lipophilic groups while introduction of non-carbohydrate polar groups generally reduced activity regardless of their size.

O6-Benzylguanine effectively inactivates the DNA-repair protein O6-alkylguanine-DNA alkyltransferase in tumor cells and has been shown to increase the cytotoxicity of chloroethylnitrosoureas. This study was undertaken to ascertain the optimal vehicle for further toxicological evaluation and eventual clinical trials of O6-benzylguanine. The solubility, metabolism, bioavailability and effectiveness of O6-benzylguanine as an adjuvant therapy with BCNU were compared using two vehicles, cremophor-EL and PEG 400. Nude mice bearing s.c. D456 MG glioblastoma xenografts were injected i.p. with 10-30 mg/kg O6-benzylguanine dissolved in either 40% PEG 400/saline or 10% cremophor-EL/saline. The number of tumor regressions noted after treatment with 10 mg/kg O6-benzylguanine followed by 12.7 mg/kg BCNU were 8/9 for the drug dissolved in PEG and 1/10 for the drug given in cremophor-EL. Using the same treatment regimen but increasing the dose of O6-benzylguanine to 30 mg/kg led to a growth delay of 45.2 and 11.5 days for the drug dissolved in PEG 400 and cremophor-EL, respectively, although the number of regressions observed were the same for both treatments. 8-[3H]-O6-Benzylguanine was more rapidly distributed to the tumor when it was delivered in PEG vehicle than when it was given in cremophor-EL. In contrast, there was a 3-fold greater amount of O6-benzylguanine in the small intestine of mice at 1 h after i.p. injection of the drug in cremophor-EL as compared with PEG 400. The rate and extent of metabolism in the liver was the same, whether the parent drug was given in PEG 400 or in cremophor-EL. These studies demonstrate that O6-benzylguanine is a more effective enhancer of the antitumor activity of BCNU when it is given in PEG 400 than when it is delivered in cremophor-EL, which may be due to a more rapid distribution of the drug to the tumor.

Human O6-alkylguanine-DNA alkyltransferase was rapidly inactivated by low concentrations of O6-benzylguanine, but the alkyltransferase from the Escherichia coli ogt gene was much less sensitive and alkyltransferases from the E. coli ada gene or from yeast were not affected. O6-Benzyl-2'-deoxyguanosine was less potent than the base, but was still an effective inactivator of the human alkyltransferase and had no effect on the microbial proteins. O6-Allylguanine was somewhat less active, but still gave complete inactivation of both the human and Ogt alkyltransferases at 200 microM in 30 min, slightly affected the Ada protein, and had no effect on the yeast alkyltransferase. O4-Benzylthymidine did not inactivate any of the alkyltransferase proteins tested. Inactivation of the human alkyltransferase by O6-benzylguanine led to the formation of S-benzylcysteine in the protein and to the stoichiometric production of guanine. The rate of guanine formation followed second-order kinetics (k = 600 M-1 s-1). Prior inactivation of the alkyltransferase by reaction with a methylated DNA substrate abolished its ability to convert O6-benzylguanine into guanine. These results indicate that O6-benzylguanine inactivates the protein by acting as a substrate for alkyl transfer and by forming S-benzylcysteine at the acceptor site of the protein. The inability of O6-benzylguanine to inactivate the microbial alkyltransferases may be explained by steric constraints at this site.(ABSTRACT TRUNCATED AT 250 WORDS)

A number of trials were conducted to determine the effect of O6-benzylguanine pretreatment on the sensitivity of human colon tumor xenografts to the antitumor effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). O6-Benzylguanine has been shown to inactivate the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), which is primarily responsible for resistance to alkylnitrosoureas including BCNU. Colon tumor xenografts carried in nude mice were analyzed for their AGT content, and tumors with low, intermediate and high levels were chosen for further study. The AGT activity of HC-1, GC-3, VRC-5 and CX-1 human colon tumor xenografts was 16, 113, 180 and 367 fmol/mg protein, respectively. Treatment of mice consisted of vehicle alone, 6.25 to 50 mg/kg BCNU administered alone or BCNU (6.25 to 25 mg/kg) 1 hr after 120 mg/kg O6-benzylguanine on days 7 and 14 post-inoculation. Toxicity studies revealed that pretreatment with O6-benzylguanine increased the toxicity of BCNU, requiring administration of about 4-fold less drug. The growth of the VRC-5 tumor at day 42 post-inoculation was inhibited by 39% following treatment with 12.5 mg/kg BCNU alone and 92% when BCNU was combined with O6-benzylguanine pretreatment. The combination of O6-benzylguanine and BCNU (12.5 mg/kg) at day 42 resulted in an inhibition of HC-1 and CX-1 tumor growth by 84 and 72%, whereas BCNU alone inhibited growth by 54 and 14%, respectively. Therefore, the degree to which the antitumor effect of BCNU was increased by O6-benzylguanine pretreatment was dependent on the AGT activity, with a greater effect in tumors of intermediate or high activity. These data suggest that there is a role for O6-benzylguanine combined with BCNU in the treatment of human colon tumors.

The overcoming effect of O6-benzylguanine on O6-methylguanine-DNA methyltransferase (MGMT)-mediated 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance in vitro was evaluated. Depletion of MGMT activity in Mer+ HeLa S3 cells by O6-benzylguanine was dose-dependent and a complete loss of MGMT activity was achieved at a concentration of 0.5 microM. The cytotoxic potential of BCNU on MGMT proficient HeLa S3 (1.10 pmol/mg of protein), SMMC-7721 (0.72 pmol/mg of protein) and Cc801 (0.39 pmol/mg of protein) was greatly enhanced when cells were exposed to 10 microM O6-benzylguanine for 1 h, but there was a lack of potentiation of BCNU sensitivity in Mer- HeLa MR cells due to its nearly undetectable level of MGMT. There existed a correlation between the extent of enhancement and the amount of MGMT activity. The intensity of enhancement expressed as dose modifying factor = IC50 (BCNU alone)/IC50 (10 microM O6-benzylguanine + BCNU) was 4.56, 3.89, 3.67 and 0.97 in HeLa S3, SMMC-7721, Cc801 and HeLa MR cells respectively. The results further demonstrated that O6-benzylguanine may have potential utility as an adjuvant in combination chemotherapy with chloroethylnitrosourea agents.

Our previous studies have indicated that O6-methyl-guanine-DNA methyltransferase (MGMT) is a key factor determining tumor cellular resistance to 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). This study describes the modulation of MGMT-mediated ACNU resistance by O6-benzylguanine pretreatment. The ACNU sensitivity of MGMT proficient human tumor HeLa S3, SMMC-7721, and Cc801 cells in tissue culture was markly enhanced by 10 mm O6-benzylguanine, and a correlation between the extent of enhancement and the level of MGMT activities was observed. A single i.p. injection of 100 mg/kg of O6-benzylguanine caused a complete inhibition of MGMT activities in HeLa S3 tumor xenografts and combination of O6-benzylguanine with ACNU (7.5 mg/kg) significantly inhibited HeLa S3 tumor growth. The results demonstrated that O6-benzylguanine could be used as a potential adjuvant in combination chemotherapy with ACNU to treat MGMT proficient tumors.

The role of rat O6-alkylguanine-DNA alkyltransferase (AGT) in modulating mutagenesis by O6-substituted guanines in the rat H-ras gene was examined. Rat4 cells were transfected with vectors carrying O6-methyl-, O6-ethyl- or O6-benzylguanine residues in place of the normal guanines at either the first, second, or both the first and second positions in codon 12 (GGA) of the H-ras coding sequence. The percentage of transformed colonies was determined for cells grown in normal medium or in medium containing O6-benzylguanine to completely deplete AGT. In parallel experiments with O6-methylguanine-containing vectors, the percentage of cellular DNA harboring codon 12 mutations was determined for normal cells and cells lacking AGT. A reasonable correspondence was observed between the percentage of mutated DNA and the percentage of transformed colonies produced in both types of cells. The results indicate that the contribution of AGT to the repair of O6-substituted guanine damage decreases as the O6 substituent is changed from methyl > ethyl > benzyl. Additionally, cellular AGT appears to repair an O6-methylguanine more readily at the first position of codon 12 than the second position. However, other repair mechanisms in these mammalian cells appear to play a major role in correcting low levels of O6-substituted guanine damage including O6-methylguanine damage.

The DNA-repair protein O6-alkylguanine-DNA alkyltransferase is known to protect tumor cells from the antitumor effects of carmustine (BCNU). This repair protein was inactivated in Copenhagen rat prostate tumors by treatment with O6-benzylguanine in attempts to increase the effectiveness of BCNU therapy. The alkyltransferase activity in the liver, kidney, lung, and prostate of Copenhagen rats was 66, 37, 65, and 122 fmol/mg protein, respectively. The activity in the Dunning R3327G rat prostate tumor was found to be 129 and 126 fmol/mg protein from intact and castrated animals, respectively. The level of this protein remained low in the tissues and tumors of rats for up to 24 h and slowly began to rise at 36 h following an i.p. injection of 80 mg/kg O6-benzylguanine. Animal survival and body weight as well as tumor volumes were monitored in rats bearing prostate tumors in the flank area that had received no treatment, O6-benzylguanine alone, BCNU alone (5.5-60 mg/kg), or 80 mg/kg O6-benzylguanine 1 h prior to BCNU (5.5 mg/kg). When O6-benzylguanine was combined with BCNU therapy, there was a regression in tumor growth that was not observed in animals treated with an equal dose of BCNU alone. A similar regression in tumor growth was observed in animals treated with a higher dose of BCNU alone (45 mg/kg); however, this regimen was more toxic than O6-benzylguanine plus BCNU (5.5 mg/kg) as determined by animal weight loss. The mean weight loss observed in animals treated with BCNU alone and in those given the combination was 24% and 6%, respectively. Histopathology revealed that animals receiving either BCNU alone or the combination had a decrease in all types of bone marrow cells, a loss of intestinal crypts, and a decreased number of lymphocytes in the spleen. The enhancement of the antitumor effect on BCNU by pretreatment with O6-benzylguanine supports a role for this therapy in the treatment of prostate cancer.

We have previously shown that O6-benzylguanine can be used to deplete cells of the DNA repair protein O6-alkylguanine-DNA alkyltransferase and to enhance the sensitivity of human glioma (SF767) and colon tumor (HT29) cells to the cytotoxic effects of alkylnitrosoureas. In the present study, the combination of O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was evaluated in vitro to determine the number of DNA interstrand cross-links formed and in vivo to compare the therapeutic index with that of BCNU alone. The number of DNA interstrand cross-links, as measured by alkaline elution, was increased in HT29 cells treated with 10 microM O6-benzylguanine for 2 h prior to BCNU exposure compared to cells treated with BCNU only. The number of single strand breaks was not increased by prior exposure to O6-benzylguanine. To evaluate the therapeutic index, HT29 and SF767 cells were grown as xenografts in nude mice and the tumor growth rate after treatment with BCNU alone was compared with the rate after treatment with O6-benzylguanine and BCNU. Treatment was administered i.p. when tumors reached 100-200 mm3. For animals bearing HT29 xenografts that were treated with 60 mg/kg O6-benzylguanine 1 h prior to 20 mg/kg BCNU, the average time for tumor volume to increase by 200% was 25 days, compared to 10 days for animals treated with 20 mg/kg BCNU alone. For animals bearing SF767 xenografts, the tumor growth of controls was not significantly different from that of animals treated with O6-benzylguanine alone or BCNU alone up to the maximally tolerated dose (50 mg/kg). For these 3 groups, the average time for tumors to reach 300 mm3 was 9-12 days. However, when animals were treated with 80 mg/kg O6-benzylguanine 1 h prior to receiving 20 mg/kg BCNU tumor size did not increase for at least 21 days. Our studies demonstrate that the therapeutic index of BCNU can be increased when given in combination with O6-benzylguanine.

The inactivation of human and Escherichia coli O6-alkylguanine-DNA alkyltransferase by O6-methylguanine and O6-benzylguanine was compared. When HT29 cell extracts or E. coli Ada protein were incubated in the presence of 200 microM O6-methylguanine for 1 h, alkyltransferase activity was reduced to 44 and 39% of control levels respectively. However, under the same conditions O6-benzylguanine completely depleted alkyltransferase activity in the extract from human cells but had virtually no effect on the Ada protein. Incubation of the HT29 cell alkyltransferase with O6-benzyl[3H]guanine resulted in a time-dependent production of [3H]guanine. No similar production of [3H]guanine was observed in the presence of the Ada protein. In CHO cells transfected with the bacterial ada gene (CHO-ada) or the human alkyltransferase cDNA (CHO-MGMT), treatment with 500 microM O6-methylguanine inhibited both alkyltransferases by greater than 85%. In contrast, 2 microM O6-benzylguanine inhibited human alkyltransferase expressed in CHO-MGMT cells by greater than 99% though concentrations as high as 25 microM for 24 h had no inhibitory effects on the bacterial alkyltransferase expressed in CHO-ada cells. This selective inhibition was also observed in vivo in transgenic mice expressing ada in the liver where O6-benzylguanine caused a decrease of only 40% in total hepatic alkyltransferase activity compared to 95% in non-transgenic mice, consistent with inhibition of only the mammalian alkyltransferase and maintenance of bacterial alkyltransferase activity in these animals. Thus, while O6-methylguanine at high concentrations inactivates both bacterial and mammalian alkyltransferases, O6-benzylguanine is a substrate only for the mammalian protein and is unable, perhaps due to steric hindrance, to inhibit the Ada protein.

Activation of the H-ras protooncogene in rats by methylating carcinogens results from a G-to-A transition mutation at the second position of codon 12 (GGA), presumably due to formation of an O6-methylguanine (m6G) at this position. A similar transition at the first position of codon 12 appears not to occur in vivo. To study the possible structural basis for this bias in mutation, we synthesized a series of 11-base H-ras sequences [e.g., 5'-d(CGCTG*G*AGGCG)-3' and two complementary strands] containing an m6G at the first, second, or both positions of codon 12 (i.e., G* = m6G). The results of solution chemical studies indicated that the individual strands formed stable hairpin structures among which that containing m6G at the second position of codon 12 was most stable. Further, the DNA duplex with m6G at the second position was significantly more stable than that with m6G at the first position, and under certain conditions, it was more stable than the unmodified duplex as well. It is possible that such a difference in stability might lead to more ready recognition of an m6G at the first position by repair proteins, and this could contribute to the apparent site specificity of mutation by methylating carcinogens at codon 12 of the H-ras gene.

Antisera raised in rabbits to three peptides corresponding to amino acid sequences found in human O6-alkylguanine-DNA alkyltransferase were used to study the fate of the alkyltransferase protein in human colon tumor cells after exposure to N-methyl-N'-nitro-N-nitrosoguanidine or to O6-benzylguanidine. Under these conditions, the alkyltransferase protein becomes inactivated, presumably by the conversion of its cysteine acceptor site to S-methylcysteine or S-benzylcysteine respectively. It was found that the protein was rapidly degraded after such inactivation both in intact cells and in cell-free extracts. It is probable that a conformational change in the protein is brought about by conversion of the alkyltransferase to the inactive form by alkylation of the cysteine acceptor site. This change may render the protein very sensitive to proteolytic degradation. The rapid degradation of the inactive form of the protein may serve as a signal for its resynthesis but in the short term ensures that its reactivation by regeneration of the cysteine acceptor site is unlikely to occur to any significant extent. The short half-life of the inactivated alkyltransferase protein makes it probable that measurement of the content of the alkyltransferase protein by immunohistochemistry, which is likely to measure the sum of the active and inactivated forms of the protein, will nevertheless yield an accurate estimation of the cellular capacity to repair O6-methylguanine provided that procedures with sufficient specificity and affinity can be developed.

The effect of O6-benzylguanine, O6-(p-chlorobenzyl)guanine, and O6-(p-methylbenzyl)guanine on the sensitivity of various human tumor cell lines to alkylating agents is evaluated. The sensitivity of human colon tumor cells, HT29, to the chloroethylating agents, 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 2-chloroethyl(methylsulfonyl) methanesulfonate (clomesone), and chlorozotocin was increased by pretreatment for 2 h with 25 microM of each analogue. O6-Benzylguanine was slightly more effective as a sensitizer in HT29 cells than the p-chlorobenzyl and p-methylbenzyl analogues. However, all analogues sensitized SF767 glioma cells to the cytotoxic effects of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, and clomesone to the same degree. Both cell lines were sensitized to the methylating agents streptozotocin and 5-(3-methyl-1-triazeno)imidazole-4-carboxamide, the active intermediate of 5-(3,3-dimethyl-1-triazenyl)imidazole-4-carboxamide, by pretreatment with 10 microM O6-benzylguanine for 2 h. The number of Raji cells surviving 50 microM clomesone decreased 3-fold upon pretreatment for 2 h with 1 microM O6-benzylguanine. The degree of enhancement was dependent on the amount of alkyltransferase protein present in cell lines. For example, HT29 cells (alkyltransferase activity, 381 fmol/mg protein) exhibited a greater degree of enhancement when treated with O6-benzylguanine than SF767 (77 fmol/mg protein) and M19-MEL melanoma (36 fmol/mg protein) cells. There was no enhancement observed in mer- cell lines, U251 (less than 2 fmol/mg protein), and BE (3 fmol/mg protein), or with alkylating agents which did not produce a cytotoxic lesion at the O6 position of guanine in DNA such as cisplatin or 4-hydroperoxycyclophosphamide. Our studies suggest that O6-benzylguanine analogues may have utility in mer+ tumors as an adjuvant to a variety of alkylating agents which produce a toxic lesion at the O6 position of guanine.