Inactivation of O6-alkylguanine-DNA alkyltransferase. 1. Novel O6-(hetarylmethyl)guanines having basic rings in the side chain

A number of novel guanine derivatives containing heterocyclic moieties at the O6-position have been synthesized using a purine quaternary salt which reacts with alkoxides under mild conditions. Initially O6-substituents were investigated in which the benzene ring of the known agent, O6-benzylguanine, was replaced by unsubstituted heterocyclic rings. The ability of these agents to inactivate the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase), both as pure recombinant protein and in the human lymphoblastoid cell line Raji, has been compared with that of O6-benzylguanine. The present paper focuses on O6-substituents with basic rings, and under standard conditions several of them proved more effective than benzyl for inactivation of both recombinant and Raji ATase. Among the pyridine derivatives, the 2-picolyl compound 7 is not very active in contrast to the 3- and 4-picolyl compounds, and this influenced our choice of isomers of other basic ring systems for study. Since halogen substitution in the thiophene ring considerably increased the activity (17 versus 6), similar modifications in the pyridine series were examined. The more polar O6-substituents in this study are on the whole compatible with the stereochemical requirements of the ATase protein, and their pharmacological properties may be valuable in subsequent in vivo investigations, particularly the thenyl (6), 5-thiazolylmethyl (12), 5-bromothenyl (17), and 2-chloro-4-picolyl (21) derivatives.     

O6-alkylguanine-DNA alkyltransferase inactivation by ester prodrugs of O6-benzylguanine derivatives and their rate of hydrolysis by cellular esterases

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.     

Reaction of O6-benzylguanine-resistant mutants of human O6-alkylguanine-DNA alkyltransferase with O6-benzylguanine in oligodeoxyribonucleotides

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.     

A single amino acid change in human O6-alkylguanine-DNA alkyltransferase decreasing sensitivity to inactivation by O6-benzylguanine

Mammalian O6-alkylguanine-DNA alkyltransferases (AGTs) are readily inactivated by incubation with the pseudosubstrate, O6-benzylguanine, but the equivalent protein from the Escherichia coli ogt gene is much less sensitive and the Saccharomyces cerevisiae and E. coli ada gene product AGTs are completely resistant to this compound. We have expressed the normal human AGT and various point mutations (C145A, W100A, and P140A) in an ada- ogt- strain of E. coli and tested these proteins against DNA substrates containing O6-methylguanine, for inactivation by O6-benzylguanine and for the ability to produce guanine from O6-benzylguanine. The C145A mutation was inactive as expected since this residue forms the methyl acceptor site. Mutants W100A and P140A were fully active against methylated DNA substrates but the P140A mutant was much less sensitive to inactivation by O6-benzylguanine and failed to form significant amounts of [3H]guanine when incubated with O6-benzyl[8-3H]-guanine. The proline at position 140 in mammalian AGTs is replaced by alanine in the Ada and yeast AGTs and by serine in the Ogt AGT. These results suggest that this proline residue affects the configuration of the active site allowing the O6-benzylguanine to enter and react with the mammalian AGT. The production of resistance to O6-benzylguanine by a single base change raises the possibility that such resistance may arise quite readily in cells of tumors treated therapeutically with the combination of O6-benzylguanine and an alkylating agent.     

Lack of evidence for a polymorphism at codon 160 of human O6-alkylguanine-DNA alkyltransferase gene in normal tissue and cancer

O6-benzylguanine (BG) is a potent, specific inactivator of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT), which enhances sensitivity to nitrosoureas in cells and tumor-bearing animals. BG is presently undergoing clinical trials for development as an agent to enhance the therapeutic index of alkylating agent chemotherapy. It has been reported that a polymorphism exists in the human agt gene, with about 15% of the Japanese population having arginine at codon 160 instead of glycine on the polypeptide (Y. Imai et al., Carcinogenesis, 16: 2441-2445, 1995). The resultant mutant AGT protein is equally effective against both methylated DNA as compared with wild type protein. However, this mutant AGT protein was less sensitive to inactivation by BG with a 20-fold increase in the ED50 value. This observation raised the possibility that a subpopulation of patients may be resistant to BG due to a single base change. We have demonstrated that this alteration also reduces the sensitivity to O6-benzyl-8-oxoguanine, an equally potent, yet much longer-lived human metabolite of BG. To test the possibility that this germ-line mutation of the agt gene might explain resistance to BG and O6-benzyl-8-oxoguanine of patients on our Phase I clinical trials, we evaluated the DNA from lymphocytes of 18 patients. The G160R mutation was not found in any of the 18 patients. To determine the frequency of this mutation in the United States population, DNA from 181 healthy individuals were investigated and, again, the mutation was not observed in this cohort. Therefore, if the mutation exists, it is in statistically <1.6% of the United States noncancerous population. To investigate the possibility that this mutation might be somatic, we evaluated genomic DNA samples from 94 human primary cancers of four different histological subtypes (brain, colon, esophageal, and head and neck). Again, none were found to have the G160R mutation.     

Enhancing effect of O6-alkylguanine derivatives on chloroethylnitrosourea cytotoxicity toward tumor cells

O6-Alkylguanine derivatives are well known as chemical modulators of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Depletion of the enzyme by these derivatives leads to increase sensitivity of tumor cells to chloroethylnitrosoureas. We tested the effect of O6-methylguanine, O6-benzylguanine, O6-(p-methylbenzyl)guanine, O6-(p-chlorobenzyl)guanine, O6-(p-methoxybenzyl)guanine, O6-methylhypoxanthine and O6-benzylhypoxanthine on the sensitivity of tumor cell lines to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU) using a colorimetric cytotoxicity assay. The sensitivity of MGMT-proficient tumor cells including HeLA S3, C6-1, C6-2/ACNU, U-138 MG and U-373 MG cells was greatly enhanced by 2 hr pretreatment of 10-100 microM O6-benzylguanine, O6-(p-methylbenzyl)guanine and O6-(p-chlorobenzyl)guanine, but not by O6-methylguanine or O6-methylhypoxanthine. O6-(p-methylbenzyl)guanine moderately sensitized the 2 cell lines, HeLa S3 and C6-1, tested in our study to ACNU cytotoxicity. O6-Benzylhypoxanthine at the high concentration (100 microM) sensitized, to some extent, 3 MGMT-proficient cell lines. Lesser degrees of enhancement by the O6-benzylguanine derivatives were noted in MGMT-deficient tumor cells. Biological effects of O6-alkylguanine derivatives on enhancing ACNU cytotoxicity of tumor cells suggest that the exocyclic 2-amino and O6-benzyl groups in O6-benzylguanine skeleton are both essential for the inhibition of MGMT activity.     

Phase I trial of O6-benzylguanine for patients undergoing surgery for malignant glioma

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.     

Repair of O6-alkylguanines in the nuclear DNA of human lymphocytes and leukaemic cells: analysis at the single-cell level

Inter-individual and cell-cell variability of repair of O6-alkylguanines (O6-AlkGua) in nuclear DNA was studied at the single-cell level in peripheral lymphocytes from healthy donors and in leukaemic cells isolated from patients with chronic lymphatic leukaemia (CLL) or acute myeloid leukaemia (AML). Cells were pulse exposed to N-ethyl- or N-(n-)butyl-N-nitrosourea in vitro, and O6-AlkGua residues in DNA were quantified using an anti-(O6-AlkGua) monoclonal antibody and electronically intensified fluorescence. The kinetics of O6-AlkGua elimination revealed considerable inter-individual differences in O6-ethylguanine (O6-EtGua) half-life (t1/2) values in DNA, ranging from 1.5 to 4.5 h (five AML patients), from 0.8 to 2.8 h (five CLL patients) and from 1.2 to 7.3 h (five healthy donors). The elimination from DNA of equimolar amounts of O6-butylguanine was generally 3-5 times slower in comparison with O6-EtGua. The t1/2 values of individual samples varied in parallel for both DNA alkylation products. Upon preincubation with O6-benzylguanine, the activity of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AT) in both lymphocytes and leukaemic blasts was reduced to < or = 1%. However, while the rate of O6-EtGua elimination from DNA was decelerated it was not abolished, suggesting the possible involvement of additional repair systems that might be co-regulated with AT. Within individual samples, no major cell subpopulations were observed whose repair kinetics would differ significantly from the remaining cells.     

The potential role of glycine-160 of human O6-alkylguanine-DNA alkyltransferase in reaction with O6-benzylguanine as determined by site-directed mutagenesis and molecular modelling comparisons

O6-Alkylguanine DNA-alkyltransferase (ATase) repairs toxic, mutagenic and carcinogenic O6-alkylguanine (O6-alkG) lesions in DNA by a highly conserved reaction involving the stoichiometric transfer of the alkyl group to the active centre cysteine residue of the ATase protein. In the Escherichia coli Ada ATase, which is effectively refactory to inhibition by O6-benzylguanine (O6-BzG), the residue corresponding to glycine-160 (G160) for the mammalian proteins of this class is replaced by a tryptophan (W). Therefore, to investigate the potential role of the G160 of the human ATase (hAT) protein in determining sensitivity to O6-BzG, site-directed mutagenesis was used to produce a mutant protein (hATG160W) substituted at position 160 with a W residue. The hATG160W mutant was found to be stably expressed and was 3- and 5-fold more sensitive than hAT to inactivation by O6-BzG, in the absence and presence of additional calf-thymus DNA respectively. A similar, DNA dependent increased sensitivity of the hATG160W mutant relative to wild-type was also found for O6-methylguanine mediated inactivation. The potential role of the W160 residue in stabilising the binding of the O6-alkG to the protein is discussed in terms of a homology model of the structure of hAT. The region occupied by G/W-160 forms the site of a putative hinge that could be important in the conformational change that is likely to occur on DNA binding. Three sequence motifs have been identified in this region which may influence O6-BzG access to the active site; YSGG or YSGGG in mammals (YAGG in E. coli Ogt, YAGS in Dat from Bacillus subtilis), YRWG in E. coli Ada and Salmonella typhimurium (but YKWS in Saccharomyces cerevisiae) or YRGGF in AdaB from B. Subtilis. Finally,conformational and stereoelectronic analysis of the putative transition states for the alkyl transfer from a series of inactivators of hAT, including O6-BzG was undertaken to rationalise the unexpected weak inhibition shown by the alpha-pi-unsaturated electrophiles.     

Investigation of the role of tyrosine-114 in the activity of human O6-alkylguanine-DNA alkyltranferase

Tyrosine-114 is one of 13 totally conserved amino acids in all known sequences of O6-alkylguanine-DNA alkyltransferase (AGT). The importance of this amino acid in repair of alkylated DNA by AGT was studied by changing it to phenylalanine (F), alanine (A), threonine (T), or glutamic acid (E) in human AGT. The activities of the mutant proteins were then compared to those of the wild type with regard to abilities to do the following: (a) protect Escherichia coli from the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); (b) repair methylated DNA in vitro; (c) bind to oligodeoxynucleotides containing O6-methylguanine; and (d) react with the low molecular weight pseudosubstrate, O6-benzylguanine. When expressed at high levels in E. coli strain GWR109, lacking endogenous AGT, the wild type and the Y114F mutant were highly effective in reducing mutations and cell killing by MNNG. The Y114A mutant had a much smaller protective effect, and mutants Y114T and Y114E were inactive. Purified preparations of all four AGT mutants showed an approximately similar degree (74-120-fold) of reduction in the rate of reaction with O6-benzylguanine. In contrast, the degree of reduction in activity toward methylated DNA substrates in vitro varied according to the mutation with the more conservative Y114F producing only a 30-fold reduction and the most drastic change of Y114E abolishing activity completely. Alteration Y114A produced a 1000-fold reduction whereas Y114T reduced activity by 10000-fold. All of the mutations affected the binding of AGT to single- or double-stranded oligodeoxynucleotides containing O6-methylguanine. The extent of increase in the Kd varied according to the amino acid with 2-5-fold (F), 7-11-fold (A), 167-200-fold (T), and 600-1000-fold (E) increases. These results are consistent with tyrosine-114 playing a role both in the binding of AGT to its DNA substrate and in facilitating the transfer of the alkyl group. It is probable that AGT resembles other DNA repair proteins in bringing about a "flipping out" of the target base from the DNA helix. Tyrosine-114 is therefore an excellent candidate for a key role in the interaction with the flipped O6-methylguanine. The results also show that when large amounts of AGT are produced in the cell, substantial decreases in the efficiency with which AGT can repair methylated DNA do not prevent the ability to protect E. coli from toxic alkylating agents. Mutant Y114F, whose activity was reduced by 30-fold, was equal to wild-type AGT in bringing about this protection.     

Study of the heterogeneity of creatine kinase-MM isoforms using western blotting

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (alkyltransferase) repairs cytotoxic DNA O6-alkylguanine adducts induced by the nitrosoureas, triazines, and tetrazines. In this study, we determined whether there was a relationship between alkyltransferase activity in colon cancer and that of adjacent normal mucosa, and whether there were demographic patient characteristics which correlated with alkyltransferase expression in either tissue. Alkyltransferase activity and expression of the alkyltransferase gene, MGMT, were measured in 49 paired primary colon cancer samples and adjacent normal appearing mucosa. Alkyltransferase activity was found in all samples. The mean activity was higher in the tumor than the mucosa (r = 0.374, P < 0. 01), although the low correlation coefficient suggested that multiple factors influence the alkyltransferase activity. MGMT mRNA could also be detected in all samples and was highly correlated with alkyltransferase activity (r = 0.64, P < 0.001). No correlation was found between alkyltransferase activity and age, or gender of the patient, or location of the tumor, although activity tended to be higher in patients with lower stage disease. Thus, alkyltransferase activity is present in most, if not all, colon cancer samples, suggesting that it could play an important role in chemotherapeutic resistance of human colon cancer. Patients with colon cancer would appear to be prime candidates for studies utilizing O6-benzylguanine to deplete alkyltransferase prior to therapy with a nitrosourea, triazine, or tetrazine.     

Thermostable archaeal O6-alkylguanine-DNA alkyltransferases

Archaea represent some of the most ancient organisms on earth, and they have relatively uncharacterized DNA repair processes. We now show, using an in vitro assay, that extracts of two Crenarchaeota (Sulfolobus acidocaldarius and Pyrobaculum islandicum) and two Euryarchaeota (Pyrococcus furiosus and Thermococcus litoralis) contain the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase). The ATase activities found in the archaea were extremely thermostable, with half-lives at 80 degreesC ranging from 0.5 hr (S. acidocaldarius) to 13 hr (T. litoralis). The temperature optima of the four proteins ranged from approximately 75 to approximately 100 degreesC, although activity was seen at 37 degreesC, the temperature optimum of the Escherichia coli and human ATases. In all cases, preincubaton of extracts with a short oligonucleotide containing a single O6-methylguanine residue caused essentially complete loss of ATase activity, suggesting that the alkylphosphotriester-DNA alkyltransferase activity seen in some prokaryotes is not present in Archaea. The ATase from Pyrobaculum islandicum had an apparent molecular mass of 15 kDa, making it the smallest of these proteins so far described. In higher organisms, ATase is responsible for the repair of toxic and mutagenic O6-alkylguanine lesions in alkylated DNA. The presence of ATase in these primitive organisms therefore suggests that endogenous or exogenous exposure to agents that generate appropriate substrates in DNA may be an early event in evolution.     

Treatment of human brain tumor xenografts with O6-benzyl-2'-deoxyguanosine and BCNU

O6-Methylguanine-DNA methyltransferase (MGMT), a constitutively expressed DNA repair protein, removes alkyl groups from the O6-position of guanine in DNA. Tumor cells with high MGMT activity are resistant to nitrosoureas and other agents that form toxic O6-alkyl adducts. O6-Benzylguanine (BG) inactivates the MGMT protein and thereby enhances the sensitivity of tumor cells to alkylating drugs. However, the therapeutic potential of BG is limited by its poor solubility and its nonspecific inactivation of MGMT in normal tissues as well as in tumor tissues. Consequently, BG analogues are being developed to identify agents that have more favorable pharmacological characteristics. We evaluated O6-benzyl-2'-deoxyguanosine (dBG), the 2'-deoxyribonucleoside analogue of BG, for its ability to inhibit MGMT and to potentiate 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a MGMT-positive human brain tumor xenograft, Daoy. When given i.p. 1 h before BCNU (25 mg/m2) to animals bearing s.c. tumors, dBG (134 mg/m2) produced a growth delay of 24.7 days, compared to 21.6 days after treatment with an equimolar dose of BG (90 mg/m2) plus BCNU and -0.6 days after treatment with BCNU alone. The combination of dBG + BCNU also increased the survival of animals bearing intracranial tumors by 65%. By increasing the dose of dBG to 300 mg/m2 (the maximum dose that could be delivered i.p. in a standard treatment volume), the growth delay of s.c. tumors increased from -0.1 days with BCNU alone to 39.3 days. dBG suppressed both tumor and liver MGMT activity to less than 1.5% of baseline, and dBG + BCNU induced extensive perivascular apoptosis. Because dBG is a 10-fold less potent MGMT inhibitor than BG in HT-29 cell extracts, these results illustrate the capacity of BG analogues to potentiate BCNU toxicity, despite less in vitro activity than the parent compound, and emphasize the importance of in vivo evaluation of BG analogues.     

Modulation of the antitumor activity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosoure a by O(6)-methyl-2'-deoxyguanosine--a new inhibitor of O(6)-alkylguanine-DNA-alkyltransferase

O6-Methyl-2'-deoxyguanosine (O6-MedG), a novel inhibitor of O6-alkylguanine-DNA alkyltransferase (O6-AGT), has been synthesized. The ability of O6-MedG to deplete the O6-AGT activity in leukemia L1210 and melanoma B16 cells in vivo has been studied. After intraperitoneal administration of O6-MedG to mice bearing leukemia L1210 or melanoma B16, the activity of O6-AGT in tumour cells decreased by 50%. Pretreatment of leukemia L1210 bearing mice with O6-MedG (200 mg/kg) 24 hours prior to ACNU (15 mg/kg) administration resulted in six out of seven 60-day survivors. Treatment of mice with ACNU (15 mg/kg) alone increased the life span by 200%. Treatment of melanoma B16 bearing mice with O6-MedG and 3 hours thereafter with ACNU resulted in a 50% inhibition of tumour growth, whereas the inhibiting effect of ACNU alone was 16%. There was no difference in leukemia growth when L1210/BCNU bearing mice were treated with O6-MedG followed by ACNU treatment. In vivo ACNU (15 mg/kg) produced a deep and prolonged inhibition of DNA, RNA and protein synthesis in leukemia L1210 cells. The DNA synthesis in leukemia L1210/BCNU cells was shown to recover more rapidly than in L1210 cells. The activities of DNA-polymerases alpha and beta and, especially, of O6-AGT were elevated in ACNU-resistant leukemia cells as compared with ACNU-sensitive cells. The activation of some repairing enzymes, such as O6-AGT, DNA-polymerases alpha and beta as well as increased levels of GSH may play a role in the development of drug resistance to ACNU.     

Pharmacokinetics and metabolism in rats of 2,4-diamino-6-benzyloxy-5- nitrosopyrimidine, an inactivator of O6-alkylguanine-DNA alkyltransferase

Inactivation of the human DNA repair protein, O6-alkylguanine-DNA-alkyltransferase (AGT), by exposure to O6-benzylguanine leads to a dramatic enhancement in the cytotoxic response of cells to chemotherapeutic alkylnitrosoureas. Benzylated pyrimidines identified as more potent inactivators than O6-benzylguanine in vitro include 2,4-diamino-6-benzyloxy-5-nitrosopyrimidine (5-nitroso-BP) and 2,4-diamino-6-benzyloxy-5-nitropyrimidine (5-nitro-BP). In efforts to determine the clinical usefulness of these benzylated pyrimidines, we examined the metabolism and pharmacokinetics of 5-nitroso-BP in Sprague-Dawley rats, together with its potency as an AGT inactivator in mice. The mean plasma half-life, clearance, and volume of distribution of 5-nitroso-BP in rats were, respectively, 3.8 min, 22 liters/hr/kg, and 2.1 liters/kg. Two metabolites were identified in rat plasma (i.e. 5-nitro-BP and 2,4,5-triamino-6-benzyloxypyrimidine) after intravenous administration of 5-nitroso-BP in rat. Reduction of 5-nitroso-BP (100 microM) occurred primarily in cytosol and was inhibited (> 95%) by 1 mM menadione. Dicumarol (100 microM), a DT-diaphorase inhibitor, did not significantly inhibit this reaction. This indicated a possible role of a dicumarol-resistant quinone reductase. At higher substrate and protein concentration, NADPH-dependent oxidation of 5-nitroso-BP to 5-nitro-BP primarily occurred in microsomes and was completely inhibited by 1-aminobenzotriazole (1 mM), a P450 inhibitor. Unfortunately, neither 5-nitroso-BP nor 5-nitro-BP was as effective as O6-benzylguanine at depleting AGT activity in mouse liver or spleen. At 1 hr after injection of 15 mg/kg O6-benzylguanine, 5-nitroso-BP, or 5-nitro-BP, AGT levels in liver fell to 1%, 66%, and 71% basal activity, respectively. Rapid cytosolic reduction of 5-nitroso-BP may explain the lack of potency of the pyrimidines in vivo.     

Retroviral transfer of a bacterial alkyltransferase gene (ada) into human bone marrow cells protects against O6-benzylguanine plus 1, 3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity

The antitumor activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is limited by the O6-alkylguanine-DNA alkyltransferase (ATase) in tumor cells and by delayed myelosuppression. Inactivation of neoplastic ATase by O6-benzylguanine (BG) improves the therapeutic index for BCNU. We have demonstrated previously that BG + BCNU-induced myelosuppression in mice is reduced by expression of the BG-resistant ATase ada in murine bone marrow. We have now generated an amphotropic retrovirus containing the ada gene and tested the effectiveness of ada expression in preventing BG + BCNU cytotoxicity in human hematopoietic progenitor cells. A retroviral producer clone with a biological titer of 6.5 x 10(4) colony-forming units/ml and 4.4 pmol ATase/mg protein was used for transduction of bone marrow. Cocultivation of these ada producer cells with progenitor cells from six normal individuals resulted in 1.9-3. 9-fold protection against BG + BCNU-induced cytotoxicity in committed progenitor cell assays. Furthermore, this cytoprotective effect was associated with a high transduction efficiency (40%) and a 2-fold increase of ATase activity in the surviving committed progenitor cell colonies. These data provide a basis for testing the clinical effectiveness of retroviral ada gene transfer into hematopoietic cells to increase the therapeutic index of BG + BCNU.     

Isolation of human O6-alkylguanine-DNA alkyltransferase mutants highly resistant to inactivation by O6-benzylguanine

The activity of O6-alkylguanine-DNA alkyltransferase (AGT) protects cells from killing by methylating or chloroethylating agents. AGT is strongly inhibited by O6-benzylguanine (ED50, 0.2 microM), and this drug is presently undergoing clinical trials to enhance chemotherapy by alkylating agents. Point mutations such as P140A (ED50, 5 microM) render AGT resistant to O6-benzylguanine (BG). Selection for such mutants may prove to be a problem in the use of BG, and a better knowledge of the factors underlying resistance to BG will enable the rational design of improved inhibitors able to inactivate these mutants. BG-resistant AGT mutants may also be valuable for expression in bone marrow stem cells to reduce myelosuppression brought about by alkylating agents, to increase the therapeutic index of therapies including BG, and for use as a selectable marker to allow other genes to be expressed in such stem cells. We have therefore set up a general screen to obtain such mutants by using the ability of AGT to protect Escherichia coli GWR109 lacking endogenous AGT from killing by N-methyl-N'-nitro-N-nitrosoguanidine. When the cells were rendered permeable to BG by mutating the lipopolysaccharide membrane component forming strain TRG8, the protection by AGT expression was abolished by treating the cells with BG. The known P140A mutant was used to test the system and was highly selected for by treatment with 50 microM BG and 40 microg/ml N-methyl-N'-nitro-N-nitrosoguanidine. The sequence coding for PVP at positions 138-140 in AGT was replaced with a random nucleotide sequence, and this library was used to transform TRG8. All of the 59 colonies analyzed having AGT activity that survived the selection from the pool of 36,000 transformants were resistant to BG. Many (69%) of these mutants contained lysine at position 140, and all of these showed the highest level of resistance with <10% loss of activity when crude cell extracts were incubated with 1.2 mM BG. This result was confirmed with three mutants (P138K/V139L/P140K, P138M/V139L/P140K, and P140K), which were purified to homogeneity. The next most common residues found at position 140 were arginine (7%) and asparagine (7%). Studies carried out with purified preparations of mutants P140R and P140N revealed that these mutations also provided resistance to BG but to a lesser extent than P140K (ED50s of 190 and 7 microM, respectively). These results indicate that: (a) this screening method can be used to evaluate BG resistance of single or multiple changes throughout the AGT sequence; and (b) replacement of proline-140 with lysine is the most effective point mutation at this site causing BG resistance and is more than 200 times more effective than replacement with alanine.     

Different cardiovascular profiles of three melanocortins in conscious rats; evidence for antagonism between gamma 2-MSH and ACTH-(1-24)

Mutants of Escherichia coli and Saccharomyces cerevisiae that lack O6-alkylguanine-DNA alkyltransferase activities have increased spontaneous mutation rates, indicating the presence of a cellular metabolite that can alkylate DNA. Bacterially catalysed nitrosation has been implicated previously in producing the endogenous alkylating agent(s). Here, nitrosated polyamines and azaserine, a model compound for nitrosated peptides, are shown to be mutagenic to E. coli ada ogt mutants deficient in O6-alkylguanine-DNA alkyltransferase activity. The mutagenicity of azaserine may be explained by its ability to methylate DNA, whereas nitrosated spermidine causes DNA damage that is susceptible to both nucleotide excision repair and O6-alkylguanine-DNA alkyltransferase activity, which indicates the generation of more bulky DNA adducts. Nitrosated peptides and polyamines are therefore potential endogenous mutagens that are harmful particularly in O6-alkylguanine-DNA alkyltransferase deficient cells.     

Torsades de pointes: a case with multiple variables

PURPOSE: The human medulloblastoma cell line D283 Med (4-HCR), a line resistant to 4-hydroperoxycyclophosphamide (4-HC), displays enhanced repair of DNA interstrand crosslinks induced by phosphoramide mustard. D283 Med (4-HCR) cells are cross-resistant to 1,3-bis(2-chloroethyl)- -nitrosourea, but partial sensitivity is restored after elevated levels of O6-alkylguanine-DNA alkyltransferase (AGT) are depleted by O6-benzylguanine (O6-BG). Studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) after AGT is depleted by O6-BG. METHODS: Limiting dilution and xenograft studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide with or without O6-BG. RESULTS: The activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) was increased after AGT depletion by O6-BG preincubation. Similar studies with Chinese hamster ovary cells, with or without stable transfection with a plasmid expressing the human AGT protein, revealed that the AGT-expressing cells were significantly less sensitive to 4-HC and 4-hydroperoxydidechlorocyclophosphamide. Reaction of DNA with 4-HC, phosphoramide mustard, or acrolein revealed that only 4-HC and acrolein caused a decrease in AGT levels. CONCLUSIONS: We propose that a small but potentially significant part of the cellular toxicity of cyclophosphamide in these cells is due to acrolein, and that this toxicity is abrogated by removal of the acrolein adduct from DNA by AGT.     

Pyridyloxobutyl adduct O6-[4-oxo-4-(3-pyridyl)butyl]guanine is present in 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone-treated DNA and is a substrate for O6-alkylguanine-DNA alkyltransferase

The lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is activated to reactive metabolites that methylate or pyridyloxobutylate DNA. Previous studies demonstrated that pyridyloxobutylated DNA interferes with the repair of O6-methylguanine (O6-mG) by O6-alkylguanine-DNA alkyltransferase (AGT). The AGT reactivity of pyridyloxobutylated DNA was attributed to (pyridyloxobutyl)guanine adducts. One potential AGT substrate adduct, 2'-deoxy-O6-[4-oxo-4-(3-pyridyl)butyl]guanosine (O6-pobdG), was prepared. This adduct was stable at pH 7.0 for greater than 13 days and to neutral thermal hydrolysis conditions (pH 7.0, 100 degrees C, 30 min). Under mild acid hydrolysis conditions (0.1 N HCl, 80 degrees C), O6-pobdG was depurinated to yield O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG). O6-pobdG was hydrolyzed to 4-hydroxy-1-(3-pyridyl)-1-butanone and guanine under strong acid hydrolysis conditions (0.8 N HCl, 80 degrees C). O6-pobG was detected in 0.1 N HCl hydrolysates of DNA alkylated with the model pyridyloxobutylating agent 4-(acetoxymethylnitrosamino)-1-(3-[5-3H]pyridyl)-1-butanone ([5-3H]NNKOAc). When [5-3H]NNKOAc-treated DNA was incubated with either rat liver or recombinant human AGT, O6-pobG was removed, presumably a result of transfer of the pyridyloxobutyl group from the O6-position of guanine to AGT's active site.