Topoisomerase I-mediated cytotoxicity of N-methyl-N'-nitro-N-nitrosoguanidine: trapping of topoisomerase I by the O6-methylguanine.

Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are known to covalently link alkyl groups at the position 6 of guanines (O6MG) in DNA. O6-alkylguanine-DNA alkyltransferase (AGT) specifically removes the methyl group of the O6MG. Using purified human topoisomerase I (Top1), we found an 8-10-fold enhancement of Top1 cleavage complexes when O6MG is incorporated in oligonucleotides at the +1 position relative to a unique Top1 cleavage site. Top1 poisoning by O6MG is attributable to a decrease of the Top1-mediated DNA religation as well as an increase in the enzyme cleavage step. Increased cleavage is probably linked to a change in the hydrogen bonding pattern, such as in the case of the 8-oxoguanine, whereas inhibition of religation could be attributed to altered base pairing, such as abasic sites or base mismatches, because incorporation of a 6-thioguanine did not affect Top1 activity. Top1-DNA covalent complexes are also induced in MNNG-treated CHO cells constitutively lacking the AGT enzyme. Conversely, no increase could be detected in CHO cells transfected with the wild-type human AGT. Moreover, we show that yeasts overexpressing the human Top1 are more sensitive to MNNG, whereas knock-out Top1 strain cells display some resistance to the drug. Altogether, these results suggest a role for Top1 poisoning by alkylated bases in the antiproliferative activity of alkylating agents as well as in the DNA lesions resulting from endogenous and carcinogenic DNA modifications.

Inactivation of human O(6)-alkylguanine-DNA alkyltransferase by modified oligodeoxyribonucleotides containing O(6)-benzylguanine.

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.

Conserved residue lysine165 is essential for the ability of O6-alkylguanine-DNA alkyltransferase to react with O6-benzylguanine.

The role of lysine(165) in the activity of the DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (AGT), and the ability of AGT to react with the pseudosubstrate inhibitor, O(6)-benzylguanine (BG), was investigated by changing this lysine to all other 19 possibilities. All of these mutants (except for K165T, which could not be tested as it was too poorly active for assay in crude cell extracts) gave BG-resistant AGTs with increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation (ED(50)) from 100-fold (K165A) to 2400-fold (K165F). Lys(165) is a completely conserved residue in AGTs from many species, and all of the mutations at this site also reduced the ability to repair methylated DNA. The least deleterious change was that to arginine, which reduced the rate constant for DNA repair by approx. 2.5-fold. Mutant K165R resembled all of the other mutants in being highly resistant to BG, with an ED(50) value for inactivation by BG>200-fold greater than wild-type. Detailed studies of purified K165A AGT showed that the rate constant for repair and the binding to methylated DNA substrates were reduced by 10-20-fold. Despite this, the K165A mutant AGT was able to protect cells from alkylating agents and this protection was not abolished by BG. These results show that, firstly, lysine at position 165 is needed for optimal activity of AGT towards methylated DNA substrates and is essential for efficient reaction with BG; and second, even if the AGT activity towards methylated DNA substrates is impaired by mutations at codon 165, such mutants can protect tumour cells from therapeutic alkylating agents. These results raise the possibility that the conservation of Lys(165) is due to the need for AGT activity towards substrates containing more bulky adducts than O(6)-methylguanine. They also suggest that alterations at Lys(165) may occur during chemotherapy with BG and alkylating agents and could limit the effectiveness of this therapy.

O(6)-methylguanine-DNA methyltransferase activity, p53 gene status and BCNU resistance in mouse astrocytes.

We observed previously that wild-type p53 rendered neonatal mouse astrocytes resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a gene dose-dependent fashion. This effect of p53 appeared to be unrelated to its cell cycle regulation or apoptotic functions. Because in many cell types O(6)-methylguanine-DNA methyltransferase (MGMT)-mediated DNA repair is an important mechanism of resistance to nitrosoureas, we measured MGMT activity in wild-type, heterozygous and p53 knockout neonatal mouse astrocytes. Wild-type p53 astrocytes had significantly greater MGMT activity than either heterozygous or p53 knockout astrocytes: MGMT activity was approximately 5-fold greater in wild-type p53 astrocytes than in p53 knockout cells. However, despite successful depletion of MGMT activity in wild-type astrocytes by O(6)-benzylguanine (BG), resistance to BCNU persisted unchanged. Moreover, we excluded the possibility that continued resistance to BCNU at the concentrations used could be explained by a compensatory induction of MGMT triggered by exposure to either BCNU or BG. Although these studies support a role for p53 regulation of MGMT in neonatal mouse astrocytes, BCNU resistance in wild-type cells appears to be mediated by a non-MGMT mechanism. Nevertheless, regulation of DNA repair by MGMT may be another mechanism by which alterations of the p53 gene promote tumor initiation or progression.

Protection of CHO cells by mutant forms of O6-alkylguanine-DNA alkyltransferase from killing by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) plus O6-benzylguanine or O6-benzyl-8-oxoguanine.

O6-Benzylguanine (BG) is an inactivator of human O6-alkylguanine-DNA alkyltransferase (AGT) currently undergoing clinical trials to enhance cancer chemotherapy by alkylating agents. Mutant forms of AGT resistant to BG in vitro were expressed in CHO cells to determine if they could impart resistance to killing by the combination of BG and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). All the BG-resistant mutant proteins tested (P140A, P140K, P138M/V139L/P140K, G156A, P140A/G160R, and G160R) showed a reduced rate of reaction with methylated DNA substrates in vitro. However, when expressed in equal amounts in CHO cells, mutants P140A, P140K, P138M/V139L/P140K, and G160R gave levels of protection from the chloroethylating agent BCNU equivalent to that of wild-type AGT. This indicates that a 10-fold reduction in rate constant did not prevent their ability to repair chloroethylated DNA in the cell. AGT activity was readily lost when CHO cells expressing wild-type AGT were exposed to BG or its 8-oxo metabolite (O6-benzyl-8-oxoguanine), but cells expressing mutants P140A or G160R required 30-fold higher concentrations and cells expressing mutants P140K or P138M/V139L/P140K were totally resistant. When cells were treated with 80 microM BCNU plus BG or 8-oxo-BG, those expressing wild-type AGT were killed when inhibitor concentrations of up to 500 microM were used, whereas cells expressing P140K or P138M/V139L/P140K showed no effect, and cells expressing P140A or G160R showed an intermediate resistance. These results suggest that: (i) appearance of BG-resistant mutant AGTs may be a problem during therapy, and (ii) the P140K mutant AGT is an excellent candidate for gene therapy approaches where expression of a BG-resistant AGT in hematopoietic cells is used to reduce toxicity.

Modulation of cyclophosphamide activity by O6-alkylguanine-DNA alkyltransferase.

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.

Point mutations in human O6-alkylguanine-DNA alkyltransferase prevent the sensitization by O6-benzylguanine to killing by N,N'-bis (2-chloroethyl)-N-nitrosourea.

Chinese hamster ovary (CHO) cells lack O6-alkylguanine-DNA alkyltransferase (AGT) activity and are sensitive to killing by N,N'-bis (2-chloroethyl)-N-nitrosourea (BCNU). Transfection of these cells with a plasmid leading to the production of wild-type human AGT rendered them resistant to BCNU but this resistance could be overcome by treatment with O6-benzylguanine, an AGT inhibitor. Transfection with plasmids expressing mutants of the AGT in which either proline140 is converted to alanine or glycine156 is converted to alanine also gave rise to CHO cells resistant to BCNU, but these mutations rendered the expressed AGT less sensitive to O6-benzylguanine, and O6-benzylguanine was therefore much less effective in restoring sensitivity to BCNU. The G156A mutation provided the greater amount of resistance to O6-benzylguanine, and the CHO cells expressing this mutant AGT were not effectively killed by the O6-benzylguanine plus BCNU combination. CHO cells expressing the mutant AGTs were also much less sensitive than those expressing the control protein with respect to loss of AGT activity and enhancement of killing by BCNU in response to the more potent AGT inhibitor, 2,4-diamino-6-benzyloxy-5-nitrosopyrimidine. Although these results raise the possibility that resistance to therapy with O6-benzylguanine and chloroethylating agents may arise by the selection for tumor cells expressing a mutated AGT, they also provide a means by which the therapeutic effectiveness of agents forming O6-alkylguanine adducts such as BCNU might be enhanced. Expression of the G156A mutant AGT in hematopoietic progenitor cells by gene therapy techniques could be used to increase their AGT activity and provide a form that was resistant to O6-benzylguanine. Such resistance would provide a way to select for cells expressing the inserted gene and would provide an increase in the therapeutic index for treatment of tumors which would have an AGT activity sensitive to O6-benzylguanine.

DNA base adducts in urine andwhite blood cells of cancer patients receiving combination chemotherapies which include N-methyl-N-nitrosourea.

Abstract Urinary 3-methyladenine (3-MeAde) excretion andlymphocyte DNA adduct formation was studied in 15 patients receiving methylnitrosourea (MNU) at several dose levels (250 mg, 300 mg and600 mg total dose, 143-385 mg m(-2)) as part of various combination chemotherapies for advanced tumours (malignant melanoma, lymphoblastic lymphosarcorna andHodgkin's disease). Urinary 3-MeAde levels were significantly increased over background in patients at all dose levels (p < 0.001) andthe increases were dose-dependent (r = 0.77, p < 0.01). There were large interindividual variations in the excretion of 3-MeAde at each dose of MNU. In a subset of patients, N7-methyl-2-deoxyguanosine (7-MedG) andO(6)-methyl-2'-deoxyguanosine (O(6)-WedG) levels in DNA from blood leucocytes showed dose-dependent increases, however there were no simple relationships between urinary methylated DNA bases andleucocyte DNA adducts. Levels of adducts in leucocyte DNA (7-MedG, < 17-217 µmol mol(-1) dG; O(6)-WedG, < 1.6-35 µmol mol(-1) dG) were comparable with those reported for other methylating chemotherapeutic drugs. Leucocyte DNA andurinary methyl adducts may be useful markers of individual responses to treatment with methylating drugs.

Alkylation and oxidative-DNA damage repair activity in blood leukocytes of smokers and non-smokers.

The levels of 3 DNA repair enzymes involved in alkylation and oxidative DNA damage repair in human peripheral blood leukocytes were measured in 20 smokers and 17 non-smokers. No differences in O6-alkylguanine-DNA-alkyltransferase (AGT) activity were found between the 2 groups and the AGT distribution within the population appeared to be unimodal. In contrast, the mean activities of both the methylpurine (MeP)- and the 2-6-diamino-4-hydroxy-5N formamidopyrimidine (FaPy)-DNA glycosylases were higher in the smokers, although only the difference between the MeP-DNA glycosylase means was statistically significant. The standard deviations of these 2 enzymes were also higher in the smokers. The MeP-DNA glycosylase activity showed a bimodal distribution when all subjects were considered. This may in part be due to the smoking habit; 83% of the subjects with enzyme activities higher than 500 fmoles/mg protein were current smokers, whilst 85% of the non-smokers had lower enzyme activities. However, if the smokers were considered separately, a bimodal distribution of this enzyme activity could still be observed. No strong correlation was observed between enzyme activity and age, although the slopes of the regression lines of enzyme activity on age were all negative. The relationship between enzyme activities was studied by bivariate distribution and a strong correlation was only found between the MeP-DNA glycosylase and the FaPy-glycosylase, with the highest values of both enzyme activities being observed in the smokers and the lowest in the non-smokers. Our results suggest that the activity of certain DNA repair enzymes can be modulated by environmental exposure.

Biomarkers of individual susceptibility to carcinogens: application for biological monitoring.

In order to develop new markers of individual susceptibility to various human carcinogens, we studied some parameters of formation and metabolism of carcinogens, as well as DNA adducts formation and DNA repair in animals and humans. Following an i.p. administration of benzo(a)pyrene (BP) to the rats, levels of urinary excretion of BP-7,8-diol correlated with tumour latency. A high correlation was found between excretion of this metabolite and BP-DNA adducts level in the liver. Healthy smokers excreted higher quantities of BP-7,8-diol, than smoking lung cancer patients, thus confirming the suggestion on existence of cancer-prone phenotype. N-nitroso compounds formed most efficiently in stomach juice of children with superficial gastritis who therefore could be at high risk of stomach cancer. N-ethyl-N'-nitro-N-nitrosoguanidine induced stomach cancer earlier in monkeys with a low level of DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (AGT) in gastric mucosa. Overall, these markers can be helpful in predicting individual susceptibility to carcinogens.

Individual levels of activity of O6-alkylguanine-DNA alkyltransferase in monkey gastric mucosa during chronic exposure to a gastrocarcinogen N-ethyl-N'-nitro-N-nitrosoguanidine.

The activity of a DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (AGT), was studied in gastric mucosa of 15 Macaca fascicularis monkeys before and during chronic oral exposure to the ethylating carcinogen N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) in order to investigate possible causes of inter-individual differences in susceptibility to its gastrocarcinogenic effect. A wide range of AGT activity (307-1903 fmol/mg protein, mean 695) was found before treatment and it decreased during the first year of exposure (means 627, 479 and 452 fmol/mg protein respectively at 6, 12 and 18 months after the beginning of the experiment). The carcinogenesis study is under way and to date four monkeys with low initial AGT level in gastric mucosa died of gastric cancer. The relevance of AGT level measurement for prediction of individual susceptibility to ENNG is discussed.

[The individual characteristics of the activity of the DNA-repair enzyme O(6)-alkylguanine-DNA alkyltransferase in the stomach of monkeys exposed to the gastric carcinogen N-ethyl-N1-nitro-N-nitrosoguanidine]. / Individual'nye osobennosti aktivnosti fermenta reparatsii DNK O(6)-alkilguanin-DNK alkiltransferazy zheludka obez'ian, podvergnutykh vozdeistviiu gastrokantserogena N-étil-N1-nitro-N-nitrozoguanidina.

Variations in the activity of a DNA repair enzyme 0(6)-alkylgianine-DNA alkyltransferase (AGT) were studied in gastric mucosa samples obtained from 15 M. fascicularis monkeys chronically exposed to a gastrocarcinogen N-ethyl-N'-nitro-N-nitrosoguanidine. Marked interindividual difference in the enzyme activity before and in the course of the exposure was observed. The value of AGT activity assay to predict individual susceptibility to alkylating carcinogens is discussed.