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O6-Alkylguanine-DNA alkyltransferase was rapidly and irreversibly inactivated by exposure to O6-benzylguanine or the p-chlorobenzyl and p-methylbenzyl analogues. This inactivation was much more rapid than with O6-methylguanine: incubation with 2.5 microM O6-benzylguanine led to more than a 90% loss of activity within 10 min, whereas 0.2 mM O6-methylguanine for 60 min was required for the same reduction. O6-Benzylguanine was highly effective in depleting the alkyltransferase activity of cultured human colon tumor (HT29) cells. Complete loss of activity was produced within 15 min after addition of O6-benzylguanine to the culture medium and a maximal effect was obtained with 5 microM. In contrast, at least 100 microM O6-methylguanine for 4 hr was needed to get a maximal effect, and this reduced the alkyltransferase by only 80%. Pretreatment of HT29 cells with 10 microM O6-benzylguanine for 2 hr led to a dramatic increase in the cytotoxicity produced by the chemotherapeutic agents 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) or 2-chloroethyl(methysulfonyl)methanesulfonate (Clomesone). Administration of O6-benzylguanine to mice at a dose of 10 mg/kg reduced alkyltransferase levels by more than 95% in both liver and kidney. These results indicate that depletion of the alkyltransferase by O6-benzylguanine may be used to investigate the role of the DNA repair protein in carcinogenesis and mutagenesis and that this treatment may be valuable to increase the chemotherapeutic effectiveness of chloroethylating agents.

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.

The activity of 8-carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazin- 4(3H)-one (temozolomide) in the treatment of a panel of xenografts derived from ependymoma, medulloblastoma, and childhood and adult high-grade glioma was evaluated in athymic nude mice bearing s.c. and intracranial tumors. Temozolomide administered daily for a total of five doses demonstrated marked activity against a panel of Mer+ xenografts despite marginal to moderate activity of 1,3-bis(2-chloroethyl)-1-nitrosourea. The growth delays produced by temozolomide in these xenografts were 1.8-7.5-fold greater than those produced by procarbazine. Although temozolomide demonstrated marginal activity against the Mer+ cell line D341 Med when a 5-day schedule was used, a high-dose 1-day schedule resulted in moderate activity. Temozolomide produced increases in median survival of 1285% (adult glioma D-54 MG), 323% (childhood glioma D-456 MG), and 68% (ependymoma D612 EP). Pretreatment of mice with O6-benzylguanine increased temozolomide-induced mortality, requiring reduction of the dosage from 1200 to 750 mg/m2 on the single-day regimen. O6-Benzylguanine pretreatment of mice bearing Mer+ D341 Med increased the growth delay of temozolomide, in duplicate experiments, from -3.1 to 4.8 and 1.1 to 4.9 days. These studies suggest that temozolomide may be active in the treatment of a broad spectrum of central nervous system cancers, including Mer+ tumors resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea.

Several 8-substituted O 6 -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, O 6 -alkylguanine-DNA alkyltransferase (AGT, alkyltransferase). Two types of compounds were identified as being significantly more effective than O 6 -benzylguanine (the prototype low molecular weight inactivator) at inactivating AGT in human HT29 colon tumor cell extracts. These were 8-substituted O 6 -benzylguanines bearing electron-withdrawing groups at the 8-position (e.g. 8-aza-O 6 -benzylguanine and O 6 -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 O 6 -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 O 6 -benzylguanine as chemotherapeutic adjuvants for enhancing the effectiveness of antitumor drugs for which the mechanism of action involves modification of the O 6 -position of DNA guanine residues

The purpose of this study was to evaluate the anti-tumor activity of sequenced administration of O6-benzylguanine (BG), streptozotocin (STZ), and 1,3-bis(2-chloroethyl)-1- nitrosourea (BCNU) in vitro and in vivo. We measured the recovery of O6-methylguanine DNA methyltransferase (MGMT) and BCNU cytotoxicity in the human glioma SF767 cell line, and anti-tumor activity against xenografts following exposure to BG, STZ or the combination of BG + STZ combined with BCNU. In SF767 cells, the combination of BG (10 microM) + STZ (0.05 mM) produced sustained inhibition of MGMT activity for at least 24 hr, and a greater potentiation of BCNU cytotoxicity than either agent alone. The combined treatment of BG + STZ increased BCNU-induced cell kill by 0.5 to 1.0 log over BG or STZ alone. The maximally tolerated doses of the combination of BG + STZ + BCNU administered to nude mice i.p. were the following: BG (80 mg/kg), STZ (100 mg/kg), and BCNU (15 mg/kg). Utilizing these doses of BG and STZ, the depletion and repletion profile of MGMT activity in SF767 xenografts was measured. STZ at 100 mg/kg did not affect xenograft MGMT activity. Subsequent to BG treatment, xenograft MGMT activity was inactivated completely for 12 hr, and the tumors gradually recovered approximately 40% of control activity by 24 hr. The combination of BG + STZ produced sustained inhibition of MGMT activity for 24 hr in the xenografts with complete recovery of MGMT activity by 48 hr. Administration of the combination of BG + BCNU to nude mice bearing SF767 tumor resulted in significant inhibition of tumor growth for 23 days. However, the addition of STZ to this combination provided no greater anti-tumor activity than that observed with BG + BCNU. The three-drug combination of BG, STZ, and BCNU produced no more than 2.4 to 13.0% weight loss with occasional lethal toxicity. Collectively, these data suggest that prolonged depletion of MGMT might be required for optimal reversal of BCNU resistance both in vitro and in vivo.

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.

We have recently suggested that optimal reversal of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance might require complete inactivation of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) for at least 24 h following BCNU administration (22). This study was undertaken to further evaluate the functional importance of the regeneration rate of MGMT activity following O6-benzylguanine (BG), O6-benzyl-2'-deoxyguanosine (dBG), and streptozotocin (STZ) in determining the potentiation of BCNU cytotoxicity in the highly resistant colon carcinoma cell line HT-29. To this end, we measured the enhancement of BCNU cytotoxicity utilizing regimens which provided complete inhibition, with partial or complete recovery of MGMT activity by 24 h. We were able to modulate the recovery rate of MGMT activity following BG or dBG administration by repeated washing of cells with complete medium. Subsequent to equally inhibitory doses of BG (100 microM) or dBG (1.0 mM) treatment without washing, MGMT activity was completely inactivated for 24 h. However, MGMT activity recovered to control levels by 24 h when cells were treated with BG or dBG and washed 4 times with complete medium. This recovery was completely inhibited for 24 h by combining BG or dBG with 2.5 mM STZ. These differential repletion profiles produced disparate potentiation of BCNU cytotoxicity. The regimens which produced complete inactivation of MGMT for 24 h produced the greatest enhancement of BCNU cytotoxicity. BG or dBG (without a wash) potentiated BCNU cytotoxicity by approximately 3 logs of synergistic cell kill. When the recovery rate of MGMT activity was markedly enhanced via washing of cells, BG-BCNU or dBG-BCNU produced less than 1 log of synergistic cell kill. The addition of STZ to BG or dBG inhibited this temporal recovery for 24 h and potentiated BCNU cytotoxicity by approximately 4 logs. These data further demonstrate that extended depletion of MGMT is required for optimal reversal of BCNU resistance. Because a three-drug combination of BG-STZ-BCNU or dBG-STZ-BCNU consistently produced greater cytotoxicity than any two-drug regimen, clinical testing of these combinations is warranted. Additionally, our data suggest that the design of clinical regimens targeting the inactivation of MGMT and the reversal of BCNU resistance should consider the functional importance of extended depletion of MGMT in order to increase the possibility of antitumor responses.

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.

This study was undertaken to ascertain the importance of prolonged depletion of O6-methylguanine DNA methyltransferase (MGMT) activity, following O6-benzylguanine (BG) and streptozotocin (STZ) exposure, in reversing 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance in vitro. We evaluated BCNU-induced cytotoxicity and measured the temporal recovery of MGMT activity in human colon carcinoma HT-29 cells following treatment with BG, STZ, or the combination of BG and STZ. The pretreatment regimens which provided the greatest potentiation of BCNU cytotoxicity were those exhibiting the greatest temporal inhibition of MGMT activity. The combination of BG (10 microM) and STZ (1.0 mM) produced sustained inhibition of MGMT activity through 24 h and potentiated BCNU cytotoxicity by at least one log greater than either agent alone. Similarly, BG (10-100 microM) produced marked reductions in MGMT activity and increased BCNU cytotoxicity in a dose-dependent fashion. A 100-microM dose of BG inhibited MGMT activity for 48 h and potentiated BCNU induced cell kill by 3 logs greater than BCNU alone. In addition, we observed that during the period of sustained inhibition of MGMT activity, no changes in the steady-state MGMT mRNA levels occurred. We conclude that prolonged inhibition of MGMT activity is an important determinant in reversing BCNU resistance and that chemotherapeutic regimens targeting the inactivation of MGMT activity should be optimized such that MGMT activity is depleted for at least 24 h following BCNU administration.

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.

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.

Experiments were carried out in mice and hamsters to determine whether the activity of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, in tissues and tumors was reduced by treatment with O6-benzylguanine in vivo. Following intraperitoneal injection of O6-benzylguanine, there was a rapid and complete loss of alkyltransferase activity in both livers and kidneys of mice and hamsters. The activity in mouse tissues was slowly restored, reaching pretreatment activities at 16 hr and 72 hr after injection of O6-benzylguanine at 10 mg/kg or 126 mg/kg, respectively. The activity in hamster liver was restored at a significantly lower rate, reaching less than 20% pretreatment activity 72 hr after treatment with 100 mg/kg of O6-benzylguanine. The efficient reduction of alkyltransferase activity by O6-benzylguanine was in sharp contrast to the inability of O6-methylguanine to bring about similar reductions. Activities dropped to about 55% of pretreatment activities in several mouse organs 4 hr after treatment with 126 mg/kg of O6-methylguanine compared to a more than 90% reduction in activity in animals after treatment with O6-benzylguanine. The sensitivity of SF767 cells to meCCNU after treatment with O6-benzylguanine was increased substantially. Furthermore, treatment of nude mice carrying SF767 tumor with 60 mg/kg of O6-benzylguanine prior to either 7.5 or 15 mg/kg of meCCNU led to significant inhibition of tumor growth. These studies indicate that O6-benzylguanine is a suitable compound for use in experiments to examine the role of the alkyltransferase protein in vivo in counteracting the effects of alkylating agents.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously shown that exposure of cells in culture to O6-methylguanine significantly reduces their level of the repair protein, O6-alkylguanine-DNA-alkyltransferase (AGT), thus rendering cells more sensitive to the cytotoxic effects of chemotherapeutic chloroethylating agents. Experiments were carried out in mice to determine whether the AGT content of tissues and tumors could be reduced by in vivo treatment with O6-methylguanine. There was a dose-dependent decrease in AGT activity in liver tissues of CD-1 mice to 24% of basal levels after four hourly intraperitoneal injections of O6-methylguanine (110 mg/kg). Although the decline in AGT activity in the liver was reversible, the activity remained at 75% of basal levels for up to 25 h after the final injection. The effect of O6-methylguanine treatment on AGT activity was measured in mouse tissues as well as human colonic carcinoma tumors (HT29 and BE) grown in Swiss athymic nude mice. The activity in the liver, kidney, and spleen of these mice decreased to 33%-35% of control levels, whereas the activity in HT29 tumors was likewise diminished to 25% of control levels after four hourly injections of O6-methylguanine (100 mg/kg). There was no enhancement of the tumoricidal effectiveness of chloroethylating agents on the HT29 tumor after O6-methylguanine treatment, probably due to a disproportionately higher level of AGT in human tissue than in murine tissue. However, these studies suggest that O6-methylguanine can be given in vivo to examine the role of the AGT protein in protecting against the toxic and carcinogenic effects of alkylating agents.

O6-Methylguanine in DNA is repaired by the action of a protein termed O6-alkylguanine-DNA alkyltransferase (AT) which transfers the methyl group to a cysteine residue in its own sequence. Since the cysteine which is methylated is not regenerated rapidly, if at all, the capacity for repair of O6-methylguanine is limited by the number of molecules of the AT available within the cell. The level and inducibility of the AT differed greatly in different mammalian cell types and species with the highest levels in human tissues and in liver and the lowest levels in brain. Only a small induction occurred in rat liver in response to exposure to alkylating agents. In E. coli such exposure increased the activity more than 100-fold. The At was not specific for methyl groups but also removed ethyl, 2-hydroxyethyl, n-propyl, isopropyl and n-butyl groups from the O6-position in DNA. The protein isolated from E. coli removed methyl groups much more rapidly than the larger alkyl groups but the mammalian AT isolated from rat liver showed much less difference in rate with adducts of different size. Ethyl and n-propyl groups were removed by the rat liver AT only three to four times more slowly than methyl groups. Another important difference between the bacterial and mammalian ATs is that the bacterial protein was also able to remove methyl groups from the O4-position of thymine in methylated DNA or poly(dT) but the AT from rat liver or human fibroblasts did not repair O4-methylthymidine.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymatic repair of the O-alkylpyrimidines (O2- and O4-alkylthymine, O2-alkylcytosine) and alkyl phosphotriesters has been studied in Escherichia coli, and the two proteins involved, a glycosylase (DNA-3-methyladenine glycosylase) and a methyltransferase (DNA-O6-methylguanine:protein-L-cysteine S-methyltransferase, EC 2.1.1.63), have been well characterized. In mammals or mammalian cells treated with carcinogenic alkylating agents, loss of these derivatives has been demonstrated repeatedly. Nevertheless, mammalian repair proteins that are analogous to those from E. coli do not detectably act on these alkyl derivatives. A variety of techniques has been used by many investigators in the United States and Europe, who conclude here that the mode of O-alkylpyrimidine and alkyl phosphotriester repair in mammalian cells differs from that in E. coli. New approaches and methods are needed to characterize these processes at the biochemical and molecular level.

Exposure of HeLa cells to 0.2 mM O6-methylguanine for 4 h or longer led to a 70-80% loss in the activity of the DNA-repair protein, O6-alkylguanine-DNA alkyltransferase. The decline in alkyltransferase activity brought about by O6-methylguanine was reversible on removing the base but at least 48 h were required for complete restoration. This loss of activity could also be brought about by other O6-alkylguanines including ethyl, n-propyl, and n-butyl, but the isopropyl and 2-hydroxyethyl derivatives were considerably less active. The rate of decline of alkyltransferase activity produced by O6-methylguanine was much more rapid than the rate of loss when protein synthesis was inhibited indicating that it was not brought about by blocking the synthesis of the protein. The loss of alkyltransferase activity was not prevented by the addition of inhibitors of nucleic acid or protein synthesis suggesting that it did not require protein synthesis or the incorporation of the O6-alkylguanine into nucleic acids. When cell free O6-alkylguanine-DNA alkyltransferase preparations were incubated in vitro with O6-methylguanine they became inactivated and when O6-[3H]methylguanine was used, [3H]guanine was produced. The inactivation was concentration dependent requiring 0.4 mM for a maximal rate and was quite slow requiring 3-4 hours for completion. These results suggest that the loss of O6-alkylguanine-DNA alkyltransferase activity is brought about by the free base acting as a very weak substrate for the protein. Exposure of mammalian cells to O6-methylguanine or O6-n-butylguanine provides a means by which the level of O6-alkylguanine-DNA-alkyltransferase can be regulated experimentally. This should enable the design of experiments to examine the role of O6-alkylguanine adducts in mutagenesis, carcinogenesis, and cell toxicity after administration of alkylating agents.

Extensive investigations of the specificity of O6-alkylguanine-DNA alkyltransferase (AAT) have been carried out. These studies have shown that: (i) the mammalian protein differs from that of Escherichia coli in lacking the ability to remove methyl groups from O4-methylthymine; (ii) the protein can remove longer alkyl groups from the O6 position but the rate of repair declines as the chain length increases; (iii) O6-methylguanine in RNA is much less active as a substrate for the protein than O6-methylguanine in double-stranded DNA; (iv) the free-base O6-alkylguanine is a very weak substrate for the protein so that reaction with it leads to the loss of alkyltransferase activity. (This property can be used to deplete AAT in cultured cells and in tissues and tumours after administration of O6-methylguanine); and (v) oligodeoxynucleotides containing O6-methylguanine are substrates for AAT. Such oligodeoxynucleotides can be labelled with 32P at very high specific activity and can be used in an ultrasensitive assay for AAT activity.

A sensitive assay procedure was developed for the measurement of the activity of mammalian O6-alkylguanine-DNA-alkyltransferase. The procedure utilized oligodeoxynucleotides containing O6-methylguanine as substrates for the reaction. The oligodeoxynucleotides were end labeled with 32P by the reaction with polynucleotide kinase and [gamma-32P]ATP and allowed to react with organ or cell extracts containing the alkyltransferase. The unmethylated product which was formed was separated from the substrate by reverse-phase high-pressure liquid chromatography. Since the repair by the alkyltransferase is bimolecular, the second order rate constants for the reaction between the labeled oligomer and repair protein from several different sources were determined. The amount of alkyltransferase present was then calculated from the amount of product formed and the appropriate second order rate constant for the reaction. Excellent agreement was obtained between the alkyltransferase levels determined in this procedure and those measured by conventional assay procedures in a variety of cell lines having both high and low activity. The method also gave results in good agreement with other assay procedures for a number of rat tissues, although a few tissues gave anomalous results owing to a high level of nuclease activity which degraded the substrate. This method should prove useful for the measurement of alkyltransferase activity in samples in which the activity is very low or the amount of material available is limited.

Previous studies have demonstrated that cell specificity exists for the alkylation of DNA from lung cells following treatment of rats with the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The concentration of the promutagenic adduct O6-methylguanine (O6MG) was found to be greatest in Clara cells followed by macrophages, type II cells and alveolar small cells. The purpose of this study was to measure the activity of the repair protein O6-methylguanine-DNA methyltransferase (O6MGMT) and to determine whether differences exist for the removal of O6MG among pulmonary cell types. Constitutive activity of O6MGMT was 2-fold greater in macrophages and type II cells than alveolar small cells and Clara cells. Treatment for 4 days with NNK (10 mg/kg/day) had no effect on O6MGMT activity in macrophages, but decreased activity in alveolar small cells and type II cells by 57 and 84%, respectively. O6MGMT activity was reduced to below limits of detection in Clara cells following treatment with NNK. The effect of NNK on O6MGMT activity was consistent with rates of removal of O6MG in macrophages and Clara cells. The loss of O6MG from DNA of macrophages followed first order kinetics (t1/2 = 48 h) while very little loss of this adduct was observed in Clara cells over an 8 day period following cessation of carcinogen treatment. Even though O6MGMT activity was reduced in alveolar small cells and type II cells, approximately 90% of the O6MG bound to DNA in these cell types was removed within 8 days after treatment was discontinued. The loss of O6MG from pulmonary cells appears to result largely from the removal of this adduct by O6MGMT since rates of cell turnover were very low (0.5-1.5%/day) in the lung and were not affected by treatment with NNK. This study indicates that the activity of O6MGMT and the rate of resynthesis of this repair enzyme differ considerably among pulmonary cells following the methylation of DNA. The high concentration of O6MG in Clara cells and the low rate of repair of this promutagenic adduct may be critical factors in the potent pulmonary carcinogenicity induced by the tobacco specific carcinogen NNK.

Exposure of HT29 cells in culture to O6-methylguanine is known to result in a reduction in O6-alkylguanine-DNA alkyltransferase (AGT) activity and an enhancement of sensitivity to the cytotoxic effects of chloroethylating agents. Since cytotoxicity of these agents may be mediated by the formation of interstrand cross-links, alkaline elution analysis was performed on HT29 cells treated with 1-(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, and Clomesone [2-chloroethyl(methylsulfonyl)methanesulfonate] in the presence or absence of O6-methylguanine pretreatment to determine if the enhanced toxicity was due to an increase in the number of cross-links formed. Interstrand cross-linking by 1-(2-chloroethyl)-1-nitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was increased by pretreatment with 0.4 mM O6-methylguanine for 24 h. Cross-linking by Clomesone was observed only in cells exposed to 0.4 mM O6-methylguanine for 24 h prior to administration of the drug and for 12 h after administration, suggesting that the resynthesis of the AGT may prevent the cross-linking by Clomesone. Complete recovery of AGT activity after reduction to 20 to 30% of the basal level upon treatment with 0.4 mM O6-methylguanine required between 8 h and 15 h in both HT29 cells and in Raji cells which were also sensitized to 1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea by exposure to O6-methylguanine. These data suggest that the enhancement of chloroethylnitrosourea toxicity after treatment with O6-methylguanine may be related to an increase in the number of DNA cross-links and that the relatively rapid rate of AGT recovery plays a role in prevention of cross-links resulting from Clomesone.

The rates of loss of O6-methylguanine and O6-ethylguanine from rat liver DNA were determined over a time period of 15 min to 4 hr after various doses (5 micrograms/kg to 2 mg/kg) of dimethylnitrosamine and diethylnitrosamine which produced total amounts of these adducts in the range of 300 to 16,000 molecules/cell. This amount is considerably less than the content of O6-alkylguanine-DNA alkyltransferase protein (approximately 60,000 molecules/hepatocyte), and during the time period studied, the adducts were found to be lost with pseudo-first order kinetics. The half-life for O6-methylguanine was 47 min. O6-Ethylguanine was removed 3.6 times more slowly with a half-life of 172 min. The ability of partially purified rat liver O6-alkylguanine-DNA alkyltransferase to remove O6-methylguanine and O6-ethylguanine from [3H]alkyl-labeled DNA substrates in vitro was measured, and it was found that O6-methylguanine was removed 3.4 times more rapidly than was O6-ethylguanine. These results are consistent with the hypothesis that most, if not all, of the repair of these adducts which occurs within the first 4 hr after treatment is due to the alkyltransferase protein. Diethylnitrosamine, which is slightly more potent as a carcinogen to rat liver, produced a total amount of O6-ethylguanine of 3.7 mumol/mol guanine/mg compared to O6-methylguanine (28 mumol/mol guanine/mg) given by dimethylnitrosamine. The slower rate of loss of the ethyl adduct is not sufficient to account for this difference, and the results, therefore, support the concept that other DNA adducts (possibly O-alkylpyrimidines) contribute to the initiation of tumors by diethylnitrosamine. Preliminary evidence that the rat liver alkyltransferase can also remove hydroxyethyl groups from DNA at a rate slower than removal of ethyl groups was also obtained. Bacterial O6-alkylguanine-DNA alkyltransferase was shown to remove methyl, ethyl, and hydroxyethyl groups from the O6 position of guanine in DNA using fluorescence detection to quantitate these adducts. The bacterial protein removed methyl groups very rapidly but was much slower than the rat liver protein on the larger adducts. These results suggest that the relative rates of repair of different alkyl groups may be species specific and must be determined experimentally in the cell of interest before conclusions concerning biological effects can be drawn.

Two human colon carcinoma cell lines which differ greatly in their content of O6-alkylguanine-DNA alkyltransferase were analyzed for their response to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 2-chloroethyl(methylsulfonyl)methanesulfonate (ClEtSoSo) before and after treatment with O6-alkylguanines. HT29 cells contained about 17 times more alkyltransferase activity than BE cells. The alkyltransferase activity of HT29 cells was reduced by 60-80% by treatment for 24 h with 0.05-0.4 mM O6-methylguanine or O6-n-butylguanine. Such pretreatment prior to exposure to CCNU or ClEtSoSo increased the sensitivity of HT29 cells to these drugs. The exposure to O6-alkylguanines gave a greater enhancement of the toxic effects of ClEtSoSo than of CCNU. There was no significant increase in the toxicity of these agents towards the BE cells which contained much lower levels of the alkyltransferase. When added alone neither O6-methylguanine nor O6-n-butylguanine showed any toxicity towards HT29 or BE cells at the doses used. These results provide strong evidence that the formation of adducts at the O6-position of guanine by these agents contributes significantly to their lethality and that this reaction is more critical for ClEtSoSo than CCNU. The enhancement of the activity of chloroethylating agents by pretreatment with nontoxic doses of O6-alkylguanines may be clinically useful in terms of increasing their therapeutic efficacy towards cells containing high levels of alkyltransferase.