QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.071316v1 311/2/585    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kaina, B. Articles by Schirrmacher, R. Search for Related Content PubMed PubMed Citation Articles by Kaina, B. Articles by Schirrmacher, R.

CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Inhibition of O⁶-Methylguanine-DNA Methyltransferase by Glucose-Conjugated Inhibitors: Comparison with Nonconjugated Inhibitors and Effect on Fotemustine and Temozolomide-Induced Cell Death

Institute of Toxicology, University of Mainz, Mainz, Germany (B.K., A.P.-S., M.C.); Institute of Nuclear Chemistry, University of Mainz, Mainz, Germany (U.M., F.R., R.S.); and Research Group Epigenetics, Deutsches Krebsforschungszentrum, Heidelberg, Germany (R.G.B.)

Received May 12, 2004; accepted July 9, 2004.

	Abstract

Top Abstract Materials and Methods Results and Discussion References

 
The DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) is an important suicide enzyme involved in the defense against O⁶-alkylating mutagens. It also plays a role in the resistance of tumors to anticancer drugs targeting the O⁶-position of guanine, such as temozolomide and fotemustine. Several potent MGMT inhibitors have been developed sensitizing cells to O⁶-alkylating agents. Aimed at targeting MGMT inhibitors to tumor cells, we synthesized MGMT inhibitory compounds conjugated with glucose to improve uptake in tumor cells. Here, we compared O⁶-benzylguanine, O⁶-2-fluoropyridinylmethylguanine (O⁶FPG), O⁶-3-iodobenzylguanine, O⁶-4-bromothenylguanine, and O⁶-5-iodothenylguanine with the corresponding C8-linker -D-glucose derivatives. All glucose conjugated inhibitors were 3- to 5-fold less effective than the corresponding nonconjugated drugs as to MGMT inhibition that was measured in cell extracts (in vitro) and cultivated HeLaS3 cells (in vivo). Except for O⁶FPG, IC₅₀ values of the guanine derivatives applied in vitro and in vivo were correlated. A similar correlation was not obvious for the corresponding glucosides, indicating differences in cellular uptake. C8--D-glucosides were less effective than -glucosides. From the newly developed glucose-conjugated inhibitors tested, O⁶-4-bromothenylguanine-C8--D-glucoside (O⁶BTG-C8-Glu) was most potent in inhibiting MGMT both in vitro and in vivo. At a concentration of 0.1 µM, it inhibited cellular MGMT to completion. It was not toxic, even when applied chronically to cells at high dose (up to 20 µM). O⁶BTG-C8-Glu strongly potentiated the killing effect of fotemustine and temozolomide, causing reversal from MGMT+ to MGMT– phenotype. Therefore, O⁶BTG-C8-Glu seems to be especially suitable for approaching MGMT inhibitor targeting in tumor therapy.

O⁶-Alkylguanine is repaired by the DNA repair protein O⁶-methylguanine-DNA methyltransferase (alkyltransferase; MGMT), which transfers the alkyl group to a cysteine in its own active center (for reviews, see Pegg et al., 1995; Gerson, 2004). Guanine in DNA is thereby restored, and MGMT gets inactivated. MGMT is unique among the repair proteins because it acts only once (suicide enzyme). Therefore, the repair capacity of a cell correlates with the amount of preexisting molecules of MGMT and the rate of its resynthesis. MGMT has a strong protective effect on reproductive survival, apoptosis, and proliferation capacity of cells upon the treatment with O⁶-alkylating agents, including antineoplastic drugs (Preuss et al., 1996b) and, therefore, it can be considered as a crucial marker of intrinsic and acquired tumor cell resistance to methylating and chloroethylating compounds (Kaina and Christmann, 2002).

MGMT is unique also from another point of view: it is highly variably expressed in normal and tumor tissues (Margison et al., 2003). For instance, in human lymphocytes it ranges in expression between 27 and 204 fmol/10⁶ cells (i.e., 7.6-fold). A given expression level in lymphocytes seems to be stable and therefore may reflect the personal situation (Janssen et al., 2001), although various environmental factors exert influence on MGMT expression (for review, see Margison et al., 2003). In tumors, MGMT is tumor type-specific, expressed with low expression in brain and malignant melanomas and high expression in ovarian and breast tumors (Preuss et al., 1996a; Hengstler et al., 1999; Margison et al., 2003). Different MGMT expression levels in tumors were related to MGMT promotor hypermethylation (Esteller et al., 1999). For brain tumors, data are available to show that the MGMT level correlates with the therapeutic response of patients treated with the O⁶-alkylating agent carmustine [N,N'-bis(2-chloroethyl)-N-nitrosourea] (Belanich et al., 1996; Jaeckle et al., 1998). MGMT was found to be inducible in rat and mouse tissues, and studies with human MGMT promoter revealed inducibility of the gene by glucocorticoids and genotoxic agents, including O⁶-alkylating drugs and X-rays (Fritz et al., 1991; Grombacher et al., 1996).

Because MGMT is a most important determinant in alkylating drug resistance and because its expression may differ dramatically in individual tumors, determination of MGMT activity would be important as a predictive indicator. Even more important, however, would be to inactivate MGMT in tumors to sensitize the tumor to an antineoplastic agent. Various highly efficient MGMT inhibitory compounds have been developed that are promising tools for tumor sensitization (Dolan et al., 1990; Pegg et al., 1995; Margison et al., 1996; Friedman et al., 1998; McElhinney et al., 1998, 2003). However, despite encouraging in vitro studies and lack of systemic side effects in patients (Friedman et al., 1998), a recent phase II trial did not reveal significant increase in the therapeutic efficacy of N,N'-bis(2-chloroethyl)-N-nitrosourea in the treatment of malignant glioblastomas (Quinn et al., 2002). The reason might be dose reduction, which is necessary because of the depletion of MGMT by the inhibitor in all organs including blood stem cells that provokes hematotoxicity. Although one might be able, in the future, to protect blood stem cells against O⁶-alkylating drug toxicity by MGMT gene transfer (Moritz et al., 1995; Hickson et al., 1998), it would be highly desirable to develop strategies of inhibitor targeting to inactivate MGMT preferably in the tumor. With this goal in mind, we approached to develop strategies of MGMT inhibitor targeting. Tumor cells are often characterized by increased glucose consumption (Arguiles and Lopez-Soriano, 1990), which is related to elevated glucose uptake due to up-regulation of glucose transporter (Yamamoto et al., 1990). Therefore, we used an approach of coupling MGMT inhibitory compounds to D-glucose. In a previous work, we reported the synthesis of glucose-conjugated MGMT inhibitors. We found them to be active when the length of the linker between D-glucose and the N9 of guanine was C >6 (Reinhard et al., 2001a,b). Here, we extended the previous study and compared various MGMT inhibitors with the corresponding C8-D-glucose derivatives as to MGMT inhibitor efficiency. Also, we determined for the most efficient glucose-conjugated inhibitor we have tested, O⁶-4-bromothenylguanine-C8--D-glucoside (O⁶BTG-C8-Glu), the cytotoxicity and the effect on fotemustine-induced cell death.

	Materials and Methods

Top Abstract Materials and Methods Results and Discussion References

 
Synthesis of Drugs. The detailed syntheses of the new compounds will be described elsewhere. In general, the synthetic strategy of Reinhard et al. (2001a) was applied for the preparation of the glucose-conjugated MGMT inhibitors (Table 1) such as 2-amino-6-(3-iodo-benzyloxy)-9-(octyl--D-glucosyl)-purine (7), 2-amino-6-(4-bromo-thiophen-2-yl-methoxy)-9-(octyl-- (10) and -D-glucosyl)-purine (9), 2-amino-6-(5-iodo-thiophen-2-yl-methoxy)-9-(octyl-- (12) and -D-glucosyl)-purine (11), and 2-amino-6-(benzyloxy)-9-(octyl--D-glucosyl)-purine (6). The fluorinated compounds 2-amino-6-(2-fluoro-pyridine-4-yl-methoxy)-9H-purine (3) and 2-amino-6-(2-fluoro-pyridine-4-yl-methoxy)-9-(octyl--D-glucosyl)-purine (8) were synthesized according to Schirrmacher et al. (2002a,b). 2-Amino-6-(benzyloxy)-9H-purine (McElhinney et al., 1998) (1), 2-amino-6-(4-bromothiophen-2-yl-methoxy)-9H-purine (Reinhard et al., 2001a) (4), 2-amino-6-(3-iodo-ben-zyloxy)-9H-purine (Vaidyanathan et al., 2000) (2), and -bromo-octyl-tetra-O-benzoyl--D-glucopyranoside (Gallo-Rodriguez et al., 1998) were obtained via published analogous procedures. 2-Amino-6-(5-iodo-thiophen-2-yl-methoxy)-9H-purine (5) was synthesized from (5-iodo-thiophen-2-yl)-methanol (D'Auria et al., 1987) and 2-amino-N,N,N-tri-methyl-9H-purine-6-yl-ammonium chloride (McElhinney et al., 1998; Schirrmacher et al., 2002b). The structural identity of all compounds was determined with ¹H, ¹³C NMR, field desorption- or electrospray ionization-mass spectroscopy, and additionally in the case of fluorinated compounds with ¹⁹F-NMR.

Preparation of Cell Extracts and MGMT Assay. Extracts were prepared from exponentially growing cells as described previously (Preuss et al., 1995). In brief, HeLa S3 cells expressing MGMT were harvested and homogenized by sonication in buffer containing 20 mM Tris-HCl, pH 8.5, 1 mM EDTA, 1 mM -mercaptoethanol, 5% glycerol, and the protease inhibitor phenylmethylsulfonyl fluoride (0.1 mM). The sonication product was centrifuged at 10,000 rpm (10 min) in the cold to remove debris, and the supernatant was snap frozen in aliquots in liquid nitrogen and stored at –80°C until use. HeLa S3 cells express MGMT at a level of 588 ± 86 fmol/mg protein (mean of 13 determinations). In each assay, an extract of HeLa MR cells deficient in MGMT served as a negative background control.

Determination of MGMT Activity. MGMT activity in cell and tissue extracts was determined essentially as reported previously (Preuss et al., 1996a). The method is based on the radioactive assay in which the transfer of a tritium labeled methyl group from the O⁶ position of guanine in DNA to protein in the cell extract is measured. For the assays, at least 100 µg of cell extract protein was used. In each assay, a negative and positive MGMT sample was included of HeLa MR and HeLa S3 cell extract, respectively. The incubation of cell extract together with [³H]methylnitrosourea-labeled calf thymus DNA containing O⁶-methylguanine (total 80,000 cpm/sample) occurred in buffer containing 70 mM HEPES-KOH (pH 7.8), 1 mM dithiothreitol, and 5 mM EDTA for 90 min. This was the optimal time span for the reaction to be completed (as demonstrated in background experiments; unpublished data). Data are expressed as femtomoles of radioactivity transferred from ³H-labeled DNA to protein per milligram of protein within the sample.

Survival Experiments. Cells of the line AT17-C3, which is a CHO-9 derivative transfected with human MGMT cDNA expression vector expressing a high amount of MGMT (Kaina et al., 1991), were seeded at a density of 300 cells/5-cm dish in F12/Dulbecco's medium containing 10% fetal calf serum. Upon incubation at 37°C and 7% CO₂ for 4 h, cells were not pretreated or pretreated with the MGMT inhibitor for the indicated times. Fotemustine treatment occurred for 60 min. Thereafter, cells were rinsed with phosphate-buffered saline, and fresh medium was added. Alternatively, cells were seeded, and 12 h later fotemustine and the inhibitor were added. Cells were incubated until colonies formed (usually 7 days). They were fixed, stained, and colony formation efficiencies were determined in relation to the mock-treated control.

	Results and Discussion

Top Abstract Materials and Methods Results and Discussion References

 
Inhibition of MGMT in Vitro and in Vivo by Various Inhibitors. First, we synthesized various guanine derivatives and checked their ability to inhibit MGMT in HeLaS3 cell extracts (in vitro) and HeLaS3 cells (in vivo). IC₅₀ values indicating the dose that inhibits MGMT activity by 50% were calculated from concentration-effect curves shown in Figs. 1 and 2 for in vitro and in vivo assays, respectively. IC₅₀ values are compiled in Table 2. Most efficient regarding MGMT inhibition both in vitro and in vivo was O⁶BTG followed by O⁶IBG, O⁶-benzylguanine, and O⁶ITG. For these drugs, the efficiency of MGMT inhibition measured in cell extracts and in intact cells was correlated (Fig. 3A). An exception was provided by O⁶FPG, which was less effective in inhibiting MGMT in vivo than in vitro, as revealed by comparison of IC₅₀ values (Fig. 3A). Although data on the uptake of the compounds are not available yet, it is supposed that O⁶-methylguanine and the other guanine derivatives harboring larger groups at the O⁶ position are able to enter the cell in a passive way. This might indeed be the case as suggested by the correlation of IC₅₀ values of MGMT inhibition determined in vitro and in vivo (Fig. 3A). The finding that O⁶FPG is less active in MGMT inhibition in vivo, however, suggests that for some derivatives diffusion into the cell and/or into the nucleus is impaired or that an active influx or efflux mechanism is at least partially involved. The findings might also be attributed to the higher electronegativity of the fluorine atom in comparison with iodine and bromine, which can alter the lipophilicity and especially the electron density in the molecule. It also indicates that the in vitro inhibitory constant of a given nonconjugated inhibitor does not necessarily reflect the inhibitor capacity it exerts in intact cells. This at the same time underlines the need for in vivo testing of the compounds as to their MGMT inhibitor capacity.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Inactivation of MGMT in vitro by various MGMT inhibitors and the corresponding C8--D-glucose derivatives. Inhibitors were added to MGMT containing HeLa S3 cell extracts in vitro 30 min before the O6-methylguanine containing radiolabeled DNA was added. Thereafter, the samples were incubated at 37°C for another 90 min. MGMT quantification was performed as described under Materials and Methods. Each assay included a determination with HeLa MR extract, which does not contain MGMT (background control). Values were related to the control (i.e., solvent without inhibitor; expression level on average 588 fmol MGMT/mg protein) and presented as percentage inhibition of MGMT activity. Data are the mean of at least three independent determinations ± S.D.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Inhibition of MGMT by glucose nonconjugated and conjugated inhibitors added to HeLa S3 cells grown in culture. Treatment of exponentially growing cells was done for 4 h. Cells were thereafter harvested, and extracts were prepared as described. MGMT was determined in the extracts and expressed in relation to the mock-treated control. Data (except E, for which a representative experiment is shown) are the mean of at least three independent experiments ± S.D.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Correlation of IC50 values that were determined in vitro (cell extracts) and in vivo (cultivated cells). IC50 values were calculated from MGMT inhibition curves shown in Figs. 1 and 2. Correlation analysis was done for the nonconjugated (A) and the glucose-conjugated inhibitors (B). Correlation coefficients (R2) were recorded without the inclusion of O6FPG and O6FPG-C8-Glu.

Inhibition of MGMT by Glucose-Conjugated Inhibitors. To convert the guanine-derived MGMT inhibitors into drugs that are subject to active transport, various MGMT inhibitors were conjugated to D-glucose that cannot enter the cell by diffusion but is taken up actively. In previous experiments, we found that conjugation of the guanine derivative with -D-glucose is most optimal if it occurs at the N9 position of guanine. We also found that a short C linker (C <6) strongly reduces the activity of the inhibitor. With a C8 spacer, however, the inhibitor activity was nearly retained. A longer spacer (C10, C12) did not improve inhibitor activity (compared with the nonconjugated parent compound), but it reduced the compound's solubility (Reinhard et al., 2001a,b). Therefore, we decided on conjugation with a C8 spacer. As shown in Figs. 1 and 2, and Table 2 for IC₅₀ values, the glucose conjugates displayed reduced IC₅₀ values compared with the parent compounds. This indicates that glucose conjugation reduces the inhibitor's ability to inhibit MGMT. This essentially confirms our previous data with a selected set of MGMT inhibitors (Reinhard et al., 2001b). Glucose conjugation (using a C8 spacer) reduced MGMT inhibition in the in vitro experiments shown here by a factor of 3 to 5.

It would be interesting to compare the inhibition of MGMT by the glucose-derivatives in vitro and in vivo. Most potent was O⁶BTG-C8-Glu followed by O⁶IBG-C8-Glu and O⁶BG-C8-Glu. O⁶ITG-C8-Glu was less efficient both in vitro and in vivo (Fig. 3B). Overall, in contrast to the nonconjugated inhibitors, the correlation between IC₅₀ (in vitro) and IC₅₀ (in vivo) of the glucose-conjugated inhibitors was not significant (Fig. 3B), which may be taken to indicate that cellular uptake and nuclear transport is different for the drugs. Again, the least efficient inhibitor was O⁶FPG-C8-Glu, notably under in vivo conditions, which makes this compound not suitable for further studies.

Because of steric reasons one might assume that a -D-glucose linker is more advantageous than the linker. Therefore, we compared two MGMT inhibitors linked to glucose by an or linker. O⁶BTG-C8--D-glucose was clearly less efficient in MGMT inhibition both in vitro and in vivo than O⁶BTG-C8--D-glucose. For O⁶ITG, the -D-glucose derivative was slightly less efficient in vitro, and, surprisingly, more efficient in vivo (Figs. 1 and 2; Table 2). In summary, the data revealed that 1) the glucose conjugates are 2- to 5-fold less effective than the nonconjugated compounds; 2) for inhibitors that were not coupled to glucose, the in vitro and in vivo MGMT inhibition constant was correlated (except for O⁶FPG); 3) for the glucose-conjugated inhibitors, the same correlation was lacking; and 4) the most efficient inhibitor, both as parent compound and glucoside in vitro and in vivo, was O⁶BTG. O⁶BTG and the corresponding derivative O⁶BTG-C8-Glu inhibit MGMT nearly to completion already at a concentration of <0.1 µM (assayed upon incubation of cells with the inhibitor for 4 h). Because of the high potency of both O⁶BTG and O⁶BTG-C8-Glu to inhibit MGMT, we decided to use O⁶BTG and the corresponding glucose derivative in further studies on cell killing.

Cytotoxicity of MGMT Inhibitors. The cytotoxicity of O⁶BTG and O⁶BTG-C8--D-glucose as well as the other inhibitors synthesized in our laboratory was checked in colony-forming assays, which is a very sensitive method for detection of killing effects on cellular level. LD₅₀ values calculated from dose-response curves (not shown) are compiled in Table 2. We should note that none of the inhibitors exerted immediate cytotoxic effects. In mass culture, cytotoxicity was not seen upon short-term treatment. Therefore, inhibition of MGMT observed in vivo cannot be due to unspecific cell killing. A reasonably good ratio between MGMT inhibition and cytotoxicity was observed for O⁶BTG and the corresponding derivative. Thus, if CHO-9 cells were chronically exposed to the agents, O⁶BTG and O⁶BTG-C8-Glu were devoid of any toxicity up to a concentration of 0.5 µM (see Fig. 4 for representative survival curves). Interestingly, O⁶BTG exerted cytotoxic effects upon chronic administration with a concentration >0.5 µM, whereas O⁶BTG-C8-Glu was not toxic even with a 10-fold higher concentration (Fig. 4; data not shown). With a concentration of up to 100 µM O⁶BTG-C8-Glu, colony formation gradually decreased to 60%. The LD₅₀ values were 0.73 and >100 µM for O⁶BTG and O⁶BTG-C8-Glu, respectively, which is far above the concentration inhibiting MGMT. If cells were treated for a short period of time (1 h), O⁶BTG and O⁶BTG-C8-Glu were even less toxic (Fig. 5); no killing effects were observed with a concentration of 5 and >100 µM, respectively (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 4. Survival of AT17-C3 cells chronically exposed to MGMT inhibitors. Cells were seeded, inhibitors were added 6 h later at the concentrations indicated, and cells were grown for 7 d until colonies formed.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Survival of AT17-C3 cells treated with O6BTG or O6BTG-C8-Glu and fotemustine as a function of inhibitor concentration. The MGMT inhibitor was added to the cells 6 h after seeding. One hour later, fotemustine was given (final concentration 40 µg/ml), and cells were incubated for another 60 min. Thereafter, the medium was removed and fresh medium was added. Cells were incubated for 7 d until colonies formed. Colony formation upon treatment with fotemustine alone was 80%.

Effect of MGMT Inhibitors on Fotemustine-Induced Cytotoxicity. To check the efficiency of O⁶BTG and O⁶BTG-C8-Glu in increasing the killing effect of an O⁶-alkylating agent on cells expressing human MGMT, we used CHO-9 cells stably transfected with human MGMT cDNA (AT17-C3 cells) as well as MGMT-expressing HeLa S3 cells. As a result of MGMT expression, these cells gained a high level of resistance to various O⁶-alkylating agents (Kaina et al., 1991; Preuss et al., 1996b). As shown in Fig. 5, O⁶BTG dramatically intensified the killing effect of the anticancer drug fotemustine (muphoran), which is an O⁶-chloroethylating agent that is in use in the treatment of malignant melanomas and glioblastomas (Boiardi et al., 2001; Tarhini and Agarwala, 2004). Whereas fotemustine at a concentration of 40 µg/ml (60-min treatment) was only very slightly toxic (80% colony formation), O⁶BTG at a concentration of 0.1 µM given 60 min before the agent reduced cell survival by >99%. Interestingly, the glucose derivative O⁶BTG-C8-Glu also intensified fotemustine-induced cell killing; however, higher concentrations of the inhibitor (up to 2 µM) were required to provoke a similar kill-intensifying effect (Fig. 5, bottom). This contrasts with MGMT inhibition experiments in which we showed that depletion of MGMT occurred already upon treatment of cells with 0.1 µM O⁶BTG-C8-Glu. The discrepancy might be explained by different half-lives of O⁶BTG and O⁶BTG-C8-Glu within the cell and the kinetics of resynthesis of MGMT. If the intracellular half-life is short, the inactivated MGMT will be replaced by newly synthesized enzyme, which may still provoke resistance before O⁶-chloroethylguanine lesions are converted into the corresponding interstrand cross-links, which are supposed to be the critical ultimate killing lesions (Erickson et al., 1980). If this is true, treatment with the inhibitor both before and after the administration of fotemustine should intensify fotemustine-induced cell kill. This was indeed the case. As shown in Fig. 6, the effect of O⁶BTG-C8-Glu on fotemustine-induced cell inactivation was clearly enhanced if it was given both before and after the pulse treatment with the antineoplastic agent.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Survival of AT17-C3 cells as a function of the concentration of O6BTG-C8-Glu in the medium. Cells were seeded, and 6 h later the inhibitor was added and left onto the plates (inhibitor). One hour later, fotemustine was given to the medium (40 µg/ml), and cells were incubated for another 60 min. Thereafter the medium was changed (inhibitorfotemustine). In a third approach, the MGMT inhibitor was again added and left onto the plates until colonies formed (inhibitorfotemustineinhibitor).

The intensifying effect of O⁶BTG and O⁶BTG-C8-Glu on cell killing induced by increasing doses of fotemustine is shown in Fig. 7. Both the parent compound (Fig. 7A) and the corresponding C8--D-glucoside (Fig. 7B) dramatically sensitized MGMT-expressing AT17-C3 cells. Thereby, they responded to fotemustine in almost the same way as MGMT-nonexpressing cells. A similar sensitizing effect of O⁶BTG-C8-Glu was found for HeLa S3 cells, which were reverted to the sensitivity of MGMT-deficient HeLa MR cells (Fig. 7C).

View larger version (22K): [in this window] [in a new window]   Fig. 7. Survival of cells as a function of the concentration of fotemustine. A, AT17-C3 cells were seeded and treated with O6BTG as described in Fig. 5. B, AT17-C3 cells were seeded and treated with O6BTG-C8-Glu 18 h later. One hour thereafter, fotemustine was given to the medium at the final concentrations indicated. Twenty-four hours thereafter, the medium was replaced by fresh medium, and cells were cultivated for 7 days until colonies formed. C, HeLa S3 cells were seeded and treated with O6BTG-C8-Glu and fotemustine as described under B. MGMT lacking CHO-9 and HeLa MR cells treated with fotemustine only served as control.

A representative of methylating agents used in tumor chemotherapy, notably in the therapy of glioblastomas and malignant melanomas, is temozolomide (Temodar), an imida-zotetrazinone that undergoes spontaneous conversion in aqueous solution to an active methylating reagent (Tsang et al., 1991). The effect of O⁶BTG-C8-Glu on temozolomide-induced cell death is shown in Fig. 8. Again, the glucose derivative was highly effective in sensitizing MGMT-expressing cells, which was shown for AT17-C3 (Fig. 8A) and HeLa S3 (Fig. 8B) cells.

View larger version (29K): [in this window] [in a new window]   Fig. 8. Survival of cells as a function of the concentration of temozolomide. A, AT17-C3 cells were seeded and treated with O6BTG-C8-Glu 18 h later. One hour thereafter, temozolomide was added to the medium at the final concentrations indicated. Twenty-four hours thereafter, the medium was replaced by fresh medium, and cells were cultivated for 7 days until colonies formed. B, HeLa S3 cells were seeded and treated with O6BTG-C8-Glu and temozolomide as described under A. CHO-9 and HeLa MR cells treated with temozolomide served as control.

O⁶BTG-C8-Glu is readily water-soluble and stable under aqueous conditions. Thus 98% of the conjugate was still intact after 24 h of incubation in 0.9% NaCl solution, as proven by high-performance liquid chromatography analysis. It is nontoxic even at high concentration and chronic exposure of cells in culture. Therefore, O⁶BTG-C8-Glu might be especially useful for targeting tumor cells that are characterized by high glucose consumption. O⁶BTG-C8-Glu is highly efficient in inactivating MGMT in vitro and inside the cell. It was also shown here to enhance the cell-killing effect of fotemustine and temozolomide, which are representatives of chloroethylating and methylating O⁶-alkylating anticancer drugs. Further studies will be performed to elucidate the mode of uptake, stability in vivo, cell type-specificity of action as well as the response of tumors that have been treated with O⁶-alkylating agents in combination with this newly developed group of MGMT inhibitors.

	Footnotes

 
This work was supported by the Deutsche Forschungsgemeinschaft Grants DFG-Ka724/12-1 and Ka724/13-1.

doi:10.1124/jpet.104.071316.

ABBREVIATIONS: MGMT, O⁶-methylguanine-DNA methyltransferase; O⁶BTG-C8-Glu, O⁶-4-bromothenylguanine-C8--D-glucoside; O⁶FPG, O⁶-2-fluoropyridinylmethylguanine; O⁶IBG, O⁶-3-iodobenzylguanine; O⁶BTG, O⁶-4-bromothenylguanine; O⁶ITG, O⁶-5-iodothenylguanine.

Address correspondence to: Dr. Bernd Kaina, Institute of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany. E-mail: kaina{at}uni-mainz.de

	References

Top Abstract Materials and Methods Results and Discussion References

 

Arguiles JM and Lopez-Soriano FJ (1990) Why do cancer cells do have such a high glycolytic rate? Med Hypothesis 32: 151–155.[CrossRef][Medline]

Belanich M, Pastor M, Randall T, Guerra D, Kibitel J, Alas L, Li B, Citron M, Wasserman P, White A, et al. (1996) Retrospective study of the correlation between the DNA repair protein alkyltransferase and survival of brain tumor patients treated with carmustine. Cancer Res 56: 783–788.[Abstract/Free Full Text]

Beranek DT (1990) Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents. Mutation Res 231: 11–30.

Boiardi A, Silvani A, Ciusani E, Watson A, Margison G, Berger E, Lucas C, and Giroux B (2001) Fotemustine combined with procarbazine in recurrent malignant gliomas: a phase I study with evaluation of lymphocyte O⁶-alkylguanine-DNA alkyltransferase activity. J Neurooncol 52: 149–156.[CrossRef][Medline]

D'Auria M, De Mico A, D'Onofrio F, and Piancatelli G (1987) Synthesis of naturally occurring bithiophenes: a photochemical approach. J Org Chem 52: 5243–5247.[CrossRef]

Dolan ME, Moschel RC, and Pegg AE (1990) Depletion of mammalian O⁶-alkylguanine-DNA alkyltransferase activity by O⁶-benzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agents. Proc Natl Acad Sci USA 87: 5368–5372.[Abstract/Free Full Text]

Erickson LC, Bradley MO, Ducore JM, Ewig RAG, and Kohn KW (1980) DNA crosslinking and cytotoxicity in normal and transformed human cells treated with antitumor nitrosoureas. Proc Natl Acad Sci USA 77: 467–471.[Abstract/Free Full Text]

Esteller M, Hamilton SR, Burger PC, Baylin SB, and Herman JG (1999) Inactivation of the DNA repair gene O⁶-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 59: 793–797.[Abstract/Free Full Text]

Friedman HS, Kokkinakis DM, Pluda J, Friedman AH, Cokgar I, Haglund MM, Ashley DM, Rich J, Dolan ME, Pegg AE, et al. (1998) Phase I trial of O⁶-benzylguanine for patients undergoing surgery for malignant glioma. J Clin Oncol 16: 3570–3575.[Abstract]

Fritz G, Tano K, Mitra S, and Kaina B (1991) Inducibility of the DNA repair gene encoding O⁶-methylguanine-DNA methyltransferase in mammalian cells by DNA-damaging treatments. Mol Cell Biol 11: 4660–4668.[Abstract/Free Full Text]

Gallo-Rodriguez C, Varela O, and de Lederkremer RM (1998) One-pot synthesis of b-D-Galf(1->4) [b-D-Galr(1–6)]-D-GlcNAc, a core trisaccharide linked O-glycosidically in glycoproteins of Trypanosoma cruzi. Carbohydrate Res 305: 163–170.[CrossRef]

Gerson SL (2004) MGMT: its role in cancer aetiology and cancer therapeutics. Nat Rev Cancer 4: 296–307.[CrossRef][Medline]

Grombacher T, Mitra S, and Kaina B (1996) Induction of the alkyltransferase (MGMT) gene by DNA damaging agents and the glucocorticoid dexamethasone and comparison with the response of base excision repair genes. Carcinogenesis 17: 2329–2336.[Abstract/Free Full Text]

Hengstler J, Tanner B, Möller L, Vydra M, Oesch F, Meinert R, and Kaina B (1999) Activity of O⁶-methylguanine-DNA methyltransferase in relation to p53 status and therapeutic response in ovarian cancer. Int J Cancer 84: 388–395.[CrossRef][Medline]

Hickson I, Fairbairn LJ, Chinnasamy N, Lashford LS, Thatcher N, Margison GP, Dexter TM, and Rafferty JA (1998) Chemoprotective gene transfer I: transduction of human haemopoietic progenitors with O⁶-benzylguanine-resistant O⁶-alkylguanine-DNA alkyltransferase attenuates the toxic effects of O⁶-alkylating agents in vitro. Gene Therapy 5: 835–841.[CrossRef][Medline]

Jaeckle KA, Eyre HJ, Townsend JJ, Schulman S, Knodson HM, Belanich M, Yarosh DB, Bearman SI, Giroux DJ, and Schold SC (1998) Correlation of tumor O⁶-methylguanine-DNA methyltransferase levels with survival of malignant astrocytoma patients treated with bis-chloroethylnitrosourea: a southwest oncology group study. J Clin Oncol 16: 3310–3315.[Abstract]

Janssen K, Grombacher U, Schlink K, Nitzsche S, Oesch F, and Kaina B (2001) Long-time expression of DNA repair enzymes MGMT and APE in human peripheral blood mononuclear cells. Arch Toxicol 75: 306–312.[CrossRef][Medline]

Kaina B and Christmann M (2002) DNA repair in resistance to alkylating anticancer drugs. Int J Clin Pharm Ther 40: 354–367.[Medline]

Kaina B, Fritz G, Mitra S, and Coquerelle T (1991) Transfection and expression of human O⁶-methylguanine-DNA methyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against the genotoxic effects of alkylating agents. Carcinogenesis 12: 1857–1867.[Abstract/Free Full Text]

Kaina B, Ziouta A, Ochs K, and Coquerelle T (1997) Chromosomal instability, reproductive cell death and apoptosis induced by O⁶-methylguanine in Mex-, Mex+ and methylation-tolerant mismatch repair compromised cells: facts and models. Mutation Res 381: 227–241.

Margison GP, Hickson I, Jelinek J, Kelly J, Elder RH, Rafferty JA, Fairbairn LJ, Dexter TM, Stocking C, et al. (1996) Resistance to alkylating agents: more or less. Anticancer Drugs 7: 109–116.[Medline]

Margison GP, Povey AC, Kaina B, and Santibanez-Koref MF (2003) Variability and regulation of O⁶-alkylguanine-DNA alkyltransferase. Carcinogenesis 24: 625–635.[Abstract/Free Full Text]

Margison GP and Santibanez-Koref MF (2002) O⁶-Alkylguanine-DNA alkyltransferase: role in carcinogenicity and chemotherapy. Bioessays 24: 255–266.[CrossRef][Medline]

McElhinney RS, Donnelly DJ, McCormick JE, Kelly J, Watson AJ, Rafferty JA, Elder RH, Middleton MR, Willington MA, McMurry BH, et al. (1998) Inactivation of O⁶-alkyguanine-DNA alkyltransferase. 1. Novel O⁶-(hetarylmethyl)guanines having basic rings in the side chain. J Med Chem 41: 5265–5271.[CrossRef][Medline]

McElhinney RS, McMurry TB, and Margison GP (2003) O⁶-Alkylguanine-DNA alkyltransferase inactivation in cancer chemotherapy. Mini Rev Med Chem 3: 471–485.[CrossRef][Medline]

Meikrantz W, Bergom MA, Memisoglu A, and Samson L (1998) O⁶-alkylguanine DNA lesions trigger apoptosis. Carcinogenesis 19: 369–372.[Abstract/Free Full Text]

Moritz T, Mackay W, Glassner BJ, Williams DA, and Samson L (1995) Retrovirus-mediated expression of a DNA repair protein in bone marrow protects hematopoietic cells from nitrosourea-induced toxicity in vitro and in vivo. Cancer Res 55: 2608–2614.[Abstract/Free Full Text]

Pegg AE, Dolan ME, and Moschel RC (1995) Structure, function and inhibition of O⁶-alkylguanine-DNA-alkyltransferase. Prog Nucleic Acid Res Mol Biol 51: 167–223.[Medline]

Preuss I, Eberhagen I, Haas S, Eibl RH, Kaufmann M, von Minckwitz G, and Kaina B (1995) O⁶-Methylguanine-DNA methyltransferase activity in breast and brain tumors. Int J Cancer 61: 321–326.[Medline]

Preuss I, Haas S, Eichhorn U, Eberhagen I, Kaufmann M, Beck T, Eibl RH, Dall P, Bauknecht T, Hengstler J, et al. (1996a) Activity of the DNA repair protein O⁶-methylguanine-DNA methyltransferase in human tumor and corresponding normal tissue. Cancer Detect Prev 20: 130–136.[Medline]

Preuss I, Thust R, and Kaina B (1996b) Protective effect of O⁶-methylguanine-DNAmethyltransferase (MGMT) on the cytotoxic and recombinogenic activity of different antineoplastic drugs. Int J Cancer 65: 506–512.[CrossRef][Medline]

Quinn JA, Pluda J, Dolan ME, Delaney S, Kaplan R, Rich JN, Friedman AH, Reardon DA, Sampson JH, Colvin OM, et al. (2002) Phase II trial of carmustine plus O(6)-benzylguanine for patients with nitrosourea-resistant recurrent or progressive malignant glioma. J Clin Oncol 20: 2277–2283.[Abstract/Free Full Text]

Reinhard J, Hull WE, von der Lieth CW, Eichhorn U, Kliem HC, Kaina B, and Wiessler M (2001a) Monosaccharide-linked inhibitors of O⁶-methylguanine-DNA methyltransferase (MGMT): synthesis, molecular modeling and structure-activity relationships. J Med Chem 44: 4050–4061.[CrossRef][Medline]

Reinhard J, Eichhorn U, Wiessler M, and Kaina B (2001b) Inactivation of O^6-methylguanine-DNA methyltransferase by glucose-conjugated inhibitors. Int J Cancer 93: 373–379.[CrossRef][Medline]

Schirrmacher R, Mühlhausen U, Wängler B, Schirrmacher E, Reinhard J, Nagel G, Kaina B, Piel M, Wiessler M, and Rösch F (2002a) Synthesis of 2-amino-6-(2-[18F]fluoro-pyridine-4-ylmethoxy)-9-(octyl-b-D-glucosyl)-purine: a novel radioligand for positron emission tomography studies of the O⁶-methylguanine-DNA methyltransferase (MGMT) status of tumor tissue. Tetrahedron Lett 43: 6301–6304.[CrossRef]

Schirrmacher R, Wängler B, Schirrmacher E, August T, and Rösch F (2002b) Dimethylpyridin-4-ylamine-catalysed alcoholysis of 2-amino-N,N,N-trimethyl-9H-purine-6-ylammonium chloride: an efficient route to O⁶-substituted guanine derivatives from alcohols with poor nucleophilicity. Synthesis 4: 538–542.[CrossRef]

Tarhini AA and Agarwala SS (2004) Management of brain metastases in patients with melanoma. Curr Opin Oncol 16: 161–166.[CrossRef][Medline]

Tsang LL, Quarterman CP, Gescher A, and Slack JA (1991) Comparison of the cytotoxicity in vitro of temozolomide and dacarbazine, prodrugs of 3-methyl-(triazen-1-yl)imidazole-4-carboxamide. Cancer Chemother Pharmacol 27: 342–346.[CrossRef][Medline]

Vaidyanathan G, Affleck DJ, Cavazos CM, Johnson SP, Shankar S, Friedman HS, Colvin MO, and Zalutsky R (2000) Radiolabeled guanine derivatives for the in vivo mapping of O⁶-alkylguanine-DNA alkyltransferase: 6-(4-[18F]fluoro-benzyloxy)-9H-purin-2-ylamine and 6-(3-[131I]iodo-benzyloxy)-9H-purin-2ylamine. Bioconjug Chem 11: 868–875.[CrossRef][Medline]

Yamamoto T, Seine Y, Fukumoto H, Koh G, Yano H, Inagaki N, Yamada Y, Inoue K, Manabe T, and Imura H (1990) Overexpression of facilitative glucose transporter genes in human cancer. Biochem Biophys Res Commun 170: 223–230.[CrossRef][Medline]

This article has been cited by other articles:

				G. J. Kitange, B. L. Carlson, M. A. Schroeder, P. T. Grogan, J. D. Lamont, P. A. Decker, W. Wu, C. D. James, and J. N. Sarkaria Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts Neuro-oncol, January 1, 2009; 11(3): 281 - 291. [Abstract] [Full Text] [PDF]

				M. Rapp, J. C. Maurizis, J. Papon, P. Labarre, T.-D. Wu, A. Croisy, J. L. Guerquin-Kern, J. C. Madelmont, and E. Mounetou A New O6-Alkylguanine-DNA Alkyltransferase Inhibitor Associated with a Nitrosourea (Cystemustine) Validates a Strategy of Melanoma-Targeted Therapy in Murine B16 and Human-Resistant M4Beu Melanoma Xenograft Models J. Pharmacol. Exp. Ther., July 1, 2008; 326(1): 171 - 177. [Abstract] [Full Text] [PDF]

				T. F. Gajewski, J. Sosman, S. L. Gerson, L. Liu, E. Dolan, S. Lin, and E. E. Vokes Phase II Trial of the O6-Alkylguanine DNA Alkyltransferase Inhibitor O6-Benzylguanine and 1,3-Bis(2-Chloroethyl)-1-Nitrosourea in Advanced Melanoma Clin. Cancer Res., November 1, 2005; 11(21): 7861 - 7865. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.071316v1 311/2/585    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kaina, B. Articles by Schirrmacher, R. Search for Related Content PubMed PubMed Citation Articles by Kaina, B. Articles by Schirrmacher, R.