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[Cancer Research 61, 53-58, January 1, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Topoisomerase I-mediated Cytotoxicity of N-Methyl-N'-nitro-N-nitrosoguanidine: Trapping of Topoisomerase I by the O6-Methylguanine1

Philippe Pourquier, Jessica L. Waltman, Yoshimasa Urasaki, Natalia A. Loktionova, Anthony E. Pegg, John L. Nitiss and Yves Pommier2

Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892 [P. P., Y. U., Y. P.]; Molecular Pharmacology Department, St. Jude Children’s Hospital, Memphis, Tennessee 38105 [J. L. W., J. L. N.]; and Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 [N. A. L., A. E. P.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
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.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Top13 is an essential enzyme in higher eukaryotic cells (1 , 2) . It regulates DNA topology during crucial processes such as replication, transcription, and chromosome condensation and segregation during mitosis (3 , 4) . Top1 reversibly cleaves one strand of the duplex DNA by forming a covalent tyrosyl-phosphodiester bond with the 3'-end of the broken DNA (5) . This intermediate is referred to as the Top1 cleavage complex. Under normal conditions, the religation step of the DNA cleavage/religation equilibrium is favored, and only a small fraction of the DNA is cleaved at any given time (3) . Top1 inhibitors such as camptothecin and its derivatives stabilize (trap) the cleavage complexes by inhibiting the religation step of the equilibrium reaction (6 , 7) . Trapping of cleavage complexes by camptothecin generates Top1-mediated DNA damage by interference with replication or transcription complexes (8, 9, 10, 11) .

Recent studies have shown that a variety of DNA alterations originating from endogenous and exogenous sources (abasic sites, uracil misincorporations, nicks, oxidized bases, UV photolesions, and carcinogenic adducts) induce the formation of Top1 cleavage complexes (reviewed in Ref. 12 ). Moreover, we recently demonstrated that Top1 trapping by the nucleoside analogue 1-ß-D-arabinofuranosylcytosine might also contribute to the cytotoxicity of this widely used anticancer agent (13) .

DNA methylation represents a cytotoxic lesion that is produced by alkylating agents such as methyl sulfates (dimethyl sulfate), methyl sulfonates, methyl nitrosamines (dimethylnitrosamine), or methyl nitrosamides (methylnitrosourea and MNNG) that are present in the environment as byproducts of industrial processes (14 , 15) . DNA methylation is also generated endogenously by nonenzymatic mechanism involving S-adenosyl-L-methionine (16) . Methylating agents are electrophilic compounds that alkylate guanines at position N3 , N7, and O6 (15) . When O6MG is generated, it tends to base pair preferentially to thymine rather than cytosine (17 , 18) , which results in G:C to A:T transition. Such mutations are probably responsible for the development of various types of tumors (19 , 20) . O6MG is specifically removed by the AGT, which catalyzes the irreversible transfer of the alkyl group of the guanine to an internal cysteine of the enzyme (Cys-145 in humans; Refs. 21, 22, 23 ). Alkylators of the O6 position guanines are highly potent drugs used in chemotherapy. Dacarbazine and temozolomide specifically methylate the O6 position of guanines and are used in the treatment of advanced melanoma, brain tumors, and lymphomas (24) . Chloroethylating agents such as 1,3-bis(2-chloroethyl)-1-nitrosourea represent standard treatments for malignant gliomas (25) and generate O6-chloroethyl adducts that are also repaired by the AGT enzyme (15) . However, the cytotoxicity of methylating agents does not seem to be attributable to their mutagenicity, and the mechanism by which alkylation of DNA leads to cell death remains unclear.

In this study, we show that the presence of O6MG in DNA can induce Top1 cleavage complexes in vitro and in MNNG-treated cells lacking the AGT enzyme. We also demonstrate that Top1 trapping by alkylated DNA may in part contribute to the cytotoxicity of MNNG.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Chemicals and Enzymes.
CPT was provided by Dr. M. E. Wall (Research Triangle Institute, Research Triangle Park, NC). MNNG was purchased from Sigma Chemical Co. (St. Louis, MO). Ten or 100 mM aliquots of drugs in DMSO were stored at -20°C, thawed, and diluted just before use. [{alpha}-32P]Cordycepin 5'-triphosphate was purchased from DuPont NEN (Boston, MA), and terminal deoxynucleotidyl transferase and T4 polynucleotide kinase were from Life Technologies, Inc., (Grand Island, NY). Polyacrylamide was purchased from Bio-Rad, Inc. (Richmond, CA). Human recombinant Top1 was purified from Sf9 cells using a baculovirus construct for the NH2-terminal truncated human Top1 cDNA as described previously (26) .

Cell Lines.
CHO cells expressing the human alkyl guanine transferase (CHO/pCMV-AGT) or transfected with the empty pCMV-neo vector (CHO/pCMV; Ref. 27 ) were cultured in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) containing 10% heat-inactivated FCS, 2 mM glutamine, and 1 mg/ml geneticin in a 5% CO2 incubator at 37°C.

Oligonucleotide Labeling and Annealing Procedures.
High-performance liquid chromatography-purified oligonucleotides were purchased from The Midland Certified Reagent Company (Midland, TX). Fig. 1ACitation shows the 36-bp duplex sequence used in standard experiments. Suicide substrates shown in Citation Fig. 3ACitation were generated by annealing a 3' end-labeled 21-mer upper strand (5'-GATCTAAAAGACTTGGAAAA(A)-3', where (A) corresponds to [{alpha}-32P]-labeled cordycepin) incorporated at the end of the oligonucleotide. 3'-Labeling and 5'-phosphorylation of single-stranded oligonucleotides and annealing were performed as described previously (26) .



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  		Fig. 1. A, sequence of the oligonucleotide derived from the Tetrahymena hexadecameric rDNA sequence (53) . *A, [32P]cordycepin label at the 3' terminus of the scissile (upper) strand. X, the +1 position where the modified base was incorporated. Arrowhead, the top1 cleavage site between T and X. B, base pairing of O6MeG and 6TG with C.

 


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  		Fig. 2. O6MG incorporation at the +1 position of a Top1 cleavage site enhances the steady-state level of Top1 cleavage complexes. A, enhancement of Top1 cleavage complexes by O6MG incorporation at the +1 position. B, effects of 6TG incorporation at the +1 position on Top1 activity. For each panel: Lanes A, DNA alone; Lanes B, + top1; Lanes D, + Top1 + 10 µM CPT; Lanes C and E, same as Lanes B and D but with further incubation in 0.5 M NaCl (final concentration) for 30 min at 25°C before addition of 0.5% SDS. X, the base at the +1 position of the Top1 cleavage site (see Fig. 1Citation ).

 


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  		Fig. 3. O6MG incorporation at the +1 position induces the formation and inhibits religation of the Top1 cleavage complexes. A, kinetics of Top1-induced DNA cleavage. Suicide oligonucleotides were incubated with Top1 for 5s, 10 s, 0.5 min, 1 min, 3 min, 5 min, 10 min, or 30 min (Lanes 1–8, respectively), and reactions were stopped with 0.5% SDS (left panel). Right panel, a control experiment to demonstrate that the cleavage complexes formed after 1 h incubation with Top1 (Lanes top1) were not reversible with an additional 30-min incubation in the presence of 0.5 M NaCl (Lanes R). Therefore, rates of cleavage could directly be calculated from the kinetics shown in the left panel. Lanes 0, DNA alone. B, O6MG incorporation inhibits the religation of Top1 cleavage complexes. Acceptors were generated from unlabeled substrates as shown in A. The unlabeled acceptors were then incubated with 10-fold excess of 3'-labeled, 23-mer donor strand containing either G or O6MG at the 5' terminus (X) for 5 s, 10 s, 0.5 min, 1 min, 3 min, 5 min, 10 min, or 30 min (Lanes 1–8, respectively). Reactions were stopped with 0.5% SDS (final concentrations). Right panel, quantitation of the gel.

 
Top1 Reactions.
DNA substrates (~50 fmol/reaction) were incubated with 5 ng of Top1 with or without CPT for the indicated times at 25°C in reaction buffer [10 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, and 15 µg/ml BSA]. Reactions were stopped by adding SDS (final concentration, 0.5%). For the reversal experiments, the SDS stop was preceded by the addition of 0.5 M NaCl. Kinetics of cleavage and religation were performed as described previously (13) . 3.3 volumes of Maxam Gilbert loading buffer (80% formamide, 0.01 M EDTA, 10 mM NaOH, 1 mg/ml xylene cyanol, and 1 mg/ml bromphenol blue) were added to reaction mixtures that were loaded on 16 or 20% denaturing polyacrylamide gels. Quantitations were performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Detection of Covalent Top1-DNA Complexes in Cells.
Top1-DNA adducts were isolated using the ICE Bioassay (13 , 28 , 29) . Briefly, 106 treated or untreated cells were pelleted and immediately lysed with 1 ml of 1% sarkosyl. After homogenization with a Dounce, cell lysates were gently layered on step gradients containing four different CsCl solutions (2 ml of each) of the following densities: 1.82, 1.72, 1.50, and 1.45 (29) . Tubes were centrifuged at 165,000 x g in a Beckman SW40 rotor for 24 h at 20°C. Half-ml fractions were collected from the bottom of the tubes. Aliquots of each fraction (100 µl) were diluted with an equal volume of 25 mM sodium phosphate buffer (pH 6.5) and applied to Immobilon-P membranes (Millipore, Bedford, MA) using a slot-blot vacuum manifold. Detection of Top1-DNA adducts was performed by Western blotting using the Top1 monoclonal antibody C21 obtained from Dr. Yung-Chi Cheng (Yale University, New Haven, CT) according to standard procedures.

Yeast Strains and Drug Sensitivity Determination.
The yeast strains used in this study are derivatives of CH335 (30) . CH335 was converted to leu2- by one-step gene disruption. A Top1- derivative of CH335leu was constructed by one-step gene disruption. The resulting strain is termed CH335top1-. Strain CH335leu was transfected with either yCP50 or pGALhTOP1. The strain carrying yCP50 served as a vector control, whereas pGALhTOP1 expresses human TOP1 under the control of the yeast GAL1 promoter (31) . Drug sensitivity determinations in yeast cells were performed as described previously (32) with the following modifications. Cells were pregrown in synthetic complete medium without uracil, with galactose as a carbon source (SC-ura/GAL). After overnight growth, cells were diluted to 2 x 106 cells/ml in fresh SC-ura/GAL, and then appropriate concentrations MNNG were added. Cells were incubated for 4 h at 30° with shaking; then aliquots were removed, and diluted samples were plated to synthetic complete agar lacking uracil, with glucose as carbon source. Survival is expressed relative to the number of viable colonies at the time of drug addition. For the comparison of the sensitivity of top1- and TOP1+ cells, the appropriate strains were grown in yeast extract/peptone/dextrose/adenine medium as described previously (32) , exposed to MNNG for 2–4 h, and then plated to yeast extract/peptone/dextrose/adenine agar to determine surviving percentages.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
O6MG Induces Top1-DNA Cleavage Complexes.
We first investigated the effects of O6MG on purified human Top1 using oligonucleotides containing a single Top1 cleavage site (Fig. 1Citation A, arrowhead). Guanine modifications were introduced at the +1 position immediately downstream to the Top1 site (Fig. 1A)Citation . Base pairing of O6MG with C results in the suppression of one hydrogen bond, whereas 6TG can still form three hydrogen bonds as in normal G:C base pairing (Fig. 1BCitation ; Ref. 33 ). As shown in Fig. 2Citation A, incorporation of O6MG at the +1 position of the Top1 cleavage site resulted in an 8–10-fold increase of cleavage complexes in the absence of camptothecin (Fig. 2Citation A, compare Lanes 2 and 7). A comparable increase was observed when O6MG was base paired to T (data not shown). By contrast, incorporation of 6TG that retains all hydrogen bonds had no effect on Top1 activity (Fig. 2B)Citation . We tested whether incorporation of O6MG at the +1 position could affect CPT activity. As shown in Fig. 2Citation A, the presence of O6MG did not prevent further increase of Top1 cleavage complexes by CPT (Fig. 2Citation A, compare Lanes 7 and 9).

Addition of salt to the Top1 reaction shifts the cleavage/religation equilibrium toward religation, probably because it prevents binding of the enzyme to the DNA once religation has taken place (3 , 34) . Under such conditions, the effect of CPT is reversed (Fig. 2Citation A, Lanes 4 and 5). Top1 trapping by O6MG in the absence of drug was also reversed in 0.5 M NaCl (Fig. 2Citation A, compare Lanes 7 and 8). Noticeably, reversal was only partial in the presence of CPT (Fig. 2Citation A, compare Lanes 9 and 10). Together, these data demonstrate that presence of O6MG immediately downstream from a Top1 cleavage site can enhance the formation of reversible Top1 cleavage complexes.

O6MG Incorporation Inhibits Religation of Top1 Cleavage Complexes and Induces Top1-mediated Cleavage.
We then investigated the mechanism by which O6MG incorporation enhances Top1 cleavage complexes. For this purpose, we used different oligonucleotide substrates to analyze separately the effects of O6MG on the induction and religation of Top1 cleavage complexes.

We first measured the effects of O6MG on the induction of the Top1-mediated cleavage using a "suicide" substrate (Fig. 3A)Citation . This type of substrate is generally used (13 , 35, 36, 37) to directly evaluate cleavage rates, assuming that the 3' end-labeled 7-mer product that is released cannot be religated because of a complete loss of annealing after cleavage (Fig. 3Citation A, compare Lanes R and Top1 in the right panel). Kinetics of cleavage revealed an ~150-fold increase in the rate of cleavage when O6MG was incorporated at the +1 position relative to the Top1 site. This enhancement was not accompanied by an increase of binding of the Top1 to DNA (data not shown).

We also investigated the effect of O6MG on the religation step of the Top1 cleavage/religation reaction. Kinetics experiments were performed using the donor-acceptor system as described in previous studies (13 , 35 , 37) . Briefly, acceptors are generated from unlabeled suicide substrates (Fig. 3A)Citation and a 10-fold excess of the 3' end-labeled complementary strand bearing either a G or an O6MG at the 5'-end (called donor) is added to the mixture. The amount of religated product (37-mer) is then measured as a function of time (Fig. 3B)Citation . We estimated an ~6-fold decrease in the initial rate of Top1-mediated religation when O6MG was incorporated at the +1 position. Thus, O6MG at the +1 position inhibits the religation step of the Top1 reaction. Together, these data show that Top1 trapping by O6MG incorporation at the +1 position of a Top1 cleavage site is attributable to a dual effect: induction of cleavage and an inhibition of religation.

Induction of Top1 Cleavage Complexes in AGT-deficient and AGT-transfected CHO Cells after MNNG Treatment.
We next investigated whether Top1 could be trapped by O6MG in cellular DNA. For this purpose, we measured Top1 cleavage complexes in CHO cells after MNNG treatment using the ICE assay (13 , 28) . CHO cells have very little or no AGT activity (27) . In the CHO/pCMV control cells, treatment with 1 µg/ml MNNG induced the formation of Top1 cleavage complexes as seen in the DNA-containing fractions 7 to 10 (Fig. 4Citation , left panels). Top1 trapping reached an optimum after 30 min of treatment and was reduced after 3 h. Conversely, there was no increase in Top1 complexes in MNNG-treated CHO/pCMV-AGT cells in which AGT activity was restored by transfection of the human repair enzyme (Fig. 4Citation , right panel). These results demonstrate that Top1 trapping by MNNG treatment is linked to the formation of O6MG, because the removal of the alkyl group by the AGT enzyme abrogated the formation of the Top1-DNA complexes.



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  		Fig. 4. Top1 cleavage complexes are induced in AGT-deficient but not in AGT-expressing CHO cells after MNNG treatment. CHO and CHO/AGT cells were treated with 1 µg/ml MNNG for the indicated times at 37°C. Approximately 106 cells were lysed in 1% sarkosyl and submitted to the ICE assay (see "Materials and Methods"). Fractions were collected from the bottom of the gradients and are indicated by numbers. Aliquots were blotted using the Top1 C21 monoclonal antibody.

 
Overexpressing Top1 in Yeast Sensitizes Cells to MNNG.
To test whether Top1 trapping by O6MG could play a role in the cytotoxicity of methylating agents, we measured the cytotoxicity of MNNG in yeast strains with different levels of Top1 expression (Fig. 5)Citation . Viability of cells was determined after plating MNNG-treated or untreated cells onto agar plates. Survival is expressed relative to the number of viable colonies at the time of drug addition. As shown in Fig. 5Citation , cells that overexpressed the human Top1 were significantly more sensitive to MNNG for concentrations <3 µg/ml (with a P = 0.0251 when 1.67 µg/ml of MNNG was used). Conversely, Top1 knockout yeasts showed the same sensitivity to MNNG as the wild-type strains. Altogether, these results suggest that Top1 poisoning is a cytotoxic mechanism for O6MG incorporation in the DNA of cells treated with monofunctional alkylating agents such as MNNG.



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  		Fig. 5. Cytotoxicity of MNNG in yeast. Control yeast ({square}) or yeast overexpressing Gal1-human Top1 ({circ}) were treated with increasing concentrations of MNNG for 4 h at 30°C. Survival is expressed relative to the number of viable colonies at the time of drug addition. Values are the means of three independent experiments; bars, SD.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
The present study demonstrates that incorporation of O6MG in DNA can poison Top1. This finding is in contrast to the modest effect of O6MG that was reported recently on Top2 (38) and suggests that Top1 and Top2 are differently affected by distortions of the DNA duplex. CPT and the DNA modifications studied thus far inhibit Top1 by inhibiting the religation step of the Top1-mediated DNA cleavage/religation equilibrium reaction (reviewed in Ref. 12 ); 13 , 26 , 39, 40, 41 ). In this study, we demonstrate that insertion of O6MG in DNA increases the cleavage rate of the Top1 reaction. We also found that enhancement of Top1 cleavage complexes by O6MG was attributable to inhibition of the religation step. This is probably because O6MG Watson-Crick base pairing is altered, such as in the case of base mismatches and abasic sites that are very efficient at slowing the religation of the Top1 cleavage complexes (39) . When the O6 position of guanine is alkylated, the N1 position is changed from a hydrogen bond donor to hydrogen bond acceptor. At physiological pH, the N1 is not protonated, and there is a suppression of the specific hydrogen bonding of O6MG to cytosine, as shown by nuclear magnetic resonance and crystal structure studies (42, 43, 44) , which favors base pairing with thymine during DNA replication (45) . Although base pairing of O6MG with C is more stable than with T (46) , the present study shows that both duplexes are able to enhance Top1 cleavage complexes in vitro (Fig. 2Citation and data not shown). Conversely, 6TG:C-containing duplex that retains normal base pairing did not affect Top1 cleavage complex formation. Together, these data confirm the importance of hydrogen bonding and subsequent alignment of the DNA 5'-hydroxyl with the tyrosyl phosphodiester bond for optimum religation and catalytic activity of Top1.

MNNG is relatively efficient at methylating the O6 position of guanine (47) . Using the ICE assay, we were able to detect the formation of Top1-DNA cleavage complexes in CHO cells treated with MNNG. CHO cells are constitutively lacking the AGT repair enzyme that specifically removes O6MG adducts. In CHO cells transfected with the human AGT, however, there was no induction of Top1 cleavage complexes, suggesting that Top1 trapping is prevented by the removal of the methyl group by AGT as O6MG adducts are formed. The absence of cleavage complexes in CHO cells expressing AGT may indicate that other DNA adducts, such as 7-methylguanine and 3-methyladenine that are formed in larger amounts than O6MG, may not function as Top1 poisons. It should be noted that we did not measure the persistence of these adducts, and that these adducts are subject to DNA repair pathways, which, unlike AGT, are not deficient in the CHO cells used. Although alkylation at the O6 position is known to play a major role in the mutagenicity and the carcinogenicity of methylating agents, little is known concerning the mechanism by which presence of O6MG in DNA leads to cell death. It has been suggested previously that cell killing by alkylating agents could be attributable to futile attempts of removing mismatched bases incorporated in the newly synthesized DNA by the mismatch repair system during replication and/or to the recognition of the damaged bases by the mismatch repair proteins directly signaling cell cycle arrest and apoptosis (48) . On the basis of our data, we propose that increased Top1 cleavage at sites of O6MG adducts could also play a role in the cytotoxicity of methylating agents. We show that yeast overexpressing human Top1 is significantly more sensitive to MNNG than the corresponding wild-type cells (see Fig. 5Citation ). Similar results were also obtained when the yeast cells overexpressed yeast Top1 (data not shown). This observation is consistent with the biochemical data indicating that Top1 can be trapped by O6MG in vitro. However, yeast cells completely lacking Top1 had the same sensitivity to MNNG as cells expressing wild-type levels of the enzyme. Certainly, MNNG is capable of killing cells by pathways that do not depend on Top1, which explains the absence of differential cytotoxicity at high-dose MNNG for the normal versus the human Top1-overexpressing yeast (Fig. 5)Citation . However, we suggest that eukaryotic cells likely have pathways that can prevent Top1-mediated cleavage at sites of DNA damage. One such pathway could be targeted degradation of Top1 or a reversible modification, such as phosphorylation. Although cells can deal with normal levels of Top1, overexpression of the enzyme saturates the cells ability to prevent enzyme cleavage at sites of DNA damage, ultimately increasing cellular sensitivity to alkylated bases. Together, our results suggest that in the context of high level of Top1 expression, such as in the case of various tumor types (49) , cells possess an efficient mechanism for MNNG-induced killing involving Top1 trapping by alkylated bases.

Interestingly, we also found that the presence of O6MG further stabilizes CPT-induced Top1 cleavage complexes (Fig. 2A)Citation . CPT binding might further destabilize the alignment of the 5'-hydroxyl of the cleaved strand downstream from the cleavage site and render religation even more difficult. This effect is similar to what we observed when nicks were present downstream from the Top1 cleavage site (positions +4 to +6; Ref. 26 ). Top1 trapping by nicks in the DNA has been proposed as a potential explanation for the synergism between ionizing radiation and CPT (28 , 50) . Such a synergism was also reported at high levels of cytotoxicity between topotecan and various alkylating agents (51) . Recently, treatment with the AGT inhibitor O6-benzylguanine has been shown to sensitize cells to various Top1 inhibitors (52) . Such a potentiation could be explained by the increase of O6MG adducts, leading to enhanced Top1 trapping and further CPT-induced irreversible DNA breaks. The presence of Top1 cleavage complexes originating from O6MG after exposure to alkylating agents may also contribute to the increase in sister chromatin exchanges and other chromatin aberrations that occurs in cells exposed to alkylating agents.

Altogether, these results suggest a role for Top I 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.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grants CA52814 and CA21765 (to J. L. W. and J. L. N.) from the National Cancer Institute and by funds from the American Lebanese Syrian Associated Charities. Back

2 To whom requests for reprints should be addressed, at Laboratory of Molecular Pharmacology, National Cancer Institute, NIH, Building 37, Room 4E28, NIH, Bethesda, MD 20892-4255. Phone: (301) 496-5944; Fax: (301) 402-0752; E-mail: pommier{at}nih.gov Back

3 The abbreviations used are: Top1, DNA topoisomerase I; O6MG, O6-methylguanine; AGT, O6-alkylguanine-DNA alkyltransferase; CPT, camptothecin; CHO, Chinese hamster ovary; ICE, in vivo complex of enzyme; 6TG, 6-thioguanine; MNNG, 1-methyl-3-nitro-1-nitrosoguanidine. Back

Received 6/30/00. Accepted 11/13/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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