[CANCERRESEARCH56, 5571-5575, December 15, 1996]

Advances in Brief

Resistance of the Human 06-Alkylguanine-DNA Alkyltransferase Containing

Arginine at Codon 160 to Inactivation by 06-Benzylguanine'

Suvarchala Edara, Sreenivas Kanugula, Karma Goodtzova, and Anthony E. Peg@Departments of Cellular and Molecular Physiology and Pharmacology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey,Pennsylvania 17033

Abstract

Inactivation of 06-alkylguanine-DNA alkyltransferase by 06-benzylguanine renders tumor cells more sensitive to kiffing by methylating andchloroethylating agents, and 06-benzylguanine is currently undergoingclinical trials for development as an agent to enhance chemotherapy. Ithas been reported recently that a polymorphism in the human 06-alkylguanine-DNA alkyltransferase gene exists, with about 15% of the populatlon studied having arginine at codon 160 instead of glycine (Y. Imai et

aL, Carcinogenesis (Lond.), 16: 2441—2445,1995). We have studied theeffects of mutations of this glycine to arginine, tryptophan, or alanine onthe interaction of human alkyltransferase with 06-benzylguanine usingdirect determination of the amount of activity remaining after incubationwith various concentrations of the inhibitor and measurement of the rate

of production of [8-3Hlguanine from 06-benzyl[8-3H]guanine as assays.These mutations had little effect on the alkyltransferase activity in repairlag O@-methylguaaine in methylated DNA. Alteration of glycine 160 totryptophan or alanine slightly increased the sensitivity to 06-benzylguamae (by up to 4-fold). However, alteration of glycine 160 to argininedrastically reduced the inactivation by O@S@benzylguaninewith at least a20-fold increase in the EDan value and a similar reduction in the produclion of guanine whether inactivation was carried out in the absence orpresence of DNA. These results raise the possibility that a subpopulationof patients may be resistant to O@-benzylguanineand that higher doses oradditional alkyltransferase inhibitors capable of inactivating this form ofthe alkyltransferase will be necessary.

Introduction

It is now well established that the presence of AGT3(EC 2. 1.1.63) is an important factor in imparting resistance to thekilling of tumor cells by methylating agents such as temozolomideand dacarbazine and by chloroethylating agents such as BCNUand 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (1, 2). One possible approach to overcoming thisresistance would be to inactivate the alkyltransferase protein, and06-benzylguanine has been developed as an inhibitor for thispurpose (3). Treatment of nude mice carrying human tumor xenografts with 06-benzylguanine has been shown to improve thetherapeutic index of BCNU at equitoxic doses (4—6)but it remainsto be seen whether these results can be duplicated in humanpatients. Phase I clinical trials are currently underway to determinean effective dose of 06-benzylguanine for alkyltransferase mactivation and the maximum tolerated dose of 06-benzylguanine andBCNU.

Although human AGT has been reported to be very sensitive to06-benzylguanine with an ED50 value of 0.2 @tM(3), the equivalentproteins from a number of other species are less sensitive despitesubstantial sequence similarity (7). There is a particularly strikingdifference with the Escherichia coli Ada and the yeast alkyltransferases which are not inhibited by 1 m@i. This difference is at least

partially explained by steric factors which limit the size of the activesite pocket in these proteins, thus excluding 06-benzylguanine. Mutations to increase the size of this pocket by introducing a prolineresidue present at position 140 in mammalian AGTs and by removinga bulky tryptophan residue allow the Ada-C protein to react with06-benzylguanine (8). However, the mutated Ada-C protein is stillsubstantially less sensitive to 06-benzylguanine than the human AGT

and other residues must also contribute to the ability to react well withthis free base.

Point mutations in the human AGT sequence at proline 138, proline140, and glycine 156 (9, 10) increase the ED50 of O@-benzylguanineby factors of 10, 20, and 240, respectively. Also, the mouse AGT,which is quite similar to the human AGT in amino acid sequence andcontains all of these residues, has been reported to be 4—5-fold(I 1) or12-fold (12) less sensitive to inactivation. These results suggest thatvariants of the human AGT having a lowered sensitivity to O6@benzylguanine may exist and be selected for during exposure to this

drug plus an alkylating agent. Such variants might be produced by themutagenic action of alkylating agents or exist naturally due to poly

morphism in the alkyltransferase gene.It is unlikely that all of the positions at which the AGT can be

altered to modify sensitivity to 06-benzylguanine have yet beenidentified, and many of the residues forming the pocket containing thecysteine acceptor site are likely candidates for such sites. It was

therefore of considerable interest that a polymorphism of codon 160was reported in which about 15% (6/40) of the subjects tested hadarginine substituted for glycine at this position (13). Although it wasreported that this alteration had little or no effect on the activity of the

alkyltransferase (when this was expressed as a bacterial fusion proteinwith glutathione S-transferase), the assays were conducted only for theability to repair methylated DNA (13). In the experiments describedbelow, we have examined the effect of alterations at codon 160 on theability to react with 06-benzylguanine. Replacement of glycine 160with arginine renders the protein much less reactive toward this drug,

whereas replacement with tryptophan or alanine increases reactivity.

Materials and MethodsReceived 8/29/96; accepted 10/29/96.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported by Grants CA-71976 and CA-57725 from the National

Cancer Institute.2 To whom requests for reprints should be addressed, at Children's Hospital, Milton S.

Hershey Medical Center, Penn State College of Medicine, 500 University Drive, Hershey,PA. 17033-0850.

3 The abbreviations used are: AGT, O@-alkylguanine-DNA alkyltransferase; BCNU,

l,3-bis(2-chloroethyl)-l-nitrosourea.

Materials. Oligodeoxynucleotides were made in the Macromolecular CoreFacility, Hershey Medical Center, by using a Milligen 7500 DNA synthesizer.

GWRIO9 cells (14) were kindly provided by Dr. L. Samson (Department ofMolecular and Cellular Toxicology, Harvard School of Public Health, Boston,MA). Restriction enzymes were purchased from Life Technologies, Inc.(Gaithersburg, MD) and New England Biolabs (Beverly, MA). The pQE-30

plasmid was obtained from Qiagen (Chatsworth, CA) and the Talon Metal

Affinity Resin was purchased from Clontech (Palo Alto, CA). Ampicillin,

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RESISTANCE TO O@-BENZYLGUAN!NE

kanamycin, isopropyl j3-c--thiogalactopyranoside, hemocyanin, and most other

biochemicals were purchased from Sigma (St. Louis, MO). N-[3H]methyl-N-nitrosourea (5.9 mCi/mmol) was obtained from Amersham Corporation (Arlington Heights, IL). Pfu DNA polymerase was purchased from Stratagene (La

Jolla, CA). O@-Benzylguanine, synthesized as described (3), was generouslyprovided by Dr. R. C. Moschel (ABL-Basic Research Program, National

Cancer Institute-Frederick Cancer Research and Development Center, Fredcrick, MD). O'@-Bearyl[8-3H}guanine(0.34 mCi/mmol) was prepared by catalytic tritium exchange of O@-benzy1guanine with tritiated water by Amersham

Corporation and was purified as described previously (7).Expression and Purification of AGT and Mutants G16OA, G16OW, and

G161A. Homogeneous preparations of recombinant human AGT were obtamed by expressing the protein from the pINAGT plasmid in GWR1O9 cellsas described previously (7, 9, 10). This plasmid contains the human AGTsequence in the E. coil expression vector pINIII-A3(Ipp―5). The means ofconstruction changes the amino terminal sequence by the addition of fiveamino acids, giving a sequence MKGGIL—in place of M—.The G16OA,G16OW, and G16IA AGT mutant proteins were prepared in the same way. Themutations were introduced into pINAGT by PCR as described previously (9,10) using the following primers (with the altered codon sequence underlined):for G16OA, 5'-GGCAACTACTCCGCAGGACTGGCC-3'; for G16OW, 5'-

GGCAACTACTCCTGGGGACTGGCC-3'; and for G161A, 5'-GGCAACTACTCCGGAGCACTGGCC-3'.

Construction of pQE3O-hAGT Plasmid for Expression and Purificationof Wild-Type AGT and Mutant G16ORProtein. The G16ORmutantAGTand a control AGT preparation were expressed and purified to homogeneityusing the pQE3O vector to place a (His)6 tag at the amino terminus of the

protein, allowing purification by immobilized metal affinity chromatography.The plasmid construction adds the 12-aminoacid sequence MRGS(H)6GS—tothe amino terminus of the protein.

The coding region of human AGT protein flanked by BamHI and KpnIsites was amplified by PCR using pGEMAGT (10) as template DNA

and the oligonucleotides 5'-GGCGATGGGGGATCCATGGACAAGGATTGT-3' with BamHI site (underlined) preceding the start codon (italics) atthe N-terminal end and 5'-GACTCTAGAGGTACCACTCAGTFTCGG-3'with a KpnI site (underlined) following the stop codon (italics) at theC-terminal end. The PCR was done using Pfu polymerase under thefollowing conditions: initial denaturation for 2 mm at 92°C,20 cycles ofdenaturation (1 mm at 92°C), annealing (1 mm at 50°C),and extension (1mm at 72°C)followed by a final extension at 72°Cfor 5 mm. The 654-bpPCR productwas gel purified and digested with BamHIand KpnIenzymesand ligated into pQE3O vector that had been digested with the sameenzymes. The ligation mixture was then electroporated into JM1O9 cells.The resulting plasmid, pQE3O-hAGT (Fig. 1), was used for expression andpurification of control AGT protein with the (His)6 tag as described below.

The 0 160R mutant AGT was produced with the same vector using athree-round PCR protocol as follows. The mutation was introduced by afirst-round PCR using as primers, 5'-GGCAACTACTCCAGAGGGCTAGCCGTG-3' (mismatch underlined) to introduce the mutation and oligo A[5'-CTACCTCAAGACTCCAG-3' matching pQE3O-hAGT plasmid fromnucleotide 861 to 845 (see Fig. 1)]. The PCR reaction was done using Pfu

polymerase with pQE3O-hAGT plasmid as template under the followingconditions: initial denaturation for 2 mm at 92°C, 15 cycles of denaturation

(1 mm at 92°C),annealing (1 mm at 50°C),and extension (1 mm at 72°C)followed by a final extension at 72°Cfor S mm. Second-round PCR withpQE3O-hAGT plasmid DNA as template was then carried out using asprimers, oligo B [5'-GATTCAATTGTGAGCGG-3' matching nucleotide55 to nucleotide 7 1 in pQE3O-hAGT plasmidi and the 246-bp product(excised from a 1% agarose gel) from the first round PCR. The PCRreaction was carried out under identical conditions as described above for

the first round PCR, except that the annealing temperature was maintainedat 46°C.The product from the second round PCR was used as template andfurther amplified under the same conditions as the first round PCR, usingoligos A and B. This third round PCR product was ethanol precipitated,

digested with BamHI and KpnI enzymes, and ligated into pQE3O vectorwhich had been digested with the same enzymes to form pQE3O-G16OR.

The entire protein coding sequences of pQE3O-hAGT and pQE3O-G16ORwere verified by sequencing to ensure that no additional mutations wereintroduced during the constructions.

@I1

1433

111586Pvu I

I 775Asel:

I 1778I 2013

Fig. 1. Plasmid used for the production of (His)6-tagged AGT and G16OR mutant.

JM1O9 cells transformed with pQE3O-hAGT or pQE3O-G16OR were grownin a 1-liter culture inoculated with an overnight grown culture at a 1:50

dilution. At a cell density equivalent to A@ of 0.5, 0.3 mai isopropyl @3-o-

thiogalactopyranoside, was added and the cells were harvested 4 h later. Thepellet was suspended in 30 ml of 20 nmi Tris-HCI (pH 8.0) and 250 mi@iNaCland disrupted using a French Press. After centrifugation at 17,000 X g for 45mm at 4°Cto remove cell debris, the supernatant was applied to a 2-mi columnof Talon IMAC resin (Clontech) equilibrated with 20 mrviTris-HC1(pH 8.0)and 250 mMNaC1,and the column was washed with this buffer containing 10mM imidazole. The AGT proteIn was then eluted using 200 mx@imidazole, and

the fractions found to contain the AGT using SDS-PAGE were pooled anddialyzed immediately against 50 mMTris-HCI (pH 7.6), 250 mittNaCI, 5 mMDTT, and 0.1 mM EDTA. The yield of protein was about 8 mg/liter of culture,and the purity was >90% as judged by SDS-PAGE.

Assay of Inactivation by O@-Benzylguanine. The purified AGT proteinand mutants were incubated with O@-benzylguaninein 0.5 ml of 50 mt@tTris-HCI (pH 7.5), 0.1 mist EDTA, and 5.0 mM DTT in the presence or absenceofcalfthymus DNA as indicated for 30 mm at 37°C.When DNA was omitted,50 i@gof hemocyanin were added instead. (Under these conditions with eitherDNA or hemocyanin added, the AGT activity was completely stable with <5%loss ofactivity in the 30-mm incubation). The residual alkyltransferase activitywas then determined by a 30-rain incubation with a 3H-labeled methylatedDNA substrate which had been methylated by reaction with N-[3H]methyl-N-nitrosourea essentially as described (3, 15). The results were expressed as thepercentage of the alkyltransferase activity remaining. The graphs of activityremaining against inhibitor concentration were used to calculate an ED50 valuerepresenting the amount of inhibitor needed to produce a 50% loss of activity.

Reaction of AGT with O@-Benzyl[8-3H]guanine.Measurements of[8-3H]guanineformation from 06-benzyl[8-3H}guaninewere carried out usingan assay mixtureconsistingof 50 nmiTris-HCI(pH 7.5), 0.1 mt@iEDTA, and5 mM Dli' in a volume of 0.25 ml in the presence or absence of DNA asindicated. The reaction was incubated for up to 2 h at 37°C,and at varioustimes, aliquots were removed and the reaction stopped by the addition of0.6—0.8ml of the same buffer containing 0.2 mMof guanine and 0.2 mistofO@-benzylguathne. These aliquots were then separated by reversed-phase high

pressure liquid chromatography to determine the amount of guanine formed(16). The results were expressed as cpm of [8-3H]guanineformed per @tgofprotein per mm. The amounts of protein and times of incubation used weresuch that [8-3H]guanine production was proportional to both the time ofincubation and to the amount of protein added.

Results

Human AGT and mutants at glycine 160 and glycine 161 wereexpressed in E. coli from both the pIN and the pQE3O vectors as

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pQE3O-hAGT4076 bp

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:@100

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Proteinp!N-AGT1.000.40.2pIN-G16OA1.080.1ND―pIN-G16OW0.960.1ND―pIN-G161A0.411.0ND―pQE3O-AGT1.000.40.2PQE3O-GI6OR0.519.04.0a

ND, not determined.

5 10 15pM 06-benzylguanine

RESISTANCE TO O6-BENZYLGUANINE

indicated in Table 1 and purified to homogeneity. There was nosignificant difference in the repair of methylated DNA or in theinactivation by 06-benzylguanine between the control preparationsisolated using the pIN and PQE3O systems, indicating that the additionof the (His)6 tag to the amino terminus of the protein by the pQE3Ovector to facilitate purification did not affect the properties of theprotein.

The mutations at glycine 160 and glycine 161 did not affect thelevel of expression and the specific activity of the purified preparations were all similar, indicating that these mutations do not destabilize the protein. They also had little or no effect on the ability of theprotein to repair methylated DNA. The rate of repair of doublestranded methylated DNA by the AGT at 37°Cis very fast and it isvery difficult to measure accurately but, as shown in Table 1, comparison of the repair of methylated DNA by very small amounts ofprotein indicated that mutants G161A and G16OR reduced the rate byat most 2-fold and mutations G16OA and G16OW had no effect at allon repair of methylated DNA. Our results with the G16OR mutantconfirm the previous report that this alteration had little or no effect onthe activity of the AGT (13).

Comparison of the mutants of AGT in which glycine 160 wasaltered to a tryptophan or an alanine residue showed that thesemutations increased the sensitivity of the protein to inactivation byO@-benzylguathne (Fig. 2A). The ED50 value was reduced by about afactor of 4 by these mutations from 0.4 to 0.1 @M(Table 1). Incontrast, the conversion of glycine 160 to an arginine residue renderedthe protein much more resistant than the control AGT (Fig. 2B),increasing the ED50 value by >20-fold to 9 @tM(Table 1). The

alteration of glycine 161 to alanine also increased the ED50 value forinactivation by 06-benzylguanine but only slightly, by 2.5-fold to 1p@M.

Previous studies have shown that the reaction of AGT with @6

benzylguanine is increased in the presence of DNA (16) and it ispossible that in vivo, the inactivation of AGT is enhanced in this way.However, as shown in Fig. 2C, the addition of DNA did not alter thelarge difference between the control and the G16OR mutant AGT,although the ED50 values were lowered to 0.2 and 4 p.M,respectively,under these conditions.

The inactivation of AGT with 06-benzylguanine is caused by theprotein recognizing the 06-benzylguanine as a substrate and converting it to guanine at the same time forming S-benzylcysteine at theactive site (7, 17). The ability of AGTs to react with 06-benzylguanine can therefore be studied directly by measuring the initial rate offormation of guanine from 06-benzylguanine under conditions wherethe inhibitor is in excess. When these studies were carried out with thecontrol and the mutant AGTs, the results confirmed that mutantsG16OA and 0160W reacted slightly more rapidly with 06-benzylguanine than the control AGT and that mutants G161A and G16ORreacted considerably more slowly (Fig. 3A). The difference wasparticularly striking for mutant G16OR where guanine production was

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020

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Fig. 2. Inactivation of control and mutant AGTs by O@-benrylguanine. A and B, resultsof incubation with O°-benzylguanine in the absence of DNA and C, results of incubationwith O@-benzylguanine in the presence of 50 @gof calf thymus DNA. A, comparesmutants G16OWand GI6OA with control AGT. B and C, compare mutants GI6OR andGI61A with control AGT.

reduced by >90% irrespective of whether the assay was carried out inthe presence (Fig. 3B) or absence of DNA (Fig. 3A). The presence ofDNA increased guanine formation by all of the AGTs tested but theincrease was less for mutants G16OA and G16OW and the DNAaddition abolished the difference between these mutants and thecontrol AGT (Fig. 3).

Table I Inactivation of recombinant control and mutant alkyltransferases by06-benzylguanine

Discussion

Our results show clearly that the presence of the basic arginineresidue at position 160 in place of the neutral glycine found in thepredominant form of human AGT substantially reduces the ability ofthe protein to react rapidly with 06-benzylguanine. This reductioncannot be attributed to a general decrease in the efficiency with whichthe protein carries out alkyl group transfer as repair of 06-methylguanine in DNA was reduced by at most a factor of 2 (Table 1). It istherefore likely to result from a decreased affinity of the protein forthe 06-benzylguanine pseudosubstrate.

Inactivation by 06-benzylguanine(ED@, @M)

Relative rate of repairof methylated DNA Assayed + hemocyanin Assayed + DNA

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RESISTANCE TO o°-BENzYLcIUANINE

Ada-C protein described above, there is a more likely explanation forthestrikingeffectof replacingglycine'@°withargimne.

This is that the charged nature of the side chain is itself responsiblefor decreasing the affinity of binding of 06-benzylguanine. There isgood evidence from comparisons of potency of inhibition by various06-benzylguanine analogues that this binding is facilitated by hydrophobic interactions between the protein and the inhibitor (2, 2 1—23).The insertion of a charged argiine residue into this environmentcould therefore greatly weaken the binding of 06-benzylguanine. Thisexplanation would also be consistent with the finding that insertion ofan alanine or tryptophan at position 160 did not reduce the interactionwith 06-benzylguanine but, in fact, slightly increased it. The fact thatthe very bulky tryptophan side chain can be tolerated suggests thatadequate space exists at this position to allow binding. The increasedsensitivity of the G16OA and 0160W mutants could be due to in

creased hydrophobic interactions.The concept that the presence of an additional charged hydrophilic

residue may reduce the ability to react with 06-benzylguanine as afree base could also provide an explanation for the decreased sensi

tivity of the mouse AGT compared to the human and rat AGT (11,24). The mouse AGT has a charged histidine residue at the positionequivalent to residue 157 in the human AGT. This position is a neutralasparagine in the more sensitive human and rat AGTs.

Irrespective of the means by which the effect is brought about, thefinding that a naturally occurring variant of the human AGT with adecreased sensitivity to O@-benzylguathne exists must be taken intoaccount in the design and interpretation of clinical trials with this drug. It

is possible that a significant fraction of the population will show adecreased response to O@-benzylguanine. Several individuals who werehomozygous for the arginine 160 form of AGT were identified by Imaiet a!. (13). They also found some subjects who were heterozygous for theG16OR polymorphism. In such heterozygous patients, tumor cells cx

pressing the resistant variant of the AGT protein may be selected by thetreatment with O@-benzylgnanine plus an alkylating agent. Our resultstherefore place additional emphasis on the synthesis and design of addi

tional inactivators of AGT active against both the control and O@benzylguanine-resistant forms of the protein.

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Fig. 3. Rate of formation of guanine from O'@-benzylguanineby control and mutantAGTs. The rate of [8-3Hlguanine formation from O'@-benzyl[8-3Hlguanine was measuredas cpm of [8-3HJguanine formed per @sgprotein per mm as described in ‘@Materialsand

Methods.― A, results for assay in the absence of DNA and B, results for assays in the

presence of 50 @sgof calf thymus DNA. Results are shown as the means for assaysinvolving four or more estimations or as the mean for assays carried out in duplicate ortriplicate. Bars. SE.

Unfortunately, at present, little is known of the manner by which

06-benzylguanine binds in the active site pocket of AGT. The onlythree-dimensional crystal structure for a DNA repair alkyltransferaseis that of the Ada-C protein of E. coli (18, 19) and this protein does

not react at all with 06-benzylguanine (7, 20); therefore, it is notpossible to either cocrystallize the inhibitor and the protein or even tomake theoretical models of the possible interaction. However, it ishighly likely that the reason for the inability of Ada-C to react is dueto the very restricted space in the active site pocket in this protein.There is experimental support for this concept. Mutations to increasethis space by removing a bulky tryptophan side chain and by introducing a proline residue do make the Ada-C protein reactive to

06-benzylguanine (8). Conversely, replacing the proline residue in thesensitive human AGT protein confers resistance to 06-benzylguanine

(9).The amino acid at position 160 is likely to form part of the active

site pocket of AGT, and it is conceivable that the presence of a

charged side chain at this position leads to interactions with otherresidues that reduce the size and thus tend to exclude 06-benzylgua

nine. However, although such a steric limitation would both explainour results and be consistent with the arguments for resistance of the

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Acknowledgments

References

We thank Dr. R. C. Moschel for the synthesis of O@-bearylguanine.

I. Gerson, S. L., Liu, L., Phillips, W. P., Zaidi, N. H., Heist, A., Markowitz, S., andWilson, J. K. V. Drug resistance mediated by DNA repair: the paradigm of O@alkylguanine DNA alkyltransferase. Proc. Am. Assoc. Cancer Ret., 35: 699—700,1994.

2. Pegg, A. E., Dolan, M. E., and Moschel, R. C. Stnicture, function and inhibition ofO'@-alkylguanine-DNAalkyltransferase. Prog. Nucleic Acid Ret. Mol. Biol., 51:167—223,1995.

3. Dolan, M. E., Motchel, R. C., and Pegg, A. E. Depletion of mammalian 0°-alkylguanine-DNA alkyltransferase activity by 0°-benzylguanine provides a means to

evaluate the role of this protein in protection against carcinogenic and therapeuticalkylating agents. Proc. Nat]. Acad. Sci. USA, 87: 5368—5372,1990.

4. Friedman, H. S., Dolan, M. E., Motchel, R. C., Pegg, A. E., Felker, G. M., Rich, J.,Bigner, D. D., and Schold, S. C. Enhancement of nitrosourea activity in medulloblastoma and glioblastoma multiforme. J. Nail. Cancer Inst., 84: 1926—1931,1992.

5. Mitchell, R. B., Moschel, R. C., and Dolan, M. E. Effect of 0°-benzylguanineon thesensitivity of human tumor xenografts to l,3-bis(2-chloroethyl)-1-nitrosourea and onDNA interstrand cross-link formation. Cancer Ret., 52: 1171—1175, 1992.

6. Gerson, S. L., Zborowska. E., Norton, K., Gordon, N. H., and Willson, J. K. V.Synergistic efficacy of 0°-benzylguanineand BCNU in a human colon cancerxenograft completely resistant to BCNU alone. Biochem. Pharmacol., 45: 483—491,1993.

7. Pegg, A. E., Boosalis, M., Samson, L., Moschel, R. C., Byers, T. L., Swenn, K., andDolan, M. E. Mechanism of inactivation of human 0°-alkylguanine-DNA alkyltransferase by 0°-benzylguanine.Biochemistry, 32: 11998—12006,1993.

8. Crone, T. M.. Kanugula, S., and Pegg, A. E. Mutations in the Ada 0°-alkylguanineDNA alkyltransferase conferring sensitivity to inactivation by 0°-benzylguanineand2,4-diamino-6-benzyloxy-5-nitrosopyrimidine. Carcinogenesis (Lond.), 16: 1687—1692, 1995.

5574

a:<@ :@ ‘— °

I— 0 0@ (0(@ @2 (0 (0 <i (@@@ C')0

z z Z@ w C')@6. @a @a. 0. 0

0.

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RESISTANCE TO 0@-BENZYLGUANlNE

9. Crone, T. M., and Pegg, A. E. A single amino acid change in human 0°-alkylguanineDNA alkyltransferase decreasing sensitivity to inactivation by 0°-benzylguanine.Cancer Res., 53: 4750—4753, 1993.

10. Crone, T. M., Cioodtzova, K., Edara, S., and Pegg, A. E. Mutations in 0°-alkylguanine-DNA alkyltransferase imparting resistance to 0°-benzylguanine.Cancer Res.,54:6221—6227,1994.

11. Elder, R. H., Margison, G. P., and Rafferty, J. A. Differential inactivation ofmammalian and E. coli 0°-alkylguanine-DNA alkyltransferase by 0°-benzylguanine.Biochem. J., 298: 231—235,1994.

12. Liu, L., Lee, K., Wasan, E., and L., G. S. Differential sensitivity ofhuman and mousealkyltransferase to 0°-benzylguanineusing a transgenic model. Cancer Ret., 56:1880—1885,1996.

13. Imai, Y., Oda, H.. Nakatturu, Y., and Ishikawa, T. A polymorphism at codon 160of human 0°-methylguanine-DNAmethyltransferase gene in young patients withadulttypecancersand functionalassay.Carcinogenesis(Land.),16: 2441—2445,1995.

14. Rebeck, G. W., and Samson, L. Increased spontaneous mutation and alkylationsensitivity of Escherichia coli strains lacking the ogs 0°-methylguanineDNA repairmethyltransferase. J. Bacteriol., 173: 2068—2076, 1991.

15. Dolan, M. E., Mitchell, R. B., Mummert, C., Moschel, R. C., and Pegg, A. E. Effectof 0°-benzylguanineanalogues on sensitivity of human tumor cells to the cytotoxiceffects of alkylating agents. Cancer Ret., 51: 3367—3372,1991.

16. Goodizova, K., Crone, T., and Pegg, A. E. Activation of human 0°-alkylguanineDNA alkyltransferase by DNA. Biochemistry, 33: 8385—8390,1994.

17. Ciocco, 0. M., Pegg, A. E., Moschel, R. C., Chae, M-Y., McLaughlin, P. J.,Zagon, I. S., and Pegg, A. E. Specific labeling of 06-alkylguanine-DNA alkyl

transferase by reaction with 0°-(p-hydroxy[3HJmethylbenzyl)guanine. CancerRet., 55: 4085—4091,1995.

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1996;56:5571-5575. Cancer Res   Suvarchala Edara, Sreenivas Kanugula, Karina Goodtzova, et al.  

-Benzylguanine6OInactivation by Alkyltransferase Containing Arginine at Codon 160 to

-Alkylguanine-DNA6OResistance of the Human

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