[CANCER RESEARCH 49, 1899-1903, April 15, 1989]

The Role of O6-Alkylguanine DNA Alkyltransferase in Limiting Nitrosourea-induced Sister Chromatid Exchanges in Proliferating Human Lymphocytes1

Joan E. Trey2 and Stanton L. Gerson3

Division of Hematology Oncology, Department of Medicine, Cleveland Metropolitan General Hospital, Cleveland, Ohio 44109 [J.E.T.] and Hematology OncologyDivision, Department of Medicine, R. L. Ireland Cancer Center, University Hospitals of Cleveland, Case Western Reserve University School of Medicine,Cleveland, Ohio 44106 [S.L.G.]

ABSTRACT

Although induction of sister chromatid exchanges (SCEs) followingnitrosourea exposure may be greater during cell proliferation, the increasecould be offset by the action of the DNA repair protein O'-alkylguanine

DNA alkyltransferase (alkyltransferase). To evaluate these factors inresting and proliferating (phytohemagglutinin stimulated) human lymphocytes, we studied the effect of changes in alkyltransferase activity onl,3-bis(2-chloroethyl)-l-nitrosourea (B( M »-induced SCEs. Phytohe

magglutinin stimulation resulted in induction of alkyltransferase activity(5.9 ±0.3 units, resting, versus 9.2 ±0.2 units, proliferating). In bothresting and proliferating lymphocytes the alkyltransferase activity wasinactivated by 85-88% after an 18-h exposure to 0.5 HIMof the modifiedbase O'methylguanine (C^mGua). However, 48 h after removal of

í/iiKíiia,proliferating lymphocytes recovered alkyltransferase activitywhile resting cells did not. In the absence of O'mGua, both resting and

proliferating lymphocytes were equally sensitive to BON l -induced SCEs.Following inactivation of the alkyltransferase by O'mGua, B( M -in

duced SCEs were markedly increased, but the increase was much greaterin resting than proliferating cells, 4-fold vs. 2.6-fold at each dose ofBCNU (/' < 0.001). The factors providing partial protection against

B( N l -induced SCEs in proliferating lymphocytes appear to include theproliferation-dependent increase in alkyltransferase activity and the abil

ity of proliferating lymphocytes to rapidly recover alkyltransferase activity after its inactivation. Thus, the alkyltransferase appears to providean important mechanism of resistance to SCE induction in human lymphocytes.

INTRODUCTION

The ultimate biological effect of DNA-damaging chemicalsis the result of a dynamic interaction between the creation ofDNA damage and the ability of the cell to repair such damage.The capacity to induce SCEs4 has been shown to be a sensitive

and quantitative method to study DNA damage (1). Althoughthe precise mechanisms by which chemicals produce SCEsremain elusive, it is likely that SCE formation is not the resultof a specific DNA lesion but rather reflects the repair of varioustypes of DNA damage. This versatility has made SCE inductionin lymphocytes an important screening test to monitor theeffects of chemicals in human exposures.

SCE formation requires DNA synthesis (2). Therefore, onlyDNA damage which is not repaired and persists during DNAreplication can give rise to an SCE (3). Rapidly dividing cellsmay be more sensitive than quiescent cells to SCE induction

Received 7/15/88; revised 12/22/88; accepted 1/20/89.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by the American Cancer Society, Ohio Division, Cuyahoga CountyBranch, and Grants CA-07912, CA-43688, P30-CA-43703 from the NationalCancer Institute and ES-00134 from the National Institute of EnvironmentalHealth Sciences.

2To whom requests for reprints should be addressed, at Cleveland Metropol

itan General Hospital, Highland View Hospital, 3395 Scranton Road, Cleveland,OH 44109.

3Recipient of a Mallinckrodt Scholar Award.'The abbreviations used are: SCE, sister chromatid exchange; BCNU, 1,3-

bis(2-chloroethyl)-l-nitrosourea; BrdUrd, 5-bromo-2'-deoxyuridine; MNU, N-nitroso-Af-methylurea; O*mGua, O'-methylguanine; PHA, phytohemagglutinin;

PBS, phosphate buffered saline.

simply because they can not effect DNA repair prior to DNAreplication. In human lymphocytes, however, the effect of mi-togen stimulation on SCE induction by alkylating carcinogensappears to depend on the class of carcinogen (4-6), suggestingthat the increased sensitivity due to rapid proliferation may insome cases be offset by concurrent induction of DNA repairmechanisms. Although the levels of many lymphocyte DNArepair proteins are known to increase with mitogen stimulation(7-14), it is not known if these increases confer protection fromchemical-induced damage. SCE induction can be a powerful

tool to study this question because an SCE represents the resultof the interaction of DNA damage and the correspondingspecific DNA repair mechanism.

Nitrosoureas are potent inducers of SCEs (1). The extent ofSCE induction by nitrosoureas can be modulated by the abilityof the cell to repair alkylation at the O6 position of guanine(15-17). This is consistent with the early suggestion that O6-

alkylguanine lesions are important in the generation of SCEs(18, 19). O6-Alkylguanine lesions are specifically repaired bythe DNA repair protein, O'-alkylguanine DNA alkyltransferase

(alkyltransferase) (20, 21), which catalyzes the covalent transferof the alkyl group from guanine to a cysteine residue in theprotein. This restores guanine and results in irreversible inactivation of the alkyltransferase (21, 22). Because of the stoichi-ometric nature of this reaction, exposure to alkylating agentscan deplete cellular stores of the alkyltransferase (21) which arerenewed only by protein synthesis (21, 22). (T'-Alkylguanine

adducts can then persist and go on to form crosslinks or beconverted to point mutations at the time of DNA synthesis. Itis the presence of the persistent adduct or crosslink which isthought to give rise to an SCE (23).

The level of alkyltransferase and thus potential protectionfrom nitrosourea-induced SCEs is not uniform among humantissues. Liver has the highest levels of alkyltransferase activitywhile bone marrow myeloid precursors have the lowest (24).The alkyltransferase in human lymphocytes is approximatelytwice that in myeloid cells, but significantly less than that ofliver (24,25). Alkyltransferase activity can be induced in humanlymphocytes during in vitro mitogen stimulation (7, 8). Becauselymphocyte alkyltransferase is intermediate relative to othertissues and because it increases during proliferation the lymphocyte is a useful target cell to study SCE induction bynitrosoureas. Nitrosourea induced SCEs in lymphocytes thusserve as a sensitive marker of damage to other human tissuesas well as a model to gauge the modulating impact of proliferation induced changes in alkyltransferase activity.

The role of the alkyltransferase in SCE formation in lymphocytes has previously been studied by exposing cells to MNUwhich forms O'-methylguanine adducts thus depleting alkyl

transferase activity during the repair reaction (8). Under theseconditions prior exposure to MNU results in potentiation ofBCNU induced SCEs in both resting and proliferating lymphocytes suggesting that alkyltransferase depletion sensitizes thecells to BCNU (26). After MNU exposure alkyltransferase

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DNA REPAIR AND NITROSOUREA INDUCED SCEs IN LYMPHOCYTES

activity recovers within 48 h in proliferating lymphocytes butdoes not recover in resting cells (8). Thus, residual MNUadducts may remain unrepaired and contribute to SCE formation. It has recently been shown that the modified base O'mGua

will inactivate the alkyltransferase specifically without directlyinteracting with DNA (27-29). This provided us with a simpleexperimental system to study the role of alkyltransferase inregulating nitrosourea induced SCEs in resting and proliferating human lymphocytes.

MATERIALS AND METHODS

Chemicals. All chemicals were obtained from Sigma (St. Louis, MO)unless otherwise specified. Lyophilized PHA (HA-15, Burroughs-Wellcome, Dartford, England) was reconstituted in sterile PBS as describedby the manufacturer and was stored at -20°C. BCNU (obtained from

the Drug Synthesis Branch. Drug Therapeutics Branch, National Cancer Institute) was stored at —¿�20"C, and reconstituted in 95% ethanoland diluted to 0.01 M in PBS. O'mGua was synthesized by Dr. Paul

Howard, Department of Environmental Health Sciences, CWRUSchool of Medicine, using the method of Demple et al. (30). Thispreparation was contaminated by less that 0.5% 6-chloroguanine asdetermined by high-performance liquid chromatography, a concentration that has no effect on alkyltransferase activity or cell growth (27).O'mGua was dissolved in 0.01 N HCI, and neutralized to pH 7 with

NaOH at a final concentration of 0.01 M. Colcemid was obtained fromGIBCO (Grand Island, New York). BrdUrd (Calbiochem, San Diego,CA) was stored at 1 mg/ml in PBS at -20°C.Hoescht 33258 Dye wasreconstituted to 0.01 M in H2O, and stored at 4°Cin the dark. Gurr

buffer (pH 6.8) and Giemsa were obtained from Biomedicai Specialties(Los Angeles, CA). [3H]Thymidine (Du Pont-New England Nuclear,Boston, MA) (2.0 Ci/mmol) was stored at 4°C.Tissue culture reagents

were obtained from Hazelton Labs (Lenexa, KS), tissue culture flasksfrom Falcon Scientific (Lincoln, NJ), and fetal bovine serum wasobtained from Hyclone Labs (Logan, UT).

Isolation and Drug Exposure of Lymphocytes for AlkyltransferaseDetermination. Lymphocytes were isolated from normal donors overficoll hypaque gradients as previously described (25), and resuspendedat 5 x 10s cell/ml in RPMI 1640 tissue culture medium supplementedwith 15% fetal bovine serum, 25 mM 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid, 100 units/ml penicillin, and 100 Mg/ml streptomycin (tissue culture medium) (25). Lymphocytes were induced toproliferate by a 24-72-h culture in the presence of 1% PHA at 37°C,

5% CQi. Resting lymphocytes were isolated from the same donor onthe day of drug exposure. Cells were counted by passage through aCoulter Counter (Model 2M) after a 1 to 20 dilution into Centrimide(0.082 Mhexadecyltrimethyl ammonium bromide (Eastman Kodak Co.,Rochester, NY), 0.16 M NaCl, 2 mM EDTA, pH 5) to disperse cellaggregates.

To determine the effect of O6-methylguanine on alkyltransferase

activity, both resting and proliferating lymphocyte populations fromthe same donor were suspended at 5 x IO5 cells/ml in tissue culturemedium and exposed to O'mGua at concentrations of 0.01-1 mM for

24 h. To determine the time course of inactivation and recovery ofalkyltransferase activity, resting and PHA-stimulated lymphocytes wereexposed to 0.5 mM O'mGua for 24 h, washed in 50 ml PBS, andresuspended in fresh tissue culture media without O'mGua and recul-tured for 72 h. At appropriate times aliquots of 20 x IO6 cells were

removed for alkyltransferase determination.Alkyltransferase Activity. Cell extracts were prepared and samples

were taken for determination of protein and DNA content using previously described methods (24). O'-Alkylguanine DNA alkyltransferaseactivity in cell extracts was measured as removal of the methyl-3Hadduci from O6-[/w?f/i>'/-1H]guaninein methyl-3H DNA alkylated withyV-[mefA}7-1H]nitrosoureaas previously described (24, 25). One unit ofalkyltransferase was defined as removal of 1 fmol of O'-methylguanine

per microgram cellular DNA. This expression adjusts for changes inDNA content of the cell that occur during the cell cycle and duringDNA synthesis (24).

Cell Culture and Drug Exposures for SCE Determination. Peripheralblood lymphocytes present in whole blood cultures from normal donorswere used for sister chromatid exchange experiments. Whole blood wasdiluted 20-fold with tissue culture medium. 1% PHA was added for24-72 h to produce proliferating lymphocytes, whereas resting cellswere maintained by incubation without PHA, or were freshly harvestedfrom the same donor on the day of BCNU exposure. 0.5 mM O'mGuawas added to the culture media 18-24 h prior to BCNU exposure.Following a 2-h incubation with 0-20 MMBCNU at 37°C,the cells

were washed with PBS, and resuspended in fresh tissue culture mediacontaining 10 Mg/ml BrdUrd. 1% PHA was added to both resting andproliferating cell cultures to facilitate SCE analysis. The cultures wereprotected from the light and incubated at 37°C,5% CO2 for 48 h

(proliferating cells at the time of drug exposure) or 72 h (resting cellsat the time of drug exposure) before harvesting for SCE determination.

Sister Chromatid Exchange. Sister chromatid exchange analysis wasperformed by a modification of the method of Perry and Wolff (31,32). After drug exposures and culture in the presence of 10 Mg/mlBrdUrd for 48 or 72 h (as above), 0.1 Mg/ml colcemid was added forthe last 45 min of culture time. The cells were harvested and chromosome preparations made using standard cytogenetic methods, andstained with the fluorescence plus Giemsa method (31, 32). Briefly, thechromosomes were stained with 0.1 mM Hoescht 33258 Dye for 15min, then covered with Macllvaine's buffer (pH 8.0), exposed to light

from two black light bulbs at a height of 5 cm for 20 min while warmedto 50°C,then stained with 2% Giemsa in Gurr buffer (pH 6.8) (32).

The differential staining pattern which results from the incorporationof BrdUrd into the DNA of replicating cells can then be used toquantitate SCEs and to indicate the number of cell divisions a particularcell has undergone during the culture period. For each data point 25intact metaphases were scored for SCEs, and 100 metaphases werescored to calculate the replication index as follows: /?/ = (! x percentageof first division cells plus 2 x percentage of second division cells plus3 x percentage of third division cells)/100) (33). SCEs and replicationindices are reported as mean ±SEM, and statistical significance isdetermined using a paired / test comparing control and drug-exposedpopulations.

RESULTS

Inactivation of the Alkyltransferase by O'mGua. To evaluate

the role of alkyltransferase activity in modulating DNA damagein resting and proliferating cells we used the modified baseO'mGua to inactivate the alkyltransferase. To determine theoptimal dose of O'mGua needed to inactivate the alklytransfer-

ase, resting and proliferating lymphocytes from normal donorswere incubated for 24 h with increasing concentrations ofO6mGua from 0.01-1 mM and the residual alkyltransferase

activity was determined (Fig. 1). The alkyltransferase activitywas significantly higher in the proliferating cells (9.2 ±0.2

[o6mGua|M

10-3

Fig. 1. Inactivation of alkyltransferase activity by i/ni(.na in lymphocytes.Lymphocytes were cultured in the presence of 1% PHA for 72 h or freshlyharvested prior to culture in tissue culture media at 5 x id" ml. O'mGua was

added for 24 h at concentrations from 0.01 to 1 mM prior to alkyltransferaseassay. Data represents mean ±SEM of four donors per point. Symbols used are:O, PHA-stimulated lymphocytes; •¿�,resting lymphocytes.

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DNA REPAIR AND NITROSOUREA INDUCED SCEs IN LYMPHOCYTES

O.SmM

PHA-stimulated

24

Hours

48

Fig. 2. Time course of regeneration of alkyltransferase activity followingO'mGua in resting and PHA-stimulated lymphocytes. Freshly obtained resting

lymphocytes or lymphocytes cultured for 3 days in PHA were cultured in tissueculture medium containing 0.5 HIMO'mGua. After 24 h the cells were washedand recultured in fresh tissue culture media. At the times indicated 20 x 10" cells

were collected and prepared for assay of alkyltransferase activity. Data representsthe mean ±SEM of four donors. Symbols used are: O, PHA-stimulated lymphocytes; •¿�,resting lymphocytes.

units) than in the resting cells (5.9 ±0.3 units). In both restingand proliferating cells there was a dose-dependent inactivationof the alkyltransferase activity reaching the same low residuallevel of activity at a dose of 0.5 mM O'mGua. This represents

88% inactivation of alkyltransferase activity in proliferatingcells and 85% inactivation of activity in resting cells.

The time course of alkyltransferase inactivation and recoveryin resting and PHA-stimulated cells is shown in Fig. 2. In bothresting and proliferating cells, maximal inactivation occurredafter 18 h of exposure to 0.5 mM O'mGua. In the PHA-stimulated cells recovery of alkyltransferase activity began 6-18 h after removal of O'mGua reaching 5.8 units at 24 h and

almost baseline at 48 h. In contrast the alkyltransferase activityin the resting cells recovered slowly and remained only 38% ofcontrol 48 h after removal of O'mGua. A similar slow recovery

in alkyltransferase activity was observed if resting cells werecultured in 1% PHA after removal of the O'mGua (data not

shown). In these cultures, regeneration of alkyltransferase ac

tivity occurred at 48 h but not at 24 h which is consistent withthe time course of induction of the alkyltransferase activity byPHA (8).

Effect of O'mGua on SCE Formation and Cell Cycle Progres

sion. To examine whether inactivation of the alkyltransferasewould alter spontaneous SCE formation in normal lymphocytes, we exposed resting and proliferating lymphocytes to 0.5mM O'mGua for 24 h prior to the addition of PHA and BrdUrd

to visualize SCEs. Results of multiple experiments of threenormal donors are shown in Table 1. There was a small butstatistically significant increase in SCEs/metaphase in proliferating lymphocytes exposed to O'mGua (P < 0.0005). Inresting lymphocytes O'mGua did not increase the number of

SCEs.We have previously shown that continuous culture in the

presence of O'mGua decreases the growth rate of a number of

cell lines (27). A similar effect on lymphocyte proliferationkinetics was observed in these experiments by calculatingchanges in the replication index under different culture conditions. A change in the replication index indicates an effect oncell cycle time. The effect of a 24-h exposure to 0.5 mM O'mGua

on the replication index in our cultures is shown in Table 1.The replication index in the proliferating cells exposed toO'mGua was decreased over the control proliferating cells (ratioof exposed/control = 0.91) (P < 0.0005) suggesting that theremay be slowing of cell cycle progression by O'mGua. The

decrease in the replication index in cells that were resting duringO'mGua exposure was not statistically significant.

The effect of O'mGua on cell cycle progression in PHA-

stimulated lymphocytes was confirmed by measuring the incorporation of [3H]thymidine in cells cultured with or without 0.5mM O'mGua using a previously described method (8). A slightdecrease in [3H]thymidine incorporation was noted that rebounded to normal stimulated values as soon as O'mGua was

removed (data not shown). In subsequent experiments, lymphocytes were exposed to O'mGua for only 18-24 h which was

sufficient to maximally inactivate the alkyltransferase activitybut minimally affect cell cycle progression and proliferation.

Role of the Alkyltransferase in BCNU-induced SCEs. BCNUcaused a dose-dependent increase in SCEs present in bothresting and proliferating lymphocytes (Fig. 3). The inductionof SCEs was linear over BCNU doses of 0-15 ^M allowing thecalculation of SCEs/metaphase/^M BCNU. At these low doses

Table 1 Effect of (f-methylguanine on SCEs and replication in resting and proliferating human lymphocytesLymphocytes were cultured for 24 h in tissue culture media with or without the addition of 1% PHA or 0.5 mM O'mGua prior to the addition of 10 pg/ml BrdUrd

and the determination of SCE as described in "Materials and Methods." For each individual experiment 25 metaphases were scored for SCEs and 100 metaphasesfor replication index as described in "Materials and Methods." The data shown represent the mean ±SD of the individual experimental means for N Z 3. Statisticalsignificance was determined using a paired I test comparing control and drug-treated populations.

Donor (no. of experiments) Condition SCE (x ±SD) Rl (x ±SD)

Restinglymphocytes1(3)control+

0.5 mMO'mGua2(2)

control+0.5 mMO'mGuaX

control+0.5 mMO'mGuaProliferating

Lymphocytes1(7)control+

0.5 mMO'mGua2(1)

control+0.5 mMO'mGua3(3)

control+0.5 mMO'mGuaX

control+0.5 mM O'mGua7.48

±2.298.14±1.028.089.527.72

±1.678.69±1.13(0.1

>/>>0.05)7.53

±1.049.03±1.869.8011.686.81

±0.167.88±0.847.82±1.348.96±1.82P<0.00052.26

±0.161.98±0.492.092.002.

19±0.241.99±0.37(0.1>A>>0.05)2.47

±0.332.05±0.542.151.562.74

±0.251.87±0.132.51±0.341.95±0.45P<

0.0005

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DNA REPAIR AND NITROSOUREA INDUCED SCEs IN LYMPHOCYTES

Resting•¿�f06mGua

PHA-Stimulated+ 06mGua

Fig. 3. Effect of alkyltransferase inactivación on BCNU-induced SCEs inresting and proliferating human lymphocytes. Lymphocytes were cultured for 24or 72 h in tissue culture medium with or without the addition of 1% PHA and0.5 HIMO*mGua prior to exposure to BCNU and determination of SCE. Eachdata point represents the mean ±SEM of two to seven experiments (50-175mctaphases) using lymphocytes from three different donors.

of BCNU, resting and proliferating lymphocytes were equallysensitive to BCNU-induced SCEs (0.76 SCEs/metaphase/MMBCNU, resting versus 0.79 SCEs/metaphase/MM BCNU, proliferating). Inactivation of the alkyltransferase by E/rnGua significantly increased the level of BCNU-induced SCEs in bothresting and proliferating cells (Fig. 3). In proliferating lymphocytes, C^mGua pretreatment resulted in a 2.6-fold enhancementof SCE formation by BCNU (2.02 SCEs/metaphase/MM BCNUwith C^mGua versus 0.79 SCEs/metaphase/MM BCNU withoutE/rnGua, P < 0.002). An identical effect was observed inlymphocytes incubated with PHA for 24, 48, or 72 h prior tof/mGua exposure (data not shown).

The effect of C^mGua in resting lymphocytes was evengreater than that seen in proliferating cells. In these cells,(/mGua pretreatment resulted in a 4-fold increase in SCEinduction by BCNU (3.08 SCEs/metaphase/MM BCNU withiAnGua versus 0.76 SCEs/metaphase/MM BCNU withoutC^mGua, P< 0.001). Thus, following inactivation of the alkyltransferase by C^mGua, both resting and proliferating lymphocytes became much more sensitive to SCE induction by BCNU.However, this effect was significantly more pronounced inresting than proliferating cells (P< 0.001). A likely explanationfor this is that proliferating lymphocytes recover alkyltransferase activity within the first 6-18 h following ¿X'mGuaexposure

whereas resting cells do not (Fig. 2).

DISCUSSION

We have evaluated the role of the DNA repair protein, O6-

alkylguanine DNA alkyltransferase, in the induction of SCEsby BCNU in resting and proliferating human lymphocytes.Mitogen stimulation of lymphocytes alters both the baselinealkyltransferase levels and the kinetics of recovery of alkyltransferase after its depletion. These alterations in alkyltransferaseactivity are a prime determinant of the ability of the nitrosoureaBCNU to induce SCEs in lymphocytes.

We have previously observed that the alkyltransferase activityin PHA-stimulated lymphocytes increases to 1.5 times controllevels 48 h after mitogen stimulation (8). Despite higher alkyltransferase activity in the proliferating cells we show here thatresting and proliferating lymphocytes are equally sensitive toSCE induction by low doses of BCNU. This suggests that thereis a threshold level of alkyltransferase activity necessary toprevent those BCNU-induced SCEs mediated by C^-alkylguan-

ine lesions much as has been observed to prevent DNA crosslinkformation following l-(2-chloroethyl)-3-cyclohexyl-l -nitrosourea (34). Normally, the alkyltransferase activity is above thisthreshold in both the resting and proliferating cells. When thealkyltransferase is inactivated by C^mGua prior to BCNU exposure, the repair capacity falls below the threshold allowing¿/-alkylguanine lesions to persist. The result is increased SCEsin both the resting and proliferating cells although a greaterincrease in SCE formation is seen in the resting cells.

There are two interpretations for the greater sensitivity ofresting versus proliferating lymphocytes to BCNU-inducedSCEs following alkyltransferase depletion by O'mGua. First,

resting lymphocytes consistently had a slightly lower level ofalkyltransferase immediately following C^mGua than proliferating cells, although this difference was not statistically significant. If the threshold hypothesis is correct, this small differencecould make the resting cells more susceptible to SCE formation.Second, a very large difference between resting and proliferatingcells emerged 6-18 h following removal of i/mGua. Proliferating cells begin to regenerate alkyltransferase activity reaching35% of pretreatment values 18 h after O'mGua is removed

whereas no change was seen during this time period in restingcells. We would suggest that this regeneration of alkyltransferase activity provides partial protection against SCE formation.Although this hypothesis could be tested by preventing regeneration of the alkyltransferase by continued exposure toO'mGua, this compound decreases the rate of DNA synthesis

and appears to slow cell cycle time when given continuously,both of which could alter the formation of SCEs and confoundthe interpretation of the data.

Our data do not allow for precise determination of the criticalthreshold value of alkyltransferase activity but do illustrate thatthe ability to regenerate alkyltransferase activity may be important in protecting cells against BCNU induced SCEs. Shouldbiochemical modulation of the alkyltransferase become an important target for cancer chemotherapy, these data indicate that¿T'mGuahas differential effects on resting and proliferating

cells. This difference could contribute both to the therapeuticefficacy and toxicity observed with the combination of C^mGuagiven to inactivate the alkyltransferase, and BCNU, given asthe therapeutic agent.

Alkyltransferase activity is known to be an important mechanism of resistance to SCE formation induced by agents whichproduce alkylation at the O6 position of guanine, especially (?-chloroethyl-alkylation which can lead to interstrand cross-links(17). Cells deficient in the alkyltransferase are more sensitiveto nitrosourea induced SCEs than cells with higher alkyltransferase activity (15-17). Inactivation of the alkyltransferase withMNU prior to exposure of human lymphocytes to BCNUresults in potentiation of SCE formation (26). Although MNUand BCNU both cause alkylation at O'-guanine, BCNU is a

more potent inducer of SCEs because it causes interstrandcross-links (23).

Brent et al. have recently elicited an additional mechanismby which cross-link precursors are repaired by the alkyltransferase (35). In his scheme, the initial 06-chloroethylguanineDNA adduci can undergo an internal rearrangement to OW-ethanoguanine both of which can either react with the alkyltransferase or go on to a cross-link reaction. In the latter case,the reaction yields an alkyltransferase DNA cross-link (35).Although this DNA-protein cross-link is likely to be less cyto-toxic and less prone to the induction of SCEs than the DNAinterstrand cross-link, repair of either adduct is important indetermining cytotoxicity and SCE formation. Data from Brent

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DNA REPAIR AND NITROSOUREA INDUCED SCEs IN LYMPHOCYTES

(35) and Kohn (36) indicate that a period of 8-12 h is requiredfor this intermediate and the subsequent cross-link to form afterBCNU exposure. Thus, changes in the level of alkyltransferaseactivity during this period would critically influence the numberof SCEs produced especially following inactivation of the alkyltransferase with i/mGua. Since proliferating cells as opposed to resting cells begin to regenerate the alkyltransferaseduring this period, alkyltransferase mediated repair of thesecross-link intermediates may be an important mechanism ofBCNU resistance.

The alkyltransferase is not the only DNA repair enzymewhich is induced in proliferating lymphocytes. The excisionrepair system (9), polymerase ß(10, 11), DNA ligase (12),uracil-DNA glycosylase (13), and poly(ADP ribose) polymerase(14) are all altered by mitogen stimulation. These repair systemsare likely to be important in modulating the extent of SCEformation by other DNA-damaging agents, and may explainthe variable effects of mitogen stimulation on SCE inductionby different chemical carcinogens. We have shown that changesin the sensitivity to SCE induction after exposure to chemicalssuch as BCNU which produce 06-alkylguanine can be explained

by corresponding changes in the levels of the alkyltransferase.As we have done with the alkyltransferase, it is possible togauge the impact of a specific DNA repair system during thetransition from the resting to the proliferating state by specifically inhibiting that system and then observing the response toDNA damage.

Monitoring human exposure to mutagenic, carcinogenic, andchemotherapeutic agents is frequently performed through theanalysis of SCE induction in human lymphocytes (1). Ourstudies indicate that a systematic analysis of the DNA repaircapacity corresponding to the type of DNA damage anticipatedas well as analysis of the state of proliferation of the cells areessential to properly interpret the relationship between toxinexposure and SCE formation.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Paul Howard for synthesis off/'mt ina. Kathleen Miller for technical assistance, and Anne LaGania

and Joyce Rusnak for help in preparation of the manuscript.

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1989;49:1899-1903. Cancer Res   Joan E. Trey and Stanton L. Gerson  Proliferating Human LymphocytesNitrosourea-induced Sister Chromatid Exchanges in

-Alkylguanine DNA Alkyltransferase in Limiting6OThe Role of

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